Generation of multidrug resistant lymphoma cell lines stably expressing P-glycoprotein

The objective of this study was to generate new P-glycoprotein (P-gp)-expressing multidrug resistant (MDR) cell lines by drug selection. Since our previous studies have been carried out with cells infected with a P-gp-containing vector, it was important to confirm our findings in cells generated by drug selection. In this report, we describe three B-lymphoma cell lines which became drug-resistant by stepwise exposure to vincristine (VCR): Raji cells resistant to 18 nM VCR (R18V), Namalwa cells resistant to 21 nM VCR (N21V) and DHL-4 cells resistant to 12 nM VCR (DHL-4/12V). Cells overexpressed P-gp and continued to express CD19, CD20 and CD22, all of which are targets for monoclonal antibody (MAb) therapy. The P-gp pump in these new cells was functional as determined by the efflux of Rhodamine 123 and DIOC2, and the three cell lines were resistant to several chemotherapeutic drugs. We further determined that their P-gp phenotype was stable in xenografted SCID mice and that the tumors were also resistant to chemotherapy. We will now use these new MDR cells to determine whether monoclonal antibodies against CD19 and -20 can reverse P-gp, as we previously demonstrated using Namalwa cells infected with a human mdr1 gene-containing retrovirus.     

No evidence of significant activity of the multidrug resistance gene product in primary human breast cancer

Background: The discovery of the multidrug resistance (MDR1) gene product P-glycoprotein (P-gp) has been widely seen as an important milestone in our understanding of the mechanisms underlying the clinical phenomenon of the emergence of resistant cells. MDR1 expression has been shown for numerous solid tumors and for virtually all hematologic malignancies. Nevertheless, results regarding MDR1/P-gp expression in human breast cancer have been controversial and the results of clinical trials on modulation of P-gp activity have not been encouraging.Patients and methods: MDR1/P-gp expression and the function of the P-gp pump were investigated in 61 tumor samples from patients with primary breast cancers by multiparameter analysis using MDR1-RT-PCR, immunohistochemistry with two MAbs (UIC2 and MRK 16) and the rhodamine 123 (Rh123) efflux assay. The cellular composition of the tumor cell suspension was analyzed by using specific MAbs against the P-gp expressing lymphocyte subsets CD4, CD8 and CD56, as well as against the HER-2/neu gene product, which was used to identify breast carcinoma cells.Results: UIC2 and MRK16 revealed a staining positivity in 72% and 75% of samples, respectively. A positive MDR1-RT-PCR signal was detected in 62% of the samples. Nevertheless, no correlation between immunohistochemistry and RT-PCR could be established. Furthermore, there was no correlation between HER-2/neu expression and MDR1-RT-PCR or P-gp immunohistochemical assays. A contamination by CD8+ and CD4+ lymphocytes was established in 100% and 84% of tumor cell suspensions, respectively. As assessed by the Rh123 efflux assay CD8+ and the CD4+ lymphocytes exhibited marked P-glycoprotein activity, whereas such activity was not detectable in a single instance for the breast carcinoma cells. In MDR1-RT-PCR positive samples, contamination by CD8 lymphocytes averaged 4.3%, while the contamination of CD8 cells in the MDR1 mRNA-negative samples was only 2.4% (P = 0.007). This signal vanished after elimination of the lymphocyte subpopulations by T-cell rosetting.Conclusions: In primary breast cancer detection of MDR1 gene expression by means of RT-PCR or immunohistochemical assays is not indicative for the MDR phenotype, since there is no evidence of significant activity of the P-gp pump.     

Hyperforin inhibits P-gp and BCRP activities in chronic lymphocytic leukaemia cells and myeloid cells

We showed previously that hyperforin (HF), a natural phloroglucinol, stimulated apoptosis in B cell chronic lymphocytic leukaemia cells (CLL) and displayed anti-angiogenic properties. In the present work, we investigated the effects of hyperforin on the activity of P-gp/MDR1, an ABC (ATP-binding cassette) transporter putatively involved in multidrug resistance (MDR). Ex vivo treatment of CLL cells with HF markedly impaired the activity of P-gp, as measured by the inhibition of the capacity of the treated cells to efflux the rhodamine 123 probe. In addition, most CLL cells expressed breast cancer resistance protein (BCRP), another ABC transporter. The activity of BCRP was also inhibited by HF, as assessed by the impaired capacity of HF-treated CLL cells to efflux the specific probe mitoxantrone. The capacity of HF to reverse P-gp and BCRP activity was confirmed in myeloid leukaemia cell lines, notably in HL-60/DNR cells selected for their resistance to daunorubicine and overexpressing P-gp. Our results therefore suggest that HF might be of interest in the therapy of CLL and other haematological malignancies through its potential capacity to revert MDR in addition to its pro-apoptotic properties.     

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。     

Chimeric, divalent and tetravalent anti-CD19 monoclonal antibodies with potent in vitro and in vivo antitumor activity against human B-cell lymphoma and pre-B acute lymphoblastic leukemia cell lines

CD19 is an attractive therapeutic target for treating human B-cell tumors. In our study, chimeric (c) divalent (cHD37) and tetravalent (cHD37-DcVV) anti-CD19 monoclonal antibodies (MAbs) were constructed, expressed and evaluated for their binding to human 19-positive (CD19(+)) tumor cell lines. They were also tested for proapoptotic activity and the ability to mediate effector functions. The antitumor activity of these MAbs was further tested in mice xenografted with the CD19(+) Burkitt's lymphoma cell line, Daudi or the pre-B acute lymphoblastic leukemia (ALL) cell line, NALM-6. The cHD37 and cHD37-DcVV MAbs exhibited specific binding and comparable proapoptotic activity on CD19(+) tumor cell lines in vitro. In addition, the cHD37 and cHD37-DcVV MAbs were similar in their ability to mediate antibody-dependent cell-mediated phagocytosis (ADCP). However, the tetravalent cHD37-DcVV MAb bound more avidly, had a slower dissociation rate, and did not internalize as well. It also had enhanced antibody-dependent cellular cytotoxicity (ADCC) with human but not murine effector cells. The cHD37 and cHD37-DcVV MAbs exhibited comparable affinity for the human neonatal Fc receptor (FcRn) and similar pharmacokinetics (PKs) in mice. Moreover, all the HD37 constructs were similar in extending the survival of mice xenografted with Daudi or NALM-6 tumor cells. Therefore, the cHD37 and cHD37-DcVV MAbs have potent antitumor activity and should be further developed for use in humans. Although not evident in mice, due to its increased ability to mediate ADCC with human but not mouse effector cells, the cHD37-DcVV MAb should have superior therapeutic efficacy in humans.     

人参皂甙Rh2逆转P-gp介导的MCF-7/ADM多药耐药性的基础研究

目的：研究人参皂甙Rh：在逆转多药耐药（MDR）方面的作用及其机理。方法：Rh：和阿霉素单独或联合作用于MCF-7及MCF-7／ADM细胞，应用MTT法确定各组细胞的IC50。罗丹明123加入各组细胞，流式细胞仪分析Rh2和维拉帕米抑制罗丹明123外排的情况。RT—PCR检测各组mdrl mRNA表达量及流式细胞仪检测各组P—gP的表达情况。结果：Rh2可以显著降低MCF-7／ADM的ADM IC50（P〈0．05），对MCF-7细胞没有影响。Rh2和维拉帕米可以增加MCF-7／ADM细胞内罗丹明123荧光表达率（P〈0．05），Rh3比维拉帕米抑制罗丹明123外排作用更强。在MCF-7细胞内Rh2和维拉帕米无此作用。Rh2对MCF-7／ADM细胞mdrl mRNA表达及P—gP表达没有影响。结论：人参皂甙Rh2可以有效逆转P—gP介导的人乳腺癌细胞耐药细胞系MCF-7／ADM的耐药性。     

Thalidomide does not interact with P-glycoprotein

Background: There is growing clinical interest in thalidomide for the treatment of various disorders due to its anti-inflammatory, immunomodulatory, and anti-angiogenic properties. In numerous clinical trials thalidomide is used as an adjunct to standard therapy. Therefore, clinicians should be aware of all possible drug–drug interactions that might occur with this drug. P-glycoprotein (P-gp), a drug efflux transporter that is expressed in many tissues, is the cause of several drug–drug interactions. P-gp induction or inhibition can lead to ineffective therapy or side-effects. In this study, we investigated thalidomide’s potential to cause drug–drug interactions on the level of P-gp. Methods: LS180 cells were incubated with thalidomide for 72 h in order to determine P-gp induction using real-time RT-PCR. A human leukaemia cell line over-expressing MDR1 (CCRF-CEM/MDR1) was used to measure uptake of rhodamine 123, a P-gp substrate, in the presence of thalidomide. Dose-dependent and bi-directional transport of thalidomide through Caco-2 cell monolayers was performed to assess site-directed permeability. Transport rates were determined using HPLC including chiral separation of the thalidomide enantiomers. Results: Thalidomide did not induce P-gp expression in LS180 cells. The uptake of rhodamine 123 in CCRF cells over-expressing MDR1 was not influenced by co-incubation with thalidomide. The transport through Caco-2 monolayers was linear and the permeability was similar for both directions. No differences between the thalidomide enantiomers were observed. Conclusions: Our study indicates that thalidomide is neither a substrate, nor an inhibitor or an inducer of P-gp. Therefore, P-gp-related drug–drug interactions with thalidomide are not likely.     

Benzquinamide inhibits P-glycoprotein mediated drug efflux and potentiates anticancer agent cytotoxicity in multidrug resistant cells.

We have previously shown that efflux of cytotoxic drugs from multidrug resistant (MDR) cell lines can be quantitated at the single cell level using a sensitive fluorescence microscopy technique. Based on the structure of compounds which inhibited the efflux of Rhodamine-123 (Rho-123) using this methodology, we hypothesized that the antiemetic, antihistaminic agent benzquinamide (BZQ) would interfere with P-glycoprotein (P-gp) mediated drug transport and potentiate the effects of anticancer agents in MDR cell lines. We show that BZQ interferes with P-gp mediated drug efflux and increases drug accumulation in MDR cells using Rho-123 as a fluorescent probe. BZQ increases the cytotoxicity of chemotherapeutic agents to both human and hamster MDR cell lines in vitro. A slight increase in cytotoxicity to chemotherapeutic agents is also observed in the parental cell lines with BZQ. BZQ increases [3H]daunorubicin accumulation and inhibits the binding of [125I]iodoaryl azidoprazosin to the P-gp in MDR cells. BZQ is a new agent to increase the cytotoxic effects of anticancer agents in MDR cells and may ultimately prove useful as an adjunct in cancer chemotherapy.     

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.     

Inhibition of P-glycoprotein transport activity in a resistant mouse lymphoma cell line by diterpenic lactones

Multidrug resistance (MDR) is believed to be a major reason for the failure of cancer treatment. It is in most cases caused by the activity of the various ABC transporters, multidrug resistance (MDR) gene-encoded p-glycoproteins that pump anticancer drugs out of the cells. P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) are the most important and widely studied members of the ABC superfamily of transporters. The ability of four diterpenic lactones isolated from Euphorbia species to modulate the transport activity of P-gp in mouse lymphoma cells was evaluated by flow cytometry. The reversion of MDR was investigated by using a standard functional assay with rhodamine 123 as a fluorescent substrate analogue of doxorubicin. Verapamil was applied as a positive control. All the compounds were able to reverse the MDR of the tested human MDR1 gene-transfected mouse lymphoma cells, in a concentration-dependent manner from 4 to 40 microg/mL, in a short-term experiment below the cytotoxic doses.     

Comparison of two functional flow cytometric assays to assess P-gp activity in acute leukemia

One of the possible causes of treatment failure in acute leukemia is the emergence of multidrug resistance caused by P-glycoprotein (P-gp) overexpression. We compared a flow cytometric assay using JC-1 with a technique using rhodamine 123 (rho123) to evaluate the P-gp function in acute leukemia. Samples from 50 acute leukemia patients were analyzed by both functional assays. The P-gp expression was assessed by an immunological flow cytometric test and the association between the P-gp status and the clinical outcome was evaluated. Of all samples, 28% showed a reversible JC-1 efflux and 36% scored positive for the rho123 assay. In two cases, the leukemic blasts showed a reversible JC-1 efflux whereas they were negative for rho123. These patients had blast cells with a very low P-gp activity. Six samples scored positive for the rho123 assay but were negative for the JC-1 test. Five of these samples did not express P-glycoprotein and were considered false positive. We found a strong correlation between the JC-1 and the rho123 test (R(s)=0.59, p<0.0001) and the JC-1 and the immunological assay (R(s)=0.29, P=0.05). There was also an association between the JC-1 status and the clinical outcome of adult patients (chi2=6.30, P=0.04). In conclusion, we recommend the JC-1 assay to study the P-gp activity in acute leukemia because it is more specific and less labor intensive than conventional functional flow cytometric tests using rhodamine 123. In addition, the JC-1 assay can be used to identify adult patients with an increased risk for adverse clinical outcome.     

Polyaromatic alkaloids from marine invertebrates as cytotoxic compounds and inhibitors of multidrug resistance caused by P-glycoprotein.

The effects of several members of the family of lamellarins, polyaromatic alkaloids isolated from tunicates belonging to the genus Didemnum, on the growth of several tumour cell lines and on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR), were investigated. Cytotoxicity experiments of lamellarins were performed on a panel of tumour cell lines, including two multidrug-resistant cell lines. Some lamellarins showed good anti-tumour activity, with similar levels of cytotoxicity against both the resistant and their corresponding parental cell lines. Two lamellarins displayed a high potency against lung carcinoma cells. Studies of the resistance modifier activity of the different lamellarins at non-toxic concentrations were also carried out in cells exhibiting MDR, and lamellarin I was selected for the highest chemosensitising activity. At non-toxic doses, verapamil and lamellarin I effectively increased the cytotoxicity of doxorubicin, vinblastine and daunorubicin in a concentration-dependent manner in multidrug-resistant cells, but the potency of lamellarin I as a MDR modulator was 9- to 16-fold higher than that of verapamil. In vitro measurements of rhodamine 123 accumulation in the multidrug-resistant Lo Vo/Dx cells suggest that lamellarin I reverses MDR by directly inhibiting the P-gp-mediated drug efflux. This work underscores the possibility of using these marine-derived compounds as a potential new source of anti-tumoral drugs active on resistant cells as well as of non-toxic modulators of the MDR phenotype.     

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。     

CD16 surface molecules regulate the cytolytic function of CD3CD16+ human natural killer cells

Monoclonal (IgG) antibodies (MAbs) directed to CD16 molecules efficiently induced lysis of the IgG-binding P815 target cells. A similar effect was observed with selected anti-CD2 MAbs. While combinations of 2 appropriate anti-CD2 MAbs were required for induction of T lymphocyte activation, single stimulatory anti-CD2 MAbs were sufficient for inducing cytolytic function in CD3- CD16+ lymphocytes. In order to study possible regulatory mechanisms existing in the process of activation and induction of the cytolytic machinery of CD3- CD16+ effector cells, we utilized the anti-CD16 OKNK MAb. Being of IgM isotype, the OKNK MAb does not allow cross-linking between CD3- D16+ lymphocytes and target cells. Pre-treatment of effector cells with OKNK MAb sharply inhibited the target cell lysis induced by either anti-CD16 (IgG) MAbs or stimulatory anti-CD2 MAb. Moreover, a strong inhibitory activity of PHA-induced target cell lysis and even of "spontaneous" lysis (at high effector:target ratio) was observed. In contrast, in CD3+ CD16+ clones, OKNK MAb selectively inhibited the cell triggering induced by anti-CD16 MAbs (but not by anti-CD3, anti-CD2 MAbs or PHA). Our data indicate that CD16 receptor molecules expressed by CD3- CD16+ lymphocytes down-regulate cell responses to anti-CD2 MAbs or PHA, and then exert a regulatory role in the cytolytic function of these cells.     

UMMS-4 enhanced sensitivity of chemotherapeutic agents to ABCB1-overexpressing cells via inhibiting function of ABCB1 transporter

Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters through efflux of antineoplastic agents from cancer cells is a major obstacle to successful cancer chemotherapy. The inhibition of these ABC transporters is thus a logical approach to circumvent MDR. There has been intensive research effort to design and develop novel inhibitors for the ABC transporters to achieve this goal. In the present study, we evaluated the ability of UMMS-4 to modulate P-glycoprotein (P-gp/ABCB1)-, breast cancer resistance protein (BCRP/ABCG2)- and multidrug resistance protein (MRP1/ABCC1)-mediated MDR in cancer cells. Our findings showed that UMMS-4, at non-cytotoxic concentrations, apparently circumvents resistance to ABCB1 substrate anticancer drugs in ABCB1-overexpressing cells. When used at a concentration of 20 μmol/L, UMMS-4 produced a 17.53-fold reversal of MDR, but showed no effect on the sensitivity of drug-sensitive parental cells. UMMS-4, however, did not significantly alter the sensitivity of non-ABCB1 substrates in all cells and was unable to reverse ABCG2- and ABCC1-mediated MDR. Additionally, UMMS-4 profoundly inhibited the transport of rhodamine 123 (Rho 123) and doxorubicin (Dox) by the ABCB1 transporter. Furthermore, UMMS-4 did not alter the expression of ABCB1 at the mRNA and protein levels. In addition, the results of ATPase assays showed that UMMS-4 stimulated the ATPase activity of ABCB1. Taken together, we conclude that UMMS-4 antagonizes ABCB1-mediated MDR in cancer cells through direct inhibition of the drug efflux function of ABCB1. These findings may be useful for the development of safer and more effective MDR modulator.     

Comparison of staurosporine and four analogues: their effects on growth, rhodamine 123 retention and binding to P-glycoprotein in multidrug-resistant MCF-7/Adr cells.

The potent kinase inhibitor staurosporine and its protein kinase C (PKC)-selective analogue CGP 41251 are known to sensitise cells with the multidrug resistance (MDR) phenotype mediated by P-glycoprotein (P-gp) to cytotoxic agents. Here four PKC-selective staurosporine cogeners, CGP 41251, UCN-01, RO 31 8220 and GF 109203X, were compared with staurosporine in terms of their MDR-reversing properties and their susceptibility towards P-gp-mediated drug efflux from MCF-7/Adr cells. Staurosporine was the most potent and the bisindolylmaleimides RO 31 8220 and GF 109203X the least potent cytostatic agents. When compared with MCF-7 wild-type cells, MCF-7/Adr cells were resistant towards the growth-arresting properties of RO 31 8220 and UCN-01, with resistance ratios of 12.6 and 7.0 respectively. This resistance could be substantially reduced by inclusion of the P-gp inhibitor reserpine. The ratios for GF 109203X, staurosporine and CGP 41251 were 1.2, 2.0 and 2.9 respectively, and they were hardly affected by reserpine. These results suggest that RO 31 8220 and UCN-01 are avidly transported by P-gp but that the other compounds are not. Staurosporine and CGP 41251 at 10 and 20 nM, respectively, decreased efflux of the P-gp probe rhodamine 123 (R123) from MCF-7/Adr cells, whereas RO 31 8220 and GF 109203X at 640 nM were inactive. CGP 41251 was the most effective and GF 109203X the least effective inhibitor of equilibrium binding of [3H]vinblastine to its specific binding sites, probably P-gp, in MCF-7/Adr cells. Overall, the results imply that for this class of compound the structural properties that determine susceptibility towards P-gp-mediated substrate transport are complex. Comparison with ability to inhibit PKC suggests that the kinase inhibitors affect P-gp directly and not via inhibition of PKC. Among these compounds CGP 41251 was a very potent MDR-reversing agent with high affinity for P-gp and least affected by P-gp-mediated resistance, rendering it an attractive drug candidate for clinical development.     

Regulation by glutathione of drug transport in multidrug-resistant human lung tumour cell lines overexpressing multidrug resistance-associated protein.

Previous studies have shown that multidrug resistance (MDR) in the doxorubicin-selected lung tumour cell lines COR-L23/R, GLC4/ADR and MOR/R is associated with overexpression of the MRP gene. In this study we report that resistance to daunorubicin, vincristine and rhodamine 123 can be partially reversed in these cell lines by exposing the cells to buthionine sulphoximine (BSO), an inhibitor of glutathione (GSH) synthesis. This effect of BSO on drug resistance was associated with an increased intracellular accumulation of daunorubicin and rhodamine 123, owing to inhibition of the enhanced drug efflux. In contrast, the accumulation of daunorubicin was not increased by BSO treatment in a P-glycoprotein (P-gp)-mediated MDR cell line. BSO treatment (25 microM, 20 h) of the cell lines resulted in 60-80% depletion of cellular GSH levels. The effects of BSO on daunorubicin accumulation in the COR-L23/R and GLC4/ADR cells were associated with cellular GSH depletion. In addition, increase of cellular GSH levels in BSO-treated COR-L23/R and GLC4/ADR cells as a result of incubation with 5 mM GSH ethyl ester restored the accumulation deficit of daunorubicin. However, the transport of daunorubicin did not increase the GSH release in any of the cell lines. These results demonstrate that drug transport in MRP- but not in P-gp-overexpressing MDR tumour cell lines can be regulated by intracellular GSH levels.     

阿帕替尼逆转P-gp转运体介导的乳腺癌化疗多药耐药性的作用及其机制

目的 探讨阿帕替尼对人乳腺癌化疗多药耐药性的逆转作用及其机制。方法 不同浓度的阿帕替尼作用于体外培养的人乳腺癌MCF-7及MCF-7/ADR细胞48 h,MTT法检测阿帕替尼对两种细胞的细胞毒性;低毒浓度的阿帕替尼与化疗药物紫杉醇及阿霉素联用,探讨阿帕替尼对两种细胞化疗耐药性的影响;采用流式细胞术检测阿帕替尼对罗丹明123在MCF-7及MCF-7/ADR细胞内蓄积的影响;采用Pgp-Glo? Assay Systems试剂盒观察阿帕替尼对多药耐药相关蛋白P-gp的ATPase活性的影响;采用Western blot法检测阿帕替尼对P-gp表达及AKT磷酸化的影响。结果 阿帕替尼可浓度依赖性地逆转乳腺癌MCF-7/ADR细胞对紫杉醇及阿霉素的多药耐药性（P<0.05）、增加罗丹明123在MCF-7/ADR细胞内的蓄积（P<0.05）及激活P-gp转运体的ATPase活性（P<0.05）,而对P-gp的表达没有影响;此外,阿帕替尼不改变AKT的磷酸化水平。结论 阿帕替尼可能通过抑制P-gp转运体的外排功能逆转P-gp转运体介导的乳腺癌化疗多药耐药性。