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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) alters levels of HIPK2–p53apoptosis signaling molecules in CRC cells. HCT-116 and HT-29 cells treated with VB extracts were probed for HIPK2, p53, p-p53, Bax, and Bcl-2 protein (A), and were compared with cells treated with both VB extracts and PFT-a (B).
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Fig6: Verbascoside (VB) alters levels of HIPK2–p53apoptosis signaling molecules in CRC cells. HCT-116 and HT-29 cells treated with VB extracts were probed for HIPK2, p53, p-p53, Bax, and Bcl-2 protein (A), and were compared with cells treated with both VB extracts and PFT-a (B).

Mentions: We next determined if expression levels of apoptosis-related proteins changed in VB-treated human CRC cells HCT-116 and HT-29. We found, after 48 h of treatment, VB increased protein expression of HIPK2, p53, p-p53, and Bax, but decreased that of Bcl-2, in a dose-dependent manner in the CRC cell lines (Figure 6A). These data both recapitulated the results we saw in the VB-treated CRC tumors in vivo, and further indicated that VB promotes apoptosis in CRC, probably through HIPK2–p53signaling axis. To verify this point, we added the p53-specific inhibitor PFT-a to the treated cells along with VB. The results showed that PFT-a rescued the cells from VB-induced apoptosis, by reducing VB-enhanced protein levels of p-p53 on Ser46, Bax, and restoring Bcl-2 protein expression, but did not affect HIPK2 protein levels (Figure 6B). These findings strongly suggest that VB-induced apoptosis is mediated by the HIPK2–p53signaling pathway.Figure 6


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) alters levels of HIPK2–p53apoptosis signaling molecules in CRC cells. HCT-116 and HT-29 cells treated with VB extracts were probed for HIPK2, p53, p-p53, Bax, and Bcl-2 protein (A), and were compared with cells treated with both VB extracts and PFT-a (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197337&req=5

Fig6: Verbascoside (VB) alters levels of HIPK2–p53apoptosis signaling molecules in CRC cells. HCT-116 and HT-29 cells treated with VB extracts were probed for HIPK2, p53, p-p53, Bax, and Bcl-2 protein (A), and were compared with cells treated with both VB extracts and PFT-a (B).
Mentions: We next determined if expression levels of apoptosis-related proteins changed in VB-treated human CRC cells HCT-116 and HT-29. We found, after 48 h of treatment, VB increased protein expression of HIPK2, p53, p-p53, and Bax, but decreased that of Bcl-2, in a dose-dependent manner in the CRC cell lines (Figure 6A). These data both recapitulated the results we saw in the VB-treated CRC tumors in vivo, and further indicated that VB promotes apoptosis in CRC, probably through HIPK2–p53signaling axis. To verify this point, we added the p53-specific inhibitor PFT-a to the treated cells along with VB. The results showed that PFT-a rescued the cells from VB-induced apoptosis, by reducing VB-enhanced protein levels of p-p53 on Ser46, Bax, and restoring Bcl-2 protein expression, but did not affect HIPK2 protein levels (Figure 6B). These findings strongly suggest that VB-induced apoptosis is mediated by the HIPK2–p53signaling pathway.Figure 6

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