Mitochondrial expression and functional activity of breast cancer resistance protein in different multiple drug-resistant cell lines.

The multidrug resistance (MDR) phenotype is characterized by the overexpression of a few transport proteins at the plasma membrane level, one of which is the breast cancer resistance protein (BCRP). These proteins are expressed in excretory organs, in the placenta and blood-brain barrier, and are involved in the transport of drugs and endogenous compounds. Because some of these proteins are expressed in the mitochondria, this study was designed to determine whether BCRP is expressed at a mitochondrial level and to investigate its function in various MDR and parental drug-sensitive cell lines. By using Western blot analysis, immunofluorescence confocal and electron microscopy, flow cytometry analysis, and the BCRP (ABCG-2) small interfering RNA, these experiments showed that BCRP is expressed in the mitochondrial cristae, in which it is functionally active. Mitoxantrone accumulation was significantly reduced in mitochondria and in cells that overexpress BCRP, in comparison to parental drug-sensitive cells. The specific inhibitor of BCRP, fumitremorgin c, increased the accumulation of mitoxantrone significantly in comparison with basal conditions in both whole cells and in mitochondria of BCRP-overexpressing cell lines. In conclusion, this study shows that BCRP is overexpressed and functionally active in the mitochondria of MDR-positive cancer cell lines. However, its presence in the mitochondria of parental drug-sensitive cells suggests that BCRP can be involved in the physiology of cancer cells.

Down-regulation of the HGF/MET autocrine loop induced by celecoxib and mediated by P-gp in MDR-positive human hepatocellular carcinoma cell line.

Many tumors are resistant to drug-induced cell-cycle arrest and apoptosis. We have reported that apoptosis can be restored in human multidrug-resistant (MDR) hepatocellular carcinoma cell lines by celecoxib. Here we show that P-glycoprotein (P-gp) mediates cell-cycle arrest and autophagy induced by celecoxib in human MDR overexpressing hepatocellular carcinoma cell line by down-regulation of the HGF/MET autocrine loop and Bcl-2 expression. Exposure of cells to a low concentration of celecoxib down-regulated the expression of mTOR and caused G1 arrest and autophagy, while higher concentration triggered apoptosis. Cell growth inhibition and autophagy were associated with up-regulation of the expression of TGFbeta1, p16(INK4b), p21(Cip1) and p27(Kip1) and down-regulation of cyclin D1, cyclin E, pRb and E2F. The role of P-glycoprotein expression in resistance of MDR cell clone to cell-cycle arrest, autophagy and apoptosis was shown in cells transfected with MDR1 small interfering RNA. These findings demonstrate that the constitutive expression of P-gp is involved in the HGF/MET autocrine loop that leads to increased expression of Bcl-2 and mTor, inhibition of eIF2alpha expression, resistance to autophagy/apoptosis and progression in the cell-cycle. Since mTor inhibitors have been proposed in treatment of "drug resistant" cancer, these data may help explain the reversing effect of mTor inhibitors.

P-glycoprotein mediates celecoxib-induced apoptosis in multiple drug-resistant cell lines.

In several neoplastic diseases, including hepatocellular carcinoma, the expression of P-glycoprotein and cyclooxygenase-2 (COX-2) are often increased and involved in drug resistance and poor prognosis. P-glycoprotein, in addition to drug resistance, blocks cytochrome c release, preventing apoptosis in tumor cells. Because COX-2 induces P-glycoprotein expression, we evaluated the effect of celecoxib, a specific inhibitor of COX-2 activity, on P-glycoprotein-mediated resistance to apoptosis in cell lines expressing multidrug resistant (MDR) phenotype. Experiments were done using MDR-positive and parental cell lines at basal conditions and after exposure to 10 or 50 micromol/L celecoxib. We found that 10 micromol/L celecoxib reduced P-glycoprotein, Bcl-x(L), and Bcl-2 expression, and induced translocation of Bax from cytosol to mitochondria and cytochrome c release into cytosol in MDR-positive hepatocellular carcinoma cells. This causes the activation of caspase-3 and increases the number of cells going into apoptosis. No effect was shown on parental drug-sensitive or on MDR-positive hepatocellular carcinoma cells after transfection with MDR1 small interfering RNA. Interestingly, although inhibiting COX-2 activity, 50 micromol/L celecoxib weakly increased the expression of COX-2 and P-glycoprotein and did not alter Bcl-x(L) and Bcl-2 expression. In conclusion, these results show that relatively low concentrations of celecoxib induce cell apoptosis in MDR cell lines. This effect is mediated by P-glycoprotein and suggests that the efficacy of celecoxib in the treatment of different types of cancer may depend on celecoxib concentration and P-glycoprotein expression.

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines.

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.

Hepatocyte growth factor and inducible nitric oxide synthase are involved in multidrug resistance-induced angiogenesis in hepatocellular carcinoma cell lines.

Based on literature, it is possible to hypothesize that multidrug resistance (MDR) and angiogenic phenotypes are linked to each other in human liver cancer cells. Our goal is to assess whether MDR cells trigger angiogenesis and to study the possible molecular mechanisms involved. Conditioned medium from parental drug-sensitive P5 cells (P5-CM) and MDR-positive P1(0.5) cells [P1(0.5)-CM] stimulated human umbilical vein endothelial cells (HUVEC) survival, proliferation, migration, and microtubular structure formation, but P1(0.5)-CM had a significantly greater effect than P5-CM. Cell implants were done in the rabbit avascular cornea to measure angiogenesis in vivo: P1(0.5) cells induced an important neovascular response in rabbit cornea after 1 week, whereas P5 cells had no effect. P1(0.5) and P5 cells produced vascular endothelial growth factor, but only P1(0.5) secreted hepatocyte growth factor (HGF) into the medium, and small interfering RNA specific for MDR1 clearly reduced HGF production in P1(0.5) cells. The transcription factor Ets-1 and the HGF receptor c-Met were up-regulated in P1(0.5) cells and in HUVEC cultured in P1(0.5)-CM. Inducible nitric oxide synthase (iNOS) seemed to play a major role in the proangiogenic effect of P1(0.5), and its inhibition by 1400W blunted the capacity of P1(0.5) cells to stimulate HUVEC proliferation, migration, and Ets-1 expression. In conclusion, these data show that development of MDR and angiogenic phenotypes are linked to each other in MDR cells. HGF production, Ets-1 and c-Met up-regulation, and iNOS expression can be part of the molecular mechanisms that enhance the angiogenic activity of the MDR-positive hepatocellular carcinoma cell line.

Differential expression proteomics of human colon cancer.

The focus of this study was to use differential protein expression to investigate operative pathways in early stages of human colon cancer. Colorectal cancer represents an ideal model system to study the development and progression of human tumors, and the proteomic approach avoids overlooking posttranslational modifications not detected by microarray analyses and the limited correlation between transcript and protein levels. Colon cancer samples, confined to the intestinal wall, were analyzed by expression proteomics and compared with matched samples from normal colon tissue. Samples were processed by two-dimensional gel electrophoresis, and spots differentially expressed and consistent across all patients were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses and by Western blot analyses. After differentially expressed proteins and their metabolic pathways were analyzed, the following main conclusions were achieved for tumor tissue: 1) a shift from beta-oxidation, as the main source of energy, to anaerobic glycolysis was observed owed to the alteration of nuclear- versus mitochondrial-encoded proteins and other proteins related to fatty acid and carbohydrate metabolism; 2) lower capacity for Na(+) and K(+) cycling; and 3) operativity of the apoptosis pathway, especially the mitochondrial one. This study of the human colon cancer proteome represents a step toward a better understanding of the metabolomics of colon cancer at early stages confined to the intestinal wall.

Vitamin E protects DNA from oxidative damage in human hepatocellular carcinoma cell lines.

Expression of multiple drug resistant (MDR) phenotype and over-expression of P-glycoprotein (P-gp) in the human hepatocellular carcinoma (HCC) cell clone P1(0.5), derived from the PLC/PRF/5 cell line (P5), are associated with strong resistance to oxidative stress and a significant (p < 0.01) increase in intracellular vitamin E content as compared with the parental cell line. This study evaluates the role of vitamin E in conferring resistance to drugs and oxidative stress in P1(0.5) cells. Parental drug-sensitive cells, P5, were incubated in alpha-tocopherol succinate (alpha-TS, 5 microM for 24 h) enriched medium to increase intracellular vitamin E content to levels comparable to those observed in P1(0.5) cells at basal conditions. Susceptibility to lipid peroxidation and oxidative DNA damage were assessed by measuring the concentration of thiobarbituric-reactive substances (TBARS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) at basal and after experimental conditions. Cell capacity to form colonies and resistance to doxorubicin were also studied. P5 cells, treated with alpha-TS, became resistant to ADP-Fe3+ and to ionizing radiation-induced lipid peroxidation as P1(0.5) cells. Exposure to ADP-Fe3+ or ionizing radiation increased TBARS and the 8-OHdG content in the P5 cells, while vitamin E enrichment abolished these effects. Irradiation doses at 5 cGy increased TBARS and 8-OHdG. They also inhibited cell capacity to form colonies in the untreated P5 cells. Incubation with alpha-TS fully reverted this effect and significantly (p < 0.01) reduced the inhibitory effect of cell proliferation induced by irradiation doses at >500 cGy. Resistance to doxorubicin was not affected by alpha-TS. These observations demonstrate the role of vitamin E in conferring protection from lipid peroxidation, ionizing radiation and oxidative DNA damage on the human HCC cell line. They also rule out any role of P-gp over-expression as being responsible for these observations in cells with MDR phenotype expression.

The MDR phenotype is associated with the expression of COX-2 and iNOS in a human hepatocellular carcinoma cell line.

The presence of multiple drug resistance (MDR1) and angiogenic phenotypes negatively affect patients' prognosis with cancer even when treated with drugs that are not transported by the MDR1 gene product. It is possible to suggest a link between the MDR1 and angiogenic phenotypes. Because prostaglandins (PGs) and nitric oxide (NO) have been proposed to be involved in angiogenesis in vivo, the production of PGs and NO and the behavior of inducible NO synthase (iNOS), cyclooxygenase 1 (COX-1), and inducible cyclooxygenase (COX-2) were studied in parental drug-sensitive (P5) liver cancer cell lines and in P5-derived MDR1 cells P1(0.5). Immunohistochemical evaluation, Northern and Western blot analysis of COX-2 and iNOS, and assessment of cell proliferation were performed in basal conditions and after the exposure to stimulants or to specific inhibitors of COX-2 and iNOS. The messenger RNA and protein levels of COX-2 and iNOS were in basal conditions higher in P1(0.5) cells than the parental P5 cells. The exposure to lipopolysaccharide (LPS) or epidermal growth factor (EGF) determined an increase of PG and NO production in both cell lines and this increase was strongly reduced by COX-2 inhibitors such as celecoxib (CLX) and nimesulide (NIME). The inhibition of NO production by COX-2 inhibitors suggests cross-talk between COX-2 and iNOS pathways. CLX and NIME also inhibited cell proliferation, but only in MDR1 cells. A specific inhibitor of iNOS, N(6)-(1-iminoethyl)-L-lysine, had only a mild effect on cell proliferation in both cell lines. In conclusion, these data support the hypothesis that the MDR1 and angiogenic phenotypes are linked to each other in human liver cancer cell lines.