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Supplementary Figures and Tables - PDF file 898K, Supplementary Table ST1: AKR1C3 over-expression in HEK-293 cells reduces IC50 of androgens. Supplementary Table ST2: Finasteride increases the testosterone formation. Supplementary Figure S1: Over-expression of AKR1C3 increases LNCaP xenograft growth in intact mice. Supplementary Figure S2: AR target FKBP51, but not AR, protein expression is increased in LNCaP-AKR1C3 xenograft tumors. Supplementary Figure S3: AKR1C3 translocation to nucleus requires AR. Top panel. NIH3T3-AKR1C3 cells infected with adenovirus LacZ. Bottom panel. NIH3T3-AKR1C3 cells infected with adenovirus AR.Supplementary Figure S4: AKR1C3 migrates with AR. Supplementary Figure S5: Duolink assay demonstrates interaction between AR and AKR1C3 in LNCaP-AKR1C3 cells. Supplementary Figure S6: AKR1C3 synergizes with SRC-2 in AR transactivation assay. Supplementary Figure S7: AKR1C3 dependent- androgen induced- AR transactivation is not cell type dependent. Transient transactivation studies conducted in COS-1 cells Supplementary Figure S8: AKR1C3-dependent increase in transactivation is selective to AR. Supplementary Figure S9: R1881 induced- AKR1C3 dependent- AR transactivation is not observed with other AKR1C. Supplementary Figure-S10: Different domains mediate the enzymatic and activator functions of AKR1C3. Supplementary Figure S11: GTx-560 is specific for AKR1C3. Supplementary Figure S12: HEK-293-AKR1C3 enzyme activity. Supplementary Figure S14: GTx-560 inhibits AKR1C3-dependent A�dione-induced AR transactivation at all concentration of AKR1C3. Supplementary Figure S15: Expression of steroidogenic enzymes in VCaP cells

Supplementary Methods - PDF file 62K, Additional methods to support the manuscript 1. Cloning and protein purification 2. AKR1C3 enzyme activity and thin layer chromatography (TLC)

Purpose: Castration-resistant prostate cancer (CRPC) may occur by several mechanisms including the upregulation of androgen receptor (AR), coactivators, and steroidogenic enzymes, including aldo keto reductase 1C3 (AKR1C3). AKR1C3 converts weaker 17-keto androgenic precursors to more potent 17-hydroxy androgens and is consistently the major upregulated gene in CRPC. The studies in the manuscript were undertaken to examine the role of AKR1C3 in AR function and CRPC.Experimental Design: LNCaP cells stably transfected with AKR1C3 and VCaP cells endogenously expressing AKR1C3 were used to understand the effect of AKR1C3 on prostate cancer cell and tumor growth in nude mice. Chromatin immunoprecipitation, confocal microscopy, and co-immunoprecipitation studies were used to understand the recruitment of AKR1C3, intracellular localization of AKR1C3 and its interaction with AR in cells, tumor xenograft, and in Gleason sum 7 CRPC tissues. Cells were transiently transfected for AR transactivation. Novel small-molecule AKR1C3-selective inhibitors were synthesized and characterized in androgen-dependent prostate cancer and CRPC models.Results: We identified unique AR-selective coactivator- and prostate cancer growth-promoting roles for AKR1C3. AKR1C3 overexpression promotes the growth of both androgen-dependent prostate cancer and CRPC xenografts, with concomitant reactivation of androgen signaling. AKR1C3 interacted with AR in prostate cancer cells, xenografts, and in human CRPC samples and was recruited to the promoter of an androgen-responsive gene. The coactivator and growth-promoting functions of AKR1C3 were inhibited by an AKR1C3-selective competitive inhibitor.Conclusions: AKR1C3 is a novel AR-selective enzymatic coactivator and may represent the first of more than 200 known nuclear hormone receptor coactivators that can be pharmacologically targeted. Clin Cancer Res; 19(20); 5613–25. ©2013 AACR.

To block the metabolically labile sites of novel tubulin inhibitors targeting the colchicine binding site based on SMART, ABI, and PAT templates, we have designed, synthesized, and biologically tested three focused sets of new derivatives with modifications at the carbonyl linker, the para-position in the C ring of SMART template, and modification of A ring of the PAT template. Structure-activity relationships of these compounds led to the identification of new benzimidazole and imidazo[4,5-c]pyridine-fused ring templates, represented by compounds 4 and 7, respectively, which showed enhanced antitumor activity and substantially improved the metabolic stability in liver microsomes compared to SMART. MOM group replaced TMP C ring and generated a potent analogue 15, which showed comparable potency to the parent SMART compound. Further modification of PAT template yielded another potent analogue 33 with 5-indolyl substituent at A ring.

Purpose: Castration-resistant prostate cancer (CRPC) may occur by several mechanisms including the upregulation of androgen receptor (AR), coactivators, and steroidogenic enzymes, including aldo keto reductase 1C3 (AKR1C3). AKR1C3 converts weaker 17-keto androgenic precursors to more potent 17-hydroxy androgens and is consistently the major upregulated gene in CRPC. The studies in the manuscript were undertaken to examine the role of AKR1C3 in AR function and CRPC. Experimental Design: LNCaP cells stably transfected with AKR1C3 and VCaP cells endogenously expressing AKR1C3 were used to understand the effect of AKR1C3 on prostate cancer cell and tumor growth in nude mice. Chromatin immunoprecipitation, confocal microscopy, and co-immunoprecipitation studies were used to understand the recruitment of AKR1C3, intracellular localization of AKR1C3 and its interaction with AR in cells, tumor xenograft, and in Gleason sum 7 CRPC tissues. Cells were transiently transfected for AR transactivation. Novel small-molecule AKR1C3-selective inhibitors were synthesized and characterized in androgen-dependent prostate cancer and CRPC models. Results: We identified unique AR-selective coactivator- and prostate cancer growth-promoting roles for AKR1C3. AKR1C3 overexpression promotes the growth of both androgen-dependent prostate cancer and CRPC xenografts, with concomitant reactivation of androgen signaling. AKR1C3 interacted with AR in prostate cancer cells, xenografts, and in human CRPC samples and was recruited to the promoter of an androgen-responsive gene. The coactivator and growth-promoting functions of AKR1C3 were inhibited by an AKR1C3-selective competitive inhibitor. Conclusions: AKR1C3 is a novel AR-selective enzymatic coactivator and may represent the first of more than 200 known nuclear hormone receptor coactivators that can be pharmacologically targeted. Clin Cancer Res; 19(20); 5613–25. ©2013 AACR.

Androgen deprivation therapy (ADT) is the mainstay of treatment for advanced prostate cancer. ADT improves overall and disease-free survival rates, but long-term therapy is associated with severe side effects of androgen and estrogen depletion including hot flashes, weight gain, depression, and osteoporosis. Effective hormone reduction can be achieved without estrogen deficiency-related side effects by using therapy with estrogenic compounds. However, cardiovascular complications induced by estrogens coupled with the availability of LHRH agonists led to discontinuation of estrogen use for primary androgen deprivation therapy in the 1980s. New treatments for prostate cancer that improve patient outcomes without the serious estrogen deficiency-related toxicities associated with ADT using LHRH analogs are needed. Herein we describe a novel nonsteroidal selective estrogen receptor-α agonist designed for first-line therapy of advanced prostate cancer that in animal models induces medical castration and minimizes many of the estrogen deficiency-related side effects of ADT. The present studies show that orally administered GTx-758 reversibly suppressed testosterone to castrate levels and subsequently reduced prostate volume and circulating prostate-specific antigen in relevant preclinical models without inducing hot flashes, bone loss, thrombophilia, hypercoagulation, or increasing fat mass.

(3-(1H-indol-2-yl)phenyl)(3,4,5-trimethoxyphenyl)methanone (I-387) is a novel synthetic compound that inhibits tubulin action and exhibits potent antitumor activity in various preclinical models. I-387 inhibited the in vitro growth of several human cancer cell lines with IC50 values in the range of 15 to 39 nmol/L. Nanomolar concentrations of the compound induced apoptosis and caused phosphorylation of the antiapoptotic protein Bcl-2. I-387 induced a strong and concentration-dependent G2-M arrest in PC-3 cells by constitutive activation of Cdc2/cyclin B1 complex and destabilized polymerization of purified tubulin in vitro by binding to the colchicine-binding site. In vivo, I-387 treatment effectively inhibited tumor growth in mice bearing PC-3 tumor xenografts. In vitro studies of nerve growth factor–dependent neurite outgrowth in PC12 pheochromocytoma cells and in vivo studies of mouse behavior showed that I-387 was less neurotoxic than vinblastine and vincristine, tubulin destabilizers with known neurotoxicity. Interestingly, multidrug-resistant cell lines that overexpressed P-glycoprotein (P-gp), multidrug resistance–associated proteins, and breast cancer resistance protein were rendered resistant to docetaxel, vinblastine, SN-38, and doxorubicin, but not to I-387. I-387 dosed at 10 mg/kg was equally effective with 76% tumor growth inhibition in xenograft models using MES-SA uterine sarcoma cells and MES-SA/DX5 cells overexpressing P-gp. In contrast, docetaxel and vinblastine were not effective in MES-SA/DX5 xenograft models. The potent in vitro and in vivo antitumor activity of I-387 suggests that it may represent a new antimitotic agent for management of various malignancies, particularly for patients with drug-resistant cancer. Mol Cancer Ther; 9(11); 2859–68. ©2010 AACR.

Nuclear factor kappaB (NF-kappaB) has been implicated in inducible chemoresistance against anthracyclines. In an effort to improve the cytotoxicity of anthracyclines while reducing their cardiotoxic effects, we have developed a novel class of extranuclear-localizing 14-O-acylanthracyclines that bind to the phorbol ester/diacylglycerol-binding C1b domain of conventional and novel protein kinase C (PKC) isoforms, thereby promoting an apoptotic response. Because PKCs have been shown to be involved in NF-kappaB activation, in this report, we determined the mechanism of NF-kappaB activation by N-benzyladriamycin-14-valerate (AD 198) and N-benzyladriamycin-14-pivalate (AD 445), two novel 14-O-acylanthracylines. We show that the induction of NF-kappaB activity in response to drug treatment relies on the activation of PKC-delta and NF-kappaB-activating kinase (NAK), independent of ataxia telengectasia mutated and p53 activities. In turn, NAK activates the IKK complex through phosphorylation of the IKK-2 subunit. We find that neither NF-kappaB activation nor ectopic expression of Bcl-X(L) confers protection from AD 198-induced cell killing. Overall, our data indicate that activation of novel PKC isoforms by cytoplasmic-targeted 14-O-acylanthracyclines promotes an apoptotic response independent of DNA damage, which is unimpeded by inducible activation of NF-kappaB.

Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.

PURPOSE The goal of our study was to evaluate the in vitro and in vivo anti-angiogenic effects of ERβ selective agonist, β-LGND2, using human retinal microvascular endothelial cell (HRMVEC) cultures and a mouse model for oxygen-induced retinopathy (OIR). METHODS The selectivity of β-LGND2 was determined using binding and transactivation assays. The effects of β-LGND2 on pathologic neovascularization were evaluated in OIR mice by histology and retinal mounts stained with isolectin B4 to quantify aberrant angiogenesis. Gene expression and protein levels were evaluated using Q-PCR, angiogenesis protein array, and Western blotting. A cell death detection ELISA kit was used to evaluate HRMVECs following hypoxic and hyperoxic conditions. In vitro angiogenesis was evaluated by growth factor-induced proliferation, tube formation, and cell migration assays. RESULTS β-LGND2-treated OIR mice had a reduced number of neovascular tufts compared to vehicle-treated animals and a significant amount of normal blood vessel maturation similar to normoxia controls. β-LGND2 inhibited in vitro hypoxia- or hyperoxia-induced cell death and the formation of endothelial tubular structures in an ERβ-specific mechanism. However, β-LGND2 did not inhibit significantly growth factor-induced HRMVEC proliferation and migration. Gene and protein studies revealed that OIR mice treated with β-LGND2 had lower levels of pro-angiogenic factors, like VEGF and HIF1α. CONCLUSIONS β-LGND2 inhibited in vitro and in vivo pathologic neovascularization in the retina in an ERβ-specific mechanism. These results show that β-LGND2, a non-steroidal ERβ selective agonist, could be a useful therapeutic for ocular diseases involving aberrant angiogenesis, like ROP, wet-AMD, and diabetic retinopathy.

We investigated the in vitro metabolism and estrogenic and antiestrogenic activity of toremifene (TOR), tamoxifen (TAM) and their metabolites to better understand the potential effects of cytochrome P-450 2D6 (CYP2D6) status on the activity of these drugs in women with breast cancer. The plasma concentrations of TOR and its N-desmethyl (NDM) and 4-hydroxy (4-OH) metabolites during steady-state dosing with TOR were also determined. Unlike TOR, TAM and its NDM metabolite were extensively oxidized to 4-OH TAM and 4-OH-NDM TAM by CYP2D6, and the rate of metabolism was affected by CYP2D6 status. 4-OH-NDM TOR concentrations were not measurable at steady state in plasma of subjects taking 80 mg of TOR. Molecular modeling provided insight into the lack of 4-hydroxylation of TOR by CYP2D6. The 4-OH and 4-OH-NDM metabolites of TOR and TAM bound to estrogen receptor (ER) subtypes with fourfold to 30-fold greater affinity were 35- to 187-fold more efficient at antagonizing ER transactivation and had antiestrogenic potency that was up to 360-fold greater than their parent drugs. Our findings suggest that variations in CYP2D6 metabolic capacity may cause significant differences in plasma concentrations of active TAM metabolites (i.e., 4-OH TAM and 4-OH-NDM TAM) and contribute to variable pharmacologic activity. Unlike TAM, the clinical benefits in subjects taking TOR to treat metastatic breast cancer would not likely be subject to allelic variation in CYP2D6 status or affected by coadministration of CYP2D6-inhibiting medications.

Phosphatidylinositol-3-kinase (PI3K)/Akt and 5'-AMP-activated protein kinase (AMPK) are attractive targets for anti-cancer drug development. Inhibition of Akt or activation of AMPK is cytotoxic to human cancer cells in vitro and in vivo. We previously demonstrated that 2-arylthiazolidine-4-carboxylic acid amides (ATCAA) are effective cytotoxic agents in prostate and melanoma cancer cell lines, with IC(50) values in the low/sub micromolar range. Using in vitro and in vivo studies, we further characterized the anti-cancer efficacy and mechanism of action of ATCAA-10, a potent lead. ATCAA-10 exhibited equal potency on both MES/SA and P-glycoprotein over-expressing multidrug resistant MES/SA/Dx5 cells, suggesting that ATCAA-10 may overcome multiple drug resistance. Cell-free kinase binding assays excluded the direct binding of ATCAA-10 to several kinases, including IGF-1R, EGFR, FGFR and PDGFR. However, in A549 and HeLa cells, ATCAA-10 effectively dephosphorylated Akt, with concomitant phosphorylation of AMPK. Determination of intracellular ATP and AMP concentrations revealed that ATCAA-10 activated AMPK by altering the intracellular AMP/ATP ratio. ATCAA-10 exhibited favorable pharmacokinetic properties in both mice and rats, including low clearance, low hepatic extraction rate, moderate volume of distribution and long half-life. In addition, ATCAA-10 inhibited A549 tumor xenograft growth with 46% tumor growth inhibition (TGI) at 20 mg/kg dose. Taken together; these results suggest that ATCAA-10 modulates the activity of two signaling pathways, PI3K/AKT/mTOR and AMPK/mTOR, resulting in the inhibition of cancer cell growth.

Phosphatidylinositol-3-kinase (PI3K)/Akt and 5'-AMP-activated protein kinase (AMPK) are attractive targets for anti-cancer drug development. Inhibition of Akt or activation of AMPK is cytotoxic to human cancer cells in vitro and in vivo. We previously demonstrated that 2-arylthiazolidine-4-carboxylic acid amides (ATCAA) are effective cytotoxic agents in prostate and melanoma cancer cell lines, with IC(50) values in the low/sub micromolar range. Using in vitro and in vivo studies, we further characterized the anti-cancer efficacy and mechanism of action of ATCAA-10, a potent lead. ATCAA-10 exhibited equal potency on both MES/SA and P-glycoprotein over-expressing multidrug resistant MES/SA/Dx5 cells, suggesting that ATCAA-10 may overcome multiple drug resistance. Cell-free kinase binding assays excluded the direct binding of ATCAA-10 to several kinases, including IGF-1R, EGFR, FGFR and PDGFR. However, in A549 and HeLa cells, ATCAA-10 effectively dephosphorylated Akt, with concomitant phosphorylation of AMPK. Determination of intracellular ATP and AMP concentrations revealed that ATCAA-10 activated AMPK by altering the intracellular AMP/ATP ratio. ATCAA-10 exhibited favorable pharmacokinetic properties in both mice and rats, including low clearance, low hepatic extraction rate, moderate volume of distribution and long half-life. In addition, ATCAA-10 inhibited A549 tumor xenograft growth with 46% tumor growth inhibition (TGI) at 20 mg/kg dose. Taken together; these results suggest that ATCAA-10 modulates the activity of two signaling pathways, PI3K/AKT/mTOR and AMPK/mTOR, resulting in the inhibition of cancer cell growth.

Obesity is an epidemic problem affecting millions of people in the Western hemisphere and costs the United States economy more than $200 billion annually. Currently, there are no effective treatments to combat obesity. Recent studies have implicated the constitutive activity of estrogen receptor (ER) β as an important regulator of metabolic diseases. However, the potential of ER-β-selective ligands to offset obesity is not clear. We evaluated the pharmacological effect of ER-β-selective ligands (β-LGNDs) in animal models of high-fat diet- and ovariectomy-induced obesity. Ligand binding, transactivation, and uterotrophic studies with β-LGNDs demonstrated selectivity for ER-β over ER-α. Animals fed a high-fat diet showed a significant increase in body weight, and this weight gain was attenuated by β-LGNDs. High-fat diet-mediated increases in serum cholesterol, leptin, glucose, and fat accumulation in organs were also reduced by β-LGNDs. In addition, MRI scanning indicated that β-LGNDs altered body composition by reducing fat mass and increasing lean body mass. Organ weights and gene expression analyses demonstrated that adipose tissue is the center of action for β-LGNDs, and the reduction in body weight is likely due to increased energy expenditure. In vitro and in vivo mechanistic studies indicated that the anti-obesity effects of β-LGNDs were due to indirect peroxisome proliferator-activated receptor γ antagonistic actions requiring the ligand binding domain of ER-β and through abrogation of the ability of PGC-1 to coactivate peroxisome proliferator-activated receptor γ. In conclusion, these studies indicate that ligand-activated ER-β is a potential therapeutic target to combat obesity and obesity-related metabolic diseases.

Microtubules are one of the most useful subcellular targets in chemotherapy. We identified a novel indole, (3-(1H-indol-2-yl)phenyl)(1H-indol-2-yl)methanone (15), that inhibits tubulin action and exhibits potent antitumor activity in various preclinical models.In vitro cancer cell growth inhibition was measured by SRB or MTT assay in human cancer cell lines. Apoptosis induced by 15 was examined in LNCaP and PC-3 cells. Effects of 15 on cell cycle distribution and tubulin were investigated via in vitro models. In vivo toxicity and xenograft efficacy studies were conducted in mice.Indole 15 inhibited the in vitro growth of a number of human cancer cell lines, including drug-resistant cell lines that over-express P-glycoprotein, multidrug resistance-associated proteins, and breast cancer resistance protein with IC(50) values in the range of 34-162 nM. Nanomolar concentrations of the compound caused down-regulation of bcl-2, induced PARP cleavage, and induced apoptosis in both LNCaP and PC-3 prostate cancer cells, as confirmed by anti-histone ELISA and DNA laddering. In vitro studies revealed that the compound inhibited polymerization of purified tubulin and induced a strong and concentration-dependent G(2)M arrest in PC-3 cells. In vivo studies in immunodeficient mice bearing PC-3 tumor xenografts showed that the compound effectively inhibited tumor growth.The potent in vitro and in vivo antitumor activities of this novel indole suggest that drugs with this novel chemical scaffold might be developed for treatment of drug-resistant prostate cancer.

Unlike nuclear-targeted anthracyclines, the extranuclear-targeted doxorubicin congener, N-benzyladriamycin-14-valerate (AD 198), does not interfere with normal topoisomerase II activity, but binds to the C1b regulatory domain of conventional and novel isoforms of protein kinase C (PKC). The resulting interaction leads to enzyme activation and rapid apoptosis in a variety of mammalian cell lines through a pathway involving mitochondrial events such as membrane depolarization (Deltapsim) and cytochrome c release. Unlike other triggers of apoptosis, AD 198-mediated apoptosis is unimpeded by the expression of Bcl-2 and Bcl-XL. We have further examined AD 198-induced apoptosis in 32D.3 mouse myeloid cells to determine how the anti-apoptotic effects of Bcl-2 are circumvented. The PKC-delta inhibitor, rottlerin, and transfection with a transdominant-negative PKC-delta expression vector both inhibit AD 198 cytotoxicity through inhibition of Deltapsim and cytochrome c release. While the pan-caspase inhibitor Z-VAD-FMK blocks AD 198-induced PKC-delta cleavage, however, it does not inhibit Deltapsim and cytochrome c release, indicating that AD 198 induces PKC-delta holoenzyme activation to achieve apoptotic mitochondrial effects. AD 198-mediated Deltapsim and cytochrome c release are also unaffected by cellular treatment with either the mitochondrial permeability transition pore complex (PTPC) inhibitor cyclosporin A or the Ca chelators EGTA and BAPTA-AM. These results suggest that AD 198 activates PKC-delta holoenzyme, resulting in Deltapsim and cytochrome c release through a mechanism that is independent of both PTPC activation and Ca flux across the mitochondria. PTPC-independent mitochondrial activation by AD 198 is consistent with the inability of Bcl-2 and Bcl-XL expression to block AD 198-induced apoptosis.

Bcl-2 inhibits apoptosis induced by numerous antitumor drugs, including doxorubicin and daunorubicin and is, thus, a major impediment to successful cancer chemotherapy. Here, we report the ability of a novel family of nonnuclear targeted anthracyclines to induce rapid apoptosis in cells despite Bcl-2 or Bcl-X(L) expression. Typified by N-benzyladriamycin-14-valerate (AD 198) and N-benzyladriamycin-14-pivalate (AD 445), this family of compounds binds to the C1 regulatory domain of protein kinase C (PKC), competitively inhibits phorbol ester binding in cell-free studies, and induces PKC translocation in intact cells. PKC-delta has an established role as a pro-apoptotic protein through the association of the holoenzyme or catalytic fragment with mitochondria. In proliferating 32D.3 myeloid cells, or in 32D.3 cells engineered to overexpress Bcl-2, substantial levels of PKC-delta are associated with mitochondria. However, after a 1-h exposure to 5 microM AD 198, cytochrome c release, caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, PKC-delta cleavage, and DNA fragmentation are observed. Pretreatment of 32D.3 cells with the selective PKC-delta inhibitor, rottlerin, but not the general PKC inhibitor, GF 109203X, or PKC-alpha and -beta inhibitor, Gö 6976, delayed the 50% cell kill to24 h for control and Bcl-2 overexpressing 32D.3 cells treated with 5 microM AD 198. Rottlerin delayed PKC-delta and PARP cleavage to20 h post-drug exposure and also delayed mitochondrial membrane depolarization. In contrast, the pan-caspase inhibitor Z-Val-Ala-Asp-CH2F blocked PKC-delta and PARP cleavage, but not mitochondrial membrane depolarization. These results suggest that AD 198 induces mitochondrial-dependent apoptosis in 32D.3 cells by activating PKC-delta holoenzyme on mitochondria, which, in turn, overrides the antiapoptotic effects of Bcl-2.

100 Background: Castration resistant prostate cancer (CRPC) may occur by several mechanisms including up-regulation of the androgen receptor (AR), coactivators, and steroidogenic enzymes, including AKR1C3. AKR1C3 (type 5 17b-hydroxysteroid dehydrogenase) converts weaker 17-keto androgenic precursors to more potent 17-hydroxy androgens and is consistently a major up-regulated gene in CRPC. The objective is to examine the role of AKR1C3 in AR function and CRPC. Methods: AR transactivation studies were performed with GRE-LUC in the presence or absence of AKR1C3 in HEK-293 cells. Gene expression patterns and prostate cancer cell and tumor growth were evaluated in LNCaP and VCaP cells. Molecular studies, including co-immunoprecipitation, chromatin immunoprecipitation and others, were performed to determine AKR1C3 localization and interaction with the AR. Novel small molecule AKR1C3 selective-inhibitors were synthesized and characterized in prostate cancer models. Results: We identified unique AR selective coactivator- and prostate cancer growth-promoting-roles for AKR1C3. AKR1C3 over-expression promotes the growth of both androgen-dependent prostate cancer and CRPC xenografts, with concomitant reactivation of androgen signaling. AKR1C3 interacted with AR in prostate cancer cells and in a human CRPC sample and was recruited to the promoter of an androgen responsive gene, PSA. The coactivator and growth promoting functions of AKR1C3 were inhibited by an AKR1C3-selective competitive inhibitor. Conclusions: AKR1C3 is a novel AR selective coactivator and may represent the first nuclear hormone receptor coactivator that can be pharmacologically targeted.

A novel indole antimitotic compound, GTx-230, was identified in cell-based screening assays that binds to the colchicine site in tubulin and destabilizes microtubules. In this study, the pharmacokinetics, antitumor efficacy, neurotoxicity, and vascular disrupting ability of GTx-230 were evaluated. GTx-230 inhibited the in vitro growth of a number of human cancer cell lines including DU-145, LNCaP, PC-3, and PPC-1 prostate cancer cells with low nanomolar IC50 values. Following a 10 mg/kg intravenous (i.v.) dose to mice, the plasma clearance (CL), volume of distribution at steady state (Vdss), and terminal half-life (T1/2) of GTx-230 were 19 ml/min/kg, 2.9 l/kg, and 1.7 h, respectively. Oral bioavailability of GTx-230 was 36% in mice. In vivo, GTx-230 (6.7 mg/kg, qd, p.o.) was effective with near 100% tumor growth inhibition in xenograft models using PC-3/TXR cells over-expressing P-glycoprotein. In contrast, docetaxel, a known antimitotic agent, was not effective in the PC-3/TXR xenograft model. In vivo studies of mouse performance on an accelerating rotarod and hotplate showed that GTx-230 did not induce any symptoms of peripheral neuropathy. In addition, we evaluated the effect of GTx-230 on in vitro vascular permeability using a transwell system with confluent HUVEC monolayers. GTx-230 resulted in increased permeability after 1 h incubation and showed potency similar to CA4. In summary, the potent in vitro and in vivo antitumor activities of GTx-230 suggest that it may represent a new antimitotic agent for management of solid malignancies, particularly for patients with drug resistant cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2788. doi:1538-7445.AM2012-2788

e13067 Background: Toremifene (TOR) and tamoxifen (TAM) are approved treatments for advanced breast cancer. It has been suggested that poor metabolizers of TAM have worse clinical outcomes than pat...