Septelia Inawati Wanandi1, Noza Hilbertina2, Nurjati Chairani Siregar2, Murdani Abdullah3, Wifanto Saditya Jeo4. 1. Department of BioChemistry and Molecular Biology, Universitas Indonesia, Jakarta, Indonesia. 2. Department of Anatomic Pathology, Universitas Indonesia, Jakarta, Indonesia. 3. Department of Internal Medician, Universitas Indonesia, Jakarta, Indonesia. 4. Department of Sergery, Universitas Indonesia, Jakarta, Indonesia.
Abstract
OBJECTIVE: The aim of this study was to investigate the effect of cancer-associated fibroblasts (CAF) secretomes on the epithelial-mesenchymal transition (EMT) of colorectal carcinoma (CRC) cells and its association with hepatocyte growth factor (HGF) signalling focussing on the HGF receptor, c-Mesenchymal epithelial transition (c-Met), and the EMT markers, vimentin and e-cadherin, in CRC cells. METHODS: Conditioned mediums (CM) containing secretomes from colorectal CAFs and their counterpart normal fibroblasts (NFs) of three CRC patients were collected and supplemented to the HT-29 CRC cells. The mRNA levels of a-smooth muscle actin (a-SMA) and HGF in both fibroblasts, as well as c-Met, vimentin, and e-cadherin in HT-29 cells after supplemented with CAF- and NF-CM were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). HGF protein level in the CM of CAFs and NFs was measured using enzyme-linked immunosorbent assay (ELISA). Vimentin and e-cadherin protein expressions were observed in HT-29 cells using immunofluorescent (IF) staining. RESULTS: Compared to the non-cancerous colon, fibroblasts from cancerous area of CRC substantially expressed higher mRNA levels of a-SMA, a CAF marker. The HGF mRNA expressions in CAFs and NFs were in line with the HGF protein level in the secretomes of both cells. CAF-CM increased c-Met and vimentin mRNA levels in HT-29 cells. Surprisingly, e-cadherin mRNA level in HT-29 cells was increased following CAF-CM supplementation. We also demonstrated the co-localization of e-cadherin and vimentin in the HT-29 cell cytoplasm. CONCLUSIONS: CAF secretomes of CRC promote a hybrid type of EMT associated with HGF signalling.
OBJECTIVE: The aim of this study was to investigate the effect of cancer-associated fibroblasts (CAF) secretomes on the epithelial-mesenchymal transition (EMT) of colorectal carcinoma (CRC) cells and its association with hepatocyte growth factor (HGF) signalling focussing on the HGF receptor, c-Mesenchymal epithelial transition (c-Met), and the EMT markers, vimentin and e-cadherin, in CRC cells. METHODS: Conditioned mediums (CM) containing secretomes from colorectal CAFs and their counterpart normal fibroblasts (NFs) of three CRC patients were collected and supplemented to the HT-29 CRC cells. The mRNA levels of a-smooth muscle actin (a-SMA) and HGF in both fibroblasts, as well as c-Met, vimentin, and e-cadherin in HT-29 cells after supplemented with CAF- and NF-CM were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). HGF protein level in the CM of CAFs and NFs was measured using enzyme-linked immunosorbent assay (ELISA). Vimentin and e-cadherin protein expressions were observed in HT-29 cells using immunofluorescent (IF) staining. RESULTS: Compared to the non-cancerous colon, fibroblasts from cancerous area of CRC substantially expressed higher mRNA levels of a-SMA, a CAF marker. The HGF mRNA expressions in CAFs and NFs were in line with the HGF protein level in the secretomes of both cells. CAF-CM increased c-Met and vimentin mRNA levels in HT-29 cells. Surprisingly, e-cadherin mRNA level in HT-29 cells was increased following CAF-CM supplementation. We also demonstrated the co-localization of e-cadherin and vimentin in the HT-29 cell cytoplasm. CONCLUSIONS: CAF secretomes of CRC promote a hybrid type of EMT associated with HGF signalling.