Efficacy_of_High-Ozonide_Oil_in_Prevention_of_Cancer
Artigo originalmente publicado em https://www.mdpi.com/2072-6694/14/5/1174
Citation: Izzotti, A.; Fracchia, E.;
Rosano, C.; Comite, A.; Belgioia, L.;
Sciacca, S.; Khalid, Z.; Congiu, M.;
Colarossi, C.; Blanco, G.; et al.
Efficacy of High-Ozonide Oil in
Prevention of Cancer Relapses
Mechanisms and Clinical Evidence.
Cancers 2022,14, 1174. https://
doi.org/10.3390/cancers14051174
Academic Editor: David
N. Danforth
Received: 12 January 2022
Accepted: 22 February 2022
Published: 24 February 2022
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cancers
Article
Efficacy of High-Ozonide Oil in Prevention of Cancer Relapses
Mechanisms and Clinical Evidence
Alberto Izzotti 1,2,* , Enzo Fracchia 3, Camillo Rosano 2, Antonio Comite 4, Liliana Belgioia 2,5 ,
Salvatore Sciacca 6, Zumama Khalid 5, Matteo Congiu 5, Cristina Colarossi 6, Giusi Blanco 6,
Antonio Santoro 7,8, Massimo Chiara 7,8 and Alessandra Pulliero 5
1Department of Experimental Medicine, University of Genoa, 16132 Genoa, Italy
2IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy; camillo.rosano@hsanmartino.it (C.R.);
liliana.belgioia@unige.it (L.B.)
3Galliera Hospital, 16128 Genoa, Italy; enzo.fracchia@galliera.it
4
Laboratory of Electron Microscopy, Department of Chemistry and Industrial Chemistry, University of Genoa,
16146 Genoa, Italy; antonio.comite@unige.it
5
Department of Health Sciences, University of Genoa, 16132 Genoa, Italy; zumama.khalid@edu.unige.it (Z.K.);
3370203@studenti.unige.it (M.C.); alessandra.pulliero@unige.it (A.P.)
6
Mediterranean Institute of Oncology (IOM), 95029 Catania, Italy; salvatore.sciacca@grupposamed.com (S.S.);
cristina.colarossi@grupposamed.com (C.C.); giusi.blanco@grupposamed.com (G.B.)
7UO Neurosurgery, Hospital Umberto I, 00161 Rome, Italy; antonio.santoro@uniroma1.it (A.S.);
massimo.chiara@uniroma1.it (M.C.)
8Department of Surgery, La Sapienza University, 00185 Rome, Italy
*Correspondence: izzotti@unige.it; Tel.: +39-010-3538522
Simple Summary:
Cancer relapses after chemo-radiotherapies arise from cancer stem cells able
to escape cell killing because of their high antioxidants level. The aim of this study was to test
the efficacy of ozonized oils to decrease the rate of cancer relapses.
In vitro
, oils at high ozonide
content penetrate inside cancer cells releasing oxygen and reactive oxygen species damaging the
thin outer membrane of inactive mitochondria. This event triggers intracellular calcium release
and activates apoptosis.
In vivo
, ozonized oil has been administered by the oral route effectively
decreasing blood antioxidants in cancer patients. This approach results in significant increase of
survival rate and decrease of relapses in 115 cancer patients (brain, lung, pancreas, colon, skin)
undergoing standard radio-chemotherapy regimens during a 4-years follow up. Obtained results
indicate that the administration of ozonized oil represents an integrated approach to decrease the risk
of radio-chemoresistance and cancer relapses in cancer patients.
Abstract:
Background: Cancer tissue is characterized by low oxygen availability triggering neo
angiogenesis and metastatisation. Accordingly, oxidation is a possible strategy for counteracting
cancer progression and relapses. Previous studies used ozone gas, administered by invasive methods,
both in experimental animals and clinical studies, transiently decreasing cancer growth. This study
evaluated the effect of ozonized oils (administered either topically or orally) on cancer, exploring
triggered molecular mechanisms. Methods:
In vitro
, in lung and glioblastoma cancer cells, ozonized
oils having a high ozonide content suppressed cancer cell viability by triggering mitochondrial
damage, intracellular calcium release, and apoptosis.
In vivo
, a total of 115 cancer patients (age
58 ±14 years
; 44 males, 71 females) were treated with ozonized oil as complementary therapy in
addition to standard chemo/radio therapeutic regimens for up to 4 years. Results: Cancer diagnoses
were brain glioblastoma, pancreas adenocarcinoma, skin epithelioma, lung cancer (small and non-
small cell lung cancer), colon adenocarcinoma, breast cancer, prostate adenocarcinoma. Survival rate
was significantly improved in cancer patients receiving HOO as integrative therapy as compared
with those receiving standard treatment only. Conclusions: These results indicate that ozonized oils
at high ozonide may represent an innovation in complementary cancer therapy worthy of further
clinical studies.
Cancers 2022,14, 1174. https://doi.org/10.3390/cancers14051174 https://www.mdpi.com/journal/cancers
Cancers 2022,14, 1174 2 of 24
Keywords:
cancer prevention; cancer therapy; ozonized oil; prevention of cancer relapses; oxidation
1. Introduction
Cancer cells differ from normal cells in several aspects, among which the blockage
of the mitochondrial function stands out. Mitochondrion is the main endogenous source
of oxidizing molecules. The mitochondrial blockage occurring in cancer cells is known
as the Warburg effect [
1
]. Recent experimental research provided evidence that cancer
cells are favored in their growth by antioxidant molecules but contrasted by pro-oxidant
molecules. Cancer stem cells, which give rise to chemo/radio resistance and relapses,
are characterized by a reducing environment, therefore being sensitive to the cytotoxic
effects of oxidative damage [
2
]. Cancer tissue is characterized by a very low level of
lipid peroxidation compared with the surrounding healthy tissues, as shown by biopsy
analysis of 120 patients with primary hepatocarcinoma [
3
]. Accordingly, today the increase
in oxidative damage represents a possible strategy for cancer therapy [
4
]. Intracellular
generation of reactive oxygen species has been proposed as a possible Trojan horse for
eliminating cancer cells [5].
Several attempts have been made in order to use ozone as a source of oxidizing species
in cancer therapy. Ozone was used as a gas [
6
] or as an ozonized aqueous solution [
7
].
These studies reported specific cytotoxic effects of ozone on cancer tissues without any
damage in healthy tissues. However, the therapeutic effect obtained was meaningful but
transitory. When used as a gas or aqueous solution, ozone, due its volatility, displays
transitory effects only in the extracellular environment. Cells are surrounded by a lipophilic
membrane hampering the entry of gas or water. Cancer cells are well equipped with antiox-
idants molecules to counteract cancer chemo/radio therapies. Accordingly, an oxidative
intracellular environment may be effective in counteracting cancer chemo/radioresistance.
Furthermore, the setting up of a high level of oxygen in the cancer mass may be useful for
preventing metastases development. Indeed, low oxygen availability (hypoxia) is the main
mechanism triggering the migration of cancer cells from the primary site. Hypoxia pro-
motes cancer invasion and metastatisation by activating the met oncogene [
8
]. Hyperbaric
ozone has been proposed as a possible tool for preventing these events [9].
In this context, microRNA-based epigenetic regulation plays an important role. Mi-
croRNA alterations drive cancer cells towards chemo/radioresistance [
10
] and modulate
oxidative stress in cancer cells [
11
]. miR-146 plays a fundamental role in lung cancer
progression and chemoresistance [12].
Increase in antioxidants such as reduced glutathione induces multi-drug resistance in
neuroblastoma [
13
]. Downregulation of miR-15 and miR-16 is associated with increased
availability of reduced glutathione in neuroblastoma cancer cells, contributing to chemore-
sistance [14].
The antioxidant environment characterizing cancer cells is related to the constitutive
overexpression of Nrf2; the pivotal transcription factor regulating the activation of antioxi-
dant response elements [
15
]. Nrf2 activity provides growth advantage by increasing cancer
chemoresistance and enhancing tumor cell growth [
16
]. Overexpression of Nrf2 in cancer
cells protects them from the cytotoxic effects of anticancer therapies, resulting in chemo-
and/or radioresistance [17].
In this context, the herein presented study aimed at evaluating the efficacy of pharma-
cological preparations composed of ozonized oils in counteracting the growth of cancer
cells. Ozonized oils are very stable molecules displaying antimicrobial properties [
18
]. The
main goal of our study was the development of high-ozonide ozonized oil (HOO) to deliver
a high amount of ozone-derived oxidizing species in a lipophilic complex able to penetrate
the cancer cells and to activate apoptosis without damaging healthy tissues. Ozonized oils
consist of unsaturated fatty acids that have been subjected to the action of ozone. The ozone
is added to the double carbon–carbon bonds, with the formation of molozonides. These
Cancers 2022,14, 1174 3 of 24
molecules quickly rearrange themselves according to the Criegee mechanism, causing the
formation of trioxanes. Ozonides are generally unstable, while trioxanes are relatively
stable but decompose under the action of reducing agents or intracellular enzymes. When
the addition of ozone to the oil reaches the saturation of the double bonds, the viscosity
increases with the progressive formation of ozonides until the oil reaches the consistency
of gelatin. The peroxides contained in the oil can be hydrolyzed, giving rise to aldehydes
and ketones with shorter chains compared with the original fatty acid. The length of the
residues is determined by the position of the double bond along with the chain that reacted
with ozone.
The goals of the herein presented experimental study were the evaluation of HOO:
(a) anticancer efficacy
in vitro
in cultured cancer cells; (b) molecular mechanism of action
at the intracellular level; (c) mechanism of action at the systemic level; (d) inability to
damage non-cancer cells; (e) anticancer efficacy and safety
in vivo
in human subjects and
cancer patients.
2. Materials and Methods
2.1. Experimental Evidence in Cultured Cancer Cells
HOO was tested in human lung adenocarcinoma cells (A549 cell line) (IRCCS Poli-
clinico San Martino, Genoa, Italy), and they were grown in D-MEM (GIBCO Invitrogen,
Milano, Italy) containing 10% fetal bovine serum (Sigma-Aldrich, Milano, Italy) at 37
◦
C in
5% CO
2
and 100% humidity. Non-ozonized sunflower oil was used as comparative sham
control. The experiment was performed in quadruplicate.
Glioblastoma U87MG cells were made available from the Biological Bank and Cell
Factory of the IRCCS Ospedale Policlinico San Martino, Genoa, Italy. They were grown in
DMEM high glucose media (Sigma-Aldrich, Milan, Italy), supplemented with 10% fetal
calf serum (Euroclone, Milan, Italy), 2 mM L-glutamine (Euroclone, Milan, Italy), and 1%
penicillin–streptomycin (Euroclone, Milan, Italy) at 37 ◦C in 5% CO2incubator.
Cells were seeded in 96-well plates at a density of 6
×
10
3
cell per well in 100 uL of
culture medium, and they were treated with 10% HOO for 2, 6, 12, and 24 h. Sham-treated
cells were used as control. After treatment, cells were washed with PBS (Euroclone, Milan,
Italy), fixed and stained with a solution of crystal violet containing methanol 20% v/v. The
following day, a solution of acetic acid 30% v/vin water was added, and samples were
read by a microplate photometer (Multiskan FC, Thermo Scientific) at 570 nm.
Cell viability was determined by Trypan blue staining (labeling dead cells) and MTT
test (labeling viable cells), as previously reported [19].
2.2. Pharmacological Mechanism. Experimental Evidence in Lung Cancer Cells
The dynamics whereby the HOO formulation induces the killing of cancer cells were
examined using a normal and trichrome fluorescence microscopy. The nucleus was stained
in blue by DAPI (Sigma), the mitochondrial membranes in green by DiOC6 (Sigma), and
calcium release into cytoplasm in red by Rhodamine2 (Sigma). HOO was stained by red
Nile dye (Sigma) to trace its penetration inside cell cytoplasm.
2.3. Field Emission Scanning Electron Microscopy and X-ray Diffraction Analyses
The HOO mechanism of killing cancer cells was also explored by a field emission
scanning electron microscope (FE-SEM, Zeiss Supra 40VP, Carl Zeiss, Germany) equipped
with energy dispersive X-ray analysis (EDX) microprobe for elemental analysis (Oxford
“INCA Energie 450
×
3”, Oxford Instruments, UK), comparatively examining sham-treated
and HOO-treated A549 cancer cells. EDX elemental analysis was performed at high magni-
fications (10,000
×
) with a spot at the middle of the cells before and after the HOO treatment.
2.4. Evaluation of Apoptosis
HOO was added to cell culture medium in order to discriminate the prevalent cell
death mechanism between necrosis and apoptosis. A549 human lung adenocarcinoma
Cancers 2022,14, 1174 4 of 24
cells were purchased from the Biological Bank and Cell Factory (IRCCS Policlinico San
Martino, Genoa, Italy). They were grown in DMEM medium (Sigma-Aldrich, Milan, Italy),
supplemented with 10% fetal calf serum (Euroclone, Milan, Italy), 2 mM L-glutamine
(Euroclone, Milan, Italy), and 1% penicillin–streptomycin (Euroclone, Milan, Italy) at 37
◦
C
in a 5% CO2incubator.
The day before the experiment, A549 cells were seeded in a 6-well plate at a density
of 8
×
10
4
cells per well in 3 mL of culture medium DMEM (Sigma-Aldrich, Milan, Italy).
After twenty-four hours of seeding, cells were treated with 10% v/vof HOO for 2 h and
4 h. Then, Muse
™
Annexin V & Dead Cell Assay was performed. Cells were dissociated
from each well to obtain single-cell suspensions, and 100
µ
L of these suspensions was
added to each tube, together with 100
µ
L of the Muse
™
Annexin V & Dead Cell Reagent
(BD Biosciences Pharmingen 2350 Qume Drive San Jose, CA, USA). The samples were
mixed thoroughly by vortexing and then stained at room temperature in dark for 20 min
before being analyzed by flow cytometry (FACS Canto II cytometer, Becton Dickinson BD,
Franklin Lakes, NJ, USA).
Microscope examination of cell morphology showed that in cancer cells treated with
ozonized oil, at 1 h cell viability is still maintained, while cell sufferance and lack of viability
is massive at 24 h. Accordingly, the mechanisms causing loss of cell viability should occur
in the 1–24 h time interval. Because the activation of apoptotic mechanisms requires at least
4 h, this was the timeline when we decided to evaluate this parameter.
2.5. Computational Bio-Structural Model Explaining the Selective Killing Cancer Cells
The 3D structure of cardiolipin, the main mitochondrion outer membrane monomer,
was reconstructed. Cardiolipin structural variations were determined according to the
presence or absence of cytochrome c binding in normal and cancer cells, respectively. The
binding between cardiolipin and HOO under all these conditions was analyzed. Cardiolipin
bilayer was built using the Membrane Builder generator from the CHARMM-GUI web
toolkit [
20
,
21
]. A bilayer was built using a deprotonate cardiolipin molecule in the same
way. We minimized both these structures by performing a short molecular dynamic (MD)
simulation using the software GROMACS [
22
]. For both systems, the pre-production
minimization and relaxation protocols were automatically generated by the CHARMM-
GUI Input Generator. They consisted of 5000 steps of energy minimization, keeping a
constant volume and a temperature of 303.15 K (NVT) using the Berendsen thermostat for
20,000 steps with a 1 fs time step. The production runs adopted a time step of 2 fs. In this
case, each MD was launched for total 1 ns of simulation.
2.6. Analysis of the Relationship between Ozonide Amount and Cancer Cell-Killing Effect
The relationship between ozonide amount and cancer cell-killing effect was examined
by analyzing comparatively 9 ozonized oils having different levels of ozonides. The
following formulations were evaluated by comparing them to the untreated control or to
control treated with non-ozonized sunflower oil (sham-control):
≤
100 ozonides (Ozone
Elite oil, Ozone cream oil 10, Oil olive O3 TuPiel),
≤
300 ozonides (VO3 active spray, Prog.
Olive oil, EMI sunflower oil, Oil Ozofarm),
≥
700 ozonides (HOO 700),
≥
1100 ozonides
(HOO 1100).
Anaplastic carcinoma cells A549 were grown in the presence of different ozonized
oils, as previously reported; their ability to induce cell death was assessed by crystal-
violet viability assay. For each formulation, the quantity of cells still vital after treatment
(percentage as compared with the untreated control bearing 100% vitality) was evaluated.
All the experiments were replicated 8 times for a total of 88 independent experimental
analyses (11 experimental conditions ×8 replicates).
2.7. Inability to Kill Normal Cells
The inability of HOO to induce cytopathic effects in healthy cells (safety) was tested
in primary differentiated human keratinocytes (Biological Bank and Cell Factory, IRCCS
Cancers 2022,14, 1174 5 of 24
Policlinico San Martino, Genoa, Italy) treated for 1–3 h with HOO, 80% v/vwith the culture
medium. The results were examined at 48 and 72 h after treatment.
2.8. Synergism with Radiotherapy
This experiment was performed to solve the problem of HOO application-timing in
relation to radiotherapy, i.e., whether a synergistic effect in killing cancer cells exists or
not. In the case of positive answer, it should be clarified whether to apply HOO before or
after the treatment with gamma radiation. To face these problems, an
in vitro
experiment
was performed in anaplastic carcinoma cells (A549) exposed to ionizing radiation (2 Gy)
undergoing HOO treatment either before or after radiation treatment. Cell survival was
evaluated by crystal violet staining, and results obtained in OHOO-treated cells compared
with those obtained in control cells exposed to radiation and treated with sunflower seed
oil (sham-control). The experiment consisted of 16 replicates in multi-well plate for a total
of 48 experiments (3 experimental conditions ×16 replicates).
2.9. Experimental Evidence in Human Subjects
The use of ozonized oil per os in human subjects as a food integrator was approved
by the Health Ministry of Malta (approval number 0075/2020 according to EC1924/2006)
issued on 17 March 2020.
Five healthy male subjects aged 49.2
±
12.7 years old were treated for 1 week, admin-
istering 12 mL of HOO per os per day. Blood samples were collected before administration
(T0) and after treatment (T1) and used for immunological analyses by FACS. The influence
of HOO on blood monocytes was performed using HLAdr monocyte activation marker. NK
and helper lymphocytes counts were performed using CD3 and CD4 markers. FACS analy-
ses were performed using a LSR Fortessa X20 (Becton and Dickinson,
Eysins, Switzerland
).
A total of 115 cancer patients (age 58
±
14 years; 44 males, 71 females) were treated with
HOO contained in cellulose pills for 8 months as complementary therapy. The treatment
was performed in parallel to standard chemo/radio therapeutic regimens performed for
each cancer type according to the international guidelines.
The cancer diagnoses were the followings: brain (glioblastoma and astrocytoma) 22,
pancreas adenocarcinoma 18, skin epithelioma (squamous and basal) 7, lung (NSCLC
and small cell lung cancer) 12, colon adenocarcinoma 13, breast cancer (estrogen receptor
positive) 24, prostate adenocarcinoma 7 (Gleason severity score >8), ovary and womb 5,
kidney and bladder 5, non-Hodgkin’s skin lymphoma 2.
Cancer status at T0 (before HOO administration) and T1 (after HOO treatment) was
examined by NMR, TAC, and PET, as performed for standard follow-up regimens according
to international guidelines. Blood analyses were performed monthly. Amounts of oxidant
(H
2
O
2
milli-equivalent per 100 mL) and antioxidant (ascorbic acid milli-equivalent per 100
mL) in blood were examined monthly by the Free Radical Analysis System using a Fras4
Evolvo System (H&D, Parma, Italy) [23,24].
3. Results
3.1. Comparative Analysis of Various Ozonized Oils
A battery of oils having different ozonide content was tested for its ability to kill A549
cancer cells by evaluating the decrease in cell viability. The results obtained are shown in
Figure 1.
Cancers 2022,14, 1174 6 of 24
Cancers 2022, 14, 6 of 25
result were the HOOs. In fact, the percentage of surviving cells was only 6.8% in HOO 700
ozonide and reached the minimum value detected of 2.7% in HOO 1100 ozonide. The
EC50 was calculated according to the exponential regression equation between ozonide
and cell viability, obtaining a value of 433 ozonides. It is noteworthy that the dose–re-
sponse effect observed for HOO depends on the amount of ozonide. This experiment
showed that the amount of ozonide is the key element of the killing effect of ozonized oils
against cancer cells.
Accordingly, HOO 1100 was selected for further analyses because of the highest level
of cancer cell killing efficacy as compared with the other ozonized oils. Indeed, in HOO,
ozonide content is equivalent to 800 meq O2/kg, corresponding to 220 mg of O3.
Figure 1. Comparative evaluation of ozonized oil effect on cancer cell viability (MTT test). Only
ozonized oils having an ozonide content >700 decrease cancer cell viability below 10% in 24 h.
3.2. Experimental Evidence in Lung Cancer Cells
Human lung cancer A549 untreated cells and cells treated with sunflower oil (sham)
rapidly grew and reached to confluency after 72 h. Conversely, A549 cells treated with
HOO showed impaired growth during first 24 h. After this time, they rapidly underwent
cell death, which culminated at 72 h. Cell death was characterized by (a) disappearance
of the cell-growth carpet; (b) presence of dead cells in the supernatant; (c) diffuse apoptotic
bodies. These results are shown in Figure 2. The lack of viability of cancer cells treated
with HOO was also demonstrated at 24 h by Trypan blue staining selectively labelling
only death cells (Figure 3).
Figure 1.
Comparative evaluation of ozonized oil effect on cancer cell viability (MTT test). Only
ozonized oils having an ozonide content >700 decrease cancer cell viability below 10% in 24 h.
Cancer cell viability was observed to be decreased in the presence of ozonized oils
compared with controls. However, ozonized oils at low ozonide were unable to reduce
the percentage of surviving cells to under 10%. The only formulations able to achieve this
result were the HOOs. In fact, the percentage of surviving cells was only 6.8% in HOO
700 ozonide and reached the minimum value detected of 2.7% in HOO 1100 ozonide. The
EC50 was calculated according to the exponential regression equation between ozonide
and cell viability, obtaining a value of 433 ozonides. It is noteworthy that the dose–response
effect observed for HOO depends on the amount of ozonide. This experiment showed
that the amount of ozonide is the key element of the killing effect of ozonized oils against
cancer cells.
Accordingly, HOO 1100 was selected for further analyses because of the highest level
of cancer cell killing efficacy as compared with the other ozonized oils. Indeed, in HOO,
ozonide content is equivalent to 800 meq O2/kg, corresponding to 220 mg of O3.
3.2. Experimental Evidence in Lung Cancer Cells
Human lung cancer A549 untreated cells and cells treated with sunflower oil (sham)
rapidly grew and reached to confluency after 72 h. Conversely, A549 cells treated with
HOO showed impaired growth during first 24 h. After this time, they rapidly underwent
cell death, which culminated at 72 h. Cell death was characterized by (a) disappearance of
the cell-growth carpet; (b) presence of dead cells in the supernatant; (c) diffuse apoptotic
bodies. These results are shown in Figure 2. The lack of viability of cancer cells treated
with HOO was also demonstrated at 24 h by Trypan blue staining selectively labelling only
death cells (Figure 3).
Cancers 2022,14, 1174 7 of 24
Cancers 2022, 14, 7 of 25
Figure 2. Time-dependent in vitro growth of A549 lung cancer cells either sham-treated with sun-
flower oil (upper row) or treated with ozonide oil >700 ozonides with a percentage v/v of ozonized
oil related to sunflower of 97% (lower row). Sham-treated cells grow rapidly reaching semi-conflu-
ence at 24 h and full confluence at 48 h. Cancer cells treated with ozonide oil already display im-
paired growth during the first 24 h. After this time, they rapidly undergo time-related increasing
cell death, culminating at 72 h.
Figure 3. Detection of non-viable cells by Trypan blue staining after 24 h since seeding of A549
cancer cells, either sham-treated with sunflower seed oil (left panels) or treated with ozonide oil
>700 ozonides (right panels). Upper panels, standard microscopy; lower panels microscopy after
Trypan blue staining. Death cells are detected only in ozonide oil treatment.
3.3. Experimental Evidence in Glioblastoma Cancer Cells
After 12 h of HOO treatment, U87MG showed 4.68% of cell viability, as evaluated by
MTT test, while A549 showed 8.43% of cell viability. After 24 h of treatment, U87MG
showed 7.69% of cell viability and A549 showed 12.16% of cell viability. Accordingly, gli-
oblastoma U87MG cells were more sensitive to HOO than lung adenocarcinoma A549
cells. This finding was well evident after 12 and 24 h of treatment. Regarding shorter time,
there was no significant time difference between the two cell lines tested. Indeed, after 2
h the percentage of live cells was 13.91% for U87MG and 13.51% for A549; after 6 h of
HOO treatment U87MG showed 6.23% of cell viability, while A549 showed 7.58%. These
results are demonstrated in Figure 4.
Figure 2.
Time-dependent
in vitro
growth of A549 lung cancer cells either sham-treated with sun-
flower oil (upper row) or treated with ozonide oil >700 ozonides with a percentage v/vof ozonized oil
related to sunflower of 97% (lower row). Sham-treated cells grow rapidly reaching semi-confluence
at 24 h and full confluence at 48 h. Cancer cells treated with ozonide oil already display impaired
growth during the first 24 h. After this time, they rapidly undergo time-related increasing cell death,
culminating at 72 h.
Cancers 2022, 14, 7 of 25
Figure 2. Time-dependent in vitro growth of A549 lung cancer cells either sham-treated with sun-
flower oil (upper row) or treated with ozonide oil >700 ozonides with a percentage v/v of ozonized
oil related to sunflower of 97% (lower row). Sham-treated cells grow rapidly reaching semi-conflu-
ence at 24 h and full confluence at 48 h. Cancer cells treated with ozonide oil already display im-
paired growth during the first 24 h. After this time, they rapidly undergo time-related increasing
cell death, culminating at 72 h.
Figure 3. Detection of non-viable cells by Trypan blue staining after 24 h since seeding of A549
cancer cells, either sham-treated with sunflower seed oil (left panels) or treated with ozonide oil
>700 ozonides (right panels). Upper panels, standard microscopy; lower panels microscopy after
Trypan blue staining. Death cells are detected only in ozonide oil treatment.
3.3. Experimental Evidence in Glioblastoma Cancer Cells
After 12 h of HOO treatment, U87MG showed 4.68% of cell viability, as evaluated by
MTT test, while A549 showed 8.43% of cell viability. After 24 h of treatment, U87MG
showed 7.69% of cell viability and A549 showed 12.16% of cell viability. Accordingly, gli-
oblastoma U87MG cells were more sensitive to HOO than lung adenocarcinoma A549
cells. This finding was well evident after 12 and 24 h of treatment. Regarding shorter time,
there was no significant time difference between the two cell lines tested. Indeed, after 2
h the percentage of live cells was 13.91% for U87MG and 13.51% for A549; after 6 h of
HOO treatment U87MG showed 6.23% of cell viability, while A549 showed 7.58%. These
results are demonstrated in Figure 4.
Figure 3.
Detection of non-viable cells by Trypan blue staining after 24 h since seeding of A549
cancer cells, either sham-treated with sunflower seed oil (left panels) or treated with ozonide oil
>700 ozonides (right panels). Upper panels, standard microscopy; lower panels microscopy after
Trypan blue staining. Death cells are detected only in ozonide oil treatment.
3.3. Experimental Evidence in Glioblastoma Cancer Cells
After 12 h of HOO treatment, U87MG showed 4.68% of cell viability, as evaluated
by MTT test, while A549 showed 8.43% of cell viability. After 24 h of treatment, U87MG
showed 7.69% of cell viability and A549 showed 12.16% of cell viability. Accordingly,
glioblastoma U87MG cells were more sensitive to HOO than lung adenocarcinoma A549
cells. This finding was well evident after 12 and 24 h of treatment. Regarding shorter time,
there was no significant time difference between the two cell lines tested. Indeed, after 2 h
the percentage of live cells was 13.91% for U87MG and 13.51% for A549; after 6 h of HOO
treatment U87MG showed 6.23% of cell viability, while A549 showed 7.58%. These results
are demonstrated in Figure 4.
Cancers 2022,14, 1174 8 of 24
Cancers 2022, 14, 8 of 25
Figure 4. Viability comparison between human lung adenocarcinoma (A549, A) and glioblastoma
(U87MG, B) cell lines treated with HOO for different times (2, 6, 12, and 24 h).
3.4. Pharmacological Mechanism. Experimental Evidence in Lung Cancer Cells. Penetration
inside Cell Cytoplasm
Sunflower seed oil and HOO penetration inside A549-treated cells was traced by mi-
croscope light scattering. Sunflower seed oil (sham-control) penetrated the cells (cyto-
plasm) only in a minimal amount and was compartmentalized (closed) into small well-
defined vacuoles. Conversely, ozonized oil (HOO) penetrated abundantly into the cyto-
plasm, likely due to its peculiar ability to oxidize cell membranes, in particular the plas-
matic membrane that delimits the cell from the external environment. Once penetrating
the cytoplasm, the ozonized oil was initially compartmentalized into vacuoles; however,
the membranes of these vacuoles were rapidly oxidized, and the oil spread into the cyto-
plasm overlapping intracellular membranes and organelles (Figure 5).
These events culminated after 24 h and were followed by death of cells treated with
HOO.
Figure 5. HOO penetration inside A549 lung cancer cells, as traced by microscope light scattering.
Figure 4.
Viability comparison between human lung adenocarcinoma (A549,
A
) and glioblastoma
(U87MG, B) cell lines treated with HOO for different times (2, 6, 12, and 24 h).
3.4. Pharmacological Mechanism. Experimental Evidence in Lung Cancer Cells. Penetration inside
Cell Cytoplasm
Sunflower seed oil and HOO penetration inside A549-treated cells was traced by mi-
croscope light scattering. Sunflower seed oil (sham-control) penetrated the cells (cytoplasm)
only in a minimal amount and was compartmentalized (closed) into small well-defined
vacuoles. Conversely, ozonized oil (HOO) penetrated abundantly into the cytoplasm, likely
due to its peculiar ability to oxidize cell membranes, in particular the plasmatic membrane
that delimits the cell from the external environment. Once penetrating the cytoplasm, the
ozonized oil was initially compartmentalized into vacuoles; however, the membranes of
these vacuoles were rapidly oxidized, and the oil spread into the cytoplasm overlapping
intracellular membranes and organelles (Figure 5).
Cancers 2022, 14, 8 of 25
Figure 4. Viability comparison between human lung adenocarcinoma (A549, A) and glioblastoma
(U87MG, B) cell lines treated with HOO for different times (2, 6, 12, and 24 h).
3.4. Pharmacological Mechanism. Experimental Evidence in Lung Cancer Cells. Penetration
inside Cell Cytoplasm
Sunflower seed oil and HOO penetration inside A549-treated cells was traced by mi-
croscope light scattering. Sunflower seed oil (sham-control) penetrated the cells (cyto-
plasm) only in a minimal amount and was compartmentalized (closed) into small well-
defined vacuoles. Conversely, ozonized oil (HOO) penetrated abundantly into the cyto-
plasm, likely due to its peculiar ability to oxidize cell membranes, in particular the plas-
matic membrane that delimits the cell from the external environment. Once penetrating
the cytoplasm, the ozonized oil was initially compartmentalized into vacuoles; however,
the membranes of these vacuoles were rapidly oxidized, and the oil spread into the cyto-
plasm overlapping intracellular membranes and organelles (Figure 5).
These events culminated after 24 h and were followed by death of cells treated with
HOO.
Figure 5. HOO penetration inside A549 lung cancer cells, as traced by microscope light scattering.
Figure 5. HOO penetration inside A549 lung cancer cells, as traced by microscope light scattering.
These events culminated after 24 h and were followed by death of cells treated
with HOO.
Cancers 2022,14, 1174 9 of 24
3.5. Mitochondrial Status and Intracellular Calcium Release
The dose-dependent release of intra-cytoplasmic calcium was detected by rhodamine
staining in cancer cells treated with HOO (Figure 6A). In the same cells, the mitochondrial
membranes were selectively stained by green dye (DiOC6), verifying the decrease in signal
intensity in HOO as compared with control cells treated with oil only (Figure 6B). This result
reflected the damage of mitochondrial membranes, as induced by ozonized oil treatment.
In cells receiving this treatment, it was also possible to observe the presence of large lipid
vacuoles mainly located in the perinuclear zone (Figure 6, arrows). We carried out the
characterization of these vacuoles by labeling them with LC3 in order to verify whether
they were autophagic vacuoles, but the results were negative. Vacuole staining performed
using dyes for lipid materials was not effective due to the rapid oxidative degradation
of the dyes (Nile red) that was observed. Therefore, it is likely that these vacuoles are
composed of oxidative lipids that cannot be catabolized by the cell. Because of this reason,
the HOO-treated cancer cell takes the characteristic appearance of a ‘foamy cell’, which is
characteristic of cells that accumulate oxidized lipids but cannot catabolize them due to the
high level of mitochondrial damage. Indeed, lipid catabolism through the beta-oxidation
biochemical pathway occurs inside mitochondria. The presence of these vacuoles in HOO-
treated cells further demonstrates that the HOO action is expressed preferentially and
directly towards the mitochondrial membranes.
Cancers 2022, 14, 9 of 25
3.5. Mitochondrial Status and Intracellular Calcium Release
The dose-dependent release of intra-cytoplasmic calcium was detected by rhodamine
staining in cancer cells treated with HOO (Figure 6A). In the same cells, the mitochondrial
membranes were selectively stained by green dye (DiOC6), verifying the decrease in sig-
nal intensity in HOO as compared with control cells treated with oil only (Figure 6B). This
result reflected the damage of mitochondrial membranes, as induced by ozonized oil
treatment. In cells receiving this treatment, it was also possible to observe the presence of
large lipid vacuoles mainly located in the perinuclear zone (Figure 6, arrows). We carried
out the characterization of these vacuoles by labeling them with LC3 in order to verify
whether they were autophagic vacuoles, but the results were negative. Vacuole staining
performed using dyes for lipid materials was not effective due to the rapid oxidative deg-
radation of the dyes (Nile red) that was observed. Therefore, it is likely that these vacuoles
are composed of oxidative lipids that cannot be catabolized by the cell. Because of this
reason, the HOO-treated cancer cell takes the characteristic appearance of a ‘foamy cell’,
which is characteristic of cells that accumulate oxidized lipids but cannot catabolize them
due to the high level of mitochondrial damage. Indeed, lipid catabolism through the beta-
oxidation biochemical pathway occurs inside mitochondria. The presence of these vacu-
oles in HOO-treated cells further demonstrates that the HOO action is expressed prefer-
entially and directly towards the mitochondrial membranes.
In Figure 6C, fluorescence in cultured cells was evaluated by opening both channels
(red and green) in order to check the overlay between mitochondrial membrane damage
(attenuated green light) and extramitochondrial calcium release (red). In the case of over-
lap, the resulting color was yellow, otherwise the green and red colors were maintained.
In HOO-treated cancer cells, the images showed a yellow staining. This result indi-
cates that the release of intracellular calcium (red) took place exactly from the mitochon-
drial membrane (green) damaged by HOO. In fact, this overlap (yellow color) did not exist
in control cells where the mitochondrial membranes were not damaged, and there was no
release of calcium from the mitochondria. Accordingly, the green color was maintained
and no red color was shown. This result indicates that in cancer cells treated with sun-
flower oil (sham-control), calcium remained compartmentalized inside mitochondria;
conversely, in HOO-treated cells, calcium was abundantly released from the mitochon-
dria and spread into the cytoplasm. This result shows that the intracellular induction of
mitochondrial damage with the consequent activation of intrinsic apoptosis was the main
mechanism underlying the anticancer action of HOO.
Figure 6. Intracellular calcium release as induced by ozonized oil in A549 lung cancer cells detected
by rhodamine staining (red) and fluorescence microscopy. Mitochondrial membranes are labeled
by DiOC6 staining (green). Red and green overlap reading both channels at the same time (right
columns) indicates that calcium is released from mitochondria (yellow). Nuclei are colored by DAPI
staining (blue).
Figure 6.
Intracellular calcium release as induced by ozonized oil in A549 lung cancer cells detected
by rhodamine staining (red) and fluorescence microscopy. Mitochondrial membranes are labeled
by DiOC6 staining (green). Red and green overlap reading both channels at the same time (right
columns) indicates that calcium is released from mitochondria (yellow). Nuclei are colored by DAPI
staining (blue).
In Figure 6C, fluorescence in cultured cells was evaluated by opening both channels
(red and green) in order to check the overlay between mitochondrial membrane damage
(attenuated green light) and extramitochondrial calcium release (red). In the case of overlap,
the resulting color was yellow, otherwise the green and red colors were maintained.
In HOO-treated cancer cells, the images showed a yellow staining. This result indicates
that the release of intracellular calcium (red) took place exactly from the mitochondrial
membrane (green) damaged by HOO. In fact, this overlap (yellow color) did not exist in
control cells where the mitochondrial membranes were not damaged, and there was no
release of calcium from the mitochondria. Accordingly, the green color was maintained and
no red color was shown. This result indicates that in cancer cells treated with sunflower oil
(sham-control), calcium remained compartmentalized inside mitochondria; conversely, in
HOO-treated cells, calcium was abundantly released from the mitochondria and spread
into the cytoplasm. This result shows that the intracellular induction of mitochondrial
Cancers 2022,14, 1174 10 of 24
damage with the consequent activation of intrinsic apoptosis was the main mechanism
underlying the anticancer action of HOO.
3.6. Field Emission Scanning Electron-Microscope and EDX Analyses
Figure 7shows the FE-SEM images of both the control and HOO-treated cells. At low
magnification (a, e), the change of morphology induced by the ozonide treatment on the
cells is evident. The HOO-treated cells underwent dramatic smoothing and rounding (b, f),
disintegration and death (c, g), and the size decreased (d, h).
The number of elements contained in cells was estimated by EDX analysis. The
carbon/oxygen ratio was high (C/O 4.3) in cancer cells, a situation envisaging the presence
of the reducing intracellular environment characterizing these cells (left panel). Conversely,
the carbon oxygen ratio became extremely low (C/O 1.5) in HOO-treated cancer cells, a
situation envisaging the occurrence of oxidative stress in the intracellular environment as
well as lipids and carbon-chain structure oxidation (right panel).
Cancers 2022, 14, 10 of 25
3.6. Field Emission Scanning Electron-Microscope and EDX Analyses
Figure 7 shows the FE-SEM images of both the control and HOO-treated cells. At low
magnification (a, e), the change of morphology induced by the ozonide treatment on the
cells is evident. The HOO-treated cells underwent dramatic smoothing and rounding (b,
f), disintegration and death (c, g), and the size decreased (d, h).
The number of elements contained in cells was estimated by EDX analysis. The car-
bon/oxygen ratio was high (C/O 4.3) in cancer cells, a situation envisaging the presence of
the reducing intracellular environment characterizing these cells (left panel). Conversely,
the carbon oxygen ratio became extremely low (C/O 1.5) in HOO-treated cancer cells, a
situation envisaging the occurrence of oxidative stress in the intracellular environment as
well as lipids and carbon-chain structure oxidation (right panel).
Figure 7. SEM images of the cells before (A–D) and after the ozonide oil (>700 ozonides) treatment
(E–H) at a magnification of ×2000 (A,E) and ×10,000.
3.7. Evaluation of Apoptosis
Annexin V & Dead Cell Assay showed that apoptosis is the main mechanism of cell
death affecting A549 cells treated with both HOO700 and 1100. Indeed, after two hours of
treatment, control cells showed 11.10% of total apoptosis, with a clear prevalence of late
apoptosis (40.90%) on early apoptosis (3.25%). Cells treated with HOO700 showed 30.35%
of total apoptosis, divided into late apoptosis (29.10%) and early apoptosis (1.25%).
HOO1100 determined 46.20% of total apoptosis (45% of late apoptosis and 1.20% of early
apoptosis). These results are shown in the upper panels of Figure 8.
Regarding 4 h of treatment, in control cells, 9.30% of total apoptosis (7.35% of late
apoptosis and 1.95% of early apoptosis) was detected, 50.80% in cells treated with
HOO700 (46.95% late and 3.85% early apoptosis) and 47.65% in cells treated with HOO
1100 (45% late apoptosis and 2.65% of early apoptosis). These results are shown in the
bottom panels of Figure 8.
Figure 7.
SEM images of the cells before (
A
–
D
) and after the ozonide oil (>700 ozonides) treatment
(E–H) at a magnification of ×2000 (A,E) and ×10,000.
3.7. Evaluation of Apoptosis
Annexin V & Dead Cell Assay showed that apoptosis is the main mechanism of cell
death affecting A549 cells treated with both HOO700 and 1100. Indeed, after two hours
of treatment, control cells showed 11.10% of total apoptosis, with a clear prevalence of
late apoptosis (40.90%) on early apoptosis (3.25%). Cells treated with HOO700 showed
30.35% of total apoptosis, divided into late apoptosis (29.10%) and early apoptosis (1.25%).
HOO1100 determined 46.20% of total apoptosis (45% of late apoptosis and 1.20% of early
apoptosis). These results are shown in the upper panels of Figure 8.
Regarding 4 h of treatment, in control cells, 9.30% of total apoptosis (7.35% of late
apoptosis and 1.95% of early apoptosis) was detected, 50.80% in cells treated with HOO700
(46.95% late and 3.85% early apoptosis) and 47.65% in cells treated with HOO 1100 (45%
late apoptosis and 2.65% of early apoptosis). These results are shown in the bottom panels
of Figure 8.
Cancers 2022,14, 1174 11 of 24
Cancers 2022, 14, 11 of 25
Figure 8. Apoptosis profile (Muse™ Annexin V & Dead Cell Assay) for A549 cells treated with 10%
v/v of OOAO 700 and OOAO 1100 and untreated cells (K). Profiles were determined 2 h (upper
panels) and 4 h (bottom panels) after treatment. Each plot has 4 quadrant markers, reflecting the
different cellular states: the upper left quadrant contains dead cells (necrosis), the upper right has
late apoptotic/dead cells (cells that are positive both for Annexin V and for cell death marker 7-
AAD, 7-Aminoactinomycin D), the lower left contains live cells, and the lower right early apoptotic
cells (cells that are positive only for Annexin V).
3.8. Computational Bio-Structural Model Explaining the Selective Killing of HOO towards Can-
cer Cells
The results of the molecular dynamic simulations suggested two different confor-
mations for the processed systems. In the case of active mitochondrion (healthy cell), the
cardiolipin bilayer was thick, tight, and symmetrical, with no breaks of continuity be-
tween the hydrophilic heads protruding into the cytoplasm. This solid structure pre-
vented the access to the hydrophobic tails of cardiolipin of the oxidizing radicals present
in cytoplasm, such as those carried by OHOO and indicated by red circles in Figure 5.
Therefore, the mitochondrion of the normal cell was resistant to the killing effects of
OHOO. This structural situation is reported in Figure 9 left panels.
Conversely, in cancer cells, cardiolipin modified its structure due to the absence of
the interaction with a functioning cytochrome c (the pivotal effector of aerobic glycolysis).
Under these circumstances, the angle of convergence of the hydrophobic legs with the
hydrophilic head was increased, a situation resulting in the divarication of the hydropho-
bic tails. This phenomenon occurred in all the cardiolipin monomers of the mitochondrial
membrane, amplifying this molecular variation on the whole mitochondrial membrane.
Thus, the mitochondrial membrane in cancer cell appeared at bioinformatic model thinner
than in normal cells. This divarication of the hydrophobic tails created breaks of continu-
ity between the hydrophilic heads of the cardiolipin that protrude into the cytoplasm,
allowing access of HOO-oxidizing radicals to the hydrophobic tails of cardiolipin. Accord-
ing to this computational model, the mitochondrion of cancer cell was specifically sensi-
tive to the damaging effects of HOO. This structural situation is reported in Figure 9 right
panels.
Figure 8.
Apoptosis profile (Muse
™
Annexin V & Dead Cell Assay) for A549 cells treated with 10%
v/vof OOAO 700 and OOAO 1100 and untreated cells (K). Profiles were determined 2 h (upper
panels) and 4 h (bottom panels) after treatment. Each plot has 4 quadrant markers, reflecting the
different cellular states: the upper left quadrant contains dead cells (necrosis), the upper right has
late apoptotic/dead cells (cells that are positive both for Annexin V and for cell death marker 7-AAD,
7-Aminoactinomycin D), the lower left contains live cells, and the lower right early apoptotic cells
(cells that are positive only for Annexin V).
3.8. Computational Bio-Structural Model Explaining the Selective Killing of HOO towards
Cancer Cells
The results of the molecular dynamic simulations suggested two different conforma-
tions for the processed systems. In the case of active mitochondrion (healthy cell), the
cardiolipin bilayer was thick, tight, and symmetrical, with no breaks of continuity between
the hydrophilic heads protruding into the cytoplasm. This solid structure prevented the
access to the hydrophobic tails of cardiolipin of the oxidizing radicals present in cytoplasm,
such as those carried by OHOO and indicated by red circles in Figure 5. Therefore, the mi-
tochondrion of the normal cell was resistant to the killing effects of OHOO. This structural
situation is reported in Figure 9left panels.
Conversely, in cancer cells, cardiolipin modified its structure due to the absence of
the interaction with a functioning cytochrome c (the pivotal effector of aerobic glycolysis).
Under these circumstances, the angle of convergence of the hydrophobic legs with the
hydrophilic head was increased, a situation resulting in the divarication of the hydrophobic
tails. This phenomenon occurred in all the cardiolipin monomers of the mitochondrial
membrane, amplifying this molecular variation on the whole mitochondrial membrane.
Thus, the mitochondrial membrane in cancer cell appeared at bioinformatic model thinner
than in normal cells. This divarication of the hydrophobic tails created breaks of continuity
between the hydrophilic heads of the cardiolipin that protrude into the cytoplasm, allowing
access of HOO-oxidizing radicals to the hydrophobic tails of cardiolipin. According to this
computational model, the mitochondrion of cancer cell was specifically sensitive to the
damaging effects of HOO. This structural situation is reported in Figure 9right panels.
Cancers 2022,14, 1174 12 of 24
Cancers 2022, 14, 12 of 25
OUTER MITOCHONDRIAL MEMBRANE.
CARDIOLIPIN MOLECULAR STRUCTURE CHANGES ACCORDING TO PROTONS AVAILABILITY
PROTONATED
(Kreb’s cycle ON)
(Normal cell)
NORMAL CELL
T
H
I
C
K
N
E
S
S
Ozonoized lipid
membrane thining (TEM)
T
H
I
C
K
N
E
S
S
HYDROPHILIC
HEAD
HYDROPHOBIC
LEGS
UNPROTONATED
(Kreb’s cycle OFF)
(Warburg effect in Cancer cells)
CANCER CELL
HYDROPHILIC
HEAD
HYDROPHOBIC
LEGS
Figure 9. Bioinformatic analysis of mitochondrial membrane in normal (left panels) and cancer
(right panels) cells. Mitochondrial membrane monomer cardiolipin undergoes a 10 A increase in
distance of hydrophobic tails when not bound with cytochrome c. This situation occurs in cancer
cells, resulting in mitochondrial membrane thinning. Accordingly, only in cancer cells can ozonized
lipid (red circles) reach the hydrophobic legs of cardiolipin, thus causing mitochondrial membrane
damage. This situation does not occur in normal cells, where ozonized lipid cannot reach this mo-
lecular target.
3.9. Inability to Kill Normal Cells
Results indicate that neither alteration of cell viability nor cytopathic effects occurred
in noncancer cells treated with HOO, as demonstrated in skin keratinocytes (Figure 10).
Figure 10. Ozonized oil (bubble oil in the culture medium 1, 2, 3 h) does not induce alterations in
normal human keratinocytes. No change in cell viability, intercellular adhesion, and substrate ad-
herence occur in ozonized oil-treated cells (Oil) as compared with untreated control.
Figure 9.
Bioinformatic analysis of mitochondrial membrane in normal (left panels) and cancer (right
panels) cells. Mitochondrial membrane monomer cardiolipin undergoes a 10 A increase in distance of
hydrophobic tails when not bound with cytochrome c. This situation occurs in cancer cells, resulting
in mitochondrial membrane thinning. Accordingly, only in cancer cells can ozonized lipid (red circles)
reach the hydrophobic legs of cardiolipin, thus causing mitochondrial membrane damage. This
situation does not occur in normal cells, where ozonized lipid cannot reach this molecular target.
3.9. Inability to Kill Normal Cells
Results indicate that neither alteration of cell viability nor cytopathic effects occurred
in noncancer cells treated with HOO, as demonstrated in skin keratinocytes (Figure 10).
Cancers 2022, 14, 12 of 25
OUTER MITOCHONDRIAL MEMBRANE.
CARDIOLIPIN MOLECULAR STRUCTURE CHANGES ACCORDING TO PROTONS AVAILABILITY
PROTONATED
(Kreb’s cycle ON)
(Normal cell)
NORMAL CELL
T
H
I
C
K
N
E
S
S
Ozonoized lipid
membrane thining (TEM)
T
H
I
C
K
N
E
S
S
HYDROPHILIC
HEAD
HYDROPHOBIC
LEGS
UNPROTONATED
(Kreb’s cycle OFF)
(Warburg effect in Cancer cells)
CANCER CELL
HYDROPHILIC
HEAD
HYDROPHOBIC
LEGS
Figure 9. Bioinformatic analysis of mitochondrial membrane in normal (left panels) and cancer
(right panels) cells. Mitochondrial membrane monomer cardiolipin undergoes a 10 A increase in
distance of hydrophobic tails when not bound with cytochrome c. This situation occurs in cancer
cells, resulting in mitochondrial membrane thinning. Accordingly, only in cancer cells can ozonized
lipid (red circles) reach the hydrophobic legs of cardiolipin, thus causing mitochondrial membrane
damage. This situation does not occur in normal cells, where ozonized lipid cannot reach this mo-
lecular target.
3.9. Inability to Kill Normal Cells
Results indicate that neither alteration of cell viability nor cytopathic effects occurred
in noncancer cells treated with HOO, as demonstrated in skin keratinocytes (Figure 10).
Figure 10. Ozonized oil (bubble oil in the culture medium 1, 2, 3 h) does not induce alterations in
normal human keratinocytes. No change in cell viability, intercellular adhesion, and substrate ad-
herence occur in ozonized oil-treated cells (Oil) as compared with untreated control.
Figure 10.
Ozonized oil (bubble oil in the culture medium 1, 2, 3 h) does not induce alterations
in normal human keratinocytes. No change in cell viability, intercellular adhesion, and substrate
adherence occur in ozonized oil-treated cells (Oil) as compared with untreated control.
Cancers 2022,14, 1174 13 of 24
Cell viability of keratinocytes treated with ozonized oils quantified by MTT test was
100% in control cells, 99.8% after 1 h, 99.4% after 2 h, 98.7% after 3 h in ozonized oil-treated
cells. Cell viability was not evaluated at times >3 h because the oil interface blocked
cell exchange with culture medium, causing cell sufferance both in sham-treated cells
(sunflower oil) and ozonized oil-treated cells, independent of oil toxicity.
3.10. Synergism with Radiotherapy
A strong radio-sensitizing effect of HOO 700 and even more of HOO 1100 was detected.
The maximum effect observed was obtained treating A549 cancer cells with HOO 1100 after
their exposure to gamma rays. From a mechanistic point of view, this effect was in line with
the activation of the intrinsic mitochondrial apoptosis activated by HOO in cancer cells
undergoing genotoxic damage induced by radiotherapy. The results obtained are shown in
Figure 11.
Cancers 2022, 14, 13 of 25
Cell viability of keratinocytes treated with ozonized oils quantified by MTT test was
100% in control cells, 99.8% after 1 h, 99.4% after 2 h, 98.7% after 3 h in ozonized oil-treated
cells. Cell viability was not evaluated at times >3 h because the oil interface blocked cell
exchange with culture medium, causing cell sufferance both in sham-treated cells (sun-
flower oil) and ozonized oil-treated cells, independent of oil toxicity.
3.10. Synergism with Radiotherapy
A strong radio-sensitizing effect of HOO 700 and even more of HOO 1100 was de-
tected. The maximum effect observed was obtained treating A549 cancer cells with HOO
1100 after their exposure to gamma rays. From a mechanistic point of view, this effect was
in line with the activation of the intrinsic mitochondrial apoptosis activated by HOO in
cancer cells undergoing genotoxic damage induced by radiotherapy. The results obtained
are shown in Figure 11.
Figure 11. Synergism between gamma ray radiation and ozonized oils (OOAO) treatments in killing
lung cancer cells.
A summary of the results obtained in vitro is reported in Table 1.
Table 1. Summary of results from in vitro experiments dealing the effect of ozonized oil at high
ozonides (HOO) in cancer cells.
Test Method Results Reference Figure
Lung cancer cells killing A549 cells, crystal violet
staining, MTT test Time-dependent cell death Figure 2
Glioblastoma cancer cells
killing
U87MG cells, crystal violet
staining
Time-dependent cell death.
Higher sensitivity than A549
cells
Figure 4
Alteration of mitochondrial
membranes
A549 cells, fluorescence mi-
croscopy, bio-informatic
analyses
Specific sensitivity to oxida-
tion of cancer cells, calcium
release
Figure 6
Figure 9
Cell morphology alteration A549 cells, scanning electron
microscope
Decreased size, rounding,
detachment from support
Figure 2
Figure 3
Figure 5
Figure 7
Oxidation of intracellular or-
ganic carbon A549 cells, X-ray diffraction Oxidation of intracellular or-
ganic carbon Figure 7
Figure 11.
Synergism between gamma ray radiation and ozonized oils (OOAO) treatments in killing
lung cancer cells.
A summary of the results obtained in vitro is reported in Table 1.
Table 1.
Summary of results from
in vitro
experiments dealing the effect of ozonized oil at high
ozonides (HOO) in cancer cells.
Test Method Results Reference Figure
Lung cancer cells killing A549 cells, crystal violet staining,
MTT test Time-dependent cell death Figure 2
Glioblastoma cancer cells killing U87MG cells, crystal
violet staining
Time-dependent cell death.
Higher sensitivity than A549 cells
Figure 4
Alteration of
mitochondrial membranes
A549 cells, fluorescence
microscopy, bio-informatic
analyses
Specific sensitivity to oxidation of
cancer cells, calcium release
Figure 6
Figure 9
Cell morphology alteration A549 cells, scanning
electron microscope
Decreased size, rounding,
detachment from support
Figure 2
Figure 3
Figure 5
Figure 7
Oxidation of intracellular
organic carbon A549 cells, X-ray diffraction Oxidation of intracellular
organic carbon Figure 7
Apoptosis A549 cells, annexin V
labeling, FACS Increase in apoptosis Figure 8
Cancers 2022,14, 1174 14 of 24
Table 1. Cont.
Test Method Results Reference Figure
Inhibition of
macrophage activation
Raw264 cells, Lps
activation, microscopy
No change in macrophage
morphology, maintenance of
rounding morphology, no
emission pf pseudopods
[25]
Inability to kill normal cells Primary differentiated
human keratinocytes
No detachment from support, no
growth inhibition Figure 10
Synergism with gamma radiation
in cancer cells killing
A549 cells, gamma radiation,
crystal violet staining
Increased cell apoptosis
and necrosis Figure 11
3.11. Experimental Evidence in Human Subjects. Healthy Subjects
Two volunteer healthy subjects (males, 55 years old) were treated with 12 mL of HOO
700 for 1 week twice per day away from meals. Cytofluorimetric analysis (FACS) was
performed to evaluate macrophages and lymphocytes pro-inflammatory activation in the
peripheral blood before (T0) and after treatment (T1). The results obtained showed a marked
decrease in macrophage activation markers (HLAdr) in both subjects, while no variations
were observed in the lymphocyte subpopulations responsible for the protective immunity.
An example of the cytofluorimetric results obtained is reported in Figure 12. Cytofluorimet-
ric analysis evaluated T-helper CD4+ and cytotoxic T lymphocytes CD8+ before and after
ozonized oil treatment without observing any variation. These results provide evidence
that HOO can induce anti-inflammatory effect without causing immuno-suppression.
Drug safety was also evaluated
in vivo
by analyzing the traditional blood chemistry
parameters of the two volunteers treated: no alterations were found in the basal physiologi-
cal state.
Cancers 2022, 14, 14 of 25
Apoptosis A549 cells, annexin V label-
ing, FACS Increase in apoptosis Figure 8
Inhibition of macrophage ac-
tivation
Raw264 cells, Lps activation,
microscopy
No change in macrophage
morphology, maintenance of
rounding morphology, no
emission pf pseudopods
[25]
Inability to kill normal cells Primary differentiated hu-
man keratinocytes
No detachment from sup-
port, no growth inhibition Figure 10
Synergism with gamma radi-
ation in cancer cells killing
A549 cells, gamma radiation,
crystal violet staining
Increased cell apoptosis and
necrosis Figure 11
3.11. Experimental Evidence in Human Subjects. Healthy Subjects
Two volunteer healthy subjects (males, 55 years old) were treated with 12 mL of HOO
700 for 1 week twice per day away from meals. Cytofluorimetric analysis (FACS) was
performed to evaluate macrophages and lymphocytes pro-inflammatory activation in the
peripheral blood before (T0) and after treatment (T1). The results obtained showed a
marked decrease in macrophage activation markers (HLAdr) in both subjects, while no
variations were observed in the lymphocyte subpopulations responsible for the protective
immunity. An example of the cytofluorimetric results obtained is reported in Figure 12.
Cytofluorimetric analysis evaluated T-helper CD4+ and cytotoxic T lymphocytes CD8+
before and after ozonized oil treatment without observing any variation. These results
provide evidence that HOO can induce anti-inflammatory effect without causing im-
muno-suppression.
Drug safety was also evaluated in vivo by analyzing the traditional blood chemistry
parameters of the two volunteers treated: no alterations were found in the basal physio-
logical state.
Figure 12. FACS analysis of blood monocytes (cd38, vertical axis). Activated monocytes are detected
by analyzing HLAdr (horizontal axis). The number of HLAdr-positive activated monocytes was
decreased (arrow) after 1 week of HOO treatment (T1, right panel) as compared with those detected
in the same subject before treatment beginning (T0, left panel).
3.12. Experimental Evidence in Human Subjects. Cancer Patients
The transferability of the results obtained in vitro and in vivo in animals was initially
tested in seven human patients affected by cancer. The results obtained are summarized
as follows.
Figure 12.
FACS analysis of blood monocytes (cd38, vertical axis). Activated monocytes are detected
by analyzing HLAdr (horizontal axis). The number of HLAdr-positive activated monocytes was
decreased (arrow) after 1 week of HOO treatment (T1, right panel) as compared with those detected
in the same subject before treatment beginning (T0, left panel).
3.12. Experimental Evidence in Human Subjects. Cancer Patients
The transferability of the results obtained
in vitro
and
in vivo
in animals was initially
tested in seven human patients affected by cancer. The results obtained are summarized
as follows.
Cancers 2022,14, 1174 15 of 24
3.13. PATIENT 1. SKIN CANCER
The patient was a 93-year-old female with malignant spino-cellular epidermoidal
carcinoma, confirmed histopathologically. The neoplasia was localized in the scalp in
the parietal region and presented ab initio a character of extreme invasiveness and rapid
progression. In fact, in only a few weeks, the cranial case was invaded with consequent
parietal osteolysis. The neoplasia continued its progression rapidly, penetrating the skull
and taking on the arachnoid. These data were revealed by computerized axial tomography
(TAC). The neoplasia had considerable size in the outer part, thus covering the whole
skull with conspicuous growth—not only endophytic (inside the skull) but also exophytic
(protrusion of the neoplastic mass outside the skull).
The cancer was highly malignant, characterized by a high level of anaplasia, rapid
progression, presence of neoplastic ulcers of significant size, high inflammation of the
peri-lesioned margins, and total absence of repair by granulomatous tissue in the areas
surrounding the neoplastic ulcer. The baseline clinical situation, as observed at the patient’s
first visit, is shown in Figure 13.
Therefore, it was decided to use HOO 1100 in a post-treatment regimen—i.e., at the
end of each radiotherapy session. Thus, at the end of the first radiotherapy session, OHOO
1100, gelled at 4
◦
C, was applied to the neoplastic lesion through a glass depositor, and the
ulcer was coated with gauze and hydrophobic bandage.
The treatment continued for eight consecutive sessions with 2 days of interval be-
tween each one. OHOO medication was renewed at the end of each radiotherapy session.
Therefore, OHOO was left to act in the pathological area for 48 h in the interval between
radiotherapy sessions.
At the second session, the presence of yellowish exudate already present before the
apposition of HOO was observed.
The treatment with radiotherapy and HOO 1100 continued for a total of eight sessions.
Changes in the neoplastic lesion following treatment are shown in Figure 13.
Microscopic examination showed a strong size decrease in the cancer mass both in
amplitude and in depth. Furthermore, the formation of a granulation repair tissue at the
margins of the lesion was observed.
The radiotherapy was then suspended, and only topical treatment with HOO 1100
continued. Medication frequency was reduced to once per week because of the impos-
sibility of the caregiver to bring the patient to the hospital more frequently. Despite the
discontinuation of the radiation treatment, the cancer did not grow but further continued
its regression, as shown in Figure 13.
These last results demonstrate the specific inhibitory effect of HOO 1100 against the
tumor, even in absence of the radiation treatment. At the end of the follow-up, patients
showed only a soft eschar of exudative material resulting from the colliquative necrosis
of the neoplastic tissue. The eschar was not cleaned because the disappearance of the
neoplastic mass left exposed the subarachnoid arteries, which were pulsating below the
eschar itself. This result showed that the disappearance of the tumor mass occurred not
only at the esophitic but also at the endophytic level. This result is remarkable because only
a preparation characterized by a high bioavailability can be able to reach even the deepest
areas of the tumor mass through a simple topical application.
In order to establish whether the topical application of OOAO had a systemic effect
on the oxidative balance, we performed free radicals’ analysis in blood plasma before (T0)
the beginning and end (T1) of HOO treatment using a Fras 4 system. At T0, the parameters
were dramatically altered with a particularly low value of oxidizing species, i.e., 38 U Carr
(normal range 250–280 U). In parallel, the antioxidants were strongly increased, with a value
of 8318 U Cor (normal range 2200–2800 U). The antioxidant/oxidant balance (UCor/UCar
ratio) was therefore 219 (normal range 7–10). These results show that the presence of
a cancer mass characterized by large size implied the strong decrease in oxidation at a
systemic level. At T1, the values were changed as follows: 201 U Carr, 7544 UCor, and
the ratio UCor/UCar was 37. Therefore, an increase of 530% of the oxidative species was
Cancers 2022,14, 1174 16 of 24
observed, a decrease of 13% of the antioxidants, a decrease of 583% of the ratio UCor/UCar.
These values indicate that the regression of the neoplastic mass was related to the variation
of the oxidative balance induced by treatment with OHOO.
At the end of the treatment the patient was in good health and no longer suffering
or feverish. During treatment, no subjective or objective side effects related to OHOO
treatment were observed.
Cancers 2022, 14, 16 of 25
species was observed, a decrease of 13% of the antioxidants, a decrease of 583% of the
ratio UCor/UCar. These values indicate that the regression of the neoplastic mass was
related to the variation of the oxidative balance induced by treatment with OHOO.
At the end of the treatment the patient was in good health and no longer suffering or
feverish. During treatment, no subjective or objective side effects related to OHOO treat-
ment were observed.
3 days 6 days 9 days 12 days 15 days
20 days 35 days 42 days
T0
Figure 13. Evolution of a radioresistant skin basal carcinoma in a female 93-year-old patient treated
with HOO.
3.14. PATIENT 2. SKIN CANCER
There was an 86-year-old female patient with a recurrent skin ulcer in the palmar
region of the right forearm. The lesion was subjected to biopsy and histopathological anal-
ysis, and then it was classified as ulcerated metatypical nodular basocellular carcinoma;
the lesion extended in depth, infiltrating the papillary and reticular derma; the presence
of surrounding chronic inflammatory infiltrates was observed. Before treatment, the le-
sion was macroscopically characterized, as reported in Figure 14.
Therefore, the lesion was definitively removed surgically. During treatment, no sub-
jective or objective side effects were observed.
Nodular basocellular carcinoma; the lesion
extended in depth infiltrating the papillary
and reticular derma
Treatment time-span: 60 days
Figure 13. Evolution of a radioresistant skin basal carcinoma in a female 93-year-old patient treated
with HOO.
3.14. PATIENT 2. SKIN CANCER
There was an 86-year-old female patient with a recurrent skin ulcer in the palmar
region of the right forearm. The lesion was subjected to biopsy and histopathological
analysis, and then it was classified as ulcerated metatypical nodular basocellular carcinoma;
the lesion extended in depth, infiltrating the papillary and reticular derma; the presence of
surrounding chronic inflammatory infiltrates was observed. Before treatment, the lesion
was macroscopically characterized, as reported in Figure 14.
Cancers 2022, 14, 16 of 25
species was observed, a decrease of 13% of the antioxidants, a decrease of 583% of the
ratio UCor/UCar. These values indicate that the regression of the neoplastic mass was
related to the variation of the oxidative balance induced by treatment with OHOO.
At the end of the treatment the patient was in good health and no longer suffering or
feverish. During treatment, no subjective or objective side effects related to OHOO treat-
ment were observed.
3 days 6 days 9 days 12 days 15 days
20 days 35 days 42 days
T0
Figure 13. Evolution of a radioresistant skin basal carcinoma in a female 93-year-old patient treated
with HOO.
3.14. PATIENT 2. SKIN CANCER
There was an 86-year-old female patient with a recurrent skin ulcer in the palmar
region of the right forearm. The lesion was subjected to biopsy and histopathological anal-
ysis, and then it was classified as ulcerated metatypical nodular basocellular carcinoma;
the lesion extended in depth, infiltrating the papillary and reticular derma; the presence
of surrounding chronic inflammatory infiltrates was observed. Before treatment, the le-
sion was macroscopically characterized, as reported in Figure 14.
Therefore, the lesion was definitively removed surgically. During treatment, no sub-
jective or objective side effects were observed.
Nodular basocellular carcinoma; the lesion
extended in depth infiltrating the papillary
and reticular derma
Treatment time-span: 60 days
Figure 14.
Evolution of an ulcerated nodular basocellular carcinoma in a female 86-year-old patient.
The lesion extended in depth, infiltrating the papillary and reticular derma; the presence of surround-
ing chronic inflammatory infiltrates was observed before treatment (T0). HOO treatment induced
cancer regression and disappearance of the surrounding inflammatory halo after 60 days.
Cancers 2022,14, 1174 17 of 24
Therefore, the lesion was definitively removed surgically. During treatment, no
subjective or objective side effects were observed.
3.15. PATIENT 3. PROSTATE CANCER
Subject was a 55-year-old male affected by prostate cancer, as confirmed by biopsy
and histopathological examination. Staging and severity were very high, with this cancer
being classified with a Gleason score 9 (max scale value 10). Cancer dimension: 3.5 cm.
Imaging (ECT) detected a trend towards invasion of the prostate capsule, peri-prostatic
adipose tissue, left seminal vesicle, and local lymph nodes. Relatively low PSA (4.5 ng/mL)
was observed due to the high anaplastic behavior and the poor differentiation. Before
surgical treatment, the patient was treated for 60 days with daily intra-rectal administration
of ozonized oil (HOO 700) and oral administration of ozonized oil 12 mL twice per day.
At surgery, the field macroscopically appeared clear and without any evident in-
flammation or defragmentation of the cancer mass. A similar situation usually did not
occur in such a high-grade malignancy characterized by high inflammation, severe infil-
tration of surrounding structures, fast growth, and extreme fragility of the cancer mass.
Accordingly, surgery removed the whole cancer, as well as surrounding tissues, seminal
vesicles, and lymph nodes. Microscope histopathological analysis did not detect any sign
of inflammation in the cancer parenchyma or surrounding tissues. The presence of tumor-
associated macrophages was not detected at all, at variance with the typical aspect of this
high-malignancy cancer (Figure 15).
Cancers 2022, 14, 17 of 25
Figure 14. Evolution of an ulcerated nodular basocellular carcinoma in a female 86-year-old patient.
The lesion extended in depth, infiltrating the papillary and reticular derma; the presence of sur-
rounding chronic inflammatory infiltrates was observed before treatment (T0). HOO treatment in-
duced cancer regression and disappearance of the surrounding inflammatory halo after 60 days.
3.15. PATIENT 3. PROSTATE CANCER
Subject was a 55-year-old male affected by prostate cancer, as confirmed by biopsy
and histopathological examination. Staging and severity were very high, with this cancer
being classified with a Gleason score 9 (max scale value 10). Cancer dimension: 3.5 cm.
Imaging (ECT) detected a trend towards invasion of the prostate capsule, peri-prostatic
adipose tissue, left seminal vesicle, and local lymph nodes. Relatively low PSA (4.5 ng/mL)
was observed due to the high anaplastic behavior and the poor differentiation. Before sur-
gical treatment, the patient was treated for 60 days with daily intra-rectal administration
of ozonized oil (HOO 700) and oral administration of ozonized oil 12 mL twice per day.
At surgery, the field macroscopically appeared clear and without any evident inflam-
mation or defragmentation of the cancer mass. A similar situation usually did not occur
in such a high-grade malignancy characterized by high inflammation, severe infiltration
of surrounding structures, fast growth, and extreme fragility of the cancer mass. Accord-
ingly, surgery removed the whole cancer, as well as surrounding tissues, seminal vesicles,
and lymph nodes. Microscope histopathological analysis did not detect any sign of in-
flammation in the cancer parenchyma or surrounding tissues. The presence of tumor-as-
sociated macrophages was not detected at all, at variance with the typical aspect of this
high-malignancy cancer (Figure 15).
Only 1 local lymph node, out of the 10 examined, was barely affected by cancer inva-
sion.
Patient subsequently underwent anti-hormone therapy and radiotherapy as sched-
uled by the standard treatment protocol. In parallel, he continued ozonized oil treatment.
After 6 months, no sign of relapse was detected.
The analysis of oxidative status in blood plasma indicated that oxidative stress was
low before ozonized oil treatment (T0) (210 U Car, normal range 250–280) as well as the
antioxidant/oxidant ratio (9.9 Ucor/UCar). After treatment, oil treatment (T1) oxidative
stress was increased (295 U Car, 7.9 Ucor/UCar ratio). No adverse effect related to the
ozonized oil treatment was detected.
Figure 15.
Prostate cancer histopathological analysis detecting poor signs of inflammation in the
cancer parenchyma and surrounding tissues despite the high malignancy grade (Gleason 9) after
HOO treatment. The presence of tumor-associated macrophages was not detected at all, at variance
with the usual aspect of this cancer type (magnification 100×).
Only 1 local lymph node, out of the 10 examined, was barely affected by cancer invasion.
Patient subsequently underwent anti-hormone therapy and radiotherapy as scheduled
by the standard treatment protocol. In parallel, he continued ozonized oil treatment. After
6 months, no sign of relapse was detected.
The analysis of oxidative status in blood plasma indicated that oxidative stress was
low before ozonized oil treatment (T0) (210 U Car, normal range 250–280) as well as the
antioxidant/oxidant ratio (9.9 Ucor/UCar). After treatment, oil treatment (T1) oxidative
Cancers 2022,14, 1174 18 of 24
stress was increased (295 U Car, 7.9 Ucor/UCar ratio). No adverse effect related to the
ozonized oil treatment was detected.
3.16. PATIENT 4. PROSTATE CANCER
There was a 76-year-old male, affected by prostate cancer (adenocarcinoma), as con-
firmed by biopsy and histopathological examination (September 2015). No local or distant
metastasis was detected. NMR revealed high metabolic rate, fast cell proliferation, and
blood vessel proliferation. Biopsy detected severe inflammation of cancer mass and sur-
rounding tissue. Intermediate malignancy: Gleason score was 6, and cancer dimension was
3.0 cm.
No results were observed in anti-hormonal therapy (November–December 2015).
Standard radiotherapy regimen was of 40 sessions (January–March 2016). In October 2016,
after radiotherapy, NMR detected cancer persistence with cell proliferation and remarkable
blood vessel proliferation.
From January 2017, HOO oral treatment (12.5 mL twice a day for 18 months) was
started. In February 2018, TAC and radioimaging demonstrated complete cancer disap-
pearance (Figure 16).
No relapses were insofar detected (ongoing follow-up, 18 months).
No adverse effect related to the ozonized oil treatment was detected.
Cancers 2022, 14, 18 of 25
Figure 15. Prostate cancer histopathological analysis detecting poor signs of inflammation in the
cancer parenchyma and surrounding tissues despite the high malignancy grade (Gleason 9) after
HOO treatment. The presence of tumor-associated macrophages was not detected at all, at variance
with the usual aspect of this cancer type (magnification 100×).
3.16. PATIENT 4. PROSTATE CANCER
There was a 76-year-old male, affected by prostate cancer (adenocarcinoma), as con-
firmed by biopsy and histopathological examination (September 2015). No local or distant
metastasis was detected. NMR revealed high metabolic rate, fast cell proliferation, and
blood vessel proliferation. Biopsy detected severe inflammation of cancer mass and sur-
rounding tissue. Intermediate malignancy: Gleason score was 6, and cancer dimension
was 3.0 cm.
No results were observed in anti-hormonal therapy (November–December 2015).
Standard radiotherapy regimen was of 40 sessions (January–March 2016). In October 2016,
after radiotherapy, NMR detected cancer persistence with cell proliferation and remarka-
ble blood vessel proliferation.
From January 2017, HOO oral treatment (12.5 mL twice a day for 18 months) was
started. In February 2018, TAC and radioimaging demonstrated complete cancer disap-
pearance (Figure 16).
No relapses were insofar detected (ongoing follow-up, 18 months).
No adverse effect related to the ozonized oil treatment was detected.
Figure 16. Disappearance of radioresistant prostate carcinoma (T0) after 1 year of HOO treatment
(T1), as detected by nuclear magnetic resonance in a 76-year-old male patient.
3.17. PATIENT 5. PROSTATE CANCER
There was a 74-year-old male affected by prostate cancer (adenocarcinoma), as con-
firmed by biopsy and histopathological examination. No metastasis was detected. High
malignancy grade (Gleason 8), PSA 9.1 ng/mL was observed. Treatment with ozonized oil
(intra-rectal once per day) and ozonized oral oil (12.5 mL twice a day) for 40 days before
therapeutic surgery was started. At surgery, despite the high cancer malignancy, no inva-
sion of prostatic capsule, surrounding adipose tissue, seminal vesicle, or lymph node was
detected; blood vessel proliferation was observed. Full eradication was observed with no
relapses after follow-up of 8 months.
Figure 16.
Disappearance of radioresistant prostate carcinoma (T0) after 1 year of HOO treatment
(T1), as detected by nuclear magnetic resonance in a 76-year-old male patient.
3.17. PATIENT 5. PROSTATE CANCER
There was a 74-year-old male affected by prostate cancer (adenocarcinoma), as con-
firmed by biopsy and histopathological examination. No metastasis was detected. High
malignancy grade (Gleason 8), PSA 9.1 ng/mL was observed. Treatment with ozonized
oil (intra-rectal once per day) and ozonized oral oil (12.5 mL twice a day) for 40 days
before therapeutic surgery was started. At surgery, despite the high cancer malignancy, no
invasion of prostatic capsule, surrounding adipose tissue, seminal vesicle, or lymph node
was detected; blood vessel proliferation was observed. Full eradication was observed with
no relapses after follow-up of 8 months.
3.18. PATIENT 6. BRAIN GLIOBLASTOMA
Female subject was a 3-year-old glioblastoma patient, as detected by TAC/NMR and
confirmed by biopsy. High malignant grade was observed. Patient underwent standard
radio/chemotherapy regimen. Ozonized oil was administered (12.5 mL twice a day for
Cancers 2022,14, 1174 19 of 24
90 days). The clinical follow-up was compared with those of three other young patients in
a similar clinical situation but devoid of ozonized oil treatment. All of these three patients
underwent fast cancer progression, and one died. Conversely, the ozonized oil led to the
arrest of cancer growth and a dramatic decrease in cancer dimension that after 3 months of
treatment was only 35% of that initially detected. A similar finding was totally unexpected
in such a fast growing cancer.
Cerebrospinal fluid was collected, and oxidative status was analyzed. The sample was
contaminated by red blood cells. For these reasons, the analysis of oxidative status was
unreliable. Conversely, after several efforts, we were successful in analyzing the antioxidant
status, whose values were 892 and 895 (replicate analyses on 15 uL
×
2) U cor (umol/L
ascorbic acid equivalent). The normal reference value was 2500, with a b max–min range of
1800, which was below the threshold. Accordingly, the observed value was extremely low
(despite the red blood cell contamination releasing antioxidant). It could be concluded that
the depletion of antioxidant due to the therapeutically induced oxidative stress (ozonized
oil treatment) had been effective on the CSF of this patient. This value (893 U cor) could be
assumed as a threshold to be reached to obtain therapeutic effects.
It is conceivable that ozonized oil, due to its high lipophilicity, is able to cross the
blood–brain barrier. Specific
in vitro
and
in vivo
tests are ongoing to further substantiate
this issue.
3.19. PATIENT 7. BRAIN GLIOBLASTOMA
Female subject was a 38-year-old affected by brain glioblastoma in left hemisphere
(diagnosis July 2014. 1st NMR, (T0)). First surgery was performed in September 2014. High
malignancy (grade III) was observed. In May 2017, relapses (2nd NMR) were detected, and
in June 2017, there was a second surgery. September 2017 radiotherapy (60 Gy) in parallel
with the start of ozonized oil therapy (oral administration, 6 mL per day) was performed.
No relapse (September 2018) was detected (3rd NMR, T1). Cancer presence at T0 as well as
its clearance at T1 are reported in Figure 17.
Cancers 2022, 14, 19 of 25
3.18. PATIENT 6. BRAIN GLIOBLASTOMA
Female subject was a 3-year-old glioblastoma patient, as detected by TAC/NMR and
confirmed by biopsy. High malignant grade was observed. Patient underwent standard
radio/chemotherapy regimen. Ozonized oil was administered (12.5 mL twice a day for 90
days). The clinical follow-up was compared with those of three other young patients in a
similar clinical situation but devoid of ozonized oil treatment. All of these three patients
underwent fast cancer progression, and one died. Conversely, the ozonized oil led to the
arrest of cancer growth and a dramatic decrease in cancer dimension that after 3 months
of treatment was only 35% of that initially detected. A similar finding was totally unex-
pected in such a fast growing cancer.
Cerebrospinal fluid was collected, and oxidative status was analyzed. The sample
was contaminated by red blood cells. For these reasons, the analysis of oxidative status
was unreliable. Conversely, after several efforts, we were successful in analyzing the an-
tioxidant status, whose values were 892 and 895 (replicate analyses on 15 uL × 2) U cor
(umol/L ascorbic acid equivalent). The normal reference value was 2500, with a b max–
min range of 1800, which was below the threshold. Accordingly, the observed value was
extremely low (despite the red blood cell contamination releasing antioxidant). It could
be concluded that the depletion of antioxidant due to the therapeutically induced oxida-
tive stress (ozonized oil treatment) had been effective on the CSF of this patient. This value
(893 U cor) could be assumed as a threshold to be reached to obtain therapeutic effects.
It is conceivable that ozonized oil, due to its high lipophilicity, is able to cross the
blood–brain barrier. Specific in vitro and in vivo tests are ongoing to further substantiate
this issue.
3.19. PATIENT 7. BRAIN GLIOBLASTOMA
Female subject was a 38-year-old affected by brain glioblastoma in left hemisphere
(diagnosis July 2014. 1st NMR, (T0)). First surgery was performed in September 2014.
High malignancy (grade III) was observed. In May 2017, relapses (2nd NMR) were de-
tected, and in June 2017, there was a second surgery. September 2017 radiotherapy (60
Gy) in parallel with the start of ozonized oil therapy (oral administration, 6 mL per day)
was performed. No relapse (September 2018) was detected (3rd NMR, T1). Cancer pres-
ence at T0 as well as its clearance at T1 are reported in Figure 17.
Figure 17. Disappearance of radioresistant brain glioblastoma (T0) after 1 year of HOO treatment
(T1), as detected by nuclear magnetic resonance in a 38-year-old female patient.
Figure 17.
Disappearance of radioresistant brain glioblastoma (T0) after 1 year of HOO treatment
(T1), as detected by nuclear magnetic resonance in a 38-year-old female patient.
After these 7 patients, a total of 115 cancer patients, 76 males and 39 females, average
age 60.1
±
17.8 years, were treated with ozonized oil. An overview of the results obtained
with regard to oxidative status in cancer patients treated with HOO is reported in Figure 18.
For reference, oxidative status was also analyzed in parallel in 40 cancer-free subjects
(22 males, 18 females, average age 58.0 ±6.4 years).
Cancers 2022,14, 1174 20 of 24
Cancers 2022, 14, 20 of 25
After these 7 patients, a total of 115 cancer patients, 76 males and 39 females, average
age 60.1 ± 17.8 years, were treated with ozonized oil. An overview of the results obtained
with regard to oxidative status in cancer patients treated with HOO is reported in Figure
18. For reference, oxidative status was also analyzed in parallel in 40 cancer-free subjects
(22 males, 18 females, average age 58.0 ± 6.4 years).
Figure 18. Analysis of oxidative status in the blood plasma of 115 cancer patients. The level of anti-
oxidant was higher in cancer patients as compared with controls (left panel, blue columns). HOO
treatment in cancer patients decreased the high level of antioxidant (left panel, red columns) moving
back their amount to the level of unaffected controls.
Standard follow-up exams included hematological analyses, blood analysis of cancer
markers (e.g., Ca-19), nuclear magnetic resonance, computerized tomography with and
without glucose tracer, echography. Clinical outcomes observed in HOO-treated cancer
patients as compared with cancer patients undergoing standard therapeutic regimens is
reported in Figure 19, referring to all cancers. Data on clinical outcomes after treatment
for each cancer type are reported in Table 2. Clinical outcomes were significantly different
(chi-square p value < 0.0001) in cancer patients receiving standard treatment only as com-
pared with those additionally receiving HOO as integrative therapy, as evaluated by chi-
square test (Figure 19).
Figure 18.
Analysis of oxidative status in the blood plasma of 115 cancer patients. The level of
antioxidant was higher in cancer patients as compared with controls (left panel, blue columns). HOO
treatment in cancer patients decreased the high level of antioxidant (left panel, red columns) moving
back their amount to the level of unaffected controls.
Standard follow-up exams included hematological analyses, blood analysis of cancer
markers (e.g., Ca-19), nuclear magnetic resonance, computerized tomography with and
without glucose tracer, echography. Clinical outcomes observed in HOO-treated cancer
patients as compared with cancer patients undergoing standard therapeutic regimens is
reported in Figure 19, referring to all cancers. Data on clinical outcomes after treatment for
each cancer type are reported in Table 2. Clinical outcomes were significantly different (chi-
square pvalue < 0.0001) in cancer patients receiving standard treatment only as compared
with those additionally receiving HOO as integrative therapy, as evaluated by chi-square
test (Figure 19).
Cancers 2022, 14, 21 of 25
.
Figure 19. Clinical outcomes observed in HOO-treated cancer patients (red columns) as compared
with cancer patients undergoing standard therapeutic regimens (blue columns), referring to all can-
cers.
Table 2. Clinical outcomes in cancer patients treated with HOO (follow-up 4 years).
Cancer Type Full Recovery Downstaging Stable
Diseases Progression Deceased Total
Brain glioblastoma 32% (n = 7) 41% (n = 9) 9% (n = 2) 5% (n = 1) 13% (n = 3 *) n = 22
Breast adenocarci-
noma 67% (n = 16) 20% (n = 5) 5% (n = 1) 0% (n = 0) 8% (n = 2 *) n = 24
Colon adenocarci-
noma 54% (n = 7) 23% (n = 3) 8% (n = 1) 8% (n = 1) 8% (n = 1 *) n = 13
Kidney/bladder can-
cer 60% (n = 3) 20% (n = 1) 0% 0% 20% (n = 1 *) n = 5
Non-Hodgkin’s skin
lymphoma 50% (n = 1) 50% (n = 1) 0% 0% 0% n = 2
Lung NSCLC 7 14% (n = 1) 43% (n = 3) 29% (n = 2) 14% (n = 1) 0 n = 7
Lung SCLC 5 0% (n = 0) 60% (n = 3) 20% (n = 1) 20% (n = 1) 0% n = 5
Ovarian/womb can-
cer 40% (n = 2) 40% (n = 2) 20% (n = 1) 0% (n = 0) 0% (n = 0) n = 5
Pancreas adenocarci-
noma 39% (n = 7) 44% (n = 8) 11% (n = 2) 6% (n = 1) 0% (n = 0) n = 18
Prostate cancer 58% (n = 4) 28% (n = 2) 14% (n = 1) 0% (n = 0) 0% (n = 0) n = 7
Skin cancer (radiore-
sistant epidermoidal
and basal carci-
noma)
28% (n = 2) 44% (n = 3) 14% (n = 1) 14% (n = 1) 0% (n = 0) n = 7
Grand total n = 50 n = 40 n = 12 n = 6 n = 7 n = 115
Average 44% 36% 10% 5% 5%
* Cancer stage at recruitment T4-N3-M1.
Figure 19.
Clinical outcomes observed in HOO-treated cancer patients (red columns) as compared
with cancer patients undergoing standard therapeutic regimens (blue columns), referring to all cancers.
Cancers 2022,14, 1174 21 of 24
Table 2. Clinical outcomes in cancer patients treated with HOO (follow-up 4 years).
Cancer Type Full Recovery Downstaging Stable
Diseases Progression Deceased Total
Brain glioblastoma 32% (n= 7) 41% (n= 9) 9% (n= 2) 5% (n= 1) 13% (n= 3 *) n= 22
Breast
adenocarcinoma 67% (n= 16) 20% (n= 5) 5% (n= 1) 0% (n= 0) 8% (n= 2 *) n= 24
Colon
adenocarcinoma 54% (n= 7) 23% (n= 3) 8% (n= 1) 8% (n= 1) 8% (n= 1 *) n= 13
Kidney/bladder
cancer 60% (n= 3) 20% (n= 1) 0% 0% 20% (n= 1 *) n= 5
Non-Hodgkin’s
skin lymphoma 50% (n= 1) 50% (n= 1) 0% 0% 0% n= 2
Lung NSCLC 7 14% (n= 1) 43% (n= 3) 29% (n= 2) 14% (n= 1) 0 n= 7
Lung SCLC 5 0% (n= 0) 60% (n= 3) 20% (n= 1) 20% (n= 1) 0% n= 5
Ovarian/womb
cancer 40% (n= 2) 40% (n= 2) 20% (n= 1) 0% (n= 0) 0% (n= 0) n= 5
Pancreas
adenocarcinoma 39% (n= 7) 44% (n= 8) 11% (n= 2) 6% (n= 1) 0% (n= 0) n= 18
Prostate cancer 58% (n= 4) 28% (n= 2) 14% (n= 1) 0% (n= 0) 0% (n= 0) n= 7
Skin cancer
(radioresistant
epidermoidal and
basal carcinoma)
28% (n= 2) 44% (n= 3) 14% (n= 1) 14% (n= 1) 0% (n= 0) n= 7
Grand total n= 50 n= 40 n= 12 n= 6 n= 7 n= 115
Average 44% 36% 10% 5% 5%
* Cancer stage at recruitment T4-N3-M1.
4. Discussion
Obtained results provide evidence that HOO is a new and interesting strategy for pre-
vention of cancer progression and relapses. Indeed, patients undergoing HOO integration,
in addition to standard therapeutic regimens, showed increased survival and decreased
rate of relapses. These clinical outcomes are justified by the mechanisms analyzed
in vitro
to shed light on the effects of HOO in cancer cells. Cancer cells are highly sensitive to
HOO oxidative effects due to their mitochondrial status. The oxidation of mitochondrial
membranes can re-activate apoptosis in cancer cells [
2
]. The selective effect of HOO in
killing cancer cells only without damaging normal cells is due to the different mitochondrial
situation. The mitochondrion is inactive in a cancer cell, both as far as concerns metabolic
(aerobic glycolysis) and pro-apoptotic function, that in normal cells is activated by the
release of cytochrome c and calcium from the mitochondrion into the cytoplasm. This situ-
ation critically differentiates cancer from healthy tissue, explaining why cancer cells cannot
die because of apoptosis while normal cells can. The outer mitochondrial membrane is
predominantly composed of phospholipids, among which the most relevant is cardiolipin.
This molecule is characterized by the presence of a hydrophilic phosphorylated head and
two hydrophobic tails. Cardiolipin is organized to form the typical phospholipidic double-
layer. However, the shape of cardiolipin is profoundly modified in relation to its binding to
cytochrome c, a fundamental component of oxidative phosphorylation [
26
]. Cardiolipin
displays its physiological structure only when bound to functioning cytochrome c or, in
other words, in the active mitochondrion characterizing normal cells but not in cancer
cells. The sensitivity to HOO is even higher for cancer stem cells that are antioxidant
addicted. These cells have been selected among cancer cell pools because of their high level
of antioxidant-based detoxifying mechanisms, allowing them to counteract the therapeutic
Cancers 2022,14, 1174 22 of 24
effects of chemo/radiotherapies. The scavenging of these antioxidants that is exerted by
HOO is an effective tool for making cancer stem cells sensitive to chemo/radiotherapies
and for overcoming their resistance. This situation explains the improved clinical efficacy
of standard therapeutic regimens in patients receiving HOO. This result may be achieved
only by using ozonized oils having very high ozonide content, extremely pure, devoid of
antioxidants, and highly bioavailable.
HOO exerts anticancer effects by activating various protective mechanisms including
(a) scavenging of antioxidants from cancer cells; (b) re-activation of intrinsic apoptosis;
(c) inhibition of the activation of tumor associated macrophages; (d) increase of oxygen
tissue availability decreasing angiogenesis and metastasis.
An additional mechanism could be the competition with the mitochondrial fat ox-
idation metabolic pathway providing energy availability in cancer cells [
27
]. HOO is a
metabolite of this pathway, but its catabolization inside the mitochondrion results in oxida-
tive stress triggering mitochondrial damage, intracellular calcium release, and apoptosis in
cancer cells. Targeting the intrinsic apoptotic pathway has been recently proposed as a new
strategy against cancer [28].
Cancer is a systemic disease. The neoplastic mass is not able to develop autonomously
in the absence of the trophic support provided by neo-angiogenesis and inflammation.
The presence of a conspicuous infiltrate of inflammatory macrophage cells is a specific
characteristic of malignant cancer. These macrophage cells are unable to counteract the
neoplastic growth, supporting it by supplying oxygen, metabolites, and neo-vessels. These
cells are referred as tumor-associated macrophages. The degree of inflammation is one of
the most predictive and prognostic indexes of the unfavorable development of a neoplasm.
Therefore, cancer should be counteracted not only at a topical level but also at the systemic
level, controlling inflammation and oxidative status in the whole organism. In this regard,
HOO could represent a significant step forward. HOO kills cancer by targeting cancer stem
cells and activating apoptosis but also by exerting anti-inflammatory effects at the systemic
level. Herein, presented results indicate that HOO induces
in vivo
anti-inflammatory
effects without causing immuno-suppression. The mechanism underlying this situation
is the inhibition of macrophage oxidative burst. Activated macrophages release oxygen-
reactive species and inflammatory cytokines in the tissue environment in order to neutralize
bacteria, if present. However, in the absence of bacteria, macrophage activation leads to
an inflammatory response, which can assume pathogenic relevance by promoting cancer
growth and progression. HOO inhibits macrophage oxidative burst through a negative
feedback mechanism; indeed, the presence of an extra-cellular environment enriched
with ozone-oxidizing species blocks the release of further oxidizing species from the
macrophages, thus inhibiting their activation and the consequent inflammation.
The trophic support to solid cancers is provided by neoangiogenesis. This process
is activated by low oxygen availability in cancer tissue, triggering hypoxia inducible
factor representing the main activator of vascular growth factor release and blood vessel
formation [
29
]. Herein, presented results provide evidence that HOO effectively releases oxygen
species inside cancer tissue, thus counteracting the hypoxic situation triggering neoangiogenesis.
5. Conclusions
In conclusion, the experimental studies performed provide evidence of the efficacy of
HOO treatment in killing cancer cells, thus integrating and potentiating the therapeutic
effects of standard therapies. This conclusion is supported by the biological plausibility of
the specific mechanisms activated by HOO in cancer cells, mainly including mitochondrial
damage and activation of apoptosis. These effects are exerted by HOO due to its peculiar
characteristics including: (a) oxidant effectiveness due to the high level of ozonide content;
(b) dose customization by evaluating oxidant/antioxidant balance in blood plasma as well
as cancer stage; (c) anti-inflammatory effects; (d) increase in oxygen tissue availability,
counteracting cancer metastasis triggered by local hypoxia; (d) capacity of penetration
inside cancer cells.
Cancers 2022,14, 1174 23 of 24
These findings provide evidence that HOO is endowed with a potential therapeutic
efficacy against cancer in the absence of detectable side effects. Due to its pharmacokinetic
and pharmacodynamic peculiarities, HOO represents an innovation in the field of comple-
mentary cancer therapy worthy of further clinical studies. Our result provides evidence
that oral administration of ozonized oils with high ozonide content is a novel strategy for
the prevention of cancer relapses and chemo/radioresistance. This approach could be used
in clinical practice to fulfill the lack of anticancer treatments occurring in intervals between
chemo/radio therapeutic regimens.
Author Contributions:
Conceptualization, A.I.; methodology, A.I., A.C.; software, C.R.; validation,
L.B.; formal analysis, A.I., S.S.; investigation, A.I., L.B.; resources, A.C.; data curation, E.F., A.S., M.C.
(Massimo Chiara); writing—original draft preparation, A.I., A.P.; writing—review and editing, Z.K.,
M.C. (Matteo Congiu); visualization, A.C.; supervision, S.S., C.C., G.B.; project administration, A.P.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
The study was conducted in accordance with the Declaration
of Helsinki, and the study was approved by the Health Ministry of Malta (approval number 0075/2020
according to EC1924/2006) issued on 17 March 2020.
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: All data are available upon request to the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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