Decreased expression of CYP11A1 influences the biosynthesis of steroid hormones and is related to several cancers. In recent years, the majority of studies have investigated the role of steroid hormones in various cancer types such as breast [13, 14], prostate , lung , liver , and colon cancer . However, the molecular mechanisms by which CYP11A1 exerts its anticancer effects on RCC are largely unknown. CYP11A1 has been demonstrated to trigger excessive oxidative stress in mitochondria against the human placental cell line originating from choriocarcinoma .
Our results suggested that CYP11A1 can suppress the EMT process by downregulating Snail and Vimentin levels. This effect was more pronounced in the Caki-1 cell line as this is a model of metastasis in RCC, which can pattern a polarized layer with biological functional and admirably-differentiated kidney tissue .
The transition from G2 to M phase was achieved by activating the cdc2/cyclin B complex by upregulation of cdc2 induced by phosphorylating cdc25c, while accreted cdc25c phosphorylation could lead to cell cycle arrest in the G2 phase . Our study found that CYP11A1 induced a remarkable increase in p-cdc25c, and a decrease in cyclin B1 and cdc2 in the Caki-1 cell line, while HEK293 cell lines exhibited reversible expression of these kinases. These results may imply that CYP11A1 is involved in G2/M phase arrest by positively suppressing the cdc2/cyclin B complex pathway with the induction of cdc25c phosphorylation. The regulation of cell cycle distribution after overexpression of CYP11A1 resulted in recovery of cell cycle distribution to normal kidney epithelial cells (HEK293). In RCC, transglutaminase 2 intersects with p53, leading to the depletion of p53 and apoptosis avoidance . Although the loss of p53 function is frequently related to induction of cell cycle arrest , we did not observe p53 expression in Caki-1 cell lines. Programmed cell death or apoptosis, is mainly regulated by intrinsic, extrinsic, and MAPK pathways, which are activated by the disruption of intracellular homeostasis, DNA damage, and stress response. The Bcl-2 family includes various pro-apoptotic proteins such as Bax, Bad, and Bim; is the main regulator of the intrinsic pathway. In RCC, incongruity in Bcl-2 expression was reported according to cancer cell type and the majority were negatively expressed . Normally, activation of Bax and Bak proteins can promote the release of cytochrome C following the extrinsic activation of caspase-3, 7, 9, and PARP, thus leading to apoptosis . Our study further observed a rapid decrease in caspase-9 and PARP expression despite the lack of any significant difference in caspase-3 and 7 after CYP11A1 transfection. These results revealed that Bcl-2 and caspase family activity was not closely related to CYP11A1-induced apoptosis. Recent studies have shown that the cellular signal transduction pathway C-Raf/MEK/ERK can act on some key effectors to regulate cell proliferation [26, 27]. ERK1/2 activation usually leads to cell proliferation, resulting in initial hallmarks of various cancer activities. Consequently, factors involved in the ERK pathway are considered prospective therapeutic targets for cancer. A large number of regulators and inhibitors of this pathway are already being used in clinical trials. Notably, ERK1/2 activation can cause cell death, resulting in pro-apoptotic functions . We found that CYP11A1-overexpressed Caki-1 cells blockaded C-Raf/ERK pathway by downregulating the phosphorylation of both C-Raf and ERK1/2. In response to oxidative stress, MAPK family members are crucial contributors to apoptosis by various signaling pathways. For example, upregulation of JNK and p38 expression was considered stress responsive and thus promote programmed cell death .
In conclusion, we demonstrated for the first time that CYP11A1 overexpression reversed the EMT process in Caki-1 cells, induced G2 phase arrest, generated ROS, and promoted apoptosis via the MAPK pathway (Fig. 4). All these findings suggest that CYP11A1 might have an anti-tumor effect on RCC, which could be an essential target leading to the removal of cancerous cells without affecting or minimalizing stress on surrounding normal cells. However, the main regulator of the CYP11A1-mediated signaling pathway suppressing cancer cell proliferation still requires further study.