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Pancreatic cancer (PanCa) is characterized by lack of early diagnosis, poor response to available therapeutic modalities and chemoresistance. Gemcitabine (GEM) is currently considered most effective therapy for PanCa; however, it shows only a marginal survival benefit of 6 months. This poor drug response has been attributed to desmoplasia, causes suboptimal drug delivery, alters tumor microenvironment (TME), which includes tumor surrounding blood vessels, fibroblasts, immune cells, extracellular matrix, and other signaling molecules and induces chemo-resistance in tumors. To overcome these existing issues associated with chemotherapy, identification and development of novel therapeutic modalities are a pressing need. Piperlongumine (PL) is a natural alkaloid isolated from the long pepper, Piper longum L., and has shown substantial cancer-preventive and therapeutic efficacy against a variety of cancers. However, delivering its effective concentration in pancreatic tumors has been challenging. We have recently engineered a multi-layered Pluronic F127 and polyvinyl alcohol stabilized and poly-L-lysine coated piperlongumine loaded poly(lactic-co-glycolic acid) nanoparticle formulation (PLGA-PL), which effectively inhibits the growth of PanCa cells. In this study, we demonstrate that PLGA-PL effectively sensitize tumor cells to GEM via decreased desmoplasia, altered TME, SHH/CXCL12/CXCR4 and immune surveillance. Our finding show that PLGA-PL synergizes with GEM in inhibiting PanCa cell (HPAF-II and Panc-1) growth, migration, and invasion compared to free PL. Mechanistically, PLGA-PL targets the TME via inhibition of sonic hedgehog (SHH) pathway and oncogenic CXCR4/CXCL12 signaling axis that inhibits bidirectional tumor-stromal cells interaction. We have also found that PLGA-PL alone and in combination with GEM targets cancer stem cells by inhibiting pluripotency maintaining stemness factors (Nanog, Sox2, c-Myc, CD133, and Oct-4) as determined by qRT-PCR, Western blotting, and immunofluorescence analysis, and further confirmed by restricting tumor sphere formation. Furthermore, PLGA-PL also effectively targets tumor-associated macrophages (TAM) by repolarizing M2 into M1 phenotype via inhibiting expression of M2 markers and an increase in M1 markers in mouse macrophage cell line RAW264.7. M2 polarization of RAW264.7 cells were induced by culture with IL-4 (20 ng/mL) in presence of PLGA-PL or vehicle control. In additions, PLGA-PL effectively increases phagocytic capacity in murine macrophages as determined by phagocytosis assay (Vybrant Phagocytosis Assay Kit). In conclusion, we observed that PLGA-PL effectively targets TME, facilitates GEM uptake by inhibiting the activation of CXCR4/CXCL12/SHH signaling, and reprograming the tumor immune surveillance. This study suggests that PLGA-PL has great potential for future clinical use in management of PanCa. Citation Format: Vivek Kumar Kashyap, Neeraj Chauhan, Mohammed Sikander, FNU Pranav, Rahul Tiwari, Bilal B. Hafeez, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Piperlongumine nanoformulation enhances gemcitabine therapeutic response in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 823.

Background: Adequate bioimaging is crucial in cancer management in many ways including screening, detection, characterization, staging and grading, therapy response, surgical guidance, and margins assessment. Indocyanine green (ICG) is one of the FDA-approved near infra-red fluorescent (NIRF) probe for cancer imaging and image-guided surgery in clinical setting. However, limitations of ICG includes poor photostability, high concentration toxicity, short circulation time, and poor cancer cell specificity. To overcome these hurdles, we engineered a nanoconstruct composed of poly(vinyl pyrrolidone) (PVP)-indocyanine green that is cloaked self-assembled with tannic acid (termed as ICG-Glow NPs) for the cancer cells/tissues specific targeting. Methods: Pursuing the novel nanotherapy approach, our lab has developed PVP-TA based ICG (PVT-ICG) fluorescent nanoparticles via self-assembly process. Our optimized PVT-ICG nanoformulation was further characterized for its physicochemical properties. An IVIS imaging system was further used to measure NIR fluorescence of novel PVT-ICG. Moreover, Human cancer (Breast, Pancreatic, Liver and Prostate) tissue microarrays (TMAs) were histochemically stained to assess cancer cell targeting/specificity of PVT-ICG. Results: PVT-ICG indicated particle size and surface charge ideal for cancer cell/tissue delivery. PVT-ICG, further, demonstrated improved photostability and fluorescent intensity. Additionally, TMA studies exhibited enhanced internalization and cancer targeting/specificity of PVT-ICG nanoparticles compared to free ICG dye in all cancers. Conclusion: Collectively, our findings suggest that this NIR fluorescent probe PVT-ICG has great potential for becoming a novel and safe imaging modality for various types of cancer cells and tumors which can result in early cancer diagnosis leading to improved disease management. Citation Format: Neeraj Chauhan, Marco Cabrera, Pallabita Chowdhury, Prashanth K. Nagesh, Anupam Dhasmana, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. Illuminating cancer cells with a novel nano fluorescent NIR probe for bioimaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 826.

Pancreatic cancer (PanCa) is the third leading cause of cancer-related deaths in the United States with limited therapeutic options available. Gemcitabine (GEM), a deoxycytidine nucleoside analog is currently considered the most effective therapy for PanCa; however, it shows only a marginal survival benefit of 6 months. PanCa cells are addicted to ribosome biogenesis (RiBi) which supports their high rate of growth and proliferation. Therefore, strategically targeting the process of RiBi could be one of the ideal strategies for the prevention and treatment of PanCa. In this study, we for the first-time report that RPA194, a catalytic subunit of RNA Pol I is differentially expressed in normal pancreatic ductal epithelial (NPDE) and cancer cells. We also observed differential expression of RPA194 in various grades of pancreatic tumor tissues and its level was increased in high-grade pancreatic tumor tissues. We tested our hypothesis that targeting RPA194 with a non-toxic pharmacological inhibitor (BMH-21) will inhibit the growth of advanced PanCa. We observed that BMH-21 inhibits the growth and induced apoptosis of PanCa cells in a dose-dependent manner and this inhibition was correlated with the expression of RPA194. We observed proteasomal mediated degradation of RPA194 in PanCa cells. BMH-21 treatment inhibited RPA194 occupancy on rDNA but did not affect other nucleolar proteins (UBF, fibrillarin, nucleolin, and nucleophosmin). Two times the effective concentration of BMH-21 (5 µM) did not show any cytotoxic effect in NPDE cells. However, BMH-21 degraded RPA 194 protein in these cells in a similar manner as in PanCa cells. Therefore, we speculate that other proteins are involved in the selective toxicity of BMH-21 in PanCa cells. These findings were translated to in vivo model systems. We observed that BMH-21 significantly (P Citation Format: Mudassier Ahmad, Carlos Perez, Andrew Massey, Vivek Kashyap, Neeraj Chauhan, Haider Ahsan, Jasmine Jasmine, Manish Tripathi, Subhash Chauhan, Bilal Hafeez. Targeting ribosome biogenesis addition is a novel strategy for pancreatic cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B035.

Background: Lung cancer is a predominant cause of morbidity and mortality across the world. Among all types of cancers, lung cancer is the leading cause of cancer mortalities in the United States. Non-small cell lung cancer is one of the dominant divisions of lung cancer that solely represents 85-90% of all lung cancer cases. Up to the present time, primary treatment regimens for this cancer include radiation therapy, chemotherapy, and surgery, with chemotherapy being the best option thus far. Despite the eminent benefits of chemotherapy, its value is offset by severe side effects such as renal and/or hepatic toxicity or insufficient amounts of drug available at the target site. Such pitfalls can be handled by employing natural compounds (polyphenols, phytochemicals, xanthonoid, etc.,) as chemopreventives or chemosensitizers which can improve chemotherapy activity while reducing its systemic side effects and drug resistance. To this end, our recent efforts demonstrated a synergistic therapeutic benefit of gambogic acid (GA) and gemcitabine (Gem) against lung cancer. However, simultaneous delivery of these two drugs at the tumor site is highly challenging. Therefore, development of an injectable nanoformulation that can effectively deliver both hydrophobic (GA) and hydrophilic (Gem) drugs in one formulation is a clinically unmet need. Methods: Pursuing the novel nanotherapy approach, our lab has developed an in-situ biodegradable and biocompatible human serum albumin (HSA) and tannic acid (TA) mediated complexed GA and Gem nanoparticles (G-G@HTA NPs) using the solvent evaporation method. G-G@HTA NPs formation was confirmed by particle size, FT-IR, and H-NMR. A superior therapeutic activity of G-G@HTA NPs was demonstrated by multiple in vitro functional assays as well as in an animal mouse model. Results: Our results confirmed that G-G@HTA NPs have the ideal particle size and surface charge for cancer cell/tissue delivery which can clearly be evident by preferential uptake of these nanoparticles in lung cancer cells. Further, G-G@HTA NPs superiorly inhibited cell proliferation and clonogenicity of NSCLC cells. Additionally, G-G@HTA NPs revealed an obvious and precise targeting of tumors in vivo. The promoted and more synergistic anti-tumor efficacy of G-G@HTA NPs was attained than that of combined treatments and single drugs treatments. These events were resulted with no apparent systemic and organ toxicities. Conclusion: In summary, this study details the design of a novel nanocarrier and provides an optimized strategy for constructing dual-loaded nanoparticles using a biodegradable, non-toxic, human serum albumin-tannic acid-based platform for GA and Gem co-delivery in the treatment of NSCLC which confirms a strong feasibility to implement such synergistic nanomedicine regimen in pre-clinical and clinical translations in future. Citation Format: Elham Hatami, Prashanth K. Nagesh, Neeraj Chauhan, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. A novel in-situ nanoparticle self-assembly for combination delivery of therapeutics to non-small cell lung cancer [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 5073.

Introduction: The tumor microenvironment is a dynamic domain and communication hub for signaling molecules, contributing to proliferative and metastatic behaviors observed in cancerous tissues. Intercellular interactions are commonly mediated through extracellular vesicles that carry vital information for growth and survival, a feature that is highly advantageous for cancer cells to hijack. Hepatocellular carcinoma (HCC) is one of the most rapidly increasing types of cancer in the United States. Often aggressive, HCC arises from the uncontrollable division of hepatocytes, which may disrupt the regular functions of the liver and other organs. Identifying biomarkers is essential to potentiate drug treatments while minimizing chances of metastasis, recurrence, and mortality. Highly proliferative cells are known to secrete exosomes containing tumor-enhancing biomolecules that contribute to cancerous progression. Thus, analyzing exosomal cargo in serum is especially attractive for early diagnostics, profiling, and therapeutic purposes. We have explored potential oncogenic lncRNAs and extracellular signaling proteins in HCC and conducted phenotypic studies to observe their mode of action. Methods: HCC cell lines were propagated in EMEM containing exosome-free FBS. Exosomes were harvested with ThermoFisher Total Exosome Isolation Reagent from cell culture media, then resuspended in PBS. Zetasizer was used to measure the sizes of harvested exosomes, and western blot analysis was used to confirm exosomal markers. UCA1 and MALAT1 were checked via RTPCR via exosomes spiked with bacterial RNA as the loading control. Bradford assays were performed on exosomes lysed with RIPA for quantifying cargo proteins. Exosomal POTE-2 was observed through western blot analysis, in addition to ELISAs to identify their presence in media. Phenotypic studies were performed using isolated exosomes. Results: The isolated exosomes were within the size range of 40-200nm, with a negative charge. The western blot of the exosomal markers confirmed their purity. RTPCR results showed LncRNAs MALAT1 and lncRNA UCA1 present in exosomes, whereas POTE-2 protein presence was confirmed with western blot. Phenotypic studies from the isolated exosomes are in progress. Conclusions: Exosomes in serum are easily accessible biomolecules making them functional, less invasive targets for profiling, diagnostics, and therapeutics via liquid biopsies. Exosome-mediated delivery of lncRNAs MALAT1 and UCA1 to primary CRC cells has been found to promote malignancy. We have verified their presence in exosomes secreted by metastatic cell lines, in addition to oncoprotein POTE-2, which hold numerous significances for our phenotypic studies. These findings reaffirm exosomes as essential contributors to disease pathogenesis and present them as promising targets for diagnostics and treatment. Citation Format: Adithya Anilkumar, Samantha Lopez, Sophia Leslie, Kyle Doxtater, Neeraj Chauhan, Bilal Hafeez, Murali Yallapu, Meena Jaggi, Subhash Chauhan, Manish Tripathi. Exosomes as nanocarriers for biomolecules and potential diagnostic targets in cancer [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 2790.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States with limited therapeutic approaches. Dysregulation of the ribosome biogenesis process was observed in various cancer including pancreatic cancer. Therefore, strategically targeting ribosome biogenesis could be a novel approach for pancreatic cancer treatment. One such approach is to target ribosome biogenesis is via small molecule inhibitors of RNA Polymerase I as these inhibitors have shown promising anti-cancer activity in pre-clinical and clinical studies. We observed differential constitutive expression of various ribosomal proteins including RPA-194 in human PanCa cells when compared with normal HPDE cells. Therefore, we hypothesized that targeting RNA pol I will inhibit the growth of pancreatic cancer. In this study, we for the first time evaluated the therapeutic effect of a novel RNA polymerase I inhibitor (BMH-21) against PanCa in pre-clinical model systems. BMH-21 significantly inhibited the ribosome biogenesis process in various pancreatic cancer cells (Panc-1, AsPC1, BxPC3, MiaPaCa-2) as determined by inhibition of rRNA synthesis and POL I occupancy on rDNA. BMH-21 treatment of pancreatic cancer cells significantly degraded RPA194 protein as determined by Western blot and confocal microscopy analysis. BMH-21 treatment (0.25-5µM) showed differential IC50 in various pancreatic cancer cells which were correlated with the expression of its target proteins RPA194 and RPA135 expression. In our flow cytometry experiment, we observed a significant G0/G1 cell cycle arrest and increase Annexin V and Annexin V7-AAD positive cells in BMH-21 treated pancreatic cancer cells as compared to the vehicle treatment group suggesting its apoptosis-inducing potential in pancreatic cancer cells. BMH-21 treatment inhibited growth and metastatic phenotypes of various PanCa cells as analyzed by various functional assays (colony formation and atomic force microscopy analyses). We also evaluated the effect of BMH-21 on various kinases, which regulate ribosome biogenesis. Interestingly, we observed BMH-21 significantly (P Citation Format: Carlos Perez, Mudassier Ahmad, Andrew Massey, Asif Shahriar, Emmanuel Anning, Vivek Kashyap, Neeraj Chauhan, Anupam Dhasmana, Manish Tripathi, Subhash Chauhan, Bilal Hafeez. Targeting ribosome biogenesis is a novel strategy to suppress the growth of pancreatic cancer [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 3950.

Background: Pancreatic cancer remains 3rd deadliest disease, with less than 7-10% survival rate. Little progress has been seen in patient’s outcome due to high desmoplasia and chemo-resistance. Immunotherapy has shown promising results in cancers, except pancreatic cancer due to their characteristic fibrotic tumor microenvironment. The therapies are unable to penetrate fibrotic tumor leading to insufficient availability of therapeutic drugs at the tumor site. A recently identified mucin, MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas, that makes it an excellent protein tumor target. This study is unique as it utilizes MUC13Ab for targeting the pancreatic tumor site and SPION nanoparticle system for delivering the stroma depleting drug (curcumin), which would help in improving immunotherapy response. Methods: The inhouse generated MUC13Ab have been conjugated with our recently developed novel patented superparamagnetic iron oxide nanoparticles (SPIONS). Conjugation efficiency of the SPION-Anti-MUC13 particles was seen through cell uptake studies, by measuring fluorescence intensity, Prussian blue staining. Invasion assay and migration assay was carried out on KPC cells. We have used female C57BL/6J black mice, orthotopic mice model for investigating targeting efficacy of MUC13-SPION-CUR. Immune checkpoint therapy (PDL-1 and CTLA-4) was administrated along with MUC13-SPION-CUR and conjugated with fluorescent indocyanine green (ICG) dye for monitoring the tumor growth. Further, immunostimulatory effect of the nano formulation was done using flow cytometry. Results: Our results showed that MUC13Ab conjugated SPIONS can efficiently internalize the PDAC cells. SPION-MUC13 using Indocyanine dye (ICG) specifically reached to the tumor site in an orthotopic syngeneic mouse model of PDAC as indicated by ICG fluorescence. Additionally, the combination formulation inhibited the tumor growth and showed more survival rate with CTLA-4. The combined treatment with CTLA-4 increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 43% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 23.8% (FoxP3+CD25+CD45+CD3+) in KrasG12D; LSL-Trp53R172H syngeneic mouse model of PDAC. Similar results were observed in SP-CUR-M13+PDL-1 group, which showed reduction in MDSCs (by 26.6%) and Tregs (by 0.1%) as compared with PDL-1 alone. Conclusion: The formulation softens up the tumors for therapies that resulted in improved response to checkpoint immunotherapies in a pancreatic orthotopic mice model. Therefore, this study indicates high significance of MUC13-SPIONS-CUR for achieving pancreatic tumor specific delivery of drugs. This study has a potential to reduce morbidity and mortality caused by the disease and improve survival in patients. Citation Format: Poornima Shaji, Ana Martinez, Melida Flores Cantu, Anupam Dhasmana, Neeraj Chauhan, Fnu Shabnam, Vincent Diego, Meena Jaggi, Stephen Behrman, Murali M. Yallapu, Subhash C. Chauhan, Sheema Khan. A novel approach to target tumor immune microenvironment and improve checkpoint immunotherapies [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 LB007.

Background: Liver cancer is the third leading cause of mortality attributed to cancer. Hepatocellular carcinoma (HCC) is the most prevalent form among various histological types accounting for 85% to 90% of primary liver cancers. As per Texas Cancer Registry, the Hispanic population had an incidence of 21.2 per 100,000 in Texas. The Rio Grande Valley, located in South Texas, is an underserved area composed highly of Hispanics. It faces multiple disparities that can attribute to increased risk factors of HCC and, thus, a higher liver cancer incidence and mortality. Due to this, there is a need to identify a new, inexpensive, and faster diagnostic biomarker in liver cancer. We have recently identified an extracellular secreted cancer antigen POTE-2, a member of the ankyrin domain-containing POTE gene family. Our preliminary data indicates high POTE-2 expression in HCC tumors. In this study, we will discuss the role of POTE-2 in HCC progression and its associated regulatory pathways. Methods: The Cancer Genome Atlas (TCGA) database of HCC patients (n=371 tumor; n=50 normal) was analyzed. Liver cancer cell lines procured from ATCC were analyzed for POTE-2 mRNA and protein expression through RT-PCR and western blot, respectively. Lentiviral-based plasmids were used for overexpression and knockdown studies for oncogenic assays. Localization of POTE-2, YAP1, and PDL1 was identified with immunofluorescence. Results: Comprehensive analysis of the TCGA database showed increased POTE-2 expression in tumors and is upregulated in all stages of HCC. Lentiviral transduction led to a change in phenotype in oncogenic assays. Modulation of POTE-2 expression led to changes in kinase activity and regulatory pathways. Immunofluorescence showed nuclear localization of YAP1 and PDL1 in the liver cancer cell lines. Conclusion: These studies will help discover novel mechanisms of POTE-2 protein function, signaling pathways, and its role in liver cancer progression. Citation Format: Samantha Lopez, Adithya Anilkumar, Kyle Doxtater, Sudhir Kotnala, Neeraj Chauhan, Murali Yallapu, Meena Jaggi, Subhash Chauhan, Manish Tripathi. Role of an ankyrin domain protein in hepatocellular carcinoma progression [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 544.

Background: Pancreatic cancer (PanCa) is expected to be the second leading cause of cancer-related death by 2030. The treatment of PanCa is highly challenging due to the extremely poor response to existing therapeutic options. Highly desmoplastic tumor microenvironment in pancreatic tumor causes suboptimal delivery of therapeutic agents in tumors that eventually resulted to chemo-resistance. Piperlongumine (PL) is a natural alkaloid isolated from the long pepper, Piper longum L., and has shown substantial cancer-preventive and therapeutic efficacy against variety of cancers. However, delivering its effective concentration in pancreatic tumors has been challenging. In this study, we have synthesized and characterized a novel nano-formulation of PL composed of a PLGA core (PLGA-PL), stabilized with polyvinyl alcohol (PVA) and coated with poly-L-lysine (PLL), and evaluated its therapeutic effects against PanCa. Methods: The various physicochemical approaches (FT-IR, DSC, TEM, TGA, and HPLC) was used to characterize the PLGA-PL formulation for particle size, chemical composition, and drug loading efficiency. Cellular uptake of PLGA-PL was achieved in incubation with PLGA-PL in PanCa cells. Further, the therapeutic efficacy of PLGA-PL was determined by using various in vitro assays (MTS, wound healing, boyden chamber, cell cycle and apoptosis assays) using PanCa cells. The effects of PLGA-PL on various key oncogenic signaling pathways were evaluated by qRT-PCR, Western blot, confocal microscopy, immunohistochemistry (IHC) analyses. Results: Our novel PLGA-PL formulation has an average size of 110 nm in dynamic light scattering and a zeta potential range of -6.52 to -7.68 mV with excellent PL loading efficiency. Cellular uptake and internalization studies show that PLGA-PL escapes lysosomal degradation, allowing for effective endosomal release into the cytosol. PLGA-PL showed superior anti-cancer activity in various PanCa cells (BxPC-3, HPAF-II, AsPC-1, Panc-1, and MIA PaCa-2) compared to free PL. Moreover, PLGA-PL showed a remarkable inhibition of the migration and invasion potential of PanCa cells. Furthermore, PLGA-PL more effectively inhibited the components of the Shh pathway and Gli targets as determined by qPCR and Western blot analysis. Additionally, PLGA-PL treatment targets cancer stem cells by regulating pluripotency, maintaining stemness factors (Oct-4, Sox2, Nanog and c-Myc), and limiting tumor sphere formation. Conclusions: Taken together, our results demonstrate that PLGA-PL nanoformulation exhibits superior anti-cancer potential than free PL against and could be used as a novel therapeutic modality for the management of PanCa. Citation Format: Vivek Kumar Kashyap, Godwin P. Darkwah, Swati Dhasmana, Neeraj Chauhan, Anupam Dhasmana, Sudhir Kotnala, Partha Laskar, Mohammed Sikander, Bilal B. Hafeez, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Novel nanoformulation of piperlongumine for pancreatic cancer therapy [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 305.

The management of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to lack of an efficient therapy and development of chemoresistance to the current standard therapy, gemcitabine. Recent studies implicate the intimate reciprocal interactions between epithelia and underlying stroma due to paracrine Sonic hedgehog (SHH) signaling in producing desmoplasia and chemoresistance in PDAC. Herein, we report for the first time that a nonsteroidal drug, ormeloxifene, has potent anticancer properties and depletes tumor-associated stromal tissue by inhibiting the SHH signaling pathway in PDAC. We found that ormeloxifene inhibited cell proliferation and induced death in PDAC cells, which provoked us to investigate the combinatorial effects of ormeloxifene with gemcitabine at the molecular level. Ormeloxifene caused potent inhibition of the SHH signaling pathway via downregulation of SHH and its related important downstream targets such as Gli-1, SMO, PTCH1/2, NF-κB, p-AKT, and cyclin D1. Ormeloxifene potentiated the antitumorigenic effect of gemcitabine by 75% in PDAC xenograft mice. Furthermore, ormeloxifene depleted tumor-associated stroma in xenograft tumor tissues by inhibiting the SHH cellular signaling pathway and mouse/human collagen I expression. Xenograft tumors treated with ormeloxifene in combination with gemcitabine restored the tumor-suppressor miR-132 and inhibited stromal cell infiltration into the tumor tissues. In addition, invasiveness of tumor cells cocultivated with TGFβ-stimulated human pancreatic stromal cells was effectively inhibited by ormeloxifene treatment alone or in combination with gemcitabine. We propose that ormeloxifene has high therapeutic index and in a combination therapy with gemcitabine, it possesses great promise as a treatment of choice for PDAC/pancreatic cancer. Cancer Res; 75(11); 2292–304. ©2015 AACR.

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.

Background: Pancreatic cancer (PanCa) is one of the leading causes of cancer-related mortality in the United States due to very limited therapeutic options. Thus, developing novel therapeutic strategies will help for the management of this disease. We recently identified VERU-111, a novel synthetic molecule which showed potent anti-cancer effect against PanCa via targeting clinically important βIII and βIV tubulin isoforms. In this study, we synthesized and characterized its novel nanoformulation (MNP-VERU) and evaluated its therapeutic effects in vitro and xenograft mouse model. Methods: MNPs were prepared by chemical precipitation method and loaded with VERU-111 using diffusion method. 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). The internalization of MNP-VERU was achieved after 6 hours incubation with MNP-VERU in PanCa cells. To determine therapeutic efficacy of MNP-VERU, we performed various in vitro (MTS, wound healing, boyden chamber real-time xCELLigence, and apoptosis assays) and in vivo (mouse tumor xenograft) studies using PanCa. Effect of MNP-VERU on various key oncogenic signaling pathways, and miRNAs was evaluated by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses respectively. Result: Our novel MNP-VERU formulation provided average size of 110 nm in dynamic light scattering (DLS) and exhibited -8.23 to -11.65 mV zeta potential with an outstanding loading efficiency (94%). Cellular uptake and internalization studies demonstrate that MNP-VERU escape lysosomal degradation, providing efficient endosomal release to cytosol. MNP-VERU showed remarkable anti-cancer potential in various PanCa cells (Panc-1, AsPC-1, HPAF-II, BxPC-3, MiaPaca) and more effectively repressed βIII and βIV tubulin isoforms via restoring the expression of miR-200c. MNP-VERU more effectively suppressed AsPC-1 cells derived xenograft tumors in athymic nude mice. Conclusions: Taken together, our results suggest that MNP-VERU has more anti-cancer potential than free VERU-111 against PanCa. MNP-VERU may reduce the toxicity and improve the bioavailability of free VERU-111 and could be used for the management of PanCa. Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Neeraj Chauhan, Prashanth K. Nagesh, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. Nanoparticle formulation of VERU 111 for pancreatic 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 3619.

Objective: Cervical cancer is one of the most common and deadly cancers among women worldwide and is associated with persistent Human Papillomavirus (HPV) infection. Human papilloma virus (HPV) expressing E6 and E7 oncoproteins involved in carcinogenesis through their interactions with the p53 and pRB pathways, respectively. Therefore, non-toxic agents that have potential to inhibit the expression of E6 and E7 oncoproteins and their regulated oncogenic signaling pathways could be used in the management of cervical cancer. Microtubule targeting agents including paclitaxel, colchicine and vinca alkaloids are widely used in the treatment of various cancers but most of these agents have toxic side effects and develop chemoresistance. Herein, we investigated the potential anti-cancer effects of a novel tubulin targeting agent (ABI-231) in in vitro and in vivo model systems. Method: ABI-231 ((2-(1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl))- methanone was synthesized in our home institution. Human cervical cancer cells (CaSki and SiHa) were used as in vitro model system. To determine the effect of ABI-231 on cell proliferation, migration and invasion, we performed MTS and wound healing assays. Effect of ABI-231 on the expression of HPV E6/E7 was determined by Western blot, qRT-PCR, and confocal microscopy. Xenograft study was performed to determine the effect of ABI-231 on cervical tumor growth. Results: ABI-231 treatment significantly (P Conclusions: Taken together, our results demonstrate the potential anti-cancer efficacy of ABI-231 in in vivo and in vivo. ABI-231 can be explored as a potent therapeutic agent for the treatment of cervical cancer. Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghai Wang, Neeraj Chauhan, Prashanth K. Nagesh, Nirnoy Dan, sonam kumari, Shabnam Malik, Saini Setua, Aditya Ganju, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan C. Chauhan. ABI-231: A novel small molecule suppresses tumor growth and metastatic phenotypes of cervical cancer cells via targeting HPV E6 and E7 [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 679.

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

Background: Human pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States. Accumulating studies have witnessed the malfunction of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), enhances patient survival by only few months. Cucurbitacins, naturally occurring dietary tetracyclic triterpenoid compounds, have shown promising anti-cancer activities. Herein, we investigated the potency and anti-cancer efficacy of a novel analogue of cucurbitacin (Cuc D) in pancreatic cancer cell lines and in a xenograft mouse model. Additionally, we determined efficacy of this analogue on MicroRNAs (miRNAs) which are important regulators of genes that have crucial roles in pancreatic tumorigenesis. Methods: The effect of Cuc D on the growth of pancreatic cancer cells was determined by cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96 h. The clonogenic potential of cancer cells was also studied using the colony formation assay. Tumor suppressor miR-145, which is downregulated in pancreatic cancer, directly target MUC13. Thus the effect of Cuc D was also investigated on the expression of miR-145 through qPCR analysis. Immunoblotting techniques were performed to study the known direct targets of miRNA-145 and its associated proteins. The anti-cancer potential of Cuc D in pancreatic cancer was also evaluated in vivo using a xenograft mouse model. Results: Our results demonstrate potent anticancer effects of Cuc D on pancreatic cancer cells. Cuc D induces dose and time dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell line models at nanomolar concentrations (100-500 nM). It also inhibits colony formation and invasiveness of pancreatic cancer cells. Furthermore, Cuc D blocks the cell cycle progression in G2/M phase and decreases the mitochondrial membrane potential in pancreatic cancer cells. Notably, Cuc D significantly increases the expression of tumor suppressor miR-145 in HPAF-II cells as observed by qPCR. Furthermore, Cuc D decreases the expression of MUC13 and its associated proteins including pAKT and HER2. In addition, it restores the expression of p53 level as studied by immunofluorescence technique. The expression of key oncogenic proteins including NF-κB, STAT3 (Tyr-705) and Mcl-1 were also downregulated. The levels of PTEN and p27 (Kip1) tumor suppressor genes were increased after Cuc D treatment. Additionally, in vivo administration of Cuc D effectively inhibited pancreatic tumor growth in xenograft mouse model. Conclusion: Overall, this study suggests that Cuc D modulates the expression of key oncogenes and tumor suppressors, thus it can be a promising therapeutic modality for pancreatic cancer prevention and treatment. Citation Format: Mohammed Sikander, Sheema Khan, Neeraj Chauhan, Mohd Saif Zaman, Murali Mohan Yallapu, Fathi T. Halaweish, Bhavin Chauhan, Shabnam Malik, Meena Jaggi, Subhash C. Chauhan. Anticancer activity of novel cucurbitacin analogue in pancreatic cancer. [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 4468. doi:10.1158/1538-7445.AM2015-4468

Background: Advanced stage of prostate cancer is difficult to treat and manage. Autophagy facilitates both disease progression and therapeutic resistance in prostate cancer. In fact, different forms of therapies such as androgen-deprivation, taxane-based chemotherapy, targeted kinase inhibition, and nutrient restriction, all induce significant cellular distress, and subsequently autophagy, which plays cyto-protective role in prostate cancer. The differential role of autophagy in prostate cancer cells is mainly dependent on the type of tumors, stage, and genetic context of cancer cells. Protein Kinase D1 is a serine-threonine kinase, a tumor-suppressor, which is down-regulated in advanced stage of prostate tumors. PKD1 is involved in many cellular functions, such as cell proliferation, apoptosis, cell adhesion and invasion. In this study we try to determine whether PKD1 causes autophagic cell death by activation of ER stress in prostate cancer cells. Methods: Prostate cancer cells expressing PKD1 (LNCaP and C4-2; C4-2-GFP; C4-2-GFP-PKD1) and null PKD1 (DU-145/PC-3) were used for the experiments. Cell viability assay was performed by MTS assay. CCL2 expression levels were quantified by ELISA. Cell migration assays were performed with cells pre-incubated with 3-Methyladenine (3-MA; autophagy inhibitor), or Salubrinal (SAL; ER stress inhibitor) or exogenously added CCL2 (100ng/mL). Immmunoblot experiments were performed using specific antibodies. Results: Our results suggest that PKD1 inhibits cell proliferation, migration and colonization, and arrest cells at G2/M phase. Our data strongly suggests that PKD1overexpressing cell activates Endoplasmic Reticulum (ER) stress-mediated autophagic cell death in prostate cancer cells compared to PKD1-null cells. Immunoblotting experiment suggested that C4-2-PKD1-GFP cells have suppressed ATG5/ATG7 and free ATG12 expression as compared to PKD1-null DU-145/PC-3 cells. These proteins are involved in the cyto-protective role of autophagy in prostate cancer cells. PKD1 also interacts and activates PERK expression, which is an initiator of Endoplasmic Reticulum stress (ER stress). PKD1-overexpressing C4-2-PKD-1-GFP and C4-2-GFP (vector control) cells inhibited nuclear translocation of oncogenic proteins such as c-Myc, ATG-5, p-NFKB-65 and β-catenin, and enhanced PERK and GSK-3β expression. Interestingly, PKD1 also suppressed the expression of p-AKT and mTOR and decreased glucose uptake in prostate cancer cells. Thus, PKD1 regulates glucose uptake with the involvement of AKT-mTOR-GSK-3β axis and decreases the nuclear translocation of c-Myc, ATG-5, p-NFKB-65 and β-catenin in prostate cancer cells. Conclusions: Our results strongly suggest that the PKD1 mediated modulation of autophagy and ER stress induced cell death can be useful for the augmentation of current chemotherapies used for prostate cancer treatment. Citation Format: Rishi K. Gara, Sonam Kumari, Sheema Khan, Neeraj Chauhan, Aditya Ganju, Subhash C. Chauhan, Meena Jaggi. Protein Kinase D1 induces autophagic cell death through activation of endoplasmic reticulum stress in prostate cancer cells. [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 1001. doi:10.1158/1538-7445.AM2015-1001

Background: Pancreatic ductal adenocarcinoma (PDAC) is an extremely fatal disease due to lack of efficient therapy and resistance to current standard therapy, gemcitabine (GEM). Numerous studies implicate the intimate reciprocal interactions due to paracrine Hedgehog (Hh) signaling between epithelia and underlying stroma that leads to desmoplasia and causes chemoresistance in PDAC. We report for the first time that a non-steroidal drug, ormeloxifene (ORM) has anticancer properties and depletes tumor-associated stromal tissue by inhibiting the Hh cellular signaling pathway in PDAC. This provoked us to investigate the combinatorial effects of ORM with GEM and we observed that ORM disrupts the stroma of pancreatic tumors, alters vascular network and thereby facilitates delivery and sensitivity of GEM. Methods: Using cell line models, Western blotting and QPCR were used to investigate the treatment effects of ORM, GEM and their combination on Hh signaling pathway and the related proteins involved in PDAC. To determine the effect of ORM on anti-tumorigenic sensitivity of GEM, we developed PDAC xenograft mice models using PanCa cells, BXPC3. Tumor tissue sections were stained for SHH (sonic hedgehog), cygb/STAP (stellate cell selective markers), α-SMA (PSC activation), FSP-1 (Fibroblast surface protein-1), collagen-1 by immunoflorescence, F4/80 (macrophage), CD31 (vasularization) through immunohistochemistry and microRNAs through in situ hybridization. ELIZA was performed for SDF1/CXCL12 and SHH secretion. Results: ORM inhibits cell proliferation and induces apoptosis in pancreatic cancer cells. ORM alone and in combination with GEM inhibits the Hh signaling pathway by downregulating both the protein and mRNA levels of SHH and its related important downstream targets: GLI1, PTCH1/2, NFκB, p-AKT and Cyclin D1. ORM potentiates the anti-tumorigenic effect of GEM by 75% in PDAC xenograft mice. Investigations of treated xenograft tumor tissues show that ORM depletes tumor-associated stromal tissue by inhibiting the Hh cellular signaling pathway and both mouse/human collagen-1, a hallmark of desmoplastic reaction. These xenograft tumors treated with ORM in combination with GEM also show the inhibition of oncogenic miR-21 and restoration of tumor suppressor, miR-132. The combination therapy inhibits stromal cell populations infiltrating into the tumor tissue and produces increased intratumoral vascular density and concentration of GEM, leading to transient stabilization of tumor growth. Tumor cells, co-cultivated with TGFβ stimulated human pancreatic stromal cells, displayed increased invasive capabilities which were inhibited on treatment with ORM alone or in combination with GEM. Conclusion: We propose that ORM has a high therapeutic index and represents a great promise for a combination therapy with GEM against stroma and neoplastic cells as a treatment of choice Citation Format: Sheema Khan, Mara Ebeling, A. Ansarullah, Neeraj Chauhan, Rishi Kumar Gara, Meena Jaggi, Haotian Zhao, Subhash C. Chauhan. A novel approach to enhance delivery and sensitivity of gemcitabine in pancreatic cancer by suppression of desmoplasia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3603. doi:10.1158/1538-7445.AM2014-3603

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in United States. Tremendous efforts have witnessed the failure of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), extends patient survival by only a few weeks. Numerous studies implicate the need for the drugs that can target multiple signaling events in pancreatic cancer. Recently, cucurbitacins, tetracyclic triterpenoid compund/molecules, belonging to a family of Cucurbitaceae have shown promising anti-cancer activities such as antiproliferation, cell cycle arrest, and apoptosis induction. Here, we report the anticancer activity of a novel analogue of cucurbitacin (Cuc D) that is able to target important signaling proteins involved in pancreatic cancer cell proliferation, invasion and metastasis. Methods: Herein, we investigated the use of Cuc D for the treatment of pancreatic cancer using a panel of pancreatic cancer cells. The effect of Cuc D on the growth of pancreatic cancer cells was determined by CellTiter 96® AQueous One Solution cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96h. The clonogenic potential of cancer cells was also studied using the colony formation assay. microRNA-21 expression levels were investigated through Real-time PCR. Immunoblotting techniques were performed to determine the effects of Cuc D at the molecular level. Results: Our results indicate the anticancer effects of Cuc D in pancreatic cancer cells. Cuc D induces concentration dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell lines at nano molar concentrations. It also inhibits colony formation and metastasis potential of pancreatic cancer cells in dose and time dependent manner. Importantly, Cuc D targets and inhibits proteins, MUC13, Bcl2 and survivin (inhibitor of apoptosis protein family member: IAP) that are involved in proliferation, metastasis and tumor progression as seen through Western blotting. Additionally, the treatment of Cuc D induces the phosphorylation of p53 (p-p53; ser-15) in pancreatic cancer cells. We also observed that Cuc D effectively inhibits miR-21 levels in pancreatic cancer, HPAF-II cells as seen through Real time PCR analysis. miR-21 is an oncogenic miRNA that is overexpressed in pancreatic cancer. Conclusion: Our findings demonstrate that Cuc D exerts multi-focal action in pancreatic cancer cells targeting multiple signaling events. Overall, this study suggests that Cuc D can be a potential and promising therapeutic modality for pancreatic cancer treatment. Citation Format: Mohammed Sikander, Mohd Saif Zaman, Neeraj Chauhan, Murali M. Yallapu, Sheema Khan, Fathi T. Halaweish, Bhavin Chauhan, Meena Jaggi, Subhash C. Chauhan. A multi-targeted approach for pancreatic cancer treatment by a novel cucurbitacin analogue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2714. doi:10.1158/1538-7445.AM2014-2714

We have successfully engineered a magnetic nanoparticles (MAG-NPs) formulation using a multi-layer approach which can be used for drug/gene/biomolecule delivery, hyperthermia, and magnetic resonance imaging (MRI) applications in cancer therapeutics. Overcoming nanoparticles clearance by the immune system remains a major challenge. This formulation is designed to provide an additional surface layer as molecular “authentication” that the body does not recognize as foreign material. The interaction between the surface of nanoparticles and plasma proteins leads to nanoparticle-protein complex which determines the rational design of clinically useful formulations for cancer therapeutics. This formulation was very effective for inhibiting tumorigenic features in in vitro and in vivo cancer models. Therefore, we further studied our MAG-NPs formulation for biologically and clinically relevant characteristic features for its clinical translation. For this, we have examined the change in particle size, zeta potential, human serum protein adsorption, hemotoxicity, uptake, targeting cancer cells, and magnetic resonance imaging of the MAG-NPs formulation. No significant change was observed in particle size and zeta potential of nanoformulation with the incubation of 0.5 to 50 wt/wt% human serum. With increase of serum concentration and particle concentration there was a clear indication that apart from serum albumin and transferrin; apolipoprotein E (Apo-E, a surrogate marker for improved circulation of nanoparticles and enhanced crossing of the blood-brain barrier) also adsorbed on the surface of nanoparticles (determined by Western blot analysis). No significant primary or secondary structural alterations were observed in serum proteins through circular dichroism study. Hemolysis assay demonstrated almost no hemolysis at the tested concentrations (up to 1 mg/mL) for nanoparticles compared to the positive control (sodium dodecyl sulphate). Particle internalization and targeting cancer cells were observed in dose dependent manner and were not influenced by protein architecture on surface of nanoparticles. Additionally, this formulation has exhibited identical T1 relaxation times (∼1.1-1.6 s) and T2 relaxation times (∼ 13-14 ms) which suggests that our nanoparticles retained MRI properties even after serum proteins adsorption and drug/biomolecular encapsulation. Based on all these superior clinical characteristics, this MAG-NPs formulation can effectively be used for the delivery of drug/siRNA/miRNAs for cancer therapy, diagnosis, and imaging. Citation Format: Murali M. Yallapu, Neeraj Chauhan, Shadi F. Othman, Vahid Khalilzad-Sharghi, Meena Jaggi, Subhash C. Chauhan. Characterization of a novel magnetic nanoparticles formulation for cancer therapeutic applications. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2014-4459

Background: Cervical cancer (CxCa) remains the fourth leading cause of cancer related death among women worldwide. Cervical cancer is mainly (∼ 99.7%) derived from high risk Human papillomavirus (HR HPV). HPV E6/E7 oncoproteins interfere with p53 and pRb (retinoblastoma) cell cycle regulatory proteins and hinder their efficacy of controlling cell growth. Advanced stage cervical cancer is difficult to treat and patients diagnosed with metastatic disease have a poor survival rate of only 16%. Therefore, there is an urgent need to develop newer treatment modalities. Ormeloxifene is a non-hormonal, anti-estrogen, oral contraceptive for human use that effectively suppresses the proliferation of decidual cells in the endometrium to inhibit egg implantation. Growing evidences also suggest that ormeloxifene has anti-cancerous properties in breast, head and neck, and chronic myeloid leukemia cancers. Hence, considering its strong anti-proliferative/cancerous effect, we hypothesized that ormeloxifene may also inhibit the growth of cervical cancer cells and may suppress the expression of E6 and E7 HPV oncoproteins. Methods: We performed in vitro anti-cancer studies with ormeloxifene (dose range: 5 µM to 25 µM) in the HPV positive cervical cancer cell lines (Caski and SiHa) by utilizing MTS and colony formation assays. To further determine the effects of ormeloxifene on the cellular and molecular level, immunoblotting, flow cytometry and real time PCR analyses were performed using the Caski cell line model. Results: Ormeloxifene effectively inhibited cervical cancer cell growth as determined by proliferation and colony formation assays in a dose dependent manner. Ormeloxifene decreased mitochondrial membrane potential and induced apoptosis as shown by flow cytometry analysis of cells stained with TMRE (tetramethylrhodamine, ethyl ester) and 7AAD/Annexin V, respectively. Apoptosis was also confirmed by immunoblotting for cleaved PARP (Poly ADP ribose polymerase), Caspase 3 and Caspase 9. Additionally, ormeloxifene treatment arrested cell cycle in G1-S transition and decreased the expression of p-Akt while total Akt remained the same. Specifically relevant to cervical cancer, ormeloxifene decreased the expression of HPV E6/E7 at mRNA and protein levels as determined by quantitative PCR analysis and immunoblotting, respectively. Interestingly, ormeloxifene restored the levels of p53, Rb and PTPN 13 (Protein tyrosine Phosphatase non receptor type 13) tumor suppressor proteins as shown by immunoblotting. Conclusion: Our findings suggest that ormeloxifene inhibits cervical cancer cell growth and decreases mitochondrial membrane potential which clearly demonstrates that apoptosis is induced via intrinsic pathway. Importantly, ormeloxifene downregulates the expression of HPV E6/E7 oncoproteins. Thus, ormeloxifene appears to be a novel treatment modality for cervical cancer. Citation Format: Neeraj Chauhan, Mohd S. Zaman, Murali M. Yallapu, Diane M. Maher, Mara C. Ebeling, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene inhibits cervical cancer cell growth through intrinsic apoptotic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2753. doi:10.1158/1538-7445.AM2014-2753

Curcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has demonstrated potent anti-cancer and cancer prevention activity in a variety of cancers. However, the clinical translation of curcumin has been significantly hampered due its extensive degradation, suboptimal pharmacokinetics and poor bioavailability. To address these clinically relevant issues, we have developed a novel curcumin loaded magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic efficacy of this novel MNP-CUR formulation in pancreatic cancer. Human pancreatic cancer cells (HPAFII and Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose dependent manner. As a result, MNP-CUR formulation effectively inhibited growth of HPAFII and Panc-1 cells in cell proliferation and colony formation assays. Similarly, the MNP-CUR formulation suppressed pancreatic tumor growth in an HPAFII xenograft mice model and improved mice survival by delaying tumor growth. The growth inhibitory effect of MNP-CUR formulation was correlated with the suppression of PCNA, Bcl-xL, Mcl-1, MUC1, collagen I and enhanced membrane β-catenin expression. Interestingly, our MNP-CUR formulation did not show any sign of hemotoxicity and stable after incubation with human serum proteins. Additionally, our MNP-CUR formulation improved serum bioavailability of curcumin in mice up to 2.5 fold as compared to curcumin in Tween-20. Furthermore, biodistribution studies of our MNP-CUR formulation demonstrate a significant amount of formulation was able to reach the pancreatic xenograft tumor(s) which suggests its clinical translational potential. In conclusion, our study suggests that our novel MNP-CUR formulation can be valuable for the treatment of pancreatic cancer. Citation Format: Murali M. Yallapu, Mara C. Ebleling, Sheema Khan, Neeraj Chauhan, Brij K. Gupta, Vasudha Sundram, Meena Jaggi, Subhash C. Chauhan. Novel curcumin loaded magnetic nanoparticles for pancreatic cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4503. doi:10.1158/1538-7445.AM2013-4503

Background: Magnetic nanoparticles (MNPs) with theranostic features (diagnosis and treatment) have attracted significant attention and will greatly improve the scope of nanomedicine in cancer applications. The aim of this study is to develop a novel MNPs formulation composed of an iron oxide core coated with α-cyclodextrin and pluronic polymer (F68), to load an anti-cancer drug (curcumin, CUR) and prevent particle aggregation, respectively. The therapeutic efficacy and imaging capabilities of this novel formulation were evaluated in breast cancer cell line models. Methods: The physico-chemical analyses of curcumin loaded MNPs (MNP-CUR) were performed using dynamic light scattering (DLS), transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) analyses. MNP-CUR internalization was evaluated after 6 hrs incubation with MDA-MB-231 cancer cells by Prussian blue stain and TEM. Anti-cancer effects of the MNP-CUR formulation was determined by a tetrazolium based dye (MTT) and colony formation assays using “triple negative” MDA-MB-231 cancer cells. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE), red mitochondrial superoxide indicator (Mitosox), and propidium iodide stains were used to evaluate the loss of mitochondrial membrane potential, reactive oxygen species (ROS) generation, and apoptosis/dead cells, respectively, to determine effects MNP-CUR on these cellular features. The imaging capability of this MNP-CUR formulation was analyzed using a magnetic resonance imaging (MRI) system. The magnetic targeting feature of the MNP-CUR formulation was evaluated using fluorescence microscopy. Results: We prepared MNP-CUR with an average aggregative particles size of ∼ 150 nm (individual particle grain size, ∼ 9-11 nm). Prussian blue stain data represent a preferential uptake of MNP-CUR in MDA-MB-231 cells in a dose dependant manner. TEM analysis demonstrates that accumulation of MNP-CUR nanoparticles in cancer cells indicate that particles are not attached on the surface of cells but internalized within the cells. The MNP-CUR formulation showed strong anti-cancer effects in breast cancer cells including “triple negative” MDA-MB-231 cancer cells compared to free curcumin. This formulation also enhanced loss of mitochondrial membrane potential, generation of ROS and apoptosis compared to free curcumin treatment. Additionally, the MNP-CUR formulation exhibits superior T2 imaging characteristics compared to T1. A significant increase of the targeting feature was observed with MNP-CUR in breast cancer cells. Conclusion: These data suggest that our novel MNP-CUR formulation exhibits potent anti-cancer activity along with imaging and magnetic targeting capabilities. This approach can be extended to pre-clinical and clinical use and may have importance in cancer treatment and cancer imaging in the future. 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 2893. doi:1538-7445.AM2012-2893

Background: Cervical cancer is one of the most common and deadly cancers among women worldwide and is strongly associated with persistent Human Papillomavirus (HPV) infection. HPV E6/E7 oncoproteins are well known to interfere with p53 and retinoblastoma (Rb) proteins and drive the development of cervical cancer. While screening for cervical cancer can reduce the incidence and mortality, treatment of advanced stage cervical cancer is difficult and often unsuccessful. Therefore, there is an urgent need for the development of additional treatment modalities. Ormeloxifene is a non-hormonal anti-estrogen oral contraceptive for human use and it effectively suppresses the highly proliferative decidual cells in the endometrium to inhibit egg implantation. Considering its strong anti-proliferative effect, we hypothesized that ormeloxifene may inhibit the growth of cervical cancer cells and may suppress the expression of HPV oncoproteins E6/E7. Methods: The effect of ormeloxifene on the proliferation of various cervical cancer cell lines (Caski, SiHa, C33A, and HT3) was determined by MTS and colony formation assay. Annexin V/7AAD and propidium iodide (PI) staining was analyzed by flow cytometry to determine ormeloxifene's effect on apoptosis and cell cycle inhibition, respectively. The expression of apoptotic proteins such as poly (ADP-ribose) polymerase (PARP), Caspase 3 and Caspase 9 was determined by immunoblotting. The potential of ormeloxifene to repress the expression of HPV E6/E7 oncogenes was determined by quantitative RT-PCR and immunoblotting analyses. Further, the efficacy of ormeloxifene to restore tumor suppressor proteins, such as p53 and Rb, was also determined by immunoblotting. Results: In all 4 cell lines, ormeloxifene effectively inhibited cervical cancer cell growth. Ormeloxifene treatment induced apoptosis in cervical cancer cells as determined by staining for Annexin V/7AAD and immunoblotting for cleaved PARP, Caspase 3, and Caspase 9. PI staining also clearly revealed that ormeloxifene treatment arrests Caski cells in the G0-G1 phase. Additionally, ormeloxifene treatment caused a marked decrease in the levels of HPV E6 and E7 oncogenes and restored the levels of p53 and Rb proteins in Caski cells. Conclusion: Our findings show that ormeloxifene significantly inhibits cervical cancer cell growth and down-regulates HPV E6/E7 oncogenes, therefore; ormeloxifene may be novel chemo-preventive and treatment method for the management of cervical 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 2882. doi:1538-7445.AM2012-2882