Tumor cells upregulate normoxic HIF-1α in response to doxorubicin

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that controls cellular homeostasis. Although its activation benefits normal tissue, HIF-1 activation in tumors is a major risk factor for angiogenesis, therapeutic resistance, and poor prognosis. HIF-1 activity is usually suppressed under normoxic conditions because of rapid oxygen-dependent degradation of HIF-1α. Here, we show that, under normoxic conditions, HIF-1α is upregulated in tumor cells in response to doxorubicin, a chemotherapeutic agent used to treat many cancers. In addition, doxorubicin enhanced VEGF secretion by normoxic tumor cells and stimulated tumor angiogenesis. Doxorubicin-induced accumulation of HIF-1α in normoxic cells was caused by increased expression and activation of STAT1, the activation of which stimulated expression of iNOS and its synthesis of nitric oxide (NO) in tumor cells. Mechanistic investigations established that blocking NO synthesis or STAT1 activation was sufficient to attenuate the HIF-1α accumulation induced by doxorubicin in normoxic cancer cells. To our knowledge, this is the first report that a chemotherapeutic drug can induce HIF-1α accumulation in normoxic cells, an efficacy-limiting activity. Our results argue that HIF-1α-targeting strategies may enhance doxorubicin efficacy. More generally, they suggest a broader perspective on the design of combination chemotherapy approaches with immediate clinical impact.

Figure 1

Doxorubicin upregulates HIF-1α. A, HIF-1α reporter activity in normoxic 4T1ODD-luc cells. B, quantification of HIF-1α reporter activity in 4T1ODD-luc cells post-treatment (n = 3, mean ± SE). *, P < 0.05 compared to control treatment, one-way ANOVA. C, Western blots: HIF-1α expression in normoxic 4T1ODD-luc and MCF-7 cells 48 hours post-treatment. Histone H1, loading control for nuclear extracts. D, HIF-1α reporter activity in orthotopic 4T1ODD-luc tumors 4 days post-treatment. E, quantification of HIF-1α reporter activity in orthotopic 4T1ODD-luc tumors (n = 7, mean ± SE). Doxorubicin (Dox) treatments were as indicated 0.1-10 μg/mL (0.58-5.8 μM). *, P < 0.01, Student t test.

Figure 2

The upregulated HIF-1α expression in doxorubicin-treated 4T1ODD-luc tumors was not due to hypoxia. A, representative immunofluorescent stainings demonstrating the distribution of HIF-1α (green) and pimonidazole (red) in two adjacent entire tumor sections 5 days post-treatment. Scale bars, 1 mm. B, HIF-1α fraction in 4T1ODD-luc tumors on days 0, 1, 4, 7, and 16 post-treatment (n = 5, mean ± SE). *, P < 0.05, **, P < 0.01, Student t test. C, pimonidazole fraction in 4T1ODD-luc tumors on days 0, 1, 4, 7, and 16 post-treatment (n = 5, mean ± SE). P > 0.05, Student t test.

Figure 3

Doxorubicin stimulates VEGF secretion by 4T1ODD-luc cells and causes a resurgent tumor angiogenesis. A, Relative viable cell numbers of all doxorubicin-treated groups were < 40% compared to the control (n = 3, mean ± SE). P < 0.05 compared to the control group, one-way ANOVA. B, mouse VEGF ELISA showing VEGF concentrations in the cell culture medium of 4T1ODD-luc cells treated with doxorubicin (0.1, 1, and 10 μg/ml) vs control treatment (n = 3, mean ± SE). *, P < 0.005, one-way ANOVA. C, representative fluorescent staining of vasculature in 4T1 ODD-luc tumors 4 days post-treatment. Scale bar, 100 μm. D, relative tumor vascular fractions in 4T1ODD-luc tumors (n = 5 in doxorubicin groups on days 0, 4, 7, and 16, n = 6 in all other groups, mean ± SE). *, P < 0.05, Student t test.

Figure 4

Doxorubicin increased intracellular NO synthesis in 4T1ODD-luc cells. A, FACS analysis: relative fluorescence intensity of NO-specific probe DAF-FM in 4T1ODD-luc cells 48 hours post-treatment (n = 3, mean ± SE). *, P < 0.05; **, P < 0.001, one-way ANOVA. Dox, doxorubicin. AU, arbitrary unit. B, representative bioluminescent images of 4T1ODD-luc cells treated with L-NAME ± Doxorubicin. C, HIF-1α reporter activity in 4T1ODD-luc cells treated with L-NAME alone (n = 3, mean ± SE). P > 0.05, one-way ANOVA. D, HIF-1α reporter activity in 4T1ODD-luc cells treated with Dox ± L-NAME (n = 3, mean ± SE). *, P < 0.05, one-way ANOVA. Dox, doxorubicin.

Figure 5

Doxorubicin upregulates and activates the STAT1-iNOS signaling pathway. A, quantitative real-time PCR: iNOS mRNA levels in 4T1ODD-luc cells 2 days post-treatment (n = 3, mean ± SE). *, P < 0.05, Student t test. B, Western blot: iNOS expression in 4T1ODD-luc cells treated with or without doxorubicin. C, Western blot: phosphorylated STAT1 (Tyr701 and Ser727) and total STAT1 in 4T1ODD-luc cells 48 hours post-treatment. D, Western blot: iNOS expression in 4T1ODD-luc cells treated with or without doxorubicin, 1400W, or EGCG. E, Western blot: the expressions of phosphorylated STAT1 (Tyr701 and Ser727) and total STAT1 in 4T1ODD-luc cells 48 hours after treatments with or without doxorubicin, 1400W, or EGCG.

Figure 6

The iNOS-specific inhibitor 1400W and the STAT1-interfering chemical EGCG suppressed the increased intracellular NO level induced by doxorubicin. Dox, doxorubicin. A - D, FACS analysis comparing intracellular NO marker DAF-FM between treated groups and the control group (n = 3, mean ± SE). *, P < 0.05, one-way ANOVA. A and C, 72 hours post-treatment. E, Western blot of nuclear extract: 1400W and EGCG suppressed the doxorubicin-induced normoxic HIF-1α accumulation in 4T1ODD-luc cells. F, Western blot of cell lysate: 1400W and EGCG suppressed the doxorubicin-induced normoxic HIF-1α accumulation in MCF-7 cells. CoCl₂, positive control for HIF-1α. G, Western blot: knockdown of STAT1 by siRNA decreased the doxorubicin-induced normoxic HIF-1α accumulation in 4T1ODD-luc cells. H, quantification of STAT1 and HIF-1α protein expression after knockdown of STAT1 by siRNA in doxorubicin-treated 4T1ODD-luc cells (n = 4, mean ± SE). *, P < 0.05 compared to the scrambled siRNA control, Student t test.

Figure 7

A, cell viability assay: 4T1ODD-luc cell viability post-treatment (n = 5, mean ± SE). *, P < 0.05 compared to the control treatment, one-way ANOVA. Dox, doxorubicin. No difference between Dox and Dox + 1400W treatment at all four time points. B, schematic diagram of the mechanisms underlying doxorubicin-induced normoxic HIF-1α accumulation, angiogenesis, and interference strategies. Gray color: other mechanisms suggested by previous studies.