| Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric complex, composed of an α and a β subunit, both belonging to the basic helix-loop-helix (bHLH)/Per-aryl hydrocarbon receptor nuclear translocator-Sim (PAS) family of transcription factors. The bHLH domain mediates dimerization and DNA binding in many transcription factors. PAS is an additional dimerization motif. Whereas HIF-1β is a common subunit of multiple bHLH proteins, HIF-1α is the unique, O2-regulated subunit that determines HIF-1 activity. Although HIF-1β is quite stable in normoxic conditions, HIF-1α is extremely unstable and quickly degraded by the ubiquitin-proteasome system. HIF-1α plays an important role in solid tumor formation in vivo by promoting angiogenesis and anaerobic metabolism. Several studies have demonstrated that HIF-1α can be detected in most types of human tumors examined, including bladder, breast, colon, glial, hepatocellular, ovarian, pancreatic, prostate, renal and oesophageal tumors. The expression of HIF-1α is inducible by oxygen concentrations below 6%, and the activation of hypoxia-responsive elements by HIF-1 has been reported to correlate with decreasing oxygen concentrations in vitro.Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is characterized by a unique structure that discriminates it from other members of the IAP family. It contains only a single BIR repeat and lacks a carboxy terminal RING finger domain. Survivin is expressed in the G2/M phase of cell cycle in a cycle-regulated manner. It directly binds to and inhibits both Caspase-3 and Caspase-7 activity, leading to arrest of apoptosis. Survivin expression is not detectable in differentiated normal adult cells of any organ, but it is abundantly expressed in embryonic tissues and in a wide range of cancer tissues including neuroblastoma, cholangiocarcinoma, colorectal, stomach, breast and uterine cervical carcinomas. It has been demonstrated recently that survivin is also frequently expressed in malignant pancreatic ductal tumors and pancreatic adenocarcinoma. Furthermore, the prognostic value of survivin expression has been reported in several human cancers. Studies demonstrating resistance of survivin-transfected cells to anticancer drug-induced apoptosis and sensitization to chemotherapy by survivin antisense treatment have shown that survivin is implicated in sensitization to chemotherapy.The integrin family of cell-surface heterodimeric glycoproteins composed of a and β subunits function primarily as receptors for extracellular matrix ligands, which regulate many aspects of cell physiology, including morphology, adhesion, migration, proliferation, and differentiation. Integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes. Many cancers show abnormalities of integrin function as a result of transformation by oncogenes. More importantly, the growth of several tumors depends on β1-integrin function. Previous studies have demonstrated that β1-integrin is a poor prognostic factor in patients with cancers. Evidences show an essential role of β1-integrins in invasion of pancreatic carcinoma cells and also suggest subtle regulatory mechanisms of cell invasion.On the other hand, the prognosis for patients with pancreatic cancer remains poor, prompting the search for new treatment strategies. Although treatments such as pre-operative chemotherapy and chemoradiation therapy are currently used for patients with advanced pancreatic cancer, their results are not satisfactory. Even in early-stage disease, we have experienced many patients who developed local recurrence of tumor or distant metastasis within a short period after operation.In pancreatic cancer, the relationship of HIF-1α, survivin and β1-integrin is not very clear at present. This study aimed to observe the effect of antisense HIF-1α on progression, metastasis and chemosensitivity of pancreatic cancer. Part 1 Expression and significance of HIF-1α in pancreatic cancer ObjectiveTo investigate the expression of HIF-1α, the relationship with clinical features, and determine the role in the development of pancreatic cancer.MethodsThe expression of HIF-1α in tumor tissues and non-cancerous adjacent pancreas tissues was detected by SP immunohistochemical technique.ResultsHIF-1α was expressed in 27 of 48 cases (56.3%). In contrast, no expression of HIF-1α in adjacent histologically noncancerous pancreas was detectable (P<0.01) . Expression of HIF-1α had no relationship with ages, sexes and tumor sizes of pancreatic cancer patients. But the status of HIF-1α was significantly correlated with metastatic status and differentiation level (P<0.05).ConclusionHigh expression of HIF-1α in pancreatic cancer suggests it may play an important role in the development of pancreatic cancer. Therefore, assessment of HIF-1α expression might be useful for predicting the prognosis of pancreatic cancer patients. Part 2 Reconstruction of eukaryotic gene expression plasmid pcDNA3.1(+) /antisense HIF-1α and its stable expression in human pancreatic cancer cell line BxPc-3ObjectiveTo further explore the role of HIF-1α, antisense plasmid of HIF-1α was constructed, and transfected into human pancreatic cancer cell line BxPc-3.MethodsAntisense HIF-1α DNA structure was amplified by PCR and inserted into eukaryotic plasmid pcDNA3.1(+). The expression plasmid pcDNA3.1(+)/antisense HIF-1α was transfected into human pancreatic cancer cell line BxPc-3 through Dosper liposomal transfection reagent. Cells stably expressing antisense HIF-1α were screened with G418. BxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O2 for 4 hours. The expression of HIF-1α was detected by RT-PCR and Western blot.ResultsThe constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two fragments at 721bp and 5.4kb, were respectively found, which was consistent with what expected. Eukaryotic plasmid was transfected into BxPc-3 cells through Dosper liposomal transfection reagent. HIF-1α mRNA (0.2367±0.0094) and protein (0.1798±0.0063) expression in the experimental group were significantly depressed by this plasmid, compared with the control (P<0.05).ConclusionBxPc-3 with stable antisense HIF-1α expression was successfully constructed, which could be applied to explore further. Part 3 An antisense plasmid targeting HIF-1α expression sensitizes pancreatic cancer cells to chemotherapyObjectiveThe apoptosis induced by chemotherapeutic agent was inhibited through HIF-1α expression. Antisense plasmid of HIF-1α was constructed and transfected into BxPc-3. The change of sensitizion to chemotherapy and the relationship with survivin was detected.MethodsBxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O2 for 4 hours. Different dosages of chemotherapy agents (5-fluorouracil, doxorubicin, and gemcitabine) were added into BxPc-3 cells after antisense HIF-1α transfected. The growth inhibition rates were measured by MTT, apoptosis rates were measured by flow cytometry and the expression of survivin was detected by RT-PCR and Western blot.ResultsHigher dosages (100, 200, and 400 mg/L of 5-fluorouracil; 0.05, 0.075, and 0.1 mg/L of doxorubicin; and 10-9, 10-8, and 10-7 mol/L of gemcitabine) caused a greater increase of inhibition in the experimental group than in control (P<0.05). survivin mRNA (0.4601±0.0122) and protein (0.3659±0.0117) expression in the experimental group were markly down-regulated compared with the control (P<0.05).ConclusionOur data demonstrate that antisense HIF-1α inhibits expressions of survivin, enhancing apoptosis in human pancreatic cancer cells and thus the sensitivity to chemotherapy was increased. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy. Part 4 An antisense plasmid targeting HIF-1α expression restrains the progression and metastasis of pancreatic cancerObjectiveTo observe the effect of antisense HIF-1α on the progression and metastasis of pancreatic cancer and the relationship with β1 integrin.MethodsBxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O2 for 4 hours. The expression of β1 integrin was detected by RT-PCR and Western blot. The migration of BxPc-3 cells was assayed using transwell cell culture chambers. Subcutaneous transplantation of BxPc-3 cells in nude mice for 8 weeks was to assess progression and metastasis of pancreatic cancer.Resultsβ1 integrin mRNA (0.2568±0.0115) and protein (0.2962±0.0167) expression in the experimental group were markly down-regulated compared with the control (P<0.05).The number of migrated BxPc-3 cells in the experimental group was 110.6±16.4, far less than in control (P<0.05). In vivo, the tumor size and weight in the experimental group were significantly lower than those in control (P<0.05).ConclusionOur data demonstrate that antisense HIF-1α inhibits expressions of β1 integrin, restraining the progression and metastasis of pancreatic cancer. Therefore, HIF-1α may play a very important role in progression and metastasis of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy. In summary, the following points of view outcome:1. High expression of HIF-1α in pancreatic cancer, but not in non-cancerous adjacent pancreas tissues, suggests it may play an important role in the development of pancreatic cancer.2. The relationship between HIF-1α expression and the clinical features of pancreatic cancer was discussed. Expression of HIF-1α had no relationship with ages, sexes and tumor sizes of pancreatic cancer patients. But the status of HIF-1α was significantly correlated with metastatic status and differentiation level. Therefore, assessment of HIF-1α expression might be useful for predicting the prognosis of pancreatic cancer patients.3. BxPc-3 with stable antisense HIF-1α expression was successfully constructed. The mRNA and protein expression of HIF-1α was successfully inhibited by the antisense HIF-1α plasmid under hypoxia.4. Under hypoxia, antisense HIF-1α inhibits expressions of survivin, enhancing apoptosis in human pancreatic cancer cells and thus the sensitivity to chemotherapy was increased. Therefore, HIF-1α may play a very important role in chemosensitivity of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy.5. Under hypoxia, antisense HIF-1α inhibits expressions of β1 integrin, restraining the progression and metastasis of pancreatic cancer. Therefore, HIF-1α may play a very important role in progression and metastasis of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy.
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