Minnelide effectively eliminates CD133+ side population in pancreatic cancer

Abstract

Background: Pancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease hallmarked by limited patient survival. Resistance to chemotherapy, a major cause of treatment failure in PDAC patients, is often attributed to Cancer Stem Cells (CSCs). Pancreatic CSCs are a small subset of quiescent cells within a tumor represented by surface markers like CD133. These cells are responsible not only for tumor recurrence, but also poor prognosis based on their "stem-like" characteristics. At present, conventional therapy is directed towards rapidly dividing PDAC cells and thus fails to target the CSC population. Methods: MIA PaCa-2, S2-013 and AsPC-1 were treated with 12.5 nM triptolide (12T cells) for 7days. The surviving cells were recovered briefly in drug-free growth media and then transferred to Cancer Stem cell Media (CSM). As a control, untreated cells were also transferred to CSM media (CSM). The 12T and CSM cells were tested for stemness properties using RNA and protein markers. Low numbers of CSM and 12T cells were implanted subcutaneously in athymic nude mice to study their tumorigenic potential. 12T and CSM cells were sorted for CD133 expression and assayed for their colony forming ability and sphere forming ability. Invasiveness of 12T cells, CSM and MIA PaCa-2 were compared using Boyden chamber assays. Results: Treated 12T cells displayed increased expression of the surface marker CD133 and the drug transporter ABCG2 compared to untreated cells (CSM cells). Both 12T and CSM cells formed subcutaneous tumors in mice confirming their tumor-initiating properties. When tested for invasion, 12T cells had increased invasiveness compared to CSM cells. CD133+ cells in both CSM and 12T showed greater colony and sphere forming ability compared to CD133- cells from each group. Consistent with these data, when injected subcutaneously in mice, CD133- cells from CSM or 12T did not form any tumors whereas CD133+ cells from both groups showed tumor formation at a very low cell number. Despite pre-exposure to triptolide in 12T CD133+ cells, treatment of tumors formed by these cells with Minnelide, a triptolide pro-drug, showed significant tumor regression. Conclusion: Our results indicated that triptolide enhanced and enriched the "stemness" in the PDAC cell lines at a low dose of 12.5 nM, but also resulted in the regression of tumors derived from these cells.