Grape seed proanthocyanidin extract reverses multidrug resistance in HL-60/ADR cells via inhibition of the PI3K/Akt signaling pathway.

BACKGROUND AND PURPOSE: Multidrug resistance (MDR) is a great challenge and obstacle in cancer treatment. It is a common problem in the treatment of acute myeloid leukemia (AML). Whether grape seed proanthocyanidin extract (GSPE) could reverse MDR in patients with AML is still unknown. The aim of this study was to investigate the MDR reverse ability of GSPE and its possible mechanism in vitro. MATERIALS AND METHODS: Human leukemia cell line HL-60 cells and HL-ADR cells were used. MTT assay were employed to identify the cytotoxic effects of different chemotherapeutic drugs and reverse ability of GSPE. Flow cytometry assays were used to verify the cell apoptosis induced by GSPE. MDR-related genes expression was tested by real-time polymerase chain reaction (Q-PCR). MDR-related protein expression was assessed by Western blotting assays. The genes and their related protein expression of multidrug resistance-associated protein 1 (MRP1), multidrug resistance protein 1 (MDR1) and lung resistance-related protein (LRP) were tested in this study. KEY RESULTS: We found that HL-60/ADR cells were resistant to a variety of chemotherapeutic drugs, including cytarabine (Ara-C), adriamycin (ADR), vincristine (VCR), daunorubicin (DNR), mitoxantrone (MTZ), pirarubicin (THP), homoharringtonine (HHT) and etoposide (VP16). Co-treatment with GSPE could significant lower the IC50 of Ara-C and ADR in HL-60/ADR cells (P < 0.01). MDR related mRNA and their protein expression of MRP1 and MDR1 were significant highly expressed in HL-60/ADR cells than HL-60 cells (P < 0.01). But only protein expression of LRP was higher in HL-60/ADR cells than HL-60 cells (P < 0.05). GSPE could induce a higher intracellular level of ADR in HL-60/ADR cells. It could also inhibit Akt phosphorylation resulted in the down regulation of MRP1, MDR1 and LRP and induce cell apoptosis. 25.0 µg/mL GSPE significant inhibited the Akt phosphorylation (P < 0.05). CONCLUSION AND IMPLICATIONS: GSPE-reversed MDR of HL-60/ADR cells might be associated with the inhibition of the PI3K/Akt signaling pathway, which resulted in the down-regulation the expression of MRP1, MDR1 and LRP. These results provide that GSPE may serve as a combination therapy in AML chemotherapy for future study.

[Protective effect of grape seed proanthocyanidin on cultured RGC-5 cells against CoCl2-induced hypoxic injury].

OBJECTIVE: To investigate the protective effects of grape seed proanthocyanidin extracts (GSPE) against CoCl2-induced hypoxic injury in cultured RGC-5 cells. METHODS: CoCl2(400 µmol/L) was used to induce hypoxic injury in cultured RGC-5 cells; the cells were pretreated with 0,100,200,400 and 800µmol/L GSPE for 24h. The cell viability was assayed by MTT; the apoptosis was detected by Hoechst 33342 staining; the intracellular reactive oxygen species (ROS) was measured by H2DCFDA oxidative reaction. The mRNA expression of Bcl-2, caspase 9 and caspase 3 was determined by real-time PCR. RESULTS: Compared to hypoxic control group, pretreatment with GSPE significantly increased viability of RGC-5 cells (P<0.001), reduced cell apoptosis (P<0 .001) and intracellular ROS(P <0 .001). In addition, GSPE significantly increased the mRNA expression of Bcl-2(P<0 .001) and decreased mRNA expression of caspase 9(P<0 .001) and caspase 3(P<0 .001) compared to hypoxic control group. CONCLUSION: GSPE may have a protective effect against CoCl2-induced hypoxic injury in cultured RGC-5 cells. The decrease of intercellular ROS, up-regulation of Bcl-2 and down-regulation of caspase 9 and caspase 3 may be involved in the mechanism of the protective effect of GSPE.

[Construction of HEK293 cell lines expressing hCysLT2 receptor and its application in screening of antagonists].

OBJECTIVE: To construct HEK293 cell lines stably expressing hCysLT(2) receptor, and to evaluate its application in screening of synthetic compounds with antagonist activity. METHODS: The recombinant plasmid pcDNA3.1(+)-hCysLT(2) was transfected into HEK293 cells using Lipofectamin 2000. The transfected HEK293 cells were selected in 96 well plates by limiting dilution with 600 µg/ml C418 for 8 weeks. The expression of human CysLT(2) receptor was detected by RT-PCR and immunofluorescence staining. In HEK293 cells stably transfected with hCysLT(2), the agonist LTD(4)-induced elevation of intracellular calcium concentration ([Ca2(+)]i) was measured as the index for screening compounds with antagonist activity. RESULT: After selection in 96 well plates by limiting dilution, 12 monoclones were obtained and 11 of them highly expressed hCysLT(2) receptor. The positive control ATP at 50 µmol/L and LTD(4) at 100 nmol/L elevated [Ca2(+)]i in hCysLT(2)-HEK293 cells. AP-2100984 inhibited LTD(4)-induced [Ca2(+)]i elevation, but selective CysLT(1) receptor antagonists did not exert such an effect. The newly synthesized compounds DXW2, DXW3, DXW4, DXW5, DXW9, DXW25, DXW26, DXW29 and DXW35 at 1 µmol/L significantly inhibited LTD(4)-induced [Ca2(+)]i elevation. The IC(50) values of DXW4 and DXW5 were 0.25 µmol/L and 7.5 µmol/L. CONCLUSION: HEK293 cell lines stably expressing hCysLT(2) receptor have been successfully constructed, and can be used to screen compounds with CysLT(2) receptor antagonist activity.

[Construction and identification of eukaryotic expression vector of rat GPR17 gene].

OBJECTIVE: To construct the eukaryotic expression vector of rat GPR17 (rGPR17) cDNA,and to identify its function in HEK293 cells. METHODS: Total RNA was extracted from rat brain tissue; full-length GPR17 cDNA was prepared by RT-PCR, and cloned into pcDNA3.1(+) plasmid. The recombinant plasmid was converted into E.coli DH5alpha and confirmed by PCR, double enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-rGPR17 was transiently transfected into HEK293 cells using Lipofectamin 2000. Expression of rGPR17 gene was confirmed by RT-PCR and immunofluorescence staining. The exogenous LTD(4) enhanced intracellular calcium was measured using Fluo-4. RESULT: RT-PCR, double enzyme digestion analysis and sequencing showed that the rGPR17 gene was cloned into recombinant vector, and the recombinant rGPR17 was expressed after transfection in HKE293 cells. LTD(4) increased intracellular calcium release in the transfected HEK293 cells. CONCLUSION: The eukaryotic expression vector of rGPR17 cDNA has been constructed; it is functionally expressed in HEK293 cells. This work provides a basis for further research of the GPR17 receptor and its antagonists.