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Triptolide Inhibits Invasion and Tumorigenesis of Hepatocellular Carcinoma MHCC-97H Cells Through NF-κB Signaling.

Wang H, Ma D, Wang C, Zhao S, Liu C - Med. Sci. Monit. (2016)

Bottom Line: To detect the effect of NF-κB on TPL-induced signal pathways, MHCC-97H cells were transfected with p65 siRNA or p65 cDNA, then treated with TPL.RESULTS We showed that TPL treatment significantly suppressed growth and induced apoptosis of MHCC-97H cells in a dose- and time-dependent manner in vitro.Overexpression of p65 restored MMP-9 activity and inhibited the TPL anti-tumor effect on MHCC-97H cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, The Affiliated Hospital of Qingdao University, Qingdao, Shangdong, China (mainland).

ABSTRACT
BACKGROUND We investigated whether the plant-derived agent triptolide (TPL) could effectively inhibit the growth and invasion of human hepatocellular carcinoma (HCC) cells. MATERIAL AND METHODS MHCC-97H cells were treated with various concentration of TPL for various times. To detect the effect of NF-κB on TPL-induced signal pathways, MHCC-97H cells were transfected with p65 siRNA or p65 cDNA, then treated with TPL. We detected cell survival and apoptosis by MTT, soft-agar colony formation assay, flow cytometry, and TUNEL assay. Cell migration and invasion was determined by Matrigel invasion and a wound-healing assay. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA); MMP-9 activity was detected by ELISA. Western blot and real-time PCR (RT-PCR) assays were used to detect p65 and MMP-9 protein and mRNA expression. A subcutaneously implanted tumor model of MHCC-97H cells in nude mice was used to assess the effects of TPL on tumorigenesis in vivo. RESULTS We showed that TPL treatment significantly suppressed growth and induced apoptosis of MHCC-97H cells in a dose- and time-dependent manner in vitro. Furthermore, TPL treatment inhibited invasion in vitro and inhibited the growth and lung metastasis of MHCC-97H cells in vivo. NF-κB and MMP-9 were inactivated with TPL treatment. Overexpression of p65 restored MMP-9 activity and inhibited the TPL anti-tumor effect on MHCC-97H cells. Knockdown of p65 blocked MMP-9 activation and enhanced TPL-induced cell apoptosis and survival inhibition, and TPL inhibition of migration and invasion in vitro. CONCLUSIONS TPL treatment inhibited MHCC-97H cell growth, invasion, and metastasis in vitro and vivo, suggesting that TPL could be developed as a potential therapeutic agent for the treatment of HCC.

No MeSH data available.


Related in: MedlinePlus

Effect of TPL on NF-κB activity and NF-κB p65 expression inMHCC-97H cell. (A) MHCC-97H cells were treated with 5, 15,or 25 μM TPL for 72 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. (B) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. (C) MHCC-97H cells were treated with 5, 15, or 25 μM TPL for 72 hours. NF-κB p65 protein was detected by western blot assay; (D) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. NF-κB p65 protein was detected by western blot assay.
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f2-medscimonit-22-1827: Effect of TPL on NF-κB activity and NF-κB p65 expression inMHCC-97H cell. (A) MHCC-97H cells were treated with 5, 15,or 25 μM TPL for 72 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. (B) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. (C) MHCC-97H cells were treated with 5, 15, or 25 μM TPL for 72 hours. NF-κB p65 protein was detected by western blot assay; (D) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. NF-κB p65 protein was detected by western blot assay.

Mentions: Since NF-κB has been shown to be involved in apoptosis and cell survival, we wanted to see whether TPL-induced apoptosis in MHCC-97H is mediated by modulation of the NF-κB pathway. In order to reveal the molecular mechanism of TPL, we first determined the concentration-dependent effect of TPL on NF-κB activity and phosphorylation of p65 in MHCC-97H cells. MHCC-97H cells treated with 5, 15, or 25 μM TPL for 72 hours resulted in a significant dose-dependent decrease in NF-κB activity. NF-κB DNA binding was measured by EMSA (Figure 2A) and phosphorylation of p65 by western blot assay (Figure 2C). Similarly, when treated with 25 μM TPL for 24–72 hours, we observed a significant time-dependent decrease in NF-κB activity and phosphorylation of p65 measured by EMSA (Figure 2B) and western blot assay (Figure 2D).


Triptolide Inhibits Invasion and Tumorigenesis of Hepatocellular Carcinoma MHCC-97H Cells Through NF-κB Signaling.

Wang H, Ma D, Wang C, Zhao S, Liu C - Med. Sci. Monit. (2016)

Effect of TPL on NF-κB activity and NF-κB p65 expression inMHCC-97H cell. (A) MHCC-97H cells were treated with 5, 15,or 25 μM TPL for 72 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. (B) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. (C) MHCC-97H cells were treated with 5, 15, or 25 μM TPL for 72 hours. NF-κB p65 protein was detected by western blot assay; (D) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. NF-κB p65 protein was detected by western blot assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920093&req=5

f2-medscimonit-22-1827: Effect of TPL on NF-κB activity and NF-κB p65 expression inMHCC-97H cell. (A) MHCC-97H cells were treated with 5, 15,or 25 μM TPL for 72 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. (B) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. (C) MHCC-97H cells were treated with 5, 15, or 25 μM TPL for 72 hours. NF-κB p65 protein was detected by western blot assay; (D) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. NF-κB p65 protein was detected by western blot assay.
Mentions: Since NF-κB has been shown to be involved in apoptosis and cell survival, we wanted to see whether TPL-induced apoptosis in MHCC-97H is mediated by modulation of the NF-κB pathway. In order to reveal the molecular mechanism of TPL, we first determined the concentration-dependent effect of TPL on NF-κB activity and phosphorylation of p65 in MHCC-97H cells. MHCC-97H cells treated with 5, 15, or 25 μM TPL for 72 hours resulted in a significant dose-dependent decrease in NF-κB activity. NF-κB DNA binding was measured by EMSA (Figure 2A) and phosphorylation of p65 by western blot assay (Figure 2C). Similarly, when treated with 25 μM TPL for 24–72 hours, we observed a significant time-dependent decrease in NF-κB activity and phosphorylation of p65 measured by EMSA (Figure 2B) and western blot assay (Figure 2D).

Bottom Line: To detect the effect of NF-κB on TPL-induced signal pathways, MHCC-97H cells were transfected with p65 siRNA or p65 cDNA, then treated with TPL.RESULTS We showed that TPL treatment significantly suppressed growth and induced apoptosis of MHCC-97H cells in a dose- and time-dependent manner in vitro.Overexpression of p65 restored MMP-9 activity and inhibited the TPL anti-tumor effect on MHCC-97H cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, The Affiliated Hospital of Qingdao University, Qingdao, Shangdong, China (mainland).

ABSTRACT
BACKGROUND We investigated whether the plant-derived agent triptolide (TPL) could effectively inhibit the growth and invasion of human hepatocellular carcinoma (HCC) cells. MATERIAL AND METHODS MHCC-97H cells were treated with various concentration of TPL for various times. To detect the effect of NF-κB on TPL-induced signal pathways, MHCC-97H cells were transfected with p65 siRNA or p65 cDNA, then treated with TPL. We detected cell survival and apoptosis by MTT, soft-agar colony formation assay, flow cytometry, and TUNEL assay. Cell migration and invasion was determined by Matrigel invasion and a wound-healing assay. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA); MMP-9 activity was detected by ELISA. Western blot and real-time PCR (RT-PCR) assays were used to detect p65 and MMP-9 protein and mRNA expression. A subcutaneously implanted tumor model of MHCC-97H cells in nude mice was used to assess the effects of TPL on tumorigenesis in vivo. RESULTS We showed that TPL treatment significantly suppressed growth and induced apoptosis of MHCC-97H cells in a dose- and time-dependent manner in vitro. Furthermore, TPL treatment inhibited invasion in vitro and inhibited the growth and lung metastasis of MHCC-97H cells in vivo. NF-κB and MMP-9 were inactivated with TPL treatment. Overexpression of p65 restored MMP-9 activity and inhibited the TPL anti-tumor effect on MHCC-97H cells. Knockdown of p65 blocked MMP-9 activation and enhanced TPL-induced cell apoptosis and survival inhibition, and TPL inhibition of migration and invasion in vitro. CONCLUSIONS TPL treatment inhibited MHCC-97H cell growth, invasion, and metastasis in vitro and vivo, suggesting that TPL could be developed as a potential therapeutic agent for the treatment of HCC.

No MeSH data available.


Related in: MedlinePlus