CYP450 2D6 and 2C19 genotypes in ADHD: not related with treatment resistance but with over-representation of 2C19 ultra-metabolizers.

OBJECTIVES: Cytochrome P450 (CYP450) is a major enzyme system involved in drug metabolism as well as regulation of brain function. Although individual variability in CYP enzymes have been studied in terms of personality traits and treatment effects, no study up to now evaluated CYP polymorphisms in children with attention deficit/hyperactivity disorder (ADHD). We aimed to define the genetic profiles of CYP2D6 and CYP2C19 relevant alleles in children with ADHD according to treatment status and compare the frequencies according to past results. METHODS: Three hundred and seventeen patients with ADHD-Combined Presentation were enrolled; symptom severity was evaluated by parents and clinicians while adverse effects of previous treatments were evaluated with parent and child reports. Reverse blotting on strip assays was used for genotyping and descriptive and bivariate analyses were conducted. A p-value was set at 0.05 (two-tailed). RESULTS: Children were divided into treatment-naïve (n=194, 61.2%) and treatment-resistant (n=123, 38.8%) groups. Within the whole sample PM, EM and UM status according to 2D6 were 3.8% (n=12), 94.3% (n=299) and 21.9% (n=6); respectively. PM, IM, EM and UM status according to 2C19 were 2.5% (n=8), 19.8% (n=63), 48.6% (n=154) and 29.0% (n=92), respectively. No relationship with treatment resistance, comorbidity or gender could be found. Importantly, CYP2C19 UMs were significantly more frequent in ADHD patients compared to previous studies in the general population. CONCLUSIONS: CYPs may be a rewarding avenue of research to elucidate the etiology and treatment of patients with ADHD.

Mitochondrial estrogen receptors alter mitochondrial priming and response to endocrine therapy in breast cancer cells.

Breast cancer is the most common cancer with a high rate of mortality and morbidity among women worldwide. Estrogen receptor status is an important prognostic factor and endocrine therapy is the choice of first-line treatment in ER-positive breast cancer. However, most tumors develop resistance to endocrine therapy. Here we demonstrate that BH3 profiling technology, in particular, dynamic BH3 profiling can predict the response to endocrine therapy agents as well as the development of acquired resistance in breast cancer cells independent of estrogen receptor status. Immunofluorescence analysis and subcellular fractionation experiments revealed distinct ER-α and ER-ß subcellular localization patterns in breast cancer cells, including mitochondrial localization of both receptor subtypes. shRNA-mediated depletion of ER-ß in breast cancer cells led to resistance to endocrine therapy agents and selective reconstitution of ER-ß in mitochondria restored sensitivity. Notably, mitochondria-targeted ER-α did not restore sensitivity, even conferred further resistance to endocrine therapy agents. In addition, expressing mitochondria-targeted ER-ß in breast cancer cells resulted in decreased mitochondrial respiration alongside increased total ROS and mitochondrial superoxide production. Furthermore, our data demonstrated that mitochondrial ER-ß can be successfully targeted by the selective ER-ß agonist Erteberel. Thus, our findings provide novel findings on mitochondrial estrogen signaling in breast cancer cells and suggest the implementation of the dynamic BH3 technique as a tool to predict acquired endocrine therapy resistance.

Kinome-wide RNAi screening for mediators of ABT-199 resistance in breast cancer cells identifies Wee1 as a novel therapeutic target.

Antiapoptotic and proapoptotic BCL-2 protein family members regulate mitochondrial apoptotic pathway. Small molecule inhibitors of antiapoptotic BCL-2 proteins including BCL-2-specific inhibitor ABT-199 (Venetoclax) are in clinical development. However, the efficiency of ABT-199 as a single agent in solid tumors is limited. We performed a high-throughput RNAi kinome screen targeting 691 kinases to identify potentially targetable kinases to enhance ABT-199 response in breast cancer cells. Our studies identified Wee1 as the primary target kinase to overcome resistance to ABT-199. Depletion of Wee1 by siRNA-mediated knockdown or inhibition of Wee1 by the small molecule Wee1 inhibitor AZD1775 sensitized SKBR3, MDA-MB-468, T47D and CAMA-1 breast cancer cells to ABT-199 along with decreased MCL1. BH3-only proteins PUMA and BIM functionally contribute to apoptosis signaling following co-targeting BCL-2 and Wee1. Suppression of Wee1 function increased mitochondrial cell death priming. Furthermore, we found that Wee1 inhibition altered MCL1 phosphorylation and protein stability, which led to HUWE1-mediated MCL1 degradation. Our findings suggest that Wee1 inhibition can overcome resistance to ABT-199 and provide a rationale for further translational investigation of BCL-2 inhibitor/Wee1 inhibitor combination in breast cancer.

RAB25 confers resistance to chemotherapy by altering mitochondrial apoptosis signaling in ovarian cancer cells.

Ovarian cancer remains one of the most frequent causes of cancer-related death in women. Many patients with ovarian cancer suffer from de novo or acquired resistance to chemotherapy. Here, we report that RAB25 suppresses chemotherapy-induced mitochondrial apoptosis signaling in ovarian cancer cell lines and primary ovarian cancer cells. RAB25 blocks chemotherapy-induced apoptosis upstream of mitochondrial outer membrane permeabilization by either increasing antiapoptotic BCL-2 proteins or decreasing proapoptotic BCL-2 proteins. In particular, BAX expression negatively correlates with RAB25 expression in ovarian cancer cells. BH3 profiling assays corroborated that RAB25 decreases mitochondrial cell death priming. Suppressing RAB25 by means of RNAi or RFP14 inhibitory hydrocarbon-stapled peptide sensitizes ovarian cancer cells to chemotherapy as well as RAB25-mediated proliferation, invasion and migration. Our data suggest that RAB25 is a potential therapeutic target for ovarian cancer.

Cytotoxic and Apoptotic Effects of Novel Pyrrolo[2,3-d]Pyrimidine Derivatives Containing Urea Moieties on Cancer Cell Lines.

BACKGROUND: Pyrrolo[2,3-d]pyrimidines have been recently reported to have anticancer activities through inhibition of different targets such as, Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, Janus Kinase (JAK), mitotic checkpoint protein kinase (Mps1), carbonic anhydrase, MDM-2. On the other hand, aryl urea moieties which are found in some tyrosine kinase inhibitors such as Sorafenib and Linifanib have aroused recent attention as responsible for anticancer activities. The aims of this paper are to synthesize pyrrolo[ 2,3-d]pyrimidine derivatives containing urea moiety and evaluate their anti-cancer activity against human lung cancer cell line (A549), prostate cancer cell line (PC3), human colon cancer cell line (SW480) and human breast cancer cell line (MCF-7). METHODS: A series of new pyrrolo[2,3-d]pyrimidines containing urea moieties have been synthesized as Scheme 1. In vitro cytotoxicity of target compounds were evaluated against, SW480, PC3, A549 and MCF-7 human cancer cell lines using a MTT assay. In order to evaluate the mechanism of cytotoxic activity of compounds 9e, 10a and 10b, having the best cytotoxic activity, Annexin V binding assay, cell cycle analysis and western blot analysis were performed. RESULTS: Among the target compounds, 10a (IC50 = 0.19 µM) was found to be the most potent derivative against PC3 cells. Compound 10b and 9e showed the strong cytotoxic activity against MCF-7 and A549 cells with IC50 value of 1.66 µM and 4.55 µM, respectively. Flow cytometry data suggest that the cytotoxic activity of the compounds on cancer cells might be mediated by apoptosis revealing a significant increase in the percentage of late apoptotic cells and causing a cell cycle arrest at different stages. Western blot analysis of apoptosis marker demonstrated that these compounds induce apoptosis through the intrinsic pathway. CONCLUSION: Compound 9e displayed the strongest cytotoxicity against A549 cancer cell line, and induced late apoptosis in A549, as confirmed by cell cycle arrest in G0/G1 phase. In addition, compound 9e reduced expression of the anti-apoptotic protein Bcl-2 and enhanced expression of the pro-apoptotic protein Bax, besides increased caspase-9 and caspase-3, as well as cleavage of PARP levels. These results suggest that compound 9e showed a cytotoxic effect in A549 cells through activation of the mitochondrial apoptotic pathway. Further studies will be undertaken in our laboratory to improve cytotoxic activity of compound 9e and to identify the biological targets of 9e which are responsible for anticancer activity.

Distinct apoptotic blocks mediate resistance to panHER inhibitors in HER2+ breast cancer cells.

Despite the development of novel targeted therapies, de novo or acquired chemoresistance remains a significant factor for treatment failure in breast cancer therapeutics. Neratinib and dacomitinib are irreversible panHER inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2-overexpressing cell lines. Here we showed that increased MCL1 and decreased BIM and PUMA mediated resistance to neratinib in ZR-75-30 and SKBR3 cells while increased BCL-XL and BCL-2 and decreased BIM and PUMA promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted antiapoptotic protein dependence and development of resistance to panHER inhibitors. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR-75-30 cells. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in neratinib-resistant SKBR3 and ZR-75-30 cells, but we did not detect a similar response in neratinib-resistant BT474 cells. Accordingly, suppression of BCL-2/BCL-XL by ABT-737 was required in addition to ERK1/2 inhibition for neratinib- or dacomitinib-induced apoptosis in neratinib-resistant BT474 cells. Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER inhibitor resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER inhibitor resistance in breast cancer cells.

Biphasic ROS production, p53 and BIK dictate the mode of cell death in response to DNA damage in colon cancer cells.

Necrosis, apoptosis and autophagic cell death are the main cell death pathways in multicellular organisms, all with distinct and overlapping cellular and biochemical features. DNA damage may trigger different types of cell death in cancer cells but the molecular events governing the mode of cell death remain elusive. Here we showed that increased BH3-only protein BIK levels promoted cisplatin- and UV-induced mitochondrial apoptosis and biphasic ROS production in HCT-116 wild-type cells. Nonetheless, early single peak of ROS formation along with lysosomal membrane permeabilization and cathepsin activation regulated cisplatin- and UV-induced necrosis in p53-null HCT-116 cells. Of note, necrotic cell death in p53-null HCT-116 cells did not depend on BIK, mitochondrial outer membrane permeabilization or caspase activation. These data demonstrate how cancer cells with different p53 background respond to DNA-damaging agents by integrating distinct cell signaling pathways dictating the mode of cell death.

AMP-activated protein kinase couples 3-bromopyruvate-induced energy depletion to apoptosis via activation of FoxO3a and upregulation of proapoptotic Bcl-2 proteins.

Most tumors primarily rely on glycolysis rather than mitochondrial respiration for ATP production. This phenomenon, also known as Warburg effect, renders tumors more sensitive to glycolytic disturbances compared to normal cells. 3-bromopyruvate is a potent inhibitor of glycolysis that shows promise as an anticancer drug candidate. Although investigations revealed that 3-BP triggers apoptosis through ATP depletion and subsequent AMPK activation, the underlying molecular mechanisms coupling AMPK to apoptosis are poorly understood. We showed that 3-BP leads to a rapid ATP depletion which was followed by growth inhibition and Bax-dependent apoptosis in HCT116 cells. Apoptosis was accompanied with activation of caspase-9 and -3 while pretreatment with a general caspase inhibitor attenuated cell death. AMPK, p38, JNK, and Akt were phosphorylated immediately upon treatment. Pharmacological inhibition and silencing of AMPK largely inhibited 3-BP-induced apoptosis and reversed phosphorylation of JNK. Transcriptional activity of FoxO3a was dramatically increased subsequent to AMPK-mediated phosphorylation of FoxO3a at Ser413. Cell death analysis of cells transiently transfected with wt or AMPK-phosphorylation-deficient FoxO3 expression plasmids verified the contributory role of AMPK-FoxO3a axis in 3-BP-induced apoptosis. In addition, expression of proapoptotic Bcl-2 proteins Bim and Bax were upregulated in an AMPK-dependent manner. Bim was transcriptionally activated in association with FoxO3a activity, while Bax upregulation was abolished in p53-null cells. Together, these data suggest that AMPK couples 3-BP-induced metabolic disruption to intrinsic apoptosis via modulation of FoxO3a-Bim axis and Bax expression. © 2015 Wiley Periodicals, Inc.