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1.  Regulation of OSU-03012 toxicity by ER stress proteins and ER stress inducing drugs 
Molecular cancer therapeutics  2014;13(10):2384-2398.
The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors, and to determine the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knock down of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012, and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase 9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase 8 inhibitor c-FLIP-s, or knock down of death receptor CD95 or the death receptor – caspase 8 linker protein FADD, suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knock down of the autophagy regulatory proteins Beclin1 or ATG5 protected cells from OSU-03012 and of [OSU-03012 + PDE5 inhibitor] toxicity. Knock down of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor] –induced JNK activation and inhibition of JNK suppressed the elevated killing caused by IRE1 knock down. Knock down of CD95 blunted JNK activation. Collectively our data demonstrates that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in GBM cells.
PMCID: PMC4185238  PMID: 25103559
2.  OSU-03012 enhances Ad.mda-7-induced GBM cell killing via ER stress and autophagy and by decreasing expression of mitochondrial protective proteins 
Cancer biology & therapy  2010;9(7):526-536.
The present studies focused on determining whether the autophagy-inducing drug OSU-03012 (AR-12) could enhance the toxicity of recombinant adenoviral delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) in glioblastoma multiforme (GBM) cells. The toxicity of a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7) was enhanced by OSU-03012 in a diverse panel of primary human GBM cells. The enhanced toxicity correlated with reduced ERK1/2 phosphorylation and expression of MCL-1 and BCL-XL, and was blocked by molecular activation of ERK1/2 and by inhibition of the intrinsic, but not the extrinsic, apoptosis pathway. Both OSU-03012 and expression of MDA-7/IL-24 increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) that correlated with increased levels of autophagy and expression of dominant negative PERK blocked autophagy induction and tumor cell death. Knockdown of ATG5 or Beclin1 suppressed OSU-03012 enhanced MDA-7/IL-24-induced autophagy and blocked the lethal interaction between the two agents. Ad.mda-7-infected GBM cells secreted MDA-7/IL-24 into the growth media and this conditioned media induced expression of MDA-7/IL-24 in uninfected GBM cells. OSU-03012 interacted with conditioned media to kill GBM cells and knockdown of MDA-7/IL-24 in these cells suppressed tumor cell killing. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.
PMCID: PMC2888700  PMID: 20107314
ROS; caspase; ER stress; CD95; cell death
3.  OSU-03012 suppresses GRP78/BiP expression that causes PERK-dependent increases in tumor cell killing 
Cancer Biology & Therapy  2012;13(4):224-236.
We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills tumor cells. OSU lethality was suppressed by knock down of PERK and enhanced by knock down of ATF6 and IRE1α. OSU treatment suppressed expression of the chaperone, BiP/GRP78, and did so through reduced stability of the protein. Knock down of BiP/GRP78 further enhanced OSU lethality. Overexpression of BiP/GRP78 abolished OSU toxicity. Pre-treatment of cells with OSU enhanced radiosensitivity to a greater extent than concomitant or sequential drug treatment with radiation exposure. Expression of a mutant active p110 PI3K, or mutant active forms of the EGFR in GBM cells did not differentially suppress OSU killing. In contrast loss of PTEN function reduced OSU lethality, without altering AKT, p70 S6K or mTOR activity, or the drug's ability to radiosensitize GBM cells. Knock down of PTEN protected cells from OSU and radiation treatment whereas re-expression of PTEN facilitated drug lethality and radiosensitization. In a dose-dependent fashion OSU prolonged the survival of mice carrying GBM tumors and interacted with radiotherapy to further prolong survival. Collectively, our data show that reduced BiP/GRP78 levels play a key role in OSU-3012 toxicity in GBM cells, and that this drug has in vivo activity against an invasive primary human GBM isolate.
PMCID: PMC3336069  PMID: 22354011
OSU-03012; BiP/GRP78; ER stress; PERK; ionizing radiation; ceramide
4.  OSU-03012 interacts with lapatinib to kill brain cancer cells 
Cancer Biology & Therapy  2012;13(14):1501-1511.
We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills brain cancer cells. OSU toxicity was enhanced by the HSP90 inhibitor 17-N-Allylamino-17-demethoxygeldanamycin (17AAG) that correlated with reduced expression of ERBB1 and ERBB2. Inhibition of the extrinsic apoptosis pathway blocked the interaction between 17AAG and OSU. OSU toxicity was enhanced by the inhibitor of ERBB1/2/4, lapatinib. Knock down of ERBB1/2/4 in a cell line specific fashion promoted OSU toxicity. Combined exposure of cells to lapatinib and OSU resulted in reduced AKT and ERK1/2 activity; expression of activated forms of AKT and to a lesser extent MEK1 protected cells from the lethal effects of the drug combination. Knock down of PTEN suppressed, and expression of PTEN enhanced, the lethal interaction between OSU and lapatinib. Downstream of PTEN, inhibition of mTOR recapitulated the effects of lapatinib. Knock down of CD95, NOXA, PUMA, BIK or AIF, suppressed lapatinib and OSU toxicity. Knock down of MCL-1 enhanced, and overexpression of MCL-1 suppressed, drug combination lethality. Lapatinib and OSU interacted in vivo to suppress the growth of established tumors. Collectively our data argue that the inhibition of ERBB receptor function represents a useful way to enhance OSU lethality in brain tumor cells.
PMCID: PMC3542242  PMID: 22990204
glioblastoma; medulloblastoma; lapatinib; OSU-03012; apoptosis; autophagy; ERBB1; PTEN
5.  Caspase-, cathepsin-, and PERK-dependent regulation of MDA-7/IL-24-induced cell killing in primary human glioma cells 
Molecular cancer therapeutics  2008;7(2):297-313.
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that correlated with inactivation of ERK1/2 and activation of JNK1-3. Activation of JNK1-3 was dependent on protein kinase R–like endoplasmic reticulum kinase (PERK), and GST-MDA-7 lethality was suppressed in PERK−/− cells. JNK1-3 signaling activated BAX, whereas inhibition of JNK1-3, deletion of BAX, or expression of dominant-negative caspase-9 suppressed lethality. GST-MDA-7 also promoted a PERK-, JNK-, and cathepsin B–dependent cleavage of BID; loss of BID function promoted survival. GST-MDA-7 suppressed BAD and BIM phosphorylation and heat shock protein 70 (HSP70) expression. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methylade-nine, expression of HSP70 or BiP/GRP78, or knockdown of ATG5 or Beclin-1 expression but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin-1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data show that GST-MDA-7 induces an endoplasmic reticulum stress response that is causal in the activation of multiple proapoptotic pathways, which converge on the mitochondrion and highlight the complexity of signaling pathways altered by mda-7/IL-24 in glioma cells that ultimately culminate in decreased tumor cell survival.
PMCID: PMC3204355  PMID: 18281515
6.  Sensitivity to the non-COX inhibiting celecoxib derivative, OSU03012, is p21WAF1/CIP1 dependent 
OSU03012 is a non-COX inhibiting celecoxib derivative with growth inhibiting and apoptotic activity in many cancer cell lines. To investigate mechanisms related to cell cycle proteins in growth inhibition and apoptosis induced by OSU03012, the primary human oral epithelial cell line, TE1177, was transformed with HPV16 E6 (TE/E6), HPV16 E7 (TE/E7) or empty vector (TE/V). TE/E6 cell lines exhibiting low levels of p53 and undetectable levels of p21WAF1/CIP1 were sensitized to the growth inhibiting and apoptotic effects of OSU03012. The TE/E7 cell lines expressing low levels of Rb and elevated levels of p53 and p21WAF1/CIP1 were resistant. OSU03012 reduced the number of cells in the S phase of the TE/E7 and TE/V cell lines with intact p53-p21WAF1/CIP1 checkpoint, but not in the checkpoint defective TE/E6 cell lines. Treatment with OSU03012 also markedly reduced the levels of cyclin A and Cdk2 in TE/E7 and TE/V, but not in TE/E6 cell lines, which had significantly enhanced basal levels of cyclin A and Cdk2. Consistent with the TE/E6 cell line, p21WAF1/CIP1−/− mouse embryo fibroblasts were more sensitive to OSU03012-induced apoptosis as evidenced by PARP and caspase 3 cleavages. These data suggest that p21WAF1/CIP1 is an important factor in the sensitivity of cells to the growth inhibiting and apoptotic effects of OSU03012.
PMCID: PMC2605165  PMID: 18798266
OSU03012; apoptosis; cell cycle; oral cancer; p21WAF1/CIP1
7.  Growth Inhibitory and Anti-Tumour Activities of OSU-03012, a Novel PDK-1 Inhibitor, on Vestibular Schwannoma and Malignant Schwannoma Cells 
Vestibular schwannomas (VS) frequently express high levels of activated AKT. Small-molecule inhibitors of AKT signaling may have therapeutic potential in suppressing the growth of benign VS and malignant schwannomas.
Primary VS and Schwann cells, human malignant schwannoma HMS-97 cells, and mouse Nf2−/− Schwann cells and schwannoma cells were prepared to investigate the growth inhibitory and anti-tumour activities of OSU-03012, a celecoxib-derived small-molecule inhibitor of phosphoinositide-dependent kinase 1. Cell proliferation assays, apoptosis, Western blot, in vivo xenograft analysis using SCID mice, and immunohistochemistry were performed.
OSU-03012 inhibited cell proliferation more effectively in both VS and HMS-97 cells than in normal human Schwann cells. The IC50 of OSU-03012 at 48 hours was approximately 3.1 μM for VS cells and 2.6 μM for HMS-97 cells, compared with the IC50 of greater than 12 μM for human Schwann cells. Similarly, mouse Nf2−/− schwannoma and Nf2−/− Schwann cells were more sensitive to growth inhibition by OSU-03012 than wild-type mouse Schwann cells and mouse schwannoma cells established from transgenic mice carrying the NF2 promoter-driven SV40 T-antigen gene. Like VS cells, malignant schwannoma HMS-97 cells expressed high levels of activated AKT. OSU-03012 induced apoptosis in both VS and HMS-97 cells and caused a marked reduction of AKT phosphorylation at both the Ser-308 and Thr-473 sites in a dose-dependent manner. In vivo xenograft analysis showed that OSU-03012 was well-tolerated and inhibited the growth of HMS-97 schwannoma xenografts by 55% after nine weeks of oral treatment. The anti-tumour activity correlated with reduced AKT phosphorylation.
OSU-03012 is a potential chemotherapeutic agent for VS and malignant schwannomas.
PMCID: PMC2692816  PMID: 19359162
Vestibular schwannoma; Malignant schwannoma HMS-97; Neurofibromatosis type 2; The Neurofibromatosis 2 (NF2) gene; Merlin; PI3K/AKT pathway; OSU-03012; Cyclooxygenase-2 inhibitor; Phosphoinositide-dependent kinase 1; Xenograft; Magnetic resonance imaging
8.  OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2 
BMC Cancer  2013;13:256.
Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms associated with enhancing the activity of lapatinib via combination with other therapies.
In the present studies, estrogen receptor (ER) positive and ER negative breast cancer cells were genetically manipulated to up- or downregulate eIF2-alpha, its phospho-mutant, Nck1, or Nck2, then treated with OSU-03012, lapatinib or the combination and assayed for cytotoxicity/cytostaticity using clonogenic assays.
Treatment of breast cancer cell lines with lapatinib and OSU-03012 (a small molecule derivative of the Cox-2 inhibitor celecoxib) induced synergistic cytotoxic/cytostatic effects. This combination therapy corresponded to an increase in the phosphorylation of eIF2-α at serine51 and a decrease in Nck1 expression. Ectopic expression of phospho-mutant eIF2-α (Ser51Ala) or downregulation of eIF2-α in addition to downregulation of the eIF2-α kinase PERK inhibited the synergistic and cytotoxic effects. Furthermore, ectopic expression of Nck1, but not Nck2 abolished the decrease in cell viability observed in combination-treated cells. Downregulation of Nck1 failed to “rescue” the ablation of the cytotoxic/cytostatic effects by the phospho-mutant of eIF2-α (Ser51Ala) demonstrating that Nck1 downregulation is upstream of eIF2-α phosphorylation in the anti-survival pathway activated by lapatinib and OSU-03012 treatment. Finally, co-immunoprecipitation assays indicated that eIF2-α dissociates from the Nck1/PP1 complex after OSU-03012 and lapatinib co-treatment.
These data indicate that OSU-03012 and lapatinib co-treatment is an effective combination therapy, which functions to enhance cell killing through the Nck1/eIF2 complex. Hence, this complex is a novel target for the treatment of metastatic breast cancer.
PMCID: PMC3674920  PMID: 23706161
Breast cancer; Lapatinib; Combination therapy; Nck; eIF2-alpha
9.  GRP78/BiP/HSPA5/Dna K is a Universal Therapeutic Target for Human Disease 
Journal of Cellular Physiology  2014;230(7):1661-1676.
The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70, HSP90, GRP94, GRP58, HSP27, HSP40 and HSP60. OSU-03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola/Marburg/Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively. Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya, Mumps, Measles, Rubella, RSV, CMV, and Influenza viruses. OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU-03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU-03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus, Dna K and bacterial phosphodiesterases are novel antibiotic targets, and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. J. Cell. Physiol. 230: 1661–1676, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
PMCID: PMC4402027  PMID: 25546329
10.  Aurora kinase A is a possible target of OSU-03012 to destabilize MYC family proteins 
Oncology Reports  2014;32(3):901-905.
OSU-03012, a 3-phosphoinositide-dependent kinase-1 (PDK1) inhibitor, destabilizes MYCN and MYC proteins in neuroblastoma cells. However, AKT phosphorylation is barely detectable in neuroblastoma cells under normal culture conditions whether treated with OSU-03012 or not. This observation suggests that PDK1 is not the main target of OSU-03012 to destabilize MYC and MYCN in neuroblastoma cells. In the present study, we explored one of the possible mechanisms by which OSU-03012 destabilizes MYC and MYCN. Since Aurora kinase A is reported to phosphorylate GSK3β, leading to its inactivation, we hypothesized that one of the targets of OSU-03012 is Aurora kinase A. Comparative analysis of OSU-03012 and VX-680, a potent and specific inhibitor of Aurora kinases, showed that both inhibitors destabilized MYC and MYCN and were significantly growth suppressive to neuroblastoma cell lines. In silico molecular docking analysis further showed that the calculated interaction energy between Aurora kinase A and OSU-03012 was −109.901 kcal/mol, which was lower than that (−89.273 kcal/mol) between Aurora kinase A and FXG, an Aurora kinase-specific inhibitor. Finally, an in vitro Aurora kinase A inhibition assay using a recombinant Aurora kinase A showed that OSU-03012 significantly inhibited Aurora kinase A, although it was weaker in potency than that of VX-680. Thus, OSU-03012 has a likelihood of binding to and inhibiting Aurora kinase A in vivo. These results suggest that OSU-03012 affects multiple cellular targets, including Aurora kinase A, to exhibit its growth suppressive and MYC and MYCN-destabilizing effects on neuroblastoma and other cancer cells.
PMCID: PMC4121397  PMID: 25017515
neuroblastoma; protein stability; phosphorylation; protein kinase inhibitor; docking simulation
11.  Adaptation by stochastic switching of a monostable genetic circuit in Escherichia coli 
Stochastic switching of a bistable genetic circuit represents a potential cost-saving strategy for adaptation to environmental challenges. This study reports that stochastic switching of a monostable circuit can be sufficient to mediate reversible adaptation in E. coli.
Stochastic switching of a monostable circuit mediated the adaptation of the engineered OSU12-hisC Escherichia coli strain to histidine starvation.The population shift of OSU12-hisC was accompanied by growth recovery and was reversible upon histidine addition. This is the first report of adaptation mediated by stochastic switching based on a monostable structure.Weak directionality in stochastic switching initiated the population shift and the fast growth of the occasionally appearing fit cells drove the later stages of adaptation.Adaptation of OSU12-hisC was resulted from the enhanced expression of the structural genes within the native His operon, along with the transcriptional reorganization of a large number of genes.
The fundamental mechanisms underlying adaptations can be divided into responsive switching and stochastic switching (Kussell and Leibler, 2005). Responsive switching is generally considered as resulting from evolved regulatory units, such as operons and regulons, which enable immediate adaptation (Jacob and Monod, 1961). However, as cells are subject to a wide range of both genetic and environmental perturbations that damage the specificity or efficiency of regulatory systems (Carroll, 2005; Crombach and Hogeweg, 2008), the limited number of regulatory units that can evolve and remain functional may not be sufficient to completely protect cell populations from the danger of extinction. Whether and how cells are able to survive external perturbations, when the corresponding regulatory units are absent or have been genetically disrupted, is an open question of great importance.
Recent studies showed the stochastic switching provided cells a huge potential for sustenance under severe conditions via a so-called ‘bet-hedging' strategy. The experimental evidence was generally based on a bistable genetic structure that fixed stochastically appearing fit state thus limiting further random switching (Kussell and Leibler, 2005; Acar et al, 2008). In contrast to bistable gene expression, monostable gene expression is much more common (Newman et al, 2006) and does not rely on a specific complex genetic architecture. Since a monostable structure has no fixation effect, the fit cells that would appear stochastically tend to return to the original steady state (i.e., unfit state). To achieve a population shift from a maladaptive state (but stable) to an adaptive state (but unstable), a significant increase in fitness (i.e., growth rate) of the fit cells is necessary. Otherwise, the random switching will mask occasionally occurring adaptive transitions and lead to an unchanged population at the stable but maladaptive state. Whether adaptation can be achieved by stochastic switching based on a monostable structure is however an open issue.
To address this question, we applied an engineered E. coli strain, OSU12-hisC, carrying a foreign gene circuit encompassing a physiologically functional gene, hisC, replaced from its native chromosomal locus (Figure 1A). Consequently, hisC in OSU12-hisC is no longer responsive to the native regulation (His operon) that senses histidine depletion. Instead, the foreign gene circuit provided a monostable structure for hisC's stochastic switching. The green fluorescent protein (gfpuv5) was co-expressed with hisC for the quantitative evaluation of HisC in single cells. The upstream regulation of TetR, whose expression level was reported by the red fluorescent protein (dsred.T4), was introduced to achieve the inducible GFP (HisC) level. The full induction of TetR by IPTG was applied to avoid any possible upstream noise that caused by the abundance of endogenous LacI.
Microscopic observation revealed that the OSU12-hisC cells showed stronger green fluorescence after histidine depletion (Figure 1B), which suggested an increased expression level of hisC. Population analysis using flow cytometry showed that the distributions of both GFP concentration and GFP bias (GFP/RFP ratio) in OSU12-hisC shifted towards a higher level in histidine-free conditions (Figure 1C and D), whereas, the depletion caused only a slight change in distributions of OSU11, a control strain carrying both the same engineered genetic circuit and an intact His operon, including the hisC gene in its native context. Repeated experiments revealed that the increases in both GFP concentration (∼2.1 folds) and GFP bias (∼1.5 folds) due to histidine depletion were highly significant (P<0.005, N=6) in OSU12-hisC. In particular, the increased GFP bias strongly suggested that the change in gene expression occurred specifically in the rewired hisC (i.e., GFP) but not in all genes (e.g., RFP). Furthermore, both the growth recovery accompanied population shift and the stress relaxation triggered restoration were clearly observed. It strongly indicated that the adaptation was mediated by stochastic switching of hisC under the monostable control.
Analysis on microcolonies' formation (Figure 4A) showed stochastic behaviour and directionality in individual cells. Variation in cellular GFP level was clearly observed in individual cells. Stochastic switching of hisC was verified according to the random changes in GFP bias along with the cell division under histidine-rich conditions (Figure 4B). On the other hand, the microcolonies formed under the histidine-free conditions tended to the higher level of GFP bias were observed (Figure 4B). The directional tendency favoured the high GFP (HisC) level was evidently detected in the first 2 h after histidine depletion, which resulted in a population shift (Figure 4C). In contrast, the distributions of microcolonies grown in histidine-rich conditions kept steady, due to the randomized directions of stochastic switching (Figure 4C). Further analysis showed that the stochastic fluctuations in the initial state had an important role not only in fate decision (i.e., whether to grow) but also in the directionality of the stochastic switch.
Microarray analysis showed the adaptation of OSU12-hisC was resulted from the enhanced expression of the structural genes within the native His operon, along with the transcriptional reorganization of a large number of genes. In summary, in contrast to bistable structures, the monostable structure used here did not fix the phenotype but allowed the cells to decide where to go. Taken together, the findings suggest that bacteria do not necessarily need to evolve signalling mechanisms to control gene expression appropriately, even for essential genes.
Stochastic switching is considered as a cost-saving strategy for adaptation to environmental challenges. We show here that stochastic switching of a monostable circuit can mediate the adaptation of the engineered OSU12-hisC Escherichia coli strain to histidine starvation. In this strain, the hisC gene was deleted from the His operon and placed under the control of a monostable foreign promoter. In response to histidine depletion, the OSU12-hisC population shifted to a higher HisC expression level, which is beneficial under starving conditions but is not favoured by the monostable circuit. The population shift was accompanied by growth recovery and was reversible upon histidine addition. A weak directionality in stochastic switching of hisC was observed in growing microcolonies under histidine-free conditions. Directionality and fate decision were in part dependent on the initial cellular status. Finally, microarray analysis indicated that OSU12-hisC reorganized its transcriptome to reach the appropriate physiological state upon starvation. These findings suggest that bacteria do not necessarily need to evolve signalling mechanisms to control gene expression appropriately, even for essential genes.
PMCID: PMC3130557  PMID: 21613982
adaptation; gene regulation; monostability; stochastic switching; transciptome
12.  Mutations in Rotavirus Nonstructural Glycoprotein NSP4 Are Associated with Altered Virus Virulence 
Journal of Virology  1998;72(5):3666-3672.
Rotaviruses are major pathogens causing life-threatening dehydrating gastroenteritis in children and animals. One of the nonstructural proteins, NSP4 (encoded by gene 10), is a transmembrane, endoplasmic reticulum-specific glycoprotein. Recently, our laboratory has shown that NSP4 causes diarrhea in 6- to 10-day-old mice by functioning as an enterotoxin. To confirm the role of NSP4 in rotavirus pathogenesis, we sequenced gene 10 from two pairs of virulent and attenuated porcine rotaviruses, the OSU and Gottfried strains. Comparisons of the NSP4 sequences from these two pairs of rotaviruses suggested that structural changes between amino acids (aa) 131 and 140 are important in pathogenesis. We next expressed the cloned gene 10 from the OSU virulent (OSU-v) and OSU attenuated (OSU-a) viruses by using the baculovirus expression system and compared the biological activities of the purified proteins. NSP4 from OSU-v virus increased intracellular calcium levels over 10-fold in intestinal cells when added exogenously and 6-fold in insect cells when expressed endogenously, whereas NSP4 from OSU-a virus had little effect. NSP4 from OSU-v caused diarrhea in 13 of 23 neonatal mice, while NSP4 from OSU-a caused disease in only 4 of 25 mice (P < 0.01). These results suggest that avirulence is associated with mutations in NSP4. Results from site-directed mutational analyses showed that mutated OSU-v NSP4 with deletion or substitutions in the region of aa 131 to 140 lost its ability to increase intracellular calcium levels and to induce diarrhea in neonatal mice, confirming the importance of amino acid changes from OSU-v NSP4 to OSU-a NSP4 in the alteration of virus virulence.
PMCID: PMC109587  PMID: 9557647
13.  Targeting ER Stress and Akt with OSU-03012 and Gefitinib or Erlotinib to Overcome Resistance to EGFR Inhibitors* 
Cancer research  2008;68(8):2820-2830.
Pre-existing and acquired resistance to epidermal growth factor receptor (EGFR) inhibitors limit their clinical usefulness in patients with advanced non-small cell lung cancer (NSCLC). This study characterizes the efficacy and mechanisms of the combination of gefitinib or erlotinib with OSU-03012, a celecoxib-derived antitumor agent, to overcome EGFR inhibitor-resistance in three NSCLC cell lines, H1155, H23, and A549. The OSU-03012/EGFR inhibitor combination induced pronounced apoptosis in H1155 and H23 cells, but not in A549 cells, suggesting a correlation between drug sensitivity and basal phospho-Akt levels independently of EGFR expression status. Evidence indicates that this combination facilitates apoptosis through both Akt signaling inhibition and upregulation of ER stress-induced, GADD153-mediated pathways. For example, ectopic expression of constitutively active Akt significantly attenuated the inhibitory effect on cell survival, and siRNA-mediated knockdown of GADD153 protected cells from undergoing apoptosis in response to drug co-treatments. Furthermore, the OSU-03012/EGFR inhibitor combination induced GADD153-mediated upregulation of death receptor 5 expression and subsequent activation of the extrinsic apoptosis pathway. It is noteworthy that the ER stress response induced by this combination was atypical in that the cytoprotective pathway was not engaged. In addition, in vivo suppression of tumor growth and modulation of intratumoral biomarkers were observed in a H1155 tumor xenograft model in nude mice. These data suggest that the concomitant modulation of Akt and ER stress pathways with the OSU-03012/EGFR inhibitor combination represents a unique approach to overcoming EGFR inhibitor resistance in NCSLC and perhaps other types of cancer with elevated basal Akt activities.
PMCID: PMC3904349  PMID: 18413750
Non-small cell lung cancer; ER stress; Akt; OSU-03012; EGFR inhibitors
14.  PDK-1/AKT pathway as a novel therapeutic target in rhabdomyosarcoma cells using OSU-03012 compound 
British Journal of Cancer  2007;97(6):785-791.
Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma including two major subtypes, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Increasing evidence suggests that oncogenesis of RMS involves multistages of signalling protein dysregulation which may include prolonged activation of serine/threonine kinases such as phosphoinositide-dependant kinase-1 (PDK-1) and AKT. To date, whether PDK-1/AKT pathway is activated in RMS is unknown. This study was to examine phosphorylation status of AKT and to evaluate a novel small molecular inhibitor, OSU-03012 targeting PDK-1 in RMS. We examined phosphorylation levels of AKT using ARMS and ERMS tissue microarray and immunohistochemistry staining. Our results showed phospho-AKTThr308 level is elevated 42 and 35% in ARMS and ERMS, respectively. Phospho-AKTSer473 level is also increased 43% in ARMS and 55% in ERMS. Furthermore, we showed that OSU-03012 inhibits cell viability and induces apoptosis in ARMS and ERMS cell lines (RH30, SMS-CTR), which express elevated phospho-AKT levels. Normal cells are much less sensitive to OSU-03012 and in which no detectable apoptosis was observed. This study showed, for the first time, that PDK-1/AKT pathway is activated in RMS and may play an important role in survival of RMS. PDK-1/AKT pathway may be an attractive therapeutic target for cancer intervention in RMS using OSU-03012.
PMCID: PMC2360380  PMID: 17848913
AKT; PDK-1; rhabdomyosarcoma; small molecular inhibitor; tissue microarray
15.  Induction of Oxidative Stress in Trypanosoma brucei by the Antitrypanosomal Dihydroquinoline OSU-40 
Dihydroquinoline derivative OSU-40 (1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate) is selectively potent against Trypanosma brucei rhodesiense in vitro (50% inhibitory concentration [IC50], 14 nM; selectivity index, 1,700) and has been proposed to cause the formation of reactive oxygen species (ROS) in African trypanosomes (J. Fotie et al., J. Med. Chem. 53:966–982, 2010). In the present study, we sought to provide further support for the hypothesis that OSU-40 kills trypanosomes through oxidative stress. Inducible RNA interference (RNAi) was applied to downregulate key enzymes in parasite antioxidant defense, including T. brucei trypanothione synthetase (TbTryS) and superoxide dismutase B (TbSODB). Both TbTryS RNAi-induced and TbSODB RNAi-induced cells showed impaired growth and increased sensitivity toward OSU-40 by 2.4-fold and 3.4-fold, respectively. Decreased expression of key parasite antioxidant enzymes was thus associated with increased sensitivity to OSU-40, consistent with the hypothesis that OSU-40 acts through oxidative stress. Finally, the dose-dependent formation of free radicals was observed after incubation of T. brucei with OSU-40 utilizing electron spin resonance (ESR) spectroscopy. These data support the notion that the mode of antitrypanosomal action for this class of compounds is to induce oxidative stress.
PMCID: PMC3346584  PMID: 22314522
16.  PERK-dependent regulation of MDA-7/IL-24-induced autophagy in primary human glioma cells 
Autophagy  2008;4(4):513-515.
Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The studies by further defines the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that were dependent on activation of JNK1-3 with subsequent activation of BAX and the induction of mitochondrial dysfunction. Activation of JNK1-3 was dependent upon protein kinase R-like endoplasmic reticulum kinase (PERK) and GST-MDA-7 lethality was suppressed in PERK-/- cells. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or of BiP/GRP78, or by knockdown of ATG5 or Beclin 1 expression, but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin 1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data demonstrate that GST-MDA-7 induces an ER stress response that, via the induction of autophagy, is causal in the activation of pro-apoptotic pathways that converge on the mitochondrion and ultimately culminate in decreased glioma cell survival.
PMCID: PMC2674579  PMID: 18299661
autophagy; caspase; ER stress; cell death
17.  OSU-CG5, a novel energy restriction mimetic agent, targets human colorectal cancer cells in vitro 
Acta Pharmacologica Sinica  2014;35(3):394-400.
Energy-restriction mimetic agents (ERMAs) are small-molecule agents that target various aspects of energy metabolism, which has emerged as a promising approach in cancer therapy. In the current study, we tested the ability of OSU-CG5, a novel ERMA, to target human colorectal cancer (CRC) in vitro.
Two human CRC cell lines (HCT-116 and Caco-2) were tested. Cell viability was assessed using MTT assay. Caspase-3/7 activities were measured using Caspase-Glo 3/7 assay kit. Western blot analysis was used to measure the expression of relevant proteins in the cells. Glucose consumption of the cells was detected using glucose uptake cell-based assay kit.
OSU-CG5 dose-dependently inhibited HCT-116 and Caco-2 cell proliferation with the IC50 values of 3.9 and 4.6 μmol/L, respectively, which were 20–25-fold lower than those of resveratrol, a reference ERMA. Both OSU-CG5 (5, 10, and 20 μmol/L) and resveratrol (50, 100, and 200 μmol/L) dose-dependently increased caspase-3/7 activity and PARP level in the cells. Furthermore, both OSU-CG5 and resveratrol induced dose-dependent energy restriction in the cells: they suppressed glucose uptake and Akt phosphorylation, decreased the levels of p-mTOR and p-p70S6K, increased the levels of ER stress response proteins GRP78 and GADD153, and increased the level of β-TrCP, which led to the downregulation of cyclin D1 and Sp1.
OSU-CG5 exhibits promising anti-cancer activity against human CRC cells in vitro, which was, at least in part, due to energy restriction and the consequent induction of ER stress and apoptosis.
PMCID: PMC4647897  PMID: 24464048
colorectal cancer; energy restriction mimetic agent; OSU-CG5; resveratrol; apoptosis; glucose uptake; Akt; mTOR; ER stress; β-TrCP
18.  Celecoxib analogues disrupt Akt signaling, which is commonly activated in primary breast tumours 
Breast Cancer Research  2005;7(5):R796-R807.
Phosphorylated Akt (P-Akt) is an attractive molecular target because it contributes to the development of breast cancer and confers resistance to conventional therapies. Akt also serves as a signalling intermediate for receptors such as human epidermal growth factor receptor (HER)-2, which is overexpressed in 30% of breast cancers; therefore, inhibitors to this pathway are being sought. New celecoxib analogues reportedly inhibit P-Akt in prostate cancer cells. We therefore examined the potential of these compounds in the treatment of breast cancer. The analogues were characterized in MDA-MB-453 cells because they overexpress HER-2 and have very high levels of P-Akt.
To evaluate the effect of the celecoxib analogues, immunoblotting was used to identify changes in the phosphorylation of Akt and its downstream substrates glycogen synthase kinase (GSK) and 4E binding protein (4EBP-1). In vitro kinase assays were then used to assess the effect of the drugs on Akt activity. Cell death was evaluated by poly(ADP-ribose) polymerase cleavage, nucleosomal fragmentation and MTS assays. Finally, tumour tissue microarrays were screened for P-Akt and HER-2 expression.
OSU-03012 and OSU-O3013 inhibited P-Akt and its downstream signalling through 4EBP-1 and GSK at concentrations well below that of celecoxib. Disruption of P-Akt was followed by induction of apoptosis and more than 90% cell death. We also noted that the cytotoxicity of the celecoxib analogues was not significantly affected by serum. In contrast, the presence of 5% serum protected cells from celecoxib induced death. Thus, the structural modification of the celecoxib analogues increased P-Akt inhibition and enhanced the bioavailability of the drugs in vitro. To assess how many patients may potentially benefit from such drugs we screened tumour tissue microarrays. P-Akt was highly activated in 58% (225/390) of cases, whereas it was only similarly expressed in 35% (9/26) of normal breast tissues. Furthermore, HER-2 positive tumours expressed high levels of P-Akt (P < 0.01), supporting in vitro signal transduction.
We determined that Celecoxib analogues are potent inhibitors of P-Akt signalling and kill breast cancer cells that overexpress HER-2. We also defined an association between HER-2 and P-Akt in primary breast tissues, suggesting that these inhibitors may benefit patients in need of new treatment options.
PMCID: PMC1242152  PMID: 16168126
19.  OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukaemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway 
British journal of haematology  2011;153(5):623-633.
Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.
PMCID: PMC3696190  PMID: 21470196
D-tyrosinol; chronic lymphocytic leukaemia; p38 mitogen-activated protein kinase (p38 MAPK); apoptosis; BIRC5
Neuro-Oncology  2014;16(Suppl 5):v198-v199.
BACKGROUND: Glioblastoma Multiforme (GBM) is the most aggressive form of malignant primary brain tumor. A subset of glioblastoma cells, known as glioblastoma stem-like cells (GSCs), are chemotherapy and radiotherapy-resistent and are hypothesized to be responsible for tumor formation, maintenance, and recurrence. The PI3K/AKT pathway is overactive in GBM and has been shown to play a role in cancer stem cell maintenance. Therefore we sought to determine the efficacy of drugs targeting the AKT pathway in multiple patient-derived GSC lines. METHODS: GSC lines were generated from patient's tumors, propagated in neurosphere media and analyzed for stem cell markers by immunocytochemistry. Effect of growth factor receptor inhibitors (erlotinib, gefetinib, sunitinib), the intracellular PI3K/AKT pathway inhibitors (GDC-0941, OSU-03012, perifosine, NVP-BEZ235, KU-0063794 and everolimus) and autophagy inhibitor (chloroquine) on cell viability was determined with MTS assay. Caspase 3/7 activity was determined using CellEvent caspase reagent. The effect of the inhibitors on cell signaling pathways was evaluated by western blot analysis. RESULTS: Growth factor receptor inhibition failed to induce significant reduction in cell viability. Intracellular inhibitors produced variable results except for dual PI3K/mTOR inhibitor NVB-BEZ235 which induced approximately 50% reduction in cell viability. While the inhibition of autophagy with chloroquine significantly increased inhibitor induced cell death in some cell lines, combined PDK1 and autophagy inhibition induced robust caspase activity and significantly reduced viability in all six GSCs examined to an average of 6.1 + 2.7% viability compared to non-treated controls. CONCLUSION: The AKT/PI3K pathway is overactive in GSCs and may contribute to stem cell maintenance as well as inhibition of apoptosis. Our data indicates that inhibiting autophagy concommitantly with PI3K/AKT pathway inhibition can potentiate their effect. Furthermore combined chloroquine and OSU-03012 resulted in a substantial decrease cell viability accross multiple GSC lines and may represent an adjuvant therapy for a disease with minimal survival.
PMCID: PMC4218604
21.  Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation 
Cancer biology & therapy  2008;7(10):1648-1662.
We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2α phosphorylation; suppression of eIF2α function abolished drug combination lethality. Cell killing was paralleled by PERK- and eIF2α-dependent lowering of c-FLIP-s protein levels and over-expression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRβ and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Over-expression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2α -↓c-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).
PMCID: PMC2674577  PMID: 18787411
Vorinostat; Sorafenib; CD95; c-FLIP-s; PDGFRβ; FLT3; autophagy; ceramide; cell death; ASMase
22.  A Novel Histone Deacetylase Inhibitor Exhibits Antitumor Activity via Apoptosis Induction, F-Actin Disruption and Gene Acetylation in Lung Cancer 
PLoS ONE  2010;5(9):e12417.
Lung cancer is the leading cause of cancer mortality worldwide, yet the therapeutic strategy for advanced non-small cell lung cancer (NSCLC) is limitedly effective. In addition, validated histone deacetylase (HDAC) inhibitors for the treatment of solid tumors remain to be developed. Here, we propose a novel HDAC inhibitor, OSU-HDAC-44, as a chemotherapeutic drug for NSCLC.
Methodology/Principal Findings
The cytotoxicity effect of OSU-HDAC-44 was examined in three human NSCLC cell lines including A549 (p53 wild-type), H1299 (p53 null), and CL1-1 (p53 mutant). The antiproliferatative mechanisms of OSU-HDAC-44 were investigated by flow cytometric cell cycle analysis, apoptosis assays and genome-wide chromatin-immunoprecipitation-on-chip (ChIP-on-chip) analysis. Mice with established A549 tumor xenograft were treated with OSU-HDAC-44 or vehicle control and were used to evaluate effects on tumor growth, cytokinesis inhibition and apoptosis. OSU-HDAC-44 was a pan-HDAC inhibitor and exhibits 3–4 times more effectiveness than suberoylanilide hydroxamic acid (SAHA) in suppressing cell viability in various NSCLC cell lines. Upon OSU-HDAC-44 treatment, cytokinesis was inhibited and subsequently led to mitochondria-mediated apoptosis. The cytokinesis inhibition resulted from OSU-HDAC-44-mediated degradation of mitosis and cytokinesis regulators Auroroa B and survivin. The deregulation of F-actin dynamics induced by OSU-HDAC-44 was associated with reduction in RhoA activity resulting from srGAP1 induction. ChIP-on-chip analysis revealed that OSU-HDAC-44 induced chromatin loosening and facilitated transcription of genes involved in crucial signaling pathways such as apoptosis, axon guidance and protein ubiquitination. Finally, OSU-HDAC-44 efficiently inhibited A549 xenograft tumor growth and induced acetylation of histone and non-histone proteins and apoptosis in vivo.
OSU-HDAC-44 significantly suppresses tumor growth via induction of cytokinesis defect and intrinsic apoptosis in preclinical models of NSCLC. Our data provide compelling evidence that OSU-HDAC-44 is a potent HDAC targeted inhibitor and can be tested for NSCLC chemotherapy.
PMCID: PMC2939045  PMID: 20856855
23.  Antitumor Effects of OSU-2S, a Non-immunosuppressive Analogue of FTY720, in Hepatocellular Carcinoma 
Hepatology (Baltimore, Md.)  2011;53(6):1943-1958.
Accumulating evidence suggests the therapeutic potential of the immunosuppressive agent FTY720 (fingolimod) in hepatocellular carcinoma (HCC). Based on our previous finding that FTY720 mediates apoptosis in HCC cells by activating reactive oxygen species (ROS)-protein kinase (PK)Cδ signaling independent of effects on sphingosine-1-phosphate (S1P) receptors, we embarked on the pharmacological exploitation of FTY720 to develop a non-immunosuppressive analogue with antitumor activity. This effort led to the development of OSU-2S, which exhibits higher potency than FTY720 in suppressing HCC cell growth through PKCδ activation. In contrast to FTY720, OSU-2S was not phosphorylated by sphingosine kinase (SphK)2 in vitro, and did not cause S1P1 receptor internalization in HCC cells or T lymphocyte homing in immunocompetent mice. Though devoid of S1P1 receptor activity, OSU-2S exhibited higher in vitro antiproliferative efficacy relative to FTY720 against HCC cells without cytotoxicity in normal hepatocytes. Several lines of pharmacological and molecular genetic evidence indicate that ROS-PKCδ-caspase-3 signaling underlies OSU-2S-mediated antitumor effects, and that differences in the antitumor activity between FTY720 and OSU-2S were attributable to SphK2-mediated phosphorylation of FTY720, which represents a metabolic inactivation of its antitumor activity. Finally, OSU-2S exhibited high in vivo potency in suppressing xenograft tumor growth in both ectopic and orthotopic models without overt toxicity. Conclusion: Using the molecular platform of FTY720, we developed OSU-2S, a novel PKCδ-targeted antitumor agent, which is devoid of S1P1 receptor activity and is highly effective in suppressing HCC tumor growth in vivo. These findings suggest that OSU-2S has clinical value in therapeutic strategies for HCC and warrants continued investigation in this regard.
PMCID: PMC3103615  PMID: 21391227
hepatocellular carcinoma; FTY720; OSU-2S; sphingosine 1-phosphate receptor; protein kinase Cδ
24.  Expression of the OSU rotavirus outer capsid protein VP4 by an adenovirus recombinant. 
Journal of Virology  1992;66(7):4407-4412.
Full-length cDNA of the VP4 gene of porcine rotavirus strain OSU was cloned into adenovirus type 5 (Ad5) downstream of the E3 promoter. The plaque-purified recombinant (Ad5-OSU VP4) expressed apparently authentic VP4 rotavirus outer capsid protein. The protein had the same molecular size (85 kDa) and electrophoretic mobility as did native OSU VP4 and was immunoprecipitated by a polyclonal antiserum raised to OSU VP4. Cotton rats that possessed prechallenge rotavirus antibodies that may have been acquired either passively or actively developed neutralizing antibodies against the OSU strain following intranasal administration of the live Ad5-OSU VP4 recombinant. The neutralizing activity was enhanced by a parenteral booster injection with baculovirus-expressed OSU VP4 antigen. In addition, a high titer of neutralizing antibodies was induced by parenteral administration of the latter antigen and subsequent intranasal administration of the Ad5-OSU VP4 recombinant. These observations indicate that the VP4 outer capsid protein of a rotavirus strain can be expressed by a recombinant adenovirus vector. This approach warrants further exploration for immunization against rotavirus disease.
PMCID: PMC241248  PMID: 1318411
25.  Regulation of autophagy by ceramide-CD95-PERK signaling 
Autophagy  2008;4(7):929-931.
The manuscripts by Park et al.1 and Zhang et al.2 were initially planned as studies to understand the regulation of cell survival in transformed cells treated with sorafenib and vorinostat, and in primary hepatocytes treated with a bile acid+MEK1/2 inhibitor. In both cell systems we discovered that the toxicity of sorafenib and vorinostat or bile acid+MEK1/2 inhibitor exposure depended on the generation of ceramide and the ligand-independent activation of the CD95 death receptor, with subsequent activation of pro-caspase 8. We noted, however, in these systems that, in parallel with death receptor–induced activation of the extrinsic pathway, CD95 signaling also promoted increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α, increased expression of ATG5, and increased processing of LC3 and vesicularization of a GFP-LC3 construct. The knockdown of ATG5 expression blocked GFP-LC3 vesicularization and enhanced cell killing. Thus ceramide-CD95 signaling promoted cell death via activation of pro-caspase 8 and cell survival via autophagy. PERK was shown to signal in a switch-hitting fashion; PERK promoted CD95-DISC formation and an eIF2α-dependent reduction in c-FLIP-s levels that were essential for cell killing to proceed, but in parallel it also promoted autophagy that was protective. The death receptor-induced apoptosis and autophagy occur proximal to the receptor rather than the mitochondrion, and the relative flow of death receptor signaling into either pathway may determine cell fate. Finally, death receptor induced apoptosis and autophagy could be potential targets for therapeutic intervention.
PMCID: PMC3292039  PMID: 18719356
Vorinostat; Sorafenib; bile acid; CD95; autophagy; ceramide; cell death; ASMase

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