UVA Irradiation Combined Brusatol Treatment Induced Human Malignant Melanoma A375 Apoptosis

Background:Melanoma is a highly malignant skin tumor. Though there are extensive studies, it’s still one of the worst skin cancers across the globe. Numerous factors contribute to its resistance to hosts of treatment regimes, such as protection from endoplasmic reticulum(ER) stress, over expression of multidrug resistance proteins, efficient DNA repair mechanisms and some other complex phenotypes. Treatment options for patients with metastatic melanoma have been very limited because of higher malignancy and epidemics. In addition, melanoma is not sensitive to chemotherapy and radiation therapy hence proven resistant type of cancers to almost all treatment types. BRAF mutations have been detected in approximately 65% of cutaneous melanomas. Most treatment regimes of melanoma focus on selective small-molecule inhibitors of V600-mutant BRAF gene(such as vemurafenib and dabrafenib). Lots of single-modality medication and treatment methods are often initially effective, but recurrence due to acquired resistance to single-modality medication treatment less than one year is a frequent clinical problem. Due to strong drug resistance of malignant melanoma, we chosen an important and widespread over-expression resistant, cytoprotective and chemopreventive transcription factors in cancer cells: NF-E2-Related Factor 2(Nrf2) as the breakthrough point. Brusatol, isolated from Brucea javanica plant, is identified as a unique inhibitor of Nrf2-ARE pathway in certain cancer cells. A little is known about the biological effects of brusatol on melanoma cells(A375), and no any study or report is in knowledge to date about brusatol alone or in combination with UVA therapy to treat melanoma cells/tumors. Objects:To examine the influence of a range of UVA doses(25, 50, 75, 100 k J/m2) and an optimum concentration(50 n M) of brusatol on proliferation of A375, and to detect Nrf2 and its target genes HO-1, NQO1, GSTP1 level along with expression of IκBα and COX2 proteins after UVA irradiation. In addition, we will perform the cell viability assay under an ascending concentration levels of brusatol(10-100 n M) to access the toxicity of the drug and detection of Endoplasmic Reticulum(ER) stress marker proteins expression viz. GRP78, ATF4 and CHOP levels at an optimum concentration(50 n M) of brusatol. Apoptotic assay will also be performed on A375 melanoma cells and Ha Ca T cells through flow cytometric assays.Initially a wide range of UVA doses(25, 50, 75, 100 k J/m2) and brusatol concentration were applied to optimize the standard dosage(75 k J/m2) and treatment level(50 n M), respectively. We treated the cells(A375 and Ha Ca T) with 50 n M brusatol after 2h post 75 k J/m2 UVA and gradually extracted the proteins at different time points. The protein extracts include the GRP78, ATF4, CHOP, Nrf2 and Bcl-2 family proteins after combined treatment. Flow cytometry was done for apoptosis array on control group, UVA irradiation group, brusatol treated group and combined treatment(UVA+Brusatol) group to measure the A375 and Ha Ca T cell apoptosis. Methods:MTS assay was used for detecting A375 cell proliferation; Western blot technique was applied to detect protein levels including GRP78, ATF4, CHOP, Nrf2 etc; Flow cytometry analysis was used to measure A375 and Ha Ca T cell apoptosis rate.Results: ① UVA effects on cell proliferation, oxidative stress, IκBα and COX2 protein level.ER stress marker proteins GRP78, ATF4, CHOP and Nrf2 with its target genes HO-1, NQO1, GSTP1 level remarkably increased in a time-dependent manner after 75 k J/m2 UVA exposure. Nrf2 protein level reached a peak at the time point 4-6 h following UVA irradiation, then gradually lessen to basal level, but IκBα declined weakly. COX2 declined in an apparent time-dependent manner after UVA irradiation, then ratchet up even significantly higher than the control group. ② The ER stress marker protein were overexpressed after 50 n M brusatol treatment. The protein level of Nrf2 and its target gene GSTP1 was inhibited. IκBα protein level has no significant changes.50 n M brusatol treatment induced ER stress in A375 cell, inhibited Nrf2-ARE pathway rather it directed to higher expression of COX-2 protein by surpassing NF-κB pathway. The working concentration(50 n M) of brusatol induce ER stress in A375 cells. The level of ER stress marker proteins viz. GRP78, ATF4 and CHOP, were significantly elevated. Total Nrf2 protein level affectively declined approximately 4 h post treatment, which led decline in its target gene-GSTP1 expression in a time-dependent manner. IκBα protein maintained at a relatively stable level while COX-2 protein was highly expressed. ② A combined treatment of 75 k J/m2 UVA irradiation and 50 n M brusatol, increased the rate of apoptosis in A375 cells when compared with UVA irradiation alone.Brusatol treatment at 2 h post UVA irradiation cause further increase of UVA-induced apoptosis, and remarkably increased the expression of pro-apoptotic Bax protein, while decreased the expression of anti-apoptotic Bcl-2 and Bcl-xl proteins in a time depended manner. Compared with independent UVA irradiation or brusatol treatment, combined treatment may lead to significant decrease in Bcl-2 and Bcl-xl proteins; Bax and Cleavage of PARP, caspase-3, caspase-7 were enhanced remarkably. Conclusion: ① 75 k J/m2 UVA irradiation has limited damage to A375 cell; This treatment activatedoxidative stress, induced Nrf2 expression. ② 50 n M brusatol treatment didn’t induce A375 cell apoptosis; This treatmentinhibited Nrf2 signaling pathway, induced ER stress and increase of COX2 level. ③ A combined treatment of 75 k J/m2 UVA irradiation and 50 n M brusatol actived mitochondria mediated apoptosis pathway in A375 cell.