Saini Setua

Restitution of Tumor Suppressor MicroRNA-145 Using Magnetic Nanoformulation for Pancreatic Cancer Therapy

IntroductionThe functional significance of lost microRNAs has been reported in several human malignancies, including pancreatic cancer (PC). Our prior work has identified microRNA-145 (miR-145) as a tumor suppressor microRNA (miRNA) in pancreatic cancer. The restoration of miR-145 downregulates a number of oncogenes including mucin MUC13, a transmembrane glycoprotein that is aberrantly expressed in pancreatic cancer, thus efficiently inhibiting tumor growth in mice. However, lack of an effective tumor-specific delivery system remains an unmet clinical challenge for successful translation of microRNAs.MethodsWe developed a miRNA-145-based magnetic nanoparticle formulation (miR-145-MNPF) and assessed its anti-cancer efficacy. Physico-chemical characterization (dynamic light scattering (DLS), transmission electron microscopy (TEM) and miR-binding efficiency), cellular internalization (Prussian blue and confocal microscopy), miR-145 restitution potential (quantitative reverse-transcription PCR (qRT-PCR), and anti-cancer efficacy (proliferation, colony formation, cell migration, cell invasion assays) of this formulation were performed using clinically relevant pancreatic cancer cell lines (HPAF-II, AsPC-1).ResultsmiR-145-MNPF exhibited optimal particle size and zeta potential which effectively internalized and restituted miR-145 in pancreatic cancer cells. miR-145 re-expression resulted in downregulation of MUC13, HER2, pAKT, and inhibition of cell proliferation, clonogenicity, migration, and invasion of pancreatic cancer cells.ConclusionsmiR-145-MNPF is an efficient system for miR-145 delivery and restitution in pancreas cancer that may offer a potential therapeutic treatment for PC either alone or in conjunction with conventional treatment.

Probing mucin interaction behavior of magnetic nanoparticles

In this study, we developed iron oxide based magnetic nanoparticles (MNPs) by precipitation of iron salts in the presence of ammonia and created four different formulations: without functionality (plain MNPs, no coating), with β-cyclodextrin (MNPs+β-CD) or pluronic 127 polymer (MNPs+F-127), and both β-cyclodextrin and pluronic 127 polymer (MNPs+β-CD-F-127) functionality for its efficient use in mucosal delivery. We studied the interaction and/or binding behavior of these MNPs formulations with porcine stomach mucin using steady-state fluorescence spectroscopy, and then quantified the bound mucin from absorption studies. Toxicity of these MNPs against cervical cancer cells and red blood cells was evaluated. Ex-vivo studies were performed using freshly collected gastrointestinal, ovarian, pancreas and colon organ tissues of pig to evaluate binding and uptake phenomenon of MNPs. Transport studies of these MNPs in mucin was evaluated using Boydens chamber assay. All these studies together suggest that the MNPs+β-CD-F-127 formulation was strongly interacted with mucin and interestingly transported through mucin compared to other MNPs formulations. Hence, MNPs+β-CD-F-127 formulation could be a good candidate for the mucoadhesive biopharmaceuticals and drug delivery system.

Antibody-Drug Conjugates for Cancer Therapy: Chemistry to Clinical Implications

Chemotherapy is one of the major therapeutic options for cancer treatment. Chemotherapy is often associated with a low therapeutic window due to its poor specificity towards tumor cells/tissues. Antibody-drug conjugate (ADC) technology may provide a potentially new therapeutic solution for cancer treatment. ADC technology uses an antibody-mediated delivery of cytotoxic drugs to the tumors in a targeted manner, while sparing normal cells. Such a targeted approach can improve the tumor-to-normal tissue selectivity and specificity in chemotherapy. Considering its importance in cancer treatment, we aim to review recent efforts for the design and development of ADCs. ADCs are mainly composed of an antibody, a cytotoxic payload, and a linker, which can offer selectivity against tumors, anti-cancer activity, and stability in systemic circulation. Therefore, we have reviewed recent updates and principal considerations behind ADC designs, which are not only based on the identification of target antigen, cytotoxic drug, and linker, but also on the drug-linker chemistry and conjugation site at the antibody. Our review focuses on site-specific conjugation methods for producing homogenous ADCs with constant drug-antibody ratio (DAR) in order to tackle several drawbacks that exists in conventional conjugation methods.

Formulation, Development, and In Vitro Evaluation of a CD22 Targeted Liposomal System Containing a Non-Cardiotoxic Anthracycline for B Cell Malignancies

Doxorubicin cardiotoxicity has led to the development of superior chemotherapeutic agents such as AD 198. However, depletion of healthy neutrophils and thrombocytes from AD 198 therapy must be limited. This can be done by the development of a targeted drug delivery system that delivers AD 198 to the malignant cells. The current research highlights the development and in vitro analysis of targeted liposomes containing AD 198. The best lipids were identified and optimized for physicochemical effects on the liposomal system. Physiochemical characteristics such as size, ζ-potential, and dissolution were also studied. Active targeting to CD22 positive cells was achieved by conjugating anti-CD22 Fab’ to the liposomal surface. Size and ζ-potential of the liposomes was between 115 and 145 nm, and −8 to−15 mV. 30% drug was released over 72 h. Higher cytotoxicity was observed in CD22+ve Daudi cells compared to CD22−ve Jurkat cells. The route of uptake was a clathrin- and caveolin-independent pathway. Intracellular localization of the liposomes was in the endolysosomes. Upon drug release, apoptotic pathways were activated partly by the regulation of apoptotic and oncoproteins such as caspase-3 and c-myc. It was observed that the CD22 targeted drug delivery system was more potent and specific compared to other untargeted formulations.

Abstract 3216: Attenuation of pancreatic tumor growth by a small molecule tubulin inhibitor

Introduction: Pancreatic cancer (PanCa) is one of the most fatal cancers and is ranked as the fourth 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 chemotherapeutic drugs. Microtubules are dynamic structures composed of α-β-tubulin heterodimers that are essential in cell division and are important targets for several clinical drugs (paclitaxel, docetaxel and vinblastine). However, clinical use of these tubulin-targeting drugs have toxicity and drug resistance issues in cancer patients. Thus, identification of more potent non-toxic inhibitors of β-tubulin is urgently required for cancer therapy purposes. In this study, we have identified a synthetic compound (ABI-231) which is a potent inhibitor of β-tubulin and evaluated its therapeutic efficacy against PanCa in vitro, and in vivo model systems. Methods: ABI-231 ((2-(1H-indol-3-yl)-1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl)) - methanone was synthesized and characterized in our department. Effect of ABI-231 on proliferation, migration and invasion of human PanCa cells (ASPC1, HPAFII, and PANC1) was performed by in vitro functional assays (MTS, wound healing, and Boyden chamber migrations). Effect of ABI-231 on the expression of β-tubulin isoforms was determined and compared with other clinical inhibitors of β-tubulin by Western blot, and qRT-PCR. Moreover, the effect of ABI-231 on the expression of β-tubulin III in PanCa cells was determined by confocal microscopy. Therapeutic efficacy of ABI-231 against PanCa was evaluated in an ectopic xenograft mouse model. Results: ABI-231 treatment inhibited cell proliferation, invasion, migration and colony formation abilities of PanCa cells in a dose-dependent manner (1-100 nM) compared to vehicle treated group. Aberrant expression of β-tubulin III is involved in aggressiveness and drug resistance of various type of cancers including PanCa. ABI-231 effectively inhibited the protein levels and mRNA expression of total β-tubulin (TBB), TBB1, TBB2c, TBB3 and TBB4 in PanCa cells via destabilization. Our confocal microscopy results further showed inhibition of β-tubulin in ABI-231 treated PanCa cells. Upregulation of micro RNA 200c (miR-200c) has been shown to inhibit the expression of β-tubulin III in cancer cells. ABI-231 treatment of PanCa cells showed significant (p Conclusion: Taken together, our results suggest that ABI-231 is a potent β-tubulin inhibitor and chemotherapeutic agent which could be used for the treatment of pancreatic cancer. Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Saini Setua, Andrew Massey, Aditya Ganju, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. Attenuation of pancreatic tumor growth by a small molecule tubulin inhibitor [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 3216. doi:10.1158/1538-7445.AM2017-3216

Abstract 1071: Mir-145 based magnetic nanoformulation for pancreatic cancer therapy

Background: Pancreatic cancer (PanCa) 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 PanCa. 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 PanCa, 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 miR-145 based nanoparticle formulation for PanCa treatment. Methods: 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 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 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. Results: miR-145 expression was progressively suppressed over the course of development from PanIN I-III to late stage poorly differentiated PDAC. 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 PanCa cells. MNPF mediated restitution of miR-145 effectively sensitizes PanCa cells for paclitaxel and TRAIL therapy. Conclusions: 1) MNP based delivery systems can be efficiently used for microRNA replacement therapy in order to restore lost microRNAs in cancer. 2) miR-145-MNPF efficiently restores miR-145 in pancreatic cancer cells and inhibits growth and invasion of PanCa. 3) miR-145 restitution using miR-145-MNPF may offer a potential therapeutic strategy for pancreatic cancer treatment alone or in combination with other therapies. Citation Format: Saini Setua, Sheema Khan, Murali Mohan Yallapu, Mohammed Sikander, Stephen W. Behrman, Meena Jaggi, Subhash C. Chauhan. Mir-145 based magnetic nanoformulation for pancreatic cancer therapy. [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 1071.