RESUMO
During cancer cell invasion, integrin undergoes constant endo/exocytic trafficking. It has been found that the recycling ability of integrin ß1 through Rab11-controlled long loop pathways is directly associated with cancer invasion. Previous studies showed that gain-of-function mutant p53 regulates the Rab-coupling protein [RCP]-mediated integrin ß1 recycling by inactivating tumor suppressor TAp63. So, we were interested to investigate the involvement of miR-205 in this process. In the current study first, we evaluated that the lower expression of miR-205 in MDA-MB-231 cell line is associated with high motility and invasiveness. Further investigation corroborated that miR-205 directly targets RCP resulting in attenuated RCP-mediated integrin ß1 recycling. Overexpression of TAp63 validates our in vitro findings. To appraise the anti-metastatic role of miR-205, we developed two in vivo experimental models- xenograft-chick embryo and xenograft-immunosuppressed BALB/c mice. Our in vivo results support the negative effect of miR-205 on metastasis. Therefore, these findings advocate the tumor suppressor activity of miR-205 in breast cancer cells and suggest that in the future development of miR-205-targeting RNAi therapeutics could be a smart alternative approach to prevent the metastatic fate of the disease.
Assuntos
Neoplasias da Mama , MicroRNAs , Animais , Embrião de Galinha , Feminino , Humanos , Camundongos , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Integrina beta1/genética , Integrina beta1/metabolismo , MicroRNAs/genética , Invasividade Neoplásica , Metástase NeoplásicaRESUMO
Tumor cells always have the need to produce an increased amount of proteins in the cells. This elevated amount of proteins increases the pressure on the organelles of the cell such as the endoplasmic reticulum and compels it to increase its protein folding efficiency. However, it is by a matter of fact, that the amount of proteins synthesized outweighs the protein folding capacity of the ER which in turn switches on the UPR pathway by activating the three major molecular sensors and other signaling cascades, which helps in cell survival instead of instant death. However, if this pathway is active for a prolonged period of time the tumor cells heads toward apoptosis. Again, interestingly this is not the same as in case of non- tumorogenic cells. This exhibit a straight natural pathway for tumor cells-specific destruction which has a great implication in today's world where hormone therapies and chemo-therapies are non-effective for various types of breast cancer, a major type being Triple Negative Breast Cancer. Thus a detailed elucidation of the molecular involvement of the UPR pathway in breast cancer may open new avenues for management and attract novel chemotherapeutic targets providing better hopes to patients worldwide.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estresse do Retículo Endoplasmático , Apoptose/genética , Resposta a Proteínas não Dobradas , Transdução de SinaisRESUMO
Although comprehensive exertions have been made in late decades for treating advanced lung cancer with inclusive therapies but efficient anti-lung cancer therapeutics are statically inadequate in the clinics. Hence, compelling novel anti-lung cancer drugs are considerably desired. This backdrop enticed us to unveil anticancer efficacy of astrakurkurol, derivative of wild edible mushroom against lung cancer, whose effects have not yet been described. Mechanistic analysis disclosed that sensitizing effect of astrakurkurol is due to cell cycle arrest at G0/G1 phase, increased level of Fas, FADD, decreased ratio of Bax/Bcl-2, and increased cleaved form of caspase 9, 8, and 3. Apart from the induction of apoptosis, it was demonstrated for the first time that astrakurkurol induced an autophagic response as evidenced by the development of acidic vesicular organelles (AVOs) with up-regulation of beclin-1, Atg7, and downregulated p62. Apoptosis and autophagy can be sparked by the same stimuli, which was as evident from the astrakurkurol-induced inactivation of PI3K/AKT signaling. The thorough scanning of the mechanism of crosstalk between apoptosis and autophagy is requisite for prosperous anticancer remedy. Triterpenoid has evidently intensified cytotoxicity, induced apoptosis and autophagy on A549 cells. Besides astrakurkurol could also curb migration and regress the size of tumor in ex ovo xenograft model. All these findings put forth astrakurkurol as a convincing novel anti-cancer agent, for scrutinizing the lung cancer therapies and as a robust contender for future in vitro and in vivo analysis.
Assuntos
Adenocarcinoma de Pulmão , Agaricales , Neoplasias Pulmonares , Células A549 , Apoptose , Autofagia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-aktRESUMO
A novel reactor was designed and implemented for water purification using deep ultraviolet light emitting diodes (LEDs). The focus was on minimizing the number of LEDs required for effective germicidal action. Simulation studies were carried out on the flow of water as well as the irradiance of UV. Variation was made in the beam divergence of the UV sources and reflectivity of optical coatings used for photon recycling. Based on optimized reactor designs, water purification was carried out both in the static and flow-through configuration. Water from various sources was spiked with a known bacterial strain, exposure studies were carried out and germicidal effect was determined. Our results indicate that under optimal design, a 3 mL volume of water shows a three order inactivation using a single UV-LED in a static reactor in 180 s. For a flow-through geometry, only three LEDs were used in the reactor implementation, and a multi-pass procedure was used to purify 150 mL of water from an Escherichia coli CFU count of 4.3 × 104/mL to 12/mL. While slow, this process requires less than 2 W, and can be powered from rechargeable sources. Faster processes can be implanted using multiple such reactor units in parallel, and can be optimized to the requirement and power levels.
Assuntos
Desinfecção , Raios Ultravioleta , Purificação da Água , Escherichia coli , Microbiologia da ÁguaRESUMO
Anoikis resistance is an essential property of cancer cells that allow the extra-cellular matrix-detached cells to survive in a suspended state in body fluid in order to metastasize and invade to distant organs. It is known that integrins play an important role in anoikis resistance, but detailed mechanisms are not well understood. Here we report that highly metastatic colon cancer cells showed a higher degree of anoikis resistance than the normal intestinal epithelial cells. These anoikis-resistant cancer cells express high-levels of integrin-α2, ß1, and activated EGFR in the anchorage-independent state than the anchorage-dependent state. In contrast, normal intestinal epithelial cells failed to elevate these proteins. Interestingly, a higher co-association of EGFR with integrin-α2ß1/-α5ß1 was observed on the surface of anoikis-resistant cells. Thus, in the absence of extra-cellular matrix, integrins in association with EGFR activates downstream effectors ERK and AKT and suppress Caspase-3 activation to induce anoikis resistance as was confirmed from the gene-ablation and pharmacological inhibitor studies. Interestingly, these anoikis-resistant cancer cells express high-level of cancer stem cell signatures (CD24, CD44, CD133, EpCAM) and pluripotent stem cell markers (OCT-4, SOX-2, Nanog) as well as drug-resistant pumps (ABCG2, MDR1, MRP1). Altogether, our findings unravel the interplay between integrin-α2ß1/-α5ß1 and EGFR in anoikis resistance and suggest that the resistant cells are cancer initiating or cancer stem cells, which may serve as a promising target to combat metastasis of cancer.
Assuntos
Anoikis , Neoplasias do Colo/metabolismo , Integrina alfa2beta1/metabolismo , Células-Tronco Neoplásicas/metabolismo , Antígeno AC133/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Anoikis/genética , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Células Epiteliais/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Integrina alfa2beta1/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteína Homeobox Nanog/metabolismo , Proteínas de Neoplasias/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismoRESUMO
Triple negative breast cancer (TNBC) tends to form aggressive tumors associated with high mortality and morbidity which urge the need for development of new therapeutic strategies. Recently, the normal metabolite Methylglyoxal (MG) has been documented for its anti-proliferative activity against human breast cancer. However, the mode of action of MG against TNBC remains open to question. In our study, we investigated the anticancer activity of MG in MDA MB 231 and 4T1 TNBC cell lines and elucidated the underlying mechanisms. MG dose-dependently caused cell death, induced apoptosis, and generated ROS in both the TNBC cell lines. Furthermore, such effects were attenuated in presence of ROS scavenger N-Acetyl cysteine. MG triggered mitochondrial cytochrome c release in the cytosol and up-regulated Bax while down-regulated anti-apoptotic protein Bcl-2. Additionally, MG treatment down-regulated phospho-akt and inhibited the nuclear translocation of the p65 subunit of NF-κB. MG exhibited a tumor suppressive effect in BALB/c mouse 4T1 breast tumor model as well. The cytotoxic effect was studied using MTT assay. Apoptosis, ROS generation, and mitochondrial dysfunction was evaluated by flow cytometry as well as fluorescence microscopy. Western blot assay was performed to analyze proteins responsible for apoptosis. This study demonstrated MG as a potent anticancer agent against TNBC both in vitro and in vivo. The findings will furnish fresh insights into the treatment of this subgroup of breast cancer.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Aldeído Pirúvico/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição RelA/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Triple negative breast cancers (TNBC) are among the most aggressive and therapy-resistant breast tumors and currently possess almost no molecular targets for therapeutic options in this horizon. In the present study we discerned the molecular mechanisms of potential interaction between the endoplasmic reticulum (ER) stress response and the MEK/ERK pathway in inducing apoptosis in TNBC cells. Here we observed that induction of ER stress alone was not sufficient to trigger significant apoptosis but simultaneous inhibition of the MEK/ERK pathway enhanced ER stress-induced apoptosis via a caspase-dependent mechanism. Our study also demonstrated nifetepimine, a dihydropyrimidone derivative as a potent anti-cancer agent in TNBC cells. Nifetepimine down-regulated the MEK/ERK pathway in MDAMB-231 and MDAMB-468 cells and resulted in blockage of ER stress-mediated GRP78 up-regulation. Detailed mechanistic studies also revealed that nifetepimine by down-regulating pERK expression also declined the promoter binding activity of TFII-I to the GRP78 promoter and in turn regulated GRP78 transcription. Studies further extended to in vivo Swiss albino and SCID mice models also revalidated the anti-carcinogenic property of nifetepimine. Thus our findings cumulatively suggest that nifetepimine couples two distinct signaling pathways to induce the apoptotic death cascade in TNBC cells and raises the possibility for the use of nifetepimine as a potent anti-cancer agent with strong immune-restoring properties for therapeutic intervention for this group of cancer bearers.
Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Pirimidinonas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Feminino , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Técnicas Imunoenzimáticas , MAP Quinase Quinase Quinases/genética , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias de Mama Triplo Negativas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The efficacy of cancer chemotherapeutics is limited by side effects resulting from narrow therapeutic windows between the anticancer activity of a drug and its cytotoxicity. Thus identification of small molecules that can selectively target cancer cells has gained major interest. Cancer cells under stress utilize the Unfolded protein response (UPR) as an effective cell adaptation mechanism. The purpose of the UPR is to balance the ER folding environment and calcium homeostasis under stress. If ER stress is prolonged, tumor cells undergo apoptosis. In the present study we demonstrated an 3,3'-(Arylmethylene)-bis-1H-indole (AMBI) derivative 3,3'-[(4-Methoxyphenyl) methylene]-bis-(5-bromo-1H-indole), named as Mephebrindole (MPB) as an effective anti-cancer agent in breast cancer cells. MPB disrupted calcium homeostasis in MCF7 cells which triggered ER stress development. Detailed evaluations revealed that mephebrindole by activating p38MAPK also regulated GRP78 and eIF2α/ATF4 downstream to promote apoptosis. Studies extended to in vivo allograft mice models revalidated its anti-carcinogenic property thus highlighting the role of MPB as an improved chemotherapeutic option.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , Fator de Iniciação 2 em Eucariotos/metabolismo , Indóis/administração & dosagem , Fator de Transcrição CHOP/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/fisiopatologia , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/genética , Feminino , Humanos , Indóis/síntese química , Indóis/química , Camundongos , Receptor Cross-Talk/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. It has been acknowledged that aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence for which E-cadherin switch is a well-established hallmark. Discerning the molecular mechanisms that regulate E-cadherin expression is therefore critical for understanding tumor invasiveness and metastasis. Here we report that SMAR1 overexpression inhibits EMT and decelerates the migratory potential of breast cancer cells by up-regulating E-cadherin in a bidirectional manner. While SMAR1-dependent transcriptional repression of Slug by direct recruitment of SMAR1/HDAC1 complex to the matrix attachment region site present in the Slug promoter restores E-cadherin expression, SMAR1 also hinders E-cadherin-MDM2 interaction thereby reducing ubiquitination and degradation of E-cadherin protein. Consistently, siRNA knockdown of SMAR1 expression in these breast cancer cells results in a coordinative action of Slug-mediated repression of E-cadherin transcription, as well as degradation of E-cadherin protein through MDM2, up-regulating breast cancer cell migration. These results indicate a crucial role for SMAR1 in restraining breast cancer cell migration and suggest the candidature of this scaffold matrix-associated region-binding protein as a tumor suppressor.
Assuntos
Neoplasias da Mama/genética , Caderinas/biossíntese , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ligação a DNA/biossíntese , Transição Epitelial-Mesenquimal/genética , Proteínas Nucleares/biossíntese , Neoplasias da Mama/patologia , Caderinas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Metástase Neoplásica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
Matrix attachment region (MAR)-binding proteins have been implicated in the transcriptional regulation of host as well as viral genes, but their precise role in HPV-infected cervical cancer remains unclear. Here we show that HPV18 promoter contains consensus MAR element in the LCR and E6 sequences where SMAR1 binds and reinforces HPV18 E6 transcriptional silencing. In fact, curcumin-induced up-regulation of SMAR1 ensures recruitment of SMAR1-HDAC1 repressor complex at the LCR and E6 MAR sequences, thereby decreasing histone acetylation at H3K9 and H3K18, leading to reorientation of the chromatin. As a consequence, c-Fos binding at the putative AP-1 sites on E6 promoter is inhibited. E6 depletion interrupts degradation of E6-mediated p53 and lysine acetyl transferase, Tip60. Tip60, in turn, acetylates p53, thereby restoring p53-mediated transactivation of proapoptotic genes to ensure apoptosis. This hitherto unexplained function of SMAR1 signifies the potential of this unique scaffold matrix-associated region-binding protein as a critical regulator of E6-mediated anti-apoptotic network in HPV18-infected cervical adenocarcinoma. These results also justify the candidature of curcumin for the treatment of HPV18-infected cervical carcinoma.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transcrição Gênica , Acetilação , Apoptose , Células HeLa , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Fator de Transcrição AP-1/metabolismoRESUMO
A biomimetic cell-based carrier system based on monocyte membranes and liposomes has been designed to create a hybrid "Monocyte-LP" which inherits the surface antigens of the monocytes along with the drug encapsulation property of the liposome. Förster resonance energy transfer (FRET) and polarization gated anisotropy measurements show the stiffness of the vesicles obtained from monocyte membranes (Mons), phosphatidylcholine membranes (LP), and Monocyte-LP to follow an increasing order of Mons > Monocyte-LP > LP. The dynamics of interface bound water molecules plays a key role in the elasticity of the vesicles, which in turn imparts higher delivery efficacy to the hybrid Monocyte-LP for a model anticancer drug doxorubicin than the other two vesicles, indicating a critical balance between flexibility and rigidity for an efficient cellular uptake. The present work provides insight on the influence of elasticity of delivery vehicles for enhanced drug delivery.
Assuntos
Antineoplásicos , Lipossomos , Lipossomos/metabolismo , Monócitos/metabolismo , Doxorrubicina , Sistemas de Liberação de MedicamentosRESUMO
Multiple mechanisms have been proposed by which tumors induce T cell apoptosis to circumvent tumor immune-surveillance. Although sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) have long been known to regulate intracellular Ca(2+) homeostasis, few studies have examined the role of SERCA in processes of T lymphocyte survival and activation. In this context it remains largely unexplored as to how tumors jeopardize SERCA function to disable T cell-mediated anti-tumor immunity. Here, we show that human CD4(+) T cells in the presence of tumor conditions manifested an up-regulation of SERCA3 expression that resulted in development of endoplasmic reticulum stress leading to CD4(+) T cell apoptosis. Prostaglandin E(2) produced by the tumor cell plays a critical role in up-regulating SERCA3 by enhancing the binding of its transcription factor Sp1. Gene manipulation and pharmacological approaches further established that an increase in SERCA expression also resulted in subsequent inhibition of PKCα and -θ and retention of NFκB in the cytosol; however, down-modulation of SERCA3 expression by a dihydropyrimidone derivative, ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate (nifetepimine), protected the CD4(+) T cells from tumor-induced apoptosis. In fact, nifetepimine-mediated restoration of PKC activity resulted in nuclear translocation of p65NFκB, thereby ensuring its survival. Studies further undertaken in a tumor-bearing mice model revalidated the immunoprotective role of nifetepimine. Our present study thus strongly suggests that imbalance in cellular calcium homeostasis is an important factor leading to CD4(+) T cell death during cancer and holds promise that nifetepimine may have the potential to be used as an immunorestoring agent in cancer bearers.
Assuntos
Neoplasias da Mama/enzimologia , Linfócitos T CD4-Positivos/metabolismo , Cálcio/metabolismo , Fatores Imunológicos/farmacologia , Proteínas de Neoplasias/metabolismo , Pirimidinonas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/biossíntese , Microambiente Tumoral/efeitos dos fármacos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linfócitos T CD4-Positivos/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Dinoprostona/genética , Dinoprostona/imunologia , Dinoprostona/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/imunologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Transplante de Neoplasias , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/imunologia , Proteína Quinase C-alfa/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/imunologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/imunologia , Fator de Transcrição Sp1/metabolismo , Transplante Heterólogo , Microambiente Tumoral/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.
Assuntos
Caspase 8/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Biflavonoides/farmacologia , Carcinoma Neuroendócrino , Caspase 8/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Citocromos c/metabolismo , DNA Complementar , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Transfecção , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Drug induced toxicity and drug resistance are the major impediments to successful application of cancer chemotherapy. Therefore, selective targeting of the key biochemical events of the malignant cells may have a great therapeutic potential in specifically kill the cancer cells. We have evaluated in vitro the cytotoxic efficacy of a previously reported copper complex viz. copper N-(2-hydroxy acetophenone) glycinate (CuNG) on different drug sensitive and resistant cancer cell lines by MTT, annexin V positivity and caspase 3 activation assays. We have also investigated the underlying signalling events in CuNG mediated apoptosis of cancer cells by Western blotting technique. We have found that CuNG preferentially induces apoptosis to malignant cells irrespective of drug sensitivity and spares the normal cells. Our studies disclose that CuNG causes cellular redox imbalance in cancer cells through depletion of intracellular GSH level. CuNG mediated depletion of intracellular GSH level induces mitochondrial superoxide generation, which detaches cyto C from mitochondrial membrane through lipid peroxidation. The detached cyto C then release into the extra mitochondrial milieu in Bax mediated pathway where CuNG facilitates the binding of Bax through dissociation of hexokinase II from mitochondrial membrane. The present study opens the possibility of developing effective chemotherapeutic drugs by synthesizing numerous chemical compounds capable of targeting cellular redox environment and thus specifically kills cancer cells of broad spectrum.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Quelantes/farmacologia , Glutationa/metabolismo , Glicina/análogos & derivados , Compostos Organometálicos/farmacologia , Animais , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quelantes/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glicina/química , Glicina/farmacologia , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Células K562 , Camundongos , Células NIH 3T3 , Compostos Organometálicos/química , Relação Estrutura-AtividadeRESUMO
One of the key biochemical features that distinguish a cancer cell from normal cells is its persistent pro-oxidative state that leads to intrinsic oxidative stress. Malignant cells have evolved sophisticated adaptation systems that involve high dependency on antioxidant functions and upregulation of pro-survival molecules to counteract the deleterious effects of reactive species and to maintain dynamic redox balance. This situation renders them vulnerable to further oxidative challenges by exogenous agents. In the present study, we advocated that pomegranate polyphenols act as pro-oxidants and trigger ROS-mediated apoptosis in cancer cells. With the help of both in vitro and in vivo models, we have established that pomegranate fruit extract (PFE) can cause a significant reduction in tumor proliferation while leaving normal tissues and cells unharmed. Administration of PFE (0.2% v/v) in Erhlich's ascites carcinoma-bearing mice for 3 weeks, inhibited the nuclear factor (erythroid-derived 2)-like 2-antioxidant response element signaling cascade, increased intracellular reactive oxygen species content, altered glutathione cycle thereby activating reactive oxygen species-induced apoptotic pathway in Erhlich's ascites carcinoma cells. Moreover, PFE mitigated epithelial to mesenchymal transition and migration in triple negative breast cancer cells (MDA-MB 231 cells) by down-regulating nuclear factor kappa light-chain-enhancer of activated B cells. Pre-treatment of tumor cells with N-acetyl cysteine protected these cells from undergoing PFE-induced apoptosis while siRNA-mediated silencing of Nuclear factor (erythroid-derived 2)-like 2 and nuclear factor kappa light-chain-enhancer of activated B cells in tumor cells increased the cytotoxic potential and pro-oxidative activity of PFE, indicating a clear role of these transcription factors in orchestrating the anticancer/pro-oxidative properties of PFE. The seminal findings provided may be exploited to develop potential therapeutic targets for selective killing of malignant cells.
Assuntos
Carcinoma , Punica granatum , Animais , Camundongos , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Antioxidantes/química , Espécies Reativas de Oxigênio/metabolismo , Frutas/química , Ascite , Polifenóis/farmacologia , Polifenóis/análise , Transição Epitelial-Mesenquimal , Estresse Oxidativo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , ApoptoseRESUMO
Cancer continues to threat mortal alongside scientific community with burgeoning grasp. Most efforts directed to tame Cancer such as radiotherapy or chemotherapy, all came at a cost of severe side effects. The plant derived bioactive compounds on the other hand carries an inevitable advantage of being safer, bioavailable & less toxic compared to contemporary chemotherapeutics. Our strategic approach employed solvent extraction of Black Seed Oil (BSO) to highlight the orchestrated use of its oil soluble phytochemicals - Thymoquinone, Carvacrol & Trans-Anethole when used in cohort. These anti-cancer agents in unbelievably modest amounts present in BSO shows better potential to delineate migratory properties in breast cancer cells as compared to when treated individually. BSO was also observed to have apoptotic calibre when investigated in MDA-MB-231 and MCF-7 cell lines. We performed chemical characterization of the individual phytochemical as well as the oil in-whole to demonstrate the bioactive oil-soluble entities present in whole extract. BSO was observed to have significant anti-cancerous properties in cumulative proportion that is reportedly higher than the individual three components. Besides, this study also reports micro-RNA regulation on BSO administration, thereby playing a pivotal role in breast cancer alleviation. Thus, synergistic action of the integrants serves better combat force against breast cancer in the form of whole extract, hence aiming at a more lucrative paradigm while significantly regulating microRNAs associated with breast cancer migration and apoptosis.
Assuntos
Neoplasias da Mama , MicroRNAs , Nigella sativa , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Nigella sativa/química , Estrutura Molecular , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêuticoRESUMO
One of the major reasons for multidrug resistance (MDR) in cancer is the overexpression of P-glycoprotein (P-gp, ABCB1), a drug efflux pump. A novel copper complex, namely, copper (II) N-(2-hydroxyacetophenone) glycinate (CuNG) previously synthesized and characterized by the authors had been tested in this study. In a cell-based assay system with human MDR1 cDNA overexpressed mouse fibroblast NIH MDR1-G185 cell line, we demonstrated that this metal complex can directly interact with this transporter. As CuNG increased cellular accumulation of doxorubicin in P-gp-expressing cells, we presumed that of CuNG may be potential to reverse P-gp-mediated drug resistance probably by lowering the P-gp expression at the protein as well as mRNA level. Interestingly, our studies on UIC2 (a conformation sensitive monoclonal antibody) binding assay indicated the direct interaction of CuNG with P-gp. However, CuNG did not compete for the substrate binding as photoaffinity labeling of P-gp with a substrate analog [(125)I] iodoarylazidoprazosin ([(125)I] IAAP) showed approximately twofold increase in [(125)I] IAAP binding in presence of CuNG. In vitro study showed that CuNG significantly stimulated P-gp-specific ATPase activity in isolated membrane preparations from NIH MDR1-G185 cells. This result further confirmed the CuNG-P-gp direct interaction. This study also demonstrated that CuNG has a drug interaction site different from verapamil-, vinblastine- and progesterone-binding sites on P-gp. Taken together, this is the first report of molecular interaction of any Schiff's base metal chelate CuNG with human P-gp. This information may be useful to design more efficacious nontoxic metal-based drugs as MDR-reversing agents.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Quelantes/farmacologia , Cobre , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Quelantes/química , Doxorrubicina/farmacologia , Fibroblastos/metabolismo , Glicina/análogos & derivados , Glicina/química , Glicina/farmacologia , Humanos , Camundongos , Compostos Organometálicos/química , Compostos Organometálicos/farmacologiaRESUMO
Astraeus hygrometricus extensively been utilized by tribal people for long time. A triterpene, astrakurkurol has been isolated from A. hygrometricus but anticancer effect of this novel triterpene has imperceptibly been investigated. Motive of this research was to scrutinize its underlying apoptotic mechanism in HepG2 cells. Cytotoxicity studies demonstrated a selective effect of astrakurkurol with towering influence in HepG2 than Thle2 cells. The exposure of these triterpene-induced marked apoptotic morphological changes enhanced the rate of cell apoptosis and arrest cell cycle at G0/G1. Furthermore, these results are aided by decline in the expression of Bcl-2, Bcl-xL with an increase in the expression of p53, Bax, Fas, FADD together with the activation of caspase cascade. Astrakurkurol also displayed a remarkable anti-migratory capacity at a lower concentration. Altogether, studies explained anti-proliferative, pro-apoptotic, and anti-migratory efficacy of astrakurkurol on HepG2, composing a gripping challenge in the advancement of novel treatments against hepatocellular carcinoma. PRACTICAL APPLICATIONS: Mushrooms, the minuscule pharmaceutical factory, bear hundreds of novel elements with incredible biological attributes. Triterpenoids from mushrooms has been proven to bear potentials of curing cancer. This study highlights the cytotoxic and anti-migratory effects of novel triterpene in vitro in HepG2 cell, an HCC cell line. Astrakurkurol mediated cell death via both extrinsic and intrinsic apoptotic signaling. Utilization of astrakurkurol will provide a non-toxic substitute of chemotherapy and also uplift the value of forsaken taxon, Astraeus and boost the rural acceptance.
Assuntos
Agaricales , Carcinoma Hepatocelular , Neoplasias Hepáticas , Apoptose , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Folclore , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genéticaRESUMO
Tumor suppressor p53 plays the central role in regulating apoptosis in response to genotoxic stress. From an evolutionary perspective, the activity of p53 has to be backed up by other protein(s) in case of any functional impairment of this protein, to trigger DNA damage-induced apoptosis in cancer cells. We adopted multiple experimental approaches to demonstrate that in p53-impaired cancer cells, DNA damage caused accumulation of p53 paralogue p73 via Chk-1 that strongly impacted Bax expression and p53-independent apoptosis. On the contrary, when p53 function was restored by ectopic expression, Chk-2 induced p53 accumulation that in turn overshadowed p73 activity, suggesting an antagonistic interaction between p53 family members. To understand such interaction better, p53-expressing cells were impaired differentially for p53 activity. In wild-type p53-expressing cancer cells that were silenced for p53 for several generations, p73 was activated, whereas no such trend was observed when p53 was transiently silenced. Prolonged p53 interference, even in functional p53 settings, therefore, leads to the "gain of cellular adaptation" in a way that alters the cellular microenvironment in favor of p73 activation by altering p73-regulatory proteins, e.g. Chk1 activation and dominant negative p73 down-regulation. These findings not only unveil a hitherto unexplained mechanism underlying the functional switchover from p53 to p73, but also validate p73 as a promising and potential target for cancer therapy in the absence of functional p53.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Apoptose/genética , Quinase 1 do Ponto de Checagem , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica/genética , Inativação Gênica , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Proteínas Nucleares/genética , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , Fatores de Tempo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Células U937 , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
It has been widely reported that cancer, along with its treatment regimens, cause severe toxicity in the host. A suitable agent having chemopreventive properties as well as capabilities of ameliorating tumor- and drug-induced toxicities is of imminent need. Pomegranate has been projected as an excellent anti-tumor, anti-inflammatory and anti-oxidant agent. In this study, for the first time, we delineated the exact signaling cascade by which dietary supplementation of pomegranate fruit extract (PFE) protects tumor-bearing mice from tumor-induced hepatotoxicity. Increased activities of serum Alanine transaminase, Aspartate transaminase, Lactate dehydrogenase and Alkaline phosphatase, as well as histological studies confirmed the establishment of a state of hepatic dysfunction in tumor-bearers. Further investigations revealed that increased hepatic reactive oxygen species content and glutathione depletion-initiated apoptosis in these hepatocytes as we observed an alteration in the apoptotic proteins. PFE supplementation in tumor-bearing mice, on the other hand, differentially modulated redox-sensitive transcription factors Nrf2 and NF-κB, ultimately decreasing tumor-induced hepatic oxidative damage and cell death. siRNA-mediated inhibition of Nrf2 and NF-κB completely abolished the hepato-protective activities of PFE while pre-treatment of tumor-conditioned hepatocytes with N-acetyl cysteine augmented the cyto-protective properties of PFE. The present study clearly identified Nrf2/NF-κB/glutathione axis as the key factor behind the hepatoprotective potential of PFE. These findings would add to the existing knowledge about cancer chemoprevention by dietary polyphenols and might lead to the application of pomegranate polyphenols as supplement to escalate the effectiveness of cancer therapy by protecting normal cells from cancer related toxicities.