AIM: We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell li...AIM: We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities. METHODS: Three different sites of SECs were constructed by PCR. K1/siRNA,K2/siRNA and K3/siRNA are located at sites 194,491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively. RESULTS: Introduction of the Kl/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the Kl/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells. CONCLUSION: Kl/siRNA and K2/siRNA can inhibit the expression of activated K-ras but K3/siRNA has no effect, demonstrating that Kl/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.展开更多
Background Pancreatic cancer is a common malignant tumor of the digestive system. It is the fourth major cause of tumor-related death and its morbidity is increasing, and hence it is imperative to develop effective fo...Background Pancreatic cancer is a common malignant tumor of the digestive system. It is the fourth major cause of tumor-related death and its morbidity is increasing, and hence it is imperative to develop effective forms of therapy for pancreatic cancer. Peptide tyrosine-tyrosine （PYY） is an important gastrointestinal peptide hormone. According to previous literatures, PYY has been shown to inhibit tumor proliferation in cellular and animal models, but there has been limited research on the detailed mechanism of PYY in pancreatic cancer. This study was to observe the effects of PYY on pancreatic cancer cell and investigate the possible mechanism. Methods The expression of Y1, Y2, and Y5 receptors on pancreatic cancer cell lines were detected by reverse transcription quantitative polymerase chain reaction （RT-qPCR）. The cytotoxicity of PYY toward the MiaPaCa-2 cell was evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay; the cell morphology and structure changes were observed under inverted microscope and transmission electron microscope respectively. Apoptosis and cell cycle were evaluated by flow cytometry. The activity of caspase-3 was determined by activity assay kits and Western blotting. The expression of survivin, vascular endothelial growth factor （VEGF）, and cyclooxygenase-2 （COX-2） were determined by RT-qPCR and Western blotting. Results Expression of Y2 receptor is the most abundant PYY receptor on pancreatic cancer cell. PYY inhibited MiaPaCa-2 cell proliferation, blocked it in G0/G1 phase, increased the proportion of apoptosis cells and caspase-3 activity, and reduced the expression of survivin, VEGF, and COX-2. Conclusions PYY weakened the ability of the pancreatic MiaPaCa-2 cell viability through cell cycle blocking and apoptosis inducing. The inhibition effect of PYY may be mediated by the Y2 receptor. The increased caspase-3 activity and reduced expression of survivin, VEGF, and COX-2 may serve as a novel mechanism in PYY inhibition effect on MiaPaCa-2 cell.展开更多
AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator prote...AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised.RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable.展开更多
文摘AIM: We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities. METHODS: Three different sites of SECs were constructed by PCR. K1/siRNA,K2/siRNA and K3/siRNA are located at sites 194,491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively. RESULTS: Introduction of the Kl/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the Kl/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells. CONCLUSION: Kl/siRNA and K2/siRNA can inhibit the expression of activated K-ras but K3/siRNA has no effect, demonstrating that Kl/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.
文摘Background Pancreatic cancer is a common malignant tumor of the digestive system. It is the fourth major cause of tumor-related death and its morbidity is increasing, and hence it is imperative to develop effective forms of therapy for pancreatic cancer. Peptide tyrosine-tyrosine （PYY） is an important gastrointestinal peptide hormone. According to previous literatures, PYY has been shown to inhibit tumor proliferation in cellular and animal models, but there has been limited research on the detailed mechanism of PYY in pancreatic cancer. This study was to observe the effects of PYY on pancreatic cancer cell and investigate the possible mechanism. Methods The expression of Y1, Y2, and Y5 receptors on pancreatic cancer cell lines were detected by reverse transcription quantitative polymerase chain reaction （RT-qPCR）. The cytotoxicity of PYY toward the MiaPaCa-2 cell was evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay; the cell morphology and structure changes were observed under inverted microscope and transmission electron microscope respectively. Apoptosis and cell cycle were evaluated by flow cytometry. The activity of caspase-3 was determined by activity assay kits and Western blotting. The expression of survivin, vascular endothelial growth factor （VEGF）, and cyclooxygenase-2 （COX-2） were determined by RT-qPCR and Western blotting. Results Expression of Y2 receptor is the most abundant PYY receptor on pancreatic cancer cell. PYY inhibited MiaPaCa-2 cell proliferation, blocked it in G0/G1 phase, increased the proportion of apoptosis cells and caspase-3 activity, and reduced the expression of survivin, VEGF, and COX-2. Conclusions PYY weakened the ability of the pancreatic MiaPaCa-2 cell viability through cell cycle blocking and apoptosis inducing. The inhibition effect of PYY may be mediated by the Y2 receptor. The increased caspase-3 activity and reduced expression of survivin, VEGF, and COX-2 may serve as a novel mechanism in PYY inhibition effect on MiaPaCa-2 cell.
基金Supported by National Institute for Health Research Liverpool Pancreatic Biomedical Research Unit and the Pancreatic Cancer Research Fund (to Nedjadi T)
文摘AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised.RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable.
