Available online on 15.10.2023 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright   © 2023 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open  Access  Full Text Article                                                                                                                            Research Article

Effect of Thymoquinone on Ovarian Carcinoma Cell Viability (OVCAR-3)

İlhan Özdemir¹* , Cenap Ekinci ¹, Cenap Ekinci¹*

¹   Dicle University Faculty of Medicine Histology and Embryology Department,Diyarbakır, Turkey.

INTRODUCTİON

Cancer is the second leading cause of death worldwide, responsible for an estimated 9.6 million deaths in 2018 ¹. Ovarian cancer is the third most common gynecological malignancy worldwide. However, it has the highest mortality rate among cancers due to its asymptomatic course, late diagnosis and recurrence ^2,3. When diagnosed, it is usually accompanied by omentum involvement, widespread malignant ascites and intraperitoneal metastasis ^4,5. According to available data, ovarian cancer also develops resistance to traditional chemotherapeutics, which contributes to recurrence ⁶. The specific etiology for ovarian cancer remains unknown, and since these cancers tend to occur in advanced stages, the early molecular events underlying development remain unknown.

Medicinal plants are important not only as therapeutic agents but also for pharmacological research and drug development. Since many features of the cell come into play in the development of cancer, treatment with a single therapy is rarely effective ⁷. For this reason, combination treatments have become preferred. Because of the different pathways targeted and the lower dosage of chemotherapeutic agents used, toxicity is significantly less ^8,9. In addition, significant side effects occur in patients with the use of chemically synthesized drugs. Therefore, the discovery and development of new drugs based on natural products has become the focus of research ^10,11. 

Thymoquinone (TQ) is one of the major bioactive components of Nigella sativa (black cumin) essential oil. It has proven its effectiveness against various diseases thanks to its many medical and pharmacological activities such as anti-inflammatory, antioxidant, antihistamine, antitumor, analgesic, anti-Alzheimer, hepatoprotector, neuroprotector, histone protein modulator, insecticidal effects, anti-ischemic, leishmanicides, radioprotectors ^{12, 13}. TQ alone has demonstrated anticancer activity in various in vitro and in vivo studies as well as in adjuvant therapy to prevent carcinogenesis or enhance the effectiveness of conventional therapeutic techniques ¹⁴.

In this study, the possible synergistic or antagonistic effect of TQ with Dox alone and in combination was investigated. In this study, the anticancer effects of TQ, one of the agents currently being studied in the current literature, were investigated in order to identify agents that would reduce the effects of chemotherapy as alternative treatments and to reveal their effectiveness. 

MATERİAL AND METHODS

Culture and passage of cells

In the study, ovarian carcinoma cell line NIH:OVCAR-3 (HTB-161™) and human skin keratinocyte cell line HaCat (RRID:CVCL 0038) were used as the healthy cell line. OVCAR-3 cell line was cultured in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin, and HaCaT cell line was cultured in DMEM medium containing the same additives, and the cells were cultured in sterile incubators at 37°C and 5% CO2. grown in an incubator containing In all studies, the study started from the 5th passage of the cell lines and ended at the 15th or 20th passage at most.

Determination of IC50 doses of Doxorubicin and Thymoquinone agents in OVCAR-3 and HaCaT cell lines

In the study, stock solutions of Dox and TQ agents were prepared using ultrapure Ethanol (Merck, USA), 5 mM stock solutions were made for Dox and 100 mM stock solutions were made for TQ, and these were portioned and stored at -20ºC. In the applications performed, the final concentration of the vehicle in the 96-well culture dish was reduced to 0.1%.

To determine Dox and TQ IC50 doses, OVCAR-3 and HaCaT cell lines were cultivated in 96-well culture plates (plates) with automatic multipipetting, 3000-5000 cells in each well, respectively. At the end of one night (approximately 16 hours), Dox was applied at 9 different concentrations obtained by serial dilution at dose ranges of 0.5-50 µM and TQ 5-500 µM, and the plates were incubated for 48 hours. In MTT cell viability analysis, the wells on the outside of the 96-well culture dish were excluded to reduce trial error, and each chemotherapy agent and vehicle control groups consisted of 6 wells. MTT test was performed for cell survival (viability) analysis after incubation. For this purpose, the "Yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)" test solution prepared at a dose of 5 mg/ml was pipetted into all wells at 20 µl/well. Then, the plates were left to incubate for 4 hours, after the incubation, the medium in the wells was completely removed and 200µl ultra puree DMSO (Merk, USA) was added to each well and kept in the incubator under dark conditions for 2-4 hours. At the end of this period, the plates were read spectrophotometrically at 492, 570 and 650 nm wavelengths with a Multiskan GO microplate reader (Thermo Scientific, USA). The value obtained from the control group applied to the vehicle was determined as a comparative viability rate based on 100% viability. IC50 results statistics for each tumor cell line and chemotherapy agents in the control and experimental groups were calculated using probit analysis with the SPSS 20.0 package program.

RESULTS

Within the scope of the study, the % cell viability obtained as a result of the MTT test in the OVCAR-3 cell series after Dox application and the IC50 value calculated using probit analysis and statistical analysis compared to the control are given in Table 1. The data obtained showed that the IC50 value was found to be 2.12 µM as a result of Dox application to the OVCAR-3 cell line for 48 hours. No IC50 value was found in 24-hour Dox application. It was observed that there were significant decreases in cell proliferation as the dose increased. The application started with the cultivation of 100,000 cells, and the number of cells was obtained as 23.31 in Dox application at 50 µM concentration (Table 1). After finding the IC50 value as a result of statistical analysis, it was determined that cell viability decreased significantly after 5 nM Dox application compared to the vehicle group (Figure 1).

Table 1. It was obtained by serial dilution in the OVCAR-3 ovarian carcinoma cell line in the concentration range of 0.5-50 µM. % cell viability, standard deviation and standard error values, 95% confidence interval and minimum maximum values obtained after Doxorubicin application at 9 different concentrations for 48 hours.

The % cell viability obtained as a result of the MTT test in the OVCAR-3 cell series after TQ application and the IC50 value calculated using probit analysis and statistical analysis compared to the control are given in Table 2. As a result of 48 hours of TQ application, the IC50 value was found to be 62.9 µM. Then, the statistical significance between the vehicle group and different concentrations of TQ was calculated using one-way ANOVA and Tukey HSD test according to P ≤ 0.05. As a result, it was observed that cell viability decreased significantly after 48 hours of TQ application and 50 µM concentration (Table 2).

Table 2. OVCAR-3 was obtained by serial dilution in the 5-500 µM concentration range in the ovarian carcinoma cell line. % cell viability, standard deviation and standard error values, 95% confidence interval and minimum and maximum values obtained after TQ application for 48 hours at 9 different concentrations. 

The effect of Dox and TQ applications on healthy cell lines was also analyzed and the % cell viability obtained from the MTT test on the HaCaT cell series after the applications of both agents and IC50 values calculated using probit analysis and statistical analysis compared to the control are given in Tables 3 and 4. .

As a result of Dox application to the HaCaT cell line for 48 hours, the IC50 value was found to be 5.32 µM. It was observed that there were significant decreases in cell proliferation as the dose increased. The application started with the cultivation of 100,000 cells, and the number of cells was obtained as 11.1679 in Dox application at 50 µM concentration (Table 3). After finding the IC50 value as a result of statistical analysis, it was determined that cell viability decreased significantly after 7.5 µM Dox application compared to the vehicle group (Figure 3).

Table 3. HaCaT was obtained by serial dilution in the concentration range of 0.5-50 µM in human healthy dermal keratinocyte cell lines. % cell viability, standard deviation and standard error values, 95% confidence interval and minimum maximum values obtained after Doxorubicin application at 9 different concentrations for 48 hours. 

The effect of TQ applications on healthy cell lines was also analyzed and the % cell viability obtained as a result of the MTT test in the HaCaT cell series and IC50 values calculated using probit analysis and statistical analysis compared to the control are given in Table 4.

No IC50 value was found as a result of 24-hour GA application to the HaCaT cell line. With 48 hours of TQ application, the IC50 affecting the viability of the HaCaT cell line was found to be 346.4 µM. It was observed that there were significant decreases in cell proliferation as the dose increased. The application started with the cultivation of 100,000 cells, and the number of cells was obtained as 39.3925 in TQ application at 500 µM concentration (Table 4). After finding the IC50 value as a result of statistical analysis, it was determined that cell viability decreased significantly after 100 µM TQ application compared to the vehicle group (Table 4).

The findings show that Thymoquinone can increase the therapeutic effect and reduce the toxicity on healthy cells when combined with chemotherapy drugs used in the treatment of ovarian cancer. It has been shown that TQ can exhibit a synergistic effect with Dox and also play a preventive role against doxorubicin toxicity.

Table 4. HaCaT was obtained by serial dilution in the concentration range of 5-500 µM in human healthy dermal keratinocyte cell lines. % cell viability, standard deviation and standard error values, 95% confidence interval and minimum maximum values obtained after TQt application at 9 different concentrations for 48 hours. 

DİSCUSSİON

Although chemotherapeutic drugs are very effective, their use is restricted due to their serious side effects and the development of drug resistance. Natural products with anticancer activity can provide solutions at this point. In this study, we found that TQ could increase the anti-cancer activity with the MTT method in combination with Dox in the OVCAR-3 cell line. Our results showed that the most promising treatment was not the use of Dox and TQ alone, but their combination, as it showed the highest cytotoxic effect among the groups by MTT analysis.

Ovarian cancer is the leading cause of gynecological cancer deaths in developed countries and often occurs at an advanced stage. ¹⁵. The current standard treatment for advanced ovarian cancer is cytoreductive surgery and platinum/taxane-based chemotherapy ^16-18. The response rate to treatment is approximately 80-90%, but many often relapse and develop resistance to chemotherapy ¹⁴. Therefore, alternative approaches to the diagnosis and treatment of ovarian cancer are needed. Over the past three decades, a number of preclinical and clinical studies have been conducted to identify potential drug candidates (as mono or combined treatments) or to improve the therapeutic efficacy of existing chemotherapy regimens against ovarian cancer ¹⁹.

New compounds derived from plants have recently gained importance as an alternative to conventional therapies due to their potent activity against carcinogenic cells with limited or negligible side effects. They are particularly focused on producing some promising cytotoxic drugs originating from natural compounds such as rosmarinic acid, alpha lipoic acid, ascorbic acid, curcumin, etc ^20-22. It is mainly aimed to reduce or even eliminate the side effects of existing chemotherapy. Antioxidant therapy can be defined as treatment that prevents or reduces the side effects of free radicals. The effectiveness of exogenous antioxidants in protecting tissues from oxidative stress in vivo is variable and depends on the type of antioxidant, its biopharmaceutical properties, its concentration at the site of action, and the nature of the oxidative stress ²².

Cancer has become a deadly disease in today's world that kills hundreds of thousands of people every year. Standard treatments such as chemotherapy, radiation therapy, or immunotherapy are successful in only a fraction of the patient population due to the heterogeneity of the disease. By targeting a single gene, gene product, or signaling pathway, only a specific group of cells in the tumor can be eliminated, while other genetically distinct variations can easily escape treatment and form tumors in the surrounding area or spread to distant locations. Therefore, in cancers, such features may ultimately lead to drug resistance ^23-25. These poor outcomes are caused by genomic instability and abnormal activation of DNA maintenance genes involved in DNA damage detection and repair. To ameliorate the failure of cancer treatments such as platinum-based chemotherapies, drug redirection (new uses of old drugs) is being implemented as sensitizers of genotoxic therapy by inhibiting the repair of DNA damage and also by causing cell death ^26,27. Based on this information, it was investigated whether the Dox-TQ combination could be a viable new cancer treatment drug. In the MTT test, the cytotoxic effect of Dox (0.5-50 µM), TQ (5-500 µM) alone or in combination on OVCAR-3 and HaCaT cells was evaluated for 24 and 48 hours. According to the results of the effects of Dox and TQ on cell growth rate, it was observed that the cell growth rate of OVCAR-3 cells decreased depending on the 48-hour period and dose. The results obtained were found to be close to the determined IC50 of TQ: 55.3 µM and 50 µM doses in pancreatic cancer lines ^28,29. After 48 hours, the most cytotoxic effect of thymoquinone alone on OVCAR-3 cell viability was found with an IC value of 62.9 µM. In another study, Samerghandien and colleagues showed that thymoquinone induces apoptosis in A549 cells by increasing the Bax/Bcl-2 ratio and positively regulates p53 expression ³⁰. After thymoquinone treatment alone in A549 cells, a slight decrease in caspase 3/7 activity was detected in late apoptosis. Spironolactone and thymoquinone compounds, each in combination with other drugs, have been shown in other studies to cause an increase in caspase 3/7 activation in cancer lines ^31,32.

The difference between the IC50 values of TQ in the OVCAR-3 cell seen in our study can be explained by the fact that the MTT test has been used in cancer research for 30 years. Since some cell lines can respond resistantly in the analysis based on absorbance measurement, different results can be obtained even in the same measurements ^33-35. A consistent IC50 value is rarely obtained for a given chemical compound against a given cancer cell line. He et al attributed this problem to differences between manufacturers and the formulas used by different laboratories. Even within the same laboratory, MTT test results reported that variable IC50 values could be obtained between different researchers and between different experiment repetitions performed by the same researcher. This discrepancy may be explained by variability in the control wells used as the basis for calculating IC50 values. These variations depend on the initial cell density and proliferation potential of the cell line ³⁶. 

In our study, TQ-induced apoptosis was induced as shown by the results of the MTT assay. To understand the role of thimoquinone in inducing apoptosis, it is necessary to examine the expression of apoptotic markers such as P53, Bcl-2 and Caspase-3.

CONCLUSİON

The current study suggests that the combination of Dox and TQ may be a promising protocol for ovarian cancer treatment and an alternative to the widely used current regimen, Paclitaxel/Carboplatin. The Dox/TQ combination offers a number of advantages over existing protocols, such as higher efficacy, lower dose and fewer side effects. More detailed mechanistic and efficacy studies in cell culture, animal models and ultimately clinical trials are recommended to evaluate Dox/TQ combination therapy.

ACKNOWLEDGEMENT

This study was a part of doctorate thesis of İlhan ÖZDEMİR"

Ethic

Ethical approval is not required because commercially available cell lines are used as an in vitro study.

Conflict of Interest: The authors declare that they have no conflict of interest.

Financial Support: 

This study was funded by Dicle University Scientific Research Platform (DÜBAP).

REFERENCES

1. Cancer. https ://www.who.int/news-room/fact-sheet s/detail/cancer. Erişim: 16.04.2023.

2. Kuroki L, Guntupalli SR. Treatment of epithelial ovarian cancer. BMJ. 2020;371:m3773. https://doi.org/10.1136/bmj.m3773 PMid:33168565

3. Barani M, Bilal M, Sabir F, Rahdar A, Kyzas GZ. Nanotechnology in ovarian cancer: Diagnosis and treatment. Life Sci. 2021;266:118914. https://doi.org/10.1016/j.lfs.2020.118914 PMid:33340527

4. Rojas V, Hirshfield KM, Ganesan S, Rodriguez-Rodriguez L. Molecular Characterization of Epithelial Ovarian Cancer: Implications for Diagnosis and Treatment. Int J Mol Sci. 2016;17(12):2113. https://doi.org/10.3390/ijms17122113 PMid:27983698 PMCid:PMC5187913

5. Chen SN, Chang R, Lin LT, Chern CU, Tsai HW, Wen ZH, Li YH, Li CJ, Tsui KH. MicroRNA in Ovarian Cancer: Biology, Pathogenesis, and Therapeutic Opportunities. Int J Environ Res Public Health. 2019;16(9):1510. https://doi.org/10.3390/ijerph16091510 PMid:31035447 PMCid:PMC6539609

6. Tarhriz V, Bandehpour M, Dastmalchi S, Ouladsahebmadarek E, Zarredar H, Eyvazi S. Overview of CD24 as a new molecular marker in ovarian cancer. J Cell Physiol. 2019;234(3):2134-2142. https://doi.org/10.1002/jcp.27581 PMid:30317611

7. Aborehab NM, Osama N. Effect of Gallic acid in potentiating chemotherapeutic effect of Paclitaxel in HeLa cervical cancer cells. Cancer Cell Int. 2019;19:154. https://doi.org/10.1186/s12935-019-0868-0 PMid:31171918 PMCid:PMC6547587

8. Gong C, Xie Y, Zhao Y, et al. Comparison of two regimens of weekly paclitaxel plus gemcitabine in patients with metastatic breast cancer: propensity score-matched analysis of real-world data. Ther Adv Drug Saf. 2022;13:20420986221146411. https://doi.org/10.1177/20420986221146411 PMid:36582188 PMCid:PMC9793024

9. Bayat Mokhtari R, Baluch N, Morgatskaya E, et al. Human bronchial carcinoid tumor initiating cells are targeted by the combination of acetazolamide and sulforaphane. BMC Cancer. 2019;19(1):864. https://doi.org/10.1186/s12885-019-6018-1 PMid:31470802 PMCid:PMC6716820

10. Frazier AL, Stoneham S, Rodriguez-Galindo C, et al. Comparison of carboplatin versus cisplatin in the treatment of paediatric extracranial malignant germ cell tumours: a report of the Malignant Germ Cell International Consortium. Eur J Cancer. 2018;98:30-7. https://doi.org/10.1016/j.ejca.2018.03.004 PMid:29859339

11. Jiang S, Pan AW, Lin TY, et al. Paclitaxel enhances carboplatin-dna adduct formation and cytotoxicity. Chem Res Toxicol. 2015;28(12):2250-2. https://doi.org/10.1021/acs.chemrestox.5b00422 PMid:26544157 PMCid:PMC4834887

12. Ahmad A, Mishra RK, Vyawahare A, Kumar A, Rehman MU, Qamar W, Khan AQ, Khan R. Thymoquinone (2-Isopropyl-5-methyl-1, 4benzoquinone) as a chemopreventive/anticancer agent: Chemistry and biological effects. Saudi Pharmaceutical Journal, 2019;27(8): 1113-1126. https://doi.org/10.1016/j.jsps.2019.09.008 PMid:31885471 PMCid:PMC6921197

13. Almajali B, Al-Jamal HAN, Taib WRW, Ismail I, Johan MF, Doolaanea AA, Ibrahim WN. (2021). Thymoquinone, as a Novel Therapeutic Candidate of Cancers. Pharmaceuticals, 2021;14(4): 369. https://doi.org/10.3390/ph14040369 PMid:33923474 PMCid:PMC8074212

14. Chowdhury FA, Hossain MK, Mostofa AGM, Akbor MM, bin Sayeed MS. Therapeutic Potential of Thymoquinone in Glioblastoma Treatment: Targeting Major Gliomagenesis Signaling Pathways. BioMed Research International, 2018;2018: 1-15. https://doi.org/10.1155/2018/4010629 PMid:29651429 PMCid:PMC5831880

15. Bhattacharya S, Muhammad N, Steele R, et al. Immunomodulatory role of bitter melon extract in inhibition of head and neck squamous cell carcinoma growth. Oncotarget. 2016;7(22):33202-9. https://doi.org/10.18632/oncotarget.8898 PMid:27120805 PMCid:PMC5078086

16. De A, De A, Sharma R, et al. Sensitization of Carboplatinum- and Taxol-Resistant High-Grade Serous Ovarian Cancer Cells Carrying p53, BRCA1/2 Mutations by Emblica officinalis (Amla) via Multiple Targets. J Cancer. 2020;11(7):1927-1939. https://doi.org/10.7150/jca.36919 PMid:32194804 PMCid:PMC7052860

17. Sourani ZM, Pourgheysari BP, Beshkar PM, et al. Gallic Acid inhibits proliferation and induces apoptosis in lymphoblastic leukemia cell line (C121). Iran J Med Sci. 2016;41(6):525-30.

18. He Z, Chen AY, Rojanasakul Y, et al. Gallic acid, a phenolic compound, exerts anti-angiogenic effects via the PTEN/AKT/HIF-1α/VEGF signaling pathway in ovarian cancer cells. Oncol Rep. 2016 Jan;35(1):291-7. https://doi.org/10.3892/or.2015.4354 PMid:26530725 PMCid:PMC4699619

19. Jiang Y, Pei J, Zheng Y, et al. Gallic Acid: A Potential Anti-Cancer Agent. Chin J Integr Med. 2022;28(7):661-671. doi: 10.1007/s11655-021-3345-2. https://doi.org/10.1007/s11655-021-3345-2 PMid:34755289

20. Park WH, Kim SH. MAPK inhibitors augment gallic acid-induced A549 lung cancer cell death through the enhancement of glutathione depletion. Oncol Rep. 2013;30(1):513-9. doi: 10.3892/or.2013.2447. https://doi.org/10.3892/or.2013.2447 PMid:23660987

21. Al Balushi N, Hassan SI, Abdullah N, et al. Addition of Gallic Acid Overcomes Resistance to Cisplatin in Ovarian Cancer Cell Lines. Asian Pac J Cancer Prev. 2022;23(8):2661-2669. doi: 10.31557/APJCP.2022.23.8.2661. https://doi.org/10.31557/APJCP.2022.23.8.2661 PMid:36037120 PMCid:PMC9741893

22. Park WH. Gallic acid induces HeLa cell death via increasing GSH depletion rather than ROS levels. Oncol Rep. 2017;37(2):1277-83. https://doi.org/10.3892/or.2016.5335 PMid:28035417

23. Housman, G., Byler, S., Heerboth, S., Lapinska, K., Longacre, M., Snyder, N., & Sarkar, S. (2014). Drug resistance in cancer: An overview. In Cancers (Vol. 6, Issue 3). https://doi.org/10.3390/cancers6031769 PMid:25198391 PMCid:PMC4190567

24. Krzyszczyk, P., Acevedo, A., Davidoff, E. J., Timmins, L. M., Marrero-Berrios, I., Patel, M., White, C., Lowe, C., Sherba, J. J., Hartmanshenn, C., O'Neill, K. M., Balter, M. L., Fritz, Z. R., Androulakis, I. P., Schloss, R. S., & Yarmush, M. L. (2018). The growing role of precision and personalized medicine for cancer treatment. TECHNOLOGY, 06(03n04), 79-100. https://doi.org/10.1142/S2339547818300020 PMid:30713991 PMCid:PMC6352312

25. Mostofa, A., Hossain, M. K., Basak, D., & bin Sayeed, M. S. (2017). Thymoquinone as a Potential Adjuvant Therapy for Cancer Treatment : Evidence from Preclinical Studies. Frontiers in Pharmacology, 8. https://doi.org/10.3389/fphar.2017.00295 PMid:28659794 PMCid:PMC5466966

26. Manolis, A. A., Manolis, T. A., Melita, H., & Manolis, A. S. (2019). Spotlight on Spironolactone Oral Suspension for the Treatment of Heart Failure : Focus on Patient Selection and Perspectives Vascular Health and Risk Management, Volume 15, 571-579. https://doi.org/10.2147/VHRM.S210150 PMid:31920323 PMCid:PMC6941679

27. Zhang, Z., Zhou, L., Xie, N., Nice, E. C., Zhang, T., Cui, Y., & Huang, C. (2020). Overcoming cancer therapeutic bottleneck by drug repurposing. Signal Transduction and Targeted Therapy, 5(1). https://doi.org/10.1038/s41392-020-00213-8 PMid:32616710

28. Jafri, M. A., Ansari, S. A., Alqahtani, M. H., & Shay, J. W. (2016b). Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies. Genome Medicine, 8(1). https://doi.org/10.1186/s13073-016-0324-x PMid:27323951 PMCid:PMC4915101

29. Zubair, H., Khan, H. Y., Sohail, A., Azim, S., Ullah, M. F., Ahmad, A., Sarkar, F. H., & Hadi, S. M. (2013). Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage and consequent cell death : putative anticancer mechanism of antioxidants. Cell Death & Disease, 4(6), e660. https://doi.org/10.1038/cddis.2013.172 PMid:23744360 PMCid:PMC3698541

30. Samarghandian, S., Azimi‐Nezhad, M., & Farkhondeh, T. (2018). Thymoquinoneinduced antitumor and apoptosis in human lung adenocarcinoma cells. Journal of Cellular Physiology, 234(7), 10421-10431. https://doi.org/10.1002/jcp.27710 PMid:30387147

31. Chu, S. C., Hsieh, Y. S., Yu, C. C., Lai, Y. Y., & Chen, P. N. (2014). Thymoquinone Induces Cell Death in Human Squamous Carcinoma Cells via Caspase Activation-Dependent Apoptosis and LC3-II Activation-Dependent Autophagy. PLoS ONE, 9(7), e101579. https://doi.org/10.1371/journal.pone.0101579 PMid:25000169 PMCid:PMC4085014

32. Warrier, N. M., Agarwal, P., & Kumar, P. (2020). Emerging Importance of Survivin in Stem Cells and Cancer : the Development of New Cancer Therapeutics. Stem Cell Reviews and Reports, 16(5), 828-852. https://doi.org/10.1007/s12015-020-09995-4 PMid:32691369 PMCid:PMC7456415

33. Präbst K, Engelhardt H, Ringgeler S, et al. Basic Colorimetric Proliferation Assays: MTT, WST, and Resazurin. Methods Mol Biol. 2017;1601:1-17. doi: 10.1007/978-1-4939-6960-9_1. https://doi.org/10.1007/978-1-4939-6960-9_1 PMid:28470513

34. Yan XX, Zhao YQ, He Y, et al. Cytotoxic and pro-apoptotic effects of botanical drugs derived from the indigenous cultivated medicinal plant Paris polyphylla var. yunnanensis. Front Pharmacol. 2023;14:1100825. https://doi.org/10.3389/fphar.2023.1100825 PMid:36778018 PMCid:PMC9911168

35. Stockert JC, Horobin RW, Colombo LL, et al. Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem. 2018;120(3):159-167. https://doi.org/10.1016/j.acthis.2018.02.005 PMid:29496266

36. He Y, Zhu Q, Chen M, et al. The changing 50% inhibitory concentration (IC50) of cisplatin: a pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer. Oncotarget. 2016;7(43):70803-21. https://doi.org/10.18632/oncotarget.12223 PMid:27683123 PMCid:PMC5342590