Impact of trehalose and hydroxychloroquine on the function of BRAF (V600E)-siRNA in the A375 melanoma cells

Document Type : Original Article

Authors

1 Department of Genetics and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Genetics and Molecular Sciences, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Genetics and Molecular biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 Department of Genetics and Molecular biology, Faculty of Medicine, Isfahan university of Medical Sciences, Isfahan, Iran

5 Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

10.22038/ijbms.2025.85017.18394

Abstract

Objective(s): Melanoma, a lethal form of skin cancer, is closely linked to mutations in melanocytes. Due to increased resistance to chemotherapy, innovative strategies, including gene and combination therapies, are being explored. This study evaluates the effects of trehalose (TRE) and hydroxychloroquine (HCQ) in enhancing the efficacy of BRAF (V600E)-siRNA in A375 cells.
Materials and Methods: The A375 cells were treated, and changes in cell viability were assessed using MTT assays. Apoptosis was evaluated using flow cytometry. Additionally, gene expression analysis of B-Raf proto-oncogene, serine/threonine kinase (BRAF), Caspase 3 (CASP3), and Phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) was performed using quantitative RT-PCR.
Results: HCQ treatment reduced cell viability and increased apoptosis compared to both control cells and cells treated with TRE. Gene expression analysis showed a significant down-regulation of BRAF expression in cells treated with HCQ and BRAF (V600E)-siRNA compared to siRNA-only treated cells. CASP3 expression was significantly up-regulated in cells treated with combined HCQ and siRNA, indicating a stronger apoptotic response. PIK3R3 expression showed no significant change in the transfected groups.
Conclusion: TRE, either alone or combined with siRNA, showed limited efficacy and may counteract the apoptotic benefits of HCQ. Conversely, HCQ, whether used alone or in combination with siRNA, enhanced apoptosis, suggesting promise as a potential treatment for melanoma.

Keywords

Main Subjects

References

1. Shain AH, Bastian BC. From melanocytes to melanomas. Nat Rev Cancer 2016; 16: 345–358.
2. Saginala K, Barsouk A, Aluru JS, Rawla P, Barsouk A. Epidemiology of melanoma. Med Sci 2021; 9: 63-71.
3. Murata T, Hibasami H, Maekawa S, Tagawa T, Nakashima K. Preferential binding of cisplatin to mitochondrial DNA and suppression of ATP generation in human malignant melanoma cells. Biochem Int 1990; 20: 949–955.
4. Seymour L, Ulbrich K, Steyger P, Brereton M, Subr V, Strohalm J, et al. Tumour tropism and anti-cancer efficacy of polymer-based doxorubicin prodrugs in the treatment of subcutaneous murine B16F10 melanoma. Br J Cancer 1994; 70: 636–641.
5. Sumimoto H, Miyagishi M, Miyoshi H, Yamagata S, Shimizu A, Taira K, et al. Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference. Oncogene 2004; 23: 6031–6039.
6. Bradish JR, Cheng L. Molecular pathology of malignant melanoma: Changing the clinical practice paradigm toward a personalized approach. Hum Pathol 2014; 45: 1315–1326.
7. Mishra H, Mishra PK, Ekielski A, Jaggi M, Iqbal Z, Talegaonkar S. Melanoma treatment: from conventional to nanotechnology. J Cancer Res Clin Oncol 2018; 144: 2283–2302.
8. Ozcan G, Ozpolat B, Coleman RL, Sood AK, Lopez-Berestein G. Preclinical and clinical development of siRNA-based therapeutics. Adv Drug Deliv Rev 2015; 87: 108–119.
9. Li L, Hou J, Liu X, Guo Y, Wu Y, Zhang L, et al. Nucleolin-targeting liposomes guided by aptamer AS1411 for the delivery of siRNA for the treatment of malignant melanomas. Biomaterials 2014; 35: 3840–3850.
10. Ihle MA, Fassunke J, König K, Grünewald I, Schlaak M, Kreuzberg N, et al. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p. V600E and non-p. V600E BRAF mutations. BMC Cancer 2014; 14: 1–13.
11. Kumar SM, Yu H, Edwards R, Chen L, Kazianis S, Brafford P, et al. Mutant V600E BRAF increases hypoxia inducible factor-1α expression in melanoma. Cancer Res 2007; 67: 3177–3184.
12. He H, Nan X, Liu S, Zhang L, Yang Z, Wu Y, et al. Anticancer effects of combinational treatment with BRAFV600E siRNA and PI3K pathway inhibitors in melanoma cell lines harboring BRAFV600E. Oncol Lett 2018; 16: 632–642.
13. Zhang Y, Zhan X, Peng S, Cai Y, Zhang YS, Liu Y, et al. Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem. Nanomedicine 2018; 14: 1679–1693.
14. White E, Mehnert JM, Chan CS. Autophagy, metabolism, and cancer. Clin Cancer Res 2015; 21: 5037–5046.
15. Guo JY, Teng X, Laddha SV, Ma S, Van Nostrand SC, Yang Y, et al. Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells. Genes Dev 2016; 30: 1704–1717.
16. Dalby K, Tekedereli I, Lopez-Berestein G, Ozpolat B. Targeting the pro-death and pro-survival functions of autophagy as novel therapeutic strategies in cancer. Autophagy 2010; 6: 322–329.
17. Zhong X, Panus D, Ji W, Wang C. Modulating polyplex-mediated gene transfection by small-molecule regulators of autophagy. Mol Pharm 2015; 12: 932–940.
18. Song W, Ma Z, Zhang Y, Yang C. Autophagy plays a dual role during intracellular siRNA delivery by lipoplex and polyplex nanoparticles. Acta Biomater 2017; 58: 196–204.
19. Dowaidar M, Gestin M, Cerrato CP, Jafferali MH, Margus H, Kivistik PA, et al. Role of autophagy in cell-penetrating peptide transfection model. Sci Rep 2017; 7: 12635-12648.
20. Hall EA, Ramsey JE, Peng Z, Hayrapetyan D, Shkepu V, O’Rourke B, et al. Novel organometallic chloroquine derivative inhibits tumor growth. J Cell Biochem 2018; 119: 5921–5933.
21. Egger ME, Huang JS, Yin W, McMasters KM, McNally LR. Inhibition of autophagy with chloroquine is effective in melanoma. J Surg Res 2013; 184: 274–281.
22. Allavena G, Del Bello B, Tini P, Volpi N, Valacchi G, Miracco C, et al. Trehalose inhibits cell proliferation and amplifies long-term temozolomide-and radiation-induced cytotoxicity in melanoma cells: A role for autophagy and premature senescence. J Cell Physiol 2019; 234: 11708–11721.
23. El-Magd MA, Khamis A, Eldeen SKN, Ibrahim WM, Salama AF. Trehalose enhances the antitumor potential of methotrexate against mice bearing Ehrlich ascites carcinoma. Biomed Pharmacother 2017; 92: 870–878.
24. Waseh S, Lee JBJFiM. Advances in melanoma: Epidemiology, diagnosis, and prognosis. Front Med 2023; 10: 1268479.
25. Mishra H, Mishra PK, Ekielski A, Jaggi M, Iqbal Z, Talegaonkar SJJocr, et al. Melanoma treatment: From conventional to nanotechnology. J Cancer Res Clin Oncol 2018; 144: 2283–2302.
26. Bradish JR, Cheng LJHP. Molecular pathology of malignant melanoma: Changing the clinical practice paradigm toward a personalized approach. Hum Pathol 2014; 45: 1315–1326.
27. Zhang Y, Zhan X, Peng S, Cai Y, Zhang YS, Liu Y, et al. Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem. Nanomedicine 2018; 14: 1679–1693.
28. Ruan W, Zhai Y, Yu K, Wu C, Xu YJIJoP. Coated microneedles mediated intradermal delivery of octaarginine/BRAF siRNA nanocomplexes for anti-melanoma treatment. Int J Pharm 2018; 553: 298–309.
29. Dalby K, Tekedereli I, Lopez-Berestein G, Ozpolat BJA. Targeting the pro-death and pro-survival functions of autophagy as novel therapeutic strategies in cancer. Autophagy 2010; 6: 322–329.
30. Song W, Ma Z, Zhang Y, Yang CJAB. Autophagy plays a dual role during intracellular siRNA delivery by lipoplex and polyplex nanoparticles. Acta Biomater 2017; 58: 196–204.
31. Zhong X, Panus D, Ji W, Wang CJMp. Modulating polyplex-mediated gene transfection by small-molecule regulators of autophagy. Mol Pharm 2015; 12: 932–940.
32. Dowaidar M, Gestin M, Cerrato CP, Jafferali MH, Margus H, Kivistik PA, et al. Role of autophagy in cell-penetrating peptide transfection model. Sci Rep 2017; 7: 12635.
33. Hall EA, Ramsey JE, Peng Z, Hayrapetyan D, Shkepu V, O’Rourke B, et al. Novel organometallic chloroquine derivative inhibits tumor growth. J Cell Biochem 2018; 119: 5921–5933.
34. Jo M-H, Kim Y-T, Park SJ. Dieckol inhibits autophagic flux and induces apoptotic cell death in A375 human melanoma cells via lysosomal dysfunction and mitochondrial membrane impairment. Int J Mol Sci 2022; 23: 14149.
35. Wang Z, Shi X, Li Y, Zeng X, Fan J, Sun Y, et al. Involvement of autophagy in recombinant human arginase-induced cell apoptosis and growth inhibition of malignant melanoma cells. Appl Microbiol Biotechnol 2014; 98: 2485–2494.
36. El-Magd MA, Khamis A, Eldeen SKN, Ibrahim WM, Salama AFJB, Pharmacotherapy. Trehalose enhances the antitumor potential of methotrexate against mice bearing Ehrlich ascites carcinoma. Biomed Pharmacother 2017; 92: 870–878.
37. Kudo T, Takeuchi K, Ebina Y-i, Nakazawa MJISRN. Inhibitory effects of trehalose on malignant melanoma cell growth: Implications for a novel topical anticancer agent on the ocular surface. ISRN Ophthalmol 2012; 2012: 1–9.
38. Tang Q, Li G, Wei X, Zhang J, Chiu J-F, Hasenmayer D, et al. Resveratrol-induced apoptosis is enhanced by inhibition of autophagy in esophageal squamous cell carcinoma. Cancer Lett 2013; 336: 325–337.
39. Al Hashmi M, Sastry KS, Silcock L, Chouchane L, Mattei V, James N, et al. Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated. Cancer Biol Ther 2020; 18: 1–9.
40. Kumar SM, Yu H, Edwards R, Chen L, Kazianis S, Brafford P, et al. Mutant V600E BRAF increases hypoxia inducible factor-1α expression in melanoma. Cancer Res 2007; 67: 3177–3184.
41.    Ozcan G, Ozpolat B, Coleman RL, Sood AK, Lopez-Berestein GJAddr. Preclinical and clinical development of siRNA-based therapeutics. Adv Drug Deliv Rev 2015; 87: 108–119.
42. Snøve Jr O, Holen TJB, communications br. Many commonly used siRNAs risk off-target activity. Biochem Biophys Res Commun 2004; 319: 256–263.
43. Qi J, Wang Ww, Chen W, Lu Wy, Shang AqJJocb. Mechanism of miR-137 regulating migration and invasion of melanoma cells by targeting PIK3R3 gene. J Cell Biochem 2019; 120: 8393–8400.
44. Jiang YL, Li SX, Liu YJ, Ge LP, Han XZ, Liu ZP. Synthesis and evaluation of trehalose-based compounds as novel inhibitors of cancer cell migration and invasion. Chem Biol Drug Des 2015; 86: 1017–1029.
45. Stevanovic D, Vucicevic L, Misirkic-Marjanovic M, Martinovic T, Mandic M, Harhaji-Trajkovic L, et al. Trehalose attenuates in vitro neurotoxicity of 6-hydroxydopamine by reducing oxidative stress and activation of MAPK/AMPK signaling pathways. Int J Mol Sci 2024; 25: 10659-10681.
46. Kuczyńska-Wiśnik D, Stojowska-Swędrzyńska K, Laskowska E. Intracellular Protective Functions and Therapeutical Potential of Trehalose. Molecules 2024; 29: 2088.
47. Imani S, Roozitalab G, Emadi M, Moradi A, Behzadi P, Kaboli PJ. The evolution of BRAF-targeted therapies in melanoma: overcoming hurdles and unleashing novel strategies. Front Oncol 2024; 14: 1504142.
48. Korch C, Hall EM, Dirks WG, Ewing M, Faries M, Varella‐Garcia M, et al. Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line. Int J Cancer 2018; 142: 561–572.
49. Kaleağasıoğlu F, Ali DM, Berger MR. Multiple facets of autophagy and the emerging role of alkylphosphocholines as autophagy modulators. Front Pharmacol 2020; 11: 547.
50. Amaravadi RK. Clinical trial results show promise of targeting autophagy BRAF mutant melanoma. Autophagy 2022; 18: 1470–1471.
51. Stein AM, Vogl FD, Sellami D. Dosage regimens of anti-lag-3 antibodies and uses thereof. US patent US20200172617A1. United States; 2020.
52. Yazd University. Abstract booklet – International J Pediatr Hematol Oncol – International Conference on Human Genetics and Genomics. Yazd, Iran: Yazd University; 2021.
Iranian Journal of Basic Medical Sciences
Volume 28, Issue 11
November 2025
Pages 1486-1494

Files

Share

How to cite

Statistics