Triptolide to induce cell death of pancreatic cancer cells via inhibition of 14-3-3γ expression.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 425-425
Author(s):  
Wei Wang ◽  
Jinbing Luo ◽  
Yinghui Liang ◽  
Yubin Chen ◽  
Wenjie Lin
Keyword(s):  
Pancreatic Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Caspase 8 ◽  
Down Regulation ◽  
Pancreatic Cancer Cells ◽  
Growth Inhibitory ◽  
Induced Apoptosis

425 Background: Pancreatic cancer is one of the malignant tumors which exhibit resistance to chemotherapy. Gemcitabine-based therapy is a standard for advanced pancreatic cancer though it brings severe side-effect and average median survival is only 6 months. Hence increasing interest has focused on new agent with targeted therapies. Here we investigated the growth-inhibitory and apoptotic effect of triptolide, a diterpenoid triepoxide, and the role of 14-3-3γ expression in the apoptotic pathway induced by triptolide in human pancreatic cancer cells (AsPC-1 and PANC-1). Methods: Cell proliferation was measured by SRB, apoptotic cells were assessed by flow cytometry for Annexin V/PI staining and western blot for cleaved caspase-8, 9, 3 and fluorescent substrate assay for activities of caspase-8, 9, 3. To explore further mechanism of triptolide triggering death receptor pathway, specific siRNA targeted for 14-3-3γ was used to knock down 14-3-3γ expression measured by ELISA. In vivo, AsPC-1 xenografts in the absence or presence of stable down-regulation of 14-3-3γ expression by RNAi were treated with triptolide for 4 weeks and the tumor growth was compared, tumor samples were tested by ELISA and western blot for 14-3-3γ level. Results: Triptolide inhibits the proliferation at extremely low concentrations (12.5-50 nM) and induces apoptosis of pancreatic cancer cells through activating the caspase cascade associated with Bid cleavage. Moreover triptolide inhibited 14-3-3γ expression at dose and time-dependent manner and 14-3-3γ down-regulation sensitized cells to triptolide-induced apoptosis. Likewise, in vivo experiment of AsPC-1 xenografts, stable down-regulation of 14-3-3γ expression by RNAi significantly enhances triptolide-induced apoptosis and tumor growth delay. Conclusions: Triptolide exerted significant growth inhibitory effects and induced apoptosis in vitro and in vivo. Triptolide may have a potential to be an effective agent against pancreatic cancer and its mechanism of action is mediated by the inhibition of 14-3-3γ expression. The role of 14-3-3γ expression involved in resistance to apoptosis pathway make it be a potential therapeutic target in pancreatic cancer.

Use of decoy receptor 3 to block gemcitabine-induced apoptosis via binding with TRAIL in pancreatic cancer.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 238-238
Author(s):  
Wei Wang ◽  
Wenjie Lin ◽  
Xin Zhuang ◽  
Qingda Wang ◽  
Jinbing Luo
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Cancer Cells ◽  
Growth Delay ◽  
Tumor Growth Delay ◽  
Down Regulation ◽  
In Vivo Experiment ◽  
Pancreatic Cancer Cells ◽  
Induced Apoptosis

238 Background: Most of human pancreatic cancer cell lines are resistant to chemotherapeutics-induced apoptosis including gemcitabine. This study is to address the role of decoy receptor3 (DcR3) in pancreatic cancer resistant to chemotherapeutics. Methods: siRNA targeted for DcR3 was used to down-regulate DcR3 expression in pancreatic cancer cells. On exploring for the pathway of DcR3 blocking Gemcitabine-induced apoptosis, up or down-regulated DcR3 level of pancreatic cancer cells were treated with Gemcitabine and apoptosis susceptibility was evaluated with apoptosis analysis. To investigate whether DcR3 is involved in TRAIL, FasL or LIGHT-mediated apoptosis, PARP and apoptotic cells staining were determined under different DcR3 level. Through ELISA-like binding analysis, immunoprecipitation and FACS, TRAIL binding with DcR3 was tested. In vivo experiment, it is to investigate whether down-regulation of DcR3 affects gemcitabine-induced apoptosis and tumor growth delay. Results: It demonstrates that DcR3 down-regulation sensitizes cells to gemcitabine-induced apoptosis and additional DcR3 level can impair gemcitabine-induced apoptosis in both of cells. It is proved that DcR3 blocks gemcitabine-induced apoptosis via binding with TRAIL which was thought not to bind with DcR3 previously. Furthermore, Regulation of DcR3 changes the amount of membrane-bound TRAIL which is correlated with gemcitabine-stimulation. In vivo experiment of AsPC-1 xenografts, stable down-regulation of DcR3 significantly enhances gemcitabine-induced apoptosis and tumor growth delay. Conclusions: DcR3 may play important role resistant to gemcitabine-induced apoptosis via binding with TRAIL in some of pancreatic cancer cells lines. Targeting DcR3 represents a promising strategy to enhance the anti-tumor activity of chemo-agent in pancreatic cancer, which has important clinical implications.

Effect of KRAS and P-STAT3 inhibition by SBT-100 on gemcitabine and pancreatic cancer growth in vivo.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e15727-e15727
Author(s):  
Sunanda Singh ◽  
Genoveva Murillo ◽  
Avani Singh ◽  
Samara Singh ◽  
Meenakshi S Parihar ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Targeted Therapy ◽  
Western Blot ◽  
Cancer Cells ◽  
Mtt Assay ◽  
Blot Analysis ◽  
Cancer Growth ◽  
Pancreatic Cancers ◽  
Pancreatic Cancer Cells

e15727 Background: Over 90% of pancreatic cancers have KRAS mutations and hyper-expression of P-STAT3 oncoproteins, which if specifically targeted may help treatment of pancreatic cancers. Singh Biotechnology’s proprietary technology engineered SBT-100, a single domain antibody that is bispecific for KRAS & STAT3, which can cross the cell membranes and bind to these intracellular oncoproteins. Combining this targeted therapy with an established chemotherapy, such as gemcitabine, may improve patient’s response to treatment. Methods: Human pancreatic cancer cells (PANC-1 and BX-PC3) were used. Biacore assay demonstrates SBT-100 binding to KRAS, KRAS (G12D), and STAT3. Immunoprecipitation (IP) and western blot analysis confirmed binding to STAT3 by SBT-100. Pancreatic cancer cells were treated at varying doses of SBT-100 ranging from 0µg/ml to 200µg/ml ± gemcitabine, and after 72 hours growth inhibition was determined by a MTT assay. PANC-1 tumors were grown in athymic nude mice, divided into four groups and staged to a range of 100-150mm3 before treatment. Groups were: vehicle only, SBT-100, gemcitabine, and SBT-100 & gemcitabine. Animals received treatments for 14 days, then monitored for 7 days. Results: Biacore study shows SBT-100 binds KRAS with an affinity of 10-9M, KRAS (G12D) with 10-8M, and STAT3 with 10-8M. IP and western blot analysis demonstrates that SBT-100 binds P-STAT3. MTT assay demonstrates SBT-100 inhibits the growth of PANC-1 and BX-PC3 (p < 0.001). In PANC1 cells a combination of SBT-100 & gemcitabine demonstrates synergism in inhibiting growth of PANC-1, even at 1/8th the gemcitabine IC50 concentration. PANC-1 xenograft study demonstrates that combination therapy of SBT-100 & gemcitabine is superior to either SBT-100 or gemcitabine alone. Compared to the vehicle group, SBT-100 & gemcitabine is far superior (p < 0.001) and gives statistically significant suppression of pancreatic cancer growth in vivo. Conclusions: Targeted therapy for KRAS and P-STAT3 expressing tumors with SBT-100 & gemcitabine is synergistic for the treatment of pancreatic cancer. This study suggests that synergism maybe achieved with lower doses of gemcitabine, thereby reducing toxicity in patients.

scholarly journals PR55α regulatory subunit of PP2A inhibits the MOB1/LATS cascade and activates YAP in pancreatic cancer cells

Oncogenesis ◽  
2019 ◽  
Vol 8 (11) ◽  
Author(s):  
Ashley L. Hein ◽  
Nichole D. Brandquist ◽  
Caroline Y. Ouellette ◽  
Parthasarathy Seshacharyulu ◽  
Charles A. Enke ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Cellular Localization ◽  
Activation Mechanism ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Anchorage Independent Growth ◽  
Pancreatic Cancer Cells

Abstract PP2A holoenzyme complexes are responsible for the majority of Ser/Thr phosphatase activities in human cells. Each PP2A consists of a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (B). While the A and C subunits each exists only in two highly conserved isoforms, a large number of B subunits share no homology, which determines PP2A substrate specificity and cellular localization. It is anticipated that different PP2A holoenzymes play distinct roles in cellular signaling networks, whereas PP2A has only generally been defined as a putative tumor suppressor, which is mostly based on the loss-of-function studies using pharmacological or biological inhibitors for the highly conserved A or C subunit of PP2A. Recent studies of specific pathways indicate that some PP2A complexes also possess tumor-promoting functions. We have previously reported an essential role of PR55α, a PP2A regulatory subunit, in the support of oncogenic phenotypes, including in vivo tumorigenicity/metastasis of pancreatic cancer cells. In this report, we have elucidated a novel role of PR55α-regulated PP2A in the activation of YAP oncoprotein, whose function is required for anchorage-independent growth during oncogenesis of solid tumors. Our data show two lines of YAP regulation by PR55α: (1) PR55α inhibits the MOB1-triggered autoactivation of LATS1/2 kinases, the core member of the Hippo pathway that inhibits YAP by inducing its proteasomal degradation and cytoplasmic retention and (2) PR55α directly interacts with and regulates YAP itself. Accordingly, PR55α is essential for YAP-promoted gene transcriptions, as well as for anchorage-independent growth, in which YAP plays a key role. In summary, current findings demonstrate a novel YAP activation mechanism based on the PR55α-regulated PP2A phosphatase.

Role of the eIF4E binding protein 4E-BP1 in regulation of the sensitivity of human pancreatic cancer cells to TRAIL and celastrol-induced apoptosis

2013 ◽  
Vol 105 (9) ◽  
pp. 414-429 ◽  
Author(s):  
Reka Chakravarthy ◽  
Michael J. Clemens ◽  
Grisha Pirianov ◽  
Nectarios Perdios ◽  
Satvinder Mudan ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Binding Protein ◽  
Human Pancreatic Cancer ◽  
Pancreatic Cancer Cells ◽  
Induced Apoptosis

scholarly journals Restore of miR-541 resensitizes pancreatic cancer cells to gemcitabine-induced apoptosis through suppression of HAX-1

2020 ◽  
Author(s):  
Hong Liu ◽  
Xuemei Gan ◽  
Jun Zhang ◽  
Xingdiao Zhang ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Potential Strategy ◽  
Induced Apoptosis ◽  
Mtt Assays

Abstract Background: MiR-541 acts as a tumor suppressor in some cancers. However, the role of miR-541 in regulating the chemosensitivity to cancer cells is still unclear. The aim of this study is to explore the effect of miR-541 on chemoresistance of pancreatic cancer (PCa) cells to gemcitabine-induced apoptosis.Methods: Gemcitabine-resistant Panc-1 and Capan-2 PCa cell lines (Panc-1/R and Capan-2/R) were established through long term exposure to gemcitabine. Effect of miR-541 on changing the sensitivity of Panc-1/R and Capan-2/R to gemcitabine-induced cytotoxicity was evaluated by MTT assays. Regulation of miR-541 on HAX-1 was confirmed by bioinformatics, western blot analysis and luciferase reporter assays. Cell apoptosis and mitochondrial membrane potential (MMP) was measured by flow cytometry analysis.Results: Comparison with Panc-1 and Capan-2, downregulation of miR-541 was observed in Panc-1/R and Capan-2/R cells. Overexpression of miR-541 was found to increase the cytotoxicity of gemcitabine to Panc-1/R and Capan-2/R cells. However, transfection with HAX-1 plasmid can abolish the effect of miR-541 on gemcitabine-induced cytotoxicity against Panc-1/R and Capan-2/R.Conclusion: Downregulation of miR-541 is responsible for development of gemcitabine resistance in PCa. Overexpression of miR-541 may represent a potential strategy to reverse the chemoresistance of PCa.

scholarly journals Agrimoniin Sensitizes Pancreatic Cancer to Apoptosis through ROS-Mediated Energy Metabolism Dysfunction

2021 ◽  
Author(s):  
Xiandong Zhu ◽  
Feixiang Duan ◽  
Yongqiang Wang ◽  
Hewei Zhang ◽  
Xiaowu Wang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Energy Metabolism ◽  
Western Blot ◽  
Cancer Cells ◽  
Mitochondrial Membrane ◽  
Cell Metabolism ◽  
Pancreatic Cancer Cells

Abstract Background Agrimoniin, a polyphenol compounds isolated from Agrimonia pilosa ledeb, has antiviral, antimicrobial, and anticancer activities in vivo and in vitro. However, its molecular mechanism in pancreatic cancer remains to be determined. Methods The proliferation was detected by colony formation, cell proliferation and toxicity, and real-time cell analysis techniques. The apoptosis was detected by flow cytometry and Western blot. Flow cytometry was used to measure the level of reactive oxygen species (ROS) and apoptosis. The level of intracellular ROS or mitochondrial membrane potential was measured with a DCFH-DA or JC-1 probe. Cell metabolism assays were analyzed and evaluated by using Agilent Seahorse Bioscience XF96 Extracellular Flux Analyzer. The target proteins were analyzed by Western blot. Subcutaneous cancer models in nude mice were established to evaluate the anticancer effects in vivo. Results Agrimoniin inhibited cell growth and promoted cell apoptosis by regulating cell metabolism in pancreatic cancer cells. Agrimoniin increased the ROS level in pancreatic cancer cells by suppressing Nrf2-dependent ROS scavenging system and disrupting normal mitochondrial membrane potential. We also found that agrimoniin significantly disrupted mitochondrial function and reduced the protein expression of mTOR/HIF-1α pathway and subsequently decreased oxygen consumption rate and extracellular acidification rate. Eventually, agrimoniin affected intracellular energy metabolism and induced apoptosis of pancreatic cancer cells. Conclusions These findings reveal the novel function of agrimoniin in promoting apoptosis of pancreatic cancer cells through mediating energy metabolism dysfunction.

scholarly journals CXCR4/Let-7a Axis Regulates Metastasis and Chemoresistance of Pancreatic Cancer Cells Through Targeting HMGA2

2017 ◽  
Vol 43 (2) ◽  
pp. 840-851 ◽  
Author(s):  
Guangfa Xiao ◽  
Xitao Wang ◽  
Yaqun Yu
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Western Blot ◽  
Cancer Cells ◽  
Molecular Mechanism ◽  
Expression Level ◽  
Pancreatic Cancer Cells

Background/Aims: Pancreatic cancer cells (PCC) is one of the most risky cancers and gemcitabine (GEM) is the standard first-line drug for treating PCC. The PCC will develop drug resistance to GEM after a period of treatment. However, the detailed molecular mechanism of pathogenesis and drug resistance remains unresolved. Methods: we employed qRT-PCR and western blot to examine the expression level of CXCR4, let-7a and HMGA2. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The expression level of epithelial marker E-cadherin and mesenthymal marker N-cadherin was detected by western blot. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Results: we first proved that CXCR4 negatively regulated let-7a in PCC. Next, let-7a was confirmed to play crucial role in tumorigenesis, metastasis and drug resistance of pancreatic cancer cells Bxpc-3 and Panc-1 in vitro and in vivo. Finally, we identified HMGA2 as important downsteam target of let-7a in PCC and overexpression of HMGA2 restores cell proliferation, metastasis and chemosensitivity of GEM inhibited by let-7a. Conlusion: Taken together, we show an important signaling pathway involved in pathogenesis and drug resistance of PCC, thereby providing deeper insight into molecular mechanism by which CXCR4/let-7a regulates tumorigenesis and drug resistance of PCC. These findings will help us develop new strategies for diagnosis and treatment of PCC.

scholarly journals Exogenous HMGB1 Promotes the Proliferation and Metastasis of Pancreatic Cancer Cells

2020 ◽  
Author(s):  
Li Zhu ◽  
Wenli Qiu ◽  
Lian Song ◽  
Tao You ◽  
Shuai Ren ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Vital Role ◽  
Enzyme Linked Immunosorbent Assay ◽  
Pancreatic Cancer Cells ◽  
Proliferation And Metastasis ◽  
Gsk 3Β ◽  
Related Proteins

Abstract Backgroud: Exogenous HMGB1 play a vital role in tumor recurrence，which reside in the tumor microenvironment. However, the mechanism of action is still unknow. We studied the proliferation and metastasis effect of exogenous HMGB1 on human SW1990 and Panc-1 cells after radiotherapy and explored the possible molecular mechanism.Materials and Methods: Residual Panc-1 cells and SW1990 cells were isolated after radiotherapy. The supernatant after radiotherapy was collected. The relative expression of HMGB1 was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). The images of normal pancreatic cancer cells and residual pancreatic cancer cells were collected by Electron microscope (EMS). The proliferation of pancreatic cancer cells which were treated with difference groups was measured by Carboxy fluorescein succinimidylester (CFSE). The migration rates were measured by wound healing assays. The expression of related proteins were detected by Western Blot. In vivo, the subcutaneous pancreatic tumor models of nude mice were created and therapeutic capabilities were tested.Results: HMGB1 was found in the supernatant of pancreatic cancer cells after radiotherapy. The results of CFSE showed that exogenous HMGB1 could promote the proliferation of pancreatic cancer cells. Meanwhile, HMGB1 also could promote the metastasis of PC cells. By western blot, HMGB1 could upregulation of p-GSK 3β, N-CA, Bcl-2, and Ki67 and down-regulation of E-CA. In vivo, EP (HMGB1 inhibitor) could inhibit the growth of tumors and HMGB1 could promote the proliferation of tumors post-radiotherapy.Conclusion: Radiotherapy could induce HMGB1 released into extracellular. Exogenous HMGB1 could promote the proliferation and metastasis of Panc-1 cells and SW1990 cells by upregulating p-GSK 3β expression which might depend on Wnt pathway.

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