Anti-Proliferative Properties Of Thymoquinone To Overcome Cisplatin-Induced Drug Resistance

In Skov-3 Section A-Research Paper

ANTI-PROLIFERATIVE PROPERTIES OF THYMOQUINONE

TO OVERCOME CISPLATIN-INDUCED DRUG RESISTANCE IN
SKOV-3
Shivani S. Tendulkar1, Aishwarya Hattiholi2, Vijay Kumbar3, Meenaz Sangolli4,
Mehul A. Shah5, Kishore Bhat6, Suneel Dodamani7*

Abstract
Background: Ovarian cancer (OvCa) malignancy is a widespread type of cancer with a poor prognosis
contributed by resistance developed against platinum-based drugs. The use of phytochemicals has been an
innovative key for the treatment of OvCa. The antiproliferative properties of Thymoquinone (TQ) have
indicated remarkable effects on targeting multiple pathways potentially leading to apoptosis. The aim of the
study augmenting the effects of TQ in overcoming cisplatin (CDDP) induced drug resistance in SKOV-3 cells.
Methods: The cells were treated with increasing doses of TQ in a time-dependent manner. The apoptotic
bodies were analyzed by DAPI while the mitochondrial membrane potential was analyzed by Rh-123. The
clonogenic assay was carried out to understand the colony formation potential. The protein expression for Bcl-
2 and p53 were assessed using western blotting.
Results: The ability of TQ to overcome CDDP-induced drug resistance with the help of TQ in SKOV-3 cell
lines. The minimum inhibition for SKOV-3 was 3 μM for CDDP and 14 μM for TQ. For the resistant SKOV-
3, the minimum inhibition was 6 μM for CDDP and 14 μM for TQ. The apoptotic bodies, mitochondrial
membrane, nuclear condensation, and nuclear fragmentation were the observed morphological changes. The
colony formation assay revealed reducing in the formation of clones. The protein expressions displayed
modifications in the levels of p53 and Bcl-2 in both cell lines.
Conclusion: TQ enhances apoptosis in SKOV-3 cells and is effective against the cells resistant to CDDP. This
indicates a major application of TQ in recurrent OvCa that are resistant to CDDP.

Keywords: Ovarian cancer, thymoquinone, cisplatin, SKOV-3, cisplatin-resistance, apoptosis

1
M.Sc PhD Scholar, Dr. Prabhakar Kore Basic Science Research Centre, KLE Academy of Higher Education
and Research, Belagavi, India. - ORCID ID: https://orcid.org/0000-0002-3936-6873
2
M.Sc, Maratha Mandal’s Central Research Laboratory NGH, Bauxite Road, Belagavi, India. ORCID ID:
https://orcid.org/0000-0003-0533-0350
3
M.Sc PhD, Dr. Prabhakar Kore Basic Science Research Centre, KLE Academy of Higher Education and
Research, Belagavi, India. ORCID ID: https://orcid.org/0000-0001-6261-1665
4
M.Sc Maratha Mandal’s Central Research Laboratory NGH, Bauxite Road, Belagavi, India. ORCID ID:
https://orcid.org/0000-0002-9861-4160
5
MDS, Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, KLE Academy of Higher
Education and Research, Belagavi, India. ORCID ID: https://orcid.org/0000-0003-3291-8716
6
MD, PhD, Maratha Mandal’s Central Research Laboratory NGH, Bauxite Road, Belagavi, India.
7
* M.Sc, PhD Dr. Prabhakar Kore Basic Science Research Centre, KLE Academy of Higher Education and
Research, Belagavi, India. ORCID ID: https://orcid.org/ 0000-0002-7463-8037

*Corresponding Author: Suneel Dodamani

*Basic Science Research Centre, KLE Academy of Higher Education and Research, Belagavi 590010, India,
Contact number: +919901943638, E-mail ID: suneelddmn18@gmail.com

DOI: - 10.48047/ecb/2023.12.si5a.0337

Eur. Chem. Bull. 2023, 12(Special Issue 5), 4329 – 4336 4329
Anti-Proliferative Properties Of Thymoquinone To Overcome Cisplatin-Induced Drug Resistance
In Skov-3 Section A-Research Paper

INTRODUCTION (10). In this study, TQ upregulated the pro-

One of the primary causes of mortality worldwide apoptotic proteins in the CDDP-resistant cells as
is cancer, estimating 18.1 million deaths and 9.6 seen by western blotting. In general, the efficacy of
million new cases. OvCa is the second most TQ was seen in resistant cells as compared to the
common and lethal gynecological malignancy, CDDP-sensitive cells through the various assays in
often diagnosed at progressive stages. Signs and the study. The effect of TQ in CDDP-resistant
symptoms generally occur at advanced stages such SKOV-3 (R_SKOV-3) has not been reported, to
as III and IV (1). There are different types of the best of our knowledge and therefore, we
reported OvCa, with epithelial OvCa being the attempt to investigate the same.
most prevalent one with a poor prognosis (2). The
symptoms and signs of OvCa are indistinct, EXPERIMENTAL
unclear, and vague. The risk factors include age, Methods & Materials
menstrual period, hormonal and infertility Chemicals and reagents
treatment, family history, genetic mutations, Cisplatin and Thymoquinone (Sigma Aldrich),
obesity, and socioeconomic status (3). After a Dulbecco’s Modified Eagle Medium (DMEM)
sequence of platinum-based treatment or radiation, (Gibco, Cat. No.-11965092), Antibiotic –
the majority of women experience a relapse of Antimycotic (100X) solution (Thermofisher
tumour or resistance to the drug and hence it is Scientific, Cat. No.-15240062), and Fetal bovine
essential to develop innovative therapeutic serum (FBS) (Gibco, Cat. No.-10270106), MTT
approaches (4). (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetra
zolium bromide (Sigma Aldrich), DAPI (4′,6-
The recurrence of tumour can be associated with diamidino-2-phenylindole) (D9542). The
various factors such as DNA influx/efflux, drug apoptotic kit was Annexin V-FITC Apoptosis
target alteration, gene mutations, autophagy, Detection Kit (R&D Systems, Cat. No. –4830-01-
epithelium-to-mesenchyme transition, tumour K). Propidium iodide (PI) (Cat. No.- P1304MP)
microenvironment, hypoxia, multi-drug resistance, was procured from ThermoFisher Scientific. The
DNA repair activation and cancer-associated primary antibodies anti-beta, B-cell leukaemia
fibroblasts (5). Various proteins involved in (Bcl-2), and p53 were purchased from Jupiter Life
pathways like the PI3K pathway promote apoptosis Sciences. The secondary antibody IgG-HRP was
in CDDP-sensitive cells that regulate proteins like procured from MERCK. Tetramethylbenzidine
Bcl-2 and Bax (6). These proteins may be (TMB) (Cat. No.- T0565) was purchased from
modulated in the resistant cells and may not Sigma Aldrich. Dimethyl sulfoxide (DMSO) and
respond to CDDP. We study a similar mechanism paraformaldehyde were procured from Qualigens,
in this investigation, as we compare the expression India. For protein isolation, RIPA lysis buffer
levels of these proteins in the SKOV-3 cells before (Cat.no. TCL131) and protease inhibitor cocktail
and after inducing CDDP resistance. We induced (ML051) were purchased from HiMedia.
resistance in the cell lines after repeated exposure
to increasing concentrations of CDDP, which was Methods
confirmed by the cytotoxic assay, and further by Cell Culture
comparing the apoptotic activities in resistant cells The SKOV-3 (human ovarian adenocarcinoma)
with the CDDP-sensitive ones. cell line was obtained from National Center for
Cell Science, Pune (India). SKOV-3 was cultured
The antiproliferative properties of TQ weaken the in McCoy’s 5A (Modified) medium with 10% of
immune system while protecting the human body FBS and 1% of the antibiotic-antimycotic solution.
from other susceptible diseases. It provides The cells were stored in a 5% CO2 incubator at
oxidative damage in the cancer cells and maintains 37°C (New Brunswick Galaxy 170R, Eppendorf
the homeostasis of normal cells. It reduces the India Private Ltd., India). The cells were sub-
expression of Bcl-2 (anti-apoptotic) on the hand cultured only after reaching 80% confluence for
and increases pro-apoptotic Bax expressions. It every experiment.
decreases the permeability of the mitochondrial
membrane, triggers ROS, and activates other pro- Cell viability assay
apoptotic signaling pathways (7). TQ has shown The cells were in the culture medium and grown
synergistic effects in cancer cells by augmenting until 80% confluency was achieved. Cells were
the cytotoxicity induced by cisplatin (8), through then washed with PBS, detached using trypsin-
the regulation of Bax and Bcl-2 (9). Moreover, TQ EDTA, and counted using a hemocytometer.
has shown sensitizing activity in resistant cells Further, 1X102 cells were seeded in a 96-well for

Eur. Chem. Bull. 2023, 12(Special Issue 5), 4329 – 4336 4330
Anti-Proliferative Properties Of Thymoquinone To Overcome Cisplatin-Induced Drug Resistance
In Skov-3 Section A-Research Paper

24h. These cells were incubated with different SKOV-3. The statistical analysis was carried out
concentrations of CDDP and TQ for 24 and 48. The using GraphPad Prism 5.1 software. The number of
cells were suspended in 100μl of MTT solution for surviving colonies by the number of cells plated
3h and incubated at 37°C. The formazan crystals was used to calculate plating efficiency (PE). The
were dissolved in 100μl of DMSO. The optical cell survival fraction (SF) was the number of
density (OD) was measured using Lisa plus colonies that arise after treatment in terms of PE.
Microtitre Reader at 490nm. Untreated cells were
used as control. All the groups were compared with Formula: SF (treated cells) = PE (treated cells) /
the control group to calculate the percentage of cell PE (control)
viability. The inhibition concentration (IC50) was
analyzed for a dose-response study that involves DAPI staining
50% cell death for each drug. The statistical The cells were seeded in a 24-well plate with
analysis was measured by the GraphPad Prism 5.1 coverslips and treated with CDDP and TQ after
software. attachment. The cells were washed with PBS after
48h and fixed using 4% PFA for 30 min. 1µg/ml of
Formula: Surviving cells (%) = Mean OD of DAPI was used to stain the cells incubated at RT
sample /Mean OD of Negative control ×100 in the dark for 5min. The cells were observed under
the fluorescence microscope with 20X
CDDP resistance development magnification and visualized using Pro RES®
The parental cell lines were plated 1X106 and Capture Pro software (Jena, Germany).
incubated at 37°C stored at 5% CO2 for 24h. The
parental cells were treated with increasing Protein isolation
concentrations of CDDP (starting from 0.2 μM) Cells were plated at 1X106 density and then treated
until the concentration was higher than the IC50 with CDDP and TQ for 48h. The cells were lysed
value (3 μM) over a period of several days. The using 500μl RIPA lysis buffer with 10μl of
method used for the development of resistant cell protease inhibitor for 1hr on ice with periodic
lines was adapted from (11) with slight vertexing. Further, the cells were transferred to 1.5
modifications. ml Eppendorf tubes and centrifuged at 12,000 rpm
for 20mins (Eppendorf centrifuge 5810 R). The
Mitochondrial membrane potential (MMP) supernatant was collected in a new tube and stored
The cells were grown in 24-well plates containing at -4°C for further use (12).
coverslips. On a subsequent day, the cells were
treated with CDDP and TQ. The cells were washed Western blotting analysis
with PBS 2-3 times after 48h. The cells were then The isolated proteins were thawed and denatured at
stained with Rhodamine-123 (Rh-123) dye for 30 100°C for 3min in a water bath. The proteins were
min. Further, the cells were washed with PBS and separated by 7.5% sodium dodecyl sulfate-
fixed using 4% PFA for 30mins. The cells were polyacrylamide gel (SDS-PAGE) for 2 ½h at
then examined under the fluorescent microscope 110V. The proteins from the gel were transferred
and the intensity was calculated using GraphPad onto a nitrocellulose membrane using a Towbin
Prism 5.1. buffer containing 25 mM Tris, 192 mM glycine,
pH 8.3 with 20% methanol (v/v) in a transfer
Colony formation assay chamber for 3h at 110V. After transfer the blot was
The cells were seeded with a cell density of 1X104 blocked using 1% Bovine Serum Albumin
in 6cms plates at 37°C in 5% CO2 overnight. After overnight at 4 °C. The blot was washed with PBS
attachment, the cells were treated with 3 μM CDDP thrice, 10 min each. The blot was treated with
and 14 μM TQ for SKOV-3, and 14 μM of TQ for primary antibodies with 1% bovine serum albumin
R_SKOV-3 according to the minimal inhibition (BSA) overnight at 4°C. The primary antibodies
concentration. After every 2 days, the culture used were anti-beta, B-cell leukemia (Bcl-2), and
medium was replaced for the cells to grow and p53. The blot was washed with PBS thrice, 10 min
incubated for 14 days at 37°C. After 14 days, the each. Further, the blot was incubated using goat
cells were washed with PBS 3 times and fixed with anti-mouse IgG-HRP with agitation for 3 h at room
4% PFA for 15min at room temperature. The temperature. To view the bands, the blot was
colonies were stained with 0.4% crystal violet for incubated in a TMB substrate for ½h. Rinse the
15min and further dried and counted. Each blot with Milli Q water to stop the reaction.
independent experiment was carried out in the
same protocol for both SKOV-3 and resistant

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Anti-Proliferative Properties Of Thymoquinone To Overcome Cisplatin-Induced Drug Resistance
In Skov-3 Section A-Research Paper

Statistical analysis (TQ24 = Treatment of SKOV-3 cells with TQ for

All the above experiments were carried out in 24h; CIS24 = Treatment of SKOV-3 cells with
triplicates. The data was characterized as mean ± CDDP for 24h; TQ48 = Treatment of SKOV-3
standard deviation for each experiment. The cells with TQ for 48h; CIS48 = Treatment of
groups were evaluated using a student's t-test for SKOV-3 cells with CDDP for 48h; RTQ24 =
both treatment and control. The significance was Treatment of CDDP-resistant-SKOV-3 cells with
established at a probability value ≤ 0.05. TQ for 24h; RCIS24 = Treatment of CDDP-
resistant-SKOV-3 cells with CDDP for 24h;
RESULTS & DISCUSSION RTQ48 = Treatment of CDDP-resistant-SKOV-3
TQ stimulates anti-proliferative effects in cells with TQ for 48h; RCIS48 = Treatment of
SKOV-3 and resistant SKOV-3 cells CDDP-resistant-SKOV-3 cells with CDDP for
This study aimed to determine the effects of CDDP 48h)
and TQ in SKOV-3 and R_SKOV-3 cells by
adapting the procedures described by Almosa et al.
(13). MTT assay was carried out in a time and
concentration-dependent manner in SKOV-3 and
R_SKOV-3 cells (Fig. 1). The IC50 values for
CDDP were found to be 2 μM and 3 μM for 24 h
and 48h, respectively. The same was observed for
TQ at 16 μM and 14 μM. The IC50 values for 48h
were used for further experiments. The SKOV-3
cells were subjected to the development of CDDP
resistance by exposing them to increasing
concentrations (Fig. 2). Minor morphological Fig. 2: Induction of CDDP resistance in SKOV-3
changes were observed over the course of this cell lines. The images indicate (1) SKOV-3 treated
exposure. The IC50 value of CDDP after the with 0.4 μM/mL (2) SKOV-3 treated with 1.2
development of resistance was observed at 6 μM μM/mL (3) SKOV-3 treated with 1.6 μM/mL (4)
while TQ at 14 μM, was efficient to inhibit 50% of SKOV-3 treated with 2 μM/mL (5) SKOV-3
R_SKOV-3 cells. All the further assays were treated with 4 μM/mL (6) SKOV-3 treated with 6
carried out in both SKOV-3 and R_SKOV-3 cell μM/mL (7) SKOV-3 treated with 8 μM/mL and (8)
lines to assess the efficiency of the apoptotic SKOV-3 treated with 10 μM/mL.
activities of the compounds.
TQ causes the mitochondrial dysfunction
The initiation of apoptosis consists of several
biochemical changes, one of them being
mitochondrial dysfunction. A change in the
mitochondrial membrane potential (MMP) (ΔΨm)
leads to membrane damage and the release of
cytochrome c thereby causing apoptosis (14).

The ΔΨm loss will be evaluated by using Rh-123

dye under a fluorescent microscope. The ΔΨm loss
is visualized by using Rh-123 dye under a
fluorescent microscope. The mitochondrial
dysfunction and organelle expansions are indicated
by the leakage of dye. Upon treatment, SKOV-3
cells showed significant changes in the MMP, with
Fig. 1: Cytotoxicity in SKOV-3 after treatment TQ inducing maximum depolarization. In
with different concentrations of CDDP and TQ R_SKOV-3 cells, the CDDP treatment did not
treatment for 24 and 48h. induce as much depolarization; while TQ was still
effective (Fig. 3) (15).

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In Skov-3 Section A-Research Paper

Fig. 3: MMP evaluation in SKOV-3 and R_SKOV-3. The images represent: (1) SKOV-3 untreated used as
control (2) SKOV-3 treated with 3 μM/mL of CDDP (3) SKOV-3 treated with 14 μM/mL of TQ (4)
R_SKOV-3 untreated (5) R_SKOV-3 treated with 6 μM/mL of CDDP (6) R_SKOV-3 treated with 14
μM/mL of TQ (7) The bar graph shows the fluorescent percentage in the above-stated groups. Data are
expressed as mean ± S.D. The significant difference is indicated as ***p < 0.0001, **p<0.01, and *p<0.05
between control cells vs treated.

Morphological changes stimulated by TQ TQ for both SKOV-3 and R_SKOV-3 were

The characteristics of early apoptosis are the observed by DAPI staining. After treatment with
nuclear and morphological modifications in cells. CDDP and TQ, nuclear condensation and
DAPI is a blue fluorescent dye that binds to DNA fragmentation, blebbing membrane, and apoptotic
minor grooves. DAPI binds to both living and dead bodies were observed. Fig. 4 indicates the changes
cells which can distinguish between the in the nuclear material further implying apoptosis
morphological transformations. The untreated cells via TQ treatment even in R_SKOV-3 when CDDP
show no or very little (due to natural cell death) showed a diminutive response.
morphological changes (16). The alterations in the
morphology after treatment with a selected dose of

Fig. 4: Representation of fluorescent images after DAPI staining. The images describe: (1) The intact nuclei
and high fluorescence intensity are shown in untreated SKOV-3 cells. (2) SKOV-3 treated with 3 μM/mL of
CDDP (3) SKOV-3 treated 14 μM/mL of TQ (4) Untreated R_SKOV-3 (5) R_SKOV-3 treated with 6
μM/ml CDDP (6) R_SKOV-3 treated with 14 μM/mL of TQ. The images indicate nuclear membrane
blebbing and apoptotic body formation (white circles), nuclear condensation (red arrows), and nuclear
fragmentation (yellow arrows).
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Anti-Proliferative Properties Of Thymoquinone To Overcome Cisplatin-Induced Drug Resistance
In Skov-3 Section A-Research Paper

Effectiveness of TQ to form colonies post- (18). The results indicate that TQ significantly
treatment declines colony formation in both groups of cells,
The response to drug treatment in clonogenic cell while multiple colonies continued to grow in the
survival indicates the efficacy of the drug (17). The presence of CDDP, in the R_SKOV-3 cells (Fig.
survival of cancer cells in the presence of the drug, 5).
and their ability to form clones from a single cell
characterizes the uncontrolled growth of cancer

Fig. 5: SKOV-3 cells representing the colony formation assay after exposure to TQ and CDDP for 14 days.
The images signify (1) Untreated SKOV-3 as control (2) SKOV-3 treated with 3 μM/mL CDDP (3) SKOV-3
treated with 14 μM/mL TQ (4) R_SKOV-3 untreated (5) R_SKOV-3 treated with 6 μM/mL CDDP (6)
R_SKOV-3 treated with 14 μM/mL TQ. (7) Illustration of colony growth in CDDP and TQ.

The colonies were counted and the graph was Bcl-2 expression significantly changed when
plotted against untreated control for both. The bar compared to the control indicating their pro-
graph represents data expressed as mean ± S.D. apoptotic effects (Fig. 6). Alternatively in
The significance difference indicated as ***p < R_SKOV-3, Bcl-2 significantly reduced after TQ
0.0001, **p<0.01, and *p<0.05. treatment. Secondly, p53 functions as a tumor-
suppressor which is down-regulated in the cancer
TQ contributes to the modulation in the protein micro-environment enabling tumor progression.
expressions The gene responsible for p53 is also found to be
The modifications in the protein expression were mutated in cancers, which enables drug resistance
analyzed for both SKOV-3 and R_SKOV-3 cells (20). In SKOV-3, we observed a slight up
post-treatment with CDDP and TQ. Bcl-2 is an regulation of p53 upon CDDP and TQ treatment.
anti-apoptotic protein, activated in various cancers In R_SKOV-3 significant increase was observed in
and closely related to chemoresistance, specifically response to TQ, which was minimal in CDDP and
studied in OvCa. The up-regulation of Bcl-2 control cells. This shows the regulation of
instigates pro-survival activity in OvCa adding to apoptotic proteins by TQ in OvCa cells and its
tumorigenesis (19). Our results revealed that after efficacy in its resistant counterpart.
the treatment with TQ and CDDP in SKOV-3, the

Fig. 6: Western blot analysis for p53 and Bcl-2 and the loading control was -actin. (1) Representation of
changes in protein expression of Bcl-2 and p53 for SKOV-3. (2) Representation of changes in protein
expression of Bcl-2 and p53 for R_SKOV-3.
reduce toxicity levels while increasing the quality
CONCLUSION of life in patients. TQ has been demonstrated as a
Naturally occurring compounds tend to promising therapeutic agent to overcome drug
synergically work with multiple pathways and resistance and alleviate adverse effects. It has
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In Skov-3 Section A-Research Paper

cytoprotective roles on drugs like CDDP, Five Platinum-Based Antitumor Agents. Front
carboplatin, paclitaxel, cyclophosphamide, etc., Pharmacol. 2020;11(March):1–17.
which prove its potential forthcoming clinical uses. 6. Stewart JJ, White JT, Yan X, Collins S,
Its effects make individuals less susceptible to Drescher CW, Urban ND, et al. Proteins
other diseases in turn protecting them from the associated with cisplatin resistance in ovarian
weakening of the immune system. Also, it shields cancer cells identified by quantitative
normal human cells from oxidative damage while proteomic technology and integrated with
decreasing mitochondrial permeability, dropping mRNA expression levels. Molecular and
Bcl-2, and enhancing Bax expression in cancer. It Cellular Proteomics. 2006;5(3):433–43.
produces excessive ROS, and DNA damage, 7. Imran M, Rauf A, Khan IA, Shahbaz M,
activating signaling pathways and further causing Qaisrani TB, Fatmawati S, et al.
apoptosis of cancer cells. It also helps to overcome Thymoquinone: A novel strategy to combat
drug resistance by obstructive the progression of cancer: A review. Vol. 106, Biomedicine and
cells. TQ can be used as broadly used as a novel Pharmacotherapy. Elsevier Masson SAS; 2018.
therapeutic in the treatment of OvCa and several p. 390–402.
other types. Hence, TQ proves to be a suitable 8. Wilson AJ, Saskowski J, Barham W, Yull F,
candidate to be taken up to clinical trials to Khabele D. Thymoquinone enhances cisplatin-
remarkably combat distinctive types of cancer. response through direct tumor effects in a
syngeneic mouse model of ovarian cancer. J
Statement and Declarations Ovarian Res. 2015 Jul 28;8(1).
Funding: No funding was involved in the project 9. Liu X, Dong J, Cai W, Pan Y, Li R, Li B. The
effect of thymoquinone on apoptosis of SK-
Conflict of Interest OV-3 ovarian cancer cell by regulation of Bcl-
The authors declare no competing interests. 2 and Bax. International Journal of
Gynecological Cancer. 2017;27(8):1596–601.
Author Contributions 10. Nessa Meher, PHILIP BEALE, CHARLES
All the authors contributed to the study design. CHAN, JUN Q. YU and, FAZLUL HUQ1.
Material preparation, data collection, and analysis Synergism from Combinations of Cisplatin and
were performed by Shivani Tendulkar and Oxaliplatin with Quercetin and Thymoquinone
Aishwarya Hattiholi. Dr Vijay Kumbar helped in Human Ovarian Tumour Models. Anticancer
with data analysis. Ms Meenaz Sangolli helped Res. 2011;31:3789–98.
with the protein analysis. Dr Suneel Dodamani, Dr 11. Govindan SV aliyaveedan, Kulsum S, Pandian
Kishore Bhat and Dr Mehul Shah commented on RS omasundara, Das D, Seshadri M, Hicks W,
previous versions of the manuscript. All authors et al. Establishment and characterization of
read and approved the manuscript. triple drug-resistant head and neck squamous
cell carcinoma cell lines. Mol Med Rep. 2015
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