| Chemotherapy is one of the major regimens for treatment of cancerous diseases including solid carcinomas and leukemia, the efficacy of which have been limited by two obstacles, that is, the toxic reactions such as cardiac lesions by doxorubicin (ADR) and the multidrug resistance (MDR) required during chemotherapy. It has been indicated that both the problems might be solved by modulation of drug redistribution in cells and tissues through regulation of the transportor of hydrophobic molecules, p-glycoprotein (p-gp). P-gp, a member of the superfamily of ATP-binding cassette (ABC) transportor, is a transmembrane (TM) protein encoded by the gene designated as mdr1. Intact p-gp consists of 1226 aa residues, with its N- and C-terminals across cell membrane. The middle segment of p-gp conform functional domain binding and hydrolysing ATP to provide energy for the trans-concentration movement of transported substrates. It has been recognized that the substrates of p-gp are featured by diversities of their structures and bioactivities, but recently it has been suggested that p-gp bind and transport hydrophobic molecules selectively by the binding sites located in TM domain and that not only anti-tumor agents but also cholesteroids be conveyed by p-gp. And verepamil, a blockor for Ca2+ channel, may be also one of the substrates of p-gp. P-gp expresses constitutively in many tissues and cells, which indicates its physiological action needed for maintaining the substantial metabolisms of the cells. On the other hand, p-gp expression can be regulated by some stimuli raised by the changes of cell environment such as stress. It is widely realized that over-expression of p-gp in tumor cells results from the long-term exposure to anti-tumor drugs and during chemotherapeutic episodes. In other way, elevation of cytosol [Ca2+] will induce activation of many genes including mdr1. The purpose of the research is focused on artificial modulation of the expression and activity of p-gp with so-called cardiotonic drugs, verapamil (VRPL) and strophan K (STPK). By inducting expression of mdr1, the gene vaccines specific for p-gp are developed and the in vivo immunoresponses induced with the vaccine are further examined. The modulation of chemosensitivity of K562 cells by STPK and VRPL MTT assay was used to detect the in vitro cell proliferation, which was considered as an analyzing system for cellular toxicity of both STPK and VRPL; for the chemosensitivity of the cells to ADR as well as modulating effects of the two cardiotonic agents. Some modifications were made in the study. The samples were centrifuged to remove the supernatants completely after incubation of the cells with MTT and isopropenol was added without HCl to dissolve the formazan precipitant. These procedures (1) greatly decreased the non-specific background, indicated by OD570 almost zero in the cell-free controls and (2) increased the test sensitivity. As seen in the study, OD570 values reached up to 2.0 when 2X105 of the cells were present in the wells. The detectable range for the number of living cells was among 2-20 X 104. 1. Cellular toxicity of STPK and VRPL to the cultured tumor cell lines: different amount of STPK (0.022-1.4μg/ml)or VRPL (0.25-25μg/ml) were respectively added into 105 cell cultures of 3 tumor cell lines, K562 cells, MCF-7 cells and SMMC 7721 cells. The cultivation was maintained for 48hrs to examine cell growth in presence or absence of the drugs. The results showed that proliferation of K562 cells was not altered compared with the control when the concentrations of STPK were less than 0.7μg/ml or those of VRPL beneath 5μg/ml, the OD570 of which were 1.06-1.18. MCF-7 cells and SMMC 7721 cells grew only when they adhere. In the culture with either STPK or VRPL, the cells detached, as the result, proliferation of the cells decreased obviously. 2. The effect of STPK and VRPL on ADR sensitivity of K562 cells: 105 of K562 cells were inoculated and cultured with 0.0625 to 4μg/ml of ADR for 24 hr... |
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