| Purpose: to investigate whether endostatin can inhibit pancreatic carcinoma growth and understand the possibility of antiangiogenic gene therapy with recombinant mouse endostatin adenovirus. Here we further investigate where endostatin combines with continuous low-dose doxorubicin can enhanced antitumor efficacy in pancreatic carcinoma model. Methods: mouse endostatin is first cloned into a transfer vector, named pCA-mEndo, pCA-mEndo was co-transformed with pBGH-3 into 293 cells, the replication-deficient recombinant adenovirus Ad-mEndo was generated efficiently by homologous recombination. Pancreatic adenocarcinoma cells(BxPC-3, AsPC-1) were infected with adenovirus Ad-mEndo. We observed the expression of endostatin mRNA and protein, supematants were analyzed for their ability to inhibit angiogenesis in CAM. In the BxPC-3 s.c. tumor model, nude mouse were treated with intratumoral injection of 1X 109pfu(plaque forming units) endostatin adenovirus(Ad-mEndo), the empty vector(Ad-GFP) or Saline(NS) and weekly administration was continued 4 times from day 14 after the implantation. Doxorubicin was injected into the peritoneal cavity of the mice. Doxorubicin at 1.2mg/kg(continuous low-dose schedule dosage) was administered twice a week for a total of 8 times. We explored their capacity of antitumor effects. In the orthotopic nude mouse model of pancreatic cancer, nude mouse were treated with intraperitoneal injection of 2 X 109pfu Ad-mEndo, NS, doxorubicin or combined Ad-mEndo with low-dose doxorubicin. Weekly intraperitoneal administration of Ad-mEndo was continued 4 times from day 2 before the implantation. Doxorubicin at 1.2mg/kg was intraperitoneal administered twice a week for a total of 8 times from 2 day after the implantation. We observed their antitumor effects, microvessel were measured by immunohistochemical analysis. Results: the pCA-mEndo was identified correctly by restriction enzyme analysis. The replication-deficient adenovirus Ad-mEndo was amplified and purified and was measured the titer. The virus titer was 6.3 X 1010pfu/ml. Adenovirus Ad-mEndo can transfer human pancreatic carcinoma cells BxPC-3, AsPC-1 in vitro with high transfer rate. The mRNA and protein of endostatin were expressed by all the infected tumor cells and angiogenesis was inhibited by supematants in CAM. In the BxPC-3 s.c. tumor model, asignificant growth inhibition was obtained by the Ad-mEndo treatment when compare with the two control treatments, ie, Ad-GFP(p<0.01), or NS(p<0.05). Treatment with low-dose doxorubicin resulted in significant growth inhibition compared with control group(NS)(P<0.01). Combined Ad-mEndo and continuous low-dose doxorubicin resulted in more effective growth inhibition than has been observed with any agent alone. Tumor growth inhibition rate of combination treatment (81.4%) was significant high compared with Ad-mEndo(46.1 %) or continuous low-dose doxorubicin(36.7%). Histological examination of xenografts showed significantly reduced microvessel density in the tumor given combined therapy compared with the tumors given either agent alone. In orthotopic nude mouse model of pancreatic cancer, Treatmeat with adenovirus Ad-mEndo or low-dose doxorubicin resulted in significant growth inhibition compared with control group(NS)(p<0.01,p<0.05). Combined Ad-mEndo and continuous low-dose doxorubicin resulted in more effective growth inhibition than has been observed with any agent alone (p<0.01). Tumor growth inhibition rate of combination treatment (92.7%) was significant high compared with Ad-mEndo(60.1%) or continuous low-dose doxorubicin(57.5%). An increased apoptotic index of tumor cells in combination treated tumors compared with the tumors given either agent alone (p<0.01). Conclusions: these result provide evidence that treatment with endostatin gene efficiently suppresses tumor angiogenesis, and inhibites pancreatic tumor growth, and combined endostatin with continuous low-dose doxorubicin obtains an enhanced inhibition of pancreatic tumor growth, our data suggested that endostatin in combination w... |
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