Ormeloxifene Research Articles

Exosomes derived from tumor adjacent fibroblasts efficiently target pancreatic tumors

The application of extracellular vesicles, particularly exosomes (EXs), is rapidly expanding in the field of medicine, owing to their remarkable properties as natural carriers of biological cargo. This study investigates utilization of exosomes derived from stromal cells of tumor adjacent normal tissues (NAF-EXs) for personalized medicine, which can be derived at the time of diagnosis by endoscopic ultrasound. Herein, we show that exosomes (EXs) derived from NAFs demonstrate differential bio-physical characteristics, efficient cellular internalization, drug loading efficiency, pancreatic tumor targeting and delivery of payloads. NAF-derived EXs (NAF-EXs) were used for loading ormeloxifene (ORM), a potent anti-cancer and desmoplasia inhibitor as a model drug. We found that ORM maintains normal fibroblast cell phenotype and renders them incompatible to be triggered for a CAF-like phenotype, which may be due to regulation of Ca2+ influx in fibroblast cells. NAF-EXs-ORM effectively blocked oncogenic signaling pathways involved in desmoplasia and epithelial mesenchymal transition (EMT) and repressed tumor growth in xenograft mouse model. In conclusion, our data suggests preferential tropism of NAF-EXs for PDAC tumors, thus imply feasibility of developing a novel personalized medicine for PDAC patients using autologous NAF-EXs for improved therapeutic outcome of anti-cancer drugs. Additionally, it provides the opportunity of utilizing this biological scaffold for effective therapeutics in combination with standard therapeutic regimen.

Synthesis and Antitumor Activity of Brominated-Ormeloxifene (Br-ORM) against Cervical Cancer.

Aberrant regulation of β-catenin signaling is strongly linked with cancer proliferation, invasion, migration, and metastasis, thus, small molecules that can inhibit this pathway might have great clinical significance. Our molecular modeling studies suggest that ormeloxifene (ORM), a triphenylethylene molecule that docks with β-catenin, and its brominated analogue (Br-ORM) bind more effectively with relatively less energy (-7.6 kcal/mol) to the active site of β-catenin as compared to parent ORM. Herein, we report the synthesis and characterization of a Br-ORM by NMR and FTIR, as well as its anticancer activity in cervical cancer models. Br-ORM treatment effectively inhibited tumorigenic features (cell proliferation and colony-forming ability, etc.) and induced apoptotic death, as evident by pronounced PARP cleavage. Furthermore, Br-ORM treatment caused cell cycle arrest at the G1-S phase. Mechanistic investigation revealed that Br-ORM targets the key proteins involved in promoting epithelial-mesenchymal transition (EMT), as demonstrated by upregulation of E-cadherin and repression of N-cadherin, Vimentin, Snail, MMP-2, and MMP-9 expression. Br-ORM also represses the expression and nuclear subcellular localization of β-catenin. Consequently, Br-ORM treatment effectively inhibited tumor growth in an orthotopic cervical cancer xenograft mouse model along with EMT associated changes as compared to vehicle control-treated mice. Altogether, experimental findings suggest that Br-ORM is a novel, promising β-catenin inhibitor and therefore can be harnessed as a potent anticancer small molecule for cervical cancer treatment.

Ormeloxifene, a nonsteroidal antifertility drug promotes megakaryocyte differentiation in leukemia cell line K562.

Ormeloxifene (ORM) (3,4-trans-2,2-dimethyl-3-phenyl-4-p-(β-pyrrolidinoethoxy) phenyl-7-methoxychroman), world's first nonsteroidal selective estrogen receptor modulatorapproved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORMcan induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan-caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c-Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h,respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells.

Abstract 952: Attenuation of β-Catenin signaling by ormeloxifene in hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer with approximately 41,000 new diagnosis and 29,000 fatalities every year only in the U.S. Severe toxicity and poor response to available chemotherapeutic agents make HCC clinical management extremely difficult. Aberrant β-catenin signaling is found to play a critical role in differentiation grades of HCC patients. Thus, attempts to find more potent and non-toxic β-Catenin inhibitors is urgently needed for cancer therapy. Notably, ormeloxifene (ORM), clinically approved selective estrogen receptor modulator with therapeutic index, has shown excellent anticancer activity, rendering it as an ideal candidate for repurposing. In this study, we investigated the anti-cancer potential of ORM against hepatocellular carcinoma. Methodology: Cell proliferation and colony formation assays were performed to assess the therapeutic activity of ORM in human hepatocellular carcinoma (HepG2, SK-HEP-1, Hep3B, and C3A) cells. Boyden chamber and Matrigel assays were carried out for investigating the effect of ORM on migration and invasion abilities of HCC cells, respectively. The effects of ORM on β-catenin and EMT associated proteins were analyzed through Western blotting and qPCR. Confocal microscopy was used to determine the β-catenin nuclear localization following ORM treatment in HCC cells. Results: As compared to vehicle-treated group, ORM treatment (2.5-20 µM) suppressed proliferation and colony formation in human hepatocellular carcinoma cells in a dose and time-dependent manner. Moreover, ORM treatment suppresses the migration and invasion of human hepatocellular carcinoma cells as shown by wound healing and Matrigel invasion assay, respectively. ORM effectively inhibited the protein levels and mRNA expression of total β-catenin. Additionally, our confocal microscopy results further showed reduced nuclear translocation of β-catenin following the ORM treatment. ORM treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), and induced the expression of pGSK3β. Experiments are also being conducted to see how ORM affects epigenetic markers linked to EMT and β-catenin signaling. Conclusion: Taken together, ORM inhibited β-catenin signaling and exhibited potent anticancer effects against HCC and could be investigated further as a novel therapeutic modality for HCC treatment. Citation Format: Mohammed Sikander, Shabnam Malik, Anyssa Rodriguez, Molly Vela, Daniel Zubieta, Vivek K. Kashyap, Anupam Dhasmana, Bilal B. Hafeez, Sheema Khan, Fathi T. Halaweish, Subhash C. Chauhan, Meena Jaggi. Attenuation of β-Catenin signaling by ormeloxifene in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 952.

Molecular spectroscopic and molecular simulation studies on the interaction of oral contraceptive drug Ormeloxifene with CT–DNA

The interaction between oral contraceptive drug Ormeloxifene (ORM) and calf thymus DNA (CT–DNA) was studied using UV–Vis, fluorescence, circular dichroism (CD) and 1H NMR spectral techniques under physiological buffer (pH 7.4). Competitive binding assays with ethidium bromide (EB) and Hoechst 33258, viscosity measurements, KI quenching studies, molecular docking and metadynamics simulation studies were also substantiated the spectroscopic results. ORM is found to binds in the minor groove of CT–DNA as evidenced by: (1) non–displacement of EB from EB/CT–DNA complex; (2) appreciable displacement of Hoechst 33258 from its CT–DNA complex; (3) slight alteration in the CD signal; (4) small shifts (Δδ < 0.033 ppm) without broadening in 1H NMR signals and (5) the nearly equal extent of quenching of fluorescence of ORM by KI in the absence and presence of CT–DNA. Negative values of both enthalpy and entropy changes pointed out that the interaction between ORM and CT–DNA is governed mainly by H–bonding and van der Waals forces. Negative free energy change suggested a spontaneous interaction between ORM and CT–DNA. The free energy landscape of the binding process was computed using metadynamics simulation. The simulation study results disclosed that ORM binds to the minor groove of DNA through H–bonding and π–π stacking interactions. The results of molecular docking and simulation studies corroborate the available experimental data.

Comparison of effectiveness of ormeloxifene with norethisterone in perimenopausal DUB treatment

Comparison of effectiveness of ormeloxifene with norethisterone in perimenopausal DUB treatment

Ormeloxifene-Hyaluronic acid microfibers for breast cancer therapy

Ormeloxifene (ORM) is a drug widely used for the treatment of breast cancer. It is a lipophilic molecule that possesses highly favourable pharmacokinetic and pharmacodynamic properties. The anti-cancer activity of ORM can be improved by developing a formulation that offers targeted delivery. This can be achieved by encapsulation using hyaluronic acid (HA). Since HA-targeted receptors (CD44) are over expressed in breast cancer, lung carcinoma and cervical cancer cells; HA has been used as a drug carrier to improve drug delivery to suppress cancer growth. Therefore we have developed a novel ORM incorporated HA microfiber (HA-ORM) formulation using a newly modified method and characterized for morphology, particle size, encapsulation efficiency and in vitro drug release. The particle size of all the formulations was in micron range. The SEM images showed that the microfibers were bead free and had smooth surface. The in vitro drug release of the microfibers in pH 6.8 suggests the release of drug in a controlled manner.

Pluronic Polymer-Based Ormeloxifene Nanoformulations Induce Superior Anticancer Effects in Pancreatic Cancer Cells.

Utilization of safe cytotoxic agents with precise anticancer activity is considered as the prime focus of cancer therapeutics research. A greater incentive for such agents arises from the molecules/drugs that are already being used for other indications. Ormeloxifene (ORM) is a nonsteroidal, nonhormonal selective estrogen receptor modulator (SERM), which has been in human use for contraception purposes. Although in the recent past, many reports have suggested its emerging role as an anticancer agent, no significant attention was paid toward generating simple and safe nanoformulation(s) for improved therapeutic activity and tumor cell-specific delivery. Our aim is to develop nanoformulation(s) of ormeloxifene to improve its targeted delivery in tumor cells. We developed ormeloxifene nanoformulation(s) by utilizing various biocompatible polymers. The optimized formulations with pluronic polymers F127 and F68 show improved nanoparticle characteristics. These formulations show enhanced cellular uptake that allows ormeloxifene’s intracellular availability. We further evaluated its improved anticancer activity by performing cell proliferation, flow cytometry, and immunoblotting assays. Overall, this study confirms possible novel nanoformulation(s) of ormeloxifene to be evolved as a new therapeutic modality for cancer treatment.

Ormeloxifene nanotherapy for cervical cancer treatment.

BackgroundCervical cancer (CxCa) ranks as the fourth most prevalent women-related cancer worldwide. Therefore, there is a crucial need to develop newer treatment modalities. Ormeloxifene (ORM) is a non-steroidal, selective estrogen receptor modulator (SERM) that is used as an oral contraceptive in humans. Recent investigations suggest that ORM exhibits potent anti-cancer activity against various types of cancers. Nanoparticulates offer targeted delivery of anti-cancer drugs with minimal toxicity and promise newer approaches for cancer diagnosis and treatment. Therefore, the nanotherapy approach is superior compared to traditional chemotherapy, which is not site-specific and is often associated with various side effects.MethodsPursuing this novel nanotherapy approach, our lab has recently developed ORM-loaded poly [lactic-co-glycolic acid] (PLGA), an FDA-approved biodegradable polymer, nanoparticles to achieve targeted drug delivery and improved bioavailability. Our optimized PLGA-ORM nanoformulation showed improved internalization in both dose- and energy-dependent manners, through endocytosis-mediated pathways in both Caski and SiHa cell lines. Additionally, we employed MTS and colony forming assays to determine the short- and long-term effects of PLGA-ORM on these cells.ResultsOur results showed that this formulation demonstrated improved inhibition of cellular proliferation and clonogenic potential compared to free ORM. Furthermore, the PLGA-ORM nanoformulation exhibited superior anti-tumor activities in an orthotopic cervical cancer mouse model than free ORM.ConclusionCollectively, our findings suggest that our novel nanoformulation has great potential for repurposing the drug and becoming a novel modality for CxCa management.

A triphenylethylene nonsteroidal SERM attenuates cervical cancer growth

Selective estrogen receptor modulator drug molecules of triphenylethylene family have gained considerable attention as anti-cancer agents. Despite recent advances in screening and development of HPV vaccines, cervical cancer remains one of the deadliest malignancies as advanced stage metastatic disease is mostly untreatable, thus warrants newer therapeutic strategies. Ormeloxifene (ORM) is a well-known SERM of triphenylethylene family that has been approved for human use, thus represents an ideal molecule for repurposing. In this study, we for the first time have demonstrated the anti-cancerous properties of ormeloxifene in cervical cancer. Ormeloxifene efficiently attenuated tumorigenic and metastatic properties of cervical cancer cells via arresting cell cycle at G1-S transition, inducing apoptosis, decreasing PI3K and Akt phosphorylation, mitochondrial membrane potential, and modulating G1-S transition related proteins (p21, cyclin E and Cdk2). Moreover, ORM repressed the expression of HPV E6/ E7 oncoproteins and restored the expression of their downstream target tumor suppressor proteins (p53, Rb and PTPN 13). As a result, ormeloxifene induces radio-sensitization in cervical cancer cells and caused potent tumor growth inhibition in orthotopic mouse model. Taken together, ormeloxifene represents an alternative therapeutic modality for cervical cancer which may have rapid clinical translation as it is already proven safe for human use.

Abstract 3636: Generation of polymeric nanoformulation of ormeloxifene for cervical cancer treatment

Abstract Background: Cervical cancer (CxCa) is one of the most common death related cancers among women around the world and associated with poor 5-year survival rate. Therefore, there is an urgent need to develop newer treatment modalities. Ormeloxifene (ORM) is a non-steroidal, Selective Estrogen Receptor Modulator (SERM) that is used as an oral contraceptive in humans. Recent investigations suggest that ORM exhibits potent anti-cancer activity against various types of cancers. Nanoparticulates offer targeted delivery of anti-cancer drugs with minimal toxicity and promise newer approaches for cancer treatment. Therefore, nanotherapy approach outstands over traditional chemotherapy which is not site specific and often associated with various side effects. Thus, pursuing this novel nanotherapy approach, we have developed ORM nanoformulation using PLGA (poly [lactic-co-glycolic acid]); an FDA approved biodegradable polymer. Methods: We generated ORM loaded PLGA nanoformulation (PLGA-ORM) employing nanoprecipitation method. PLGA-ORM was characterized for its physicochemical properties such as particle size, FT-IR, DSC and drug loading. We next performed cellular uptake studies in Caski and SiHa cell lines at different concentrations (5, 10, 20 and 25 µM) and temperatures (4° and 37° C) utilizing fluorescent microscope, flow cytometer and TEM. To evaluate the cellular uptake mechanism of PLGA-ORM, we did another flow cytometer experiment using various endocytosis pathways’ inhibitors. Next, we determined anti-proliferative activities of PLGA-ORM by performing MTS and colony formation assays. Furthermore, we investigated anti-tumoral functions of PLGA-ORM in an orthotopic mice model of cervical cancer. Results: Our optimized PLGA-ORM showed particle size around 250 nm measured by DLS. ORM was completely miscible in this formulation as indicated by FT-IR and DSC spectra, which resulted in excellent drug loading of about 80% done by HPLC. PLGA-ORM nanoformulation exhibited improved internalization in dose, time and energy dependent manner through endocytosis mediated pathways in both Caski and SiHa cell lines. Additionally, we employed MTS and colony forming assays to determine the short- and long-term effects of PLGA-ORM on these cells, results showed that this formulation had an improved inhibition of cellular proliferation and clonogenic potential compared to free ORM. Furthermore, PLGA-ORM nanoformulation exhibited superior anti-tumorous activities in an orthotropic cervical cancer mouse model compared to free ORM. Conclusion: Our findings suggest that, this PLGA-ORM nanoformulation has great potential for repurposing the drug and becoming a novel modality for cervical cancer management which needs to be developed as a lead therapy approach with appropriate clinical investigations. Citation Format: Neeraj Chauhan, Murali M. Yallapu, Diane M. Maher, Bilal B. Hafeez, Mohammed Sikander, Meena Jaggi, Subhash Chauhan. Generation of polymeric nanoformulation of ormeloxifene for cervical cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3636.

Abstract 4809: Bromo-ormeloxifene inhibits epithelial mesenchymal transition via targeting β-catenin signaling pathways in cervical cancer cells

Abstract Background: Cervical cancer (CxC) is a leading cause of mortality and morbidity among women worldwide. Current chemotherapeutic agents for CxC have shown systemic toxicity in CxC patients. Ormeloxifene (ORM) is a non-toxic and non-steroidal drug with well-defined pharmacokinetic and pharmacodynamic properties in humans. Studies have shown its anti-cancer potential in various pre-clinical mouse models. Here, we have synthesized and characterized a novel analogue of ormeloxifene, Bromo-ormeloxifene (Br-ORM), which showed more therapeutic efficacy against CxC in vitro and in vivo model systems. Methodology: The effect of Br-ORM on CxC cells (CaSki and SiHa) growth and proliferation was determined by colony formation and MTS assays. Molecular docking of Br-ORM with β-catenin was done by AutoDock4 software. Effect of Br-ORM on the expression of epithelial-to mesenchymal (EMT) markers (N-cadherin, slug, snail), MMPs (MMP2 and MMP3) and miR-200a was analyzed by Western blot and qPCR analyses respectively. Apoptosis analysis was done by Annexin-V staining kit. Effect of Br-ORM on β-catenin cellular localization in CxC cells was analyzed by immunofluorescence analysis. The anti-tumor efficacy of Br-ORM was investigated in orthotopic xenograft mouse model of CxC. Results: Br-ORM (10-20 µM) effectively inhibited growth and proliferation of CxC cells in a dose and time-dependent manner as compared to ORM. Br-ORM efficiently suppressed metastatic phenotypes of CxC cells as determined by significant (P&amp;lt;0.05) decrease in invasion and migration potential of CxC cells. Moreover, Br-ORM showed increased apoptosis, which was observed by enhanced Annexin-V staining and PARP protein cleavage. Br-ORM markedly reduced the EMT process as evident by repression of N-cadherin, slug, snail, MMPs (MMP2 and MMP3) and β-catenin/TCF-4 transcriptional activity. Br-ORM potently reduced the translocation of β-catenin in the nucleus. Bioinformatic analysis revealed that Br-ORM proficiently binds into active site of β-catenin with a minimum energy -7.6 kcal/mol. Br-ORM treatment replenished the expression of miR-200a, which directly targets β-catenin in CxC cells. Br-ORM treatment (250 µg/mouse, three times a week) significantly (P&amp;lt;0.01) regressed the cervical tumor growth in orthotopic xenograft mouse model. Similar molecular effects of Br-ORM were observed in excised tumor tissues. Conclusion: These results suggest that Br-ORM inhibits the metastatic phenotypes of CxC cells via targeting β-catenin signaling pathway. Br-ORM could be used as a novel therapeutic modality for the treatment of CxC. Citation Format: Mohammed Sikander, Shabnam Malik, Bilal B. Hafeez, Sonam Kumari, Sheema Khan, John Apraku, Hassan Mandil, Andrew E. Massey, Aditya Ganju, Parvez Khan, Fathi T. Halaweish, Subhash C. Chauhan, Meena Jaggi. Bromo-ormeloxifene inhibits epithelial mesenchymal transition via targeting β-catenin signaling pathways in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4809.

Abstract 5777: Ormeloxifene augments the therapeutic response of enzalutamide via targeting androgen receptor splice variant 7

Abstract Enzalutamide (ENZ) is a second generation anti-androgen drug in clinical use for the treatment of advanced prostate cancer (PrCa). Use of ENZ has some limitations because of its toxic side effect and developing chemoresistance. It has been shown that expression of androgen receptor splice variant 7 (ARV7) in advanced PrCa is involved in ENZ therapy resistance. It is noteworthy that ENZ does not target AR splice variants because these lack the ligand binding domain of AR. Thus, identification of non-toxic agents which can target ARV7 can improve the enzalutamide therapy response and could be used for the management of advanced stage PrCa. Herein, we identified a clinically approved selective estrogen receptor modulator ormeloxifene (ORM), which augments ENZ therapy response via suppression of ARV7 expression in PrCa cells. We used androgen-independent human prostate cancer cells (22Rv1), which express ARV7 as a model system to study the effect of ORM alone or in combination with ENZ. ORM treatment showed a more potent effect in 22Rv1 cells as compared to ENZ alone. ORM treatment also sensitized the effects of ENZ in 22Rv1 cells. ORM treatment effectively inhibited colony formation ability of 22Rv1 cells; this effect was additive in combination with ENZ. We next performed Western blot analysis of ARV7 in control and ORM treated 22Rv1 cells. ORM effectively inhibited the protein level of AR V7 at 12 hrs post-treatment in 22Rv1 cells; however, no effect was observed with the treatment of ENZ. ORM treatment also inhibited the promoter activity of the AR target gene PSA as determined by luciferase assay. To translate these finding to an in vivo model system, we investigated the chemo-sensitization potential of ORM in a 22Rv1 cell-derived xenograft mouse model. ORM treatment (200 µg/mouse three times a week for 8 consecutive weeks) significantly (P&amp;lt;0.01) inhibited prostate tumor growth. However, we did not observe any significant tumor growth inhibition with oral administration of ENZ (10 mg/kg body weight). ORM treatment improved the therapeutic response of ENZ in 22Rv1 cells derived xenograft mouse model as monitored by significant (P&amp;lt;0.01) decreases in both tumor volume and tumor weight as compared to ENZ treatment alone. Our findings indicate that ORM alone or in combination with ENZ could be an effective therapeutic approach for the treatment of advanced PrCa. Citation Format: Bilal B. Hafeez, Andrew E. Massey, Vivek K. Kashyap, Mohammed Sikander, Advit Shetty, Mehdi Chaib, Hassan Mandil, Shabnam Malik, Mohan M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Ormeloxifene augments the therapeutic response of enzalutamide via targeting androgen receptor splice variant 7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5777.

Abstract 2110: Therapeutic intervention for pancreatic cancer using autologous exosomes

Abstract Objective: Pancreatic cancer (PanCa) is the third deadliest cancer in United States with a poor survival rate. Despite extensive research efforts, there is not any substantial progress in cancer therapeutics due to impediments against intracellular drug delivery. Thus, novel delivery vehicles are required that are biocompatible and non-immunogenic. This is possible by utilizing an autologous biological material as delivery vehicles that can be applied as a personalized medicine. Towards this, our lab has optimized an exosomes based therapeutic approach, which utilizes exosomes isolated from the cultured tumor adjacent normal (NAT) fibroblast cells. We utilized this scaffold for safe and effective delivery of therapeutic payload. Methods: Following surgical resection, freshly collected, matched tumor adjacent normal tissues were utilized to isolate fibroblasts. Exosomes were purified from the isolated fibroblasts in presence of exosome-depleted FBS media. Physico-chemical characterization (DLS, TEM), presence of exosomal marker (CD63; immunoblotting), protein concentration (Bradford assay), cellular internalization (confocal microscopy) and miR-145 expression (qRT-PCR) in the exosomes were performed. Efficacy of exosomes to deliver therapeutics was investigated utilizing our recently identified drug, ormeloxifene (ORM). Drug encapsulation and loading efficiency was performed using UPLC. Functional assays, such as, proliferation were performed using clinically relevant PanCa cell lines (HPAF-II/AsPC-1). Results: NAT derived exosomes show effective size and zeta potential (size: 44.12 ± 0.89; Zeta potential: -14.9 mV), which is ideal for drug delivery purposes. The purification of exosomes was confirmed by analyzing the expression of exosomal markers, such as CD63. Immunofluorescence for CD63 expression confirmed the efficient delivery of exosomes in PanCa cells. Our results indicated high drug loading capacity of NAT derived exosomes as demonstrated though UPLC. ORM loaded exosome treatment efficiently delivered ORM into the cancer cells and inhibited tumorigenic features such as, proliferation of PanCa cells. Additionally, NAT derived exosomes showed enhanced expression of tumor suppressor microRNA, miR-145, suggestive of their therapeutic importance. We observed restoration of lost miR-145 levels in PanCa cells on incubation with NAT derived exosomes. This further indicates relevance for their utilization in the development of novel therapeutic modalities. Conclusion: Our observations offer importance of the utilization of NAT derived exosomes for personalized medicine as a therapeutic strategy in PanCa treatment. Citation Format: Saini Setua, Sheema Khan, Murali Yallapu, Sonam Kumari, Meena Jaggi, Subhash C. Chauhan. Therapeutic intervention for pancreatic cancer using autologous exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2110.

Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

Autophagy, a regulated nutrient recycling program can affect both cell survival and cell death. Here, we show that Ormeloxifene (ORM), a selective estrogen receptor modulator approved for oral contraceptive use induces autophagic flux in ovarian cancer cells, which is activated by an ER stress response upstream of autophagy. The ER stress response is characterized by activation of IRE1α, PERK and ATF6 and is under regulation of JNK. Pharmacological inhibition of either autophagy or ER stress increased cell survival, as did silencing of autophagy proteins LC3 and Beclin 1, implying that ORM-induced autophagy is pro-death in nature. Ultrastructural observations of treated cells confirmed stages of autophagic maturation. Caspase-dependent apoptosis succeeded these events and was characterized by generation of reactive oxygen species and disruption of mitochondrial membrane potential. A concomitant inhibition of the Akt/mTOR axis was also observed with possible regulation of Akt by ORM. ORM inhibited tumor growth in ovarian xenograft model and displayed autophagic activity. In summary, in vitro and in vivo results reveal that ORM induces autophagy-associated cell death to attenuate proliferation of ovarian cancer cells. Our results demonstrate that using ORM in combination with ER stress and autophagy modulators could offer better therapeutic outcome in ovarian cancer.

Abstract 2101: Ormeloxifene suppresses the growth of prostate tumor via inhibition of β-catenin induced AR signaling

Abstract Background: Prostate cancer (PrCa) first manifests as an androgen-dependent disease and can be treated with androgen-deprivation therapy. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy displays by progression to hormone refractory (androgen-independent) advanced stage PrCa which is primary cause of patient’s death. Main underlying cause for the onset of hormone refractory cancer is ligand independent activation of AR signaling in PrCa cells. It has been shown that β-catenin acts as a non-androgen activator of AR which enhances AR transactivation in PrCa cells. Thus, identification of agents with excellent pharmacokinetics and pharmacodynamics parameters that can inhibit ligand independent activation of AR signaling might be highly useful for the treatment of advanced stage PrCa. Herein, we identified a synthetic molecule, ormeloxifene (ORM), which efficiently represses β-catenin mediated ligand independent activation of AR signaling, thus, inhibits growth and metastatic features of PrCa cells. Methods: Androgen-refractory but AR positive PrCa cell (C4-2) was used as an in vitro and in vivo model systems. Effect of ORM on AR and PSA protein levels was determined by Western blot analysis. Effect of ORM treatment was analyzed on AR and PSA luciferase activities by transiently transfecting the C4-2 cells by AR and PSA luciferase plasmids. Renilla construct was used as an internal control. C4-2 cells nuclear and cytoplasmic lysates were prepared using Active Motif kit. Immunoprecipitation analysis was performed to determine if ORM inhibits physical interaction of β-catenin with AR. Therapeutic efficacy of ORM was evaluated in cell lines and PrCa xenograft mouse models. Results: ORM dose-dependently (10, 15 and 20 µM) inhibited the protein levels of AR and its downstream target protein PSA. ORM (10 and 20μM) treatment also inhibited AR transactivation as determined by decreased promoter activities of AR and its target gene PSA. ORM (10 and 20μM) treatment inhibited protein levels of nuclear β-catenin and physical interaction of β-catenin with AR in PrCa cells. ORM administration dose- dependently (intra-peritoneal; 100 and/or 500µg/mouse; thrice/week) significantly (P&amp;lt;0.01) inhibited growth of C4-2 cells derived xenograft tumors in athymic nude mice. ORM treatment significantly (P&amp;lt;0.01) inhibited the expressions of nuclear AR and β-catenin expressions in xenograft tumor tissues. These ORM treated mice did not show any apparent toxicity in our study. Conclusion: Our study demonstrates that ORM is a potent inhibitor of β-catenin-mediated activation of AR signaling. Based on its safety profile, ORM might be an ideal candidate for repurposing to treat advanced stage PrCa alone or in combination with other therapies. Citation Format: Aditya Ganju, Bilal Bin Hafeez, Mohammed Sikander, Vivek Kumar Kashyap, Murali Mohan Yallapu, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene suppresses the growth of prostate tumor via inhibition of β-catenin induced AR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2101. doi:10.1158/1538-7445.AM2017-2101

Abstract 3862: Ormeloxifene, a novel pharmacological activator of PKD1 enhances docetaxel sensitivity in prostate cancer

Abstract Background: Prostate cancer (PrCa) is the second leading cause of cancer-related deaths in American Men. Docetaxel (DTX) is a standard first-line treatment for metastatic castration-resistant PrCa after the failure of hormone therapy. However, most PrCa patients who receive DTX experience only transient benefits and rapidly develop incurable drug resistance. Protein Kinase D1 (PKD1), one of the serine threonine kinases from PKD family is highly expressed in normal prostate tissues and is suppressed during PrCa progression. Accumulative evidence suggest a tumor suppressive role of PKD1 in PrCa, while other isoforms of PKD (PKD2 and PKD3) act as oncogene. In this study, we identified pharmacological agent Ormeloxifene (ORM) which selectively activates PKD1 and inhibits metastasis associated protein 1 (MTA1), thus induces sensitivity to DTX treatment in PrCa cells. Materials and Methods: We have used androgen-independent human PrCa cells (C4-2) which show low PKD1 expression compared to other PrCa cell lines. Cells were treated with 10 and 15 μM doses of ORM for 24 hrs and various functional assays (cell proliferation, colony formation, motility and invasion) were performed. In a parallel experiment, cells were treated with ORM (10 and 15 μM) for 24 hrs protein and RNA samples isolation. Protein lysates were used to investigate the effect of ORM on PKD1, PKD2, PKD3 and MTA1 protein levels. qRT-PCR was performed to investigate the effect of ORM on PKD1, PKD2 and PKD3 expression at mRNA levels. To investigate if ORM treatment sensitizes the effects of DTX, cells were treated with ORM and DTX alone or in combination. In-silico docking studies were performed to determine the putative molecular interaction of ORM with MTA1. Results: ORM treatment inhibits proliferation and clonogenic potential of C4-2 cells. We observed that ORM significantly induces PKD1 expression at protein and mRNA level in C4-2 cells. To determine whether this PKD1 inducing effects of ORM in PrCa cells is specific, we examined the effects of ORM on PKD2 and PKD3 at mRNA and protein levels. Interestingly, we observed that ORM treatment inhibits expression of oncogenic PKD3 isoform, however, no effect PKD2 was observed. MTA1 is involved in DTX resistance and ORM treatment effectively inhibited the expression of MTA1. However, there was no effect of DTX treatment on the expression of MTA1. We also observed that ORM treatment significantly potentiates the effect of DTX on cell viability and colony formation of C4-2 cells. In-silico docking studies between ORM and MTA1 showed four potential binding sites with best score at serine 270. Conclusion: Overall, our study defines ORM as a novel PKD1 activator/modulator which also inhibits a key metastasis associated protein, MTA1 and sensitizes the PrCa cells to DTX. Based on these results, it appears that ORM may be a novel therapeutic modality for advanced stage metastatic PrCa alone or in combination with DTX. Citation Format: Aditya Ganju, Bilal Bin Hafeez, Fathi Halaweish, Wei Li, Man Mohan Singh, Murali Mohan Yallapu, Subhash Chauhan, Meena Jaggi. Ormeloxifene, a novel pharmacological activator of PKD1 enhances docetaxel sensitivity in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3862.

Abstract 4392: Generation of a novel ormeloxifene nanoparticle formulation for pancreatic cancer treatment

Abstract Pancreatic cancer (PanCa) is the fourth leading cancer with 85% mortality rate in the United States. Therefore, there is an utmost need to discover new modalities that can result in enhanced therapeutic efficacy with low to minimal side effects. Ormeloxifene (ORM) is a synthetic molecule which is widely used as an oral contraceptive in humans. Numerous studies indicate that ORM exhibits potent anti-cancer effects through inhibition of important oncogenic signaling events in several cancers. Our studies also demonstrate the anticancer effects of ORM in various cancer cells. However, the ORM effects can be improved by encapsulating in a nanoformulation which provides targeted delivery of ORM to the tumors. Therefore, in order to increase the therapeutic efficacy of ORM, we have developed a novel ORM encapsulated poly(lactic-co-glycolic acid) formulation (PLGA-ORM NPs). This formulation has been characterized for particle size, zeta potential, chemical composition, drug loading efficiency using various physico-chemical methods such as DLS, TEM, FT-IR, DSC, and TGA. Because of its facile composition (PLGA core, PVA, PLL and PEG-linker) this novel formulation is compatible for antibody/aptamer conjugation to achieve tumor specific targeting. The PLGA-ORM formulations indicate efficient encapsulation of ORM. The particle size of PLGA-ORM formulation (∼ 100 nm) indicates that this formulation can preferentially reach and accumulate in tumors by the Enhanced Permeation and Retention (EPR) effect. The uptake and internalization studies demonstrate that PLGA-ORM NPs escape lysosomal degradation providing its efficient endosomal release to cytosol in PanCa cells. PLGA-ORM NPs showed superior anti-cancer potential in various pancreatic cancer cells (HPAF-II, BXPC-3, Panc-1, MiaPaca) and in BXPC-3 xenograft mice. PLGA-ORM NPs suppressed pancreatic xenograft tumor growth and improved the mice survival. In addition, PLGA-ORM NPs also reduce the metastasis potential. PLGA-ORM NPs inhibit tumorigenic and metastatic phenotypes via suppression of AKT phosphorylation and inhibition of key oncogenes involved in pancreatic progression such as MUC1, HER2 and CD31. Additionally, PLGA-ORM NPs treated xenograft tumors showed reduced staining of the proliferating cell nuclear antigen (PCNA), cytokeratin-19 (CK19), MUC1, HER2 and CD31 in immunohistochemical analysis. In conclusion, this study suggests that PLGA-ORM formulation is highly efficient for the inhibition of pancreatic tumor growth and can be valuable for the treatment of pancreatic cancer in future. Citation Format: Sheema Khan, Neeraj Chauhan, Murali M. Yallapu, Mara C. Ebeling, Swathi Balakrishna, Robert T. Ellis, Paul A. Thompson, Stephen W. Behrman, Nadeem Zafar, Man M. Singh, Fathi T. Halaweish, Meena Jaggi, Subhash C. Chauhan. Generation of a novel ormeloxifene nanoparticle formulation for pancreatic cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4392. doi:10.1158/1538-7445.AM2015-4392

Rapid quantitative analysis of ormeloxifene and its active metabolite, 7-desmethyl ormeloxifene, in rat plasma using liquid chromatography-tandem mass spectrometry.

Ormeloxifene (ORM) is a non-steroidal, selective estrogen receptor modulator used as a once a week oral contraceptive and is intended for long-term clinical use by females. Therefore, a simple, sensitive and rapid LC-MS/MS method was developed and validated for simultaneous quantification of ORM and its active metabolite (7-desmethyl ormeloxifene, 7-DMO) in rat plasma. Also, the method was partially validated in human plasma. Chromatographic separation was achieved on Discovery HS C-18 column (5μm, 50×4.6mm) with mobile phase [acetonitrile: aqueous ammonium acetate (0.01M) buffer (85: 15, %v/v)] at a flow rate of 0.8mL/min. The calibration curve was linear (r≥0.99) for a concentration range of 0.78-100ng/mL for both the analytes. The precision (%RSD; ORM, 1.3 to 13.4; 7-DMO, 3.1 to 15.0) and accuracy (%bias; ORM, -13.8 to 12.5; 7-DMO, -10.6 to 6.8) in both rat and human plasma were within the acceptable limits. The recovery for ORM and 7-DMO was always >79.1% and >72.9%, respectively. Both the analytes were found stable in rat plasma even after 30 days of storage at -80°C and on being subjected to three freeze-thaw cycles. The method has not only short run time (3.5min) but requires a low plasma sample volume (20μL) and is the first reported LC-MS/MS method for simultaneous quantification of the marketed drug known as centchroman (INN: ormeloxifene) and the metabolite 7-DMO in plasma. The method was applied to evaluate drug-drug interaction of ORM with the commonly prescribed antidepressant drug sertraline using serial sampling in rats.

Nanoparticle formulation of ormeloxifene for pancreatic cancer

Pancreatic cancer is the fourth most prevalent cancer with about an 85% mortality rate; thus, an utmost need exists to discover new therapeutic modalities that would enhance therapy outcomes of this disease with minimal or no side effects. Ormeloxifene (ORM), a synthetic molecule, has exhibited potent anti-cancer effects through inhibition of important oncogenic and proliferation signaling pathways. However, the anti-cancer efficacy of ORM can be further improved by developing its nanoformulation, which will also offer tumor specific targeted delivery. Therefore, we have developed a novel ORM encapsulated poly(lactic-co-glycolic acid) nanoparticle (NP) formulation (PLGA-ORM NP). This formulation was characterized for particle size, chemical composition, and drug loading efficiency, using various physico-chemical methods (TEM, FT-IR, DSC, TGA, and HPLC). Because of its facile composition, this novel formulation is compatible with antibody/aptamer conjugation to achieve tumor specific targeting. The particle size analysis of this PLGA-ORM formulation (∼100 nm) indicates that this formulation can preferentially reach and accumulate in tumors by the Enhanced Permeability and Retention (EPR) effect. Cellular uptake and internalization studies demonstrate that PLGA-ORM NPs escape lysosomal degradation, providing efficient endosomal release to cytosol. PLGA-ORM NPs showed remarkable anti-cancer potential in various pancreatic cancer cells (HPAF-II, AsPC-1, BxPC-3, Panc-1, and MiaPaca) and a BxPC-3 xenograft mice model resulting in increased animal survival. PLGA-ORM NPs suppressed pancreatic tumor growth via suppression of Akt phosphorylation and expression of MUC1, HER2, PCNA, CK19 and CD31. This study suggests that the PLGA-ORM formulation is highly efficient for the inhibition of pancreatic tumor growth and thus can be valuable for the treatment of pancreatic cancer in the future.