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1.  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
2.  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
3.  PERK–Dependent Regulation of Ceramide Synthase 6 and Thioredoxin Play a Key Role in mda-7/IL-24–Induced Killing of Primary Human Glioblastoma Multiforme Cells 
Cancer research  2010;70(3):1120-1129.
Melanoma differentiation associated gene-7(mda-7) encodes IL-24, a cytokine that can selectively trigger apoptosis in transformed cells. Recombinant mda-7 adenovirus (Ad.mda-7) effectively kills glioma cells, offering a novel gene therapy strategy to address deadly brain tumors. In this study, we defined the proximal mechanisms by which Ad-mda-7 kills glioma cells. Key factors implicated included activation of the endoplasmic reticulum stress kinase protein kinase R–like endoplasmic reticulum kinase (PERK), Ca++ elevation, ceramide generation and reactive oxygen species (ROS) production. PERK inhibition blocked ceramide or dihydroceramide generation, which were critical for Ca++ induction and subsequent ROS formation. Activation of autophagy and cell death relied upon ROS formation, the inhibition of which ablated Ad.mda-7–killing activity. In contrast, inhibiting TRX induced by Ad.MDA-7 enhanced tumor cytotoxicity and improved animal survival in an orthotopic tumor model. Our findings indicate that mda-7/IL-24 induces an endoplasmic reticulum stress response that triggers production of ceramide, Ca2+, and ROS, which in turn promote glioma cell autophagy and cell death.
doi:10.1158/0008-5472.CAN-09-4043
PMCID: PMC2890071  PMID: 20103619
4.  Mechanism by Which Mcl-1 Regulates Cancer-Specific Apoptosis Triggered by mda-7/IL-24, an IL-10-Related Cytokine 
Cancer research  2010;70(12):5034-5045.
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a cytokine belonging to the IL-10 family, selectively induces apoptosis in cancer cells without harming normal cells by promoting an endoplasmic reticulum (ER) stress response. The precise molecular mechanism by which the ER stress response culminates in cell death requires further clarification. The present study shows that in prostate carcinoma cells, the mda-7/IL-24-induced ER stress response causes apoptosis by translational inhibition of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1). Forced expression of Mcl-1 blocked mda-7/IL-24 lethality, whereas RNA interference or gene knockout of Mcl-1 markedly sensitized transformed cells to mda-7/IL-24. Mcl-1 downregulation by mda-7/IL-24 relieved its association with the proapoptotic protein Bak, causing oligomerization of Bak and leading to cell death. These observations show the profound role of the Bcl-2 protein family member Mcl-1 in regulating cancer-specific apoptosis induced by this cytokine. Thus, our studies provide further insights into the molecular mechanism of ER stress-induced cancer-selective apoptosis by mda-7/IL-24. As Mcl-1 is overexpressed in the majority of prostate cancers, mda-7/IL-24 might provide an effective therapeutic for this disease.
doi:10.1158/0008-5472.CAN-10-0563
PMCID: PMC3171699  PMID: 20501829
5.  Enhancing mda-7/IL-24 therapy in renal carcinoma cells by inhibiting multiple protective signaling pathways using sorafenib and by Ad.5/3 gene delivery 
Cancer Biology & Therapy  2010;10(12):1290-1305.
We have determined whether an adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, more effectively infects and kills renal carcinoma cells (RCCs) compared to a serotype 5 virus, Ad.5-mda-7. RCCs are a tumor cell type that generally does not express the receptor for the type 5 adenovirus; the coxsakie and adenovirus receptor (CAR). Ad.5/3-mda-7 infected RCCs to a much greater degree than Ad.5-mda-7. MDA-7/IL-24 protein secreted from Ad.5/3-mda-7-infected RCCs induced MDA-7/IL-24 expression and promoted apoptosis in uninfected “bystander” RCCs. MDA-7/IL-24 killed both infected and bystander RCCs via CD95 activation. Knockdown of intracellular MDA-7/IL-24 in uninfected RCCs blocked the lethal effects of conditioned media. Infection of RCC tumors in one flank, with Ad.5/3-mda-7, suppressed growth of infected tumors and reduced the growth rate of uninfected tumors implanted on the opposite flank. The toxicity of the serotype 5/3 recombinant adenovirus to express MDA-7/IL-24 was enhanced by combined molecular or small molecule inhibition of MEK1/2 and PI3K; inhibition of mTOR, PI3K and MEK1/2; or use of the multi-kinase inhibitor sorafenib. In RCCs, combined inhibition of cytoprotective cell signaling pathways enhanced the MDA-7/IL-24-induction of CD95 activation, with greater mitochondrial dysfunction due to loss of MCL-1 and BCL-XL expression and tumor cell death. Treatment of RCC tumors in vivo with sorafenib also enhanced Ad.5/3-mda-7 toxicity and prolonged animal survival. Future combinations of these approaches hold promise for developing a more effective therapy for kidney cancer.
doi:10.4161/cbt.10.12.13497
PMCID: PMC3047088  PMID: 20948318
ERK; JNK; PI3K; AKT; MDA-7/IL-24; sorafenib; PERK; MAPK; interleukin; RCC; kidney
6.  Infection of Schistosomiasis japanicum is likely to enhance proliferation and migration of human breast cancer cells: mechanism of action of differential expression of MMP2 and MMP9 
Objective
To study whether the infection of Schistosomiasis japanicum (S. japanicum) is related to enhanced proliferation and migration of cancer cells, and the molecular mechanism pertains to cancer cell metastasis in human host.
Methods
The gene of S. japanicum glutathione transferase (sjGST) cloned from S. japanicum was expressed, purified and applied in a series of assays to explore the effect of sjGST on proliferation and migration of MDA-MB-435S, and the expression of MMP2 and MMP9. Immunofluorescence assay for the binding of sjGST to MDA-MB-435S was also carried out.
Results
Results showed that sjGST enhanced proliferation and migration in human breast cancer cell MDA-MB-435S signifycantly at 50-200 nM, but did not enhance them in human lung cancer cell A549. Immunofluorescence assay for the binding of sjGST to MDA-MB-435S and A549 showed that GST was readily bound to the breast cancer cells, but showed almost no binding to human lung cancer cells. The assays for gelatinase activity showed that both MMP2 and MMP9 activities were increased significantly in the presence of sjGST (50-200 nM) in MDA-MB-435S, but they were not significant in A549.
Conclusions
Our current results show strongly that S. japanicum GST binds to MDA-MB-435S probably via its receptor, and enhances proliferation and migration of the cancer cells by up-regulatory expression of MMP2 and MMP9.
doi:10.1016/S2221-1691(11)60063-4
PMCID: PMC3609145  PMID: 23569720
Infection; Schistosomiasis japanicum; Glutathione s-transferase; Proliferation; Gelatinase; MMP2 and MMP9; Migration; Breast cancer
7.  Adenoviral ER-targeted mda-7/IL-24 vector enhances human cancer cell killing 
Molecular cancer therapeutics  2008;7(8):2528-2535.
We developed several adenoviral vectors designed to target MDA-7 expression to different subcellular compartments (ie, ER, mitochondria, nucleus, and cytosol) and evaluated their ability to enhance apoptosis. Adenoviral ER-targeted mda-7/IL-24 vector (Ad-ER-mda7) selectively and effectively inhibited the growth and proliferation of lung (A549 and H1299) and esophageal (Seg1 and Bic1) cancer cells by enhancing cell killing. Both Ad-mda7 and Ad-ER-mda7 activated a novel pathway of ER stress-induced apoptosis characterized by unregulated expression of phosphorylated JNK (p-JNK), phosphorylated cJun (p-cJun), and phosphorylated RNA-dependent protein kinase (p-PKR). Caspase-4 activation mediated Ad-mda7- and Ad-ER-mda7-induced cell death. In addition, Ad-mda7- and Ad-ER-mda7-mediated growth inhibition correlated with activation of ER molecular markers PKR and JNK both in vitro (in Ad-mda7- or Ad-ER-mda7-treated lung cancer cells) and in vivo. These findings suggest that vectors targeting the endoplasmic reticulum (Ad-ER-mda7) may be more effective in cancer gene therapy possibly through more effective induction or ER stress pathways.
doi:10.1158/1535-7163.MCT-08-0083
PMCID: PMC2597048  PMID: 18723497
Apoptosis; MDA-7; adenovirus; gene therapy
8.  MDA-7 results in downregulation of AKT concomitant with apoptosis and cell cycle arrest in breast cancer cells 
Cancer gene therapy  2011;18(7):10.1038/cgt.2011.20.
The melanoma differentiation-associated gene-7 (mda-7) is a known mediator of apoptosis in cancer cells but not in normal cells. We hypothesized that MDA-7 interferes with the prosurvival signaling pathways that are commonly altered in cancer cells to induce growth arrest and apoptosis. We also identified the cell signaling pathways that are antagonized by MDA-7 leading to apoptosis. Using an adenoviral expression system, mda-7 was introduced into the breast cancer cell lines SKBr3, MCF-7 and MDA-MB-468, each with a different estrogen receptor (ER) and HER-2 receptor status. Downstream targets of MDA-7 were assessed by reverse phase protein array analysis, western blot analysis and immunofluorescence confocal microscopy. Our results show that MDA-7-induced apoptosis was mediated by caspases in all cell lines tested. However, MDA-7 modulates additional pathways in SKBr3 (HER-2 positive) and MCF-7 (ER positive) cells including downregulation of AKT-GSK3β and upregulation of cyclin-dependent kinase inhibitors in the nucleus. This leads to cell cycle arrest in addition to apoptosis. In conclusion, MDA-7 abrogates tumor-promoting pathways including the activation of caspase-dependent signaling pathways ultimately leading to apoptosis. In addition, depending on the phenotype of the breast cancer cell, MDA-7 modulates cell cycle regulating pathways to mediate cell cycle arrest.
doi:10.1038/cgt.2011.20
PMCID: PMC3875403  PMID: 21546925
MDA-7; IL-24; breast cancer and AKT
9.  Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells 
Cancer Biology & Therapy  2013;14(11):1039-1049.
In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca2+. Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda-7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.
doi:10.4161/cbt.26110
PMCID: PMC3925659  PMID: 24025359
MDA-7/IL-24; HDACI; ceramide; apoptosis; bystander; cytokine; ROS; caspase; animal study
10.  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
11.  Glutathione S-transferases in human liver cancer. 
Gut  1991;32(12):1546-1549.
An immunohistochemical study of glutathione S-transferase (GST) expression in hepatocellular carcinoma and cholangiocarcinoma is described. Unlike most animal models of hepatic malignancy pi class GST was not consistently overexpressed in hepatocellular carcinoma. This tumour type either predominantly expressed alpha class GST or failed to express GST. By contrast, cholangiocarcinoma always expressed pi class GST, presumably reflecting the tissue of origin, since in human biliary epithelium pi class GST is the predominant GST. The variable expression of pi class GST which was observed in hepatocellular carcinoma may reflect transformation of hepatocytes damaged by toxins, since this GST can be induced after a chemical insult such as alcohol. As well as indicating the biochemical heterogeneity of hepatocellular carcinoma with respect to GST, this study indicates the need for further study of the nature of inherent drug resistance in these tumour types.
Images
PMCID: PMC1379260  PMID: 1663474
12.  Mechanism of autophagy to apoptosis switch triggered in prostate cancer cells by antitumor cytokine mda-7/IL-24 
Cancer research  2010;70(9):3667-3676.
mda-7/IL-24 is a unique member of the IL-10 gene family, which displays a broad range of antitumor properties including induction of cancer-specific apoptosis. Adenoviral mediated delivery by Ad.mda-7 invokes an endoplasmic reticulum stress response that is associated with ceramide production and autophagy in some cancer cells. Here we report that Ad.mda-7-induced ER stress and ceramide production triggers autophagy in human prostate cancer cells, but not normal prostate epithelial cells, through a canonical signaling pathway that involves Beclin-1, atg5 and hVps34. Autophagy occurs in cancer cells at early times after Ad.mda-7 infection but a switch to apoptosis occurs by 48 hr post-infection. Inhibiting autophagy with 3-methyladenosine increases Ad.mda-7-induced apoptosis, suggesting that autophagy may be initiated first as a cytoprotective mechanism. Inhibiting apoptosis by overexpression of anti-apoptotic proteins Bcl-2 or Bcl-xL increased autophagy after Ad.mda-7 infection. During the apoptotic phase, the MDA-7/IL-24 protein physically interacted with Beclin-1 in a manner that could inhibit Beclin-1 function culminating in apoptosis. Conversely, Ad.mda-7 infection elicited calpain-mediated cleavage of the autophagic protein ATG5 in a manner that could facilitate switch to apoptosis. Our findings reveal novel aspects of the interplay between autophagy and apoptosis in prostate cancer cells that underlie the cytotoxic action of mda-7/IL-24, possibly providing new insights in the development of combinatorial therapies for prostate cancer.
doi:10.1158/0008-5472.CAN-09-3647
PMCID: PMC2874885  PMID: 20406981
mda-7/IL-24; protective autophagy; apoptosis; Beclin-1; atg5
13.  Determination of oxidative stress markers and their importance in early diagnosis of uremia-related complications 
Indian Journal of Nephrology  2009;19(1):8-12.
The existence of oxidative stress and the higher incidence of cardiovascular diseases in association with uremia is well proved. The uremic status of serum copper, ceruloplasmin (CP), protein thiols, malonyldialdehyde (MDA), and glutathione S-transferase (GST) levels was studied. The study was carried out on 51 chronic renal failure (CRF) patients who were not on hemodialysis therapy and on 42 healthy controls. Serum urea, creatinine, and MDA levels were found to be significantly increased (P < 0.001), and total protein, albumin, protein thiols, and copper levels were found to be significantly decreased in CRF patients compared to normal controls (P < 0.001). Ceruloplasmin levels were decreased significantly (P < 0.05), and there was no significant change in serum GST levels in CRF patients compared to normal controls. In conclusion, the significant increase in levels of MDA, and the decrease in levels of protein thiols, CP, and copper in uremia patients when compared to controls, reconfirms the presence of stress in this patient population. In view of the changes in other markers of oxidative stress, this absence of any significant change in the activity of GST in uremia patients compared to controls, warrants further study.
doi:10.4103/0971-4065.50673
PMCID: PMC2845196  PMID: 20352004
Cardiovascular diseases; ceruloplasmin; copper; glutathione S-transferase; malonyldialdehyde; uremia
14.  A novel method for screening the glutathione transferase inhibitors 
BMC Biochemistry  2009;10:6.
Background
Glutathione transferases (GSTs) belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST) inhibitors, a novel method was designed.
Results
Our results showed that two fragments of GST, named F1 peptide (GYWKIKGLV) and F2 peptide (KWRNKKFELGLEFPNL), can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB) and glutathione as substrates) was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction.
Conclusion
It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes.
doi:10.1186/1471-2091-10-6
PMCID: PMC2662877  PMID: 19291299
15.  Hepatoprotective and Antioxidant Effect of Bauhinia hookeri Extract against Carbon Tetrachloride-Induced Hepatotoxicity in Mice and Characterization of Its Bioactive Compounds by HPLC-PDA-ESI-MS/MS 
BioMed Research International  2014;2014:245171.
The hepatoprotective and antioxidant activity of Bauhinia hookeri ethanol extract (BHE) against CCl4-induced liver injury was investigated in mice. BHE was administered (500 and 1000 mg/kg/day) along with CCl4 for 6 weeks. The hepatic marker enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were determined in the serum. The antioxidant parameters: glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST), and malondialdehyde (MDA) were estimated in the liver homogenate. BHE treatment significantly inhibited the CCl4-induced increase in ALT (44 and 64%), AST (36 and 46%), ALP (28 and 42%), and MDA (39 and 51%) levels at the tested doses, respectively. Moreover, BHE treatment markedly increased the activity of antioxidant parameters GSH, GPx, GR, GST, and SOD. Histological observations confirmed the strong hepatoprotective activity. These results suggest that a dietary supplement of BHE could exert a beneficial effect against oxidative stress and various liver diseases by enhancing the antioxidant defense status, reducing lipid peroxidation, and protecting against the pathological changes of the liver. The hepatoprotective activity of BHE is mediated, at least in part, by the antioxidant effect of its constituents. The active constituents of BHE were identified by HPLC-PDA-ESI/MS/MS.
doi:10.1155/2014/245171
PMCID: PMC4053259  PMID: 24955350
16.  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
17.  Glutathione-S-transferases in lung and sputum specimens, effects of smoking and COPD severity 
Respiratory Research  2008;9(1):80.
Background
Oxidative stress plays a potential role in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). Glutathione S-transferases (GSTs) detoxify toxic compounds in tobacco smoke via glutathione-dependent mechanisms. Little is known about the regulation and expression of GSTs in COPD lung and their presence in airway secretions.
Methods
GST alpha, pi and mu were investigated by immunohistochemistry in 72 lung tissue specimens and by Western analysis in total lung homogenates and induced sputum supernatants from non-smokers, smokers and patients with variable stages of COPD severity.
Results
GST alpha was expressed mainly in the airway epithelium. The percentage of GST alpha positive epithelial cells was lower in the central airways of patients with very severe (Stage IV) COPD compared to mild/moderate COPD (p = 0.02). GST alpha by Western analysis was higher in the total lung homogenates in mild/moderate COPD compared to cases of very severe disease (p < 0.001). GST pi was present in airway and alveolar epithelium as well as in alveolar macrophages. GST mu was expressed mainly in the epithelium. Both GST alpha and pi were detectable in sputum supernatants especially in patients with COPD.
Conclusion
This study indicates the presence of GST alpha and pi especially in the epithelium and sputum supernatants in mild/moderate COPD and low expression of GST alpha in the epithelium in cases of very severe COPD. The presence of GSTs in the airway secretions points to their potential protective role both as intracellular and extracellular mediators in human lung.
doi:10.1186/1465-9921-9-80
PMCID: PMC2654438  PMID: 19077292
18.  Apoptotic Sphingolipid Signaling by Ceramides in Lung Endothelial Cells 
The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate–chain length (C8:0) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-α–generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C8:0 ceramide– and TNF-α (plus cycloheximide)–induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C16:0 ceramide in response to C8:0 and TNF-α treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-α–stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.
doi:10.1165/rcmb.2007-0274OC
PMCID: PMC2396244  PMID: 18192502
apoptosis; lung; cytokines; signaling; sphingolipids
19.  Honey Supplementation in Spontaneously Hypertensive Rats Elicits Antihypertensive Effect via Amelioration of Renal Oxidative Stress 
Oxidative stress is implicated in the pathogenesis and/or maintenance of elevated blood pressure in hypertension. This study investigated the effect of honey on elevated systolic blood pressure (SBP) in spontaneously hypertensive rats (SHR). It also evaluated the effect of honey on the amelioration of oxidative stress in the kidney of SHR as a possible mechanism of its antihypertensive effect. SHR and Wistar Kyoto (WKY) rats were randomly divided into 2 groups and administered distilled water or honey by oral gavage once daily for 12 weeks. The control SHR had significantly higher SBP and renal malondialdehyde (MDA) levels than did control WKY. The mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione S-transferase (GST) were significantly downregulated while total antioxidant status (TAS) and activities of GST and catalase (CAT) were higher in the kidney of control SHR. Honey supplementation significantly reduced SBP and MDA levels in SHR. Honey significantly reduced the activities of GST and CAT while it moderately but insignificantly upregulated the Nrf2 mRNA expression level in the kidney of SHR. These results indicate that Nrf2 expression is impaired in the kidney of SHR. Honey supplementation considerably reduces elevated SBP via amelioration of oxidative stress in the kidney of SHR.
doi:10.1155/2012/374037
PMCID: PMC3270456  PMID: 22315654
20.  The metabolic bioactivation of caffeic acid phenethyl ester (CAPE) mediated by tyrosinase selectively inhibits glutathione S-transferase 
Chemico-biological interactions  2011;192(3):243-256.
Glutathione S-transferase (GST) and multidrug resistance-associated proteins (MRPs) play major roles in drug resistance in melanoma. In this study, we investigated caffeic acid phenethyl ester (CAPE) as a selective GST inhibitor in the presence of tyrosinase, which is abundant in melanoma cells. Tyrosinase bioactivates CAPE to an o-quinone, which reacts with glutathione to form CAPE-SG conjugate. Our findings indicate that 90% CAPE was metabolized by tyrosinase after a 60-min incubation. LC–MS/MS analyses identified a CAPE-SG conjugate as a major metabolite. In the presence of tyrosinase, CAPE (10–25 µM) showed 70–84% GST inhibition; whereas in the absence of tyrosinase, CAPE did not inhibit GST. CAPE-SG conjugate and CAPE-quinone (25 µM) demonstrated ≥85% GST inhibition via reversible and irreversible mechanisms, respectively. Comparing with CDNB and GSH, the non-substrate CAPE acted as a weak, reversible GST inhibitor at concentrations >50 µM. Furthermore, MK-571, a selective MRP inhibitor, and probenecid, a non-selective MRP inhibitor, decrease the IC50 of CAPE (15 µM) by 13% and 21%, apoptotic cell death by 3% and 13%, and mitochondrial membrane potential in human SK-MEL-28 melanoma cells by 10% and 56%, respectively. Moreover, computational docking analyses suggest that CAPE binds to the GST catalytic active site. Caffeic acid, a hydrolyzed product of CAPE, showed a similar GST inhibition in the presence of tyrosinase. Although, as controls, 4-hydroxyanisole and l-tyrosine were metabolized by tyrosinase to form quinones and glutathione conjugates, they exhibited no GST inhibition in the absence and presence of tyrosinase. In conclusion, both CAPE and caffeic acid selectively inhibited GST in the presence of tyrosinase. Our results suggest that intracellularly formed quinones and glutathione conjugates of caffeic acid and CAPE may play major roles in the selective inhibition of GST in SK-MEL-28 melanoma cells. Moreover, the inhibition of MRP enhances CAPE-induced toxicity in the SK-MEL-28 melanoma cells.
doi:10.1016/j.cbi.2011.03.015
PMCID: PMC3706206  PMID: 21458432
Melanoma; Quinone; Glutathione; GST; Caffeic acid phenethyl ester; MRP
21.  Autophagy switches to apoptosis in prostate cancer cells infected with melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) 
Autophagy  2011;7(9):1076-1077.
MDA-7/IL-24 has noteworthy potential as an anticancer therapeutic because of its diversity of antitumor properties, its lack of toxicity toward normal cells and tissues, and its safety and efficacy as evidenced in a phase I clinical trial. In a recent study, we document that Ad.mda-7-induced ER stress and ceramide production leads to early autophagy that subsequently switches to apoptosis in human prostate cancer cells. During the apoptotic phase, the MDA-7/IL-24 protein physically interacts with Beclin 1 and this interaction might inhibit Beclin 1 function culminating in apoptosis. Conversely, Ad.mda-7 infection leads to calpain-mediated cleavage of the Atg5 protein that might also facilitate a biochemical switch from autophagy to apoptosis. Our recent paper reveals novel aspects of the interplay between autophagy and apoptosis that underlie the cytotoxic action of MDA-7/IL-24 in prostate cancer cells. These new insights into MDA-7/IL-24 action provide intriguing leads for developing innovative combinatorial approaches for prostate cancer therapy.
doi:10.4161/auto.7.9.16163
PMCID: PMC3210317  PMID: 21610321
mda-7/IL-24; protective autophagy; apoptosis; Beclin 1; Atg5
22.  Regulation of the FOXO3a/Bim signaling pathway by 5,7-dihydroxy-8-nitrochrysin in MDA-MB-453 breast cancer cells 
Oncology Letters  2012;5(3):929-934.
We previously demonstrated that 5,7-dihydroxy-8-nitrochrysin (NOC), a novel synthetic chrysin analog, preferentially inhibits HER-2/neu-overexpressing MDA-MB-453 breast cancer cell growth by inducing apoptosis; however, the precise molecular mechanism was unclear. In this study, we demonstrated that NOC significantly induces apoptosis of MDA-MB-453 cells and that this is primarily mediated through a mitochondrial death cascade. This was presented as a loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9. NOC induces a significant increase in levels of the BH3-only protein Bim. Small interfering RNA-mediated knockdown of Bim markedly attenuated NOC-induced apoptosis. An upstream transcriptional regulator of Bim, forkhead box O3a transcription factor (FOXO3a), experienced a decrease in phosphorylation and nuclear translocation. Silencing of FOXO3a resulted in a marked attenuation in the expression of Bim, as well as protection against NOC-mediated apoptosis. Furthermore, NOC-induced activation and nuclear localization of FOXO3a was associated with reduced levels of Akt phosphorylation. These results suggest that NOC induces apoptosis in MDA-MB-453 human breast cancer cells via caspase activation and modulation of the Akt/FOXO3a pathway.
doi:10.3892/ol.2012.1077
PMCID: PMC3576196  PMID: 23425937
breast cancer; chrysin; 5,7-dihydroxy-8-nitrochrysin; Akt; forkhead box O3a; Bim
23.  Neuroprotective activity of Wedelia calendulacea on cerebral ischemia/reperfusion induced oxidative stress in rats 
Indian Journal of Pharmacology  2011;43(6):676-682.
Objective:
This study was undertaken to evaluate the neuroprotective activity of Wedelia calendulacea against cerebral ischemia/reperfusion induced oxidative stress in the rats.
Materials and Methods:
The global cerebral ischemia was induced in male albino Wistar rats by occluding the bilateral carotid arteries for 30 min followed by 1 h and 4 h reperfusion. At various times of reperfusion, the histopathological changes and the levels of malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione–s–transferase (GST), and hydrogen peroxide (H2O2) activity and brain water content were measured.
Results:
The ischemic changes were preceded by increase in concentration of MDA, hydrogen peroxide and followed by decreased GPx, GR, and GST activity. Treatment with W. calendulacea significantly attenuated ischemia–induced oxidative stress. W. calendulacea administration markedly reversed and restored to near normal level in the groups pre-treated with methanolic extract (250 and 500 mg/kg, given orally in single and double dose/day for 10 days) in dose-dependent way. Similarly, W. calendulacea reversed the brain water content in the ischemia reperfusion animals. The neurodegenaration also conformed by the histopathological changes in the cerebral-ischemic animals.
Conclusion:
The findings from the present investigation reveal that W. calendulacea protects neurons from global cerebral–ischemic injury in rat by attenuating oxidative stress.
doi:10.4103/0253-7613.89825
PMCID: PMC3229784  PMID: 22144773
Brain edema; global cerebral ischemia; histopathology; oxidative stress; Wedelia calendulacea
24.  Oxidative Stress Kills Human Primary Oligodendrocytes Via Neutral Sphingomyelinase: Implications for Multiple Sclerosis 
Multiple sclerosis (MS) is the most common human demyelinating disease of the central nervous system where oxidative stress has been proposed to play an important role in oligodendroglial death. However, molecular mechanisms that couple oxidative stress to the loss of oligodendrocytes are poorly understood. This study underlines the importance of neutral sphingomyelinase–ceramide pathway in mediating oxidative stress-induced apoptosis and cell death of human primary oligodendrocytes. Various oxidative stress-inducing agents, such as, superoxide radical produced by hypoxanthine and xanthine oxidase, hydrogen peroxide, aminotriazole capable of inhibiting catalase and increasing intracellular level of H2O2, or reduced glutathione-depleting diamide induced the activation of neutral sphingomyelinase and the production of ceramide. It is interesting to note that antisense knockdown of neutral but not acidic sphingomyelinase ablated oxidative stress-induced apoptosis and cell death in human primary oligodendrocytes. This study identifies neutral but not acidic sphingomyelinase as a target for possible therapeutic intervention in MS.
doi:10.1007/s11481-007-9066-2
PMCID: PMC2131733  PMID: 18040843
oligodendrocytes; cell death; oxidative stress; ceramide; neutral sphingomyelinase; antisense knockdown
25.  JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas 
Neurosurgery  2012;70(2):497-510.
Background
Glutathione S-transferases (GSTs) control multidrug-resistance and are upregulated in many cancers including malignant gliomas. The diazeniumdiolate JS-K generates nitric oxide (NO) on enzymatic activation by glutathione and GST, showing promising NO-based anticancer efficacy.
Objective
To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo.
Methods
U87 glioma cells and primary glioblastoma cell lines were exposed to JS-K and a variety of inhibitors to study cell death by necrosis, apoptosis and other mechanisms. GST-expression was evaluated by immunocytochemistry, PCR and Western blot and NO release from JS-K using a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo.
Results
Dose-dependent inhibition of cell proliferation was observed in human U87 glioma cells and primary glioblastoma cells in vitro. Cell death was partially induced by caspase-dependent apoptosis which could be blocked by Z-VAD-FMK and Q-VD-OPH. GST-inhibition by sulfasalazine, cGMP inhibition by ODQ and MEK 1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signalling pathways. Response to JS-K correlated with mRNA and protein expression of GST and the amount of NO released by the glioma cells. Growth of U87 xenografts was significantly reduced, with immunohistochemical evidence for increased necrosis, apoptosis and reduced proliferation.
Conclusion
Our data for the first time show the potent antiproliferative effect of JS-K in gliomas in vitro and in vivo. These findings warrant further investigation of this novel NO-releasing prodrug in gliomas.
doi:10.1227/NEU.0b013e31823209cf
PMCID: PMC3253212  PMID: 21849924
glioma; GST; JS-K; nitric oxide (NO); U87

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