Study On The Mechanisms Of Shikonin-induced Human Choriocarcinoma Cell Apoptosis In Vitro

To find highly effective and low toxic natural anticancer drugs has been a hotspot in recent years. With the rapid development of cancer chemotherapy, new anticancer drugs are emerging constantly. Shikonin has been confirmed with wide pharmaceutical effects on tumor cells and less toxicity to the normal human cells.Although studies have shown that shikonin can inhibit growth and proliferation of tumor cells, there are few reports about its inhibition on human choriocarcinoma cells in vitro.In this research we studied the molecular mechanisms of apoptosis induced by shikonin in human choriocarcinoma cells. Results presented here indicated that shikonin can activate apotosis signal pathways in JEG-3 cells, and then induce JEG-3 cell apoptosis. Further studies showed that the activation of Caspase-3 was required for shikonin-induced apoptosis in JEG-3 cells, and importantly, activation of MAPKs were inhibited during the process.Methods1,MTT assay was applied to determine the half inhibitory concentration of effects on human choriocarcinoma JEG-3,JAR cells treated with shikonin.2,Light microscope was utilized to observe morphological changes of human choriocarcinoma JEG-3 cells treated with shikonin.3,Hochest33258 fluorescence staining was used to distinguish the apoptotic morphological change.4,Cell apoptosis was measured by both propidium iodide(PI) staining and Annexin-V/PI staining. 5,Western Blot was used to detect the expression of apoptosis-related proteins: PARP, Caspase-3, and activation of other signal proteins including p-ERK, p-JNK, p38, p-AKT, pSTAT3, AR.ResultsWe showed that shikonin can remarkablely inhibit the growth of JEG-3 and JAR cells with a time-and concentration-dependent manner, and the IC50 values were 6.3±0.6μM and 9.2±0.8μM respectively, whereas the inhibition rate of shikonin on HEK-293 and L-02 cells were all less than 1%. After JEG-3 cells were treated with different concentrations of shikonin for 24 hours, cells turned round , condensed, and detached from the bottom.Furthermore, 48 hours later, infected cells blebed and died, lots of cell fragments were observed. Results of MTT assay showed that proliferation of JEG-3 and JAR cells were significantly decreased by shikonin treatment, especially for the JEG-3 cells. In addition, FACS results confirmed that shikonin can inhibit cell growth in a concentration - and time- dependent manner. Hochest33258 fluorescence staining assay showed that shikonin induced apoptosis in JEG-3 cells. Infected cells shrinked and chromatin condensed at 24 hours. PI staining assay showed the apoptosis peak, which significantly increased with the increase of the concentration and time. Annexin-V/PI staining detection indicated that apoptotic rates of JEG-3 cells were 7.3%, 29.4%, 48.8% after treated with 0μM, 5μM, 10μM shikonin for 12 hours respectively.To further elucidate the apoptosis pathway induced by shikonin, expressions of Caspase-3, PARP, p-JNK, p-AKT, p-ERK, p38, pSTAT3, AR were detected with Western blot. Results indicated that shikonin could activate Caspase-3 , inactivate p-JNK, p-ERK p-AKT, p-STAT3, and surpress the expression of AR.Furthermore, a 86KD fragment of PARP was observed ,which indicates that Caspase-3 was activated as PARP was one of the substrates of Caspase-3. These results suggest that shikonin induce apoptosis of tumor cells through caspase pathways and inactivate the mitogen-activated protein kinase (MAPK) passway. Moreever, growth, prolification and suvival of the tumor cells were inhibited significantly.Conclusion1,Shikonin could significantly inhibit the proliferation of JEG-3 cells by inducing apoptosis in a time-and-cncentration-dependent manner.2,Shikonin could induce part of cell to apoptosis through the Caspase-3 pathway, and effect some signal pathways(eg. MAPK pathway,AKT pathway,STAT3 pathway).