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1.  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
2.  The development of MDA-7/IL-24 as a cancer therapeutic 
Pharmacology & therapeutics  2010;128(2):375-384.
The cytokine melanoma differentiation associated gene 7 (mda-7) was identified by subtractive hybridization as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Based on conserved structure, chromosomal location and cytokine-like properties, MDA-7, was classified as a member of the interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and rapid cell death. In addition, MDA-7/IL-24 has been noted to radiosensitize tumor cells which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress and suppress protein translation. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, strongly arguing that MDA-7/IL-24 could have significant therapeutic value. This review describes what is presently known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.
doi:10.1016/j.pharmthera.2010.08.001
PMCID: PMC2947573  PMID: 20732354
MDA-7; IL-24; Apoptosis; Autophagy; Ceramide; ROS; Ca2+; Clinical trial; Signal transduction; PERK; ER stress; MCL-1
3.  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.
doi:10.1158/1535-7163.MCT-07-2166
PMCID: PMC3204355  PMID: 18281515
4.  mda-7/IL-24: A Unique Member of the IL-10 Gene Family Promoting Cancer-Targeted Toxicity 
Cytokine & growth factor reviews  2010;21(5):381-391.
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that displays nearly ubiquitous cancer-specific toxicity, with no harmful effects toward normal cells or tissues. mda-7/IL-24 was cloned from human melanoma cells by differentiation induction subtraction hybridization (DISH) and promotes endoplasmic reticulum (ER) stress culminating in apoptosis or toxic autophagy in a broad-spectrum of human cancers, when assayed in cell culture, in vivo in human tumor xenograft mouse models and in a Phase I clinical trial in patients with advanced cancers. This therapeutically active cytokine also induces indirect anti-tumor activity through inhibition of angiogenesis, stimulation of an anti-tumor immune response, and sensitization of cancer cells to radiation-, chemotherapy- and antibody-induced killing.
doi:10.1016/j.cytogfr.2010.08.004
PMCID: PMC3164830  PMID: 20926331
mda-7/IL-24; apoptosis; autophagy; bystander antitumor activity; cancer terminator virus
5.  MDA-7/IL-24 as a cancer therapeutic: from bench to bedside 
Anti-cancer drugs  2010;21(8):725-731.
The novel cytokine melanoma differentiation associated gene-7 (mda-7) was identified by subtractive hybridization in the mid-1990s as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Based on conserved structure, chromosomal location and cytokine-like properties, MDA-7, has now been classified as a member of the expanding interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and ultimately cell death. In addition, MDA-7/IL-24 has been noted to be a radiosensitizing cytokine, which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, which strongly argues that MDA-7/IL-24 may have significant therapeutic value. This review describes what is known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.
doi:10.1097/CAD.0b013e32833cfbe1
PMCID: PMC2915543  PMID: 20613485
MDA-7: melanoma differentiation associated gene 7
6.  MDA-7/IL-24–induced cell killing in malignant renal carcinoma cells occurs by a ceramide/CD95/PERK–dependent mechanism 
Molecular cancer therapeutics  2009;8(5):1280-1291.
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 clarifying the mechanism(s) by which glutathione S-transferase (GST)-MDA-7 altered cell survival of human renal carcinoma cells in vitro. GST-MDA-7 caused plasma membrane clustering of CD95 and the association of CD95 with procaspase-8. GST-MDA-7 lethality was suppressed by inhibition of caspase-8 or by overexpression of short-form cellular FLICE inhibitory protein, but only weakly by inhibition of cathepsin proteases. GST-MDA-7–induced CD95 clustering (and apoptosis) was blocked by knockdown of acidic sphingomyelinase or, to a greater extent, ceramide synthase-6 expression. GST-MDA-7 killing was, in parallel, dependent on inactivation of extracellular signal–regulated kinase 1/2 and on CD95-induced p38 mitogen-activated protein kinase and c-jun NH2-terminal kinase-1/2 signaling. Knockdown of CD95 expression abolished GST-MDA-7–induced phosphorylation of protein kinase R–like endoplasmic reticulum kinase. GST-MDA-7 lethality was suppressed by knockout or expression of a dominant negative protein kinase R–like endoplasmic reticulum kinase that correlated with reduced c-jun NH2-terminal kinase-1/2 and p38 mitogen-activated protein kinase signaling and maintained extracellular signal–regulated kinase-1/2 phosphorylation. GST-MDA-7 caused vacuolization of LC3 through a mechanism that was largely CD95 dependent and whose formation was suppressed by knockdown of ATG5 expression. Knockdown of ATG5 suppressed GST-MDA-7 toxicity. Our data show that in kidney cancer cells GST-MDA-7 induces ceramide-dependent activation of CD95, which is causal in promoting an endoplasmic reticulum stress response that activates multiple proapoptotic pathways to decrease survival.
doi:10.1158/1535-7163.MCT-09-0073
PMCID: PMC2889018  PMID: 19417161
7.  MEK1/2 inhibitors and 17AAG synergize to kill human GI tumor cells in vitro via suppression of c-FLIP-s levels and activation of CD95 
Molecular cancer therapeutics  2008;7(9):2633-2648.
Prior studies have noted that inhibitors of MEK1/2 enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered survival in carcinoma cells. MEK1/2 inhibitors (PD184352; AZD6244 (ARRY-142886)) interacted in a synergistic manner with geldanamycins (17AAG, 17DMAG) to kill hepatoma and pancreatic carcinoma cells that correlated with inactivation of ERK1/2 and AKT and with activation of p38 MAPK; p38 MAPK activation was ROS-dependent. Treatment of cells with MEK1/2 inhibitors and 17AAG reduced expression of c-FLIP-s that was mechanistically connected to loss of MEK1/2 and AKT function; inhibition of caspase 8 or over-expression of c-FLIP-s abolished cell killing by MEK1/2 inhibitors and 17AAG. Treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent plasma membrane clustering of CD95 without altering the levels or cleavage of FAS ligand. In parallel, treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent association of caspase 8 with CD95. Inhibition of p38 MAPK or knock down of BID, FADD or CD95 expression suppressed MEK1/2 inhibitor and 17AAG lethality. Similar correlative data were obtained using a xenograft flank tumor model system. Our data demonstrate that treatment of tumor cells with MEK1/2 inhibitors and 17AAG induces activation of the extrinsic pathway and that suppression of c-FLIP-s expression is crucial in transduction of the apoptotic signal from CD95 to promote cell death.
doi:10.1158/1535-7163.MCT-08-0400
PMCID: PMC2585522  PMID: 18790746
CD95; caspase; extrinsic; FLIP
8.  Vorinostat and sorafenib synergistically kill tumor cells via FLIP suppression and CD95 activation 
Purpose and Design
Mechanism(s) by which the multi-kinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal and pancreatic adenocarcinoma cells have been defined.
Results
Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal and pancreatic adenocarcinoma cells in multiple short term viability (24–96h) and in long term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase 8, and to a lesser extent by inhibition of caspase 9. Twenty four hours after exposure, the activities of ERK1/2, AKT and NFκB were only modestly modulated by sorafenib and vorinostat treatment. However, 24h after exposure, sorafenib and vorinostat- treated cells exhibited markedly diminished expression of c-FLIP-s, full length BID, BCL-2, BCLXL, MCL-1, XIAP, increased expression of BIM, and increased activation of BAX, BAK and BAD. Expression of eIF2α S51A blocked sorafenib and vorinostat –induced suppression of c-FLIP-s levels and over-expression of c-FLIP-s abolished lethality. Sorafenib and vorinostat treatment increased surface levels of CD95 and CD95 association with caspase 8. Knock down of CD95 or FADD expression significantly reduced sorafenib / vorinostat -mediated lethality.
Conclusions
These data demonstrate that combined exposure of epithelial tumor cell types to sorafenib and vorinostat diminishes expression of multiple anti-apoptotic proteins, promotes activation of the CD95 extrinsic apoptotic and the lysosomal protease pathways, and that suppression of c-FLIP-s expression represents a critical event in transduction of the pro-apoptotic signals from CD95 to promote mitochondrial dysfunction and death.
doi:10.1158/1078-0432.CCR-08-0469
PMCID: PMC2561272  PMID: 18765530
Vorinostat; Sorafenib; CD95; c-FLIP-s; caspase 8; cathepsin; cell death
9.  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
10.  OSU-03012 Stimulates PKR-Like Endoplasmic Reticulum-Dependent Increases in 70-kDa Heat Shock Protein Expression, Attenuating Its Lethal Actions in Transformed Cells 
Molecular pharmacology  2008;73(4):1168-1184.
We have further defined mechanism(s) by which 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide [OSU-03012 (OSU)], a derivative of the cyclooxygenase-2 (COX2) inhibitor celecoxib but lacking COX2 inhibitory activity, kills transformed cells. In cells lacking expression of protein kinase R-like endoplasmic reticulum kinase (PERK-/-), the lethality of OSU was attenuated. OSU enhanced the expression of Beclin 1 and ATG5 and cleavage of pro-caspase 4 in a PERK-dependent fashion and promoted the Beclin 1- and ATG5-dependent formation of vacuoles containing LC3, followed by a subsequent caspase 4-dependent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B was activated and released into the cytosol and genetic suppression of caspase 4, cathepsin B, or apoptosis-inducing factor function significantly suppressed cell killing. In parallel, OSU caused PERK-dependent increases in 70-kDa heat shock protein (HSP70) expression and decreases in 90-kDa heat shock protein (HSP90) and Grp78/BiP expression. Changes in HSP70 expression were post-transcriptional. Knockdown or small-molecule inhibition of HSP70 expression enhanced OSU toxicity, and overexpression of HSP70 suppressed OSU-induced low pH vesicle formation and lethality. Our data demonstrate that OSU-03012 causes cell killing that is dependent on PERK-induced activation of multiple toxic proteases. OSU-03012 also increased expression of HSP70 in a PERK-dependent fashion, providing support for the contention that OSU-03012-induced PERK signaling promotes both cell survival and cell death processes.
doi:10.1124/mol.107.042697
PMCID: PMC2674576  PMID: 18182481

Results 1-10 (10)