RESUMO
Autophagy is typically activated in cancer cells as a rescue strategy in response to cellular stress (e.g., chemotherapy). Herein, we found that Berbamine Hydrochloride (Ber) can act as an effective inhibitor of the late stage of autophagic flux, thereby potentiating the killing effect of chemotherapy agents. Lung carcinoma cells exposed to Ber exhibited increased autophagosomes, marked by LC3-II upregulation. The increased level of p62 after Ber treatment indicated that the autophagic flux was blocked at the late stage. The lysosome staining assay and cathepsin maturation detection indicated impaired lysosomal acidification. We found that Nox2 exhibited intensified co-localization with lysosomes in Ber-treated cells. Nox2 is a key enzyme for superoxide anion production capable of transferring electrons into the lysosomal lumen, thereby neutralizing the inner protons; this might explain the aberrant acidification. This hypothesis is further supported by the observed reversal of lysosomal cathepsin maturation by Nox2 inhibitors. Finally, Ber combined with cisplatin exhibited a synergistic killing effect on lung carcinoma cells. Further data suggested that lung carcinoma cells co-treated with Ber and cisplatin accumulated excessive reactive oxygen species (ROS), which typically activated MAPK-mediated mitochondria-dependent apoptosis. The enhanced anti-cancer effect of Ber combined with cisplatin was also confirmed in an in vivo xenograft mouse model. These findings indicate that Ber might be a promising adjuvant for enhancing the cancer cell killing effect of chemotherapy via the inhibition of autophagy. In this process, Nox2 might be a significant mediator of Ber-induced aberrant lysosomal acidification.
Assuntos
Antineoplásicos , Carcinoma , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Cisplatino/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia , Apoptose , Lisossomos/metabolismo , Pulmão/metabolismo , Concentração de Íons de Hidrogênio , Catepsinas/metabolismo , Catepsinas/farmacologia , Catepsinas/uso terapêutico , Carcinoma/tratamento farmacológico , Carcinoma/metabolismoRESUMO
Although artesunate has been reported to be a promising candidate for colorectal cancer (CRC) treatment, the underlying mechanisms and molecular targets of artesunate are yet to be explored. Here, we report that artesunate acts as a senescence and autophagy inducer to exert its inhibitory effect on CRC in a reactive oxygen species (ROS)-dependent manner. In SW480 and HCT116 cells, artesunate treatment led to mitochondrial dysfunction, drastically promoted mitochondrial ROS generation, and consequently inhibited cell proliferation by causing cell cycle arrest at G0/G1 phase as well as subsequent p16- and p21-mediated cell senescence. Senescent cells underwent endoplasmic reticulum stress (ERS), and the unfolded protein response (UPR) was activated via IRE1α signaling, with upregulated BIP, IRE1α, phosphorylated IRE1α (p-IRE1α), CHOP, and DR5. Further experiments revealed that autophagy was induced by artesunate treatment due to oxidative stress and ER stress. In contrast, N-Acetylcysteine (NAC, an ROS scavenger) and 3-Methyladenine (3-MA, an autophagy inhibitor) restored cell viability and attenuated autophagy in artesunate-treated cells. Furthermore, cellular free Ca2+ levels were increased and could be repressed by NAC, 3-MA, and GSK2350168 (an IRE1α inhibitor). In vivo, artesunate administration reduced the growth of CT26 cell-derived tumors in BALB/c mice. Ki67 and cyclin D1 expression was downregulated in tumor tissue, while p16, p21, p-IRE1α, and LC3B expression was upregulated. Taken together, artesunate induces senescence and autophagy to inhibit cell proliferation in colorectal cancer by promoting excessive ROS generation.
Assuntos
Neoplasias Colorretais , Endorribonucleases , Animais , Apoptose , Artesunato/farmacologia , Autofagia , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases , Espécies Reativas de Oxigênio/metabolismoRESUMO
Melanoma is the most lethal type of skin cancer. Despite the breakthroughs in the clinical treatment of melanoma using tumor immunotherapy, many patients do not benefit from these immunotherapies because of multiple immunosuppressive mechanisms. Therefore, there is an urgent need to determine the mechanisms of tumor-immune system interactions and their molecular determinants to improve cancer immunotherapy. In this study, combined analysis of microarray data and single-cell RNA sequencing data revealed the key interactions between immune cells in the melanoma microenvironment. First, differentially expressed genes (DEGs) between normal and malignant tissues were obtained using GEO2R. The DEGs were then subjected to downstream analyses, including enrichment analysis and protein-protein interaction analysis, indicating that these genes were associated with the immune response of melanoma. Then, the GEPIA and TIMER databases were used to verify the differential expression and prognostic significance of hub genes, and the relationship between the hub genes and immune infiltration. In addition, we combined single cell analysis from GSE123139 to identify immune cell types, and validated the expression of the hub genes in these immune cells. Finally, cell-to-cell communication analysis of the proteins encoded by the hub genes and their interactions was performed using CellChat. We found that the CCL5-CCR1, SELPLG-SELL, CXCL10-CXCR3, and CXCL9-CXCR3 pathways might play important roles in the communication between the immune cells in tumor microenvironment. This discovery may reveal the communication basis of immune cells in the tumor microenvironment and provide a new idea for melanoma immunotherapy.
Assuntos
Melanoma , Neoplasias Cutâneas , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/patologia , Prognóstico , Neoplasias Cutâneas/genética , Transcriptoma , Microambiente Tumoral/genéticaRESUMO
Dioscin (Dio), steroid saponin, exists in several medicinal herbs with potent anticancer efficacy. This study aimed to explore the effect of Dio on the immune-related modulation and synergistic therapeutic effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV-Tk/GCV) suicide gene therapy system in murine melanoma, thereby providing a research basis to improve the potential immunomodulatory mechanism underlying combination therapy. Using both in vitro and in vivo experiments, we confirmed the immunocidal effect of Dio-potentiated suicide gene therapy on melanoma. The results showed that Dio upregulated connexin 43 (Cx43) expression and improved gap junction intercellular communication (GJIC) in B16 cells while increasing the cross-presentation of antigens by dendritic cells (DCs), eventually promoting the activation and antitumor immune killing effects of CD8+ T lymphocytes. In contrast, inhibition or blockade of the GJIC function (overexpression of mutant Cx43 tumor cells/Gap26) partially reversed the potentiating effect. The significant synergistic effect of Dio on HSV-Tk/GCV suicide gene therapy was further investigated in a B16 xenograft mouse model. The increased number and activation ratio of CD8+ T lymphocytes and the levels of Gzms-B, IFN-γ, and TNF-α in mice reconfirmed the potential modulatory effects of Dio on the immune system. Taken together, Dio targets Cx43 to enhance GJIC function, improve the antigens cross-presentation of DCs, and activate the antitumor immune effect of CD8+ T lymphocytes, thereby providing insights into the potential immunomodulatory mechanism underlying combination therapy.
Assuntos
Conexina 43 , Melanoma , Animais , Comunicação Celular , Conexina 43/genética , Conexina 43/metabolismo , Apresentação Cruzada , Diosgenina/análogos & derivados , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Junções Comunicantes/metabolismo , Terapia Genética/métodos , Humanos , Melanoma/tratamento farmacológico , Melanoma/terapia , Camundongos , Simplexvirus/genética , Simplexvirus/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismo , Timidina Quinase/farmacologiaRESUMO
Cancer cells have developed chemoresistance and have improved their survival through the upregulation of autophagic mechanisms that protect mitochondrial function. Here, we report that the traditional Chinese anticancer agent tubeimoside I (Tub), which is a potent inhibitor of autophagy, can promote mitochondria-associated apoptosis in lung cancer cells. We found that Tub disrupted both mitochondrial and lysosomal pathways. One of its mechanisms was the induction of DRP1-mediated mitochondrial fragmentation. Another mechanism was the blocking of late-stage autophagic flux via impairment of lysosomal acidification through V-ATPase inhibition; this blocks the removal of dysfunctional mitochondria and results in reactive oxygen species (ROS) accumulation. Excessive ROS accumulation causes damage to lysosomal membranes and increases lysosomal membrane permeability, which leads to the leakage of cathepsin B. Finally, cathepsin B upregulates Bax-mediated mitochondrial outer membrane permeability and, subsequently, cytosolic cytochrome C-mediated caspase-dependent apoptosis. Thus, the cancer cell killing effect of Tub is enhanced through the formation of a positive feedback loop. The killing effect of Tub on lung cancer cells was verified in xenografted mice. In summary, Tub exerts a dual anticancer effect that involves the disruption of mitochondrial and lysosomal pathways and their interaction and, thereby, has a specific and enhanced killing effect on lung cancer cells.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Catepsina B/metabolismo , Catepsina B/farmacologia , Linhagem Celular Tumoral , China , Humanos , Neoplasias Pulmonares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Medicina Tradicional Chinesa/métodos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Permeabilidade , Espécies Reativas de Oxigênio/metabolismo , Saponinas/metabolismo , Triterpenos/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Autophagy inhibition has been demonstrated to increase the efficacy of conventional chemotherapy. In this study, we identified hederagenin, a triterpenoid derived from Hedera helix, as a potent inhibitor of autophagy and then hypothesized that hederagenin might synergize with chemotherapeutic drugs (e.g., cisplatin and paclitaxel) to kill lung cancer cells. Firstly, we observed that hederagenin induced the increased autophagosomes in lung cancer cells concomitantly with the upregulation of LC3-II and p62, which indicated the impairment of autophagic flux. The colocalization assay indicated hederagenin could not block the fusion of lysosomes and autophagosomes, whereas the lysosomal acidification might be inhibited by hederagenin as revealed by the reduced staining of acidity-sensitive reagents (i.e., Lysotracker and acridine orange). The aberrant acidic environment then impaired the function of lysosome, which was evidenced by the decrease of mature cathepsin B and cathepsin D. Lastly, hederagenin, in agree with our hypothesis, promoted pro-apoptotic effect of cisplatin and paclitaxel with the accumulation of reactive oxygen species (ROS); while the synergistic effect could be abolished by the ROS scavenger, N-acetyl-L-cysteine. These data summarily demonstrated hederagenin-induced accumulation of ROS by blocking autophagic flux potentiated the cytotoxicity of cisplatin and paclitaxel in lung cancer cells.
Assuntos
Autofagia/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias Pulmonares/patologia , Ácido Oleanólico/análogos & derivados , Paclitaxel/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/ultraestrutura , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Dioscin, a natural glycoside derived from many plants, has been proved to exert anti-cancer activity. Several studies have found that it reverses TGF-ß1-induced epithelial-mesenchymal transition (EMT). Whether dioscin can reverse EMT by pathways other than TGF-ß is still unknown. METHODS: We used network-based pharmacological methods to systematically explore the potential mechanisms by which dioscin acts on lung cancer. Cell Counting Kit-8 assay, scratch healing, Transwell assay, Matrigel invasion assay, immunofluorescence assay, and Western blotting were employed to confirm the prediction of key targets and the effects of dioscin on EMT. RESULTS: Here, using network-based pharmacological methods, we found 42 possible lung cancer-related targets of dioscin, which were assigned to 98 KEGG pathways. Among the 20 with the lowest p-values, the PI3K-AKT signaling pathway is involved and significantly related to EMT. AKT1 and mTOR, with high degrees (reflecting higher connectivity) in the compound-target analysis, participate in the PI3K-AKT signaling pathway. Molecular docking indicated the occurrence of dioscin-AKT1 and dioscin-mTOR binding. Functional experiments demonstrated that dioscin suppressed the proliferation, migration, invasion, and EMT of human lung adenocarcinoma cells in a dose-dependent manner, without TGF-ß stimulation. Furthermore, we determined that dioscin downregulated p-AKT, p-mTOR and p-GSK3ß in human lung adenocarcinoma cells without affecting their total protein levels. The PI3K inhibitor LY294002 augmented these changes. CONCLUSION: Dioscin suppressed proliferation, invasion and EMT of lung adenocarcinoma cells via the inactivation of AKT/mTOR/GSK3ß signaling, probably by binding to AKT and mTOR, and inhibiting their phosphorylation.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos/farmacologia , Diosgenina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Diosgenina/química , Diosgenina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
PURPOSE: To investigate the synergistic effect of resveratrol on the bystander effect of TK/GCV suicide gene system in melanoma cells. METHODS: The effect of resveratrol on the growth of B16 cells and the synergistic effect of resveratrol with or without GCV were detected by MTT assay and high content screening assay. The effect of resveratrol on GJIC function was detected by flow cytometry combined with fluorescence tracer and fluorescence microscope, and the expression of gap junction protein was detected by western blotting. Synergistic killing effect of resveratrol plus TK/GCV was tested in vivo using transplanted melanoma model. RESULTS: In vitro, resveratrol can enhanced GJ function and upregulated Cx32 and Cx43 protein expression in B16 cells. Resveratrol synergized with GCV to kill mixed B16 melanoma cells (20% TK+ cells and 80% TK- cells) and to improve apoptosis rate of TK- cells (the bystander effect of TK system), and the synergistic action was reversed by the GJ inhibitor AGA. In vivo, when B16 cells were mixed with 30% TK+ B16 cells, significantly reduced tumor weight and volume were observed after combinational treatment with resveratrol plus GCV as compared with GCV or resveratrol treatment alone. CONCLUSIONS: Resveratrol could synergistically enhance the killing effect of TK/GCV suicide gene system in melanoma B16 cells and transplanted melanoma. It might be a promising adjuvant of TK/GCV therapy.
Assuntos
Ganciclovir/uso terapêutico , Terapia Genética , Melanoma Experimental/terapia , Resveratrol/uso terapêutico , Timidina Quinase/genética , Animais , Efeito Espectador , Linhagem Celular Tumoral , Sinergismo Farmacológico , Genes Transgênicos Suicidas , Melanoma Experimental/tratamento farmacológico , CamundongosRESUMO
Melanoma is a global concern and accounts for the major mortality of skin cancers. Herpes simplex virus thymidine kinase gene with ganciclovir (HSV-TK/GCV) is a promising gene therapy for melanoma. Despite its low efficiency, it is well known for its bystander effect which is mainly mediated by gap junction. In this study, we found that curcumin reduced B16 melanoma cell viability in both time- and dose-dependent manner. Further study showed that curcumin improved the gap junction intercellular communication (GJIC) function, and upregulated the proteins essential to gap junction, such as connexin 32 and connexin 43, indicating the potential role in enhancing the bystander effect of HSV-TK/GCV. By co-culturing the B16TK cells, which stably expressed TK gene, with wildtype B16 (B16WT) cells, we found that co-treatment of curcumin and GCV synergistically inhibited B16 cell proliferation, but the effect could be eliminated by the gap junction inhibitor AGA. Moreover, curcumin markedly increased apoptosis rate of B16WT cells, suggesting its effect in enhancing the bystander effect of HSV-TK/GCV. In the in-vivo study, we established the xenografted melanoma model in 14 days by injecting mixture of B16TK and B16WT cell in a ratio of 3:7. The result demonstrated that, co-administration of curcumin and GCV significantly inhibited the xenograft growth, as indicated by the smaller size and less weight. The combinational effect was further confirmed as a synergistic effect. In conclusion, the results demonstrated that curcumin could enhance the killing effect and the bystander effect of HSV-TK/GCV in treating melanoma, which might be mediated by improved gap junction. Our data suggested that combination of HSV-TK/GCV with curcumin could be a potential chemosensitization strategy for cancer treatment.
RESUMO
The main mechanistic function of most chemotherapeutic drugs is mediated by inducing mitochondria-dependent apoptosis. Tumor cells usually respond to upregulate autophagy to eliminate impaired mitochondria for survival. Hypothetically, inhibiting autophagy might promote mitochondria-dependent apoptosis, thus enhancing the efficacy of chemotherapeutic therapies. We previously identified N-methylparoxetine (NMP) as an inducer of mitochondrial fragmentation with subsequent apoptosis in non-small cell lung cancer (NSCLC) cells. We discovered that ROS was accumulated in NMP-treated NSCLC cells, followed by c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38) activation. This was reversed by the application of a reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), leading to a reduction in apoptosis. Our data suggested that NMP induced apoptosis in NSCLC cells by activating mitogen-activated protein kinase (MAPK) pathway. We further speculated that the remarkable increase of ROS in NMP-treated NSCLC cells might result from an inhibition of autophagy. Our current data confirmed that NMP blocked autophagy flux at late stage wherein lysosomal acidification was inhibited. Taken together, this study demonstrated that NMP could exert dual apoptotic functions-mitochondria impairment and, concomitantly, autophagy inhibition. NMP-related excessive ROS accumulation induced apoptosis by activating the MAPK pathway in NSCLC cells.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Paroxetina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estrutura Molecular , Paroxetina/análogos & derivados , Paroxetina/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The protein EPHB4 plays a vital role in various tumor types. However, few studies into the function of circ-EPHB4 (hsa_circ_0001730) in tumors have been conducted. This study aimed to investigate the functions of circ-EPHB4 and the underlying mechanism of circ-EPHB4 in regulating hepatocellular carcinoma (HCC). The expression of circ-EPHB4 was found to be downregulated in HCC tumor tissues, whereas circ-EPHB4 overexpression suppressed cell viability, induced apoptosis, and inhibited cell migration and invasion in Huh7 and HepG2 cells. circ-EPHB4 levels were negatively correlated with tumor weight, size, and metastasis foci in nude mouse models, suggesting circ-EPHB4 inhibits tumorigenesis, tumor development, and metastasis. In addition, HIF-1α and PI3K-AKT pathways were markedly affected by circ-EPHB4 overexpression. HIF-1α could potentially be the target of circ-EPHB4. By overexpressing both HIF-1α and circ-EPHB4, the antitumor effect of circ-EPHB4 should be most probably correlated with HIF-1α. In conclusion, circ-EPHB4 is a tumor inhibitor in HCC and functions by inhibiting HIF-1α expression.
Assuntos
Carcinoma Hepatocelular/patologia , Perfilação da Expressão Gênica/métodos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/patologia , RNA/genética , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA CircularRESUMO
ViceninII is a naturally flavonoid glycoside extracted from Dendrobium officinale, a precious Chinese traditional herb, has been proven to be valuable for cancer treatment. Transforming growth factor-ß1 (TGF-ß1), promotes the induction of epithelialâ»mesenchymal transition (EMT), a process involved in the metastasis of cells that leads to enhanced migration and invasion. However, there is no previously evidence that ViceninII has an inhibitory effect on cancer metastasis, specifically on the TGF-ß1-induced EMT process in lung adenocarcinoma cells. In this experiment, we used UV, ESIMS, and NMR to identify the structure of ViceninII.A549 and H1299 cells were treated with TGF-ß1 in the absence and presence of ViceninII, and subsequent migration and invasion were measured by wound-healing and transwell assays. The protein localization and expressions were detected by immunofluorescence and Western blotting. The results indicated that TGF-ß1 induced spindle-shaped changes, increased migration and invasion, and upregulated or downregulated the relative expression of EMT biomarkers. Meanwhile, these alterations were significantly inhibited when co-treated with ViceninII and inhibitors LY294002 and SB431542. In conclusion, ViceninII inhibited TGF-ß1-induced EMT via the deactivation of TGF-ß/Smad and PI3K/Akt/mTOR signaling pathways.This is the first time that the anti-metastatic effects of ViceninII have been demonstrated, and their molecular mechanisms provided.
Assuntos
Apigenina/farmacologia , Dendrobium/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucosídeos/farmacologia , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Apigenina/isolamento & purificação , Benzamidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Dioxóis/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Quimioterapia Combinada , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucosídeos/isolamento & purificação , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Estrutura Molecular , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Despite its low transfer efficiency, suicide gene therapy with HSV-TK is known for its bystander killing effect. The connexin-based gap junction is believed to mediate the bystander effect. Recently, we found that resveratrol, a polyphenol compound, increased the expression of Cx26 and Cx43, which are connexins and important constituents of gap junctions, in murine hepatoma cells. Hypothetically, the resveratrol-induced upregulation of gap junctions may improve the bystander effect that HSV-TK/GCV has on hepatoma cells. Our present investigation revealed that resveratrol could enhance intercellular communication at the gap junctions in CBRH7919 hepatoma cells and thereby enhance the bystander killing effect of GCV on CBRH7919TK cells. However, inhibition of gap junction using its long-term inhibitor alpha-glycyrrhetinic acid had a negative influence on the bystander effect of gene therapy with HSV-TK/GCV. In addition, combined resveratrol and GCV treatment in tumor-bearing mice with CBRH7919TK and CBRH7919WT cells at a ratio of 2:3 resulted in a significant decrease in the volume and weight of the tumor in comparison to GCV or only resveratrol. The present results demonstrate that resveratrol can enhance the bystander effect exerted by the HSV-TK/GCV system by enhancing connexin-mediated gap junctional communication.
Assuntos
Antioxidantes/farmacologia , Ganciclovir/farmacologia , Terapia Genética/métodos , Neoplasias Hepáticas Experimentais/terapia , Resveratrol/farmacologia , Proteínas do Envelope Viral/farmacologia , Animais , Sinergismo Farmacológico , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Timidina Quinase/genética , Timidina Quinase/metabolismo , TransfecçãoRESUMO
Cancer cells are characterized by malignant proliferation and aberrant metabolism and are thereby liable to the depletion of nutrients and accumulation of metabolic waste. To maintain cellular homeostasis, cancer cells are prone to upregulating the canonical autophagy pathway. Here, we identified paroxetine hydrochloride (Paxil) as a late autophagy inhibitor and investigated its killing effect on lung cancer cells and with a xenograft mouse model in vivo. Upregulated LC3-II and p62 expression indicated that Paxil inhibited autophagy. Acid-sensitive dyes (e.g., LysoTracker and AO staining) indicated reduced lysosomal acidity following Paxil treatment; consequently, the maturation of the pH-dependent hydroxylases (e.g., cathepsin B and D) substantially declined. Paxil also induced the fragmentation of mitochondria and further intensified ROS overproduction. Since the autophagy pathway was blocked, ROS rapidly accumulated, which activated JNK and p38 kinase. Such activity promoted the localization of Bax, which led to increased mitochondrial outer membrane permeability. The release of Cytochrome c with the loss of the membrane potential triggered a caspase cascade, ultimately leading to apoptosis. In contrast, the clearance of ROS by its scavenger, NAC, rescued Paxil-induced apoptosis accompanied by reduced p38 and JNK activation. Thus, Paxil blocked the autophagic flux and induced the mitochondria-dependent apoptosis via the ROS-MAPK pathway.
RESUMO
Chemoresistance is a major limiting factor that impairs the outcome of non-small cell lung cancer (NSCLC) chemotherapy. Paclitaxel (Tax) induces protective autophagy in NSCLC cells, leading to the development of drug resistance. We recently identified a new autophagy inhibitor (alpha-hederin) and hypothesized that it may promote the killing effect of Tax on NSCLC cells. We found that alpha-hederin (α-Hed) could block late autophagic flux in NSCLC cells by altering lysosomal pH and inhibiting lysosomal cathepsin D maturation. Combination treatment of α-Hed and Tax synergistically reduced NSCLC cell proliferation and increased NSCLC cell apoptosis compared with treatment with α-Hed or Tax alone. Furthermore, α-Hed plus Tax enhanced the accumulation of intracellular reactive oxygen species (ROS) in NSCLC cells, while the ROS inhibitor N-acetylcysteine reversed the inhibitory effect of the combination treatment. Our findings suggest that α-Hed can increase the killing effect of Tax on NSCLC cells by promoting ROS accumulation, and that combining α-Hed with classical Tax represents a novel strategy for treating NSCLC.
Assuntos
Autofagia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Ácido Oleanólico/análogos & derivados , Paclitaxel/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saponinas/metabolismo , Apoptose/efeitos dos fármacos , Autofagossomos/metabolismo , Catepsinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Ácido Oleanólico/metabolismo , Paclitaxel/química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologiaRESUMO
BACKGROUND Rho-associated coiled-coil containing protein kinases 2 (ROCK2) is one of the best characterized targets for the small GTPase Rho. It has been reported that ROCK2 is critical for cancer cell migration and invasion. The objective of this study was to investigate the association of ROCK2 expression with clinicopathological features and overall survival of breast cancer patients. MATERIAL AND METHODS The expression of ROCK2 in breast cancer and paired adjacent normal tissues was detected and compared by immunohistochemical staining of tissue array. ROCK2 mRNA expression and clinicopathological information was extracted from the TCGA breast cancer dataset. The association of ROCK2 expression with the clinicopathological characteristics of patients with breast cancer was evaluated using univariate and multivariate Cox proportional hazards models. Overall survival was analyzed using the Kaplan-Meier method. RESULTS Immunohistochemistry showed that ROCK2 expression was significantly higher in tumor tissues than in paired adjacent normal tissues [immunoreactivity score (IRS): tumor, 5.25±2.10, n=40 vs. adjacent normal 3.83±1.06, n=40, P<0.01]. The IRS was correlated to breast cancer staging. Similarly, the mRNA level of ROCK2 was correlated to tumor stage. Notably, ROCK2 mRNA expression (hazard ratio [HR] 1.665 and 95% confidence interval [CI] 1.115-2.488, P=0.013) were also associated with overall survival in a multivariate analysis. CONCLUSIONS Upregulation of ROCK2 was associated with the progression of breast cancer. High expression of ROCK2 may predict poor overall survival rates for breast cancer patients.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Progressão da Doença , Quinases Associadas a rho/metabolismo , Mama/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
Aleuritolic acid (AA) is a triterpene that is isolated from the root of Croton crassifolius Geisel. In the present study, the cytotoxic effects of AA on hepatocellular carcinoma cells were evaluated. AA exerted dose- and time-dependent cytotoxicity by inducing mitochondria-dependent apoptosis in the hepatocellular carcinoma cell line, HepG2. Meanwhile, treatment with AA also caused dysregulation of autophagy, as evidenced by enhanced conversion of LC3-I to LC3-II, p62 accumulation, and co-localization of GFP and mCherry-tagged LC3 puncta. Notably, blockage of autophagosome formation by ATG5 knockdown or inhibitors of phosphatidylinositol 3-kinase (3-MA or Ly294002), significantly reversed AA-mediated cytotoxicity. These data indicated that AA retarded the clearance of autophagic cargos, resulting in the production of cytotoxic factors and led to apoptosis in hepatocellular carcinoma cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Ácidos Palmíticos/farmacologia , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
BACKGROUND Adenylyl cyclase 9 (ADCY9) is an enzyme that modulates signal transduction by producing the second messenger, cyclic adenosine monophosphate (cAMP). The aim of the present study was to investigate the association of ADCY9 expression with clinicopathological features and disease-free survival of colon cancer patients. MATERIAL AND METHODS Immunohistochemistry staining with ADCY9 antibody was performed on a tissue microarray. Immunoreactivity scores (IRS) were recorded and applied for association analysis. ADCY9 mRNA expression and clinicopathogical information were also extracted from TCGA colon cancer dataset and analyzed using univariate and multivariate Cox proportional hazards models. RESULTS ADCY9 IRS was significantly higher (P=0.002) in tumor tissues (6.40±1.26, n=200) than in adjacent normal samples (4.13±0.83, n=8). The IRS and mRNA expression of ADCY9 were correlated to colon cancer TNM staging. Longer disease-free survival was observed in patients with lower ADCY9 expression (P=0.001). In the multivariate models, ADCY9 expression level (hazard ratio [HR] 5.495, 95% confidence interval [CI] 1.753-17.227, P=0.003), and distant metastasis (HR 4.329, 95% CI 1.374-13.636, P=0.012) were still associated with disease-free survival. CONCLUSIONS High ADCY9 expression is a poor prognostic factor for disease-free survival in colon cancer.
Assuntos
Adenilil Ciclases/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias do Colo/enzimologia , Adulto , Idoso , Neoplasias do Colo/patologia , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Análise Serial de Tecidos/métodosRESUMO
Senescence-associated secretory phenotype (SASP) factors, such as IL-6 and IL-8, are extremely critical in tissue microenvironment. Senescent human fibroblasts facilitate epithelial-mesenchymal transition (EMT) in premalignant epithelial cells mainly through the secretion of SASP factors. Meanwhile, premalignant human HaCaT Keratinocyte (HaCaT) cells as immortal epithelial cells are susceptible to malignant transformation. Paeonol, an herbal phenolic component found in peonies, exerts anti-aging and anti-tumor efficacies, while the molecular mechanisms of paeonol on EMT in premalignant HaCaT cells induced by SASP factors are unclear. In this study, we first established a senescent human fetal lung fibroblast MRC-5 cell model using hydrogen peroxide evaluated by senescence-associated ß-galactosidase assay. Upon paeonol treatment, intracellular reactive oxygen species levels in aging MRC-5 cells were significantly decreased via regulation of nuclear translocation of Nrf2. Then we curiously studied whether the aging MRC-5 cell-conditioned medium could induce EMT in premalignant HaCaT cells, and the results showed that paeonol significantly reduced the clonogenic, migratory, and invasive capacities of premalignant HaCaT cells potentially induced by IL-6 and IL-8. Moreover, we found that paeonol notably altered pluripotency of EMT-associated markers via the modulation of ERK and TGF-ß1/Smad pathway in premalignant HaCaT cells. These findings suggest that paeonol may be used as an adjuvant therapy for SASP factor-mediated EMT in premalignant lesion.
Assuntos
Acetofenonas/farmacologia , Senescência Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/citologia , Humanos , Queratinócitos/citologiaRESUMO
Connexin 43 (Cx43), a vital gap junction protein in tumor microenvironment (TME), is a novel molecular target for melanoma chemotherapeutics due to its tumor suppressive function. Dioscin, an herbal steroidal saponin, exerts anti-tumor effects while the underlying mechanism is unclear. Using WB, FACS, and immunofluorescence methodologies, we found dioscin significantly activated the transcription and translation of Cx43 via the retinoid acid signaling pathway and simultaneously enhanced the transporting function of Cx43. Through stimulating Cx43, dioscin remarkably suppressed the migratory and invasive capacities of B16 cells, and notably decreased pluripotency markers of cancer stem cells and epithelial-to-mesenchymal transition in B16 cells and animal tumor tissues. Conversely, dioscin improved the secretion of pro-inflammatory cytokines (IL-6, TNFα, and IL-1ß), and the phagocytic capacity of tumor-associated macrophages by increasing M2-to-M1 phenotype transition. More strikingly, even in Cx43 functional deficient B16 and RAW264.7 cells, dioscin still dramatically reversed the aggravated tumor malignancy and reduced macrophage phagocytic activity. Two classical metastasis animal models were utilized in vivo and results showed that dioscin showed significant anti-metastatic effects, which is closely related to the expression of Cx43 either in in situ tumor or metastatic lung nodes. In conclusion, dioscin targets Cx43 to suppress the tumor cell malignancy and activate macrophage sensitivity, thereby targeting melanoma microenvironment.