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Objective: To investigate the effect of oxymatrine (OM) on the expression of Gli2 in LTC-14 cells and PANC-1 cells induced by TGF-β1. Methods: LTC-14 cells and PANC-1 cells were divided into four groups:control group, TGF-β1 group, TGF-β1 + OM group, and OM group. The protein was extracted after 12 h, and the expressions of related proteins were detected by Western blot. Results: Compared with control group, the expression of Gli2 in LTC-14 cells was significantly decreased induced by TGF-β1, while the expressions of α-SMA, FN, and CoL-I were significantly increased. The expressions of Gli2, FN, CoL-I, and α-SMA were increased significantly induced by TGF-β1when compared with those of control group in PANC-1 cells, and the expression of Gli2 was inhibited by OM pretreatment in TGF-β1 + OM group compared with TGF-β1 group. OM pretreatment can increase the expression of Gli2 before stimulating with TGF-β1 in TGF-β1 + OM group in comparison with TGF-β1 group. Conclusion: OM plays an protected role in pancreatic fibrosis through promoting the expression of Gli2 in LTC-14 cells.
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Objective: To investigate the effect of oxymatrine (OM) on expression of related molecules in Smad3, Gli1 signaling pathway in PANC-1 cells induced by TGF-β1. Methods: TGF-β1-induced PANC-1 cells were used to establish the pancreatic fibrosis model in vitro, and observe the effects of OM pretreatment on the related molecular expression of Smad3/Gli1. Gli1 and Smad3 RNA interference plasmids were transfected into PANC-1 cells. The protein expression levels of Smad3, Gli1 and α-SMA were measured by Western blotting. The levels of fibronectin (FN) and type I collagen (CoL-I) in the supernatant of cell culture were detected by ELISA. Results: Compared with the control group, the protein expressions of Smad3, Gli1 and α-SMA increased significantly in PANC-1 cells after treated with TGF-β1. The expressions of Gli1, α-SMA, FN, and CoL-I in PANC-1 cells decreased significantly after Gli1 RNA interference plasmid transfection compared with TGF-β1 induced group. The expression of Smad3, Gli1, α-SMA, FN, and CoL-I also decreased significantly in PANC-1 cells after Smad3 RNA interference plasmid transfection compared with TGF-β1 group. Conclusion: OM could prevent pancreatic fibrosis by regulating TGF-β1/Smad3/Gli1 signaling pathway in PANC-1 cells.
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OBJECTIVE:To study the effects of ginsenoside CK combined with 5-fluorouracil (5-FU) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of human pancreatic cancer PANC-1 cells. METHODS:PANC-1 cells of logarithmic growth phase were randomly divided into blank control group,ginsenoside CK group (30 mg/L),5-FU group (25 mg/L)and combination group(ginsenoside CK 30 mg/L+5-FU 25 mg/L). MTT method was used to detect the cell proliferation in-hibition rate in each group after 24,48,72 h;flow cytometry was used to detect the cell apoptosis rate after 48 h;enzyme-linked immunosorbent assay was used to detect the fibronectin expression in cells after 24,48,72,96 h;and Western blot was used to detect the expressions of vimentin,N-cadherin,E-cadherin,protein kinase(Akt)and phosphorylated Akt(p-Akt)protein in cells after 48 h. RESULTS:Compared with blank control group,the cell proliferation inhibition rate,early and late apoptotic rates,pro-tein expression level of E-cadherin in ginsenoside CK group,5-FU group and combination group were obviously increased (P<0.05),while the protein expression levels of fibronectin,vimentin,N-cadherin,and p-Akt/Akt levels were obviously decreased (P<0.05);the effects of above-mentioned indexes in combination group were superior to ginsenoside CK group and 5-FU group (P<0.05). CONCLUSIONS:Both ginsenoside CK and 5-FU can inhibit the proliferation of PANC-1 cells,induce their apoptosis and inhibit EMT,which may be associated with inhibiting phosphatidylinositol 3-kinase/Akt pathway. In addition,the combination of ginsenoside CK and 5-FU can produce a better effect.
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AIM: To observe the effect of receptor-interacting protein 2 (Rip2) overexpression on human pan-creatic cancer cell line Panc-1.METHODS: pEGFP-C2 and pEGFP-Rip2 plasmids were respectively transfected into the Panc-1 cells using JetPRIME reagent.The cells were divided into control group, pEGFP-C2 group and pEGFP-Rip2 group. The apoptosis in the cells was detected 48 h after transfection by flow cytometry.Rip2 level and the expression of apoptosis-related proteins, Bax, cytoplasmic cytochrome c ( Cyt-c) and Bcl-2, were analyzed by Western blot.The activity of caspase-3 was measured by colorimetric method.RESULTS: Rip2 protein expression significantly increased in the cells transfected with control and pEGFP-C2 plasmids.The apoptotic rate in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group, whereas no significant difference of apoptotic rate was observed between control group and pEGFP-C2 group.The protein expression of Bax and cytoplasmic Cyt-c was remarkably increased and the protein expression of Bcl-2 was obviously decreased in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group.The activity of caspase-3 in pEGFP-Rip2 group was obviously increased as compared with control group and pEGFP-C2 group.CON-CLUSION: Overexpression of Rip2 is able to induce apoptosis in the Panc-1 cells, and the mechanism may be related to the up-regulation of Bax and cytoplasmic Cyt-c protein expression, down-regulation of Bcl-2 protein expression and en-hancement of caspase-3 activity, thus activating intrinsic apoptotic pathway.
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Objective To investigate the effects of β-Elemene on the apoptosis of human pancreatic cancer Panc-1 cells; To discuss its mechanism of action. Methods β-Elemene (10, 20, 40, 80, 160 μg/mL) were incubated to the Panc-1 cells in vitro cultured for 24 h, 48 h and 72h, and trypan blue refusal method was used to detect cell inhibition rate;Apoptosis rate was measured by TUNEL; Hoechst33258 fluorescent staining was used to observe the changes of the nucleus. The activity of Caspase-3, 8 and 9 were detected by ELISA. Western blot was used to detect the expressions of Fas, FasL and Cyt c and AIF. Results The activity of Panc-1 cells was obviously inhibited time/concentration dependent inhibition (P<0.05, P<0.01), and the apoptosis rate increased after incubated with β-Elemene (P<0.01, P<0.001) after incubated with β-Elemene for 24 h, 48 h and 72 h; After giving β-Elemene 72 h, Panc-1 cells nucleus were broken obviously, and chromatin condensed and showed strong blue fluorescence, along with of apoptotic bodies; After incubated with β-Elemene for 48 h, Caspase-3, 8 and 9 activity significantly increased (P<0.05, P<0.01); protein expressions of Fas, FasL, Cyt c and AIF were significantly enhanced (P<0.05, P<0.01, P<0.001). Conclusion β-Elemene can inhibit Panc-1 cell proliferation, induce apoptosis, and the mechanism may be related to activating cell death receptor pathway and mitochondrial apoptosis pathway to play anti-tumor effects.
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The stiffness of cancer cells is attributable to intermediate filaments such as keratin. Perinuclear reorganization via phosphorylation of specific serine residue in keratin is implicated in the deformability of metastatic cancer cells including the human pancreatic carcinoma cell line (PANC-1). 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter and protein kinase C (PKC) activator. However, its effects on phosphorylation and reorganization of keratin 8 (K8) are not well known. Therefore, we examined the underlying mechanism and effect of TPA on K8 phosphorylation and reorganization. TPA induced phosphorylation and reorganization of K8 and transglutaminase-2 (Tgase-2) expression in a time- and dose-dependent manner in PANC-1 cells. These effects peaked after 45 min and 100 nM of TPA treatment. We next investigated, using cystamine (CTM), Tgase inhibitor, and Tgase-2 gene silencing, Tgase-2's possible involvement in TPA-induced K8 phosphorylation and reorganization. We found that Tgase-2 gene silencing inhibited K8 phosphorylation and reorganization in PANC-1 cells. Tgase-2 gene silencing, we additionally discovered, suppressed TPA-induced migration of PANC-1 cells and Tgase-2 overexpression induced migration of PANC-1 cells. Overall, these results suggested that TPA induced K8 phosphorylation and reorganization via Tgase-2 expression in PANC-1 cells.
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Humans , Cell Line , Cystamine , Gene Silencing , Intermediate Filaments , Keratin-8 , Phosphorylation , Protein Kinase C , SerineABSTRACT
Objective To investigate the combination effect of curcumin and γ-ray irradiation on PANC-1 cells in vitro.Methods PANC-1 cells were exposed to γ-rays in the presence or absence of curcumin.MTT assay was performed to evaluate cell viability.The expression of P21 was evaluated with RT-PCR and Western blot.Cell cycle distribution and apoptosis were tested by flow cytometry.Results Compared with the γ-ray irradiation group,combination treatment of curcumin and irradiation decreased the cell viability (t =6.72,P ＜ 0.01) and increased the percentage of cells in S-phase (t =4.78,P ＜ 0.05),apoptosis rate (t =6.58,P ＜ 0.01),P21 protein and mRNA expression (t =5.72,5.63,P ＜ 0.01) in PANC-1 cells.Conclusions Curcumin increases the radiosensitivity of PANC-1 cells,which may have clinical implication on radiotherapy of pancreatic cancer.
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Objective To investigate the effect of resveratrol on the proliferation and invasion of human pancreatic cancer PANC-1 cells. Methods Five groups including blank control group, 0. 1% dimethylsulfoxide (DMSO) group and resveratrol groups (50, 100, 200 μmol/L) were established. The proliferation of PANC-1 cells was detected by MTT assay. The apoptosis and cell cycle change were analyzed by flow cytometry. The invasive ability of PANC-1 cells was observed with a Transwell cell culture chamber. The expressions of Bax, Bcl-2,matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) of the PANC-1 cells were assayed by real-time quantitative PCR and Western blot. All data were analyzed using the analysis of variance. Results ( 1 ) The inhibition rate of resveratrol on the proliferation of PANC-1 cells was 0 in the blank control group, 3.25% ±0.42% in the 0. 1% DMSO group, 13.23% ± 1.68% in the 50 μmol/L of resveratrol group, 42.25% ± 3.20% in the 100 μmol/L of resveratrol group, and 56.94% ±5.31% in the 200 μmol/L of resveratrol group. There was a significant difference in the inhibition rate among the five groups (F=460. 10, P<0.05). (2) The apoptosis rate was 0.05% ±0.03% in the blank control group, 3.39% ± 1.77% in the 0. 1% DMSO group, 6.92% ± 1.85% in the 50 μmol/L of resveratrol group, 19.05% ± 2.01% in the 100 μmol/L of resveratrol group, and 27. 17% ±6.43% in the 200 μmol/L of resveratrol group. There was a significant difference in the apoptosis rate among the five groups (F = 38.84, P < 0.05). (3) There was no significant effect of 0. 1% DMSO on the cell cycle of PANC-1 cells. The number of PANC-1 cells in the G0/G1 and S phase was increased. (4) The average number of invading PANC-1 cells was 61 ± 13 in the blank control group, 54 ± 13 in the 0. 1% DMSO group, 48 ± 15 in the 50 μmol/L of resveratrol group, 23 ±6 in the 100 μ mol/L of resveratrol group and 18 ±7 in the 200 μmol/L of resveratrol group. There was a significant difference in the number of invading PANC-1 cells among the five groups (F = 69.08, P < 0.05 ). (5) There were up-regulated mRNA and protein expressions of Bax and down-regulated mRNA and protein expressions of Bcl-2, and the expressions of MMP-2 and MMP-9 of the PANC-1 cells were inhibited in the resveratrol groups. The changes of the protein expressions of Bax, Bcl-2, MMP-2, MMP-9 were consistent with the changes of the mRNA expressions of the four indexes. Conclusion Resveratrol can significantly inhibit the proliferation and invasion, as well as induce apoptosis of PANC-1 cells in vitro.