Decreased Autophagic Activity in Triple-Negative Breast Cancer Cells upon Hydroxychloroquine and Thymoquinone Combination Treatment

Year 2024, Volume: 6 Issue: 3, 246 - 256, 14.10.2024

Tuğcan Korak , Merve Gulsen Bal Albayrak , Sevinc Yanar , Gürler Akpınar , Murat Kasap

https://doi.org/10.52827/hititmedj.1472196

Abstract

Objective: Autophagy plays a significant role in breast cancer tumorigenesis, including triple-negative breast cancer. Research indicates that hydroxychloroquine and thymoquinone modulate autophagy, potentially suppressing its activity. However, their combined effects on autophagy in triple-negative breast cancer remain unexplored. In this study, we investigated the potential anti-cancer and autophagy-modulating effects of hydroxychloroquine-thymoquinone combination on triple-negative breast cancer cells in vitro.
Material and Method: The viability of MDA-MB-231 cells was evaluated after treatment with hydroxychloroquine (10-210 µM) and thymoquinone (5-45 µM) for 24 and 48 hours using the WST-1 assay. Combination effects were analyzed using the Chou-Talalay method with CompuSyn (v.10). Autophagic vesicles were visualized using an Autophagy Detection Kit and fluorescence microscopy to investigate their role in the decrease in cell viability. Statistical analysis was performed with GraphPad Prism (v.8.3.0).
Results: At both 24- and 48-hour intervals post-treatment, a significant decrease in viability was observed for both hydroxychloroquine and thymoquinone treatments individually (p<0.0001). The combination of these drugs revealed pronounced synergistic effects at 24 hours, whereas antagonistic effects were noted at 48 hours (combination index>1). At 24 hours, favorable dose reduction effects were evident (dose reduction index >1), while the 48-hour results showed an unfavorable reduction (dose reduction index<1). Consequently, the 24-hour synergistic effects resulted in a reduction in autophagic vesicles (p<0.0001).(p<0.0001).
Conclusion: This study revealed, for the first time, a time-dependent decrease in triple-negative breast cancer cell viability via the autophagy mechanism induced by hydroxychloroquine and thymoquinone, highlighting their novel implications for triple-negative breast cancer treatment and autophagy modulation.

Keywords

Autophagy, breast cancer, drug-combination, hydroxychloroquine, thymoquinone

Ethical Statement

Not applicable.

Triple Negatif Meme Kanseri Hücrelerinde Hidroksiklorokin ve Timokinon Kombinasyon Uygulaması Sonrası Otofajik Aktivitenin Azalması

Abstract

Amaç: Otofaji, triple negatif meme kanseri alt tipi dahil olmak üzere meme kanserinde tümör oluşumunda etkili bir mekanizmadır. Araştırmalar, hidroksiklorokin ve timokinon’un otofajiyi düzenleyerek aktivitesini potansiyel olarak baskıladığını göstermektedir. Ancak, bu maddelerin kombine uygulanmasının triple negatif meme kanserinde otofaji üzerindeki etkileri henüz aydınlatılmamıştır. Bu çalışma, hidroksiklorokin ve timokinon kombinasyonunun triple negatif meme kanseri hücrelerinde hücrelerinde antikanser ve otofajik etkilerini in vitro olarak incelemeyi hedeflemektedir.
Gereç ve Yöntem: Hidroksiklorokin (10-210 µM) ve timokinon (5-45 µM)’nun 24 ve 48 saat boyunca MDA-MB-231 hücrelerine uygulanması sonucu hücrelerin canlılığı WST-1 testi ile değerlendirildi. Kombinasyonlarının etkileri, CompuSyn (v.10) ile kombinasyon indeksi ve doz azaltma indeksi hesaplanarak Chou-Talalay yöntemiyle analiz edildi. Hücre canlılığının azalmasındaki otofaji etkisini gözlemlemek adına, otofajik veziküllerin tanımlanması ve görüntülenmesi için “Autophagy Detection Kit” ve floresan mikroskobu kullanıldı. İstatistiksel analizler, GraphPad Prism (v.8.3.0) kullanılarak gerçekleştirildi (p<0,0001).
Bulgular: Hidroksiklorokin ve timokinon’un 24 ve 48 saatlik uygulamaları sonucu hücre canlılığında anlamlı bir azalma gözlemlendi (p<0,0001). Kombinasyon uygulamaları sonucu sinerjistik etkileri 24 saatte belirgin olarak görülürken, 48 saat uygulanmaları sonucu ise antagonistik etkiler elde edildi (kombinasyon indeksi>1). Doz azaltmada olumlu etkiler 24 saatte belirgin iken (doz azaltma indeksi >1), 48 saatte tam tersi etkiler elde edildi (doz azaltma indeksi<1). Sonuç olarak, kombinasyonun 24 saatlik uygulamasıyla karşılaşılan sinerjistik etkilerin otofajik vezikülleri azalttığı gösterildi (p<0,0001).
Sonuç: Bu çalışma, hidroksiklorokin ve timokinon tarafından indüklenen otofaji mekanizması aracılığıyla triple negatif meme kanseri hücrelerinde zamanla bağımlı canlılık azalmasını ilk kez ortaya koymuştur. Bu kombinasyon ileriki çalışmalarla birlikte triple negatif meme kanseri tedavisi ve otofaji modülasyonunda etkin rol oynama potansiyeli taşımaktadır.

Keywords

Hidroksiklorokin, ilaç kombinasyonu, meme kanseri, otofaji, timokinon

Ethical Statement

Etik kurul iznine gerek yoktur.

References

• Leon-Ferre RA, Goetz MP. Advances in systemic therapies for triple negative breast cancer. BMJ 2023;381:e071674.
• Dass SA, Tan KL, Rajan RS, et al. Triple Negative Breast Cancer: A Review of Present and Future Diagnostic Modalities. Medicina 2021;57:62.
• Kim HM, Koo JS. The Role of Autophagy in Breast Cancer Metastasis. Biomedicines 2023;11:618.
• Barsoum RS, Bassily S, Baligh OK, et al. Renal disease in hepatosplenic schistosomiasis: a clinicopathological study. Trans R Soc Trop Med Hyg 1977;71:387–391.
• Hashemi M, Paskeh MDA, Orouei S, et al. Towards dual function of autophagy in breast cancer: A potent regulator of tumor progression and therapy response. Biomed Pharmacother 2023;161:114546.
• Wu Q, Sharma D. Autophagy and Breast Cancer: Connected in Growth, Progression, and Therapy. Cells 2023;12:1156.
• Liu LQ, Wang SB, Shao YF, et al. Hydroxychloroquine potentiates the anti-cancer effect of bevacizumab on glioblastoma via the inhibition of autophagy. Biomed Pharmacother 2019;118:109339.
• Abdel-Aziz AK, Saadeldin MK, Salem AH, et al. A Critical Review of Chloroquine and Hydroxychloroquine as Potential Adjuvant Agents for Treating People with Cancer. Futur Pharmacol 2022;2:431–443.
• Cocco S, Leone A, Roca MS, et al. Inhibition of autophagy by chloroquine prevents resistance to PI3K/AKT inhibitors and potentiates their antitumor effect in combination with paclitaxel in triple negative breast cancer models. J Transl Med 2022;20:290.
• Korak T, Ergül E, Sazci A. Nigella sativa and Cancer: A Review Focusing on Breast Cancer, Inhibition of Metastasis and Enhancement of Natural Killer Cell Cytotoxicity. Curr Pharm Biotechnol 2020;21:1176–1185.
• Racoma IO, Meisen WH, Wang QE, Kaur B, Wani AA. Thymoquinone Inhibits Autophagy and Induces Cathepsin-Mediated, Caspase-Independent Cell Death in Glioblastoma Cells. PLoS ONE 2013;8:e72882.
• Gomathinayagam R, Ha JH, Jayaraman M, Song YS, Isidoro C, Dhanasekaran DN. Chemopreventive and Anticancer Effects of Thymoquinone: Cellular and Molecular Targets. J Cancer Prev 2020;25:136–151.
• Ünal TD, Hamurcu Z, Delibaşı N, et al. Thymoquinone Inhibits Proliferation and Migration of MDA-MB-231 Triple Negative Breast Cancer Cells by Suppressing Autophagy, Beclin-1 and LC3. Anti-Cancer Agents Med Chem 2020;21:355–364.
• Chou T, Talalay P. Generalized Equations for the Analysis of Inhibitions of Michaelis‐Menten and Higher‐Order Kinetic Systems with Two or More Mutually Exclusive and Nonexclusive Inhibitors. Eur J Biochem 1981;115:207–216.
• Zhou N, Liu Q, Wang X, et al. The combination of hydroxychloroquine and 2-deoxyglucose enhances apoptosis in breast cancer cells by blocking protective autophagy and sustaining endoplasmic reticulum stress. Cell Death Discov 2022;8:286.
• Verbaanderd C, Maes H, Schaaf MB, et al. Repurposing Drugs in Oncology (ReDO)—chloroquine and hydroxychloroquine as anti-cancer agents. ecancermedicalscience 2017;11:781.
• Pezzani R, Salehi B, Vitalini S, et al. Synergistic Effects of Plant Derivatives and Conventional Chemotherapeutic Agents: An Update on the Cancer Perspective. Medicina 2019;55:110.
• Rahim R, Strobl JS. Hydroxychloroquine, chloroquine, and all-trans retinoic acid regulate growth, survival, and histone acetylation in breast cancer cells. Anti-Cancer Drugs 2009;20:736–745.
• Wang F, Zhang Z, Leung WT, et al. Hydroxychloroquine reverses the drug resistance of leukemic K562/ADM cells by inhibiting autophagy. Mol Med Rep 2019;20:3883–3892.
• Peng X, Zhang S, Jiao W, et al. Hydroxychloroquine Synergizes With The PI3K Inhibitor BKM120 to Exhibit Antitumor Efficacy Independent of Autophagy. 2021;40:374.
• Schlotter CM, Vogt U, Allgayer H, Brandt B. Molecular targeted therapies for breast cancer treatment. Breast Cancer Res 2008;10:211.
• Peleg L, Ashkenazi IE, Carlebach R, Chaitchik S. Time‐dependent toxicity of drugs used in cancer chemotherapy: Separate and combined administration. Int J Cancer 1989;44:273–275.
• Alagkiozidis I, Facciabene A, Tsiatas M, et al. Time-dependent cytotoxic drugs selectively cooperate with IL-18 for cancer chemo-immunotherapy. J Transl Med 2011;9:77.
• Abrahams B, Gerber A, Hiss DC. Combination Treatment with EGFR Inhibitor and Doxorubicin Synergistically Inhibits Proliferation of MCF-7 Cells and MDA-MB-231 Triple-Negative Breast Cancer Cells In Vitro. Int J Mol Sci 2024;25:3066.
• Sun L, Mi K, Hou Y, et al. Pharmacokinetic and Pharmacodynamic Drug–Drug Interactions: Research Methods and Applications. Metabolites 2023;13:897.
• Sandritter TL, McLaughlin M, Artman M, Lowry J. The Interplay between Pharmacokinetics and Pharmacodynamics. Pediatr Rev 2017;38:195–206.
• Wright DFB, Winter HR, Duffull SB. Understanding the time course of pharmacological effect: a PKPD approach. Br J Clin Pharmacol 2011;71:815–823.
• Adinew GM, Messeha SS, Taka E, Badisa RB, Antonie LM, Soliman KFA. Thymoquinone Alterations of the Apoptotic Gene Expressions and Cell Cycle Arrest in Genetically Distinct Triple-Negative Breast Cancer Cells. Nutrients 2022;14:2120.
• Chen J, Pan Q, Bai Y, Chen X, Zhou Y. Hydroxychloroquine Induces Apoptosis in Cholangiocarcinoma via Reactive Oxygen Species Accumulation Induced by Autophagy Inhibition. Front Mol Biosci 2021;8:720370.
• Chou TC. The mass-action law based algorithms for quantitative econo-green bio-research. Integr Biol 2011;3:548–559.
• Khazaei MR, Bozorgi M, Khazaei M, Moradi A, Bozorgi A. Computational and in vitro analyses on synergistic effects of paclitaxel and thymoquinone in suppressing invasive breast cancer cells. Mol Biol Rep 2024;51:388.
• Soni P, Kaur J, Tikoo K. Dual drug-loaded paclitaxel–thymoquinone nanoparticles for effective breast cancer therapy. J Nanoparticle Res 2015;17:18.
• Şakalar Ç, İzgi K, İskender B, et al. The combination of thymoquinone and paclitaxel shows anti-tumor activity through the interplay with apoptosis network in triple-negative breast cancer. Tumor Biol 2016;37:4467–4677.
• Thabet NA, El‐Khouly D, Sayed‐Ahmed MM, Omran MM. Thymoquinone chemosensitizes human colorectal cancer cells to imatinib via uptake/efflux genes modulation. Clin Exp Pharmacol Physiol 2021;48:911–920.
• Gómez-Virgilio L, Silva-Lucero M del C, Flores-Morelos DS, et al. Autophagy: A Key Regulator of Homeostasis and Disease: An Overview of Molecular Mechanisms and Modulators. Cells 2022;11:2262.