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.

Effect of nicotine on chemoresistant phenotype as mediated through Src-dependent Id1 expression in pancreatic adenocarcinoma cells.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 216-216
Author(s):  
J. G. Trevino ◽  
S. R. Pillai ◽  
S. P. Chellappan
Keyword(s):  
Pancreatic Cancer ◽  
Western Blot ◽  
Cell Viability ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pancreatic Tumor ◽  
Pancreatic Cancer Cells ◽  
Dependent Pathway

216 Background: The signaling pathways contributing to DNA-binding protein inhibitor Id1 expression and chemoresistance in pancreatic cancer remain unknown. Id1 plays a role in pancreatic tumor progression with tumor-promoting effects of nicotine regulating protein tyrosine kinase Src activation and Id1 expression, both associated with chemoresistance in other systems. We hypothesize Id1 expression regulates chemoresistance in pancreatic cancer through a nicotine-promoting Src-dependent pathway. Methods: We probed pancreatic cancer cell lines (L3.6pl, PANC-1, Mia-PaCa-2) for innate gemcitabine chemoresistance with cell viability MTT assay and western blot analysis of PARP cleavage programmed cell death. Gemcitabine-sensitive cells were exposed to rising gemcitabine concentrations to establish a resistant subtype, L3.6plGemRes. Protein analysis and mRNA expression were determined by western blot analysis and RT-PCR respectively. Induction of Src phosphorylation or Id1 expression was performed with nicotine (1 μM). Results: Inhibition of c-Src expression was performed with short-interfering RNA (siRNA). Nicotine-induced Src phosphorylation and Id1 expression. Inhibition of Src by siRNA resulted in decreased nicotine-induced Id1 expression. Inhibition of Src and Id1 expression by siRNA in innate or established gemcitabine resistant pancreatic cancer cells resulted in gemcitabine sensitization. To determine if nicotine contributes to gemcitabine chemoresistance, we exposed gemcitabine-sensitive cells to nicotine with subsequent exposure to gemcitabine IC50, 250 ng/ml, and cell viability assays confirmed a 2-fold increase in cell prolilferation and a 4.5-fold reduction in apoptosis. Further, nicotine induced phosphorylation of key signaling enzymes involved in proliferation and apoptosis, Erk1/2 and Akt respectively. Conclusions: In summary, we demonstrate that Id1, through a nicotine-promoting Src-dependent pathway, is necessary for establishment of a chemoresistant phenotype in pancreatic cancer cells. Understanding the signaling pathways involved in pancreatic tumor chemoresistance will lead to therapies resulting in improved tumor responses. No significant financial relationships to disclose.

scholarly journals DDX31: A Novel Functional Oncogene Promotes Migration and Proliferation of Pancreatic Cancer

2021 ◽  
Author(s):  
Yang Liu ◽  
Yongjie Xie ◽  
Jinsheng Ding ◽  
Liangliang Wu
Keyword(s):  
Pancreatic Cancer ◽  
Western Blot ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ductal Adenocarcinoma ◽  
Rna Helicases ◽  
Pancreatic Cancer Cells

Abstract Purpose: Pancreatic cancer is one of the most malignant cancers with rapid disease progression. Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% in exocrine pancreatic cancer. DDX31 is one of the Asp-Glu-Ala-Asp (DEAD)-box RNA helicases (DDX) family member, which has never been reported in pancreatic ductal adenocarcinoma. Through comprehensive analysis of bioinformatics screening, clinical pathological data and experiment results verification, we found DDX31 may be a promising oncogene.Patients and methods: The potential correlation between DDX3 expression and clinical feature of PDAC was analyzed, which revealed that patients with high DDX31 expression may have a poor prognosis. Elevated expression levels of DDX31 in PDAC compared with adjacent normal tissues were determined by immunohistochemical and Western blot analyses. Western blot analysis of N-cadherin, Snail, transwell, and wound healing assays was carried out to evaluate the pro-metastasis effects of DDX31 in PDAC. In vitro experiments included colony formation assay. Edu labeling assay, CCK-8, western blot analysis of Ki67, PCNA, and an in vivo subcutaneous mouse model were used to analyze the role of DDX31 in PDAC proliferation.Results: In our research, integrated bioinformatics analysis of the TCGA and GEO databases was performed to identify the metastasis and proliferation-related differentially expressed genes (DEG). DDX31 predicts strong metastasis and proliferation capacity of PDAC, was finally screened. Then, the clinical data identified that highexpression-DDX31 was correlated with pancreatic tumor size, pathological grade, and lymph node metastasis. The in vitro and vivo experiments revealed that overexpression-DDX31 promoted the migration, proliferation and cell viability of pancreatic cancer cells, these functions of DDX31 had also been proved in the knockdown results. Moreover, the EMT related markers and proliferation markers were identified to be positively regulated by DDX31 in pancreatic cancer cells.Conclusion: Thus, our work uncovered that DDX31 promotes migration and proliferation in PDAC and might be a promising therapeutic target in pancreatic cancer.

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.

scholarly journals Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells

2011 ◽  
Vol 16 (1) ◽  
Author(s):  
Amit Khanna ◽  
Kulandaivelu Mahalingam ◽  
Debarshi Chakrabarti ◽  
Giridharan Periyasamy
Keyword(s):  
Pancreatic Cancer ◽  
Protein Expression ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Target Genes ◽  
Fold Increase ◽  
Pancreatic Cancer Cells

AbstractGemcitabine, a novel pyrimidine nucleoside analog, has become the standard chemotherapeutic agent for pancreatic cancer patients. The clinical impact of gemcitabine remains modest owing to the high degree of inherent and acquired resistance. There are various lines of evidence that confirm the role of Ets-1, a proto-oncoprotein, in tumor invasion, progression, and chemoresistance. This study examines a hypothesis that implicates Ets-1 in the development of gemcitabine-resistance in pancreatic cancer cells. Ets-1 protein expression was assessed in parental pancreatic cancer cells and their gemcitabine-resistant clones. Western blot analysis revealed elevated levels of Ets-1 protein expression in gemcitabine-resistant PANC1GemRes (4.8-fold increase; P < 0.05), MIA PaCa2GemRes (3.2-fold increase; P < 0.05), and Capan2GemRes (2.1-fold increase; P < 0.05) cells as compared to their parental counterparts. A time course analysis was conducted to determine the change in Ets-1 expression in the parental cells after incubation with gemcitabine. Reverse transcriptase quantitative real-time PCR (RT-qPCR) and Western blot analysis revealed a significant increase in Ets-1 expression. All the three parental cells incubated with gemcitabine showed elevated Ets-1 protein expression at 6 h. By 24 h, the expression level had decreased. Using small interfering RNA (siRNA) against Ets-1 in gemcitabine-resistant cells, we demonstrated a reversal in gemcitabine chemosensitivity and also detected a marked reduction in the expression of the Ets-1 target genes MMP1 and uPA. Our novel finding demonstrates the significance of Ets-1 in the development of gemcitabine chemoresistance in pancreatic cancer cells. Based on these results, a new siRNA-based therapeutic strategy targeting the Ets-1 genes can be designed to overcome chemoresistance.

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.

In Vitro and In Vivo Antitumor Efficacy of Docetaxel and Sorafenib Combination in Human Pancreatic Cancer Cells

2010 ◽  
Vol 999 (999) ◽  
pp. 1-11
Author(s):  
P. Ulivi ◽  
C. Arienti ◽  
W. Zoli ◽  
M. Scarsella ◽  
S. Carloni ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Antitumor Efficacy ◽  
Human Pancreatic Cancer ◽  
Pancreatic Cancer Cells

scholarly journals Cyathus striatus Extract Induces Apoptosis in Human Pancreatic Cancer Cells and Inhibits Xenograft Tumor Growth In Vivo

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2017
Author(s):  
Lital Sharvit ◽  
Rinat Bar-Shalom ◽  
Naiel Azzam ◽  
Yaniv Yechiel ◽  
Solomon Wasser ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Pancreatic Cancer Cell Line ◽  
Cancer Cell Line ◽  
Culture Liquid ◽  
Human Pancreatic Cancer ◽  
P53 Signaling ◽  
Pancreatic Cancer Cells

Pancreatic cancer is a highly lethal disease with limited options for effective therapy and the lowest survival rate of all cancer forms. Therefore, a new, effective strategy for cancer treatment is in need. Previously, we found that a culture liquid extract of Cyathus striatus (CS) has a potent antitumor activity. In the present study, we aimed to investigate the effects of Cyathus striatus extract (CSE) on the growth of pancreatic cancer cells, both in vitro and in vivo. The proliferation assay (XTT), cell cycle analysis, Annexin/PI staining and TUNEL assay confirmed the inhibition of cell growth and induction of apoptosis by CSE. A Western blot analysis demonstrated the involvement of both the extrinsic and intrinsic apoptosis pathways. In addition, a RNAseq analysis revealed the involvement of the MAPK and P53 signaling pathways and pointed toward endoplasmic reticulum stress induced apoptosis. The anticancer activity of the CSE was also demonstrated in mice harboring pancreatic cancer cell line-derived tumor xenografts when CSE was given for 5 weeks by weekly IV injections. Our findings suggest that CSE could potentially be useful as a new strategy for treating pancreatic cancer.

scholarly journals NR5A2 transcriptional activation by BRD4 promotes pancreatic cancer progression by upregulating GDF15

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Feng Guo ◽  
Yingke Zhou ◽  
Hui Guo ◽  
Dianyun Ren ◽  
Xin Jin ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Transcriptional Activation ◽  
Ductal Adenocarcinoma ◽  
Pancreatic Cancer Cells ◽  
Gene Sets ◽  
And Migration

AbstractNR5A2 is a transcription factor regulating the expression of various oncogenes. However, the role of NR5A2 and the specific regulatory mechanism of NR5A2 in pancreatic ductal adenocarcinoma (PDAC) are not thoroughly studied. In our study, Western blotting, real-time PCR, and immunohistochemistry were conducted to assess the expression levels of different molecules. Wound-healing, MTS, colony formation, and transwell assays were employed to evaluate the malignant potential of pancreatic cancer cells. We demonstrated that NR5A2 acted as a negative prognostic biomarker in PDAC. NR5A2 silencing inhibited the proliferation and migration abilities of pancreatic cancer cells in vitro and in vivo. While NR5A2 overexpression markedly promoted both events in vitro. We further identified that NR5A2 was transcriptionally upregulated by BRD4 in pancreatic cancer cells and this was confirmed by Chromatin immunoprecipitation (ChIP) and ChIP-qPCR. Besides, transcriptome RNA sequencing (RNA-Seq) was performed to explore the cancer-promoting effects of NR5A2, we found that GDF15 is a component of multiple down-regulated tumor-promoting gene sets after NR5A2 was silenced. Next, we showed that NR5A2 enhanced the malignancy of pancreatic cancer cells by inducing the transcription of GDF15. Collectively, our findings suggest that NR5A2 expression is induced by BRD4. In turn, NR5A2 activates the transcription of GDF15, promoting pancreatic cancer progression. Therefore, NR5A2 and GDF15 could be promising therapeutic targets in pancreatic cancer.

Sign in / Sign up

Export Citation Format

Share Document