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. 2012:2012:390957.
doi: 10.1155/2012/390957. Epub 2012 Nov 29.

Inhibition of Hypoxia Inducible Factor Alpha and Astrocyte-Elevated Gene-1 Mediates Cryptotanshinone Exerted Antitumor Activity in Hypoxic PC-3 Cells

Hyo-Jeong Lee  1 , Deok-Beom Jung, Eun Jung Sohn, Hanna Hyun Kim, Moon Nyeo Park, Jae-Hwan Lew, Seok Geun Lee, Bonglee Kim, Sung-Hoon Kim
Affiliations

Inhibition of Hypoxia Inducible Factor Alpha and Astrocyte-Elevated Gene-1 Mediates Cryptotanshinone Exerted Antitumor Activity in Hypoxic PC-3 Cells

Hyo-Jeong Lee et al. Evid Based Complement Alternat Med. 2012.

Erratum in

  • Evid Based Complement Alternat Med. 2013;2013:267352

Abstract

Although cryptotanshinone (CT) was known to exert antitumor activity in several cancers, its molecular mechanism under hypoxia still remains unclear. Here, the roles of AEG-1 and HIF-1α in CT-induced antitumor activity were investigated in hypoxic PC-3 cells. CT exerted cytotoxicity against prostate cancer cells and suppressed HIF-1α accumulation and AEG-1 expression in hypoxic PC-3 cells. Also, AEG-1 was overexpressed in prostate cancer cells. Interestingly, HIF-1α siRNA transfection enhanced the cleavages of caspase-9,3, and PAPR and decreased expression of Bcl-2 and AEG1 induced by CT in hypoxic PC-3 cells. Of note, DMOG enhanced the stability of AEG-1 and HIF-1α during hypoxia. Additionally, CT significantly reduced cellular level of VEGF in PC-3 cells and disturbed tube formation of HUVECs. Consistently, ChIP assay revealed that CT inhibited the binding of HIF-1α to VEGF promoter. Furthermore, CT at 10 mg/kg suppressed the growth of PC-3 cells in BALB/c athymic nude mice by 46.4% compared to untreated control. Consistently, immunohistochemistry revealed decreased expression of Ki-67, CD34, VEGF, carbonic anhydrase IX, and AEG-1 indices in CT-treated group compared to untreated control. Overall, our findings suggest that CT exerts antitumor activity via inhibition of HIF-1α, AEG1, and VEGF as a potent chemotherapeutic agent.

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Figures

Figure 1
Figure 1
Effect of cryptotanshinone (CT) on hypoxia-induced HIF-1α activation in PC-3 cells. (a) Chemical structure of CT. (b) Effect of CT on the viability of prostate cancer cells under normoxia and hypoxia. (c) Effect of HIF-1α accumulation in hypoxic PC-3 cells. Cells were treated with CT (10 μM) for 0.5, 2, 4, 6, and 24 h under hypoxia and Western blotting was performed. (d) Effect of CT on HIF-1α transcriptional activity in hypoxic PC-3 cells by ELISA. Cells were treated with or without CT (5 or 10 μM) under normoxia or hypoxia for 6 h and HIF-1α transcriptional activity was evaluated by TransAM HIF-1 transcriptional factor assay kit. Data represent means ± S.D. ## P < 0.01 versus normoxia control. *P < 0.05 or **P < 0,01 versus hypoxia control. (e) Effect of CT on HIF-1α nuclear translocation in hypoxic PC-3 cells. Cells treated with or without CT (10 μM) under hypoxia for 6 h were fixed with 10% formalin for immunocytochemistry fluorescence staining. Green color was detected for HIF-1α in PC-3 cells, while nuclei were counterstained with blue color using DAPI.
Figure 2
Figure 2
CT attenuates the expression of AEG-1 and HIF-1α in hypoxic PC-3 cells. (a) Expression of AEG-1 in various prostate cancer cells and normal prostate cells under hypoxia. Cell lysates were prepared and subjected to Western blotting. (b) Effect of hypoxia (left panel) or CT (right panel) on the expression of AEG-1 and HIF-1α in hypoxic PC-3 cells. (c) Effect of AEG-1 or HIF-1α siRNA transfection on AEG-1 expression by Western blotting (left panel) or HIF-1α activity by ELISA (right panel). (d) Effect of DMOG on the expression of AEG-1 and HIF-1α in hypoxic PC-3 cells. (e) Effect of PI3K inhibitor wortmannin (20 μM) on AEG-1 or HIF-1α expression under normoxia and hypoxia by Western blotting.
Figure 3
Figure 3
HIF-1α siRNA transfection attenuates AEG-1 expression and the viability of PC-3 cells under hypoxia, but DMOG enhances the expression of HIF-1α and AEG-1 in hypoxic PC-3 cells. (a) Effect of HIF-1α siRNA transfection or DMOG on HIF-1α stability and AEG-1 expression in hypoxic PC-3 cells. Cells were transfected with either control siRNA vector, HIF-1α siRNA, or AEG-1 siRNA and after 24 h were incubated in hypoxia for an additional 24 h. For DMOG treatment, Cells treated with DMOG for 24 hours under hypoxia were fixed with 10% formalin for immunocytochemistry fluorescence staining. Detection of HIF-1α (red) and AEG-1 (green) in PC-3 cells. Nuclei (blue) were counterstained using DAPI. (b) Effect of HIF-1α siRNA transfection on the viability of hypoxic PC-3 cells. Cells transfected with either non-_target siRNA, HIF-1α siRNA were incubated under hypoxia for 48 h and the viability of the cells was evaluated by using BrdU assay. (c) Effect of cryptotanshinone on cell death in PC-3 cells. Cells were treated with cryptotanshinone (5, 10 μM) for 48 h. (d) HIF-1α inhibition is necessary for CK-induced apoptotic cell death in PC-3 cells. PC-3 cells were transfected with either siRNA control or siRNA HIF-1α for 24 h and exposed to 10 μM cryptotanshinone for 48 h.
Figure 4
Figure 4
CT exerts antiangiogenic activity via inhibition of HIF-1α under hypoxia. (a) Effect of CT on the protein expression of VEGF in hypoxic PC-3 cells. Cell lysates were prepared and subjected to Western blotting to determine VEGF expression. (b) Effect of CT on the secreted production of VEGF in hypoxic PC-3 cells. VEGF levels in the culture supernatants were measured by using a Quantikine VEGF ELISA kit. Data represent means ± S.D. ### P < 0.001 versus normoxia control. **P < 0,01 versus hypoxia control. (c) Effect of CT on tube formation in HUVECs treated with supernatant of PC-3 culture media. HUVECs were treated with VEGF (20 ng/mL) or the supernatant from PC-3 culture media with or without CT under hypoxia for 24 h. Tube formation assay was performed using growth factor-reduced matrigel. Cells were fixed with Diff-Quick solution, photographed randomly under an Axiovert S 100 light microscope at ×100 magnification. (d) Effect of CT on the binding of HIF-1α to VEGF promoter. Cells were treated with or without CT (5 or 10 μM) under hypoxia for 6 h. The immunoprecipitated DNA with HIF-1α antibody was amplified by PCR analysis for VEGF promoter using ChIP primers.
Figure 5
Figure 5
CT inhibits tumor growth in PC-3 xenograft model. (a) Effect of CT on the growth of PC-3 cells in BALB/c athymic nude mice. From 3 days after inoculation, CT (10 mg/kg body wt) was given by i.p. injection with 2% Tween-80 as vehicle every other days. Tumor growth was monitored twice a week. (b) Effect of CT on final tumor weight after sacrifice. Values represent means ± SD. n = 5. *P < 0.05 compared with untreated control. (c) Photographs of dissected tumors. (d) Effect of CT on body weights of mice at various time points. Values represent means ± SD. n = 5.
Figure 6
Figure 6
Effect of CT on the biomarkers of Ki-67, AEG-1, VEGF, CD34, and CAIX by immunohistochemistry. Immunohistochemistry was performed in tumor sections with antibodies of Ki-67 for proliferation, VEGF for angiogenesis, CD34 for microvessel density, CAIX for hypoxia, and AEG-1. The photographs were taken at ×200 magnification.
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