Cell Survival Signaling Pathways Research Articles

DOX-Vit D, a Novel Doxorubicin Delivery Approach, Inhibits Human Osteosarcoma Cell Proliferation by Inducing Apoptosis While Inhibiting Akt and mTOR Signaling Pathways.

Doxorubicin (DOX) is a very potent and effective anticancer agent. However, the effectiveness of DOX in osteosarcoma is usually limited by the acquired drug resistance. Recently, Vitamin D (Vit-D) was shown to suppress the growth of many human cancer cells. Taken together, we synthesized DOX-Vit D by conjugating Vit-D to DOX in order to increase the delivery of DOX into cancer cells and mitigate the chemoresistance associated with DOX. For this purpose, MG63 cells were treated with 10 µM DOX or DOX-Vit D for 24 h. Thereafter, MTT, real-time PCR and western blot analysis were used to determine cell proliferation, genes and proteins expression, respectively. Our results showed that DOX-Vit D, but not DOX, significantly elicited an apoptotic signal in MG63 cells as evidenced by induction of death receptor, Caspase-3 and BCLxs genes. Mechanistically, the DOX-Vit D-induced apoptogens were credited to the activation of p-JNK and p-p38 signaling pathway and the inhibition of proliferative proteins, p-Akt and p-mTOR. Our findings propose that DOX-Vit D suppressed the growth of MG63 cells by inducing apoptosis while inhibiting cell survival and proliferative signaling pathways. DOX-Vit D may serve as a novel drug delivery approach to potentiate the delivery of DOX into cancer cells.

Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patients

ObjectiveRegenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Whereas little is known about human diabetic MSCs (dMSCs), particularly those from patients with PAD, dMSCs have a limited expansion capacity but can be improved with human platelet lysate (PL) supplementation. PL is rich in many growth factors, including epidermal growth factor (EGF), which is known to be important to cell proliferation and survival signaling pathways. We hypothesize that dMSCs have a reversible defect in EGF receptor pathways. The objective of this work was to test this hypothesis using dMSCs from PAD patients. MethodsThe secretome expression of EGF and prominent angiogens was characterized from bone marrow (BM)-derived and adipose tissue-derived (ATD) dMSCs from five patients (six limbs) undergoing major amputation. Western blot was used to characterize the AKT and extracellular signal-regulated protein kinases 1 and 2 expression in dMSCs under standard culture (5% fetal bovine serum plus fibroblast growth factor 2 [FGF2]), 5% human PL, or 5% fetal bovine serum plus EGF. Healthy donor MSCs were control cells. The angiogenic activity of BM- and ATD-dMSCs was tested on human umbilical vein endothelial cells (ECs). Paired t-test, analysis of variance, and Kruskal-Wallis tests were used as appropriate. ResultsBoth BM- and ATD-dMSCs had typical MSC surface marker expression and similar expansion profiles, and they did not express EGF in their secretome. PL supplementation of dMSCs improved AKT signaling, but they were resistant to FGF2 activation of extracellular signal-regulated protein kinases 1 and 2. EGF supplementation led to similar AKT expression as with PL, but PL had greater phosphorylation of AKT at 30 and 60 minutes. The conditioned media from both BM- and ATD-dMSCs had robust levels of prominent angiogens (vascular endothelial growth factor, monocyte chemoattractant protein 1, hepatocyte growth factor), which stimulated EC proliferation and migration, and the co-culture of dMSCs with ECs led to significantly longer EC sprouts in three-dimensional gel than EC-alone pellets. ConclusionsPL and EGF supplementation improves AKT expression in dMSCs over that of FGF2, but PL improved pAKT over that of EGF. Thus, PL supplementation strategies may improve AKT signaling, which could be important to MSC survival in cellular therapies. Furthermore, BM- and ATD-dMSCs have similar secretomes and robust in vitro angiogenic activity, which supports pursuing dMSCs from both reservoirs in regenerative medicine strategies.

Abstract 2448: Chemotherapy drug-induced AXL activation and cell survival signaling via reactive oxygen species that can be inhibited to enhance drug efficacy in mesothelioma

Abstract Cellular oxidative stress is characterized by elevated reactive oxygen species (ROS), and is a frequent phenotype of cancer cells. The consequences of ROS are complex, where ROS can promote or inhibit tumorigenesis. In mesothelioma, asbestos exposure to serous membranes induces ROS through iron content and chronic inflammation, and ROS promote cell survival signaling in mesothelioma. On the other hand, a current chemotherapy regime for mesothelioma consisting of a platinum and antifolate agent combination also induce ROS. Mesothelioma is notoriously chemotherapy-resistant, and we propose that ROS induced by cisplatin and pemetrexed may promote cell survival signaling pathways, which ultimately may contribute to chemotherapy resistance. In The Cancer Genome Atlas datasets, we found AXL kinase expression is relatively high in mesothelioma compared to other cancer samples. We showed that ROS induce the phosphorylation of AXL, which was blocked by the selective inhibitor BGB324 in VMC40 and P31 mesothelioma cells. We also showed that cisplatin and pemetrexed induce the phosphorylation of AXL and Akt, which was also blocked by BGB324 as well as by N-acetylcysteine antioxidant. AXL knockdown in these cells enhances sensitivity to cisplatin and pemetrexed. Similarly, AXL inhibitor BGB324 also enhances sensitivity to cisplatin and pemetrexed. Finally, higher synergy was observed when cells were pretreated with BGB324 before adding chemotherapy. These results demonstrate cisplatin and pemetrexed induce ROS that activate AXL, and blocking AXL activation enhances the efficacy of cisplatin and pemetrexed. These results suggest AXL inhibition may represent an effective strategy to enhance the efficacy of chemotherapy in mesothelioma. We further hypothesize AXL signaling may be part of an adaptive response to chemotherapeutic agents, providing transient resistance to cisplatin and pemetrexed that may ultimately contribute to acquired chemotherapy resistance. Citation Format: Derek B. Oien, Tamás Garay, Sarah Eckstein, Jeremy Chien. Chemotherapy drug-induced AXL activation and cell survival signaling via reactive oxygen species that can be inhibited to enhance drug efficacy in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2448.

Sulfiredoxin-1 enhances cardiac progenitor cell survival against oxidative stress via the upregulation of the ERK/NRF2 signal pathway

Cardiac stem/progenitor cells (CPCs) have recently emerged as a potentially transformative regenerative medicine to repair the infarcted heart. However, the limited survival of donor cells is one of the major challenges for CPC therapy. Our recent research effort on preconditioning human CPCs (hCPCs) with cobalt protoporphyrin (CoPP) indicated that sulfiredoxin-1 (SRXN1) is upregulated upon preconditioning aldehyde dehydrogenase bright hCPCs (ALDHbr-hCPCs) with CoPP. Further studies demonstrated that overexpressing SRXN1 enhanced the survival capacity for ALDHbr-hCPCs. This was associated with the up-regulation of anti-apoptotic factors, including BCL2 and BCL-xL. Meanwhile, overexpressing SRXN1 decreased the ROS generation and mitochondrial membrane potential, concomitant with the up-regulated primary antioxidant systems, such as PRDX1, PRDX3, TXNRD1, Catalase and SOD2. It was also observed that overexpressing SRXN1 increased the migration, proliferation, and cardiac differentiation of ALDHbr-hCPCs. Interestingly, SRXN1 activated the ERK/NRF2 cell survival signaling pathway, which may be the underlying mechanism through which overexpressing SRXN1 lead to protection of hCPCs against oxidative stress-induced apoptosis. Taken together, these results provide a rationale for the exploration of SRXN1 as a novel molecular target that can be used to enhance the effectiveness of cardiac stem/progenitor cell therapy for ischemic heart disease.

OP-41 - Nitroxide radical-containing nanoparticles impairs the tumorigenic potential of triple negative breast cancer

Persistent elevated intracellular reactive oxygen species (ROS), requisite for cancerous phenotypes, has been known to affect cancers in a positive feedback manner, by playing critical roles in survival, proliferation and metastasis processes. Hence, scavenging of ROS to abrogate cell survival signaling pathways may therefore offer a potential therapeutic regime to mitigate the progression of various cancers. We therefore synthesized ROS scavenging nitroxide radical-containing nanoparticles (RNPs); pH insensitive RNPO and pH sensitive RNPN, to impede the proliferative and metastatic characteristics of the triple negative breast cancer cell line, MDA-MB-231, both in vitro and in vivo. RNPs significantly inhibited proliferation, colony forming potential, migration and invasion of MDA-MB-231 cells, accompanied by decrease in ROS level. Furthermore, anti-tumor and anti-metastatic potential of RNPs was observed in an MDA-MB-231 xenograft mouse model via downregulation of ROS-sensitive transcriptional factor, NF-κB and migration regulator, MMP-2 with negligible adverse effect compared to low molecular weight TEMPOL and paclitaxel. Conclusively, our findings provide insights into the use of nanoparticle over low molecular weight antioxidants in the redox-based cancer therapeutics.

Targeting EGFR-mediated autophagy as a potential strategy for cancer therapy.

Autophagy is a naturally occurring programed cellular catabolic process stimulated by cellular stress for energy homeostasis maintenance and elimination of harmful substances. It mostly works as pro-survival mechanism but on the other hand deregulation of autophagy has been linked to non-apoptotic cell death known as "type II programed cell death." Emerging evidences indicate that EGFR (epidermal growth factor receptor)-mediated RAS/RAF/MEK/ERK signaling pathway plays a critical role in the induction of autophagy in various tumors. It has further been established that this signaling pathway is also involved in several other anti-proliferative events such as apoptosis and senescence. However, the signaling pathway activity and effects are highly dependent on the cell type and the stimulus. It is currently being evident that autophagy induction by RAS/RAF/MEK/ERK pathway through small molecules may be a potential therapeutic strategy for cancer. However, to our best knowledge, the role of EGFR-mediated RAS/RAF/MEK/ERK signaling pathway in autophagy-mediated cell death and survival have not previously been reviewed. In this review, we discuss the current state of knowledge on how RAS/RAF/MEK/ERK signaling pathway regulates autophagy and the role of this EGFR-mediated autophagy in diseases. We further examine the cross-talk between this EGFR-mediated autophagy and apoptosis as well as how this process is currently being utilized for cancer treatment and suggest promoting autophagy-related cell death by small molecules may be exploited to design better therapeutic strategies for early stage and locally advanced tumors.

VDAC1 as Pharmacological Target in Cancer and Neurodegeneration: Focus on Its Role in Apoptosis.

Cancer and neurodegeneration are different classes of diseases that share the involvement of mitochondria in their pathogenesis. Whereas the high glycolytic rate (the so-called Warburg metabolism) and the suppression of apoptosis are key elements for the establishment and maintenance of cancer cells, mitochondrial dysfunction and increased cell death mark neurodegeneration. As a main actor in the regulation of cell metabolism and apoptosis, VDAC may represent the common point between these two broad families of pathologies. Located in the outer mitochondrial membrane, VDAC forms channels that control the flux of ions and metabolites across the mitochondrion thus mediating the organelle's cross-talk with the rest of the cell. Furthermore, the interaction with both pro-apoptotic and anti-apoptotic factors makes VDAC a gatekeeper for mitochondria-mediated cell death and survival signaling pathways. Unfortunately, the lack of an evident druggability of this protein, since it has no defined binding or active sites, makes the quest for VDAC interacting molecules a difficult tale. Pharmacologically active molecules of different classes have been proposed to hit cancer and neurodegeneration. In this work, we provide an exhaustive and detailed survey of all the molecules, peptides, and microRNAs that exploit VDAC in the treatment of the two examined classes of pathologies. The mechanism of action and the potential or effectiveness of each compound are discussed.

Expression of Hypoxia Inducible Factor 1alpha Is Protein Kinase A-dependent in Primary Cortical Astrocytes Exposed to Severe Hypoxia

The hypoxia inducible factor 1 (HIF-1) and the cyclic AMP-responsive element binding protein (CREB) are two transcription factors that have been studied in the context of neuronal survival and neurodegeneration. HIF-1 upregulation and CREB activation have been observed not only in neurons but also in astrocytes under conditions of hypoxia. We hypothesized that activation of CREB regulate HIF-1α expression in the nucleus of cortical astrocytes under in vitro ischemic condition. To test the hypothesis, we determined the effects of inhibiting the CREB activation pathway on the expression of HIF-1α protein in astrocytes exposed to CoCl2 and severe hypoxia (near anoxia, 0.1% O2). The results demonstrated that inhibition of CaMKII and CaMKIV had no effect on both HIF-1α and pCREB expression in cortical astrocytes exposed to CoCl2 and anoxia. In contrast, PKA inhibition lowered the expression of HIF-1α and pCREB expression. Furthermore, the inhibition of PKA but not CaMKII or CaMKIV increased cell death of astrocytes exposed to near anoxia. The results suggest that PKA plays an important role in the cell survival signaling pathways in astrocytes.

Calcium-activated SK potassium channels are key modulators of the pacemaker frequency in locus coeruleus neurons

The physiological, intrinsic activity of noradrenergic locus coeruleus (LC) neurons is important for the control of sleep/wakefulness, cognition and autonomous body functions. Dysregulations of the LC-noradrenergic network contribute to the pathogenesis of psychiatric disorders and are key findings in early stages of neurodegenerative diseases. Therefore, identifying ion channels mediating the intrinsic pacemaking mechanism of LC neurons, which is in turn directly coupled to Ca2+ homeostasis and cell survival signaling pathways, can help to foster our understanding of the vulnerability of these neurons in neurodegenerative diseases. Small-conductance Ca2+-activated K+ (SK) channels regulate the intrinsic firing patterns in different central neurons and are essential regulators of the intracellular Ca2+ homeostasis. However, the role of SK channels for the intrinsic pacemaking of LC neurons in mice is still unclear. Therefore we performed qPCR expression analysis as well as patch clamp recordings of in vitro brainstem slices, for instance testing SK channel blockers and activators like apamin and NS309, respectively. Although we found a transcriptional expression of SK1, SK2 and SK3 channels, SK2 was the predominantly expressed subunit in mouse LC neurons. Using perforated-patch clamp experiments, we found that SK channels are essential regulators of the intrinsic pacemaking of LC neurons, mediating a large fraction of the afterhyperpolarization (AHP) in these cells. Consistent with a previous observation that a concerted action of L- and T-type Cav channels is essential for the pacemaking of LC neurons, we found that SK channel activation, and the respective AHP amplitude, is primarily coupled to Ca2+ influx via these types of Ca2+ channels. Our study identified SK2 channels as drug targets for the tuning of the pacemaker frequency in disorders involving a dysregulation of the LC.

Cisplatin and Pemetrexed Activate AXL and AXL Inhibitor BGB324 Enhances Mesothelioma Cell Death from Chemotherapy.

Reactive oxygen species (ROS) can promote or inhibit tumorigenesis. In mesothelioma, asbestos exposure to serous membranes induces ROS through iron content and chronic inflammation, and ROS promote cell survival signaling in mesothelioma. Moreover, a current chemotherapy regimen for mesothelioma consisting of a platinum and antifolate agent combination also induce ROS. Mesothelioma is notoriously chemotherapy-resistant, and we propose that ROS induced by cisplatin and pemetrexed may promote cell survival signaling pathways, which ultimately may contribute to chemotherapy resistance. In The Cancer Genome Atlas datasets, we found AXL kinase expression is relatively high in mesothelioma compared to other cancer samples. We showed that ROS induce the phosphorylation of AXL, which was blocked by the selective inhibitor BGB324 in VMC40 and P31 mesothelioma cells. We also showed that cisplatin and pemetrexed induce the phosphorylation of AXL and Akt, which was also blocked by BGB324 as well as by N-acetylcysteine antioxidant. AXL knockdown in these cells enhances sensitivity to cisplatin and pemetrexed. Similarly, AXL inhibitor BGB324 also enhances sensitivity to cisplatin and pemetrexed. Finally, higher synergy was observed when cells were pretreated with BGB324 before adding chemotherapy. These results demonstrate cisplatin and pemetrexed induce ROS that activate AXL, and blocking AXL activation enhances the efficacy of cisplatin and pemetrexed. These results suggest AXL inhibition combined with the current chemotherapy regimen may represent an effective strategy to enhance the efficacy of chemotherapy in mesothelioma. This is the first study, to our knowledge, on chemotherapy-induced activation of AXL and cell survival pathways associated with ROS signaling.

Oncogenic RAC1 and NRAS drive resistance to endoplasmic reticulum stress through MEK/ERK signalling

Cancer cells are able to survive under conditions that cause endoplasmic reticulum stress (ER-stress), and can adapt to this stress by upregulating cell-survival signalling pathways and down-regulating apoptotic pathways. The cellular response to ER-stress is controlled by the unfolded protein response (UPR). Small Rho family GTPases are linked to many cell responses including cell growth and apoptosis. In this study, we investigate the function of small GTPases in cell survival under ER-stress. Using siRNA screening we identify that RAC1 promotes cell survival under ER-stress in cells with an oncogenic N92I RAC1 mutation. We uncover a novel connection between the UPR and N92I RAC1, whereby RAC1 attenuates phosphorylation of EIF2S1 under ER-stress and drives over-expression of ATF4 in basal conditions. Interestingly, the UPR connection does not drive resistance to ER-stress, as knockdown of ATF4 did not affect this. We further investigate cancer-associated kinase signalling pathways and show that RAC1 knockdown reduces the activity of AKT and ERK, and using a panel of clinically important kinase inhibitors, we uncover a role for MEK/ERK, but not AKT, in cell viability under ER-stress. A known major activator of ERK phosphorylation in cancer is oncogenic NRAS and we show that knockdown of NRAS in cells, which bear a Q61 NRAS mutation, sensitises to ER-stress. These findings highlight a novel mechanism for resistance to ER-stress through oncogenic activation of MEK/ERK signalling by small GTPases.

Villin-1 and Gelsolin Regulate Changes in Actin Dynamics That Affect Cell Survival Signaling Pathways and Intestinal Inflammation

Cell stress signaling pathways result in phosphorylation of the eukaryotic translation initiation factor 2 subunit alpha (EIF2S1 or EIF2A), which affects regulation of protein translation. Translation reprogramming mitigates stress by activating pathways that result in autophagy and cell death, to eliminate damaged cells. Actin is modified during stress and EIF2A is dephosphorylated to restore homeostasis. It is not clear how actin affects EIF2A signaling. We studied the actin-binding proteins villin 1 (VIL1) and gelsolin (GSN) in intestinal epithelial cells (IECs) to determine whether they respond to cell stress response and affect signaling pathways. We performed studies with mice with disruptions in Vil1 and Gsn (double-knockout mice). Wild-type (WT) mice either were or were not (controls) exposed to cell stressors such as tumor necrosis factor and adherent-invasive Escherichia coli. Distal ileum tissues were collected from mice; IECs and enteroids were cultured and analyzed by histology, immunoblots, phalloidin staining, immunohistochemistry, electron microscopy, and flow cytometry. HT-29 cells were incubated with cell stressors such as DTT, IFN, and adherent-invasive E coli or control agents; cells were analyzed by immunoblots and quantitative polymerase chain reaction. Green fluorescent protein and green fluorescent protein tagged mutant EIF2A were expressed from a lentiviral vector. The mouse immunity-related GTPase (IRGM1) was overexpressed in embryonic fibroblasts from dynamin1 like (DNM1L) protein-knockout mice or their WT littermates. IRGM1 was overexpressed in embryonic fibroblasts from receptor interacting serine/threonine kinase 1-knockout mice or their WT littermates. Human IRGM was overexpressed in human epithelial cell lines incubated with the DNM1L-specific inhibitor Mdivi-1. Mitochondria were analyzed by semi-quantitative confocal imaging. We performed immunohistochemical analyses of distal ileum tissues from 6-8 patients with Crohn's disease (CD) and 6-8 individuals without CD (controls). In IECs exposed to cell stressors, EIF2A signaling reduced expression of VIL1 and GSN. However, VIL1 and GSN were required for dephosphorylation of EIF2A and recovery from cell stress. In mouse and human IECs, prolonged, unresolved stress was accompanied by continued down-regulation of VIL1 and GSN, resulting in constitutive phosphorylation of EIF2A and overexpression of IRGM1 (or IRGM), which regulates autophagy. Overexpression of IRGM1 (or IRGM) induced cell death by necroptosis, accompanied by release of damage-associated molecular patterns (DAMPs). In double-knockout mice, constitutive phosphorylation of EIF2A and over-expression of IRGM1 resulted in spontaneous ileitis that resembled human CD in symptoms and histology. Distal ileum tissues from patients with CD had lower levels of VIL1 and GSN, increased phosphorylation of EIF2A, increased levels of IRGM and necroptosis, and increased release of nuclear DAMPs compared with controls. In studies ofintestinal epithelial tissues from patients with CD and embryonic fibroblasts from mice, along with enteroids and human IEC lines, we found that induction of cell stress alters the cytoskeleton in IECs via changes in the actin-binding proteins VIL1 and GSN. Acute changes in actin dynamics increase IEC survival, whereas long-term changes in actin dynamics leadto IEC death and intestinal inflammation. IRGM regulates necroptosis and release of DAMPs to induce gastrointestinal inflammation, linking IRGM activity with CD.

MiR-596 Modulates Melanoma Growth by Regulating Cell Survival and Death

Tumors grow because cancer cells lack the ability to balance cell survival and death signaling pathways. miR-596, a microRNA located at the 8p23.3 locus, has been shown by the TCGA-Assembler to be deleted in a significant number of melanoma samples. Here, we also validated the low levels of miR-596 in melanoma compared to tissue nevi, and Kaplan-Meier curve analysis revealed that low miR-596 expression was associated with worse overall survival. Moreover, we showed that miR-596 overexpression effectively inhibited MAPK/ERK signaling, cell proliferation, migration, and invasion and increased the cell apoptosis of melanoma cells. In addition, we found that miR-596 directly targets MEK1 and two apoptotic proteins, MCL1, and BCL2L1, in melanoma cells. Our findings indicated that miR-596 is an important miRNA that both negatively regulates the MAPK/ERK signaling pathway by targeting MEK1 and modulates the apoptosis pathway by targeting MCL1 and BCL2L1, suggesting that miR-596 could be a therapeutic candidate for treating melanoma, and a prognostic factor for melanoma patients.

APE1 modulates cellular responses to organophosphate pesticide-induced oxidative damage in non-small cell lung carcinoma A549 cells.

Monocrotophos (MCP) and chlorpyrifos (CP) are widely used organophosphate pesticides (OPPs), speculated to be linked with human pathologies including cancer. Owing to the fact that lung cells are most vulnerable to the environmental toxins, the development and progression of lung cancer can be caused by the exposure of OPPs. The present study investigates the oxidative DNA damage response evoked by MCP and CP in human non-small cell lung carcinoma A549 cells. A549 cells were exposed to MCP and CP; cytotoxicity and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether MCP and CP can initiate the DNA repair and cell survival signalling pathways in A549 cells, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes and transcription factors (TFs) involved in cell survival mechanisms. A significant increase in cell viability and ROS generation was observed when exposed to low and moderate doses of MCP and CP at different time points (24, 48 and 72h) studied. A549 cells displayed a dose-dependent accumulation of apurinic/apyrimidinic (AP) sites after 24h exposure to MCP advocating for the activation of AP endonuclease-mediated DNA BER-pathway. Cellular responses to MCP- and CP-induced oxidative stress resulted in an imbalance in the mRNA and protein expression of BER-pathway enzymes, viz. PARP1, OGG1, APE1, XRCC1, DNA pol β and DNA ligase III α at different time points. The treatment of OPPs resulted in the upregulation of TFs, viz. Nrf2, c-jun, phospho-c-jun and inducible nitric oxide synthase. Immunofluorescent confocal imaging of A549 cells indicated that MCP and CP induces the translocation of APE1 within the cytoplasm at an early 6h time point, whereas it promotes nuclear localization after 24h of treatment, which suggests that APE1 subcellular distribution is dynamically regulated in response to OPP-induced oxidative stress. Furthermore, nuclear colocalization of APE1 and the TF c-jun was observed in response to the treatment of CP and MCP for different time points in A549 cells. Therefore, in this study we demonstrate that MCP- and CP-induced oxidative stress alters APE1-dependent BER-pathway and also mediates cell survival signalling mechanisms via APE1 regulation, thereby promoting lung cancer cell survival and proliferation.

NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis.

Therapeutic targeting of chemoresistant and recurrent glioblastoma stem cells with a proapoptotic variant of oncolytic herpes simplex virus.

Temozolomide (TMZ) chemotherapy, in combination with maximal safe resection and radiotherapy, is the current standard of care for patients with glioblastoma (GBM). Despite this multimodal approach, GBM inevitably relapses primarily due to resistance to chemo-radiotherapy, and effective treatment is not available for recurrent disease. In this study we identified TMZ resistant patient-derived primary and previously treated recurrent GBM stem cells (GSC), and investigated the therapeutic activity of a pro-apoptotic variant of oHSV (oHSV-TRAIL) in vitro and in vivo. We show that oHSV-TRAIL modulates cell survival and MAP Kinase proliferation signaling pathways as well as DNA damage response pathways in both primary and recurrent TMZ-resistant GSC. Utilizing real time in vivo imaging and correlative immunohistochemistry, we show that oHSV-TRAIL potently inhibits tumor growth and extends survival of mice bearing TMZ-insensitive recurrent intracerebral GSC tumors via robust and selective induction of apoptosis-mediated death in tumor cells, resulting in cures in 40% of the treated mice. In comparison, the anti-tumor effects in a primary chemoresistant GSC GBM model exhibiting a highly invasive phenotype were significant but less prominent. This work thus demonstrates the ability of oHSV-TRAIL to overcome the therapeutic resistance and recurrence of GBM, and provides a basis for its testing in a GBM clinical trial.

Abstract 3224: Cucurbitacin D inhibits prostate tumor growth via targeting glucose metabolism

Abstract Background: Emergence of hormone-refractory PrCa (HRPC) after the anti-androgen therapy, cancer metastasis and chemo-resistance are the major hurdle for the treatment of prostate cancer (PrCa) patients. Accumulative evidence suggests that altered glucose metabolism is one of the mechanisms for metastatic PrCa cell survival and chemo-resistance. Therefore, identification and generation of natural/or synthetic pharmacological agents that can limit altered glucose metabolism might be highly useful for the treatment of metastatic and chemo-resistant PrCa. Cucurbitacins have shown potent anti-cancer and glucagonostic activities along with severe liver toxicity. Thus, a focus on developing different analogues of cucurbitacin is being pursued by scientific community. Herein, we report that Cucurbitacin D, an analogue of cucurbitacin, suppresses the growth of metastatic PrCa cells in vitro and in vivo via targeting glucose metabolism and its associated molecular targets. Methods: HRPC cells (PC3 and DU145) was used as a model system. Effect of Cucurbitacin D on PrCa cell proliferation and apoptosis was performed by MTS, xCELLigence and Annexin V assays. Effect of Cucurbitacin D on clonogenic potential of PrCa cells was examined by colony formation assay. In silico analysis was performed to study if Cucurbitacin D interacts with GLUT1 receptor. Effect of Cucurbitacin D on glucose metabolism of PrCa cells was performed by metabolic shift, glucose and lactate uptake assays. Effect of Cucurbitacin D treatment on key molecules of cell survival and glucose metabolism signaling pathways in PrCa cells was analyzed by Western blot and qRT-PCR analyses. Therapeutic efficacy of Cucurbitacin D against PrCa was evaluated in an ectopic PrCa xenograft mouse model. Results: Cucurbitacin D (0.1 to 1 µM) treatment significantly (P<0.01) inhibited the growth and metastatic potential of PrCa cells in a dose-dependent manner. Cucurbitacin D effectively induced apoptosis in PrCa cells as shown by enhanced Annexin V staining and PARP protein cleavage and arrested cells in G2/M phase via modulation of cell cycle regulatory proteins (inhibition of cyclin D1/E and Mcl-1 and induction of p21 and p27). Cucurbitacin D treatment dose-dependently decreased the lactate production, glucose uptake in PrCa cells which was correlated by suppressed expression of GLUT1 and its proficient docking with GLUT1 (with binding energy for this complex is -8.5 kcal mol-1). It has been reported that miR-132 targets the GLUT1, interestingly, Cucurbitacin D replenished the expression of miR-132 in PrCa cells. These growth inhibitory effects of Cucurbitacin D were confirmed in PrCa xenograft mouse model while administered intra-tumorally (1 mg/kg body weight thrice/week). Conclusion: Our results suggest that Cucurbitacin D is a novel modulator of glucose metabolism which could be a promising therapeutic modality alone or in combination of conventional chemotherapeutic agents for PrCa. Citation Format: Mohammed Sikander, Bilal Bin Hafeez, Shabnam Malik, Aditya Ganju, Fathi T. Halaweish, Murali Mohan Yallapu, Subhash C. Chauhan, Meena Jaggi. Cucurbitacin D inhibits prostate tumor growth via targeting glucose metabolism [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 3224. doi:10.1158/1538-7445.AM2017-3224

Abstract 2238: Novel sylibin analogues target ovarian cancer EMT-Wnt/β-catenin resistance pathways

Abstract Ovarian cancer (OC) is one of most lethal malignancies in woman reproductive tract. The OC diagnosed in advanced stage (III-IV) patients is highly aggressive and relapses back in over 80% patient after initial response to chemotherapy. Currently used chemotherapies are limited due to significant adverse/toxic effects, a narrow therapeutic index, and development of multidrug resistance mediated by the epithelial-to-mesenchymal transition (EMT) and cancer cell membrane efflux transporters. It is presumed that an ideal anti-OC drug that has diverse mechanisms, would inhibit several key cell-survival signaling and resistance pathways when combined with conventional chemotherapy (paclitaxel and cisplatin). Silybin, a polyphenolic flavonoid, has previously shown to inhibit metastasis by inhibition of EMT pathways and bypass other drug resistance factors i.e. efflux transporters in ovarian cancer both in vitro and in vivo. However, clinical use of silybin is limited due to their poor absorption and low bioavailability and poor potency. To overcome this, we designed and synthesized 11 silybin derivatives using molecular modeling, computer aided drug design, structure activity relationship, natural product lead optimization and ring disjunction approaches. In our preliminary findings, we found the lead molecule (15k) to have a cytotoxicity (IC50&lt1µM) with potency more than 200-fold compared to silybin (IC50~150µM). The 15k was found to be around 10-fold selective in OC cells (OV2008, A2780) compared to normal ovarian cells. Additionally, compared to other cancer cells belonging to prostate, breast, lung, 15k was (3-6)-fold selective in OC cells (OV2008, A2780). The 15k produced synergistic activity and potentiated the cytotoxic effects of platinum (cisplatin), anthracyclines (doxorubicin) and taxane (paclitaxel) anticancer agents. While 15k also reversed the drug resistance mediated by ABC-efflux transporters to paclitaxel and doxorubicin. Mechanistically, the cytotoxic effect of lead compound 15k, as determined by using molecular biology assays, was found to be due to inhibition of apoptosis, tubulin inhibition, and inhibition of stemness pathways (Wnt-β-catenin-EMT). The 15k had significant anti-metastatic effects as shown by their inhibition of migration and invasion potential on OC cells. In silico findings suggest that pharmacokinetic [PK] profiles of these compounds are favorable compared to silybin. Further in vivo PK/pharmacodynamic studies are underway to establish the clinical use of lead molecule in advanced metastasized, drug resistant OC patients. Citation Format: Haneen A. Amawi, Noor Hussein, Aubry Fetcenkoa, Rawan Alnafisah, Karthikeyan Chandrabose, Elangovan Manivannan, Piyush Trivedi, Amit K. Tiwari. Novel sylibin analogues target ovarian cancer EMT-Wnt/β-catenin resistance pathways [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 2238. doi:10.1158/1538-7445.AM2017-2238

Different Sequences of Fractionated Low-Dose Proton and Single Iron-Radiation-Induced Divergent Biological Responses in the Heart.

Deep-space travel presents risks of exposure to ionizing radiation composed of a spectrum of low-fluence protons (1H) and high-charge and energy (HZE) iron nuclei (e.g., 56Fe). When exposed to galactic cosmic rays, each cell in the body may be traversed by 1H every 3-4 days and HZE nuclei every 3-4 months. The effects of low-dose sequential fractionated 1H or HZE on the heart are unknown. In this animal model of simulated ionizing radiation, middle-aged (8-9 months old) male C57BL/6NT mice were exposed to radiation as follows: group 1, nonirradiated controls; group 2, three fractionated doses of 17 cGy 1H every other day (1H × 3); group 3, three fractionated doses of 17 cGy 1H every other day followed by a single low dose of 15 cGy 56Fe two days after the final 1H dose (1H × 3 + 56Fe); and group 4, a single low dose of 15 cGy 56Fe followed (after 2 days) by three fractionated doses of 17 cGy 1H every other day (56Fe + 1H × 3). A subgroup of mice from each group underwent myocardial infarction (MI) surgery at 28 days postirradiation. Cardiac structure and function were assessed in all animals at days 7, 14 and 28 after MI surgery was performed. Compared to the control animals, the treatments that groups 2 and 3 received did not induce negative effects on cardiac function or structure. However, compared to all other groups, the animals in group 4, showed depressed left ventricular (LV) functions at 1 month with concomitant enhancement in cardiac fibrosis and induction of cardiac hypertrophy signaling at 3 months. In the irradiated and MI surgery groups compared to the control group, the treatments received by groups 2 and 4 did not induce negative effects at 1 month postirradiation and MI surgery. However, in group 3 after MI surgery, there was a 24% increase in mortality, significant decreases in LV function and a 35% increase in post-infarction size. These changes were associated with significant decreases in the angiogenic and cell survival signaling pathways. These data suggest that fractionated doses of radiation induces cellular and molecular changes that result in depressed heart functions both under basal conditions and particularly after myocardial infarction.

Activator protein-1 (AP-1): a bridge between life and death in lung epithelial (A549) cells under hypoxia.

Activator protein-1 (AP-1) transcription factor plays a central role in hypoxia to modulate the expression of genes that decides the fate of the cell. The aim of the present study was to explore the role of AP-1 subunits in lung epithelial (A549) cells under hypoxia. Cell cycle studies by flow cytometry indicated that cell viability was unaffected by the initial hypoxia exposure (0.5% O2 at 37°C) for 6 and 12h. However, both transient cell cycle arrest and cell death was detected at 24 and 48h. Flow cytometry and spectrofluorometry data confirmed the increase in ROS levels. Elevated ROS and calcium levels activated the stress-related MAPK signaling cascade. ERK and JNK were activated in early hypoxic exposure (within 6h), whereas p38 were activated in 48h of hypoxia. These subtypes further stimulated the subunits of AP-1 at different times of hypoxia exposure to orchestrate different genes responsible for cell proliferation (6 and 12h) and apoptosis (24and 48h). Our results clearly depict the role of AP-1 heterodimer, i.e., p-c-jun/c-fos, p-c-jun/fosB, junD/c-fos, and junD/fosB in cell proliferation/survival by regulating the expression of Bcl-2 and cyclins (D1 and B1) at 6h and 12h of hypoxia, whereas junB/Fra-1 heterodimer have important role in apoptosis by regulating the expression of p53, Bax, and cyclin-dependent kinase inhibitors (p16, p21, p27) at 24h and 48h of hypoxia. Also, the cell survival signaling pathway NO-AKT interrupted at 24h and 48h of hypoxia indicating cell death. In conclusion, hypoxia for different time points activated different subunits of AP-1 that combined to form different heterodimers. These dimers regulated the expression of genes responsible for cell proliferation and apoptosis. Since, AP-1 plays a role in the decisive phenomenon of the cell to choose between proliferation and apoptosis; thus, its subunits or dimers could be a good therapeutic target for many diseases.