Advances in progenitor cell therapy using scaffolding constructs for central nervous system injury.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality in the United States. Current clinical therapy is focused on optimization of the acute/subacute intracerebral milieu, minimizing continued cell death, and subsequent intense rehabilitation to ameliorate the prolonged physical, cognitive, and psychosocial deficits that result from TBI. Adult progenitor (stem) cell therapies have shown promise in pre-clinical studies and remain a focus of intense scientific investigation. One of the fundamental challenges to successful translation of the large body of pre-clinical work is the delivery of progenitor cells to the target location/organ. Classically used vehicles such as intravenous and intra arterial infusion have shown low engraftment rates and risk of distal emboli. Novel delivery methods such as nanofiber scaffold implantation could provide the structural and nutritive support required for progenitor cell proliferation, engraftment, and differentiation. The focus of this review is to explore the current state of the art as it relates to current and novel progenitor cell delivery methods.

ECHOGENIC LIPOSOMES FOR NITRIC OXIDE DELIVERY AND BREAST CANCER TREATMENT

Liposomes, also known as nontoxic, biodegradable, and non-immunogenic therapeutic delivery vehicles, have been proposed as a carrier for drugs and antitumor agents in cancer chemotherapy. Echogenic liposomes (ELIP) have the potential to entrap air or bioactive gas to enhance acoustic reflectivity in ultrasound and are used as a contrast agent. The innovative part of this study is based on a novel concept to encapsulate nitric oxide (NO) gas into ELIP, deliver it to breast cancer cells, and control its release via direct ultrasound exposure. Studies on the effect of NO in tumor biology have shown that a high levels of NO (> 300 nM) leads to cytostasis or apoptosis by decreasing the translation of several cell cycle proteins and stimulating cancer cell death by activating the p53 pathway. The central hypothesis is that NO gas can be packaged and delivered through a delivery methodology to breast cancer cells to facilitate tumor regression with minimal systemic toxicity. The primary goal of this thesis is to develop an echogenic liposomal solution that has the ability to encapsulate NO, to release NO locally upon ultrasound exposure, and to induce breast cancer cell death. NO-containing echogenic liposomes (NO-ELIP) were prepared by the freezing-under-pressure method previously developed in our laboratory. It was necessary to evaluate stability of NO-ELIP and release of NO from NO-ELIP by measuring echogenicity using intravascular ultrasound images. Breast cancer cell lines, MDA-MB-231 and MDA-MB-468, were selected to investigate the cytotoxic effects of NO liberated from NO-ELIP and their response to NO concentration. Ultrasound-triggered NO release from NO-ELIP using ultrasound activation was studied. It was demonstrated that NO-ELIP remained stable for 5 hours in bovine serum albumin. Delivery of NO using NO-ELIP induced cytotoxicity and programmed cell death of MDA-MB-231 and MDA-MB-468 after 5 hours of incubation. Enhancement of the NO-ELIP effect for therapeutic application was observed with ultrasound activation. This work demonstrates that NO-ELIP can incorporate and deliver NO to breast cancer cells providing increased NO stability and ultrasound-controlled NO release. Improved therapeutic effect with the use of NO-ELIP is expected to be found for breast cancer treatment.

Antisense-mediated inhibition of papillomavirus type-18 in human squamous cell carcinoma

Numerous co-factors, genetic, environmental and physical, play an important role in development and prognosis of cancer. Each year in the USA, more than 31,000 cases of oral and 13,000 cases of cervical cancer are diagnosed. Substantial epidemiological data supports a high correlation between development of these cancers and the presence of specific types of human papillomaviruses (HPV). Molecular biological studies show that not only are several of the viral genes necessary and sufficient to cause transformation but they also function synergistically with other co-factors. Evidence suggests that prevention of infection or inhibition of viral gene expression may alter the course of malignant transition. The main objective of this project was to test the hypothesis that some human carcinoma cells, containing HPV, behave in malignant manner because the viral genes function in the maintenance of some aspect of the transformed phenotype.^ The specific aims were (1) to select oral and cervical cancer cell lines which were HPV-negative or which harbored transcriptionally active HPV-18, (2) to construct and determine the effects of recombinant sense or antisense expressing vectors, (3) to test the effects of synthetic antisense oligodeoxynucleotides on the transformed behavior of these cells.^ To screen cells, we performed Southern and Northern analysis and polymerase chain reactions. When antisense-expressing vectors were used, cells harboring low numbers of HPV-18 where unable to survive transfection but they were readily transfected with all other constructs. Rare antisense transfectants obtained from HPV-positive cells showed significantly altered characteristics including malignant potential in nude mice. The HPV-negative cells showed no differences in transfection efficiencies or growth characteristics with any construct.^ In addition, treatment of the HPV-positive cells with antisense, but not random oligodeoxynucleotides, resulted in decreased cell proliferation and even cell death. These effects were dose-dependent, synergistic and HPV-specific.^ These results suggest that expression of viral genes play an important role in the maintenance of the transformed phenotype which implies that inhibition of expression, by antisense molecules, may be therapeutic in HPV-induced tumors. ^

Identification of a novel tumor suppressor gene on human chromosome 3p14-p12 involved in renal cell carcinoma

Nonpapillary renal cell carcinoma (RCC) is an adult cancer of the kidney which occurs both in familial and sporadic forms. The familial form of RCC is associated with translocations involving chromosome 3 with a breakpoint at 3p14-p13. Studies focused on sporadic RCC have shown two commonly deleted regions at 3p14.3-p13 and 3p21.3. In addition, a more distal region mapping to 3p26-p25 has been linked to the Von Hippel Lindau (VHL) disease gene. A large proportion of VHL patients develop RCC. The short arm of human chromosome 3 can, therefore, be dissected into three distinct regions which could encode tumor suppressor genes for RCC. Loss or inactivation of one or more of these loci may be an important step in the genesis of RCC.^ I have used the technique of microcell-mediated chromosome transfer to introduce an intact, normal human chromosome 3 and defined fragments of 3p, dominantly marked with pSV2neo, into the highly malignant RCC cell line SN12C.19. The introduction of chromosome 3 and of a centric fragment of 3p, encompassing 3p14-q11, into SN12C.19 resulted in dramatic suppression of tumor growth in nude mice. Another defined deletion hybrid contained the region 3p12-q24 of the introduced human chromosome and failed to suppress tumorigenicity. These data define the region 3p14-p12, the most proximal region of high frequency allele loss in sporadic RCC as well as the region containing the translocation breakpoint in familial RCC, to contain a novel tumor suppressor locus involved in RCC. We have designated this locus nonpapillary renal cell carcinoma-1 (NRC-1). Furthermore, we have functional evidence that NRC-1 controls the growth of RCC cells by inducing rapid cell death in vivo. ^

Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

p75 receptor-mediated neurotrophism: A new mechanism of melanoma cell survival and invasion

Trophism as a "clonal dominance" support mechanism for tumor cells is an unexplored area of tumor progression. This report presents evidence that the human melanoma low-affinity neurotrophin receptor (p75) can signal independently of its high-affinity tyrosine kinase counterparts, the TRK family of kinases. Signaling may be accomplished by a p75-associated purine-analog-sensitive kinase and results in enhanced invasion into a reconstituted basement membrane with a corresponding stimulation of matrix metalloproteinase-2 expression. Additionally, a "stress culture" survival assay was developed to mimic the growth limiting conditions encountered by melanoma cells in a rapidly growing primary tumor or metastatic deposit prior to neoangiogenesis. Under these conditions, p75, promotes the survival of high p75 expressing brain-colonizing melanoma cells. Extensive 70W melanoma cell-cell contact, which downregulates p75, immediately precedes the induction of cell death associated with diminished production of two key cell survival factors, bcl-2 and the p85 subunit of phosphoinositol-3-kinase, and an elevation in apoptosis promoting intracellular reactive oxygen species (ROSs). Since one function of bcl-2 may be to control the generation of ROSs via the antioxidant pathway, these cells may receive a apoptosis-prompting "double hit". 70W melanoma cell death occurred by an apoptotic mechanism displaying classical morphological changes including plasma membrane blebbing, loss of microvilli and redistribution of ribosomes. 70W apoptosis could be pharmacologically triggered following anti-p75 monoclonal antibody-mediated clustering of p75 receptors. 70W cells fluorescently sorted for high-p75 expression (p75$\sp{\rm H}$ cells) exhibited an augmented survival potential and a predilection to sort with the S + G2/M growth phase, relative to their low p75 expressing, p75$\sp{\rm L}$ counterparts. Apoptosis is significantly delayed by p75$\sp{\rm H}$ cells, whereas p75$\sp{\rm L}$ cells are exquisitely prone to initiate apoptosis. Importantly, the p75$\sp{\rm L}$ cells that survive apoptosis, highly re-expressed p75 and were remarkably responsive to exogenous NGF.^ These are the first data to implicate p75-mediated neurotrophism as an invasion and survival support mechanism employed by brain-metastatic cells. In particular, these results may have implications in little understood phenomena of tumor progression, such as the emergence of "clonal dominance" and tumor dormancy. ^

Mechanisms of liposomal all-{\it trans\/} retinoic acid and vitamin D3 induced inhibition of cell proliferation and stimulation of apoptosis in aggressive B-cell non-Hodgkin's lymphoma

The Non-Hodgkin's Lymphoma (NHLs) are neoplasms of the immune system. Currently, less than 1% of the etiology of the 22,000 newly diagnosed lymphoma cases in the U.S.A. every year is known. This disease has a significant prevalence and high mortality rate. Cell growth in lymphomas has been shown to be an important parameter in aggressive NHL when establishing prognosis, as well as an integral part in the pathophysiology of the disease process. While many aggressive B cell NHLs respond initially to chemotherapeutic regimens such as CHOP-bleo (adriamycin, vincristine and bleomycin) etc., relapse is common, and the patient is then often refractory to further salvage treatment regimens.^ To assess their potential to inhibit aggressive B cell NHLs and induce apoptosis (also referred to as programmed cell death (PCD)), it was proposed to utilize the following biological agents-liposomal all-trans retinoic acid (L-ATRA) which is a derivative of Vitamin A in liposomes and Vitamin D3. Preliminary evidence indicates that L-ATRA may inhibit cell growth in these cells and may induce PCD as well. Detailed studies were performed to understand the above phenomena by L-ATRA and Vitamin D3 in recently established NHL-B cell lines and primary cell cultures. The gene regulation involved in the case of L-ATRA was also delineated. ^

T cell lipid peroxidation induces ferroptosis and prevents immunity to infection

The selenoenzyme glutathione peroxidase 4 (Gpx4) is a major scavenger of phospholipid hydroperoxides. Although Gpx4 represents a key component of the reactive oxygen species-scavenging network, its relevance in the immune system is yet to be defined. Here, we investigated the importance of Gpx4 for physiological T cell responses by using T cell-specific Gpx4-deficient mice. Our results revealed that, despite normal thymic T cell development, CD8(+) T cells from T(ΔGpx4/ΔGpx4) mice had an intrinsic defect in maintaining homeostatic balance in the periphery. Moreover, both antigen-specific CD8(+) and CD4(+) T cells lacking Gpx4 failed to expand and to protect from acute lymphocytic choriomeningitis virus and Leishmania major parasite infections, which were rescued with diet supplementation of high dosage of vitamin E. Notably, depletion of the Gpx4 gene in the memory phase of viral infection did not affect T cell recall responses upon secondary infection. Ex vivo, Gpx4-deficient T cells rapidly accumulated membrane lipid peroxides and concomitantly underwent cell death driven by ferroptosis but not necroptosis. These studies unveil an essential role of Gpx4 for T cell immunity.

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.

Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth.

Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.

The liver stage of Plasmodium berghei inhibits host cell apoptosis.

Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite-host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei-infected cells and compared their level of expression and their distribution to that of non-infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non-infected HepG2 cells, whereas P. berghei-infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)-alpha/D-galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non-treated control mice as well as in TNF-alpha-treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death.

4HPR-X radiation mechanisms of interactions in non-small cell lung cancer (NSCLC) cells in vitro

Lung cancer is the leading cause of cancer death. However, poor survival using conventional therapies fuel the search for more rational interventions. The objective of this study was to design and implement a 4HPR-radiation interaction model in NSCLC, employing a traditional clinical modality (radiation), a relatively new, therapeutically unexplored agent (4HPR) and rationally combining them based on molecular mechanistic findings pertaining to their interactions. To test the hypothesis that 4HPR sensitizes cells to radiation-induced cell death via G2+M accumulation, we designed a working model consisting of H522 adenocarcinoma cells (p53, K-ras mutated) derived from an NSCLC patient; 4HPR at concentrations up to 10 μM; and X radiation up to 6 Gy generated by a patient-dedicated Phillips RT-250 X ray unit at 250 KV, 15 mA, 1.85 Gy/min. We found that 4HPR produced time- and dose-dependent morphological changes, growth inhibition, and DNA damage-inducing enhancement of reactive oxygen species. A transient G2+M accumulation of cells maximal at 24 h of continuous 4HPR exposure was used for irradiation time scheduling. Our data demonstrated enhanced cell death (both apoptotic and necrotic) in irradiated cells pre-treated with 4HPR versus those with either stressor alone. 4HPR's effect of increased NSCLC cells' radioresponse was confirmed by clonogenic assay. To explore these practical findings from a molecular mechanistic perspective, we further investigated and showed that levels of cyclin B1 and p34cdc2 kinase—both components of the mitosis promoting factor (MPF) regulating the G2/M transition—did not change following 4HPR treatment. Likewise, cdc25C phosphatase was not altered. However, enhanced p34cdc2 phosphorylation on its Thr14Tyr15 residues—indicative of its inactivation and increased expression of MPF negative regulators chk1 and wee1 kinases—were supportive of explaining 4HPR-treated cells' accumulation. Hence, p34cdc2 phosphorylation, chk1, and wee1 warrant further evaluation as potential molecular targets for 4HPR-X radiation combination. In summary, we (1) demonstrated that 4HPR not only induces cell death by itself, but also increases NSCLC cells' subsequent radioresponse, indicative of potential clinical applicability, and (2) for the first time, shed light on deciphering 4HPR-X radiation molecular mechanisms of interaction, including the finding of 4HPR's role as a p34cdc2 inactivator via Thr14Tyr15 phosphorylation. ^

Dimethylsphingosine evokes diverse cellular death modes in HL60

Among the barriers to successful cancer treatment is the acquired resistance that tumors undergo due to clonal evolution. Non-cross resistant drugs could add to the current options of chemotherapeutic drugs. In order to improve tumor response, investigators have been identifying defective death pathways acquired by specific cancer types, so to target directly those pathways. Sphingolipids have emerged as potential drugs for tumor-targeted therapy, and among them, Dimethylsphingosine (DMS), known to be a competitive inhibitor of Sphingosine Kinase (SK). DMS actions have been documented by several investigators, but the mechanisms by which DMS exerts cytotoxicity have not been fully investigated. We evaluated the cytotoxicity of DMS against human leukemia cell lines and against blasts isolated from leukemia patients. Cell line viability decreased proportionally to DMS concentration and treatment time. Resistant and MDR positive cell lines were the most sensitive, indicating DMS efficacy against human leukemia MDR. Importantly, leukemia samples showed a similar sensitivity to DMS, the first demonstration of DMS activity against fresh human leukemia specimens. Mechanistically we have demonstrated that DMS efficacy is due to its ability to induce cytotoxicity by inducing necrosis, apoptosis or both concomitantly, revealing a mixed-feature cell death mode never described before for DMS. Further, we have shown evidence suggesting pathways cross-talk, since apoptosis inhibition led to accelerated rate of necrosis. DMS diverse killing mechanisms and the high expression of SK in leukemias could explain DMS potent cytotoxicity. DMS-based regimens may increase response rates and therefore, improve leukemia treatment. ^

Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

Anticancer activity of OSW-1: Induction of apoptotic pathway in leukemia and autophagic death in pancreatic cancer through a calcium mediated mechanism

OSW-1 is a natural compound found in the bulbs of Orninithogalum saudersiae, a member of the lily family. This compound exhibits potent antitumor activity in vitro with the IC50 values in the low nanomolar concentration range and demonstrating its ability to kill drug resistant cancer cells. In an effort to discover the unknown mechanism of action of this novel compound as a potential anticancer agent, the main objective of this research project was to test the cytotoxicity of OSW-1 against various cancer lines, and to elucidate the biochemical and molecular mechanism(s) responsible for the anticancer activity of OSW-1. My initial investigation revealed that OSW-1 is effective in killing various cancer cells including pancreatic cancer cells and primary leukemia cells resistant to standard chemotherapeutic agents, and that non-malignant cells were less sensitive to this compound. Further studies revealed that in leukemia cells, OSW-1 causes a significant increase in cytosolic calcium and activates rapid calcium-dependent apoptosis by the intrinsic pathway. Additionally, OSW-1 treatment leads to the degradation of the ER chaperone GRP78/BiP implicated in the survival of cancer cells. Meanwhile, it shows a reduced sensitivity in respiration-deficient sub-clones of leukemia cells which had higher basal levels of Ca2+. Mechanistically, it was further demonstrated that cytosolic Ca2+ elevations were observed together with Na+ decreases in the cytosol, suggesting OSW-1 caused the calcium overload through inhibition of the Na+/Ca 2+exchanger (NCX). Although similar calcium disturbances were observed in pancreatic cancer cells, mechanistic studies revealed that autophagy served as an initial pro-survival mechanism subsequent to OSW-1 treatment but extended autophagy caused inevitable cell death. Furthermore, combination of OSW-1 with autophagy inhibitors significantly enhances the cytotoxicity against pancreatic cancer cells. Taken together, this study revealed the novel mechanism of OSW-1 which is through inhibition of the Na+/Ca2+ exchanger and provides a basis for using this compound in combination with other agents for the treatment of pancreatic cancer which is resistant to available anticancer drugs. ^