Browsing by Person "Wallace, J."

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Physiological levels of soy isoflavones inhibit the growth of oestrogen receptor β-positive but not α-positive breast cancer cells
(Society for Endocrinology, 2012-03) Wallace, J.; Gow, Iain F.; Warnock, Mary
The biphasic effect of soy isoflavones (genistein and daidzein) on oestrogen receptor (ER)α+ breast cancer cell proliferation is well documented, with concentrations of ≤10 µM promoting proliferation, and growth inhibitory and apoptotic effects seen at concentrations ≥10 µM. However genistein concentrations as low as 1nM may inhibit the proliferation of some ERα-/β+ cells. This is at odds with epidemiological evidence which suggests that high serum levels of soy isoflavones (around 1 µM) are associated with reduced breast cancer risk, particularly for ERα+ and premenopausal cancers. It is possible that interactions with endogenous oestrogens may be behind this discrepancy. The object here was to investigate combinations of genistein and daidzein at levels achievable in the serum through diet alone, at pre- and postmenopausal oestrogen levels, on breast cancer cell proliferation and apoptosis. ERα+ MCF7 and ERα-/β+ MDA-MB-231 cells were treated with genistein, daidzein and 17β-oestradiol at physiologically relevant levels. Physiological levels of soy isoflavones promoted but not inhibit the proliferation of MCF7 cells, whilst simultaneously inducing low level apoptosis. E2 acted synergistically on proliferation with the isoflavones, increasing proliferation further, but did not impact upon their apoptotic capacity. All concentrations of genistein and daidzein resulted in a slight reduction in MDA-MB-231 proliferation, with this dropping dramatically at the highest dose of 31.6 µM. Overall this suggests that physiological levels of isoflavones may be of benefit as chemotherapeutic agents against ERα-/β+ breast cancer. However they are not capable of reducing the proliferation of ERα+ MCF7 cells, or inhibiting oestrogenic promotion of growth. Other factors must be involved, not reflected by this model. Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.
Physiological levels of soy isoflavones reduce proliferation and promote apoptosis in the ER_-positive breast cancer cell line MDA-MB-231
(Cambridge University Press, 2012-04) Wallace, J.; Warnock, Mary; Gow, Iain F.
Dietary soy, particularly the high levels consumed as part of traditional Eastern-Asian diets, may be protective against certain subtypes of breast cancer(1). This effect is partly related its high isoflavone phytoestrogen content (genistein and daidzein). However much in vitro evidence suggests a contradictory, growth promoting impact of physiological (serum) levels of isoflavones in oestrogen receptor alpha positive (ERa+) breast cancer cell lines(2). This has led to the contraindication of isoflavone supplement use in post menopausal breast cancer survivors. The inconsistency between epidemiological and in vitro evidence may relate to the relative expression levels of ERa and its counterpart ERb. Unlike in cell lines predominantly expressing ERa, the proliferation of ERb-dominant cell lines can be inhibited by concentrations of genistein as low as 1 nM(3,4). This information is relevant as the ERa - /b+ subgroup represent approximately 18% of all breast tumours(5) but the beta-receptor is not routinely tested for upon diagnosis. We assessed the impact of physiologically relevant concentrations (serum levels achievable through diet: 0.01nM to 31.6 mM) of the soy isoflavones genistein and daidzein on proliferation (MTT assay) and apoptosis with the Annexin V-Cy32 Apoptosis Detection Kit and DAPI staining/Nuclear Area Factor (NAF) calculation in the ERa - /b+ breast cancer cell line MDA-MB-231. Low doses of genistein or daidzein (- 0.01 nM) inhibited MDA-MB-231 proliferation by around 20%, although this frequently failed to achieve statistical significance. The highest concentrations used (31.6 mM) resulted in a dramatic decrease in percent proliferation compared with a vehicle only control (meanSD : 48.512.6, p = 0.004 and 48.413.1, p<0.001 for genistein and daidzein respectively; n = 3). 17b-oestradiol (E2; the major circulating oestrogen) also slightly reduced proliferation at its pre- and post-menopausal serum concentrations (1nM and 1 pM respectively). This reduction in proliferation was associated with an observed increase in cell death at 10 and 31.6 mM genistein and daidzein, which was at least partly explained by an increase in the percentage of apoptotic cells. While the combination of E2 and isoflavones failed to show any synergistic growth inhibitory, or pro-apoptotic effects, some inhibition of proliferation was still documented. This implies that the growth inhibitory effects of genistein, daidzein and E2 may not be regulated by the same mechanisms. However, more importantly, the apoptotic cell death observed with soy isoflavone treatment was not abrogated by the addition of physiological E2 concentrations. Overall, our data suggests that at concentrations achievable through the diet ( 10 mM) soy isoflavones may be potentially beneficial as chemotherapeutic agents against ERa - /b+ breast cancers, through their ability to reduce proliferation and promote apoptosis, even in the presence of physiological E2 levels. Routine determination of the expression levels of ERb in addition to ERa in breast cancer would be an invaluable biomarker to indicate the potential efficacy of this cheap and readily available treatment, and indicates a possible pharmacological target. 1. Wu AH, Yu MC, Tseng CC, Pike MC (2008) Brit J Cancer 98:9-14. 2. Hwang CS, Kwak HS, Lim HJ, et al. (2006) J Steroid Biochem Mol Bio 101:246-53. 3. Rajah TT, Du N, Drews N, Cohn R (2009) Pharmacology 84:68-73. 4. Kang X, Jin S, Zhang Q (2009) J Food Sci 74:H237-H242. 5. Skliris GP, Leygue E, Watson PH, Murphy LC (2008) J Steroid Biochem Mol Bio 109:1-10.
The effect of 17-β oestradiol, resveratrol, and genistein on Na+/H+ exchange in breast cancer cells lines
(Cambridge University Press, 2012-03) Wallace, J.; Foy, Daniel; Yousuf, Huma; Warnock, Mary; Gow, Iain F.
The tumour environment is more acidic than normal (pH 6.8 vs 7.3)(1), and this may aid tumour progression or affect the uptake of drugs. The extracellular pH may be partly-regulated by cellular sodium/hydrogen exchangers (NHEs), and NHE activation may in turn be regulated by hormones such as oestradiol(2). Some breast cancers possess receptors for oestradiol, and stimulation or blockade of these receptors may modulate the cell's ability to regulate pH. We studied this by investigating the effect of oestradiol on the ability of breast cancer cell lines to regulate pH by NHE. We used MCF-7 (expresses oestrogen receptors a and b) and MDA-MB-231 (expresses only oestrogen receptor b) cell lines. Given the interaction of phytoestrogens with the oestrogen receptor(3), we also looked at the effect of resveratrol (RSV) or genistein (Gen). MCF-7 and MDA-MB-231 cells were used either as suspensions, or on coverslips, in the absence of added oestradiol. Cells were loaded with H+ -sensitive fluorescent probe BCECF, then washed to remove excess dye. Suspended cells were allowed to adhere to the glass bottom of the 35mm perfusion chamber, coverslips with cells were added to the chamber directly. Cells were perfused (1 ml/min) at 37C with HEPES-buffed Tyrode (pH 7.4) to which drugs were added as required. Acidification was induced by the NH4Cl pre-pulse technique, recovery was achieved by returning to normal Tyrode. Cells were illuminated by alternating wavelengths of 440 and 503 nm light (2 Hz sample rate), and fluorescent light (>535 nm) was captured by a CCD camera. The fluorescence ratios are directly proportional to the H+ concentration. Drugs were added directly to the Tyrode, and since RSV and Gen were dissolved in DMSO, DMSO (0.01% final) was added to the pre-perfusing Tyrode in those experiments, and used as a control in the absence of drugs. Results are mean ratio valuesSEM or slopes of rate of change of ratio/minSEM for three separate experimental days, except for the DMSO controls where n = 6. Differences were assessed by Student's paired or unpaired t tests. A brief (4 min) exposure of breast cancer cells to NH4Cl (20mM) caused a transient alkalisation (decrease in 440/503 ratio, i.e. decrease in [H + ]i) followed by an acidification and recovery after ammonium removal. In MDA cells, addition of 17b-oestradiol (E2; 1 pM final concentration) had no effect on the rate of recovery from acidification (Control: - 3.44E-041.34E-04; MDA: - 2.26E-040.69E-04). In MCF-7 cells, perfusing with Tyrode containing 1 pM E2 significantly increased the rate of recovery from acidification (Control: - 1.61E-040.22E-04; MCF-7: - 3.66E- 040.18E-04; p<0.05). Inclusion of DMSO alone in the medium perfusing MCF-7 cells significantly increased the basal ratio compared with cells in Tyrode alone (Control: 1.240.18; DMSO: 1.710.09; p<0.05), and also increased the rate of recovery (Control: - 1.61E- 040.22E-04; DMSO: - 3.97E-040.52E-04; p<0.02). Inclusion of RSV (1 mM) in the perfusate with MCF-7 cells also caused an increase in the rate of recovery relative to the DMSO-only control (DMSO: - 3.97E-040.52E-04; RSV: - 6.84E-040.56E-04; p<0.02). Gen (1 mM) had no significant effects in MCF-7 cells, though all three recovery rates were numerically higher than the DMSO control. In summary, we have shown that E2 and RSV at physiological levels can increase the rate of recovery from acidification in a breast cancer cell line expressing oestrogen receptors a and b. Given the potential importance of cytosolic and extracellular pH regulation in both the ability of cytotoxic drugs to cross the membrane or the cell to proliferate, the physiological effects of foodborne phytohormones on pathways regulating cell pH clearly requires further investigation. 1. Raghunand N, He X, van Sluis R, Mahoney B, Baggett B, Taylor CW, Paine-Murrieta G, Roe D, Bhujwalla ZM, Gillies RJ (1999) Br J Cancer 80: 1005-1011. 2. Kilic A, Jaradov S, and Karmazyn M (2009) J Mol Cell Cardiol 46:360-36. 3. Hwang CS, Kwak HS, Lim HJ, Lee SH, Kang YS, Choe TB, Hur HG & Han KO (2006) J Steroid Biochem Mol Biol 101: 246-253.
The life and death of breast cancer cells: proposing a role for the effects of oestrogen and phytoestrogens on potassium channels
(Springer, 2011-07-15) Wallace, J.; Gow, Iain F.; Warnock, Mary; QMU Bursary
Changes in the regulation of potassium channels are increasingly implicated in the altered activity of breast cancer cells. Increased or reduced expression of a number of K+ channels have been identified in numerous breast cancer cell lines and cancerous tissue biopsy samples, compared to normal tissue, and are associated with tumor formation and spread, enhanced levels of proliferation, and resistance to apoptotic stimuli. Through knockout or silencing of K+ channel genes, and use of specific or more broad pharmacologic K+ channel blockers, the growth of numerous cell lines, including breast cancer cells, has been modified. In this manner it has been proposed that in MCF7 breast cancer cells proliferation appears to be regulated by the activity of a number of K+ channels, including the Ca2+ activated K+ channels, and the voltage-gated K+ channels hEAG and Kv1.1. The effect of phytoestrogens on K+ channels has not been extensively studied but yields some interesting results. In a number of cell lines the phytoestrogen genistein inhibits K+ current through several channels including Kv1.3 and hERG. Where it has been used, structurally similar daidzein has little or no effect on K+ channel activity. Since many K+ channels have roles in proliferation and apoptosis in breast cancer cells, the impact of K+ channel regulation by phytoestrogens is of potentially great relevance.