Human breast cancer cell lines MDA-MB-231, MDA-MB-435, and MCF-7 were generously provided by Dr. Salem Chouaib (Institut Gustave Roussy, Villejuif, France) and were maintained in complete DMEM supplemented with 10% FCS (Hyclone-GE Healthcare life Sciences, Pittsburg, PA, USA), as previously described (7). The MCF-10A breast epithelial cell line (22) was the generous gift of Dr. Joan Brugge (Harvard Medical School, Boston, MA, USA) provided through the laboratory of Dr. Raif Geha (Boston Children’s Hospital, Boston, MA, USA). MCF-10A cells were maintained in DMEM-F12 medium supplemented with 5% horse serum (Invitrogen), 20 ng/ml EGF (Peprotech), 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin (Sigma) (St. Louis, MO, USA) and penicillin–streptomycin (Hyclone). Paclitaxel (hereafter referred to as taxol) was purchased from Sigma and MH (UMF^® 16+) from ApiHealth (Auckland, New Zealand). As a control for MH, we used a sugar solution (designated sugar control or SC) containing equivalent concentrations of the three major sugars in honey (38.2% fructose, 31.3% glucose, and 1.3% sucrose) (23). For all reagents, appropriate dilutions to the desired concentrations were made fresh in culture medium before addition to the cells in culture.

Cell viability was determined as previously detailed (7) using CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI, USA). This assay quantifies the amount of ATP present as a correlate of the number of metabolically viable cells in culture. Briefly, MDA-MB-231 tumor cells (5 × 10³ cells/well) or MCF-10A cells (2 × 10⁴ cells/well) were cultured in 96-well plate and exposed to different concentrations of MH (range 0.25–2%; w/v) for different time periods (range 24–72 h), as indicated. At the end of culture, a cell lysis solution, containing a luciferin derivative, Ultra-Glo™ Recombinant Luciferase and Mg²⁺, was added and this converts the luciferin derivative into a luminescent signal proportional to the amount of ATP present. Luminescence was measured using a Glomax Luminometer (Promega) and normalized to control. The data are presented as percent cell viability of experimental groups compared to that of control, untreated cells.

The activity of caspase-3/7, caspase-6, caspase-8, and caspase-9 were assayed using specific Caspase-Glo^® assay kits (Promega), essentially following manufacturer’s instructions (7). Briefly, MDA-MB-231 or MCF-7 cells were seeded in a 96-well U bottom plate (5 × 10³ cells/well) and treated with MH (1–5% w/v, final concentrations) or Taxol (50 ng/ml) for 24 h. Subsequently, cell lysis solution, containing a luciferase substrate derivative, Ultra-Glo™ Recombinant Luciferase and Mg²⁺, was added for 2 h at room temperature. For all assays, duplicate plates were set up to quantify cell viability. Luminescence was measured using a Glomax Luminometer. The luminescent signal was normalized to control, as per manufacturer’s protocol, and adjusted relative to the extent of cell viability in each group. All determinations were done in duplicate for each experimental group. The data are reported as the mean of relative fold increase compared to untreated cells.

Detailed procedures for immunoblotting have been published previously (24, 25). MDA-MB-231 cells (4 × 10⁶ cells/well) were seeded overnight in 20 × 100 mm culture dish in DMEM containing 2% FBS. Cells were then gently washed and incubated in DMEM/5% FBS in the presence of MH (0.03–1% final concentration) for different times (range 15 min–12 h). At the end of incubation, cells were lysed using RIPA buffer (25 mM Tris-Cl, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors) and lysates clarified by centrifugation at 10,000 rpm and stored at −80°C until use. Aliquots (60–80 µg) of total proteins were resolved on 10–12% SDS-PAGE, transferred to a nitrocellulose membrane and blocked with 5% non-fat milk for 1 h. Membranes were then probed with appropriate primary antibodies (1/1,000 dilution) overnight at 4°C. The antibodies used were specific to STAT3 [Cell Signaling Technology (CST), MA, USA], pY-STAT3 (Tyr705) (CST), cytochrome c (CST), Bcl-2 (Santa Cruz Biotechnology, CA, USA), Bax (Santa Cruz), phosphotyrosine (mAb clone 4G10) (Upstate Biotechnology, NY, USA), VDAC (CST) or β-actin (CST). Blots were then washed and exposed to appropriate anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (1:2,000; CST) and developed using the ECL Plus substrate system (Pierce-Thermo Fisher Scientific, MA, USA). The chemiluminescent band signal was detected by Typhoon FLA 9500 biomolecular imager (GE Healthcare Life Sciences). Densitometric analysis of band intensity on blots was done using ImageJ (National Institutes of Health, USA).

Lysates of cytoplasmic and mitochondrial fractions were prepared using the Qproteome Mitochondria Isolation Kit (Qiagen), according to the manufacturer’s protocol. Briefly, MDA-MB-231 cells (5 × 10⁶ cells/well) were seeded overnight in 20 × 100 mm culture dish in DMEM containing 2% FBS. Cells were then gently washed and incubated in DMEM/5% FBS in the presence of MH or SC (1% final concentration) for 24, 48, and 72 h. After treatment, cell were harvested, washed in 0.9% NaCl, and incubated for 10 min at 4°C in lysis buffer on a shaker. The homogenate was centrifuged at 1,000 × g for 10 min at 4°C; this supernatant was designated as cytosolic fraction. The pellet was resuspended in disruption buffer, passed through a 26-G needle 15 times. The lysate was centrifuged at 1,000 × g for 10 min at 4°C and carefully transferred supernatant to a clean 1.5 ml tube. To obtain the enriched mitochondrial fraction, the supernatant was centrifuged at 6,000 × g for 20 min at 4°C. All buffers were supplemented with protease inhibitors at 1:100, provided within the kit. The isolated mitochondria were lysed using RIPA buffer (25 mM Tris-Cl, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors) and lysates clarified by centrifugation at 16,000 rpm and stored at −80°C until use.

MDA-MB-231 cells (4 × 10⁶ cells/well) were seeded overnight in 2% FBS DMEM growth medium. Cells were then gently washed and incubated in DMEM/5% FBS in the presence of 1 or 5% MH for 4.5 h. Brefeldin A (CST) was added at 1 mg/ml concentration for the final 4 h of incubation to block protein egress from the endoplasmic reticulum. Total cell extracts were then prepared, as described above, resolved on 10% SDS-PAGE and blots were probed with anti-IL-6 rabbit mAb (clone D3K2N; CST). The effect of MH treatment on IL-6 secretion was also analyzed. MDA-MB-231 or MCF-7 cells were seeded overnight in 2% FBS DMEM in 6-well plates (2 × 10⁶/well), and then incubated in 5% FBS medium in the presence of indicated concentrations of MH for different times (2–12 h). Cell-free culture supernatants were then collected and analyzed for IL-6 content by a specific ELISA (Biolegend, CA, USA).

Intracellular production of ROS was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (DCF-DA) as a fluorescent probe. Cells (5 × 10⁵ cells/well) were treated with the indicated concentrations of MH (0.3–5%) or hydrogen peroxide (as a positive control) for 24 h, then washed with PBS and stained with 5 µM (DCF-DA; Sigma, MO, USA) for 30 min at 37°C in the dark. DCF-DA is a fluorogenic dye that measures ROS within intact cells. The cells were then washed, resuspended in PBS, and analyzed on a FACSCanto II (BD Biosciences, San Jose, CA, USA). The mean fluorescence intensity was quantified using CellQuest software (BD Biosciences).

The detailed procedure for this assay has been published (26). Briefly, a layer of Noble agar (2.4%; 1 ml) was poured into wells of a 6-well plate and allowed to set. A second layer (2.9 ml) containing 0.3% low melting Noble agar dissolved in growth media containing cells (5 × 10³ cells/ml) was placed on top of the first layer and allowed to set at 4°C for 5 min followed by incubation at 37°C for 30–60 min. Cells were incubated for 2 weeks to form colonies. Growth medium (2 ml) containing MH at different concentrations was then layered on top of the second layer and cells were incubated in a humidified incubator at 37°C for 1 week. At the end of the experiment, colonies were stained with 2% Giemsa stain, photographed and counted. All colonies were counted based on their size with the number of large colonies (>200 μm in diameter) being used for calculating the effect of MH on colony formation.

This was carried out following a previously described protocol (27). Cells were grown in 6-well plate until confluence, incubated for 10 min with Moscona buffer following which a scrape was made through the confluent monolayer with a plastic pipette tip of 1 mm diameter. The tissue culture plates were then washed and incubated at 37°C in supplemented growth medium in the presence or absence of MH and/or drug. At the bottom side of each dish, two arbitrary places were marked where the width of the wound was measured with an inverted microscope (objective ×4) (Olympus 1X71, Japan). Motility is expressed as the average ± SEM of the difference between the measurements at time 0 and subsequent determinations after 6 h.

The extent of invasiveness of cancer cells was determined using a Matrigel Invasion Chamber (8-µm pore size; BD Biosciences), as described previously (27). Briefly, cells (1 × 10⁵ cells in 0.5 ml of media plus treatment) were seeded into the upper chamber of the Matrigel system. The bottom chamber in the system was filled with RPMI supplemented with 10% FBS as a chemo-attractant and then incubated at 37°C for 24 h. Cells that have migrated through the Matrigel were fixed with 4% formaldehyde, stained with DAPI and counted in 25 random fields under a microscope.

The effect of the various treatments on angiogenic activity was assessed using an in vitro assay whereby the formation of capillary-like structures was measured using human umbilical vein endothelial cells (HUVECs) plated on Matrigel-coated plates, as previously described (27). The Matrigel matrix was thawed, gently mixed to homogeneity using cooled pipettes, and diluted v/v with the EndoGRO™-MV-VEGF Complete Media Kit medium (Millipore, Temecula, CA, USA). Matrigel, supplemented with angiogenic peptides and other effectors, was then used to coat the wells of a 96-well plate (50 µl/well) and allowed to solidify prior to treatment. HUVECs (4 × 10⁴ cells/well) were added and incubated for 8 h at 37°C in 0.1 ml of EndoGRO™-MV-VEGF Complete Media Kit medium (Millipore, Temecula, CA, USA) in the presence or absence of different concentrations of MH. The cells were photographed using an inverted phase contrast photomicroscope. The tubular network growth area was compared in control and MH-treated cells. Tube formation was quantified by determining the length of tube-like structures formed in each well.

Statistical significance between control and treated groups was analyzed using the unpaired, two-tailed Student’s t-test, using the statistical program of GraphPad Prism version 6 software (San Diego, CA, USA). For multiple comparisons, we used one-way ANOVA with post hoc Tukey’s test (GraphPad Prism). Differences between experimental groups were considered significant when p-values were <0.05.

We first investigated the effect of exposure to different concentrations of MH (range 0.3–5.0%) on breast cancer cells following incubation for 24–72 h. As a control, we used a solution containing the three major sugars (fructose, glucose, and sucrose) at equivalent concentrations to those found in MH and designated SC solution (23). Incubation of MDA-MB-231 (Figures 1A,B) or MCF-7 (Figures 1C,D) breast cancer cells with MH resulted in significant loss of viability, which was dependent on time of exposure and concentration of MH used in culture. At 24 h, significant loss of cell viability was observed after culture with 2.5 or 5% MH in both cell lines and this was further increased after 72 h of culture. Incubation of cancer cells with equivalent concentrations of sugars (2.5 or 5% SC) also led to decreased viability, although to a lesser extent than that observed with MH. Interestingly, incubation of cancer cells with lower concentrations (0.6 and 1.25%) of MH also resulted in significantly decreased viability, which was clearly evident after 72 h of culture (Figures 1B,D). Importantly, cells incubated with equivalent SC solutions showed no significant reduction in proliferation at any time point. Moreover, we also investigated the effect of MH on the non-neoplastic MCF-10A breast epithelial cell line (Figures 1E,F). Although high concentrations (2.5 and 5%) of MH or SC were also toxic to MCF-10A cells, no significant loss of viability was observed when lower concentrations (0.6 and 1.25%) were used, even after 72 h of incubation (Figure 1F). These data demonstrate that concentrations of MH up to 1.25% were generally non-toxic to non-neoplastic cells. Furthermore, exposure of human breast cancer cells to MH resulted in a significant loss of viability that was clearly dose and time dependent. Our results suggest that MCF-7 cells were relatively more resistant to MH-induced growth inhibition compared to MDA-MB-231 cells. This is illustrated by the comparative susceptibility of both cell lines to MH at 48 h (Figure 1G). The calculated MH IC₅₀ values for MDA-MB-231 cells at 48 and 72 h are 3.0 and 2.6%, respectively. The corresponding IC₅₀ values for MCF-7 cells are 4.0 and 3.2%, respectively.

Previously, we demonstrated the capacity of MH to induce apoptosis in a melanoma cell line through the activation of the intrinsic pathway involving caspase 9 and caspase 3 (7). To assess the activation of caspase pathway in MDA-MB-231 and MCF-7 cells, they were incubated with up to 5% MH (the higher concentration is required to observe apoptosis over a 24 h period). Exposure of MDA-MB-231 cells to 5% MH solution resulted in the activation of both initiator caspases 9 and 8 (3.6- and 6.9-fold increase, respectively) (Figures 2A,B). As far as the executioner caspases, our findings show that incubation of MDA-MB-231 cells with MH led to the activation of caspase 3/7 and caspase 6 (13.9- and 3.1-fold increase, respectively) (Figures 2C,D), indicative of an accelerated caspase activation leading to apoptosis. The activation of initiator and executioner caspases was dependent on the concentration of MH used, as incubation of MDA-MB-231 cells with 1% solution for 24 h had a minimal effect on caspase activity (Figures 2B,D). By contrast, treatment of MDA-MB-231 cells with paclitaxel (taxol; 50 ng/ml) led to a small but significant increase in caspase 8 (1.7-fold) activity (Figures 2B,F), but no evidence for the activation of caspase 9 could be observed (Figure 2A), consistent with previous findings (7, 28). Although maximal caspase 3/7 and 8 activation was observed at 24 h, the activity of both caspases was still significantly elevated after 72 h of exposure to MH (Figures 2E,F). These findings demonstrate the capacity of MH to induce apoptosis in MDA-MB-231 cells through the activation of multiple initiator and executioner caspases. Human MCF-7 breast cancer cells have been reported to be caspase 3-deficient (29). The fact that the growth of these cells was still susceptible to inhibition by MH prompted further investigation of the exact caspase(s) involved in this cell line. The data clearly show that, unlike the case with MDA-MB-231 cells, exposure of MCF-7 cells to MH induced the activation of only caspase 9 and caspase 6 (Figures 2G–J). This suggests that MH-induced apoptosis in caspase 3-deficient cancer cells occurs through the induction of the intrinsic pathway and is mediated by the executioner caspase 6.

Increased synthesis of reactive oxygen and nitrogen radicals is indicative of a heightened state of oxidative stress and may be associated with enhanced apoptosis. We, therefore, examined whether the ability of MH to inhibit the growth of MDA-MB-231 or MCF-7 cancer cells is associated with increased production of ROS. Incubation with MH using a range of concentrations (0.3–5%) for 24 h failed to induce any intracellular ROS formation in MDA-MB-231 (Figures 3A–G) or MCF-7 (data not shown) cells. A similar analysis was carried out after 48 h of incubation with MH, but ROS production remained undetectable in both MDA-MB-231 and MCF-7 cells (data not shown). As a positive control, incubation with H₂O₂ led to 98% of the cells expressing ROS (Figure 3B). The lack of effect of MH on ROS production in MDA-MB-231 and MCF-7 breast cancer cells is not universal. Exposure of a human melanoma cancer cell line, MDA-MB-435, to even low concentrations (0.6–1.25%) of MH induced ROS production in 34.7 and 38.1% of the cells, respectively, in comparison to control (Figures 3H–J). Thus, MH exhibits differential ROS-inducing capacity in different cancer cells and induces apoptosis in MDA-MB-231 and MCF-7 cells independent of ROS production.

Selective permeabilization of the mitochondrial outer membrane, which is controlled by Pro- and anti-apoptotic members of the Bcl-2 family, is a critical factor in the induction of apoptosis (30). Upon induction of apoptosis, the pro-apoptotic protein Bax translocates to mitochondrial membranes, where it inserts and mediates the release of cytochrome c from the intermembrane space into the cytosol, thereby triggering the activation of the caspase pathway (31, 32). Incubation of MDA-MB-231 cells with MH resulted in a gradual, and dose-dependent, decrease in the level of Bcl-2 that was first evident after 48 h of treatment (Figure 4A). The extent of the reduction in Bcl-2 level was approximately 60 and 74% after 48 or 72 h of culture with 1% MH, respectively (Figures 4A,B). However, there was no alteration in Bcl-2 levels in cells cultured with 1% SC solution for up to 72 h (Figures 4A,B). In contrast to the reduction in Bcl-2, MH induced a significant increase in the levels of Bax protein at all time points (24–72 h) tested (Figures 4C,D). We further determined the changes in Bax and cytochrome c levels over 24–72 h in cytosolic and mitochondrial lysates of MDA-MB-231 cells after exposure to 1% MH or SC solution. The results demonstrate that incubation with MH, but not SC, induced the translocation of Bax from the cytosol to the mitochondria which, in turn, triggers the translocation of cytochrome c in the opposite direction, namely from the mitochondria to the cytosol (Figures 4E–G). These findings highlight the involvement of the mitochondria in MH-induced death of breast cancer cells.

Cell migration and invasion together with the capacity to form colonies at distant sites are critical properties that define all metastatic tumors, such as MDA-MB-231 cancer cells. Therefore, we next addressed the effect of MH treatment on these essential functions using specific in vitro assays. These studies were carried out on MDA-MB-231 cells only since MCF-7 cells are known to have low migratory and invasive properties (33). The clonogenic cell survival assay determines the ability of cancer cells to proliferate indefinitely and form colonies. Treatment of MDA-MB-231 cells with MH led to a significant inhibition in colony formation in a concentration-dependent manner (Figures 5A,B). The extent of the observed inhibition was 34, 38, and 57% at MH concentrations of 1.25, 2.5, and 5%, respectively (Figure 5A). It should be noted that in this assay MH treatment had no impact on the total number of the preformed colonies (Figure 5B). Rather, exposure to MH led a significant decrease in the percentage of large colonies (>200 μm in diameter) in comparison with the control group (Figure 5A), which reflects an inhibitory effect on the colonies’ growth. These results demonstrate that MH exerted a significant inhibitory effect on long-term colony formation capacity of MDA-MB-231 cells.

Cancer cell migration was examined using the wound-healing assay (34). MDA-MB-231 cells were incubated for 6 h in the presence or absence of MH (0.6 or 1.25% final concentration). These concentrations were chosen because they are known not to cause any significant apoptosis in these cells over this time period (Figure 1A). The results of the wound-healing assay show that exposure to 1.25% solution of MH led to a significant inhibition in cell migration (up to 26% within 6 h; Figures 6A,B). Exposure to 0.6% solution also led to a small (14%) but insignificant level of inhibition. Cancer cell invasion was also investigated by determining the extent of cell migration through a layer of matrigel (35). The results of this assay, shown in Figure 6C, demonstrate the level of susceptibility of this critical cancer cell function to MH exposure. MDA-MB-231 cells incubated in the presence of exceedingly small doses of MH (0.15–1.25%) were significantly inhibited in their cell invasion ability. The degree of inhibition was proportional to the concentration of MH used in the assay, with concentrations of 0.15, 0.3, 0.6, and 1.25% resulting in 9, 29, 45, and 68% inhibition, respectively (Figure 6C). These results demonstrate the heightened susceptibility of two of the critical properties of metastatic cancer cells, namely cell migration and invasion, to inhibition by exceedingly low concentrations of MH.

Proliferation and metastatic spread of cancer cells is dependent on the formation of an intricate network of new blood vessels within the tumor, a process known as angiogenesis (36). The potential effect of MH on tumor angiogenesis has not been explored previously. Therefore, we tested the consequence of exposure to MH on the ability of HUVECs to form vascular tubes, an in vitro correlate assay for angiogenesis. Incubation of HUVEC with MH at 0.6 and 1.25% final concentrations led to significant reduction (17 and 36% inhibition, respectively) in vascular tube formation (Figures 7A,C). Importantly, the observed inhibition was not due to any decrease in the viability of HUVECs (Figure 7B). Thus, we conclude that MH may also be able to influence in vivo tumor growth via a disruption of the angiogenic potential of cancer cells.

We next investigated the underlying mechanism of how MH decreases the viability of cancer cells. Of particular interest is the identification of the earliest molecular targets of MH in breast cancer cells. MDA-MB-231 cells are known to express constitutively activated STAT3 pathway, principally through the engagement of an IL-6/IL-6R autocrine loop (37), and this pathway has been implicated in breast cancer oncogenesis (38). Therefore, we examined the consequence of MH exposure on STAT3 phosphorylation. As early as 15 min after incubation with 1% (w/v) MH solution, the level of active pY-STAT3 was reduced by 65% compared to the level observed in untreated cells, and by 1 h the level of inhibition was >80% (Figures 8A,B). The inhibition of pY-STAT3 persisted for at least 6 h and returned to normal levels by 12 h, presumably due to continuous cell growth and utilization of the inhibitory factors in MH. Importantly, MH treatment had no significant effect on total STAT3 protein levels at any of the time points examined (Figure 8A). Next, we determined the sensitivity of pY-STAT3 inhibition following 1 h-exposure to differing concentrations (range 1–0.03%) of MH or SC solution (Figure 8C). Significant inhibition of pY-STAT3 was evident following culture with MH at all doses, with the pY-STAT3 levels ranging from 19.0 ± 3.0 to 52.7 ± 10.1% (mean ± SEM) of control following exposure to 1 or 0.03% MH concentration, respectively (Figures 8C,D). Importantly, incubation of MDA-MB-231 cells with SC solution resulted in only a marginal reduction (69.0 ± 2.8% of control) in pY-STAT3 levels which was observed only at the highest concentration (1%) of SC used (Figures 8C,D). These findings demonstrate that incubation of MDA-MB-231 cells with MH at concentrations as low as 0.03% results in a significant and specific reduction in pY-STAT3 levels. Tyrosine phosphorylation of cytosolic proteins is a widely utilized mechanism for the regulation of cell cycle and signal transduction pathways in eukaryotic cells (39, 40). Therefore, we wished to examine the global pattern of tyrosine-phosphorylated proteins in cancer cells following treatment with MH. The results of this analysis revealed no evidence that the total protein tyrosine phosphorylation pattern is grossly altered in MDA-MB-231 cells exposed to 1 (data not shown) or 5% MH (Figure 8E), suggesting that the targeted effect on pY-STAT3 is specific.

MCF-7 cells are known to express low levels of constitutive pY-STAT3 (41, 42). Nevertheless, a similar analysis of the effect of MH on pY-STAT3 in MCF-7 cells revealed very similar findings to those in MDA-MB-231 cells (Figure 9). Within 30 min of exposure to 1.25% MH, the level of pY-STAT3 was reduced by ~30% compared to the level observed in untreated cells, with maximal inhibition (80.3%) occurring at 1 h of incubation (Figures 9A,B). Despite a partial recovery in pY-STAT3 levels by 4 h, they were still significantly inhibited (55% of normal level) even after 12 h of treatment (Figures 9A,B). Similar to the results in MDA-MB-231 cells, MH treatment had no significant effect on total STAT3 protein levels within this time frame (Figure 9A). The sensitivity of pY-STAT3 inhibition following a 1 h-exposure to differing concentrations (range 1.25–0.075%) of MH or SC solution (Figure 9C) was also examined. The levels of pY-STAT3, but not total STAT3, were dramatically reduced following exposure to MH in a dose-dependent manner (Figure 9C). The degree of inhibition ranged from 90% at 1.25% MH to 54% at the lowest MH concentration of 0.075% (Figure 9C). Moreover, incubation of MCF-7 cells with SC solution had no effect on pY-STAT3 levels at the same concentration range (Figure 9C).

Since IL-6/IL-6R signaling is thought to be primarily responsible for the constitutive pY-STAT3 in MDA-MB-231 cells, we next sought to determine the effect of MH on IL-6 synthesis. Cells were incubated with MH (at 1.25 or 5% final concentration) for 4.5 h in the presence of Brefeldin A, an inhibitor of protein egress from the endoplasmic reticulum. The presence of IL-6 in total cell extracts was then determined by Western blots. The data, illustrated in Figure 10A, demonstrate that treatment with MH inhibits IL-6 synthesis in a dose-dependent manner. Relative to untreated control cells, the amount of detectable IL-6 protein was reduced by 59 and 83% in cells incubated with 1.25 or 5% MH solution, respectively (Figure 10A). Inhibition of IL-6 production was also observed at the level of IL-6 protein in culture supernatants, as determined by ELISA. The addition of MH to MDA-MB-231 cells resulted in a significant, dose-dependent, inhibition of IL-6 secretion after overnight incubation (Figure 10B). Moreover, significant suppression of IL-6 secretion (~60%) was observed as early as 2–4 h following cell culture in the presence of MH (Figure 10C), demonstrating the rapid kinetics of this inhibition. Furthermore, we investigated the potential effect of SC solution on IL-6 secretion. Unlike MH, incubation of MDA-MB-231 cells with up to 2.5% SC solution had no significant effect on IL-6 secretion (Figure 10D). Finally, we analyzed the effect of MH treatment on IL-6 production in MCF-7 cells. Similar to MDA-MB-231 cells, culture of MCF-7 cells in the presence of MH resulted in dose-dependent inhibition of IL-6 secretion, ranging from 28% inhibition at 0.3% MH to 62% at 2.5% MH (Figure 10E). We conclude that exposure of MDA-MB-231 and MCF-7 breast cancer cells to MH leads to inhibition of IL-6 production as well as secretion. This inhibition is quite independent of the sugar content of MH.