3,4-dihydroxy-5,4’-dimethoxybibenzyl sensitizes non-small cell lung cancer cells to cisplatin-induced apoptosis via p53 upregulation

Abstract

Lung cancer is a leading fatal malignancy with the highest number of cancer deaths. Targeting a specific protein regulating cancer progression and metastasis has been attracting much attention for the development of cancer therapy. Thus, this study was aimed to investigate the effect of 3,4-dihydroxy-5,4′-dimethoxybibenzyl (DS-1) in targeting MDM2-attenuating p53 function and inhibition EMT in lung cancer cells. The efficacy of DS-1 or combined with cisplatin in lung cancer cells was determined by MTT, nuclear staining, and Annexin V/PI assays. The expression of apoptosis- and EMT-related proteins was determined by western blot analysis. Metastatic behaviors were evaluated by cell shape characterization, migration, invasion, and anchorage-independent cell growth assay. To investigate the role of DS-1 on stabilization and degradation of p53, cycloheximide chasing assay and immunoprecipitation were conducted, and the active form of p53 was investigated by immunofluorescent staining assay. To confirm and demonstrate the site interaction between DS-1 and MDM2 protein, the in silico computational analysis was performed. The results showed that DS-1 exhibited a cytotoxic effect and sensitized lung cancer cells to cisplatin-induced apoptosis. DS-1 caused a significant increase in cellular level of p53 protein, while the active form of p53 (phosphorylation at Ser15) was insignificant change. DS-1 combined with cisplatin could enhance p-p53 (Ser15) and p53 downstream signaling (Bax, Bcl-2, and Akt), leading to higher level of apoptosis. Immunoprecipitation analysis revealed that DS-1 decreased p53-ubiquitin complex, a prerequisite step of p53 proteasomal degradation. Molecular docking simulation further evidenced that DS-1 interacts with MDM2 within p53-binding domain by carbon-hydrogen bond interaction at Lys27, π-Alkyl interactions at Ile37, and Leu30, and Van der Waals interactions at Ile75, Val51, Val69, Phe67, Met38, Tyr43, Gly34, Phe31, and Lys27. Treatment of DS-1 and cisplatin in patient-derived primary lung cancer cells showed the consistent effects of increasing cisplatin sensitivity. For cancer metastasis, DS-1 significantly inhibited the proliferation of lung cancer cells compared to the control group. The aggressive behavior of cancer cells including migration and invasion ability was significantly reduced in the DS-1-treated cells. Besides, anchorage-independent growth analysis provides evidence that DS-1 could suppress the growth of the cancer cell in detached condition indicated by the significant reduction in cell colony size and number. For mechanisms, we found that DS-1 suppressed EMT indicated by the reduction of EMT markers namely N-cadherin, Snail, and Slug, while increasing epithelial maker of E-cadherin. Also, DS-1 was shown to decrease the cellular levels of integrin β1. Our findings provide evidence of DS-1 as an MDM2 inhibitor and metastasis prevention through integrin and FAK suppression, which may benefit the development of this compound for lung cancer treatment.