Apoptosis-related Cell Death Research Articles

HDAC/NAMPT dual inhibitors overcome initial drug-resistance in p53-null leukemia cells

The occurrence of cancer is closely related to metabolism and epigenetics. Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression as epigenetic regulators, while nicotinamide phosphoribosyltransferase (NAMPT) is significantly involved in maintaining cellular metabolism. In this study, we rationally designed a series of novel HDAC/NAMPT dual inhibitors based on the structural similarity between HDAC and NAMPT inhibitors. The representative compounds 39a and 39h exhibit significant selective inhibitory activity on HDAC1-3 with IC50 values of 0.71–25.1 nM, while displaying modest activity against NAMPT. Compound 39h did not exhibit inhibitory activity against 370 kinases, demonstrating its target specificity. These two compounds exhibit potent anti-proliferative activity in multiple leukemia cell lines with low nanomolar IC50s. It is worth noticing that the dual inhibitors 39a and 39h overcome the primary resistance of HDAC or NAMPT single target inhibitor in p53-null AML cell lines, with the induction of apoptosis-related cell death. NMN recovers the cell death induced by HDAC/NAMPT dual inhibitors, which indicates the lethal effects are caused by the inhibition of NAD biosynthesis pathway as well as HDAC. This research provides an effective strategy to overcome the limitations of HDAC inhibitors in treating p53-null leukemia.

Use of Deep-Learning Assisted Assessment of Cardiac Parameters in Zebrafish to Discover Cyanidin Chloride as a Novel Keap1 Inhibitor Against Doxorubicin-Induced Cardiotoxicity.

Doxorubicin-induced cardiomyopathy (DIC) brings tough clinical challenges as well as continued demand in developing agents for adjuvant cardioprotective therapies. Here, a zebrafish phenotypic screening with deep-learning assisted multiplex cardiac functional analysis using motion videos of larval hearts is established. Through training the model on a dataset of 2125 labeled ventricular images, ZVSegNet and HRNet exhibit superior performance over previous methods. As a result of high-content phenotypic screening, cyanidin chloride (CyCl) is identified as a potent suppressor of DIC. CyCl effectively rescues cardiac cell death and improves heart function in both in vitro and in vivo models of Doxorubicin (Dox) exposure. CyCl shows strong inhibitory effects on lipid peroxidation and mitochondrial damage and prevents ferroptosis and apoptosis-related cell death. Molecular docking and thermal shift assay further suggest a direct binding between CyCl and Keap1, which may compete for the Keap1-Nrf2 interaction, promote nuclear accumulation of Nrf2, and subsequentially transactivate Gpx4 and other antioxidant factors. Site-specific mutation of R415A in Keap1 significantly attenuates the protective effects of CyCl against Dox-induced cardiotoxicity. Taken together, the capability of deep-learning-assisted phenotypic screening in identifying promising lead compounds against DIC is exhibited, and new perspectives into drug discovery in the era of artificial intelligence are provided.

A Brief Review on Chemoresistance; Targeting Cancer Stem Cells as an Alternative Approach

The acquisition of resistance to traditional chemotherapy and the chemoresistant metastatic relapse of minimal residual disease both play a key role in the treatment failure and poor prognosis of cancer. Understanding how cancer cells overcome chemotherapy-induced cell death is critical to improve patient survival rate. Here, we briefly describe the technical approach directed at obtaining chemoresistant cell lines and we will focus on the main defense mechanisms against common chemotherapy triggers by tumor cells. Such as, the alteration of drug influx/efflux, the enhancement of drug metabolic neutralization, the improvement of DNA-repair mechanisms, the inhibition of apoptosis-related cell death, and the role of p53 and reactive oxygen species (ROS) levels in chemoresistance. Furthermore, we will focus on cancer stem cells (CSCs), the cell population that subsists after chemotherapy, increasing drug resistance by different processes such as epithelial-mesenchymal transition (EMT), an enhanced DNA repair machinery, and the capacity to avoid apoptosis mediated by BCL2 family proteins, such as BCL-XL, and the flexibility of their metabolism. Finally, we will review the latest approaches aimed at decreasing CSCs. Nevertheless, the development of long-term therapies to manage and control CSCs populations within the tumors is still necessary.

Synergetic effects of thymoquinone-loaded porous PVPylated Fe3O4 nanostructures for efficient pH-dependent drug release and anticancer potential against triple-negative cancer cells.

Porous iron oxide nanostructures have attracted increasing attention due to their potential biomedical applications as nanocarriers for cancer and many other therapies as well as minimal toxicity. Herbal anti-cancer agent thymoquinone loaded on Fe3O4 nanoparticles is envisaged to offer solution towards cancer treatment. The purpose of the present study was to investigate the efficacy of thymoquinone-loaded PVPylated Fe3O4 magnetic nanoparticles (TQ-PVP-Fe3O4 NPs) against triple-negative breast cancer (TNBC) cells. The porous PVPylated Fe3O4 NPs were prepared by a simple solvothermal process, whereas the thymoquinone drug was loaded via the nanoprecipitation method. Fourier transform infrared (FTIR) spectroscopic analysis confirmed the molecular drug loading, and surface morphological observation further confirmed this. The quantity of thymoquinone adsorbed onto the porous PVPylated Fe3O4 NPs was studied by thermogravimetric analysis (TGA). The positive surface charge of TQ-PVP-Fe3O4 NPs facilitates the interaction of the NPs with cancer (MDA-MB-231) cells to enhance the biological functions. In addition, the anticancer potential of NPs involving cytotoxicity, apoptosis induction, reactive oxygen species (ROS) generation, and changes in the mitochondrial membrane potential (ΔΨm) of TNBC cells was evaluated. TQ-PVP-Fe3O4 NP-treated cells effectively increased the ROS levels leading to cellular apoptosis. The study shows that the synthesized TQ-PVP-Fe3O4 NPs display pH-dependent drug release in the cellular environment to induce apoptosis-related cell death in TNBC cells. Hence, the prepared TQ-PVP-Fe3O4 NPs may be a suitable drug formulation for anticancer therapy.

Abstract 3231: Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma

Abstract Background: Osteosarcoma (OS) is the most frequent primary malignant tumor of bone in children and adolescents. Although immune checkpoint inhibitor, anti-programmed death protein 1 (PD-1) antibody, has dramatically improved the clinical outcome in some cancer patients, OS patients are less sensitive to PD-1 blockade due to poor immune responses. Recently, oncolytic virotherapy has been shown to stimulate the immune system through induction of immunogenic cell death (ICD). We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with PD-1 blockade and OBP-502 in OS cells. Methods: We used 2 murine OS cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. We analyzed the in vitro antitumor effect of OBP-502 using XTT assay and western blot analysis. Virus-induced ICD was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of oncolytic immunotherapy, we investigated the in vivo antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+, CD4+ and Foxp3+ T cells was analyzed by immunohistochemistry. Results: Flow cytometric analysis demonstrated that K7M2 and NHOS cells had the expression of PD-L1 and integrin, but not CAR. XTT assay showed that OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. Western blot analysis revealed that OBP-502 induced the apoptosis-related cell death in K7M2 and NHOS cells. ELISA assay demonstrated that OBP-502 significantly increased the level of extracellular ATP and HMGB1 in K7M2 and NHOS cells. In vivo experiment using a subcutaneous K7M2 xenograft tumor model showed that combination of anti-PD-1 antibody and OBP-502 significantly reduced tumor growth compared to mock treatment or monotherapy. Immunohistochemical analysis revealed that OBP-502 significantly increased the number of tumor-infiltrating CD8+ T cells compared to mock treatment or monotherapy. By contrast, OBP-502 did not affect the number of CD4+ and Foxp3+ T cells in tumor tissues. Conclusion: These results suggest that oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus is a promising antitumor strategy to promote the therapeutic potential of PD-1 blockade in OS through stimulation of antitumor immune response. Citation Format: Koji Demiya, Hiroshi Tazawa, Yusuke Mochizuki, Miho Kure, Joe Hasei, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma [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 3231.

PARP inhibitors affect growth, survival and radiation susceptibility of human alveolar and embryonal rhabdomyosarcoma cell lines

PurposePARP inhibitors (PARPi) are used in a wide range of human solid tumours but a limited evidence is reported in rhabdomyosarcoma (RMS), the most frequent childhood soft-tissue sarcoma. The cellular and molecular effects of Olaparib, a specific PARP1/2 inhibitor, and AZD2461, a newly synthesized PARP1/2/3 inhibitor, were assessed in alveolar and embryonal RMS cells both as single-agent and in combination with ionizing radiation (IR).MethodsCell viability was monitored by trypan blue exclusion dye assays. Cell cycle progression and apoptosis were measured by flow cytometry, and alterations of specific molecular markers were investigated by, Real Time PCR, Western blotting and immunofluorescence experiments. Irradiations were carried out at a dose rate of 2 Gy (190 UM/min) or 4 Gy (380 UM/min). Radiosensitivity was assessed by using clonogenic assays.ResultsOlaparib and AZD2461 dose-dependently reduced growth of both RH30 and RD cells by arresting growth at G2/M phase and by modulating the expression, activation and subcellular localization of specific cell cycle regulators. Downregulation of phospho-AKT levels and accumulation of γH2AX, a specific marker of DNA damage, were significantly and persistently induced by Olaparib and AZD2461 exposure, this leading to apoptosis-related cell death. Both PARPi significantly enhanced the effects of IR by accumulating DNA damage, increasing G2 arrest and drastically reducing the clonogenic capacity of RMS-cotreated cells.ConclusionsThis study suggests that the combined exposure to PARPi and IR might display a role in the treatment of RMS tumours compared with single-agent exposure, since stronger cytotoxic effects are induced, and compensatory survival mechanisms are prevented.

Organosulfur compounds induce cytoprotective autophagy against apoptosis by inhibiting mTOR phosphorylation activity in macrophages.

Organosulfur compounds (OSCs) are the bioactive components of garlic. Some OSCs have apoptotic or autophagy-inducing effects. Autophagy plays roles in both cytoprotection and apoptosis-related cell death, and the interaction between autophagy and apoptosis is important in the modulation of immune responses. The mechanism of an OSC-mediated effect via the interaction of autophagy and apoptosis is unknown. In this study, the effects of five OSC compounds on autophagy in the macrophage cell line RAW264.7 and primary macrophages were investigated. We found that S-allylcysteine (SAC), diallyl disulde (DADS) and diallyl tetrasulfide (DTS) treatment increased the number of autophagosomes of RAW264.7 cells, inhibited the phosphorylation of ribosomal protein S6 kinase beta-1 (p70S6K/S6K1) which is a substrate of mammalian target of rapamycin (mTOR), and significantly enhanced autophagy flux. The induction of autophagy by SAC, DADS and DTS was inhibited by stably knocking down the expression of autophagy-related gene 5 (ATG5) with short hairpin RNA (shRNA). Further experiments confirmed that SAC, DADS and DTS also induced apoptosis in RAW264.7 cells. The induction of apoptosis and Caspase 3 activity by SAC, DADS and DTS were increased by stably knocking down of ATG5 expression with shRNA in RAW264.7 cells or treating with 5 mM 3-MA in primary macrophages. Our results suggest that SAC, DADS and DTS induce both autophagy and apoptosis. The autophagy induction protects macrophages from apoptosis by inhibiting mTOR phosphorylation activity to maintain the mass of immune cells.

Identification of Synergistic Interaction Between Cannabis-Derived Compounds for Cytotoxic Activity in Colorectal Cancer Cell Lines and Colon Polyps That Induces Apoptosis-Related Cell Death and Distinct Gene Expression.

Introduction: Colorectal cancer remains the third most common cancer diagnosis and fourth leading cause of cancer-related mortality worldwide. Purified cannabinoids have been reported to prevent proliferation, metastasis, and induce apoptosis in a variety of cancer cell types. However, the active compounds from Cannabis sativa flowers and their interactions remain elusive.Research Aim: This study was aimed to specify the cytotoxic effect of C. sativa-derived extracts on colon cancer cells and adenomatous polyps by identification of active compound(s) and characterization of their interaction.Materials and Methods: Ethanol extracts of C. sativa were analyzed by high-performance liquid chromatography and gas chromatograph/mass spectrometry and their cytotoxic activity was determined using alamarBlue-based assay (Resazurin) and tetrazolium dye-based assay (XTT) on cancer and normal colon cell lines and on dysplastic adenomatous polyp cells. Annexin V Assay and fluorescence-activated cell sorting (FACS) were used to determine apoptosis and cell cycle, and RNA sequencing was used to determine gene expression.Results: The unheated cannabis extracts (C2F), fraction 7 (F7), and fraction 3 (F3) had cytotoxic activity on colon cancer cells, but reduced activity on normal colon cell lines. Moreover, synergistic interaction was found between F7 and F3 and the latter contains mainly cannabigerolic acid. The F7 and F7+F3 cytotoxic activity involved cell apoptosis and cell cycle arrest in S or G0/G1 phases, respectively. RNA profiling identified 2283 differentially expressed genes in F7+F3 treatment, among them genes related to the Wnt signaling pathway and apoptosis-related genes. Moreover, F7, F3, and F7+F3 treatments induced cell death of polyp cells.Conclusions: C. sativa compounds interact synergistically for cytotoxic activity against colon cancer cells and induce cell cycle arrest, apoptotic cell death, and distinct gene expression. F3, F7, and F7+F3 are also active on adenomatous polyps, suggesting possible future therapeutic value.

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication.

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5′-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5′-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans.

Vitamin K2 Supports Hematopoiesis through Acting on Bone Marrow Mesenchymal Stromal/Stem Cells

[Background] Myelodysplastic syndrome is an intractable disorder characterized by ineffective hematopoiesis. Although allogeneic hematopoietic stem cell transplantation is the only curative therapy for eligible patients, hematopoiesis-supportive pharmacotherapy is practically important for transplant-ineligible patients to overcome transfusion dependency and infections. Vitamin K2 (VK2, menatetrenone) is a drug used to aim at improvement of hematopoiesis in MDS patients (Leukemia 14: 1156, 2000). However, the exact mechanism how VK2 improves hematopoiesis remains largely unknown. It was reported that VK2 induces MDS cells to undergo apoptosis (Leukemia 13: 1399, 1999). Here, we investigated our hypothesis that VK2 exerts its hematopoiesis-supportive effects through acting on mesenchymal stem/stromal cells (BM-MSCs) in the bone marrow microenvironment.[Methods] Normal bone marrow (BM) samples from healthy adult volunteers were purchased from AllCells (Emeryville, CA). BM-CD34+ cells were isolated from BM-mononuclear cells using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Human BM-MSCs were isolated according to our previously published methods (Stem Cells 32:2245, 2014). In co-culture experiments, BM-MSCs with or without VK2 treatment were seeded on a 24-well culture plate. BM-CD34+ cells were applied on the MSC-grown plate and co-cultured in SFEM (StemCell Technologies, Vancouver, Canada) supplemented with 100 ng/mL SCF, 100 ng/mL Flt-3 ligand, 50 ng/mL TPO and 20 ng/mL IL-3. After 10 days of co-culture, the number and surface marker expression of the expanded hematopoietic cells were examined by flow cytometric analysis.[Results] We first tested the direct effect of VK2 on BM-CD34+ cells. BM-CD34+ cells were treated with VK2 at various concentrations ranged from 0 µM to 10 µM for 24 hours and then cultured in SFEM in combinations with cytokines. Surprisingly, viable hematopoietic cells were hardly detected in the expansion culture of BM-CD34+ cells treated with 10 µM VK2. Even with 1 µM treatment, the number of CD45+ cells was decreased, as compared to that of expansion culture of untreated BM-CD34+ cells. The apoptosis analysis showed that the percentage of AnnexinV+ PI+ cells in the expanded hematopoietic cells is increased by VK2 treatment. We next examined the effect of VK2 on the hematopoiesis-supportive capability of BM-MSCs. BM-MSCs were pretreated with VK2 at various concentrations and then co-cultured with BM-CD34+ cells. The numbers of CD34+ cells and CD45+ cells were increased in a VK2 dose-dependent manner. These results demonstrated that VK2 shows different effects on distinct stem/progenitor cells: the induction of apoptosis in BM-CD34+ cells and the enhancement of hematopoiesis-supportive capability of BM-MSCs. We then investigated whether apoptosis-related cell death of BM-CD34+ cells by VK2 treatment is ameliorated in the presence of BM-MSCs. Both BM-CD34+ cells and BM-MSCs were treated with VK2 for 24 hours, and then co-cultured. The number of CD34+ cells was not decreased significantly in contrast to its severe decrease in single culture of VK2-treated BM-CD34+ cells.We further analyzed the effect of VK2 on BM-MSCs. Subpopulation analysis in co-culture of CD34+ cells with VK2-treated BM-MSCs showed that the expansion efficacy of CD34+CD38+ cells is higher in comparison to that of CD34+CD38- cells. In addition, the percentages of CD34-CD33+ cells and CD34-CD13+ cells were higher than those in co-cultures with untreated BM-MSCs. Therefore, VK2-treated BM-MSCs supported the expanded CD34+ cells to skew their phenotype toward myeloid lineage. The presence of a transwell in the co-culture system was unrelated to the expansion pattern of CD34+ cells, which suggested the involvement of soluble factors with respect to the underlining mechanism. We therefore compared the levels of hematopoiesis-supporting cytokine mRNA expression in VK2-treated and untreated BM-MSCs: VK2-treated BM-MSCs showed lower expression of CXCL12/SDF-1 mRNA and a trend toward higher expression of GM-CSF mRNA.[Summary] VK2 acted on BM-MSCs to support their ability to enhance expansion and myeloid differentiation of BM-CD34+ cells probably via altered GM-CSF and CXCL12/SDF-1 expression in MSCs. These findings may help to identify the mechanisms of therapeutic effects of VK2 in patients with MDS (Figure). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SapC-DOPS nanovesicles as targeted therapy for lung cancer.

Lung cancer is the deadliest type of cancer for both men and women. In this study, we evaluate the in vitro and in vivo efficacy of a biotherapeutic agent composed of a lysosomal protein (Saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS), which can be assembled into nanovesicles (SapC-DOPS) with selective antitumor activity. SapC-DOPS targets phosphatidylserine, an anionic phospholipid preferentially exposed in the surface of cancer cells and tumor-associated vasculature. Because binding of SapC to phosphatidylserine is favored at acidic pHs, and the latter characterizes the milieu of many solid tumors, we tested the effect of pH on the binding capacity of SapC-DOPS to lung tumor cells. Results showed that SapC-DOPS binding to cancer cells was more pronounced at low pH. Viability assays on a panel of human lung tumor cells showed that SapC-DOPS cytotoxicity was positively correlated with cell surface phosphatidylserine levels, whereas mitochondrial membrane potential measurements were consistent with apoptosis-related cell death. Using a fluorescence tracking method in live mice, we show that SapC-DOPS specifically targets human lung cancer xenografts, and that systemic therapy with SapC-DOPS induces tumor apoptosis and significantly inhibits tumor growth. These results suggest that SapC-DOPS nanovesicles are a promising treatment option for lung cancer.

Beta-Lapachone Suppresses Non-small Cell Lung Cancer Proliferation through the Regulation of Specificity Protein 1.

Lung cancer is the leading cause of cancer-related death worldwide, and non-small cell lung cancer (NSCLC) is the most common pathological type with a reported frequency of about 85% of all cases. Despite recent advances in therapeutic agents and targeted therapies, the prognosis for NSCLC remains poor, and therefore it is important to identify the biological targets of this complex disease since a blockade of such targets would affect multiple downstream signaling cascades. β-Lapachone (β-Lap) is an antiproliferative agent that selectively induces apoptosis-related cell death in a variety of human cancer cells. However, the mechanisms of its action require further investigation. In this study, we show that treatment with β-lap triggers apoptosis and cell-cycle arrest in two NSCLC cell lines: H1299 and NCI-H358. The transcription factor specificity protein 1 (Sp1) was markedly inhibited by β-lap in a dose- and time-dependent manner. Furthermore, β-lap modulated the protein expression levels of the Sp1 regulatory genes, including cell-cycle regulatory proteins and antiapoptotic proteins, resulting in apoptosis. Taken together, our results indicate that β-lap may be a potential antiproliferative agent candidate by inducing apoptotic cell death in NSCLC tissue through downregulation of Sp1.

Arsenic-induced Aurora-A activation contributes to chromosome instability and tumorigenesis

Arsenic may cause serious environmental pollution and is a serious industrial problem. Depending on the dosage, arsenic may trigger the cells undergoing either proliferation or apoptosis-related cell death. Because of lack of the proper animal model to study arsenic induced tumorigenesis, the accurate risk level of arsenic exposure has not been determined. Arsenic shows genotoxic effect on human beings who uptake water contaminated by arsenic. Chromosome aberration is frequently detected in arsenic exposure-related diseases and is associated with increased oxidative stress and decreased DNA repairing activity, but the underlying mechanism remains elusive.Aurora-A is a mitotic kinase, over-expression of Aurora-A leads to centrosome amplification, chromosomal instability and cell transformation. We revealed that Aurora-A is over-expressed in the skin and bladder cancer patients from blackfoot-disease endemic areas. Our cell line studies reveal that arsenic exposure between 0.5μM and 1μM for 2–7days are able to induce Aurora-A expression and activation based on promoter activity, RNA and protein analysis. Aurora-A overexpression further increases the frequency of unsymmetrical chromosome segregation through centrosome amplification followed by cell population accumulated at S phase in immortalized keratinocyte (HaCaT) and uroepithelial cells (E7). Furthermore, Aurora-A over-expression was sustained for 1–4weeks by chronic treatment of immortalized bladder and skin cells with NaAsO2. Aurora-A promoter methylation and gene amplification was not detected in the long-term arsenic treated E7 cells. Furthermore, the expression level of E2F1 transcription factor (E2F1) is increased in the presence of arsenic, and arsenic-related Aurora-A over-expression is transcriptionally regulated by E2F1. We further demonstrated that overexpression of Aurora-A and mutant Ha-ras or Aurora-A and mutant p53 may act additively to trigger arsenic-related bladder and skin cancer formation, respectively. It indicates that from chromosome instability proceeding to tumorigenesis, the simultaneous action of Aurora-A with activated oncogenic factor or inactivated tumor suppressor is required.In summary, we hypothesize that low concentration (0.5–1μM) of arsenic-induced E2F1-Aurora-A signaling pathway results in aberrant chromosome distribution during cell mitosis, the abnormal mitotic cells proceed to cancer cells only after acquiring additional tumorigenic factors. Our studies suggest that inhibition of low concentration of arsenic induced Aurora-A expression may provide a new theraputical strategy for the prevention and treatment of arsenic-related cancers.

Bromelain-Induced Apoptosis in GI-101A Breast Cancer Cells

Bromelain is a proteolytic enzyme extracted from the stems and the immature fruits of pineapple that was found to be antitumorigenic in different in vitro models. Bromelain has been reported to promote apoptosis, particularly in breast cancer cells, with the up-regulation of c-Jun N-terminal kinase and p38 kinase. Our study was designed to determine if bromelain could induce apoptosis in GI-101A breast cancer cells. GI-101A cells were treated with increasing concentrations of bromelain for 24 hours. The effect of bromelain for inducing cell death via activation of the apoptosis mechanism in GI-101A cells was further determined by using caspase-9 and caspase-3 assays along with the M30-Apoptosense assay to measure cytokeratin 18 (CK18) levels in the cytoplasm of the cultured cancer cells. A dose-dependent increase in the activities of caspase-9 and caspase-3 coinciding with elevation of CK18 levels was found in bromelain-treated cells compared with control cells. Furthermore, the apoptosis induction by bromelain was confirmed by DNA fragmentation analysis and 4,6'-diamino-2-phenylindole dihydrochloride fluorescence staining of the nucleus. Our results indicate an increase in apoptosis-related cell death in breast cancer cells with increasing concentrations of bromelain.

Thyroid Hormones Antagonize and Tetrac, a Deaminated T4 Analog, Sensitizes Bortezomib Action in Multiple Myeloma Cells

Abstract 2867 Background:Multiple myeloma (MM) is a highly resistant hematological neoplasia that remains an incurable disease. A leading drug in MM treatment is bortezomib, a selective proteasome inhibitor. Current treatment protocols have extended the overall survival of patients with MM, however, ultimately the disease becomes refractory to all forms of treatments and therefore drugs with new mechanisms of action are urgently needed.Experimental and clinical observations suggest that thyroid hormones (T3 and T4) modulate neoplastic cells and activate MAPK pathway through binding to αv β3 integrin, commonly overexpressed in cancer. Tetraiodothyroacetic acid (tetrac), a non-agonist T4 analog, selectively blocks T3 and T4 binding to αv β3 receptor site. MM cells interact with αv β3 for invasion/growth and thyroid diseases were associated with increased MM risk. We recently demonstrated the thyroid hormones- αv β3-MAPK axis in myeloma cells. In the current study we further show that T3 and T4 antagonize bortezomib action via MAPK activation and that in the presence of tetrac bortezomib action is significantly enhanced. Methods:Cell lines: MM cell lines (RPMI-8226, ARK, ARP-1, U266 and CAG) are cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS/antibiotics. Before addition of T3 or T4, cells are grown for 48 hours without serum. Bone marrow (BM) aspirates were obtained from patients with MM treated at the Meir Medical Center. Signed institutional review board–approved written informed consent was obtained from all patients. Primary MM cells were separated on Ficoll gradient and were cultured in RPMI 1640. Reagents and chemicals: T3, T4, tetrac, MAPK inhibitor (U0126), autophagy inhibitor (3MA) and pan caspase inhibitor, Z-VAD. Cells were treated with T3 or T4 (1nM-100nM and 1μM) in the presence/absence of tetrac (100nM and 1μM) and/or bortezomib (25nM) and tested by several methods: Cell number. Cell proliferation assay: WST-1 (10% final concentration) is incubated at 37°C for 2 h and read using microELISA reader at 440nm. Cell cycle: Cells are harvested, fixed and stained with DNA propidium iodide (PI) (50 μg/ml) /RNAse A (10mg/ml) and analyzed for DNA content by FACS. Analysis of apoptosis/necrosis: Cells (105) are incubated with 5 μl Annexin V (FITC conjugated)/5 μl PI and analyzed by FACS. Expression of apoptotic genes (real-time PCR). Results were repeated 2–3 times in triplicates and were analyzed using unpaired students t test.Results and discussion: Results demonstrate that T3 and T4 at near physiological and supra physiological levels, increased myeloma cell viability by 15–50% and cell number by 30%-60%. This increased viability was blocked by U0126, indicating involvement of the MAPK pathway. In parallel a 20–25% reduction in cell death and of pro-apoptotic genes expression was documented following treatment with the hormones. Co-treatment of myeloma cell lines with T3 or T4 reduced bortezomib cytotoxicity and increased cell survival in a MAPK-dependent manner. Pretreatment of MM cell lines and primary cells from MM patients with tetrac, 48 hours before the addition of bortezomib, resulted in a synergistic cytotoxic effect. The effect of tetrac was blocked using a pan-caspase inhibitor (Z-VAD) but not by an autophagy inhibitor (3MA), suggesting apoptosis-related cell death. Conclusions:We present here novel data demonstrating that T3 and T4 may oppose bortezomib action via MAPK activation. Blocking the thyroid hormones- αv β3 axis using tetrac, promotes bortezomib cytotoxicity, suggesting this approach as a promising adjunct therapy in MM. Disclosures:No relevant conflicts of interest to declare.

Different effect of protein kinase B/Akt and extracellular signal-regulated kinase inhibition on trichostatin A-induced apoptosis in epithelial ovarian carcinoma cell lines

Histone deacetylase inhibitor-induced apoptosis in cancer cells may be mediated by the Ras/Raf/MEK/ERK and protein kinase B/Akt signaling pathways. However, inhibition of ERK and Akt activity has different effects on proliferation and apoptosis in cancer cells. We assessed and compared the inhibitory effects of Akt and ERK pathways on the apoptotic effect of trichostatin A using the human epithelial carcinoma cell lines OVCAR-3 and SK-OV-3. Trichostatin A induced nuclear damage, decrease in Bid and Bcl-2 protein levels, increase in Bax levels, cytochrome c release, activation of caspases (8, 9, and 3) and increase in tumor suppressor p53 levels. Akt inhibitor potentiated trichostatin A-induced apoptosis-related protein activation and cell death, whereas ERK inhibitor exhibited an additive toxic effect. These results suggest that the Akt and ERK inhibitors may have a differential effect on trichostatin A-induced apoptosis in human epithelial ovarian carcinoma cell lines. Akt inhibitor may potentiate the apoptotic effect of trichostatin A on ovarian carcinoma cell lines by increasing the activation of the caspase-8-dependent pathway and the mitochondria-mediated cell death pathway, leading to caspase activation. In contrast, ERK inhibitor may exhibit an additive toxic effect on trichostatin A toxicity by increasing apoptosis-related protein activation.

The Kinetics and Redox State of Nitric Oxide Determine the Biological Consequences in Lung Adenocarcinoma

Few studies have explored the mechanistic basis for the apparent paradoxical effects of nitric oxide and its interrelated redox species (NO_X) in cancer biology. Our aim was to determine the differential effects of the redox state and kinetics of nitrosative species on the key cancer processes of apoptosis. Therefore, a murine lung adenocarcinoma cell line was exposed to various NO_X donor compounds differing in redox state and delivery kinetics. DNA strand breaks (DSBs) were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay) and correlated with cell viability by the MTT and soft agar colony assays, while caspase enzymatic activity was measured using an in vitro fluorogenic caspase assay. Finally, cDNA microarrays defined apoptosis-related gene expression alterations resultant from these NO_X donors. Exogenous NO_X differentially influences DSBs, and apoptosis-related cell death and expression based on the redox state and kinetics of NO_X delivery. In our murine lung adenocarcinoma model we have demonstrated differential effects of NO_X based on the mode of delivery and redox state. These data suggest that the development of NO_X-based cancer chemotherapy must consider the redox state and kinetics of delivery into their logical design.

PROTECTION FROM HEPATIC ISCHEMIA/REPERFUSION INJURY AND IMPROVEMENT OF LIVER REGENERATION BY ??-LIPOIC ACID

The aim of this study was to characterize the in vivo action of lipoic acid (LA) in hepatic ischemia/reperfusion injury (IRI) and its effects on liver regeneration involving the investigation of mechanisms of action and effects on animal survival. Two groups of rats were compared: one group received 500 micromol alpha-LA injected via the inferior vena cava 15 min before the induction of 90 min of selective ischemia. The untreated group received vehicle. Influence of LA on IRI of the liver was determined in short- and long-term experiments. Cellular damage was decreased under preconditioning conditions with LA. Caspase 3, 8, and 9 activities were significantly lower in the LA group accompanied by a decrease in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive hepatocytes. Electron micrographs in the untreated group showed massive mitochondrial damage. The survival rate as end point of liver function was markedly increased after pretreatment with LA. Increased levels of tumor necrosis factor alpha was shown by enzyme-linked immunosorbent assay as well as real-time reverse transcription-polymerase chain reaction in the LA group accompanied by increased mitotic index and Ki-67 staining in liver tissue. Attenuation of IRI of the rat liver in vivo by LA is accompanied by reduction of necrosis and apoptosis-related cell death, whereas liver regeneration is increased.

Different Protection Mechanisms after Pretreatment with Glycine or α-Lipoic Acid in a Rat Model of Warm Hepatic Ischemia

Background/Aim: α-Lipoic (LA) acid pretreatment has previously been described to reduce ischemia/reperfusion injury (IRI) after warm liver ischemia, whereas glycine pretreatment has been shown to be protective mostly in models of cold hepatic ischemia. The aim of this study was to determine whether glycine decreases IRI after warm hepatic ischemia. Furthermore we investigated whether doses of LA other than those used previously are also protective against IRI after warm hepatic ischemia. Methods: Selective liver ischemia was maintained over a period of 90 min. In long-term as well as short-term experiments we studied IRI in several groups comparing animal survival as the pivotal endpoint. Results: Animal survival was improved by glycine and 5,000 µmol LA, whereas all animals died within 3 days after pretreatment with 50 µmol LA. In the glycine group we observed a tendency towards decreased apoptosis-related cell death measured by the activity of caspase-3 in liver tissue and the percentage of TUNEL-positive hepatocytes in comparison to the untreated group. Serum α-glutathione S-transferase, lipid peroxidation, and caspase-3 activity as well as the percentage of TUNEL-positive hepatocytes and the percentage of liver necrosis were only significantly decreased by 5,000 µmol LA pretreatment. Liver tissue levels of tumor necrosis factor (TNF)α were reduced only in the glycine group whereas TNFα was increased in the untreated as well as the LA group. Levels of TNFα mRNA were upregulated in both the glycine- and LA-pretreated groups. Conclusion: Our data show that increased animal survival by glycine was accompanied by a reduced TNFα content in liver tissue. Protection by glycine is likely to result from a reduction in adverse TNFα effects. Administration of high-dose LA on the other hand led to a significant reduction in necrosis- and apoptosis-related cell death in IRI of the liver without a reduction in liver TNFα.

Effects of dexamethasone on apoptosis-related cell death after spinal cord injury.

The purpose of this study was to analyze the expression of F7-26 (Apostain) in injured spinal cord tissue, and the modifying effects of dexamethasone administration. A total of 56 adult female Wistar rats were subjected to traumatic spinal cord injury (SCI) to induce complete paraplegia. These rats were divided into two groups according to whether they received dexamethasone (doses of 1 mg/kg daily) post-SCI. Injured spinal cord tissue was studied by means of conventional histological techniques, and Apostain expression was determined by immunohistochemical analysis at 1, 4, 8, 24, and 72 hours, and at 1 and 2 weeks after SCI in all the animals. Apostain-positive cells, mainly neurons and glial cells, were detected 1 hour after injury, peaking at 8 hours, after which the number decreased. One week after injury, apoptosis was limited to a few glial cells, mainly oligodendrocytes, and 2 weeks after injury there was no evidence of Apostain-positive cells. In the group of paraplegic rats receiving post-SCI intraperitoneal dexamethasone, there was a significant decrease in the number of Apostain-positive cells. Analysis of the results indicated that apoptosis plays a role in the early period after SCI and that administration of dexamethasone decreases apoptosis-related cell death in the injured spinal cord tissue.