Evaluation of the effect of inhibition of HSP70 in induction of apoptosis by polymeric nanocurcumin in A-375 melanoma cell line

Abstract

Cancers are considered as one of the most important cause of death in the world and annually 10 million people all around the world die due to cancers. Melanoma is a cancer that origins from the melanocyte cells of the skin and accounts for about 2.1% of all people diagnosed with cancers in the world. Various kinds of treatments are used for melanoma and one of the suggested treatments that we hope to be widely used in future for melanoma patients is polymeric nanocurcumin. Several researches have shown that polymeric nanocurcumin can play a role in the treatment of cancers like melanomas. Heat shock proteins are a group of proteins that are expressed under cellular stress conditions and facilitate the folding of proteins and remove misfolded proteins. One group of the HSP proteins that have 70 kilodaltons weight named heat shock proteins 70. One hypothesis is that curcumin causes the destruction of proteins in order to induce apoptosis, and cancer cells increase the expression of heat shock proteins to resist its effect. On the other hand, another hypothesis is proposed that increasing the expression of heat shock proteins act in line with curcumin induction of apoptosis.The aim of the study is to determine whether the expression level of HSP70 will be changed in A-375 melanoma cell line by polymeric nanocurcumin treatment and this change will be in line with inducing apoptosis or cellular resistance. Methods: The present laboratory study was performed on A-375 melanoma cell lines in DMEM culture medium containing 20% FBS and 10% penstrep. MTT Test was performed and 20000 and 7000 A-375 cells were cultured in the culture medium at 24 and 48 hours , respectively. Nanocurcumin solution was prepared and after MTT test, A375 cells were treated with 13 microliter of nanocurcumin solution at 24 hour and 12 microliter of nanocurcumin solution at 48 hour, and in the control group, only PBS was added to the cells. After RNA extraction, a pair of primers was prepared and then cDNAs were synthesized from RNAs. The expression level of heat shock protein 70 (A1 and A2 types) was investigated by Real Time PCR. Apoptosis test was performed and the number of apoptotic cells were checked by flow cytometry. Results: As a result, it was shown that the treatment of melanoma cells by polymeric nanocurcumin reduced the expression of heat shock protein 70 (A1 and A2 types) and this reduction of the expression is in the direction of inducing apoptosis in this cell lines.