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Verbascoside promotes apoptosis by regulating HIPK2-p53 signaling in human colorectal cancer.

Zhou L, Feng Y, Jin Y, Liu X, Sui H, Chai N, Chen X, Liu N, Ji Q, Wang Y, Li Q - BMC Cancer (2014)

Bottom Line: In vitro effects of VB on CRC cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry; HIPK2, p53, p-p53, Bax, and Bcl-2 were measured by western blot.In summary, VB treatment significantly enhanced the protein expression of pro-apoptotic HIPK2, p53, p-p53, Bax, and decreased anti-apoptotic Bcl-2 expression in CRC cells.We also found that VB effectively activated the HIPK2-p53 signaling pathway, resulting in increased CRC cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. lzwf@hotmail.com.

ABSTRACT

Background: We investigated the role of the HIPK2-p53 signaling pathway in tumorigenesis and resistance to the drug Verbascoside (VB) in colorectal cancer (CRC), using in vivo and in vitro experiments.

Methods: Primary human CRC samples and normal intestinal tissues from patients were analyzed for HIPK2 expression by immunohistochemistry (IHC) and its expression was correlated against patients' clinicopathological characteristics. Human CRC HCT-116 cells were implanted in BALB/c nude mice; mice with xenografted tumors were randomly administrated vehicle (control), 20, 40, or 80 mg/mL VB, or 1 mg/mL fluorouracil (5-FU). HIPK2, p53, Bax, and Bcl-2 expression in these tumors were determined by IHC. In vitro effects of VB on CRC cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry; HIPK2, p53, p-p53, Bax, and Bcl-2 were measured by western blot.

Results: IHC analysis for 100 human CRC tumor samples and 20 normal intestinal tissues, showed HIPK2 expression to inversely correlate with Dukes stage and depth of invasion in CRC (P<0.05). In vivo, the inhibition rates of 20, 40, and 80 mg/mL VB on CRC xenograft tumor weight were 42.79%, 53.90%, and 60.99%, respectively, and were accompanied by increased expression of HIPK2, p53, and Bax, and decreased Bcl-2 expression in treated tumors. In vitro, VB significantly inhibited proliferation of CRC cell lines HCT-116, HT-29, LoVo, and SW620, in a time- and dose-dependent manner. The apoptosis rates of 25, 50, and 100 μM VB on HCT-116 cells were 10.83±1.28, 11.25±1.54, and 20.19±2.87%, and on HT-29 cells were 18.92±6.12, 21.57±4.05, and 25.14±6.73%, respectively. In summary, VB treatment significantly enhanced the protein expression of pro-apoptotic HIPK2, p53, p-p53, Bax, and decreased anti-apoptotic Bcl-2 expression in CRC cells.

Conclusions: HIPK2 protein modulates the phosphorylation status of p53, and levels of Bax and Bcl-2 in CRC. We also found that VB effectively activated the HIPK2-p53 signaling pathway, resulting in increased CRC cell apoptosis.

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Verbascoside (VB) promoted apoptosis via p53 in human CRC cells. CRC HCT-116 and HT-29 cells were treated by VB at indicated doses and duration, and then analyzed for apoptosis by flow cytometry. Inhibition at 25, 50, and 100 μM of VB to HCT-116 cells was 20.20 ± 4.08%, 43.28 ± 9.80%, and 56.79 ± 9.11% (A), and HT-29 cells, 4.36 ± 3.39%, 18.22 ± 3.94%, and 37.01 ± 6.98%, respectively (B). HCT-116 apoptosis rate after being treated with 25, 50, and 100 μM of VB was 10.83 ± 1.28%, 11.25 ± 1.54%, and 20.19 ± 2.8%, and the HT-29 apoptosis rate, 18.92 ± 6.12%, 21.57 ± 4.05%, and 25.14 ± 6.73%, respectively (C). HCT-116 and HT-29 apoptosis rate was 11.25 ± 1.54% and 21.57 ± 4.05% after being treated with 50 μM Verbascoside; and 5.03 ± 2.77% and 3.11 ± 1.16% after being treated with FPT-a (p53-specific inhibitor); and 5.02 ± 0.73% and 3.18 ± 1.82% after being treated with FPT-a and 50 μM VB respectively (D).
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Fig5: Verbascoside (VB) promoted apoptosis via p53 in human CRC cells. CRC HCT-116 and HT-29 cells were treated by VB at indicated doses and duration, and then analyzed for apoptosis by flow cytometry. Inhibition at 25, 50, and 100 μM of VB to HCT-116 cells was 20.20 ± 4.08%, 43.28 ± 9.80%, and 56.79 ± 9.11% (A), and HT-29 cells, 4.36 ± 3.39%, 18.22 ± 3.94%, and 37.01 ± 6.98%, respectively (B). HCT-116 apoptosis rate after being treated with 25, 50, and 100 μM of VB was 10.83 ± 1.28%, 11.25 ± 1.54%, and 20.19 ± 2.8%, and the HT-29 apoptosis rate, 18.92 ± 6.12%, 21.57 ± 4.05%, and 25.14 ± 6.73%, respectively (C). HCT-116 and HT-29 apoptosis rate was 11.25 ± 1.54% and 21.57 ± 4.05% after being treated with 50 μM Verbascoside; and 5.03 ± 2.77% and 3.11 ± 1.16% after being treated with FPT-a (p53-specific inhibitor); and 5.02 ± 0.73% and 3.18 ± 1.82% after being treated with FPT-a and 50 μM VB respectively (D).

Mentions: Based on the cell proliferation inhibition data, we selected 48-htreatment of CRC HCT-116 and HT-29 as the optimal time frame for apoptosis experiments. We used drug doses of 25, 50, and 100 μM of VB to treat cells for 48 h (Figure 5A, B), and used FITC Annexin-V/PI method to measure apoptosis induced by VB. Our data showed the apoptosis rate to be significantly increased by VB in a dose-dependent manner (Figure 5C). Interestingly, this pro-apoptotic effect by VB was countered by a p53-specific inhibitor, FPT-a (Figure 5D). This suggests that VB promotes apoptosis in CRC cells through ap53-dependent mechanism.Figure 5


Verbascoside promotes apoptosis by regulating HIPK2-p53 signaling in human colorectal cancer.

Zhou L, Feng Y, Jin Y, Liu X, Sui H, Chai N, Chen X, Liu N, Ji Q, Wang Y, Li Q - BMC Cancer (2014)

Verbascoside (VB) promoted apoptosis via p53 in human CRC cells. CRC HCT-116 and HT-29 cells were treated by VB at indicated doses and duration, and then analyzed for apoptosis by flow cytometry. Inhibition at 25, 50, and 100 μM of VB to HCT-116 cells was 20.20 ± 4.08%, 43.28 ± 9.80%, and 56.79 ± 9.11% (A), and HT-29 cells, 4.36 ± 3.39%, 18.22 ± 3.94%, and 37.01 ± 6.98%, respectively (B). HCT-116 apoptosis rate after being treated with 25, 50, and 100 μM of VB was 10.83 ± 1.28%, 11.25 ± 1.54%, and 20.19 ± 2.8%, and the HT-29 apoptosis rate, 18.92 ± 6.12%, 21.57 ± 4.05%, and 25.14 ± 6.73%, respectively (C). HCT-116 and HT-29 apoptosis rate was 11.25 ± 1.54% and 21.57 ± 4.05% after being treated with 50 μM Verbascoside; and 5.03 ± 2.77% and 3.11 ± 1.16% after being treated with FPT-a (p53-specific inhibitor); and 5.02 ± 0.73% and 3.18 ± 1.82% after being treated with FPT-a and 50 μM VB respectively (D).
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Fig5: Verbascoside (VB) promoted apoptosis via p53 in human CRC cells. CRC HCT-116 and HT-29 cells were treated by VB at indicated doses and duration, and then analyzed for apoptosis by flow cytometry. Inhibition at 25, 50, and 100 μM of VB to HCT-116 cells was 20.20 ± 4.08%, 43.28 ± 9.80%, and 56.79 ± 9.11% (A), and HT-29 cells, 4.36 ± 3.39%, 18.22 ± 3.94%, and 37.01 ± 6.98%, respectively (B). HCT-116 apoptosis rate after being treated with 25, 50, and 100 μM of VB was 10.83 ± 1.28%, 11.25 ± 1.54%, and 20.19 ± 2.8%, and the HT-29 apoptosis rate, 18.92 ± 6.12%, 21.57 ± 4.05%, and 25.14 ± 6.73%, respectively (C). HCT-116 and HT-29 apoptosis rate was 11.25 ± 1.54% and 21.57 ± 4.05% after being treated with 50 μM Verbascoside; and 5.03 ± 2.77% and 3.11 ± 1.16% after being treated with FPT-a (p53-specific inhibitor); and 5.02 ± 0.73% and 3.18 ± 1.82% after being treated with FPT-a and 50 μM VB respectively (D).
Mentions: Based on the cell proliferation inhibition data, we selected 48-htreatment of CRC HCT-116 and HT-29 as the optimal time frame for apoptosis experiments. We used drug doses of 25, 50, and 100 μM of VB to treat cells for 48 h (Figure 5A, B), and used FITC Annexin-V/PI method to measure apoptosis induced by VB. Our data showed the apoptosis rate to be significantly increased by VB in a dose-dependent manner (Figure 5C). Interestingly, this pro-apoptotic effect by VB was countered by a p53-specific inhibitor, FPT-a (Figure 5D). This suggests that VB promotes apoptosis in CRC cells through ap53-dependent mechanism.Figure 5

Bottom Line: In vitro effects of VB on CRC cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry; HIPK2, p53, p-p53, Bax, and Bcl-2 were measured by western blot.In summary, VB treatment significantly enhanced the protein expression of pro-apoptotic HIPK2, p53, p-p53, Bax, and decreased anti-apoptotic Bcl-2 expression in CRC cells.We also found that VB effectively activated the HIPK2-p53 signaling pathway, resulting in increased CRC cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. lzwf@hotmail.com.

ABSTRACT

Background: We investigated the role of the HIPK2-p53 signaling pathway in tumorigenesis and resistance to the drug Verbascoside (VB) in colorectal cancer (CRC), using in vivo and in vitro experiments.

Methods: Primary human CRC samples and normal intestinal tissues from patients were analyzed for HIPK2 expression by immunohistochemistry (IHC) and its expression was correlated against patients' clinicopathological characteristics. Human CRC HCT-116 cells were implanted in BALB/c nude mice; mice with xenografted tumors were randomly administrated vehicle (control), 20, 40, or 80 mg/mL VB, or 1 mg/mL fluorouracil (5-FU). HIPK2, p53, Bax, and Bcl-2 expression in these tumors were determined by IHC. In vitro effects of VB on CRC cell proliferation and apoptosis were measured by CCK-8 assay and flow cytometry; HIPK2, p53, p-p53, Bax, and Bcl-2 were measured by western blot.

Results: IHC analysis for 100 human CRC tumor samples and 20 normal intestinal tissues, showed HIPK2 expression to inversely correlate with Dukes stage and depth of invasion in CRC (P<0.05). In vivo, the inhibition rates of 20, 40, and 80 mg/mL VB on CRC xenograft tumor weight were 42.79%, 53.90%, and 60.99%, respectively, and were accompanied by increased expression of HIPK2, p53, and Bax, and decreased Bcl-2 expression in treated tumors. In vitro, VB significantly inhibited proliferation of CRC cell lines HCT-116, HT-29, LoVo, and SW620, in a time- and dose-dependent manner. The apoptosis rates of 25, 50, and 100 μM VB on HCT-116 cells were 10.83±1.28, 11.25±1.54, and 20.19±2.87%, and on HT-29 cells were 18.92±6.12, 21.57±4.05, and 25.14±6.73%, respectively. In summary, VB treatment significantly enhanced the protein expression of pro-apoptotic HIPK2, p53, p-p53, Bax, and decreased anti-apoptotic Bcl-2 expression in CRC cells.

Conclusions: HIPK2 protein modulates the phosphorylation status of p53, and levels of Bax and Bcl-2 in CRC. We also found that VB effectively activated the HIPK2-p53 signaling pathway, resulting in increased CRC cell apoptosis.

Show MeSH
Related in: MedlinePlus