Methotrexate combination effects with genistein and daidzein on MDA-MB-231 breast cancer cell viability

Aims: To investigate the effect of methotrexate and soy isoflavones genistein and daidzein on MDA-MB-231 breast cancer cell viability. Study Design: In-vitro study using cultured cells


INTRODUCTION
Methotrexate is used frequently for treating leukemia, solid tumors and rheumatoid arthritis [1]. Acquired resistance to methotrexate [2] prompts high-dose therapy leading to a likelihood of toxic side-effects [3]. Combination therapy with antioxidant phytochemicals was proposed to mitigate the toxic side effects associated with methotrexate but the impact on drug effectiveness remains controversial [4, 5,6]. Observational studies showed there were low rates of breast cancer incidence in Asian women linked to the consumption of soy products [7]. A variety of soybean products were found to prevent methotrexate gastro-intestinal toxicity, but the anti-apoptotic components appeared to be relatively high molecular weight (>10 kd) compounds [8]. Soy isoflavones possess anticancer activity [9,10]. Genistein produces synergisms with other anticancer agents due to a chemo-sensitizing effect [10,11].
To our knowledge no investigation of the effect of soy isoflavones on methotrexate cytotoxicity towards breast cancer cells has been published. The aim of this study was to investigate the effect of methotrexate combined with soy isoflavones genistein and daidzein on MDA-MB-231 breast cancer cell viability. The approach conforms to a variable ratio method for assessing interactions using the median effect model detailed by Chou and co-workers [12].

Cell Culture
MDA-MB-231 breast cancer cells were cultured using DMEM medium supplemented with +10% FBS and 1% pen step. Culture flasks and 96-well micro-plates were incubated at 37°C in a 5% CO 2 atmosphere (LEEC Research CO 2 Incubator, LEEC Ltd., Nottingham, UK). Cells were trypsinized, counted using a NucleoCounter (model NC-3000, ChemoMetec, Allerod, Denmark) and seeded at 10,000 cells/well in 96 well plates with 50 µl media per well. Cells were incubated overnight at 37°C to allow attachment.
Stock solutions of methotrexate, genistein, and daidzein (100 mM) were prepared with DMSO, diluted 10 fold with DMEM and sterilized with 0.2 µm cellulose acetate filters before use. Sterilized solutions were serially diluted to achieve 2x target concentration (max; 200 µM with <0.05% DMSO final concentration). Cells were treated with methotrexate, genistein or daidzein alone and incubated for 72 hours. Cell viability was determined using the MTT assay (Section 2.2.). These tests were analyzed to find EC50 values and such data were used for the design of combination studies.

MTT Assay
Microwell plates were washed x 2-fold with ice cold PBS (100 µl) with a third wash remaining in the wells. MTT reagent 20 µl (5 mg/ ml in PBS buffer) was added per well and plates were incubated at 37°C for 2 hours. Blue formazan crystals formed were dissolved by adding 100 µl isopropanol (with 0.04N HCl) and incubating for one hour. Absorbances were measured at 570 nm using a microplate reader (VersaMax™ ELISA microplate reader, Molecular Devices, Sunnyvale, CA, USA.).Data were expressed as mean ± standard error of means (SEM) of 2independent experiments with six-replicate (microwells) per treatment-concentration (n=12).

Combination Studies
Samples containing (4x target concentration) methotrexate (25 µl) were added to microplate wells containing 10,000 seeded cells and 50 µl of culture media. Then 25 µl of genistein or daidzein (4x target concentrations) were added to achieve a final concentration equal to one-half of EC50 for each isoflavone. The final range of methotrexate concentrations were 0, 1, 2, 5, 10, 20 and 100 µM. The cells were incubated for 72 hours and subjected to MTT assay as describe above. Combination test involved 3-independent experiments with 6 microwells per each treatment concentration (n=18).

Data and Statistical Analysis
Experimentally values for cell viability were fitted by non-linear regression (NLR) to the Logistics function shown below, where PRED is the predicted response, C = minimum response, and M= maximum response. E is 50% effect dose or drug-dose, which produces a 50% response (E50), and B is the steepness of the curve [13].
PRED =C+ ((M-C)/(1+EXP(B*Ln(D/E)))) NLR was implemented using SPSS software (IBM SPSS v21.). Estimates for EC50 were subjected to isobologram analysis. Doseresponse data were also analyzed median effect model. The fraction of affected cells (Fa) and unaffected cells (Fu) are calculated for different doses of drug (Fa = 100-% viable cells/100 and Fu = 1-Fa). A plot of log (Fa/Fu) versus drug concentration was fitted to a straight-line graph (Y = mx +c) to determine the median effect dose (Dm) and slope (m) using CompuSyn™ software [12]. Values for the combination Index (CI) were calculated either manually or via CompuSyn™. To determine CI manually, we used the relations below, where d 50 MTX and d 50 Gen are the 50% effect dose from combination studies, and EC50 1 and EC50 2 are values for each agent alone.
The size of CI is indication whether combination therapies produce synergism (CI<1.0), antagonism (CI>1.0) or additive behavior (CI=1.0) [13]. Table 1 and Fig. 1 show dose-effect parameters for MDA-MB-231 cells treated with methotrexate, genistein or daidzein determined using a logistic function to fit experimental data. There was a good fit for NLR predictions with observed points (R2 = 0.98-0.99). Table 2 shows dose-effect parameters arising from CompuSyn™ analysis of the same data. The median dose (Dm) corresponds to the 50% effect dose (EC50) and slope (m) is a measures of the steepness of the plot of log (Fa/Fu) versus drug concentration.   The combination index (CI) value using data from Table 1 was CI = 1.9 for methotrexate and CI = 1.1 for methotrexate combination at the concentrations corresponding to 50% effect. The values for CI were predicted to increase from 1.1 to 5.0 ( Fig. 4) with decreasing methotrexate: isoflavone ratio (Fig. 4).

Fig. 4. A plot of combination index (CI) values for treatment of MDA-MB-231 breast cancer cells with methotrexate (1-100 µM) and 30 daidzein or genistein
Data generated using CompuSyn analysis

DISCUSSION
Methotrexate is an anticancer agent [1 Consumption of soy isoflavones is thought to reduce the risk of breast cancer [8,9, Methotrexate inhibits cell proliferation by blocking the enzyme dihydrofolate reductase (DHFR), depleting the intracellular pool of fo restriction of methyl-group availability for DNA synthesis [1]. In addition, exposure to methotrexate increases intracellular oxidative stress due to the inhibition of NAD(P)H reductases [14,15]. Soy isoflavones show estrogen receptor activation, tyrosine kinase inhibition, induction of cell-cycle arrest, anti inflammatory action, and general antioxidant activity [10,16,17]. For the preceding reasons, we tested the hypothesis that combination of methotrexate with isoflavones would produce enhanced anti-cancer activity compared with each agent alone.
EC50 values from this study ( The combination index (CI) value using data from methotrexate-genistein and CI = 1.1 for methotrexate-daidzein combination at the concentrations corresponding to 50% effect. The values for CI were predicted to increase from 1.1 to 5.0 (Fig. 4) with decreasing methotrexate: isoflavone ratio

Fig. 4. A plot of combination index (CI) values 231 breast cancer 100 µM) and 30-µM daidzein or genistein
Data generated using CompuSyn analysis is an anticancer agent [1][2][3]14]. Consumption of soy isoflavones is thought to he risk of breast cancer [8,9,10]. Methotrexate inhibits cell proliferation by blocking the enzyme dihydrofolate reductase (DHFR), depleting the intracellular pool of folate, and by group availability for DNA synthesis [1]. In addition, exposure to methotrexate increases intracellular oxidative stress due to the inhibition of NAD(P)H-liked reductases [14,15]. Soy isoflavones show tivation, tyrosine kinase cycle arrest, antiinflammatory action, and general antioxidant activity [10,16,17]. For the preceding reasons, we tested the hypothesis that combination of methotrexate with isoflavones would produce cancer activity compared with EC50 values from this study (Table 1) are in broad agreement with previous reports, taking in assay conditions, e.g. different culture medium, and drug exposure The EC50 was 80 µM for methotrexate 231 cultured with MEM media and a drug exposure time of 24 hrs [18]. By comparison, EC50 was 18.5 µM methotrexate with low protein (5% FBS) medium [19]. Tests using genistein and MDA-MB-231 cells sho that EC50 ranges from 46.8 µM [20] to 50 [21,22] depending on the exposure time and other assay conditions.
There was antagonism between methotrexate and soy isoflavones in this study. The EC50 for methotrexate increased from 44.7 µM to 57.6 µM in the presence of genistein with CI>1.0 indicating antagonism. The EC50 value for methotrexate with daidzein present (29.7 µM) was closer to values (20-22 µM) predicted by isobologram analysis (Fig. 3) for additive response meaning neither positive nor negative interaction (Table 1 and Fig. 3.) but here also CI >1.0. For both isoflavones CI> 1.0 indicating antagonism. The degree antagonism between methotrexate and genistein seem to be greater than those with daidzein. Co predictions showed decreasing antagonism ( Fig. 4) with increasing methotrexate: isoflavones ratio.
Relating in-vitro data to human exposure conditions requires that EC50s are subjected to in-vitro, in vivo extrapolation (IVIVE) by step correction for the effects of absorption, serum protein binding, liver metabolism and renal clearance [23,24]. Past literature, data may be useful also for addressing IVIVE issues. Briefly, it is known that methotrexate is bioavailable oral doses of < 20 mg/m 2 (<0.54 methotrexate are 50-95% absorbed leading to peak plasma concentrations of 300 within 1.5-3 hours and a half-life of elimination of 4-6h [3]. Most of the circulating methotrexate is excreted via the kidneys in an intact form with <10% metabolism in the liver to form 7 methotrexate [3]. On the other hand, the plasma concentration profile for soy isoflavones is influenced by a host of factors, e.g. relative proportion of aglycone and glycosylated forms, type of food matrix used for administration, and forms of processing [25]. Using the dose for genistein or daidzein frequently used in human trials (45-56 mg/ day), the fractional excretion rate (apparent bioavailability) was 20 peak plasma concentration of 2-5 µM some 4 8hrs after intake [26]; reviewed by [ basis of such data [3,26,27], a typical exposure to 20 mg methotrexate + 56mg genistein would produce plasma concentrations for methotrexate that are 60-400 times higher compared to peak plasma concentration for isoflavone. Moreover, the peak concentrations for isoflavone would occur after the peak for methotrexate [3,26,27]. According to the present paper high ; Article no.JALSI.35206 with low protein (5% FBS) medium [19]. Tests 231 cells showed that EC50 ranges from 46.8 µM [20] to 50-90 µM [21,22] depending on the exposure time and There was antagonism between methotrexate and soy isoflavones in this study. The EC50 for increased from 44.7 µM to 57.6 µM in the presence of genistein with CI>1.0 indicating antagonism. The EC50 value for methotrexate with daidzein present (29.7 µM) 22 µM) predicted by 3) for additive nse meaning neither positive nor negative Fig. 3.) but here also CI >1.0. For both isoflavones CI> 1.0 indicating antagonism. The degree antagonism between methotrexate and genistein seem to be greater than those with daidzein. CompuSyn Tm predictions showed decreasing antagonism 4) with increasing methotrexate: isoflavones data to human exposure are subjected to , in vivo extrapolation (IVIVE) by stepwise correction for the effects of absorption, serum protein binding, liver metabolism and renal . Past literature, data may be useful also for addressing IVIVE issues. Briefly, it is known that methotrexate is bioavailable and (<0.54 mg/Kg) 95% absorbed leading to peak plasma concentrations of 300-2000 µM life of elimination of 6h [3]. Most of the circulating methotrexate is s in an intact form with <10% metabolism in the liver to form 7-OH methotrexate [3]. On the other hand, the plasmaconcentration profile for soy isoflavones is influenced by a host of factors, e.g. relative proportion of aglycone and glycosylated forms, pe of food matrix used for administration, and ]. Using the dose for genistein or daidzein frequently used in human 56 mg/ day), the fractional excretion rate (apparent bioavailability) was 20-50% with a 5 µM some 4-]; reviewed by [27]. On the ], a typical exposure to 20 mg methotrexate + 56mg genistein would produce plasma concentrations for methotrexate 400 times higher compared to the peak plasma concentration for isoflavone. Moreover, the peak concentrations for isoflavone would occur after the peak for methotrexate ]. According to the present paper high methotrexate: isoflavone ratios are not conducive for antagonism.
Evidence is emerging that antioxidant phytochemicals can reduce toxicity to healthy cells, whilst not affecting efficacy [4-7] depending on changes to absorption, metabolism, distribution and excretion (ADME) characteristics [15,28,29,30]. Research using leukemic cells showed that genistein inhibits methotrexate uptake by the reduced folate transporter owing to its role as tyrosine kinase inhibitor [31,32,33]. Genistein was found to promote the transcription of efflux transporter (ABCC1/MRP1) protein for MDA-MB-231 cells with no net effect onmitoxantrone toxicity owing to the simultaneous inhibition of the same transporter [34]. Many polyphenols from beverages were also reported to inhibit methotrexate and folate uptake at low pH involving the proton coupled folate transporter [35][36][37][38][39]; genistein had no effect on the low-pH uptake of methotrexate and folate by CaCo2 cells [35]. Interestingly, genistein was reported to moderate genes from MDA-MB-231 cells suppressed by epigenetic mechanism [21] thereby increasing the cell sensitivity to therapeutic drugs. In general, methotrexate toxicity would be enhanced by phytochemicals that increase uptake, promote methotrexate modification to polyglutamated forms, and / or decrease methotrexate efflux [15]. For example, genistein (and its metabolites) were found to be inhibitors for breast cancer resistant protein (ABCG2/BCRP) efflux transporter [40,41]. Methotrexate and 7-OH methotrexate were also identified as substrates for ABCC2 (MRP2), ABCC3 (MRP3), and ABCG2/BCRP) and found to have an enormous impact on drug concentration profile [29].

CONCLUSION
In conclusion, a diverse range of potential interactions may occur between genistein and methotrexate that go to produce antagonism with regard to cytoxicity for MDA-MB-231 cells. The results from this study show a rising tolerance of breast cancer cells towards methotrexate in the presence of genistein. However, we speculate that antagonism is unlikely where concentrations for methotrexate are much higher than genistein. More research is needed also to consider methotrexate interactions with isoflavones in terms of changes to ADME and the consequences for other health outcomes. The different levels of antagonism observed for methotrexate and genistein or daidzein is interesting and worthy of further study. There is scope also to consider the possible role of soy isoflavone/ MTX therapy on immune responses or rheumatoid arthritis [1,3,8,14,35,36]