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. 2019 May 24:12:4109-4118.
doi: 10.2147/OTT.S195615. eCollection 2019.

Grape seed procyanidin B2 promotes the autophagy and apoptosis in colorectal cancer cells via regulating PI3K/Akt signaling pathway

Ruijuan Zhang  1 , Qianyun Yu  2 , Wenqiang Lu  1 , Jun Shen  1 , Dongqing Zhou  1 , Yingjue Wang  1 , Shurong Gao  1 , Zhijun Wang  1
Affiliations

Grape seed procyanidin B2 promotes the autophagy and apoptosis in colorectal cancer cells via regulating PI3K/Akt signaling pathway

Ruijuan Zhang et al. Onco Targets Ther. .

Erratum in

Abstract

Aim: Colorectal cancer (CRC) is a major malignancy in China, which is the critical risk of people health. Many natural herbs extracts have been found to exhibit good therapeutic effect on CRC. Our previous study found that grape seed procyanidins B2 (PB2) would induce CRC cell death. However, the molecular mechanism underlying its anti-tumor effect on CRC remains unclear. Thereby, this study aimed to investigate the anti-tumor mechanism of PB2 on CRC. Methods: CCK-8, western blotting, flow cytometry, qRT-PCR and animal study were used in the current study. Results: The in vitro and in vivo data demonstrated that PB2 could promote the apoptosis of CRC cells in a dose-dependent manner, which was significantly reversed by caspase 3 inhibitor. Meanwhile, PB2 dose-dependently induced autophagy in CRC cells, which was markedly attenuated by autophagy inhibitor 3-MA. In addition, PB2 dose-dependently inhibited the expressions of p-PI3K, p-Akt and p-mTOR in the cells. Conclusion: PB2 dose-dependently induced apoptosis and autophagy in CRC cells via downregulation of PI3K/Akt pathway. This study provided the experimental basis for further development of PB2 as a new effective anticancer drug for the patients with CRC.

Keywords: PI3K/Akt/mTOR signaling pathway; apoptosis; autophagy; colorectal cancer; grape seed procyanidin extract.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Inhibitory effect of PB2 on the proliferation of CRC cells. (A) Inhibitory effects of PB2 on the proliferation of LoVo and HT29 cells were determined by CCK-8 assay. LoVo or HT29 cells were treated with PB2 (6, 12 or 24 μM) for 12, 24 and 48 hrs, respectively. Then, CCK-8 assay was performed to investigate the growth inhibitory effect of PB2 on cells. (B) Morphological observation of LoVo or HT29 cells after treating with PB2 (6, 12 or 24 μM) for 12, 24 and 48 hrs, respectively.
Figure 2
Figure 2
PB2 induced autophagy in CRC cells. (A, B) LoVo cells were treated with 6, 12 or 24 μM PB2 for 48 hrs. Then, the formation of autophagosomes was observed by TEM and acridine orange (AO, Amresco, Inc) staining. (C) LoVo cells transfected by ptfLC3 expressing plasmid were treated with 6, 12 or 24 μM PB2 for 48 hrs. Then, GFP-LC3 expression was observed by fluorescence microscopy. (D) LoVo cells were treated with 6, 12 or 24 μM PB2 for 48 hrs. Then, western blot was performed to detect the effect of PB2 on the autophagy-associated proteins including Beclin1, Atg5, LC3 I and LC3 II. Abbreviation: TEM, Transmission Electron Microscope.
Figure 3
Figure 3
PB2 induced apoptosis in CRC cells. (A, B) Apoptosis analysis by flow cytometry in LoVo cells treated with 6, 12 or 24 μM PB2 for 48 hrs. **P<0.01, vs Ctrl. (C) Western blot analysis was used to measure the Bax, Bcl-2 and Cleaved Caspase-3 in LoVo cells treated with 6, 12 or 24 μM PB2 for 48 hrs.
Figure 4
Figure 4
PB2-induced apoptosis in CRC cells was reversed by autophagy inhibitor 3-MA. (A) LoVo cells were treated as follow: Control, 24 μM PB2, 3-MA, 3-MA+PB2 for 48 hrs. Then, CCK-8 assay was performed to investigate the survival of LoVo cells. **P<0.01, vs Ctrl. #P<0.05, vs PB2. (B) Western blot analysis was used to measure the protein expressions of Beclin1, Atg5, LC3 I, LC3 II, Bax, Bcl-2 and Cleaved Caspase-3. (C, D) Apoptosis in LoVo cells treated as PB2 or/and 3-MA was detected by flow cytometry. **P<0.01, vs Ctrl. ##P<0.01, #P<0.01, vs PB2.
Figure 5
Figure 5
PB2 induced autophagy and apoptosis in CRC cells via regulation of PI3K/Akt signaling pathway. (A) LoVo cells were treated as follow: Control, PB2-L, PB2-M, PB2-H, LY294002+PB2-H, LY294002 for 48 hrs. Then, western blot analysis was used to measure the protein expressions of p-PI3K, p-Akt, p-mTOR, PI3K, Akt and mTOR in cells. (B) LoVo cells were treated as follow: Control, PB2-H, LY294002+PB2-H, LY294002 for 48 hrs. Then, western blot analysis was used to measure the protein expressions of Beclin1, Atg5, LC3 I, LC3 II, Bax, Bcl-2 and Cleaved Caspase-3 in cells. (C) LoVo cells were treated as follow: Control, PB2-H, LY294002+PB2-H, LY294002 for 48 hrs. Then, CCK-8 assay was performed to investigate the survival of LoVo cells. *P<0.05, **P<0.01, vs Ctrl. #P<0.05, vs PB2. (D, E) Apoptosis in LoVo cells treated as PB2 or/and LY294002 was detected by flow cytometry. *P<0.05, **P<0.01, vs Ctrl. #P<0.05, ##P<0.01, vs PB2. Abbreviations: CRC, colorectal cancer; 5-Fu, 5-fluorouracil.
Figure 6
Figure 6
Anti-tumor effect of PB2 on the colorectal cancer (CRC) in vivo. (A) LoVo cells were used to establish the model of orthotopic transplantation tumor. Two weeks later, the mice were orally administrated PB2 with different doses and 5-FU every day. After 28 days treatment, the nude mice were sacrificed. The primary tumors were isolated and photographed. (B) The volume of the tumors was measured every 7 days. (C) The tumor weight from different groups was calculated lastly. (D) Western blot was performed to detect the effect of PB2 on proteins expressions including Beclin1 and Atg5, Bax, Bcl-2 and p-Akt. PB2-L: PB2 low-dose (25 mg/kg/day); PB2-M: PB2 middle-dose (50 mg/kg/day); PB2-H: PB2 high-dose (100 mg/kg/day). *P<0.05 and **P<0.01, vs control group.
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