Pancreatic Cancer Cell Line Patu8988 Research Articles

Abnormal expression of PTEN and PIK3CA in pemetrexed-resistant human pancreatic cancer cell line Patu8988.

The aim of this study was to investigate the expression of PTEN and PIK3CA in the pemetrexed-resistant human pancreatic cancer cell line Patu8988, and to evaluate their effects on the biological behavior of pancreatic cancer cells. PTEN and PIK3CA gene and protein expressions were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot, respectively, in a pemetrexed-resistant pancreatic cancer cell line and in the parent strain of the pancreatic cancer cells. The discrepancies between the two types of cell lines were detected by a transwell test. RT-PCR and western blot analyses revealed that PTEN and PIK3CA were overexpressed in the pemetrexed-resistant pancreatic cancer cell line. PTEN and PIK3CA were shown to be upregulated by 89 and 76% (western blot), respectively, in the pemetrexed-resistant cell line, compared to the normal pancreatic cancer cell line. The migratory and invasive abilities of the pemetrexed-resistant pancreatic cancer cell were significantly reduced compared to those of the parent strain (P < 0.05; transwell assay). Both PTEN and PIK3CA expression was abnormally enhanced in the pemetrexed-resistant cell line Patu8988; the co-existence of high levels of PTEN and PIK3CA in the pemetrexed-resistant pancreatic cancer line cells induced a significant decrease in their migratory and invasive capacities. This suggested that the mechanism of pemetrexed resistant may be affected by PTEN and PIK3CA, and that these may alter the biological behavior of cancer cells.

Regulation of CX3CR1 expression in pancreatic cancer cells by hypoxia inducible factor lα

Objective: This study aimed to investigate the effect of hypoxia inducible factor 1α (HIF-1α) expression on CX3CR1 and its regulatory mechanism in pancreatic cancer cell line Patu8988. Methods: The highly expressed plasmid pcDNA3.1 HIF-1α and siRNA HIF-1α were initially constructed.After the plasmid was separately transfected to the pancreatic cancer cells, CX3CR1 and HIF-1α expressions were assayed by western blot analysis and real-time quantitative reverse transcriptase-polymerase chain reaction. The relationship between HIF-1α and CX3CR1 promoter was determined by chromatin immunoprecipitation and luciferase technology. Results: The overexpressed HIF-1α could upregulate the CX3CR1 expression in pancreatic cancer cells. The CX3CR1 expression was significantly reduced when HIF-1 α was knocked down. Chromatin immunoprecipitation assay demonstrated that HIF-1α could be directly bound to the hypoxia-response element (5'-A/GCGTG-3') of the CX3CR1 promoter. This binding activity was significantly enhanced under hypoxic condition. CX3CR1 promoter-induced HIF-1α overexpression could significantly upregulate the expression of luciferase reporter genes in pancreatic cancer cells (P<0.01). Conclusion: HIF-1α could regulate CX3CR1 expression in pancreatic cancer cells.

Lentivirus-mediated shRNA interference targeting vascular endothelial growth factor inhibits angiogenesis and progression of human pancreatic carcinoma

Angiogenesis is known to be essential to the survival, growth, invasion and metastasis of cancer cells. Vascular endothelial growth factor(VEGF) is an important factor regulating tumor angiogenesis. In the present study, we analyzed the effect of lentivirus-mediated shRNA interference targeting vascular endothelial growth factor(VEGF) on angiogenesis and progression in the pancreatic cancer cell line Patu8988 invitro and invivo. The study aimed to construct a recombinant lentivirus carrying targeted VEGF shRNA(LV-RNAi) to be used to transfect Patu8988 cells, and we investigated its anti-angiogenic and growth inhibitory effects on pancreatic cancer. VEGF expression was measured by RQ-PCR, western blotting and enzyme-linked immunosorbent assay(ELISA). In subcutaneous transplantation models, tumor volumes were determined, and the expression levels of VEGF and CD34 were assessed by immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) was used to determine apoptosis. In the orthotopic transplantation models, tumor volume and liver metastasis were determined. We successfully constructed LV-RNAi and confirmed that it knocked down the VEGF gene at the mRNA and protein levels in Patu8988 cells. In the subcutaneous transplantation models, tumors with low levels of VEGF expression exhibited reduced pancreatic carcinoma angiogenesis and growth, and the apoptotic index was significantly higher. In the orthotopic transplantation models, tumors with low levels of VEGF expression exhibited significantly reduced pancreatic carcinoma growth, but no significant difference was observed between the three mouse groups, LV-RNAi, LV-NC and the control, in regards to liver metastasis. In summary, lentivirus-mediated RNAi silencing of VEGF inhibited tumor angiogenesis and growth, and increased apoptosis of the pancreatic cancer cell line Patu8988. VEGF targeted gene silencing approach has the potential to serve as a novel treatment for pancreatic cancer.

B7-H3 overexpression in pancreatic cancer promotes tumor progression

B7-H3, a member of the B7-family molecules, plays an important role in adaptive immune responses. In addition, B7-H3 is also expressed in several types of human cancers and is correlated with the poor outcome of cancer patients. However, its exact role in cancer is not known. In the present study, we compared B7-H3 expression in normal pancreas and pancreatic cancer tissue specimens, and determined the effects of low B7-H3 expression on the human pancreatic cancer cell line Patu8988 using lentivirus-mediated RNA interference. B7-H3 expression in pancreatic specimens was determined by enzyme-linked immunosorbent assay (ELISA). A Patu8988 cell line with low B7-H3 expression was established by lentivirus-mediated RNA interference to investigate the effect of B7-H3 on cell proliferation, migration and invasion in vitro. By establishing subcutaneous transplantation tumor and orthotopic transplantation pancreatic cancer mouse models, the effect of B7-H3 on cell proliferation, migration and invasion was studied in vivo. B7-H3 in tissue samples was significantly higher in the pancreatic cancer group than in the normal pancreas group (mean ± SD, 193.6±9.352 vs. 87.74±7.433 ng/g; P<0.0001). B7-H3 knockdown by RNA interference decreased cell migration and Transwell invasion up to 50% in vitro. No apparent impact was observed on cell proliferation in vitro. In the subcutaneous transplantation tumor mouse model, the tumor growth rate was reduced by the knockdown of B7-H3. In the orthotopic transplantation pancreatic cancer mouse model, the effect of inhibiting metastasis by knocking down B7-H3 was assessed in terms of the average postmortem abdominal visceral metastatic tumor weight. This demonstrated that inhibition of B7-H3 expression reduced pancreatic cancer metastasis in vivo. In conclusion, B7-H3 is aberrantly expressed in pancreatic cancer. In addition to modulating tumor immunity, B7-H3 may have a novel role in regulating pancreatic tumor progression.

Metformin inhibits proliferation of human pancreatic cancer cell line Patu8988

The proliferation of Patu8988 cells pre-treated with metformin for 72h was measured by CCK-8 assay.Cell cycle was analyzed by flow cytometry.The expression of related genes was detected by RT-PCR.Metformin suppressed the proliferation of Patu8988 cells in a dose-dependent manner( r=0.994,P＜0.05 ).The proportion of cells at G0/G1 stage was increased while that of G2/M stage decreased (P＜0.05 ).The expressions of MMP-3,cyclinD1,p53 were down regulated and that of Bax was up regulated.The results show that metformin may inhibit the proliferation of Patu8988 cells,via blocking the cell cycle and affecting the expression of related genes. Key words: Metformin; Pancreatic neoplasms; Proliferation; Cell cycle

Effects of histone deacetylase 1 on proliferation and apoptosis of pancreatic cancer cell line

Objective To investigate effects of histone deacetylase 1 (HDAC1) gene silencing on proliferation and apoptosis of pancreatic cancer cell line PaTu8988. Methods The PaTu8988 cells were cultured and divided into control group (untreated), negative control group (treated with 30 nmol/L negative siRNA), low HDAC1 group (treated with 15 nmol/L HDAC1 siRNA) and high HDAC1 group (treated with 30 nmol/L HDAC1 siRNA). The real-time PCR and Western blot were used to detect the efficiency of HDAC1 gene silencing on mRNA and protein levels ,respectively, at 48 hours after transfection of HDAC1 siRNA. Cell proliferation and apoptosis were evaluated using cell counting kit and flow cytometry, respectively. Results Forty-eight hours after transfection of HDAC1 siRNA, the expression of HDAC1 mRNA level in PaTu8988 cells was 46.1%±6.1% in low HDAC1 group and 32.3%±1.4% in high HDACI group, which were lower than that in control group (100.0%±3.4%) and negative control group (87.4%±28.3%,P<0.05). The expression of HDAC1 protein was higher in control and negative control groups than in low and high HDAC1 groups. Cell survival rate was 100.0%±17.1% in control group, 87.1%±5.0% in negative control group, 68.7%±4.7% in low HDAC1 group and 61.6%±2.0% in high HDAC1 group with significant difference (P<0.05). Meanwhile, the cell apoptic rate in control (4.20%±0.95%) and negative control (4.59%±1.26%) groups was lower than that in low (10.09%±1.36%) and high (11.19%±6.07%) HDACI groups (P<0.05). Conclusions HDACI siRNA can effectively and specifically inhibit the expression of HDAC1 and proliferation of PaTu8988 cells and induce cell apoptosis. Key words: Pancreatic neoplasms, RNA interference, Therapy

Enhanced therapeutic effects for human pancreatic cancer by application K-ras and IGF-IR antisense oligodeoxynucleotides

To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specific to GTT point mutation at codon 12 and type I insulin-like growth factor receptor (IGF-IR) antisense oligodeoxynucleotide (IGF-IR ASODN) on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras mutation ASODN specific to K-ras point mutation at codon 12 was designed and composed. After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively, the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming assay and transmission electron microscopy; the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction (RT-PCR) and flow cytometry respectively; apoptosis was determined by flow cytometry. The combined antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. The results of PCR-SSP and sequence analysis showed that the human pancreatic cancer cell line Patu8988 had point mutation at codon 12, and the mutation style was GGT-->GTT. 2-32 microg/mL K-ras ASODN and 2-32 microg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth, induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone. However, there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone. In tumor bearing mice, the combination of K-ras ASODN and IGF-IR ASODN showed a significant inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically significant difference compared with each alone. It has been found that K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the growth of Patu8988 cells, and induce their apoptosis via reinforcing specific down regulation of K-ras and IGF-IR mRNA and protein expression.