The Apoptotic Effect Of Tanshinone ⅡA On Human Renal Cell Carcinoma Cells And The Molecular Mechanism Of Its Actions

[Background]Renal cell carcinoma (RCC), the most common cancer arising from the renalparenchyma, causes prevalent deaths worldwide and its incidence continues to rise. Althoughseveral therapeutic approaches have been applied to the treatment of RCC, including surgery,immunological therapies and vaccine treatment, further improvements are still needed.Therefore, it is extremely necessary to explore and develop new anti-cancer drugs. Notably,Traditional Chinese Medicine (TCM) draws much attention due to its dramatic roles in cancertherapy with the characteristics of low-toxicity and specificity, etc. Tanshinone ⅡA (Tan ⅡA),one of the phytochemical compounds isolated from the Chinese medicinal herb Danshen (rootof Salvia miltiorrhiza Bunge), possesses antioxidant and anti-aging properties. At present, TanⅡA has become a research hotspot, because it’s anti-cancer activity. However, the effect ofTan ⅡA on human RCC and its molecular mechanism need to be clarified.[Objective]Therefore, we aim to investigate the apoptotic effect of Tan ⅡA on human RCC786-Ocells and unveil the molecular mechanism by which it functions. Specifically, the viability of786-O cells following Tan ⅡA treatment was examined at first. Further, we delved into thesignaling pathways plus molecular mechanism by which Tan ⅡA induced786-O cell cyclearrest and apoptosis.[Methods]The viability of the786-O cells treated with Tan ⅡA (0μg/mL,1μg/mL,2μg/mL,4μg/mL and8μg/mL) was measured by MTT assay. As a comparison, human embryonickidney (HEK)293A cells were also treated with Tan ⅡA（0μg/mL,1μg/mL,2μg/mL,4μg/mL,8μg/mL,10μg/mL,20μg/mL,30μg/mL and40μg/mL）for24h and then subjectedto MTT assay. To demonstrate the growth inhibition of786-O cells induced by Tan ⅡA, thecell cycle distribution of786-O cells treated with and without Tan ⅡA was analyzed usingFlow cytometry (PI staining). To confirm cell cycle profile, the level of cyclin A proteinwhich is active in the S phase was subjected to western blot (WB). Moreover, to illustrate the molecular mechanism underlying cell cycle arrest, the expression levels of p21and p53whichare related with cell cycle checkpoints were determined by WB. On the other hand, TanⅡA-induced apoptosis was assessed through Flow cytometry (Annevin V/PI staining).Thereafter, the expression levels apoptosis-related molecules including Fas and bax which aretranscriptionally activated by p53and caspase-3which is an apoptotic executor wereexamined by WB. In order to reveal the molecular mechanism of p53up-regulation, the levelof MDM2protein which is a p53negative regulator was tested by WB. At last, a plasmidcontaining GFP-tagged NPM was constructed, and its sub-cellular localization was checkedby confocal microscope after its transfection in786-O cells. The physical interaction betweenNPM and p53was confirmed via co-immnunoprecipitation.[Results](1) The MTT assay indicated that Tan ⅡA treatment for24h induced a marked inhibition ofthe growth of786-O cells. The half maximal inhibitory concentration (IC50) value forTan ⅡA treated786-O were estimated to be2μg/mL, whereas that for the Tan ⅡAtreated293A was8μg/mL.(2) When786-O cells were treated with Tan ⅡA (0,2,4and8μg/ml) for24h, Flowcytometry analysis (PI stain) showed that the percentages of786-O cells in S-phasewere10.0%,11.5%,20.4%and23.3%, respectively. WB indicated that Tan ⅡA induceda dose-dependent upregulation of cyclin A. At the same time, p53and its downstreamgene p21were also increased in Tan ⅡA treated786-O cells, in a dose-dependentmanner.(3) Following the treatment of786-O cells for24h with Tan ⅡA (2,4and8μg/mL), thenumber of early apoptotic cells increased from8.6(control) to27.2,27.8and28.8%,respectively. While the number of late apoptotic cells respectively increased from2.9(control) to9.2,12.5and13.9%. The total percentage of apoptotic cells was directlyrelated to the Tan ⅡA concentration, increased from11.5%(control) to36.4,40.3and42.7%. Tan ⅡA treatment resulted in a significant dose-dependent increase in the levelof bax and caspase-3.(4) WB showed no change in the level of MDM2protein which is a negative regulator ofp53. Confocal microscopy showed that Tan ⅡA treatment resulted in a nucleoplasmictranslocation of GFP-tagged NPM from nucleolus. Co-immunoprecipitation tested thatthere was physical interaction between NPM and p53. [Conclusion](1) Tan ⅡA treatment can cause significant dose-dependent inhibition of786-O cell growth,while Tan ⅡA treatment results in minor cytotoxicity on293A cells.(2) Tan ⅡA treatment can induce S phase cell cycle arrest of786-O cells. The molecularmechanism can be illustrated by the upregulation of p53and its transcriptional targetgene p21, which are involved in S phase cell cycle arrest of786-O cells induced by TanⅡA treatment.(3) Tan ⅡA treatment can further induce human RCC786-O cell apoptosis. In the apoptotic786-O cells treated with Tan ⅡA, the level of Fas proteins expression shows no change,in contrast, the expression level of bax protein increases. As a result, there is anupregulation of the apoptotic executor caspase-3. These data indicate that bax isinvolved in Tan ⅡA-induced cell apoptosis; Tan ⅡA-induced apoptosis is through amitochondrial pathway rather than Fas pathway.(4) The translocation of Nucleolar protein NPM from nucleolus to nucleoplasm in786-Ocells after Tan ⅡA treatment and the physical interaction between NPM and p53indicates that NPM may be involved in the regulation of p53stability at apost-translational level, and ensuing upregulation of p21.Our present study demonstrates that Tan ⅡA treatment can cause an effective growthinhibition and cell cycle arrest followed by apoptosis in human RCC786-O cells. Tan ⅡAtreatment can also trigger the translocation of NPM from nucleolus to nucleoplasm.Subsequently, Nucleoplasmic NPM can enhance p53stability via direct interaction betweenthe two, as a result, the downstream targeted genes including p21and bax are upregulated. Itis remarkable that Tan ⅡA has reduced cycotoxicity on HEK293A cells comparing with786-O cells, which suggests that Tan ⅡA has mild side effect on normal cells. Our researchwill provide promising guideline for the tumor therapy.