The Effect Of Norcantharidin On Migration And Invasion Of Hepatoma SMMC-7721Cell

Hepatocellular carcinoma(HCC) is a very common cancer, and also has avery high degree of malignancy.It has become the third leading cause of cancerdeath in the world, second only to lung cancer and stomach cancer, which is agreat threat to human health. The main factors affecting the prognosis of livercancer is recurrence and metastasis.Although the surgical resection is still thefirst choice of treatment for liver cancer, most patients have clinical ormicroscopic metastasis when diagnosed. so that the prognosis of HCC remainspoor. High metastasis recurrence of liver cancer is a major obstacle toimproving the survival rate of patients. Local tumor invasion and metastasis isthe leading cause of death in patients with liver cancer.So the key treatment ofliver cancer is to stop its invasion and metastasis.The invasion of hepatoma cells to basement membrane(BM) is animportant factor leading to tumor growth, invasion and metastasis. The maincomponent of BM is the extracellular matrix(ECM). Therefore, whether thehepatoma cell can break through ECM is the key factor of local invasion anddistant metastasis. ECM degradation is an extremely complex process, whichhas a number of factors participated in. The matrix metalloproteinases (MMPs)and their inhibitors, tissue inhibitor of metalloproteinase(TIMPs), play a keyrole in this process.MMPs can degrade ECM,and TIMPs can inhibit thedegradation of ECM by inhibiting the activity of MMPs.Cantharidin is the active ingredient of the blister beetle, and is an activeingredient of the traditional Chinease herbal medicine Mylabris. Norcantha-ridin(NCTD) is a derivative of cantharidin, and synthesized from maleic acidand amine furan Diels-Alder. It is a new anticancer drug,which has stronganti-tumor activity and the unique role to elevate leukocyte.This drug ismainly used for the treatment of liver cancer, esophageal cancer, stomach cancer and so on. A number of studies show that NCTD antitumor mechanismsinclude induction of apoptosis, inhibition of cell proliferation, invasion andmetastasis prevented, antiangiogenic and/or anti-angiogenic mimicry.But thevariety biological behaviors of NCTD to tumor cells and its mechanism needfurther study.We study the effect of NCTD on migration and invasion ofhepatoma cell and its mechanism.Objective: In this experiment,we use different concentrations of NCTDto intervent hepatoma cell SMMC-7721to study the effect on invasion andmetastasis.To investigate the role of MMPs and TIMPs and the mechanism inthis process by the expressions of MMP-2and TIMP-2protein and mRNA.Methods: Human hepatoma cells SMMC-7721were cultured and bedivided into a control group and different concentrations(1.0μg/ml、2.0μg/ml、4.0μg/ml) of NCTD intervented group. Cell migration and invasion ofSMMC-7721effected by NCTD was respectively detected by the woundclosure assay and Transwell chamber assay. The MMP-2and TIPM-2proteinexpressions were investigated by immunocytochemistry.The mRNA ofMMP-2and TIMP-2were examined through real-time fluorescencequantitative polymerase chain reaction (RTFQ-PCR).Results:1The result of the scratch assay showed that After24hours the controlgroup cells had already grown to91.52%. In contrast,cells of1.0μg/ml groupgrew to77.63%,and2.0μg/ml group grew to58.90%,4.0μg/ml group only to32.24%.The migration of drug intervened groups was statistically declinedcompared to the control group,P<0.05. Moreover, with the increase of drugconcentration, the inhibition of cells migration was more obvious.Theinhibitory effect of NCTD on SMMC-7721cells was significantly indoes-dependent manner.2The cell invasion assay revealed that compared with control group theinvasive ability significantly reduced in1.0μg/ml drug intervened group(91.00±12.87vs129.23±17.58)，P<0.05.We also observed that the invasivecells of the2.0μg/ml drug intervened was decreased compared with the 1.0μg/ml group(57.35±15.94vs91.00±12.87)，P<0.05.The invasive cells of2.0μg/ml drug intervened group was the least,and the invasive ability furtherdeclined compared with2.0μg/ml group(32.00±8.02vs57.35±15.94)，P<0.05.3The expression of MMP-2and TIMP-2protein in SMMC-7721cellsintervened with NCTD was detected by immunocytochemistry.The resultrevealed that the expression of MMP-2protein was gradually declined in1.0μg/ml、2.0μg/ml、4.0μg/ml NCTD group compared with that of the controlgroup（0.0899±0.0073,0.0686±0.0034,0.0423±0.0036vs0.1331±0.0060），P<0.05; Inversely, the expression of TIMP-2protein was significantlyincreased with the increase of NCTD concentration(0.0431±0.0031,0.0709±0.0042,0.1021±0.0063vs0.0162±0.0040）, P<0.05. The difference wasstatistically significant, and also has a dose-dependent manner.(4) NCTDimpact the expression of MMP-2and TIMP-2mRNA in SMMC-7721cells.The result of RTFQ-PCR revealed that with the increase of NCTDconcentration (1.0μg/ml、2.0μg/ml、4.0μg/ml), the expression of MMP-2mRNA gradually reduced compared with that of control group（0.89±0.15，0.71±0.10，0.52±0.14vs1.00±0.14，P<0.05）.On the contrary, the expressionof TIMP-2mRNA was significantly increased compared with that of controlgroup（1.31±0.17，1.60±0.17，1.83±0.14vs1.00±0.10，P<0.05）. The differencewas statistically significant, and also has a dose-dependent manner.Conclusion: NCTD can inhibit the migration and invasion of humanhepatoma cells SMMC-7721, and this phenomenon positively correlated withthe drug concentrations. The mechanism may be related to the expression ofMMP-2and TIMP-2.