The Inhibition And The Mechanisms Of Cochinchina Momordica Seed On B16 Cells

Objective: To confirm the anti-tumor activity and anti-differentiation effects of cochinchina momordica seed extract on melanoma B16 cells and the underlying mechanisms. To investigate the effect of MAPKs associated protein in CMSEE-induced melanoma B16 cells differentiation. To investigate the effects of ethanol extract of cochinchina momordica seed (CMSEE) on the invasion and metastasis of melanoma B16 cells in vitro and in vivo, as well as its probable mechanism.Methods:1 Abstraction ethanol extract and water extract of Cochinchina Momordica Seed, the growth-inhibitory effect of ethanol extract and water extract of Cochinchina Momordica Seed (CMSEE and CMSWE) on MDA-MB-231, B16, EC109, K562, A549 tumor cell lines and normal cell were examined by MTT assay, morphological changes of tumor cells were analyzed by light microscope.2 Clone formation assay was used to assess the effect of CMSEE(40, 20, 10μg/ml)on the colony formation ability of B16 cells.3 Cell cycle and apoptosis rate of B16 cell were examined by flow cyto- -metry (FCM) in four experimental groups(0, 40, 100μg/ml CMSEE ) for 24 hours.4 The inhibitory effect of CMSEE (10, 20, 40, 100μg/mL)on B16 cell was detected by MTT assay. The effect of CMSEE on melanin production and tyrosinase activity of B16 cells were assessed by colorimetry. morphological changes of B16 cells and melanosome were observed by light microscope and transmission electron microscopy.5 The effects of MAPKs associated protein (t-proteins, p-ERK1/2, p-JNK, p-p38) in CMSEE-induced differentiation of melanoma B16 cells were investigated by Western blot.6 Effects of CMSEE(20, 25, 30μg/mL) on the invasion and metastasis of B16 cells were detected by scratch and transwell assays.7 The transcriptional levels of matrix metalloproteinases (MMP)-9 and MMP-2 in B16 cells treated with different dose of CMSEE(20, 25, 30μg/mL)for various times (0, 6, 12, 24, 36, 48 h) were detected by RT-PCR. Expressions of MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 proteins in B16 cells treated with CMSEE were analyzed by Western blotting.8 B16-implanted mouse model was established, and the metastasis and invasion of B16-implanted tumors were observed after treatment with CMSEE.Results:1 CMSEE could significantly inhibit the proliferation of tumor cell lines in a dose-dependent manner ( P<0. 01), but CMSWE had no such effect. Both CMSEE and CMSWE showed no obvious influence on growth of normal cell. The inhibory effect of CMSEE is more obvious than that of CMSWE on proliferation of B16 cell (P<0.01).2 CMSEE(10,20,40μg/ml)can significantly inhibit the growth of B16 cells in vitro. With the concentration increasing, the number of clones and cells grew down(P<0.05).3 After treatment with 100μg/mL CMSEE, B16 cells showed apoptosis morphologic changes while treated with 40μg/mL have no changes. Meanwhile the proportion of B16 cells in the G0/G1 phase was elevated significantly( P<0. 01) .4 The greatest inhibit rate was 70.06% observed in the treatment group of B16 cells by 100μg/mL CMSEE for 72 hours. It suggested that CMSEE could induced the differentiation of melanoma cell B16, which was characterized by the enhancement of dendrites-like outgrowth, melanogenesis, melanin production and tyrosinase activity.5 Western blot analyses showed the sustaining phosphorylation of p38- -MAPK in B16 cells was accompanied by the increasing of the dephosphorylation of ERK1/2 and JNK after CMSEE treatment. Moreover, ERK1/2 and JNK specific inhibitors gave rise to enhancement of B16 cell differentiation while p38MAPK inhibitor blocked the CMSEE-induced differentiation. Taken together, our results suggested that alteration of MAPKs activity was involved in the differentiation of CMSEE-induced melanoma B16 cells.6 After treatment of 20~30μg/mL CMSEE, the diameters of scratch became broader than that of the control group(P<0.05), and the number of B16 cells through the transwell chambers was decreased significantly in a dose-dependent manner(P<0.05).7 CMSEE could down-regulated the expression levels of MMP-9 and MMP-2 mRNAs and proteins in a dose-dependent manner. Expressions of TIMP-1 and TIMP-2 proteins in B16 cells were not changed.8 CMSEE effectively inhibited the metastasis and invasion of B16 -implanted tumors in vivo.Conclusions:1 CMSEE significantly inhibited tumor cell growth in vitro especially for B16 cells. CMSEE could induce differentiation of B16 cells in low concentration and apoptosis in high, and the mechanism of apoptosis might be related to cell cycle arrest.2 The ethanol extract of cochinchina momordica seed inhibited the growth of B16 cells through inducing differentiation, and the effect was closely associated with MAPKs (p38, ERK1/2, JNK) activation, which provided a potentially novel agent for the therapy of melanoma.3 CMSEE could inhibit the invasion and metastasis of melanoma B16 cells in vitro and in vivo, and the mechanism might relate with down-regulating the expression of MMP-2 and MMP-9 mRNAs and proteins.