The Anti-melanoma Effects And The Possible Mechanisms Of Paeonol And Triptolide

Cutaneous malignant melanoma (CMM) is considered one of the most aggressive and lethal malignancies because of its propensity to metastasize and resistance to cytotoxic agents. It also presents significant morbidity and a high mortality rate through all the years. Unfortunately, the increase in incidence has not been paralleled by the development of ideal therapeutic agents. Therefore, a search for new therapeutic approaches is necessary. As traditional Chinese medicine monomers with various pharmacological properties, triptolide and paeonol have been indicated to have anti-tumor effects. In this research, we investigated the anti-melanoma effects of these two monomers and explored the potential mechanisms involved in this process.Effect of paeonol and triptolide on inhibiting melanoma cells growthThe inhibition of cell proliferation was measured by using CCK8assay, and then, cell cycle distribution was evaluated by flow cytometric analysis of PI (Propidium Iodide). Results showed that0.5mM,1mM,2mM,4mM and8mM paeonol for24,48, and72h inhibited the proliferation of A375cells, with the IC50of5.19mM,1.50mM and1.09mM at24,48and72h, respectively. The IC50of paeonol on M14cells was4.11mM,1.60mM and1.17mM at24,48and72h, respectively. Paeonol arrested cell cycle at G2/M phase.12.5nM,25nM,50nM and100nM triptolide for24,48and72h can also inhibit the proliferation of A375cells, with the IC50of84.46nM,33.00nM and8.53nM at different time span. The IC50of triptolide on M14cells was130.90nM,35.90nM and6.50nM at24,48and72h. But triptolide arrested cell cycle at S phase.Effect of paeonol and triptolide on inducing melanoma cells apoptosisWe observed cells stained with Hoechst33258, and analyzed the apoptosis rate of melanoma cells by flow cytometry. After cells were treated by5mM paeonol for24h or30nM triptolide for48h, both A375and M14cells displayed apoptotic morphological alterations. After treated by0mM,1.25mM,2.5mM and5mM paeonol for24h, the early apoptosis rate of A375cells was3.11%±0.53%,13.74%±1.73%,25.95%±0.57%and46.44%±0.81%, while the figure of M14cells was1.00%±0.08%,2.00%±0.01%,2.99%±0.29%and14.73%±0.94%,respectively, with statistical significance (P<0.05). After treated by0nM,10nM,20nM and30nM triptolide for48h, the apoptosis rate of A375cells was4.20%±0.09%,19.50%±0.32%,36.57%±3.85%and58.68%±2.03%, while the figure of M14cells was2.92%±0.17%.20.99%±0.40%,34.28%±2.04%and 63.38%±0.71%, respectively, with statistical significance (P<0.05). The effects of inducing apoptosis was in a dose-dependent way.Mechanisms in apoptosis induced by paeonol and triptolide on melanoma cellsWe chose A375cells to investigate the possible regulatory mechanisms of apoptosis induced by paeonol and triptolide. In this section, we detected the activity of caspase3, caspase8and caspase9. Meanwhile, the protein level of p53, NF-κB and some of their downregulation genes was evaluated. The results showed that the activity of caspase3, caspase8and caspase9elevated with statistical significance after cells were treated by2.5mM and5mM paeonol. The protein level of p53and Bax was upregulated with dose going higher, and the protein level of NF-κB, Bcl-2and Bcl-XL was downregulated. These results showed that paeonol induced apoptosis through not only caspase and NF-κB pathway, but also p53pathway. After A375cells were treated by10nM,20nM and30nM triptolide for48h, capsase3and caspase9was activated, but the activity of caspase8decreased. The protein level of NF-κB,Bcl-2and Bcl-XL was also downregulated with dose increasing, which means triptolide induces A375cells apoptosis in a caspase and NF-κB dependent pathway.Effect of paeonol and triptolide on inhibiting melanoma cells invasion and its mechanismsInvasion and migration are the most essential characteristics of malignant tumor. In order to observe the effect of paeonol and triptolide on inhibiting melanoma cells invasion and migration, we used transwell test and cell scratch assay. Results indicated both of the two traditional Chinese medicine monomers can inhibit the invasion of A375and M14cells. After treated by1.25mM paeonol for24h, the migration distance of A375cells decresed from197.00±11.14to46.67±5.69, and the result of M14also decresed, from172.67±9.29to33.00±6..24, both with statistical significance (P<0.01), After treated with0mM,1.25mM,2.5mM or5mM paeonol for24h, the number of A375cells on basement membrane was180.67±13.65,131.67±10.41,74.33±3.21and33.33±4.16separately, and the number of M14cells was decreased from306.00±7.55to253.67±14.01,150.33±9.6and55.33±5.69, all with statistical significance (P<0.01). After treated by10nM triptolide for48h, the migration distance of A375cells decresed from286.67±17.62to185.67±7.09, and the result of M14also decresed from415.67±19.76to213.33±10.02, both with statistical significance (P<0.01), After treated with OnM,10nM,20nM or30M triptolide for48h, the number of A375cells on basement membrane was183.00±10.82,143.67±7.77,60.67±4.04and27.33±2.52separately, and the number of M14cells was decreased from129.67±7.23to80.33±2.51,65.00±5.00and20.33±1.53, all with statistical significance (P<0.01).Real-time PCR and Western blot were carried out to detect the gene and protein expression of VEGF and uPA, and approach the mechanisms involved in this process. After cells were treated by paeonol, the gene expression was upregulated. Result of control,1.25mM,2.5mM and5mM group, was:0.20±0.02,0.28±0.01,0.38±0.01and1.97±0.21for uPA;0.59±0.06,0.79±0.04,2.27±0.02and3.61±0.21for VEGF. Protein level was downregulated after paeonol treated. Result of control,1.25mM,2.5mM and5mM group, was:1.01,0.89,0.68and0.46for uPA;1.13,1.00,0.92and0.52for VEGF. After cells were treated by triptolide, the gene expression of uPA and VEGF was also upregulated. Result of control,10nM,20nM and30nM group, was:0.20±0.02,0.41±0.00,0.95±0.05and5.84±0.16for uPA;0.59±0.06,0.45±0.11,0.89±0.08and1.00±0.03for VEGF. Protein level was downregulated after triptolide treated. Result of control,10nM,20nM and30nM group, was:1.01,0.81,0.44and0.15for uPA;1.13,1.46,1.34and0.96for VEGF. Based on these results, we suppose that paeonol and triptolide inhibit the invasion and migration of melanoma cells by downregulation of the protein level of VEGF and uPA.ConclusionPaeonol and triptolide can both inhibit the proliferation of melanoma A375and M14cells, the mechanism of paeonol may be arresting melanoma cell cycle at G2/M phase, inhibiting cell mitotic diversion. For triptolide, the mechanism would be arresting cell cycle at S phase, inhibiting the duplication of DNA.Both of the two Chinese traditional medicine monomers can induce melanoma A375and M14cells apoptosis in a dose-dependent way. Paeonol and triptolide can induce A375cells apoptosis through caspase-dependent way and NF-KB-mediated manner. Besides, paeonol also regulates cell apoptosis through a p53-dependent way.Meanwhile, paeonol and triptolide inhibited melanoma cells invasion and migration through reducing the protein level of VEGF and uPA.In conclusion, paeonol and triptolide both have anti-melanoma effects, possessing the bright future of exploring as the new therapeutic agents in the treatment of cutaneous malignant melanoma.