Iranian
Biomedical Journal
Volume 29, Issue 5 (9-2025)
IBJ 2025, 29(5): 294-308 |
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Babazadeh S M, Zolfaghari* M R, Zargar M, Baesi K, Ghaemi A. Chloroquine Enhances Mda-7- Induced Apoptosis via miR-7 and HSP70 Modulation in Glioblastoma. IBJ 2025; 29 (5) :294-308
URL: http://ibj.pasteur.ac.ir/article-1-5131-en.html
URL: http://ibj.pasteur.ac.ir/article-1-5131-en.html
Abstract:
Background: Melanoma differentiation-associated gene-7 (Mda-7) selectively suppresses growth and induces apoptosis in various tumor cells without harming normal cells. Inhibition of autophagy has been shown to enhance the efficacy of many cancer therapies. However, its effect on the anticancer activity of Ad/Mda-7 in GBM has remained unclear. This study investigated the combined effect of an autophagy inhibitor (CQ) and Mda-7 in U87 cancer cells.
Methods: Cell proliferation was assessed using the MTT assay. Apoptosis rates, autophagy induction, and ROS levels were measured using flow cytometry. Caspase-9 and β-actin protein levels were analyzed by Western blotting. ELISA was employed to quantify HSP70 and TRAIL level in the culture medium. Real-time PCR evaluated the expression levels of cell death-related genes (P38 MAPK, Bax, and TRAIL) and specific miRNAs (miR-7, miR-122, and miR-21) in treated cells.
Results: Combined treatment with Ad/Mda-7 and the autophagy inhibitor CQ significantly reduced cell viability and proliferation. Ad/Mda-7 induced LC3-II protein expression in U87 cancer cells, which was further increased by autophagy inhibition through CQ. The combination treatment also increased apoptosis rates, elevated ROS levels, and decreased HSP70 protein expression, highlighting its synergistic anticancer effects. Increasing the expression of miR-7 and miR-122 indicated that the elevated levels of these endogenous miRNAs may help improve the treatment process.
Conclusion: Our findings indicate that the combination of Ad/Mda-7 and CQ synergistically could inhibit U87 cancer cell growth and could serve as a promising approach for treating human GBM.
Methods: Cell proliferation was assessed using the MTT assay. Apoptosis rates, autophagy induction, and ROS levels were measured using flow cytometry. Caspase-9 and β-actin protein levels were analyzed by Western blotting. ELISA was employed to quantify HSP70 and TRAIL level in the culture medium. Real-time PCR evaluated the expression levels of cell death-related genes (P38 MAPK, Bax, and TRAIL) and specific miRNAs (miR-7, miR-122, and miR-21) in treated cells.
Results: Combined treatment with Ad/Mda-7 and the autophagy inhibitor CQ significantly reduced cell viability and proliferation. Ad/Mda-7 induced LC3-II protein expression in U87 cancer cells, which was further increased by autophagy inhibition through CQ. The combination treatment also increased apoptosis rates, elevated ROS levels, and decreased HSP70 protein expression, highlighting its synergistic anticancer effects. Increasing the expression of miR-7 and miR-122 indicated that the elevated levels of these endogenous miRNAs may help improve the treatment process.
Conclusion: Our findings indicate that the combination of Ad/Mda-7 and CQ synergistically could inhibit U87 cancer cell growth and could serve as a promising approach for treating human GBM.
Type of Study: Full Length/Original Article |
Subject:
Molecular Microbiology
References
1. Jin HF, Wang JF, Shao M, Zhou K, Ma X, Lv XP. Down-regulation of miR-7 in gastric cancer is associated with elevated LDH-A expression and chemoresistance to cisplatin. Front Cell Dev Biol. 2020:8:555937. [DOI:10.3389/fcell.2020.555937]
2. Tan Y, Sanders AJ, Zhang Y, Martin TA, Owen S, Ruge F, et al. Interleukin-24 (IL-24) expression and biological impact on HECV endothelial cells. Cancer Genomics Proteomics. 2015;12(5):243-50.
3. Persaud L, Mighty J, Zhong X, Francis A, Mendez M, Muharam H, et al. IL-24 promotes apoptosis through cAMP-dependent PKA pathways in human breast cancer cells. Int J Mol Sci. 2018;19(11):3561. [DOI:10.3390/ijms19113561]
4. Fuchs Y, Steller H. Programmed cell death in animal development and disease. Cell. 2011;147(4):742-58. [DOI:10.1016/j.cell.2011.10.033]
5. Lefranc F, Kiss R. Autophagy, the trojan horse to combat glioblastomas. Neurosurg Focus. 2006;20(4):7. [DOI:10.3171/foc.2006.20.4.4]
6. Marino G, Niso-Santano M, Baehrecke EH, Kroemer G. Self-consumption: The interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol. 2014;15(2):81-94. [DOI:10.1038/nrm3735]
7. Moloudi K, Neshasteriz A, Hosseini A, Eyvazzadeh N, Shomali M, Eynali S, et al. Synergistic effects of arsenic trioxide and tadiation: Triggering the intrinsic pathway of apoptosis. Iran Biomed J. 2017;21(5):330-7. [DOI:10.18869/acadpub.ibj.21.5.330]
8. Wu ST, Sun GH, Cha TL, Kao C-C, Chang -Y, Kuo S-C, et al. CSC-3436 switched tamoxifen-induced autophagy to apoptosis through the inhibition of AMPK/mTOR pathway. J Biomed Sci. 2016;23(1):60. [DOI:10.1186/s12929-016-0275-y]
9. Amaravadi RK, Thompson CB. The roles of therapy-induced autophagy and necrosis in cancer treatment. Clin Cancer Res. 2007;13(24):7271-9. [DOI:10.1158/1078-0432.CCR-07-1595]
10. Tasdemir E, Galluzzi L, Maiuri MC, Criollo A, Vitale I, Hangen E, et al. Methods for assessing autophagy and autophagic cell death. Methods Mol Biol. 2008:445:29-76. [DOI:10.1007/978-1-59745-157-4_3]
11. Rabinowitz JD, White E. Autophagy and metabolism. Science. 2010;330(6009):1344-8. [DOI:10.1126/science.1193497]
12. White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer. 2012;12(6):401-10. [DOI:10.1038/nrc3262]
13. Zanotto-Filho A, Braganhol E, Klafke K, Figueiro F, Terra SR, Paludo FJ, et al. Autophagy inhibition improves the efficacy of curcumin/temozolomide combination therapy in glioblastomas. Cancer Lett. 2015;358(2):220-31. [DOI:10.1016/j.canlet.2014.12.044]
14. Golden EB, Cho H-Y, Jahanian A, Hofman FM, Louie SG, Schonthal AH, et al. Chloroquine enhances temozolomide cytotoxicity in malignant gliomas by blocking autophagy. Neurosurg Focus. 2014;37(6):12. [DOI:10.3171/2014.9.FOCUS14504]
15. Mei L, Chen Y, Wang Z, Wang J, Wan J, Yu C, et al. Synergistic anti-tumour effects of tetrandrine and chloroquine combination therapy in human cancer: A potential antagonistic role for p21. Br J Pharmacol. 2015;172(9):2232-45. [DOI:10.1111/bph.13045]
16. Lefort S, Joffre C, Kieffer Y, Givel A-M, Bourachot B, Zago G, et al. Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers. Autophagy. 2014;10(12):2122-42. [DOI:10.4161/15548627.2014.981788]
17. Farrow JM, Yang JC, Evans CP. Autophagy as a modulator and target in prostate cancer. Nat Rev Urol. 2014;11(9):508-16. [DOI:10.1038/nrurol.2014.196]
18. Yang A, Kimmelman AC. Inhibition of autophagy attenuates pancreatic cancer growth independent of TP53/TRP53 status. Autophagy. 2014;10(9):1683-4. [DOI:10.4161/auto.29961]
19. Babazadeh SM, Zolfaghari MR, Zargar M, Baesi K, Hosseini SY, Ghaemi A. Interleukin-24-mediated antitumor effects against human glioblastoma via upregulation of P38 MAPK and endogenous TRAIL-induced apoptosis and LC3-II activation-dependent autophagy. BMC Cancer. 2023;23(1):519. [DOI:10.1186/s12885-023-11021-y]
20. Amin W, Enam SA, Sufiyan S, Naeem S, Laghari AA, Bajwa MH, et al. Autophagy-associated markers LC3-II, ULK2 and microRNAs miR-21, miR-126 and miR-374 as a potential prognostic indicator for glioma patients. J Biol Life Sci. 2023:10:20944. [DOI:10.21203/rs.3.rs-3347646/v1]
21. Yazdi AH, Zarrinpour V, Moslemi E, Forghanifard MM. A signature of three microRNAs is a potential diagnostic biomarker for glioblastoma. Iran Biomed J. 2022;26(4):301-12. [DOI:10.52547/ibj.3671]
22. Liou G-Y, Storz P. Reactive oxygen species in cancer. Free Radical Res. 2010;44(5):479-96. [DOI:10.3109/10715761003667554]
23. Park EJ, Min K-J, Choi KS, Kubatka P, Kruzliak P, Kim DE, et al. Chloroquine enhances TRAIL-mediated apoptosis through up-regulation of DR5 by stabilization of mRNA and protein in cancer cells. Sci Rep. 2016:6:22921. [DOI:10.1038/srep22921]
24. Zhou N, Wei ZX, Qi ZX. Inhibition of autophagy triggers melatonin-induced apoptosis in glioblastoma cells. BMC Neurosci. 2019;20(1):63. [DOI:10.1186/s12868-019-0545-1]
25. Kumar S, Yedjou CG, Tchounwou PB. Arsenic trioxide induces oxidative stress, DNA damage, and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells. J Exp Clin Cancer Res. 2014;33(1):42. [DOI:10.1186/1756-9966-33-42]
26. Li J, Yang D, Wang W, Piao S, Zhou J, Saiyin W, et al. Inhibition of autophagy by 3-MA enhances IL-24-induced apoptosis in human oral squamous cell carcinoma cells. J Exp Clin Cancer Res. 2015;34(1):97. [DOI:10.1186/s13046-015-0211-0]
27. Goncalves RM, Agnes JP, Delgobo M, de Souza PO, Thome MP, Heimfarth L, et al. Late autophagy inhibitor chloroquine improves efficacy of the histone deacetylase inhibitor SAHA and temozolomide in gliomas. Biochem Pharmacol. 2019:163:440-50. [DOI:10.1016/j.bcp.2019.03.015]
28. Zhang J, Chen H, Chen C, Liu H, He Y, Zhao J, et al. Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying IL-24/endostatin enhances glioma therapy. Cancer Lett. 2021:509:26-38. [DOI:10.1016/j.canlet.2021.03.027]
29. Rasoolian M, Kheirollahi M, Hosseini SY. MDA-7/interleukin 24 (IL-24) in tumor gene therapy: Application of tumor penetrating/homing peptides for improvement of the effects. Expert Opin Biol Ther. 2019;19(3):211-23. [DOI:10.1080/14712598.2019.1566453]
30. Bhoopathi P, Lee N, Pradhan AK, Shen X-N, Das SK, Sarkar D, et al. mda-7/IL-24 induces cell death in neuroblastoma through a novel mechanism involving AIF and ATM. Cancer Res. 2016;76(12):3572-82. [DOI:10.1158/0008-5472.CAN-15-2959]
31. Menezes ME, Shen X-N, Das SK, Emdad L, Guo C, Yuan F, et al. MDA-7/IL-24 functions as a tumor suppressor gene in vivo in transgenic mouse models of breast cancer. Oncotarget. 2015;6(35):36928-42. [DOI:10.18632/oncotarget.6047]
32. Ma YF, Ren Y, Wu CJ, Zhao XH, Xu H, Wu DZ, et al. Interleukin (IL)-24 transforms the tumor microenvironment and induces anticancer immunity in a murine model of colon cancer. Mol Immunol. 2016:75:11-20. [DOI:10.1016/j.molimm.2016.05.010]
33. Menezes ME, Bhoopathi P, Pradhan AK, Emdad L, Das SK, Guo C, et al. Role of MDA-7/IL-24 a multifunction protein in human diseases. Adv Cancer Res. 2018:138:143-82. [DOI:10.1016/bs.acr.2018.02.005]
34. Cai Y, Cai J, Ma Q, Xu Y, Zou J, Xu L, et al. Chloroquine affects autophagy to achieve an anticancer effect in EC109 esophageal carcinoma cells in vitro. Oncol Lett. 2017;15(1):1143-8. [DOI:10.3892/ol.2017.7415]
35. Chen HM, Lai ZQ, Liao HJ, Xie JH, Xian Y-F, Chen YL, et al. Synergistic antitumor effect of brusatol combined with cisplatin on colorectal cancer cells. Int J Mol Med. 2018;41(3):1447-54. [DOI:10.3892/ijmm.2018.3372]
36. Soeda A, Lathia J, Williams BJ, Wu Q, Gallagher J, Androutsellis-Theotokis A, et al. The p38 signaling pathway mediates quiescence of glioma stem cells by regulating epidermal growth factor receptor trafficking. Oncotarget. 2017;8(20):33316-28. [DOI:10.18632/oncotarget.16741]
37. Mariani SM, Krammer PH. Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage. Eur J Immunol. 1998;28(3):973-82.
https://doi.org/10.1002/(SICI)1521-4141(199803)28:03<973::AID-IMMU973>3.0.CO;2-T [DOI:10.1002/(SICI)1521-4141(199803)28:033.0.CO;2-T]
38. Monma H, Iida Y, Moritani T, Okimoto T, Tanino R, Tajima Y, et al. Chloroquine augments TRAIL-induced apoptosis and induces G2/M phase arrest in human pancreatic cancer cells. Plos One. 2018:13(3):0193990. [DOI:10.1371/journal.pone.0193990]
39. Li S, Li Z, Chen S, Zhu Y, Li Y, Yin X, et al. Apoptotic and autophagic cell death induced in cervical cancer cells by a dual specific oncolytic adenovirus. Anticancer Drugs. 2023;34(3):361-72. [DOI:10.1097/CAD.0000000000001452]
40. Samadi M, Mokhtari-Azad T, Nejati A, Norooz-Babaei Z, Foroushani AR, Haghshenas MR, et al. The antitumor effect of oncolytic respiratory syncytial virus via the tumor necrosis factor-alpha induction and ROS-bax-mediated mechanisms. BMC Cancer. 2023;23(1):803. [DOI:10.1186/s12885-023-11326-y]
41. Evdonin A, Kinev A, Tsupkina N, Guerriero V, Raynes DA, Medvedeva N. Extracellular HspBP1 and Hsp72 synergistically activate epidermal growth factor receptor. Biol Cell. 2009;101(6):351-60. [DOI:10.1042/BC20080069]
42. Song J, Takeda M, Morimoto RI. Bag1-Hsp70 mediates a physiological stress signalling pathway that regulates Raf-1/ERK and cell growth. Nat Cell Biol. 2001;3(3):276-82. [DOI:10.1038/35060068]
43. Park YH, Seo JH, Park JH, Lee HS, Kim KW. Hsp70 acetylation prevents caspase-dependent/independent apoptosis and autophagic cell death in cancer cells. Int J Oncol. 2017;51(2):573-8. [DOI:10.3892/ijo.2017.4039]
44. Ferretti GD, Quaas CE, Bertolini I, Zuccotti A, Saatci O, Kashatus JA, et al. HSP70-mediated mitochondrial dynamics and autophagy represent a novel vulnerability in pancreatic cancer. Cell Death Differ. 2024;31(7):881-96. [DOI:10.1038/s41418-024-01310-9]
45. Wuchty S, Arjona D, Li A, Kotliarov Y, Walling J, Ahn S, et al. Prediction of associations between microRNAs and gene expression in glioma biology. Plos One. 2011;6(2):14681. [DOI:10.1371/journal.pone.0014681]
46. Lages E, Guttin A, El Atifi M, Ramus C, Ipas H, Dupre I, et al. MicroRNA and target protein patterns reveal physiopathological features of glioma subtypes. Plos One. 2011;6(5):20600. [DOI:10.1371/journal.pone.0020600]
47. Morales-Martínez M, Vega MI. Role of microRNA-7 (MiR-7) in cancer physiopathology. Int J Mol Sci. 2022;23(16):9091. [DOI:10.3390/ijms23169091]
48. Ahsani Z, Mohammadi-Yeganeh S, Kia V, Karimkhanloo H, Zarghami N, Paryan M. WNT1 gene from WNT signaling pathway is a direct target of miR-122 in hepatocellular carcinoma. Appl Biochem Biotechnol. 2017;181(3):884-97. [DOI:10.1007/s12010-016-2256-8]
49. Wang B, Wang H, Yang Z. MiR-122 inhibits cell proliferation and tumorigenesis of breast cancer by targeting IGF1R. Plos One. 2012;7(10):47053. [DOI:10.1371/journal.pone.0047053]
50. Tang Y, Zhao S, Wang J, Li D, Ren Q, Tang Y. Plasma miR-122 as a potential diagnostic and prognostic indicator in human glioma. Neurol Sci. 2017;38(6):1087-92. [DOI:10.1007/s10072-017-2912-y]
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