内容
【摘要】 目的 以k562/dox和mcf7/dox细胞为对象，探讨异汉防己碱对化疗药物阿霉素（dox）的增敏作用及其作用机制。 方法 采用mtt法检测异汉防己碱的内在细胞毒性及其对阿霉素的增敏作用，并以rf值评价其增敏效果。应用流式细胞术（fcm）检测细胞膜上pgp的表达以及细胞内dox和罗丹明123（rh123）的蓄积量。结果 异汉防己碱在10μg/ml的无毒剂量可明显增强dox的细胞毒性。k562/dox和mcf7/dox细胞膜上pgp均呈强阳性表达，但异汉防己碱对该pgp表达水平无明显影响。异汉防己碱可使k562/dox和mcf7/dox细胞内dox和rh123的荧光密度（fi）均明显增加，由此证明异汉防己碱可有效抑制pgp的功能。结论 异汉防己碱可通过抑制pgp的功能而增强阿霉素的敏感性，从而有效逆转肿瘤细胞的多药耐药性（mdr），它可能成为有效多药耐药逆转剂的候选药物。 【关键词】 异汉防己碱；阿霉素；肿瘤细胞；多药耐药性 abstract：objective to explore the effect and mechanism of isotetrandrine to enhance doxorubicin (dox) sensitivity of k562/dox and mcf7/dox cells. methods the activity of isotetrandrine to enhance doxorubicin cytotoxicity was tested using mtt [3(4, 5dimethylthiazol)2,5diphenyltetrazolium bromide] assay and evaluated by the reversal fold (rf) values. the level of pglycoprotein (pgp) expression and intracellular accumulation of doxorubicin and rhodamine123 (rh123) were assessed by flow cytometry (fcm). results the doxorubicininduced cytotoxicity was significantly potentiated by isotetrandrine with the concentration of 10μg/ml. pgp was expressed in both k562/dox cells and mcf7/dox cells, but the level of pgp expression was not distinct difference at the absence or presence of isotetrandrine. the intracellular accumulation of dox and rh123 was increased in the presence of isotetrandrine, which indicated that the function of pgp was effectively inhibited. conclusion isotetrandrine exhibited potent effect in the reversal of tumor multidrug resistance (mdr) by inhibiting the function of pgp in vitro, suggesting that it may become a candidate of effective mdr reversing agents in cancer chemotherapy. key words：isotetrandrine; doxorubicin; tumor cells; multidrug resistance introduction cancer multidrug resistance (mdr) is one of the major obstacles for the success of chemotherapy. it is related to a 170kda plasma membrane protein, pglycoprotein (pgp)[1]. pgp functions as an atpdependent drugs transporter which unilaterally transports the intracellular drugs out of the cells，thereby reducing drug cytotoxicity. so chemotherapy drugs in conjunction with a pgp inhibitor at the time of tumor treatment will be the effective way to overcome mdr. accordingly, a variety of drugs have been reported as agents for overcoming mdr. however, side effects of many drugs make them incapable effectively applied in the clinic. therefore, development of safe and effective mdr reversing agents is eagerly required. isotetrandrine, a bisbenzylisoquinoline alkaloid extracted from traditional chinese drug mahonia, is diastereoisomer of tetrandrine(tet)[2]. previous reports have shown that tet could exhibit reversal effect on tumor mdr [35]. in this study, we were to investigate the reversal effect of isotetrandrine on mdr in k562/dox and mcf7/dox cells. 1 materials and methods 1.1 drugs and reagents isotetrandrine was extracted and prepared by our project group; doxorubicin(dox) was purchased from jintairong medicine co. ltd., guangdong, china; 3(4,5dimethylthiazol)2,5diphenyltetrazolium bromide(mtt) and rhodamine 123 (rh123) were purchased from sigma co., usa; antihumanpgp mouse mab and fitcmarked goatantimouse igg were purchased from maixin biotechnology co. ltd.,fuzhou, china; fetal calf serum and rpmi1640 medium were purchased from gibco, usa. 1.2 cell lines and cell culture human leukemia (k562) cells and human breast cancer (mcf7) cells and their doxresistant (k562/dox and mcf7/dox) cells were purchased from institute of haematology, chinese academy of medical sciences. they were cultured in rpmi1640 medium with 10% fetal calf serum at 37℃ in a humidified 5% co2 atmosphere. k562/dox and mcf7/dox cells were maintained in culture medium with 1.0 μg/ml dox and incubated in doxfree medium for 2 weeks before the planned experiment. 1.3 drugs cytotoxicity assay the intrinsic cytotoxicity of isotetrandrine and the ability of it to potentiate dox cytotoxicity in all kinds of cells were determined with mtt assay [68]. cells were seeded into 96well plates at 2×104/well. various concentrations of dox and isotetrandrine were subsequently added and incubated for 48h. then 5μg/ml mtt was added and incubated for 4h later, the culture fluid was eliminated and dmso was added. after formazan in the cells was dissolved by dmso, the absorbance at 570nm was detected and inhibitor rates of cells were obtained. by regression analysis, ic50 which was the concentration of the drug causing 50% inhibition of cells growth was obtained. the reversal fold (rf), the ratio of ic50 of cytotoxic drug (dox) alone and that of cytotoxic drug (dox) in the presence of modulator (isotetrandrine), embodied the effect of isotetrandrine to enhance doxorubicin cytotoxicity and reversal potency of isotetrandrine. 1.4 detection of expression level of pgp antihumanpgp mouse mab was added in all kinds of cells in logarithmic phase at the absence or presence of isotetrandrine and incubated for 30min at 4℃, then added fitcmarked goatantimouse igg and incubated for 30min at 4℃ again, the expression of pgp was determined by fcm. 1.5 accumulation and efflux of rh123 assay rh123 is the favorable and specific substrate of pgp, so the accumulation and efflux of rh123 assay is classical method of determining pgp function. at the accumulation assay, cells (1×106/ml) in logarithmic phase were incubated with medium containing 2μg/ml rh123 in the presence or absence of 10μg/ml isotetrandrine at 37℃ for 45 min. after two washes with icecold pbs, the intracellular rh123associated fluorecence intensity (fi) was measured with fcm. at the efflux assay, cells (1×106/ml) in logarithmic phase were first incubated with medium containing 6μg/ml rh123 at 37℃ for 45min, washed three times with serumfree rpmi1640 medium, and then incubated in the presence or absence of 10μg/ml isotetrandrine at 37℃ for 45min. after two washes with icecold pbs, the intracellular rh123associated fi was measured with fcm. 1.6 detection of intracellular dox concentration the k562/dox and mcf7/dox cells (1×106/ml) in logarithmic phase were exposed to 10μg/ml dox in the presence or absence of 10μg/ml isotetrandrine for 90 min at 37℃. after two washes with icecold pbs, intracellular doxassociated fi was measured with fcm. 1.7 data analysis all data were presented as ±s and analyzed using spss statistic software. 2 results 2.1 resistance of k562/dox and mcf7/dox cells to dox by mtt assay and regression analysis, ic50 of dox to inhibit the reproduction of mcf7 cells was 0.22μg/ml and ic50 of dox to mcf7/dox cells was 45.25μg/ml, thereby the resistant fold was 205.68; ic50 of dox to inhibit the reproduction of k562 cells was 1.48μg/ml and ic50 of dox to k562/dox cells was 75.25μg/ml, thereby the resistant fold was 50.84. their resistant folds were over 20, so the k562/dox and mcf7/dox cells had phenotype of mdr. 2.2 intrinsic cytotoxicity of isotetrandrine as shown in tab1, inhibitory rate of isotetrandrine to cells growth took on dependence of concentrations. along with the rise of isotetrandrine concentration, inhibitory rates of cells were gradually increased. when concentration of isotetrandrine was 20μg/ml, inhibitory rate of cells growth were about 10%. so the concentrations under 20μg/ml were its noncytotoxic dose. we chose 10μg/ml dose of isotetrandrine to study the effect of it enhancing dox sensitivity.tab 1 inhibitory rate of isotetrandrine on au kinds of cells表1 异汉防己碱对各种细胞的抑制率 2.3 effect of isotetrandrine on dox cytotoxicity the activity of isotetrandrine to enhance doxorubicin cytotoxicity in k562/dox and mcf7/dox cells was shown in tab 2. isotetrandrine exhibited a distinct reversal effect at 10μg/ml concentration. no such activity was found in k562 and mcf7 cells. 2.4 effect of isotetrandrine on expression level of pgp in isotetrandrineuntreated group, positive expression rates of pgp in k562/dox and mcf7/dox cells were （75.28±4.36）% and （73.25±3.15）% respectively. in isotetrandrinetreatedtab 2 effect of isotetrandrine on dox表2 异汉防己碱对阿霉素细胞毒性的影响 group, positive expression rates of pgp in k562/dox and mcf7/dox cells were （72.48±3.45）% and （71.55±3.37）% respectively, no distinctive difference with isotetrandrineuntreated group (p 0.05). the results indicated that isotetrandrine hardly affected expression level of pgp. 2.5 effect of isotetrandrine on pgp function no matter at rh123 accumulation experiment or at rh123 efflux experiment, intracellular rh123 concentration (rh123associated fi) of doxresistant cells in isotetrandrinetreated group was significantly higher than that in isotetrandrineuntreated group(p 0.05), although it did not reach the level of rh123 concentration in doxsensitive cells, suggesting that isotetrandrine could promote rh123 accumulation and inhibit rh123 efflux and therefore inhibiting pgp function of “drug efflux bump”, as shown in tab 3.tab 3 effect of isotetrandrine on intracellular rh123表3 异汉防己碱对细胞内rh123浓度的影响 a: significantly different from isotetrandrineuntreated group (p 0.05)2.6 effect of isotetrandrine on intracellular accumulation of dox at the absence of isotetrandrine, intracellular doxassociated fi in k562/dox and mcf7/dox cells was (30.47±0.25) and (33.25±0.12) respectively. at the presence of 10μg/ml isotetrandrine, intracellular doxassociated fi in k562/dox and mcf7/dox cells was rised to (65.48±0.47) and ( 65.38±0.35) respectively. this also explained why isotetrandrine enhanced dox cytotoxicity in doxresistant cells. 3 discussion mdr is a common problem in cancer chemotherapy. it is a phenomenon that tumor cells resistant to a kind of anticancer drug were also resistant to a variety of in functionally and(or) structurally unrelated anticancer agents. mdr is related to the actions of one or more membrane transport proteins. among them, pgp that promote the expulsion of anticancer drugs is classical mdr mechanism. a number of studies have tried to find mdr modulators which increase anticancer drug accumulation in cancer cells [911]. but so far, the modulators have not been successfully applied in the clinic because of their toxicity and side effects. so the search for new drugs with low toxicity is in progress to satisfy an urgent need for clinical applications. in this study, the ability of isotetrandrine to enhance dox cytotoxicity in doxresistant cells was discussed. the present results showed that isotetrandrine of 10μg/ml (noncytotoxic dose) concentration could make ic50 of dox to k562/dox and mcf7/dox cells distinctly decreased. the intracellular doxassociated fi was significantly increased in the presence of isotetrandrine of 10μg/ml concentration. the results suggested that isotetrandrine could enhance dox cytotoxicity, therefore effectively reversing mdr of k562/dox and mcf7/dox cells. in the present report, we further investigated the mechanism of isotetrandrine. by fcm assay, isotetrandrine hardly affected the expression level of pgp in k562/dox and mcf7/dox cells. so we further investigated the inhibitory effects of isotetrandrine on pgp function.the accumulation and efflux experiment of rh123 is an important method of determining pgp function in drugresistant cell lines expressing pgp[12], because the efflux of fluorescent dye rh123 is pgpdependent. in this study, after cells were incubated with rh123, the intracellular rh123associated fi in doxresistant cells was lower than that in doxsensitive cells and intracellular rh123associated fi in doxresistant cells was distinctly increased by isotetrandrine. so isotetrandrine could inhibit pgp efflux function and therefore reversing mdr of doxresistant cells. in a word, isotetrandrine possessed potent reversal effect on tumor mdr. it may become a candidate of tumor mdr reversing agents. 【参考文献】 ［1］ gottesman mm, pastan i. biochemistry of multidrug resistance mediated by the multidrug transporter[j]. annu rev biochem, 1993, 62:385427. 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