Tanshinone IIA Could Inhibit Pancreatic Cancer BxPC-3 Cells through Increasing PERK, ATF6, Caspase-12 and CHOP Expression to Induce Apoptosis

Tanshinone IIA (Tan-IIA) is extracted from Dan-Shen. Tan-IIA could inhibit human pancreatic cancer BxPC-3 cells through decreasing TCTP, Mcl-1 and Bcl-xl expression in vitro. Our previous study showed that Tan-IIA can inhibit hepatocellular carcinoma hep-J5 cells and human breast cancer BT-20 cells through inducing endoplasmic reticulum (ER) stress. In the present study, we investigated the ER stress related protein expressions in human pancreatic cancer BxPC3 cells were treated with Tan-IIA. The ER stress related protein expressions in human pancreatic cancer BxPC-3 cells were evaluated by western blotting. The results showed that Tan-IIA can increase the protein expressions of PERK, ATF6, Caspase-12 and CHOP, but decrease Bip, PDI, Calnexin, Calreticulin and Bcl-2 expression. These findings indicated that Tan-IIA can inhibit human pancreatic cancer BxPC-3 cells by inducing ER stress to induce apoptosis.

Pancreatic stellate cells promote proliferation and invasiveness of human pancreatic cancer cells via galectin-3

AIM: To investigate the role of pancreatic stellate cells (PSCs) and galectin-3 (GAL-3) in the proliferation and infiltration of pancreatic cancer cell line SW1990. METHODS: Human pancreatic cancer cell line SW1990 and PSCs were cultured in vitro . Supernatant fluid of cultured PSCs and SW1990 cells was collected. Expression of GAL-3 in SW1990 cells and PSCs was detected by ELISA, RT-PCR and Western blotting. Proliferation of cultured PSCs and SW1990 cells was measured by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Infiltration of SW1990 cells was detected by a cell infiltration kit. RESULTS: SW1990 cells expressed GAL-3 and this was up-regulated by the supernatant fluid of cultured PSCs. PSCs did not express GAL-3. SW1990 cells stimulated proliferation of PSCs via GAL-3. GAL-3 antibody inhibited SW1990 cell proliferation, while the supernatant fluid of PSCs stimulated proliferation of SW1990 cells through interaction with GAL-3 protein. The supernatant fluid of PSCs enhanced the invasiveness of SW1990 cells through interaction with GAL-3. CONCLUSION: GAL-3 and PSCs were involved in the proliferation and infiltration process of pancreatic cancer cells.

Dendritic cells engineered to secrete anti-Dc R3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro

AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a Cr-51 releasing test. T cell responses induced by RNAloaded DCs were analyzed by measuring cytokine levels, including IFN-gamma, IL-10, IL4, TNF-alpha and IL-12. RESULTS The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DCtumor- anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-gamma and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.

Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro

AIM:To elucidate the role of dickkopf3(Dkk3)in human pancreatic cancer cell growth.METHODS:Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymerase chain reaction(realtime RTPCR),Western blotting and immunofluorescence.Methylation of the Dkk3 promoter sequence was examined by methylationspecific polymerase chain reaction(MSP)and Dkk3 mRNA expression was determined by realtime RTPCR after 5aza2'deoxycytidine(5azadC)treatment.The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96?AQueous One Solution Cell Proliferation Assay(MTS)after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells.The expression ofβcatenin,phosphorylated extracellular signalregulated protein kinases(pERK)and extracellular signalregulated protein kinases(ERK)was also examined by realtime RTPCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells.RESULTS:The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested.The Dkk3 promoter sequence was methylated in the MIA PaCa2 and AsPC1 cell lines,which showed reduced Dkk3 expression.These two cell lines,which initially had a methylated Dkk3 promoter,showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent,5azadC,treatment(P<0.05 or P<0.01).When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa2 cells,the ability of cells to proliferate decreased(P<0.01),and the expression ofβcatenin and pERK was downregulated(P<0.01).Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmidtransfected cells.CONCLUSION:Our findings,for the first time,implicate Dkk3 as a tumor suppressor in human pancreatic cancer,through the downregulation ofβcatenin expression via the ERKmediated pathway.

ROLE OF PANCREATIC STELLATE CELLS AND GALECTIN-3 ON PROLIFERATION AND INFILTRATION OF HUMAN PANCREATIC CANCER CELL LINE SW1990

Objective To investigate the role of pancreatic stellate cells (PSCs) and galectin-3 (GAL-3) on the proliferation and infiltration of pancreatic cancer cell line SW1990. Methods Human pancreatic cancer cell line SW1990 and PSCs were cultured in vitro. Supernatant of cultured PSCs and SW1990 cells was collected. Expressions of GAL-3 in SW1990 cells and PSCs were detected by ELISA, RT-PCR and Western blot. The proliferation of those cultured PSCs and SW1990 cells were measured by MTT assay and flowcytometry. Infiltration of SW1990 cells was detected by cell infiltration kit. Results SW1990 cells expressed GAL-3 and the expression was up-regulated by the supernatant fluid of cultured PSCs. PSCs did not express GAL-3. SW1990 cells could stimulate the proliferation of PSCs via GAL-3. GAL-3 antibody could inhibit SW1990 cells proliferation and infiltration, which indicated that supernatant of PSCs might stimulate the proliferation of SW1990 cells through the interaction with GAL-3 protein. The supernatant fluid of PSCs could enhance the invasiveness of SW1990 cells through the interaction with GAL-3. Conclusion GAL-3 and PSCs was involved in the proliferation and infiltration process of pancreatic cancer.

Effects of ginsenoside Rh2 on growth and migration of pancreatic cancer cells

AIM:To investigate the effects of ginsenoside Rh2 on the human pancreatic cancer cell line Bxpc-3.METHODS:The human pancreatic cancer cell line Bxpc-3 was cultured in vitro and treated with or without ginsenoside Rh2.Growth rates for Bxpc-3 cells were assessed by methyl thiazolyl tetrazolium(MTT) and colony formation assays.Cell cycle changes were analyzed by flow cytometry.Apoptosis was measured by flow cytometry and Hoechst 33258 fluorescence staining.A scratch assay and a Matrigel invasion assay were used to detect cell migration and invasion.Expression of Bax,Bcl-2,survivin,cyclin D1,matrix metalloproteinase(MMP)-2,MMP-9,cleaved caspase-3,caspase-8,and caspase-9 mRNA were determined by reverse transcriptase-polymerase chain reaction(RT-PCR).Bax,Bcl-2,survivin,cyclin D1,cleaved caspase-3,caspase-8 and caspase-9 protein levels were examined by western blotting.Expression of MMP-2 and MMP-9 proteins in culture supernatants were determined by enzymelinked immunosorbent assay(ELISA).RESULTS:Rh2 significantly inhibited Bxpc-3 cell proliferation in a dose-and time-dependent manner,as evaluated by the MTT(P < 0.05) and colony formation assays(P < 0.05).Compared to the control group,Rh2 significantly increased the percentage of Bxpc-3 cells in the G 0 /G 1 phase from 43.32% ± 2.17% to 71.32% ± 1.16%,which was accompanied by a decrease in S phase(from 50.86% ± 1.29% to 28.48% ± 1.18%) and G 2 /M phase(from 5.81% ± 1.19% to 0.20% ± 0.05%) in a dose-dependent manner(P < 0.05),suggesting that Rh2 arrested cell cycle progression at the G 0 /G 1 phase,as measured by flow cytometry.Compared to the control group,cells treated with Rh2 showed significantly higher apoptosis ratios in a dosedependent manner(percentage of early apoptotic cells:from 5.29% ± 2.28% to 38.90% ± 3.42%(F = 56.20,P < 0.05);percentage of late apoptotic cells:from 4.58% ± 1.42% to 36.32% ± 2.73%(F = 86.70,P < 0.05).Rh2 inhibited Bxpc-3 cell migration and invasion,as detected by scratch wound healing assay and Matrigel invasion assay [percentages of scratch wound healing for 12 h,24 h and 48 h(control vs experimental group):37.3% ± 4.8%vs 18.30% ± 1.65%,58.7% ± 3.5% vs 38.00% ± 4.09% and 93.83% ± 4.65% vs 65.50% ± 4.09%,respectively;t = 6.489,t = 6.656 and t = 7.926,respectively,P < 0.05;the number of cells invading at various concentrations(0 μmol/L,35 μmol/L,45 μmol/L and 55 μmol/L):81.10 ± 9.55,46.40 ± 6.95,24.70 ± 6.88 and 8.70 ± 3.34,respectively(F = 502.713,P < 0.05)].RT-PCR,western blotting or ELISA showed that mRNA and protein expression of Bax,cleaved caspase-3 and caspase-9 were upregulated(P < 0.05),while mRNA and protein expression of Bcl-2,survivin,cyclin D1,MMP-2 and MMP-9 were downregulated(P < 0.05).CONCLUSION:Ginsenoside Rh2 inhibits proliferation,migration and invasion and induces apoptosis of the human pancreatic cancer cell line Bxpc-3.

Cis-hydroxyproline-induced inhibition of pancreatic cancer cell growth is mediated by endoplasmic reticulum stress

AIM： To investigate the biological effects of cishydroxyproline （CHP） on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms. METHODS： The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase （FAK） was characterized by Western blotting and immunofluorescence. Induction of endoplasmic reticulum （ER） stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 （GRP78） and growth arrest and DNA inducible gene （GADD153）. Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly（ADP-ribose） polymerase （PARP） as well as DNA laddering. RESULTS： In addition to inhibition of proliferation, incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions, followed by a loss of cell adherence. Simultaneously, we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage.CONCLUSION： Our data suggest that the initiation of ER stress machinery by CliP leads to an activation of intracellular proteolytic processes, including caspaseindependent FAK degradation, resulting in damaging pancreatic carcinoma cells.

山姜素介导NF-κB信号通路对胰腺癌细胞BXPC-3增殖、侵袭和炎症反应的影响

目的探讨山姜素介导核因子-κB(NF-κB)信号通路对胰腺癌细胞BXPC-3增殖、侵袭能力和炎症反应的影响。方法将人胰腺癌细胞BXPC-3分为对照组(0μmol/L山姜素)和不同浓度(25、50、100、200、400μmol/L)山姜素处理组,细胞计数试剂盒-8(CCK-8)法、克隆形成实验检测细胞增殖能力,流式细胞术检测细胞周期,Transwell小室法检测细胞侵袭,蛋白免疫印迹法检测相关蛋白表达,酶联免疫吸附(ELISA)法检测炎症因子表达情况。结果CCK-8实验结果显示,与对照组比较,不同浓度(25、50、100、200、400μmol/L)山姜素处理后,胰腺癌细胞BXPC-3的细胞活力显著下降(P<0.05);且山姜素浓度越高、处理时间越长,对BXPC-3细胞活性的抑制作用越明显(P<0.05)。与对照组比较,经50、100、200μmol/L山姜素溶液处理后,细胞的克隆形成率、细胞侵袭能力、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、神经-钙黏素(N-cadherin)、纤维连接蛋白(Fibronectin)、NF-κB亚基p65、基质金属蛋白酶-9(MMP-9)、细胞周期蛋白D1(cyclin D1)蛋白表达水平均明显下降,转化生长因子-β(TGF-β)、IL-10、上皮-钙黏素(E-cadherin)及NF-κB抑制蛋白(IκBα)蛋白表达水平明显上升(P<0.05)。加入激活剂脂多糖(LPS)后,细胞活力、细胞侵袭能力、TNF-α、IL-6、p65、MMP-9、cyclin D1蛋白表达水平明显上升,TGF-β、IL-10、IκBα蛋白表达水平明显下降(P<0.05);而加入NF-κB通路抑制剂Sulfasalazine后上述指标变化相反。结论山姜素可抑制胰腺癌细胞BXPC-3的增殖、侵袭能力和炎症反应,其机制可能与介导NF-κB信号通路有关。

Novel therapeutic targets for pancreatic cancer

Pancreatic cancer has become the fourth leading cause of cancer death in the last two decades. Only 3%-15% of patients diagnosed with pancreatic cancer had 5 year survival rate. Drug resistance, high metastasis, poor prognosis and tumour relapse contributed to the malignancies and difficulties in treating pancreatic cancer. The current standard chemotherapy for pancreatic cancer is gemcitabine, however its efficacy is far from satisfactory, one of the reasons is due to the complex tumour microenvironment which decreases effective drug delivery to target cancer cell. Studies of the molecular pathology of pancreatic cancer have revealed that activation of KRAS, overexpression of cyclooxygenase-2, inactivation of p16INK4A and loss of p53 activities occurred in pancreatic cancer. Co-administration of gemcitabine and targeting the molecular pathological events happened in pancreatic cancer has brought an enhanced therapeutic effectiveness of gemcitabine. Therefore, studies looking for novel targets in hindering pancreatic tumour growth are emerging rapidly. In order to give a better understanding of the current findings and to seek the direction in future pancreatic cancer research; in this review we will focus on targets suppressing tumour metastatsis and progression, KRAS activated downstream effectors, the relationship of Notch signaling and Nodal/Activin signaling with pancreatic cancer cells, the current findings of non-coding RNAs in inhibiting pancreatic cancer cell proliferation, brief discussion in transcription remodeling by epigenetic modifiers (e.g., HDAC, BMI1, EZH2) and the plausible therapeutic applications of cancer stem cell and hyaluronan in tumour environment.

Targeting tight junctions during epithelial to mesenchymal transition in human pancreatic cancer

Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and-18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin(CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1,-7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition(EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.

Pancreatic cancer organotypics: High throughput, preclinical models for pharmacological agent evaluation

Pancreatic cancer carries a terrible prognosis,as the fourth most common cause of cancer death in the Western world.There is clearly a need for new therapies to treat this disease.One of the reasons no effective treatment has been developed in the past decade may in part,be explained by the diverse influences exerted by the tumour microenvironment.The tumour stroma cross-talk in pancreatic cancer can influence chemotherapy delivery and response rate.Thus,appropriate preclinical in vitro models which can bridge simple 2D in vitro cell based assays and complex in vivo models are required to understand the biology of pancreatic cancer.Here we discuss the evolution of 3D organotypic models,which recapitulare the morphological and functional features of pancreatic ductal adenocarcinoma(PDAC).Organotypic cultures are a valid high throughput preclinical in vitro model that maybe a useful tool to help establish new therapies for PDAC.A huge advantage of the organotypic model system is that any component of the model can be easily modulated in a short timeframe.This allows new therapies that can target the cancer,the stromal compartment or both to be tested in a model that mirrors the in vivo situation.A major challenge for the future is to expand the cellular composition of the organotypic model to further develop a system that mimics the PDAC environment more precisely.We discuss how this challenge is being met to increase our understanding of this terrible disease and develop novel therapies that can improve the prognosis for patients.

CYP3A5 unexpectedly regulates glucose metabolism through the AKT-TXNIP-GLUT1 axis in pancreatic cancer

CYP3A5 is a cytochrome P450(CYP)enzyme that metabolizes drugs and contributes to drug resistance in cancer.However,it remains unclear whether CYP3A5 directly influences cancer progression.In this report,we demonstrate that CYP3A5 regulates glucose metabolism in pancreatic ductal adenocarcinoma.Multi-omics analysis showed that CYP3A5 knockdown re-sults in a decrease in various glucose-related metabolites through its effect on glucose trans-port.A mechanistic study revealed that CYP3A5 enriches the glucose transporter GLUT1 at the plasma membrane by restricting the translation of TXNIP,a negative regulator of GLUT1.Notably,CYP3A5-generated reactive oxygen species were proved to be responsible for atten-uating the AKT-4EBP1-TXNIP signaling pathway.CYP3A5 contributes to cell migration by maintaining high glucose uptake in pancreatic cancer.Taken together,our results,for the first time,reveal a role of CYP3A5 in glucose metabolism in pancreatic ductal adenocarcinoma and identify a novel mechanism that is a potential therapeutic target.

COX-2抑制剂联合survivin反义寡核苷酸抗胰腺癌BxPC-3细胞的效应

目的:探讨COX-2抑制剂和survivin反义寡核苷酸联合应用对胰腺癌BxPC-3细胞的抗肿瘤效应及其可能机制.方法:应用胰腺癌BxPC-3细胞进行研究,将BxPC-3细胞分为4组:A组(对照组),B组(Celecoxib 80μmol/L)组,C组(300 nmol/L survivin ASODN),D组(80μmol/L celecoxib+300 nmol/L survivin ASODN).采用MTT检测细胞增殖.流式细胞仪检测细胞凋亡率,caspase-3试剂盒检测caspase-3活性,并用RT-PCR检测Bcl-2、survivin和Mcl-1的mRNA国的变化.结果:将80μmol/L celecoxib和300 nmol/L survivin反义寡核苷酸单独或联合作用于胰腺癌BxPC-3细胞24h和48h,D组细胞的存活率明显低于B,C组(24h:41.0%±0.4% vs 71.0%±2.2%,63.3%±4.5%;48h:34.2%±1.1% vs 61.6%±1.7%,55.0%±3%;P<0.01).作用24h后,流式细胞仪检测细胞凋亡率显示,D组细胞的凋亡率明显高于B,C组(30.33%±3.49% vs 11.93%±1.17%,22.07%±0.93%;P<0.01).caspase-3活性在B,C,D组明显高于对照组(0.04867±0.0021,0.02967±0.0021,0.08767±0.0042 vs 0.007±0.0001;P<0.01),D组细胞的caspase-3活性明显高于B,C组(0.08767±0.0042 vs 0.04867±0.0021,0.02967±0.0021;P<0.01).用100μmol/L塞来昔布作用于胰腺癌BxPC-3细胞24h后,survivin/β-actin和Mcl-1/β-actin的mRNA的比值明显低于对照组(0.68±0.05 vs 1.05±0.06,P<0.01),而Bcl-2/β-actin的mRNA的比值无明显变化(0.99±0.02 vs 1.07±0.06,P>0.05).结论:联合应用COX-2抑制剂塞来昔布和survivin反义寡核苷酸可明显诱导胰腺癌细胞的凋亡,抑制细胞增殖,并能够明显提高caspase-3活性.COX-2抑制剂塞来昔布诱导细胞凋亡可能通过survivin和Mcl-1途径,而非Bcl-2途径.

冬凌草甲素对胰腺癌BxPC-3细胞Cyclin D/Rb/p16信号蛋白的影响研究

[目的]通过观察冬凌草甲素对胰腺癌BxPC-3细胞形态和DNA变化的影响,研究其对Cyclin D/Rb/p16信号通路中基因表达的调节作用,分析冬凌草甲素对胰腺癌BxPC-3细胞的诱导与Cyclin D/Rb/p16信号通路之间的内在联系,为冬凌草甲素抗肿瘤提供可能的途径,为临床使用提供有利数据。[方法]冬凌草甲素作用BxPC-3细胞后,经瑞氏染色和Hoechst 33258荧光染色观察细胞形态,RT-PCR和实时荧光定量法(realtime PCR)检测Cyclin D/Rb/p16信号通路基因的表达变化;甲基化专一性PCR试剂盒检测p16基因的甲基化。[结果]冬凌草甲素(32μg·mL-1)作用于BxPC-3细胞后,瑞氏染色可见细胞出现典型的凋亡小体,Hoechst33258荧光染色可见BxPC-3细胞出现特征性凋亡变化,冬凌草甲素(32μg·mL-1)作用BxPC-3细胞36 h时,CDK4的表达量达到最低,但p16的表达量达到最高;p16基因有甲基化。[结论]1、冬凌草甲素能诱导BxPC-3细胞形态改变。2、冬凌草甲素在一定程度下调了CDK4基因的表达,上调了p16基因的表达,不能改变p16基因的甲基化,可能通过影响Cyclin D/Rb/p16信号通路而抑制胰腺癌的生长周期。

丁酸钠对胰腺癌BxPC-3细胞凋亡机制的影响

目的:探讨丁酸钠对胰腺癌BxPC-3细胞凋亡的影响及其作用机制。方法:采用MTT法观察不同浓度丁酸钠对BxPC-3细胞增殖的抑制作用;应用流式细胞仪检测丁酸钠作用于BxPC-3细胞后的凋亡情况;TRAP-ELISA法检测细胞端粒酶活性的变化;RT-PCR技术分析人端粒酶逆转录酶(hTERT)、bcl-2 mRNA的表达水平。结果:丁酸钠作用BxPC-3细胞能明显抑制细胞增殖,其作用具有时间和剂量依赖性。丁酸钠处理72 h的BxPC-3细胞凋亡率明显提高(P<0.01),S期细胞比例显著下降(P<0.05),G0/G1期细胞比例显著升高(P<0.05)。经丁酸钠处理的实验组端粒酶活性为0.96±0.12,未经丁酸钠处理的对照组端粒酶活性为4.18±0.34,二者之间差异有显著性(P<0.05)。丁酸钠处理的实验组hTERT mRNA水平为0.168±0.045,与对照组0.685±0.141比较差异有统计学意义(P<0.01),bcl-2实验组水平为0.138±0.034,与对照组0.715±0.026比较有统计学差异。结论:丁酸钠具有强烈诱导胰腺癌BxPC-3细胞凋亡的作用,其发生机制与丁酸钠下调hTERT Bcl-2 mRNA表达水平直接相关。

人胰腺癌细胞株HPAC和BxPC-3中Src/MAPK信号转导途径中pp60c-Src和pERK1/2表达的差异

目的研究人胰腺癌细胞株HPAC和BxPC-3中Src/MAPK信号转导系统,探寻不同类型胰腺癌信号转导通路的差异,为研究靶点药物提供基础。方法将1640培养的HPAC和BxPC-3经细胞计数相同后各分为4组,分别为对照组、TGF-α组、TGF-α+PP2组和TGF-α+PD组。细胞贴壁并达70%融合后,换无血清培养48h。无血清处理作对照,用无血清培养基溶解终浓度为7nmol/L的TGF-α刺激TGF-α组2h,TGF-α+PP2组先加入终浓度为10μmol/L的PP2刺激细胞20min后加入7nmol/L的TGF-α刺激2h,TGF-α+PD组先加入30μmol/L的PD98059刺激细胞20min后加入终浓度为7nmol/L的TGF-α刺激2h。用蛋白印迹方法分析HPAC和BxPC-3的4组细胞pp60c-Src和pERK1/2的表达。结果HPAC细胞pp60c-Src的表达:对照组显著高于TGF-α组、TGF-α+PD组(P<0.05)和TGF-α+PP2组(P<0.01);HPAC细胞pERK1/2的表达:TGF-α组显著高于对照组和TGF-α+PD组(P<0.01),与TGF-α+PP2组相比较差异无统计学意义(P>0.05)。BxPC-3细胞pp60c-Src的表达:TGF-α组显著高于对照组(P<0.05)和TGF-α+PP2组(P<0.01),与TGF-α+PD组相比较差异无统计学意义(P>0.05);BxPC-3细胞pERK1/2的表达:TGF-α组显著高于对照组、TGF-α+PD组和TGF-α+PP2组(P<0.01)。结论HPAC和BxPC-3细胞中Src/MAPK信号途径存在差异。推测在BxPC-3细胞株中c-Src是活化Ras/Raf/MAPK途径的关键信号分子,应进一步研究在HPAC细胞株中c-Src的作用。

辛伐他汀对胰腺癌BxPC-3细胞株Shh相关蛋白的影响

目的探讨辛伐他汀对胰腺癌Bx PC-3细胞株Shh相关蛋白的影响。方法胰腺癌Bx PC-3细胞株随机分成对照组,5-氟尿嘧啶(5-FU)组(50μmol/L),辛伐他汀高、中、低剂量组(20,10,5μmol/L)。采用台盼蓝染色法和MTT法检测胰腺癌Bx PC-3细胞株的生长曲线和增殖抑制率;采用细胞划痕法检测胰腺癌Bx PC-3细胞株运动能力;采用免疫组织化学、RT-PCR法检测胰腺癌Bx PC-3细胞株Shh、Ptch1、Smo、Gli1水平。结果与对照组比较,5-FU组,辛伐他汀高、中剂量组胰腺癌Bx PC-3细胞OD值、运动能力和Shh、Ptch1、Smo、Gli1水平明显降低(P<0.05)。结论辛伐他汀通过抑制Bx PC-3细胞株Shh、Ptch1、Smo、Gli1水平在胰腺癌治疗中发挥积极作用。

生长抑素联合吉西他滨对人胰腺癌BxPC-3细胞增殖抑制作用的研究

目的:初步探讨生长抑素(SST)、吉西他滨(Gem)单药或联合用药对人原位胰腺癌细胞BxPC-3细胞增殖的影响。方法:(1)体外培养人原位胰腺癌细胞BxPC-3。使用不同浓度生长抑素干预24h、48h、72h;不同浓度吉西他滨干预72h;计算出生长抑素及吉西他滨半数抑制浓度(IC50),联合用药进行干预。具体分组:A组:空白对照组,B组:生长抑素IC50浓度,C组:吉西他滨IC50浓度,D组:1/2生长抑素IC50浓度+1/2吉西他滨IC50浓度,干预72h;(2)MTT法检测各浓度药物对细胞增殖抑制的影响。结果:生长抑素及吉西他滨均可抑制人原位胰腺癌细胞BxPC-3的增殖,且与药物的浓度和时间正相关;联合用药组比单药组的抑制作用明显增强。结论:生长抑素及吉西他滨均可抑制人原位胰腺癌细胞BxPC-3的增殖,两者联合作用效应更强。

二甲双胍对胰腺癌BxPC-3细胞恶性表型的影响

目的:探索二甲双胍对胰腺癌BxPC-3细胞恶性表型的影响。方法:以Smad4基因天然缺失型人胰腺癌BxPC-3细胞为对象,采用CCK-8法和流式细胞术分别检测不同剂量二甲双胍(5、10、20 mmol/L)作用24 h后的细胞增殖和凋亡情况,并计算细胞存活率和凋亡率;采用Transwell迁移试验检测不同剂量二甲双胍(10、20 mmol/L)作用24 h后的细胞迁移情况,记录迁移细胞数;采用实时定量聚合酶链反应法和Western blotting法分别检测细胞中钙黏着蛋白E(E-cadherin)、波形蛋白(Vimentin)、补体反应基因32(RGC-32)的mRNA及蛋白表达情况。结果:与对照组和5 mmol/L二甲双胍组比较,10、20 mmol/L二甲双胍组细胞的存活率均显著降低,凋亡率均显著升高,且20 mmol/L二甲双胍组细胞的凋亡率显著高于10 mmol/L二甲双胍组(P<0.05)。与对照组比较,10、20 mmol/L二甲双胍组迁移细胞数均显著减少,且20 mmol/L二甲双胍组显著少于10 mmol/L二甲双胍组(P<0.05);10、20 mmol/L二甲双胍组细胞中E-cadherin mRNA及其蛋白的相对表达量均显著升高,且20 mmol/L二甲双胍组E-cadherin mRNA的相对表达量显著高于10 mmol/L二甲双胍组;10 mmol/L二甲双胍组细胞中Vimentin mRNA,20 mmol/L二甲双胍组细胞中Vimentin mRNA及其蛋白以及10、20 mmol/L二甲双胍组细胞中RGC-32 mRNA及其蛋白的相对表达量均显著降低,且20 mmol/L二甲双胍组Vimentin mRNA及其蛋白、RGC-32 mRNA的相对表达量均显著低于10 mmol/L二甲双胍组(P<0.05或P<0.01)。结论:二甲双胍可剂量依赖性地通过Smad4非依赖性通路抑制BxPC-3细胞的增殖和迁移,促进其凋亡,这可能与胰腺癌细胞上皮间质转化过程以及RGC-32表达受到抑制有关。

细梗香草皂苷联合吉西他滨对胰腺癌细胞BxPC-3抑制作用的研究

目的观察细梗香草皂苷(LC)和吉西他滨(GEM)单药及联合用药对人胰腺癌Bx PC-3细胞增殖和凋亡的影响,探究LC抗胰腺癌的作用机制。方法培养人胰腺癌Bx PC-3细胞时加入不同浓度梯度的LC与GEM,采用MTS法检测细胞增殖抑制率,计算两种药物的半抑制浓度(IC_(50))和联合作用指数(CI)。将细胞分为4组,A组(对照空白)、B组(GEM 1μmol/L)、C组(LC15μg/ml)和D组(GEM 1μmol/L+LC 15μg/ml联合用药),培养后采用AnnexinⅤ-FITC/PI法观察LC与GEM对Bx PC-3细胞的诱导凋亡作用;Western blot法检测抗多聚(ADP-核糖)聚合酶(PARP)、抗半胱氨酸蛋白酶-3(Caspase-3)的表达。结果8~64μg/ml的LC及1.25~20μmol/L的GEM均对Bx PC-3细胞的增殖有抑制作用,且呈浓度依赖性,其IC_(50)分别为16.55μg/ml和1.27μmol/L。15μg/ml的LC与各个浓度的的GEM协同作用最强,CI值为0.53~0.65。B、C、D组的早、晚期凋亡率与A组比较差异均有统计学意义(均P<0.01),且D组与B、C组的早期凋亡率比较差异均有统计学意义(均P<0.01)。C、D组与A组比较,PARP蛋白表达水平明显降低(均P<0.05);B、C、D组与A组比较,Caspase-3蛋白表达水平增加(均P<0.05)。结论 LC有抑制胰腺癌细胞生长的作用,与GEM联合作用效果更好;其作用机制可能是通过激活Caspase-3表达,分解PARP,从而诱导细胞凋亡。