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. 2015 Nov 23:14:200.
doi: 10.1186/s12943-015-0470-6.

Minnelide effectively eliminates CD133(+) side population in pancreatic cancer

Alice Nomura  1 , Olivia McGinn  1 , Vikas Dudeja  1 , Veena Sangwan  1 , Ashok K Saluja  1   2 , Sulagna Banerjee  3   4
Affiliations

Minnelide effectively eliminates CD133(+) side population in pancreatic cancer

Alice Nomura et al. Mol 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 (12 T cells) for 7 days. 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 12 T and CSM cells were tested for stemness properties using RNA and protein markers. Low numbers of CSM and 12 T cells were implanted subcutaneously in athymic nude mice to study their tumorigenic potential. 12 T and CSM cells were sorted for CD133 expression and assayed for their colony forming ability and sphere forming ability. Invasiveness of 12 T cells, CSM and MIA PaCa-2 were compared using Boyden chamber assays.

Results: Treated 12 T cells displayed increased expression of the surface marker CD133 and the drug transporter ABCG2 compared to untreated cells (CSM cells). Both 12 T and CSM cells formed subcutaneous tumors in mice confirming their tumor-initiating properties. When tested for invasion, 12 T cells had increased invasiveness compared to CSM cells. CD133(+) cells in both CSM and 12 T 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 12 T 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 12 T 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.

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Figures

Fig. 1
Fig. 1
a Treatment of pancreatic cancer cell lines MIA PaCa-2, S2-013 and AsPC1 with 12.5 nM triptolide for 7 days resulted in 20 % viable cells. b These cells (12 T) had increased expression of ABCG2 gene when compared to untreated CSM cells. c Increased expression of ABCG2 correlated with increased DNA dye efflux in MIA PaCa-2 12 T cells. d Quantitation of inhibition of DNA dye efflux on treatment with Verapamil, an inhibitor of ABC transporter in 12 T and CSM cells. The error bars represent SEM (n = 4; * = p < 0.05)
Fig. 2
Fig. 2
a 12 T cells showed higher percentage of CD133+ cells in all three cell lines tested: MIA PaCa-2, S2-013, AsPC1. b 12 T cells also showed increased CD24+CD44+ESA+ cells compared to CSM cells. CSM and 12 T cells showed increased expression of stemness proteins like Jagged, Notch1 and Gli1 (c) Tumor progression in athymic nude mice from (d) CSM cells and (e) 12 T cells. The error bars represent SEM (n = 4; * = p < 0.05)
Fig. 3
Fig. 3
CD133+ cells from both CSM and 12 T cells showed a increased colony forming ability and b increased sphere forming ability when compared with CD133− cells. Tumor volume over time from c 500 CSM CD133+ cells and d 500 12 T CD133+ cells formed tumors in athymic mice while the CD133- cells did not form tumors in either group. e H&E staining and immunofluorescent staining of ALDH1 of the CSM-CD133+ and 12 T-CD133+ derived tumors. The error bars represent SEM (n = 4; * = p < 0.05)
Fig. 4
Fig. 4
a Boyden chamber assay showing increased invasion of 12 T cells compared to CSM cells. RNA Expression of transcription factors involved in b EMT and c cytoskeletal re-corganization gene expression in 12 T cells compared to CSM cells. d Protein expression showing positive correlation of CD133, ABCG2 and Vimentin in CSM CD133+ and 12 T CD133+ tumor bearing animals and e immunofluorescent staining of Vimentin tumor expression. The error bars represent SEM (n = 4; * = p < 0.05)
Fig. 5
Fig. 5
Tumor progression after implantation of a CSM-CD133+ cells and b 12 T-CD133+ cells and treatment with Minnelide (0.42 mg/kg body weight) in athymic nude mice. Tumor weight of c CSM-CD133+ tumors and d 12 T-CD133+ tumors and tumor volume of e CSM-CD133+ tumors and f 12 T-CD133+ tumors after treatment with Minnelide. The error bars represent SEM (n = 4; * = p < 0.05)
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