RESUMO
This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the authors. This study provided an explanation for why loss of FoxP3 in inducible regulatory T cells results in reduced expression of interleukin (IL)-10 despite the absence of FoxP3 binding sites in the IL-10 promoter. STAT3 binding sites do exist in the promoter, and evidence for a direct molecular interaction between FoxP3 and STAT3 proteins was provided as an explanation of the effect of loss of FoxP3. As supporting evidence, we reported modeling of a structural interaction between these two transcription factors in Figure 4D. As the N-terminal region of FoxP3, which consists of the Exon-2 region, had not been solved at structural resolution, we mistakenly used what we deduced to be a FoxP3 related transcription factor, NFAT, in the modeling. The model depicted in Figure 4D therefore did not represent a putative interaction between FoxP3 and STAT3 as labeled, but rather a putative interaction between NFAT and STAT3. Given the incorrect labeling of Figure 4D, the lack of documentation in the paper describing exactly how the modeling was performed, the lack of evidence shown in the paper for the choice of NFAT as the modeling partner, and the limited supporting evidence for a cooperative interaction between FoxP3 and STAT3, the editors have concluded with the corresponding author that the appropriate course of action is to retract the paper. We apologize for any confusion and inconvenience caused to readers.
Assuntos
Neoplasias da Mama/fisiopatologia , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Modelos Moleculares , Fatores de TranscriçãoRESUMO
BACKGROUND: Cancer metastasis is one of the most common causes of treatment failure and death in cancer patients. It has been acknowledged that aberrant activation of epithelial-to-mesenchymal transition (EMT) program, endows cancer cells with metastatic competence for which E-cadherin switch is a well-established hallmark. Suppression of E-cadherin by its transcriptional repressor Slug is thus a determining factor for EMT. Here, we aimed at discerning (i) the molecular mechanisms that regulate Slug/E-cadherin axis in oncogenic K-ras-expressing non-small cell lung carcinoma (NSCLC) cells, and (ii) the effect of aspirin in modulating the same. METHODS: The migratory behaviour of NSCLC cell line A549 were deciphered by wound healing assay. Further assessment of the molecular mechanisms was done by western blotting, RT-PCR, confocal microscopy, chromatin immunoprecipitation and small interfering RNA (siRNA)-mediated gene silencing. RESULTS: Here we report that in oncogenic K-ras-expressing A549 cells, Ras/ERK downstream Elk-1 forms p-Elk-1-p300 complex that being directly recruited to SLUG promoter acetylates the same to ensure p65NFκB binding for transcriptional up-regulation of Slug, a transcriptional repressor of E-cadherin. Aspirin inhibits EMT and decelerates the migratory potential of A549 cells by down-regulating Slug and thereby up-regulating E-cadherin. Aspirin impedes activation and nuclear translocation of p65NFκB, essential for this transcription factor being available for SLUG promoter binding. As a consequence, Slug transcription is down-regulated relieving A549 cells from Slug-mediated repression of E-cadherin transcription, thereby diminishing the metastatic potential of these oncogenic Ras-expressing NSCLC cells. CONCLUSIONS: Cumulatively, these results signify a crucial role of the anti-inflammatory agent aspirin as a novel negative regulator of epithelial-to-mesenchymal transition thereby suggesting its candidature as a promising tool for deterring metastasis of highly invasive K-ras-expressing NSCLC cells.
Assuntos
Aspirina/administração & dosagem , Caderinas/biossíntese , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Fatores de Transcrição/biossíntese , Caderinas/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição da Família Snail , Fator de Transcrição RelA/biossíntese , Fator de Transcrição RelA/genética , Fatores de Transcrição/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
Tumour progression is associated with immune-suppressive conditions that facilitate the escape of tumour cells from the regimen of immune cells, subsequently paralysing the host defence mechanisms. Induction of CD4(+) CD25(+) FoxP3(+) T regulatory (Treg) cells has been implicated in the tumour immune escape mechanism, although the novel anti-cancer treatment strategies targeting Treg cells remain unknown. The focus of this study is to define the interaction between tumour and immune system, i.e. how immune tolerance starts and gradually leads to the induction of adaptive Treg cells in the tumour microenvironment. Our study identified hyperactivated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -signalling as a potential target for reversing Treg cell augmentation in breast cancer patients. In more mechanistic detail, pharmacological inhibitors of MEK/ERK signalling inhibited transforming growth factor-ß (TGF-ß) production in tumour cells that essentially blocked TGF-ß-SMAD3/SMAD4-mediated induction of CD25/interleukin-2 receptor α on CD4(+) T-cell surface. As a result high-affinity binding of interleukin-2 on those cells was prohibited, causing lack of Janus kinase 1 (JAK1)/JAK3-mediated signal transducer and activator of transcription 3 (STAT3)/STAT5 activation required for FoxP3 expression. Finally, for a more radical approach towards a safe MEK inhibitor, we validate the potential of multi-kinase inhibitor curcumin, especially the nano-curcumin made out of pure curcumin with greater bioavailability; in repealing tumour-shed TGF-ß-induced Treg cell augmentation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Curcumina/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Comunicação Parácrina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Química Farmacêutica , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Evasão Tumoral/efeitos dos fármacosRESUMO
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
BACKGROUND: Complementary medicines, including homeopathy, are used by many patients with cancer, usually alongside with conventional treatment. However, the molecular mechanisms underneath the anti-cancer effect, if any, of these medicines have still remained unexplored. To this end we attempted to evaluate the efficacy of calcarea carbonica, a homeopathic medicine, as an anti-cancer agent and to delineate the detail molecular mechanism(s) underlying calcerea carbonica-induced tumor regression. METHODS: To investigate and delineate the underlying mechanisms of calcarea carbonica-induced tumor regression, Trypan blue dye-exclusion test, flow cytometric, Western blot and reverse transcriptase-PCR techniques were employed. Further, siRNA transfections and inhibitor studies were used to validate the involvement of p53 pathway in calcarea carbonica-induced apoptosis in cancer cells. RESULTS: Interestingly, although calcarea carbonica administration to Ehrlich's ascites carcinoma (EAC)- and Sarcoma-180 (S-180)-bearing Swiss albino mice resulted in 30-35% tumor cell apoptosis, it failed to induce any significant cell death in ex vivo conditions. These results prompted us to examine whether calcarea carbonica employs the immuno-modulatory circuit in asserting its anti-tumor effects. Calcarea carbonica prevented tumor-induced loss of effector T cell repertoire, reversed type-2 cytokine bias and attenuated tumor-induced inhibition of T cell proliferation in tumor-bearing host. To confirm the role of immune system in calcarea carbonica-induced cancer cell death, a battery of cancer cells were co-cultured with calcarea carbonica-primed T cells. Our results indicated a "two-step" mechanism of the induction of apoptosis in tumor cells by calcarea carbonica i.e., (1) activation of the immune system of the host; and (2) induction of cancer cell apoptosis via immuno-modulatory circuit in p53-dependent manner by down-regulating Bcl-2:Bax ratio. Bax up-regulation resulted in mitochondrial transmembrane potential loss and cytochrome c release followed by activation of caspase cascade. Knocking out of p53 by RNA-interference inhibited calcarea carbonica-induced apoptosis thereby confirming the contribution of p53. CONCLUSION: These observations delineate the significance of immuno-modulatory circuit during calcarea carbonica-mediated tumor apoptosis. The molecular mechanism identified may serve as a platform for involving calcarea carbonica into immunotherapeutic strategies for effective tumor regression.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carbonato de Cálcio/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/química , Neoplasias da Mama , Carbonato de Cálcio/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer cells often develop multiple mechanisms of drug resistance during tumor progression, which is the major reason for the failure of breast cancer therapy. High constitutive activation of NFκB has been found in different cancers, creating an environment conducive for chemotherapeutic resistance. Here we report that doxorubicin-induced SMAR1-dependent transcriptional repression and SMAR1-independent degradation of IkBα resulted in nuclear translocation of p65NFκB and its association with p300 histone acetylase and subsequent transcription of Bcl-2 to impart protective response in drug-resistant cells. Consistently SMAR1-silenced drug-resistant cells exhibited IkBα-mediated inhibition of p65NFκB and induction of p53-dependent apoptosis. Interestingly, curcumin pretreatment of drug-resistant cells alleviated SMAR1-mediated p65NFκB activation and hence restored doxorubicin sensitivity. Under such anti-survival condition, induction of p53-p300 cross-talk enhanced the transcriptional activity of p53 and intrinsic death cascade. Importantly, promyelocyte leukemia-mediated SMAR1 sequestration that relieved the repression of apoptosis-inducing genes was indispensable for such chemo-sensitizing ability of curcumin. A simultaneous decrease in drug-induced systemic toxicity by curcumin might also have enhanced the efficacy of doxorubicin by improving the intrinsic defense machineries of the tumor-bearer. Overall, the findings of this preclinical study clearly demonstrate the effectiveness of curcumin to combat doxorubicin-resistance. We, therefore, suggest curcumin as a potent chemo-sensitizer to improve the therapeutic index of this widely used anti-cancer drug. Taken together, these results suggest that curcumin can be developed into an adjuvant chemotherapeutic drug.
Assuntos
Antineoplásicos/farmacologia , Curcumina/metabolismo , Proteína p300 Associada a E1A/metabolismo , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/genética , Fator de Transcrição RelA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Apoptose , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Camundongos , Transplante de Neoplasias , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
The most common alterations found in breast cancer are inactivation or mutation of tumor suppressor gene p53. The present study revealed that theaflavins induced p53-mutated human breast cancer cell apoptosis. Pharmacological inhibition of caspase-8 or expression of dominant-negative (Dn)-caspase-8/Fas-associated death domain (FADD) partially inhibited apoptosis, whereas caspase-9 inhibitor completely blocked the killing indicating involvement of parallel pathways that converged to mitochondria. Further studies demonstrated theaflavin-induced Fas upregulation through the activation of c-jun N-terminal kinase, Fas-FADD interaction in a Fas ligand-independent manner, caspase-8 activation and t-Bid formation. A search for the parallel pathway revealed theaflavin-induced inhibition of survival pathway, mediated by Akt deactivation and Bcl-xL/Bcl-2-associated death promoter dephosphorylation. These well-defined routes of growth control converged to a common process of mitochondrial transmembrane potential loss, cytochrome c release and activation of the executioner caspase-9 and -3. Overexpression of either constitutively active myristylated-Akt (Myr-Akt) or Dn-caspase-8 partially blocked theaflavin-induced mitochondrial permeability transition and apoptosis of p53-mutated cells, whereas cotransfection of Myr-Akt and Dn-caspase-8 completely abolished theaflavin effect thereby negating the possibility of existence of third pathways. These results and other biochemical correlates established the concept that two distinct signaling pathways were regulated by theaflavins to induce mitochondrial death cascade, eventually culminating to apoptosis of p53-mutated human breast cancer cells that are strongly resistant to conventional therapies.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Biflavonoides/farmacologia , Neoplasias da Mama/patologia , Catequina/farmacologia , Mutação/genética , Proteína Supressora de Tumor p53/genética , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Caspase 9/metabolismo , Inibidores de Caspase , Citocromos c/metabolismo , Proteína de Domínio de Morte Associada a Fas/antagonistas & inibidores , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Proteína de Morte Celular Associada a bcl/antagonistas & inibidores , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo , Receptor fas/antagonistas & inibidores , Receptor fas/genética , Receptor fas/metabolismoRESUMO
The long-term survival of osteosarcoma patients with metastatic or recurrent disease remains dismal, and new therapeutic options are urgently needed. The purpose of our study was to compare the efficacy of CD47 mAb plus doxorubicin combination therapy in mouse models of osteosarcoma with CD47 mAb and doxorubicin monotherapy. Forty-eight NOD scid gamma (NSG) mice with intratibial MNNG/HOS tumors received CD47 mAb, doxorubicin, combination therapy, or control IgG treatment. Twenty-four mice (n = 6 per group) underwent pre- and post-treatment magnetic resonance imaging (MRI) scans with the macrophage marker ferumoxytol, bioluminescence imaging, and histological analysis. Tumor ferumoxytol enhancement, tumor flux, and tumor-associated macrophages (TAM) density were compared between different groups using a one-way ANOVA. Twenty-four additional NSG mice underwent survival analyses with Kaplan-Meier curves and a log-rank (Mantel-Cox) test. Intratibial osteosarcomas demonstrated significantly stronger ferumoxytol enhancement and significantly increased TAM quantities after CD47 mAb plus doxorubicin combination therapy compared to CD47 mAb (P = 0.02) and doxorubicin monotherapy (P = 0.001). Tumor-bearing mice treated with CD47 mAb plus doxorubicin combination therapy demonstrated significantly reduced tumor size and prolonged survival compared to control groups that received CD47 mAb (P = 0.03), doxorubicin monotherapy (P = 0.01), and control IgG (P = 0.001). In conclusion, CD47 mAb plus doxorubicin therapy demonstrates an additive therapeutic effect in mouse models of osteosarcomas, which can be monitored with an immediately clinically applicable MRI technique.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Antígeno CD47/imunologia , Doxorrubicina/uso terapêutico , Osteossarcoma/tratamento farmacológico , Animais , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/imunologia , Linhagem Celular Tumoral , Óxido Ferroso-Férrico/análise , Humanos , Luminescência , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Imageamento por Ressonância Magnética , Camundongos Endogâmicos NOD , Imagem Óptica , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/imunologia , Fagocitose/efeitos dos fármacosRESUMO
CD47 monoclonal antibodies (mAbs) activate tumor-associated macrophages (TAMs) in sarcomas to phagocytose and eliminate cancer cells. Though CD47 mAbs have entered clinical trials, diagnostic tests for monitoring therapy response in vivo are currently lacking. Ferumoxytol is an FDA-approved iron supplement which can be used "off label" as a contrast agent: the nanoparticle-based drug is phagocytosed by TAM and can be detected with magnetic resonance imaging (MRI). We evaluated if ferumoxytol-enhanced MRI can monitor TAM response to CD47 mAb therapy in osteosarcomas. Forty-eight osteosarcoma-bearing mice were treated with CD47 mAb or control IgG and underwent pre- and post-treatment ferumoxytol-MRI scans. Tumor enhancement, quantified as T2 relaxation times, was compared with the quantity of TAMs as determined by immunofluorescence microscopy and flow cytometry. Quantitative data were compared between experimental groups using exact two-sided Wilcoxon rank-sum tests. Compared to IgG-treated controls, CD47 mAb-treated tumors demonstrated significantly shortened T2 relaxation times on ferumoxytol-MRI scans (p < 0.01) and significantly increased F4/80+CD80+ M1 macrophages on histopathology (p < 0.01). CD47 mAb-treated F4/80+ macrophages demonstrated significantly augmented phagocytosis of ferumoxytol nanoparticles (p < 0.01). Thus, we conclude that ferumoxytol-MRI can detect TAM response to CD47 mAb in mouse models of osteosarcoma. The ferumoxytol-MRI imaging test could be immediately applied to monitor CD47 mAb therapies in clinical trials.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígeno CD47/genética , Imunoterapia/métodos , Macrófagos/metabolismo , Imageamento por Ressonância Magnética/métodos , Nanopartículas/metabolismo , Osteossarcoma/genética , Animais , Humanos , Camundongos , Osteossarcoma/patologiaRESUMO
Despite considerable progress with our understanding of glioblastoma multiforme (GBM) and the precise delivery of radiotherapy, the prognosis for GBM patients is still unfavorable with tumor recurrence due to radioresistance being a major concern. We recently developed a cross-linked iron oxide nanoparticle conjugated to azademethylcolchicine (CLIO-ICT) to target and eradicate a subpopulation of quiescent cells, glioblastoma initiating cells (GICs), which could be a reason for radioresistance and tumor relapse. The purpose of our study was to investigate if CLIO-ICT has an additive therapeutic effect to enhance the response of GBMs to ionizing radiation. Methods: NSG™ mice bearing human GBMs and C57BL/6J mice bearing murine GBMs received CLIO-ICT, radiation, or combination treatment. The mice underwent pre- and post-treatment magnetic resonance imaging (MRI) scans, bioluminescence imaging (BLI), and histological analysis. Tumor nanoparticle enhancement, tumor flux, microvessel density, GIC, and apoptosis markers were compared between different groups using a one-way ANOVA and two-tailed Mann-Whitney test. Additional NSG™ mice underwent survival analyses with Kaplan-Meier curves and a log rank (Mantel-Cox) test. Results: At 2 weeks post-treatment, BLI and MRI scans revealed significant reduction in tumor size for CLIO-ICT plus radiation treated tumors compared to monotherapy or vehicle-treated tumors. Combining CLIO-ICT with radiation therapy significantly decreased microvessel density, decreased GICs, increased caspase-3 expression, and prolonged the survival of GBM-bearing mice. CLIO-ICT delivery to GBM could be monitored with MRI. and was not significantly different before and after radiation. There was no significant caspase-3 expression in normal brain at therapeutic doses of CLIO-ICT administered. Conclusion: Our data shows additive anti-tumor effects of CLIO-ICT nanoparticles in combination with radiotherapy. The combination therapy proposed here could potentially be a clinically translatable strategy for treating GBMs.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Desoxiadenosinas/uso terapêutico , Glioblastoma/tratamento farmacológico , Nanomedicina Teranóstica , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/radioterapia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Desoxiadenosinas/química , Desoxiadenosinas/farmacologia , Portadores de Fármacos/química , Feminino , Compostos Férricos/química , Glioblastoma/mortalidade , Glioblastoma/radioterapia , Humanos , Estimativa de Kaplan-Meier , Imageamento por Ressonância Magnética , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microvasos/fisiologiaRESUMO
Elevated cyclin D1 (CCND1) expression levels in mantle cell lymphoma (MCL) are associated with aggressive clinical manifestations related to chemoresistance, but little is known about how this important proto-oncogene contributes to the resistance of MCL. Here, we showed that RNA interference-mediated depletion of CCND1 increased caspase-3 activities and induced apoptosis in the human MCL lines UPN-1 and JEKO-1. In vitro and xenotransplant studies revealed that the toxic effect of CCND1 depletion in MCL cells was likely due to increase in histone H2AX phosphorylation, a DNA damage marker. DNA fiber analysis suggested deregulated replication initiation after CCND1 depletion as a potential cause of DNA damage. Finally, in contrast to depletion or inhibition of cyclin-dependent kinase 4, CCND1 depletion increased chemosensitivity of MCL cells to replication inhibitors hydroxyurea and cytarabine. Our findings have an important implication for CCND1 as a potential therapeutic target in MCL patients who are refractory to standard chemotherapy.
Assuntos
Ciclina D1/metabolismo , Dano ao DNA , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Animais , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Replicação do DNA , Modelos Animais de Doenças , Xenoenxertos , Humanos , Linfoma de Célula do Manto/patologia , Camundongos , Proto-Oncogene Mas , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Glioblastoma (GBM) has a dismal prognosis. Evidence from preclinical tumor models and human trials indicates the role of GBM-initiating cells (GIC) in GBM drug resistance. Here, we propose a new treatment option with tumor enzyme-activatable, combined therapeutic and diagnostic (theranostic) nanoparticles, which caused specific toxicity against GBM tumor cells and GICs. The theranostic cross-linked iron oxide nanoparticles (CLIO) were conjugated to a highly potent vascular disrupting agent (ICT) and secured with a matrix-metalloproteinase (MMP-14) cleavable peptide. Treatment with CLIO-ICT disrupted tumor vasculature of MMP-14-expressing GBM, induced GIC apoptosis, and significantly impaired tumor growth. In addition, the iron core of CLIO-ICT enabled in vivo drug tracking with MR imaging. Treatment with CLIO-ICT plus temozolomide achieved tumor remission and significantly increased survival of human GBM-bearing mice by more than 2-fold compared with treatment with temozolomide alone. Thus, we present a novel therapeutic strategy with significant impact on survival and great potential for clinical translation. Mol Cancer Ther; 16(9); 1909-21. ©2017 AACR.
Assuntos
Neoplasias Encefálicas/genética , Expressão Gênica , Glioblastoma/genética , Metaloproteinase 14 da Matriz/genética , Nanomedicina Teranóstica , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Modelos Animais de Doenças , Compostos Férricos/química , Citometria de Fluxo , Glioblastoma/diagnóstico , Glioblastoma/mortalidade , Glioblastoma/terapia , Humanos , Imageamento por Ressonância Magnética , Espectrometria de Massas , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Nanopartículas/química , Temozolomida , Nanomedicina Teranóstica/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-ß receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies.
RESUMO
With phytochemicals executing a plethora of anti-tumor mechanisms, targeting the 'guardian angel' p53 appears to be a critical strategy to energize the process of cancer therapeutics. Regulation of anti-tumor p53 functions by dietary plant polyphenols particularly black tea and its active component theaflavins has gained immense recognition from the point of view of both efficacy and safety. This review highlights the complexities of p53 functions, molecular mechanisms of its inactivation in cancer, and therapeutic strategies for rescuing p53 dysfunction in tumors using theaflavins. It describes how theaflavins, by steering a single molecular target - p53, regulate multiple hallmarks of carcinogenesis i.e., tumor glycolysis, angiogenesis, metastasis, apoptosis and drug resistance. Additionally, considering the rising of the current concept of cancer stem cells (CSCs), the sole participant in tumor evolution, the review discusses about the possible role of theaflavin-p53 cross talk in targeting CSCs. Such attempts to target the complexities of p53 functions during neogenesis will be of immense help in developing a "new" strategy for successful cancer prevention and therapy by theaflavins.
Assuntos
Biflavonoides/farmacologia , Biflavonoides/uso terapêutico , Catequina/farmacologia , Catequina/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Humanos , Neoplasias/patologiaRESUMO
Despite major advances in understanding the mechanisms of tumor immunity, its successful translation into effective tumor immunotherapy is hindered by the ability of tumors to foster a tolerant microenvironment and to activate a plethora of immunosuppressive mechanisms. Among different strategies employed by tumors to thwart immune responses, shedding of immunosuppressive molecules, such as sialic acid-containing glycosphingolipids, gangliosides, by the tumor is one important strategy. Aberrant and elevated expression of gangliosides has been demonstrated on the surface of cancer cells. Here we discuss about the molecular mechanisms underneath the contribution of tumor gangliosides in targeting multiple steps of T cell response. We shall also underscore the contribution of T-regulatory, NK and dendritic cells in this immunosuppressive network. Inhibitory effects of gangliosides ultimately converge to T cell apoptosis in receptor-dependent and -independent manners via IL-2 deprivation, ROS production, cytochrome c release, NFkappaB inhibition and caspase activation. Current wealth of information promises a future scenario in which synchronized blockade of immunosuppressive mechanisms and removal of inhibitory signals might be effective in overcoming immunological tolerance and promoting tumor regression.
Assuntos
Gangliosídeos/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Comunicação Celular/imunologia , Humanos , Tolerância Imunológica , Imunoterapia , Neoplasias/terapiaRESUMO
The present study demonstrates that theaflavins exploit p53 to impede metastasis in human breast cancer cells. Our data suggest that p53-dependent reactive oxygen species (ROS) induce p53-phosphorylation via p38MAPK in a feedback loop to inhibit IkappaBalpha-phosphorylation and NF-kappaB/p65 nuclear translocation, thereby down-regulating the metastatic proteins metalloproteinase (MMP)-2 and MMP-9. When wild-type p53-expressing MCF-7 cells are transfected with p53 short-interfering RNA, or treated with a pharmacological inhibitor of ROS, theaflavins fail to inhibit NF-kappaB-mediated cell migration. On the other hand, NF-kappaB over-expression bestows MCF-7 cells with resistance to the anti-migratory effect of theaflavins. These results indicate that inhibition of NF-kappaB via p53-ROS crosstalk is a pre-requisite for theaflavins to accomplish the anti-migratory effect in breast cancer cells.