• The Biological Characterization of Galeterone Analogs VNPT-178 and VNLG-74A for the Treatment of Prostate Cancer

      McCarty, David; Njar, Vincent (2017)
      The expression of truncated androgen receptor spice-variants (AR-Vs) presents a significant clinical challenge for the treatment of advanced stages of prostate cancer and confers complete resistance to abiraterone and enzalutamide. Targeted depletion of these receptors in addition to antagonism of the androgen signaling axis has been actively evaluated as a superior treatment paradigm but has thus far been unsuccessful in clinical trials. Based on our studies of galeterone and its analogs, we believe these compounds possess a specific activity that is both independent and overlapping of their antagonism of the androgen receptor (AR). In this study, we perform basic biochemical techniques attempting to better delineate the actions of galeterone analogs VNPT-178 and VNLG-74A and identify a rational, common target for improved study of their utility against all stages of prostate cancer development. Direct comparisons of VNPT-178 and VNLG-74A to vehicle or equimolar concentrations of abiraterone and enzalutamide reveal both compounds exhibit improved antiproliferative activities in multiple prostate cancer cell models of androgen and AR dependence. VNPT-178 and VNLG-74A directly antagonize the full-length AR in LNCaP and CWR22Rv1 (22Rv1) cells while promoting the depletion of full-length and truncated receptors with dose- and time-dependency. Activation of the unfolded protein response is rapid and sustained, long preceding appreciable antagonism of the AR in 22Rv1 cells, and followed by CHOP upregulation and PARP cleavage - an effect also seen in PC-3 cells albeit with slower kinetics. Molecular docking of VNPT-178 and VNLG-74A reveals greater potential for binding the ATPase domains of BiP/Grp78 and Hsp70-1A compared to the AR's ligand-binding domain. Severe or sustained activation of the unfolded protein response can induce apoptosis. Taken together, our data suggest that galeterone analogs VNPT-178 and VNLG-74A directly modulate the substrate interactions of BiP promoting endoplasmic reticulum stress and apoptosis independent of AR antagonism. Depletion of AR proteins is possibly the result of similar activities against Hsp70. The sensitivity of 22Rv1 cells to VNPT-178- and VNLG-74A-induced apoptosis may underlie a failure of this cell model to adequately represent the clinical challenge of AR-V-expressing prostate cancers.
    • Characterization of novel retinoic acid metabolism blocking agents (RAMBAs) in breast cancer model systems

      Patel, Jyoti B.; Njar, Vincent (2005)
      All-trans-retinoic acid (ATRA) and other retinoids, potent modulators of a variety of important cellular functions are effective in prevention and therapy of many cancers including breast cancer. Increased metabolism of ATRA by ATRA inducible cytochrome P450 enzymes present in different cells including cancer cells is one of the reasons for the emergence of ATRA resistance and hampering its clinical use. Additionally, high therapeutic doses of ATRA have adverse effects and are not recommended.Retinoic acid metabolism blocking agents (RAMBAs) prevent rapid metabolism of ATRA. Use of RAMBAs is an emerging approach to increase the endogenous levels and thus potentiate the effect of ATRA without administering high exogenous doses. The objective of this project was to investigate the in vitro and in vivo role of novel C-4 azolyl RAMBAs rationally developed in our laboratory, against breast cancer; determine how they would potentiate the effectiveness of ATRA as well as study the molecular mechanisms involved. Novel RAMBAs (VN/14-1, VN/50-1, VN/66-1 and VN/69-1), structural analogs of ATRA/13-cis-retinoic acid, inhibited ATRA metabolism in hormone dependent MCF-7 cells and T47D cells; and microsomes with IC50 values in nanomolar range as determined by HPLC analysis. RAMBAs (0.0001--10 muM) had dose dependent antiproliferative action, suggesting that they have retinoidal activity as well. VN/50-1 and VN/14-1 enhanced ATRA's antiproliferative action in MCF-7 cells. RAMBAs induced differentiation in these cell lines as determined by analysis of differentiation markers cytokeratin 8/18 and E-cadherin. Induction of apoptosis by RAMBAs was determined by TUNEL assay and Western blotting of apoptosis related proteins. Cell cycle analysis indicated that RAMBAs arrest cells in the G1 phase and reduce cells in the S phase. RAMBAs reduced the expression of Cyclin D1 in these cell lines. VN/14-1 like ATRA increased the expression of estrogen receptor-alpha in MCF-7 cells. VN/14-1 besides being a potent RAMBA, has pleiotropic role, is well tolerated and significantly suppressed MCF-7 tumor growth (∼90.35% vs. control, p < 0.0005; at 66.0 mumole/kg/day dose) in female ovariectomized nude mice. Promising RAMBAs endowed with multiple biological activities should be suitable for further clinical investigations for chemoprevention and therapy of breast cancer.
    • Characterization of the Anti-Cancer Activities of Clinical Candidate Galeterone (VN/124-1) and its Novel Analogs in Human Prostate and Pancreatic Cancer Models In Vitro and In Vivo

      Kwegyir-Afful, Andrew Kenneth; Njar, Vincent; 0000-0002-6852-0972 (2016)
      Cancer is a "moving target" disease, as the condition progresses, critical driving oncogenes evolve. Effective treatment for cancers, such as prostate cancer (PC) and pancreatic ductal adenocarcinoma (PDAC) are based on targeting multiple oncogenes implicated in disease progression and drug resistance. Our group pioneered the development of galeterone (gal, originally designated VN/124-1); a small molecule inhibitor that impedes both AR positive and negative tumor growth, suggesting its ability to modulate multiple oncogenes. The goal of this thesis was to assess the ability of gal and novel analogs to modulate multiple oncogenic targets in a variety of clinically relevant human PC and PDAC models to inhibit cell viability, tumor growth and metastatic potential. Our studies show that gal and VNPT55 induce proteasomal degradation of AR/AR-V7, implicating Mdm2/CHIP E3 ligase. Gal/VNPT55 also degraded ARv567es, with VNPT55 exhibiting additional effects on AR-V7 mRNA. We also observed significant gal/VNPT55-induced increase in Bax/Bcl-2 ratio preceding cytochrome c release with a concomitant cleavage of caspase 3 and PARP, culminating in apoptosis. Gal and analogs also repressed pathways implicated in invasiveness (NF-KB, Twist1 and Mnk1/2-eIF4E axis), depleted expression of epithelial-mesenchymal-transition markers (N-Cadherin, MMP-2/-9 and Snail) and down-regulated putative stem cell factors (CD44, Oct-4 and BMI-1). These effects resulted in inhibition of cellular biological activities (migration and invasion) in vitro. Furthermore, gal/analogs effectively decreased viability of several drug-resistant PC cells. Strong anti-proliferative activity on gal-resistant cells by the novel analogs unraveled the lack of cross resistance. Additionally, anti-tumor activity of gal and VNPT55 on CWR22Rv1 xenograft (CRPC model) recapitulated in vitro activities of the compounds, with no apparent host toxicity. Finally, based on the findings that these agents effectively inhibited pathways associated with PDAC disease progression and drug-resistance, we assessed the potential of the agents as PDAC therapy. Gal/analogs alone or in combination with gemcitabine significantly decreased viability of gemcitabine-naive/resistant PDAC cells. Gal/analogs also inhibited transcription factors, prominent in PDAC disease progression. These agents also caused strong growth inhibition of MiaPaCa-2 xenografts. Together, these data demonstrate that gal and novel analogs possess multiple anti-cancer properties, providing a strong rationale for their development in prostate and pancreatic cancer therapy.
    • Development of Novel Agent VN/124-1 for the Treatment of Advanced Prostate Cancer

      Bruno, Robert D.; Njar, Vincent (2009)
      Prostate cancer (PCA) is the second leading cause of cancer-related deaths in men in the U.S. The standard of care for advanced disease remains androgen ablation, but despite strong initial responses in most cases, therapies inevitably fail in nearly all patients. Our group has designed a novel therapeutic, designated VN/124-1, which offers a distinct advantage over current therapies in that it globally inhibits androgen production and inhibits the androgen receptor directly. To test whether or not VN/124-1 also had an effect on androgen independent PCA (AIPC), the AIPC cell lines PC-3 and DU-145 were treated with increasing concentrations of VN/124-1, and it was found that the compound inhibits the growth of both cell lines at concentrations previously shown to be physiologically relevant. Microarray technology was employed to analyze gene expression changes in PC-3 cells treated with VN/124-1 and revealed the up-regulation of genes involved in cellular stress and amino acid metabolism as well as the down-regulation of genes involved in DNA replication and cell cycle progression. These results led to the hypothesis that VN/124-1 was inhibiting cell growth via induction of the endoplasmic reticulum stress response (ERSR). Quantitative real-time PCR and western blot analyses confirmed that VN/124-1 induced the expression and activation of genes involved in the ERSR in PC-3 cells. Activation of the ERSR resulted in the growth arrest of PC-3 cells in the G1/G0 phase of the cell cycle. VN/124-1 was found to induce the ERSR by causing disruptions in ER Ca2+ homeostasis. In-vivo studies using PC-3 xenografts demonstrated VN/124-1 also significantly inhibits the growth of androgen-independent tumors. VN/124-1's diverse mechanisms of action may offer an advantage over other androgen ablative therapies, demonstrated by its improved efficacy over leading lyase inhibitor, abiraterone, in mouse LAPC-4 xenograft models. VN/124-1 was also compared to the compounds VNLG/21-1 (3-fluoro VN/124-1) and VN/124-1 Sulfamate which were designed to increase the stability and oral bioavailability of VN/124-1, respectively in-vivo. However, these molecules failed to demonstrate any clear advantage over VN/124-1 treatment. Together these studies demonstrate the potential efficacy of VN/124-1 and aid in the understanding of the molecule's complex pharmacology.
    • Mechanisms of Action and Efficacy Modulation of Novel Retinoic Acid Metabolism Blocking Agent (RAMBA) VN/12-1 in Estrogen Receptor-α Negative Breast Cancer Model Systems: Targeting Autophagy

      Godbole, Abhijit Moreshwar; Njar, Vincent (2011)
      Breast cancer (BC) is the most common cancer and the second leading cause of cancer related deaths among women in the United States. Despite important advances in treatment, breast cancer remains incurable due to the emergence of alternative pathways adopted by cancer cells to overcome the effects of anti-cancer therapy. This molecular heterogeneity in the cellular response to anti-cancer therapy challenges the development of effective therapeutic strategies. To overcome this issue, a rational approach would be to concomitantly target clinically relevant cellular abnormalities with combination therapy or to use a potent multi-targeted agent. Autophagy is a critical physiological process of cancer cell survival in the presence of chemotherapeutic stress and is an important cause of resistance to anti-cancer therapy. Drugs that inhibit autophagy e.g. chloroquine-(CHL) deprive the cells of this protective mechanism and therefore are promising agents in combination chemotherapy of breast cancer. All-trans retinoic acid (ATRA), a potent differentiation inducing agent, has been tested as anticancer agent for a number of cancers including breast cancer, but its rapid metabolism to inactive metabolites is a major problem in its clinical use. VN/12-1, a novel retinoic acid metabolism blocking agent (RAMBA) developed in our laboratory, showed improved anti-proliferative activity over ATRA against estrogen receptor α (ER-α) positive and negative breast cancer cell lines. Human epidermal growth factor receptor-2 (Her-2) is a critical protein involved cell proliferation and metastasis. Her-2 has been involved in increasing the frequency of tumor stem cells and it has also been implicated in the development of drug resistance. Hence, for our current studies, we primarily focused on a cell line which is deficient in ER-α and which overexpresses Her-2 (SKBR-3 cells). We showed here that VN/12-1 inhibits G1-S phase cell cycle transition. However, short treatment of VN/12-1 induced autophagy in these cells and protected cells from apoptosis. VN/12-1-induced autophagy was preceded by ER stress (ERS). We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of Beclin-1 enhanced cell death induced by VN/12-1 in SKBR-3 cells. The combination of VN/12-1with autophagy inhibitor - CHL resulted in synergistic effects in that it enhanced apoptosis in vitro and in vivo which was caspase dependent. Importantly, VN/12-1 (5 mg/kg twice weekly) and VN/12-1 (5 mg/kg twice weekly) + chloroquine (50 mg/kg twice weekly) significantly suppressed established SKBR-3 tumor growth by 81.4% (p < 0.001 vs. control) and 96.2% (p < 0.001 vs control), respectively without incurring overt signs of toxicity or significant loss of body weight. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of RAMBAs in the treatment of breast cancer. It is hoped that this combination therapy would solve the problem of the development of acquired resistance due to autophagy that typically follows the chemotherapeutic treatment of breast cancer. We envision further advanced preclinical development of this combination therapy as potential therapy for breast cancer.
    • Multi-mechanistic inhibitors of androgen action for the treatment of prostate cancer

      Vasaitis, Tadas Sean; Brodie, Angela; Njar, Vincent (2007)
      Prostate cancer (PC) is a major cause of mortality among males in the USA. The prominent treatment strategy for advanced cases involves inhibition of the effects of androgens through orchidectomy, LHRH agonists/antagonists, anti-androgens, and inhibitors of androgen synthesis. Despite these treatments, nearly all patients will experience recurrence, in which they fail to satisfactorily respond to endocrine-based treatments. However, recent evidence increasingly implicates a continued role of the androgen receptor (AR), in all disease stages. Therefore, improved strategies for targeting the AR signaling axis are needed. On this basis, we are developing novel agents with the ability to block multiple targets within the androgen signaling pathway. The major targets are androgen biosynthesis, ligand-AR binding, and AR expression. The rationally designed compounds VN/125-1, VN/124-1, VN/85-1, VN/87-1, VN/108-1 and VNLG/82-1 were evaluated for inhibition of CYP17 (a key enzyme in androgen biosynthesis), and compared with the clinically used ketoconazole. For these studies, we utilized our cell-based radiometric acetic acid releasing assay. Among the effective compounds, IC50 values ranged from 0.5 to 112 nM. In competitive binding and transactivation studies, VN/124-1, VN/125-1, and VN/85-1 prevented binding of radiolabeled ligand to both the mutant LNCaP AR and the wild-type AR, and reduced AR mediated transactivation by 90-99% at 10 muM. In proliferation studies, VN/124-1, VN/125-1 and VN/85-1 inhibited the growth of DHT-stimulated LNCaP and LAPC4 cells with IC50 values in the low micromolar range (i.e., <10 muM). They also inhibited AR-negative PC3 cell proliferation, suggesting mechanisms additional to modulation of androgen signaling. Through western blot technique and immunofluorescence microscopy, the effects of VN/124-1 on AR expression and translocation were observed. VN/124-1 dramatically down-regulated AR expression, both in vitro and in vivo, which corresponded to its superior anti-tumor activity previously observed in our LAPC4 xenograft model. VN/124-1 also induced apoptosis in LNCaP cells as shown by annexin/FITC staining and caspase 3/7 activity assays. These finding suggest that combined inhibition of androgen biosynthesis, AR-mediated transactivation, and AR expression represents a new class of agents with exciting potential for PC treatment.
    • Retinoids and retinoic acid metabolism blocking agents in combination with histone deacetylase inhibitors for prostate cancer therapy

      Khandelwal, Aakanksha; Njar, Vincent (2007)
      All-trans-retinoic acid (ATRA), a metabolite of vitamin A, is 5 to 8 times lower in prostate carcinoma tissue when compared to a normal prostate and benign prostatic hyperplasia. ATRA plays a major role in a number of biological processes and is metabolized by cytochrome P450 enzymes into inactive polar metabolites. The anticancer effects of a novel class of agents called atypical retinoic acid metabolism blocking agents (RAMBAs) were examined in combination with histone deacetylase inhibitors (HDACIs). The purpose of this study was to select combinations of agents which would have potent anti-cancer activities in a hormone insensitive prostate cancer model. One particular RAMBA, VN/66-1 which displays favorable pharmacokinetics and minimal toxicity was found to be highly potent in inhibiting PC-3 cell growth. Two HDACIs, SAHA and MS-275 were also found to be potent in inhibiting PC-3 cell viability. The combinations of VN/66-1 + MS-275 and VN/66-1 + SAHA synergistically inhibited PC-3 cell growth caused cytotoxicity/cytostaticity. Treatment of PC-3 xenografts with VN/66-1 (10 mg/kg/day) + MS-275 (2.5 mg/kg/day) for 18 days resulted in an 85% reduction in final mean tumor volume compared with control. The combination of VN/66-1 + SAHA, however, did not reduce tumor growth as effectively as the two agents individually most likely due to too low doses. The data obtained suggest that the mechanism of action of the combination of VN/66-1 + MS-275 is through apoptosis and DNA damage-induced p21 WAF1/CIP1 activation. Active p21 is involved in the inhibition of the cdc2-cyclin B1 complex resulting in eventual G2/M phase arrest. This induction of p21 is also in part due to the enhancement of acetylation of histones H3 and H4 which is also responsible for the activation of tumor suppressor gene RARbeta2, the latter which also plays a role in mediating cell death. Up-regulation of Bad and down-regulation of Bcl-2 as well as PARP cleavage indicated that apoptosis via the intrinsic pathway is also contributing to cell death. Together, these results suggest that VN/66-1 or its combination with MS-275 may be a novel therapy for the treatment of hormone refractory prostate carcinoma.