Abstract
Introduction: Obesity is a significant risk factor in the development of endometrial cancer and poses a serious health threat. Furthermore, morbidities related to obesity are associated with a significantly increased risk of mortality for women who develop endometrial cancer. Obesity is associated with altered levels of many hormones including leptin, insulin, insulin-like growth factor-1 (IGF-I) and adiponectin. The role of these hormones in the progression of endometrial cancer is largely unknown. In this study, it was hypothesised that these obesity-related hormones including as leptin, insulin and IGF-I would promote cancer growth, while adiponectin would inhibit cancer growth.
Objectives: The aim of this study was to use a number of selected cells lines to determine if obesity-related hormones leptin, insulin, IGF-I and adiponectin have effects on cell proliferation, secretion of vascular endothelial growth factor (VEGF) which is a protein associated with the development of blood vessels used to support the growth of cancers, and the expression of specific proteins associated with different cell signalling cascades. These are the phosphatidylinositol 3-kinase (PI-3K)/ Protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway (PI-3K/Akt/mTOR), Janus kinase family and signal transducers and activators of transcription-3 (JAK/STAT3) and 5' adenosine monophosphate-activated protein kinase (AMPK) protein.
Materials and Methods: To examine these hypotheses, two endometrial cell lines, Ishikawa and HEC-1A, a breast cancer cell line, MFC-7 and an ovarian cancer cell line, SKOV-3 were used in this study. Cell lines were cultured and the cells exposed to selected range of leptin, insulin, IGF-I and adiponectin concentrations, both individually and in combinations. In some culture conditions, the addition of pharmacological inhibitors were used to investigate the hormone-stimulated cellular responses including proliferation, secretion of VEGF, and activation of selective signalling molecules using Western blots, flow cytometry and immunofluorescent imaging with an optical sectioning microscope.
Results: In chapter 3, the aim of the experiments was to investigate the effects of leptin, insulin, IGF-I and adiponectin on cell growth and secretion of VEGF in the endometrial cancer cell line, Ishikawa. It was shown that leptin (20-100 ng/ml), insulin (116-2900 ng/ml), and IGF-I (20-500 ng/ml) increased the proliferation of the Ishikawa endometrial cancer cells. On the other hand, adiponectin inhibited cell proliferation at high concentrations (50 - 100 ng/ml) while increasing it at a lower concentration (20 ng/ml). The combinations of leptin, insulin or IGF-I with adiponectin showed different responses, and there was a lack of evidence for additive growth effects in the combinations. For instance, addition of adiponectin (20 ng/ml) to leptin or insulin increased cell numbers. However, the combination of adiponectin (20 ng/ml) and IGF-I reduced cell numbers to the control (no growth factor) levels. These findings suggest that IGF-I uses a different pathway to that used by leptin and insulin to increase cell proliferation.
In chapter 3, the secretion of VEGF was also investigated. Ishikawa cells did not increase VEGF secretion in response to leptin, insulin or IGF-I compared to control cells. In contrast, the VEGF secretion was increased by adiponectin (20-100 ng/ml).
In chapter 4, the aim of the experiments was to compare the effects of these hormones on alternative cancer cell lines with endometrial cancer cell line Ishikawa. The selected cell lines were the endometrial cancer cell line, HEC-1A, a breast cancer cell line MCF-7 and an ovarian cancer cell line, SKOV-3. Cell proliferation, level of apoptosis and VEGF secretion were determined in these cell lines, and the results showed that the effects of the hormones on these parameters were cell line-dependent. For example, cell numbers were increased by leptin, insulin and IGF-I in HEC-1A cells, but insulin did not increase cell numbers in the MCF-7 cell line. In addition, none of these three hormones affected SKOV-3 cell numbers.
In chapter 5, experiments aimed to investigate the mechanism by which leptin stimulates proliferation of Ishikawa cells. Leptin was shown to trigger oncogenic signalling proteins including PI-3K/Akt/mTOR, JAK/STAT3 and AMPK, thus suggesting that leptin stimulated cell proliferation by triggering PI-3K and JAK activation. This was confirmed by the inhibition of these two proteins using specific inhibitors. The results also suggested that there is a cross-talk between JAK/STAT3 and PI-3K/Akt/mTOR pathways, and that JAK is the upstream protein for these two pathways. Leptin’s ability to reduce the secretion of VEGF was not affected by these two inhibitors. Thus, these results also suggest that cell proliferation and VEGF secretion are independently regulated by leptin because leptin increased cell proliferation using JAK/PI-3K pathways and reduced VEGF secretion by reduction in AMPK pathway. It is interesting to note that levels of AMPK and phosphorylated AMPK (p-AMPK), which are often associated with or have been reported to be responsible for VEGF secretion in cancer cells, did not show a significant change in leptin-treated cells. This may explain the reduction in VEGF secretion in the presence of leptin.
In chapter 6, experimental aims were to investigate the effects of two concentrations (20 ng/ml and 100 ng/ml) of adiponectin on cell proliferation and secretion of VEGF from the Ishikawa cell line. The lower concentration of adiponectin increased cell proliferation and VEGF secretion. Furthermore, it also increased the levels of Akt, p-Akt, STAT3, p-STAT3, AMPK, and p-AMPK as detected by Western blotting. These results suggest that low concentrations of adiponectin stimulated cell proliferation in a similar manner to leptin except for the AMPK protein expressions, and that adiponectin was triggering the PI-3K/Akt and JAK/STAT3 pathways. Moreover, that these low concentrations of adiponectin increased AMPK and p-AMPK proteins expression and also increased the VEGF secretion, which was unexpected. However, this increase in VEGF secretion was produced only in 20 ng/ml adiponectin, stimulating conditions. This may suggest that 20 ng/ml adiponectin is a more highly tumorigenic stimulator than leptin. Higher concentration of adiponectin (100 ng/ml) reduced Ishikawa cell proliferation. The reduction of cell proliferation was not via the inhibition of any of the stimulatory pathways. This suggests that high adiponectin concentration may exert its inhibitory effects via different pathways other than the stimulatory pathways.
In chapter 7, the aim of the experiments was to investigate the stimulatory effects on growth of insulin and IGF-I in Ishikawa cells. Results showed that both insulin and IGF-I increased cell numbers but reduced secretion of VEGF. The level of Akt and p-Akt were elevated but AMPK expression was not changed, and p-AMPK was significantly reduced. Collectively, these results suggest that insulin and IGF-I activate cell proliferation through a PI-3K/Akt pathway. Again, it was interesting to note that low AMPK was linked to the low secretion of VEGF that was observed in the presence of insulin and IGF-I.
Conclusions: Initially, it was hypothesised that leptin, insulin and IGF-I promote cancer growth, while adiponectin inhibits it. The results were generally consistent with the initial hypothesis in some cases. For example, all of the so-called tumorigenic hormones, (that is, leptin, insulin and IGF-I) increased proliferation in Ishikawa cells, however, these hormones reduced the secretion of angiogenic signal VEGF, moreoever, responses to these hormones varied between cell lines, with the hormones stimulating proliferation in the endometrial cell line and the breast cell line but not in the ovarian cell line.
On the other hand, in Ishikawa cells it was found that while higher concentrations of adiponectin reduced proliferation of tumour cells and increased VEGF secretion, as expected, lower concentrations of adiponectin resulted in a surprising increase in cell proliferation as well as an increase in VEGF secretion.
Investigation of the expression of certain intracellular mediators in Ishikawa cells would suggest that leptin-induced cell proliferation but not VEGF secretion is stimulated via the PI-3K/Akt/mTOR pathway and that the JAK/STAT3 pathway is an important mediator of this. Leptin, insulin and IGF-I all increased proliferation and decreased VEGF secretion from Ishikawa cancer cells. While it was demonstrated that activation of the PI-3K/Akt/mTOR pathway occurred, there were some differences in response, especially when in combination with adiponectin, that suggested the utilisation of different pathways or receptors.
From these experiments, it is impossible to determine the effect of these hormones in vivo. While it is somewhat unexpected that VEGF secretion was normally reduced with increasing cell proliferation, these findings are consistent with the hypothesis that obesity-related hormones, and particularly the combination of higher levels of leptin combined with lower levels of adiponectin, have an impact on tumour growth in obese women with low grade endometrial cancer.
Further in vivo and clinical studies are required to determine the clinical importance of these relationships and to explore the therapeutic potential of targets within the identified pathways.
New observations in this study; While it is recognised that there is a significant literature of the potential impact of leptin and adiponectin on cancer growth, I have attempted to perform a systematic study of the chosen hormones on Ishikawa cells and to compare these findings with those of other cell lines. The studies performed for this thesis contribute to an understanding of this subject in the following areas.
• Using Ishikawa cells, I have described the contradictory increase in proliferation but decrease in VEGF secretion under the influence of leptin, insulin and IGF-I. Using these cells, I have also described how low concentrations of adiponectin increase cell proliferation while higher concentrations inhibit proliferation but increase VEGF secretion. I have also described how these effects compare with the effects of these hormones in a selection of other cell lines.
• I have documented the presence of the receptors of leptin (Ob-R), insulin (InsR), IGF-I (IGFR) and adiponectin (AdipoR) in the cell membrane, cytoplasm and nucleus.
• I also described serine phosphorylation of STAT3 in Ishikawa cells in the presence of leptin and low adiponectin and provided evidence that cross-talk occurred between PI-3K/Akt/mTOR and JAK/STAT3 pathways in Ishikawa endometrial cancer cells. In addition, an increase in AMPK and p-AMPK in association with increased VEGF secretion with adiponectin was described.