| Objective: To explore the potential anti-cancer mechanism of magnesium cantharidate, which is the inhibitor of protein phosphatase 2A(PP2A), the effect of magnesium cantharidate on the mitogen-activated protein kinase(MAPK) signaling pathway and cell cycle-related proteins was studied. In addition, the okadaic acid, which is also the inhibitor of PP2 A, was studied as a parallel control.Methods: 1. The sulforhodamine B(SRB) staining was performed to detect the inhibiting proliferation effect of magnesium cantharidate with different concentrations on hepatocellular carcinoma cells SMMC-7721.2. SRB staining was used to detect the promoting proliferation effect of OA at different low concentrations on hepatocellular carcinoma cells SMMC-7721 in vitro. Also, the immunofluorescence staining was used to detect the effect of OA at different low concentrations on the expression of SMMC-7721 proliferating cell nuclear antigen(PCNA).3. The activity assay kit for PP2 A was used to detect the effects on PP2 A activity by treatment with magnesium cantharidate and OA.4. The effects of magnesium cantharidate and OA on the expression of ERK1, ERK2, p38 MAPK, JNK1 and JNK2 gene in SMMC-7721 cells were detected by RT-q PCR.5. The expression levels of protein and protein phosphorylation of ERK1/2, p38 MAPK, and JNK were detected by Western blot in the magnesium cantharidate and OA treated hepatocellular carcinoma cells SMMC-7721.6. The expression of cyclin Cdc25 C, Cdc2 and the protein phosphorylation expression levels of Cdc2 in hepatocellular carcinoma cells SMMC-7721 were detected by Western blot.Results: 1. Magnesium cantharidate has a significant inhibitory effect on hepatocellular carcinoma cells SMMC-7721. The inhibition rate increased with the increasing concentration, which showed a dose-dependent fashion, and its IC50 is 2.266 mmol/L.2. When the OA concentration was 5.9 nmol/L, it still had an inhibiting effect on the proliferation of hepatocellular carcinoma cells SMMC-7721. However, as the gradual decline of OA concentration, it began to have a promotion effect on the proliferation of hepatocellular carcinoma cells, but at even lower concentrations, its proliferation promotion effect was gradually decreased, especially when the concentration reached 0.01475 nmol/L, there was no promotion effect on proliferation at all. The expression change tendency of PCNA was consistent with that of SRB.3. When the concentration of magnesium cantharidate is 0.283 mmol/L, there is no obvious inhibition(P>0.05) effect, but when the concentration is 0.567mmol/L, magnesium cantharidate can significantly inhibit PP2A(P<0.01). With the increasing concentration, the inhibitory effect becomes more obvious. In addition, 0.059 nmol/L OA can also significantly inhibit PP2A(P<0.01).4. Compared to the control group, at the concentration 0.283mmol/L of magnesium cantharidate, the expression of ERK1, ERK2 had no significant change(P>0.05). However, when the concentration of magnesium cantharidate is 0.567mmol/L, expression of ERK1, ERK2 decreased significantly, with the increasing concentration, it decreased more obviously. On the contrary, when OA is 0.059 nmol/L, the expression of ERK1, ERK2 become significantly higher(P<0.01). Under different concentrations of magnesium cantharidate and OA, the expression of p38 MAPK, JNK1, JNK2 all become significantly higher(P<0.01).5. The expression pattern of total protein of ERK1/2, p38 MAPK, JNK were consistent with that of the gene expression. Compared to the control group, when the concentration of magnesium cantharidate is 0.283mmol/L, the ERK1/2 phosphorylation level had no significant change(P>0.05), when the concentration of magnesium cantharidate is 0.567mmol/L, phosphorylation level of ERK1/2 decreased significantly, with the increasing concentration, it decreased more obvious. However, when OA is 0.059 nmol/L, the phosphorylation level of ERK1/2 become significantly higher(P<0.01). Under differentconcentrations of magnesium cantharidate and OA, the phosphorylation level of p38 MAPK, JNK all become significantly higher(P<0.01).6. Compared to the control group, the expression of cyclin Cdc25 C, Cdc2 and the protein phosphorylation expression levels of Cdc2 has no obvious change at the concentration of 0.283?mol/L magnesium cantharidate(P>0.05), when the concentration increased 0.567?mol/L, the expression of Cdc25 C, Cdc2 decreased significantly(P<0.01). With the increasing concentration, it decreased more obviously. However, the phosphorylation level of Cdc2 is significantly increased(P<0.01), it gradually increased along with the increasing concentration. OA has an opposite effect on magnesium cantharidate on the expression of Cdc25 C, Cdc2 and phosphorylation level of Cdc2.Conclusion: 1.Magnesium cantharidate significantly inhibit the proliferation of hepatocellular carcinoma cells SMMC-7721, while low concentration OA can promote hepatocellular carcinoma cells SMMC-7721 proliferation; 2.Contrary to OA, we speculated magnesium cantharidate inhibit the ERK1/2 signalling pathway by inhibit the activity of the PP2 A firstly, then down-regulate Cdc25 C, which resulted the activity of Cdc2 phosphorylation sites decreased, and the cells arrested at G2/M phase. All of above changes ultimately induced apoptosis of hepatocellular carcinoma cells SMMC-7721. |
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