Cepharanthine hydrochloride reverses P­glycoprotein-mediated multidrug resistance in human ovarian carcinoma A2780/Taxol cells by inhibiting the PI3K/Akt signaling pathway.

Ovarian cancer has the highest mortality rate among gynecologic malignant tumors. The major obstacle to treatment success is multidrug resistance (MDR) to chemotherapy drugs. Cepharanthine hydrochloride (CH), a natural alkaloid-derived compound, has shown MDR reversal potency in several tumor cell lines; however, the molecular mechanism is not entirely known. In the present study, we assessed whether CH sensitized malignant cells to chemotherapy drugs in ovarian cancer and explored the relevant mechanism. We found that CH reduced the IC50 value of paclitaxel and increased intracellular rhodamine-123 accumulation in human ovarian cancer A2780/Taxol cells in a concentration-dependent manner. Reverse transcription polymerase chain reaction and western blot assay demonstrated that CH inhibited MDR1 expression as indicated by reduced mRNA and protein levels in A2780/Taxol cells. In addition, the inhibitory effect was strengthened after CH was combined with the specific PI3K/Akt signaling pathway inhibitor LY294002. Furthermore, p­Akt expression decreased gradually with the concentration of CH (2, 4 and 8 µM). Taken together, these findings indicated that CH reversed P­glycoprotein-mediated MDR in A2780/Taxol cells by inhibiting the PI3K/Akt signaling pathway.

Dexmedetomidine improves early postoperative cognitive dysfunction in aged mice.

Postoperative cognitive dysfunction (POCD) is a frequent complication following major surgery in the elderly. However, the exact pathogenic mechanisms are still unknown. Dexmedetomidine, a selective alpha 2 adrenal receptor agonist, was revealed anesthesia and brain protective role. The present study aimed to examine whether dexmedetomdine protects against POCD induced by major surgical trauma under general anesthesia in aged mice. In the present study, cognitive function was assessed by Y-maze. Proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF-α), apoptosis-related factor caspase-3 and Bax were detected by real-time PCR, Western blot or immunohistochemistry. The results showed that anesthesia alone caused weak cognitive dysfunction on the first day after general anesthesia. Cognitive function in mice with splenectomy under general anesthesia was significantly exacerbated at the first and third days after surgery, and was significantly improved by dexmedetomidine administration. Splenectomy increased the expression of IL-1ß, TNF-α, Bax and caspase-3 in hippocampus. These changes were significantly inversed by dexmedetomidine. These results suggest that hippocampal inflammatory response and neuronal apoptosis may contribute to POCD, and selective alpha 2 adrenal receptor excitation play a protective role.

FNC, a novel nucleoside analogue, blocks invasion of aggressive non-Hodgkin lymphoma cell lines via inhibition of the Wnt/ß-catenin signaling pathway.

Chemotherapy is the primary therapy for malignant lymphoma (ML). However, the clinical outcome is still far from satisfactory. Consequently, an understanding of the mechanism of modulating cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. FNC, 2'- deoxy- 2' -ß- fluoro -4'- azidocytidine, a novel cytidine analogue, has demonstrated significantly inhibitory effects on proliferation of several non-Hodgkin lymphoma (NHL) cell lines. A previous study indicated that FNC effectively inhibited the growth of Raji and JeKo-1 cells in dose-time dependent effects with IC50 values of 0.2µM and 0.097µM, respectively. This study was focused on investigating the anti-invasive properties of FNC on NHL cells and its potential mechanisms of action. Cell adhesion and transwell chamber assays were utilized to investigate the anti-invasive effects of FNC on Raji and JeKo-1 cells. Real-time PCR and Western blotting were employed to qualify the expression of ß-catenin, the glycogen synthase kinase-3 beta (GSK-3ß), E-cadherin vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The results revealed that FNC remarkably inhibited the adhesion, migration and invasion of two human aggressive non-Hodgkin lymphoma cell lines in a dose dependent manner. Furthermore, ß-catenin, MMP-2, MMP-9, VEGF mRNA and protein levels were decreased after FNC treatment, while GSK-3ß and E-cadherin increased. Our studies thus provide evidence and a rationale that FNC may offer an effective chemotherapeutic agent by regulating the invasion and metastasis of aggressive non-Hodgkin lymphoma via inhibition of the Wnt/ß-catenin signaling pathway.

Anti-hepatitis B virus activity of α-DDB-DU, a novel nucleoside analogue, in vitro and in vivo.

α-DDB-DU, 2'-deoxy-3'-(4,4'-dimethoxy-2'-methoxycarbonyl-5,6,5',6'-bis(methylenedioxy)-1,1'-biphenyl-2-carboxyl)uridine, is a novel nucleoside analogue accomplished by linking α-DDB (α-dimenthoxy dicarboxylate biphenyl) and DU (2'-deoxyuridine) via an ester bond. In the current study, the anti-HBV activity and hepatoprotective effect of this compound were investigated both in vitro and in vivo. In the human HBV-transfected liver cell line HepG2.2.15, α-DDB-DU effectively suppressed the secretion of the HBV antigens in a dose-dependent manner, with inhibition rate of 42.31% for HBsAg and 31.52% for HBeAg at 5 µM on day 9. In addition, it could inhibit the viral DNA replication effectively at the concentration of 5 µM, with 81.18% intracellular inhibition and 88.55% extracellular inhibition, respectively, on day 9. In the duck hepatitis B virus (DHBV) infected model, DHBV DNA levels were markedly reduced after treatment with the α-DDB-DU at the dosages of 0.8 mg/kg day, 4 mg/kg day and 20 mg/kg day. The inhibition rate of α-DDB-DU at the dose of 20 mg/kg day reached 93.75% and 89.43%, in duck serum and liver, respectively, on day 10. Furthermore, the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in both serum and livers were notably reduced on day 10 and histopathological evaluation of the animals' livers indicated significant improvement. In conclusion, α-DDB-DU possesses significant inhibitory activity against HBV replication and ameliorates hepatic pathology significantly.

[Relationship between methylation status of promoter and expression of XAF1 gene in papillary thyroid carcinoma].

OBJECTIVE: To explore the relationship between methylation status of XAF1 (X-linked inhibitor of apoptosis protein associated factor-1) gene promoter and its protein expression in papillary thyroid carcinoma (PTC). METHODS: Methylation-specific polymerase chain reaction (MSP) and immunohistochemical substance P (SP) technique were used to detect the methylation status of XAF1 gene promoter and its protein expression in 70 PTC cases and their matched adjacent non-cancerous epithelium (NCE). RESULTS: In NCE, there was no promoter methylation of XAF1 gene while the rate was 35.7% (25/70) in PTC (χ(2) = 27.206, P < 0.01). And it was correlated with tumor TNM stage, pathological grade and lymph node metastasis (P < 0.05). The positive rates of XAF1 protein expression in NCE and PTC were 100% (70/70) and 55.7% (39/70) respectively. And there was significant difference (χ(2) = 36.458, P < 0.01). In PTC, the positive rates of XAF1 protein expression in Grades I and II were 67.5% (27/40) and 40.0% (12/30) respectively. And they were 35.7% (10/28) and 69.0% (29/42) in the lymph node metastasis and non-metastasis groups respectively. And there were significant differences between two groups (P < 0.05). Futhermore, there was distinct correlation between methylation of XAF1 gene promoter and its protein expression (χ(2) = 8.864, P < 0.01). CONCLUSION: Methylation of promoter may be one of the important inactivating factors of XAF1 gene. And it plays an important role in the carcinogenesis and progression of PTC.

[Correlation between reversing effect of cepharanthine hydrochloride on multidrug resistance and P-glycoprotein expression and function of K562/ADR cells].

In this study, cepharanthine hydrochloride (CH) was tested for its potential ability to modulate the expression and function of P-glycoprotein (P-gp) in the multidrug-resistant human chronic myelogenous leukemia cell line K562/ADR. Cytotoxicity of adriamycin (ADR) alone or in combination with CH or verapamil (VER) in K562 and K562/ADR cells was determined by MTT assay. Based on flow cytometric technology, the effect of CH or VER on the uptake and efflux of rhodamine123 (Rho123) and the accumulation of ADR in these cells was detected by measuring Rho123 or ADR-associated mean fluorescence intensity (MFI). The effects of CH and VER on P-glycoprotein (P-gp) expression in K562 and K562/ADR cells were also measured using a flow cytometry with PE-conjugated P-glycoprotein antibody. The results show that CH significantly enhanced the sensitivity of K562/ADR cells to ADR, 4 micromol x L(-1) of CH enhanced the sensitivity of K562/ADR cells to ADR by 7.43 folds, the reversal activity was 3.19 times higher than that of verapamil. However, CH had no effect on drug-sensitive K562 cells (P < 0.05). CH increased Rho123 and ADR accumulation in a concentration-dependent manner (2-8 micromol x L(-1)) and inhibited the efflux of Rho123 from these cells, but did not affect the accumulation and efflux of Rho123 from the wild-type drug-sensitive K562 cells. The inhibition effect of CH on P-gp expression in K562/ADR cells is in a time- and concentration-dependent manner. The reversal activity of CH is possibility related to inhibition of P-gp function and expression, which lead to an increased intracellular accumulation of anticancer drugs.

Increased expression and function of P-glycoprotein in peripheral blood CD56+ cells is associated with the chemoresistance of non-small-cell lung cancer.

PURPOSE: Chemoresistance is common among non-small-cell lung cancer (NSCLC), P-glycoprotein (P-gp), encoded by the human multi-drug-resistant MDR1 gene, and multidrug-resistance protein 1 (MRP1) might be major contributors. The aim of the present study was to develop an effective method to investigate the expression and function of P-gp in the peripheral CD56+ cells in order to clarify their correlation with the chemoresistance in NSCLC. METHODS: Using microbead technology and a RT-qPCR methodology, we evaluated the expression levels of P-gp and MRP1 in the purified CD56+ cells in the chemoresistance and chemo-naive NSCLC patients compared with that in the healthy volunteers. Flow cytometric analysis was used to investigate the changes of P-gp function in the CD56+ cells between the three cohorts. RESULTS: The MDR1 gene expression was elevated markedly (twofold-tenfold), and P-gp function was increased in the chemoresistance cohort compared with the chemo-naive and the healthy cohorts; whereas there was only about two times averagely elevated for the MRP1 gene expression. No statistical significance (p > 0.05) was seen with respect to the expression of MDR1 and MRP1, the function of P-gp between the chemo-naive and the healthy cohorts. CONCLUSIONS: P-gp in peripheral CD56+ cells demonstrated possible clinical relevance as predictive biomarkers for the identification of chemoresistance in NSCLC, while MRP1 may not play a significant role in the drug resistance in NSCLC. The potential applications for this finding are provided evidence to screen the potential P-gp reversors and to diagnose and manage the chemoresistance in NSCLC patients.

In vitro activity of cepharanthine hydrochloride against clinical wild-type and lamivudine-resistant hepatitis B virus isolates.

Hepatitis B virus (HBV) infection causes major public health problems worldwide. The clinical limitation of current antiviral drugs for HBV, such as lamivudine, is the emergence of drug-resistant viral strains during prolonged antiviral therapy. Cepharanthine hydrochloride (CH), a natural alkaloid-derived compound, has been reported to possess potent activity against various viruses. The present study was performed to evaluate the in vitro activity of CH against clinical wild-type and lamivudine-resistant HBV isolates in transiently transfected cells. HBV DNA was extracted from serum samples collected both before lamivudine therapy and at the time of viral breakthrough and was amplified by polymerase chain reaction (PCR). The amplicons were cloned into a novel expression vector, pHY106, which can initiate the intracellular HBV replication cycle after cell transfection. Following transfection of the cloned amplicon into HepG2 cells, a drug susceptibility assay was performed. The level of viral antigen, HBeAg, was determined by enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR (Q-PCR) was used for determining the amount of intracellular HBV DNA. Heat stress cognate 70 (Hsc70), a host protein required for HBV replication, was also analyzed by reverse transcription PCR (RT-PCR) to explore the possible antiviral mechanism of CH. The results showed that CH inhibited replication and HBeAg production by either wild-type or lamivudine-resistant HBV clinical isolates in a dose-dependent manner. The Hsc70 mRNA was also downregulated significantly. In conclusion, CH is active against both wild-type and lamivudine-resistant HBV clinical isolates, and its activity may be associated with its inhibition of host Hsc70.

[Methylation and expression of RUNX3 gene promoter in papillary thyroid carcinoma].

OBJECTIVE: To study the relationship between status of methylation of human runt-related transcription factor 3 (RUNX3) gene promoter in papillary thyroid carcinoma (PTC). METHODS: Methylation-specific PCR and immunohistochemical SP technique were used to detect the methylation of RUNX3 gene promoter and expression of its protein in 56 cases of PTC and their matched adjacent non-carcinous epithelium (NCE). RESULTS: In NCE, there was no methylation of RUNX3 gene promoter, while in PTC the methylation rate was 35.7%(20/56), which was related to the tumor TNM stage, pathological grade and lymph node metastasis (P < 0.05). The positive rates of RUNX3 protein expression in NCE and PTC were 100.0% and 60.7%, respectively, with a significant difference (χ(2) = 27.378, P < 0.05). In PTC, the positive rates of RUNX3 protein expression in gradeI and grade II were 70.0% and 37.5%, respectively (P < 0.05); the rates were 46.7% and 76.9% in lymph node metastasis group and no metastasis group, respectively (P < 0.05). Moreover, there was a distinct correlation between methylation of RUNX3 gene promoter and expression of its protein (χ(2) = 21.62, P < 0.01). CONCLUSIONS: Methylation of promoter might be one of the important factors of inactivation of RUNX3 gene, and might play an important role in carcinogenesis and progression of PTC.

[Correlation of mRNA and protein expressions of Runx3 gene in papillary thyroid carcinoma].

OBJECTIVE: To explore the mRNA and protein expressions of Runx3 gene in papillary thyroid carcinoma (PTC). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the Runx3 mRNA and protein levels in 67 human PTC specimens and their matched adjacent non-cancerous epithelium (NCE). RESULTS: The relative expression value of Runx3 mRNA was 0.31 ± 0.07 in PTC versus 0.92 ± 0.08 in NCE (t = 38.251, P < 0.05). In PTC, it was correlated with the tumor pathological grade and lymph node metastasis (χ(2) = 5.511, 6.492, P < 0.05). The integral optical density value of Runx3 protein confirmed was 1012 ± 221 in PTC versus 1993 ± 199 in NCE (t = 18.413, P < 0.05). In PTC, it was correlated with the tumor TNM stage, pathological grade and lymph node metastasis (χ(2) = 5.550, 9.678, 5.070, P < 0.05). Furthermore there was a distinct correlation between the mRNA and protein expressions of Runx3 gene (χ(2) = 42.699, P < 0.05). CONCLUSION: The mRNA and protein expressions of Runx3 gene in PTC were lower than those in NCE. A lower expression of Runx3 gene may play an important role in the carcinogenesis and progression of PTC.

[Expression of Piwil2 and its relationship with tumor invasion and metastasis in papillary thyroid carcinoma].

OBJECTIVE: To study the expressions of Piwil2 protein and mRNA in papillary thyroid carcinoma (PTC) and the relationship between Piwil2 and the invasion and metastasis of PTC. METHODS: Immunohistochemistry and in situ hybridization were used to detect the expression of Piwil2 protein and mRNA in 60 cases of PTC with the matched adjacent non-cancerous epithelium (NCE). RESULTS: The positive rates of Piwil2 protein expression in PTC and NCE were 88.3% (53/60) and 10.0% (6/60) respectively, with significant difference (χ² = 73.654, P < 0.01). The positive rates of Piwil2 mRNA expression in PTC and NCE were 85.0% (51/60) and 6.7% (4/60) respectively, also with significant difference (χ(2) = 74.148, P < 0.01). Up-regulated expressions of Piwil2 protein and mRNA were related to the invasion and metastasis of PTC (P < 0.05). CONCLUSIONS: Piwil2 may play a role in the invasion and metastasis of PTC.

Determination of SNX-2112, a selective Hsp90 inhibitor, in plasma samples by high-performance liquid chromatography and its application to pharmacokinetics in rats.

A sensitive and specific reversed-phase high-performance liquid chromatography method with ultraviolet detection has been developed and validated for the identification and quantification of SNX-2112 in rat plasma. Following sample preparation using liquid-liquid extraction, the analytes were separated by the mobile phase acetonitrile-water (40:60, v/v) with an Agilent RP-HPLC column (ZORBAX SB-C18, 5 microm, 4.6 mm x 250 mm) at a flow rate of 1 ml/min, column temperature of 30 degrees C and detection wavelength of 251 nm. The retention time of SNX-2112 was 11.2 min. A good linear relationship was obtained in the concentration range studied (0.07-21 microg/ml, R(2)>0.9982), and the LLOD and LLOQ for SNX-2112 were 0.02 and 0.07 microg/ml, respectively. The mean absolute recovery of SNX-2112 in plasma ranged from 88.58 to 99.61% at the studied concentrations. The intra- and inter-batch relative standard deviations were 1.7-3.5 and 1.9-4.4%, respectively. This method was successfully applied to pharmacokinetic studies in rats after intravenous administration of SNX-2112.

Phyllaemblicin B inhibits Coxsackie virus B3 induced apoptosis and myocarditis.

Coxsackie virus B3 (CVB3) is believed to be a major contributor to viral myocarditis since virus-associated apoptosis plays a role in the pathogenesis of experimental myocarditis. In this study, we investigated the in vitro and in vivo antiviral activities of Phyllaemblicin B, the main ellagitannin compound isolated from Phyllanthus emblica, a Chinese herb medicine, against CVB3. Herein we report that Phyllaemblicin B inhibited CVB3-mediated cytopathic effects on HeLa cells with an IC(50) value of 7.75+/-0.15microg/mL. In an in vivo assay, treatment with 12mgkg(-1)d(-1) Phyllaemblicin B reduced cardiac CVB3 titers, decreased the activities of LDH and CK in murine serum, and alleviated pathological damages of cardiac muscle in myocarditic mice. Moreover, Phyllaemblicin B clearly inhibited CVB3-associated apoptosis effects both in vitro and in vivo. These results show that Phyllaemblicin B exerts significant antiviral activities against CVB3. Therefore, Phyllaemblicin B may represent a potential therapeutic agent for viral myocarditis.

Transcriptomic and proteomic approach to studying SNX-2112-induced K562 cells apoptosis and anti-leukemia activity in K562-NOD/SCID mice.

SNX-2112, a novel inhibitor of Hsp90 currently used as an anti-tumor drug, induces apoptosis in multiple tumor cell lines. It destabilizes specific client proteins, but the molecular mechanism of the apoptosis effect of SNX-2112 is poorly understood. Here, we analyzed the apoptotic effect of SNX-2112 on human chronic myeloid leukemia (CML) K562 cells. Transcriptomic and proteomic approaches further revealed that caspase signals originated from mitochondria dysfunction, mediated by Akt signaling pathway inactivity. Additionally, SNX-2112 prolonged the survival time of NOD/SCID mice inoculated with K562 tumor cells. Our results demonstrated the therapeutic potential of SNX-2112 against human CML.

[The experimental research about the effect of Prunella vulgaris L. on Raji cells growth and expression of apoptosis related protein].

OBJECTIVE: To investigate the anti-lymphoma effect of Prunella vulgaris L. in order to offer exprimental data for the treatment of lymphoma with Prunella vulgaris L. in clinic. METHODS: Effect of Prunella vulgaris L. injection on inhibition ratio of cell growth of Raji cells and IC50 were tested by MTT assay. The growth curve line of Raji cells was drawn also by MTT assay. The cellular morphology was observed by invert microscope, Giemas staining and MTT assay. The expression of apoptosis related protein bcl-2, bax was measured by immunocytochemistry and the quantitative analysis was made with figure analysis system. RESULTS: 1. Prunella vulgaris L. could obviously suppress the cell proliferation of Raji cells in a concentration-dependent manner (r = 0.97). The IC50 was 0.118 mg/ml. 2. After Raji cells was reacted with injection of Prunella vulgaris L. (50 mg/ml) , the morphlogical of apoptosis were observed by invert microscope, Giemsa staining and MTT assay. RESULTS: The results of immunocytochemistry showed that after Raji cells were treated by the injection of Prunella vulgaris L. (50 mg/ml) for 48 hours, the expression of bcl-1 was up-regulated, and the expression of bax was down-regulated. The differences between process group and control group were significant (P < 0.01). CONCLUSION: Prunella vulgaris L. can suppress the proliferation of Raji cells and may be a new anti-lymphoma drug. Inducing the apoptosis of Raji cells maybe one of anti-lymphoma mechanisms.

[Reversal of multidrug resistance in drug-resistant cell line EAC/ADR by cepharanthine hydrochloride and its mechanism].

AIM: To investigate the correlation between reversal effect of cepharanthine hydrochloride (CH) on multidrug resistance (MDR) in drug-resistant cell line EAC/ADR and the nuclear transcription factor-KB (NF-KB). METHODS: Cytotoxicity was determined by the tetrazolium (MTT) assay in vitro. An EAC/ADR cell homograft model was established to investigate the effect of CH on reversing MDR in vivo. The constitutive activity and activation of NF-KB by drugs were measured by Dot-Enzyme-linked Immune Sorbent Assay (Dot-ELISA). RESULTS: CH was shown to potentiate the cytotoxicity of ADR, a 13- fold reversal effect of resistance was achieved in vitro. In mice bearing EAC/ADR cell homografts, CH was found to prolong the survival time of animals bearing tumor. Increase in life span over control was 75. 37%. In addition, the constitutive activity of NF-KB and activation of NF-KB by chemotherapy were lowered by CH. CONCLUSION: The findings suggest that CH is able to reverse drug resistance and its mechanism may be related to suppressing the constitutive activity and activation of NF-KB by drugs.

[Regulation of telomerase activity and cell cycle of K562 cells by oridonin].

AIM: To investigate the effect of oridonin (ORI) on telomerase activity and cell cycle of human leukemic cell line K562 cells. METHODS: Immunohistochemistry (IHC) technique was used to determine the expression of hTERT or C-myc. Telomerase activity was detected with TRAP-PCR-ELISA assay. In addition, the percentages of K562 cells in different cell cycle were determined by flow cytometry (FCM) at 24th and 48th hours separately after adding the different concentrations of ORI. RESULTS: After the K562 cells were treated with ORI at 3.43 micromol x L(-1) for 48 h, the expression of hTERT and C-myc decreased obviously. There was statistical significant (P < 0.05) difference between experimental groups and the normal controls. In addition, the telomerase activity of K562 cells was significantly inhibited by ORI at the dose of 3.43 micromol x L(-1) for 48 h. At the same time, the cell cycle distribution changed, the percentage of G0/G1 or G2/M stages cells increased and that of the S stage cells decreased after ORI was added. CONCLUSION: ORI can effectively inhibit telomerase activity in K562 cells. Arresting cell cycle and decreasing the expression of hTERT and C-myc may be the mechanism of action.