A Study Of Integrin β₁ Antisense Oligodeoxynucleotides On Pancreatic Cancer Therapy

Pancreatic cancer is one of the most lethal human cancers. Pancreaticoduodene-ctomy offers the only possibility for cure. Because of local spread or metastatic disease at the time of diagnosis, fewer than 20% of patients are eligible for surgical resection,yet the overall 5-year survival is less than 4%. Conventional chemotherapy has shown only a minimal survival benefit due to the poor response of pancreatic cancer to current chemotherapeutic regimens. The lethality of this cancer is related to its propensity to invade adjacent organs and metastasize. Along with the in-depth understanding of the molecular biology of pancreatic cancer, specific therapies targeted to factors that facilitate invasion may increase the overall prognosis of pancreatic cancer.There are more evidences suggesting that cell adhesion molecules may play a pivotal role in the development and progression of the malignant phenotype in a range of tumor types. Integrins are the most important family of cell surface adhesion molecules that mediate interactions between cells and the extracellular matrix (ECM). They are heterodimeric transmembrane receptors consisting of a andβ subunits. Each subunit is a glycoprotein with a large extracellular domain and a relatively small cytoplasmic domain. The different integrin subfamilies are determined by the β subunits, for example, the β1 subunit associates with different a subunits to form the integrin β1 subfamily. There are currently 18 a and 9 β subunits identified, and they combine to form more than 28 different α-β heterodimers with distinct binding specificities. Members of the integrins β1 subfamily primarily bind to components of the extracellular matrix, such as fibronectin, collagens, and laminins. Among the different integrin heterodimers, integrins β1 appear to play a crucial role in regulating tumor cell proliferation, differentiation, adhesion, motility, invasion, and metastasis in various human tumor including pancreatic cancer.Antisense oligodeoxynucleotides (ASODN) are designed to bind to their complementary mRNA sequence. Once they get inside the cells, hybridization of ASODN to their target mRNA can activate RNase H, which then causes cleavage of the target mRNA, eventually leading to inhibition of expression of the protein encoded by the mRNA. ASODN have a higher specifically compared with conventional drugs. Variety of genes known to be key regulators which are associated with the malignant phenotype of cancer cells, have been validated as molecular targets for antisense therapy.Here we intended to investigate the effects of integrin β₁ ASODN on invasive ability and cell growth, apoptosis and chemosensitivity of pancreatic carcinoma cell line BXPC-3 in vitro, moreover, researched the effect of integrin β₁ ASODN treatment in human pancreatic carcinoma transplanted subcutaneously in nude mice. Part I The expression of integrin β₁ and protein kinase Ca in pancreatic carcinoma and their significanceObjective To investigate the expression of integrin β₁ and protein kinase Cα (PKCα) in pancreatic carcinoma and their significance.Methods The expression of integrin β₁ and PKCα in 56 pancreatic carcinomas and 23 adjacent noncancer tissures were studied by immunohistochemistry, and their relationship to clinical pathology were analyzed.Results1. Increased integrin β₁ expression was 71.43% in pancreatic carcinomas and 43.48% in adjacent noncancer tissures(P<0.05 ) ;Increased PKCα expression in pancreatic carcinomas was 62.50%while it was 21.74 % in adjacent noncancer tissures(P<0.05) .2. Increased integrin β₁ expression was associated with TNM stage(P＜0.05) and lymph node metastasis(P<0.05 ) .3. Increased PKCα expression was related to differentiated degree(P<0.05) , TNM stage(P<0.05 ), and lymph node metastasis(P<0.05 ) .4. In pancreatic carcinomas, positive correlation was found between the expression of integrin β₁ and that of PKCα(r=0.653, P<0.05).Conclusions1. The intimate relation of integrin β₁ and PKCα may play animportant role in the tumorigenesis and tumor progression in pancreatic carcinoma.2. Integrin β₁ and PKCα may be useful diagnostic parameters for the invasion and metastasis pancreatic cancer. Part II Effect of integrin β₁ antisense oligodeoxyn-ucleotides on invasive ability of pancreatic carcinomacell line BXPC-3 in vitroObjective To investigate the effects of integrin β₁ antisense oligodeoxynucleotides (ASODN) on invasive ability of pancreatic carcinoma cell line BXPC-3 in vitroMethods Integrin β₁ ASODN was transfected into BXPC-3 cells with liposome. The expression of integrin β₁ mRNA and protein were determined by RT-PCR and Western-blot. The invasive ability was measured by the number of cells to penetrate polycarbonates coated with matrigel.Results1. The expression of integrin β₁ mRNA and protein of BXPC-3 cells by transfected with concentration ranging from 32mg/L to 128mg/L ASODN was significantly lower than that of control group(P<0.05).The inhibitive effect of 64 mg/L and 128mg/L integrin pi ASODN was the strongest(P<0.01).2. Invasive tests in vitro also showed that the invasive ability of BXPC-3 cells were inhibited by transfected with 64 mg/L integrin β₁ ASODN.Conclusions1. In this cell line model, integrin β₁ ASODN can effectively inhibit the expression of integrin β₁ gene, thus inhibited the protein synthesis.2. Integrin β₁ ASODN can decrease the ability of invasion of BXPC-3 cells. Part III Effect of integrin β₁ antisense oligodeoxynucleo-tides on cell growth, apoptosis and chemosensitivity of pancreatic carcinoma cell line BXPC-3Objective To investigate the effects of integrin β₁ antisense oligodeoxynucleotides (ASODN) on cell growth, apoptosis and chemosensitivity of pancreatic carcinoma cell line BXPC-3.Methods Integrin β₁ ASODN was transfected into BXPC-3 cells with liposome. The viability rate was assessed by MTT method. Annexin V-FITC/PI flow cytometer method to detect the induced apoptotic condition. Different concentrations of 5-FU, Adriamycin(ADM), Gemcitabine were given 24 h after cells transfected with integrin β₁ ASODN. The dose-effects responses were observed and IC₅₀ s were calculated. Results1. Transfecting BXPC-3 cells with 32mg/L～128mg/L integrin β₁ ASODN inhibited growth significantly.2. Treated with 64mg/L integrin β₁ ASODN for 24h, Annexin V-FITC/PI flow cytometer method determined the percentage of early apoptoic cells was 20.17±2.04%.3. Integrin β₁ ASODN increased the chemosensitivity of BXPC-3 cells to 5-FU, ADM, and Gemcitabine. The IC₅₀ s of different chemotherapy groups were significantly lower than that of control groups.Conclusions1. Integrin β₁ ASODN can effectively inhibite BXPC-3 cells growth.2. Integrin β₁ ASODN can induce BXPC-3 cells apoptosis.3. Integrin β₁ ASODN can enhance the chemosensitivity in pancreatic carcinoma cell line BXPC-3. Part IV Effect of integrin β₁ antisense oligdexynucleo-tides on human pancreatic carcinoma transplantedsubcutaneously in nude miceObjective To investigate the effect of integrin β₁ antisense oligodeoxynucleotides(ASODN) treatment in human pancreatic carcinoma transplanted subcutaneously in nude mice.Methods The models of human pancreatic carcinoma transplanted subcutaneously were established in nude mice, then divided randomly into 3 groups and different treatment were given respectively(control group, random oligodeoxynucleotides group and ASODN group). The weight of nude mice and tumor volume were observed, the tumor growth inhibitory rate and the tumor response rate calculated, too. The expression of integrin β₁ mRNA and protein in tumor tissue were determined by RT-PCR and Western-blot.Results1. The tumor growth inhibitory in the random oligodeoxynucleotides group and the ASODN group were 1.49% and 71.55%, respectively. The tumor response rate of the ASODN group was 15.36%. There was no statistical difference in nude mice weight among 3 groups.2. The expression level of integrin β₁ mRNA and protein was decreased in the ASODN group, whereas the expression of random oligodeoxynucleotides group there was no significant difference in contrast with that of control group.Conclusions1. Integrin β₁ ASODN can effectively inhibit the expression of integrin β₁ gene and protein in vivo. 2. Integrin β₁ ASODN can results in marked inhibit human pancreatic carcinoma - growth in nude mice.3. Integrin β₁ ASODN may be a safe and effective treatment approach for human pancreatic carcinoma.