Chemosensitizing effect of peptides from lentinus squarrosulus on cisplatin-induced apoptosis in human lung cancer cells

Cisplatin, platinum-based chemotherapy, is one of the standard chemotherapies for the treatment of lung cancer. However, its usefulness is limited due to the adverse effects and susceptibility to chemoresistance which lead to therapeutic failure. Nowadays, natural chemosensitizers, which can enhance the efficacy and decrease the effective dose of chemo-drugs, have been found to be interested in novel therapeutic. Recently, the anticancer activity of peptides extracted from Lentinus squarrosulus Mont. in human lung cancer cells has been reported. The novel potential of L. squarrosulus peptides as a chemotherapeutic adjuvant to increase cisplatin-induced apoptosis was uncovered in this study. Crude peptides extracted from fresh fruiting bodies of L. squarrosulus Mont. were further purifies by fast protein liquid chromatography on HiTrap, DEAE FF column. The bound peptides were eluted with stepwise increase salt concentration (0.1, 0.2, 0.3, 0.4 and 0.5 M NaCl) in phosphate buffer (pH 7.4). The peptide fraction with highest purity (eluted with 0.4 M NaCl in phosphate buffer, pH 7.4) was used for further experiment. The maximum non-toxic concentration of 0.4 M NaCl eluted peptides was found to be 5 µg/mL in lung cancer H460 cells, dermal papilla (DP) and proximal renal (HK-2) cells. Flow cytometry, cell viability and co-staining assays confirmed that pre-treatment with peptide increased cisplatin-induced apoptosis in H460 cells in comparison to only cisplatin treated group. Western blot analysis result showed that peptide activated p53, a tumor suppressor and pro-apoptosis BAX protein as well as decreased anti-apoptotic protein, Mcl-1 and Bcl-2. Moreover, treatment of H460 cells with peptide down-regulated the level of integrins (α5, αV, β1, β3, β5) and down-stream survival molecules (pFAK/FAK, pSrc/Src, pAkt/Akt) with time-dependent manner. In addition, peptide selectively sensitized cisplatin toxicity on lung cancer H23, H292 and A549 cells without affecting DPs and HK-2 cells. However, 0.4 M NaCl eluted peptide pretreatment did not increase the viability of 25 uM cisplatin-treated human renal cells. Interestingly, peptides from 0.5 M NaCl fraction could preserve the viability of 25 uM cisplatin-treated HK-2 cells with time-dependent manner. Augmentation of the anti-apoptosis proteins, Mcl-1 and Bcl-2, as well as key survival signaling proteins, Akt and its active form pAkt, were shown after 24 h treatment with 5 µg/mL of this peptide fraction on HK-2 cells. The novel findings herein encourage the development of peptides from Lentinus squarrosulus Mont. as potential chemotherapeutic adjuvant peptides.