Curcumins Reverse Resistance To Cisplatin In Lung Cancer Cell By Mechanism FA/BRCA Pathway

Background and ObjectiveFA/BRCA pathway plays a pivotal role in repair of DNA cross-linking damage induced by DNA interstrand cross-link (ICL) agents and greatly influences drug response in cancer treatment. The studies have demonstrated that inhibition of the FA/BRCA pathway can enhance the cytotoxic effects of ICL-inducing anticancer drugs and can reduce anticancer drug resistance. But the mechanism for relationship between the FA/BRCA pathway function status and chemoresistance to cisplatin (DDP) in lung cancer cells is poorly understood. Also it is not clear whether FA/BRCA pathway inhibitor, such as curcumin, can enhance sensitivity to DDP in A549/DDP cell, a DDP-resistant lung cancer cell line. Thus, the purposes of this study are to explore the FA/BRCA pathway mechanism of resistance to DDP in lung cancer cells, and investigate the efficiency of curcumin in potentiating the cytotoxic effects of DDP in A549/DDP cells, and explore the feasibility of curcumin as DDP-resistant reversal agent in treatment of lung cancer.Methods1. The proliferation inhibition rates of lung cancer cell lines A549and Calu-1treated with variant concentration of DDP were measured using the CCK-8assay. The expression levels of FANCC, FANCF, FANCL and FANCD2mRNA were detected by real-time fluorescent quantitative PCR (RFQ-PCR). The expressions of FANCC, FANCF, FANCL and FANCD2proteins were determined by Western blotting. FANCD2monoubiquitination level was defined as the ratio of FANCD2-L and FANCD2-S (L/S).2. After treatment with different concentrations of curcumin, DDP alone or combination of curcumin with DDP, the FANCD2monoubiquitination levels were measured by Western blotting in lung cancer cell lines A549and A549/DDP, respectivly. The formation of FANCD2nuclear foci were determined by immunofluorescence staining. The rate of cell proliferation inhibition was detected by CCK-8assay. Apoptosis was assessed by DAPI staining. Apoptosis rate was measured by flow cytometry using Annexin V/PI methods. Results1. After treatment with DDP, the rates of proliferation inhibiting of A549and Calu-1lung cancer cell lines were increased gradually with a dose-dependent manner (P<0.05) and a time-dependent manner (P<0.05). The50%inhibitory concentrations of DDP were significantly lower in A549cells than in Calu-1cells. FANCF and FANCL mRNA expression levels in A549cells were markedly lower than those in Calu-1cells. The FANCD2mRNA levels were increased in A549cells at24H after treatment of DDP (in2.5-5μg/ml and20μg/ml DDP respectively) and were decreased at48H after treatment of DDP, while the FANCD2mRNA expression were significantly increased in Calu-1cells at24H and48H after treatment of DDP (except for DDP concentration of20μg/ml at two time points). Meantime, the expressions of FANCC, FANCF and FANCL protein in A549cells were significantly decreased during the treatment of5-20μg/ml DDP as compared with control group. After treatment of5-10μg/ml and20μg/ml DDP, however, FANCC and FANCF mRNA levels in Calu-1cells were initially increased and then decreased after treatment of variant concentrations DDP. The FANCD2monoubiquitination levels in Calu-1cells were significantly higher than those in A549cells.2. A549/DDP cell lines were moderately resistant to DDP. The FANCD2monoubiquitination levels in A549/DDP cells were significantly higher than those in A549cells following treatment with different concentrations of curcumin, DDP alone or combination of curcumin and DDP. In the concentration range of0-20μg/ml DDP, the addition of curcumin can significantly reduce FANCD2monoubiquitination levels in A549/DDP cells. FANCD2monoubiquitination levels in A549/DDP cells treated with combination of DDP and curcumin were significantly lower than those in these cells treated with curcumin or DDP alone. After treatment with combination of curcumin and DDP, the fluorescence nuclear foci expressions were significantly decreased in A549/DDP cells than in A549cells, implying that curcumin can inhibite FANCD2monoubiquitination levels and the formation of nuclear foci induced by DDP in these DDP-resistant lung caner cells. Proliferation inhibition rate in A549cells treated with curcumin puls DDP was significantly higher than that in these cells treated with curcumin or cisplatin alone, but synergism of curcumin and DDP on proliferation inhibition is not found in the DDP-sensitive cells (q value<1.15). Proliferation inhibition rate in A549/DDP cells treated with curcumin puls cisplatin was also significantly higher than that in these cells treated with single drug, moreover, there is a synergistic effect both curcumin and cisplatin on proliferation inhibition in the DDP-resistant cells(q values>1.15). The inhibitory concentrations of50%of DDP in A549/DDP cells were decreased significantly after treatment with cisplatin plus DDP. In addition, after treatment with curcumin plus DDP in A549/DDP cells, DAPI stained showed that their nuclear chromatin condensation which formed a lot of particulate matter, the nucleus breaks down into fragments, and apoptotic bodies can be seen. Apoptosis rate was markedly higher in A549cells than in A549/DDP cells after treatment with single drug. However, apoptosis rate in A549/DDP cells was significantly higher than that in A549when treatment with combination of curcumin and DDP of10-20μg/ml concentration. These results indicate that the activity of FA/BRCA pathway DNA damage repair function induced by DDP is inhibited effectively by curcumin.ConclusionsFA/BRCA pathway is involved in the mechanism of resistant to DDP in lung cancer cells, FANCF and FANCC protein of this pathway may play important roles in DNA damage signaling and repair induced by DDP in Calu-1cells. A high level of FANCD2monoubiquitination in lung cancer cells is a feature of resistance to cisplatin. FANCD2L/S ratio may be a useful indicator to predict the sensitivity to platinum-based chemotherapy in patients with lung cancer. Curcumin inhibits FANCD2monoubiquitination induced by DDP in A549/DDP cells, reduces nuclear foci formation, increases cell proliferation inhibition rates, and induces cell apoptosis, thereby reverse resistance to DDP in the DDP resistant-drug lung cancer cells. As a FA/BRCA pathway inhibitor, curcumin can inhibit FA/BRCA pathway DNA damage repair function, reverse resistance to DDP in lung cancer cell, suggesting that curcumin may serve as a chemosensitizer to ICL-inducing anticancer drugs.