| Hepatocellular carcinoma(HCC) is the fifth most common malignancyworldwide with a rising incidence, high malignancy, hided onset and poorprognosis. Operative resection and liver transplantation are the only curableway to treat HCC, and suitable for approximate15%patients. Most patientslose the opportunity to surgery due to intrahepatic or extrahepatic metastasis.The effect of surgical treatment is poor due to the high recurrence rate.Invasion and metastasis of hepatoma cells is the significant reason ofmetastasis and recurrence. So invasion and metastasis are the major obstacleto HCC treatment. Drug therapy is the main way of HCC treatment. But theeffect of drugs in use is unsatisfactory.Researchers found inhibition effect of cantharidin and ramification withvarious mechanisms to many malignant tumors. Sodium Cantharidinate andVitamin B6is the active ingredient of cantharidin and often used to treatadvanced malignancy such as live cancer, gastric cancer, lung cancer,pancreatic cancer, breast cancer, nasopharynx cancer and bladder cancer.Cantharidinate and Vitamin B6have a strong effect of anti-tumor and alsoenhance immunity. Researches show Sodium Cantharidinate and Vitamin B6inhibited proliferation of hepatoma cells and induced cell apoptosis. But theeffect of Sodium Cantharidinate and Vitamin B6on invasion of hepatomacells is still unknown.Ras/Raf/MEK/ERK pathway play an important role in HCC metastasis.And HCC metastasis can be suppressed by inhibiting ERK1/2and p-ERK1/2phosphorylation. This research is about to study the effect of SodiumCantharidinate and Vitamin B6on invasion of hepatoma cells, and whether theeffect is related to Ras/Raf/MEK/ERK pathway.Objective: To study the effect of Sodium Cantharidinate and Vitamin B6 on invasion of SMMC-7721cells, and whether the effect be concerned withRas/Raf/MEK/ERK pathway.Method: Cultivate Hepatocellular carcinoma cell line SMMC-7721invitro. Cells were treated with different concentrations of SodiumCantharidinate and Vitamin B6(0.25μmol/L,0.5μmol/L,0.75μmol/L,1.0mol/L,1.5mol/L and2.0mol/L) for24h,48h and72hours. The cellsproliferation was detected by MTT. The effect of invasion of cells treated withSodium Cantharidinate and Vitamin B6(0.5μmol/L,1.0mol/L,1.5mol/L and2.0mol/L) was observed by Transwell invasion assay. Expression of ERK1/2and p-ERK1/2in SMMC-7721cells treated with different concentrations ofSodium Cantharidinate and Vitamin B6(0.5μmol/L,1.0mol/L,1.5mol/L and2.0mol/L) was tested by Western-Blot.Result: MTT assay showed OD values of experimental groups weresignificantly lower than OD values of control group after24h in cells treatedwith Sodium Cantharidinate and Vitamin B6(P＜0.05). The inhibitory rateswere4.93%,5.86%,13.9%,14.38%,20.14%,31.16%as drug concentrationsrising. The differences were statistically significant among experimentalgroups(P＜0.05) except0.25μmol/L with0.5μmol/L and0.75μmol/L with1.0μmol/L. After48h, OD values of experimental groups were significantlylower than OD values of control group(P＜0.05) except0.25μmol/L group.The inhibitory rates were1.58%,14.8%,23.88%,25.93%,35.15%,49.05%.The differences were statistically significant among experimental groups(P＜0.05) except0.75μmol/L with1.0μmol/L group. After72h, OD values ofexperimental groups were significantly lower than OD values of controlgroup(P＜0.05). The inhibitory rates were12.5%,14.58%,14.6%,16.92%,18.42%,27.11%. The differences were statistically significant between2.0μmol/L group and other experimental groups(P＜0.05), and no differencesamong other experimental groups.The SMMC-7721cell number passed through matrigel which treatedwith Sodium Cantharidinate and Vitamin B6in0.5μmol/L,1.0μmol/L,1.5μmol/L,2.0μmol/L for24h were significantly lower than control group(P ＜0.05). The inhibitory of invasion were21.73%,31.14%,34.61%,46.44%asdrug concentrations rising. The difference were statistically significantbetween0.5μmol/L and2.0μmol/L group(P＜0.05). After48h, the cell numberin experimental groups were significantly lower than control group(P＜0.05).The inhibitory of invasion were26.68%,36.19%,42.24%,53.13%. Thedifference were statistically significant between0.5μmol/L and2.0μmol/Lgroup(P＜0.05). After72h, the cell number in1.5μmol/L and2.0μmol/Lgroups were significantly lower than control group(P＜0.05), but nodifference between1.5μmol/L and2.0μmol/L. The inhibitory of invasion were14.56%,18.34%,31.48%,40.01%.The OD value ratios(targets/β-actin) of ERK1/2in SMMC-7721cellstreated with Sodium Cantharidinate and Vitamin B6for24h and48h showedno difference between experimental groups and control groups. But the ODvalue ratios of ERK1/2in experimental group were significantly lower thancontrol group after24h(P＜0.05). The OD value ratio of p-ERK1/2were0.925±0.038,0.941±0.02,0.7±0.017,0.565±0.031in experimental groups asdrug concentrations rising and1.185±0.146in control group. The differenceswere statistically significant among experimental groups(P＜0.05) except0.5μmol/L with1.0μmol/L. After48h, the OD value ratios in experimentalgroups were significantly lower than control group(P＜0.05). The OD valueratios of p-ERK1/2were1.558±0.155,1.555±0.094,1.397±0.014,0.796±0.019in experimental groups and2.109±0.1in control group. Thedifference were statistically significant between2.0μmol/L and otherexperimental groups(P＜0.05).Conclusion: Sodium Cantharidinate and Vitamin B6can inhibit theproliferation of SMMC-7721cells with a dose-dependent manner.Sodium Cantharidinate and Vitamin B6can inhibit the invasion ofSMMC-7721cells with a dose-dependent manner. And the effect was peakedin2μmol/L by48h.The expression of p-ERK1/2was strongly suppressed by SodiumCantharidinate and Vitamin B6with a dose-dependent manner. But Sodium Cantharidinate and Vitamin B6had no effect on ERK1/2expression. Theresult declared Sodium Cantharidinate and Vitamin B6can inhibit theproliferation and invasion of SMMC-7721cells by suppress thephosphorylation of ERK1/2protein. |
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