Brusatol inhibits HIF-1 signaling pathway and suppresses glucose uptake under hypoxic conditions in HCT116 cells

Abstract

Hypoxia-inducible factor-1 (HIF-1) is an important transcription factor that induces adaptive responses upon low oxygen conditions in human cancers and triggers off a poor prognostic outcome of conventional treatments. In this study, we discovered for the first time that brusatol (BRU), a quassinoid extracted from Brucea Esters, has the capability to inhibit HIF-1 signaling pathway. We found that BRU concentration-dependently down-regulated HIF-1α protein levels under hypoxia or CoCl₂-induced mimic hypoxia in HCT116 cells without causing significant cytotoxicity. Besides, the transactivation activity of HIF-1 was suppressed by BRU under hypoxic conditions, as well as the expression of HIF-1 target genes, including VEGF, GLUT1, HK2 and LDHA. In addition, BRU can also decrease glucose consumption under hypoxia through inhibition of HIF-1 signaling pathway. Further studies revealed that the inhibitory effect of BRU on HIF-1 signaling pathway might be attributed to promoting degradation of HIF-1α. Interestingly, intracellular reactive oxygen species (ROS) levels and mitochondrial ROS level were both decreased by BRU treatment, indicating the involvment of mitochondrial ROS regulation in the action of BRU. Taken together, these results provided clear evidence for BRU-mediated HIF-1α regulation and suggested its therapeutic potential in colon tumors.

Figure 1. BRU inhibits proliferation of HCT116 cells.

(A) HCT116 cells were exposed to different concentrations of BRU for 24 h, followed by MTT assay to measure cell viability. Data was presented as means ± S.D. (n = 6). ^***p < 0.001 versus 0 nM BRU-treated group. (B) HCT116 cells were treated with DMSO and BRU (60 nM) for 12 h and 24 h and amount of living cells were detected by trypan blue exclusion assay. (C) HCT116 cells were exposed to different concentrations of BRU for 24 h and images were captured under phase contrast microscopy to observe cell morphology. Scale bars, 100 μm.

Figure 2. BRU down-regulates HIF-1α protein levels in hypoxic conditions.

(A) HCT116 cells were treated with hypoxia (1% O₂) for different time and the quantity of HIF-1α protein was analyzed by Western blot. (B) The relative quantity of HIF-1α protein described in (A). (C) HCT116 cells were treated with 200 μM CoCl₂-induced mimic hypoxia for different time and the quantity of HIF-1α protein was analyzed by Western blot. (D) The relative quantity of HIF-1α protein described in (C). (E) HCT116 cells were treated with various concentrations of BRU for 4 h under hypoxia (1% O₂) and the quantity of HIF-1α protein was analyzed by Western blot. (F) The relative quantity of HIF-1α protein described in (E). Data was presented as means ± S.D. (n = 3). ^***p < 0.001 and ^*p < 0.05 versus the hypoxia alone group. (G) HCT116 cells were treated with various concentrations of BRU for 4 h under 200 μM CoCl₂-induced mimic hypoxia and the quantity of HIF-1α protein was analyzed by Western blot. (H) The relative quantity of HIF-1α protein described in (G). Data was presented as means ± S.D. (n = 3). ^***p < 0.001 and ^**p < 0.01 versus the mimic hypoxia alone group.

Figure 3. BRU does not affect the transcriptional level of HIF-1α in hypoxic conditions.

(A) HCT116 cells were cultured under normoxia or hypoxia (1% O₂) in the presence of various concentrations of BRU for 12 h. The mRNA levels of HIF-1α were analyzed by qRT-PCR and normalized with β-actin mRNA levels. (B) HCT116 cells were cultured under normoxia or 200 μM CoCl₂-induced mimic hypoxia in the presence of various concentrations of BRU for 12 h. The mRNA levels of HIF-1α were analyzed by qRT-PCR and normalized with β-actin mRNA levels.

Figure 4. BRU promotes degradation of HIF-1α protein in hypoxic conditions.

(A) HCT116 cells were cultured under normoxia or hypoxia (1% O₂) in the presence or absence of 10 μM MG132, with or without 60 nM BRU, for 4 h and the quantity of HIF-1α protein was analyzed by Western blot. (B) HCT116 cells were cultured under normoxia or 200 μM CoCl₂-induced mimic hypoxia in the presence or absence of 10 μM MG132, with or without 60 nM BRU, for 4 h and the quantity of HIF-1α protein was analyzed by Western blot. (C) and (D) HCT116 cells were firstly incubated with MG132 in normoxia. Then, CHX (50 μg/ml)-containing fresh medium was added into cells, and the cells were further incubated in hypoxia for different time in the presence or absence of 60 nM BRU. At each time point, cells were harvested and the quantity of HIF-1α protein was analyzed by Western blot. (E) and (F) HCT116 cells were firstly incubated with MG132 in normoxia. Then, CHX (50 μg/ml)-containing fresh medium was added into cells, and the cells were further incubated in CoCl₂-induced mimic hypoxia for different time in the presence or absence of 60 nM BRU. At each time point, cells were harvested and the quantity of HIF-1α protein was analyzed by Western blot. (G) The relative change in the HIF-1α protein levels at each time point described in (C) and (D). Data was presented as means ± S.D. (n = 3). ^**p < 0.01 and ^*p < 0.01 versus the hypoxia alone group. (H) The relative change in the HIF-1α protein levels described in (E) and (F). Data was presented as means ± S.D. (n = 3). ^**p < 0.01 and ^*p < 0.01 versus the mimic hypoxia alone group.

Figure 5. BRU inhibits the transactivation function of HIF-1 in hypoxic conditions.

HCT116 cells were transiently cotransfected with the HRE-luciferase plasmid and an internal control vector pRL-TK for 24 h and then treated with or without BRU for 6 h under hypoxia (A) or under CoCl₂-induced mimic hypoxia (B), and then luciferase activity was quantitated. Data was presented as means ± S.D. (n = 6). ^***p < 0.001, ^**p < 0.01 and ^*p < 0.05 versus the hypoxia alone group in (A). ^***p < 0.001 versus the mimic hypoxia alone group in (B).

Figure 6. BRU down-regulates the expression of VEGF in HCT116 cells in hypoxic conditions.

(A) HCT116 cells were treated with various concentrations of BRU for 12 h under normoxia or hypoxia (1% O₂). The mRNA levels of VEGF were analyzed by qRT-PCR and normalized with β-actin mRNA levels. Data was presented as means ± S.D. (n = 3). ^***p < 0.001 versus the hypoxia alone group. (B) HCT116 cells were treated with various concentrations of BRU for 12 h under 200 μM CoCl₂-induced mimic hypoxia. The mRNA levels of VEGF were analyzed by qRT-PCR and normalized with β-actin mRNA levels. Data was presented as means ± S.D. (n = 3). ^**p < 0.01 and ^*p < 0.05 versus the mimic hypoxia alone group. (C) HCT116 cells were treated with various concerntrations of BRU for 24 h under hypoxia and the quantity of VEGF protein was analyzed by Western blot. (D) HCT116 cells were treated with various concerntrations of BRU for 24 h under 200 μM CoCl₂-induced mimic hypoxia and the quantity of VEGF protein was analyzed by Western blot. (E) The relative quantity of VEGF protein described in (C). Data was presented as means ± S.D. (n = 3). ^***p < 0.001 versus the hypoxia alone group. (F) The relative quantity of VEGF protein described in (D). Data was presented as means ± S.D. (n = 3). ^*p < 0.05 versus the mimic hypoxia alone group.

Figure 7. BRU inhibits expression of glycolytic enzymes and glucose consumption in HCT116 cells under hypoxia.

(A) HCT116 cells were treated with various concentrations of BRU for 12 h under hypoxia (1% O₂). (B), (C) and (D) The mRNA levels of GLUT1, HK2 and LDHA were analyzed by reverse transcription-PCR and normalized with β-actin mRNA levels. Data was presented as means ± S.D. (n = 3). ^***p < 0.001 versus the hypoxia alone group (0 nM BRU-treated group). (E) HCT116 cells were treated with various concentrations of BRU for 24 h under hypoxia (1% O₂) and then glucose consumption was measured with commercial kit. Data was presented as means ± S.D. (n = 4). ^***p < 0.001 versus the hypoxia alone group (0 nM BRU-treated group).

Figure 8. BRU decreases intracellular ROS and mitochondrial ROS levels in HCT116 cells.

(A) HCT116 cells were treated with 60 nM BRU for 3 h and intracellular ROS generation was analyzed by DCFH-DA using confocal microscope (magnification, 200×). (B) HCT116 cells were treated with various concentrations of BRU for 3 h and intracellular ROS generation was analyzed by DCFH-DA using flow cytometry. (C) The fluorescence intensity of DCF described in (B) was quantitated. Data was presented as means ± S.D. (n = 3). ^***p < 0.001 and ^**p < 0.01 versus the control group. (D) HCT116 cells were treated with 60 nM BRU for 3 h under hypoxia (1% O₂) and intracellular ROS generation was analyzed by DCFH-DA using flow cytometry. (E) The fluorescence intensity of DCF described in (C) was quantitated. Data was presented as means ± S.D. (n = 3). ^***p < 0.001 versus the normoxia group, ^##p < 0.01 versus the hypoxia group. (F) HCT116 cells were treated with 60 nM BRU for 3 h under hypoxia (1% O₂) and mitochondrial ROS generation was analyzed by mitoSOX Red using flow cytometry. (G) The fluorescence intensity of mitoSOX described in (F) was quantitated. Data was presented as means ± S.D. (n = 3). ^***p < 0.001 versus the normoxia group, ^#p < 0.05 versus the hypoxia group.