RESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most malignant tumors and the fourth leading cause of cancer-related death worldwide. Sorafenib is currently acknowledged as a standard therapy for advanced HCC. However, acquired resistance substantially limits the clinical efficacy of sorafenib. Therefore, further investigations of the associated risk factors are highly warranted. METHODS: We analysed a group of 78 HCC patients who received sorafenib treatment after liver resection surgery. The expression of SCAP and its correlation with sorafenib resistance in HCC clinical samples were determined by immunohistochemical analyses. Overexpression and knockdown approaches in vitro were used to characterize the functional roles of SCAP in regulating sorafenib resistance. The effects of SCAP inhibition in HCC cell lines were analysed in proliferation, apoptosis, and colony formation assays. Autophagic regulation by SCAP was assessed by immunoblotting, immunofluorescence and immunoprecipitation assays. The combinatorial effect of a SCAP inhibitor and sorafenib was tested using nude mice. RESULTS: Hypercholesterolemia was associated with sorafenib resistance in HCC treatment. The degree of sorafenib resistance was correlated with the expression of the cholesterol sensor SCAP and consequent deposition of cholesterol. SCAP is overexpressed in HCC tissues and hepatocellular carcinoma cell lines with sorafenib resistance, while SCAP inhibition could improve sorafenib sensitivity in sorafenib-resistant HCC cells. Furthermore, we found that SCAP-mediated sorafenib resistance was related to decreased autophagy, which was connected to decreased AMPK activity. A clinically significant finding was that lycorine, a specific SCAP inhibitor, could reverse acquired resistance to sorafenib in vitro and in vivo. CONCLUSIONS: SCAP contributes to sorafenib resistance through AMPK-mediated autophagic regulation. The combination of sorafenib and SCAP targeted therapy provides a novel personalized treatment to enhance sensitivity in sorafenib-resistant HCC.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Autofagia , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Colesterol , Resistencia a Antineoplásicos , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Sorafenib/farmacología , Sorafenib/uso terapéuticoRESUMEN
PURPOSE: To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms. MATERIALS AND METHODS: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of ß-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for ß-catenin, HDAC1and HDAC3 was tested by q-PCR. ß-Catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit. RESULTS: Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/ G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was 6.22±0.25%, which increased to 7.17±0.20% and 18.1±0.42% in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of ß-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of ß-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of ß-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased. CONCLUSIONS: TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/ß-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasas/química , Ácidos Hidroxámicos/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismo , Acetilación , Apoptosis/efectos de los fármacos , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , beta Catenina/genéticaRESUMEN
OBJECTIVE: To investigate the effects of the 20(S)-ginsenoside Rh2 [Rh2(S)]on cell proliferation, histone deacetylase 1 (HDAC1) and HDAC2 activity, and expression of cyclin in human erythroleukemia K562 cells. METHODS: The K562 cells were treated with Rh2(S) at various concentrations (10-80 µmol/L). Cell proliferation activity was detected by CCK-8 assay. Flow cytometry (FCM) was used to detect cell cycle and apoptotic changes. The HDAC activity of cells was measured by chemical colorimetry. The protein expressions of HDAC1, HDAC2, cyclin D1, CDK4, p16INK4A and p21 after 48 hour-treatment of Rh2 (S) (10, 20, 40, 60 µmol/L) were examined by Western blotting. RESULTS: The proliferation of K562 cells was inhibited by Rh2 (S) (20-80 µmol/L) in dose-and time-dependent manner. FCM analyses revealed that the number of the K562 cells treated with 60 µmol/L Rh2(S) was arrested in G0/G1 phase. The apoptosis rates of K562 cells were respectively (8.09±0.86)%, (9.44±0.53)% and (22.80±2.16)% after induced by 20, 40, 60 µmol/L Rh2(S), which showed statistically significant difference (P<0.05) compared with the control group (2.63±0.14)%. HDAC activity of the cells treated with Rh2(S) (40, 60 µmol/L) was reduced. Western blotting showed that the expressions of HDAC1, HDAC2, cyclin D1 and CDK4 decreased after induced by Rh2(S), and p16INK4A, p21 proteins were enhanced significantly. CONCLUSION: The Rh2(S) can inhibit the proliferation of K562 cells and induce its cycle arrest and apoptosis through inhibiting HDAC1 and HDAC2 activity, down-regulating the expression of cyclin D1 and activating p16INK4A and p21.
Asunto(s)
Ciclinas/metabolismo , Ginsenósidos/farmacología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/farmacología , Citometría de Flujo , Humanos , Células K562 , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , Factores de TiempoRESUMEN
OBJECTIVE: To investigate the combined effect of evodiamine (EVO) and norcantharidin (NCTD) on HepG2 cells. METHODS: HepG2 cells were treated with EVO or/and NCTD at different concentrations in vitro, with blank culture medium as the negative control. The inhibitory effect of EVO or/and NCTD was determined by MTT assay. Cell cycle and cell apoptosis were assessed by flow cytometry (FCM) combined with PI staining and annexin V-FITC/PI, respectively. The expressions of Bax and Bcl-2 were examined by Western blotting. RESULTS MTT assay showed that NCTD and EVO had restraining effect on the growth of HepG2 cells. The combination of NCTD and EVO had significant synergistic inhibition effect (CI<1). The arrest rate of G2/M phase was (36.13±1.63)% in the EVO induced group and (10.67±0.89)% in the NCTD induced group, and the combination of EVO and NCTD up-regulated the rate to (73.42±1.92)% (P<0.05). In addition, the apoptosis rate of HepG2 cells treated by two-drug combination increased significantly from (15.78 ± 3.08)% in the EVO group and (11.47 ± 1.60)% in the NCTD group to (21.86±2.70)% in the EVO combined with NCTD group (P<0.05). Western blotting demonstrated that the expression level of Bax protein went up significantly (P<0.05), meanwhile Bcl-2 protein dropped significantly (P<0.05) in the combined induced group. CONCLUSION: EVO combined with NCTD showed synergetic effect on anti-proliferation and pro-apoptosis in HepG2 cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proliferación Celular/efectos de los fármacos , Quinazolinas/farmacología , Western Blotting , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Citometría de Flujo , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células Hep G2 , Hepatoblastoma/genética , Hepatoblastoma/metabolismo , Hepatoblastoma/patología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
OBJECTIVE: To investigate the inhibitory effect of trichostatin A (TSA) on the proliferation of HepG2 cells and explore the underlying mechanism. METHODS: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72 h were examined for cell growth inhibition using a cell counting kit, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under inverted microscope. The expressions of beta-catenin, HDAC1, HDAC3, H3K9, cyclinD1 and Bax proteins in the exposed cells were detected by Western blotting, and the expressions of HDAC1 and HDAC3 mRNAs by quantitative fluorescent PCR. RESULTS: Exposure to TSA caused significant dose- and time-dependent inhibition of HepG2 cell proliferation (P<0.05) and resulted in increased cell percentage in G0/G1 and G2/M phases and decreased cell percentage in S phase. The apoptotic index in the control group was (6.22 ± 0.25)%, which increased to (7.17 ± 0.20)% and (18.14 ± 0.42)% after exposure to 250 and 500 nmol/L TSA, respectively. Exposure to 250 and 500 nmol/L TSA also caused cell morphology changes with numerous floating cells. The expressions of beta-catenin, H3K9 and Bax proteins were significantly increased and CyclinD1, HDAC1, and HDAC3 protein expressions decreased in TSA-treated cells, but the expressions of HDAC1 and HDAC3 mRNAs showed no significant changes. CONCLUSIONS: TSA can inhibit the proliferation of HepG2 cells and induce cell cycle arrest and apoptosis by inhibiting HDAC activity, promoting histone acetylation, and activating Wnt/beta-catenin signaling pathway.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Acetilación , Apoptosis , Puntos de Control del Ciclo Celular , Ciclina D1/metabolismo , Células Hep G2/efectos de los fármacos , Histona Desacetilasa 1/metabolismo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Vía de Señalización Wnt , Proteína X Asociada a bcl-2/metabolismo , beta Catenina/metabolismoRESUMEN
In previous experiments, ginsenoside Rh2 induced apoptosis and cell cycle arrest, which indicates a potential role for ginsenoside Rh2 in anticancer treatment. The effect of ginsenoside Rh2 on cancer is marked and ginsenoside Rh2 has been shown to inhibit pancreatic tumor migratory ability. In the present study, Transwell chambers were used in order to investigate whether ginsenoside Rh2 inhibits the migratory ability of HepG2 liver carcinoma cells. Furthermore, to analyze activator protein 1 (AP-1) transcription factor expression following Rh2 treatment, ten plasmids encoding Renilla luciferase coupled to the transcription factors were transiently transfected into the HepG2 cells and luciferase was detected by the Luciferase Reporter Assay system reagent. The results indicated that ginsenoside Rh2 inhibited HepG2 cell migratory ability. The expression levels of AP-1 transcription factors were increased in HepG2 cells following induction by phorbol 12-myristate 13-acetate, but ginsenoside Rh2 suppressed this induced AP1 expression. AP-1 transcription factors recruit histone deacetylase (HDAC)4 and affect its transcription, thus, the expression levels of HDAC4 were also analyzed, and these were found to be increased in the Rh2 treatment group. Matrix metalloproteinase 3 (MMP3), a gene downstream of AP-1, was then investigated, and the treatment group expressed reduced levels of MMP3 gene and protein. Therefore, the inhibitory effect of ginsenoside Rh2 on the migratory ability of HepG2 may be presumed to occur by the recruitment of HDAC and the resulting inhibition of AP1 transcription factors, in order to reduce the expression levels of MMP3 gene and protein.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Ginsenósidos/toxicidad , Factor de Transcripción AP-1/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Células Hep G2 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/genéticaRESUMEN
OBJECTIVE: To investigate the effect of ginsenoside Rh2 on leukemia KG1-α cells. METHODS: KG1-α cells were cultured in 96-well plate and harvested in the exponential phase. The cells were induced in the presence of different concentrations of Rg1, Rb1, Rh2 respectively for 24, 48, 72 hours. The conventional culture was performed in blank control group, and cytarabine hydrochloride as the positive control. The inhibitory effects of ginsenoside Rb1, Rg1, Rh2 on the growth of KG1-α cells were tested by CCK-8 assay. Then the most efficient ginsenoside was chosen for the further study by IC50. The effects of the ginsenoside Rh2 on cell cycle and apoptosis were detected by flow cytometry (FCM) combined with PI staining and annexin V-FITC/PI, respectively. The expressions of P53, P21, cyclin D1 and cleaved-caspase-3 were examined by Western blotting. RESULTS: CCK-8 assay results showed that IC50 of ginsenoside Rh2, ginsenoside Rb1, ginsenoside Rg1 and cytarabine were 75, 207, 268, 1058 µmol/L, respectively. Compared with those in blank control group, the KG1-α cells after treated with 75 µmol/L ginsenoside Rh2 showed up-regulated apoptosis rates from (5.37±0.02)% to (8.37±0.015)% at 24 hours and to (33.22±1.67)% at 48 hours (P<0.05). In addition, the percentage of cells in G0/G1 phase increased significantly from (26.78±3.14)% to (29.26±2.31)% at 24 hours and to (44.77±2.26)% at 48 hours, and the percentage in S phase decreased significantly from (65.43±2.22)% to (51.46±0.57)% and (48.29±1.80)%, respectively. The expression levels of cleaved-caspase 3, P53 and P21 proteins went up significantly when the cells were treated with 75 µmol/L Rh2(P<0.05), meanwhile cyclin D1 protein dropped significantly (P<0.05). CONCLUSION: Ginsenoside Rh2 could inhibit the proliferation of KG1-α cell and prompt its apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Ginsenósidos/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Leucemia/metabolismo , Leucemia/patología , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Ginsenoside Rg1 is one effective anticancer and antioxidant constituent of total saponins of Panax ginseng (TSPG), which has been shown to have various pharmacological effects. Our previous study demonstrated that Rg1 had anti-tumor activity in K562 leukemia cells. The aim of this study was designed to investigate whether Rg1 could induce apoptosis in TF-1/Epo cells and further to explore the underlying molecular mechanisms. Here we found that Rg1 could inhibit TF-1/Epo cell proliferation and induce cell apoptosis in vitro in a concentration and time dependent manner. It also suppressed the expression of EpoR on the surface membrane and inhibited JAK2/STAT5 pathway activity. Rg1 induced up-regulation of Bax, cleaved caspase-3 and C-PAPR protein and down-regulation of Bcl-2 and AG490, a JAK2 specific inhibitor, could enhance the effects of Rg1. Our studies showed that EpoR-mediated JAK2/STAT5 signaling played a key role in Rg1-induced apoptosis in TF-1/Epo cells. These results may provide new insights of Rg1 protective roles in the prevention a nd treatment of leukemia.
Asunto(s)
Apoptosis/efectos de los fármacos , Ginsenósidos/farmacología , Janus Quinasa 2/metabolismo , Leucemia/patología , Receptores de Eritropoyetina/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Fármacos del Sistema Nervioso Central/farmacología , Eritropoyetina/metabolismo , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Eritropoyetina/antagonistas & inhibidores , Receptores de Eritropoyetina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/genética , Células Tumorales CultivadasRESUMEN
AIMS AND BACKGROUND: Ginsenoside Rh2, which exerts the potent anticancer action both in vitro and in vivo, is one of the most well characterized ginsenosides extracted from ginseng. Although its effects on cancer are significant, the underlying mechanisms remain unknown. In this study, we sought to elucidate possible links between ginsenoside Rh2 and phosphoglucose isomerase/autocrine motility factor (PGI/AMF). METHODS: KG1α, a leukemia cell line highly expressing PGI/AMF was assessed by western blot analysis and reverse transcription- PCR (RT-PCR) assay after transfection of a small interfering (si)-RNA to silence PGI/AMF. The effect of PGI/ AMF on proliferation was measured by typan blue assay and antibody array. A cell counting kit (CCK)-8 and flow cytometry (FCM) were adopted to investigate the effects of Rh2 on PGI/AMF. The relationships between PGI/AMF and Rh2 associated with Akt, mTOR, Raptor, Rag were detected by western blot analysis. RESULTS: KG1α cells expressed PGI/AMF and its down-regulation significantly inhibited proliferation. The antibody array indicated that the probable mechanism was reduced expression of PARP, State1, SAPK/JNK and Erk1/2, while those of PRAS40 and p38 were up-regulated. Silencing of PGI/AMF enhanced the sensibility of KG1α to Rh2 by suppressing the expression of mTOR, Raptor and Akt. CONCLUSION: These results suggested that ginsenoside Rh2 suppressed the proliferation of KG1α, the same as down-regulation of PGI/AMF. Down-regulation of PGI/ AMF enhanced the pharmacological effects of ginsenoside Rh2 on KG1α by reducing Akt/mTOR signaling.