Abstract
Shikonin (SHK) is a pleiotropic agent with remarkable cell growth inhibition activity against various cancer types, especially non–small cell lung cancer (NSCLC), but its molecular mechanism is still unclear. Our previous study found that miR-628-3p could inhibit the growth of A549 cells and induce its apoptosis. Bioinformatics analysis predicted that miR-628-3p promoter sequence contained p53 binding sites. Considering the regulatory effect of SHK on p53, we speculate that SHK may inhibit the growth and induce apoptosis of NSCLC cells by up-regulating miR-628-3p. CCK-8 and EdU assay confirmed the inhibitory effect of SHK on A549 and PC-9 cells. Meanwhile, quantitative reverse transcription–polymerase chain reaction and Western blot showed that SHK could promote the expression of p53 and miR-628-3p in a dose-dependent manner. Overexpression of p53 or miR-628-3p can inhibit the growth and promote apoptosis of A549 and PC-9 cells, while silencing p53 or miR-628-3p has the opposite effect. Dual luciferase reporting assay and ChIP (chromatin immunoprecipitation) assay further verified the direct interaction between p53 and the promoter of miR-628-3p. Gene knockdown for p53 or miR-628-3p confirmed that SHK inhibits the growth and induces apoptosis of A549 and PC-9 cells at least partly by up-regulating p53/miR-628-3p signaling pathway. Therefore, these novel findings provide an alternative approach to target p53/miR-628-3p axis and could be used for the development of new treatment strategies for NSCLC.
Highlights
Lung cancer is the most often diagnosed cancer and the leading cause of cancer death worldwide (18.4% of overall cancer mortality) (Bray et al, 2018); it places a heavy burden on the health care system and causes a significant challenge to clinicians and patients
The results revealed a dose-dependent decrease in proliferation of A549 and PC-9 cells after SHK treatment, and the inhibition rate of concentration greater than 2.0 μM was significantly different from that of the untreated group (Figure 1A)
The inhibition rate of SHK on A549 and PC-9 cells treated for 48 h was higher than that of corresponding concentration treatment for 24 h, but there was no significant difference observed in both cell lines except the 2.0 μM treatment on PC-9 cells, so did the situation in IC50 concentration of SHK in A549 cells at 48 and 24 h, but the significant difference was detected on PC-9 cells
Summary
Lung cancer is the most often diagnosed cancer (approximately 11.6% of all cancer cases) and the leading cause of cancer death worldwide (18.4% of overall cancer mortality) (Bray et al, 2018); it places a heavy burden on the health care system and causes a significant challenge to clinicians and patients. Novel Mechanism of Shikonin Anti-Cancer chemotherapy for NSCLC (such as anaplastic lymphoma kinase inhibitors, tyrosine kinase inhibitors, and epidermal growth factor receptor inhibitors) has shown better clinical outcome than that for SCLC (Wang et al, 2020). These results indicated that the development of small molecule drugs for the treatment of NSCLC is still of great significance. The exact antitumor molecular mechanism of SHK remains to be elucidated
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