The Experimental Studies Of Heparanase Antisense Oligodeoxynucleotide Inhibiting Invasive Ability Of Human Pancreatic Cancer Cell

Pancreatic cancer has a poor prognosis due to its aggressive biological behaviour. In the majority of cases, diagnosis is only established late in the course of the disease, when local invasive growth and distant metastasis have already occurred. At that point, the opportunity for curative therapy, which is surgery at an early stage, has already passed. Even though complete resection may still be achieved, the rate of relapses is high. Adjuvant combination therapy may offer a small survival advantage but is associated with treatment related morbidity. It therefore is not unequivocally accepted. The cytotoxic agent gemcitabine is the only drug currently known to be effective for palliation. Nevertheless, the absolute gain in survival is marginal.On the molecular level, several events including proto-oncogene mutations such as K-ras, tumour suppressor gene mutations in the p16INK4a, DPC4, and p53 genes, and overexpression of a variety of growth factors and their receptors have been described in the carcinogenesis of pancreatic cancer. The underlying causes for the aggressiveness of this cancer are still not fully understood. Invasion of tumour cells into the surrounding tissue requires loosening of cell - cell adhesion, invasion of the basement membrane and disassembly of the extracellular matrix. Chief components of basement membrane (BM) and extracellular matrix (ECM) such as collagens, fibronectin and laminin are substrates of metalloproteinases and cysteine and serine proteases, which are known to be upregulated in metastatic cancers. Recently, another ECM degrading enzyme, mammalian heparanase (HPSE), has been cloned from human placenta tissue and platelets. The enzyme cleaves another key component of BM and ECM, namely heparan sulphate proteoglycans (HSPG). In addition to the enzymatic degradation of the ECM heparanase acts by releasing growth factors such as bFGF, heparan sulphate (HS) fragments and other enzymes such as lipoprotein lipase from the ECM. Heparanase expression has been studied in human primary tumours and tumour cell lines. Its expression correlates with tumour progression and invasiveness.Antisense oligodeoxynucleotides (AS-ODNs) are designed to bind to their complementary mRNA sequence. Once they get inside the cells, hybridization of AS-ODNs 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. AS-ODNs have more specific effects than conventional drugs. In the present study, we transfected human pancreatic cancer cell line Panc-1 with HPSE AS-ODN, then examined HPSE gene expression and protein level. The viability rate was assessed by MTT, and a modified Boyden chamber assay was performed to evaluate the invasive ability of Panc-1 cells after AS-ODN transfection in vitro. Moreover, the models of human pancreatic cancer subcutaneous xenograft in nude mice were established. The effects of HPSE AS-ODN on HPSE gene expression and tumour growth of human pancreatic cancer transplanted subcutaneously in nude mice were investigated. We hope that our results may provide experimental evidences of clinic therapy with HPSE AS-ODN for the pancreatic cancer. Part I The clinical significance of heparanase expression in pancreatic cancerObjectiveTo investigate the clinical significance of heparanase expression in pancreaticcancer. Methods1. Fifty pancreatic cancer tissue samples were obtained from 36 male and 14 female patients (median age, 63 years; range, 41-77 years) in Tongji Hospital, According to the classification of UICC, there were 5 stage I, 10 stage II, 27 stage III, and 8 stage IV pancreatic cancers. Two tumours were well differentiated (grade 1), 35 tumours were moderately differentiated (grade 2), and 13 tumours were poorly differentiated (grade 3). Of the 50 tumour patients, there were 32 lymph node positive vs 18 negative tumours.2. The expression of heparanase in 50 pancreatic cancers were studied by immunohistochemistry, and the relationship between heparanase expression and clinicopathological parameters was analyzed.Results1. Heparanase showed high expression in pancreaticl cancers (76%, 38 / 50).2. There was no correlation between UICC stage or grade of disease and heparanase expression.3. There was a trend towards heparanase expression in lymph node positive vs negative tumours (P =0.06).Conclusions1. There was no correlation between UICC stage of disease and heparanase expression. The result suggested that heparanase expression may occur in early stage of disease, which may relate to invasion and metastasis of the pancreatic cancer.2. There was no correlation between grade of disease and heparanase expression. The result suggested that heparanase expression may occur in well differentiated tumour, so invasion and metastasis of the pancreatic cancer should not be ignored in the well differentiated tumours.3. Although there was no statistical difference between lymph node positive and negative tumours, there was a trend towards heparanase expression in lymph node positive tumours.4. Heparanase may play an important role in tumour progression of pancreatic cancer. Heparanase may be useful parameter for invasion and metastasis of the pancreatic cancer. Part II Effects of heparanase antisense oligodeo-xynucleotide on invasive ability ofpancreatic cancer cells in vitroObjectiveTo investigate the effects of heparanase (HPSE) antisense oligodeoxynucleotide (AS-ODN) on HPSE gene expression, cell growth and invasive ability of human pancreatic cancer cell line Panc-1 in vitro. To provide experimental evidences of clinic therapy with HPSE AS-ODN for the pancreatic cancer. Methods1. Cell Culture: Panc-1, a human pancreatic cancer cell line, was obtained from Union Hospital of Beijing.2. AS-ODN Synthesis: AS-ODN against HPSE with sequence 5' -GGC TTC GAG CGC AGC AGC AT-3' , nonsense oligodeoxynucleotide (NS-ODN) as control.3. Cultured Panc-1 cells were divided into three groups: AS-ODN group (transfected AS-ODN with liposome), NS-ODN group (transfected NS-ODN with liposome), blank control group (no transfection).4. RT-PCR was performed to measure the expressions of HPSE mRNA.5. Western-blot was performed to measure the expressions of HPSE protein.6. The viability rate was assessed by MTT.7. The invasive ability in vitro was evaluate by invasion chamber assay. Results1. The expressions of HPSE mRNA and protein of Panc-1 cells treated with AS-ODN were significantly inhibited, and there was a marked drop of the viability rate in AS-ODN group (61.90±1.66%). The average number of invasion cells of AS-ODN group in vitro was 60.00 ± 9.3095. There was statistical difference between AS-ODN group and control group.2. The expressions of HPSE mRNA and protein of Panc-1 cells treated with NS-ODN were not significantly inhibited, and there was no marked drop of the viability rate in NS-ODN group (93.70±4.68%). The average number of invasion cells of NS-ODN group in vitro was 240.75 ± 9.3586. There was no statistical difference between NS-ODN group and control group. Conclusion1. Our results suggested that HPSE AS-ODN may inhibit HPSE mRNA and protein expression.2. Our results suggested that HPSE AS-ODN may reduce cell growth of Panc-1 in vitro.3. Our results suggested that HPSE AS-ODN may decrease the invasive ability of Panc-1 in vitro.4. Our findings provide experimental evidences of clinic therapy with HPSE AS-ODN for the pancreatic cancer. Part III The inhibitory effects of heparanase antisenseoligdexynucleotides on human pancreatic cancertransplanted subcutaneously in nude miceObjectiveTo investigate the effects of heparanase (HPSE) antisense oligodeoxyn-ucleotide (AS-ODN) on HPSE gene expression and tumour growth of human pancreatic cancer transplanted subcutaneously in nude mice. To provide experimental evidences of clinic therapy with HPSE AS-ODN for the pancreatic cancer. Methods1. Cell Culture: Panc-1, a human pancreatic cancer cell line, was obtained from Union Hospital of Beijing.2. AS-ODN Synthesis: AS-ODN against HPSE with sequence 5' -GGC TTC GAG CGC AGC AGC AT-3' , nonsense oligodeoxynucleotide (NS-ODN) as control.3. The models of human pancreatic cancer transplanted subcutaneously were established in nude mice: 15 male BALB/C nude mice, 6-8 weeks old and 18-22 g weight, were supplied by Hubei Center for Disease Control and Prevention. 1× 10~8 tumour cells in 0.2 ml of DMEM were subcutaneously injected into flank of every mouse.4. The models of nude mice were divided randomly into AS-ODN group (AS-ODN injected into subcutaneous xenograft, 10mg/kg), NS-ODN group (NS-ODN injected into subcutaneous xenograft, 10mg/kg), control group. The injection was given once every two days, ten times in total.5. After treatment, the tumour weight was measured and tumour growth inhibitory rate was calculated.6. RT-PCR and Western-blot were performed to measure the expressions of HPSE mRNA and protein levels, respectively. Results1. Tumour growth of AS-ODN group was significantly inhibited, and the average weihgt of subcutaneous xenografts in AS-ODN group was 1.860 ± 0.5052 (g). The tumour growth inhibitory rate of AS-ODN group was 36.9 %. The expressions of HPSE mRNA and protein in subcutaneous xenografts treated with AS-ODN were significantly inhibited. There was statistical difference between AS-ODN group and control group.2. Tumour growth of NS-ODN group was not significantly inhibited, and the average weihgt of subcutaneous xenografts in NS-ODN group was 2.768 ± 0.6154 (g). The tumour growth inhibitory rate of NS-ODN group was 6.2 %. The expressions of HPSE mRNA and protein in subcutaneous xenografts treated with NS-ODN were not significantly inhibited. There was no statistical difference between NS-ODN group and control group.Conclusions1. Our findings suggested that HPSE AS-ODN results in marked inhibition of HPSE gene expression and tumour growth of human pancreatic cancer transplanted subcutaneously in nude mice.2. HPSE AS-ODN may be a novel treatment approach for human pancreatic cancer. Our results provide experimental evidences of clinic therapy with HPSE AS-ODN for the pancreatic cancer.