Pilar Garcia-Morales

Histone deacetylase inhibitors induced caspase-independent apoptosis in human pancreatic adenocarcinoma cell lines.

The antitumor activity of the histone deacetylase inhibitors was tested in three well-characterized pancreatic adenocarcinoma cell lines, IMIM-PC-1, IMIM-PC-2, and RWP-1. These cell lines have been previously characterized in terms of their origin, the status of relevant molecular markers for this kind of tumor, resistance to other antineoplastic drugs, and expression of differentiation markers. In this study, we report that histone deacetylase inhibitors induce apoptosis in pancreatic cancer cell lines, independently of their intrinsic resistance to conventional antineoplastic agents. The histone deacetylase inhibitor–induced apoptosis is due to a serine protease–dependent and caspase-independent mechanism. Initially, histone deacetylase inhibitors increase Bax protein levels without affecting Bcl-2 levels. Consequently, the apoptosis-inducing factor (AIF) and Omi/HtrA2 are released from the mitochondria, with the subsequent induction of the apoptotic program. These phenomena require AIF relocalization into the nuclei to induce DNA fragmentation and a serine protease activity of Omi/HtrA2. These data, together with previous results from other cellular models bearing the multidrug resistance phenotype, suggest a possible role of the histone deacetylase inhibitors as antineoplastic agents for the treatment of human pancreatic adenocarcinoma.

Post-transcriptional Regulation of P-Glycoprotein Expression in Cancer Cell Lines

The present study of inhibitors shows that the histone deacetylase–induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5′ end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5′ end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5′ untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA. (Mol Cancer Res 2007;5(6):641–53)

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines.

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G(1) arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G(1) arrest. This G(1) arrest was associated with up-regulation of p27(kip1), whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G(1) arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 ΔEGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

Cyclin D3 is down-regulated by rapamycin in HER-2-overexpressing breast cancer cells

Rapamycin and its analogues are being tested as new antitumor agents. Rapamycin binds to FKBP-12 and this complex inhibits the activity of FRAP/mammalian target of rapamycin, which leads to dephosphorylation of 4EBP1 and p70 S6 kinase, resulting in blockade of translation initiation. We have found that RAP inhibits the growth of HER-2-overexpressing breast cancer cells. The phosphorylation of mammalian target of rapamycin, p70 S6 kinase, and 4EBP1 is inhibited by rapamycin and cells are arrested in the G1 phase, as determined by growth assays, fluorescence-activated cell sorting analysis, and bromodeoxyuridine incorporation studies. Rapamycin causes down-regulation of cyclin D3 protein, retinoblastoma hypophosphorylation, loss of cyclin-dependent kinase (cdk) 4, cdk6, and cdk2 activity. The half-life of cyclin D3 protein decreases after rapamycin treatment, but not its synthesis, whereas the synthesis or half-life of cyclin D1 protein is not affected by the drug. Additionally, rapamycin caused accumulation of ubiquitinated forms of cyclin D3 protein, proteasome inhibitors blocked the effect of rapamycin on cyclin D3, and rapamycin stimulated the activity of the proteasome, showing that the effect of rapamycin on cyclin D3 is proteasome proteolysis dependent. This effect depends on the activity of HER-2 because Herceptin, a neutralizing antibody against HER-2, is able to block both the induction of proteasome activity and the cyclin D3 down-regulation due to rapamycin. Furthermore, inhibition of HER-2 gene expression by using small interfering RNA blocked the rapamycin effects on cyclin D3. These data indicate that rapamycin causes a G1 arrest in HER-2-overexpressing breast cancer cells that is associated with a differential destabilization and subsequent down-regulation of cyclin D3 protein. [Mol Cancer Ther 2006;5(9):2172–81]

Susceptibility of multidrug resistance tumor cells to apoptosis induction by histone deacetylase inhibitors.

The main goal of our study has been to analyze the efficiency of new anticancer drugs, specifically histone deacetylase inhibitors, in tumor cells bearing a multidrug resistance phenotype. We report that the histone deacetylase inhibitors, Trichostatin A and Suberoylanilide Hydroxamic Acid (SAHA), dramatically reduce cell viability and promote apoptosis in different drug‐resistant cells, affecting in a much lesser extent to their parental drug‐sensitive counterparts. The differential effects induced by Trichostatin A and SAHA between drug‐sensitive and drug‐resistant cells are reflected on the main characteristics of the resistant phenotype. Thus, reverse transcription‐PCR and Western immunoblots confirm that both histone deacetylase inhibitors promote endogenous down‐regulation of P‐glycoprotein, which is overexpressed in the drug‐resistant cells. Transfection of drug‐sensitive cells with the P‐glycoprotein cDNA ruled out the a priori possible association between apoptosis and down‐regulation of P‐glycoprotein induced by the histone deacetylase inhibitors. The results suggest a therapeutic potential of histone deacetylase inhibitors in the treatment of cancers with acquired resistance.

Cholinergic stimulation of ion fluxes in pancreatic islets

Cholinergic agents are known to stimulate the hydrolysis of polyphosphoinositides in pancreatic islets. The effect of carbamylcholine upon ion fluxes in the islet cells was investigated. Carbamylcholine provoked a rapid but poorly sustained increase in 45Ca and 86Rb outflow from perifused islets. Such a cationic response was observed at different glucose concentrations (zero to 16.7 mM), at three concentrations of carbamylcholine (10 microM, 100 microM and 1.0 mM), and in the absence or presence of extracellular Ca2+. It coincided with a biphasic stimulation of insulin release, both the cationic and secretory responses being abolished in the presence of atropine (10 microM). At variance with nutrient secretagogues, carbamylcholine failed to affect the net production of cyclic AMP and caused a transient decrease in 32P outflow from islets prelabelled with [32P]phosphate. It is proposed that cholinergic agents mobilize Ca2+ from intracellular sites, possibly through generation of inositol, 1,4,5-triphosphate from phosphatidylinositol 4,5-bisphosphate. The intracellular redistribution of Ca2+ does not appear sufficient, however, to account fully for the secretory response, which may also involve activation of protein kinase C by diacylglycerol.

Inhibitory effect of clonidine upon adenylate cyclase activity, cyclic AMP production, and insulin release in rat pancreatic islets.

Conflicting opinions were recently expressed concerning the possible effect of α2-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca2+), NaF, GTP,, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable , in part at least, to inhibition of adenylate cyclase.

The coupling of metabolic to secretory events in pancreatic islets. The possible role of glutathione reductase

The participation of glutathione reductase in the process of nutrient-stimulated insulin release was investigated in rat pancreatic islets exposed to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU caused a time-and dose-related, irreversible inhibition of glutathione reductase activity. This coincided with a fall in both GSH/GSSG ratio and the thiol content of the islets. Pretreatment of the islets with BCNU inhibited the oxidation of glucose and its stimulant action upon both 45Ca net uptake and insulin release. Although BCNU (up to 0.5 mM) failed to affect the oxidation of L-leucine and L-glutamine, it also caused a dose-related inhibition of insulin release evoked by the combination of these two amino acids. The latter inhibition was apparently not fully accounted for by the modest to negligible effects of BCNU upon 45Ca uptake, 45Ca efflux, 86Rb efflux and cyclic AMP production. Since BCNU failed to inhibit insulin release evoked by the association of Ba2+ and theophylline, these results support the view that glutathione reductase participates in the coupling of metabolic to secretory events in the process of nutrient-stimulated insulin release. However, the precise modality of such a participation, for example the control of intracellular Ca2+ distribution, remains to be elucidated.

Impairment of insulin release by methylation inhibitors

The possible participation of enzymatic methylation reactions in the process of insulin release was investigated in rat pancreatic islets. The combination of 3-deazaadenosine and DL-homocysteine impaired the incorporation of 3H-methyl from L-[methyl-3H]methionine into endogenous islet proteins and phospholipids, but failed to affect turnover in the phosphatidylinositol cycle. The inhibitors of methylation decreased insulin release evoked by D-glucose or the combinations of D-glucose and gliclazide, L-leucine and L-glutamine, or Ba2+ and theophylline. The inhibitors of methylation did not impair either the oxidation of D-glucose or affect its capacity to decrease K+ conductance, stimulate Ca2+ inflow and provoke 45Ca accumulation in pancreatic islets. It is proposed that, in the process of insulin secretion, a methyl acceptor protein and/or phospholipid play(s) a limited modulatory role in the coupling of cytosolic Ca2+ accumulation to exocytosis.

Differentiation and drug resistance relationships in leukemia cells

It is well established that the effectiveness of anticancer drugs may result from combined cytotoxic and differentiation activities on tumor cells. Also, differentiating agents are able to alter the susceptibility of cancer cells to antineoplastic drug therapy. However, the acquisition and/or development of drug resistance that frequently appears in anticancer treatment can impair these interactions between differentiation agents and cytotoxic drugs. In the present study, we report that the acquisition of resistance to anthracyclines in two humans, promyeolocytic leukemia HL‐60 and eythroleukemia K562 cell lines, results in a restricted maturation process induced by differentiating agents with respect to that exhibited by their corresponding drug‐sensitive counterparts. Interestingly, differentiating agents are able to decrease the overexpression of drug‐efflux pumps as it is the case of MRP1 in the resistant HL‐60 cells, thus increasing the sensitivity of cells to drug treatment. In addition, susceptibility of the drug‐sensitive cells to certain apoptotic stimuli is significantly reduced after differentiation. The results here reported indicate complex interactions between cytotoxic (drug therapy) and non‐cytotoxic (differentiation) cancer treatments, which should be taken into account to improve therapeutic efficiency.