Effects Of Snake Venom Cystatin Combined With Snake Venom BJ46a On High Metastatic Hetatocarcinomal Cells

Hepatocellular carcinoma(HCC) is one of the most common malignancy worldwide.HCC is associated with a high potential for vascular invasion,metastasis, and recurrence even after surgical resection,leading to poor prognosis.HCC cells excreting proteinases,which breakdown extracellular matrix(ECM),accounts for invasion and metastasis.Recent years,many biotherapy studies for HCC focus on ECM-associated proteinases,especially some active components of snake venom. Previously we cloned the genes of sv-Cystatin and BJ46a and initially testified their individual function of inhibiting invasion and metastasis of B16 melanoma cells. Considering sv-Cystatin and BJ46a targeting to different protein,we assume their synergetic function when simultaneous expression in the same cells.We transducted sv-Cystatin and BJ46a into MHCC97H cells(a high metastasis hepatocellular carcinomal cell) by a two-gene eukaryotic expression vector,investigated their new or synergetic effects on MHCC97H cells in vitro and in vivo and further analyzed the potential molecule mechanism.Finally,we observed the therapeutical effects in vivo using recombinate sv-Cystatin and BJ46a adenovirus.1.Establishment of MHCC97H cells with expression of sv-Cystatin or/and BJ46aRecombinate eukaryotic expression vectors pBudCE4.1/sv-Cystatin, pBudCE4.1/BJ46a and pBudCE4.1/sv-Cystatin+Bj46a were constructed by molecular cloning technique.MHCC97H/sv-Cystatin,MHCC97H/BJ46a and MHCC97H/ sv-Cystatin+Bj46a cells were established and testfied to express or co-express sv-Cystatin and BJ46a by westernblot.These cells could be used for subsequent research.2.Effects of sv-Cystatin or/and BJ46a expression on the growth,invasion and metastasis of MHCC97H cellsThe cell growth assay and tumor weight in nude mouse inoculated subcutaneously with MHCC97H cells showed that expression of sv-Cystatin could suppress the reproductive activity of MHCC97H cells markly,but co-expression of sv-Cystatin and BJ46a could not influence the growth of MHCC97H cells synergistically.MHCC97H cells with expression of sv-Cystatin were found to grow aggregately by tight cell-cell junction and to move slower compared with parent cells. Transwell-matrigel test and pulmonary metastasis nude mouse model inoculated subcutaneously with hepatocarcinomal cells revealed that expression of sv-Cystatin and BJ46a could inhibit invasion and metastasis of MHCC97H cells in vitro and in vivo,respectively and synergistically.3.Molecular mechanism of the effect of sv-Cystatin or/and BJ46a expression on the growth,invasion and metastasis of MHCC97H cellsFlow cytometric analysis,electron microscope and TUNEL test observation demonstrated that induction of apoptosis resulted in growth inhibition of MHCC97H cells with expression of sv-Cystatin.The potential mechanism is that the interaction between sv-Cystatin and Twist,which reduce the expression of Twist and further lead to dismissing suppression of the p53 pathway during cell apoptosis,increase of P53 and its target molecule,Bax trigger the apoptosis cascade reaction in chondrosome path.However,no synergistic effect of apoptosis was found in MHCC97H cells with co-expression of sv-Cystatin and BJ46a due to the role of BJ46a.Measurement of Cathepsin B activity by fluorescence-luminosity,MMPs expression by gelatin zymography,activity of transcription factor Twist by EMSA and expression of E-cadherin and N-cadherin by real-time PCR and westernblot etc certified that:1) Decrease of MMP2 and MMP9 are the direct reason for suppression of invasion and metastasis of MHCC97H cells with expression of BJ46a.2) Simultaneous decrease of CathepsinB,MMP2 and MMP9,and weakness of Twist activity that lead to up-regulation of E-cadherin and down-regulation of N-cadherin contributed to the inhibition of invasion and metastasis of MHCC97H cells with expression of sv-Cystatin.3) The above multiple effects lead to the most severe suppression of invasion and metastasis of MHCC97H cells with co-expression of sv-Cystatin and BJ46a. 4.Therapeutical effects of sv-Cystatin and BJ46a mediated by adenovirus on metastatic hepatic carcinomaRecombinate sv-Cystatin and BJ46a adenovirus were produced respectively using pShuttle-IRES-hrGFP-1 vector.Intratumoral injection of pulmonary metastasis nude mouse inoculated subcutaneously with MHCC97H cells revealed that Ad/sv-Cystatin could reduce the rate of pulmonary metastasis of MHCC97H cells by 30.4%,and Ad/sv-Cystatin combinded with Ad/BJ46a could just decrease the rate by 28%with no synergistic effect.In addition,the growth of MHCC97H cells in vivo decreased to some extent by intratumoral injection of Ad/sv-Cystatin.