Study For The Effect Of Compound Extracted From Cochinchina Momordica Seed On Mouse Melanoma B16Cells Differentiation

Objective: To study the effects of nine compounds （p-hydroxylcinnamaldehyde, coniferylaldehyde, p-hydroxylbenzaldehyde3-o-Methoxyaniline-p-hydroxylbenzaldehyde, ligballinol and so on）extracted from cochinchina momordica seed on the growth of mousemelanoma B16cells, and filter out the most effective sorts; To observe theinduction of differentiation effects of p-hydroxylcinnamaldehyde on B16cellsand the underlying mechanisms, providing the reference theory for p-hydroxylcinnamaldehyde as a new anti-tumor drug.Methods:1The growth-inhibitory effects of nine compounds of cochinchinamomordica seed (p-hydroxylcinnamaldehyde, coniferylaldehyde,p-hydroxylbenzaldehyde,3-o-Methoxyaniline-p-hydroxylbenzaldehyde,ligballinol and so on) on B16cell were examined by SRB assay,morphological changes of tumor cells were analyzed by light microscope.2After treated by PHB (2,4,6μg/ml) for24,48,72h, the growth-inhibitoryeffect was detected by SRB assay.3The morphological changes of B16cells were observed by Wright-Giemsa staining after treated by PHB (2,4,6μg/ml) for72h.4The effects of PHB (2,4,6μg/ml) on melanin production and tyrosinaseactivity of B16cells were assessed by colorimetry.5The effects of PHB (6μg/ml) on cell growth of B16cells were assessed bygrowth curve.6Clone formation assay was used to assess the effect of PHB (6μg/ml) onthe colony formation ability of B16cells.7The effects of PHB (6μg/ml) on the invasion were assessed by scratchassays.8Cell cycle and apoptosis rate in B16cell were examined by Flow cytometry(FCM) after treated with6μg/ml PHB for defferent time (0h,6h,12h,24h and48h).9The effects of MAPKs associated protein（t-proteins, p-p38, p-JNK andp-ERK1/2） in PHB-induced differentiation of melanoma cells wereinvestigated by Western blot.10The transcriptional levels of differentiation associated gene c-myc, Tyr,Trp1, Trp2in B16cells treated with PHB(6μg/ml) for different time（6h,12h,24h）were detected by Real-time.Results:1The nine compounds p-hydroxylcinnamaldehyde, coniferylaldehyde,p-hydroxylbenzaldehyde,3-o-Methoxyaniline-p-hydroxylbenzaldehyde,ligballinol and so on) extracted from cochinchina momordica seed couldinhibit the proliferation of B16cell to some extent. The inhibory effect of PHBis the most obvious compared with other group (P <0.05).2Compared to the controll group, PHB (2,4,6μg/ml) can significantlyinhibit the growth of B16cells in vitro, the difference was significant (P<0.05).3Giemsa staining showed that cells grow into bigger and fibrous shapearranged in beam in B16cells treated with PHB for72h.with the increasingconcentration, the cells show the dendrite-like construction, which is thetypical symbol of differentiation. While the cells in control group wereobserved appearance irregularity, growth intensively and polygon shapes.4Compared to the controll group, PHB(2,4,6μg/ml) can significantlyelevate the melanin amount and tyrosinase activity of B16cells after treatedwith PHB for48h in vitro, with the concentration increasing, the differencewas significant (P <0.05).5After treated with PHB for48h in vitro, the growth ability of B16cellsdecreased significantly compared to the control group, the difference wassignificant (P <0.05).6After treated with PHB for48h in vitro, the cell clonality of B16cellsreduced significantly, both the size and the number of the clone is smaller compared to the control group, the difference was significant (P <0.05).7After treated with PHB for48h in vitro, the diameters of scratch becamebroader that of the control group (P <0.05).8FCM analyses showed that the apoptosis rate of B16cell changed a littleafter treated with PHB(6μg/ml) for different times(6h,12h,24h,48h), butcompared to the controll group, the proportion of B16cells in the G0/G1phase was elevated while the proportion of B16cells in the S and G2/M phasereduced in a time depended manner, the difference was significant (P <0.05).9Western blot analyses showed PHB can also up-regulate the expression ofp-P38and p-JNK, but show no significant effect on the level of p-ERK,compared to the control group, the difference was significant (P>0.05).10Real time analyses showed that PHB could up-rdgulate the expression ofc-myc, Tyr, Trp1, Trp2, which further indicated that the increased amount ofmelain induced by PHB is via the elevated expression of differentiationassociated gene Tyr, Trp1, Trp2, while the melain quality is one of symbol ofdifferentiation.Conclusion:1p-hydroxylcinnamaldehyde （PHB） is the most effective compoundextracted from cochinchina momordica seed in anti-tumor aspect, which couldsignificantly inhibit B16cell growth.2Not only could PHB induced melanoma cells differentiation in cell shapebut also in function, it can also decrease the proliferation ability, clonality andinvation and induced melanoma cells into normal cells.3PHB could significantly up-regulate the expression of p-P38and P-JNK,but show no effect on the expression of P-ERK, which confirm that themechanism of melanoma cells induced by PHB may be related with theMAPK-associated pathway.