Figure 1.
(
A) Transcriptional induction of
MT-1X in vascular endothelial cells after treatment with organoantimony compounds shown in
Table 1. Human brain microvascular endothelial cells were incubated with the organoantimony compounds at 10 µM each for 3 h, and the expression of
MT-1X mRNA was determined by real-time RT-PCR; (
B) The map of MRE and ARE regions in the bovine MT promoter.
Figure 1.
(
A) Transcriptional induction of
MT-1X in vascular endothelial cells after treatment with organoantimony compounds shown in
Table 1. Human brain microvascular endothelial cells were incubated with the organoantimony compounds at 10 µM each for 3 h, and the expression of
MT-1X mRNA was determined by real-time RT-PCR; (
B) The map of MRE and ARE regions in the bovine MT promoter.
Figure 2.
Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Structure of Sb35; (B) Sb35-induced transcription of MT. Vascular endothelial cells were incubated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (upper panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (lower panels). MT-1A, MT-1E, and MT-2A mRNA expression was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. * p < 0.05 and ** p < 0.01 indicate significantly different from the corresponding control.
Figure 2.
Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Structure of Sb35; (B) Sb35-induced transcription of MT. Vascular endothelial cells were incubated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (upper panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (lower panels). MT-1A, MT-1E, and MT-2A mRNA expression was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. * p < 0.05 and ** p < 0.01 indicate significantly different from the corresponding control.
Figure 3.
The MTF-1–MRE pathway mediates Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) MRE-driven transcriptional activity. Vascular endothelial cells transfected with an MRE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and MRE-driven transcriptional activity was determined by performing the MRE-directed reporter assay; (B) siRNA-mediated MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h, and expression of MTF-1 mRNA was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three samples. ** p < 0.01 indicates significantly different from the corresponding siControl; (C) Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
Figure 3.
The MTF-1–MRE pathway mediates Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) MRE-driven transcriptional activity. Vascular endothelial cells transfected with an MRE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and MRE-driven transcriptional activity was determined by performing the MRE-directed reporter assay; (B) siRNA-mediated MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h, and expression of MTF-1 mRNA was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three samples. ** p < 0.01 indicates significantly different from the corresponding siControl; (C) Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.

Figure 4.
Expression of Nrf2 and its downstream proteins, Sb35-induced ARE-driven transcriptional activity, and Sb35-induced proteasome inhibition in vascular endothelial cells. (A) Expression of Nrf2, HO-1, and GCLM. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (left panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (right panels), and expression of Nrf2, HO-1, and GCLM was determined by performing Western blotting; (B) ARE-driven transcriptional activity. Vascular endothelial cells transfected with an ARE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and ARE-driven transcriptional activity was determined by performing ARE-directed reporter assay. Data are represented as mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding control; (C) Sb35-induced proteasome inhibition. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 24 h, and ubiquitinated proteins were determined by performing Western blotting.
Figure 4.
Expression of Nrf2 and its downstream proteins, Sb35-induced ARE-driven transcriptional activity, and Sb35-induced proteasome inhibition in vascular endothelial cells. (A) Expression of Nrf2, HO-1, and GCLM. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (left panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (right panels), and expression of Nrf2, HO-1, and GCLM was determined by performing Western blotting; (B) ARE-driven transcriptional activity. Vascular endothelial cells transfected with an ARE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and ARE-driven transcriptional activity was determined by performing ARE-directed reporter assay. Data are represented as mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding control; (C) Sb35-induced proteasome inhibition. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 24 h, and ubiquitinated proteins were determined by performing Western blotting.

Figure 5.
The Nrf2–ARE pathway mediates Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Expression of Nrf2. Vascular endothelial cells transfected with control or Nrf2 siRNA were treated with or without 10, 50, or 100 µM Sb35 for 24 h, and expression of Nrf2 was determined by performing Western blotting; (B) Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after Nrf2 knockdown. Vascular endothelial cells transfected with control or Nrf2 siRNA were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
Figure 5.
The Nrf2–ARE pathway mediates Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Expression of Nrf2. Vascular endothelial cells transfected with control or Nrf2 siRNA were treated with or without 10, 50, or 100 µM Sb35 for 24 h, and expression of Nrf2 was determined by performing Western blotting; (B) Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after Nrf2 knockdown. Vascular endothelial cells transfected with control or Nrf2 siRNA were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
Figure 6.
As35 or P35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Structures of As35 and P35; (B) Endothelial MT-1A, MT-1E, and MT-2A expression after As35 (upper panels) or P35 (lower panels) treatment. Vascular endothelial cells were treated with or without 10, 20, 50, or 100 µM As35 for 12 h (upper panels) or 5, 10, 20, or 30 µM P35 or 12 h (lower panels). The cells were also treated with 100 µM Sb35, which served as a comparative control. MT-1A, MT-1E, and MT-2A mRNA expression was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicate significantly different from the corresponding control.
Figure 6.
As35 or P35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Structures of As35 and P35; (B) Endothelial MT-1A, MT-1E, and MT-2A expression after As35 (upper panels) or P35 (lower panels) treatment. Vascular endothelial cells were treated with or without 10, 20, 50, or 100 µM As35 for 12 h (upper panels) or 5, 10, 20, or 30 µM P35 or 12 h (lower panels). The cells were also treated with 100 µM Sb35, which served as a comparative control. MT-1A, MT-1E, and MT-2A mRNA expression was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicate significantly different from the corresponding control.
Figure 7.
The MTF-1–MRE pathway mediates P35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Vascular endothelial cells transfected with an MRE reporter vector were treated with or without 1, 5, 10, 20, or 30 µM P35 for 12 h, and MRE-driven transcriptional activity was determined by performing the MRE-directed reporter assay. ** p < 0.01 indicates significantly different from the corresponding control; (B) P35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 10 or 30 µM P35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
Figure 7.
The MTF-1–MRE pathway mediates P35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Vascular endothelial cells transfected with an MRE reporter vector were treated with or without 1, 5, 10, 20, or 30 µM P35 for 12 h, and MRE-driven transcriptional activity was determined by performing the MRE-directed reporter assay. ** p < 0.01 indicates significantly different from the corresponding control; (B) P35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 10 or 30 µM P35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
Figure 8.
The Nrf2–ARE pathway mediates P35-induced transcription of endothelial MT-2A. (A,B) ARE-driven transcriptional activity. Vascular endothelial cells transfected with an ARE reporter vector were treated with or without 1, 5, 10, 20, or 30 µM P35 for 12 h, and ARE-driven transcriptional activity was determined by performing the ARE-directed reporter assay. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding control; (C) P35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after Nrf2 knockdown. Vascular endothelial cells transfected with control or Nrf2 siRNA were treated with or without 10 or 30 µM P35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
Figure 8.
The Nrf2–ARE pathway mediates P35-induced transcription of endothelial MT-2A. (A,B) ARE-driven transcriptional activity. Vascular endothelial cells transfected with an ARE reporter vector were treated with or without 1, 5, 10, 20, or 30 µM P35 for 12 h, and ARE-driven transcriptional activity was determined by performing the ARE-directed reporter assay. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding control; (C) P35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after Nrf2 knockdown. Vascular endothelial cells transfected with control or Nrf2 siRNA were treated with or without 10 or 30 µM P35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
Table 1.
Organoantimony compounds used in this study.
Table 1.
Organoantimony compounds used in this study.
No. | Molecular Formula |
---|
Sb13 | C23H20NSb | N-Methyl-Sb-phenylethynyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb14 | C24H22NSb | N-Ethyl-Sb-phenylethynyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb15 | C25H24NSb | N-iso-propyl-Sb-Phenylethynyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb16 | C26H26NSb | N-2-Methylpropyl-Sb-phenylethynyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb17 | C28H28NSb | N-Cyclohexyl-Sb-phenylethynyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb18 | C28H22NSb | N-Phenyl-Sb-phenylethynyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb19 | C21H20NSb | N-Methyl-Sb-phenyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb20 | C24H26NSb | N-t-Butyl-Sb-phenyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb22 | C16H18NSb | N-Methyl-Sb-methyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb23 | C19H26NSb | N-Methyl-Sb-trimethylsilylmethyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb24 | C19H24NSb | N-t-Butyl-Sb-methyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine |
Sb26 | C21H21O3Sb | Tris(4-methoxylphenyl)stibane |
Sb29 | C21H21Sb | Tris(4-methylphenyl)stibane |
Sb30 | C21H21Sb | Tris(3-methylphenyl)stibane |
Sb31 | C21H21Sb | Tris(2-methylphenyl)stibane |
Sb32 | C27H33Sb | Tris(2,4,6-trimethylphenyl)stibane |
Sb33 | C18H12F3Sb | Tris(4-fluorophenyl)stibane |
Sb34 | C18H12Cl3Sb | Tris(4-chlorophenyl)stibane |
Sb35 | C18F15Sb | Tris(pentafluorophenyl)stibane |
Sb37 | C24H15S3Sb | Tris(1-benzothiophen-2-yl)stibane |
Sb38 | C24H15O3Sb | Tris(2-benzofuranyl)stibane |
Sb40 | C21H12F9Sb | Tris[(4-trifluoromethyl)phenyl]stibane |
Sb41 | C27H27O6Sb | Tris(4-ethoxycarbonylphenyl)stibane |
Sb42 | C27H36N3Sb | Tris[2-(N,N-dimethylaminomethyl)phenyl]stibane |
Sb43 | C24H27O3Sb | Tris[2-(methoxymethyl)phenyl]stibane |
Sb44 | C24H27S3Sb | Tris[2-(methylsulfanylmethyl)phenyl]stibane |
Sb46 | C18H15Cl2Sb | Triphenylantimony dichloride |
Sb48 | C22H21O4Sb | Triphenylantimony diacetate |