AAV-HGFK1 And Ad-p53 Cocktail Therapy Prolongs Survival Of Mice With Colon Cancer

Background & Objective Colorectal carcinoma is the third leading cause of cancer-related deaths worldwide and the second most frequent cancer in all developed nations.Liver metastasis of colorectal cancer is the major cause of death,and occurs in over 60%of colon cancer patients.Since no therapy at present has significant effect on hepatic metastasis of colorectal cancer,it is necessary to develop alternative treatment strategies for liver metastasis.Anti-angiogenic therapy is promising for hepatic metastasis of colorectal cancer.A growing number of anti-angiogenic molecules have been discovered,some of which are used in clinical treatment of metastasis.In gene therapy,vectors expressing anti-angiogenic genes are used to produce anti-angiogenic proteins within the cells themselves.It has been shown that the hairpin loop and the first kringle domain(K1) has been shown to have anti-angiogenic activity and to be indispensable for receptor binding.Recently we reported a simple and feasible strategy using a combined rAAV-BMP2 and Ad-BMP2 vector system to increase in vivo bone induction and rAAV expressing HGFK1(rAAV-HGFK1) has antiangiogenic and antitumor cell effects on hepatocellular carcinoma.This study tried to evaluate the application of recombinant adeno-associated virus rAAV-HGFK1 as a new anti-angiogenic cancer gene therapy tool for treating colorectal carcinoma.Its efficacy in combination with recombinant adenovirus(Adv) carrying p53 gene(Ad-p53) was also evaluated.Methods CT26 colon carcinoma cells derived from BALB/c mice,Lovo human colon carcinoma cells,MILE SVEN 1(MS1) mouse pancreatic islet endothelial cells transduced with a SV40 large T antigen(tsA-58-3) and SVEC4-10EE2(SV10) tumorigenic endothelial cells transformed with SV40 were cultured.Their cDNA template was amplified by PCR for EGFR and HGFR.rAAV particles were produced by a helper virus-free system.The recombinant adenoviruses (Ad) were produced by transfecting 293A cells with recombinant adenovirus vectors. After the four kinds of cells were infected by rAAV-EGFP+Ad-null, rAAV-EGFP+Ad-p53,rAAV-HGFK1+ Ad-null or rAAV-HGFK1+Ad-p53 and incubated for 48h,the cells were seeded for 12 hours and incubated in 100μL media with or without HGF or EGF for an additional 72h,MTT assayed to determine growth inhibition of cells that infected by the viruses.The cells were infected at the same way as above described and stimulated with different concentrations of HGF or EGF in DMEM for an additional 24 hours,total proteins were extracted for Western blot assayed EGFR,P-EGFR,c-Met(HGFR),P-c-Met andβ-actin.Three days after infection of MS1 or SV10 cells with the virus,confluent cells grown in 6-well plates were cultured in 2%FBS DMEM.The monolayers were mechanically wounded to obtain a denuded area of 1 mm wide,and observed under a 100×microscope 20 hours after incubation.Lovo or CT26 colon cancer were injected into the spleens of BALB/c and nude mouse to establish models and the mice were treated with rAAV-HGFK1 alone or in combination with Ad-p53 in vivo.The mice were randomly assigned to 4 groups for cocktail therapy survival study(6 each):(A) rAAV-EGFP control,(B) rAAV-HGFK1, (C) Ad-p53,and(D) rAAV-HGFK1+Ad-p53.Viruses were injected through the tail vein 1 day after injection of CT26 cells or Lovo cells.The survival time of each mouse was recorded.Another CT26 BALB/c model(10 mice each group) was treated and on days 12 and 20 after inoculation,5 mice per group were sacrificed with their pancrease,livers and spleens excised,weighted.The sections were stained with H&E, or an anti-EGFP antibody to observe the efficacy of rAAV infection in vivo.For assessing angiogenesis,liver tissue sections were stained with an anti-HGFK1 antibody and an anti-CD31 antibody and were counter-stained with hematoxylin. Apoptosis was detected by TUNEL with an in situ cell death detection kit. Quantification of apoptotic cells was expressed as a mean of the ratio of apoptotic cells to the total number of tumor cells in 10 random fields containing distinct TUNEL-positive cells at 200×magnification.Morphologically necrotic areas were excluded from all analyses.Results By adding Ad into rAAV-EGFP,the transduction efficiency of rAAV-EGFP on CT26 and Lovo cells is significantly improved.At both rAAV doseage,the addition of Ad-null could enhance EGFP expression in CT26 and Lovo cell lines.The expression enhancement was most obvious when Lovo cells were transfected with 10000vg/cell rAAV-EGFP,thus the addition of Ad-null increased the EGFP expression by 8-10 folds.By RT-PCR and Western blot,we found Lovo expressed EGFR and HGFR while CT26 didn't.SV10 expressed EGFR and HGFR,and MS 1 expressed low-level HGFR and EGFR.And interestingly,when the activation statuses of HGF receptor c-Met and EGF receptor EGFR in SV10 cells were checked,it was clear that rAAV-HGFK1 and rAAV-HGFK1+Ad-p53 strongly inhibited the phsophorylation of EGFR.We observed a mild decrease in phosphorylation of Met and a strong inhibition of phosphorylation of EGFR in Lovo cells treated with rAAV-HGFK1+Ad-EGFP or rAAV-HGFK1+Ad-p53,suggesting that HGFK1 may inhibit Lovo cells proliferation partially through acting as a HGF antagonist.Proliferation of SV10 cells but not MS1 cells increased in response to increasing doses of EGF or HGF(rAAV-EGFP+Ad-EGFP control).This increase in proliferation was completely negated by treatments of rAAV-HGFK1+Ad-EGFP,or rAAV-HGFK1+Ad-p53.We found that MS1 cells did not show any proliferation response to HGF.Virus treatments did not alter the response of the cells towards HGF,but if the cells had been treated with EGF,rAAV-HGFK1+Ad-p53 combinatorial treatment siginificantly reduced the proliferation rate.This may due to the fact that rAAV-HGFK1+Ad-p53 could strongly inhibit EGFR phosphorylation in MS1 cells. The unresponsiveness of MS1 cells towards HGF could be attributed to the low expression level of HGF receoptr in the cells.CT26 proliferation was unresponsive to HGF.This unresponsiveness may due to the fact that the expression of Met in CT26 was undetectable.And we did not observe any significant effect on CT26 proliferation by rAAV-EGFP+Ad-EGFP,rAAV-EGFP+Ad-p53,rAAV-HGFK1+Ad-EGFP or rAAV-HGFK1+Ad-p53 treatment.CT26 is also mildly responsive to EGF, thus treatment of increasing concentration of EGF led to mild increase of growh ratio. rAAV-EGFP+Ad-EGFP,rAAV-EGFP+Ad-p53,rAAV-HGFK1+Ad-EGFP could all inhibit this increase of proliferation.Proliferation of Lovo cells,on the other hand, increased remarkably in response to increasing dose of HGF or EGF.These increases were inhibited by rAAV-EGFP+Ad-p53,rAAV-HGFK1+Ad-EGFP or rAAV-HGFK1+Ad-p53 treatment.The relative ability of cell migration was determined by wound healing assay.In both MS1 and SV10 systems,the wounds in rAAV-EGFP+Ad-EGFP group nearly completed healing in 20 hours,rAAV-HGFK1+Ad-p53 cells migrated the slowest. The speeds of wound healing of rAAV-HGFK1+Ad-EGFP and rAAV-EGFP+ Ad-p53 groups were intermediate between rAAV-EGFP+Ad-EGFP and rAAV-HGFK1+Ad-p53.These findings suggested that Ad-p53 or rAAV-HGFK1 has an inhibitory effect on endothelial cell migration.In survival experiment,control(rAAV-EGFP treated) mice succumbed to tumor burden quickly,with median survival of 21.5 days(CT26) and 33 days(Lovo). rAAV-HGFK1 or Ad-p53 treatments could mildly prolong the survival of the mice. The median survival for rAAV-HGFK1 treated mice were 27 days(CT26) and 36.5 days(Lovo)(Both p＜0.05,compare with rAAV-EGFP,log-rank test).The median survival for Ad-p53 treated mice were 27.5 days(CT26) and 39.5 days(Lovo)(Both p＜0.05,compare with rAAV-EGFP,log-rank test).Interestingly,we found that rAAV-HGFK1+Ad-p53 viral cocktail could remarkably prolong survival of the mice. The median survival of rAAV-HGFK1+Ad-p53 treated mice were 63 days(CT26) and 60.5 days(Lovo)(Both p＜0.01,compare with rAAV-HGFK1 or Ad-p53, log-rank test).Furthermore,one mouse inoculated with CT26 and two mice inoculated with Lovo in the rAAV-HGFK1+Ad-p53 group survived through the window of observation(90+ days) and did not develop tumors in their livers and spleens.The weights of spleens and livers of mice in rAAV-HGFK1+Ad-p53 group compared with rAAV-HGFK1 or Ad-p53 groups did not differ significantly at day 12. But at day 20 the weights of spleens and livers from rAAV-HGFK1+Ad-p53 viral cocktail treatment group were significantly(p＜0.05) lower than rAAV-HGFK1 or Ad-p53 group.This suggested the growth of tumor at primary and secondary site of rAAV-HGFK1+Ad-p53 group was slowed.When the tumors in CT26 injected mice were examined histochemically,it was found that the rAAV-HGFK1+Ad-p53 combinatorial treatment group induced large area of tumor necrosis in primary tumors and liver metastases.We checked the expression of HGFK1 in liver concentrated in vascular epithelia.And in vivo,HGFK1 expression could be enhanced by Ad-p53. Furthermore,TUNEL staining revealed that whereas rAAV-HGFK1 or Ad-p53 alone could mildly induce apoptosis in the tumors,rAAV-HGFK1+Ad-p53 combinatorial treatment caused much more apoptosis in the tumors.It was found that tumors in rAAV-EGFP,rAAV-EGFP+Ad-p53,and rAAV-HGFK1 treatment groups were heavily infiltrated with endothelial cells in anti-CD31 staining section.On the other hand,rAAV-EGFP+ Ad-p53 treated tumor contained very limited number of endothelial cells.To exclude the possibility that the viral cocktail may downregulate CD31 expression instead of inhibiting angiogenesis, we stained the tissue for another endothelial marker vWF.We found that the relative abundance of vWF positive cells arcoss the treatment groups were similar to CD31 positive cells.This indicated that rAAV-EGFP+Ad-p53 treatment could inhibit tumor angiogenesis.Conclusion rAAV-HGFK1 could antagonize EGF and HGF signal and rAAV-HGFK1+Ad-p53 combinatorial treatment significantly reduced the proliferation rate and migraion of MS1 and SV10 cells.Adding Adv into rAAV can increase the efficacy of treatment with rAAV for human and mouse colon cancer cell lines in vitro and in vivo.More importantly,rAAV-HGFK1 and Ad-p53 exhibited mild therapeutic effect on their own,but when administered together they synergistically prolonged survival, reduced tumor growth,and induced tumor cell death,rAAV plus Adv cocktail gene therapy is a novel treatment for colorectal cancer.