| ObjectiveGecko is a traditional Chinese medicine,which has clear curative antineoplastic effect in clinic applications.Treating malignant tumor with differentiation-inducing therapy has become an attractive method because of its low toxicity and prominent effect.Our previous results showed that AG(aqueous extracts of fresh gecko),M-AG(macromolecular fractions separated from fresh gecko by Sephadex G-25 column;Peak 1),LZ(fractions obtained from M-AG through DEAE-cellulose DE-52 ion-exchange column chromatography),SS(fractions obtained through penyl sepharose fast flow)all had strong anti-tumor effect in vivo.And AG could induce the differentiation of Bel-7402 human hepatocellular carcinoma cells.Further on this basis,we indended to investigate the molecular mechanism of the active fractions obtained from fresh gecko which had obvious anti-tumor effect in vivo.In addition,we tried to get the anti-tumor active monomers from SS by combining the methods of morphological changes observation and anti-proliferative activity detection in vitro.Methods1.Morphological analysis:Morphological changes were observed directly or after Gimesa staining using an inverted microscrope.2.In vitro assay for anti-proliferative activity:Anti-proliferative activity was determined by the MTT assay.3.Biochemical indexes analysis:B16 mouse melanoma cells:melanin release was measured by colorimetric method using a 96 well plate.Bel-7402 hepatocellular carcinoma cell:AFP(alpha fetoprotein)was detected by radioimmunoassay.ALB(albumin),ALP(alkaline phosphatase)and y-GT(y-glutamyl transpeptidase)were detected by biochemical methods with commercial kits.4.The expression level of the proteins involved in cell cycle(CDC2),MAPK(mitogen-activated protein kinase)signal pathway(P38,P-P38,ERK1/2,P-ERK1/2,JNK1/2,P-JNK1/2)and Nuclear Factor-KB(NF-kB)which closely related with cellular differentiation were assessed by Western blot.Results1.The molecular mechanism of aqueous extracts of fresh gecko(AG)inducing differentiation of Bel-7402 cellsMorphological analysis indicated that both B16 cells and Bel-7402 cells treated with AG showed differentiation-inducing morphological changes compared with the normal controls.B16 cells treated with AG presented as dispersed grovwth and morphological alteration towards a more elongated dendrite-like shape.As for Bel-7402 cells treated with AG,the number of clones reduced and cells were relatively bigger,elongated,irregular or presented as dispersed growth,and these features were similar with the group of cells induced by ATRA.Data showed that AFP secretion of the cells obviously decreased,ALB secretion obviously increased,and the activity of ALP and y-GT markedly decreased at all the three concentrations of AG,and those effects were all in a dose-dependent manner.Nevertheless,when using the colorimetric method with a 96 well plate for melanin release,the drug itself had a great impact on the absorbance proved by multiply times of tries.AG showed obviously anti-proliferative activity on Bel-7402 cells in vitro dose-dependently.AG could reduced the protein expression of CDC2,but the change was not obvious(p>0.05).The total ERK1/2,P38 and JNK1/2 proteins did not show any significant changes after AG treatment.The expression levels of P-P38 and P-JNK1/2 proteins in Bel-7402 cells were too low to be detected,and there were no difference between groups treated or untreated with AG.However,the activation of ERK1/2 appeared at 5 min or 15 min after being treated with AG.The activation of ERK1/2 was delayed and sustained till 30 min,even the significant expression of P-ERK1/2 in Bel-7402 cells could be detected after treated with AG for 72 h.In addition,U0126 significantly decreased the ability of MEK1/2 to phosphorylate ERK1/2.Therefore,we suggested that AG-induced differentiation depended on the activation of ERK1/2,but not the activation of P38 or JNK1/2.In conclusion,AG showed significantly dose-dependently anti-tumor effect in vitro.Morphological and biochemical indexes suggested that AG had obvious differentiation-inducing effects on Bel-7402 cells,and the mechanism involved the activation of ERK1/2.2.The molecular mechanism of M-AG(macromolecular fractions separated from fresh gecko by Sephadex G-25 column;Peak 1)inducing differentiation of Bel-7402 cellsAccording to the result of AG differentiation-inducing effect,we chose Bel-7402 cells as the cell model.All the mophalogical analysis,biochemical indexes and cytotoxic activity results showed that M-AG had the similar effect on Bel-7402 cells.Therefore,results in vitro indicated that M-AG had differentiation-inducing effect on Bel-7402 cells.Western blot analysis of protein expressions involved in cell cycle and MAPK signal pathway showed that M-AG had no obvious impact on the expression of CDC2,P38,ERK1/2,JNK1/2,P-P38 and P-JNK1/2.But M-AG could significantly active ERK1/2,and the expression the P-ERK1/2 was delayed and sustained.Further study indicated that M-AG had no obvious influence on the expression of NF-kB too.3.The anti-tumor effect of LZ(fractions obtained from M-AG through DEAE-cellulose DE-52 ion-exchange column chromatography)in vitroResults showed that both freeze-dried LZ and non-freeze-dried LZ had dose-dependently anti-proliferative activity on Bel-7402 cells in vitro,but the effect of freeze-dried LZ was better than non-freeze-dried LZ.However,the in vitro results were not satisfactory when compared with the effect of AG or M-AG.On one hand,it needed higher concentration of LZ to have a same inhibitory rate with AG or M-AG.On the other hand,the morphological changes of Bel-7402 cells were not as obvious as cells treated AG or M-AG.Therefore,we didn't explore the biochemical indexes changes and molecular mechanism.4.The molecular mechanism of fractions obtained through penyl sepharose fast flow(SS)inducing differentiation of Bel-7402 cellsAccording to the result of AG differentiation-inducing effect,we chose Bel-7402 cells as the cell model.All the mophalogical analysis,biochemical indexes and cytotoxic activity results showed that SS had the similar effect on Bel-7402 cells.Therefore,results in vitro indicated that SS had differentiation inducing effect on Bel-7402 cells.Western blot analysis of protein expressions involved in MAPK signal pathway showed that SS could significantly active ERK1/2,and the expression the P-ERK1/2 was delayed and sustained.5.Seperation of anti-tumor active monomers from SSWith the methods of morphological changes observation and anti-proliferative activity detection in vitro,we further intended to get the anti-tumor active monomers from SS.The first two experiments showed that fraction ? obtained through HPLC was the most effective fraction,but later repeated experiments didn't have the same results and the reason may be the instability of methods or PEG reducing the activity of fraction?.Furthermore,we enriched the fractions by freeze-drying method and the results indicated that fraction ? was still most effective,but the difference was small.We guess that it was not an ideal way to get the active monomers through HPLC.SS treated with 70 ? or 100? showed similar anti-tumor effect in vitro with SS.Therefore,the anti-tumor active components involved in SS were heat resistant.ConclusionAll AG,M-AG and SS inhibit Bel-7402 cell proliferation in vitro through inducing cell differentiation,and the mechanism involves the sustained activation of ERK1/2.LZ could inhibit the growth of Bel-7402 cells in vitro,and the effect of freeze-dried LZ was better than non-freeze-dried LZ.It was not stable to get anti-tumor active monomers from SS through HPLC. |
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