Levels Of Bcl-2 Family Proteins Research Articles

560 Saikosaponin A induces HEKa cell apoptosis via ROS generation

Background: Psoriasis is a common, chronic and noninfectious skin disease with the complex pathogenesis which is reported as excessive proliferation and aberrant apoptosis of keratinocytes. Saikosaponin A (SSA) is a kind of triterpenoid saponin extracted from Chinese traditional medicine Radix bupleuri, possessing various biological functions such as anti-inflammatory, immune regulation and anti-tumor. But the effect of SSA on keratinocytes is unclear. Objective: To explore the effect of SSA on keratinocytes and to analyze the corresponding mechanism. Methods: 1. The cell viability was detected by MTT assay and the apoptosis was examined using the annexin V-FITC/PI flow cytometry double staining method. Apoptosis-related protein were determined by Western blot. 2. After stimulation with SSA, DCFH-DA and JC-1 were used to detect intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential. MitoSOX Red and MitoTracker Green were used to observe generation and distribution of ROS in mitochondria by confocal laser scanning microscope; ROS-related protein was detected by Western blot. 3. Antioxidants N-acetyl-cysteine (NAC, 10mM) was added to HEKa cells 1h before SSA(20μM) treatment to identify whether SSA induced the apoptosis of HEKa cells through ROS production. Results: 1. Compared with the control group, SSA significantly inhibited the viability and induced the apoptosis of HEKa cells with changes in BCL-2 family protein levels. 2. SSA promoted ROS production of mitochondria and decreased Δψ, which caused changes in ROS-related protein levels and oxidative stress damage to cells. 3. SSA-treated HEKa cells produced more ROS, but this effect was inhibited by the antioxidants NAC. Furthermore, NAC treatment attenuated HEKa cell apoptosis and proliferation suppression of SSA. Conclusion: SSA can inhibit proliferation and induce apoptosis of HEKa cells through ROS generation, making SSA a potential promising candidate drug for future research.

Time-restricted feeding ameliorates maternal high-fat diet-induced fetal lung injury

Maternal inflammation ensuing from high-fat diet (HFD) intake during pregnancy is related to spontaneous preterm birth and respiratory impairment among premature infants. Recently, a circadian aligned dietary intervention referred to as Time-restricted feeding (TRF) has been reported to have beneficial metabolic effects. This study aimed to assess the effects of maternal TRF on fetal lung injury caused by maternal HFD intake. Female Wistar rats were kept on following three dietary regimens; Ad libitum normal chow diet (NCD-AL), Ad libitum HFD (HFD-AL) and Time-restricted fed HFD (HFD-TRF) from 5 months before mating and continued through pregnancy. Fetal lung samples were collected on the embryonic day 18.5, and apoptotic and inflammatory markers were assessed using TUNEL assay, western blotting, and qRT-PCR. Our results showed that TRF considerably prevented maternal HFD-induced apoptosis in fetal lung tissue that corroborated with a reduction in caspase activation and increased levels of anti-apoptotic BCL2 family proteins together with a lower level of ER-stress and autophagy markers including ATF6, CHOP and LC3-II. Besides, fetal lungs from HFD-TRF dams exhibited reduced expression of inflammatory genes that correlated with reduction and apoptotic injury throughout fetal development. Our results thus put forth TRF as a unique non-pharmacological approach to boost perinatal health beneath metabolic stress.

Loss of BIM in T cells results in BCL-2 family BH3-member compensation but incomplete cell death sensitivity normalization.

BIM is the master BH3-only BCL-2 family regulator of lymphocyte survival. To understand how long-term loss of BIM affects apoptotic resistance in T cells we studied animals with T cell-specific deletion of Bim. Unlike CD19CREBimfl/fl animals, LCKCREBimfl/fl mice have pronounced early lymphocytosis followed by normalization of lymphocyte counts over time. This normalization occurred in mature T cells, as thymocyte development and apoptotic sensitivity remained abnormal in LCKCREBimfl/fl mice. T cells from aged mice experienced normalization of their absolute cell numbers and responses against various apoptotic stimuli. mRNA expression levels of BCL-2 family proteins in CD4+ and CD8+ T cells from young and old mice revealed upregulation of several BH3-only proteins, including Puma, Noxa, and Bmf. Despite upregulation of various BH3 proteins, there were no differences in anti-apoptotic BCL-2 protein dependency in these cells. However, T cells had continued resistance to direct BIM BH3-induced mitochondrial depolarization. This study further highlights the importance of BIM in cell death maintenance in T cells and provides new insight into the dynamism underlying BH3-only regulation of T cell homeostasis versus induced cell death and suggests that CD4+ and CD8+ T cells compensate differently in response to loss of Bim.

Synthesis, characterization, apoptosis, ROS, autophagy and western blotting studies of cyclometalated iridium(III) complexes

Three iridium(III) complexes [Ir(ppy)2(DQPC)](PF6) (Ir-1), [Ir(bzq)2(DQPC)](PF6) (Ir-2) and [Ir(piq)2(DQPC)](PF6) (Ir-3) were synthesized and characterized. The cytotoxicity in vitro of the ligand DQPC and complexes Ir-1, Ir-2 and Ir-3 against cancer cells HeLa, A549, HepG2 and normal cell NIH3T3 was assessed by MTT methods. These complexes exhibit significant inhibitory effect on HeLa cells growth with an IC50 value of 2.4 ± 0.2, 2.4 ± 0.1 and 0.5 ± 0.1 µM for Ir-1, Ir-2 and Ir-3, respectively. Further studies indicated that complexes Ir-1-Ir-3 can induce apoptosis in HeLa cells and enhance the levels of intracellular ROS and Ca2+. The release of cyto-c and the changes of mitochondrial membrane potential were investigated by fluorescent microscope and flow cytometry. The effects of the complexes on DNA damage, autophagy and cell invasion were investigated under fluorescent microscope. The proportion of apoptotic and cell cycle arrest were assayed by flow cytometry. In addition, the expression levels of caspase-3 and Bcl-2 family proteins were investigated by western blot. The results show that the complexes induce apoptosis in HeLa through ROS-mediated mitochondrial dysfunction pathway.

Apoptosis contributes to the cytotoxicity induced by amodiaquine and its major metabolite N-desethylamodiaquine in hepatic cells

Amodiaquine (ADQ), an antimalarial drug used in endemic areas, has been reported to be associated with liver toxicity; however, the mechanism underlying its hepatoxicity remains unclear. In this study, we examined the cytotoxicity of ADQ and its major metabolite N-desethylamodiaquine (NADQ) and the effect of cytochrome P450 (CYP)-mediated metabolism on ADQ-induced cytotoxicity. After a 48-h treatment, ADQ and NADQ caused cytotoxicity and induced apoptosis in HepG2 cells; NADQ was slightly more toxic than ADQ. ADQ treatment decreased the levels of anti-apoptotic Bcl-2 family proteins, which was accompanied by an increase in the levels of pro-apoptotic Bcl-2 family proteins, indicating that ADQ-induced apoptosis was mediated by the Bcl-2 family. NADQ treatment markedly increased the phosphorylation of JNK, extracellular signal-regulated kinase (ERK1/2), and p38, indicating that NADQ-induced apoptosis was mediated by MAPK signaling pathways. Metabolic studies using microsomes obtained from HepG2 cell lines overexpressing human CYPs demonstrated that CYP1A1, 2C8, and 3A4 were the major enzymes that metabolized ADQ to NADQ and that CYP1A2, 1B1, 2C19, and 3A5 also metabolized ADQ, but to a lesser extent. The cytotoxicity of ADQ was increased in CYP2C8 and 3A4 overexpressing HepG2 cells compared to HepG2/CYP vector cells, confirming that NADQ was more toxic than ADQ. Moreover, treatment of CYP2C8 and 3A4 overexpressing HepG2 cells with ADQ increased the phosphorylation of JNK, ERK1/2, and p38, but not the expression of Bcl-2 family proteins. Our findings indicate that ADQ and its major metabolite NADQ induce apoptosis, which is mediated by members of the Bcl-2 family and the activation of MAPK signaling pathways, respectively.

Polymeric nanoparticles encapsulating α-mangostin inhibit the growth and metastasis in colorectal cancer

α-Mangostin, the most abundant xanthone extracted from pericarp of mangosteen fruits, has been shown potential treatment effects in various cancer cell types. This led us to explore the anti-tumor activity and related mechanisms of α-mangostin in colorectal cancer. In this study, poly(ethylene glycol)–poly(e-caprolactone)–poly(ethylene glycol) (PEG–PCL–PEG, PECE) nanoparticles was used to encapsulate free α-mangostin, creating α-mangostin loaded PECE (α-M/PECE) nanoparticles. When compared with free α-mangostin, α-M/PECE induced a significantly more apoptosis of both CT26 and HCT116 cells in vitro, leading to an obviously more loss of mitochondrial membrane potential (Δψm) in HCT116 cells. Results of western blot assay and caspase-inhibiting assay showed that cell apoptosis induced by α-mangostin might via activating both death receptor-mediated caspase-8 pathway and mitochondrial dependent cytochrome c/caspase-9, with simultaneously regulating expression levels of Bcl-2 family proteins, and ultimately activating caspase-3. Moreover, the administration of α-M/PECE nanoparticles also exhibited significantly stronger inhibition in tumor growth, tumor angiogenesis, tumor cell proliferation as well as inducted more tumor cell apoptosis in subcutaneous xenograft models. Especially, α-M/PECE nanoparticles showed excellent anti-metastasis efficacy in both pulmonary metastasis model and abdominal metastasis model of colorectal cancer. The safety assessment also showed no obvious side effects were detected in mice which were treated with free α-mangostin or α-M/PECE nanoparticles. These results suggested the potential of α-M/PECE nanoparticles as an excellent intravenously injectable formulation of free α-mangostin for the treatment of colorectal cancer.

Abstract 2889: The Yes-Associated Protein plays a role in tumor formation, stemness, and therapy response in high risk neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood. Despite intensive multimodal therapy, >50% of high risk (HR) NB patients relapse with chemotherapy-resistant disease. RAS/RAF/MAPK pathway mutations and increased Yes-Associated Protein (YAP) transcriptional activity are more prevalent in relapsed NB compared to diagnostic tumors. YAP is a transcriptional co-activator, regulated by the Hippo pathway that binds with TEAD in the nucleus to activate genes promoting cell growth, self-renewal, and survival. YAP aberrant transcription has been implicated in many tumor types, yet the role for YAP in NB is unknown. YAP protein is heterogeneously expressed in HR NB cell lines. We used human derived NB cell lines stably transduced to express (NGPYAP) or inhibit (SK-N-ASshYAP, NLFshYAP) YAP to further characterize YAP’s role in NB. While TEAD expression increases with YAP genetic inhibition, expression of YAP paralog TAZ did not change. SK-N-ASshYAP xenografts in NSG mice showed significantly delayed time to tumor formation and slower growth compared to empty vector transduced xenografts. Unmodified NB cells grown as neurospheres in neural basal media showed increased YAP expression and stemness markers, OCT4 and SOX2, by RT-qPCR. OCT4 and SOX2 were dependent on YAP expression as they did not increase in NLFshYAP neurospheres. Furthermore, NLFshYAP had decreased number and size of neurospheres compared to control. Given YAP’s role in tumor stemness, we treated unmodified NB cells with 13-cis-retinoic acid (RA) to see if YAP-induced stemness would overcome RA-induced differentiation. Interestingly, YAP expression and downstream targets, OCT4 and SOX2, increased in response to RA treatment. This suggests that RA paradoxically induces YAP transcriptional activity and may be selecting for a more chemoresistant tumor initiating phenotype. Lastly, YAP knockdown sensitized NRAS- and NF-1-mutated NBs to the MEK inhibitor, Trametinib, and to chemotherapy in vitro. In adult cancers, YAP’s role in therapy resistance is due to its regulation of anti-apoptotic Bcl-2 family proteins. However, YAP knockdown or induced expression in NBs did not change Bcl-2 family protein levels, nor did it affect Bim binding patterns to Mcl-1 or Bcl-2. Thus, investigations are ongoing to understand how YAP negatively regulates Trametinib response. We treated NB cells with Verteporfin (VP), an FDA-approved drug used to treat macular degeneration that inhibits YAP-TEAD interactions in other cancers. While VP inhibited YAP expression in NBs, it also had significant off target effects, potently killing YAP null NBs via light-activated generation of reactive oxygen species. Given the relevance of YAP in relapsed NBs and its effects on NB tumor formation, stemness, and therapy resistance, the need for targeted and specific YAP inhibitors is critical to reinstating therapy sensitivity at relapse. Citation Format: Jenny Shim, Yoo Joo Lee, Kaitlyn Janssen, Alexa Fritz, Kelly C. Goldsmith. The Yes-Associated Protein plays a role in tumor formation, stemness, and therapy response in high risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2889.

Activation of LXR Receptors and Inhibition of TRAP1 Causes Synthetic Lethality in Solid Tumors.

Cholesterol is a pivotal factor for cancer cells to entertain their relentless growth. In this case, we provide a novel strategy to inhibit tumor growth by simultaneous activation of liver-X-receptors and interference with Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1). Informed by a transcriptomic and subsequent gene set enrichment analysis, we demonstrate that inhibition of TRAP1 results in suppression of the cholesterol synthesis pathway in stem-like and established glioblastoma (GBM) cells by destabilizing the transcription factor SREBP2. Notably, TRAP1 inhibition induced cell death, which was rescued by cholesterol and mevalonate. Activation of liver X receptor (LXR) by a clinically validated LXR agonist, LXR623, along with the TRAP1 inhibitor, gamitrinib (GTPP), results in synergistic reduction of tumor growth and cell death induction in a broad range of solid tumors, which is rescued by exogenous cholesterol. The LXR agonist and TRAP1 inhibitor mediated cell death is regulated at the level of Bcl-2 family proteins with an elevation of pro-apoptotic Noxa. Silencing of Noxa and its effector BAK attenuates cell death mediated by the combination treatment of LXR agonists and TRAP1 inhibition. Combined inhibition of TRAP1 and LXR agonists elicits a synergistic activation of the integrated stress response with an increase in activating transcription factor 4 (ATF4) driven by protein kinase RNA-like endoplasmic reticulum kinase (PERK). Silencing of ATF4 attenuates the increase of Noxa by using the combination treatment. Lastly, we demonstrate in patient-derived xenografts that the combination treatment of LXR623 and gamitrinib reduces tumor growth more potent than each compound. Taken together, these results suggest that TRAP1 inhibition and simultaneous activation of LXR might be a potent novel treatment strategy for solid malignancies.

Venetoclax Synergistically Enhances the Anti-leukemic Activity of Vosaroxin Against Acute Myeloid Leukemia Cells Ex Vivo.

The survival rate for acute myeloid leukemia remains unacceptably low, in large part owing to resistance to chemotherapy and high rates of relapse. There is an urgent need to develop new therapeutic modalities, in particular such that are tolerated by patients over the age of 60years, who form the bulk of new acute myeloid leukemia diagnoses. Vosaroxin (SNS-595), a second-generation topoisomerase II inhibitor and DNA intercalating agent, shows promising preclinical and clinical activity against acute myeloid leukemia. Venetoclax (ABT-199), a selective Bcl-2 inhibitor, was recently approved for the treatment of acute myeloid leukemia. The objective of this study was to determine the anti-leukemic activity and the underlying molecular mechanisms for the combination of venetoclax and vosaroxin in acute myeloid leukemia cell lines and primary patient samples ex vivo. Using both acute myeloid leukemia cell lines and primary patient samples, annexin V/propidium iodide staining and flow cytometry analyses were used to quantify apoptosis induced by venetoclax or vosaroxin, alone or in combination, with subsequent western blotting analyses to assess levels of Bcl-2 family proteins. Alkaline comet assays were performed to quantify DNA damage induced by the two agents and to determine the effect of venetoclax on DNA repair. Finally, colony-forming assays were conducted on normal human CD34+ cord blood cells and primary acute myeloid leukemia patient samples to determine the effect of venetoclax and vosaroxin on normal hematopoietic and leukemic progenitor cells. We found that venetoclax and vosaroxin synergistically induced apoptosis in multiple acute myeloid leukemia cell lines. Although vosaroxin could partially abrogate the increase of Mcl-1 protein induced by venetoclax, it could not abrogate the increased binding of Bim to Mcl-1 induced by venetoclax. Cooperative induction of DNA damage occurred within 8h of treatment with venetoclax plus vosaroxin. Moreover, repair of DNA damage induced by vosaroxin was significantly attenuated by venetoclax. The combination also synergistically induced apoptosis in primary acute myeloid leukemia patient samples and significantly reduced the colony formation capacity of acute myeloid leukemia progenitor cells, while sparing normal hematopoietic progenitor cells. Vosaroxin and venetoclax synergistically induce apoptosis in acute myeloid leukemia cells and cooperatively target acute myeloid leukemia progenitor cells while sparing normal hematopoietic progenitor cells. Our results support the clinical testing of vosaroxin in combination with venetoclax for treating patients with acute myeloid leukemia, especially in the elderly population.

Abstract PD7-07: Synergistic targeting of CDK4/6 and BCL-2 pathways in estrogen receptor positive breast cancer

Abstract Background: Despite incremental advances in chemotherapy and endocrine therapy, survival outcomes for patients with ER-positive (ER+) metastatic breast cancer (MBC) remain poor. The majority of relapsing tumors exhibit deregulation of the cyclin-dependent kinase 4 and 6 (CDK4/6)/cyclin D1 (CCND1)/Rb signaling pathway. CDK4/6 inhibitors (such as palbociclib) in combination with endocrine therapy have been shown to significantly improve progression free survival in patients who are in 1st or 2nd line relapse, although overall survival benefit has yet to be demonstrated. This may reflect their largely cytostatic mechanism of action, with minimal induction of tumor cell death. Thus, combinatorial strategies that also induce apoptosis could be beneficial. Notably, the pro-survival protein BCL-2 is overexpressed in the majority of ER+ tumors and the potent and specific BCL-2 inhibitor venetoclax (ABT-199) has been found to synergize with endocrine therapy in patient derived xenograft (PDX) models. Promising activity has also been observed in an early phase clinical trial. We therefore investigated dual targeting of the CDK4/6 and BCL-2 pathways in pre-clinical models of ER+ and BCL-2+ breast cancer. Results: We first examined endocrine sensitive or resistant cell-lines and found that pro-survival BCL-2 proteins were upregulated in resistant cells. BCL-2 family protein levels were also found to be elevated in palbociclib resistant cells, suggesting that BCL-2 could represent a therapeutic target. We next determined whether venetoclax improved response to dual therapy comprising the selective estrogen receptor degrader fulvestrant and palbociclib. In clonogenic assays of endocrine sensitive breast cancer cell lines, triple therapy containing venetoclax significantly reduced the number and size of colonies, when compared to double therapy. The addition of venetoclax to fulvestrant/palbociclib also augmented cell death in tumor organoid models derived from either ER+ BCL-2+ primary tumors or PDX models. Moreover, triple therapy improved tumor response and overall survival in mice bearing ER+ BCL-2+ PDX tumors. Mechanistically, this was accompanied by increased apoptosis and reduced cellular proliferation (as determined by cleaved caspase-3 and Ki67 levels, respectively). As CDK4/6 inhibitors have recently been shown to promote anti-tumor immunity, we evaluated immune modulation using the ER+ 67NR cell line in a syngeneic (BALB/c) mouse mammary tumor model. Similar to the PDX models, triple therapy comprising fulvestrant, palbociclib and venetoclax was more effective than double therapy comprising either fulvestrant/palbociclib or fulvestrant/venetoclax. Flow cytometric analysis of tumors revealed that this was accompanied by a reduced intratumoral FOXP3+:cytotoxic CD8 T-cell ratio. Conclusions: The addition of the BCL-2 inhibitor venetoclax to conventional therapy comprising endocrine therapy and a CDK4/6 inhibitor augments tumor response and elicits a favorable intratumoral immune profile. Collectively, these findings support investigation of combination therapy in the clinic for patients with ER+ BCL-2+ MBC. Citation Format: Whittle JR, Vaillant F, Policheni AN, Liu K, Pal B, Giner G, Fernandez K, Gray DH, Caldon CE, Smyth GK, Visvader JE, Lindeman GJ. Synergistic targeting of CDK4/6 and BCL-2 pathways in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-07.

Arsenic Trioxide Induces Apoptosis and Regulatory Functions of Plasmacytoid Dendritic Cells

IntroductionArsenic trioxide (As2O3), a well-established drug for acute promyelocytic leukemia, has been recently shown to have therapeutic potential in a number of mouse models of autoimmune diseases such as, systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and chronic graft-versus-host disease. However, the therapeutic mechanism remains largely unknown.Plasmacytoid dendritic cells (pDCs) are a unique subset of dendritic cells specialized in secreting high level of type I interferons and promote anti-viral responses. Clinically, pDCs have also been reported to be implicated in the pathogenesis of a series of autoimmune diseases characterized by a type I IFN signature, such as SSc.Given the pathogenetic and therapeutic role of As2O3 and pDCs in the same spectrum of diseases, we examined the effects of As2O3 on pDCs viability and functions to better understand the therapeutic mechanism of As2O3 on pDC-related autoimmune diseases.Materials and MethodspDCs cells were isolated from fresh peripheral blood mononuclear cells (PBMCs) by magnetic selection. pDCs were cultured in presence of different concentrations of As2O3 to check for viability and functional changes. pDCs viability and apoptosis were checked by flow cytometry with Fixable Viability Dye-Annexin V staining. Phenotype evaluation was done with surface staining. Bcl-2 and Bax protein expressions, reactive oxygen species (ROS) level, and cytokine production of pDCs were measured by intracellular staining. IRF7 and phospho-IRF7 protein levels were measured with cell signaling staining. Cytokine concentrations in supernatants were quantified by ELISA, and IRF7 expression in pDCs was analyzed by real-time RT-PCR.Naïve CD4+ T and CD19+ B cells were isolated from PBMCs. A pDC/T cell co-culture system was constructed to test As2O3 effect on pDCs in inducing T cell proliferation and polarization. A pDC/B cell co-culture system was used to test As2O3 effect on pDCs in inducing B cell differentiation.ResultsWe first investigated the effect of As2O3 on the viability of human pDCs. We observed that As2O3 decreased dose-dependently the percentages of viable pDCs by inducing cell apoptosis. We then checked the protein expression levels of the apoptosis related Bcl-2 family proteins. As2O3 induced a dramatic upregulation of Bax, while slightly down-regulated the expression of Bcl-2, leading to a significantly decreased Bcl-2 to Bax ratio, which determined cell apoptosis.As2O3 is a well-known inducer of oxidative stress, which is one of the major upstream mediators of cell apoptosis. We first demonstrated that As2O3 induced reactive oxygen species (ROS) within pDCs. However, the anti-oxidant NAC pre-treatment only slightly decreased the percentages of apoptotic cells, indicating that As2O3 could induce pDCs apoptosis independently of ROS generation.Then, we investigated the functional effects of As2O3 on pDCs, and observed that culture for 6h with 5μM of As2O3 almost totally inhibited production of IFN-α from gated pDCs within PBMCs (IFN-α positive cells: 28.56±9,99% without As2O3 vs 1.43±1.65% with 5μM As2O3, p<0.05, n=4). ELISA results proved these results and revealed that As2O3 also restrained the secretion of TNF-α, IL-6 and CXCL10 from pDCs. We also demonstrated that As2O3 inhibited IFN-α secretion from pDCs of 12 untreated SSc patients (IFN-α positive cells: 22.48±12.71% without As2O3 vs 1.65±1.24% with 5μM As2O3, p=0.0001, n=12).Further studies revealed that As2O3 inhibited the secretion of IFN-α from activated pDCs by potently down-regulating the total protein and mRNA expression, and also phosphorylation level of inteferon regulatory factor 7 (IRF7), a key adaptor of the type I inteferon pathway.Finally, As2O3 inhibited pDCs maturation by decreasing the expressions of CD80, CD86 and HLA-DR, and increasing PD-L1 expression. As2O3 also down-regulated pDCs capacity to induce CD4+ T cell proliferation, Th1/Th22 polarization, and B cell differentiation towards plasmablasts.ConclusionOur study demonstrated that As2O3, at different concentrations, negatively affects the viability and functions of human pDCs, suggesting that pDCs could be a target for As2O3 treatment efficacy in autoimmune diseases with type-I interferon signature. We are currently running a prospective multicenter clinical trial testing As2O3 in the setting of chronic GVHD (ClinicalTrials.gov Identifier: NCT02966301). DisclosuresMohty:MaaT Pharma: Consultancy, Honoraria.

Abstract A075: Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis?

Abstract Cancer is a disease of accumulated genetic mutations that alter a cell’s function. One of the common cellular functions affected in cancer cells is the apoptotic pathway. Apoptosis, also known as programmed cell death, is a regulatory biologic function controlled by protein interactions within the BCL-2 protein family. Normally, cells use this pathway to induce cell death when they detect an error in the cell’s functioning that cannot be fixed. In multiple types of cancers, including prostate cancer, there is often an excess of pro-survival BCL-2 proteins. Consequently, these cells have an inability to induce apoptosis, leading to an increase in cancer cell survival, sometimes even in the presence of chemotherapeutic chemicals. This research investigates the potential difference in BCL-2 proteins between metastatic prostate cancer cells and premetastatic prostate cancer cells. Specifically, changes in cancer cell response to chemotherapy after inhibiting antiapoptotic BCL-2 proteins via small-molecule inhibitors are being evaluated. We hypothesize that chemoresistant cancer cells are sensitized to BCL-2 inhibitors by first priming their mitochondria with a traditional chemotherapeutic agent. “Mitochondrial priming” is bringing the antiapoptotic BCL-2 proteins to their apoptotic threshold (1). To accomplish these aims, metastatic prostate cancer cell lines PC3, LnCap C4/2, and DU145 as well as a normal prostate cancer cell line, PNT2, were used. We first sought to compare the level of BCL-2 family proteins among the different cell types. PC3 cells have significantly higher levels of BCL-2, BCL-xL, and BAD as detected by Western blot and quantified by densitometry, corroborating findings from other labs. Next, cells were treated with 5uM etoposide or 100nM docetaxel for 24 hours or 48 hours, and viability was assessed by MTT and apoptosis via Annexin V-FITC. We found that cell viability was significantly decreased in LnCap and DU145 cells at 24 hours, which can be attributed specifically to apoptosis. Moreover, increasing amounts of apoptosis occurred after 48 hours of treatment. Comparatively, PNT2 and PC3 cells have significantly less apoptosis induced by either etoposide or docetaxel treatment. Apoptosis was further confirmed by caspase-3 activation as well as the ability to block apoptosis by pretreatment with the caspase inhibitor Boc-D-fmk in LnCap cells. Additionally, overexpression of BCL-xL in LnCap cells abrogated etoposide-induced apoptosis. Therefore BCL-xL is a potential target for priming. Next, we focused on enhancing the apoptotic effects of etoposide and docetaxel by combing their use with ABT-737, which is a pan BCL-2 antiapoptotic inhibitor. Evaluating the effects of these drugs on various prostate cancerous cell lines in comparison to the effects on a normal prostate cell line may provide insight into their clinical usefulness and relevance in combination treatments. Cells were treated with 0, 100nM, 500nM, 1uM, 2.5uM, or 5uM ABT-737 for 24 hours and apoptosis was assessed via Annexin V-FITC. Preliminary results suggest that on its own, ABT-737 has little effect on LnCap cells (~10%) and no effect on PC3, DU-145, or PNT2 cells. However, pretreating LnCap cells for 24 hours with etoposide as low as 100nM followed by 5 hours exposure to 500nM ABT-737 can induce twice as much apoptosis. These results suggest that etoposide “primes” cells and then the BCL-2 inhibitor pushes them over the edge towards apoptosis. Since less etoposide (100nM) in the combined treatment was effective, as opposed to 500nM when given alone, it may be possible to administer lower amounts of clinically established chemotherapeutics. However, preliminary results also show a ~30% increase in apoptosis in DU145 cells and 50% increase in the normal prostate cell line (PNT2) after combination treatment of 50nM docetaxel for 48 hours followed by 5 hours exposure to 1uM ABT-737. These results suggest that even though lower levels of chemotherapeutics may be effective at killing prostate cancer cells when paired with ABT-737, this combination treatment may be just as harmful to healthy prostate cells. Ongoing studies are also focused on combination treatments with ABT-199, which is selective for antiapoptotic BCL-xL. Reference: 1. Chonghalie TN, et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 2011:334(6059):1129-33. Citation Format: Kaitlyn Walsh, Rachel Wilson, Mary E. McManamy, Victoria Del Gaizo Moore. Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis? [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A075.

Nutritionally Available Selenocysteine Derivative Antagonizes Cisplatin-Induced Toxicity in Renal Epithelial Cells through Inhibition of Reactive Oxygen Species-Mediated Signaling Pathways.

Discovery of nutritionally available agents that could antagonize cisplatin-induced nephrotoxicity is of great significance and clinical application potential. 3,3'-Diselenodipropionic acid (DSePA) is a seleno-amino acid derivative that exhibits strong antioxidant activity. Therefore, this study aimed to examine the protective effects of DSePA on cisplatin-induced renal epithelial cells damage as well as the molecular mechanisms. The results revealed that DSePA effectively inhibited cell apoptosis induced by cisplatin through suppressing the caspase activation and poly(ADP-ribose) polymerase cleavage. In addition, DSePA blocked the cisplatin-induced mitochondrial dysfunction, as evidenced by the loss of mitochondrial membrane potential and reduction of mitochondrial mass. The results of western blot analysis showed that DSePA reversed the expression level of Bcl-2 family proteins altered by cisplatin. The cisplatin-activated AKT pathway was also modulated by DSePA. Moreover, these results indicate that DSePA could protect HK-2 cells from cisplatin-induced toxicity in renal epithelial cells by inhibiting intracellular reactive oxygen species-mediated apoptosis while showing an unobvious effect on its anticancer efficacy. Taken together, this study demonstrates that selenocysteine could be further developed as novel nutritionally available agents to antagonize cisplatin-induced nephrotoxicity during cancer therapy.

Tetracaine induces apoptosis through a mitochondrion-dependent pathway in human corneal stromal cells in vitro

Purpose: Tetracaine is a local anesthetic widely used in ocular diagnosis and ophthalmic surgery and may lead to some adverse effects and complications at a clinical dose. To assess the cytotoxicity and molecular toxicity mechanisms of tetracaine, we used human corneal stromal (HCS) cells as an in vitro model to study the effects of tetracaine on HCS cells.Materials and methods: The cytotoxicity of tetracaine on HCS cells was investigated by examining the changes of cell growth, morphology, viability and cell cycle progressing when HCS cells were treated with tetracaine at concentrations from 10 g/L to 0.078125 g/L. To prove the hypothesis that the cytotoxicity of tetracaine on HCS cells was related with apoptosis induction, we further detected multiple changes in HCS cells, including plasma membrane (PM) permeability, phosphatidylserine (PS) orientation, genomic DNA integrality, and cell ultrastrcuture after treated with tetracaine. Furthermore, the pro-apoptotic signalling pathway induced by tetracaine was explored through detecting the activation of various caspases, the changes of mitochondrial transmembrane potential (MTP), the expression level of Bcl-2 family proteins and the amount of mitochondria-released apoptosis regulating proteins in cytoplasm.Results: Tetracaine at concentrations above 0.15625 g/L had a dose- and time-dependent cytotoxicity to HCS cells, which resulted cell growth inhibition, proliferation retardation, morphological abnormalities and decreased viability. Meanwhile, we found that the HCS cells treated with tetracaine had typical features associated with apoptosis, including an increase in PM permeability, PS externalization, DNA fragmentation and apoptotic body formation. Tetracaine not only resulted in caspase-3, caspase-8 and caspase-9 activation and disruption of MTP but also downregulated Bcl-2 and Bcl-xL and upregulated Bad and Bax, along with the upregulation of cytoplasmic cytochrome c (Cyt. c) and apoptosis-inducing factor (AIF).Conclusions: These results suggested that tetracaine-induced apoptosis might be triggered through Fas death receptors and mediated by Bcl-2 family proteins in the mitochondria-dependent pathway. Our findings identified the cytotoxicity and molecular mechanisms of tetracaine, which could provide a reference value for the safety of this medication and prospective therapeutic interventions in eye clinic.

ABT-737 ameliorates docetaxel resistance in triple negative breast cancer cell line.

PurposeThis study aimed to validate the synergistic effect of ABT-737 on docetaxel using MDA-MB-231, a triple negative breast cancer (TNBC) cell line overexpressing B-cell lymphoma-2 (Bcl-2).MethodsWestern blot analysis was performed to assess expression levels of Bcl-2 family proteins and caspase-related molecules. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution was determined by flow cytometry analysis. Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) was used for pretreatment to assess the role of caspases.ResultsCell viability of MDA-MB-231 after combination treatment with ABT-737 and docetaxel was significantly lower than that after docetaxel or ABT-737 monotherapy based on MTT assay (both P < 0.001), with a combination index of 0.41. The proportion of sub-G1 population after combination treatment was significantly higher than that after docetaxel or ABT-737 monotherapy (P = 0.001, P = 0.003, respectively). Pretreatment with z-VAD-fmk completely restored cell viability of MDA-MB-231 from apoptotic cell death induced by combination therapy (P = 0.001). Although pro-caspase-8 or Bid did not show significant change in expression level, pro-casepase-9 showed significantly decreased expression after combination treatment. Cleaved caspase-3 showed increased expression while poly (ADP-ribose) polymerase cleavage was induced after combination treatment. However, hypoxia-inducible factor 1-alpha and aldehyde dehydrogenase 1 totally lost their expression after combination treatment.ConclusionCombination of ABT-737 with docetaxel elicits synergistic therapeutic effect on MDA-MB-231, a TNBC cell line overexpressing Bcl-2, mainly by activating the intrinsic pathway of apoptosis. Therefore, adjunct of ABT-737 to docetaxel might be a new therapeutic option to overcome docetaxel resistance of TNBCs overexpressing Bcl-2.

ABT737 induces mitochondrial pathway apoptosis and mitophagy by regulating DRP1-dependent mitochondrial fission in human ovarian cancer cells

The anti-apoptotic BCL2 family of proteins elicits a broad cell survival program mainly by promoting cell migration, invasion, and metastasis. High expression level of BCL2 family proteins is a characteristic feature of cancer cells, especially in cisplatin-resistant cancer cells. Recent studies have shown that BCL2 family proteins play a housekeeping role in modulating mitochondrial dynamics. However, it is not clear whether BCL2 family proteins are relevant to mitochondrial fission and fusion in cisplatin-resistant ovarian cancer cells. Here, we report that the BCL2/BCLXL inhibitor ABT737 induced apoptosis more potently in cisplatin-resistant SKOV3/DDP ovarian cancer cells than in cisplatin-sensitive SKOV3 ovarian cancer cells. ABT737 significantly increased levels of DRP1 in mitochondria and increased rates of mitochondrial fission, and then induced cytochrome C release from mitochondria and mitophagy in SKOV3/DDP cells. Mdivi-1, a selective inhibitor of DRP1, weakened ABT737-induced mitochondrial fission, intrinsic apoptotic pathways, and mitophagy in SKOV3/DDP cells. Taken together, these results demonstrate a novel function of ABT737 in inducing DRP1-dependent apoptotic mitochondrial fission and highlight that targeting anti-apoptotic BCL2 family proteins may be an emerging therapeutic strategy for patients with cisplatin-resistant ovarian cancer.

Study on the effect of reactive oxygen species-mediated oxidative stress on the activation of mitochondrial apoptosis and the tenderness of yak meat

This study investigated the effect of reactive oxygen species-mediated oxidative stress on activation of mitochondrial apoptosis and tenderness of yak meat during postmortem ageing. Oxidative stress degree, Ca2+ levels, membrane permeability transition pore opening, mitochondrial membrane potential, apoptotic factors and the shear force were examined. Results showed that the ROS generated by H2O2 significantly increased mitochondrial oxidative stress by decreasing the activities of superoxide dismutase, catalase and glutathione peroxidase, and increasing lipid peroxidation. Furthermore, oxidative stress enhanced Ca2+ production and cytochrome c release, changed the levels of Bcl-2 family proteins and activated caspase-9 and -3 activities. Ultimately, oxidative stress increased the apoptosis rate and tenderness of yak meat. These observations confirmed that ROS-mediated oxidative stress participates in the activation of the apoptotic cascade reaction involving Ca2+ and Bcl-2 family proteins. The results further suggested that ROS-mediated oxidative stress plays a significant role in meat tenderization through the mitochondrial apoptotic pathway.

Gefitinib Enhances Mitochondrial Biological Functions in NSCLCs with EGFR Mutations at a High Cell Density.

Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and has been approved for the treatment of non-small cell lung cancers (NSCLCs) with EGFR mutations. Here we demonstrated that gefitinib induced a significantly enhanced biological activity of succinate-tetrazolium reductase (STR) in mitochondria and mitochondrial membrane potential in HCC827 cells (EGFR mutation NSCLCs, sensitive to gefitinib) at a high cell density. We assessed the biological activity (STR, mitochondrial membrane potential, expression level of Bcl-2 family proteins) of gefitinib on NSCLCs at different cell densities. The 3D cell culture experiments showed the enhanced mitochondrial biological activity in clustered cell culture treated with gefitinib. Interestingly, the expression levels of Bcl-xL and Bax, were affected by the cellular number and gefitinib treatment. We also found that gefitinib prevented additive anticancer activity in the combinational treatment with doxorubicin, which induces mitochondria-dependent apoptotic cell death. Our results indicate that gefitinib may work as a mitochondrial protector against combinational treatment with mitochondria-dependent anticancer agents in high-cell-density.

RETRACTED ARTICLE: Dioscin induces prostate cancer cell apoptosis through activation of estrogen receptor-\u03b2

Recent researches have shown that estrogen receptor-β (ERβ) activator may be a potent anticancer agent for prostate cancer (PCa), and our previous study also indicated that dioscin can upregulate the expression of ERβ in MC3T3-E1 cell. In the present work, the activity and mechanism of dioscin, a natural product, against PCa were investigated. The results showed that dioscin markedly inhibited cell viability, colony formation, motility and induced apoptosis in PC3 cells. Moreover, dioscin disrupted the formation of PC3 cell-derived mammospheres and reduced aldehyde dehydrogenase (ALDH) level and the CD133+/CD44+ cells, indicating that dioscin had a potent inhibitory activity on prostate cancer stem cells (PCSCs). In vivo results also showed that dioscin significantly suppressed the tumor growth of PC3 cell xenografts in nude mice. Furthermore, mechanism investigation showed that dioscin markedly upregulated ERβ expression level, subsequently increased prolyl hydroxylase 2 level, decreased the levels of hypoxia-inducible factor-1α, vascular endothelial growth factor A and BMI-1, and thus induced cell apoptosis by regulating the expression levels of caspase-3 and Bcl-2 family proteins. In addition, transfection experiment of ERβ-siRNA further indicated that diosicn showed excellent activity against PCa in vitro and in vivo by increasing ERβ expression level. The co-immunoprecipitation (Co-IP) results further suggested that dioscin promoted the interaction of c-ABL and ERβ, but did not change c-ABL expression. Moreover, the molecular docking assay showed that dioscin processed powerful affinity toward to ERβ mainly through the strong hydrogen bonding and hydrophobic effects, and the actions of dioscin on ERβ activation and tumor cells inhibition were significantly weakened in the mutational (Phe-336, Phe-468) PC3 cells. Collectively, these findings proved that dioscin exerted efficient anti-PCa activity via activation of ERβ, which should be developed as an efficient candidate in clinical for treating this cancer in the future.

Abstract 4033: Combined MEK and BCL-2/XL inhibition as a potential drug combination for the treatment of high-grade serous ovarian cancer

Abstract High-grade serous ovarian cancer (HGSOC) accounts for 70-80% of ovarian cancer deaths. Despite an initial response to platinum-based chemotherapy, treatment resistance eventually occurs in most patients. The overall aim of our studies is to identify synergistic drug combinations for the treatment of HGSOC and biomarkers that predict sensitivity for future translation in clinical trials. In particular, this study focuses on the vulnerabilities of patient-derived ovarian cancer cells to combined inhibition of the MEK pathway and the anti-apoptotic proteins BCL-2 and BCL-XL. The Ras/MAPK pathway is activated in a subset of HGSOC by gene copy alterations. However, MEK inhibitors have not been evaluated in HGSOC. In this study, we used a collection of 14 primary samples derived from ascites cells of platinum-resistant HGSOC patients (Liu JF et al. 2016). We investigated the sensitivity of these tumor cells to the MEK inhibitor cobimetinib (GDC-0973). Treatment with GDC-0973 had no effects on cell viability in vitro in any of the patient-derived models. To investigate the response to MEK inhibition, we performed Reverse Phase Protein Array of the tumor cells treated with 0.3μM of GDC-0973 in vitro. We found that MEK inhibition by GDC-0973 reduced cell cycle progression markers and upregulated the pro-apoptotic protein BIM. Since more than half of the models express high levels of anti-apoptotic BCL-2 family proteins which neutralize BIM, we examined the effects of antagonism of BCL-2 pro-survival proteins in combination with GDC-0973. Treatment with a combination of GDC-0973 and the BCL-2/XL antagonist, navitoclax (ABT-263) significantly reduced cell number and increased cell death in 10 out of 14 patient-derived models. Protein levels of BIM following treatment with GDC-0973 correlated with sensitivity to the drug combination (R2=0.8 p&amp;lt;0.0001). Interestingly, the BIM levels after MEK inhibition correlate with BIM protein levels before the treatment, suggesting that baseline BIM levels could be used as biomarker of sensitivity. Levels of the pro-survival protein MCL-1 also played a critical role in patient-derived cells that were resistant to the drug combination. Indeed, treatment with a specific MCL-1 inhibitor, A-1210477, in combination with GDC-0973 and ABT-263 led to dramatic tumor cell killing in vitro compared to dual combination of GDC-0973 and ABT-263. Finally, we tested the combination of GDC-0973 and ABT-263 in vivo and preliminary results indicate that the drug combination is well tolerated and is able to significantly reduce tumor growth in patient-derived xenograft models (n=4). Our studies provide significant evidence that combined inhibition of MEK and BCL-2/XL may be an effective drug combination for treatment of HGSOC and that the pro-apoptotic protein BIM may serve as a predictive biomarker to stratify patients that can benefit from these targeted therapies Citation Format: Claudia Iavarone, Ioannis Zervantonakis, Laura M. Selfors, Sangeetha Palakurthi, Joyce F. Liu, Ursula A. Matulonis, Ronny I. Drapkin, Gordon B. Mills, Joel D. Leverson, Deepak Sampath, Joan S. Brugge. Combined MEK and BCL-2/XL inhibition as a potential drug combination for the treatment of high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2017-4033