Browsing by Author "Tarkiainen, Susanna"

The motivation of this study was to find new treatment options for the rare cancer pseudomyxoma peritonei (PMP). PMP is a slowly progressing mucinous adenocarcinoma that originates from the appendix and disseminates into the peritoneum where the cancer cells secrete large amounts of MUC2, the main component of intestinal mucus, into the peritoneum. The disulfide isomerase AGR2 helps MUC2 with forming the correct intramolecular disulfide bonds prior secretion, and is essential to MUC2 protein production. The mucus build-up into the peritoneum causes stress on vital organs, and eventually death. Therefore, inhibiting MUC2 production in PMP cancer cells might slow down the disease progression significantly. MAPK/ERK and cAMP/PKA signaling pathways stimulate MUC2 production, and activating mutations in KRAS and GNAS of these pathways are common in PMP. The aim of this study was to elucidate how MUC2 and AGR2 affect each other’s expression levels, and how the MAPK/ERK pathway and the cAMP/PKA pathway targeting substances caffeine, theophylline, cromolyn, fudosteine, octreotide, and lanreotide, affect MUC2 expression in vitro. This study was conducted on human colorectal adenocarcinoma cell lines LS174T, LoVo, and HT29 that all produce large amounts of MUC2. In addition, LS174T and LoVo cell lines carry activating heterozygous mutations in their KRAS genes. MUC2 and AGR2 expression levels were measured on mRNA level with real-time quantitative reverse transcriptase polymerase chain reaction. The effects of MUC2 on AGR2 expression, and vice versa, were tested by silencing each at a time with the appropriate siRNA. MUC2 siRNA suppressed both MUC2 and AGR2 mRNA levels down to 50 % in LS174T cells and down to 40 % in LoVo cells in respect to their control groups. AGR2 siRNA suppressed AGR2 mRNA levels down to 50 % in LS174T cells and even down to 30 % in LoVo cells in respect to their control groups, while there were no statistically significant changes in MUC2 mRNA levels. Caffeine and theophylline inhibit phosphodiesterase of the cAMP/PKA signaling pathway, and in consequence, the hydrolysis of the secondary messenger cAMP, prolonging the activation of the pathway. Caffeine stimulated MUC2 production in LS174T and LoVo cells. In addition, AGR2 mRNA levels increased in LoVo cells, in which the fold change in MUC2 mRNA levels was much greater. Theophylline, a compound found in tea, and used for the treatment of asthma, did not affect MUC2 production. PMP cancer cells express protein S100P. Cromolyn is a pharmaceutical substance used for the treatment of asthma, and it inhibits S100P, and thereby S100P/RAGE signaling -dependent activation of MAPK/ERK pathway. Fudosteine, on the other hand, is a mucoactive pharmaceutical substance that lowers the production of MUC5AC, the main component of lung mucus. Since activating GNAS mutation stimulates the production of both MUC2 and MUC5AC in HT29 cells, the expression of both must be at least partially controlled by same mechanisms. In addition, Somatostatin analogues octreotide and lanreotide inhibit ERK1/2 of the MAPK/ERK pathway, as well as protein kinase A of the cAMP/PKA pathway, and hence cAMP production. However, none of these four tested pharmaceutical substances were able to inhibit MUC2 production in the cell lines used this study in vitro.