RESUMEN
TRAIL, an apoptosis inducing cytokine currently in phase II clinical trial, was investigated for its capability to induce apoptosis in six different human tumor cell lines out of which three cell lines showed resistance to TRAIL induced apoptosis. To investigate whether Anacardic acid (A1) an active component of Anacardium occidentale can sensitize the resistant cell lines to TRAIL induced apoptosis, we treated the resistant cells with suboptimal concentration of A1 and showed that it is a potent enhancer of TRAIL induced apoptosis which up-regulates the expression of both DR4 and DR5 receptors, which has been observed in the cellular, protein and mRNA levels. The death receptors upregulation consequent to A1 treatment was corroborated by the activation of p53 as well as phosphorylation of p38 and JNK MAP kinases and concomitant inactivation of NFκß and ERK signaling cascades. Also, A1 modulated the expression of key apoptotic players like Bax, Bcl-2 and CAD along with the abatement of tumor angiogenesis in vivo in EAT mouse model. Thus, post A1 treatment the TRAIL resistant cells turned into TRAIL sensitive cells. Hence our results demonstrate that A1 can synergize TRAIL induced apoptosis through the upregulation of death receptors and downregulation of anti-apoptotic proteins in cancer context.
Asunto(s)
Ácidos Anacárdicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ácidos Anacárdicos/administración & dosificación , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Liposomas , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , ARN Mensajero/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Approximately 15% of globally diagnosed breast cancers are designated as triple negative breast cancer (TNBC). In this study, we investigated the effect of the natural compound, Bis(2- ethyl hexyl) 1H-pyrrole-3,4-dicarboxylate (TCCP), purified from Tinospora cordifolia on MDA-MB-231, a TNBC cell line. The pro-apoptotic nature of TCCP on MDA-MB-231 was determined by assessing various apoptotic markers. ROS generation, intracellular calcium, mitochondrial membrane potential (ΔΨm), MPTP, cardiolipin peroxidation and caspase activity were determined fluorometrically. BAX, BCL-2, cytochrome c, caspases, and p53 protein expressions were determined by immunoblotting. Further, the effect of TCCP on DNA and cell death was determined by DNA fragmentation assay, annexin-V staining, and cell cycle analysis. TCCP treatment caused endogenous ROS generation, increase in intracellular calcium and phosphorylation of p53 in a concentration-dependent manner, which was reverted upon pre-treatment with pifithrin-µ. This led to the downstream altered expression of Bcl-2 and Bax proteins, mitochondrial membrane depolarization, MPTP, and cardiolipin peroxidation. TCCP induced cytochrome c release into the cytosol, caspase activation, ultimately resulting in DNA fragmentation. Further, induction of apoptosis and morphological alterations were evident from the phosphatidylserine externalization and increase in sub G1 population. The in vivo Ehrlich ascites tumor (EAT) mouse study revealed the effectiveness of TCCP in reducing the tumor burden and resulted in a ~2 fold increase in mice survival with minimal hepato-renal toxicity. Overall, TCCP was shown to be efficient in inducing ROS and mitochondrial-mediated apoptosis by restoring p53 activity in MDA-MB-231â¯cells and also induced EAT cell death in vivo thereby inhibiting tumor proliferation.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Pirroles/química , Especies Reactivas de Oxígeno/metabolismo , Tinospora/química , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Peroxidación de Lípido/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tasa de Supervivencia , Tinospora/metabolismoRESUMEN
Heat shock proteins (HSPs), molecular chaperones, are crucial for the cancer cells to facilitate proper functioning of various oncoproteins involved in cell survival, proliferation, migration, and tumor angiogenesis. Tumor cells are said to be "addicted" to HSPs. HSPs are overexpressed in many cancers due to upregulation of transcription factor Heat-shock factor 1 (HSF-1), the multifaceted master regulator of heat shock response. Therefore, pharmacological targeting of HSPs via HSF-1 is an effective strategy to treat malignant cancers like triple negative breast cancer. In the current study, we evaluated the efficacy of a pyrrole derivative [bis(2-ethylhexyl)1H-pyrrole-3,4-dicarboxylate], TCCP, purified from leaves of Tinospora cordifolia for its ability to suppress heat shock response and angiogenesis using MDA-MB-231 cells and the murine mammary carcinoma: Ehrlich ascites tumor model. HSP90 was downregulated by TCCP by inactivation of HSF-1 resulting in inhibition of tumor cell proliferation, VEGF-induced cell migration, and concomitant decrease in tumor burden and neo-angiogenesis in vivo. The mechanism of suppression of HSPs involves inactivation of PI3K/Akt and phosphorylation on serine 307 of HSF-1 by the activation of ERK1. HSF-1 and HSP90 and 70 localization and expression were ascertained by immunolocalization, immunoblotting, and qPCR experiments. The anti-angiogenic effect of TCCP was studied in vivo in tumor-bearing mice and ex vivo using rat corneal micro-pocket assay. All the results thus corroborate the logic behind inactivating HSF-1 using TCCP as an alternative approach for cancer therapy.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Pirroles/toxicidad , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Córnea/efectos de los fármacos , Córnea/metabolismo , Femenino , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Factores de Transcripción del Choque Térmico/antagonistas & inhibidores , Humanos , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Patológica/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirroles/química , Pirroles/aislamiento & purificación , Pirroles/uso terapéutico , Ratas , Ratas Wistar , Tinospora/química , Tinospora/metabolismo , Trasplante HeterólogoRESUMEN
Synthesis of 3-(4-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)piperazin-1-yl) benzoisothiazole derivatives (5a-i), which constitute a new class of isoxazolines, has been accomplished in regio-selective manner. These derivatives have been prepared by employing the reaction between substituted aldoximes (4a-i) and 3-(4-Allylpiperazin-1-yl) benzoisothiazole in presence of chloramine-T which afforded in good yields. These compounds were screened for cytotoxic activity on tumor cells. Four among the nine synthesized compounds were found to exhibit potent cytotoxic and antineoplastic activities in comparison to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) protein in mammalian cancer cells. The rest of the derivatives showed moderate activity.