Effects of thymoquinone in the expression of mucin 4 in pancreatic cancer cells: implications for the development of novel cancer therapies

Abstract

Pancreatic cancer is one of the most lethal cancers in the world, as it continues to be resistant to any therapeutic approaches. The high molecular weight glycoprotein mucin 4 (MUC4) is aberrantly expressed in pancreatic cancer and contributes to the regulation of differentiation, proliferation, metastasis, and the chemoresistance of pancreatic cancer cells. The absence of its expression in the normal pancreatic ductal cells makes MUC4 a promising target for novel cancer therapeutics. Natural products have been widely investigated as potential candidates in cancer therapies, and thymoquinone (TQ), extracted from the seeds of Nigella sativa, has shown excellent antineoplastic properties in some systems. In the present study, we evaluated the effect of TQ on pancreatic cancer cells and specifically investigated its effect on MUC4 expression. The MUC4-expressing pancreatic cancer cells FG/COLO357 and CD18/HPAF were incubated with TQ, and in vitro functional assays were done. The results obtained indicate that treatment with TQ downregulated MUC4 expression through the proteasomal pathway and induced apoptosis in pancreatic cancer cells by the activation of c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase pathways. In agreement with previous studies, the decrease in MUC4 expression correlated with an increase in apoptosis, decreased motility, and decreased migration of pancreatic cancer cells. MUC4 transient silencing studies showed that c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase pathways are activated in pancreatic cancer cells, indicating that the activation of these pathways by TQ is directly related to the MUC4 downregulation induced by the drug. Overall, TQ has potential for the development of novel therapies against pancreatic cancer.

Figure 1

Effect of TQ in cell viability and MUC4 expression on FG/COLO357 cells. A, cytotoxicity of TQ in FG/COLO357 cells after being incubated with the drug for 24 h. Points, mean of quadruplicate values; bars, SE. B, Western blot analysis of MUC4 expression in FG/COLO357 cells after being incubated with different doses of TQ and different time intervals. Protein lysates (20 μg) were resolved on 2% SDS agarose gels. β-Actin was used as the loading control. C, confocal microscopy images of FG/COLO357 cells after being incubated with different concentrations of TQ. Cells were stained with anti-MUC4 monoclonal antibody and FITC-conjugated secondary antibody. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 5 μm.

Figure 2

Downregulation of MUC4 expression on FG/COLO357 cells by TQ. A, measurement of MUC4 transcripts of cells incubated with TQ by RT-PCR. The housekeeping gene β-actin was used as a control. B, quantification of MUC4 transcripts of cells incubated with TQ by real-time PCR. Columns, fold difference of the MUC4 mRNA level in untreated cells (0 μmol/L) and TQ-treated cells (50 μmol/L); bars, SE. The housekeeping gene β-actin was used as an internal control. Statistical analysis was done and samples were not significantly different. C, Western blot analysis of MUC4 expression after being incubated with TQ in the presence of the PrI MG132. Experimental samples included media only, PrI only, TQ only, and PrI with TQ. Protein lysates (20 μg) were resolved in 2% SDS agarose gels. β-Actin was used as the loading control. D, Western blot analysis of MUC4 expression after being incubated with TQ in the presence of the GGTI garcinol. Experimental samples included media only, GGTI only, TQ only, and GGTI with TQ. Protein lysates (20 μg) were resolved in 2% SDS agarose gels. β-Actin was used as the loading control. E, Western blot analysis of total STAT1 and its phosphorylated form (pSer-STAT1) of cells incubated with TQ. Protein lysates (40 μg) were resolved in 10% SDS-PAGE gels.

Figure 3

Effect of TQ on the motility and migration of FG/COLO357 cells. A, optical microscopy images (×4) of the wound-healing assay of FG/COLO357 cells after being incubated with different doses of TQ for 24 h. Dashed yellow lines indicate the migration progress of the cells in the 24-h period. B, quantification of FG/COLO357 cells that migrated through the 8-μm pore size PET membrane after being incubated with TQ. Columns, mean number of cells in 10 different random fields (×10); bars, SE. *, P < 0.005 versus control. Representative optical microscope images of the PET membranes are shown.

Figure 4

Effect of TQ on the cytoskeleton of FG/COLO357. A, confocal microscopy images of FG/COLO357 cells after being incubated with different concentrations of TQ. Actin filaments in the cells were visualized after staining with fluorescent phallotoxins. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 5 μm. B, Western blot analysis of MUC4 and HER2 expression in FG/COLO357 cells after treatment with TQ. Protein lysates (20 μg) were resolved in 2% SDS agarose gels. β-Actin was used as the loading control. C, Western blot analysis of FAK and its phosphorylated form in FG/COLO357 cells after TQ treatment. Protein lysates (40 μg) were resolved in 10% SDS-PAGE gels.

Figure 5

Effect of TQ (50 μmol/L) in the apoptosis and necrosis of FG/COLO357 cells. A, flow cytometry results after staining cells with Annexin V and PI. Triplicate samples were analyzed after 4 and 24 h of incubation, and these were classified as healthy (Annexin V⁻, PI⁻), apoptotic (Annexin V⁺, PI⁻), and necrotic (Annexin V⁺, PI⁺). Columns, mean of triplicate samples at each time point; bars, SE. Representative dot plot data are shown. *, P < 0.05 versus healthy cells (24 h). B, confocal microscopy images of cells incubated with TQ (0 and 50 μmol/L) for 24 h stained with Annexin V and PI. Scale bars, 10 μm.

Figure 6

Correlation of MUC4 downregulation and the induction of apoptosis in TQ-treated FG/COLO357 cells. A, Western blot analysis of functional proteins in downstream signaling pathways in cells incubated with TQ. Protein lysates (40–60 μg) were resolved in 10% SDS-PAGE electrophoresis, and the expression of phospho-JNK (pJNK), total JNK, phospho-p38 (pp38), total p38, Bax, Bcl-xL, cleaved caspase-9, caspase-3, and β-actin was analyzed. B, Western blot analysis of functional proteins expressed in MUC4 transiently knocked down FG/COLO357 cells. After doing transient knockdown of MUC4 in FG/COLO357 for 48 h, protein lysates were analyzed and compared with MUC4-expressing cells. C, model of TQ-mediated cytotoxicity in FG/COLO357 cells. TQ induces the apoptosis of cancer cells by different pathways: (1) direct downregulation of MUC4 posttranscriptionally, which leads to apoptosis by the activation of JNK and p38 MAPK pathways; (2) as MUC4 is associated with HER2, the migration of cancer cells is decreased by the inhibition of FAK; (3) TQ might also induce the activation of the TGF-β pathway that can lead to the activation of JNK and p38 MAPK pathways directly or by its induced posttranscriptional downregulation of MUC4; and (4) TQ might sensitize pancreatic cancer cells to Fas-mediated apoptosis through the generation of ROS that activate JNK pathway.