NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.

Inhibition of Deubiquitinase Activity and Ubiquitination of Jak2 Blocks Cytokine Signaling and Induces Tumor Cell Apoptosis

The Jak-stat pathway is critical for cellular proliferation and is commonly found to be deregulated in many solid tumors as well as hematological malignancies. Such findings have spurred the development of novel therapeutic agents that specifically inhibit Jak2 kinase, thereby suppressing tumor cell growth. Tyrphostin AG490, the first described Jak2 inhibitor, displays poor pharmacology and requires high concentrations for anti-tumor activities. Our research group screened a small library of AG490 structural analogues and identified WP1130 as a potent inhibitor of Jak2 signaling. However, unlike AG490, WP1130 did not directly inhibit Jak2 kinase activity. Our results show that WP1130 induces rapid ubiquitination and subsequent re-localization of Jak2 into signaling incompetent aggresomes. In addition to Jak2, WP1130 also induces accumulation of other ubiquitinated proteins without inhibiting 20S proteasome activity. Further analysis of the mechanism of action of WP1130 revealed that WP1130 acts as a partly selective DUB inhibitor. It specifically inhibits the deubiquitinase activity of USP9x, USP5, USP14 and UCH37. WP1130 mediated inhibition of tumor-associated DUBs resulted in down-regulation of anti-apoptotic and up-regulation of pro-apoptotic proteins, such as MCL-1 and p53 respectively. Our results demonstrate that chemical modification of a previously described Jak2 inhibitor results in the unexpected discovery of a novel compound which acts as a DUB inhibitor, suppressing Jak-Stat signaling by a novel mechanism.

TEMOZOLOMIDE AND BEVACIZUMAB THERAPY IN ADVANCED HEMANGIOPERICYTOMA/ SOLITARY FIBROUS TUMOR

Tumors comprising the spectrum of hemangiopericytoma/ malignant solitary fibrous tumor (HPC/SFT) are thought to arise from fibroblasts and represent a small subset of soft tissue sarcomas. Surgery is typically the treatment of choice for localized disease, with reported 10-year overall survival rates of 54-89% after complete surgical resection. However, for the approximately 20% of HPC/SFT patients who eventually develop local recurrences and/or distant metastases, options for effective treatment are limited and are poorly defined. Alternative therapeutic options are therefore needed for improved palliation and disease control. We hypothesize that HPC/SFT are a spectrum of soft tissue tumors with unique clinical, pathological, and molecular makeup and clinical behavior. HPC/SFT respond to unique therapeutic agents that specifically target aberrations specific to these tumors. We retrospectively reviewed the characteristics and the clinical outcomes for all HPC/SFT patients whose tumor specimens have been reviewed at the MD Anderson Cancer Center from January 1993 to June 2007 by a MD Anderson pathologist and were treated at the institution with available electronic medical records. We identified 128 patients, 79 with primary localized disease and 49 with recurrent and/or metastatic disease. For the 23 patients with advanced HPC/SFT who received adriamycin-based, gemcitabine based, or paclitaxel chemotherapy as first- or second-line therapy, the overall RECIST response rate was 0%. Most patients achieved a brief duration of disease stabilization on chemotherapy, with median progression-free survival (PFS) period of 4.6 months. For the 14 patients with advanced HPC/SFT who received temozolomide and bevacizumab systemic therapy, the overall RECIST response rate was 14%, with the overall Choi response rate of 79%. The median PFS for the cohort was 9.7 months with a median 6-month progression free rate of 78.6%. The most frequently observed toxic effect of temzolomide-bevacizumab therapy was myelosuppression. We have designed a phase II study to evaluate the safety and efficacy of temozolomide-bevaciumab in locally advanced, recurrent, and metastatic HPC/SFT in a prospective manner. Combination therapy with temozolomide and bevacizumab may be a potentially clinically beneficial regimen for advanced HPC/SFT patients.

ROLE OF GSH METABOLISM IN MEDIATING STROMAL-LEUKEMIA INTERACTION AND PROMOTING CELL SURVIVAL AND DRUG RESISTANCE IN CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The interaction between CLL cells and the bone marrow stromal environment is thought to play a major role in promoting the leukemia cell survival and drug resistance. My dissertation works proved a novel biochemical mechanism by which the bone marrow stromal cells exert a profound influence on the redox status of primary CLL cells and enhance their ability to sustain oxidative stress and drug treatment. Fresh leukemia cells isolated from the peripheral blood of CLL patients exhibited two major redox alterations when they were cultured alone: a significant decrease in cellular glutathione (GSH) and an increase in basal ROS levels. However, when cultured in the presence of bone marrow stromal cells, CLL cells restored their redox balance with an increased synthesis of GSH, a decrease in spontaneous apoptosis, and an improved cell survival. Further study showed that CLL cells were under intrinsic ROS stress and highly dependent on GSH for survival, and that the bone marrow stromal cells promoted GSH synthesis in CLL cells through a novel biochemical mechanism. Cysteine is a limiting substrate for GSH synthesis and is chemically unstable. Cells normally obtain cysteine by uptaking the more stable and abundant precursor cystine from the tissue environment and convert it to cysteine intracellularly. I showed that CLL cells had limited ability to take up extracellular cystine for GSH synthesis due to their low expression of the transporter Xc-, but had normal ability to uptake cysteine. In the co-culture system, the bone marrow stromal cells effectively took up cystine and reduced it to cysteine for secretion into the tissue microenvironment to be taken up by CLL cells for GSH synthesis. The elevated GSH in CLL cells in the presence of bone marrow stromal cells significantly protected the leukemia cells from stress-induced apoptosis, and rendered them resistant to standard therapeutic agents such as fludarabine and oxaliplatin. Importantly, disabling of this protective mechanism by depletion of cellular GSH using a pharmacological approach potently sensitized CLL cells to drug treatment, and effectively enhanced the cytotoxic action of fludarabine and oxaliplatin against CLL in the presence of stromal cells. This study reveals a key biochemical mechanism of leukemia-stromal cells interaction, and identifies a new therapeutic strategy to overcome drug resistance in vivo.

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^

Design, synthesis and biological evaluation of 5$\sp\prime$-mononucleotide prodrugs: Strategies to introduce nucleotides into cells

The neutral bis ((pivaloyloxy)methyl) (PIV$\sb2\rbrack$ derivatives of FdUMP, ddUMP, and AZTMP were synthesized as potential membrane-permeable prodrugs of FdUMP, ddUMP, and AZTMP. These compounds were designed to enter cells by passive diffusion and revert to the parent nucleotides after removal of the PIV groups by hydrolytic enzymes. These prodrugs were prepared by condensation of FUdR, ddU, and AZT with PIV$\sb2$ phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV$\sb2$-FdUMP, PIV$\sb2$-ddUMP, and PIV$\sb2$-AZTMP were stable in the pH range 1.0-4.0 (t$\sb{1/2} = {>}$100 h). They were also fairly stable at pH 7.4 (t$\sb{1/2} = {>}$40 h). In 0.05 M NaOH solution, however, they were rapidly degraded (t$\sb{1/2} < 2$ min). In the presence hog liver carboxylate esterase, they were converted quantitatively to the corresponding phosphodiesters, PIV$\sb1$-FdUMP, PIV$\sb1$-ddUMP, and PIV$\sb1$-AZTMP; after 24 h incubation, only trace amounts of FdUMP, ddUMP, and AZTMP (1-5%) were observed indicating that the PIV$\sb1$ compounds were poor substrates for the enzyme. In human plasma, the PIV$\sb2$ compounds were rapidly degraded with half-lives of less than 5 min. The rate of degradation of the PIV$\sb2$ compounds in the presence of phosphodiesterase I was the same as that in buffer controls, indicating that they were not substrates for this enzyme. In the presence of phosphodiesterase I, PIV$\sb1$-FdUMP, PIV$\sb1$-ddUMP, and PIV$\sb1$-AZTMP were converted quantitatively to FdUMP, ddUMP, and AZTMP.^ PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP were effective at controlling HIV type 1 infection in MT-4 and CEM tk$\sp-$ cells in culture. Mechanistic studies demonstrated that PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP were taken up by the cells and converted to ddUTP and AZTTP, both potent inhibitors of HIV reverse transcriptase. However, a potential shortcoming of PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP as clinical therapeutic agents is that they are rapidly degraded (t$\sb{1/2}$ = approx. 4 minutes) in human plasma by carboxylate esterases. To circumvent this limitation, chemically-labile nucleotide prodrugs and liposome-encapsulated nucleotide prodrugs were investigated. In the former approach, the protective groups bis(N, N-(dimethyl)carbamoyloxymethyl) (DM$\sb2$) and bis (N-(piperidino)carbamoyloxymethyl) (DP$\sb2$) were used to synthesize DM$\sb2$-ddUMP and DP$\sb2$-ddUMP, respectively. In aqueous buffers (pH range 1.0-9.0) these compounds were degraded with half-lives of 3 to 4 h. They had similar half-lives in human plasma demonstrating that they were resistant to esterase-mediated cleavage. However, neither compound gave rise to significant concentrations of ddUMP in CEM or CEM tk$\sp-$ cells. In the liposome-encapsulated nucleotide prodrug approach, three different liposomal formulations of PIV$\sb2$-ddUMP (L-PIV$\sb2$-ddUMP) were investigated. The half-lifes of these L-PIV$\sb2$-ddUMP preparations in human plasma were 2 h compared with 4 min for the free drug. The preparations were more effective at controlling HIV-1 infection than free PIV$\sb2$-ddUMP in human T cells in culture. Collectively, these data indicate that PIV$\sb2$-FdUMP, PIV$\sb2$-ddUMP, and PIV$\sb2$-AZTMP are effective membrane-permeable prodrugs of FdUMP, ddUMP, and AZTMP. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

Roles of mismatch repair proteins in the recognition and processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) are among the most toxic type of damage to a cell. Many ICL-inducing agents are widely used as therapeutic agents, e.g. cisplatin, psoralen. A bettor understanding of the cellular mechanism that eliminates ICLs is important for the improvement of human health. However, ICL repair is still poorly understood in mammals. Using a triplex-directed site-specific ICL model, we studied the roles of mismatch repair (MMR) proteins in ICL repair in human cells. We are also interested in using psoralen-conjugated triplex-forming oligonucleotides (TFOs) to direct ICLs to a specific site in targeted DNA and in the mammalian genomes. ^ MSH2 protein is the common subunit of two MMR recognition complexes, and MutSα and MutSβ. We showed that MSH2 deficiency renders human cell hypersensitive to psoralen ICLs. MMR recognition complexes bind specifically to triplex-directed psoralen ICLs in vitro. Together with the fact that psoralen ICL-induced repair synthesis is dramatically decreased in MSH2 deficient cell extracts, we demonstrated that MSH2 function is critical for the recognition and processing of psoralen ICLs in human cells. Interestingly, lack of MSH2 does not reduce the level of psoralen ICL-induced mutagenesis in human cells, suggesting that MSH2 does not contribute to error-generating repair of psoralen ICLs, and therefore, may represent a novel error-free mechanism for repairing ICLs. We also studied the role of MLH1, anther key protein in MMR, in the processing of psoralen ICLs. MLH1-deficient human cells are more resistant to psoralen plus UVA treatment. Importantly, MLH1 function is not required for the mutagenic repair of psoralen ICLs, suggesting that it is not involved in the error-generating repair of this type of DNA damage in human cells. ^ These are the first data indicating mismatch repair proteins may participate in a relatively error-free mechanism for processing psoralen ICL in human cells. Enhancement of MMR protein function relative to nucleotide excision repair proteins may reduce the mutagenesis caused by DNA ICLs in humans. ^ In order to specifically target ICLs to mammalian genes, we identified novel TFO target sequences in mouse and human genomes. Using this information, many critical mammalian genes can now be targeted by TFOs.^

The Role Of IL-8-Mediated Src Family Kinase Activation In Tumor-Tumor And Tumor-Stromal Interactions In Metastasis Of Prostate Cancer

Men with localized prostate cancer (PCa) have a 100% five-year survival rate, but this rate drops to 33% for men with metastatic disease. A better understanding of the metastatic process is needed to develop better therapies for PCa. Aberrant activation of protein tyrosine kinases, including Src Family Kinases (SFKs) contribute to metastasis through numerous functions, one of which leads to increased expression of cytokines, such as IL-8. However, the relationship between Src activity and IL-8 regulation is not completely understood. In cell line models, I determined that IL-8 activates Src and in turn Src activates IL-8 demonstrating a feed forward loop contributing to the migration and invasion of PCa cells. However, IL-8 is also produced by tumor-associated stromal cells. In bone marrow derived stromal cells (HS5), I demonstrated a feed forward loop occurs as was observed in tumor cells. HS5 conditioned media increased Src activity in PCa cells. By silencing IL-8 in HS5 cells, Src activity was decreased to control levels in PCa cells as was migration and invasion. Thus, stromal cells producing IL-8 contribute to metastatic properties of PCa by a paracrine mechanism. To examine the effect of stromal cells on tumor growth and metastatic potential of PCa in vivo, I mixed HS5 and PCa cells and co-injected them intraprostatically. I determined that tumor growth and metastases were increased. By silencing IL-8 in HS5 cells and co-injecting them with PCa cells intraprostatically, tumor growth and metastases were still increased relative to injection of PCa cells alone, but decreased relative to co-injections with PCa cells and HS5 cells. These studies demonstrated: (1) a feed forward loop in both tumor and stromal cells, whereby IL-8 activates Src, derepressing IL-8 expression in PCa cells in vitro; (2) stromal produced IL-8 activates Src and contributes to the migration and invasion of PCa cells in vitro; and (3) stromal produced IL-8 is responsible, in part, for increases in PCa tumor growth and metastatic potential. Together, these studies demonstrated that IL-8-mediated Src activity increases the metastatic potential of PCa and therapeutic agents interfering with the IL-8/SFK signaling axis may be useful for prevention and treatment of metastases.

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.

The two -state model of receptor activation: The agonist and the efficacy

The Two State model describes how drugs activate receptors by inducing or supporting a conformational change in the receptor from “off” to “on”. The beta 2 adrenergic receptor system is the model system which was used to formalize the concept of two states, and the mechanism of hormone agonist stimulation of this receptor is similar to ligand activation of other seven transmembrane receptors. Hormone binding to beta 2 adrenergic receptors stimulates the intracellular production of cyclic adenosine monophosphate (cAMP), which is mediated through the stimulatory guanyl nucleotide binding protein (Gs) interacting with the membrane bound enzyme adenylylcyclase (AC). ^ The effects of cAMP include protein phosphorylation, metabolic regulation and transcriptional regulation. The beta 2 adrenergic receptor system is the most well known of its family of G protein coupled receptors. Ligands have been scrutinized extensively in search of more effective therapeutic agents at this receptor as well as for insight into the biochemical mechanism of receptor activation. Hormone binding to receptor is thought to induce a conformational change in the receptor that increases its affinity for inactive Gs, catalyzes the release of GDP and subsequent binding of GTP and activation of Gs. ^ However, some beta 2 ligands are more efficient at this transformation than others, and the underlying mechanism for this drug specificity is not fully understood. The central problem in pharmacology is the characterization of drugs in their effect on physiological systems, and consequently, the search for a rational scale of drug effectiveness has been the effort of many investigators, which continues to the present time as models are proposed, tested and modified. ^ The major results of this thesis show that for many b2 -adrenergic ligands, the Two State model is quite adequate to explain their activity, but dobutamine (+/−3,4-dihydroxy-N-[3-(4-hydroxyphenyl)-1-methylpropyl]- b -phenethylamine) fails to conform to the predictions of the Two State model. It is a weak partial agonist, but it forms a large amount of high affinity complexes, and these complexes are formed at low concentrations much better than at higher concentrations. Finally, dobutamine causes the beta 2 adrenergic receptor to form high affinity complexes at a much faster rate than can be accounted for by its low efficiency activating AC. Because the Two State model fails to predict the activity of dobutamine in three different ways, it has been disproven in its strictest form. ^

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

A major goal of chemotherapy is to selectively kill cancer cells while minimizing toxicity to normal cells. Identifying biological differences between cancer and normal cells is essential in designing new strategies to improve therapeutic selectivity. Superoxide dismutases (SOD) are crucial antioxidant enzymes required for the elimination of superoxide (O2·− ), a free radical produced during normal cellular metabolism. Previous studies in our laboratory demonstrated that 2-methoxyestradiol (2-ME), an estradiol derivative, inhibits the function of SOD and selectively kills human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The present work was initiated to examine the biochemical basis for the selective anticancer activity of 2-ME. Investigations using two-parameter flow cytometric analyses and ROS scavengers established that O2·− is a primary and essential mediator of 2-ME-induced apoptosis in cancer cells. In addition, experiments using SOD overexpression vectors and SOD knockout cells found that SOD is a critical target of 2-ME. Importantly, the administration of 2-ME resulted in the selective accumulation of O 2·− and apoptosis in leukemia and ovarian cancer cells. The preferential activity of 2-ME was found to be due to increased intrinsic oxidative stress in these cancer cells versus their normal counterparts. This intrinsic oxidative stress was associated with the upregulation of the antioxidant enzymes SOD and catalase as a mechanism to cope with the increase in ROS. Furthermore, oxygen consumption experiments revealed that normal lymphocytes decrease their respiration rate in response to 2-ME-induced oxidative stress, while human leukemia cells seem to lack this regulatory mechanism. This leads to an uncontrolled production of O2·−, severe accumulation of ROS, and ultimately ROS-mediated apoptosis in leukemia cells treated with 2-ME. The biochemical differences between cancer and normal cells identified here provide a basis for the development of drug combination strategies using 2-ME with other ROS-generating agents to enhance anticancer activity. The effectiveness of such a combination strategy in killing cancer cells was demonstrated by the use of 2-ME with agents/modalities such as ionizing radiation and doxorubicin. Collectively, the data presented here strongly suggests that 2-ME may have important clinical implications for the selective killing of cancer cells. ^

Therapeutic Targeting of ATP7B in Ovarian Carcinoma.

PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

The effect of body mass index on therapeutic response to bacterial vaginosis in pregnancy.

Our objective was to determine the effect of body mass index (BMI) on response to bacterial vaginosis (BV) treatment. A secondary analysis was conducted of two multicenter trials of therapy for BV and TRICHOMONAS VAGINALIS. Gravida were screened for BV between 8 and 22 weeks and randomized between 16 and 23 weeks to metronidazole or placebo. Of 1497 gravida with asymptomatic BV and preconceptional BMI, 738 were randomized to metronidazole; BMI was divided into categories: < 25, 25 to 29.9, and > or = 30. Rates of BV persistence at follow-up were compared using the Mantel-Haenszel chi square. Multiple logistic regression was used to evaluate the effect of BMI on BV persistence at follow-up, adjusting for potential confounders. No association was identified between BMI and BV rate at follow-up ( P = 0.21). BMI was associated with maternal age, smoking, marital status, and black race. Compared with women with BMI of < 25, adjusted odds ratio (OR) of BV at follow-up were BMI 25 to 29.9: OR, 0.66, 95% CI 0.43 to 1.02; BMI > or = 30: OR, 0.83, 95% CI 0.54 to 1.26. We concluded that the persistence of BV after treatment was not related to BMI.

The effect of body mass index on therapeutic response to bacterial vaginosis in pregnancy.

Our objective was to determine the effect of body mass index (BMI) on response to bacterial vaginosis (BV) treatment. A secondary analysis was conducted of two multicenter trials of therapy for BV and TRICHOMONAS VAGINALIS. Gravida were screened for BV between 8 and 22 weeks and randomized between 16 and 23 weeks to metronidazole or placebo. Of 1497 gravida with asymptomatic BV and preconceptional BMI, 738 were randomized to metronidazole; BMI was divided into categories: < 25, 25 to 29.9, and > or = 30. Rates of BV persistence at follow-up were compared using the Mantel-Haenszel chi square. Multiple logistic regression was used to evaluate the effect of BMI on BV persistence at follow-up, adjusting for potential confounders. No association was identified between BMI and BV rate at follow-up ( P = 0.21). BMI was associated with maternal age, smoking, marital status, and black race. Compared with women with BMI of < 25, adjusted odds ratio (OR) of BV at follow-up were BMI 25 to 29.9: OR, 0.66, 95% CI 0.43 to 1.02; BMI > or = 30: OR, 0.83, 95% CI 0.54 to 1.26. We concluded that the persistence of BV after treatment was not related to BMI.