Study Of The Yuanhuacine's Anti-melanoma Effect And Its Mechanism

Background.Yuanhuacine (YHC) was isolated from D. genkwa and has been reported to have activity in the termination of early pregnancy by combination with testosterone propionate. YHC has been shown to be active against the growth of P-388 lymphocytic leukemia cells, L-1210 lympHoid leukemia, human KB carcinoma cell lines. In addition, YHC also suppresses DNA, RNA and protein synthesis of some tumor cells. Recent research showed that YHC has weak effect on tumor necrosis factor (TNF-a) and interleukin-1 (IL-la, IL-lb) biosynthesis, but exhibited potent inhibitory activities against DNA topo I. In the experiments in vivo, YHC significantly inhibited the growth of P-388 lympHocytic leukemia cells and suppressed tumor growth of Lewis lung carcinoma in BDF1 mice and in C57BL/6 mice.However, although the antiproliferative effects of YHC have been well- documented, the cellular mechanisms underlying the antiproliferative activity of YHC are unclear. In present study, we demonstrated that yuanhuacine inhibited the growth of human malignant melanoma A 375 cells in a dose- and time-dependent manner. This growth arrest was shown to be because of cell cycle inhibition at G₂/M, apoptosis induction and activation of protein kinase C (PKC). The experiments in vivo confirmed that yuanhuacine suppressed the growth of melanoma. These results indicate that yuanhuacine can inhibit cancer cell growth not only in vitro but also in vivo, therefore it may ultimately be proved to be useful as a new potential antitumor medicine for malignant melanoma.Methods.Survival/proliferation of cell lines treated with YHC was determined by trypan blue dye exclusion assay or CCK-8 assay. [³H]-thymidine incorporation assay was performed to assess DNA synthesis of cells. Cell cycle distribution was determined by flow cytometric analysis of DNA content of nuclei of cells following staining with propidium iodide. Apoptosis induction by YHC in A375 cells was assessed by quantification of phosphatidylserine exposure using annexin V-FITC kit. Alterations in mitochondrial membrane potential (△Ψm) were analyzed by flow cytometry using the△Ψm-Sensitive dye rhodamine123. Western blot was performed to investigate the expression of cell cycle and apoptosis related proteins. Cytosolic calcium level of cells after short-term and long-term YHC exposure was measured using a calcium probe fluo-3·AM by laser confocal microscopy and flow cytometry. Intracellular or purled PKC activity was determined by ³²p radioisotope incorporation. Confocal microscopy was used to observe fluorescence disposition to evaluate the translocation of PKCγ-EGFR The inhitition of tumor growth was confirmed by C57BL/6 melanoma modeling.Results.1. The treatment of A375 cells with 0.01-10μM of YHC resulted in a dose-dependent decrease in cell survival with an IC₅₀ of 6.78±1.74μM for 24 h and 1.23±0.29μM for 48 h.2. Viability of the cells was inhibited significantly upon treatment with YHC in a dose- and a time-dependent manner. The inhibition rate of cell viability was increased to 32.2±6.52%, 43.51±4.69%, 46.1±5.26% and 55±2.28% respectively which were treated with YHC for 12, 24, 36 and 48h in concentration of 10μM, and IC₅₀ was 4.89μM for 48 h.3. [³H]-thymidine incorporation into A375 cells was decreased to 51.6±2.57%, 38.8±5.49%, 32.3±6.53%, 4.2±3.96% (as a percentage of control) respectively, after treatment with YHC in concentrations of 0.1, 1, 10 and 50 mM for 48h, and IC₅₀ was 0.23μM.4. Exposure of A375 cells to YHC for increasing time intervals produced a concentration and time-dependent G₂/M pHase cell cycle arrest. For example, after treatment with 0.1, 1 and 10μM YHC for 48 h, the percentage of cells in the G₂/M pHase increased to 11.42±2.49%, 15.74±2.94% and 22.4±2.48%, respectively, showing significant difference compared with the control group (8.47±1.34%). After incubation with 1μM YHC for 12 h, 24 h and 48 h, the percentage of cells in the G₂/M pHase increased to 10.62±1.96%, 10.82±2.83%and 15.7±2.94%, respectively.5. When cells were treated with 0.1, 1 and 10μM YHC for 48 h, CyclinB 1 and cdc2 protein expression levels were decreased significantly. 6. Flow cytometric quantification of apoptotic A375 cells stained with annexin V-FITC/PI showed that YHC treatment increased the percentage of apoptotic cells to 4.8%, 8.5% and 24.6%, respectively in concentration of 0.1, 1 and 10μM for 48 h.7. The△Ψm of A375 cell was reduced from 9.99±0.85 to 8.02±1.45, 6.1±0.15 and 4.23±0.7 by exposure to 0.1, 1 and 10μM YHC.8. Treatment with yuanhuacine resulted in cytochrome c releasing from mitochondria and activation of caspase-3, accompanied with significant down-regulation of the Bcl-2 and mdm-2 expression, up-regulation of Bax, p53 and p21 expression.9. The cytosolic calcium level was 1.23 folds higher compared with control after treatment with 1μM YHC for 48 h, and the level's upregulation was observed within 25 min.10. YHC induced protein kinase C activation both in cell and in tube, and this effect independented on Ca²⁺. PKC activity was 1.78 and 3.53 folds higher compared with or without Ca²⁺/PMA.11. YHC induced PKCγtranslocation from cytoplasm to plasma membrane and showed synergistic effect with PMA.12. YHC suppressed the growth of malignant melanoma in C57BL/6, the inhibition rate was 31% for animal which were application with YHC, subcutaneous injection in dose of 0.8mg/kg induced 47% decrease and intraperitoneal injection 0.4mg/kg YHC resulted in the reduction of 50.2%.Conclusion.YHC inhibited A375 cell growth in a dose- and a time-dependent manner, and arrested cell cycle at the G₂/M phase through decreased expression of cyclin B1 and cdc2 and activation of p53. Simultaneously, our data also provide evidence for the induction of apoptotic cell death of YHC, which may be attributable to the dissipation of△Ψm, the up-regulation of Bax and down-regulation of Bcl-2, accompanied with cytochrome c releasing and caspase-3 cleavage. In addition, p53, p21 and mdm2 contribute to this process. YHC also induces cytosolic calcium increasing and PKC activation. Furthermore, YHC inhibits the growth of malignant melanoma in vivo. These results indicate that as an extraction from DapHne genkwae, YHC may ultimately prove useful for malignant melanoma.