KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex.

The KRAS oncogene, present in over 90% of pancreatic ductal adenocarcinomas, is most frequently the result of one of three gain-of-function substitution mutations of codon 12 glycine. Thus far, RAS mutations have been clinically refractory to both direct and selective inhibition by systemic therapeutics. This report presents the results of pre-clinical assessment of a lipoplex comprising a plasmid-encoded, modular bi-functional shRNA (bi-shRNA), which executes selective and multi-mutant allelic KRASG12mut gene silencing, encased within a fusogenic liposome systemic delivery vehicle. Using both a dual luciferase reporter system and a Restriction Fragment Length Polymorphism (RFLP) assay, selective discrimination of KRASG12mut from KRASwt was confirmed in vitro in PANC1 cells. Subsequently, systemic administration of the bi-shRNAKRAS fusogenic lipoplex into female athymic Nu/Nu mice bearing PANC1 xenografts demonstrated intratumoral plasmid delivery, KRASG12mut knockdown, and inhibition of tumor growth, without adverse effect. Clinical trials with the bi-shRNA lipoplex have been implemented.

The NQO1 bioactivatable drug, ß-lapachone, alters the redox state of NQO1+ pancreatic cancer cells, causing perturbation in central carbon metabolism.

Many cancer treatments, such as those for managing recalcitrant tumors like pancreatic ductal adenocarcinoma, cause off-target toxicities in normal, healthy tissue, highlighting the need for more tumor-selective chemotherapies. ß-Lapachone is bioactivated by NAD(P)H:quinone oxidoreductase 1 (NQO1). This enzyme exhibits elevated expression in most solid cancers and therefore is a potential cancer-specific target. ß-Lapachone's therapeutic efficacy partially stems from the drug's induction of a futile NQO1-mediated redox cycle that causes high levels of superoxide and then peroxide formation, which damages DNA and causes hyperactivation of poly(ADP-ribose) polymerase, resulting in extensive NAD+/ATP depletion. However, the effects of this drug on energy metabolism due to NAD+ depletion were never described. The futile redox cycle rapidly consumes O2, rendering standard assays of Krebs cycle turnover unusable. In this study, a multimodal analysis, including metabolic imaging using hyperpolarized pyruvate, points to reduced oxidative flux due to NAD+ depletion after ß-lapachone treatment of NQO1+ human pancreatic cancer cells. NAD+-sensitive pathways, such as glycolysis, flux through lactate dehydrogenase, and the citric acid cycle (as inferred by flux through pyruvate dehydrogenase), were down-regulated by ß-lapachone treatment. Changes in flux through these pathways should generate biomarkers useful for in vivo dose responses of ß-lapachone treatment in humans, avoiding toxic side effects. Targeting the enzymes in these pathways for therapeutic treatment may have the potential to synergize with ß-lapachone treatment, creating unique NQO1-selective combinatorial therapies for specific cancers. These findings warrant future studies of intermediary metabolism in patients treated with ß-lapachone.

Preclinical Justification of pbi-shRNA EWS/FLI1 Lipoplex (LPX) Treatment for Ewing's Sarcoma.

The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency. We initiated development of pbi-shRNA EWS/FLI1 lipoplex (LPX) for the treatment of type 1 Ewing's sarcoma. Clinical-grade plasmid was manufactured and both sequence and activity verified. Target protein and RNA knockdown of 85-92% was demonstrated in vitro in type 1 human Ewing's sarcoma tumor cell lines with the optimal bi-shRNA EWS/FLI1 plasmid. This functional plasmid was placed in a clinically tested, liposomal (LP) delivery vehicle followed by in vivo verification of activity. Type 1 Ewing's sarcoma xenograft modeling confirmed dose related safety and tumor response to pbi-shRNA EWS/FLI1 LPX. Toxicology studies in mini-pigs with doses comparable to the demonstrated in vivo efficacy dose resulted in transient fever, occasional limited hypertension at low- and high-dose assessment and transient liver enzyme elevation at high dose. These results provide the justification to initiate clinical testing.

Tumor-selective use of DNA base excision repair inhibition in pancreatic cancer using the NQO1 bioactivatable drug, ß-lapachone.

Base excision repair (BER) is an essential pathway for pancreatic ductal adenocarcinoma (PDA) survival. Attempts to target this repair pathway have failed due to lack of tumor-selectivity and very limited efficacy. The NAD(P)H: Quinone Oxidoreductase 1 (NQO1) bioactivatable drug, ß-lapachone (ARQ761 in clinical form), can provide tumor-selective and enhanced synergy with BER inhibition. ß-Lapachone undergoes NQO1-dependent futile redox cycling, generating massive intracellular hydrogen peroxide levels and oxidative DNA lesions that stimulate poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation. Rapid NAD(+)/ATP depletion and programmed necrosis results. To identify BER modulators essential for repair of ß-lapachone-induced DNA base damage, a focused synthetic lethal RNAi screen demonstrated that silencing the BER scaffolding protein, XRCC1, sensitized PDA cells. In contrast, depleting OGG1 N-glycosylase spared cells from ß-lap-induced lethality and blunted PARP1 hyperactivation. Combining ß-lapachone with XRCC1 knockdown or methoxyamine (MeOX), an apyrimidinic/apurinic (AP)-modifying agent, led to NQO1-dependent synergistic killing in PDA, NSCLC, breast and head and neck cancers. OGG1 knockdown, dicoumarol-treatment or NQO1- cancer cells were spared. MeOX + ß-lapachone exposure resulted in elevated DNA double-strand breaks, PARP1 hyperactivation and TUNEL+ programmed necrosis. Combination treatment caused dramatic antitumor activity, enhanced PARP1-hyperactivation in tumor tissue, and improved survival of mice bearing MiaPaca2-derived xenografts, with 33% apparent cures. SIGNIFICANCE: Targeting base excision repair (BER) alone has limited therapeutic potential for pancreatic or other cancers due to a general lack of tumor-selectivity. Here, we present a treatment strategy that makes BER inhibition tumor-selective and NQO1-dependent for therapy of most solid neoplasms, particularly for pancreatic cancer.

Targeting glutamine metabolism sensitizes pancreatic cancer to PARP-driven metabolic catastrophe induced by ß-lapachone.

BACKGROUND: Pancreatic ductal adenocarcinomas (PDA) activate a glutamine-dependent pathway of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH) production to maintain redox homeostasis and support proliferation. Enzymes involved in this pathway (GLS1 (mitochondrial glutaminase 1), GOT1 (cytoplasmic glutamate oxaloacetate transaminase 1), and GOT2 (mitochondrial glutamate oxaloacetate transaminase 2)) are highly upregulated in PDA, and among these, inhibitors of GLS1 were recently deployed in clinical trials to target anabolic glutamine metabolism. However, single-agent inhibition of this pathway is cytostatic and unlikely to provide durable benefit in controlling advanced disease. RESULTS: Here, we report that reducing NADPH pools by genetically or pharmacologically (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) or CB-839) inhibiting glutamine metabolism in mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) PDA sensitizes cell lines and tumors to ß-lapachone (ß-lap, clinical form ARQ761). ß-Lap is an NADPH:quinone oxidoreductase (NQO1)-bioactivatable drug that leads to NADPH depletion through high levels of reactive oxygen species (ROS) from the futile redox cycling of the drug and subsequently nicotinamide adenine dinucleotide (NAD)+ depletion through poly(ADP ribose) polymerase (PARP) hyperactivation. NQO1 expression is highly activated by mutant KRAS signaling. As such, ß-lap treatment concurrent with inhibition of glutamine metabolism in mutant KRAS, NQO1 overexpressing PDA leads to massive redox imbalance, extensive DNA damage, rapid PARP-mediated NAD+ consumption, and PDA cell death-features not observed in NQO1-low, wild-type KRAS expressing cells. CONCLUSIONS: This treatment strategy illustrates proof of principle that simultaneously decreasing glutamine metabolism-dependent tumor anti-oxidant defenses and inducing supra-physiological ROS formation are tumoricidal and that this rationally designed combination strategy lowers the required doses of both agents in vitro and in vivo. The non-overlapping specificities of GLS1 inhibitors and ß-lap for PDA tumors afford high tumor selectivity, while sparing normal tissue.

Phase 1 Trial of Bi-shRNA STMN1 BIV in Refractory Cancer.

Stathmin1 (STMN1) is a microtubule modulator that is expressed in multiple cancers and correlates with poor survival. We previously demonstrated in vivo safety of bifunctional (bi) shRNA STMN1 bilamellar invaginated vesicle (BIV) and that systemic delivery correlated with antitumor activity. Patients with superficial advanced refractory cancer with no other standard options were entered into trial. Study design involved dose escalation (four patients/cohort) using a modified Fibonacci schema starting at 0.7 mg DNA administered via single intratumoral injection. Biopsy at baseline, 24/48 hours and resection 8 days after injection provided tissue for determination of cleavage product using next-generation sequencing (NGS) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), 5' RLM rapid amplification of cDNA ends (RACE) assay. Serum pharmacokinetics of circulating plasmid was done. Twelve patients were entered into three dose levels (0.7, 1.4, 7.0 mg DNA). No ≥ grade 3 toxic effects to drug were observed. Maximum circulating plasmid was detected at 30 seconds with less than 10% detectable in all subjects at 24 hours. No toxic effects were observed. Predicted cleavage product was detected by both NGS (n = 7/7 patients analyzed, cohorts 1, 2) and RLM RACE (n = 1/1 patients analyzed cohort 3). In conclusion, bi-shRNA STMN1 BIV is well tolerated and detection of mRNA target sequence-specific cleavage product confirmed bi-shRNA BIV mechanism of action.

ATM regulates insulin-like growth factor 1-secretory clusterin (IGF-1-sCLU) expression that protects cells against senescence.

Downstream factors that regulate the decision between senescence and cell death have not been elucidated. Cells undergo senescence through three pathways, replicative senescence (RS), stress-induced premature senescence (SIPS) and oncogene-induced senescence. Recent studies suggest that the ataxia telangiectasia mutant (ATM) kinase is not only a key protein mediating cellular responses to DNA damage, but also regulates cellular senescence induced by telomere end exposure (in RS) or persistent DNA damage (in SIPS). Here, we show that expression of secretory clusterin (sCLU), a known pro-survival extracellular chaperone, is transcriptionally up-regulated during both RS and SIPS, but not in oncogene-induced senescence, consistent with a DNA damage-inducible mechanism. We demonstrate that ATM plays an important role in insulin-like growth factor 1 (IGF-1) expression, that in turn, regulates downstream sCLU induction during senescence. Loss of ATM activity, either by genomic mutation (ATM-deficient fibroblasts from an ataxia telangiectasia patient) or by administration of a chemical inhibitor (AAI, an inhibitor of ATM and ATR), blocks IGF-1-sCLU expression in senescent cells. Downstream, sCLU induction during senescence is mediated by IGF-1R/MAPK/Egr-1 signaling, identical to its induction after DNA damage. In contrast, administration of an IGF-1 inhibitor caused apoptosis of senescent cells. Thus, IGF-1 signaling is required for survival, whereas sCLU appears to protect cells from premature senescence, as IMR-90 cells with sCLU knockdown undergo senescence faster than control cells. Thus, the ATM-IGF-1-sCLU pathway protects cells from lethality and suspends senescence.

Prodrug strategy to achieve lyophilizable, high drug loading micelle formulations through diester derivatives of ß-Lapachone.

ß-Lap prodrug micelle strategy improves the formulation properties of ß-lap therapeutics. The resulting micelles yield apparent high ß-lap solubility (>7 mg mL(-1) ), physical stability, and ability to reconstitute after lyophilization. In the presence of esterase, ß-lap prodrugs are efficiently converted into parent drug (i.e., ß-lap), resulting in NQO1-dependent lethality of NSCLC cells.

Superparamagnetic iron oxide nanoparticles: amplifying ROS stress to improve anticancer drug efficacy.

Superparamagnetic iron oxide nanoparticles (SPION) are an important and versatile nano- platform with broad biological applications. Despite extensive studies, the biological and pharmacological activities of SPION have not been exploited in therapeutic applications. Recently, ß-lapachone (ß-lap), a novel anticancer drug, has shown considerable cancer specificity by selectively increasing reactive oxygen species (ROS) stress in cancer cells. In this study, we report that pH-responsive SPION-micelles can synergize with ß-lap for improved cancer therapy. These SPION-micelles selectively release iron ions inside cancer cells, which interact with hydrogen peroxide (H(2)O(2)) generated from ß-lap in a tumor-specific, NQO1-dependent manner. Through Fenton reactions, these iron ions escalate the ROS stress in ß-lap-exposed cancer cells, thereby greatly enhancing the therapeutic index of ß-lap. More specifically, a 10-fold increase in ROS stress was detected in ß-lap-exposed cells pretreated with SPION-micelles over those treated with ß-lap alone, which also correlates with significantly increased cell death. Catalase treatment of cells or administration of an iron chelator can block the therapeutic synergy. Our data suggest that incorporation of SPION-micelles with ROS-generating drugs can potentially improve drug efficacy during cancer treatment, thereby provides a synergistic strategy to integrate imaging and therapeutic functions in the development of theranostic nanomedicine.

Application of a proapoptotic peptide to intratumorally spreading cancer therapy.

Bit1 is a proapoptotic mitochondrial protein associated with anoikis. Upon cell detachment, Bit1 is released into the cytoplasm and triggers caspase-independent cell death. Bit1 consists of 179 amino acids; for the C-terminal, two thirds of the molecule functions as a peptidyl-tRNA hydrolase, whereas the N-terminus contains a mitochondrial localization signal. Here, we localize the cell death domain (CDD) to the N-terminal 62 amino acids of Bit1 by transfecting cells with truncated Bit1 cDNA constructs. CDD was more potent in killing cells than the full-length Bit1 protein when equivalent amounts of cDNA were transfected. To develop Bit1 CDD into a cancer therapeutic, we engineered a recombinant protein consisting of the CDD fused to iRGD, which is a tumor-specific peptide with unique tumor-penetrating and cell-internalizing properties. iRGD-CDD internalized into cultured tumor cells through a neuropilin-1-activated pathway and triggered cell death. Importantly, iRGD-CDD spread extensively within the tumor when injected intratumorally into orthotopically implanted breast tumors in mice. Repeated treatment with iRGD-CDD strongly inhibited tumor growth, resulting in an average reduction of 77% in tumor volume and eradication of some tumors. The caspase independence of Bit1-induced cell death makes CDD a potentially attractive anticancer agent, because tumor resistance to the main mechanisms of apoptosis is circumvented. Using iRGD to facilitate the spreading of a therapeutic agent throughout the tumor mass may be a useful adjunct to local therapy for tumors that are surgically inoperable or difficult to treat systemically.

PDRG1, a novel tumor marker for multiple malignancies that is selectively regulated by genotoxic stress.

We have previously cloned and characterized a novel p53 and DNA damage-regulated gene named PDRG1. PDRG1 was found to be differentially regulated by ultraviolet (UV) radiation and p53. In this study, we further investigated stress regulation of PDRG1 and found it to be selectively regulated by agents that induce genotoxic stress (DNA damage). Using cancer profiling arrays, we also investigated PDRG1 expression in matching normal and tumor samples representing various malignancies and found its expression to be upregulated in multiple malignancies including cancers of the colon, rectum, ovary, lung, stomach, breast and uterus when compared to their respective matched normal tissues. Western blot and immunohistochemical analyses were also performed on select specimen sets of colon cancers and matching normal tissues and the results also indicated PDRG1 overexpression in tumors relative to normal tissues. To gain insight into the function of PDRG1, we performed PDRG1 knockdown in human colon cancer cells and found its depletion to result in marked slowdown of tumor cell growth. These results suggest that PDGR1 may be linked to cell growth regulation. Yeast two-hybrid screen also led to the identification of PDCD7, CIZ1 and MAP1S as PDRG1-interacting proteins that are involved in apoptosis and cell cycle regulation which further implicate PDRG1 in controlling cell growth regulation. Taken together, our results indicate that PDRG1 expression is increased in multiple human malignancies suggesting it to be a high-value novel tumor marker that could play a role in cancer development and/or progression.

DOC45, a novel DNA damage-regulated nucleocytoplasmic ATPase that is overexpressed in multiple human malignancies.

In this article, we report the characterization of a novel DNA damage-regulated gene, named DNA damage-regulated overexpressed in cancer 45 (DOC45). Our results indicate that DNA damage-inducing agents, including doxorubicin (adriamycin), etoposide, and ionizing and UV radiation, strongly downregulate DOC45 expression, whereas endoplasmic reticulum stress-inducing agents do not. Our results also indicate that DOC45 is overexpressed in several human malignancies, including cancers of the colon, rectum, ovary, lung, stomach, and uterus. DOC45 harbors conserved nucleotide triphosphate-binding motifs and is capable of ATP hydrolysis, findings that highlight its function as a novel ATPase. Although predominantly cytoplasmic, DOC45 exhibits a characteristic nucleocytoplasmic distribution and, on inhibition of nuclear export, predominantly accumulates in the nucleoli. These results suggest that DOC45 may shuttle between nucleus and cytoplasm to carry out its function. Our results also indicate that DOC45 expression is enhanced during oncogenic Ras-mediated transformation and that its expression is linked to phosphoinositide 3-kinase signaling pathway. Furthermore, short hairpin RNA-mediated knockdown of DOC45 in human colon cancer cells inhibits their proliferation and enhances cellular sensitivity to doxorubicin-induced cell death, suggesting that DOC45 plays an important role in cell proliferation and survival. Collectively, our results indicate that DOC45 is a novel ATPase that is linked to cellular stress response and tumorigenesis, and may also serve as a valuable tumor marker.

Anoikis effector Bit1 negatively regulates Erk activity.

Bcl-2 inhibitor of transcription (Bit1) is a mitochondrial protein that functions as a peptidyl-tRNA hydrolase, but, when released into the cytoplasm, it elicits apoptosis. The proapoptotic function is uniquely counteracted by integrin-mediated cell attachment. We generated a conditional KO mouse of the Bit1 gene by using the Cre-LoxP recombination system. Bit1-null mice were born alive but with some developmental abnormalities. They developed a runting syndrome after birth and died within the first 2 weeks. Cultured fibroblasts from the Bit1-null embryos [mouse embryo fibroblasts (MEFs)] were more resistant to cell death induced by loss of attachment to extracellular matrix (anoikis) than cells from the wild-type or heterozygous littermates. MEFs and tissues from Bit1 KO mice displayed a marked increase in Erk phosphorylation. Knocking down Bit1 expression in cultured cells resulted in increased Erk activation, and partially knocking down Erk reversed the increased anoikis resistance of Bit1 knockdown. The enhanced Erk activation was associated with decreased Erk phosphatase activity. These studies establish the physiological significance of Bit1 activity and begin to delineate a Bit1 signaling pathway that acts through Erk regulation.

Cloning and characterization of a p53 and DNA damage down-regulated gene PIQ that codes for a novel calmodulin-binding IQ motif protein and is up-regulated in gastrointestinal cancers.

We have identified a p53 and DNA damage-regulated gene that encodes a novel IQ motif protein, which we have named p53 and DNA damage-regulated IQ motif protein (PIQ). PIQ has two isoforms, long (PIQ-L) and short (PIQ-S), and both bind to calmodulin in the presence and absence of calcium. PIQ expression is down-regulated by p53 and DNA damage-inducing agents, whereas PIQ itself represses the expression of p53 up-regulated modulator of apoptosis that is a key mediator of p53-induced apoptosis. Thus, PIQ is a novel protein that may function to bridge a crosstalk between p53 and calmodulin-regulated cellular processes. We further show that PIQ expression is up-regulated in a number of primary colorectal and gastric tumors when compared with matching normal tissues, suggesting that PIQ may be involved in tumorigenesis and could serve as a valuable diagnostic/prognostic marker for gastrointestinal tumors.

Genotoxic and endoplasmic reticulum stresses differentially regulate TRB3 expression.

TRB3 has recently been identified as a potential pro-apoptotic protein that may modulate the Akt/PKB-dependent signaling pathway. Here we report that TRB3 expression is strongly upregulated by endoplasmic reticulum (ER) stress-inducing agents that (1) promote ER Ca2+ pool depletion or (2) disrupt protein trafficking. Genotoxic stress (DNA damage)-inducing agents, by contrast, downregulate TRB3 expression and appear to do so through both p53-dependent and -independent mechanisms. To the best of our knowledge, TRB3 is the first gene that is upregulated by ER stress and downregulated following genotoxic stress. Collectively, these findings highlight the importance of stress-specific signaling cascades as well as point out the seemingly divergent roles that TRB3 may play in the cellular stress response.

Cloning and characterization of a novel gene PDRG that is differentially regulated by p53 and ultraviolet radiation.

We report the cloning and characterization of a novel p53 and DNA damage-regulated gene (PDRG). The human and mouse PDRG sequences are highly homologous and contain open reading frames of 133 amino acids each with molecular masses of 15.5 and 15.3 kDa, respectively. PDRG codes for a novel protein that does not show similarity to any known protein in the databases. Human PDRG is predominantly expressed in normal testis and exhibits reduced but detectable expression in other organs. GFP-tagged PDRG was predominantly detected as aggregates that appeared to reside in a distinct subcellular compartment. PDRG mRNA was upregulated by ultraviolet radiation (UV) but downregulated by tumor suppressor p53. UV is known to transcriptionally upregulate the expression of certain genes by activating the transcription factor Oct-1, while p53 has been reported to suppress transcription of certain genes by directly binding to a novel head-to-tail response element. Cloning and sequence analysis of PDRG promoter revealed the presence of Oct-1-binding element and a putative head-to-tail-type p53-binding site. Indeed, UV as well as exogenous Oct-1 independently increased PDRG promoter activity, suggesting that UV could mediate PDRG upregulation via Oct-1. Exogenous wild-type p53 was found to downregulate the PDRG promoter activity indicating that wild-type p53 transcriptionally suppresses the expression of PDRG and may mediate its effect via the putative head-to-tail response element. Furthermore, stable expression of exogenous PDRG was found to decrease the clonogenic survival after UV irradiation, which highlights the significance of PDRG in facilitating UV-induced killing.

Apo2L/TRAIL differentially modulates the apoptotic effects of sulindac and a COX-2 selective non-steroidal anti-inflammatory agent in Bax-deficient cells.

The nonsteroidal anti-inflammatory drugs (NSAIDs) are believed to mediate their anticancer effects by inducing apoptosis but the molecular mechanisms of their apoptotic effects remain largely unknown. Here we report that two different NSAIDs, sulindac sulfide and SC-'236 engage the death receptor 5 (DR5) and mitochondrial pathways to mediate apoptosis in human colon cancer cells. We show that sulindac sulfide and SC-'236-induced apoptosis is coupled with upregulation of DR5, caspase 8 activation and Bid cleavage. Thus, a cross talk appears to exist between the DR5 and mitochondrial pathways during apoptosis induced by these NSAIDs. We further show that sulindac sulfide and SC-'236-induced DR5 upregulation occurs independent of the COX inhibitory effects of these NSAIDs. Using Bax-proficient (Bax+/-) and Bax-deficient (Bax-/-) HCT116 human colon cancer cells, we further demonstrate that Apo2L/TRAIL differentially modulates the apoptotic effects of sulindac sulfide and SC-'236. For example, sulindac sulfide upregulates DR5 in both Bax-deficient and proficient cells, but Apo2L/TRAIL efficiently potentiates sulindac sulfide-induced apoptosis as well as activation of caspase-8, -9 and -3 only in Bax-proficient cells. SC-'236 also upregulates DR5 in both Bax-proficient and Bax-deficient cells but Apo2L/TRAIL potentiates SC-'236-mediated apoptosis and caspases-8 and -3 activation in both Bax-proficient and Bax-deficient cells. Further, in Bax-deficient cells, neither sulindac sulfide nor SC-'236 in combination with Apo2L/TRAIL effectively promotes the release of cytochrome c from mitochondria into cytosol and caspase-9 activation. Collectively, our results suggest that unlike sulindac sulfide, SC-'236 in combination with Apo2L/TRAIL can overcome Bax deficiency to induce apoptosis. These results have important clinical implications in that the tumors harboring Bax mutations are likely to develop resistance to sulindac but not to SC-'236-like NSAIDs. In conclusion, the data presented herein form the basis of future in-depth studies to further explore the utility of Apo2L/TRAIL and NSAIDs, in combination, as a novel cancer preventive/therapeutic strategy.

Endoplasmic reticulum calcium pool depletion-induced apoptosis is coupled with activation of the death receptor 5 pathway.

Thapsigargin (TG), by inducing perturbations in cellular Ca(2+) homeostasis, has been shown to induce apoptosis. The molecular mechanisms of Ca(2+) perturbation-induced apoptosis are not fully understood. In this study, we demonstrate for the first time that TG-mediated perturbations in Ca(2+) homeostasis are coupled with activation of the death receptor 5 (DR5)-dependent apoptotic pathway in human cancer cells. TG selectively upregulated DR5 but had no effect on the expression of the other TRAIL receptor, DR4. TG also upregulated the expression of the DR5 ligand TRAIL (tumor necrosis factor-related apoptosis inducing ligand), albeit in a cell-type specific manner. TG-induced apoptosis has been shown to be associated with activation of the mitochondrial pathway. We found that TG upregulation of DR5 and TRAIL was coupled with caspase 8 activation and Bid cleavage, suggesting that the TG-regulated DR5 pathway could be linked to the mitochondrial pathway. TG enhanced not only DR5 mRNA stability but also increased induction of the DR5 genomic promoter-reporter gene. The TG-induced increase in DR5 expression appeared to occur as a consequence of TG-induced endoplasmic reticulum (ER) Ca(2+) pool depletion. Thus, we report our novel findings that ER Ca(2+) pool depletion-induced apoptotic signals are mediated, at least in part, via a DR5-dependent apoptotic pathway and there appears to be a cross-talk between the death receptor and mitochondrial pathways.