Double-edged sword of chemosensitizer: increase of multidrug resistance protein (MRP) in leukemic cells by an MRP inhibitor probenecid

The multidrug resistance protein (MRP) is a drug efflux membrane pump conferring multidrug resistance to tumor cells. Clinical trials have been undertaken to improve the effectiveness of chemotherapy by adding an MRP inhibitor to the treatment regimen. This study attempted not only to determine novel resistance mechanisms in MRP-overexpressing AML cells (AML-2/DX100) by chronic exposure to doxorubicin in the presence of an MRP inhibitor probenecid but also to find out whether probenecid could increase MRP levels. AML-2/DXPBA cultured in the presence of probenecid (600 microM) and doxorubicin (100 ng/ml) showed a higher level of the multidrug resistance (MDR) phenotype when compared to AML-2/DX100. AML-2/DXPBA showed increased levels of MRP compared to those of AML-2/DX100. Probenecid increased the MRP levels without an increase in MRP mRNA in AML-2/WT in both a time- and dose-dependent manner. Of the MRP inhibitors including probenecid, ofloxacin, erythromycin, and rifampicin used in this study, only probenecid showed a marked chemosensitizing effect in AML-2/DX100 but not in HL-60/Adr, suggesting that the chemosensitizing effects of the MRP inhibitors vary according to the type of resistant cells. The maximum noncytotoxic concentrations of these MRP inhibitors increased the MRP levels to various degrees in both AML-2/WT and HL-60/WT. However, the chemosensitizing effects of the MRP inhibitors were not correlated with their MRP-increasing effects. Altogether, MRP inhibitors such as probenecid have been shown to function as a double-edged sword, indicating that they are not only an effective chemosensitizer of MRP-associated MDR tumor cells but also an MRP activator. Therefore caution should be taken whenever using MRP inhibitors to reverse MRP-mediated multidrug resistance in clinical cancer chemotherapy as well as when used to inhibit MRP expression in vitro.     

A novel system for evaluation of drug mixtures for potential efficacy in treating multidrug resistant cancers

Multidrug resistant (MDR) cancer is difficult to treat. Chemicals that are effective MDR modulators have never exited clinical trials as FDA approved products due to side effects. It has been hypothesized that using a combination of chemotherapeutics with a mixture of MDR modulators (each with different side effects) may lead to useful treatment strategies. Because the experimental space for combination treatments can be large, this space may be impracticable to explore using animal studies. Here we describe an in vitro system based on microfabrication and cell culture that can potentially be used to explore large experimental spaces efficiently. The Microscale Cell Culture Analog (µCCA) concept mimics the body's response using interconnected compartments that represent various tissues or organs. A µCCA is based on the structure of an appropriate physiologically based pharmacokinetic (PBPK) model and emulates the body's dynamic response to exposure to various drugs and chemicals. For this problem we have chosen a µCCA with living cells representing the liver (HepG2/C3A), bone marrow (MEG‐01), uterine cancer (MES‐SA), and a MDR variant of uterine cancer (MES‐SA/DX‐5). In proof of concept experiments we found in 24 h “acute” exposures and 72 h treatments that the µCCA system predicts combining the chemotherapeutic, doxorubicin, with cyclosporine and nicardipine, as MDR modulators will have greater efficacy than using doxorubicin by itself or with either modulator alone. This combined strategy is selective in inhibiting MES‐SA/DX‐5 cell proliferation and may prove to be advantageous in vivo by specifically targeting MDR cancer with acceptable side‐effects. This cell specific synergy was not observed in traditional 96‐well plate assays. By combining the µCCA with a PBPK model, appropriate drug doses and area under the curve exposure for in vivo trials can be extrapolated directly from the results obtained with this device. This device and approach should be useful in screening potential drug/modulator combinations to determine candidate treatments for MDR cancer. Indeed this approach may be useful for in vitro evaluation of human response to a wide range of exposures to mixtures of chemicals or drugs. Biotechnol. Bioeng. 2009;103: 187–198. © 2008 Wiley Periodicals, Inc.     

P-glycoprotein subcellular localization and cell morphotype in MDR1 gene-transfected human osteosarcoma cells.

Efflux of chemotherapy agents by P-glycoprotein at the plasma membrane is thought to be a major cause of cancer multidrug-resistance (MDR). However, the mechanism underlying the cellular accumulation and distribution of cytotoxic drugs is still poorly defined. We have recently found that P-glycoprotein is expressed also in the nucleus of MDR cell lines selected in doxorubicin (DXR), suggesting the possible involvement of this protein in the direct extrusion of the drug from the nucleus of resistant cells. In this study, we analyzed the subcellular localization of P-glycoprotein, in a series of U-2 OS osteosarcoma cell clones transfected with MDR1 gene in order to verify whether the nucleus is a constant site for the localization and functional activity of P-glycoprotein, and in which way some aspects of cell morphology related to MDR depend on the subcellular P-glycoprotein localization rather than on the exposure to the selective drug. Our results indicate that to achieve a subcellular drug distribution prevailing in the cytoplasm but not in the nucleus, a significant increase in the expression of P-glycoprotein at the different cellular compartments, including the plasma membrane, the cytoplasm, and the nucleus, is needed, although the in vitro drug resistance appears to be mainly dependent on the expression of P-glycoprotein at the cell surface. With regard to the morphological characteristics of MDR cells involving the cell surface and the chromatin arrangement, the influence of DXR appears to be prevalent, although P-glycoprotein overexpression cannot be excluded.     

Mitochondrial alterations induced by serum amine oxidase and spermine on human multidrug resistant tumor cells

Summary. Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H₂O₂ and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H₂O₂. However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.     

A strong glutathione S-transferase inhibitor overcomes the P-glycoprotein-mediated resistance in tumor cells. 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) triggers a caspase-dependent apoptosis in MDR1-expressing leukemia cells

The new glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines, i.e. CEM-VBL10, CEM-VBL100, and U-2 OS/DX580. The mechanism of cell death triggered by NBDHEX has been deeply investigated in leukemia cell lines. Kinetic data indicate a similar NBDHEX membrane permeability between multidrug resistance cells and their sensitive counterpart revealing that NBDHEX is not a substrate of the P-glycoprotein export pump. Unexpectedly, this molecule promotes a caspase-dependent apoptosis that is unusual in the P-glycoprotein-overexpressing cells. The primary event of the apoptotic pathway is the dissociation of glutathione S-transferase P1-1 from the complex with c-Jun N-terminal kinase. Interestingly, leukemia MDR1-expressing cells show lower LC50 values and a higher degree of apoptosis and caspase-3 activity than their drug-sensitive counterparts. The increased susceptibility of the multidrug resistance cells toward the NBDHEX action may be related to a lower content of glutathione S-transferase P1-1. Given the low toxicity of NBDHEX in vivo, this compound may represent an attractive basis for the selective treatment of MDR1 P-glycoprotein-positive tumors.     

Activation of a transfected FGFR-1 receptor in Madin-Darby epithelial cells results in a reversible loss of epithelial properties

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine-kinase family of receptors. We have found that Madin-Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse-derived cDNA encoding FGF receptor-1 (FGFR-1) in MDCK cells resulted in the acquisition of a fibroblast-like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF-induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR-1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR-1 -expressing MDCK cells formed monolayers tht displayed high electrical resistance. Incubation of monolayers of FGFR-1-transfected cells with bFGF resulted in the loss of trans-epithelial resitance. Monolayers of parental MDCK cells did not lose their trans-epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans-epithelial resistance, indicating that the effects on the trans-epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans-epithelial resistance is separable from the morphological transition. © 1995 Wiley-Liss, Inc.     

The drug resistance proteins, multidrug resistance-associated protein and P-glycoprotein, do not confer resistance to Fas-induced cell death

BACKGROUND: Multidrug resistance (MDR) is mediated by the drug resistance proteins, the multidrug resistance-associated protein (MRP) and P-glycoprotein, both of which confer resistance by the active efflux of chemotherapeutic drugs from the cell. Reduced Fas (CD95/APO-1) expression and resistance to Fas-mediated apoptosis have also been correlated with P-glycoprotein-mediated MDR. METHODS: We investigated cell surface Fas expression (using anti-Fas monoclonal antibody DX2.1) in a series of MRP-expressing drug-resistant leukemia sublines, and P-glycoprotein-expressing leukemia sublines, and their susceptibility to apoptosis induced by anti-Fas treatment (CH-11 monoclonal antibody). Caspase-3 activation was detected by Western blot and apoptosis was determined by flow cytometry with 7-aminoactinomycin D (7-AAD) staining of cells. RESULTS: Fas expression was not reduced in either the MRP- or P-glycoprotein-expressing drug-resistant cell lines, although expression was reduced by 15% in one low-level drug-resistant subline. Expression of MRP or P-glycoprotein did not confer resistance to caspase-3 activation or to anti-Fas-induced cell death. CONCLUSIONS: MDR mediated by the drug transport proteins MRP and P-glycoprotein does not correlate with resistance to Fas-mediated cell death or resistance to caspase-3 activation.     

Are altered pHi and membrane potential in hu MDR 1 transfectants sufficient to cause MDR protein-mediated multidrug resistance?

Multidrug resistance (MDR) mediated by overexpression of the MDR protein (P-glycoprotein) has been associated with intracellular alkalinization, membrane depolarization, and other cellular alterations. However, virtually all MDR cell lines studied in detail have been created via protocols that involve growth on chemotherapeutic drugs, which can alter cells in many ways. Thus it is not clear which phenotypic alterations are explicitly due to MDR protein overexpression alone. To more precisely define the MDR phenotype mediated by hu MDR 1 protein, we co-transfected hu MDR 1 cDNA and a neomycin resistance marker into LR73 Chinese hamster ovary fibroblasts and selected stable G418 (geneticin) resistant transfectants. Several clones expressing different levels of hu MDR 1 protein were isolated. Unlike previous work with hu MDR 1 transfectants, the clones were not further selected with, or maintained on, chemotherapeutic drugs. These clones were analyzed for chemotherapeutic drug resistance, intracellular pH (pHi), membrane electrical potential (Vm), and stability of MDR 1 protein overexpression. LR73/hu MDR 1 clones exhibit elevated pHi and are depolarized, consistent with previous work with LR73/mu MDR 1 transfectants (Luz, J.G. L.Y. Wei, S. Basu, and P.D. Roepe. 1994. Biochemistry. 33:7239-7249). The extent of these perturbations is related to the level of hu MDR 1 protein that is expressed. Cytotoxicity experiments with untransfected LR73 cells with elevated pHi due to manipulating percent CO2 show that the pHi perturbations in the MDR 1 clones can account for much of the measured drug resistance. Membrane depolarization in the absence of MDR protein expression is also found to confer mild drug resistance, and we find that the pHi and Vm changes can conceivably account for the altered drug accumulation measured for representative clones. These data indicate that the MDR phenotype unequivocally mediated by MDR 1 protein overexpression alone can be fully explained by the perturbations in Vm and pHi that accompany this overexpression. In addition, MDR mediated by MDR protein overexpression alone differs significantly from that observed for MDR cell lines expressing similar levels of MDR protein but also exposed to chemotherapeutic drugs.     

2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one derivatives, novel type of cytotoxic agents active on multidrug-resistant cell lines. Synthesis and biological evaluation

We have earlier postulated that the presence of a pyridazone ring fused with an anthracenedione moiety resulted in the analog's ability to overcome multidrug resistance of tumor cells [J. Med. Chem.1999, 42, 3494]. High cytotoxic activity of obtained anthrapyridazones [Bioorg. Med. Chem.2003, 11, 561] toward the resistant cell lines, prompted us to synthesize the similarly modified acridine compounds. A series of pyridazinoacridin-3-one derivatives (2b-h) were prepared from the reaction of 9-oxo-9,10-dihydroacridine-1-carboxylate with POCl(3), followed by addition of the appropriate (alkylamino)alkylhydrazines. In vitro cytotoxic activity toward sensitive and resistant leukemia cell lines: L1210, K562, K562/DX, HL-60, HL-60/VINC, and HL-60/DX, with various type of multidrug resistance (MDR and MRP) was determined. The compounds studied exhibited in comparison to the reference cytostatics (DX, MIT) desirable very low resistance indexes (RI). Variations have been observed depending upon the substituent and the type of drug exporting pump. The cytotoxic activities of examined compounds, as well as of model anthrapyridazone derivative PDZ, were lower than those of reference drugs (DX, MIT) due to their diminished affinity to DNA.     

miR‐495 sensitizes MDR cancer cells to the combination of doxorubicin and taxol by inhibiting MDR1 expression

MDR1 is highly expressed in MDR A2780DX5 ovarian cancer cells, MDR SGC7901R gastric cancer cells and recurrent tumours. It pumps cytoplasmic agents out of cells, leading to decreased drug accumulation in cells and making cancer cells susceptible to multidrug resistance. Here, we identified that miR‐495 was predicted to target ABCB1, which encodes protein MDR1. To reduce the drug efflux and reverse MDR in cancer cells, we overexpressed a miR‐495 mimic in SGC7901R and A2780DX cells and in transplanted MDR ovarian tumours in vivo. The results indicated that the expression of MDR1 in the above cells or tumours was suppressed and that subsequently the drug accumulation in the MDR cells was decreased, cell death was increased, and tumour growth was inhibited after treatment with taxol‐doxorubicin, demonstrating increased drug sensitivity. This study suggests that pre‐treatment with miR‐495 before chemotherapy could improve the curative effect on MDR1‐based MDR cancer.     

Quinoline derivative KB3-1 potentiates paclitaxel induced cytotoxicity and cycle arrest via multidrug resistance reversal in MES-SA/DX5 cancer cells

AIMS: The resistance to chemotherapeutic drugs is a major problem for successful cancer treatment. Multidrug resistance (MDR) phenotype is characterized by over-expression of P-glycoprotein (P-gp) on the cancer cell plasma membrane that extrudes drugs out of the cells. Therefore, novel MDR reversal agents are desirable for combination therapy to reduce MDR and enhance anti-tumor activity. Thus, the present study was aimed to evaluate the potent efficacy of novel quinoline derivative KB3-1 as a potent MDR-reversing agent for combined therapy with TAX. MAIN METHODS: MDR reversing effect and TAX combined therapy were examined by Rhodamine accumulation and efflux assay and Confocal immunofluorescence microscopy, Western blotting, TUNEL assay, and cell cycle analysis. KEY FINDINGS: The discovery of quinoline-3-carboxylic acid [4-(2-[benzyl-3[-(3,4-dimethoxy-phenyl)-propionyl]-amino]-ethyl)-phenyl]-amide (KB3-1) as a novel MDR-reversal agent. KB3-1 significantly enhanced the accumulation and retention of a P-gp substrate, rhodamine-123 in the P-gp-expressing MES-SA/DX5 uterine sarcoma cells but not in the P-gp-negative MES-SA cells at non-toxic concentrations of 1 microM and 3 microM. Similarly, fluorescence microscopy observation revealed that KB3-1 reduced the effluxed rhodamine-123 expression on the membrane of MES-SA/DX5 cells. Consistent with decreased P-gp pumping activity, confocal microscopic observation revealed that KB3-1 effectively diminished the expression of P-gp in paclitaxel (TAX)-treated MES-SA/DX-5 cells. Furthermore, Western blotting confirmed that KB3-1 reduced P-gp expression and enhanced cytochrome C release and Bax expression in TAX treated MES-SA/DX-5 cells. In addition, KB3-1 enhanced TAX-induced apoptotic bodies in MES-SA/DX5 cells by TdT-mediated-dUTP nick-end labeling (TUNEL) staining assay aswell as potentiated TAX- induced cytotoxicity, G2/M phase arrest and sub-G1 apoptosis in MES-SA/DX5 cells but not in MES-SA cells. Interestingly, KB3-1 at 3 microM had comparable MDR-reversal activity to 10 microM verapamil, a well-known MDR- reversal agent. SIGNIFICANCE: KB3-1 can be a MDR-reversal drug candidate for combination chemotherapy with TAX.     

Drug concentration-dependent expression of multidrug resistance-associated protein and P-glycoprotein in the doxorubicin-resistant acute myelogenous leukemia sublines.

The multidrug resistance of cancer cells can be mediated by an overexpression of the human MDR1 and MRP genes, which encode the transmembrane efflux pumps, the 170 kDa P-glycoprotein (Pgp) and the 190 kDa multidrug resistance-associated protein (MRP), respectively. In this study, we investigate which protein is preferentially overexpressed in the function of doxorubicin concentrations in the acute myelogenous leukemia cell line (OCI/AML-2). Multidrug-resistant AML-2 sublines were isolated in doxorubicin concentrations of 20, 100, 250, and 500 ng/ml. MRP was at first expressed at low concentrations of less than 5 x IC50 (100 ng/ml) of doxorubicin followed by the overexpression of Pgp with concentrations of more than 12.5 x IC50 (250 ng/ml) of doxorubicin. In addition, it appeared that increased amounts of MRP and its mRNA in AML-2/DX20 and /DX100 decreased gradually in both AML-2/DX250 and /DX500 overexpressing Pgp. In conclusion, it is thought that the overexpression of MRP or Pgp is dependent upon drug concentrations. It could be implicated that the overexpression of MRP might be negatively related to that of Pgp.     

Altered Expression of Proliferation-Inducing and Proliferation-Inhibiting Genes Might Contribute to Acquired Doxorubicin Resistance in Breast Cancer Cells

This study was designed to investigate the molecular changes that may develop during exposure of breast cancer cells to anticancer agents and that may lead to acquired resistance. We used two breast cancer cell lines, a parental (MCF7/WT) and a doxorubicin-resistant (MCF7/DOX) one. Cell survival, cell cycle distribution and RT-PCR expression level of genes involved in DNA damage response, MDR1, GST and TOPOIIα were measured. MCF7/DOX cells were five-fold more resistant to doxorubicin (DOX) than the MCF7/WT cells. DOX treatment causes arrest of MCF7/DOX cells in G1 and G2 phases of cell cycle whereas MCF7/WT cells were arrested in S-phase. The molecular changes in both cell lines due to DOX treatment could be classified into: (1) the basal level of p53, p21, BRCA1, GST and TOPOIIα mRNA was higher in MCF7/DOX than MCF7/WT. During DOX treatment, the expression level of these genes decreased in both cell lines but the rate of down-regulation was faster in MCF7/WT than MCF7/DOX cells. (2) The expression level of MDR1 was the same in both cell lines but 48 and 72 h of drug treatment, MDR1 disappeared in MCF7/WT but still expressed in MCF7/DOX. (3) There was no change in the expression level of BAX, FAS and BRCA2 in both cell lines. Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIα could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.     

Doxorubicin induces the acetylation of histone H1 in a human colon cancer cell line (LoVo/DX) selected for resistance to the drug, but not in the sensitive parental line (LoVo)

The effect of doxorubicin (DX) treatment on H1 synthesis and acetylation was studied in two human colon adenocarcinoma cell lines, sensitive (LoVo) and resistant (LoVo/DX) to this drug. Histone variants were resolved by a high resolution two-dimensional gel electrophoresis system coupled to fluorography for the detection of radioactive incorporation. The relative synthesis of H1.4 and H1.5 variants was slightly reduced by DX. This is probably related to the inhibition of DNA synthesis consequent to drug treatment. The main effect is that DX induces the acetylation of H1 isoproteins in the LoVo/DX resistant line but not in the parental line, which is 30 times more sensitive to anthracyclines. The different behavior of the two cell lines cannot be attributed to different cellular drug retention since the DX doses chosen (1.25 for LoVo and 40 micrograms/ml for LoVo/DX cells) correspond to similar intracellular drug concentrations. H1 acetylation persisted after exposure to cycloheximide in DX treated LoVo/DX cells, indicating that it is a postranslational event. The induction of H1 acetylation appears rather specific since no increase was found in 3H-acetate incorporation on the total cellular TCA-precipitable fraction. In addition DX treatment did not modify the acetylation of core histones in either LoVo or LoVo/DX cell lines.     

Lymphokine-activated killer cell susceptibility and adhesion molecule expression of multidrug resistant breast carcinoma

Reports showing susceptibility of multidrug resistant (MDR) cancer cells to immune effectors, together with P-glycoprotein (P-gp) expression in immune effector subsets, including immature natural killer (NK) cells, and some activated T cells, suggest P-gp or some changes associated with it, have implications in immune-mediated mechanisms. A series of experiments were done to determine the nature of alterations associated with susceptibility to immune effector cells of MDR tumor cells. A cell line isolated from the malignant pleural effusion of a breast cancer patient was transfected with human and murine MDR1 genes, and four variants with different levels of MDR were obtained. Lymphokine-activated killer (LAK) activity was measured by a ⁵¹Chromium release, and conjugate formation assays. MDR1 transfectant P-gp⁺ breast carcinoma lines had increased LAK susceptibility compared to their parent line. Some part of the increased LAK susceptibility of drug-resistant cell lines was at the binding/recognition level as shown by conjugate formation assays. This suggests that differences may exist between paired cell lines with respect to the expression of cell adhesion molecules (CAMs). Monoclonal antibodies (mAbs) to CAMs and flow cytometry were used to quantitate these antigens. The CAMs studied were those previously found to be upregulated by stimulating NK cells with (interleukin-2) IL-2; ICAM-1 (CD54), LFA-3 (CD58), N-CAM (CD56), and the β chain of LFA-1 (CD18). Although no differences in these CAMs were found between the breast carcinoma line and its MDR1-transfected variants, the target susceptibility results given above suggest that IL-2 treatment could be effective in combination with current protocols using chemotherapeutics, monoclonal antibodies (mAbs) and stem cell transplantation.     

Mechanisms of altered sequestration and efflux of chemotherapeutic drugs by multidrug-resistant cells

This review considers the mechanisms associated with the pleiotropic resistance of cancer cells to chemotherapeutic drugs, and more particularly those related to intracellular pH (pHi). The multidrug resistance (MDR) phenomenon responsible for the decreased accumulation and increased efflux of cytotoxic drugs is generally associated with excess levels of P-glycoproteins (Pgps) encoded by MDR genes and/or the multidrug resistance-associated protein (MRP). MDR cell lines, derived from normal or tumor cells, frequently exhibit abnormally elevated pHi and changes in the production of various proteins. Recent studies have suggested that, in addition to the impact of the ATP-dependent membrane transporters Pgp and MRP on drug transport, other mechanisms linked to pHi changes in MDR cells may play an important role in drug resistance. We have shown that alkalinization of the acidic compartments (endosomes and lysosomes) by lysosomotropic agents could stimulate the efflux of vinblastine from drug-resistant mouse renal proximal tubule cells. The fact that weak base chemotherapeutic drugs can be sequestered within the acidic organelles of MDR cells sheds new light on the cellular mechanisms of drug resistance. This revised version was published online in July 2006 with corrections to the Cover Date.     

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

Complexation study and anticellular activity enhancement by doxorubicin-cyclodextrin complexes on a multidrug-resistant adenocarcinoma cell line.

Ability of molecular complexes of [Doxorubicin (DX)-cyclodextrin (Cd)] to enhance the anticellular activity of antineoplastic drug Doxorubicin and to reverse its multidrug resistance has been investigated. A spectroscopic study of the alpha, beta, and gamma-[DX-Cds] complexes has been investigated in relation to their biological effects on a multidrug resistant (MDR) human rectal adenocarcinoma cell line (HRT-18). A ten fold enhancement of DX anticellular activity in presence of beta-cyclodextrin alone was detected.     

Electron microscopic study of the combined action of ampicillin and cephalexin on E. coli]

The data of electron microscopy study of morphological variation of E. coli, strain 423 in the logarithmic phase after exposure to ampicillin (2 gamma/ml) and cephalexin (4 gamma/ml) are presented. Pronounced ultrastructural changes not only in the cell wall but also in the cytoplasm were found. After exposure to ampicillin alone changes of the same type were observed. However, after exposure to the combination of the 2 antibiotics these changes were more pronounced and observed in the predominating part of the cells. Examination of ultrathin slices of the strain treated with cephalexin revealed no ultrastructural changes. The morphological changes in the cells of E. coli, strain 423 after its treatment with ampicillin and cephalexin combination were due mainly to ampicillin effect, while cephalexin increased the level of the changes.