Isoform specific changes in calcium transporters in colon and breast cancer cell lines

Calcium transporters play vital roles in the transport of calcium ions across cells of the mammary gland and the intestine. One such transporter is the plasma membrane Ca2+-ATPase (PMCA), of which there are 4 different genes (PMCA1-4). In these studies we investigated the hypothesis that the expression of PMCA is altered in HT-29 colon cancer cells during sodium butyrate and post-confluence mediated differentiation. We also investigated if PMCA expression is altered in breast cancer cell lines in an isofrom specific manner. Our results indicate isoform specific changes in PMCA mRNA and protein levels in HT-29 cells during differentiation, using real time RT-PCR and western blotting, respectively. We also observed pronounced alterations in the mRNA levels of the PMCA isoform linked to lactation (PMCA2) in a bank of breast cancer cell lines compared to normal cell lines. Changes in other isoforms were less pronounced. To further study the role of specific calcium transporters we have optimised conditions for the reverse transfection of MCF-7 breast cancer cells using NeoFX (Ambion). Using real time RT-PCR we have confirmed gene knockdown for specific isoforms and have studied the time course of knockdown over 96 hours. We see approximately 68 % inhibition at 24 hours increasing to 84 % 96 hours post-reverse transfection. Our studies suggest that the expression of specific calcium transporter isoforms can be significantly altered in cancer cell lines and that isoform specific inhibition of calcium transporters is possible using reverse transfection of siRNA

The role of tissue transglutaminase (TG2) in regulating the tumour progression of the mouse colon carcinoma CT26

The multifunctional enzyme tissue transglutaminase (TG2) is reported to both mediate and inhibit tumour progression. To elucidate these different roles of TG2, we established a series of stable-transfected mouse colon carcinoma CT26 cells expressing a catalytically active (wild type) and a transamidating-inactive TG2 (Cys277Ser) mutant. Comparison of the TG2-transfected cells with the empty vector control indicated no differences in cell proliferation, apoptosis and susceptibility to doxorubicin, which correlated with no detectable changes in the activation of the transcription factor NF-?B. TG2-transfected cells showed increased expression of integrin ß3, and were more adherent and less migratory on fibronectin than control cells. Direct interaction of TG2 with ß3 integrins was demonstrated by immunoprecipitation, suggesting that TG2 acts as a coreceptor for fibronectin with ß3 integrins. All cells expressed the same level of TGFß receptors I and II, but only cells transfected with active TG2 had increased levels of TGFß1 and matrix-deposited fibronectin, which could be inhibited by TG2 site-directed inhibitors. Moreover, only cells transfected with active TG2 were capable of inhibiting tumour growth when compared to the empty vector controls. We conclude that in this colon carcinoma model increased levels of active TG2 are unfavourable to tumour growth due to their role in activation of TGFß1 and increased matrix deposition, which in turn favours increased cell adhesion and a lowered migratory and invasive behaviour.

Polyunsaturated fatty acid requirements of murine colon adenocarcinomas

The polyunsaturated fatty acid (PUFA) requirements of three transplantable murine colon adenocarcinomas, the MAC13, MAC16 and MAC26, were evaluated in vitro and in vivo. When serum concentrations became growth limiting in vitro, proliferation of the MAC13 and MAC26 cell lines was stimulated by linoleic acid (LA) at 18μM and arachidonic acid (AA) at 16 or 33μM respectively. This was not demonstrated by the MAC16 cell line. MAC13 and MAC26 cells were found to be biochemically fatty acid deficient as measured by the formation of Mead acid (20:3 n-9), but the MAC16 cells were not. In vivo the growth of the MAC26 tumour was stimulated by daily oral administration of LA between 0.4-2.0g/kg. There was a threshold value of 0.4g/kg for the stimulation of MAC26 tumour growth, above which there was no further increase in tumour growth, and below which no increase in tumour growth was observed. This increased tumour growth was due to the stimulation of tumour cell proliferation in all areas of the tumour, with no effect on the cell loss factor. The growth of the MAC13, MAC16, and MAC26 cell lines in vitro were more effectively inhibited by lipoxygenase (LO) inhibitors than the cyclooxygenase inhibitor indomethacin. The specific 5-LO inhibitor Zileuton and the leukotriene D4 antagonist L-660,711 were less effective inhibitors of MAC cell growth in vitro than the less specific LO inhibitors BWA4C, BWB70C and CV6504. Studies of the hyroxyeicosatetraenoic acids (HETEs) produced from exogenous AA in these cells, suggested that a balance of eicosanoids produced from 5-LO, 12-LO and 15-LO pathways was required for cell proliferation. In vivo BWA4C, BWB70C and CV6504 demonstrated antitumour action against the MAC26 tumour between 20-50mg/kg/day. CV6504 also inhibited the growth of the MAC 13 tumour in vivo with an optimal effect between 5-10mg/kg/day. The antitumour action against the MAC16 tumour was also accompanied by a reduction in the tumour-induced host body weight loss at 10-25mg/kg/day. The antitumour action of CV6504 in all three tumour models was partially reversed by daily oral administration of 1.0g/kg LA. Studies of the AA metabolism in tumour homogenates suggested that this profound antitumour action, against what are generally chemoresistant tumours, was due to inhibition of eicosanoid production through LO pathways. As a result of these studies, CV6504 has been proposed for stage I./II. clinical trials against pancreatic cancer by the Cancer Research Campaign. This will be the first LO inhibitor entering the clinic as a therapeutic agent.

A study of the growth and differentiation of the human adenocarcinoma of the colon cell line HT-29

The HT-29 human colon adenocarcinoma cell line, like many epithelial cells, displays an undifferentiated phenotype when cultured on plastic substrata. Biochemical markers of differentiation, such as brush border associated enzymes and carcinoembryonic antigen were expressed at very low levels. The differentiation-inducing effects of the culture of HT-29 cells on collagen type I gels were evaluated, and were assessed by morphological appearance, brush border associated enzyme activities and the secretion of CEA. The effect that this more physiological environment had on their chemosensitivity to a panel of chemotherapeutic agents was determined, so as to indicate whether this system could be used to improve the selectivity of screening for novel anticancer agents. Initial studies were performed on HT-29 cells derived from cells seeded directly from plastic substrata onto the collagen gels (designated Non-PPC gels). Their time of exposure to the collagen was limited to the time course of a single experiment and the results suggested that a longer, more permanent exposure might produce a more pronounced differentiation. HT-29 cells were then passaged continuously on collagen gels for a minimum of 10 passages prior to experimentation (designated PPC gels). The same parameters were measured, and compared to those for the cells grown on plastic and on the non-passaged collagen gels (Non-PPC) from the original studies. Permanently passaged cells displayed a similar degree of morphological differentiation as the non-passaged cells, with both culture conditions resulting in a more pronounced differentiation than that achieved by culture on plastic. It was noted that the morphological differentiation observed was very heterogeneous, a situation also seen in xenografted tumours in vivo. The activity of alkaline phosphatase and the production of CEA was higher in the cells passaged on collagen (PPC) than the cells cultured on non-passaged collagen gel (Non-PPC) and plastic. The biochemical determination of aminopeptidase activity showed that collagen gel culture enhanced the activity in both non-passaged and passaged HT-29 cells above that of the cells cultured on plastic. However, immunocytochemical localization of aminopeptidase and sucrase-isomaltase of samples of cells grown on the various substrata for 7, 14, 21 and 28 days showed a reduction in both enzymes in the cells grown on collagen gels when compared to cells grown on plastic. The reason for the discrepancy between the two assays for aminopeptidase is at this stage unexplained. Although, there was evidence to suggest that the culture of HT-29 cells on collagen gels was capable of inducing morphological and biochemical markers of enterocytic differentiation, there were no differences in the chemosensitivity of the different cell groups to a panel of anticancer agents. Preliminary studies suggested that the ability of the cells to polarize by their culture on porous filter chambers without any exogenous ECM was sufficient to enhance HT-29 differentiation and the onset of differentiation was probably correlated with the production of ECM by the cells themselves.

A novel role for the adipokine visfatin/pre-B cell colony-enhancing factor 1 in prostate carcinogenesis

Adipose tissue is now well established as an endocrine organ and multiple hormones termed ‘adipokines’ are released from it. With the rapidly increasing obese population and the increased risk mortality from prostate cancer within the obese population we looked to investigate the role of the adipokine visfatin in LNCaP and PC3 prostate cancer cell lines. Using immunohistochemistry and immunocytochemistry we demonstrate visfatin expression in LNCaP (androgen-sensitive) and PC3 (androgen-insensitive) human prostate cancer cell lines as well as human prostate cancer tissue. Additionally, we show that visfatin increases PC3 cell proliferation and demonstrate the activation of the MAPKs ERK-1/2 and p38. Moreover we also demonstrate that visfatin promotes the expression and activity of MMP-2/9 which are important proteases involved in the breakdown of the extracellular matrix, suggesting a possible role for visfatin in prostate cancer metastases. These data suggest a contributory and multifunctional role for visfatin in prostate cancer progression, with particular relevance and emphasis in an obese population.