RESUMEN
Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with a poor prognosis for metastasis and recurrent disease. Large-scale sequencing endeavors demonstrate that Rhabdomyosarcomas have a dearth of precisely targetable driver mutations. However, IGF-2 signaling is known to be grossly altered in RMS. The insulin receptor (IR) exists in two alternatively spliced isoforms, IR-A and IR-B. The IGF-2 signaling molecule binds both its innate IGF-1 receptor as well as the insulin receptor variant A (IR-A) with high affinity. Mitogenic and proliferative signaling via the canonical IGF-2 pathway is, therefore, augmented by IR-A. This study shows that RMS patients express increased IR-A levels compared to control tissues that predominantly express the IR-B isoform. We also found that Hif-1α is significantly increased in RMS tumors, portraying their hypoxic phenotype. Concordantly, the alternative splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a potential hypoxia-responsive element, which is also the binding site for the RNA-binding protein CUG-BP1 (CELF1). We designed Splice Switching Oligonucleotides (SSO) against this binding site to decrease IR-A levels in RMS cell lines and, consequently, rescue the IR-B expression levels. SSO treatment resulted in a significant reduction in cell proliferation, migration, and angiogenesis. Our data shows promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. It is evident that Rhabdomyosarcomas use IR alternative splicing as yet another survival strategy that can be exploited as a therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.
RESUMEN
Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine in vitro synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells in vitro. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects in vivo. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leiomiosarcoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Benzamidas/administración & dosificación , División Celular/efectos de los fármacos , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Humanos , Leiomiosarcoma/patología , Pirimidinas/administración & dosificación , GemcitabinaRESUMEN
Interleukins-6 (IL-6)/GP130 signaling pathway represents a promising target for cancer therapy due to its critical role in survival and progression of multiple types of cancer. We have identified Bazedoxifene, a Food and Drug Administration (FDA)-approved drug used for the prevention of postmenopausal osteoporosis, with novel function as inhibitor of IL-6/GP130 interaction. In this study, we investigate the effect of Bazedoxifene in rhabdomyosarcoma and evaluate whether inhibiting IL-6/GP130 signaling is an effective therapeutic strategy for rhabdomyosarcoma. The inhibitory effect of Bazedoxifene was assessed in rhabdomyosarcoma cell lines in vitro and RH30 xenograft model was used to further examine the suppressive efficacy of Bazedoxifene on tumor growth in vivo. Rhabdomyosarcoma cells showed their sensitivity to GP130 inhibition using gene knockdown or neutralized antibody, suggesting IL-6/GP130 as therapeutic target in rhabdomyosarcoma cells. Bazedoxifene decreased the signal transducer and activator of transcription3 (STAT3) phosphorylation, blocked STAT3 DNA binding, and down-regulated the expression of STAT3 downstream genes. Bazedoxifene also induced cell apoptosis, reduced cell viability, and inhibited colony formation in rhabdomyosarcoma cells. The inhibition of colony formation, STAT3 phosphorylation, or cell viability following Bazedoxifene treatment was partially reversed by addition of excess IL-6 or overexpression of constitutive STAT3, respectively, supporting Bazedoxifene acted through IL-6/GP130 signaling. In addition, Bazedoxifene repressed cell invasion and angiogenesis in vitro. Furthermore, oral administration of Bazedoxifene significantly suppressed tumor growth and expression of STAT3 phosphorylation in nude mice bearing established human rhabdomyosarcoma xenograft. Taken together, these findings validate IL-6/GP130 signaling as therapeutic target in rhabdomyosarcoma and provide first evidence that Bazedoxifene may serve as a novel promising drug targeting IL-6/GP130 for treatment of rhabdomyosarcoma.
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Receptor gp130 de Citocinas/metabolismo , Indoles/farmacología , Interleucina-6/metabolismo , Rabdomiosarcoma/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Desnudos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although activation of the STAT3 pathway has been associated with tumor progression in a wide variety of cancer types (including ovarian cancer), the precise mechanism of invasion and metastasis due to STAT3 are not fully delineated in ovarian cancer. We found that pSTAT3 Tyr705 is constitutively activated in patient ascites and ascites-derived ovarian cancer cells (ADOCCs), and the range of STAT3 expression could be very high to low. In vivo transplantation of ADOCCs with high pSTAT3 expression into the ovarian bursa of mice resulted in a large primary tumor and widespread peritoneal metastases. In contrast, ADOCCs with low STAT3 expression or ADOCCs with STAT3 expression knockdown, led to reduced tumor growth and an absence of metastases in vivo. Cytokines derived from the ADOCC culture medium activate the interleukin (IL)-6/STAT pathway in the STAT3 knockout (KO) cells, compensating for the absence of inherent STAT3 in the cells. Treatment with HO-3867 (a novel STAT3 inhibitor at 100 p.p.m. in an orthotopic murine model) significantly suppressed ovarian tumor growth, angiogenesis and metastasis by targeting STAT3 and its downstream proteins. HO-3867 was found to have cytotoxic effects in ex vivo cultures of freshly collected human ovarian cancers, including those resistant to platinum-based chemotherapy. Our results show that STAT3 is necessary for ovarian tumor progression/metastasis and highlight the potential for targeting STAT3 by HO-3867 as a therapeutic strategy for ovarian cancer.
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Ascitis/patología , Neoplasias Ováricas/patología , Factor de Transcripción STAT3/metabolismo , Regulación hacia Arriba , Animales , Ascitis/metabolismo , Movimiento Celular , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Piperidonas/administración & dosificación , Piperidonas/farmacología , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: Epithelioid sarcoma is a rare neoplasm uniquely comprised of cells exhibiting both mesenchymal and epithelial features. Having propensity for local and distant recurrence, it poses a diagnostic dilemma secondary to pathologic complexity. Patients have dismal prognosis due to lack of effective therapy. HDAC inhibitors (HDACi) exhibit marked antitumor effects in various malignancies. The studies here demonstrate that pan-HDAC inhibitors constitute novel therapeutics versus epithelioid sarcoma. Human ES cells (VAESBJ, HS-ES, Epi-544) were studied in preclinical models to evaluate HDACi effects. Immunoblot and RT-PCR were used to evaluate expression of acetylated tubulin, histones H3/H4, EZH2 upon HDACi. MTS and clonogenic assays were used to assess the impact of HDACi on cell growth. Cell culture assays were used to evaluate the impact of HDACi and EZH2-specific siRNA inhibition on cell-cycle progression and survival. Unbiased gene array analysis was used to identify the impact of HDACi on epithelioid sarcoma gene expression. Xenografts were used to evaluate epithelioid sarcoma tumor growth in response to HDACi. HDAC inhibition increased target protein acetylation and abrogated cell growth and colony formation in epithelioid sarcoma cells. HDACi induced G(2) cell-cycle arrest and marked apoptosis, and reduced tumor growth in xenograft models. HDACi induced widespread gene expression changes, and EZH2 was significantly downregulated. EZH2 knockdown resulted in abrogated cell growth in vitro. IMPLICATIONS: The current study suggests a clinical role for HDACi in human epithelioid sarcoma, which, when combined with EZH2 inhibitors, could serve as a novel therapeutic strategy for epithelioid sarcoma patients. Future investigations targeting specific HDAC isoforms along with EZH2 may potentially maximizing treatment efficacy.
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Antineoplásicos/administración & dosificación , Epigénesis Genética/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Sarcoma/tratamiento farmacológico , Sarcoma/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: HDAC isoform-specific inhibitors may improve the therapeutic window while limiting toxicities. Developing inhibitors against class I isoforms poses difficulties as they share high homology among their catalytic sites; however, HDAC8 is structurally unique compared to other class I isoforms. HDAC8 inhibitors are novel compounds and have affinity for class I HDAC isoforms demonstrating anti-cancer effects; little is known about their activity in malignant peripheral nerve sheath tumors (MPNST). Recently, we demonstrated anti-MPNST efficacy of HDAC8i in human and murine-derived MPNST pre-clinical models; we now seek to consider the potential therapeutic inhibition of HDAC8 in MPNST. METHODS: Four Human MPNST cell lines, a murine-derived MPNST cell line, and two HDAC8 inhibitors (PCI-34051, PCI-48012; Pharmacyclics, Inc. Sunnyvale, CA) were studied. Proliferation was determined using MTS and clonogenic assays. Effects on cell cycle were determined via PI FACS analysis; effects on apoptosis were determined using Annexin V-PI FACS analysis and cleaved caspase 3 expression. In vivo growth effects of HDAC8i were evaluated using MPNST xenograft models. 2D gel electrophoresis and mass spectrometry were used to identify potential HDAC8 deacetylation substrates. RESULTS: HDAC8i induced cell growth inhibition and marked S-phase cell cycle arrest in human and murine-derived MPNST cells. Relative to control, HDAC8i induced apoptosis in both human and murine-derived MPNST cells. HDAC8i exhibited significant effects on MPNST xenograft growth (p=0.001) and tumor weight (p=0.02). Four potential HDAC8 substrate targets were identified using a proteomic approach: PARK7, HMGB1, PGAM1, PRDX6. CONCLUSIONS: MPNST is an aggressive sarcoma that is notoriously therapy-resistant, hence the urgent need for improved anti-MPNST therapies. HDAC8 inhibition may be useful for MPNST by improving efficacy while limiting toxicities as compared to pan-HDACis.
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Apoptosis , Histona Desacetilasas/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neurilemoma/enzimología , Proteínas Represoras/biosíntesis , Animales , Línea Celular Tumoral , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Humanos , Ratones , Ratones SCID , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Neurilemoma/tratamiento farmacológico , Neurilemoma/genética , Neurilemoma/patología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Vacuna BCG/administración & dosificación , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/mortalidad , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/mortalidad , Animales , Femenino , Humanos , MasculinoRESUMEN
Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated and could contribute to tumorigenesis of medulloblastoma. Numerous studies have demonstrated that inhibition of the persistent STAT3 signaling pathway results in decreased proliferation and increased apoptosis in human cancer cells, indicating that STAT3 is a viable molecular target for cancer therapy. In this study, we investigated a novel non-peptide, cell-permeable small molecule, named LY5, to target STAT3 in medulloblastoma cells. LY5 inhibited persistent STAT3 phosphorylation and induced apoptosis in human medulloblastoma cell lines expressing constitutive STAT3 phosphorylation. The inhibition of STAT3 signaling by LY5 was confirmed by down-regulating the expression of the downstream targets of STAT3, including cyclin D1, bcl-XL, survivin, and micro-RNA-21. LY5 also inhibited the induction of STAT3 phosphorylation by interleukin-6 (IL-6), insulin-like growth factor (IGF)-1, IGF-2, and leukemia inhibitory factor in medulloblastoma cells, but did not inhibit STAT1 and STAT5 phosphorylation stimulated by interferon-γ (IFN-γ) and EGF, respectively. In addition, LY5 blocked the STAT3 nuclear localization induced by IL-6, but did not block STAT1 and STAT5 nuclear translocation mediated by IFN-γ and EGF, respectively. A combination of LY5 with cisplatin or x-ray radiation also showed more potent effects than single treatment alone in the inhibition of cell viability in human medulloblastoma cells. Furthermore, LY5 demonstrated a potent inhibitory activity on cell migration and angiogenesis. Taken together, these findings indicate LY5 inhibits persistent and inducible STAT3 phosphorylation and suggest that LY5 is a promising therapeutic drug candidate for medulloblastoma by inhibiting persistent STAT3 signaling.
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Aminopiridinas/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Factor de Transcripción STAT3/antagonistas & inhibidores , Sulfonamidas/farmacología , Transporte Activo de Núcleo Celular , Aminopiridinas/síntesis química , Aminopiridinas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular , Cisplatino/farmacología , Ciclina D/genética , Ciclina D/metabolismo , Citocinas/farmacología , Regulación hacia Abajo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Meduloblastoma/metabolismo , MicroARNs/genética , Fosforilación , Transducción de Señal , Sulfonamidas/síntesis química , Sulfonamidas/química , Survivin , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND: Medulloblastoma is the most common type of pediatric brain tumor. Although numerous factors influence patient survival rates, more than 30% of all cases will ultimately be refractory to conventional therapies. Current standards of care are also associated with significant morbidities, giving impetus for the development of new treatments. We have previously shown that oncolytic measles virotherapy is effective against medulloblastoma, leading to significant prolongation of survival and even cures in mouse xenograft models of localized and metastatic disease. Because medulloblastomas are known to be highly vascularized tumors, we reasoned that the addition of angiogenesis inhibitors could further enhance the efficacy of oncolytic measles virotherapy. Toward this end, we have engineered an oncolytic measles virus that express a fusion protein of endostatin and angiostatin, two endogenous and potent inhibitors of angiogenesis. METHODS: Oncolytic measles viruses encoding human and mouse variants of a secretable endostatin/angiostatin fusion protein were designed and rescued according to established protocols. These viruses, known as MV-hE:A and MV-mE:A respectively, were then evaluated for their anti-angiogenic potential and efficacy against medulloblastoma cell lines and orthotopic mouse models of localized disease. RESULTS: Medulloblastoma cells infected by MV-E:A readily secrete endostatin and angiostatin prior to lysis. The inclusion of the endostatin/angiostatin gene did not negatively impact the measles virus' cytotoxicity against medulloblastoma cells or alter its growth kinetics. Conditioned media obtained from these infected cells was capable of inhibiting multiple angiogenic factors in vitro, significantly reducing endothelial cell tube formation, viability and migration compared to conditioned media derived from cells infected by a control measles virus. Mice that were given a single intratumoral injection of MV-E:A likewise showed reduced numbers of tumor-associated blood vessels and a trend for increased survival compared to mice treated with the control virus. CONCLUSIONS: These data suggest that oncolytic measles viruses encoding anti-angiogenic proteins may have therapeutic benefit against medulloblastoma and support ongoing efforts to target angiogenesis in medulloblastoma.
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Inhibidores de la Angiogénesis/uso terapéutico , Angiostatinas/antagonistas & inhibidores , Endostatinas/antagonistas & inhibidores , Virus del Sarampión/fisiología , Meduloblastoma/terapia , Viroterapia Oncolítica/efectos adversos , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Virus del Sarampión/genética , Meduloblastoma/patología , Ratones , Neoplasias Experimentales , Virus Oncolíticos/genética , Células Vero , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: c-Myc is a well-known oncogene frequently up-regulated in different malignancies, whereas liver-specific microRNA (miR)-122, a bona fide tumor suppressor, is down-regulated in hepatocellular cancer (HCC). Here we explored the underlying mechanism of reciprocal regulation of these two genes. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and northern blot analysis demonstrated reduced expression of the primary, precursor, and mature miR-122 in c-MYC-induced HCCs compared to the benign livers, indicating transcriptional suppression of miR-122 upon MYC overexpression. Indeed, chromatin immunoprecipitation (ChIP) assay showed significantly reduced association of RNA polymerase II and histone H3K9Ac, markers of active chromatin, with the miR-122 promoter in tumors relative to the c-MYC-uninduced livers, indicating transcriptional repression of miR-122 in c-MYC-overexpressing tumors. The ChIP assay also demonstrated a significant increase in c-Myc association with the miR-122 promoter region that harbors a conserved noncanonical c-Myc binding site in tumors compared to the livers. Ectopic expression and knockdown studies showed that c-Myc indeed suppresses expression of primary and mature miR-122 in hepatic cells. Additionally, Hnf-3ß, a liver enriched transcription factor that activates miR-122 gene, was suppressed in c-MYC-induced tumors. Notably, miR-122 also repressed c-Myc transcription by targeting transcriptional activator E2f1 and coactivator Tfdp2, as evident from ectopic expression and knockdown studies and luciferase reporter assays in mouse and human hepatic cells. CONCLUSION: c-Myc represses miR-122 gene expression by associating with its promoter and by down-regulating Hnf-3ß expression, whereas miR-122 indirectly inhibits c-Myc transcription by targeting Tfdp2 and E2f1. In essence, these results suggest a double-negative feedback loop between a tumor suppressor (miR-122) and an oncogene (c-Myc).
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Carcinoma Hepatocelular/fisiopatología , Proteínas de Unión al ADN/fisiología , Factor de Transcripción E2F1/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Hepáticas/fisiopatología , MicroARNs/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Factores de Transcripción/fisiología , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Factor de Transcripción E2F1/genética , Regulación Neoplásica de la Expresión Génica/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , MicroARNs/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Wrightia tomentosa Roem. & Schult. (Apocynaceae) is known in the traditional medicine for anti-cancer activity along with other broad indications like snake and scorpion bites, renal complications, menstrual disorders etc. However, the anti-cancer activity of this plant or its constituents has never been studied systematically in any cancer types so far. AIM OF THE STUDY: To evaluate the anti-cancer activities of the ethanolic extract of W. tomentosa and identified constituent active molecule(s) against breast cancer. MATERIAL AND METHODS: Powdered leaves of W. tomentosa were extracted with ethanol. The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa were tested for its anti-proliferative and pro-apoptotic effects in breast cancer cells MCF-7 and MDA-MB-231. RESULTS: The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa inhibited the proliferation of human breast cancer cell lines, MCF-7 and MDA-MB-231. The fraction F-4 obtained from hexane fraction inhibited proliferation of MCF-7 and MDA-MB-231 cells in concentration and time dependent manner with IC50 of 50 µg/ml and 30 µg/ml for 24 h, 28 µg/ml and 22 µg/ml for 48 h and 25 µg/ml and 20 µg/ml for 72 h respectively. The fraction F-4 induced G1 cell cycle arrest, reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and subsequent apoptosis. Apoptosis is indicated in terms of increased Bax/Bcl-2 ratio, enhanced Annexin-V positivity, caspase 8 activation and DNA fragmentation. The active molecule isolated from fraction F-4, oleanolic acid and urosolic acid inhibited cell proliferation of MCF-7 and MDA-MB-231 cells at IC50 value of 7.5 µM and 7.0 µM respectively, whereas there is devoid of significant cell inhibiting activity in non-cancer originated cells, HEK-293. In both MCF-7 and MDA-MB-231, oleanolic acid and urosolic acid induced cell cycle arrest and apoptosis as indicated by significant increase in Annexin-V positive apoptotic cell counts. CONCLUSION: Our results suggest that W. tomentosa extracts has significant anti-cancer activity against breast cancer cells due to induction of apoptosis pathway. Olenolic and urosolic acid are important constituent molecules in the extract responsible for anti-cancer activity of W. tomentosa.
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Antineoplásicos/farmacología , Apocynaceae , Ácido Oleanólico/farmacología , Extractos Vegetales/farmacología , Triterpenos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Etanol/química , Células HEK293 , Humanos , L-Lactato Deshidrogenasa/metabolismo , Hojas de la Planta , Solventes/química , Ácido UrsólicoRESUMEN
BACKGROUND: Cytokines are known as important regulators of the entire gamut of cancer from initiation, invasion and metastasis. This fact and plethora of gene polymorphism data prompted us to investigate cytokine gene polymorphisms in breast cancer (BC) patients. METHODS: Selected polymorphisms in the IL-1ß [-511 T>C (rs16944) and +3954 C>T (rs1143634)]; IL-6 [-174 G>C (rs1800795)]; IL-10 [-1082 A>G (rs1800896), -819 T>C (rs1800871) and -592 A>C (rs1800872)] genes were genotyped in 200 BC patients and 200 healthy volunteers in a case-control study using PCR-RFLP and direct DNA sequencing techniques. Peripheral cytokine levels were measured using ELISA. Allele and genotype data were analyzed for significance of differences between cases and controls using Chi-Square [χ(2)] test. Two sided P-values of less than 0.05 were considered to be statistically significant. RESULTS: Peripheral level of all three cytokines did not show any significant difference between cases and controls. Allele and genotype frequency of IL-1ß [-511 T>C (rs16944)] did not show any difference between cases and controls. On the other hand mutant allele and genotype at IL-1ß [+3954 C>T (rs1143634)] associated with increased risk of BC. This was also true for pre-menopausal cases and for mutant genotype in post-menopausal cases. Mutant allele and genotypes at IL-6 [-174 G>C (rs1800795)] appeared to be protective in nature such that controls had a higher frequency of both mutant alleles and genotypes. None of the three SNPs in IL-10 gene associated with risk of BC, except significant association of mutant allele and genotypes of -1082 A>G (rs1800896) polymorphism with postmenopausal BC. CONCLUSIONS: Mutant allele and genotype at IL-1ß [+3954 C>T (rs1143634)] site associated with increased BC risk, while mutant allele and genotypes at IL-6 [-174 G>C (rs1800795)] polymorphism appeared to be protective. Also, there was significant association of mutant allele and genotypes of IL-10 [-1082 A>G (rs1800896)] with postmenopausal BC. None of the other polymorphisms investigated appear to affect BC risk.
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Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad/genética , Interleucina-10/genética , Interleucina-1beta/genética , Interleucina-6/genética , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Neoplasias de la Mama/sangre , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Frecuencia de los Genes , Genotipo , Humanos , Interleucina-10/sangre , Interleucina-1beta/sangre , Interleucina-6/sangre , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Factores de Riesgo , Adulto JovenRESUMEN
In this first report on the association of an IL-10 promoter polymorphism with type 2 diabetes mellitus in a North Indian population, the -592A/C SNP (rs1800872) was genotyped by PCR-RFLP and the IL-10 level measured using ELISA. Although no significant difference was observed in the genotypic frequencies (P = 0.657), diabetes patients carried a significantly higher number of A alleles at the -592 position, 25.6% (P < 0.001, odds ratio 0.887, 95% CI 0.670-1.184). Significant correlations were detected in postprandial glucose levels of CC-genotype patients and controls (P = 0.025), age and waist-hip ratio of CA patients and controls (P ≤ 0.001), and fasting glucose (P = 0.045) and low-density lipoprotein (P = 0.049) in all patients and controls. The serum IL-10 level was significantly higher in patients than in controls (P = 0.033). The polymorphism was significantly associated with disease incidence and its biochemical manifestations.
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Diabetes Mellitus Tipo 2/genética , Interleucina-10/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Población Blanca/genética , Adulto , Secuencia de Bases , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/sangre , Femenino , Genotipo , Humanos , India , Interleucina-10/sangre , Masculino , Persona de Mediana Edad , Datos de Secuencia MolecularRESUMEN
BACKGROUND & OBJECTIVES: The greater tendency to diabetes in Indians may be due to genetic factors in addition to environment and diet. CD36, a class B scavenger cell surface receptor mediates internalization of oxidized low density lipoprotein (Ox-LDL) leading to the formation of macrophage foam cells. CD36 deficiency is related to phenotypic expression of the metabolic syndrome, frequently associated with atherosclerotic cardiovascular diseases resulting in raised levels of glucose thereby contributing to type 2 diabetes (T2DM). Therefore, the association of human CD36 gene mutation to T2DM needs investigation. We undertook this study to investigate CD36 gene status in north Indian subjects by screening for the deletion of exons 3, 4 and 5 and certain polymorphisms. METHODS: Clinical characteristics were compared between 300 T2DM patients and 100 healthy controls. Deletion analysis was carried out for exons 3, 4 and 5 of CD36 gene in 300 T2DM patients using PCR and agarose gel electrophoresis. Genotype analysis for two polymorphisms 478C>T and delAC in exons 4 and 5 respectively was carried out using PCR-RFLP method. RESULTS: Biochemical parameters such as fasting and post-prandial glucose levels, total cholesterol, LDL-cholesterol and blood pressure were slightly raised in the T2DM patients when compared with controls with lowered HDL-cholesterol. No exonic deletion was observed in the 300 patients and 100 controls screened. All individuals were found to be homozygous (CC and -/-) for the two polymorphisms studied. INTERPRETATION & CONCLUSIONS: Although no exonic deletion was found in T2DM patients, our study suggests that all 15 exons need to be screened for mutations which lead to CD36 deficiency. Genotyping studies of the two SNPs in the CD36 gene confirmed the absence of exons 4 and 5 deletion. This is perhaps the first report from India suggesting that CD36 is one of the several important genes that need to be explored in relation to T2DM.
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Antígenos CD36/genética , Diabetes Mellitus Tipo 2/genética , Adulto , Glucemia/análisis , Presión Sanguínea , LDL-Colesterol/sangre , Exones , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , India , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Eliminación de SecuenciaRESUMEN
TNF-α and -ß, the multi-functional pro-inflammatory cytokines, are known to play important roles in both tumor progression and destruction based on their concentrations. Growth factors and various stimuli such as cytokines regulate proliferation of the breast epithelial cells. Therefore, the polymorphisms in the genes encoding these signaling molecules could affect the risk of breast cancer. We have investigated selected genetic polymorphisms in TNF-α promoter (rs1800629, -308 G>A and rs361525, -238 G>A) and TNF-ß intron 1 (rs909253, +252 A>G) in ethnically two different case-control groups from India. The study included 200 cases and 200 controls from an Indo-European (North Indian) group, and 265 cases and 237 controls from a Dravidian (South Indian) group. Genotyping of a total of 902 individuals was done by direct DNA sequencing. None of the polymorphisms showed significant association with breast cancer in the Indo-European group; however, all the three polymorphisms showed strong association with breast cancer in the Dravidian group. Further, sub-group analysis in the Indo-European group showed no significant difference between pre-menopausal cases and controls or between post-menopausal cases and controls at any of the loci analyzed. However, all the polymorphisms in the Dravidian group were significantly associated with pre-menopausal but not with post-menopausal breast cancer. In conclusion, TNF-α and -ß polymorphisms are strongly associated with breast cancer in the Dravidian but not in the Indo-European group.
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Neoplasias de la Mama/etnología , Neoplasias de la Mama/genética , Variación Genética , Linfotoxina-alfa/genética , Polimorfismo Genético , Factor de Necrosis Tumoral alfa/genética , Adulto , Anciano , Anciano de 80 o más Años , Mama/metabolismo , Estudios de Casos y Controles , Etnicidad , Femenino , Genotipo , Humanos , India , Menopausia , Persona de Mediana Edad , RiesgoRESUMEN
Several genetic studies worldwide have recommended VDR as a candidate gene for determining risk of benign prostate hyperplasia (BPH). We investigated the association between VDR gene polymorphisms and the risk of BPH in an Indian male population. Three polymorphic sites of VDR gene, viz., Fok-I, Taq-I and Bsm-I were genotyped in 160 BPH patients and 160 controls. Logistic regression models were used to determine the genetic effects using SPSS statistical software. A statistically significant association between VDR genotype (Taq-I and Bsm-I) and BPH (p=0.02 and 0.03) was obtained. In exploratory analyses, we also examined the association with responder and non-responder subgroups of patients for association of VDR (Taq-I) genotype with drug responsiveness. Our results established that Taq-I and Bsm-I genetic variants of VDR gene influence susceptibility BPH in Indian population. VDR genotypes specifically, Taq-I polymorphic variant is significantly associated with the improvement of BPH patients with standard drug therapy.
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Predisposición Genética a la Enfermedad , Hiperplasia Prostática/genética , Receptores de Calcitriol/genética , Anciano , Alelos , Frecuencia de los Genes , Genotipo , Humanos , India , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Hiperplasia Prostática/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Glutathione S-transferases may be over expressed in benign prostate hyperplasia (BPH) but association of GST polymorphism with susceptibility to the disease is unclear. The objective of this study was to determine relationships between polymorphisms in the GSTM1, T1 and P1 genes with risk of symptomatic BPH and response to standard therapy. The study population comprised 160 symptomatic BPH patients with BPE (benign prostatic enlargement) and LUTS (lower urinary tract symptoms) and 200 age-matched controls. Patient inclusion criteria were: age>50 years; prostate size>30 cm3; AUA (American Urological Association) score>7; and PVR volume≤200 ml. Patients were treated with alpha-adrenergic blockers and 5alpha-reductase inhibitors for 6 months and subdivided based on significant improvement in parameters between pre and post combined therapy. The GSTT1 and GSTM1 variants genotyped with multiplex-PCR, whereas GSTP1 polymorphisms were determined with PCR-RFLP (polymerase chain reaction- restriction fragment length polymorphism). We observed a lack of any association with GSTT1 (p=0.45, OR=2.25, 95% CI=1.71-2.22) and GSTP1 (p=0.92 and 0.99) genes. There was a significant positive association with null alleles of the GSTM1 (p=0.000, OR=2.24, 95%CI =1.46-3.42) gene. Combined analysis of the three genotypes demonstrated further increase in the risk of symptomatic BPH (p=0.009, OR=8.31 95%CI=1.71-40.4). Polymorphisms of GST genes were not associated with rates for responders and non-responders. GSTM1 deletion is significantly associated with the increased risk of symptomatic BPH, but none of the GST polymorphisms appears associated with response to standard BPH therapy.