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Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide.

Zhou ZL, Luo ZG, Yu B, Jiang Y, Chen Y, Feng JM, Dai M, Tong LJ, Li Z, Li YC, Ding J, Miao ZH - Mol. Cancer (2010)

Bottom Line: Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells.However, triptolide was found to increase the levels of HIF-1α mRNA.The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Background: Hypoxia-inducible factor-1α (HIF-1α), a critical transcription factor to reduced O2 availability, has been demonstrated to be extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis. Thus it has been considered as a potential anticancer target. Triptolide is the main principle responsible for the biological activities of the Traditional Chinese Medicine tripterygium wilfordii Hook F. Triptolide possesses great chemotherapy potential for cancer with its broad-spectrum anticancer, antiangiogenesis, and drug-resistance circumvention activities. Numerous biological molecules inhibited by triptolide have been viewed as its possible targets. However, the anticancer action mechanisms of triptolide remains to be further investigated. Here we used human ovarian SKOV-3 cancer cells as a model to probe the effect of triptolide on HIF-1α.

Results: Triptolide was observed to inhibit the proliferation of SKOV-3 cells, and meanwhile, to enhance the accumulation of HIF-1α protein in SKOV-3, A549 and DU145 cells under different conditions. Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells. However, triptolide was found to increase the levels of HIF-1α mRNA. Unexpectedly, the HIF-1α protein induced by triptolide appeared to lose its transcriptional activity, as evidenced by the decreased mRNA levels of its target genes including VEGF, BNIP3 and CAIX. The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene. Moreover, the silencing of HIF-1α partially prevented the cytotoxicity and apoptosis triggered by triptolide.

Conclusions: The potent induction of HIF-1α protein involved in its cytotoxicity, together with the suppression of HIF-1 transcriptional activity, indicates the great therapeutic potential of triptolide as an anticancer drug. Meanwhile, our data further stress the possibility that HIF-1α functions in an unresolved nature or condition.

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Triptolide decreased the transcription activity of HIF-1α protein. A. SKOV-3 cells were cultured under normoxic or hypoxic conditions in the presence or absence of triptolide for 12 h. The mRNA levels of VEGF, BNIP3 and CAIX were analyzed by real-time PCR and normalized with β-actin mRNA expression. B. ELISA assays were done for VEGF secretion from the SKOV-3 cells treated as in A. C. Triptolide inhibited new microvessel outgrowth arising from rat aorta rings. The representative images were from three separate experiments with similar results. D. MCF-7 cells were transiently transfected with the HRE-luciferase and renilla-luciferase reporter plasmids and then cultured at normoxia or hypoxia in the presence or absence of triptolide for 12 h followed by assays for luciferase activity. Data shown in A, B and D were expressed as mean ± SD, n = 3. The significant difference between triptolide-treated groups and hypoxia-control groups was analyzed by Student t test. * P < 0.05.
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Figure 4: Triptolide decreased the transcription activity of HIF-1α protein. A. SKOV-3 cells were cultured under normoxic or hypoxic conditions in the presence or absence of triptolide for 12 h. The mRNA levels of VEGF, BNIP3 and CAIX were analyzed by real-time PCR and normalized with β-actin mRNA expression. B. ELISA assays were done for VEGF secretion from the SKOV-3 cells treated as in A. C. Triptolide inhibited new microvessel outgrowth arising from rat aorta rings. The representative images were from three separate experiments with similar results. D. MCF-7 cells were transiently transfected with the HRE-luciferase and renilla-luciferase reporter plasmids and then cultured at normoxia or hypoxia in the presence or absence of triptolide for 12 h followed by assays for luciferase activity. Data shown in A, B and D were expressed as mean ± SD, n = 3. The significant difference between triptolide-treated groups and hypoxia-control groups was analyzed by Student t test. * P < 0.05.

Mentions: HIF-1α protein functions as a critical transcription factor in adaptive response to hypoxia [1]. To determine whether triptolide also increases the transcriptional activity of HIF-1α protein when enhancing its accumulation, we examined the expression levels of its several target genes including vascular endothelial growth factor (VEGF), BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and carbonic anhydrase IX (CAIX) [30,31] in the triptolide-treated SKOV-3 cells. Unexpectedly, however, the results revealed that the mRNA levels of the three genes did not increase but decreased typically in a concentration-dependent manner (Fig. 4A). Moreover, the secretion of VEGF protein, a critical angiogenesis factor, also reduced (Fig. 4B). Triptolide was further revealed to obviously inhibit the sprout outgrowth from the rat aorta rings (Fig. 4C), indicating its antiangiogenesis capability as previously reported [10,11,21].


Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide.

Zhou ZL, Luo ZG, Yu B, Jiang Y, Chen Y, Feng JM, Dai M, Tong LJ, Li Z, Li YC, Ding J, Miao ZH - Mol. Cancer (2010)

Triptolide decreased the transcription activity of HIF-1α protein. A. SKOV-3 cells were cultured under normoxic or hypoxic conditions in the presence or absence of triptolide for 12 h. The mRNA levels of VEGF, BNIP3 and CAIX were analyzed by real-time PCR and normalized with β-actin mRNA expression. B. ELISA assays were done for VEGF secretion from the SKOV-3 cells treated as in A. C. Triptolide inhibited new microvessel outgrowth arising from rat aorta rings. The representative images were from three separate experiments with similar results. D. MCF-7 cells were transiently transfected with the HRE-luciferase and renilla-luciferase reporter plasmids and then cultured at normoxia or hypoxia in the presence or absence of triptolide for 12 h followed by assays for luciferase activity. Data shown in A, B and D were expressed as mean ± SD, n = 3. The significant difference between triptolide-treated groups and hypoxia-control groups was analyzed by Student t test. * P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2958983&req=5

Figure 4: Triptolide decreased the transcription activity of HIF-1α protein. A. SKOV-3 cells were cultured under normoxic or hypoxic conditions in the presence or absence of triptolide for 12 h. The mRNA levels of VEGF, BNIP3 and CAIX were analyzed by real-time PCR and normalized with β-actin mRNA expression. B. ELISA assays were done for VEGF secretion from the SKOV-3 cells treated as in A. C. Triptolide inhibited new microvessel outgrowth arising from rat aorta rings. The representative images were from three separate experiments with similar results. D. MCF-7 cells were transiently transfected with the HRE-luciferase and renilla-luciferase reporter plasmids and then cultured at normoxia or hypoxia in the presence or absence of triptolide for 12 h followed by assays for luciferase activity. Data shown in A, B and D were expressed as mean ± SD, n = 3. The significant difference between triptolide-treated groups and hypoxia-control groups was analyzed by Student t test. * P < 0.05.
Mentions: HIF-1α protein functions as a critical transcription factor in adaptive response to hypoxia [1]. To determine whether triptolide also increases the transcriptional activity of HIF-1α protein when enhancing its accumulation, we examined the expression levels of its several target genes including vascular endothelial growth factor (VEGF), BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and carbonic anhydrase IX (CAIX) [30,31] in the triptolide-treated SKOV-3 cells. Unexpectedly, however, the results revealed that the mRNA levels of the three genes did not increase but decreased typically in a concentration-dependent manner (Fig. 4A). Moreover, the secretion of VEGF protein, a critical angiogenesis factor, also reduced (Fig. 4B). Triptolide was further revealed to obviously inhibit the sprout outgrowth from the rat aorta rings (Fig. 4C), indicating its antiangiogenesis capability as previously reported [10,11,21].

Bottom Line: Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells.However, triptolide was found to increase the levels of HIF-1α mRNA.The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Background: Hypoxia-inducible factor-1α (HIF-1α), a critical transcription factor to reduced O2 availability, has been demonstrated to be extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis. Thus it has been considered as a potential anticancer target. Triptolide is the main principle responsible for the biological activities of the Traditional Chinese Medicine tripterygium wilfordii Hook F. Triptolide possesses great chemotherapy potential for cancer with its broad-spectrum anticancer, antiangiogenesis, and drug-resistance circumvention activities. Numerous biological molecules inhibited by triptolide have been viewed as its possible targets. However, the anticancer action mechanisms of triptolide remains to be further investigated. Here we used human ovarian SKOV-3 cancer cells as a model to probe the effect of triptolide on HIF-1α.

Results: Triptolide was observed to inhibit the proliferation of SKOV-3 cells, and meanwhile, to enhance the accumulation of HIF-1α protein in SKOV-3, A549 and DU145 cells under different conditions. Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells. However, triptolide was found to increase the levels of HIF-1α mRNA. Unexpectedly, the HIF-1α protein induced by triptolide appeared to lose its transcriptional activity, as evidenced by the decreased mRNA levels of its target genes including VEGF, BNIP3 and CAIX. The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene. Moreover, the silencing of HIF-1α partially prevented the cytotoxicity and apoptosis triggered by triptolide.

Conclusions: The potent induction of HIF-1α protein involved in its cytotoxicity, together with the suppression of HIF-1 transcriptional activity, indicates the great therapeutic potential of triptolide as an anticancer drug. Meanwhile, our data further stress the possibility that HIF-1α functions in an unresolved nature or condition.

Show MeSH
Related in: MedlinePlus