The Mechanism Of Ginsenoside Rg3 Anti-melanoma Effects Correlated With Regulation Of HDAC3 And FUT4 Expression

I. ObjectiveMalignant melanoma is the leading cause of skin cancer related death(80%) worldwide, the median survival time of patients with stage IV melanoma is less than 1 year and 5-year survival rate of less than 10%. According to the World Health Organization(WHO), the incidence of melanoma is rising faster than that of any other type of cancer worldwide. The short-lived therapeutic response, tumor resistance, and adverse effects of traditional drugs used in melanoma therapy, including dacarbazine, temozolomide and interleukin-2, along with recently approved targeted therapeutic agents, such as vemurafenib and ipilimumab, have limited their use due to low responsive rates and/or high toxicities. Therefore, the development of novel therapeutic strategies and targets are needed for the treatment of melanoma.Ginseng, is a well-known herbal medicine, has been used for thousands of years alone or in combination with other herbal ingredients, such as invigorant, cardiotonic, and drugs for anti-inflammatory, anti-tumor and immune stimulation. 20(R)-Ginsenoside Rg3 is a monomer extracted from ginseng, has a wide range of pharmacological and therapeutic effects. Previous reports showed that Rg3 had anticancer effect in gastric, breast, colon and hepatocellular cancers,Rg3 enhances the chemosensitivity of patientsto docetaxel and cisplatin with prostate and colon cancer. Moreover, Rg3 has been shown to inhibit tumor angiogenesis and induce cancer cell apoptosis in liver carcinomas. Currently, Shen Yi capsule which contains Rg3 as the main ingredient is used clinically to treat late-stage non-small cell lung cancer(NSCLC) in China. However, the anti-tumor mechanism of Rg3 on melanoma remains unclear.Acetylation and deacetylation are epigenetic processes that are in the regulation of gene expression. Previous studies suggested that abnormal expression of HDACs was associated with tumor growth, invasion, and metastasis. HDAC6 is highly expressed in human pancreatic and breast cancers, overexpression of HDAC2 has been found in cervical and gastric cancers, and high levels of HDAC8 expression have been reported in childhood neuroblastoma. Overexpression of HDAC3 is reported in many cancer types, such as lung, colorectal and gastric cancer, as well as in prostate cancer. However, the interplay between Rg3, HDAC3, and melanoma growth remains unclear.Protein glycosylation plays an important role in pathophysiological steps of tumor progression, including tumor proliferation, invasion, haematogenous metastasis and angiogenesis. Fucosylation is one of the important steps in protein glycosylation. Fucosyltransferases(FUTs) are the key enzymes catalyzing the synthesis of fucosylated glycans. The 1, 3-fucosylation of Le Y is catalyzed by fucosyltransferase IV(FUT4). Increased FUT4 expression has been reported in many cancers, such as gastric, colorectal, and lung cancer. However, the mechanism of Rg3 on melanoma cell proliferation, and its potential role in regulation of FUT4 and Le Y expression on cell proliferation have not been reported.In the present study, we demonstrated the antitumor effect of Rg3 against melanoma correlated with regulation HDAC3 and FUT4 expression. Analyzed the effects of Rg3 on anti-melanoma cell proliferation, induced apoptosis and regulated some signal pathways. In order to provide the scientific basis for the clinical treatment of melanoma.II. Methods1. Immunohistochemistry method was used to detect the expression of HDAC3 in 32 cases of paraffin histopathological human melanoma tissues. SPSS16.0 statisticalsoftware was used for statistical analysis, and p-value less than 0.05 were considered significant. The correlation between the two indicators and the major clinical characteristics of melanoma were analyzed by chi-square test.2. The cell proliferation was detected by CCK-8 and colony forming assay.3. The HDAC3 si RNA、FUT4 si RNA were transient transfection into the melanoma cells to down-regulate HDAC3 and FUT4 expression.4. The gene expression of HDAC3,PCNA,FUT4 �'� p65 were analyzed by Realtime PCR.5. The expressions of HDAC3,Ac-p53(k373/k382),FUT4,Le Y,EGFR,p EGFR,ERK,p ERK,PARP,Caspase-3,-8,-9,Bcl-2,Bax,Survivn,NF-κB and EGFR/MAPK signaling pathway proteins were detected by Western Blot.6. The expression of HDAC3, FUT4, PCNA and p65 were checked by immuofluorenscence and immunohistochemistry.7. The cell cycle and apoptosis were analysis by flow cytometer.8. The DNA binding activities of NF-κB were detected by EMSA.9. The p53 and NF-κB luciferase activity were analysis luciferase reporter assay.10. The binding activities of NF-κB to FUT4 promoter were detected by Ch IP assay.III. Results1. Ginsenoside Rg3 inhibits melanoma cell proliferation through down-regulation of histone deacetylase 3(HDAC3) and increase p53 acetylation(1) Increased expression of HDAC3 correlates with lymph node metastasis and clinical stage of melanoma.(2) Rg3 inhibits melanoma cell growth.(3) Rg3 decreases HDAC3 expression.(4) Down-regulating HDAC3 expression inhibits melanoma cell proliferation.(5) Rg3 and down-regulating HDAC3 expression increase p53 acetylation and transcription activity.(6) Down-regulation HDAC3 by Rg3 alters cell cycle protein expression.(7) Rg3 inhibits the growth of melanoma xenograft tumors in vivo.2. Ginsenoside Rg3-induced EGFR/MAPK pathway deactivation inhibits melanoma cell proliferation by decreasing FUT4/Le Y expression(1) Rg3 inhibits cell growth in A375 melanoma cells.(2) Rg3 decreases the expression of FUT4 and Le Y.(3) Down-regulating FUT4 expression inhibits A375 cell proliferation and decreases the expression of Le Y.(4) Down-regulating FUT4 expression decreases the tyrosine phosphorylation of EGFmediated EGFR/MAPK.(5) Rg3 inhibits the growth of melanoma xenograft tumors in vivo.3. Ginsenoside Rg3 suppresses FUT4 expression through targeting NF-κB/p65 signaling pathway and induces melanoma cell death(1) Rg3 inhibited human melanoma cell proliferation.(2) Rg3 induced human melanoma cell apoptosis.(3) Rg3 inhibited the activation of NF-κB signaling pathway.(4) Rg3 decreased FUT4 expression and inhibited NF-κB binging to FUT4 promoter.(5) Rg3 induced apoptosis by inhibiting both NF-κB signaling pathway and FUT4 expression.(6) Rg3 suppressed the growth of human melanoma cancer xenograft.IV. Conclusions1. Ginsenoside Rg3 inhibits melanoma cell proliferation through down-regulation of histone deacetylase 3(HDAC3) and increase p53 acetylation(1) The antitumor effect of Rg3 against melanoma both in vitro and in vivo.(2) Rg3 decreases HDAC3 expression.(3) Down-regulating HDAC3 expression inhibits melanoma cell proliferation.(4) Rg3 inhibits melanoma proliferation through down-regulating HDAC3 expression,increase p53 acetylation and transcription activity.2. Ginsenoside Rg3-induced EGFR/MAPK pathway deactivation inhibits melanoma cell proliferation by decreasing FUT4/Le Y expression(1) Rg3 inhibits cell growth correlated with down-regulation of FUT4 and Le Yexpression.(2) Down-regulating FUT4 expression decreases the expression of Le Y.(3) Rg3 inhibits cell proliferation by down-regulating EGFR/MAPK signaling pathway through reducing FUT4/Le Y expression.3. Ginsenoside Rg3 suppresses FUT4 expression through targeting NF-κB/p65 signaling pathway and induces melanoma cell death(1) Rg3 inhibited human melanoma cell proliferation and induced apoptosis both in vitro and in vivo.(2) Rg3 induced apoptosis through inhibition of the NF-κB signaling pathway proteins expression and NF-κB DNA binding and transcription activity.(3) NF-κB binged to FUT4 promoter and regulated FUT4 expression.(4) Rg3 induces tumor cell apoptosis correlated with its inhibitory effect on NF-κB signaling pathway mediated FUT4 expression.