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PloS One 2013MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the...
BACKGROUND
MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown.
METHODS
We utilized isogenic HCT116 (MLH1-/MSH3-) cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3) and also MSH3 by chromosome 5 (HCT116+3+5). We generated HCT116+3+5, SW480 (MLH1+/MSH3+) and SW48 (MLH1-/MSH3+) cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU), SN-38, oxaliplatin, or the histone deacetylase (HDAC) inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed.
RESULTS
MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB) repair. We then utilized PCI-24781 that interferes with homologous recombination (HR) indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone.
CONCLUSION
MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1 status. PCI-24781-mediated impairment of HR enhanced oxaliplatin sensitivity, suggesting that reduced DSB repair capacity may be contributory.
Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Benzofurans; Camptothecin; Cell Death; Cell Nucleus; Cell Survival; Colonic Neoplasms; DNA Damage; DNA Mismatch Repair; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Fluorescent Antibody Technique; Fluorouracil; Gene Knockdown Techniques; HCT116 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Irinotecan; MutL Protein Homolog 1; MutS Homolog 3 Protein; Nuclear Proteins; Organoplatinum Compounds; Oxaliplatin; Rad51 Recombinase
PubMed: 23724141
DOI: 10.1371/journal.pone.0065369 -
Journal of Hepatology Jul 2012Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat, demonstrating the need for more effective therapeutic strategies. In...
BACKGROUND & AIMS
Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat, demonstrating the need for more effective therapeutic strategies. In our current study, we examined the therapeutic effects of the histone deacetylase (HDAC) inhibitor PCI-24781 against GBCa that developed in BK5.erbB2 mice.
METHODS
PCI-24781 [50 mg/kg/day] and control solutions were administered to BK5.erbB2 mice for 4 weeks. The therapeutic effect of PCI-24781 was evaluated by ultrasound biomicroscopy (USBM) throughout the experiment and histological analyses at the end of the experiment. To investigate potential mechanisms underlining the therapeutic effects of PCI-24781 on GBCa in BK5.erbB2 mice, PCI-24781-treated gallbladders were subjected to Western blot and RT-PCR analysis. The inhibitory effect of PCI-24781 on the growth of BTC cells was compared to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and gemcitabine. To study the role of miRNAs in GBCa tumorigenesis, the expression profile of 368 miRNAs in GBCas from BK5.erbB2 (both treated and untreated) and wild type mice was analyzed.
RESULTS
Treatment of BK5.erbB2 mice with PCI-24781 for 1 month prevented 79% of GBCa cases from progression and showed a clinical effect in 47% of cases. We also confirmed a potent inhibitory effect on tumor cell growth in human BTC cell lines treated with PCI-24781. This effect was associated with downregulation of ErbB2 mRNA and ErbB2 protein/activity and upregulation of acetylated histone and acetylated tubulin. Treatment with PCI-24781 resulted in decreased expression of Muc4, an intramembrane ligand for ErbB2, in BTC cells. PCI-24781 had more effects on growth inhibition of BTC cells than SAHA. In addition, PCI-24781 effectively inhibited the growth of gemcitabine-resistant cells. miRNA profiling revealed that the expression of several miRNAs was significantly altered in GBCa in the BK5.erbB2 mouse compared to normal gallbladder, including upregulated miR21, which was downregulated by PCI-24781.
CONCLUSIONS
These results indicate that PCI-24781 potently inhibits the growth of BTC cells by decreasing ErbB2 expression and activity as well as regulating altered miRNA expression. PCI-24781 may have a potential value as a novel chemotherapeutic agent against human BTC in which ErbB2 is overexpressed.
Topics: Animals; Antimetabolites, Antineoplastic; Benzofurans; Carcinoma; Carrier Proteins; Cell Division; Cell Line, Tumor; Deoxycytidine; Disease Models, Animal; Gallbladder; Gallbladder Neoplasms; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Mice; Mice, Mutant Strains; Mucin-4; Phosphorylation; RNA, Messenger; Gemcitabine
PubMed: 22326466
DOI: 10.1016/j.jhep.2012.01.018 -
Cancer Biology & Therapy Jun 2011The concept of cancer stem cells is generally accepted in different malignancies. We have previously shown that the MDA-MB231 breast cancer cells were more radiation...
The concept of cancer stem cells is generally accepted in different malignancies. We have previously shown that the MDA-MB231 breast cancer cells were more radiation resistant when sorted for the two stem cell markers CD24 and ESA. In this study, we examined a possible mechanism that might underlie this phenotype by looking at cell cycle profile and the effect this has on DNA repair pathways. The cell cycle profile showed that there were more CD24(-) ESA(+) sorted MDA-MB231 cells in the S- and G(2)-phases compared with the unsorted cells, 60 and 38% respectively. Cyclin D and E protein levels supported the cell cycle profile and highlighted the possible involvement of homologous recombination (HR) repair in the radioresistant phenotype. To further support this, CD24(-) ESA(+) sorted MDA-MB231 cells demonstrated statistically significant more RAD51 and less γ-H2AX foci 2 h post 4Gy ionising radiation, compared with the unsorted population. Inhibition of the HR pathway effectively sterilised the CD24(- ) ESA(+) sorted MDA-MB231 cells but had no effect on the unsorted cells or MDA468 control breast cancer cell line. Although the changes we saw were specific to MDA-MB231, these results merit further investigation and can be crucial in identifying a mechanism responsible for cancer stem cells treatment resistance in primary tumors.
Topics: Benzofurans; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplastic Stem Cells; Rad51 Recombinase; Radiation Tolerance; Recombination, Genetic
PubMed: 21558789
DOI: 10.4161/cbt.11.12.15699 -
Anticancer Research Apr 2011The antitumor activity of histone deacetylase inhibitors (HDACI) on multidrug-resistant sarcoma cell lines has not been previously described. Treatment of...
The antitumor activity of histone deacetylase inhibitors (HDACI) on multidrug-resistant sarcoma cell lines has not been previously described. Treatment of multidrug-resistant sarcoma cell lines with HDACI PCI-24781 resulted in dose-dependent accumulation of acetylated histone, p21 and poly(ADP-ribose)polymerase (PARP) cleavage products. Growth of these cell lines was inhibited by PCI-24781 at IC(50) of 0.43 to 2.7. When we looked for synergy of PCI-24781 with chemotherapeutic agents, we found that PCI-24781 reverses drug resistance in all four multidrug-resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/-7 activity. Expression of RAD51 (a marker for DNA double-strand break repair) was inhibited and the expression of GADD45α (a marker for growth arrest and DNA-damage) was induced by PCI-24781 in multidrug-resistant sarcoma cell lines. In conclusion, HDACI PCI-24781 synergizes with chemotherapeutic drugs to induce apoptosis and reverses drug resistance in multidrug-resistant sarcoma cell lines.
Topics: ATP Binding Cassette Transporter, Subfamily B; Antineoplastic Agents; Apoptosis; Benzofurans; Blotting, Western; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Nuclear Proteins; Poly(ADP-ribose) Polymerases; Sarcoma; ATP-Binding Cassette Sub-Family B Member 4
PubMed: 21508354
DOI: No ID Found -
Cancer Chemotherapy and Pharmacology Feb 2011To better understand the mechanisms of cytotoxicity and cell death induced by HDACI PCI-24781 in bone sarcoma cells.
PURPOSE
To better understand the mechanisms of cytotoxicity and cell death induced by HDACI PCI-24781 in bone sarcoma cells.
METHODS
Four bone sarcoma cell lines were treated with PCI-24781, and the cytotoxicity was investigated. Further, accumulation of acetylated histones, p21, and PARP cleavage were evaluated in PCI-24781-treated cells. The synergistic effect of PCI-24781 to doxorubicin and its mechanism was investigated in bone sarcoma cells.
RESULTS
MTT assay demonstrated that the growth of bone sarcoma cells was inhibited after treatment with PCI-24781. Accumulation of acetylated histones, p21, and PARP cleavage were found in PCI-24781-treated cells. Expression of DNA repair protein RAD51 was inhibited, and the expression of apoptosis protein GADD45α was induced by PCI-24781 in bone sarcoma cells. Bone sarcoma cells treated with PCI-24781 become more sensitive to doxorubicin. The caspase-3/7 activity was increased with doxorubicin and PCI-24781 treatment in these cells.
CONCLUSIONS
HDACI PCI-24781 has a synergistic effect on doxorubicin-induced apoptosis in bone sarcoma cells.
Topics: Acetylation; Apoptosis; Benzofurans; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Chondrosarcoma; Doxorubicin; Drug Synergism; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Nuclear Proteins; Osteosarcoma; Rad51 Recombinase
PubMed: 20461381
DOI: 10.1007/s00280-010-1344-7 -
Clinical Cancer Research : An Official... May 2009We investigated the cytotoxicity and mechanisms of cell death of the broad-spectrum histone deacetylase (HDAC) inhibitor PCI-24781, alone and combined with bortezomib in...
PURPOSE
We investigated the cytotoxicity and mechanisms of cell death of the broad-spectrum histone deacetylase (HDAC) inhibitor PCI-24781, alone and combined with bortezomib in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines and primary lymphoproliferative (CLL/SLL) cells.
EXPERIMENTAL DESIGN
Apoptosis, mitochondrial membrane potential, cell cycle analysis, and reactive oxygen species (ROS) were measured by flow cytometry, whereas caspase activation was determined by Western blot. Nuclear factor kappaB (NF-kappaB)-related mRNAs were quantified by reverse transcription-PCR, NF-kappaB-related proteins by Western blotting, and NF-kappaB DNA-binding activity by electromobility shift assay. Finally, gene expression profiling was analyzed.
RESULTS
PCI-24781 induced concentration-dependent apoptosis that was associated with prominent G(0)/G(1) arrest, decreased S-phase, increased p21 protein, and increased ROS in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Dose-dependent apoptosis with PCI-24781 was also seen among primary CLL/SLL cells. PCI-24781-induced apoptosis was shown to be ROS- and caspase-dependent. Combined PCI-24781/bortezomib treatment resulted in strong synergistic apoptosis in all non-Hodgkin lymphoma lines (combination indices, 0.19-0.6) and was additive in Hodgkin lymphoma and primary CLL/SLL cells. Further, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone acetylation compared with either agent alone. Gene expression profiling showed that PCI-24781 alone significantly down-regulated several antioxidant genes, proteasome components, and NF-kappaB pathway genes, effects that were enhanced further with bortezomib. Reverse transcription-PCR confirmed down-regulation of NF-kappaB1 (p105), c-Myc, and IkappaB-kinase subunits, where NF-kappaB DNA binding activity was decreased.
CONCLUSION
We show that PCI-24781 results in increased ROS and NF-kappaB inhibition, leading to caspase-dependent apoptosis. We also show that bortezomib is synergistic with PCI-24781. This combination or PCI-24781 alone has potential therapeutic value in lymphoma.
Topics: Aged; Antineoplastic Agents; Apoptosis; Benzofurans; Blotting, Western; Boronic Acids; Bortezomib; Caspases; Cell Cycle; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Female; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lymphoma; Lymphoma, Non-Hodgkin; Male; Membrane Potential, Mitochondrial; Middle Aged; NF-kappa B; Pyrazines; Reactive Oxygen Species; Tumor Cells, Cultured
PubMed: 19417023
DOI: 10.1158/1078-0432.CCR-08-2365 -
Clinical Cancer Research : An Official... May 2009Histone deactylase inhibitors (HDACi) are a promising new class of anticancer therapeutics; however, little is known about HDACi activity in soft tissue sarcoma (STS), a...
PURPOSE
Histone deactylase inhibitors (HDACi) are a promising new class of anticancer therapeutics; however, little is known about HDACi activity in soft tissue sarcoma (STS), a heterogeneous cohort of mesenchymal origin malignancies. Consequently, we investigated the novel HDACi PCI-24781, alone/in combination with conventional chemotherapy, to determine its potential anti-STS-related effects and the underlying mechanisms involved.
EXPERIMENTAL DESIGN
Immunoblotting was used to evaluate the effects of PCI-24781 on histone and nonhistone protein acetylation and expression of potential downstream targets. Cell culture-based assays were utilized to assess the effects of PCI-24781 on STS cell growth, cell cycle progression, apoptosis, and chemosensitivity. Quantitative reverse transcription-PCR, chromatin immunoprecipitation, and reporter assays helped elucidate molecular mechanisms resulting in PCI-24781-induced Rad51 repression. The effect of PCI-24781, alone or with chemotherapy, on tumor and metastatic growth was tested in vivo using human STS xenograft models.
RESULTS
PCI-24781 exhibited significant anti-STS proliferative activity in vitro, inducing S phase depletion, G(2)/M cell cycle arrest, and increasing apoptosis. Superior effects were seen when combined with chemotherapy. A PCI-24781-induced reduction in Rad51, a major mediator of DNA double-strand break homologous recombination repair, was shown and may be a mechanism underlying PCI-24781 chemosensitization. We showed that PCI-24781 transcriptionally represses Rad51 through an E2F binding-site on the Rad51 proximal promoter. Although single-agent PCI-24781 had modest effects on STS growth and metastasis, marked inhibition was observed when combined with chemotherapy.
CONCLUSIONS
In light of these findings, this novel molecular-based combination may be applicable to multiple STS histologic subtypes, and potentially merits rigorous evaluation in human STS clinical trials.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzofurans; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cisplatin; Dose-Response Relationship, Drug; Doxorubicin; Exonucleases; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunohistochemistry; Luciferases; Luminescent Measurements; Mice; Mice, SCID; Reverse Transcriptase Polymerase Chain Reaction; S Phase; Sarcoma, Experimental; Tumor Burden; Xenograft Model Antitumor Assays
PubMed: 19417021
DOI: 10.1158/1078-0432.CCR-08-2714 -
Proceedings of the National Academy of... Dec 2007Histone deacetylase (HDAC) inhibitors such as the phenyl hydroxamic acid PCI-24781 have emerged recently as a class of therapeutic agents for the treatment of cancer....
Histone deacetylase (HDAC) inhibitors such as the phenyl hydroxamic acid PCI-24781 have emerged recently as a class of therapeutic agents for the treatment of cancer. Recent data showing synergy of HDAC inhibitors with ionizing radiation and other DNA-damaging agents have suggested that HDAC inhibitors may act, in part, by inhibiting DNA repair. Here we present evidence that HDAC enzymes are important for homologous recombinational repair of DNA double-strand breaks. Combination studies of PCI-24781 with the poly(ADP-ribose) polymerase inhibitor PJ34, an agent thought to produce lesions repaired by homologous recombination (HR), resulted in a synergistic effect on apoptosis. Immunofluorescence analysis demonstrated that HDAC inhibition caused a complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigations revealed that inhibition of HDAC enzymes by PCI-24781 led to a significant reduction in the transcription of genes specifically associated with HR, including RAD51. RAD51 protein levels were significantly decreased after 24 h of drug exposure both in vitro and in vivo. Consistent with inhibition of HR, treatment with PCI-24781 resulted in a decreased ability to perform homology directed repair of I-SceI-induced chromosome breaks in transfected CHO cells. In addition, an enhancement of cell killing was observed in Ku mutant cells lacking functional nonhomologous end joining compared with WT cells. Together these results demonstrate that HDAC enzymes are critically important to enable functional HR by controlling the expression of HR-related genes and promoting the proper assembly of HR-directed subnuclear foci.
Topics: Animals; Apoptosis; Benzofurans; Cell Line, Tumor; DNA Repair; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Rad51 Recombinase; Radiation Tolerance; Radiation-Sensitizing Agents; Recombination, Genetic; Transcription, Genetic; Up-Regulation
PubMed: 18042714
DOI: 10.1073/pnas.0707828104 -
Clinical Cancer Research : An Official... Nov 2007PCI-24781 is a novel broad spectrum histone deacetylase inhibitor that is currently in phase I clinical trials. The ability of PCI-24781 to act as a radiation sensitizer...
PURPOSE
PCI-24781 is a novel broad spectrum histone deacetylase inhibitor that is currently in phase I clinical trials. The ability of PCI-24781 to act as a radiation sensitizer and the mechanisms of radiosensitization were examined.
EXPERIMENTAL DESIGN
Exponentially growing human SiHa cervical and WiDr colon carcinoma cells were exposed to 0.1 to 10 micromol/L PCI-24781 in vitro for 2 to 20 h before irradiation and 0 to 4 h after irradiation. Single cells and sorted populations were analyzed for histone acetylation, H2AX phosphorylation, cell cycle distribution, apoptotic fraction, and clonogenic survival.
RESULTS
PCI-24781 treatment for 4 h increased histone H3 acetylation and produced a modest increase in gammaH2AX but negligible cell killing or radiosensitization. Treatment for 24 h resulted in up to 80% cell kill and depletion of cells in S phase. Toxicity reached maximum levels at a drug concentration of approximately 1 micromol/L, and cells in G(1) phase at the end of treatment were preferentially spared. A similar dose-modifying factor (DMF(0.1) = 1.5) was observed for SiHa cells exposed for 24 h at 0.1 to 3 micromol/L, and more radioresistant WiDr cells showed less sensitization (DMF(0.1) = 1.2). Limited radiosensitization and less killing were observed in noncycling human fibroblasts. Cell sorting experiments confirmed that depletion of S-phase cells was not a major mechanism of radiosensitization and that inner noncycling cells of SiHa spheroids could be sensitized by nontoxic doses. PCI-24781 pretreatment increased the fraction of cells with gammaH2AX foci 24 h after irradiation but did not affect the initial rate of loss of radiation-induced gammaH2AX or the rate of rejoining of DNA double-strand breaks.
CONCLUSIONS
PCI-24781 shows promise as a radiosensitizing agent that may compromise the accuracy of repair of radiation damage.
Topics: Benzofurans; Cell Line, Tumor; DNA Breaks, Double-Stranded; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Radiation Tolerance; Radiation-Sensitizing Agents
PubMed: 18006784
DOI: 10.1158/1078-0432.CCR-07-1126 -
Molecular Cancer Therapeutics May 2006CRA-024781 is a novel, broad spectrum hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and...
CRA-024781 is a novel, broad spectrum hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and is under evaluation in phase I clinical trials for cancer. CRA-024781 inhibited pure recombinant HDAC1 with a K(i) of 0.007 mumol/L, and also inhibited the other HDAC isozymes HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-024781 resulted in the accumulation of acetylated histone and acetylated tubulin, resulting in an inhibition of tumor cell growth and the induction of apoptosis. CRA-024781 parenterally administered to mice harboring HCT116 or DLD-1 colon tumor xenografts resulted in a statistically significant reduction in tumor growth at doses that were well tolerated as measured by body weight. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells, and an alteration in the expression of many genes in the tumors, including several involved in apoptosis and cell growth. These results reveal CRA-024781 to be a novel HDAC inhibitor with potent antitumor activity.
Topics: Acetylation; Animals; Antineoplastic Agents; Benzofurans; Biomarkers, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Drug Design; Enzyme Inhibitors; Female; HCT116 Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; In Vitro Techniques; Mice; Mice, Inbred BALB C; Poly(ADP-ribose) Polymerases; Transcription, Genetic; Tumor Cells, Cultured
PubMed: 16731764
DOI: 10.1158/1535-7163.MCT-05-0442