Abstract
The combination of platinum and gemcitabine is one of the standard regimens in the treatment of advanced lung squamous carcinoma (LSC). Resistance to gemcitabine is main barrier to the successful treatment of LSC. In this study, we showed that suppression of the Fanconi anemia (FA) pathway increased the sensitivity of two LSC cell lines SK-MES-1 and KLN205 to gemcitabine. Moreover, we found that the CHK1 pathway and the FA pathway are functionally compensatory in the repair of DNA damage in the LSC cell lines. Inactivation of one of the two pathways led to DNA damage, triggering compensatory activation of other pathway. Furthermore, we demonstrated that FANCD2 depletion combined with CHK1 inhibitor MK-8776 significantly potentiated the cytotoxicity of gemcitabine to the two LSC cell lines, compared to individual FANCD2 depletion or MK-8776 treatment. The enhanced effect of gemcitabine-chemosensitization was accompanied by loss of DNA repair function and accumulation of DNA single strand breaks and double strand breaks, in parallel with obvious increase of caspase-3 dependent apoptosis. Our results indicate that the enhancement effect of FANCD2 depletion combined with CHK1 inhibitor in sensitizing the LCS cells to gemcitabine supports the FA pathway and CHK1 as two therapeutic targets for improvement of anti-tumor regimens in treatment of LSC.
Highlights
Lung cancer is the top cause of cancer-related death[1]
It is noteworthy that the sensitization effect by depleting FANCL, FANCD2 or CHK1 in SK-MES-1 cells was more remarkable than in LKN205 cells, for instance, the IC50 of gemcitabine in the SK-MEK-1 cells decreased from 20.56 ± 6.83 to 5.14 ± 2.27 and 2.86 ± 0.78 after FANCD2 and CHK1 depletion respectively, whereas the IC50 in the KLN205 cells decreased from 8.56 ± 3.45 to 3.77 ± 1.52, and 1.85 ± 0.39 with same treatment, respectively (Fig. 1C,D)
The degree of sensitization to cisplatin by depleting the Fanconi anemia (FA) pathway factors was more significant than that by silencing CHK1 (Fig. 1E,F). These results suggest that the FA pathway and CHK1 are implicated in gemcitabine resistance in SK-MES-1 cells
Summary
Lung cancer is the top cause of cancer-related death[1]. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer and more than 60% of NSCLC patients are diagnosed in locally advanced and advanced stage[2,3]. Gemcitabine can be incorporated into the growing DNA strand, and induces chain termination after the addition of the nucleotide[11] These perturbations of DNA metabolism prevent complete replication and trigger the DNA damage response (DDR) pathways[12]. We hypothesized that co-inhibition of CHK1 and the FA pathway might further sensitize LSC cells to gemcitabine compared to the suppression of CHK1 or the FA pathway alone. Combination of MK-8776 and the FA pathway suppression by siRNA produced significant gemcitabine-sensitization as compared with CHK1 inhibitor treatment and the FA pathway suppression alone, which was accompanied by markedly elevated phosphorylations of CHK1 (S345), CHK2 (T68), and H2AX (S139), and obvious inhibition of the RAD51-mediated repair of gemcitabine-induced DSBs, demonstrating a enhancement effect between CHK1 inhibitor and suppression of the FA pathway in sensitizing LSC cells to gemcitabine
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