Effect of Chloroquine on Expression of Apoptosis and Autophagy Genes in MOLT-3 and IMR-32 Cells

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Аннотация

The goal of the study was to compare an influence of chloroquine (CQ) on expression of apoptosis and autophagy genes in the cells of two tumor cells – leukemia MOLT-3 and neuroblastoma IMR-32 cultured in complete growth and serum-free RPMI-1640 and DMEM media, respectively, for 24 and 48 hours. The viability of cells was evaluated by MTT method, gene expression – by real time PCR. For MTT test, the cells were incubated with 10–100 µМ CQ. The expression of apoptosis (CASP3, BAX, BCL2) and autophagy (ULK1, BECN1, MAP1LC3B) genes was studied using 30 and 50 µМ CQ, which exerted considerable inhibitory effect on viability of cells of both lines, but did not promote their complete death. The sensitivity of both cell lines to CQ was higher in serum-free medium, however, the expression of apoptosis and autophagy genes substantially differed between them. In MOLT-3 cells, mRNA levels of pro-apoptotic genes CASP3 and BAX increased after 24-h incubation in serum-free medium, whereas in IMR-32 cells the expression of these genes increased only after 48-h in the presence of higher CQ concentration. In the cells of both lines 24-h CQ treatment resulted in enhanced expression of anti-apoptotic gene BCL2. In MOLT-3 cells, the absence of nutrients different combinations stimulated the genes of all three autophagy stages ULK1, BECN1 и MAP1LC3B, but none of applied treatment schemes did not affect the expression of ULK1 and MAP1LC3B genes in IMR-32 cells. Overall, 24-h culture with CQ under conditions of serum starvation appears to be more optimal for modulation of autophagy in MOLT-3 cells. In IMR-32 cells, CQ does not exert considerable influence on expression of autophagy genes, and their decreased viability is associated with activation of other mechanisms.

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Авторлар туралы

E. Prokopenko

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS; Saint Petersburg State University

Хат алмасуға жауапты Автор.
Email: nagalak@mail.ru
Ресей, Saint Petersburg; Saint Petersburg

T. Sokolova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS

Email: nagalak@mail.ru
Ресей, Saint Petersburg

O. Nadey

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS

Email: nagalak@mail.ru
Ресей, Saint Petersburg

A. Trubnikova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS

Email: nagalak@mail.ru
Ресей, Saint Petersburg

N. Agalakova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS

Email: nagalak@mail.ru
Ресей, Saint Petersburg

Әдебиет тізімі

  1. Chern YJ, Tai IT (2020) Adaptive response of resistant cancer cells to chemotherapy. Cancer Biol Med 17(4): 842–863. https://doi.org/10.20892/j.issn.2095-3941.2020.0005
  2. Weng X, Zeng WH, Zhong LY, Xie LH, Ge WJ, Lai Z, Qin Q, Liu P, Cao DL, Zeng X (2024) The molecular mechanisms of chemotherapeutic resistance in tumors (Review). Oncol Rep 52(5): 157. https://doi.org/10.3892/or.2024.8816
  3. Fong W, To KKW (2021) Repurposing chloroquine analogs as an adjuvant cancer therapy. Recent Pat. Anticancer Drug Discov 16(2): 204–221. https://doi.org/10.2174/1574892815666210106111012
  4. Mohsen S, Sobash PT, Algwaiz GF, Nasef N, Al-Zeidaneen SA, Karim NA (2022) Autophagy agents in clinical trials for cancer therapy: A brief review. Curr Oncol 29(3): 1695–1708. https://doi.org/10.3390/curroncol29030141
  5. Agalakova NI (2024) Chloroquine and chemotherapeutic compounds in experimental cancer treatment. Int J Mol Sci 25(2): 945. https://doi.org/10.3390/ijms25020945
  6. De Sanctis JB, Charris J, Blanco Z, Ramírez H, Martínez GP, Mijares MR (2023) Molecular mechanisms of chloroquine and hydroxychloroquine used in cancer therapy. Anticancer Agents Med Chem 23(10): 1122–1144. https://doi.org/10.2174/1871520622666220519102948
  7. Hassan AM, Zhao Y, Chen X, He C (2024) Blockage of autophagy for cancer therapy: A comprehensive review. Int J Mol Sci. 25(13): 7459. https://doi.org/10.3390/ijms25137459
  8. Noguchi M, Hirata N, Tanaka T, Suizu F, Nakajima H, Chiorini JA (2020) Autophagy as a modulator of cell death machinery. Cell Death Dis 11: 517. https://doi.org/10.1038/s41419-020-2724-5
  9. Russell RC, Guan KL (2022) The multifaceted role of autophagy in cancer. EMBO J 41(13): e110031. https://doi.org/10.15252/embj.2021110031
  10. Hama Y, Ogasawara Y, Noda NN (2023) Autophagy and cancer: Basic mechanisms and inhibitor development. Cancer Sci 114(7): 2699–2708. https://doi.org/10.1111/cas.15803
  11. He L, Zhang J, Zhao J, Ma N, Kim SW, Qiao S, Ma X (2018) Autophagy: The Last Defense against Cellular Nutritional Stress. Adv Nutr 9(4): 493–504. https://doi.org/10.1093/advances/nmy011
  12. Prerna K, Dubey VK (2022) Beclin1-mediated interplay between autophagy and apoptosis: New understanding. Int J Biol Macromol 204: 258–273. https://doi.org/10.1016/j.ijbiomac.2022.02.005
  13. Skrzeszewski M, Maciejewska M, Kobza D, Gawrylak A, Kieda C, Waś H (2024) Risk factors of using late-autophagy inhibitors: Aspects to consider when combined with anticancer therapies. Biochem Pharmacol 225: 116277. https://doi.org/10.1016/j.bcp.2024.116277
  14. Trubnikova AD, Prokopenko ES, Sokolova TV, Nadei OV, Agalakova NI (2023) Expression of apoptosis and autophagy genes in HeLa and Hek 293 cells under conditions of nutrient deprivation. J Evol Biochem Phys 59: 2304–2314. https://doi.org/10.1134/S0022093023060315
  15. Racz GA, Nagy N, Tovari J, Apati A, Vertessy BG (2021) Identification of new reference genes with stable expression patterns for gene expression studies using human cancer and normal cell lines. Sci Rep 11: 19459. https://doi.org/10.1038/s41598-021-98869-x
  16. Zhang T, Yu J, Cheng S, Zhang Y, Zhou CH, Qin J, Luo H (2023) Research progress on the anticancer molecular mechanism of targets regulating cell autophagy. Pharmacology 108(3): 224–237. https://doi.org/10.1159/000529279
  17. Rahman MA, Saikat AS, Rahman MS, Islam M, Parvez MA, Kim B (2023) Recent update and drug target in molecular and pharmacological insights into autophagy modulation in cancer treatment and future progress. Cells 12(3): 458. https://doi.org/10.3390/cells12030458
  18. Mulcahy Levy JM, Zahedi S, Griesinger AM, Morin A, Davies KD, Aisner DL, Kleinschmidt-DeMasters BK, Fitzwalter BE, Goodall ML, Thorburn J, Amani V, Donson AM, Birks DK, Mirsky DM, Hankinson TC, Handler MH, Green AL, Vibhakar R, Foreman NK, Thorburn A (2017) Autophagy inhibition overcomes multiple mechanisms of resistance to BRAF inhibition in brain tumors. Elife 6: e19671. https://doi.org/10.7554/eLife.19671
  19. Müller A, Weyerhäuser P, Berte N, Jonin F, Lyubarskyy B, Sprang B, Kantelhardt SR, Salinas G, Opitz L, Schulz-Schaeffer W, Giese A, Ella L, Kim EL (2023) Concurrent Activation of Both Survival-Promoting and Death-Inducing Signaling by Chloroquine in Glioblastoma Stem Cells: Implications for Potential Risks and Benefits of Using Chloroquine as Radiosensitizer. Cells 12(9): 1290. https://doi.org/10.3390/cells12091290
  20. Kazakova D, Shimamura M, Kurashige T, Hamada K, Nagayama Y (2022) Re-evaluation of the role of autophagy in thyroid cancer treatment. Endocr J 69(7): 847–862. https://doi.org/10.1507/endocrj.EJ22-0017
  21. Mauthe M, Orhon I, Rocchi C, Zhou X, Luhr M, Hijlkema KJ, Coppes RP, Engedal N, Mari M, Reggiori F (2018) Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy 48: 1435–1455. https://doi.org/10.1080/15548627.2018.1474314
  22. Choi DS, Blanco E, Kim YS, Rodriguez AA, Zhao H, Huang TH, Chen CL, Jin G, Landis MD, Burey LA, Qian W, Granados SM, Dave B, Wong HH, Ferrari M, Wong ST, Chang JC (2014) Chloroquine eliminates cancer stem cells through deregulation of Jak2 and DNMT1. Stem Cells 32: 2309–2323. https://doi.org/10.1002/stem.1746
  23. Duarte D, Vale N (2016) New trends for antimalarial drugs: Synergism between antineoplastics and antimalarials on breast cancer cells. Biomolecules 10: 1623. https://doi.org/10.3390/biom10121623
  24. Liang DH, Choi DS, Ensor JE, Kaipparettu BA, Bass BL, Chang JC (2016) The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair. Cancer Lett 376: 249–258. https://doi.org/10.1016/j.canlet.2016.04.002
  25. El-Gowily AH, Loutfy SA, Ali EM, Mohamed TM, Mansour MA (2021) Tioconazole and chloroquine act synergistically to combat doxorubicin-induced toxicity via inactivation of PI3K/AKT/mTOR signaling mediated ROS-dependent apoptosis and autophagic flux inhibition in MCF-7 breast cancer cells. Pharmaceuticals (Basel) 14(3): 254. https://doi.org/10.3390/ph14030254
  26. Pagotto A, Pilotto G, Mazzoldi EL, Nicoletto MO, Frezzini S, Pastò A, Amadori A (2017) Autophagy inhibition reduces chemoresistance and tumorigenic potential of human ovarian cancer stem cells. Cell Death Dis 8(7): e2943. https://doi.org/10.1038/cddis.2017.327
  27. Fukuda T, Oda K, Wada-Hiraike O, Sone K, Inaba K, Ikeda Y, Miyasaka A, Kashiyama T, Tanikawa M, Arimoto T, Kuramoto H, Yano T, Kawana K, Osuga Y, Fujii T (2015) The anti-malarial chloroquine suppresses proliferation and overcomes cisplatin resistance of endometrial cancer cells via autophagy inhibition. Gynecol Oncol 137(3): 538–545. https://doi.org/10.1016/j.ygyno.2015.03.053
  28. Lin YC, Lin JF, Wen SI, Yang SC, Tsai TF, Chen HE, Chou KY, Hwang TI (2017) Chloroquine and hydroxychloroquine inhibit bladder cancer cell growth by targeting basal autophagy and enhancing apoptosis. Kaohsiung J Med Sci 33(5): 215–223. https://doi.org/10.1016/j.kjms.2017.01.004
  29. Fauzi YR, Nakahata S, Chilmi S, Ichikawa T, Nueangphuet P, Yamaguchi R, Nakamura T, Shimoda K, Morishita K (2021) Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells. PLoS One 16(8): e0256320. https://doi.org/10.1371/journal.pone.0256320
  30. Kim EL, Wüstenberg R, Rübsam A, Schmitz-Salue C, Warnecke G, Bücker EM, Pettkus N, Speidel D, Rohde V, Schulz-Schaeffer W, Deppert W, Giese A (2010) Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells. Neuro-Oncol 12(4): 389–400. https://doi.org/10.1093/neuonc/nop046
  31. Hu T, Li P, Luo Z, Chen X, Zhang J, Wang C, Chen P, Dong Z (2016) Chloroquine inhibits hepatocellular carcinoma cell growth in vitro and in vivo. Oncol Rep 35(1): 43–49. https://doi.org/10.3892/or.2015.4380
  32. Lakhter AJ, Sahu RP, Sun Y, Kaufmann WK, Androphy EJ, Travers JB, Naidu SR (2013) Chloroquine promotes apoptosis in melanoma cells by inhibiting BH3 domain-mediated PUMA degradation. J Invest Dermatol 133(9): 2247–2254. https://doi.org/10.1038/jid.2013.56
  33. Balic A, Sørensen MD, Trabulo SM, Sainz B Jr, Cioffi M, Vieira CR, Miranda-Lorenzo I, Hidalgo M, Kleeff J, Erkan M, Heeschen C (2014) Chloroquine targets pancreatic cancer stem cells via inhibition of CXCR4 and hedgehog signaling. Mol Cancer Ther 13(7): 1758–1771. https://doi.org/10.1158/1535-7163.MCT-13-0948
  34. Niemann B, Puleo A, Stout C, Markel J, Boone BA (2022) Biologic functions of hydroxychloroquine in disease: from COVID-19 to cancer. Pharmaceutics 14: 2551. https://doi.org/.3390/ pharmaceutics14122551
  35. Nakano K, Masui T, Yogo A, Uchida Y, Sato A, Kasai Y, Nagai K, Anazawa T, Kawaguchi Y, Uemoto S (2020) Chloroquine induces apoptosis in pancreatic neuro‐endocrine neoplasms via endoplasmic reticulum stress. Endocr Relat Cancer 27: 431–439. https://doi.org/10.1530/ERC-20-0028
  36. Zalckvar E, Berissi H, Eisenstein M, Kimchi A (2009) Phosphorylation of Beclin 1 by DAP-kinase promotes autophagy by weakening its interactions with Bcl-2 and Bcl-XL. Autophagy 5(5): 720–722. https://doi.org/10.4161/auto.5.5.8625
  37. Ye J, Zhang J, Zhu Y, Wang L, Jiang X, Liu B, He G. (2023) Targeting autophagy and beyond: Deconvoluting the complexity of Beclin-1 from biological function to cancer therapy. Acta Pharm Sin B. 13(12): 4688–4714. https://doi.org/10.1016/j.apsb.2023.08.008
  38. Mattiolo P, Yuste VJ, Boix J, Ribas J (2015) Autophagy exacerbates caspase-dependent apoptotic cell death after short times of starvation. Biochem Pharmacol 98(4): 573–586. https://doi.org/10.1016/j.bcp.2015.09.021

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2. Fig. 1. Viability of MOLT-3 and IMR-32 cells after culturing with 10-100 μM CQ in complete and serum-free media for 24 and 48 h. Mean values ± SE (n = 8 - 15) for each cell line are presented. The viability level of cells growing under control conditions was taken as 100% and is shown as a red dashed line. CQ (10-100) - Chloroquine at different concentrations. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control (one-factor ANOVA analysis for multiple comparisons with Dunnett's a posteriori test).

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3. Fig. 2. Effect of 30 and 50 μM CQ on relative CASP3 gene expression in MOLT-3 and IMR-32 cells in complete and serum-free RPMI-1640 and DMEM media, respectively, after 24- and 48-h culturing. Data were normalised relative to the CNOT4 reference gene. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05, ** p ˂ 0.01 compared to control (Kraskell-Wallis test for multiple comparisons with Dunn's a posteriori test).

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4. Fig. 3. Changes in BAX gene expression in MOLT-3 and IMR-32 cells after 24- and 48-h incubation with different concentrations of CQ in media with and without serum. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05, ** p < 0.01 compared to control (Kraskell-Wallis test with Dunn's posterior test).

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5. Fig. 4. Effect of CQ on BCL2 gene expression in MOLT-3 and IMR-32 cell lines after 24- and 48-h incubation in complete and serum-free media. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05, ** p < 0.01 compared to control for cells of each line (Kraskell-Wallis test with Dunn's posterior test).

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6. Fig. 5. ULK1 autophagy gene mRNA levels after treatment of MOLT-3 and IMR-32 cells with different concentrations of CQ in media with and without serum for 24 and 48 h. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05 compared to control (Kraskell-Wallis test with Dunn's posterior test).

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7. Fig. 6. Effect of 30 and 50 μM CQ on BECN1 gene expression in MOLT-3 and IMR-32 cell lines after 24- and 48-h incubation in complete and serum-free media. Medians and interquartile ranges are shown (n = 5 - 7). Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p ˂ 0.05, ** p < 0.01 compared to control for cells of each line (Kraskell-Wallis test with Dunn's posterior test).

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8. Fig. 7. MAP1LC3B autophagy gene expression in MOLT-3 and IMR-32 cells after incubation with different concentrations of CQ in different media. Medians and interquartile ranges (n = 5 - 7) are shown. Cont - control, CQ30 - 30 μM chloroquine, CQ50 - 50 μM chloroquine. * p < 0.05, ** p < 0.01 compared to control for cells of each line (Kraskell-Wallis test with Dunn's posterior test).

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