Recommbiant MeaslesVirus Hu 191 Alone And Combined With Cisplatin Induced Oncolysis Of Gastric Cancer And Its Underlying Mechanisms

Objective:Gastric cancer(GC)is one of the most common cause of cancer death worldwide.Despite a decreasing cancer mortality,the 5-year overall survival is still low.Therefore,it is urgent to identify effective therapeutics for enhancing the poor survival of GC patients.Many different classes of oncolytic viruses have been explored as novel cancer therapeutics previously.The Chinese measles vaccines are widely used in china and have an excellent safety record for more than 50 years.Previous studies have shown that therapeutic effects of mv-hu191 for the treatment of lung carcinoma.However,whether or not a live-attenuated measles virus especially Chinese Hu-191 strain can be an attractive candidate for the treatment of GC remains unknown.And the underlying mechanisms of the oncolytic effect need to be further clarified.In this study,the antitumor effect of r-MV-Hul91,a recombinant Chinese Hu191 MV generated previously in our laboratory by efficient reverse genetics system,was evaluated in gastric cancer(GC).We analyzed its anti-tumor apoptosis mechanism,revealed a connection between oncolytic virus-induced apoptosis and specialized functional biomembrane domains lipid rafts,and explored new approaches to cancer treatment.Methods:1?r-MV-Hul91 was amplified on Vero cells.All cells and culture media were collected when more than 90%of Vero cells had cytopathic effects.r-MV-Hul91 was collected through repeated freeze-thaw and low temperature centrifugation Virus titers of r-MV-Hul91 were detected by plaque forming unit(PFU)assay on Vero cells.Immunofluorescence of MV-P protein was performed to detect presence of virus in both BGC-823 and SGC-7901 cells after infection with r-MV-Hu191 for 24-48 hours.To determine the proliferation kinetics of r-MV-Hu191 on GC cells,cells and supernatant were collected after infection with r-MV-Hu191 for 24-96 hours and virus titers were detected by tissue culture infective dose(TCID 50)assay on Vero cells.2?BGC-823 and SGC-7901 cells were incubated with r-MV-Hu191 at a certain multiplicity of infection(MOI)for 24h-96h.Cytopathic effects and syncytium formation of GC cells were observed using phase contrast microscope.Cell viabilities of GC cells were determined by CCK-8 assay.3?BGC-823 and SGC-7901 cells were incubated with r-MV-Hu191 for 24h-96h.Cells were collected,incubated with Annexin V-fluorescein isothiocyanate(FITC)and propidium iodide(PI)reagents and analyzed on a flow cytometer.BGC-823 and SGC-7901 cells were incubated with r-MV-Hu191 at a certain multiplicity of infection(MOI)for 24h-96h.All the proteins well collected,and the expression of the activation of caspase 3 and PARP was examined by Western blotting assay.After co-treatment with Z-VAD(a pan-caspase inhibitor)and r-MV-Hu191 for 72 hours,the activation of caspase 3 and PARP was examined by Western blotting assay and the apoptosis rate was measured by flow cytometry analysis.4?BGC-823 and SGC-7901 cells were treated with 5 mM M?CD for 2 hours at 37?in fresh maintenance media,then all cells were collected to examine the expression of flotillin 1 by Western blotting assay.The titers of total measles virus in both BGC-823 and SGC-7901 cells and the expression of MV-P were determined after 24h infected with r-MV-Hu191.5?To disrupt lipid rafts prior to virus infection,cells were treated with 5 mM M?CD for 2 hours at 37? in fresh maintenance media,then cells were incubation with virus-containing culture medium.To disrupt lipid rafts after virus infection,cells were incubated with virus-containing culture medium for 2 hours,followed by treatment with M?CD(5mM)for another 2 hours before maintenance in fresh medium.After treatments described as above,cells were collected and subjected to Western blotting assay or flow cytometry analysis.6?GC tumor models were established in mice by subcutaneous injection of SGC-7901 cell in the right flank of nude mice.On post-implantation day 7,mice were randomly divided into two groups,10 in each group.From this day,mice from the r-MV-Hu191 treatment group received intratumoral injections 6 times,while the mice from the mock treated group were injected with equal volume of Opti-MEM.On post-implantation day 15,3 mice from each group were sacrificed to collect tumor tissues for detection of in situ apoptosis and virus replication.Proteins were also extracted from fresh tissues and subjected to Western blotting.The other 7 mice from each group were housed till day of death or euthanasia.Results:1?BGC-823 and SGC-7901 cells could be successfully infected by r-MV-Hu191 with MOI of 0.1,as proved by visualization of virus particles by MV-P protein expression.Furthermore,r-MV-Hul91 replicated in both cell lines,reaching a peak titer at 48 hours after infection in BGC-823 cells,and 72 hours in SGC-7901 cells.2?In both cell lines,reduction of viability was first observed within 48 hours after infection,and the antitumor potency of r-MV-Hu191 demonstrated a dose and time dependent manner.Infection of r-MV-Hu191 at MOIs of 0.1 and 1 elicited dramatic cytopathic effects(CPEs)in a dose and time dependent manner in both BGC-823 and SGC-7901 cells.3?After BGC-823 and SGC-7901 cells were infected with r-MV-Hu191 at a MOI of 0.5,the proportion of apoptotic cells was significantly increased in a time-dependent manner.In BGC-823 cells,the apoptotic rate after r-MV-Hu191 infection was 3%,23%,28%and 43%at 24,48,72 and 96 hours respectively;however,the apoptosis rate at the mock treated group was only 2%.In SGC-7901 cells,the apoptotic rate after r-MV-Hu191 infection was 1%,6%,19%and 26%at 24,48,72 and 96 hours respectively;however,the rate was only 1%in mock treated group.To elucidate apoptosis induced by r-MV-Hu191 was a caspase cascade,the expression of the activation of caspase 3 and PARP was examined.Upon infection with r-MV-Hu191,the activation of caspase 3 and PARP,indicated by the expression of the cleaved forms,was significant increased in a time and dose-dependent manner.Moreover,co-treatment with Z-VAD for 72 hours suppressed activation of caspase 3 and PARP in both cell lines.Densitometry analysis confirmed highly significant decrease of caspase 3 activation(P<0.01),and significant decrease of PARP activation(P<0.05)in both cell lines.Flow cytometry revealed that the increase in apoptosis was partially reversed in the presence of Z-VAD.Taken together,these data suggest that r-MV-Hu191 induced the caspase-dependent apoptosis in human GC cells.4?After lipid rafts were disrupted by dissolving cholesterol from the plasma membranes using MPCD before infection,the titers of total r-MV-Hu191 virus particles were significantly reduced in both BGC-823 and SGC-7901 cells.Decrease in MV-P protein expression further confirmed decrease in the amount of virus following lipid raft disruption.Densitometry analysis confirmed highly significant decrease of MV-P expression in the infected cells(P<0.01).Although lipid raft disruption hampered virus replication,it did not affect the initial virus binding.From titration of the virus-containing culture medium,the amount of unbound virus remained the same in cells either with or without M?CD treatment.Consistently,virus titration of lysates from infected cells showed that the amount of bound virus did not vary with or without pre-treatment of M?CD.In addition,validity of use of M?CD to impair lipid raft integrity was verified by lack of flotillin 1 in the buoyant density fractions after treatment.5?When lipid raft integrity was impaired by M?CD prior to viral infections,the expression of protein markers for activation of the cellular apoptosis pathway was decreased.Consistently,the ratio of apoptotic cells decreased from 35%to 7%in BGC-823 cells,and from 22%to 3%in SGC-7901 cells when pre-treated of M?CD.On the other hand,if M?CD was administered after viral infection for 2 hours,allowing sufficient time for viral attachment to the plasma membrane,the ratio of apoptotic cells were not decreased by lipid rafts disruption in both GC cell lines.In fact,the ratio of apoptotic cells was even increased when lipid raft integrity was impaired following viral binding.6?The in vivo tumor-suppressive effect of r-MV-Hu191 was first detected on days 10 after tumor implantation,which was 3 days after treatment.The therapeutic efficacy increased over time,resulting in effective suppression of tumor growth from post-implantation day 10 to day 16,and most significantly on day 16 after 6 injections.r-MV-Hu191 treatment resulted in a significant increase in the survival rate.The median survival of r-MV-Hu191 treated group was 30 days,as compared with 17 days in the mock treated group.The median survival of r-MV-Hu191-treated animals,with a 1.76-fold increase,was significantly longer than that of the mock treated group(P<0.01).Moreover,no significant difference in body weight between the two groups was observed.MV-P protein was detected in the tumor sections treated with r-MV-Hu191,but not in the mock treated group.The CPE manifested by formation of syncytia was detected only in r-MV-Hul 91-treated tumors.Furthermore,induction of apoptosis in tumor tissues was confirmed by analysis of both cleaved caspase 3 expression and in situ apoptosis.Increase of cleaved caspase 3 expression was also confirmed in protein extracts from tumor tissues and quantitative analysis(P<0.01).Conclusion:1?r-MV-Hu191 induced cytopathic effects and inhibited tumor proliferation in vitro.2?r-MV-Hu191 supressed tumors growth and prolonged survial in vivo.3?Replication rather than binding of r-MV-Hu191 required lipid rafts.4?r-MV-Hul91-induced apoptosis was dependent on lipid raft integrity prior to viral binding.5?r-MV-Hu191 induced caspase-dependent apoptosis both in vitro and in vivo.Recommbiant MeaslesVirus hu 191 Combined with Cisplatin Treated Gastric Cancer and Its Underlying MechanismsObjective:Combining therapeutic modalities has an ability to produce additive or synergistic effect exceeding either approach alone.In this study,we analyzed the anti-tumor effect of r-MV-Hu191 a recombinant Chinese Hul91 measles virus(MV)with cisplatin(DDP)in human GC both in vitro and in vivo with aim to find a safe and effective adjuvant therapy against GC to achieve better patient tolerability and to delay drug resistance based on the previous part of the study.Methods:1?r-MV-Hu191 was amplified on Vero cells.All cells and culture media were collected when more than 90%of Vero cells had cytopathic effects.r-MV-Hu191 was collected through repeated freeze-thaw and low temperature centrifugation.Virus titers of r-MV-Hu191 were detected by plaque forming unit(PFU)assay on Vero cells.2?r-MV-Hu191 and DDP were co-treated on BGC-823 and SGC-7901 cells for 72h.Cell viabilities of GC cells were determined by CCK-8 assay.Data from individual dose response experiments was used to calculate the ZIP scores.BGC-823 and SGC-7901 cells were treated with r-MV-Hu191 and DDP at the optimum doses for combination therapy as determined above for 24-72h.Cell viabilities of GC cells were determined by CCK-8 assay.3?For virus replication assays,r-MV-Hu191 and DDP were co-treated on BGC-823 and SGC-7901 cells for 24-72h.DDP was added at indicated dose post 24h infection.After different time intervals,cells and supernatant were collected,and subjected to titrate for MV by TCID50 assay on Vero cells.BGC-823 and SGC-7901 cells were co-treated with r-MV-Hu191 and DDP for 0-24h,viral P protein expression was examined by immunofluorescence assay.4?r-MV-Hu191?DDP and Z-VAD were co-treated on BGC-823 and SGC-7901 cells for 96h.Cells were collected,incubated with Annexin V-fluorescein isothiocyanate(FITC)and propidium iodide(PI)reagents and analyzed on a flow cytometer.All the proteins well collected,and the expression of the activation of caspase 3 and PARP was examined by Western blotting assay.5?GC tumor models were established in nude mice by subcutaneous injection of BGC-823 cells in the right flank.Mice were randomly divided into four groups,10 in each group.Mice that received r-MV-Hu191 treatment were injected intratumorally with virus suspension 6 times.Mice that received DDP were intraperitoneally injected DDP 3 times.In animals,control groups that were not scheduled to receive r-MV-Hul91 and/or DDP,sham injections of PBS or Opti-MEM medium were administered.On post-implantation day 13,3 mice from each group were sacrificed,tumor tissues were collected for analysis of in situ apoptosis and angular vein blood were collected for analysis of liver and kidney functions.Proteins were also extracted from fresh tissues and subjected to Western blotting.The other 7 mice from each group were housed till day of death or euthanasia.Results:1?Compared to use of r-MV-Hu191 or DDP alone,the combination of r-MV-Hu191 with DDP induced significantly higher cytotoxicity in both cell lines.Furthermore,ZIP analysis confirmed that the interaction between r-MV-Hu191 and DDP was almost universally synergistic over all the dose-response matrix in both BGC-823 and SGC-7901 cells.For BGC-823 cells,the strongest synergistic effect was found within the region of dose combinations where the dose of r-MV-Hu191 was fixed at 0.1 MOI with average ZIP synergy scores 12.101.As for SGC-7901,the strongest synergistic effect was found within the region of dose combinations where the dose of DDP was fixed at 2.8 ?M with average ZIP synergy scores 6.953.To evaluate whether the synergistic effect is dependent on treatment time.The synergistic effect of r-MV-Hu191 and DDP was first detected at 48hours of combinational treatment,and this efficacy was increased over time,resulting in synergistic suppression of cell growth in both BGC-823 and SGC-7901 cells.2?In SGC-7901 cells,DDP only resulted in marginally lower levels of viral replication and viral P protein expression at 24 h,but not on later time;as for BGC-823 cells,DDP induced no inhibition on virus replication and viral P protein expression all the time points.3?Compared with the control group,the proportion of apoptotic cells was significantly increased upon treatment with r-MV-Hu191 and DDP alone or in combination in both cell lines(P<0.001).In addition,the combination of r-MV-Hul91 and DDP induced more significant apoptosis than r-MV-Hu191 or DDP treatment alone(P<0.001).Moreover,in the presence of Z-VAD,the increase in apoptosis was partially reversed.The activation of caspase 3 and PARP were upregulated after combinational treatment of r-MV-Hu191 and DDP compared with r-MV-Hu191 or DDP treatment alone at 24 h and 48 h.And we confirmed these observations at 72 h of treatment,when Z-VAD reversed caspase 3 and PARP activation in the combinational and r-MV-Hu191 treatment groups.4?Compared with the mock treated groups,tumor-suppressive effect was observed in mice upon r-MV-Hu191(P<0.05)or DDP treatments alone(P<0.001),and upon combinational treatment(P<0.001)on day 11,when the first mouse was euthanized.In addition,compared with r-MV-Hu191 or DDP treatment alone,tumor volumes after combinational treatment were significantly decreased(P<0.001 compared with r-MV-Hu191 group,P<0.01 compared with DDP group).Administration of r-MV-Hu191 or combinational therapy resulted in significant increase of survival compared with mock therapy(median survival 18 days in mock vs 23 days in r-MV-Hu191,P<0.01;and 33 days in combinational therapy,P<0.01).However,comparison between the DDP group and the mock showed no significant differences in survival rates(median survival 18 days in mock vs 17 days in DDP,P>0.05).Although there was no significant difference in survival rates between the combinational and DDP treatment groups(P>0.05),survival time was more extended by combinational treatment compared with virus alone(P<0.01).There was higher level of cleaved caspase 3 from tumor tissues undergoing combinational treatment,compared with r-MV-Hu191 and DDP treated groups.H&E staining showed that tumor cells from the mock group had high cell density,hyperchromatic nuclei and well-defined cell borders.In contrast,tumors from mice treated with r-MV-Hu191 alone showed lower cell density.Tumors from mice treated with DDP alone showed loss of nuclear staining,increased cytoplasmic eosinophilia,and loss of cellular detail and cell borders compared with mock group.In addition,combinational treatment led to enhanced cell death and symptoms of necrosis in the tumor mass compared to single r-MV-Hu191 or DDP treatment.In IHC analysis,increased expression of cleaved caspase 3 was observed in the combinational treatment group compared with the r-MV-Hu191 and DDP groups.TUNEL analysis demonstrated that the combination of r-MV-Hu191 and DDP induced more cell apoptosis than that of r-MV-Hul 91 or DDP alone.5?Weight loss was first observed in DDP and combination treated mice relative to mock treated groups on day 11(the first mouse was sacrificed on that day).In addition,the body weight of the combination group was slightly heavier than that of the DDP group and indicated that the toxicity of DDP could be reduced by combination with r-MV-Hul91.There was no significant difference in levels of blood Alb,Glb,AST,ALT,BUN,and Cr between treatment and control groups.Conclusion:1?r-MV-Hu191 in combination with DDP synergistically suppressed human GC cells.2?MV replication was not affected by DDP chemotherapy.3?r-MV-Hul91 and DDP combinational treatment significantly increased apoptosis in GC cells.4?r-MV-Hu191 and DDP combinational treatment was effective in vivo.5?Side effect induced by r-MV-Hu191 and DDP combinational treatment in vivo was trivial.