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.

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.

Cyclooxygenase-2 activation mediates the proangiogenic effect of nitric oxide in colorectal cancer.

PURPOSE: Up-regulation of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes has been reported in colorectal cancer. We aimed at evaluating the possible interaction between the nitric oxide and COX-2 pathways, and its effect on promoting tumor angiogenesis. EXPERIMENTAL DESIGN: Expression of iNOS, COX-2, vascular endothelial growth factor (VEGF), and CD31 was analyzed in tumor samples and corresponding normal mucosa obtained from 46 surgical specimens. We also evaluated iNOS activity, prostaglandin E(2) (PGE(2)), cyclic GMP and cyclic AMP production in the same specimens. Nitrite/nitrate levels, and PGE(2) and VEGF production were assessed in HCT116 and HT29 colon cancer cell lines after induction and selective inhibition of the two enzyme pathways. RESULTS: A significant correlation was found between iNOS and COX-2 immunohistochemical expression. PGE(2) production significantly correlated with iNOS activity and cGMP levels. A significant correlation was also found among PGE(2) production, microvessel density, and VEGF expression. Coinduction of both iNOS and COX-2 activities occurred after lipopolysaccharide (LPS) and epidermal growth factor (EGF) treatment in HCT116 and HT29 cells. Inhibition of iNOS by 1400W significantly reduced both LPS- and EGF-induced PGE(2) production. Treatment with LPS, EGF, and arachidonic acid significantly increased VEGF production in the iNOS-negative/COX-2-positive HT29 cells. This effect was completely reversed by treatment with the selective COX-2 inhibitor celecoxib. CONCLUSIONS: Our data showed a prominent role of nitric oxide in stimulating COX-2 activity in colorectal cancer. This interaction is likely to produce a cooperative effect in promoting angiogenesis through PGE(2)-mediated increase in VEGF production.

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.

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.

A HERG current sustains a cardiac-type action potential in neuroblastoma S cells.

From the adrenergic SH-SY5Y human neuroblastoma clone, we isolated a subclone (21S) endowed with a glial-oriented phenotype. At difference from the parental clone, 21S cells responded to depolarizing stimuli with overshooting action potentials, whose repolarization phase was composed of an initial rapid episode, followed by a long-lasting plateau and a slow return to the resting potential (V(REST)). The action potential depolarization phase was sustained by a TTX-sensitive Na(+) current, while the first repolarizing episode was produced by the scanty delayed rectifier potassium current (I(KDR)) expressed in 21S cells. The bulk of repolarization, including the after-hyperpolarization, was sustained by the human eag related (HERG) potassium current (I(HERG)) that also governs V(REST) in 21S cells. This double role of I(HERG), together with the poor expression of I(KDRs), represents a novel finding in electrophysiology, as well as gives a clue to identify a new excitable element of the complex cellular population of neuroblastoma.