Glucose deprivation reduces proliferation and motility, and enhances the anti-proliferative effects of paclitaxel and doxorubicin in breast cell lines in vitro

Abstract

Background: Breast cancer chemotherapy with high dose alkylating agents is severely limited by their collateral toxicity to crucial normal tissues such as immune and gut cells. Taking advantage of the selective dependence of cancer cells on high glucose and combining glucose deprivation with these agents could produce therapeutic synergy.

Methods: In this study we examined the effect of glucose as well as its deprivation, and antagonism using the non-metabolized analogue 2-deoxy glucose, on the proliferation of several breast cancer cell lines MCF7, MDA-MB-231, YS1.2 and pII and one normal breast cell line, using the MTT assay. Motility was quantitatively assessed using the wound healing assay. Lactate, as the end product of anaerobic glucose metabolism, secreted into culture medium was measured by a biochemical assay. The effect of paclitaxel and doxorubicin on cell proliferation was tested in the absence and presence of low concentrations of glucose using MTT assay.

Results: In all cell lines, glucose supplementation enhanced while glucose deprivation reduced both their proliferation and motility. Lactate added to the medium could substitute for glucose. The inhibitory effects of paclitaxel and doxorubicin were significantly enhanced when glucose concentration was decreased in the culture medium, requiring 1000-fold lesser concentration to achieve a similar degree of inhibition to that seen in glucose-containing medium.

Conclusion: Our data show that a synergy was obtained by combining paclitaxel and doxorubicin with glucose reduction to inhibit cancer cell growth, which in vivo, might be achieved by applying a carbohydrate-restricted diet during the limited phase of application of chemotherapy; this could permit a dose reduction of the cytotoxic agents, resulting in greater tolerance and lesser side effects.

Fig 1. Effect of glucose starvation on cell proliferation.

pII (panel A) and YS1.2 (panel B) cell density was determined, using the MTT assay, at seeding day (day 0, hatched bars), and at days 1 and 4 (D 1 and D4) after culture in medium containing glucose (open bars) or without glucose (solid bars). The degree of proliferation of MDA-MB-231 and MCF10A as indicated in panels C-D was determined at day 4 (D 4) after culture in medium with (open bars) or without (closed bars) glucose. Panel E (MCF10A) and panel F (pII), show growth of cells (number of cells were measured using hemocytometer) cultured in + glucose medium (black line),—glucose medium (red line), or these two media alternated every 72 h (green line). Histobars represent means ± SEM of at least 3 independent determinations. * denotes significant difference from cells cultured in + glucose medium with p<0.05.

Fig 2. Effect of glucose concentration on breast cancer cell proliferation.

Proliferation of pII (panel A), MDA-MB-231 (panel B), YS1.2 (panel C), and MCF10A (panel D) cells after culture for 4 days in + glucose medium (open bars) or–glucose medium supplemented with various concentrations of glucose as indicated (solid bars), was determined using the MTT assay. Histobars represent means ± SEM of at least 3 independent determinations. * denotes significant difference from cells cultured in—glucose medium, with p<0.05.

Fig 3. Effect of glucose concentration on breast cancer cell motility.

Cells were cultured to confluency in + glucose medium. The medium was then changed to–glucose medium + glucose additions as indicated. A scratch was made through the cell monolayer and the width measured immediately and after further 24h incubation. Panels A-B for MDA-MB-231 cells, panels C-D for pII cells, and panels E-F for YS1.2 cells. Histobars represent means ± SEM for each condition. * denotes significant difference from cells cultured in—glucose medium, with p<0.05.

Fig 4. Effect of glucose starvation on lactate levels.

Extracellular lactate level in pII cells upon culture in + glucose medium (open bar), or—glucose medium (solid bar) for 1 day was determined as described in Methods. Histobars represent means ± SEM of at least 3 independent determinations. * denotes significant difference from cells cultured in + glucose medium, with p<0.05.

Fig 5. Effect of glucose starvation on pII cell motility.

The degree of pII cell motility upon culture in + glucose medium (panel A),—glucose medium (panel B), -glucose medium for 24 h followed by culture in +glucose medium for another 24 h, or -glucose medium in the presence of 20 mM L-lactate was determined (panels B-C) as described in Methods. Histobars represent means ± SEM of at least 3 independent determinations. * denotes significant difference with p<0.05.

Fig 6. Effect of 2-deoxy glucose on cell proliferation.

Proliferation of the cell lines indicated was measured after 4 days of exposure to either vehicle or various concentrations of 2-DG, using the MTT assay. Histobars represent means ± SEM of at least 3 independent experiments. * denotes significant difference from vehicle-treated cells, with p<0.05.

Fig 7. Effect of 2-deoxy glucose on cell motility.

Motility of the YS1.2 (A), pII (B), and MDA-MB-231 (C) cells in response to treatment with various concentrations of 2-DG or vehicle, was measured using the wound healing assay. Histobars represent means ± SEM of at least 3 independent experiments. * denotes significant difference from vehicle treated cells, with p<0.05.

Fig 8. Effect of glucose depletion on the anti-proliferative effects of paclitaxel in breast cancer cell lines.

The effect of paclitaxel on cell proliferation upon culture in +glucose medium (black line, taken as 100%) or -glucose medium plus 5 mM (red line), or 1.7 mM glucose (blue line) was determined at day 4 using the MTT assay. Histobars represent means ± SEM of at least 3 independent determinations. * denotes significant difference from cells treated with vehicle (normal saline), with p<0.05.

Fig 9. Effect of glucose depletion on the anti-proliferative effects of doxorubicin in breast cancer cell lines.

The effect of doxorubicin on cell proliferation upon culture in +glucose medium (black line, taken as 100%) or—glucose medium plus 5 mM (red line), or 1.7 mM glucose (blue line) was determined at day 4 using the MTT assay. Histobars represent means ± SEM of at least 3 independent determinations. * denotes significant difference from cells treated with vehicle (normal saline), with p<0.05.