Shikonin reactivates TSGs GADD45B and PPP3CC to block NSCLC cell proliferation and migration through JNK/P38/MAPK signaling pathways

Shikonin, a natural naphthoquinone compound extracted from the Chinese traditional herbal medicine “Lithospermum erythrorhizon”, possesses antitumor activity against various cancer types. Tumor-suppressor genes (TSGs) negatively regulate cell growth, proliferation, and differentiation, thereby inhibiting tumor formation. However, the molecular mechanism of action of shikonin on TSGs in non–small-cell lung cancer (NSCLC) remains unclear. The inhibitory effect of shikonin on the proliferation and migration abilities of lung cancer cells were measured by Cell Counting Kit 8 (CCK8) and wound healing assays. The alteration of genes by shikonin treatment was detected by mRNA high-throughput sequencing and further confirmed by qPCR and western blotting experiments. The dominant functions of the upregulated genes were analyzed by GO and KEGG profiling. Shikonin inhibited the proliferation and migration of A549 and H1299 NSCLC cells in a dose-dependent manner. mRNA high-throughput sequencing revealed a total of 1794 upregulated genes in shikonin-treated NSCLC cells. Moreover, bioinformatic analysis of GO and KEGG profiling revealed that the up-regulated genes were mostly involved in the JNK/P38/MAPK signaling pathway, among which the expression of GADD45B and PPP3CC was significantly enhanced. Finally, we confirmed that GADD45B and PPP3CC were indeed upregulated in JNK/P38/MAPK pathway. Taken together, these results suggested that shikonin might affect the expression of GADD45B and PPP3CC through the JNK/P38/MAPK pathway, therefore exerting an inhibitory effect on the proliferation and migration of cancer cells. To our knowledge, this is the first study reporting the role of shikonin in upregulating TSGs to activate the JNK/P38/MAPK signaling pathways in NSCLC.