Inhibition of checkpoint kinase 1 potentiates anticancer activity of gemcitabine in bladder cancer cells.
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ABSTRACT:
SUBMITTER: Isono M
PROVIDER: S-EPMC8119486 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.
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Project description:The protein kinase checkpoint kinase 1 (Chk1) has been implicated as a key regulator of cell cycle progression and DNA repair, and inhibitors of Chk1 (e.g., UCN-01 and EXEL-9844) potentiate the cytotoxic actions of chemotherapeutic drugs in tumor cells. We have examined the ability of PD-321852, a small-molecule Chk1 inhibitor, to potentiate gemcitabine-induced clonogenic death in a panel of pancreatic cancer cell lines and evaluated the relationship between endpoints associated with Chk1 inhibition and chemosensitization. Gemcitabine chemosensitization by minimally toxic concentrations of PD-321852 ranged from minimal (<3-fold change in survival) in Panc1 cells to >30-fold in MiaPaCa2 cells. PD-321852 inhibited Chk1 in all cell lines as evidenced by stabilization of Cdc25A; in combination with gemcitabine, a synergistic loss of Chk1 protein was observed in the more sensitized cell lines. Gemcitabine chemosensitization, however, did not correlate with abrogation of the S-M or G2-M checkpoint; PD-321852 did not induce premature mitotic entry in gemcitabine-treated BxPC3 or M-Panc96 cells, which were sensitized to gemcitabine 6.2- and 4.6-fold, respectively. In the more sensitized cells lines, PD-321852 not only inhibited gemcitabine-induced Rad51 focus formation and the recovery from gemcitabine-induced replication stress, as evidenced by persistence of gamma-H2AX, but also depleted these cells of Rad51 protein. Our data suggest the inhibition of this Chk1-mediated Rad51 response to gemcitabine-induced replication stress is an important factor in determining gemcitabine chemosensitization by Chk1 inhibition in pancreatic cancer cells.
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Project description:Drug repositioning is an emerging approach to developing novel cancer treatments. Vorinostat is a histone deacetylase inhibitor approved for cancer treatment, but it could attenuate its anticancer activity by activating the mTOR pathway. The HMG-CoA reductase inhibitor fluvastatin reportedly activates the mTOR inhibitor AMP-activated protein kinase (AMPK), and we thought that it would potentiate vorinostat's anticancer activity in renal cancer cells. The combination of vorinostat and fluvastatin induced robust apoptosis and inhibited renal cancer growth effectively both in vitro and in vivo. Vorinostat activated the mTOR pathway, as evidenced by the phosphorylation of ribosomal protein S6, and fluvastatin inhibited this phosphorylation by activating AMPK. Fluvastatin also enhanced vorinostat-induced histone acetylation. Furthermore, the combination induced endoplasmic reticulum (ER) stress that was accompanied by aggresome formation. We also found that there was a positive feedback cycle among AMPK activation, histone acetylation, and ER stress induction. This is the first study to report the beneficial combined effect of vorinostat and fluvastatin in cancer cells.
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Project description:Checkpoint kinase 2 (CHK2) plays pivotal function as an effector of cell cycle checkpoint arrest following DNA damage. Recently, we found that co-treatment of NSC109555 (a potent and selective CHK2 inhibitor) potentiated the cytotoxic effect of gemcitabine (GEM) in pancreatic cancer MIA PaCa-2 cells. Here, we further examined whether NSC109555 could enhance the antitumour effect of GEM in pancreatic adenocarcinoma cell lines. In this study, the combination treatment of NSC109555 plus GEM demonstrated strong synergistic antitumour effect in four pancreatic cancer cells (MIA PaCa-2, CFPAC-1, Panc-1 and BxPC-3). In addition, the GEM/NSC109555 combination significantly increased the level of intracellular reactive oxygen species (ROS), accompanied by induction of apoptotic cell death. Inhibition of ROS generation by N-acetyl cysteine (NAC) significantly reversed the effect of GEM/NSC109555 in apoptosis and cytotoxicity. Furthermore, genetic knockdown of CHK2 by siRNA enhanced GEM-induced apoptotic cell death. These findings suggest that inhibition of CHK2 would be a beneficial therapeutic approach for pancreatic cancer therapy in clinical treatment.
| S-EPMC4159025 | biostudies-literature
Project description:There is an unmet need to develop new agents or strategies against therapy resistant pancreatic cancer (PanCA). Recent studies from our laboratory showed that STAT3 negatively regulates NF-κB and that inhibition of this crosstalk using Nexrutine® (Nx) reduces transcriptional activity of COX-2. Inhibition of these molecular interactions impedes pancreatic cancer cell growth as well as reduces fibrosis in a preclinical animal model. Nx is an extract derived from the bark of Phellodendron amurense and has been utilized in traditional Chinese medicine as antidiarrheal, astringent, and anti-inflammatory agent for centuries. We hypothesized that "Nx-mediated inhibition of survival molecules like STAT3 and NF-κB in pancreatic cancer cells will improve the efficacy of the conventional chemotherapeutic agent, gemcitabine (GEM)." Therefore, we explored the utility of Nx, one of its active constituents berberine and its derivatives, to enhance the effects of GEM. Using multiple human pancreatic cancer cells we found that combination treatment with Nx and GEM resulted in significant alterations of proteins in the STAT3/NF-κB signaling axis culminating in growth inhibition in a synergistic manner. Furthermore, GEM resistant cells were more sensitive to Nx treatment than their parental GEM-sensitive cells. Interestingly, although berberine, the Nx active component used, and its derivatives were biologically active in GEM sensitive cells they did not potentiate GEM activity when used in combination. Taken together, these results suggest that the natural extract, Nx, but not its active component, berberine, has the potential to improve GEM sensitivity, perhaps by down regulating STAT3/NF-κB signaling. © 2016 Wiley Periodicals, Inc.
| S-EPMC5800424 | biostudies-literature
Project description:Background: Tyrosine kinase inhibitors (TKIs) are widely used for treating solid and hematologic malignancies. However, their efficacy is frequently short lived, warranting the search for safe potentiation strategies. Short courses of fasting were shown to sensitize cancer cells to chemo- and radiotherapy while increasing the resistance of healthy tissues to the same agents. The purpose of this study was to assess the potential of fasting to increase the efficacy of TKIs. Methods: starvation-mimicking culture conditions were studied for their ability to potentiate the effects of Epidermal Growth Factor Receptor (EGFR), Human Epidermal Growth Factor Receptor 2 (HER2), Anaplastic Lymphoma Kinase (ALK) and multitarget TKIs in terms of cancer cell growth, signaling cascades inhibition, and changes in gene expression profile in TKI-sensitive cancer cells. In vivo, the activity of crizotinib or regorafenib, weekly cycles of fasting, or their combination was compared in tumor xenografts models. Results: In vitro, starvation-mimicking culture conditions increased the ability of TKIs to block cancer cell growth and to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway. At the gene expression profile level, starvation and crizotinib led to similar changes, but their combination strengthened Rb-, MYC-, and E2F-dependent transcription inhibition. In vivo, both TKIs and cycles of fasting slowed tumor growth, but, when combined, they were significantly more effective than either type of treatment alone. Conclusions: Cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use. RNA was collected from H3122 cells in 4 different conditions: serum, starvation, treated with Crizotinib, starvation and treated with Crizotinib. Each condition was run in quadruplicate
2016-07-03 | E-GEOD-62663 | biostudies-arrayexpress
Project description:Pancreatic cancer is one of the most lethal solid malignancies with little treatment options. We have recently shown that expression of protease activated receptor (PAR)-1 in the tumor microenvironment drives progression and induces chemoresistance of pancreatic cancer. As thrombin is the prototypical PAR-1 agonist, here we addressed the effect of the direct thrombin inhibitor dabigatran on pancreatic cancer growth and drug resistance in an orthotropic pancreatic cancer model. We show that dabigatran treatment did not affect primary tumor growth whereas it significantly increased tumor dissemination throughout the peritoneal cavity. Increased dissemination was accompanied by intratumoral bleeding and increased numbers of aberrant and/or collapsed blood vessels in the primary tumors. In combination with gemcitabine, dabigatran treatment limited primary tumor growth, did not induce bleeding complications and prevented tumor cell dissemination. Dabigatran was however not as efficient as genetic ablation of PAR-1 in our previous study suggesting that thrombin is not the main PAR-1 agonist in the setting of pancreatic cancer. Overall, we show that dabigatran potentiates gemcitabine-induced growth inhibition of pancreatic cancer but does not affect primary tumor growth when used as a monotherapy.
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Project description:In a recent phase II clinical trial, low-dose (100 mg/m(2)) gemcitabine showed promise as a radiosensitizer in bladder cancer, but underlying mechanisms lack elucidation. Here, we investigated the mechanism of radiosensitization by low-dose gemcitabine in bladder cancer cell lines.Four bladder cancer cell lines were screened for radiosensitization by low-dose gemcitabine using clonogenic assay, and gemcitabine-resistant RT112gem and CALgem cells created by exposure to increasing gemcitabine doses. Four key gemcitabine-regulatory genes were knocked down by transient siRNA. Nude mice carrying CALgem subcutaneous xenografts were exposed to 100 mg/kg gemcitabine ± ionizing radiation (IR) and response assessed by tumor growth delay.Gemcitabine was cytotoxic in the low nanomolar range (10-40 nmol/L) in four bladder cancer cell lines and radiosensitized all four lines. Sensitizer enhancement ratios at 10% survival were: RT112 1.42, CAL29 1.55, T24 1.63, and VMCUB1 1.47. Transient siRNA knockdown of deoxycytidine kinase (dCK) significantly reduced radiosensitization by gemcitabine (P = 0.02). RT112gem and CALgem cells displayed robust decreases of dCK mRNA and protein levels; reexpression of dCK restored gemcitabine sensitivity. However, CALgem xenografts responded better to combination gemcitabine/IR than either treatment alone (P < 0.001) with dCK strongly expressed in the tumor vasculature and stroma.Gemcitabine resistance in bladder cancer cell lines was associated with decreased dCK expression, but gemcitabine-resistant xenografts were responsive to combination low-dose gemcitabine/IR. We propose that dCK activity in tumor vasculature renders it gemcitabine sensitive, which is sufficient to invoke a tumor response and permit tumor cell kill in gemcitabine-resistant tumors.
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Project description:Claudin-low breast cancer is an aggressive subtype that confers poor prognosis and is found largely within the clinical triple-negative group of breast cancer patients. Here, we have shown that intrinsic and immune cell gene signatures distinguish the claudin-low subtype clinically as well as in mouse models of other breast cancer subtypes. Despite adaptive immune cell infiltration in claudin-low tumors, treatment with immune checkpoint inhibitory antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death receptor 1 (PD-1) were ineffective in controlling tumor growth. CD4+FoxP3+ Tregs represented a large proportion of the tumor-infiltrating lymphocytes (TILs) in claudin-low tumors, and Tregs isolated from tumor-bearing mice were able to suppress effector T cell responses. Tregs in the tumor microenvironment highly expressed PD-1 and were recruited partly through tumor generation of the chemokine CXCL12. Antitumor efficacy required stringent Treg depletion combined with checkpoint inhibition; delays in tumor growth were not observed using therapies that modestly diminished the number of Tregs in the tumor microenvironment. This study provides evidence that the recruitment of Tregs to the tumor microenvironment inhibits an effective antitumor immune response and highlights early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint blockade antibodies in specific subtypes of cancer that are heavily infiltrated with adaptive immune cells.
| S-EPMC5669567 | biostudies-literature
Project description:Immune checkpoint blockade (ICB) induces durable response in approximately 20% of patients with advanced bladder urothelial cancer (aUC). Over 50% of aUCs harbor genomic alterations along the phosphoinositide 3-kinase (PI3K) pathway. The goal of this project was to determine the synergistic effects and mechanisms of action of PI3K inhibition and ICB combination in aUC. Alterations affecting the PI3K pathway were examined in The Cancer Genome Atlas (TCGA) and the Cancer Dependency Map databases. Human and mouse cells with Pten deletion were used for in vitro studies. C57BL/6 mice carrying syngeneic tumors were used to determine in vivo activity, mechanisms of action and secondary resistance of pan-PI3K inhibition, ICB and combination. Alterations along the PI3K pathway occurred in 57% of aUCs in TCGA. CRISPR (clustered regularly interspaced short palindromic repeats) knockout of PIK3CA induced pronounced inhibition of cell proliferation (p=0.0046). PI3K inhibition suppressed cancer cell growth, migration and colony formation in vitro. Pan-PI3K inhibition, antiprogrammed death 1 (aPD1) therapy and combination improved the overall survival (OS) of syngeneic mice with PTEN-deleted tumors from 27 days of the control to 48, 37, and 65 days, respectively. In mice with tumors not containing a PI3K pathway alteration, OS was prolonged by the combination but not single treatments. Pan-PI3K inhibition significantly upregulated CD80, CD86, MHC-I, and MHC-II in dendritic cells, and downregulated the transforming growth factor beta pathway with a false discovery rate-adjusted q value of 0.001. Interferon alpha response was significantly upregulated with aPD1 therapy (q value: <0.001) and combination (q value: 0.027). Compared with the control, combination treatment increased CD8+ T-cell infiltration (p=0.005), decreased Treg-cell infiltration (p=0.036), and upregulated the expression of multiple immunostimulatory cytokines and granzyme B (p<0.01). Secondary resistance was associated with upregulation of the mammalian target of rapamycin (mTOR) pathway and multiple Sprr family genes. The combination Pan-PI3K inhibition and ICB has significant antitumor effects in aUC with or without activated PI3K pathway and warrants further clinical investigation. This combination creates an immunostimulatory tumor milieu. Secondary resistance is associated with upregulation of the mTOR pathway and Sprr family genes.
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