R.I. Mahato, A.K. Chaudhary, G. Mondal, V. Kumar, K. Kattel, D. Chitkara, A. Mittal
University of Nebraska Medical Center,
Keywords: microRNA-205, gemcitabine, polymeric micelles, pancreatic cancer
Summary:Despite enormous progress made over the decades, the survival rate for pancreatic cancer patients still remains poor. Although gemcitabine (GEM) is effective in treating pancreatic cancer, its rapid metabolism into inactive form reduces its therapeutic potential. Therefore, we synthesized methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-gemcitabine-graft-dodecanol (mPEG-b-PCC-g-GEM-g-DC), which self-assembled into micelles and inhibited tumor growth, with high accumulation in the tumor compared to free GEM. Since pancreatic cancer overexpress EGFR, we conjugated EGFR targeting GE11-peptide to malemido-poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol (GE11-PEG-PCD) and prepared mixed micelles with mPEG-b-PCC-g-GEM-g-DC for active targeting. These GE11-linked mixed micelles showed significant inhibition of tumor growth in orthotopic pancreatic tumor bearing mice. miRNA target multiple genes which influence cell proliferation, migration and invasion, and promotes chemoresistance. We found downregulation of miR-205 in clinical pancreatic cancer tissues and cell lines (MAI PaCa-2, HPAF-II, BXPC3 and MIA PaCa-2R), but its level was significant low in gemcitabine resistant cells. miR-205 was highly downregulated in the pancreatic patient tumor tissues with highest metastastatic sites and lowest with least metastasis sites. Considering miR-205 to resensitize GEM-resistant pancreatic cancer, we stably overexpressed miR-205 in MIA PaCa-2R cells which resulted in its sensitization to GEM. Surprisingly, miR-205 alone was sufficient in inhibiting MIA PaCa-2R cell growth, CSCs and tumor size in subcutaneous tumor bearing mice. However, the combination of miR-205 and GEM was more effective in reducing cell migration and invasion, CSC proliferation, 3D spheroids and induction of apoptosis. There was significant reduction in the expression of CSC, EMT and chemoresistance markers after miR-205 overexpression. Similarly, a significant reduction in orthotopic pancreatic tumor size in mice overexpressing miR-205 after intravenous administration of GEM-conjugated polymeric micelles was achieved. Moreover, immunohistochemical analysis of tumor tissues from orthotopic tumor bearing mice showed significant reduction in oncogenic drug resistance protein Caveolin-1 and cell proliferation marker Ki-67 after combination treatment compared to the control. Since the use of miR-205 stably overexpressing cell line is not a feasible strategy to treat pancreatic cancer in the clinic, we developed GEM-conjugated cationic copolymers, poly(ethyleneglycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graftcationic ligands) (mPEG-b-PCC-g-GEM-g-DC-g-CAT) to form complex with miR-205 mimic to co-deliver miR-205 and GEM using mixed micelles decorated with EGFR targeting antibody, cetuximab for site-specific delivery to the tumor after systemic admisntartion. Cetuximab-linked mixed micellar polyplex containing miR-205 and GEM showed EGFR mediated enhanced transfection and cellular uptake in EGFR expressing MIA PaCa-2R cells. In orthotopic pancreatic tumor model, cetuximab-linked mixed micellar polyplex containing miR-205 and GEM treated mice showed enhanced accumulation in the tumor after systemic administration. There was significant reduction in tumor growth due to apoptosis of tumor cells with reduced tumor cell proliferation as evident from immuno-histochemical analysis. These finding suggest, miR-205 helps in sensitizing GEM-resistant pancreatic cancer cells and act as tumor suppressor. Further, targeted delivery of payloads enhanced the accumulation of drugs to tumor site reducing undesired toxicity.