Dihydroorotate dehydrogenase Inhibitors Target c-Myc and Arrest Melanoma, Myeloma and Lymphoma cells at S-phase

Abstract

Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. Inhibition of this enzyme impedes cancer cell proliferation but the exact mechanisms of action of these inhibitors in cancer cells are poorly understood. In this study, we showed that cancer cells, namely melanoma, myeloma and lymphoma overexpressed DHODH protein and treatment with A771726 and Brequinar sodium resulted in cell cycle arrest at S-phase. Transfection with DHODH shRNA depleted DHODH protein expression and impeded the proliferation of melanoma cells. shRNA knockdown of DHODH in combination with DHODH inhibitors further reduced the cancer cell proliferation, suggesting that knockdown of DHODH had sensitized the cells to DHODH inhibitors. Cell cycle regulatory proteins, c-Myc and its transcriptional target, p21 were found down- and up-regulated, respectively, following treatment with DHODH inhibitors in melanoma, myeloma and lymphoma cells. Interestingly, knockdown of DHODH by shRNA had also similarly affected the expression of c-Myc and p21 proteins. Our findings suggest that DHODH inhibitors induce cell cycle arrest in cancer cells via additional DHODH-independent pathway that is associated with p21 up-regulation and c-Myc down-regulation. Hence, DHODH inhibitors can be explored as potential therapeutic agents in cancer therapy.

Keywords: A771726; Brequinar; DHODH shRNA; Ramos; p21..

Figure 1

DHODH expression in melanocytes, PBMCs (normal cells) and A375, H929, Ramos (cancer cells). Equal amount of protein lysate was used for Western blot analysis with human primary DHODH antibody. Tubulinβ served as protein loading control. Data represents mean ± SE of 3 independent experiments. *, P<0.05 and ***, P < 0.001.

Figure 2

DHODH inhibitors cause a dose-dependent decrease in the proliferation of human melanoma, myeloma and lymphoma cell lines. A375, H929 and Ramos cells were treated with increasing concentrations of A771726 and BQR for 72 hours. Inhibition of proliferation was measured by XTT assay. Values shown are mean ± SE of 3 independent experiments. A, EC₅₀ of A771726, A375 - 14.52 μM; H929 - 45.78 μM; Ramos - 5.36 μM. B, EC₅₀ of BQR, A375 - 0.14 μM; H929 - 0.24 μM; Ramos - 0.054 μM. C, Uridine rescue with DHODH inhibitors. *, P<0.05.

Figure 3

DHODH inhibitors affect DHODH enzyme activity but not the DHODH protein expression in A375 cells. A, DHODH enzymatic assay with A771726 and BQR using pure human DHODH enzyme. B, DHODH enzymatic assay with A771726 and BQR using A375, H929 and Ramos cells lysates. C, A375 cells were incubated with 10, 30, 100 and 200 µM A771726 and 0.016, 0.05, 0.15 and 0.45 µM of BQR for 24 and 48 hours and cell viability was determined by Trypan blue stain. D, Western blot of A375 cell lysate after treatment with A771726 and BQR. Cells were harvested at 48 hours after treatment for Western blot analysis. Tubulinβ served as protein loading control. Values shown are mean ± SE of 3 independent experiments. *, P < 0.05, **, P < 0.01 and ***, P <0.001.

Figure 4

Cell cycle distribution of melanoma, myeloma and lymphoma cell lines after treatment with DHODH inhibitors. A771726 and BQR: Induce S and G₂/M phase arrest in A375 cells (A, B, C and D); Induce S-phase arrest in Ramos cells (E, F, G and H); Induce G₀/G₁ and S-phase arrest in H929 cells (I and J). Values shown are mean ± SE of 3 independent experiments. *, P <0.05, **, P <0.01, ***, P <0.001 and ****, P <0.0001.

Figure 4

Cell cycle distribution of melanoma, myeloma and lymphoma cell lines after treatment with DHODH inhibitors. A771726 and BQR: Induce S and G₂/M phase arrest in A375 cells (A, B, C and D); Induce S-phase arrest in Ramos cells (E, F, G and H); Induce G₀/G₁ and S-phase arrest in H929 cells (I and J). Values shown are mean ± SE of 3 independent experiments. *, P <0.05, **, P <0.01, ***, P <0.001 and ****, P <0.0001.

Figure 4

Cell cycle distribution of melanoma, myeloma and lymphoma cell lines after treatment with DHODH inhibitors. A771726 and BQR: Induce S and G₂/M phase arrest in A375 cells (A, B, C and D); Induce S-phase arrest in Ramos cells (E, F, G and H); Induce G₀/G₁ and S-phase arrest in H929 cells (I and J). Values shown are mean ± SE of 3 independent experiments. *, P <0.05, **, P <0.01, ***, P <0.001 and ****, P <0.0001.

Figure 4

Cell cycle distribution of melanoma, myeloma and lymphoma cell lines after treatment with DHODH inhibitors. A771726 and BQR: Induce S and G₂/M phase arrest in A375 cells (A, B, C and D); Induce S-phase arrest in Ramos cells (E, F, G and H); Induce G₀/G₁ and S-phase arrest in H929 cells (I and J). Values shown are mean ± SE of 3 independent experiments. *, P <0.05, **, P <0.01, ***, P <0.001 and ****, P <0.0001.

Figure 4

Cell cycle distribution of melanoma, myeloma and lymphoma cell lines after treatment with DHODH inhibitors. A771726 and BQR: Induce S and G₂/M phase arrest in A375 cells (A, B, C and D); Induce S-phase arrest in Ramos cells (E, F, G and H); Induce G₀/G₁ and S-phase arrest in H929 cells (I and J). Values shown are mean ± SE of 3 independent experiments. *, P <0.05, **, P <0.01, ***, P <0.001 and ****, P <0.0001.

Figure 5

DHODH silencing potentiates cancer cell sensitivity to DHODH inhibitors in A375 cells. A, Cells were transfected with DHODH siRNA (4 nM) or control siRNA (4 nM) and harvested after 48 hours. B, Cells were transfected with DHODH shRNA (2 μg) or control shRNA (2 µg) and harvested after 48 hours. Equal amounts of protein lysates were used for Western blot analysis with human primary DHODH antibody. β-actin and anti-GAPDH served as protein loading controls. C, Prolonged depletion of DHODH caused growth retardation. D and E, Growth curve of DHODH suppressed and non-suppressed cells (prolonged and transient) after 72 hours of treatment with A771726 and BQR. Cell count was determined by Trypan blue exclusion assay as described in Materials and Methods. Values shown are mean ± SE of 3 independent experiments. *, P <0.05, **, P <0.01 and ***, P <0.001.

Figure 6

DHODH inhibitors target c-Myc and p21 signaling proteins in cancer cells. Equal amounts of protein lysates were used for Western blot analysis with the antibodies indicated. Tubulin-β and GAPDH served as protein loading control. A, A375 cells were treated with DMSO or DHODH inhibitors and cells were harvested at the indicated time point. B, H929 cells were treated with DMSO or DHODH inhibitors and cells were harvested at 48 hours after treatment. C, Ramos cells were treated with DMSO or DHODH inhibitors and cells were harvested at 48 hours after treatment. D, A375 cells plated in 6-well plates were transfected with DHODH shRNA (2 μg) or the control shRNA (2 μg) for 48 hours before immunoblotted with antibodies against c-Myc and p21. E, A375 cells plated in 6-well plates were transfected with DHODH siRNA (4 nM) or the control siRNA (4 nM) for 48 hours before harvesting for Western blot assays as described above. Data represents mean ± SE of 3 independent experiments. *, P <0.05, **, P <0.01, ***, P <0.001 and ****, P<0.0001.