ABSTRACT
INTRODUCTION: FLT3-ITD mutations occur in approximately 25% of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Despite initial efficacy, short duration of response and high relapse rates limit clinical use of selective FLT3 inhibitors. Combination approaches with other targeted therapies may achieve better clinical outcomes. MATERIALS AND METHODS: Anti-leukemic activity of multikinase inhibitor olverembatinib (HQP1351), alone or in combination with BCL-2 inhibitor lisaftoclax (APG-2575), was evaluated in FLT3-ITD mutant AML cell lines in vitro and in vivo. A patient-derived FLT3-ITD mutant AML xenograft model was also used to assess the anti-leukemic activity of this combination. RESULTS: HQP1351 potently induced apoptosis and inhibited FLT3 signaling in FLT3-ITD mutant AML cell lines MV-4-11 and MOLM-13. HQP1351 monotherapy also significantly suppressed growth of FLT3-ITD mutant AML xenograft tumors and prolonged survival of tumor-bearing mice. HQP1351 and APG-2575 synergistically induced apoptosis in FLT3-ITD mutant AML cells and suppressed growth of MV-4-11 xenograft tumors. Combination therapy improved survival of tumor bearing-mice in a systemic MOLM-13 model and showed synergistic anti-leukemic effects in a patient-derived FLT3-ITD mutant AML xenograft model. Mechanistically, HQP1351 downregulated expression of myeloid-cell leukemia 1 (MCL-1) by suppressing FLT3-STAT5 (signal transducer and activator of transcription 5) signaling and thus enhanced APG-2575-induced apoptosis in FLT3-ITD mutant AML cells. CONCLUSIONS: FLT3 inhibition by HQP1351 downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 to potentiate cellular apoptosis in FLT3-ITD mutant AML. Our findings provide a scientific rationale for further clinical investigation of HQP1351 combined with APG-2575 in patients with FLT3-ITD mutant AML.
ABSTRACT
Despite the high initial response rates to PARP inhibitors (PARPi) in BRCA-mutated epithelial ovarian cancers (EOC), PARPi resistance remains a major challenge. Chemical modifications of RNAs have emerged as a new layer of epigenetic gene regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of mRNA, yet the role of m6A modification in PARPi resistance has not previously been explored. Here, we show that m6A modification of FZD10 mRNA contributes to PARPi resistance by upregulating the Wnt/ß-catenin pathway in BRCA-mutated EOC cells. Global m6A profile revealed a significant increase in m6A modification in FZD10 mRNA, which correlated with increased FZD10 mRNA stability and an upregulation of the Wnt/ß-catenin pathway. Depletion of FZD10 or inhibition of the Wnt/ß-catenin sensitizes resistant cells to PARPi. Mechanistically, downregulation of m6A demethylases FTO and ALKBH5 was sufficient to increase FZD10 mRNA m6A modification and reduce PARPi sensitivity, which correlated with an increase in homologous recombination activity. Moreover, combined inhibition of PARP and Wnt/ß-catenin showed synergistic suppression of PARPi-resistant cells in vitro and in vivo in a xenograft EOC mouse model. Overall, our results show that m6A contributes to PARPi resistance in BRCA-deficient EOC cells by upregulating the Wnt/ß-catenin pathway via stabilization of FZD10. They also suggest that inhibition of the Wnt/ß-catenin pathway represents a potential strategy to overcome PARPi resistance. SIGNIFICANCE: These findings elucidate a novel regulatory mechanism of PARPi resistance in EOC by showing that m6A modification of FZD10 mRNA contributes to PARPi resistance in BRCA-deficient EOC cells via upregulation of Wnt/ß-catenin pathway.
Subject(s)
Adenosine/metabolism , Drug Resistance, Neoplasm/genetics , Frizzled Receptors/genetics , Ovarian Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , AlkB Homolog 5, RNA Demethylase/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Animals , BRCA2 Protein/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Female , Frizzled Receptors/metabolism , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Methylation , Mice, SCID , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Phthalazines/pharmacology , Piperazines/pharmacology , RNA, Messenger/metabolism , Wnt Signaling Pathway/drug effects , Wnt Signaling Pathway/genetics , Xenograft Model Antitumor Assays , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
CARM1 is an arginine methyltransferase that asymmetrically dimethylates protein substrates on arginine residues. CARM1 is often overexpressed in human cancers. However, clinically applicable cancer therapeutic strategies based on CARM1 expression remain to be explored. Here, we report that EZH2 inhibition is effective in CARM1-expressing epithelial ovarian cancer. Inhibition of EZH2 activity using a clinically applicable small molecule inhibitor significantly suppresses the growth of CARM1-expressing, but not CARM1-deficient, ovarian tumors in two xenograft models and improves the survival of mice bearing CARM1-expressing ovarian tumors. The observed selectivity correlates with reactivation of EZH2 target tumor suppressor genes in a CARM1-dependent manner. Mechanistically, CARM1 promotes EZH2-mediated silencing of EZH2/BAF155 target tumor suppressor genes by methylating BAF155, which leads to the displacement of BAF155 by EZH2. Together, these results indicate that pharmacological inhibition of EZH2 represents a novel therapeutic strategy for CARM1-expressing cancers.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/metabolism , Neoplasms, Glandular and Epithelial/enzymology , Ovarian Neoplasms/enzymology , Protein-Arginine N-Methyltransferases/metabolism , Animals , Carcinoma, Ovarian Epithelial , Cell Line, Tumor , Enhancer of Zeste Homolog 2 Protein/genetics , Female , Humans , Mice , Neoplasms, Glandular and Epithelial/genetics , Neoplasms, Glandular and Epithelial/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Protein-Arginine N-Methyltransferases/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
BACKGROUND: Multi-drug resistance (MDR) remains a major impediment in cancer therapy. A major goal for scientists is to discover more effective compounds that are able to circumvent MDR and simultaneously have minimal adverse side effects. OBJECTIVE: In the present study, we aim to determine the anti-MDR effects of pyramidatine (Z88), a cinnamic acid-derived bisamide compound isolated from the leaves of Aglaia perviridis, on KB/VCR (vincristineresistant human oral cancer cells) and MCF-7/ADR (adriamycin-resistant human breast adenocarcinoma) cells. METHODS: Cell viability and average resistant fold (RF) of Z88 were examined by Cell Counting Kit-8 (CCK-8) assay. Flow cytometry, western blot, RT-PCR, Rhodamine 123 accumulation assay and P-glycoprotein (P-gp) ATPase assay were used to demonstrate the anti-MDR activity and mechanism of Z88. RESULTS: The average RF of Z88 is 0.09 and 0.51 in KB/VCR and MCF-7/ADR cells. A CCK-8 assay showed that Z88 could enhance the cytotoxicity of VCR toward KB/VCR cells. A FACS analysis revealed that Z88 could enhance the VCR-induced apoptosis as well as G2/M arrest in a dose-dependent manner in KB/VCR cells. Western blot results showed that the expression levels of PARP, Bax, and cyclin B1 all increased after treatment with 0.2 µmol/L (µM) of VCR combined with 10 µM of Z88 for 24 h in KB/VCR cells. Z88 also could enhance the accumulation of rhodamine 123. Further studies showed that Z88 could inhibit the verapamil stimulated Pgp ATPase activity. Additionally, qPCR detection and western blot assays revealed that Z88 could decrease the expression of P-gp at both RNA and protein level. CONCLUSION: Z88 exerted potent anti-MDR activity in vitro and its mechanisms are associated with dualinhibition of the function and expression of P-gp. These findings encourage efforts to develop more effective reversal agents to circumvent MDR based on Z88.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Amides/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Mouth Neoplasms/drug therapy , Vincristine/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Amides/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Molecular Structure , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Structure-Activity Relationship , Tumor Cells, Cultured , Vincristine/chemistryABSTRACT
PARP inhibition is known to be an effective clinical strategy in BRCA mutant cancers, but PARP inhibition has not been applied to BRCA-proficient tumors. Here, we show the synergy of BET bromodomain inhibition with PARP inhibition in BRCA-proficient ovarian cancers due to mitotic catastrophe. Treatment of BRCA-proficient ovarian cancer cells with the BET inhibitor JQ1 downregulated the G2-M cell-cycle checkpoint regulator WEE1 and the DNA-damage response factor TOPBP1. Combining PARP inhibitor Olaparib with the BET inhibitor, we observed a synergistic increase in DNA damage and checkpoint defects, which allowed cells to enter mitosis despite the accumulation of DNA damage, ultimately causing mitotic catastrophe. Moreover, JQ1 and Olaparib showed synergistic suppression of growth of BRCA-proficient cancer in vivo in a xenograft ovarian cancer mouse model. Our findings indicate that a combination of BET inhibitor and PARP inhibitor represents a potential therapeutic strategy for BRCA-proficient cancers.
Subject(s)
Azepines/pharmacology , Ovarian Neoplasms/metabolism , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Proteins/antagonists & inhibitors , Triazoles/pharmacology , Animals , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Cell Cycle/drug effects , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA Damage , Drug Synergism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Nuclear Proteins/metabolism , Ovarian Neoplasms/genetics , Poly(ADP-ribose) Polymerases/metabolism , Protein-Tyrosine Kinases/metabolism , Proteins/metabolismABSTRACT
Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor ß (Tgf-ß) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity.
Subject(s)
Epigenetic Repression/immunology , Gene Expression Regulation/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Matrix Attachment Region Binding Proteins/biosynthesis , Programmed Cell Death 1 Receptor/biosynthesis , Animals , Enzyme-Linked Immunospot Assay , Humans , Immunoprecipitation , Lymphocyte Activation/immunology , Matrix Attachment Region Binding Proteins/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms/immunology , Neoplasms/metabolismABSTRACT
Senescent cells exhibit dramatic changes in protein post-translational modifications. Here, we describe a method, stable isotope labeling with amino acids in cell culture (SILAC) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS), to identify changes in the ubiquitinome in cells that have undergone oncogene-induced senescence.
Subject(s)
Cellular Senescence , Oncogenes/genetics , Proteome , Proteomics , Ubiquitins/metabolism , Cell Line , Cellular Senescence/genetics , Chromatography, Affinity , Chromatography, Liquid/methods , Fibroblasts/metabolism , Genes, ras , Humans , Isotope Labeling , Protein Processing, Post-Translational , Proteomics/methods , Tandem Mass Spectrometry/methods , Ubiquitins/isolation & purificationABSTRACT
The emergence of tumor cells with certain stem-like characteristics, such as high aldehyde dehydrogenase (ALDH) activity due to ALDH1A1 expression, contributes to chemotherapy resistance and tumor relapse. However, clinically applicable inhibitors of ALDH activity have not been reported. There is evidence to suggest that epigenetic regulation of stem-related genes contributes to chemotherapy efficacy. Here, we show that bromodomain and extraterminal (BET) inhibitors suppress ALDH activity by abrogating BRD4-mediated ALDH1A1 expression through a super-enhancer element and its associated enhancer RNA. The clinically applicable small-molecule BET inhibitor JQ1 suppressed the outgrowth of cisplatin-treated ovarian cancer cells both in vitro and in vivo Combination of JQ1 and cisplatin improved the survival of ovarian cancer-bearing mice in an orthotopic model. These phenotypes correlate with inhibition of ALDH1A1 expression through a super-enhancer element and other stem-related genes in promoter regions bound by BRD4. Thus, targeting the BET protein BRD4 using clinically applicable small-molecule inhibitors, such as JQ1, is a promising strategy for targeting ALDH activity in epithelial ovarian cancer. Cancer Res; 76(21); 6320-30. ©2016 AACR.
Subject(s)
Aldehyde Dehydrogenase/antagonists & inhibitors , Azepines/pharmacology , Neoplasms, Glandular and Epithelial/drug therapy , Nuclear Proteins/antagonists & inhibitors , Ovarian Neoplasms/drug therapy , Transcription Factors/antagonists & inhibitors , Triazoles/pharmacology , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase 1 Family , Animals , Carcinoma, Ovarian Epithelial , Cell Cycle Proteins , Cell Line, Tumor , Cisplatin/pharmacology , Female , Humans , Mice , Neoplasms, Glandular and Epithelial/enzymology , Nuclear Proteins/physiology , Ovarian Neoplasms/enzymology , Retinal Dehydrogenase , Transcription Factors/physiologyABSTRACT
Restoration of anti-tumor immunity by blocking PD-L1 signaling through the use of antibodies has proven to be beneficial in cancer therapy. Here, we show that BET bromodomain inhibition suppresses PD-L1 expression and limits tumor progression in ovarian cancer. CD274 (encoding PD-L1) is a direct target of BRD4-mediated gene transcription. In mouse models, treatment with the BET inhibitor JQ1 significantly reduced PD-L1 expression on tumor cells and tumor-associated dendritic cells and macrophages, which correlated with an increase in the activity of anti-tumor cytotoxic T cells. The BET inhibitor limited tumor progression in a cytotoxic T-cell-dependent manner. Together, these data demonstrate a small-molecule approach to block PD-L1 signaling. Given the fact that BET inhibitors have been proven to be safe with manageable reversible toxicity in clinical trials, our findings indicate that pharmacological BET inhibitors represent a treatment strategy for targeting PD-L1 expression.
Subject(s)
Azepines/pharmacology , B7-H1 Antigen/genetics , Immunity/drug effects , Neoplasms/immunology , Neoplasms/metabolism , Proteins/antagonists & inhibitors , Triazoles/pharmacology , Animals , B7-H1 Antigen/metabolism , Cell Cycle Proteins , Cell Line, Tumor , Disease Progression , Female , Humans , Mice, Inbred C57BL , Neoplasms/pathology , Nuclear Proteins/metabolism , Proteins/metabolism , T-Lymphocytes, Cytotoxic/drug effects , Time Factors , Transcription Factors/metabolism , Transcription, Genetic/drug effectsABSTRACT
Natural products have become sources of developing new drugs for the treatment of cancer. To seek candidate compounds that inhibit the growth of liver cancer, components of Chloranthus serratus were tested. Here, we report that shizukaol D, a dimeric sesquiterpene from Chloranthus serratus, exerted a growth inhibition effect on liver cancer cells in a dose- and time-dependent manner. We demonstrated that shizukaol D induced cells to undergo apoptosis. More importantly, shizukaol D attenuated Wnt signalling and reduced the expression of endogenous Wnt target genes, which resulted in decreased expression of ß-catenin. Collectively, this study demonstrated that shizukaol D inhibited the growth of liver cancer cells by modulating Wnt pathway.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Liver Neoplasms/drug therapy , Liver/drug effects , Triterpenes/pharmacology , Wnt Signaling Pathway/drug effects , Antineoplastic Agents, Phytogenic/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Liver/metabolism , Liver/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Magnoliopsida/chemistry , Triterpenes/chemistryABSTRACT
The SPOP gene, which encodes an E3 ubiquitin ligase adaptor, is frequently mutated in a number of cancer types. However, the mechanisms by which SPOP functions as a tumor suppressor remain poorly understood. Here, we show that SPOP promotes senescence, an important tumor suppression mechanism, by targeting the SENP7 deSUMOylase for degradation. SPOP is upregulated during senescence. This correlates with ubiquitin-mediated degradation of SENP7, which promotes senescence by increasing HP1α sumoylation and the associated epigenetic gene silencing. Ectopic wild-type SPOP, but not its cancer-associated mutants, drives senescence. Conversely, SPOP knockdown overcomes senescence. These phenotypes correlate with ubiquitination and degradation of SENP7 and HP1α sumoylation, subcellular re-localization, and its associated gene silencing. Furthermore, SENP7 is expressed at higher levels in prostate tumor specimens with SPOP mutation (n = 13) compared to those with wild-type SPOP (n = 80). In summary, SPOP acts as a tumor suppressor by promoting senescence through degrading SENP7.
Subject(s)
Endopeptidases/metabolism , Nuclear Proteins/metabolism , Prostatic Neoplasms/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Cells, Cultured , Cellular Senescence , Chromobox Protein Homolog 5 , Endopeptidases/genetics , Gene Silencing , HEK293 Cells , Humans , Male , Molecular Sequence Data , Nuclear Proteins/genetics , Proteolysis , Repressor Proteins/genetics , SumoylationABSTRACT
Although cellular senescence is accompanied by global alterations in genome architecture, how the genome is restructured during the senescent processes is not well understood. Here, we show that the hCAP-H2 subunit of the condensin II complex exists as either a full-length protein or an N-terminus truncated variant (ΔN). While the full-length hCAP-H2 associates with mitotic chromosomes, the ΔN variant exists as an insoluble nuclear structure. When overexpressed, both hCAP-H2 isoforms assemble this nuclear architecture and induce senescence-associated heterochromatic foci (SAHF). The hCAP-H2ΔN protein accumulates as cells approach senescence, and hCAP-H2 knockdown inhibits oncogene-induced senescence. This study identifies a novel mechanism whereby condensin drives senescence via nuclear/genomic reorganization.
Subject(s)
Adenosine Triphosphatases/physiology , Cellular Senescence/physiology , DNA-Binding Proteins/physiology , Multiprotein Complexes/physiology , Serine Endopeptidases/physiology , Base Sequence , HCT116 Cells , HeLa Cells , Humans , Molecular Sequence DataABSTRACT
Oncogene-induced senescence (OIS) is an important tumor suppression mechanism preventing uncontrolled proliferation in response to aberrant oncogenic signaling. The profound functional and morphological remodelling of the senescent cell involves extensive changes. In particular, alterations in protein ubiquitination during senescence have not been systematically analyzed previously. Here, we report the first global ubiquitination profile of primary human cells undergoing senescence. We employed a well-characterized in vitro model of OIS, primary human fibroblasts expressing oncogenic RAS. To compare the ubiquitinome of RAS-induced OIS and controls, ubiquitinated peptides were enriched by immune affinity purification and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified 4,472 ubiquitination sites, with 397 sites significantly changed (>3 standard deviations) in senescent cells. In addition, we performed mass spectrometry analysis of total proteins in OIS and control cells to account for parallel changes in both protein abundance and ubiquitin levels that did not affect the percentage of ubiquitination of a given protein. Pathway analysis revealed that the OIS-induced ubiquitinome alterations mainly affected 3 signaling networks: eIF2 signaling, eIF4/p70S6K signaling, and mTOR signaling. Interestingly, the majority of the changed ubiquitinated proteins in these pathways belong to the translation machinery. This includes several translation initiation factors (eIF2C2, eIF2B4, eIF3I, eIF3L, eIF4A1) and elongation factors (eEF1G, eEF1A) as well as 40S (RPS4X, RPS7, RPS11 and RPS20) and 60S ribosomal subunits (RPL10, RPL11, RPL18 and RPL35a). In addition, we observed enriched ubiquitination of aminoacyl-tRNA ligases (isoleucyl-, glutamine-, and tyrosine-tRNA ligase), which provide the amino acid-loaded tRNAs for protein synthesis. These results suggest that ubiquitination affects key components of the translation machinery to regulate protein synthesis during OIS. Our results thus point toward ubiquitination as a hitherto unappreciated regulatory mechanism during OIS.
Subject(s)
Cellular Senescence , Oncogene Protein p21(ras)/metabolism , Amino Acid Sequence , Cells, Cultured , Chromatography, High Pressure Liquid , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factor-4F/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Isotope Labeling , Oncogene Protein p21(ras)/genetics , Peptides/analysis , Peptides/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Tandem Mass Spectrometry , UbiquitinationABSTRACT
Aurora B is a serine/threonine kinase that has a key role in mitosis and is overexpressed in cancer cells. Aberrations in Aurora B are highly correlated with tumorigenesis and cancer development, so many studies have focused on the development of Aurora B kinase inhibitors. Based on one of our previous high-throughput screening studies, we identified lead compound S6, a small-molecule benzofuran derivative that binds Aurora B and inhibits its kinase activity in vitro. S6 also displayed high selectivity for Aurora B inhibition. The cytotoxicity of S6 was assessed against a panel of 21 cancer cell lines. The cervical cancer cell line HeLa, liver cancer cell line HepG2 and colon cancer cell line SW620 were the most sensitive to S6 treatment. We found that S6 decreased the proliferation and colony formation of these three cell lines and elevated their percentages of cells in the G2/M phase of the cell cycle. S6 also inhibited phospho-histone H3 on Ser 10, a natural biomarker of endogenous Aurora B activity. The growth suppression of liver cancer QGY-7401 xenograft tumors was observed in nude mice after S6 administration, and this effect was accompanied by the in vivo inhibition of phospho-histone H3 (Ser 10). Taken together, we conclude that targeting Aurora B with compound S6 may be a novel strategy for cancer treatment, and additional studies are warranted.
Subject(s)
Antineoplastic Agents/chemical synthesis , Aurora Kinase B/antagonists & inhibitors , Benzofurans/chemical synthesis , Liver Neoplasms, Experimental/drug therapy , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Benzofurans/chemistry , Benzofurans/pharmacology , Benzofurans/therapeutic use , Blotting, Western , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , High-Throughput Screening Assays , Humans , Liver Neoplasms, Experimental/enzymology , Liver Neoplasms, Experimental/pathology , Mice, Nude , Molecular Structure , Surface Plasmon Resonance , Xenograft Model Antitumor AssaysABSTRACT
Multidrug resistance (MDR) to anticancer drugs is a major obstacle to successful chemotherapy in the treatment of cancers. Identification of natural compounds capable of circumventing MDR with minimal adverse side effects is an attractive goal. Here, we found that H6, a gypenoside aglycon from Gynostemma pentaphyllum, displayed potent anti-MDR activity. Average resistant fold (RF) of H6 is 1.03 and 1.04 in KB/VCR and MCF-7/ADR cells compared to their parental cells. H6 alone ranging from 2 µmol/l to 40 µmol/l (µM) did not display a significant anti-proliferative effect on KB/VCR cells and other cells, while the compound at these concentrations enhanced the cytotoxicity of vincristine (VCR) to KB/VCR cells. H6 showed a significant synergistic effect in combination with VCR. By quantification of sub-G(1) fraction cells, H6 also enhanced the VCR-induced apoptosis in a dose-dependent manner. The short time treatment with H6 increased the intracellular accumulation of rhodamine 123 (Rho123) and 5(6)-carboxyfluorescein diacetate (CFDA) in KB/VCR cells. Further studies showed that H6 treatment resulted in the decrease of the RNA transcript level of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). H6 inhibited the function of P-gp by stimulating P-gp ATPase activity and decreased MRP1 expression with a blockade of STAT3 phosphorylation. These findings suggest that H6, a multi-targets reversal agent with no significant toxic effect, may be a potential candidate to circumvent the P-gp and MRP1-mediated MDR.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Drug Resistance, Neoplasm/drug effects , Gynostemma , Sapogenins/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cell Line, Tumor , Humans , Mouth Neoplasms , Multidrug Resistance-Associated Proteins/metabolism , VincristineABSTRACT
Cyclophilin A (CypA) is a member of peptidyl prolyl isomerases (PPIases), which catalyze the cis/trans isomerization of prolyl peptide bonds on the NH-terminal side of Pro residues in peptide chains. Altered expression of CypA has been reported in hepatocellular carcinoma (HCC), but the biological functions of CypA in HCC remain unknown. We found that the level of CypA expression correlated with the metastatic capability of two HCC cell lines, MHCC97-L and MHCC97-H. Stable expression of ectopic CypA in SK-Hep1 cells promotes cell adhesion, motility, chemotaxis, and in vivo lung metastasis, without affecting cell proliferation. We further analyzed microarray results to identify target genes controlled by CypA. Twenty-one genes related to metastasis were altered by CypA over-expression. A member of matrix metalloproteinase, MMP3, was identified by microarray analysis. The regulation of MMP3 and its homologue MMP9 by CypA were further confirmed by quantitative real-time RT-PCR and zymography assay. Taken together, our data suggest that CypA promotes HCC cell metastasis at least partially through up-regulation of MMP3 and MMP9.
Subject(s)
Carcinoma, Hepatocellular/pathology , Cyclophilin A/metabolism , Liver Neoplasms/pathology , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 9/metabolism , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Adhesion/genetics , Cell Line, Tumor , Cell Proliferation , Cyclophilin A/genetics , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 9/genetics , Mice , Mice, Nude , Microarray Analysis , Neoplasm Invasiveness/genetics , Neoplasm MetastasisABSTRACT
Sedlin is an evolutionarily conserved and ubiquitously expressed protein that is encoded by the gene SEDL. Mutations in the latter are known to be causative for spondyloepiphyseal dysplasia tarda. However, the mechanism underlying this remains unclear. We have previously shown that Sedlin interacts with the intracellular chloride channel proteins CLIC1 and CLIC2 in the cytoplasm. In this report we show that Sedlin is also physically associated with protein associated with MRG 14 kDa (PAM14), a nuclear protein that interacts with the transcription factor MORF4-related gene on chromosome 15 (MRG15). This was suggested by yeast two-hybrid screening and was confirmed with GST pull-down and immunoprecipitation assays. Moreover, we demonstrate that the C-terminus of Sedlin and the N-terminus of PAM14 are critical for their interaction. Together, these results suggest that nucleus-localized Sedlin may play a role in regulation of transcriptional activities of the MRG family of transcription factors via binding to PAM14.