Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

OBJECTIVE: Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. METHODS: Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. RESULTS: Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. CONCLUSION: Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

OBJECTIVE: Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. METHODS: Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. RESULTS: Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. CONCLUSION: Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Resveratrol inhibits oral squamous cell carcinoma cells proliferation while promoting apoptosis through inhibition of CBX7 protein.

As a natural compound, resveratrol (Res) is confirmed to be promising drug for the treatment of malignant tumors. Therefore, our study aimed to observe the impacts of Res on the proliferation and apoptosis of oral squamous cell carcinoma cells (HSC-3 cells) as well as the mechanism involving chromobox protein homolog 7 (CBX7) signal transduction. HSC-3 cells were treated with Res, Akt agonist (AL3818) and p16 inhibitor (SC79), and transfected with CBX7 mimics and inhibitor plasmids. The CCK-8 assay was used to detect cell proliferation, flow cytometry was performed to assess cell cycle and apoptosis, and cell colonies and histone DNA level were also measured. Western blot analysis was used to determine the expression levels of related proteins. HSC-3 cells showed decreased cell proliferation, colonies, BrdU-counled cells and increased apoptosis, histone DNA level, the activities of caspase-3 and caspase-9 when treated with Res. Western blot analysis revealed elevated Cle-PARP and Cle-caspase 3 expression and reduced t-PARP expression in HSC-3 cells treated with Res compared with control. AL3818 and SC79 could decrease the inhibitory effects of Res on the growth of HSC-3 cells. Furthermore, CBX7 overexpression could also partly reverse the roles of Res in the growth of HSC-3 cells, and Akt and p16 signal transduction. Our results demonstrate that Res suppresses the proliferation, and induces the apoptosis of oral squamous cell carcinoma cells through the inhibition of CBX7/Akt and the activation of p16 cascades.

Inhibitory effects of DDPH on two components of delayed rectifier potassium current in guinea pig ventricular cells.

AIM: To study the effects of 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxy-phenyl-ethylamino) propane hydro-chloride (DDPH) on the rapidly activating component (I(Kr)), and the slowly activating component (I(Ks)) of the delayed rectifier potassium current (I(K)) in guinea pig ventricular myocytes. METHODS: Whole-cell patch clamp recording techniques. RESULTS: DDPH (0.1-100 micromol/L) blocked the I(Kr) in a concentration-dependent manner. The IC50 (micromol/L) was 6.1 (95 % confidence limits: 2.8-13.5). IC50 (micromol/L) of DDPH blocking I(Ks) was 12.5 (95 % confidence limits: 4.8-32.2). DDPH (10 micromol/L) did not affect activation time constants and the voltage-dependent activation of both I(Kr) and I(Ks), the half-activation voltage (V1/2, mV) and slope factor (k, mV) were I(Kr): -23.5+/-2.4 and 8.1+/-2.2 [in presence of DDPH, P >0.05, compared with control, V1/2 (-21.7+/-0.8) and k (5.9+/-0.8)]; I(Ks): 27.1+/-0.7 and 16.6+/-0.8 [in presence of DDPH, P >0.05, compared with control, V1/2 (27.0+/-0.8) and k (14.9+/-0.9)]. DDPH slightly increased the deactivation time-constant of I(Kr) ( r) and I(Ks) ( s) at low concentration (<10 micromol/L). The inactivation of I(Kr) was significantly accelerated by DDPH. CONCLUSIONS: DDPH inhibited both I(Kr) and I(Ks). The blockade was not due to its influence on activation, but the process of deactivation. The blocking of I(Kr) by DDPH was further associated with its acceleration the channel inactivation.

Dauricine-induced changes in monophasic action potentials and effective refractory period of rabbit left ventricle in situ.

AIM: To study the effects of dauricine and sotalol on monophasic action potentials and effective refractory period of the rabbit heart in situ. METHODS: Monophasic action potentials recording and programmed electrical stimulation techniques. RESULTS: Iv injection of dauricine 0.5 mg . kg-1 . min-1 for 24 min produced a decrease of the amplitude of monophasic action potentials from (17+/-6) mV to (7.1 +/- 1.5) mV (P < 0.05) and increased the duration of 50 % and 90 % repolarization of monophasic action potentials and effective refractory period from (130 +/- 26), (167 +/- 25), (128 +/- 12) ms to (198 +/- 33), (235 +/- 34), (185 +/- 25) ms (P < 0.05), respectively. Sotalol had the similar effects on monophasic action potentials and effective refractory period to that of dauricine. Dauricine and sotalol did not change the ratio between effective refractory period and the duration of 90 % repolarization. CONCLUSION: Dauricine and sotalol decreased the amplitude of monophasic action potentials and increased the duration of 50 % and 90 % repolarization and effective refractory period.