Palbociclib has no clinically relevant effect on the QTc interval in patients with advanced breast cancer.

The aim of this study was to assess the potential effects of palbociclib in combination with letrozole on QTc. PALOMA-2, a phase 3, randomized, double-blind, placebo-controlled trial, compared palbociclib plus letrozole with placebo plus letrozole in postmenopausal women with estrogen receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer. The study included a QTc evaluation substudy carried out as a definitive QT interval prolongation assessment for palbociclib. Time-matched triplicate ECGs were performed at 0, 2, 4, 6, and 8 h at baseline (Day 0) and on Cycle 1 Day 14. Additional ECGs were collected from all patients for safety monitoring. The QT interval was corrected for heart rate using Fridericia's correction (QTcF), Bazett's correction (QTcB), and a study-specific correction factor (QTcS). In total, 666 patients were randomized 2 : 1 to palbociclib plus letrozole or placebo plus letrozole. Of these, 125 patients were enrolled in the QTc evaluation substudy. No patients in the palbociclib plus letrozole arm of the substudy (N=77) had a maximum postbaseline QTcS or QTcF value of ≥ 480 ms, or a maximum increase from clock time-matched baseline for QTcS or QTcF values of ≥ 60 ms. The upper bounds of the one-sided 95% confidence interval for the mean change from time-matched baseline for QTcS, QTcF, and QTcB at all time points and at steady-state Cmax following repeated administration of 125 mg palbociclib were less than 10 ms. Palbociclib, when administered with letrozole at the recommended therapeutic dosing regimen, did not prolong the QT interval to a clinically relevant extent.

Inhibition of MCL-1 by obatoclax sensitizes Sp2/0-Ag14 hybridoma cells to glutamine deprivation-induced apoptosis.

For several cancer cell types, the lack of an adequate supply of the amino acidl-glutamine (Gln) triggers apoptosis, a phenomenon termed Gln addiction. In this report, we examined the role of the anti-apoptotic proteins of the B-cell lymphoma 2 (BCL-2) protein family in the survival of Sp2/0-Ag14 (Sp2/0) mouse hybridoma cells, a cell line that undergoes apoptosis within minutes of Gln deprivation. Western blot analysis revealed that myeloid cell leukaemia 1 (MCL-1) was expressed at much higher levels than BCL-2, B-cell lymphoma extra-large and BCL-2-like protein 2 making it the prominent pro-survival BCL-2 family member in this hybridoma. Gln deprivation triggered a progressive decrease in MCL-1 protein levels, which coincided with the decrease in Sp2/0 cell survival. Moreover, Sp2/0 cells were much more sensitive to the broad Bcl-2 homology domain-3 (BH3) mimetic obatoclax (which targets MCL-1) than to the more selective drug ABT-737 (which does not target MCL-1). Finally, we show that obatoclax sensitizes Sp2/0 cells to apoptosis following Gln starvation. All together, the data presented here reveal that modulation of the pro-survival protein MCL-1 is an important step in the sequence of events leading to the initiation of apoptosis in Gln-starved Sp2/0 cells. Cancer cells require an adequate supply ofl-glutamine for their survival. Using a mouse hybridoma cell line that is exquisitely sensitive to glutamine starvation, we show that the levels of the pro-survival BCL-2 family protein MCL-1 decrease upon glutamine starvation in a manner that correlates with the loss of cell viability. Moreover, inhibiting MCL-1 with the drug obatoclax sensitizes hybridoma cells to glutamine starvation. Thus, in some cancer cells, glutamine starvation triggers the inactivation of pro-survival proteins. Our data suggest that the combined inhibition of glutamine biosynthesis pathways and BCL-2 proteins may prove effective against some cancers.

Control of late apoptotic events by the p38 stress kinase in L-glutamine-deprived mouse hybridoma cells.

L-Glutamine (Gln) starvation rapidly triggers apoptosis in Sp2/0-Ag14 (Sp2/0) murine hybridoma cells. Here, we report on the role played by the stress-activated kinase p38 mitogen-activated protein kinase (MAPK) in this process. p38 activation was detected 2 h after Gln withdrawal and, although treatment with the p38 inhibitor SB203580 did not prevent caspase activation in Gln-starved cells, it reduced the occurrence of both nuclear condensation/fragmentation and apoptotic body formation. Similarly, transfection of Sp2/0 cells with a dominant negative p38 MAPK reduced the incidence of nuclear pyknosis and apoptotic body formation following 2 h of Gln starvation. Gln withdrawal-induced apoptosis was blocked by the overexpression of the anti-apoptotic protein Bcl-xL or by the caspase inhibitor Z-VAD-fmk. Interestingly, Bcl-xL expression inhibited p38 activation, but Z-VAD-fmk treatment did not, indicating that activation of this MAPK occurs downstream of mitochondrial dysfunction and is independent of caspases. Moreover, the anti-oxidant N-acetyl-l-cysteine prevented p38 phosphorylation, showing that p38 activation is triggered by an oxidative stress. Altogether, our findings indicate that p38 MAPK does not contribute to the induction of apoptosis in Gln-starved Sp2/0 cells. Rather, Gln withdrawal leads to mitochondrial dysfunction, causing an oxidative stress and p38 activation, the latter contributing to the formation of late morphological features of apoptotic Sp2/0 cells.

Investigation of a cyanine dye assay for the evaluation of the biocompatibility of magnesium alloys by direct and indirect methods.

Magnesium and its alloys are promising candidates for a new generation of biodegradable metals in orthopaedic applications due to their excellent biocompatibility, biodegradability, and mechanical properties that are similar to natural bone. However, direct in vitro assessment of these materials in the presence of cells is complicated by degradation products from the alloy that lead to a false positive for the most commonly used cell adhesion and cell proliferation assays. In this paper, a cyanine dye was used to quantitatively evaluate the in vitro biocompatibility of a Mg AZ31 alloy by both direct and indirect methods. The cytotoxicity of the corrosion products was evaluated via an indirect method; a 25% decrease in cell viability compared to control samples was observed. Moreover, direct assessment of cell adhesion and proliferation showed a statistically significant increase in cell number at the surface after 72 h. In addition, the degradation rate and surface characteristics of the Mg AZ31 alloy were evaluated for both direct and indirect tests. The degradation rate was unaffected by the presence of cells while evidence of an increase in calcium phosphate deposition on the magnesium alloy surface in the presence of cells was observed. This study demonstrates that a cyanine dye based assay provides a more accurate assessment of the overall in vitro biocompatibility of biodegradable metals than the more commonly used assays reported in the literature to date.

GADD153 expression does not necessarily correlate with changes in culture behavior of hybridoma cells.

BACKGROUND: The acute sensitivity of some hybridoma cell lines to culture-related stresses severely limits their productivity. Recent developments in the characterization of the stress signals modulating the cellular phenotype revealed that the pro-apoptotic transcription factor Gadd153 could be used as a marker to facilitate the optimization of mammalian cell cultures. In this report, we analyzed the expression of Gadd153 in Sp2/0-Ag14 murine hybridoma cells grown in stationary batch culture and subjected to two different culture optimization paradigms: L-glutamine supplementation and ectopic expression of Bcl-xL, an anti-apoptotic gene. RESULTS: The expression of Gadd153 was found to increase in Sp2/0-Ag14 cells in a manner which coincided with the decline in cell viability. L-glutamine supplementation prolonged Sp2/0-Ag14 cell survival and greatly suppressed Gadd153 expression both at the mRNA and protein level. However, Gadd153 levels remained low after L-glutamine supplementation even as cell viability declined. Bcl-xL overexpression also extended Sp2/0-Ag14 cell viability, initially delayed the induction of Gadd153, but did not prevent the increase in Gadd153 protein levels during the later phase of the culture, when cell viability was declining. Interestingly, L-glutamine supplementation prevented Gadd153 up-regulation in cells ectopically expressing Bcl-xL, but had no effect on cell viability. CONCLUSION: This study highlights important limitations to the use of Gadd153 as an indicator of cell stress in hybridoma cells.

Influence of mixed organosilane coatings with variable RGD surface densities on the adhesion and proliferation of human osteosarcoma Saos-2 cells to magnesium alloy AZ31.

In the last decade, the use of magnesium and its alloys as biodegradable implant materials has become increasingly accepted. However, surface modification of these materials to control the degradation rate in the early stages of healing and improve their biocompatibility is crucial to the successful implementation of magnesium alloy implants in medicine. Cell adhesion and proliferation at the implant surface is a vital factor for successful integration of a biomaterial within the body. Cells accomplish this task by binding to ligands such as the arginine-glycine-aspartic acid peptide sequence (RGD) commonly found on adhesive proteins present in the extracellular matrix. In this paper, we report a biomimetic surface modification strategy involving deposition of a mixed organosilane layer on Mg AZ31 followed by covalent immobilization of RGD peptides through a heterobifunctional cross-linker molecule. Our results indicate that with optimized deposition conditions uniform organosilane coatings were successfully deposited on the Mg AZ31 substrate. Furthermore, we have demonstrated that the surface density of immobilized RGD can be varied by depositing organosilane layers from solutions containing two different organosilanes in specified ratios. Increases in cell adhesion and cell proliferation were observed on the surface modified substrates.

Oxidative stress is not required for the induction of apoptosis upon glutamine starvation of Sp2/0-Ag14 hybridoma cells.

L-glutamine (Gln) withdrawal rapidly triggers apoptosis in the murine hybridoma cell line Sp2/0-Ag14 (Sp2/0). In this report, we examined the possibility that Gln deprivation of Sp2/0 cells triggers an oxidative stress which would contribute to the activation of apoptotic pathways. Gln withdrawal triggered an oxidative stress in Sp2/0 cells, as indicated by an increased accumulation of reactive oxygen species (ROS) and an increase in the intracellular content in protein carbonyl groups. Gln starvation also caused a decrease in the intracellular levels of glutathione (GSH). However, a decrease in GSH was not sufficient to induce Sp2/0 cell death since reducing GSH levels with DL-buthionine-[S,R]-sulfoximine did not affect cell viability. The antioxidant N-acetyl-L-cysteine (NAC), while effective in inhibiting ROS accumulation and oxidative stress, did not prevent the loss in cell viability or the processing and activation of caspase-3 triggered by Gln starvation. On the other hand, NAC did reduce the formation of apoptotic bodies in dying cells. Altogether these results indicate that in Sp2/0 cells, Gln deprivation leads to the induction of an oxidative stress which, while involved in the formation of apoptotic bodies, is not essential to the activation of the cell death program.

Ammonium ions improve the survival of glutamine-starved hybridoma cells.

BACKGROUND: As a consequence of a reprogrammed metabolism, cancer cells are dependent on the amino acid l-glutamine for their survival, a phenomenon that currently forms the basis for the generation of new, cancer-specific therapies. In this paper, we report on the role which ammonium ions, a product of glutaminolysis, play on the survival of l-glutamine-deprived Sp2/0-Ag14 mouse hybridoma cells. RESULTS: The supplementation of l-glutamine-starved Sp2/0-Ag14 cell cultures with either ammonium acetate or ammonium chloride resulted in a significant increase in viability. This effect did not depend on the ability of cells to synthesize l-glutamine, and was not affected by the co-supplementation with α-ketoglutarate. When we examined the effect of ammonium acetate and ammonium chloride on the induction of apoptosis by glutamine deprivation, we found that ammonium salts did not prevent caspase-3 activation or cytochrome c leakage, indicating that they did not act by modulating core apoptotic processes. However, both ammonium acetate and ammonium chloride caused a significant reduction in the number of l-glutamine-starved cells exhibiting apoptotic nuclear fragmentation and/or condensation. CONCLUSION: All together, our results show that ammonium ions promote the survival of l-glutamine-deprived Sp2/0-Ag14 cells and modulate late-apoptotic events. These findings highlight the complexity of the modulation of cell survival by l-glutamine, and suggest that targeting survival-signaling pathways modulated by ammonium ions should be examined as a potential anti-cancer strategy.

Peptabody-EGF: a novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells.

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

Rapid induction of the intrinsic apoptotic pathway by L-glutamine starvation.

While the amino acid L-glutamine is known to play a role in the survival of several cell types, the underlying molecular mechanisms are still poorly defined. We show in this report that L-glutamine starvation rapidly triggered apoptosis in Sp2/0-Ag14 hybridoma cells. This process involved the activation of both caspases-9 and -3, suggesting that L-glutamine deprivation initiated an intrinsic apoptotic pathway in Sp2/0-Ag14 cells. Supporting this idea, the cytosolic release of the mitochondrial proteins SMAC/DIABLO and cytochrome c (Cyt c) was observed, with an initial limited leakage occurring during the first 30 min of L-glutamine deprivation, followed by a greater release after 60 min. The latter occurred simultaneously with the translocation of the pro-apoptotic protein Bax to the mitochondria. Finally, a decline in XIAP levels and the activation of caspases-3 and -9 were observed. Thus, L-glutamine deprivation of Sp2/0-Ag14 cells rapidly triggers intracellular events, which target the mitochondria, leading to the cytosolic release of apoptogenic factors, the activation of caspases-9 and -3, and the commitment to the death program. This work introduces the Sp2/0Ag14 hybridoma as a unique model for the study of the molecular events underlying the pro-survival function of L-glutamine.

Induction of cellular necrosis by the glutathione peroxidase mimetic ebselen.

The selenium-based compound ebselen is a powerful antioxidant, a potent anti-inflammatory agent and a potential neuroprotective compound. Several studies have demonstrated that part of the biological effect of ebselen is the result of the inhibition of apoptosis. We show in this report that ebselen induced the necrotic cell death of Sp2/0-Ag14 hybridoma cells. This process was rapid, with over 90% of the cells being dead after a 2 h exposure to 50 microM ebselen. The toxic effect of ebselen could not be prevented by the caspase inhibitor Z-VAD-fmk but could be blocked with thiol-containing compounds. Interestingly, ebselen addition completely prevented caspase activation in cycloheximide-treated Sp2/O-Ag14 cells, indicating that this antioxidant interferes with the apoptotic machinery. Our results indicate that some cell types are acutely sensitive to the toxic effect of ebselen, and that ebselen-induced cell death interferes with apoptotic processes. These observations are of particular importance since ebselen is currently used in clinical trials for possible use as therapeutic agent for stroke.

Bcl-xL expression interferes with the effects of L-glutamine supplementation on hybridoma cultures.

While feeding protocols and ectopic expression of anti-apoptotic genes have been used to improve the viability of hybridoma cell lines, the effect of the expression levels of survival genes on the behavior of hybridomas following nutrient supplementation is unknown. In this study, we compared the behavior of the Sp2/0-Ag14 hybridoma (Bcl-xL(low)) and the P3x63-Ag8.653 myeloma (Bcl-xL(high)) following culture supplementation with the amino acid L-glutamine (L-Gln). Our data revealed that L-Gln addition substantially increased Sp2/0-Ag14 cell viability and total cell density, concomitant with a decrease in the rate of cell death. This effect was not seen when other amino acids or D-glucose (D-Glc) replaced L-Gln. The improvement in the culture behavior of Sp2/0-Ag14 cells was attributed to a reduction in the rate of accumulation of apoptotic cells. On the other hand, L-Gln supplementation had only a limited effect on the growth of the P3x63-Ag8.653 cells. Interestingly, Sp2/0-Ag14 cells over-expressing Bcl-xL showed a culture behavior upon L-Gln complementation that was similar to the P3x63-Ag8.653 myeloma. These results suggest that the anti-apoptotic gene expression profile of hybridoma cells can markedly impact on the beneficial effects afforded by nutrient supplementation.