RESUMEN
Background and Aim: Bromodomain and extraterminal domain (BET) family proteins are epigenetic regulators involved in human malignances. Targeting BET proteins for degradation using proteolysis-targeting chimera (PROTAC) recently has drawn increasing attention in the field of cancer therapeutics. BET proteins have been found to be overexpressed in HCC cells and tumor tissues. However, the biological activity of BET-PROTACs in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated anti-HCC activity of BETd-260, a BET-PROTAC molecule using in vitro and in vivo models. Methods: BETd-260-mediated anti-HCC activity was investigated by cell viability, apoptosis assays. Efficacy was examined with a cell lines-derived HCC xenograft model in mice. Anticancer mechanism was investigated by RT-PCR, western blotting and immunohistochemical staining. Results: BETd-260 potently suppressed cell viability and robustly induced apoptosis in HCC cells. BETd-260 reciprocally modulated the expression of several apoptotic genes in HCC cells, i.e., suppressing the expression of anti-apoptotic Mcl-1, Bcl-2, c-Myc, and X-linked inhibitor of apoptosis (XIAP), whereas increasing the expression of pro-apoptotic Bad. BETd-260 treatment led to disruption of mitochondrial membrane integrity, and triggered apoptosis via intrinsic signaling in HCC cells. BETd-260 triggered apoptosis in HCC xenograft tissue and profoundly inhibited the growth of HCC xenograft tumors in mice. Conclusion: Our data suggest that pharmacological targeting of BET for degradation may be a novel therapeutic strategy for the treatment of HCC.
RESUMEN
Myeloid cell leukemia-1 (Mcl-1) is highly expressed in tumor tissues and cells of hepatocellular carcinoma (HCC), yet the role of Mcl-1 in cancer stem-like cells (CSLCs) remains largely unclear. Herein, we showed that knockdown of Mcl-1 significantly inhibited HCC cells to form spheres under ultra-low attachment condition in serum-free medium, and also attenuated clone formation. Inhibition of Mcl-1 by specific inhibitors S63845 or A-1210477 hindered secondary sphere formation, triggered apoptosis signaling and reduced the level of stem cell transcription factor Nanog, Sox2 and KLF4 in HCC spheroids cells. This study suggests that Mcl-1 is an essential factor for the survival and self-renewal of HCC CSLCs.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Hepatocelular/patología , Autorrenovación de las Células/efectos de los fármacos , Neoplasias Hepáticas/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Células Madre Neoplásicas/efectos de los fármacos , Apoptosis , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Indoles/farmacología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/efectos de los fármacos , Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína Homeótica Nanog/efectos de los fármacos , Proteína Homeótica Nanog/metabolismo , Pirimidinas/farmacología , Factores de Transcripción SOXB1/efectos de los fármacos , Factores de Transcripción SOXB1/metabolismo , Sulfonamidas/farmacología , Tiofenos/farmacologíaRESUMEN
Bromodomains and extra-terminal (BET) proteins inhibitors are promising cancer therapeutic agents. However, tumor cells often develop resistance to BET inhibitors, greatly limiting their therapeutic potential. To study the mechanism underlying the resistance of BET inhibitors in hepatocellular carcinoma (HCC) cells, we herein investigated the impact of BET inhibitor JQ1 on the gene expression of Bcl-2 family members by RNA sequencing analysis, and found that acute treatment with JQ1 triggered upregulation of Mcl-1 in HCCLM3 and BEL7402â¯cell lines. This JQ1-triggered Mcl-1 upregulation was further confirmed by quantitative reverse transcription polymerase chain reaction and western blotting analysis, both at mRNA and protein levels. Inhibition of Mcl-1 by RNA interference dramatically enhanced JQ1-triggered caspase-3 activation, cleavage of poly (ADP-ribose) polymerase and apoptotic cell death induction in multiple HCC cell lines. Moreover, JQ1 in combination with cyclin-dependent kinase inhibitor flavopiridol at a subtoxic concentration that reduced expression of Mcl-1, triggered massive apoptotic cell death in HCCLM3 and BEL7402â¯cell lines. Together, these data suggest that Mcl-1 is a major contributor to BET inhibitor-resistance in HCC cells, and that combining drugs capable of down-regulating Mcl-1 may promote therapeutic potential in human HCC.
Asunto(s)
Apoptosis/efectos de los fármacos , Azepinas/administración & dosificación , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Triazoles/administración & dosificación , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Células Hep G2 , Humanos , Proteínas/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hepatocellular carcinoma (HCC) ranks the sixth most common cancer and the third cause of cancer-related mortality worldwide. Recent studies identified that circ-ITCH Suppresses mutiple cancers proliferation via inhibiting the Wnt/beta-Catenin pathway. In current study, conducted a genetic association study together with epidemiological follow-up study to delineate the role of circ-ITCH in the development and progression of HCC. we found rs10485505 (adjusted OR =1.18; 95% CI=1.06-1.31; P value =3.1×10-3) and rs4911154 (adjusted OR =1.27; 95% CI=1.14-1.43; P value =3.7×10-5) were significantly associated with increased HCC risk. The expression level of circ-ITCH was significantly lower in HCC tissues, compared with that in adjacent tissues (P value < 0.001). Cox regression analysis indicated that high expression of circ-ITCH was associated with favorable survival of HCC (HR=0.45; 95% CI=0.29-0.68; P value < 0.001). These results indicate that circ-ITCH may have an inhibitory effect on HCC, and could serve as susceptibility and prognostic biomarkers for HCC patients.
Asunto(s)
Biomarcadores de Tumor , Carcinogénesis/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Polimorfismo Genético , ARN , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas/genética , Adulto , Anciano , Alelos , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Biología Computacional/métodos , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo de Nucleótido Simple , ARN Circular , Proteínas Represoras/sangre , Factores de Riesgo , Ubiquitina-Proteína Ligasas/sangreRESUMEN
Bromodomain and Extra-Terminal Domain (BET) inhibitors, such as JQ1 have emerged as novel drug candidates and are being enthusiastically pursued in clinical trials for the treatment of cancer. However, many solid cancers are resistance to BET inhibitors. To explore methods for improving the therapeutic potential of BET inhibitors, we investigated the combinational activity of JQ1 with Oridonin, a bioactive molecules derived from Traditional Chinese Medicine in hepatocellular carcinoma (HCC) cells. Our results showed that Oridonin synergistically enhanced the abilities of JQ1 to inhibit cell viability in HCC cells and, significantly augmented JQ1-triggered apoptosis in HCC cells and in HCC cancer stem-like cells. Moreover, Oridonin dose-dependently inhibited the expression of several anti-apoptotic proteins, such as Bcl-2, Mcl-1, and x-linked inhibitor of apoptosis (xIAP) in HCC cells. Cell fractionation and western blotting analysis showed that the enhancement of apoptosis by Oridonin was associated with cytochrome c release, activation of caspase-9, -3 and cleavage of PARP, indicating the activation of mitochondrial apoptosis pathway. Altogether, our findings demonstrate that Oridonin may be used to effectively enhance the sensitivity of BET inhibitors in HCC therapy via downregulation of the expression of multiple anti-apoptotic proteins.
RESUMEN
Neddylation is a post-translational protein modification process associated with carcinogenesis and cancer development. MLN4924, a pharmaceutical neddylation inhibitor, induces potent anti-cancer effects in multiple types of cancers. In this study, we investigated the effects of MLN4924 on human osteosarcoma (OS). Levels of both NEDD8 activating enzyme E1 (NAE1) and ubiquitin-conjugating enzyme E2M (Ube2M), two critical components of the neddylation pathway, were much higher in OS tissues and cells than in normal osseous tissues and cells. MLN4924 treatment led to DNA damage, reduced cell viability, senescence and apoptosis in OS cells. Moreover, MLN4924 inhibited OS xenograft tumor growth in mice. Mechanistically, MLN4924 blocked the neddylation of cullins and induced accumulation of several tumor-suppressive substrates of Cullin-RING E3 ubiquitin ligases (CRLs), including CDT1, Wee1, p21, p27, Noxa, and p16. These results suggest clinical studies investigating the utility of MLN4924 for the treatment of OS are warranted.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Óseas/tratamiento farmacológico , Ciclopentanos/farmacología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Osteosarcoma/tratamiento farmacológico , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Pirimidinas/farmacología , Animales , Neoplasias Óseas/patología , Senescencia Celular/efectos de los fármacos , Proteínas Cullin/metabolismo , Daño del ADN , Humanos , Ratones , Osteosarcoma/patología , Enzimas Activadoras de Ubiquitina/análisis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bromodomain 4 (BRD4) is an epigenetic regulator that, when inhibited, has anti-cancer effects. In this study, we investigated whether BRD4 could be a target for treatment of human hepatocellular carcinoma (HCC). We show that BRD4 is over-expressed in HCC tissues. Suppression of BRD4, either by siRNA or using JQ1, a pharmaceutical BRD4 inhibitor, reduced cell growth and induced apoptosis in HCC cell lines while also slowing HCC xenograft tumor growth in mice. JQ1 treatment induced G1 cell cycle arrest by repressing MYC expression, which led to the up-regulation of CDKN1B (P27). JQ1 also de-repressed expression of the pro-apoptotic BCL2L11 (BIM). Moreover, siRNA knockdown of BIM attenuated JQ1-triggered apoptosis in HCC cells, suggesting an essential role for BIM in mediating JQ1 anti-HCC activity.
Asunto(s)
Azepinas/farmacología , Proteína 11 Similar a Bcl2/metabolismo , Carcinoma Hepatocelular/prevención & control , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas/prevención & control , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2/genética , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oridonin is an orally available drug isolated from Traditional Chinese Medicine. Previous studies with oridonin have demonstrated broad-spectrum anticancer activity in a variety of cancer types. However, the effect of oridonin in uveal melanoma has not been addressed. In this study, we aimed to investigate whether oridonin elicited anticancer activity and its underlying mechanism in human uveal melanoma cells. We demonstrated that oridonin potently reduced cell viability, induced apoptosis and inhibited clonogenic survival and growth with single digit micromolar concentrations in uveal melanoma OCM-1 and MUM2B cell lines. We found that oridonin markedly increased the expression of proapoptotic Bcl-2 family protein Bim in uveal melanoma cells, and knockdown Bim by small interfering RNA significantly attenuated oridonin-induced cell death, indicating an essential role of Bim in oridonin-mediated anticancer activity. Additionally, we observed that oridonin suppressed Fatty Acid Synthase (FAS) expression in uveal melanoma cells, and enforced FAS expression by insulin partially rescued the cells from oridonin-induced apoptosis, showing that inhibition of FAS also contributed to oridonin-mediated apoptosis. Taken together, we reported that oridonin displays potent anticancer effect against uveal melanoma cells through upregulation of Bim and inhibition of FAS. Since oridonin is a popular anticancer agent, our study therefore may have translational implication on the management of patients with uveal melanoma.
Asunto(s)
Apoptosis/efectos de los fármacos , Diterpenos de Tipo Kaurano/farmacología , Regulación hacia Abajo/efectos de los fármacos , Ácido Graso Sintasas/metabolismo , Melanoma/enzimología , Melanoma/patología , Regulación hacia Arriba/efectos de los fármacos , Neoplasias de la Úvea/enzimología , Neoplasias de la Úvea/patología , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Proteína 11 Similar a Bcl2 , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Proteínas de la Membrana , Proteínas Proto-Oncogénicas , Ensayo de Tumor de Célula MadreRESUMEN
Neuroblastoma is an aggressive childhood disease. Even with intensive conventional treatments, the long term survival rate for children with neuroblastoma remains less than 40%, highlighting the importance of finding new therapies. Bcl-2 family proteins play crucial roles in survival, proliferation and chemotherapeutic resistance of neuroblastoma cells. Therefore, targeting Bcl-2 with small molecule inhibitor ABT-263 could be a novel strategy for treatment of neuroblastoma. However, previous studies indicated that most neuroblastoma cell lines are resistant to ABT-263-mediated apoptosis. Thus, it is crucial to discover approaches that could overcome ABT-263 resistance. In this study, we examined the anticancer activity of ABT-263 in combination with norcantharidin (NCTD), a small-molecule anticancer drug derived from a traditional Chinese medicine, in human malignant neuroblastoma cells. We found that NCTD substantially enhanced ABT-263-mediated apoptosis induction, cell viability inhibition, and clonal formation inhibition in neuroblastoma SH-SY5Y and CHLA-119 cell lines. Moreover, the combination anticancer activity was accompanied by upregulation of Noxa, and was associated with characteristics of mitochondrial apoptosis signaling, such as cytosolic release of cytochrome c, activation of caspase-9,-3, and cleavage of PARP. Notably, we observed that knockdown of Noxa significantly attenuated cell death induction by cotreatment with ABT-263 and NCTD, indicating Noxa essentially contributes to the combination anticancer effect. Collectively, our study demonstrated that NCTD could overcome ABT-263-resistance in neuroblastoma cells, and suggested that combinational treatment of ABT-263 with NCTD might be a novel therapeutic option for children with neuroblastoma.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Neuroblastoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Mitocondrias/efectos de los fármacos , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Small-molecule Bcl-2/Bcl-xL inhibitor Navitoclax represents a promising cancer therapeutic since preclinical and clinical studies with Navitoclax have demonstrated strong anticancer activity in several types of cancers. However, because Navitoclax has a low binding affinity to Mcl-1, anticancer activity by Navitoclax is often attenuated by the elevated expression of Mcl-1 in hepatocellular carcinoma (HCC) and other cancers, posing a serious problem for its potential clinical utilities. Therefore, approaches that suppress the expression of Mcl-1 are urgently needed to overcome Navitoclax-resistance in these cancers. Here, we reported that aspirin markedly suppressed Mcl-1 expression, and significantly enhanced Navitoclax-mediated cell viability inhibition and apoptosis induction in HCC cells. We further showed that aspirin robustly enhanced Navitoclax-triggered cytosolic cytochrome c release, activation of initiator caspase-9 and effector caspase-3, and cleavage of PARP. Importantly, the cell death induction by the combination could be rescued by a cell-permeable caspase-9 inhibitor Z-LEHD-FMK, indicative of an indispensable role of mitochondrial apoptosis pathway during the combination effect. Taken together, our study suggests that aspirin can be used to enhance Navitoclax-mediated anticancer activity via suppression of Mcl-1. Since aspirin is one of the most commonly used medicines, our findings therefore have translational impacts on Navitoclax-based therapy for HCC.
Asunto(s)
Compuestos de Anilina/farmacología , Aspirina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/farmacología , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Oligopéptidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARNRESUMEN
BACKGROUND: The members of inhibitor of apoptosis proteins (IAPs) family are key negative regulators of apoptosis. Overexpression of IAPs are found in hepatocellular carcinoma (HCC), and can contribute to chemotherapy resistance and recurrence of HCC. Small-molecule Second mitochondria-derived activator of caspases (Smac) mimetics have recently emerged as novel anticancer drugs through targeting IAPs. The specific aims of this study were to 1) examine the anticancer activity of Smac mimetics as a single agent and in combination with chemotherapy in HCC cells, and 2) investigate the mechanism of anticancer action of Smac mimetics. METHODS: Four HCC cell lines, including SMMC-7721, BEL-7402, HepG2 and Hep3B, and 12 primary HCC cells were used in this study. Smac mimetic SM-164 was used to treat HCC cells. Cell viability, cell death induction and clonal formation assays were used to evaluate the anticancer activity. Western blotting analysis and a pancaspase inhibitor were used to investigate the mechanisms. RESULTS: Although SM-164 induced complete cIAP-1 degradation, it displayed weak inhibitory effects on the viability of HCC cells. Nevertheless, SM-164 considerably potentiated Apo2 ligand or TNF-related apoptosis-inducing ligand (APO2L/TRAIL)- and Doxorubicin-mediated anticancer activity in HCC cells. Mechanistic studies demonstrated that SM-164 in combination with chemotherapeutic agents resulted in enhanced activation of caspases-9, -3 and cleavage of poly ADP-ribose polymerase (PARP), and also led to decreased AKT activation. CONCLUSIONS: Smac mimetics can enhance chemotherapeutic-mediated anticancer activity by enhancing apoptosis signaling and suppressing survival signaling in HCC cells. This study suggests Smac mimetics are potential therapeutic agents for HCC.
Asunto(s)
Antineoplásicos/administración & dosificación , Biomimética , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular , Proteínas Mitocondriales , Triazoles/administración & dosificación , Desaminasas APOBEC , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citidina Desaminasa/metabolismo , Doxorrubicina/administración & dosificación , Humanos , Péptidos y Proteínas de Señalización Intracelular/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular/síntesis química , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Proteínas Mitocondriales/administración & dosificación , Proteínas Mitocondriales/síntesis química , Proteínas Musculares/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
Small-molecule cell-permeable Bcl-2/Bcl-xL antagonist ABT-737 has recently emerged as a novel cancer therapeutic agent because it potently induces apoptosis in certain cancer cells. However, since ABT-737 binds to Mcl-1 with low affinity, ABT-737-mediated apoptosis signaling is inhibited in hepatocellular carcinoma (HCC) cells and other solid cancer cells due to the elevated expression of Mcl-1. Accordingly, strategies that target Mcl-1 are explored for overcoming ABT-737-resistance. In this study, we reported that Norcantharidin (NCTD), a small-molecule anticancer drug derived from Chinese traditional medicine blister beetle (Mylabris), induced transcriptional repression of Mcl-1 and considerably enhanced ABT-737-triggered cell viability inhibition and apoptosis in multiple HCC cell lines. Moreover, we observed that the enhancement of ABT-737-mediated apoptosis by NCTD was associated with activation of mitochondrial apoptosis signaling pathway, which involved cytosolic release of cytochrome c, cleavage of caspase-9 and caspase-3. Additionally, knockdown of Bax/Bak, the key effectors permeabilizing mitochondrial outer membrane significantly attenuated the enhancement, indicating mitochondrial apoptosis pathway played an essential role in the execution of the apoptosis. Finally, knockdown of Mcl-1 substantially potentiated ABT-737-mediated apoptotic cell death, confirming the potency of Mcl-1 repression by NCTD in enhancing ABT-737-induced apoptosis. These results therefore suggest that combination treatment with NCTD can overcome ABT-737 resistance and enhance ABT-737 therapeutic efficacy in treating human HCC.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Nitrofenoles/farmacología , Sulfonamidas/farmacología , Antineoplásicos/química , Compuestos de Bifenilo/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neoplasias Hepáticas/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Nitrofenoles/química , Piperazinas/química , Piperazinas/farmacología , Sulfonamidas/química , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
OBJECTIVE: To elucidate the molecular mechanism of norcantharidin (NCTD) in inducing apoptosis of liver cancer cells so as to provide basic rationales for its application in liver cancer treatment. METHODS: Liver cancer cell lines of SMMC-7721 and BEL-7402 were treated with NCTD. The cell growth inhibition was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cell death detected by trypan blue exclusion assay and apoptosis examined by Annexin V/PI staining and flow cytometry. The cleavage of caspase-9, -3 and PARP, and the expression of the anti-apoptotic Bcl-2 proteins were analyzed by Western blot. RESULTS: The MTT results showed that, after a treatment with NCTD for 24, 48 and 72 h, the IC50 of NCTD in SMMC-7721 cell line was 12, 6 and 1.6 µg/ml respectively; in BEL-7402, the IC50 10, 4 and 2 µg/ml respectively. Trypan blue exclusion assay showed that NCTD mediated substantial cell death in two cancer cell lines. Apoptosis assay showed that, after a 12 h treatment with 10 µg/ml NCTD, 27% of SMMC-7721 cells were induced to undergo apoptosis, an increment of 20% over the untreated control cells (7%); 30% of BEL-7402 cells became apoptotic, an increment of 22% over the untreated control cells (8%). Western blot analysis showed that NCTD treatment potently induced the activation of caspase-9, -3 and the cleavage of PARP, and markedly down-regulated the expression of Bcl-2, Bcl-X(L) and Mcl-1. CONCLUSION: NCTD strongly inhibits liver cancer cell growth and potently induces apoptotic cell death in two liver cancer cell lines. The strong anticancer activity of NCTD may be induced through targeting multiple Bcl-2 anti-apoptotic family members.