FG020327逆转肿瘤多药耐药性的作用及其机制

背景与目的肿瘤细胞过量表达P鄄糖蛋白穴P-glycoprotein.P-gp雪导致多药耐药穴multidrug resistance,MDR雪是目前肿瘤化疗的一大障碍,使用多药耐药逆转剂与抗癌药物联合化疗是克服临床多药耐药的重要方法。本研究对一种新的多芳基取代咪唑化合物FG020327的体外逆转活性及其逆转机制进行了探讨。方法以MTT法检测FG020327对多药耐药肿瘤细胞MCF-7/ADR及KBv200的耐药逆转活性;以荧光分光光度计法检测FG020327对MCF-7/ADR细胞内抗癌药物阿霉素累积的影响;以罗丹明蓄积实验检测该化合物对P-gp功能的影响。结果FG020327在体外具有较强的逆转活性,在5μmol/L浓度下使多药耐药细胞KBv200对长春新碱的敏感性增加44.9倍,逆转活性是公认的强逆转剂维拉帕米的3倍熏但它对敏感株对抗癌药物的敏感性基本无影响。2.5、5和10μmol/L的FG020327使MCF-7/ADR细胞中阿霉素的累积分别增加2.3、2.7和3.7倍,但是在敏感株MCF鄄7细胞却观察不到阿霉素累积的增加。FG020327浓度依赖性增加KBv200细胞内的罗丹明蓄积,但对敏感株KB细胞内的罗丹明蓄积无影响。结论FG020327具有较强的体外逆转MDR的活性,它可能通过抑制P鄄gp功能及增加MDR细胞内抗癌药物的累积逆转MDR。     

Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine

Purpose The present study aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), in vitro and in vivo.Methods Drug sensitivity was determined using the MTT assay. The in vivo effect of Tet was investigated using nude mice grafted with sensitive and resistant KB human epidermoid cancer cells. Doxorubicin (Dox) accumulation was analyzed by fluorospectrophotometry and the protein and mRNA levels of P-glycoprotein (P-gp) were determined by immunocytochemistry and RT-PCR, respectively.Results BrTet at 0.25, 0.5 and 1 M reversed Dox resistance in MDR human breast cancer MCF-7/Dox cells dose-dependently and its potency was greater than that of Tet at the same concentrations. BrTet reversed vincristine (VCR), Dox and paclitaxel resistance in MDR human oral epidermoid carcinoma KBv200 cells as well as innate VCR and Dox resistance in human hepatocellular carcinoma Bel₇₄₀₂ cells. However, BrTet showed no effect on the IC₅₀ values of the above-mentioned anticancer drugs in sensitive MCF-7 and KB cells. No reversal effect of BrTet on the cytotoxicity of 5-fluorouracil and cisplatin, non-P-gp substrates, was observed. In nude mice bearing KBv200 xenografts on the left flank and KB xenografts on the right flank, i.p. injection of 5 mg/kg and 10 mg/kg BrTet significantly enhanced the antitumor activity of Dox against KBv200 xenografts with inhibitory rates of 33.0% and 39.2%, while Dox alone inhibited the growth of KBv200 xenografts by only 11.6%. No enhancement by BrTet was seen in KB xenografts. Moreover, BrTet at 5 mg/kg reversed paclitaxel resistance in KBv200 xenografts. Fluorospectrophotometric assay showed that BrTet significantly increased the intracellular accumulation of Dox in MCF-7/Dox cells in a dose-dependent manner. BrTet also inhibited the overexpression of P-gp in MCF-7/Dox cells, but had no effect on mdr1 expression.Conclusions BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs. BrTet may be a promising MDR modulator for eventual assessment in the clinic.     

The multidrug resistance of tumour cells was reversed by tetrandrine in vitro and in xenografts derived from human breast adenocarcinoma MCF-7/adr cells

Multidrug resistance (MDR) is one of the main obstacles limiting the efficacy of chemotherapy treatment of tumours. One of the main causes of MDR is linked to the overexpression of P-glycoprotein (P-gp). This study aimed to characterise tetrandrine (Tet), a potent inhibitor of P-gp mediated MDR. Cytotoxicity was determined by the tetrazolium (MTT) assay. A MCF-7/adr cell xenograft model was established to investigate the effect of Tet on reversing MDR in vivo. Mechanistic experiments were conducted to examine the uptake, efflux and accumulation of doxorubicin (Dox) and Fura-2, and to assess lipid membrane fluidity. Tet potentiated the cytotoxicity of Dox; a 20.4-fold reversal of resistance was achieved in the presence of 2.5 micromol/l of Tet. Accumulation and efflux studies with the P-gp substrates, Dox and Fura-2, demonstrated that Tet inhibited the P-gp-mediated drug efflux. In addition, Tet lowered cell membrane fluidity in a concentration-dependent manner. In mice bearing the MDR MCF-7/adr cell xenografts, coadministration of Tet potentiated the antitumour activity of doxorubicin without a significant increase in toxicity. Tet was an extremely potent MDR modulator both in vitro and in vivo, without apparently enhancing the toxicity of the co-administered drugs. Hence, Tet holds great promise as a MDR modulator for the treatment of P-gp-mediated MDR cancers.     

The synergistic reversal effect of multidrug resistance by quercetin and hyperthermia in doxorubicin-resistant human myelogenous leukemia cells

Purpose: This study aimed to evaluate the multidrug resistance (MDR) reversal activity of quercetin (Que) in combination with hyperthermia (HT) in human myelogenous leukemia cells K562/A.

Methods: The cytotoxicity of Que alone and the effect of Que and HT to doxorubicin (Dox) cytotoxicity were determined using MTT assay in K562 and K562/A cells. K562/A cells was heated with or without Que pretreatment, and the protein and mRNA levels of heat shock protein 70 (HSP70) and P-glycoprotein (P-gp) were determined by flow cytometry (FCM) and RT-PCR, respectively. Intracellular accumulation of Dox, cell cycle and apoptosis were monitored with FCM.

Results: Que alone inhibited cell growth in a dose-dependent manner in K562 and K562/A cells. Either Que or HT alone had a weak reversal effect on Dox resistance, however, combination HT and Que showed a much more significant reversal effect on Dox resistance (reverse fold 9.49). The elevated protein expression and mRNA level of HSP70 and P-gp in response to HT were inhibited by Que. Pretreatment with Que caused the cells to accumulate Dox 8.3-fold higher than in control cells. In addition, Que induced apoptosis and G2/M arrest in a dose-dependent manner, and the combination of Que and HT was found to have a synergistic efeect on apoptosis.

Conclusions: Que pretreatment could significantly inhance the MDR reversal activity of HT in resistant cell line, by sensitizing the cell to reversing MDR activity of HT.     

The synergistic reversal effect of multidrug resistance by quercetin and hyperthermia in doxorubicin-resistant human myelogenous leukemia cells

PURPOSE: This study aimed to evaluate the multidrug resistance (MDR) reversal activity of quercetin (Que) in combination with hyperthermia (HT) in human myelogenous leukemia cells K562/A. METHODS: The cytotoxicity of Que alone and the effect of Que and HT to doxorubicin (Dox) cytotoxicity were determined using MTT assay in K562 and K562/A cells. K562/A cells was heated with or without Que pretreatment, and the protein and mRNA levels of heat shock protein 70 (HSP70) and P-glycoprotein (P-gp) were determined by flow cytometry (FCM) and RT-PCR, respectively. Intracellular accumulation of Dox, cell cycle and apoptosis were monitored with FCM. RESULTS: Que alone inhibited cell growth in a dose-dependent manner in K562 and K562/A cells. Either Que or HT alone had a weak reversal effect on Dox resistance, however, combination HT and Que showed a much more significant reversal effect on Dox resistance (reverse fold 9.49). The elevated protein expression and mRNA level of HSP70 and P-gp in response to HT were inhibited by Que. Pretreatment with Que caused the cells to accumulate Dox 8.3-fold higher than in control cells. In addition, Que induced apoptosis and G2/M arrest in a dose-dependent manner, and the combination of Que and HT was found to have a synergistic effect on apoptosis. CONCLUSIONS: Que pretreatment could significantly enhance the MDR reversal activity of HT in resistant cell line, by sensitizing the cell to reversing MDR activity of HT.     

Altered activity of MDR-reversing agents on KB3-1 cells transfected with Gly(185)-->Val human P-glycoprotein

P-glycoprotein (P-gp) is a transmembrane glycoprotein that confers multidrug resistance (MDR). It has been demonstrated that the Gly185 residue within the cytoplasmic loop between predicted transmembrane portions 2 and 3 plays an important role in substrate specificity of human P-gp. Derivatives of cyclosporin interact with and reverse the ability of P-gp to act as a drug efflux pump. To determine if the Gly185 residue of human P-gp is also important for the interaction of P-gp with closely related cyclosporin derivatives, we examined the effect of PSC-833 and CsA on P-gp in KB3-1 cells transfected with human wild-type P-gp (GSV-2) or with the mutant P-gp (VSV-1) that habored the Gly185-->Val substitution. While the ability of CsA to sensitize VSV-1 cells to anticancer agents was enhanced, no changes in the potency of PSC-833 against cells transfected with either the wild-type or mutant P-gp were observed. In addition, VSV-1 transfected cells were more sensitive to CsA inhibition of verapamil-stimulated ATPase activity than cells transfected with wild-type P-gp. Furthermore, the intracellular accumulation of CsA was low in GSV-2 P-gp-expressing cells, compared with its accumulation in VSV-1 cells and it was found to be as high as in non-P-gp expressing KB3-1 cells. These results indicated an enhanced sensitivity of Val185-P-gp expressing cells to CsA that correlated with increased intracellular accumulation in these cells. In contrast, no significant difference in the accumulation of PSC-833 was observed among the parental, wild-type or resistant cells. Since PSC-833 was found to be more potent than CsA, these studies provided insight into the effects of the structure of MDR modulators in mediating sensitivity to anticancer drugs.     

载体表达小干扰RNA逆转卵巢癌的多药耐药

目的:探讨载体表达的小干扰RNA(smallinterferingRNA,siRNA)逆转卵巢癌细胞多药耐药的可行性。方法:浓度梯度诱导法建立人卵巢癌阿霉素耐药细胞株OVCAR/AR;脂质体介导将MDR1特异性siRNA的表达载体(pSN/mdr1a和pSN/mdr1b)转染OVCAR/AR细胞;实时定量RT-PCR检测MDRlmRNA的表达;流式细胞术检测P-gp的表达,罗丹明试验检测P-gp的药物转运功能;MTT法检测OVCAR/AR细胞对化疗药的抵抗性。结果:转染pSN/mdr1a和pSN/mdr1b后,OVCAR/AR细胞的MDR1mRNA和P-gp的表达均显著下降,P-gp的转运功能减少,OVCAR/AR细胞对阿霉素、泰素的耐药性逆转。结论:载体表达的siRNA可持久有效地抑制卵巢癌耐药细胞MDR1mRNA和P-gp的表达,并逆转其多药耐药。     

Development of a syngeneic in vivo tumor model and its use in evaluating a novel P-glycoprotein modulator, PGP-4008

Multidrug resistance (MDR) is a phenomenon by which tumor cells develop reduced sensitivity to anticancer drugs, which often leads to the failure of cancer chemotherapy. A prominent mechanism of MDR is the overexpression of the multidrug efflux pump, P-glycoprotein (P-gp), that decreases the intracellular accumulation of many anticancer drugs, leading to increased tumor growth. Intensive efforts are under way to develop clinically useful MDR modulators that inhibit the function of P-gp for use in combination with established anticancer drugs. Our goal was to develop an improved in vivo solid tumor model utilizing immunocompetent animals to examine the efficacy of P-gp-specific MDR modulators. Using in vitro cytotoxicity and drug accumulation assays, two transformed murine cell lines, JC and TIB-75, were found to demonstrate the P-gp-mediated MDR phenotype. In contrast, two similar lines did not express functional P-gp. Western blot analyses confirmed the expression of P-gp and the lack of expression of the closely related drug efflux protein MRP1 in the JC and TIB-75 cell lines. The JC cell line displayed excellent tumorigenicity and consistent growth kinetics when implanted into immune-competent Balb/c mice. Animals treated with a combination of a known MDR modulator, cyclosporin A, and a cytotoxic drug, doxorubicin, exhibited significantly reduced tumor growth compared with untreated controls or animals treated with either cyclosporin A or doxorubicin alone. Similarly, a novel P-gp-specific MDR modulator, PGP-4008, in combination with doxorubicin showed inhibition of tumor growth. However, in contrast with the significant loss of body weight observed in the animals treated with the combination of cyclosporin A and doxorubicin, those treated with PGP-4008 plus doxorubicin did not experience weight loss. Therefore, this syngeneic solid tumor model provides a new in vivo system that can be used to evaluate the efficacy of P-gp inhibitors in an immune-competent host. This should allow improved prediction of the clinical utility of these compounds.     

In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576

The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs. Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs. XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines. XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576. Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date. Accumulation and efflux studies with the P-gp substrates, [3H]daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium. This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp. Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by [3H]azidopine implying a direct interaction with the protein. In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.     

异汉防己碱增强多药耐药肿瘤细胞对阿霉素的敏感性及其机制

目的:以K562/DOX和MCF-7/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P-gp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MCF-7/DOX细胞膜上P-gp均呈强阳性表达,但异汉防己碱对该P-gp表达水平无明显影响。异汉防己碱可使K562/DOX和MCF-7/DOX细胞内DOX和123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P-gp的功能。结论:异汉防己碱可通过抑制P-gp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。     

Reversal of P-glycoprotein-mediated multidrug resistance of cancer cells by five schizandrins isolated from the Chinese herb Fructus Schizandrae

Purpose   Fructus Schizandrae (FS) is commonly used as a tonic in traditional Chinese medicine. Recently, FS was found to significantly improve liver dysfunction in chronic hepatitis patients. The present study was to assess the reversal effect of five schizandrins and crude extract from FS (named LCC) on multidrug resistance (MDR) of cancer cells, both in vitro and in vivo. Chemically, the five schizandins are derivatives of dibenzo-(a, c)-cyclooctene lignan with distinct structures differing from any known MDR reversal agents. Methods  A panel of sensitive and resistant cancer cell lines were treated with various concentrations of LCC and schizandrins. Drug sensitivity, accumulation of Doxorubicin (Dox), expression of P-glycoprotein and protein kinase C (PKC), and apoptosis were determined in vitro. The in vivo effect was tested in nude mice grafted with sensitive and resistant human epidermal cancer cell line to vincristine (VCR) (KB, KBv200). Results  The tested five compounds at 25 μM showed various levels of MDR reversal activity, of which, schizandrin A (Sin A) was the most potent one. Sin A reversed VCR resistance in KBv200 cells, MCF-7/Dox cells and Bel7402 cells by 309-, 38-, and 84-folds, respectively. Also, Sin A reversed the resistance of Dox in the above cancer cell lines. LCC at 25 μg/ml reversed VCR resistance by 619-folds in KBv200, 181-folds in MCF-7/Dox cell line, and 1,563-folds in innate resistance of human hepatic cellular carcinoma Bel7402 cells to VCR. Furthermore, LCC and its active component Sin A potently reversed the cross-resistance to paclitaxel in those cell lines. Both Sin A and LCC markedly increased intracellular Dox accumulation and enhanced apoptosis, down-regulated Pgp protein and mRNA and total PKC expression in MDR cells. Coadministration of LCC (p.o.) significantly potentiated the inhibitory effect of VCR (i.p.) on tumor growth in nude mice bearing KBv200 xenograft. Conclusions  The LCC and its active component Sin A have remarkable reversal effect on MDR in cancer cells by inhibition of both the function and expression of Pgp and total PKC. Min Huang and Jing Jin have contributed equally to this work.     

Modulation of resistance to idarubicin by the cyclosporin PSC 833 (valspodar) in multidrug-resistant cells

Idarubicin (IDA) is an anthracycline anticancer drug utilized in the treatment of acute leukemias. There are conflicting data published with regard to the cross-resistance of IDA in multidrug-resistant (MDR) cells expressing P-glycoprotein (P-gp). We evaluated the cytotoxicity and cellular accumulation of IDA in a panel of anthracycline-selected MDR cell lines. Leukemia K562/R7 cells and sarcoma MES-SA/Dx5 cells expressing high levels of the MDR1 (ABCB1) gene were resistant to IDA (42-fold and 150-fold, respectively). In both of these cell lines, resistance to IDA was equivalent to that for doxorubicin, the drug used to select for the MDR variants. The P-gp inhibitor PSC 833 (valspodar) at 2 microM completely restored sensitivity to IDA. IDA accumulation was decreased 12-fold in MES-SA/Dx5 cells vs parental cell line, and drug uptake was restored to control levels by PSC 833. Reduced intracellular IDA was correlated with P-gp content by flow cytometry. Experiments in NIH3T3 murine cells transfected with the human MDR1 gene substantiated the findings of cross-resistance to IDA and reversal of resistance by PSC 833. Our data indicate that IDA is a high-affinity substrate for P-gp.     

EGCG对人耐药口腔表皮样癌细胞株耐药逆转的实验

 目的 研究EGCG对人多药耐药口腔癌细胞KBV200的细胞毒增敏作用及裸鼠移植瘤的抑瘤作用。方法 MTT法检测药物对细胞的毒性作用，流式细胞术分别检测细胞P糖蛋白的表达，HPLC检测细胞内VCR浓度，采用KB和KBV200细胞分别种植同一裸鼠左、右腋下，观察用药后体重、抑瘤率的改变。RT-PCR检测瘤组织mdr1的表达。结果 EGCG在100mg·L^-1以下剂量对两株肿瘤细胞的抑制率均小于10％，EGCG与VCR联合应用可明显提高VCR的细胞毒作用；EGCG联合VCR作用后KBV200细胞内VCR浓度升高，P糖蛋白的表达下降；EGCG可增加VCR对KBV200的抑瘤作用，可降低瘤组织MDR1的表达量。结论 EGCG可增强VCR对多药耐药肿瘤细胞KBV200的细胞毒作用，机制可能与降低MDRI-mRNA、P-gp表达，提高细胞内药物浓度有关。     

Vitamin E reverses multidrug resistance in vitro and in vivo

Multidrug resistance (MDR) is a major obstacle to successful and effective chemotherapeutic treatments of cancers. This study explored the reversal effects of vitamin E on MDR tumor cells in vitro and in vivo, elucidating the potential mechanism of this reversal. VE at a concentration of 50 μM exhibited a significant reversal of the MDR effect (compared to only PTX in DMSO, p < 0.05) in two human MDR cell lines (H460/taxR and KB-8-5). The MDR cell xenograft model was established to investigate the effect of VE on reversing MDR in vivo. Mice intravenously injected with Taxol (10 mg/kg) with VE (500 mg/kg, IP) showed an ability to overcome the MDR. VE and its derivatives can significantly increase intracellular accumulation of rhodamine 123 and doxorubicin (P-gp substrate), but not alter the levels of P-gp expression. These treatments also did not decrease the levels of intracellular ATP, but were still able to inhibit the verapamil-induced ATPase activity of P-gp. The new application of VE as an MDR sensitizer will be attractive due to the safety of this treatment.     

The study of innate drug resistance of human hepatocellular carcinoma Bel7402 cell line

The innate drug resistance of human hepatocellular carcinoma (HCC) Bel7402 cell line was studied in vitro. MTT assay showed that Bel7402 cells were innately resistant to doxorubicin (Dox), and even more resistant to vincristine (VCR). This resistance could be effectively reversed by verapamil (Ver), one of the classical multidrug resistance (MDR) modulating agents. However, the differences in 5-fluorouracil (5-FU) toxicity between these two cell lines is much less and the resistance of Bel7402 cells could only be slightly reversed by Ver, which may be experimental noise. Immunocytochemical staining using anti-p-glycoprotein monoclonal antibody JSB-1 indicated that the expression of the P-glycoprotein (P-gp) in the innate Bel7402 cells was elevated compared with the sensitive KB cells. The accumulation of Dox in innate resistant Bel7402 cells was 50.7% lower than that in sensitive KB cells by using spectrofluometric analyses, and the accumulation of Dox increased 1.6 fold in Bel7402 cells in the presence of Ver. The susceptibility of Dox-induced apoptosis was also increased in the presence of Ver by using flow cytometric assay and DNA fragmentation quantitative assay as well as by Hoechst 33258 staining. It appears that the innate Bel7402 cells might be useful in screening new antitumour drugs or new chemosensitisers which could overcome the innate or acquired resistant mechanism, and the toxicity and reversal effects with 5-FU are different from those known to be P-gp substrates such as VCR, Dox, and taxol.     

LY294002与化疗药物联用对白血病K562细胞多药耐药的逆转作用

背景与目的: 传统逆转白血病多药耐药的药物由于不良反应大而限制了其在临床中的应用,进一步研究白血病多药耐药产生机制和有效逆转靶点成为攻克白血病多药耐药的关键.为此,本研究探讨LY294002[磷脂酰肌醇-3-激酶(P13-K/Akt)通路抑制剂]对人类白血病K562细胞多药耐药的逆转作用.方法: 锥虫蓝拒染法测定细胞生长增殖.Western印迹榆测K562/S和K562/D细胞中P-gp及p-Akt的表达.流式细胞术检测细胞内药物积聚.结果: K562/D细胞对柔红霉素(DNR)、多柔比星(ADR)、长春新碱(VCR)、依托泊苷(VP16)交叉耐药,相对其亲本细胞的耐药倍数分别为65、52、134和50.DNR诱导了K562/D细胞的P-gp、p-Akt过度表达.LY294002使K562/D细胞内药物积聚增加,部分逆转了K562/D细胞对DNR、ADR、VCR、VP16的耐药性(相对耐药倍数降至23、21、63和29),而对敏感细胞K562/S的耐药性无影响. 结论:LY294002部分逆转K562/D细胞的多药耐药,可能于DNR诱导K562/D细胞的P-gp、p-Akt过度表达而LY294002抑SUP13-K/Akt信号转导通路有关.     

Reversal of MDR1/P-glycoprotein-mediated multidrug resistance by vector-based RNA interference in vitro and in vivo

Overexpression of P-glycoprotein (P-gp) encoded by MDR1 gene in cancer cells results in multidrug resistance (MDR) to structurally and mechanistically different chemotherapeutic drugs, which is a major cause for cancer chemotherapy failures to cancer patients. Recently, there were several reports showing that expression of siRNAs targeting MDR1 gene is able to reverse the P-gp mediated MDR, however, the in vivo reversal effects for MDR have still not been identified. We developed a novel MDR reversal system using RNA interference technique in human epidermoid carcinoma KBv200 cells. The stably expressing MDR1 shRNA cells (KBv200/MDR1sh) were established with transfection of vector pEGFPC2-H1-MDR1shDNA containing MDR1-V siRNA expression cassette, and we found that more than 90% of MDR1 mRNA and P-gp were reduced. KBv200/MDR1sh cells simultaneously showed stably expressing EGFP and kept low MDR1 expression beyond ten passages. Compared KBv200/MDR1sh cells with KBv200 cells, resistance to vincristine and doxorubicin decreased from 62.4-fold to 10.5-fold and from 74.5-fold to 9.5-fold respectively, and intracellular doxorubicin accumulation enhanced from 0.30 +/- 0.08 nmoles/10(6) cells to 0.86 +/- 0.16 nmoles/10(6) cells, and the fluorescence intensity of intracellular Rhodamine 123 accumulation increased from 3.58 +/- 1.63/10(6) cells to 13.96 +/- 3.07/10(6) cells. In the nude mice xenografts, vincristine (0.2 mg/kg of body weight) inhibited the growth of KBv200/MDR1sh solid tumors by 42.0%, but the same dose of vincristine didn't inhibit the growth of KBv200 solid tumors significantly. These results suggest that administration of RNAi targeted MDR1 gene can effectively reverse MDR both in vitro and in vivo models.     

Potentiation of the reversal activity of SDZ PSC833 on multi-drug resistance by an anti-p-glycoprotein monoclonal antibody MRK-16

SDZ PSC833 (PSC833), an analogue of cyclosporines, is one of the most potent modulators of multi-drug resistance (MDR). We previously reported that MRK-16, an anti-P-glycoprotein MAb, enhanced MDR reversal activity of cyclosporin A (CsA) through inhibition of P-glycoprotein-mediated CsA transport. We have examined here whether MRK-16 can enhance MDR reversal activity of PSC833. We found that MRK-16 potentiated the MDR reversal activity of PSC833, and of CsA, in MDR sublines of human myelocytic leukemia K562 and human ovarian cancer A2780 cells. Like MRK-16 combined with CsA, MRK-16 enhanced the effect of a sub-optimum dose of PSC833 on vincristine accumulation in MDR cells. However, MRK-16 could not increase cellular accumulation of PSC833 in MDR tumor cells, yet it could increase cellular accumulation of CsA. P-glycoprotein could not transport PSC833 but could transport CsA. Our results indicate that MRK-16 potentiates the MDR reversal activity of both PSC833 and CsA, yet also suggest that the molecular mechanism of the potentiation differs between the two substances. © 1996 Wiley-Liss, Inc.     

Reversal effect of tyroservatide (YSV) tripeptide on multi-drug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU

Tyroservatide (YSV) is an active, low-molecular-weight polypeptide that has been shown to have antitumor effects on human hepatocellular carcinoma BEL-7402 cells in vitro and in vivo. Multi-drug resistance (MDR) represents a major obstacle to the success of cancer chemotherapy. To enhance the chemosensitivity of tumor cells, attention has been focused on MDR modulators. In this study, we evaluated the reversal effect of YSV on MDR, and explored its mechanism of action in vitro. Administration of YSV reversed the multi-drug resistance of human hepatocellular carcinoma BEL-7402/5-FU cells significantly. The intracellular accumulation of doxorubicin and Rhodamine-123 (Rh123) were increased, which implied that the function of the P-glycoprotein (P-gp) efflux pump was inhibited by YSV. Moreover, the mRNA and protein expression of multi-drug resistance gene (MDR1) were also decreased by YSV. We observe that lung-resistance protein (LRP) and multi-drug resistance-associated protein (MRP1) each contribute to MDR in BEL-7402/5-FU cells as well. The mRNA and protein expression of LRP were decreased by YSV. No significant change was observed in mRNA expression of MRP1. However, we observe that the MRP1 protein level was reduced after treatment with YSV. These data demonstrate that YSV effectively reverses MDR in BEL-7402/5-FU cells, and that its mechanism of action is associated with the down-regulation of MDR1, MRP1 and LRP expression, as well as the inhibition of P-gp function.