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1.
Biotechniques ; 65(6): 322-330, 2018 12.
Article in English | MEDLINE | ID: mdl-30477327

ABSTRACT

We describe a novel automated cell detection and counting software, QuickCount® (QC), designed for rapid quantification of cells. The Bland-Altman plot and intraclass correlation coefficient (ICC) analyses demonstrated strong agreement between cell counts from QC to manual counts (mean and SD: -3.3 ± 4.5; ICC = 0.95). QC has higher recall in comparison to ImageJauto, CellProfiler and CellC and the precision of QC, ImageJauto, CellProfiler and CellC are high and comparable. QC can precisely delineate and count single cells from images of different cell densities with precision and recall above 0.9. QC is unique as it is equipped with real-time preview while optimizing the parameters for accurate cell count and needs minimum hands-on time where hundreds of images can be analyzed automatically in a matter of milliseconds. In conclusion, QC offers a rapid, accurate and versatile solution for large-scale cell quantification and addresses the challenges often faced in cell biology research.


Subject(s)
Cell Count/methods , Image Processing, Computer-Assisted/methods , Software , Animals , Cell Count/economics , Cell Line , Cell Line, Tumor , Humans , Image Processing, Computer-Assisted/economics , Mice , Microscopy/economics , Microscopy/methods , Time Factors , Workflow
2.
Phytomedicine ; 39: 33-41, 2018 Jan 15.
Article in English | MEDLINE | ID: mdl-29433681

ABSTRACT

BACKGROUND: The CXCR4-RhoA and PI3K-mTOR signaling pathways play crucial roles in the dissemination and tumorigenesis of oral squamous cell carcinoma (OSCC). Activation of these pathways have made them promising molecular targets in the treatment of OSCC. Zerumbone, a bioactive monocyclic sesquiterpene isolated from the rhizomes of tropical ginger, Zingiber zerumbet (L.) Roscoe ex Sm. has displayed promising anticancer properties with the ability to modulate multiple molecular targets involved in carcinogenesis. While the anticancer activities of zerumbone have been well explored across different types of cancer, the molecular mechanism of action of zerumbone in OSCC remains largely unknown. PURPOSE: Here, we investigated whether OSCC cells were sensitive towards zerumbone treatment and further determined the molecular pathways involved in the mechanism of action. METHODS: Cytotoxicity, anti-proliferative, anti-migratory and anti-invasive effects of zerumbone were tested on a panel of OSCC cell lines. The mechanism of action of zerumbone was investigated by analysing the effects on the CXCR4-RhoA and PI3K-mTOR pathways by western blotting. RESULTS: Our panel of OSCC cells was broadly sensitive towards zerumbone with IC50 values of less than 5 µM whereas normal keratinocyte cells were less responsive with IC50 values of more than 25 µM. Representative OSCC cells revealed that zerumbone inhibited OSCC proliferation and induced cell cycle arrest and apoptosis. In addition, zerumbone treatment inhibited migration and invasion of OSCC cells, with concurrent suppression of endogenous CXCR4 protein expression in a time and dose-dependent manner. RhoA-pull down assay showed reduction in the expression of RhoA-GTP, suggesting the inactivation of RhoA by zerumbone. In association with this, zerumbone also inhibited the PI3K-mTOR pathway through the inactivation of Akt and S6 proteins. CONCLUSION: We provide evidence that zerumbone could inhibit the activation of CXCR4-RhoA and PI3K-mTOR signaling pathways leading to the reduced cell viability of OSCC cells. Our results suggest that zerumbone is a promising phytoagent for development of new therapeutics for OSCC treatment.


Subject(s)
Carcinoma, Squamous Cell/drug therapy , Mouth Neoplasms/drug therapy , Receptors, CXCR4/metabolism , Sesquiterpenes/pharmacology , TOR Serine-Threonine Kinases/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Phosphatidylinositol 3-Kinases/metabolism , Receptors, CXCR4/antagonists & inhibitors , Signal Transduction/drug effects , rhoA GTP-Binding Protein/metabolism
3.
BMC Genomics ; 18(Suppl 1): 934, 2017 01 25.
Article in English | MEDLINE | ID: mdl-28198666

ABSTRACT

BACKGROUND: The drug discovery and development pipeline is a long and arduous process that inevitably hampers rapid drug development. Therefore, strategies to improve the efficiency of drug development are urgently needed to enable effective drugs to enter the clinic. Precision medicine has demonstrated that genetic features of cancer cells can be used for predicting drug response, and emerging evidence suggest that gene-drug connections could be predicted more accurately by exploring the cumulative effects of many genes simultaneously. RESULTS: We developed DeSigN, a web-based tool for predicting drug efficacy against cancer cell lines using gene expression patterns. The algorithm correlates phenotype-specific gene signatures derived from differentially expressed genes with pre-defined gene expression profiles associated with drug response data (IC50) from 140 drugs. DeSigN successfully predicted the right drug sensitivity outcome in four published GEO studies. Additionally, it predicted bosutinib, a Src/Abl kinase inhibitor, as a sensitive inhibitor for oral squamous cell carcinoma (OSCC) cell lines. In vitro validation of bosutinib in OSCC cell lines demonstrated that indeed, these cell lines were sensitive to bosutinib with IC50 of 0.8-1.2 µM. As further confirmation, we demonstrated experimentally that bosutinib has anti-proliferative activity in OSCC cell lines, demonstrating that DeSigN was able to robustly predict drug that could be beneficial for tumour control. CONCLUSIONS: DeSigN is a robust method that is useful for the identification of candidate drugs using an input gene signature obtained from gene expression analysis. This user-friendly platform could be used to identify drugs with unanticipated efficacy against cancer cell lines of interest, and therefore could be used for the repurposing of drugs, thus improving the efficiency of drug development.


Subject(s)
Computational Biology/methods , Drug Design , Drug Repositioning , Gene Expression Regulation/drug effects , Software , Algorithms , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Databases, Genetic , Gene Expression Profiling , Humans , Inhibitory Concentration 50 , Protein Kinase Inhibitors/pharmacology , Reproducibility of Results , Transcriptome , Web Browser , Workflow
4.
Oncotarget ; 7(36): 57633-57650, 2016 Sep 06.
Article in English | MEDLINE | ID: mdl-27192118

ABSTRACT

Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.


Subject(s)
Breast Neoplasms/metabolism , Fibroblast Growth Factors/metabolism , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Breast Neoplasms/diagnosis , Cell Line, Tumor , Cell Survival , Doxorubicin/chemistry , Female , Gene Silencing , Humans , MCF-7 Cells , Phosphorylation , Prognosis , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/metabolism , Signal Transduction
5.
Oncotarget ; 7(19): 27802-18, 2016 May 10.
Article in English | MEDLINE | ID: mdl-27050151

ABSTRACT

Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC.


Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Molecular Targeted Therapy , Mouth Neoplasms/drug therapy , Mouth Neoplasms/genetics , Animals , Antineoplastic Agents/pharmacology , Areca/adverse effects , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , DNA Copy Number Variations , Female , Gene Amplification , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genes, Neoplasm/genetics , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Mouth Neoplasms/pathology , Mutation , Sequence Analysis, RNA , Sequence Deletion , Transcriptome , Xenograft Model Antitumor Assays
6.
Phytomedicine ; 21(12): 1645-50, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-25442272

ABSTRACT

This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC50 values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC50 values of 50.8 and 40.2 µM, while the other active compounds did not significantly affect CYP1A2 activity with IC50 values more than 100 µM. Ki values further determined for eupatorin and sinensetin were 46.4 and 35.2 µM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations.


Subject(s)
Cytochrome P-450 CYP1A2 Inhibitors/pharmacology , Cytochrome P-450 CYP1A2/metabolism , Flavonoids/pharmacology , Plant Extracts/pharmacology , Humans , Inhibitory Concentration 50 , Molecular Structure
7.
PLoS One ; 9(1): e86230, 2014.
Article in English | MEDLINE | ID: mdl-24475091

ABSTRACT

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6 16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6 16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.


Subject(s)
Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Aryl Hydrocarbon Hydroxylases/genetics , Genetic Variation , Methoxsalen/pharmacology , Models, Molecular , Structure-Activity Relationship , Aryl Hydrocarbon Hydroxylases/chemistry , Cytochrome P-450 CYP2A6 , Escherichia coli , Humans , Inhibitory Concentration 50 , Isoenzymes/genetics , Molecular Structure , Mutation/genetics , Protein Binding , Protein Conformation
8.
J Nat Med ; 68(2): 402-6, 2014 Apr.
Article in English | MEDLINE | ID: mdl-23881640

ABSTRACT

Eurycomanone, an active constituent isolated from Eurycoma longifolia Jack, was examined for modulatory effects on cytochrome P450 (CYP) isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 using in vitro assays. The IC50 value was determined to assess the potencies of modulation for each CYP isoform. Our results indicated that eurycomanone did not potently inhibit any of the CYP isoforms investigated, with IC50 values greater than 250 µg/ml. Hence there appears to be little likelihood of drug-herb interaction between eurycomanone or herbal products with high content of this compound and CYP drug substrates via CYP inhibition.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Plant Extracts/pharmacology , Quassins/pharmacology , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP2A6 , Cytochrome P-450 CYP2C19 , Cytochrome P-450 CYP2C8 , Cytochrome P-450 CYP2C9 , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 CYP3A/metabolism , Humans , Isoenzymes/metabolism
9.
Apoptosis ; 18(12): 1447-68, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23900974

ABSTRACT

The fibroblast growth factor receptors (FGFRs) regulate important biological processes including cell proliferation and differentiation during development and tissue repair. Over the past decades, numerous pathological conditions and developmental syndromes have emerged as a consequence of deregulation in the FGFRs signaling network. This review aims to provide an overview of FGFR family, their complex signaling pathways in tumorigenesis, and the current development and application of therapeutics targeting the FGFRs signaling for treatment of refractory human cancers.


Subject(s)
Neoplasms/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Signal Transduction , Animals , Antineoplastic Agents/therapeutic use , Apoptosis , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/physiopathology , Receptors, Fibroblast Growth Factor/genetics
10.
Cancer Lett ; 336(1): 127-39, 2013 Aug 09.
Article in English | MEDLINE | ID: mdl-23612072

ABSTRACT

6-Shogaol has been shown to possess many antitumor properties including inhibition of cancer cell growth, inhibition of cancer metastasis, induction of apoptosis in cancer cells and induction of cancer cell differentiation. Despite its prominent antitumor effects, the direct molecular target of 6-shogaol has remained elusive. To identify the direct targets of 6-shogaol, a comprehensive antitumor profile of 6-shogaol (NSC752389) was tested in the NCI-60 cell line in an in vitro screen. The results show that 6-shogaol is COMPARE negative suggesting that it functions via a mechanism of action distinct from existing classes of therapeutic agents. Further analysis using microarray gene profiling and Connectivity Map analysis showed that MCF-7 cells treated with 6-shogaol display gene expression signatures characteristic of peroxisome proliferator activated receptor γ (PPARγ) agonists, suggesting that 6-shogaol may activate the PPARγ signaling pathway for its antitumor effects. Indeed, treatment of MCF-7 and HT29 cells with 6-shogaol induced PPARγ transcriptional activity, suppressed NFκB activity, and induced apoptosis in breast and colon cancer cells in a PPARγ-dependent manner. Furthermore, 6-shogaol is capable of binding to PPARγ with a binding affinity comparable to 15-delta prostaglandin J2, a natural ligand for PPARγ. Together, our findings suggest that the antitumor effects of 6-shogaol are mediated through activation of PPARγ and imply that activation of PPARγ might be beneficial for breast and colon cancer treatment.


Subject(s)
Breast Neoplasms/metabolism , Catechols/pharmacology , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , PPAR gamma/metabolism , Antineoplastic Agents/pharmacology , Apoptosis , Breast Neoplasms/pathology , Cell Line, Tumor , Colonic Neoplasms/pathology , Dose-Response Relationship, Drug , Enzyme Activation , Female , Humans , Inhibitory Concentration 50 , Ligands , MCF-7 Cells , NF-kappa B/metabolism , Oligonucleotide Array Sequence Analysis , Prostaglandin D2/analogs & derivatives , Prostaglandin D2/metabolism , Signal Transduction , Transcription, Genetic
11.
J Ethnopharmacol ; 143(2): 586-91, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-22885070

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Labisa pumila (LP), popularly known with its local name, Kacip Fatimah, is a well known herb grown in Indochina and Southeast Asia and is traditionally used to regain energy after giving birth in women. The propensity of LP to cause drug-herb interaction via cytochrome P450 (CYP) enzyme system has not been investigated. AIM OF THE STUDY: To evaluate the in vitro inhibitory effects of various LP extracts (aqueous, ethanol, dichloromethane (DCM) and hexane) on cytochrome P450 2C8 (CYP2C8), CYP2C9, CYP2C19 and CYP3A4 activities. MATERIALS AND METHODS: Probe substrate-based high performance liquid chromatography (HPLC) methods were established for CYP2C9, CYP2C19 and CYP3A4 whereas a fluorescence-based enzyme assay was established for CYP2C8. The metabolite formations were examined after incubation of probe substrate with respective CYP isoform in the present or absent of LP extracts. The inhibitory effect of LP was characterized with kinetic parameters IC(50) and K(i) values. RESULTS: LP extracts showed differential effect of CYP activities with the order of inhibitory potency as follows: dichloromethane>hexane>ethanol>aqueous. This differential effect was only observed in CYP2C isoforms but not CYP3A4. Both the hexane and DCM extracts exhibited moderate to potent inhibition towards CYP2C activities in different modes including non-competitive, competive and mixed-type. The DCM effect was notably strong for CYP2C8 and CYP2C9 showing K(i) values of below 1 µg/ml. The selectivity of LP for CYP2C isoforms rather than CYP3A4 may be attributed to the presence of relatively small, lipophilic yet slightly polar compounds within the LP extracts. CONCLUSIONS: The results of our study revealed that phytoconstituents contained in LP, particularly in hexane and dichloromethane extracts, were able to selectively inhibit CYP2C isoforms. The inactivation was characterized by low K(i) values, in particular, in CYP2C8 and CYP2C9. These in vitro data indicate that LB preparations contain constituents that can potently inhibit CYP2C activities and suggest that this herb should be examined for potential pharmacokinetic drug interactions in vivo.


Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Plant Extracts/pharmacology , Primulaceae , Cytochrome P-450 Enzyme System/metabolism , Ethanol/chemistry , Hexanes/chemistry , Methylene Chloride/chemistry , Solvents/chemistry , Water/chemistry
12.
Cancer Lett ; 314(2): 166-75, 2012 Jan 28.
Article in English | MEDLINE | ID: mdl-22033244

ABSTRACT

The efficacy of cisplatin for treating nasopharyngeal carcinoma (NPC) is limited by the dose-related toxicities and the development of resistance to cisplatin. Recent studies have shown that B cell lymphoma-2 (BCL-2) is overexpressed and confers chemoresistance in NPC. Thus, targeted therapy against BCL-2 may enhance the antitumour effects of chemotherapy by sensitizing the tumor cells to undergo apoptosis. This study evaluated the combined effects of BCL-2 inhibition and cisplatin in NPC cells. Our results demonstrate that inhibition of BCL-2 by small-hairpin RNA (shRNA) or the BCL-2 inhibitor YC137, synergizes cisplatin sensitivity in NPC cells that overexpress BCL-2. We also show that YC137 enhance cisplatin-induced apoptosis in HK1 and CNE1 cells through suppression of BCL-2 protein expression, induction of mitochondrial depolarization and activation of caspase 9 and caspase 3/7. These findings suggest that the combination of BCL-2 inhibition and cisplatin represents a promising strategy for treating NPC.


Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Nasopharyngeal Neoplasms/drug therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Thiazoles/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Humans , Nasopharyngeal Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/analysis
13.
Mol Cancer Ther ; 10(10): 1982-92, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21831963

ABSTRACT

Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F-203; NSC 703786) and 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (GW-610; NSC 721648) are antitumor agents with novel mechanism(s). Previous studies have indicated that cytochrome (CYP) P450 1A1 is crucial for 5F-203 activity. In the present study, we investigated the functional role of 2 newly identified CYP P450 enzymes, CYP2S1 and CYP2W1, in mediating antitumor activity of benzothiazole compounds. We generated isogenic breast cancer (MDA-MB-468, MCF-7) and colorectal cancer (CRC; KM12 and HCC2998) cell lines depleted for CYP1A1, CYP2S1, or CYP2W1. The sensitivity of these cells to 5F-203 and GW-610 was then compared with vector control cells. 5F-203 exhibited potent activity against breast cancer cells, whereas GW-610 was effective against both breast and colorectal cancer cells. CYP1A1 was induced in both breast cancer and CRC cells, while CYP2S1 and CYP2W1 were selectively induced in breast cancer cells only following treatment with 5F-203 or GW-610. Depletion of CYP1A1 abrogated the sensitivity of breast cancer and CRC cells to 5F-203 and GW-610. Although depletion of CYP2S1 sensitized both breast cancer and CRC cells toward 5F-203 and GW-610, CYP2W1 knockdown caused marked resistance to GW-610 in CRC cells. Our results indicate that CYP-P450 isoforms, with the exception of CYP1A1, play an important role in mediating benzothiazole activity. CYP2S1 appears to be involved in deactivation of benzothiazoles, whereas CYP2W1 is important for bioactivation of GW-610 in CRC cells. Because CYP2W1 is highly expressed in colorectal tumors, GW-610 represents a promising agent for CRC therapy.


Subject(s)
Benzothiazoles/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/enzymology , Cytochrome P-450 Enzyme System/metabolism , Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Cell Growth Processes/drug effects , Cell Line, Tumor , Colorectal Neoplasms/pathology , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P450 Family 2 , Enzyme Induction/drug effects , Female , Humans , Male , Thiazoles/pharmacology
14.
Breast Cancer Res Treat ; 128(2): 301-13, 2011 Jul.
Article in English | MEDLINE | ID: mdl-20686837

ABSTRACT

Recent gene expression profiling studies have identified five breast cancer subtypes, of which the basal-like subtype is the most aggressive. Basal-like breast cancer poses serious clinical challenges as there are currently no targeted therapies available to treat it. Although there is increasing evidence that these tumors possess specific sensitivity to cisplatin, its success is often compromised due to its dose-limiting nephrotoxicity and the development of drug resistance. To overcome this limitation, our goal was to maximize the benefits associated with cisplatin therapy through drug combination strategies. Using a validated kinase inhibitor library, we showed that inhibition of the mTOR, TGFßRI, NFκB, PI3K/AKT, and MAPK pathways sensitized basal-like MDA-MB-468 cells to cisplatin treatment. Further analysis demonstrated that the combination of the mTOR inhibitor rapamycin and cisplatin generated significant drug synergism in basal-like MDA-MB-468, MDA-MB-231, and HCC1937 cells but not in luminal-like T47D or MCF-7 cells. We further showed that the synergistic effect of rapamycin plus cisplatin on basal-like breast cancer cells was mediated through the induction of p73. Depletion of endogenous p73 in basal-like cells abolished these synergistic effects. In conclusion, combination therapy with mTOR inhibitors and cisplatin may be a useful therapeutic strategy in the treatment of basal-like breast cancers.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/drug therapy , Carcinoma, Basal Cell/drug therapy , Cell Proliferation/drug effects , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Apoptosis , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinoma, Basal Cell/genetics , Carcinoma, Basal Cell/metabolism , Cell Line, Tumor , Cisplatin/administration & dosage , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Drug Synergism , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sirolimus/administration & dosage , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Tumor Protein p73 , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
15.
Drug Metab Dispos ; 38(5): 745-51, 2010 May.
Article in English | MEDLINE | ID: mdl-20139165

ABSTRACT

Variation in CYP2A6 levels and activity can be attributed to genetic polymorphism and, thus, functional characterization of allelic variants is necessary to define the importance of CYP2A6 polymorphism in humans. The aim of the present study was to investigate the reported alleles CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22, in terms of the functional consequences of their mutations on the enzyme catalytic activity. With use of the wild-type CYP2A6 cDNA as template, site-directed mutagenesis was performed to introduce nucleotide changes encoding K194E substitution in CYP2A6*15, R203S substitution in CYP2A6*16, K476R substitution in CYP2A6*21, and concurrent D158E and L160I substitutions in CYP2A6*22. Upon sequence verification, the CYP2A6 wild-type and mutant constructs were individually coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. A kinetic study using a coumarin 7-hydroxylase assay indicated that CYP2A6*15 exhibited higher V(max) than the wild type, whereas all mutant constructs, except for variant CYP2A6*16, exhibited higher K(m) values. Analysis of the V(max)/K(m) ratio revealed that all mutants demonstrated 0.85- to 1.05-fold differences from the wild type, with the exception of variant CYP2A6*22, which only portrayed 39% of the wild-type intrinsic clearance. These data suggested that individuals carrying the CYP2A6*22 allele are likely to have lower metabolism of CYP2A6 substrate than individuals expressing CYP2A6*15, CYP2A6*16, CYP2A6*21, and the wild type.


Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Polymorphism, Single Nucleotide/physiology , Amino Acid Substitution/physiology , Base Sequence , Biocatalysis , Cell Membrane/metabolism , Coumarins/metabolism , Cytochrome P-450 CYP2A6 , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Kinetics , Mutagenesis, Site-Directed , Spectrophotometry , Transformation, Genetic , Umbelliferones/metabolism
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