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Lysosomal alpha-glucosidase: cell-specific processing and altered maturation in HT-29 colon cancer cells.

ABSTRACT: We have previously described the abnormal localization of resident Golgi proteins and O-glycans in the rough endoplasmic reticulum of mucin-secreting HT-29 M6 colon cancer cells, suggesting altered protein trafficking in these cells [Egea, Francí, Gambús, Lesuffleur, Zweibaum and Real (1993) J. Cell Sci. 105, 819-830]. In the present work, we have chosen lysosomal alpha-glucosidase as a reporter to examine the intracellular traffic of glycoproteins in M6 cells. We have compared the synthesis and processing of alpha-glucosidase in mucin-secreting M6 cells and in Caco-2 colon cancer cells, the latter resembling normal absorptive intestinal epithelium. Our results show that alpha-glucosidase processing and secretion is markedly delayed in M6 cells as compared to Caco-2 cells or normal fibroblasts, and this delay is caused by an accumulation of alpha-glucosidase precursor form in the trans-Golgi network. Furthermore, treatment in Caco-2 cells with brefeldin A led to changes in alpha-glucosidase maturation similar to those observed in untreated M6 cells. To determine whether altered processing occurs in other cultured cells, a panel of cancer cell lines and cultures from normal exocrine pancreas were examined. In pancreas-derived cultures, alpha-glucosidase showed a processing pattern different from that described until now. Only HT-29 cells and HT-29-derived subpopulations displayed a defect in alpha-glucosidase maturation. In conclusion, alpha-glucosidase processing is more diverse than has previously been described; this finding may have tissue-specific functional implications.

SUBMITTER: Franci C

PROVIDER: S-EPMC1217045 | biostudies-other | 1996 Feb

REPOSITORIES: biostudies-other

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Project description:In the present study, we evaluated the antitumor effect of two synthetic analogs of vitamin D, namely PRI-2191 [(24R)-1,24-dihydroxyvitamin D3] and PRI-2205 (5,6-trans calcipotriol), in combined human colon HT-29 cancer treatment with 5-fluorouracil (5-FU). Mice bearing HT-29 tumors transplanted subcutaneously or orthotopically were injected with vitamin D analogs and 5-FU in various schedules. A statistically significant inhibition of subcutaneous or orthotopic tumor growth was observed as a result of combined therapy. In HT-29 tumors and in cells from in vitro culture, we observed increased vitamin D receptor (VDR) expression after treatment with either PRI-2205 or 5-FU alone, or in combination. Moreover, PRI-2205 decreased the percentage of cells from intestinal tumors in G2/M and S stages and increased sub-G1. Increased VDR expression was also observed after combined treatment of mice with 5-FU and PRI-2191. Moreover, our docking studies showed that PRI-2205 has stronger affinity for VDR, DBP and CAR/RXR ligand binding domains than PRI-2191. PRI-2191 analog, used with 5-FU, increased the percentage of subcutaneous tumor cells in G0/G1 and decreased the percentage in G2/M, S and sub-G1 populations as compared to 5-FU alone. In in vitro studies, we observed increased expression of p21 and p-ERK1/2 diminution via use of both analogs as compared to use of 5-FU alone. Simultaneously, PRI-2191 antagonizes some pro-apoptotic activities of 5-FU in vitro. However, in spite of these disadvantageous effects in terms of apoptosis, the therapeutic effect expressed as tumor growth retardation by PRI-2191 is significant. Our results suggest that the mechanism of potentiation of 5-FU antitumor action by both analogs is realized via increased p21 expression and decreased p-ERK1/2 level which may lead to diminution of thymidylate synthase expression. Higher binding affinity for VDR, DBP, but also for CAR\RXR ligand binding domain of PRI-2205 may, in part, explain its very low toxicity with sustained anticancer activity.

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Lysosomal alpha-glucosidase: cell-specific processing and altered maturation in HT-29 colon cancer cells.

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