scholarly journals pHLARE: A New Biosensor Reveals Decreased Lysosome pH in Cancer Cells

2020 ◽  
pp. mbc.E20-06-0383
Author(s):  
Bradley A. Webb ◽  
Francesca M. Aloisio ◽  
Rabab A. Charafeddine ◽  
Jessica Cook ◽  
Torsten Wittmann ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Longitudinal Studies ◽  
Neurodegenerative Disorders ◽  
Therapeutic Target ◽  
Dynamic Range ◽  
Mammalian Target Of Rapamycin ◽  
Inhibiting Activity ◽  
Promising Therapeutic Target ◽  
Intracellular Compartments

Many lysosome functions are determined by a lumenal pH of ∼5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is often increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or used for in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE (pHLysosomal Activity REporter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1), in breast, pancreatic, colon, and glioblastoma cancer cells compared with untransformed cells, and with the activated oncogenes H-RasV12 and R-RasV12. pHLARE is a new tool to accurately measure pHlys, for improved understanding of lysosome dynamics that could be a promising therapeutic target.

scholarly journals pHLARE: A Genetically Encoded Ratiometric Lysosome pH Biosensor

2020 ◽  
Author(s):  
Bradley A. Webb ◽  
Jessica Cook ◽  
Torsten Wittmann ◽  
Diane L. Barber
Keyword(s):  
Pancreatic Cancer ◽  
Neurodegenerative Disorders ◽  
Dynamic Range ◽  
Mammalian Target Of Rapamycin ◽  
Inhibiting Activity ◽  
Pancreatic Cancer Cells ◽  
Promising Therapeutic Target ◽  
Intracellular Compartments ◽  
Summary Statement

AbstractMany lysosome functions are determined by a lumenal pH of ~5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE (pH Lysosomal Activity REporter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1), in breast and pancreatic cancer cells compared with tissue-matched untransformed cells, and with the activated oncogene H-RasV12. pHLARE is a new tool to accurately measure pHlys, for improved understanding of lysosome dynamics that could be a promising therapeutic target.Summary StatementMost lysosome functions require a low lumenal pH, which is dysregulated in many human diseases. We report a new genetically biosensor to accurately measure lysosome pH dynamics, which we use to show decreased lysosome pH in cancer cell lines.

scholarly journals Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Xiao-ren Zhu ◽  
Shi-qing Peng ◽  
Le Wang ◽  
Xiao-yu Chen ◽  
Chun-xia Feng ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Therapeutic Target ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Human Pancreatic Cancer ◽  
Migration And Invasion ◽  
Potential Therapeutic Target ◽  
Protein Levels ◽  
Pancreatic Cancer Cells

AbstractPancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.

scholarly journals ERα-Targeted Therapy in Ovarian Cancer Cells by a Novel Estradiol-Platinum(II) Hybrid

Endocrinology ◽  
2013 ◽  
Vol 154 (7) ◽  
pp. 2281-2295 ◽  
Author(s):  
K. Brasseur ◽  
V. Leblanc ◽  
F. Fabi ◽  
S. Parent ◽  
C. Descôteaux ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
High Efficiency ◽  
Mammalian Target Of Rapamycin ◽  
Ovarian Cancer Cells ◽  
New Family ◽  
17Β Estradiol ◽  
Apoptosis Inducing Factor

Abstract As we previously showed, we have synthesized a new family of 17β-estradiol-platinum(II) hybrids. Earlier studies revealed the VP-128 hybrid to show high efficiency compared with cisplatin toward hormone-dependent breast cancer cells. In the present research, we have studied the antitumor activity of VP-128 in vitro and in vivo against ovarian cancer. In nude mice with ovarian xenografts, VP-128 displayed selective activity toward hormone-dependent tumors and showed higher efficiency than cisplatin to inhibit tumor growth. Similarly, in vitro, transient transfection of estrogen receptor (ER)-α in ERα-negative A2780 cells increased their sensitivity to VP-128-induced apoptosis, confirming the selectivity of VP-128 toward hormone-dependent tumor cells. In agreement, Western blot analysis revealed that VP-128 induced higher caspase-9, caspase-3, and poly (ADP-ribose) polymerase cleavage compared with cisplatin. The activation of caspase-independent apoptosis was also observed in ERα-negative A2780 cells, in which VP-128 rapidly induced the translocation of apoptosis-inducing factor to the nucleus. Conversely, subcellular localization of apoptosis-inducing factor was not modified in ERα-positive Ovcar-3 cells. We also discovered that VP-128 induces autophagy in ovarian cancer cells because of the formation of acidic vesicular organelles (AVOs) and increase of Light Chain 3B-II protein responsible for the formation of autophagosomes; pathways related to autophagy (AKT and mammalian target of rapamycin) were also down-regulated, supporting this mechanism. Finally, the inhibition of autophagy using chloroquine increased VP-128 efficiency, indicating a possible combination therapy. Altogether these results highlight the beneficial value of VP-128 for the treatment of hormone-dependent ovarian cancers and provide preliminary proof of concept for the efficient targeting of ERα- by 17β-estradiol-Pt(II)-linked chemotherapeutic hybrids in these tumors.

Preclinical evaluation of SC0191, a small molecule inhibitor of Wee1 kinase.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15637-e15637
Author(s):  
Chundao Yang ◽  
Zhengwei Li ◽  
Qi Li ◽  
Yuanfeng Xia ◽  
Chi-Chung Chan ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Cancer Cells ◽  
Mouse Models ◽  
Small Molecule ◽  
Tp53 Mutation ◽  
Inhibiting Activity ◽  
In Vivo Antitumor Activity ◽  
Molecule Inhibitor ◽  
Wee1 Kinase

e15637 Background: TP53 mutation is common in cancer cells, especially in small cell lung cancer (SCLC) and pancreatic cancer (PC). The G1 checkpoint function is lost in these cancer cells, which more rely on G2 checkpoint to repair damaged DNA. Wee1 is a serine/threonine kinase, which phosphorylates the Tyr15 locus of CDC2, inhibits the activity of CDC2, and stays in the G2 phase. Wee1 inhibition can promote the cells with DNA damage into the M phase, thus driving the TP53 mutant cancer cells to apoptosis. The purpose of this study was to investigate the in vitro and in vivo antitumor activity of SC0191, a small molecule inhibitor of Wee1 kinase, in preclinical models of SCLC and PC with TP53 mutation. Methods: The kinase inhibiting activity of SC0191 was determined using the Wee1 kinase assays. The cellular anti-proliferative activity was evaluated with TP53 mutant NCI-H446 SCLC and BxPC-3 PC cell lines. The in vivo antitumor activity of SC0191 was evaluated in the NCI-H446 and BxPC-3 cells-derived xenograft (CDX) mouse models, respectively. Results: SC0191 displayed potent kinase inhibiting activity for Wee1 with IC50 22.3 nM. SC0191 significantly inhibited tumor cell proliferation in TP53 mutant NCI-H446 cells with IC50 231.1 nM and BxPC-3 cells with IC50 223.8 nM. Moreover, SC0191 oral administration showed significant antitumor efficacy which was better than AZD1775, in the NCI-H446 and BxPC-3 CDX mouse models, respectively. Conclusions: A novel Wee1 inhibitor, SC0191, has demonstrated excellent antitumor efficacy in TP53 mutant solid cancer preclinical studies, and represents a promising clinical candidate for treating solid tumors, such as SCLC and PC.

scholarly journals LncRNA HANR Promotes Tumorigenesis and Increase of Chemoresistance in Hepatocellular Carcinoma

2017 ◽  
Vol 43 (5) ◽  
pp. 1926-1938 ◽  
Author(s):  
Jia Xiao ◽  
Yi Lv ◽  
Fujun Jin ◽  
Yingxia Liu ◽  
Yi Ma ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Therapeutic Target ◽  
Mice Model ◽  
Over Expression ◽  
In Vivo Experiments ◽  
Non Coding Rna ◽  
Promising Therapeutic Target ◽  
Induced Apoptosis ◽  
Pathway Analyses

Background/Aims: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world and the third leading cause of cancer-related death. Critical roles for long non-coding RNAs (lncRNAs) have recently been demonstrated for a variety of cancers, including hepatocellular carcinoma. However, the effect and mechanism of lncRNAs in HCC tumorigenesis and chemoresistance have not been extensively characterized. Methods: In the current study, we have identified a HCC-expressed lncRNA termed as HANR (HCC associated long non-coding RNA). We identified HANR by microarray analysis and validated its up-regulated expression by quantitative PCR. RNA pull-down and pathway analyses were conducted to evaluate physical and functional interactions with HANR. In vivo experiments were performed to assess tumorigenesis and increase of chemoresistance. In addition, the HANR expression in HCC specimens was detected by FISH. Xenograft and orthotopic mice model was constructed to observe the effect of HANR on tumorigenesis and chemoresistance in vivo. Results: HANR was demonstrated to be up-regulated in HCC patients and HCC cell lines. Increased HANR expression in HCC predicted short survival of patients. Knock-down of HANR markedly retarded cell proliferation, suppressed HCC xenograft/orthotopic tumor growth, induced apoptosis and enhanced chemosensitivity to doxorubicin, while over-expression of HANR showed the opposite effects. It was found that HANR bind to GSKIP for regulating the phosphorylation of GSK3β in HCC. Conclusion: Our results demonstrate that HANR contributes to the development of HCC and is a promising therapeutic target for chemosensitization of HCC cells to doxorubicin, which may represent a promising therapeutic target in the future.

scholarly journals HAT1 Promotes Gemcitabine Resistance by Regulating the PVT1/EZH2 Complex in Pancreatic Cancer

2020 ◽  
Author(s):  
Yan Sun ◽  
Jian Shen ◽  
Dianyun Ren ◽  
Yingke Zhou ◽  
Jingyuan Zhao ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Statistical Significance ◽  
Gemcitabine Resistance ◽  
Development Of Resistance ◽  
Pancreatic Cancer Cells ◽  
Promising Therapeutic Target ◽  
The Relationship

Abstract Background: The poor prognosis of pancreatic cancer is primarily due to the development of resistance to therapies, including gemcitabine. PVT1 has been shown to interact with EZH2, promoting gemcitabine resistance in pancreatic cancer. Methods: In this study, we assessed the ability of PVT1/EZH2 targeting to reverse resistance to gemcitabine in pancreatic cancer cells. MTS assay, colony formation assay, and mouse xenotransplantation experiments were used to evaluate the anti-tumor effects of gemcitabine in HAT1 knockdown or overexpressing pancreatic cancer cells. The relationship between HAT1 and PVT1 in pancreatic cancer was determined by RNA sequencing, quantitative real-time PCR, and chromatin immunoprecipitation. Co-immunoprecipitation, pull-downs, western blotting, and immunohistochemistry were used to assess the relationship between HAT1 and EZH2 in pancreatic cancer. Chitosan (CS)-tripolyphosphate (TPP)-siHAT1 nanoparticles were developed to evaluate their effects on the anti-tumor potential of gemcitabine in pancreatic cancer. Student’s t-test, one-way analysis of variance (ANOVA), or two-way ANOVA was used to evaluate statistical significance. P-values <0.05 were considered statistically significant. All values were expressed as means ± SD. Results: Our results showed that the aberrant HAT1 expression promoted gemcitabine resistance in pancreatic cancer cells. We also found that HAT1 enhanced the binding of BRD4 to the PVT1 promoter, thereby promoting PVT1 transcription. Moreover, we found that HAT1 prevented EZH2 degradation by interfering with UBR4 binding to the N-terminal domain of EZH2, thus maintaining EZH2 protein stability. Finally, we showed that CS-TPP-siHAT1 nanoparticles augmented the anti-tumor effects of gemcitabine in pancreatic cancer cells in vitro and in vivo. Conclusions: Our findings suggest that by increasing the levels of the PVT1/EZH2 complex, HAT1 promotes gemcitabine resistance in pancreatic cancer. Therefore, HAT1 is a promising therapeutic target for pancreatic cancer.

scholarly journals Tapping into a phospholipid-LRH-1 axis yields a powerful anti-inflammatory agent with in vivo activity against colitis

2020 ◽  
Author(s):  
Suzanne G. Mays ◽  
Emma H. D’Agostino ◽  
Autumn R. Flynn ◽  
Xiangsheng Huang ◽  
Guohui Wang ◽  
...  
Keyword(s):  
Weight Loss ◽  
Therapeutic Target ◽  
Transcriptional Activity ◽  
Hormone Receptors ◽  
Binding Pocket ◽  
Inflammatory Agent ◽  
Promising Therapeutic Target ◽  
Hybrid Molecules ◽  
Anti Inflammatory

AbstractAs ligands for nuclear hormone receptors (NRs), phosphatidylcholines are powerful signaling molecules. Here, we demonstrate that mimicking phosphatidylcholine-NR interactions is a robust strategy for improving agonists of liver receptor homolog-1 (LRH-1), a promising therapeutic target for diabetes and colitis. Conventional LRH-1 modulators only occupy part of the binding pocket, leaving vacant a region important for phosphatidylcholine binding and allostery. Therefore, we constructed a set of hybrid molecules that incorporate elements of natural phosphatidylcholines into the scaffold of a synthetic LRH-1 agonist. The phosphatidylcholine-mimicking group increases LRH-1 binding affinity and transcriptional activity via formation of productive interactions with residues that coordinate the phosphatidylcholine headgroup. In organoid and in vivo models of colitis, the best new agonist upregulates LRH-1-controlled anti-inflammatory genes and significantly improves colonic histopathology and disease-related weight loss. This is the first evidence of in vivo efficacy for an LRH-1 modulator in colitis, a leap forward in agonist development.

scholarly journals DUSP28 is a novel biomarker responsible for aggravating malignancy via the autocrine signaling pathways in metastatic pancreatic cancer

2018 ◽  
Author(s):  
Jungwhoi Lee ◽  
Jungsul Lee ◽  
Chulhee Choi ◽  
Jae Hoon Kim
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Umbilical Vein ◽  
Metastatic Pancreatic Cancer ◽  
Autocrine Signaling ◽  
Pancreatic Cancer Cells ◽  
Promising Therapeutic Target ◽  
Umbilical Vein Endothelial Cells

AbstractPancreatic cancer remains one of the most dangerous cancers with a grave prognosis. We previously reported that pancreatic cancer cells can secrete dual specificity phosphatise 28 (DUSP28) to the cultured medium. However, its biological function is poorly understood. Here, we have identified the function of DUSP28 in human metastatic pancreatic cancer. Treatment with recombinant DUSP28 (rDUSP28) significantly increased the migration, invasion, and viability of metastatic pancreatic cancer cells through the activation of CREB, AKT, and ERK1/2 signaling pathways. Furthermore, rDUSP28 acted as an oncogenic reagent through the interaction with integrin α1 in metastatic pancreatic cancer cells. In addition, rDUSP28 induced pro-angiogenic effects in human umbilical vein endothelial cells (HUVECs). Administration of rDUSP28 also produced tumor growth in vivo. Notably, sDUSP28 can easily be detected by immunoassay. The results establish the rationale for sDUSP28 as a promising therapeutic target and biomarker for metastatic pancreatic cancer patients.

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