| Cancer, known medically as malignant neoplasm, is a disease caused by the malfunction of cell growth and proliferation control. The incidence of hepatocellular carcinoma(HCC) in China accounts for 55% of patients with liver cancer around the world. It has become a major killer in our nation, which is one of the serious threatens to the health and lives. Metastasis of HCCis the main reason leading to the death of patients with HCC. The abnormal apoptosis pathway in HCC cells to evade apoptosis further promotes HCC metastasis and recurrence. It is important and necessary to explore a new method for targeted therapy of HCC.HAb18G/CD147 is a novel hepatoma-associated antigen, cloned by our laboratory previously. As one of members of CD147 family, HAb18G/CD147 is highly expressed in tumor tissues and on the membrane of tumor cells. Previous studies have proved that the interaction of HAb18G/CD147 with integrin can activate the downstream signaling pathway of PI3K-Akt, which increases matrix metalloproteinase(MMPs) secretion of tumor cells, thereby promoting tumor cell invasion and metastasis. HAb18 antibody could specifically block the HAb18G/CD147, then inhibited tumor cell invasion and migration. Humanized antibody h HAb18 derived from murine antibody HAb18 had been constructed in our laboratory previously. Our study aimed to construct recombinant Newcastle disease virus(NDV) which expressed hHAb18 antibody to explore the combined effects of antibody targeted therapy and oncolytic viral therapy in HCC. Furthermore, we compared the antibody affinity to HAb18G/CD147 among the humanized antibody hHAb18, the murine antibody HAb18 and antibody CHO-h HAb18, and determine the specificity binding of hHAb18 antibody with HAb18G/CD147 molecule. We illustrated the molecular mechanism of hHAb18 antibody inhibiting tumor cell invasion and metastasis, and its anti-tumor effect withan orthotopic xenograft HCC tumor model.The study composed of four parts: PartⅠConstruction and rescue of the recombinant Newcastle disease virus carrying humanized antibody hHAb18To construct the recombinant NDV carring hHAb18 antibody, we cloned the gene of hHAb18 mAb from engineered cell line CHO-TS28 and followed by inserting into the region between HN and F genes in pBR-rNDV which containing the full-length genome of NDV. The recombinant plasmid was named p BR-rNDV-18 HL. By co-transfection of helper plasmids pcDNA-NP, pcDNA-P, and pcDNA-L with pBR-rNDV-18 HL, we obtained the recombinant virus and named rNDV-18 HL. Expression of hHAb18 antibody was detected by western blot in the BSR-T7/5 cells which infected rNDV-18 HL. The release of h HAb18 antibody in the supernantant of BSR-T7/5 cells was detected by ELISA after rNDV-18 HL infection. The highest concentration of hHAb18 antibody reached 7 μg/ml on the third day of post-infection.Our results demonstrated that: 1) Reverse genetics technology was used to construct and rescue recombinant Newcastle disease virus named rNDV-18 HL which expressed hHAb18; 2) The recombinant virus rNDV-18 HL could successfully expressed the exogenous gene hHAb18. PartⅡ Comparison of antibody affinity to HAb18G/CD147 between humanized antibody hHAb18 and murine antibody HAb18We used surface plasmon resonance(SPR) technique to compare antibody affinity to HAb18G/CD147 among humanized antibody hHAb18, murine antibody HAb18 and CHO-hHAb18 antibody. SPR analysis demonstrated that the hHAb18 affinity was similar with HAb18 affinity and CHO-hHAb18, and the KD values were 4.15 × 10-10 mol/L, 2.73 × 10-10 mol/L and 2.66 × 10-10 mol/L, respectively. Western blot and immunofluorescence showed that both hHAb18 and HAb18 could bind specifically to the membrane molecule of HAb18G/CD147 in three human HCC cell lines, SMMC-7721, HepG2, and Huh-7. By trypan blue exclusion, MTT and BrdU assay,hHAb18 antibody at concentrations of 0.01, 0.05 and 0.1 mg/ml exerted no effects on the proliferation of HCC cells. Moreover, AnnexinV-FITC/PI and TUNEL assay showed that hHAb18 antibody could not cause apoptosis at the highest concentration, observed with the laser scanning confocal microscope and inverted microscope.Our results demonstrated that: 1) hHAb18, HAb18 and CHO-hHAb18 antibody showed equal affinity with HAb18G/CD147; 2) A specific binding of hHAb18 to membrane HAb18G/CD147 was observed in HCC cells; 3) hHAb18 exerted no effects on cellproliferationandapoptosis at doses of 0.01, 0.05 and 0.1 mg/ml. Part Ⅲ Inhibition of humanized antibody hHAb18 on HCC cell invasion and involving molecular mechanismHAb18G/CD147 has been confirmed to be closely associated with tumor invasion and metastasis. In order to prove that the humanized antibody hHAb18 inhibited the invasion and metastasis of tumor cells by specifically blocking HAb18G/CD147 in tumor cells, and also clarify the molecular mechanism, firstly we used transwell chamber assay to measure the effect of hHAb18 antibody on tumor cells invasion. Results showed that hHAb18 antibody significantly inhibited the invasive ability of SMMC-7721 cells. Zymography and real-time PCR showed that secretion of MMPs levels in human acute monocytic leukemia cells, THP-1 was significantly downregulated by hHAb18 antibody, and the relative mRNA levels of MMPs in SMMC-7721 cells were also significantly decreased(compared with control group, P < 0.01). Wound healing assay proved that hHAb18 antibody inhibited the migration of SMMC-7721 cells and SMMC-7721-CD147/GFP cells at 24 h and 48 h, respectively, and this inhibition did not work after addition of MMP inhibitor, Marimatat. Meanwhile, hHAb18 antibody had no inhibitory effects on the invasion and metastasis of CD147 gene knockout cell line, SMMC-K7721, as well as the relative mRNA levels of MMPs.The formation of F-actin stress fiber and lamellipodia in SMMC-7721 cells was significantly weakened at 24 h and 48 h after the treatment of hHAb18 antibody. This phenomenon was similar with the distribution of F-actin in SMMC-K7721 cells. Then western blot was used to detect protein levels and their corresponding phosphorylation levels of FAK-PI3K-Akt-Girdin signaling pathways in the tumor cells. Results showed that hHAb18 antibody inhibited the phosphorylation level of FAK, PI3 K, Akt, and Girdin within 2-5 hours. Orthotopic transplantation model with SMMC-7721 cells in nude mice was established. On the fifth day after surgery, hHAb18 antibody was intraperitoneally injected into nude mice twice a week for three consecutive weeks. Until the mice died of tumor burden, the number of intrahepatic metastases was calculated and the survival time of mouse in each group was analyzed. Our study discovered that hHAb18 antibody significantly reduced the number of intrahepatic metastases in mice with dose-dependent manner(P = 0.004). The survival time of 10 mg/kg antibody-treated mice was significantly longer than the control group(P < 0.001).Our results demonstrated that: 1) hHAb18 antibody inhibited the invasion and metastasis of HCC cells both in vitro and in vivo; 2) hHAb18 antibody specifically binded with membrane molecule HAb18G/CD14 in tumor cells, and inhibited tumor cell invasion by downregulating the secretion of MMPs; 3) hHAb18 antibody modified cytoskeletal rearrangements and reduced tumor cells migration via inhibiting the phosphorylation level of FAK, PI3 K, Akt, and Girdin. Part Ⅳ Characteristicsof rNDV-18 HL on propagation and anti-tumor activities in vitroTo study the characteristics of rNDV-18 HL on virus propagation and anti-tumor activities in vitro, four cell lines including BSR-T7/5, SMMC-7721, HepG2, and Huh-7 were infected with rNDV-18 HL and wild-type NDV Italien, respectively. The viral proliferation rate and cell viability were measured by TCID50 and MTT, respectively. Comparing with NDV Italien, the insertion and expression of exogenous antibody gene did not alter the characteristics of virus propagation and virus cytopathic ability(P > 0.05). The recombinant rNDV-18 HL induced cell-cell fusion widely and rapidly, and the syncytia were detached subsequently. To select the sensitive cell line of amplification of rNDV-18 HL for the preclinical application, four engineered cell lines, including 293-T, PLAT-E, X, and Y were infected with rNDV-18 HL. The proliferation rate was measured by TCID50 and cell viability was measured by MTT. The propagation of rNDV-18 HL in X cells showed significant higher than the other three cell lines(P < 0.01). The results indicate that X was a host cell line for amplification in large-scale of rNDV-18 HL.Our results demonstrated that: 1) rNDV-18 HLexpressed hHAb18 and shared no difference with its parental virus strain NDV Italien in proliferation and virulence; 2) X could be the host cell line for rNDV-18 HL productionin large-scale.All the above results demonstrated that reverse genetics technology and rescue system were used to construct Newcastle disease virus carrying hHAb18 antibody, and the recombinant virus rNDV-18 HL successfully expressed the exogenous gene hHAb18. The humanized antibody h HAb18 could specifically block the HAb18G/CD147, which expressed on the membrane of tumor cells, and then downregulated the secretion of MMPs, thereby inhibited tumor cell invasion. The tumor cell migration was associated with HAb18G/CD147 which regulating the phosphorylation level of each molecule in FAK-PI3K-Akt-Girdin signaling pathway. The blocking effect of hHAb18 antibody on its target molecule significantly inhibited the formation of cytoskeleton F-actin stress fiber and lamellipodia, which decreased tumor cell migration. Insertion of exogenous antibody gene exerted no significant effects on the propagation and cytopathic ability of the virus. The recombinant virus rNDV-18 HL successfully expressed the exogenous gene hHAb18, and showed the inhibition of HCC cell proliferation in vitro. This study has paved a new way to treatment of tumor with the humanized antibody combined with the oncolytic virotherapy. |
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