We investigated the mechanism of toxicity and resistance development of small molecule tyrosine kinase inhibitor lapatinib in HCT 116 colon cancer cells. Lapatinib mediated cell death in HCT 116 cells was caspase independent and involved cytosolic release of apoptosis inducing factor. Treatment of HCT 116 cells with 10µM Lapatinib lead to the outgrowth of lapatinib resistant HCT 116 cells. Our studies show that alterations in the expression and activation of Bcl-2 family proteins allow lapatinib resistant HCT 116 cells to resist cytotoxic effects of lapatinib as well as of other commonly used chemotherapeutic agents. In hepatoma and pancreatic cancer cells, the effects of combining multi-kinase inhibitor sorafenib with histone deacetylase inhibitors (HDACIs) namely, vorinostat and sodium valproate were investigated. It was found that sorafenib synergizes with HDACIs resulting in enhanced cell death compared to death induced by the drugs individually. The mechanism of action of sorafenib and vorinostat combination treatment as well as sorafenib and sodium valproate combined treatment was shown to involve activation of the CD95 death receptor pathway. Alterations in the CD95 pathway can render cancer cells resistant to chemotherapeutic agents. Hence, we combined sorafenib+sodium valproate with a BH-3 domain mimetic named obatoclax (GX-15-070) which resulted in enhanced toxicity to cancer cells. More importantly, knock-down of CD95 (to mimic non-functional CD95 pathway) reduced cell death induced by sorafenib+sodium valproate combined but failed to protect cells from cell death induced by sorafenib+sodium valproate+obatoclax combined. This suggests that combining sorafenib+HDACI with obatoclax may not only enhance toxicity to cancer cells but may also reduce chances of resistance development via alterations in the CD95 pathway. These studies enhance our knowledge of existing treatment strategies for cancer as well as throw light on how current approaches can be improved in order to better diagnose and treat cancer. Understanding mechanisms of drug action as well as resistance development will allow us to combine existing therapies effectively in order best target cancer cells as well as provide us with information that can help us design new cancer treatment strategies.

ENZYMATIC REGULATION OF OPIOID ANTINOCICEPTION AND TOLERANCE By Lynn C. Hull, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2009 Director: William L. Dewey, Ph.D. Department of Pharmacology and Toxicology The involvement of kinases in opioid actions has long been established. The acute actions of opioids, through the Gi/Go G-proteins, cause the inhibition of adenylyl cyclase and therefore a decrease in protein kinase A (PKA) activation. Additionally, acute opioid administration may cause the G-protein to activate the phospholipase C (PLC)-mediated cascade leading to the activation of protein kinase C (PKC). The phosphorylation of the MOR which can lead to both desensitization by uncoupling of the G-protein coupled receptors (GPCRs) from the G-proteins and to internalization by recruitment of β-arrestins has long been identified as a key process in tolerance. Phosphorylation by PKA and PKC leads primarily to uncoupling of the receptor from the G-proteins. Phosphorylation of the receptor by G-protein coupled receptor kinase (GRK) leads to the recruitment of β-arrestins and internalization of the receptor. Many in vitro studies have come to the conclusion that GRK induced internalization plays a more central role in the tolerance to high efficacy opioids and a lesser role in low- and moderate-efficacy opioid tolerance. In fact it has been hypothesized that morphine, a moderate-efficacy opioid, causes no internalization at all, while the desensitization of the receptor via phosphorylation by PKA and PKC plays a more central role in low- and moderate-efficacy opioid tolerance. We sought to test these in vitro findings in an in vivo model of opioid tolerance. Animals were made tolerant to one of a number of opioids of varying efficacy (low-efficacy meperidine, moderate-efficacy morphine and fentanyl, and high-efficacy [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO)) over an 8 hour period and then were administered one of the kinases’ inhibitors. Tolerance reversal was determined by challenging these mice with the same opioids to which they were tolerant. Calcium is known to play an important role in the acute antinociceptive actions of opioids as well as in opioid tolerance. Therefore it is important to determine how opioids are affecting the regulation of intracellular calcium. Our laboratory has previously shown that Calcium Induced Calcium Release (CICR), the ryanodine receptor and intracellular microsomal Ca2+ pools all play a role in opioids’ actions. It is also well known that mammalian ADP-ribosyl cyclase, CD38’s, product cADPR acts on the ryanodine receptor to cause Ca2+ release into the intracellular space. We chemically and genetically altered CD38 and then tested the acute effect of morphine as well as what effect these treatments had on morphine tolerance to determine what role if any, that CD38 may play in the acute actions of morphine antinociception as well as in morphine tolerance. Together, studies focusing on the role of an ADP-ribosyl cyclase, CD38, and 3 separate kinases, PKA, PKC and GRK, in opioids’ actions were performed in order to better understand the roles of these enzymes’ pathways in the actions of opioid-induced antinociception and subsequent development of tolerance. It is hoped that the results herein add useful knowledge to the general understanding of this drug class, and will one day be of use in the development of future analgesics and in the clinical treatment of pain and reduction in tolerance.

Tobacco dependence is the leading cause of preventable death in the United States. Despite currently available smoking cessation therapies, there is a high rate of relapse in smoking among those attempting to quit. While the somatic signs of nicotine withdrawal (insomnia, increased appetite, weight gain) contribute to the continuation of smoking behavior, it has been hypothesized that the affective signs (depression, anxiety, craving, irritability) are greater motivators of relapse and continued tobacco use. There are few studies that assess the molecular and receptor-mediated mechanisms of nicotine withdrawal; therefore, our studies focus on identifying the nicotinic acetylcholine receptor (nAChR) subtypes and post-receptor calcium-dependent mechanisms involved in nicotine withdrawal behaviors. Using precipitated, spontaneous, and conditioned place aversion (CPA) models, we measured physical and affective signs of nicotine withdrawal in mice. Our data show that major nAChR subtypes have differential roles in nicotine withdrawal. Additionally, our results suggest a behavioral relevance for L-type calcium channels in physical nicotine withdrawal signs, while calcium/calmodulin dependent protein kinase II (CaMKII) appears to be involved in both physical and affective withdrawal behaviors. Additionally, we conducted biochemical studies in the ventral tegmental area (VTA) and nucleus accumbens (NAc) to examine the relationship between altered withdrawal behavioral responses and calcium-dependent molecular mechanisms that contribute to nicotine withdrawal behaviors. Our results suggest an important role for β2-containing nAChRs in nicotine-withdrawal induced decreases in CaMKII and synapsin I function in the NAc. Overall, our studies implicate a critical role for the α4α6β2* nAChR subtype in the behavioral and molecular aspects of nicotine withdrawal, thus aiding in the elucidation of nAChR subunits and mechanisms that contribute to nicotine withdrawal behaviors. The current studies are imperative for generating more successful smoking cessation therapies.

Tyrosine nitration results in altered function of smooth muscle voltage-gated L-type calcium channel. We explored the possibility that smooth muscle contains denitrase activity to allow functional recovery of the calcium channel without requiring synthesis of new channel proteins. Following peroxynitrite treatment of mouse colonic smooth muscle strips, CaCl2 (1 mM)-induced smooth muscle contraction was significantly reduced by 67% (P ≤ 0.05), which reversed by approximately 86% upon periodic washing within 2 hr period (P ≤ 0.001). The effect of the c-Src kinase inhibitor, PP2, on muscle contraction was also restored after 2 hr post-peroxynitrite treatment consistent with the thesis that recovery from tyrosine nitration allows for tyrosine phosphorylation of the calcium channel. In addition, sodium orthovanadate prevented nitration-induced inhibition of muscle contraction by approximately 90%. Moreover, denitration of nitrated proteins was observed by western blots in smooth muscle cells over 2 hr. Since nitrotyrosine formation interferes with tyrosine kinase pathways involved in cell signaling, the presence of denitrase activity in smooth muscle cells may have profound and important effects in restoring the function of nitrated proteins involved in cell signaling processes.

Radiation therapy is a widely used tool in cancer therapy and is frequently offered as the first line of treatment for cancers of the breast. While radiotherapy is often initially effective in killing tumor cells or suppressing their growth, there are factors that confer tumor cell resistance to irradiation. Development of resistance may lead to disease recurrence despite the use of surgery, chemotherapy and radiation therapy. A primary goal of the studies in Dr. Gewirtz’s laboratory is to develop strategies to overcome resistance to radiation (and chemotherapy) in breast cancer, with the ultimate goal of preventing or attenuating disease recurrence. One of these approaches involves combining the active form of vitamin D, 1,25-di hydroxy vitamin D3 or its analogs with radiotherapy. Our proposed studies were designed to build upon and extend previous work from this laboratory focused on determining the nature of cell death when vitamin D3 is combined with ionizing radiation in breast tumor cells. Studies were extended to the wild type p53, estrogen receptor positive, ZR-75-1 breast cancer cell line. We were able to validate that vitamin D3 does in fact, sensitize ZR-75-1 breast cancer cells to radiation therapy and substantiate that autophagy is the mode of sensitization by vitamin D3. Interestingly, our experimental system demonstrated that autophagy can actually have dual roles. Specifically, inhibition of autophagy both enhanced sensitivity to radiation and attenuated radiation sensitization by 1,25D3. Moreover, this experimental model proved to be a useful tool in trying to distinguish the factors involved in cytoprotective and cytotoxic autophagy, as we were able to demonstrate a potential role of 5' adenosine monophosphate-activated protein kinase in the sensitization of breast tumor cells to radiation by vitamin D3 as well as cytotoxic autophagy.

Mounting evidence indicates an involvement of inflammation in the pathogenesis of Alzheimer’s disease. While there are other mechanisms involved, it is this role of inflammatory processes that we wish to investigate. Triptolide is the major constituent in the Chinese herb, Tripterygium wilfordii Hook F, and has been used for centuries as part of Chinese herbal medicine. The four ringed structure has close homology to drugs of the steroid class and it has been shown to be beneficial as an anti-inflammatory for rheumatoid arthritis and for treatment of certain cancers. The aim of this study was to evaluate the potential therapeutic effect of Triptolide on the neuropathology and deficits of spatial 6 learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) doubletransgenic mice, a well established Alzheimer’s disease (AD) mouse model. After treatment of APP/PS1 mice with Triptolide (40μg/kg, three times weekly,), initiated when the mice were 5 months old, for as little as 8 weeks, significant decrease in β-amyloid (Aβ) deposition and microglia activation was observed. Moreover, Triptolide treatment robustly rescued spatial memory deficits observed in APP/PS1 mice. However, APP processing, tau hyperphosphorylation, and the activities of the two major kinases involved in tau hyperphosphorylation, cyclin dependent kinase 5 (cdk5) and glycogen synthase kinase 3β (GSK3β) were not affected by the Triptolide treatment. Based on the recent finding for the inhibitory effect of Triptolide on Aβ-induced production of pro-inflammatory cytokines from microglia, we propose that Triptolide treatment may have beneficial properties in halting glial activation and help restore an immune system that fights plaque deposition. Although the exact mechanism of action has yet to be deduced, the increase in APP CTFs while having a significant decrease in amyloid plaque deposition suggests that alterations in gamma secretase activity may be a possible answer. Currently, these results support the use of Triptolide as an effective therapeutic to prevent the progression of Alzheimer’s disease.

Morphine and Δ9-THC are drugs that produce analgesia and rewarding effects. However, chronic treatment with morphine and Δ9-THC produces problematic side-effects including tolerance and physical dependence. The cellular mechanisms underlying opioid and cannabinoid antinociceptive tolerance have been studied for years. Research has demonstrated that the expression of morphine and Δ9-THC antinociceptive tolerance may be mediated through intracellular signaling pathways, such as the adenylyl cyclase /Protein Kinase A (PKA) cascade. The present study investigated the role of PKA in the expression of morphine and Δ9-THC antinociceptive tolerance. Male Swiss Webster mice were treated chronically with morphine or Δ9-THC and the warm-water tail-flick test was used to assess antinociception. These studies revealed that the level and the duration of morphine antinociceptive tolerance both influenced whether PKA activity was increased in mouse brain and spinal cord. Cytosolic PKA activity was increased in the thalamus of 3-day morphine-tolerant mice expressing a 45-fold level of tolerance, but not in mice that expressed a 10-fold level of tolerance. In addition, cytosolic PKA activity was increased in the lumbar spinal cord (LSC) of 15-day morphine-tolerant mice. However, chronic treatment with A9-THC had no effect on neuronal PKA activity even in mice that expressed a high level of antinociceptive tolerance. The absence of an effect of chronic treatment with A9-THC on neuronal PKA activity was supported by the development of a positive control in which the PKA activator Sp-8-Br-cAMPS was administered intracerebroventricularly (i.c.v.) and intrathecally (i.t.) in drug-naYve mice and increases in PKA activity were observed in several brain regions and LSC. Finally, the i.c.v. injection of two peptide fragments of native Protein Kinase A inhibitor (PKI) peptide, PKI-(6-22)-amide and PKI-(Myr- 14-22)- amide, significantly reversed antinociceptive tolerance in mice treated chronically with morphine. PKI-(6-22)-amide (i.c.v.) also inhibited PKA activity in brain regions (thalamus, periaqueductal gray (PAG), and medulla) and LSC, which studies have shown play a role in morphine-induced analgesia. Moreover, PKI-(6-22)-amide reduced the increase in PKA activity in thalamus and LSC observed with chronic morphine treatment. Overall, these studies provide evidence that PKA plays a role in morphine tolerance, but not Δ9-THC tolerance at the doses and times tested.

ROLE OF MAGL INHIBITION IN NICOTINE WITHDRAWAL AND REWARD. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. by Pretal Ishvarlal Patel Muldoon Director: M. Imad Damaj, PhD Professor, Department of Pharmacology and Toxicology Tobacco use is one of the leading causes of preventable deaths worldwide. Nicotine, the main psychoactive component of tobacco, sustains and initiates tobacco addiction. Cessation of nicotine induces a dependence withdrawal syndrome. Recent in vivo studies indicate that the endocannabinoid (EC) system modulates both nicotine reward and withdrawal. The purpose of this proposal is to investigate the role of enhancing endogenous 2-arachidonoylglycerol (2-AG) and by blocking its degradative enzyme, monoacylglycerol lipase (MAGL) enzyme, in nicotine reward and dependence. The selective MAGL inhibitor JZL184 dose-dependently reduced both precipitated and spontaneous somatic and aversive withdrawal signs in mice. These effects were blocked by rimonabant indicating a CB1 receptor mechanism. Furthermore, repeated administration of JZL184 for 6 days did not produce tolerance to the alleviation of withdrawal and the treatment did not induce alterations in CB1 receptor levels or receptor-mediated G-protein activity in various brain regions. In addition, a decrease in 2-AG levels was found in the nucleus accumbens in nicotine-dependent mice undergoing precipitated withdrawal, suggesting that a dysregulation of this EC signaling system occurs during nicotine withdrawal. Lastly, we tested the effectiveness of a combination of low-dose JZL184 and high dose of the FAAH inhibitor PF-3845 on spontaneous nicotine withdrawal. Indeed, the combination of low-dose JZL184 and PF-3845 significantly attenuated nicotine spontaneous withdrawal signs. MAGL inhibition by JZL184 dose-dependently caused a significant blockade of nicotine reward as measured in the mouse conditioned place preference (CPP). In contrast to withdrawal, JZL184’s effect on nicotine CPP was not CB1 mediated. In addition, JZL184 treatment did not cause significant alterations in CB1 receptor levels or receptor-mediated G-protein activity in several brain regions involved in nicotine reward. The effects of JZL184 on nicotine CPP was selective since the drug failed to alter food-induced CPP and LiCl-induced conditioned place aversion in the mouse. Interestingly, active doses of JZL184 did not only cause an increases in 2-AG levels but also induced a concomitant decrease in arachidonic acid (AA) levels in various brain regions suggesting an AA cascade dependent-mechanism. In line of these changes, a cox-2 inhibitor, valdecoxib, dose-dependently blocked nicotine preference.

Cannabinoids produce antinociception in many preclinical models of acute and chronic pain. In contrast, cannabinoids produce inconsistent analgesia in humans, showing little or no efficacy in treating acute pain, with modest efficacy in treating chronic inflammatory pain. This discrepancy may reflect an overreliance on preclinical assays of pain-stimulated behaviors, defined as behaviors that increase in rate or intensity following delivery of a noxious stimulus. In these assays, antinociception is indicated by a reduction in pain-stimulated behaviors, and antinociception is produced either by a reduction in sensory sensitivity to the noxious stimulus (i.e. true analgesia) or by false positive motor impairment. This dissertation addresses this weakness by complementing cannabinoid effects in conventional assays of pain-stimulated behavior with their effects in novel assays of pain-depressed behavior. Pain-depressed behaviors are defined as behaviors that decrease in rate or intensity following presentation of a noxious stimulus. Motor impairment does not produce false positive antinociception in assays of pain-depressed behavior, because antinociception is indicated by a blockade or reversal of pain-induced behavioral depression. In this dissertation, an intraperitoneal (IP) injection of lactic acid served as an acute noxious stimulus to stimulate stretching (pain-stimulated behavior) or depress intracranial self-stimulation (ICSS) (pain-depressed behavior), whereas, IP injection(s) of lipopolysaccharide (LPS) served as a chronic/acute inflammatory-related noxious stimulus to stimulate mechanical allodynia (pain-stimulated behavior) or depress ICSS (pain-depressed behavior). Cannabinoids tested in the assays of acid-stimulated stretching and acid-depressed ICSS included: mixed CB1R/CB2R agonists THC and CP55940, drugs that modulate levels of the endogenous cannabinoid agonist anandamide (URB597 and PF3845), and a selective CB2R agonist, GW405833. THC was also tested in assays of LPS-stimulated mechanical allodynia and LPS-depressed ICSS. In general, mixed CB1R/CB2R agonists were ineffective or exacerbated pain-depressed behavior regardless of noxious stimulus. Contrastingly, URB597 and GW405833 produced antinociception in the assay of acid-depressed ICSS; however their effects were not mediated by CBRs. All compounds produced antinociception in the assay of pain-stimulated behavior, except for PF3845. These results suggest that assays of pain-depressed behavior may be useful for development of cannabinoid analgesic medications, but that further research is needed to determine mechanisms underlying cannabinoid-mediated antinociception in these assays.

A majority of Americans regularly consume alcohol, but the risk factors leading to excessive drinking and alcohol abuse are unevenly distributed throughout the population. Genetic differences can account for only 40-60% of this variability. While variations in ethanol preference drinking in rodent models have been reported, the neurobiological factors underlying these behaviors are still not completely understood. Thus, these studies were designed to determine behavioral and molecular factors associated with the initiation of ethanol drinking preference in an inbred mouse model. We harnessed the power of inter-individual variation of ethanol drinking within an inbred mouse strain to essentially eliminate genetic variability and focus on environmental factors. Our studies have characterized robust, persistent individual variability in ethanol intake in C57 mice using a two-bottle choice paradigm. Ethanol intake differences were not due to litter effects or differences in taste preference. Social rank nor basal anxiety phenotypes could account for ethanol preference. Based on the shared co-morbidity of anxiety and alcoholism, and that alcoholics report anxiety and stress reduction as major motivational factors for drinking, we used an ethologically-relevant social defeat model to investigate stress-influences on ethanol drinking. We found that social defeat has bidirectional effects on ethanol drinking. Mice with a low predilection for ethanol tend to increase drinking following social stress while high preference mice decrease drinking. Even though social defeat produced a measurable physiological response in mice, defeat stress did not alter anxiety measures in the light-dark box. Thus, the current findings did not fully support the tension-reduction hypothesis of alcoholism. In order to determine the molecular factors underlying these differences in ethanol preference drinking, we employed genome-wide expression profiling to identify gene networks altered in ethanol-preferring and ethanol-avoiding mice. Genes involved in synaptic vesicle release, glutamate and BDNF signaling were differentially altered in drinking mice. Following stress-influenced ethanol drinking, expression profiling identified transcripts involved in dopamine signaling, the extra-hypothalamic stress response and alterations in steroid and glucocorticoid synthesis. Most importantly, these expression studies and behavioral analysis following histone deacetylase inhibition may be the first to implicate epigenetic factors involving chromatin acetylation and/or methylation as contributing to environmental modulation of ethanol intake.