Simultaneously targeting tissue transglutaminase and kidney type glutaminase sensitizes cancer cells to acid toxicity and offers new opportunities for therapeutic intervention - PubMed (original) (raw)

. 2015 Jan 5;12(1):46-55.

doi: 10.1021/mp500405h. Epub 2014 Dec 10.

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Simultaneously targeting tissue transglutaminase and kidney type glutaminase sensitizes cancer cells to acid toxicity and offers new opportunities for therapeutic intervention

William P Katt et al. Mol Pharm. 2015.

Abstract

Most cancer cells undergo characteristic metabolic changes that are commonly referred to as the Warburg effect, with one of the hallmarks being a dramatic increase in the rate of lactic acid fermentation. This leads to the production of protons, which in turn acidifies the microenvironment surrounding tumors. Cancer cells have acquired resistance to acid toxicity, allowing them to survive and grow under these detrimental conditions. Kidney type glutaminase (GLS1), which is responsible for the conversion of glutamine to glutamate, produces ammonia as part of its catalytic activities and has been shown to modulate cellular acidity. In this study, we show that tissue, or type 2, transglutaminase (TG2), a γ-glutamyl transferase that is highly expressed in metastatic cancers and produces ammonia as a byproduct of its catalytic activity, is up-regulated by decreases in cellular pH and helps protect cells from acid-induced cell death. Since both TG2 and GLS1 can similarly function to protect cancer cells, we then proceeded to demonstrate that treatment of a variety of cancer cell types with inhibitors of each of these proteins results in synthetic lethality. The combination doses of the inhibitors induce cell death, while individual treatment with each compound shows little or no ability to kill cells. These results suggest that combination drug treatments that simultaneously target TG2 and GLS1 might provide an effective strategy for killing cancer cells.

Keywords: 968; cancer; glutaminase; tissue transglutaminase.

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Figures

Figure 1

Figure 1

Inhibitors of GLS1 and TG2. 968 and BPTES are reversible allosteric regulators of GLS1. MDC is a reversible inhibitor of TG2, which serves as an amine-bearing substrate. T101 and Z-Don are irreversible inhibitors of TG2, which bind at the catalytic site. All inhibitors are cell permeable except for T101.

Figure 2

Figure 2

MDC treatment lowers the pH of cancer cell culture media. T98G cells (black circles), MCF-7 cells (gray triangles), or U-87 MG cells (white circles) were cultured in DMEM supplemented with 10% FBS and the indicated amounts of MDC. Six days later, the pH of the media was determined. Increasing the concentration of MDC caused the pH of the culturing medium from each of the cell lines to decrease steadily until a maximum change of 0.3–0.5 pH units was reached. Error bars represent the standard deviation of three separate experiments.

Figure 3

Figure 3

Media acidification induces TG2 expression. T98G and SK-BR-3 cells were cultured in standard growth media (pH 7.65), supplemented with or without 25 μM retinoic acid (RA) or in growth media at pH 6.15. The relative amounts of TG2 expression detected for each condition were quantitated with respect to vinculin expression by densiometric analysis. The vertical line indicates a portion of the blot that was spliced out.

Figure 4

Figure 4

Proliferation of MDA-MB-231 cells in the presence of different amounts of 968 and/or MDC. (A) MDA-MB-231 cells were cultured in the presence of 968 (black circles) or MDC (white circles) for 6 days and then counted. Dose curves were determined using SigmaPlot. (B) Histogram depicting specific data point from the dose curves for 968, MDC, or 968 and MDC treatments in MDA-MB-231 cells. The _Y_-axis represents the number of cells present in the culture after 6 days of drug treatment, while the _X_-axis is positioned at the starting number of cells. Values indicated with * were calculated from dose curves in panel A, and error bars represent the standard deviation from the nearest experimental measurement. (C) Combination index (CI) values calculated for 968 and MDC when used to treat MDA-MB-231 cells as described above, at a ratio of 4.2 μM 968 to 60 μM MDC. The CI was calculated at regular intervals that represent a specific fraction (5%) of normal cell growth. Plots were determined considering the two drugs as either mutually exclusive (black circles) or mutually nonexclusive (white circles) in their binding. Error bars in panels A and B represent the standard deviation of three independent measurements.

Figure 5

Figure 5

Combining 968 and MDC induces cell death in MCF-7 breast cancer cells and U-87 MG brain cancer cells. (A,B) Histograms showing key data points collected from dose curves for 968, MDC, or 968 and MDC treatments in MCF-7 (A) or U-87 MG (B) cells. The _Y_-axes represent the number of cells in culture after 6 days of drug treatment, while the _X_-axes are positioned at the starting number of cells. Values indicated with * were calculated from dose curves, and their error bars represent the standard deviation from the nearest experimental measurement. (C,D) Combination index (CI) values calculated for 968 and MDC when used to treat MCF-7 (C) or U-87 MG (D) cells, used at a ratio of 12 μM/60 μM for MCF-7 cells and 4.2 μM/60 μM for U-87 MG cells. The CI was calculated at regular intervals that represent a specific fraction (5%) of normal cell growth. Plots were determined considering the two drugs as either mutually exclusive (black circles) or mutually nonexclusive (white circles) in their binding. Error bars in panels A and B represent the standard deviation of three independent measurements.

Figure 6

Figure 6

Proliferation of MDA-MB-231 cells in the presence of various concentrations and combinations of MDC, BPTES, and 968. (A) Histograms showing key data points collected from dose curves for MDA-MB-231 cells treated with BPTES, MDC, or BPTES and MDC. The _Y_-axis represents the number of cells in culture after 6 days of the indicated drug treatments, while the _X_-axis is positioned at the starting number of cells. Values indicated with * were calculated from dose curves, and error bars represent the standard deviation from the nearest experimental measurement. (B) Effects of 968 and MDC (black bars), 968, MDC, and 6.6 mM dimethyl-α-ketoglutarate (white bars), or MDC alone (gray bars) on the growth of MDA-MB-231 cells. Cells were treated for 6 days with 968 and/or MDC added at the indicated fractions of their IC50 values (4.2 μM for 968 and 60 μM for MDC).

Figure 7

Figure 7

Cancer cells treated with 968 and the TG2 inhibitors Z-Don and T101. (A) MDA-MB-231 cells were cultured in the presence of 968, Z-Don, or a combination of 968 and Z-Don (at a ratio of 4.2 μM 968/37.5 μM Z-Don) for 6 days and then counted. The _Y_-axis represents the number of cells in culture after 6 days of drug treatment, while the _X_-axis is positioned at the starting number of cells. Values marked with * were calculated from dose curves, and error bars represent the standard deviation from the nearest experimental measurement. (B) Effects of 968 with or without T101, upon the growth of Mia-PaCa-2 (black circles, 968; white circles, 968 and T101) or U-87 MG (gray triangles, 968; white squares, 968 and T101) cells. Cells were treated with the indicated amounts of 968, with or without 10 μM T101, and after 6 days of growth the cells were counted. Error bars represent the standard deviation of three independent measurements.

Figure 8

Figure 8

Increasing the acidity of cell culturing medium enhances the potency of MDC or 968. T98G cells were cultured in normal (pH = 7.65; black circles) or acidic (pH = 6.15; white circles) media for 6 days in the presence of varying amounts of either (A) MDC or (B) 968 and then counted. Error bars represent the standard deviation of three independent measurements.

Figure 9

Figure 9

Cartoon representing the implications of TG2 or GAC inhibition on intracellular acidity levels and cell viability. Cancer cells normally maintain a healthy, neutral pH (upper left). Inhibition of either GAC (with 968) or TG2 (with MDC) causes the pH in cells to decrease and limits cell growth (upper right and lower left). However, inhibiting TG2 and GLS1 simultaneously decreases the pH to an intolerable level (lower right) and kills cells. In this way, a cotherapy of MDC and 968 could potentially be beneficial for the treatment of a broad range of cancer cell types.

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