Dr. Chauhan has completed his PhD at the age of 27 years from Central Drug Research Institute (CDRI), Lucknow, India and received postdoctoral training from University of Nebraska Medical Center (UNMC), Omaha, NE. Currently he is serving as a Professor of Pharmaceutical sciences at University of Tennessee Health Science Center (UTHSC), Memphis, TN, a premier educational institution. He has published more than 85 papers in reputed journals and has been serving as an editorial board member for several journals of international repute.

Pancreatic cancer (PanCa) has a dismal prognosis despite current multimodality therapies. Chemo-resistance to commonly utilized chemotherapeutic agents remains a major clinical challenge. The prognosis of patients with pancreatic ductal adenocarcinoma is extremely poor, and current therapies such as, Nab paclitaxel and gemcitabine result in only marginal survival rates for patients. Therefore, the research efforts are underway to discover new therapeutic options to treat PanCa and overcome resistance to available therapies. Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL), receptor agonists are promising cancer therapeutics with selective killing ability toward malignant cells. Recently, we have identified microRNA-145 (miR-145) as a tumor suppressor in PanCa regulating oncogenic signaling pathways and have developed novel nanoparticle mediated mechanisms that effectively restores lost miR-145 levels in PanCa. The aim of this study is to identify mechanisms to counteract trail resistance in PanCa and sensitize tumors towards chemotherapies. Our results demonstrate that miR-145 restoration increases TRAIL sensitivity in pancreatic cancer cells and renders them highly responsive towards Nab-pactitaxel. We observed increased activation of TRAIL induced death receptor pathway as depicted by the activation of DR5, FLIP. This triggers the cells towards apoptotic cell death as indicated through activation of caspases, increased sub-G0 population and decreased cell proliferation. Additionally, TRAIL treatment in presence of miR-145 showed tremendous responsiveness of cells towards nab-paclitaxel induced cell death. In conclusion, these observations suggest that the miR-145 restoration counteracts chemo-resistance mechanisms in pancreatic cancer that may lead to novel combination therapies for PanCa treatment.

Docetaxel (DTX) is a standard first-line treatment for metastatic castration-resistant Prostate Cancer after the failure of hormone therapy. However, most Prostate Cancer (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 suggests 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. 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 were performed to determine the putative molecular interaction of ORM with MTA1, showed four potential binding sites with maximum binding potential at Serine 270. Overall, our study defines ORM as a novel PKD1 activator/modulator which also inhibits a key metastasis associated protein, MTA1 and sensitizes PrCa cells to DTX, which could be a novel therapeutic modality for PrCa treatment alone or in combination with DTX.

Pancreatic cancer (PanCa) is one of the most fatal of all cancers and is ranked as the fourth most common cause of cancer related deaths among both men and women in the US. The management of PanCa, is exceptionally difficult due to the extremely poor response to available therapeutic modalities. Highly desmoplastic microenvironment in pancreatic tumor causes suboptimal drug delivery and increases chemo-resistance. Plumbagin (PL), a naturally occurring napthoquinone derived from the root of Plumbago zeylanica L., has showed potent cancer preventive and therapeutic activity against variety of cancers. However, the clinical translation of PL has been significantly hampered due to its toxicity and suboptimal bioavailability. To address these clinically relevant issues, we have developed a novel PL-loaded magnetic nanoparticle (MNP-PL) formulation and evaluated its therapeutic efficacy in ectopic xenograft mouse model. This MNP-PL formulation was prepared using Magnetic nanoparticles (MNPs) composed of an iron oxide core which has distinct advantages in i) bio/hemo-compatibility, ii) biodegradation, iii) higher drug loading capacity and iv) improved bioavailability. Our novel MNP-PL formulation provided average size of 100 nm in dynamic light scattering (DLS) and exhibited –9.42 to –10.79 mV zeta potential with an outstanding PL loading efficiency (96%). We have evaluated anti-cancer potential of MNP-PL formulation in human PanCa cells (HPAF-II, AsPc1 and Panc-1). We first performed MTS and colony formation assays to determine the effects of free PL and MNP-PL formulation on growth of PanCa cells. In this experiment, cells were treated with various concentrations of free PL (1-15 μM) or MNP-PL (1-15 μM) for 24 hrs. Results exhibited efficient internalization of the MNP-PL formulation in a dose-dependent manner. As a result, the MNP-PL formulation showed four fold dose advantage over free PL. IC50 of free PL was recorded 10 μM which was significantly reduced to 2.5 μM in MNP-PL.MNP-PL also showed four fold inhibition in colony formation compared to free PL. MNP-PL treatment more efficiently inhibited oncogenic CXCL12/CXCR4 signaling pathway in both PanCa and patient derived stromal fibroblast cells. MNP-PL treatment also showed decreased expression of CXCR4 protein levels in PanCa cells. Moreover, MNP-PL treatment inhibited stromal derived factor 1 (SDF-1)/CXCL12 expression in stromal fibroblasts. Intra-tumoral administration of MNP-PL (50 μg/mouse) for 5 weeks inhibited established ectopic xenograft tumors in athymic nude mice compared to vehicle treated mice. This effect was more pronounced compared to free PL. These results suggest that our novel MNP-PL formulation has more anti-cancer potential than free PL against PanCa. In conclusion, MNP-PL formulation may reduce the toxicity and improve the bioavailability of free PL and could be used for the treatment of PanCa.

Cervical cancer is one of the leading causes of mortality among women in US. Naturally occurring dietary compounds have gained increasing attention for their anticancer efficacy. Cucurbitacins, tetracyclic triterpenoid compound, belong to a family of Cucurbitaceae have shown promising anticancer activity. Herein, we investigated the potential anticancer effects of a novel analogue of cucurbitacin D (Cuc D) against cervical cancer in in vitro and in a xenograft mouse model. In our study, we used human cervical cancer cells (CaSki and SiHa). Cells were treated with Cuc D (0.05 to 1μM) for 48 and 72 hrs. MTS and colony formation assays were performed to investigate the effects of Cuc D on cell viability and proliferation. Western Blot analysis was performed to investigate the effects of Cuc D on cell proliferation and apoptotic markers. To determine the therapeutic effects of Cuc D, we used female athymic nude mice and injected CaSki cells (4 x 106) into the cervix to develop orthotopic xenograft tumors. Cuc D (1 mg/kg body weight) was administered through intra-tumoral injection four week post-tumor cell injection. Tumor volume in these mice were recorded biweekly. Cuc D inhibited cell viability of cervical cancer cells in a dose dependent manner. IC50 of Cuc D was observed 400 nM and 250 nM in Caski and SiHa cells, respectively. Cuc D treatment effectively inhibited growth of cervical cancer cells which was determined by decreased cell proliferation and colony formation assays. Cuc D treatment induced apoptosis in cervical cancer cells as measured by enhanced Annexin V staining. Western blot result also illustrated cleavage in PARP protein in Cuc D treated cells which further confirms apoptosis induction. Cuc D treatment also inhibited PI3K and c-Myc protein levels and phosphorylation of STAT3 and Rb proteins. In addition, Cuc D treatment induced protein levels of p21, p27 and PTEN. Interestingly, Cuc D treatment also induced the expressions of tumor suppressor microRNAs, (miR145, miR-143 and miR-34a) as determined by qRT-PCR. In an orthotopic tumor xenograft mouse model, Cuc D treatment effectively inhibited cervical cancer cells derived xenograft tumors in athymic nude mice. Taken together, our results demonstrate potent anticancer efficacy of Cuc D in cervical cancer cells via modulation of key onco/tumor suppressor proteins. Thus, our result demonstrate that Cuc D could be a useful therapeutic agent for cervical cancer treatment.

Background: Cervical cancer (CxCa) is the fourth leading cause of cancer related death among women worldwide. CxCa 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 hamper their efficacy of regulating cellular 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 (ORM) is a non-steroidal Selective Estrogen Receptor Modulator (SERM) used as an oral contraceptive in humans. Recent investigations suggest that Ormeloxifene exhibits potent anti-cancer activity against various types of cancers. Nanoparticulates offer targeted delivery of anti-cancer drugs and promise newer approaches for cancer diagnosis and treatment. Tumors due to their enhanced permeability and retention (EPR) effect allow nanoparticles specifically to enter and accumulate in them. Site specific delivery of anti-cancer drugs using nanotechnology provides very minimal or no toxicity. 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, our lab recently has developed ORM loaded PLGA (poly [lactic-co-glycolic acid]); an FDA approved biodegradable polymer, nanoparticle(s) to achieve the targeted drug delivery and improved bioavailability. Methods: ORM encapsulated PLGA nanoparticles (PLGA-ORM) were developed employing nanoprecipitation method utilizing various concentration of the drug ormeloxifene (range: 5mg -25mg). Poly vinyl alcohol (PVA) and Poly (L-lysine) were used as a stabilizer for nanoparticle preparation. Physiochemical characterization of PLGA-ORM nanoformulation was determined by analyzing particle size, drug loading and drug release by employing dynamic light scattering (DLS), transmission electron microscopy (TEM) and high performance liquid chromatography (HPLC) techniques. Additionally, anti-cancer efficacy of PLGA-ORM nanoformulation was tested in SiHa and Caski HPV 16 positive cervical cancer cells. Results: PLGA-ORM has a drug concentration dependent particle size varying between 210nm to 278.6nm with almost 98% drug loading efficacy and with a sustained drug release profile. PLGA-ORM showed dose, time and energy dependent cellular uptake through endocytosis mediated pathways in both SiHa and Caski cells. Moreover, PLGA-ORM had an improved inhibition of cellular proliferation and clonogenic potential when compared with free ORM. Further studies are in the progress for preclinical evaluation of PLGA-ORM using an orthotropic mouse model for cervical cancer. Conclusion: Taking together, our findings suggest that PLGA-ORM nanoformulation has a great potential for repurposing the drug; as a novel modality for cervical cancer treatment and needs tto be developed as a lead therapy approach with appropriate pre-clinical/clinical investigations.

Objectives: Prostate cancer (PrCa) is the ranks among second in cancer mortalitity rates among men in the United States. Chemotherapy (Docetaxel, Dox) is frequently employed as first-line therapeutic option. However, toxicity and chemo-resistance issues of docetaxel limit its clinical application. Improving docetaxel targeted delivery and enhancing its activity at the tumor site is emerging therapeutic strategy through targeted nanoparticle system for PrCa therapy. Prostate Specific Membrane Antigen (PSMA) is highly overexpressed in PrCa cells, which is a highly attractive molecular target for PrCa treatment. In this study, we developed and determined anti-cancer efficacy of a novel docetaxel loaded, PSMA targeted super paramagnetic iron oxide nanoparticles (PSMA-SPION-Dox) formulation for PrCa therapy. Methods: Docetaxel loaded super paramagnetic iron oxide (SPION-Dox) formulation is composed of an iron oxide core encrusted with cyclodextrin (for drug loading) and F127 polymer (for particle stability and chemosensitization) layers. Therapeutic efficacy of this unique nanoformulation was evaluated using clinically relevant prosate cell lines through proliferation and colony formation assays. Molecular effects of this formulation on apoptosis, anti-apotosis, and drug resistance associated proteins were evaluated using western blot studies. Contrast imaging property of SPION-Dox formulation was examined using Phantom Gel MR imaging model. For active targeting, PSMA antibody conjugation to this formulation was achieved through N-hydroxysuccinimide group containing PEG polymer. Active targeting potential of this formulation was evaluated in PSMA+ (C4-2) and PSMA– (PC-3) cell lines, C4-2 generated tumor xenografts.Results: SPION-Dox formulation displayed ideal particle size and zeta potential that can effectively internalized in PrCa cells. This nanoformulation showed enhanced anti-cancer efficacy in prostate cancer cells. Additionally, it induces the expression of apoptosis associated proteins, Bax and Bad, cleaved PARP, and caspase 3, and downregulated the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL. Likewise, it also inhibited the expression of chemoresistance associated proteins (PSMA and MDR1). Our anti-PSMA functionalized SPION-Dox formulation exhibited a intense uptake pattern in PSMA+ cells (C4-2) compared to PSMA null (PC-3)- cells, annotating its targeting potential. Also similar targeting potential was also observed in ex-vivo studies while using C4-2 tumor xenografts. Conclusion: Anti-PSMA functionalized SPION-Dox formulation can efficiently target PSMA + PrCa cells and deliver docetaxel into prostate tumors. This targeted drug delivery system could reduce the docetaxel dose in annihilating cancer cells, thus curtailing docetaxel associated undesirable effects and chemo-resistance.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA, accounting for about 40,000 deaths annually. Unfortunately, little progress has been seen in the outcomes of patients with PDAC as it is unable to be diagnosed at an early stage and tumor develops high desmoplasia and chemo-resistance to chemotherapeutic drugs, such as gemcitabine (gem). Potent anticancer, chemopreventive, chemo-sensitization properties have been reported for curcumin (cur). However, the major clinical challenge is its clinical translation that has been significantly hampered due to its low solubility, high rate of metabolism, poor bioavailability and pharmacokinetics. Therefore, we have developed a magnetic nanoparticle formulation of curcumin (Mag-cur) that targets the tumor as well as the surrounding microenvironment resulting in its efficient and targeted delivery. Investigations for the treatment effects of M agcur, gem and their combination on Hh signaling pathway in PDAC, were performed using Western blotting and qPCR analysis. We also studied the antitumor effects using PanCa xenograft mice . Imunohistochemistry/immunofluorescence analysis was performed in the excised tumor tissue sections from mice for the important proteins in sonic hedgehog (SHH) pathway, such as Gli-1, SMO, CXCR-4, patch1/2, sufu, markers for stellate cells (cygb/STAP) and myofibroblast (α-SMA) and macrophages (F4/80). The microRNAs were investigated using insitu hybridization. Our results demonstrate that Mag-cur alone or in combination with gem inhibits pluripotency maintaining factors (Nanog, Sox- 2, c-Myc and Oct-4) in human pancreatic cancer stem cells and the formation of stumorspheres. We found that Mag-cur inhibits PDAC proliferation, migration, invasion and tumor growth in xenograft mouse model by suppression of the sonic hedgehog (SHH) signaling pathway and CXCR4/CXCL12 signaling axis that inhibit bidirectional tumor-stromal cells interaction. Mag-cur treatment alone or in combination with CXCR4 antagonist (amd3100) inhibits CXCL12 induced up regulation of CXCR4, SHH and NFĸB-65 in cells. Further, qRT-PCR results showed that Mag-cur treatment increases gem sensitivity by up regulating the human nucleoside transporter genes (hENT, hCNT) and blocking ribonucletide reductase subunits (RRM1/RRM2) which further supports the use of Mag-cur for suppression of PanCa growth. Immunostaining of tumor sections from treated pancreatic xenograft mice revealed that MNP-cur treatment efficiently inhibits key proteins of SHH signaling such as SHH and Gli-1. Mag-cur also disrupts the stroma of fibrotic pancreatic tumors by inhibiting the proliferating stellate and myeloid cells. This was indicated by the decreased staining of αSMA, macrophages and cygb/STAP in tumor tissues. Additionally, the tumor tissues from Mag-cur treated mice showed extensive downregulation of oncogenic miR-21 expression which has been associated with cancer progression and drug resistance. Therefore, these results suggest that Mag-cur has a great potential for future clinical use in the treatment of PDAC.

Pancreatic cancer (PC) is the fourth leading cause of cancer related deaths in the USA, with a 5-year survival rate of less than 5%. MicroRNAs have been identified as attractive targets for therapeutic intervention. The functional significance of lost microRNAs have been reported in several human malignancies, including PC. Therefore, restoring lost miRNA function can provide a potential therapeutic benefit. Prior work has identified microRNA-145 (miR-145) as a tumor suppressor miRNA in pancreatic cancer. The restoration of miR-145 downregulates a number of oncogenes including mucin MUC13, a glycoprotein that is aberrantly expressed in PC, and efficiently inhibits tumor growth in mice. The main challenge for successful translation of microRNAs into clinical practice remains an effective in vivo delivery system. The focus of this study was to develop and assess the efficacy of a miRNA delivery method for PC treatment. Magnetic nanoparticle (MNP) based nanoformulation of miR-145 (miR-145-MNPF) was developed for the intracellular delivery and sustained release of miR-145. The positively charged polyethyleneimine (PEI) molecules were used to increase the loading efficiency of miR-145. MUC13 expressing pancreatic ductal adenocarcinoma cell lines (HPAF-II and AsPC-1) were used for the study. Following transient transfection of miR-145-MNPF, Western blotting and immunofluorescence techniques were used to investigate the effects of miR-145 restoration on number of proteins including MUC13. Additionally, functional studies of the effects of miR-145 restitution using miR-145-MNPF included cell proliferation, colony formation, cell migration, and cell invasion assays. Treatment of cells with miR-145-MNPF led to efficient intracellular delivery of miR-145 mimics as observed through prussian blue staining. This led to the simultaneous upregulation of miR-145 levels in cells as confirmed by qRT-PCR. miR-145 restitution resulted in significant downregulation of target oncogenes including MUC13, HER2, P-AKT and p53 as observed through Western blotting and immunofluorescence techniques. miR-145-MNPF inhibited cell proliferation, clonogenicity, migration, and invasion of PC cells. These results suggest that the MNP based delivery systems can be efficiently used for microRNA replacement therapy in order to restore the lost microRNAs in cancer. miR-145 restitution using miR-145-MNPF may offer a potential therapeutic strategy for pancreatic cancer treatment alone or in combination with other therapies.

Hypoxia-inducible factor 1 α (HIF-1α) plays an important role in reprogramming of cancer cell metabolism by activating the transcription of genes which encode glucose transporters and enzymes involved in glycolysis. MUC13, is a transmembrane mucin which is aberrantly expressed in pancreatic cancer, while an altered glucose metabolism is known to facilitate cancer cell survival and proliferation. Herein, we investigated the effects of MUC13 expression on glucose metabolism and elucidated underlying signaling mechanisms that might be involved in this process. MUC13 null pancreatic cancer Panc-1 cells were used for the study. Glucose and Lactate assay were performed in Panc-1 cells stably expressing MUC13 (Panc-1-M13) and vector (Panc-1-V). Cell culture media after 48 hours was collected to measure the amount of unused glucose and L-lactate concentration using glucose and lactate assay kits. Immunoblot and semi-quantitative PCR analyses were performed to check the expression of protein and mRNA levels, respectively. Our results demonstrate that MUC13 acts as a modulator of the glucose metabolism in pancreatic cancer cells by regulating the expression and activity of hypoxia inducible factor-1α (HIF-1α) and its downstream targets. We observed increased amount of L-lactate production and glucose consumption in Panc-1-M13 cells as compared to Panc-1-V (wild type) cells. This facilitates metabolic alterations and help tumor cells survive and proliferate under these conditions as indicated by increased cellular growth pattern in Panc-1-M13 cells. Our results demonstrate increased expression of the downstream targets of HIF-1α, such as cell regulatory (c-Myc), and cell survival (Bcl-2) proteins, while decreased expression of tumor suppressor/cell cycle inhibitor (p-27) proteins in Panc-1-M13 cells. Additionally, an increase in the expression of a critical downstream target of HIF-1α, Glut-1 was observed in Panc-1-M13 cells. Our studies show that MUC13 acts as a key regulator of the metabolic process and facilitates metabolic alterations in the non-hypoxic environment that help tumor cells survive and proliferate under these conditions.