PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (67)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
Document Types
1.  Development of Selective Inhibitors for Aldehyde Dehydrogenases based on Substituted Indole-2,3-diones 
Journal of medicinal chemistry  2014;57(3):714-722.
Aldehyde dehydrogenases (ALDH) participate in multiple metabolic pathways and have been indicated to play a role in several cancerous disease states. Our laboratory is interested in developing novel and selective ALDH inhibitors. We looked to further work recently published by developing a class of isoenzyme selective inhibitors using a similar indole-2,3-diones that exhibit differential inhibition of ALDH1A1, ALDH2 and ALDH3A1. Kinetic and X-ray crystallography data suggest these inhibitors are competitive against aldehyde binding, forming direct interactions with active site cysteine residues. The selectivity is precise in that these compounds appear to interact directly with the catalytic nucleophile, Cys243, in ALDH3A1, but not in ALDH2. In ALDH2, the 3-keto group is surrounded by the adjacent Cys301/303. Surprisingly, the orientation of the interaction changes depending on the nature of the substitutions on the basic indole ring structure and correlates well with the observed structure-activity relationships for each ALDH isoenzyme.
doi:10.1021/jm401377v
PMCID: PMC3954746  PMID: 24444054
2.  Development of Selective Inhibitors for Aldehyde Dehydrogenases Based on Substituted Indole-2,3-diones 
Journal of Medicinal Chemistry  2014;57(3):714-722.
Aldehyde dehydrogenases (ALDH) participate in multiple metabolic pathways and have been indicated to play a role in several cancerous disease states. Our laboratory is interested in developing novel and selective ALDH inhibitors. We looked to further work recently published by developing a class of isoenzyme-selective inhibitors using similar indole-2,3-diones that exhibit differential inhibition of ALDH1A1, ALDH2, and ALDH3A1. Kinetic and X-ray crystallography data suggest that these inhibitors are competitive against aldehyde binding, forming direct interactions with active-site cysteine residues. The selectivity is precise in that these compounds appear to interact directly with the catalytic nucleophile, Cys243, in ALDH3A1 but not in ALDH2. In ALDH2, the 3-keto group is surrounded by the adjacent Cys301/303. Surprisingly, the orientation of the interaction changes depending on the nature of the substitutions on the basic indole ring structure and correlates well with the observed structure–activity relationships for each ALDH isoenzyme.
doi:10.1021/jm401377v
PMCID: PMC3954746  PMID: 24444054
3.  Translational Medicine: Proceed at Your Own Risk 
Nature reviews. Drug discovery  2013;12(5):327-328.
Although investigators are encouraged to translate their laboratory research to impact the care of patients, there is an unappreciated downside to participating in “T1 translation” from the standpoint of the investigator if their translational efforts do not yield positive results in pivotal clinical trials.
doi:10.1038/nrd4005
PMCID: PMC4283600  PMID: 23629490
4.  Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain 
Science translational medicine  2014;6(251):251ra118.
Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase 2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R2=0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than wild-type mice. Finally, Alda-1 treatment was also beneficial when given even after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians’ apparent lower pain tolerance.
doi:10.1126/scitranslmed.3009539
PMCID: PMC4234033  PMID: 25163478
5.  Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system 
Science translational medicine  2014;6(255):255ra130.
Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.
doi:10.1126/scitranslmed.3009027
PMCID: PMC4215699  PMID: 25253673
6.  PKCε ACTIVATION PROMOTES FGF-2 EXOCYTOSIS AND INDUCES ENDOTHELIAL CELL PROLIFERATION AND SPROUTING 
Journal of molecular and cellular cardiology  2013;63:10.1016/j.yjmcc.2013.07.006.
Protein kinase C epsilon (PKCε) activation controls fibroblast growth factor-2 (FGF-2) angiogenic signaling. Here, we examined the effect of activating PKCε on FGF-2 dependent vascular growth and endothelial activation. ψεRACK, a selective PKCε agonist induces pro-angiogenic responses in endothelial cells, including formation of capillary like structures and cell growth. These effects are mediated by FGF-2 export to the cell membrane, as documented by biotinylation and immunofluorescence, and FGF-2/FGFR1 signaling activation, as attested by ERK1/2-STAT-3 phosphorylation and de novo FGF-2 synthesis. Similarly, vascular endothelial growth factor (VEGF) activates PKCε in endothelial cells, and promotes FGF-2 export and FGF-2/FGFR1 signaling activation. ψεRACK fails to elicit responses in FGF-2−/− endothelial cells, and in cells pretreated with methylamine (MeNH2), an exocytosis inhibitor, indicating that both intracellular FGF-2 and its export toward the membrane are required for the ψεRACK activity. In vivo ψεRACK does not induce angiogenesis in the rabbit cornea. However, ψεRACK promotes VEGF angiogenic responses, an effect sustained by endothelial FGF-2 release and synthesis, since anti-FGF-2 antibody strongly attenuates VEGF responses. The results demonstrate that PKCε stimulation promotes angiogenesis and modulates VEGF activity, by inducing FGF-2 release and autocrine signaling.
doi:10.1016/j.yjmcc.2013.07.006
PMCID: PMC3812807  PMID: 23880610
Protein Kinase C ε; Endothelial cells; Fibroblast Growth Factor-2; Vascular Endothelial Growth Factor; Angiogenesis; Exocytosis
7.  Nociceptive-induced Myocardial Remote Conditioning Is Mediated By Neuronal Gamma Protein Kinase C 
Basic research in cardiology  2013;108(5):10.1007/s00395-013-0381-x.
Deciphering the remote conditioning molecular mechanism may provide targets to develop therapeutics that can broaden the clinical application. To further investigate this, we tested whether two protein kinase C isozymes, the ubiquitously expressed epsilon PKC (εPKC) and the neuronal specific gamma PKC (γPKC), mediate nociceptive-induced remote myocardial conditioning.
Male Sprague-Dawley rats were used for both in vivo and ex vivo myocardial ischemia-reperfusion protocols. For the in vivo studies, using a surgical abdominal incision for comparison, applying only to the abdomen either bradykinin or the εPKC activator (ψεRACK) reduced myocardial infarct size (45±1%, 44±2%, respectively, versus incision: 43±2%, and control: 63±2%, P < 0.001). Western blot showed only εPKC, and not γPKC, is highly expressed in the myocardium. However, applying a selective γPKC inhibitor (γV5-3) to the abdominal skin blocked remote protection by any of these strategies.
Using an ex vivo isolated heart model without an intact nervous system, only selective εPKC activation, unlike a selective classical PKC isozyme activator (activating α, β, βII and γ), reduced myocardial injury. Importantly, the classical PKC isozyme activator given to the abdomen in vivo (with an intact nervous system including γPKC) during myocardial ischemia reduced infarct size as effectively as an abdominal incision or ψεRACK (45±1% versus 45±2% and 47±1%, respectively). The classical PKC activator-induced protection was also blocked by spinal cord surgical transection.
These findings identified potential remote conditioning mimetics, with these strategies effective even during myocardial ischemia. A novel mechanism of nociceptive-induced remote conditioning, involving γPKC, was also identified.
doi:10.1007/s00395-013-0381-x
PMCID: PMC3856950  PMID: 23982492
infarct size; remote; incision; protein kinase C; gamma; epsilon
8.  A Novel Aldehyde Dehydrogenase-3 Activator (Alda-89) Protects Submandibular Gland Function from Irradiation without Accelerating Tumor Growth 
Purpose
To determine the effect of Alda-89 (an ALDH3 activitor) on (1) the function of irradiated (RT) submandibular gland (SMG) in mice, (2) its toxicity profile and (3) its effect on the growth of head and neck cancer (HNC) in vitro and in vivo.
Experimental Design
Adult mice were infused with Alda-89 or vehicle before, during and after RT. Saliva secretion was monitored weekly. Hematology, metabolic profile and post-mortem evaluation for toxicity were examined at the time of sacrifice. Alda-89 or vehicle was applied to HNC cell lines in vitro, and SCID mice transplanted with HNC in vivo with or without radiation; HNC growth was monitored. The ALDH3A1 and ALDH3A2 protein expression was evaluated in 89 HNC patients and correlated to freedom from relapse (FFR) and overall survival (OS).
Results
Alda-89 infusion significantly resulted in more whole saliva production and a higher percentage of preserved acini after RT compared to vehicle control. There was no difference in the complete blood count, metabolic profile, and major organ morphology between the Alda-89 and vehicle groups. Compared to vehicle control, Alda-89 treatment did not accelerate HNC cell proliferation in vitro, nor did it affect tumor growth in vivo with or without RT. Higher expression of ALDH3A1 or ALDH3A2 was not significantly associated with worse FFR or OS in either HPV-positive or HPV-negative group.
Conclusion
Alda-89 preserves salivary function after RT without affecting HNC growth or causing measurable toxicity in mice. It is a promising candidate to mitigate RT-related xerostomia.
doi:10.1158/1078-0432.CCR-13-0127
PMCID: PMC3745542  PMID: 23812668
ALDH3A1; ALDH3A2; Alda-89; xerostomia; radiation; head and neck cancer
9.  In vivo measurement of ALDH2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1-13C]pyruvate 
NMR in biomedicine  2012;26(6):607-612.
To date, measurements of the activity of aldehyde dehydrogenase-2 (ALDH2), a critical mitochondrial enzyme for eliminating certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in magnetic resonance spectroscopy (MRS) of hyperpolarized 13C-labeled substrates now provide a method to detect and image in vivo metabolic pathways with signal-to-noise ratio gains greater than 10,000 fold over conventional MRS techniques. However aldehydes, due to their toxicity and short T1 relaxation times, are generally poor targets for such 13C-labeled studies. In this work, we show that dynamic magnetic resonance spectroscopic imaging of hyperpolarized [1-13C]pyruvate and its conversion to [1-13C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH, a co-factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in 13C-lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R2=0.76).
doi:10.1002/nbm.2897
PMCID: PMC3634870  PMID: 23225495
hyperpolarized 13C; ALDH2 activity; liver; ethanol; pyruvate; lactate; NADH
10.  The challenge in translating basic research discoveries to treatment of Huntington disease 
Rare Diseases  2014;2:e28637.
Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion of a polyglutamine repeats close to the N-terminal of the huntingtin protein. Although unequivocal genetic tests have been available for about 20 years, current pharmacological treatments do not prevent or slow down disease progression. Recent basic research identified potential novel drug targets for the treatment of Huntington disease. However, there are clear challenges in translating these discoveries into treatment strategies for these patients. The following is a brief discussion of these challenges using our recent experience as an example.
doi:10.4161/rdis.28637
PMCID: PMC4091548  PMID: 25054095
Huntington disease; neurodegeneration; mitochondria; polyglutamine; animal model; protein-protein interactions; P110 peptide inhibitor; Drp1
11.  Peripheral Sensitization Increases Opioid Receptor Expression and Activation by Crotalphine in Rats 
PLoS ONE  2014;9(3):e90576.
Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids.
doi:10.1371/journal.pone.0090576
PMCID: PMC3942445  PMID: 24594607
12.  Selective activation of PKC epsilon in mitochondria is neuroprotective in vitro and reduces focal ischemic brain injury in mice 
Journal of neuroscience research  2013;91(6):799-807.
Activation of PKCε confers protection against neuronal ischemia/reperfusion. Since activation of PKCε leads to its translocation to multiple intracellular sites, a mitochondrial-selective PKCε activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKCε. PKCε can regulate key cytoprotective mitochondrial functions including electron transport chain activity, reactive oxygen species (ROS) generation, mitochondrial permeability transition, and detoxification of reactive aldehydes. We tested the ability of mitochondrial selective activation of PKCε to protect primary brain cell cultures or mice subjected to ischemic stroke. Pre-treatment with either general PKCε activator peptide, ψεRACK, or mitochondrial-selective PKCε activator, ψεHSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both ψεRACK and ψεHSP90 were blocked by the PKCε antagonist, εV1–2, indicating protection requires PKCε interaction with its anchoring protein, εRACK. Further supporting a mitochondrial mechanism for PKCε, neuroprotection by ψεHSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, ψεHSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hours of reperfusion. Thus selective activation of mitochondrial PKCε preserves mitochondrial function in vitro and improves outcome in vivo, suggesting potential therapeutic value clinically when brain ischemia is anticipated, including neurosurgery and cardiac surgery.
doi:10.1002/jnr.23186
PMCID: PMC3905808  PMID: 23426889
mitochondria; astrocytes; acute stroke; cell culture; animal models
13.  Inhibition of mitochondrial fragmentation diminishes Huntington’s disease–associated neurodegeneration 
The Journal of Clinical Investigation  2013;123(12):5371-5388.
Huntington’s disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell–derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell–derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT–like inhibitor may prevent or slow the progression of HD.
doi:10.1172/JCI70911
PMCID: PMC3859413  PMID: 24231356
14.  Acute Inhibition of Excessive Mitochondrial Fission After Myocardial Infarction Prevents Long‐term Cardiac Dysfunction 
Background
Ischemia and reperfusion (IR) injury remains a major cause of morbidity and mortality and multiple molecular and cellular pathways have been implicated in this injury. We determined whether acute inhibition of excessive mitochondrial fission at the onset of reperfusion improves mitochondrial dysfunction and cardiac contractility postmyocardial infarction in rats.
Methods and Results
We used a selective inhibitor of the fission machinery, P110, which we have recently designed. P110 treatment inhibited the interaction of fission proteins Fis1/Drp1, decreased mitochondrial fission, and improved bioenergetics in three different rat models of IR, including primary cardiomyocytes, ex vivo heart model, and an in vivo myocardial infarction model. Drp1 transiently bound to the mitochondria following IR injury and P110 treatment blocked this Drp1 mitochondrial association. Compared with control treatment, P110 (1 μmol/L) decreased infarct size by 28±2% and increased adenosine triphosphate levels by 70+1% after IR relative to control IR in the ex vivo model. Intraperitoneal injection of P110 (0.5 mg/kg) at the onset of reperfusion in an in vivo model resulted in improved mitochondrial oxygen consumption by 68% when measured 3 weeks after ischemic injury, improved cardiac fractional shortening by 35%, reduced mitochondrial H2O2 uncoupling state by 70%, and improved overall mitochondrial functions.
Conclusions
Together, we show that excessive mitochondrial fission at reperfusion contributes to long‐term cardiac dysfunction in rats and that acute inhibition of excessive mitochondrial fission at the onset of reperfusion is sufficient to result in long‐term benefits as evidenced by inhibiting cardiac dysfunction 3 weeks after acute myocardial infarction.
doi:10.1161/JAHA.113.000461
PMCID: PMC3835263  PMID: 24103571
cardiac myocytes; Drp1; heart; mitochondria; protein‐protein interaction inhibitor
15.  Sustained inhibition of PKCα reduces intravasation and lung seeding during mammary tumor metastasis in an in vivo mouse model 
Oncogene  2010;30(3):323-333.
Metastasis is the major reason for breast cancer-related deaths. Although there is a host of indirect evidence for a role of PKCα in primary breast cancer growth, its role in the molecular pathways leading to metastasis have not been comprehensively studied. By treating mice with αV5-3, a novel peptide inhibitor selective for PKCα, we were able to determine how PKCα regulates metastasis of mammary cancer cells using a syngeneic and orthotopic model. The primary tumor growth was not affected by αV5-3 treatment. However, the mortality rate was reduced and metastasis in the lung decreased by more than 90% in the αV5-3-treated mice relative to the control-treated mice. αV5-3 treatment reduced intravasation by reducing MMP-9 activities. αV5-3 treatment also reduced lung seeding of tumor cells and decreased cell migration, effects that were accompanied by a reduction in NFκB-activity and cell surface levels of the CXCL12 receptor, CXCR4. αV5-3 treatment caused no apparent toxicity in non-tumor bearing naïve mice. Rather, inhibiting PKCα protected against liver damage and increased the number of immune cells in tumor-bearing mice. Importantly, αV5-3 showed superior efficacy relative to anti-CXCR4 antibody in reducing metastasis, in vivo. Together, these data show that pharmacological inhibition of PKCα effectively reduces mammary cancer metastasis by targeting intravasation and lung seeding steps in the metastatic process and suggest that PKCα-specific inhibitors, such as αV5-3, can be used to study the mechanistic roles of PKCα specifically and may provide a safe and effective treatment for the prevention of lung metastasis of breast cancer patients.
doi:10.1038/onc.2010.415
PMCID: PMC3767436  PMID: 20856202
bioluminescence; mammary cancer; metastasis and protein kinase C
16.  δPKC inhibition or εPKC activation repairs endothelial vascular dysfunction by regulating eNOS post-translational modification 
The balance between endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) and reactive oxygen species (ROS) production determines endothelial-mediated vascular homeostasis. Activation of protein kinase C (PKC) has been linked to imbalance of the eNOS/ROS system, which leads to endothelial dysfunction. We previously found that selective inhibition of delta PKC (δPKC) or selective activation of epsilon PKC (εPKC) reduces oxidative damage in the heart following myocardial infarction. In this study we determined the effect of these PKC isozymes in the survival of coronary endothelial cells (CVEC). We demonstrate here that serum deprivation of CVEC increased eNOS-mediated ROS levels, activated caspase-3, reduced Akt phosphorylation and cell number. Treatment with either the δPKC inhibitor, δV1-1, or the εPKC activator, ψεRACK, inhibited these effects, restoring cell survival through inhibition of eNOS activity. The decrease in eNOS activity coincided with specific de-phosphorylation of eNOS at Ser1179, and eNOS phosphorylation at Thr497 and Ser116. Furthermore, δV1-1 or ψεRACK induced physical association of eNOS with caveolin-1, an additional marker of eNOS inhibition, and restored Akt activation by inhibiting its nitration. Together our data demonstrate that 1) in endothelial dysfunction, ROS and reactive nitrogen species (RNS) formation result from uncontrolled eNOS activity mediated by activation of δPKC or inhibition of εPKC 2) inhibition of δPKC or activation of εePKC correct the perturbed phosphorylation state of eNOS, thus increasing cell survival. Since endothelial health ensures better tissue perfusion and oxygenation, treatment with a δPKC inhibitor and/or an εPKC activator in diseases of endothelial dysfunction should be considered.
doi:10.1016/j.yjmcc.2009.11.002
PMCID: PMC3760592  PMID: 19913548
17.  Protein kinase C, an elusive therapeutic target? 
Nature reviews. Drug discovery  2012;11(12):937-957.
Preface
Protein kinase C (PKC) has been a tantalizing target for drug discovery ever since it was first identified as the receptor for the tumor promoter phorbol ester in 19821. Although initial therapeutic efforts focused on cancer, additional diseases, including diabetic complications, heart failure, myocardial infarction, pain and bipolar disease were targeted as researchers developed a better understanding of the roles that PKC’s eight conventional and novel isozymes play in health and disease. Unfortunately, both academic and pharmaceutical efforts have yet to result in the approval of a single new drug that specifically targets PKC. Why does PKC remain an elusive drug target? This review will provide a short account of some of the efforts, challenges and opportunities in developing PKC modulators to address unmet clinical needs.
doi:10.1038/nrd3871
PMCID: PMC3760692  PMID: 23197040
18.  δPKC mediates microcerebrovascular dysfunction in acute ischemia and in chronic hypertensive stress in vivo 
Brain research  2007;1144:146-155.
Maintaining cerebrovascular function is a priority for reducing damage following acute ischemic events such as stroke, and under chronic stress in diseases such as hypertension. Ischemic episodes lead to endothelial cell damage, deleterious inflammatory responses, and altered neuronal and astrocyte regulation of vascular function. These, in turn, can lead to impaired cerebral blood flow and compromised blood–brain barrier function, promoting microvascular collapse, edema, hemorrhagic transformation, and worsened neurological recovery. Multiple studies demonstrate that protein kinase C (PKC), a widely expressed serine/threonine kinase, is involved in mediating arterial tone and microvascular function. However, there is no clear understanding about the role of individual PKC isozymes. We show that intraperitoneal injection of δV1-1–TAT47–57 (0.2 mg/kg in 1 mL), an isozymespecific peptide inhibitor of δPKC, improved microvascular pathology, increased the number of patent microvessels by 92% compared to control-treated animals, and increased cerebral blood flow by 26% following acute focal ischemia induced by middle cerebral artery occlusion in normotensive rats. In addition, acute delivery of δV1-1–TAT47–57 in hypertensive Dahl rats increased cerebral blood flow by 12%, and sustained delivery δV1-1–TAT47–57 (5 uL/h, 1 mM), reduced infarct size by 25% following an acute stroke induced by MCA occlusion for 90 min. Together, these findings demonstrate that δPKC is an important therapeutic target for protection of microvascular structure and function under both acute and chronic conditions of cerebrovascular stress.
doi:10.1016/j.brainres.2007.01.113
PMCID: PMC3742377  PMID: 17350602
Cerebral blood flow; Hypertension; Microvasculature; Protein kinase C; Stroke; Vasculature
19.  Mitigation of Radiation-Induced Dermatitis by Activation of Aldehyde Dehydrogenase 2 Using Topical Alda-1 in Mice1 
Radiation research  2012;178(1):69-74.
Ning, S., Budas, G. R., Churchill, E. N., Chen, C., Knox, S. J. and Mochly-Rosen, D. Mitigation of Radiation-Induced Dermatitis by Activation of Aldehyde Dehydrogenase 2 Using Topical Alda-1 in Mice.
Radiation-induced dermatitis is a debilitating clinical problem in cancer patients undergoing cancer radiation therapy. It is also a possible outcome of exposure to high levels of radiation due to accident or hostile activity. We report that activation of aldehyde dehydrogenase 2 (ALDH2) enzymatic activity using the allosteric agonist, Alda-1, significantly reduced 4-hydroxynonenal adducts accumulation, delayed the onset of radiation dermatitis and substantially reduced symptoms in a clinically-relevant model of radiation-induced dermatitis. Importantly, Alda-1 did not radioprotect tumors in mice. Rather, it increased the sensitivity of the tumors to radiation therapy. This is the first report of reactive aldehydes playing a role in the intrinsic radiosensitivity of normal and tumor tissues. Our findings suggest that ALDH2 represents a novel target for the treatment of radiation dermatitis without reducing the benefit of radiotherapy.
PMCID: PMC3417825  PMID: 22404739
21.  Protein kinase C epsilon is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes 
Oncogene  2011;31(20):2593-2600.
Protein kinase C (PKC) ε, a member of the novel PKC family, plays key roles in mitogenesis and survival in normal and cancer cells. PKCε is frequently overexpressed in epithelial cancers, particularly in lung cancer. Using a shRNA approach, here we established that PKCε is required for non-small cell lung carcinoma (NSCLC) growth in vitro as well as tumor growth when inoculated into athymic mice. Moreover, sustained delivery of a PKCε selective inhibitor peptide, εV1-2, reduced xenograft growth in mice. Both RNAi depletion and pharmacological inhibition of PKCε caused a marked elevation in the number of apoptotic cells in NSCLC tumors. PKCε-depleted NSCLC cells show elevated expression of pro-apoptotic proteins of the Bcl-2 family, caspase recruitment domain (CARD)-containing proteins, and TNF ligands/receptor superfamily members. Moreover, a Gene Set Enrichment Analysis (GSEA) revealed that a vast majority of the genes changed in PKCε-depleted cells were also deregulated in human NSCLC. Our results strongly suggest that PKCε is required for NSCLC cell survival and maintenance of NSCLC tumor growth. Therefore, PKCε may represent an attractive therapeutic target for NSCLC.
doi:10.1038/onc.2011.428
PMCID: PMC3432976  PMID: 21996750
PKCε; non-small cell lung carcinoma; tumorigenesis; cell survival; apoptotic genes
22.  Ethanol for cardiac ischemia: the role of protein kinase c 
The physiological effects of ethanol are dependent upon the amount and duration of consumption. Chronic excessive consumption can lead to diseases such as liver cirrhosis, and cardiac arrhythmias, while chronic moderate consumption can have therapeutic effects on the cardiovascular system. Recently, it has also been observed that acute administration of ethanol to animals prior to an ischemic event provides significant protection to the heart. This review focuses on the different modalities of chronic vs. acute ethanol consumption and discusses recent evidence for a protective effect of acute ethanol exposure and the possible use of ethanol as a therapeutic agent.
doi:10.1177/1753944708094735
PMCID: PMC3600863  PMID: 19124442
PKC; ethanol; ischemic preconditioning; ischemia; reperfusion; cardiac protection
23.  ALDH2 Activator Inhibits Increased Myocardial Infarction Injury by Nitroglycerin Tolerance 
Science translational medicine  2011;3(107):107ra111.
Nitroglycerin, which helps impaired cardiac function as it is converted to nitric oxide, is used worldwide to treat patients with various ischemic and congestive cardiac diseases, including angina pectoris. Nevertheless, after continuous treatment, the benefits of nitroglycerin are limited by the development of tolerance to the drug. Nitroglycerin tolerance is a result of inactivation of aldehyde dehydrogenase 2 (ALDH2), an enzyme essential for cardioprotection in animals subjected to myocardial infarction (MI). Here we tested the hypothesis that the tolerance that develops as a result of sustained nitroglycerin treatment increases cardiac injury by subsequent MI. In a rat model of MI, 16 hours of prior, sustained nitroglycerin treatment (7.2 mg/kg/day) resulted in infarcts that were twice as large as those in untreated control animals and in diminished cardiac function at 3 days and 2 weeks after the MI. We also sought to identify a potential treatment to protect against this increased cardiac damage. Nitroglycerin inhibited ALDH2 activity in vitro, an effect that was blocked by Alda-1, an activator of ALDH2. Co-administration of Alda-1 (16 mg/kg/day) with the nitroglycerin prevented the nitroglycerin-induced increase in cardiac dysfunction after MI in rats, at least in part by enhancing metabolism of reactive aldehyde adducts that impair normal protein functions. If our animal studies showing that nitroglycerin tolerance increases cardiac injury upon ischemic insult are corroborated in humans, activators of ALDH2 such as Alda-1 may help to protect MI patients from this nitroglycerin-induced increase in cardiac injury, while maintaining the cardiac benefits of the increased nitric oxide concentrations produced by nitroglycerin.
doi:10.1126/scitranslmed.3002067
PMCID: PMC3547591  PMID: 22049071
24.  A Novel Aldehyde Dehydrogenase-3 Activator Leads to Adult Salivary Stem Cell Enrichment In Vivo 
Purpose
To assess aldehyde dehydrogenase (ALDH) expression in adult human and murine submandibular gland (SMG) stem cells and to determine the effect of ALDH3 activation in SMG stem cell enrichment.
Experimental Design
Adult human and murine SMG stem cells were selected by cell surface markers (CD34 for human and c-Kit for mouse) and characterized for various other stem cell surface markers by flow cytometry and ALDH isozymes expression by quantitative reverse transcriptase PCR. Sphere formation and bromodeoxyuridine (BrdUrd) incorporation assays were used on selected cells to confirm their renewal capacity and three-dimensional (3D) collagen matrix culture was applied to observe differentiation. To determine whether ALDH3 activation would increase stem cell yield, adult mice were infused with a novel ALDH3 activator (Alda-89) or with vehicle followed by quantification of c-Kit+/CD90+ SMG stem cells and BrdUrd+ salispheres.
Results
More than 99% of CD34+ huSMG stem cells stained positive for c-Kit, CD90 and 70% colocalized with CD44, Nestin. Similarly, 73.8% c-Kit+ mSMG stem cells colocalized with Sca-1, whereas 80.7% with CD90. Functionally, these cells formed BrdUrd+ salispheres, which differentiated into acinar- and ductal-like structures when cultured in 3D collagen. Both adult human and murine SMG stem cells showed higher expression of ALDH3 than in their non–stem cells and 84% of these cells have measurable ALDH1 activity. Alda-89 infusion in adult mice significantly increased c-Kit+/CD90+ SMG population and BrdUrd+ sphere formation compared with control.
Conclusion
This is the first study to characterize expression of different ALDH isozymes in SMG stem cells. In vivo activation of ALDH3 can increase SMG stem cell yield, thus providing a novel means for SMG stem cell enrichment for future stem cell therapy.
doi:10.1158/1078-0432.CCR-11-0179
PMCID: PMC3544360  PMID: 21998334
25.  PKCδ activation mediates angiogenesis via NADPH oxidase activity in PC-3 prostate cancer cells 
The Prostate  2010;71(9):946-954.
Background
PKCδ is generally known as a pro-apoptotic and anti-proliferative enzyme in human prostate cancer cells.
Methods
Here, we investigated the role of PKCδ on the growth of PC-3 human prostate cancer cells in vivo and in vitro.
Results
We found that sustained treatment with a specific PKCδ activator (ψδ receptor for active C kinase, ψδRACK) increased growth of PC-3 xenografts. There was increased levels of HIF-1α, vascular endothelial growth factor and CD31-positive cells in PC-3 xenografts, representative of increased tumor angiogenesis. Mechanistically, PKCδ activation increased the levels of reactive oxygen species (ROS) by binding to and phosphorylating NADPH oxidase, which induced its activity. Also, PKCδ-induced activation of NADPH oxidase increased the level of HIF-1α.
Conclusions
Our results using tumors from the PC-3 xenograft model suggest that PKCδ activation increases angiogenic activity in androgen-independent PC-3 prostate cancer cells by increasing NADPH oxidase activity and HIF-1α levels and thus may partly be responsible for increased angiogenesis in advanced prostate cancer.
doi:10.1002/pros.21310
PMCID: PMC3544470  PMID: 21541971
angiogenesis; HIF-1α; NADPH oxidase; prostate cancer; protein kinase C

Results 1-25 (67)