Gemcitabine sensitivity can be induced in pancreatic cancer cells through modulation of miR-200 and miR-21 expression by curcumin or its analogue CDF

Abstract

Curcumin induces cancer cell growth arrest and apoptosis in vitro, but its poor bioavailability in vivo limits its antitumor efficacy. We have previously evaluated the bioavailability of novel analogues of curcumin compared with curcumin, and we found that the analogue CDF exhibited greater systemic and pancreatic tissue bioavailability. In this study, we evaluated the effects of CDF or curcumin alone or in combination with gemcitabine on cell viability and apoptosis in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer (PC) cell lines. Mechanistic investigations revealed a significant reduction in cell viability in CDF-treated cells compared with curcumin-treated cells, which were also associated with the induction of apoptosis, and these results were consistent with the downregulation of Akt, cyclooxygenase-2, prostaglandin E(2), vascular endothelial growth factor, and NF-kappaB DNA binding activity. We have also documented attenuated expression of miR-200 and increased expression of miR-21 (a signature of tumor aggressiveness) in gemcitabine-resistant cells relative to gemcitabine-sensitive cells. Interestingly, CDF treatment upregulated miR-200 expression and downregulated the expression of miR-21, and the downregulation of miR-21 resulted in the induction of PTEN. These results prompt further interest in CDF as a drug modality to improve treatment outcome of patients diagnosed with PC as a result of its greater bioavailability in pancreatic tissue.

Figure 1

Growth inhibition of BxPC-3 cells treated with 0.1 to 4.0 μmol/L of CDF/curcumin (A); BxPC-3, MIAPaCa-E, and MIAPaCa-M cells treated with 1 to 4 μmol/L of CDF/curcumin, 10 nmol/L of gemcitabine, and the combination (B); and MIAPaCa-E and MIAPaCa-M cells treated with 10 μmol/L of CDF/curcumin, 10 nmol/L of gemcitabine, and the combination (C) were evaluated by the MTT assay. D, clonogenic assay of MIAPaCa-E cells were treated with 4 μmol/L of CDF/curcumin, 10 nmol/L of gemcitabine, and the combinations. The P values represent comparisons between cells treated by either of the drugs and their combinations by using the paired t test.

Figure 2

A, light photomicrographs of BxPC-3, MIAPaCa-E, and MIAPaCa-M cell line. MIAPaCa cells were exposed to gemcitabine, and the paired cell line was called MIAPaCa-E and MIAPaCa-M based on their changes in morphology from epithelial-like to mesenchymal-like phenotype. B, induction of apoptosis in BxPC-3, MIAPaCa-E, and MIAPaCa-M cells treated with 1 to 4 μmol/L of CDF/curcumin, 10 nmol/L of gemcitabine, and their combinations. C, induction of apoptosis in MIAPaCa-E and MIAPaCa-M cells treated with 10 μmol/L of CDF/curcumin, 10 nmol/L of gemcitabine, and the combination. The P values represent comparisons between cells treated by either of the drugs and their combinations by using the paired t test.

Figure 3

A, the expression of COX-2, E-cadherin, PTEN, pAkt, tropomyosin, and β-actin in BxPC-3, MIAPaCa-E, and MIAPaCa-M cell lines treated with 1 to 4 μmol/L CDF/curcumin, 10 nmol/L gemcitabine, or the combination for 72 h. B, comparative expression analysis of miR-21 in BxPC-3, MIAPaCa-E, and MIAPaCa-M cells by real-time miRNA RT-PCR. Cells were treated with 1 to 4 μmol/L CDF/curcumin, 10 nmol/L gemcitabine, or their combinations for 72 h. The expression of PTEN, pAkt, and NF-κB in MIAPaCa-E cells after transfection with miR-21 antisense oligo (C) and transfection with PTEN cDNA (D), followed by gemcitabine treatment for 48 h.

Figure 4

DNA binding activity of NF-κB in nuclear protein extracts of BxPC-3, MIAPaCa-E, and MIAPaCa-M cells as assessed by electrophoretic mobility shift assay. Cells were treated with 1 to 4 μmol/L CDF/curcumin, 10 nmol/L gemcitabine, or their combinations for 72 h.

Figure 5

PGE₂ ELISA (A) and VEGF ELISA (B) were performed using conditioned medium collected from BxPC-3, MIAPaCa-E, and MIAPaCa-M cells. Cells were treated with 1 to 4 μmol/L CDF/curcumin, 10 nmol/L gemcitabine, or their combinations for 24 h. Please note that the basal level of PGE₂ in BxPC-3 cells was 10 times higher than MIAPaCa-E and MIAPaCa-M cells.

Figure 6

A, comparative expression of miR-200b and miR-200c in BxPC-3, MIAPaCa-E, and MIAPaCa-M cells as assessed by real-time miRNA RT-PCR. Cells were treated with 1 to 4 μmol/L CDF/curcumin, 10 nmol/L gemcitabine, or their combinations. Expression of miR-200b (B) and expression of miR-200c (C) are shown.