Effect Of Recombinant Adenovirus Mediated NK4on The Biological Behaviors In Human Multiply Myeloma RPMI8226Cells

Hepatocyte growth factor (HGF) is a multifunctional protein. c-Met is thereceptor for HGF, a protein product of a proto-oncogene. It is a transmembranetyrosine kinase with structural and functional features of a growth factor receptor. Onbinding with its ligand, autophosphorylation of the receptor stimulates its intrinsictyrosine kinase activity with resultant changes in cellular motility, growth andinvasion. c-Met overexpression has been found in many human tumors. HGF/c-Methas been proved to be involved in malignancy development and progression. Elevatedlevels of HGF has been found in serum and BM of multiple myeloma (MM) patientscompared with that of healthy controls and higher serum level of HGF at time ofdiagnosis has a strong negative prognostic impact. Previous studies have shown thatc-Met and its ligand HGF are overexpressed in human myeloma cell lines andprimary human multiple myeloma cells. In addition, HGF has been reported topromote MM cell growth and migratrion in vitro.In the light of these results, we hypothesize that interfering with the HGF/c-Metsignaling pathway of MM cells might have therapeutic value for MM. AnHGF-antagonist, NK4, consisting of the N-terminal hairpin domain and four kringledomains, has been identified. NK4binds to c-MET, and competitively antagonizesHGF-induced tyrosine phosphorylation of c-MET, resulting in inhibition ofHGF-mediated cell proliferation, migration and invasion. In addition, NK4also hasinhibitory effects on angiogenesis driven by basic fibroblast growth factor (BFGF)and vascular endothelial growth factor (VEGF). This anti-angiogenic activity of NK4other than its HGF antagonistic activity presumably resides in its four kringle domainswhich are structurally similar to angiostatin. The multifunctional properties of NK4on tumor cells and tumor vasculature suggest its potential therapeutic value in MM. Inthis study, we examined the biological effects of NK4on MM cell line RPMI8226invitro and explore its possible mechanisms with the aim of providing theoretical rationale and experimental evidence for its potential clinical application of NK4inMM treatment.Part1. Constructing a recombinant adenovirus vector pAd-NK4using Gateway technologyAIM: To construct a recombinant adenovirus vector pAd-NK4by Gatewaytechnology. METHODS: The NK4gene was amplified by PCR with plasmidcontaining HGF/NK4as template, and then cloned into adenovirus adshuttle vectorpYr-adshuttle-6to acquire recombinant plasmid pYr-adshuttle-6-NK4. From this, theexpression cassette of NK4-IRES-EGPF was transfered to pAD/BL-DEST to acquirerecombinant adenovirus plasmid pAd-NK4-IRES-EGFP, using LR recombinantreaction. The correct clone was linearized and transfected into293A cells for packingand amplifying adenovirus pAd-NK4was obtained and identified and the titer wasdetermined by TCID50. The transfection efficiency was observed under thefluorescent microscope. RESULTS: The recombinant adenovirus vector containinghuman NK4gene was constructed successfully with the titer6.3×108PFU/ml. Thisvector was able to infect HEK293cells efficiently. CONCLUSION: Therecombinant adenovirus expressing NK4gene and green fluorescence has beensuccessfully constructed using Gateway technology, on which further study could bebased.PART2. Anti-proliferation activities of Ad-NK4on human multiplymyeloma RPMI8226cellsAIM: The aim of this study was to confirm the antiproliferative activity of Ad-NK4on human multiply myeloma RPMI8226cells and to elucidate the mechanism of itsactivity. METHODS:1. Western blot and RT-PCR analysis were used to evaluatethe expression of NK4mRNA and protein in RPMI8226cells transfected withAd-NK4.2. MTT assay was used to estimate the antiproliferative activity ofAd-NK4on RPMI8226cells.3. Flow cytometric analysis of PI-stained cells wasused to analyze the effect of Ad-NK4on the apoptosis of RPMI8226cells.4. DNAagarose gel electrophoresis assay was used to observe the apoptosis-inducing effectsof Ad-NK4on RPMI8226cells.5. Western blot analysis was performed to measure the expression of the downstream effector proteins of Akt/mTOR pathway,the cell cycle regulatory proteins, and apoptosis-associated proteins. RESULTS:1.The results of Western blot and RT-PCR showed the expression of NK4mRNA andprotein was obviously higer in infected Ad-NK4RPMI8226cells compared withcontrol.2. The proliferation capacity of RPMI8226cells transfected with Ad-NK4was inhibited to33.5%±1.21%(p<0.05),52.8%±1.63%(p<0.05) and54.8%±1.77%(p<0.05) at24,48and72h, respectively.3. The result of flow cytometricanalysis of PI-stained cells indicated that the apoptotic rate in RPMI8226cellstransfected with Ad-NK4was21.2%, much higher than that (3.52%) in the control.4. DNA agarose gel electrophoresis showed a characteristic "ladder" ofinternucleosomal DNA fragmentation in RPMI8226cells transfected with Ad-NK4.5. The expression of the cell cycle regulatory proteins CDK4and Cyclin D1weredownregulated,while the protein expression of P27was upregulated. The expressionof anti-apoptosis protein Bcl-2was decreased,while the expression of pro-apoptosisproteins Bax, Cleaved caspase9and Cleaved caspase3were upregulated.Accordingly,the ratio of Bax/Bcl-2was increased. The expression of the downstreameffector proteins of Akt/mTOR pathway including P-AKT, P-mTOR, P-70S6K andP-4BEP-1were all downregulated, while the protein expression of T-AKT was notinfluenced. CONCLUSION: Our data indicated that Ad-NK4inhibited theproliferation of human multiply myeloma RPMI8226cells, which could be attributedin part to its apoptosis-inducing by increasing the ratio of Bax/Bcl-2and caspaseactivation. In addition,Akt/mTOR pathway may be involved in the process ofapoptosis.PART3. Anti-adhesion activities of Ad-NK4on human multiplymyeloma RPMI8226cellsAIM: The aim of this study was to confirm the anti-adhesion activity of Ad-NK4onhuman multiply myeloma RPMI8226cells and to explore possible mechanism.METHODS:1. The adhesion assay of cells to the extracellularmatrix was used toestimate the adhesion activity of Ad-NK4on RPMI8226cells.2. The adhesionassay of RPMI8226cells to ECV304cells was used to estimate the adhesion activityof Ad-NK4on RPMI8226cells.3. Western blot was performed to measure the expression of the adhesion and invasion-associated proteins(MMP2\MMP3\MMP7\VEGF). RESULTS:1. The adhesion assay of RPMI8226cells to the extracellularmatrix (Matrigel) indicated that the inhibitory rates of celladhesion in Ad-NK4group (62.6%), UO126group (47.8%), RAPA group (15.7%)and AG490group (50.5%) were significantly higher than in Ad-GFP group (0.4%)(P<0.05). In addition, the adhesion assay of RPMI8226cells to the extracellularmatrix(FN) indicated that the inhibitory rates of cell adhesion in Ad-NK4group (46.2%),UO126group (59.4%) and AG490group (55.8%) were significantly higher than inAd-GFP group (0). No significant differences could be found in the inhibitory rates ofcell adhesion between RAPA group (7.7%) and Ad-GFP group (0), nor betweenmTOR group and untreated group (0)(P>0.05).2. The adhesion assay ofRPMI8226cells to ECV304cells indicated that the inhibitory rates of cell adhesion inAd-NK4group (62.6%), UO126group (47.8%), RAPA group (15.7%) and AG490group (50.5%) were significantly higher than in Ad-GFP group (0.4%)(P<0.05). Nosignificant differences could be found in the inhibitory rates of cell adhesion betweenAd-GFP group and untreated group (P>0.05).3. The expression level of MMP-2,MMP-3and VEGF were decreased in Ad-NK4group, compared with untreated groupor Ad-GFP group (P<0.05). However, in our present study, no significant differencescould be found in the expression of MMP-7proteins in Ad-NK4group compared withuntreated group or Ad-GFP group (P>0.05). There was no significant difference inthe expression level of the above protein between Ad-GFP group and untreated group(p>0.05). CONCLUSION: Our data indicated that Ad-NK4inhibited the adhesionof human multiply myeloma RPMI8226cells. In addition, Erk pathway andJAK/STAT pathway may be involved in the process.PART4. Effects of Ad-NK4combined with bortezomib onapoptosis of multiple myeloma RPMI8226cellsAIM: This study was purposed to investigate the effect of Ad-NK4andbortezomib(BOR) on multiple myeloma RPMI8226cells and itsmechanisms． METHODS:1. MTT assay was used to estimate the antiproliferativeactivity of Ad-NK4and (or) BOR on RPMI8226cells.2. Flow cytometric analysisof PI-stained cells was used to analyze the effect of Ad-NK4and (or) BOR on the apoptosis of RPMI8226cells.3. Western blot analysis was performed to measurethe expression of Cleaved caspase-3, Bax and Bcl-2. RESULTS:1. RPMI8226cells were treated with Ad-GFP, Ad-NK4, BOR, Ad-GFP+BOR and Ad-NK4+BOR,respectively and cell proliferation was measured at24,48and72hours using MTTassay. The inhibitory rates of the cell proliferation for24h were6.65%,37.4%,27.1%,21.2%and68.5%, respectively. When treated for48h, the inhibitory rates were0,52.6%,29.7%,34.6%and59.7%, respectively. When treated for72h, the inbitoryrate was4.9%,59.7%,50.7%,41.3%and74.1%, respectively. At the differenttime point, the inhibitory rates of the proliferation in cells treated with combination ofAd-NK4and BOR was significantly higher than BOR alone.2. The apoptoic rate incells treated with combination of Ad-NK4and BOR (47.5%) was significantly higherthan Ad-NK4(12.1%) or BOR (14.5%) alone.3. The result of western blotindicated the level of Cleaved caspase-3and Bax in cells treated with combination ofAd-NK4and BOR was significantly higher than bortezomib or NS-398alone.However, Bcl-2protein expression was significantly lower. Meanwhile, the ratio ofBax/Bcl-2was increased. CONCLUSION: It is concluded that Ad-NK4canenhance the apoptosis-inducing effect of BOR on multiple myeloma cell lineRPMI8226,which is associated with higher ratio of Bax/Bcl-2and higher activity ofCaspase-3．...