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In vitro interactions between rodent hepatic stellate cells and metastatic and non-metastatic human colorectal cancer cell lines

S Bennaser, N C Bird, D Canovas
Published in : HAMDAN MEDICAL JOURNAL ; Vol 6, No 3 (2013)
DOI : 10.7707/hmj.v6i3.235

Abstract


Hepatic stellate cells (HSCs) are involved in the invasiveness of cancer cells through transdifferention into myofibroblast-like cells, which are characterized by the expression of alpha-smooth muscle actin [α-SMA (Abcam, Cambridge, UK)]. The aim of the present study is to examine whether the HT-29 metastatic colon cancer cell line and Colo-741 non-metastatic colon cancer cell line influence the activation, transdifferentiation and survival of HSCs. Quiescent HSCs (qHSCs) were isolated and identified by vitamin A autofluorescence, Oil red O lipid staining and glial fibrillary acidic protein (GFAP) expression. The duration of qHSC transdifferention was determined using immunocytochemistry via sequential staining of α-SMA and Oil red O on plastic cultures at days 1, 3 and 5. Immunocytochemistry was performed to quantitatively examine the expression of α-SMA in the metastatic and non-metastatic colon cancer cell lines. α-SMA expression was observed for a period of 10 days and the Ki-67 proliferation assay was investigated on day 1 of qHSCs co-culture with HT-29 or Colo-741 conditioned medium. In addition, we examined a caspase-cleaved fragment of cytokeratin (CK)-18 in the qHSCs of these co-cultures. The duration of qHSC transdifferention into myofibroblast-like cells was detected by α-SMA on day 5, whereas lipid droplets, which represent the quiescent phase, were detected up to day 3 only. We found expression of α-SMA at day 1 in the HT-29 co-culture, which increased consistently throughout the duration of the experiment. In contrast, α-SMA was observed only on days 7 and 10 in the Colo-741 co-culture. Using the Ki67 proliferated assay, we could determine that the stellate cells had proliferated in the HT-29 conditioned medium, whereas the Colo-741 conditioned medium did not show any changes. However, by using CK18, it was noted that Colo-741 were found to be significantly proapoptotic. These findings show that the metastatic cell line accelerates transdifferentiation whereas the non-metastatic cell line retards it. In addition, the non-metastatic cell line induced higher levels of apoptosis in myofibroblast-like cells, providing further evidence for a positive role of stellate cells in the metastatic process rather than simply acting to provide a pseudocapsule between the tumour and liver parenchyma, as previously thought.


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