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. 2020 Sep 18:11:576547.
doi: 10.3389/fphar.2020.576547. eCollection 2020.

Repurposing Ziyuglycoside II Against Colorectal Cancer via Orchestrating Apoptosis and Autophagy

Affiliations

Repurposing Ziyuglycoside II Against Colorectal Cancer via Orchestrating Apoptosis and Autophagy

Can Bai et al. Front Pharmacol. .

Abstract

Effective chemotherapy drugs for colorectal cancer remain a challenge. In this research, Ziyuglycoside II (Ziyu II), exhibits considerable antitumor activity against CRC cells both in vitro and in vivo. The results showed that Ziyu II induced apoptosis through the accumulation of reactive oxygen species (ROS), which was necessary for Ziyu II to inhibit colorectal cancer cells. Intriguingly, The treatment of Ziyu II triggered complete autophagic flux in CRC cells. Inhibition of autophagy partially reversed Ziyu II-induced growth inhibition, demonstrating a cytotoxic role of autophagy in response to Ziyu II-treated. Mechanism indicated that Ziyu II-induced autophagy by inhibiting Akt/mTOR pathway. Akt reactivation partially reduced Ziyu II-induced LC3-II turnover and LC3 puncta accumulation. Especially, Ziyu II improves the sensitivity of 5-fluorouracil which is the first-line chemotherapy drug in colorectal cancer cells. This research provides novel insight into the molecular mechanism of Ziyu II's anti-proliferation, including apoptosis and autophagy, and lays a foundation for the potential application of Ziyu II in clinical CRC treatment.

Keywords: Akt; Ziyuglycoside II; autophagy; cancer therapy; colorectal cancer.

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Figures

Figure 1
Figure 1
Ziyuglycoside II inhibits colorectal cancer cells proliferation both in vitro and in vivo. (A). Chemical structure of Ziyu II. MTT assay of colorectal cancer lines incubated with the designated concentrations of Ziyu II for 24 h (B). EdU incorporation (C), LDH release assay in cells incubated as in (F). Colony formation assay of DLD-1 and HCT116 cells intervented with the designated concentrations of Ziyu II for 2 weeks. Characteristic images (D) and quantification of colonies (E) were shown. G–J, NOD-SCID mice were injected with DLD-1 cells and intervented with Ziyu II or vehicle. Tumor volumes were measured at designated time points (I). Photograph of dissected tumors derived from control or Ziyu II-treated mice (G). Tumor weights at time of sacrifice (H).The expression of Ki67 was detected by IHC in tumor xenografts (J), and relative immunohistochemical scores were shown (K) Scale bar, 50 μm. All data are means ± SD. **p < 0.01; ***p < 0.001.
Figure 2
Figure 2
Ziyuglycoside II induces apoptosis in colorectal cancer cells in vitro. DLD-1 and HCT116 cells were subjected to Ziyu II Dox 10uM for 24 h, and flow cytometric analysis of apoptosis (A). (B) MTT assay of DLD-1 and HCT116 cells indicated with the presence or absence of Ziyu II, and in combination with or without ZVAD for 24 h. Colony formation assay of DLD-1 and HCT116 cells indicated with the presence or absence of Ziyu II, and in combination with or without ZVAD for 2 weeks. The characteristic images (C) and count of colonies (D) were shown. (E) Flow cytometric analysis of ROS in DLD-1 and HCT116 cells treated with the indicated concentration of Ziyu II for 24 h. (F). MTT assay of DLD-1 and HCT116 cells indicated with NAC for 24 h. All data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 3
Figure 3
Ziyuglycoside II induces autophagy in colorectal cancer cells both in vitro and in vivo. (A) The protein of LC3, Atg7, and ATG5 of DLD-1 and HCT116 cells were detected by Western blot after 24 h of treatment with Ziyu II. (B) Immunoblotting of LC3 in DLD-1 and HCT116 cells indicated the presence or absence of Ziyu II, and in combination with or without 3 MA for 24 h (C) Formation of endogenous LC3 puncta in cells exposed to DMSO or Ziyu II for 24 h and (E) total number of endogenous LC3 puncta per cell. (D), the formation of exogenous GFP-LC3 puncta in cells treated with DMSO or Ziyu II for 24 h and (F) the total number of exogenous LC3 puncta per cell. Scale bars: 10 μm. LC3 expression in orthotopic xenografts was examined by IHC. Representative images were provided as indicated (G), and relative immunohistochemical scores were shown (H). Scale bar, 50 μm. All data are means ± SD. **p < 0.001.
Figure 4
Figure 4
Ziyuglycoside II promotes autophagy flux in colorectal cancer cells. (A) Immunoblotting of LC3 in cells treated with or without the Ziyu II for 24 h. (B, C) The colocalization of endogenous LC3 with LAMP1 was detected by immunofluorescent analysis after treatment of Ziyu II and CQ (10 mM) for 24 h, respectively. (D–F) DLD-1 and HCT116 cells were transiently transfected with an RFP-GFP tandem fluorescent-tagged LC3 (RFP-GFP-LC3) and indicated with Ziyu II and 10 mmol/L chloroquine (CQ) for 24 h, respectively. The formation of autophagosome (RFP-positive; GFP-positive) and autophagolysosome (RFP-positive; GFP-negative) were detected and quantified by ImageJ. Scale bars: 10 μm. All data are means ± SD. **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
Ziyuglycoside II induces autophagy by inhibiting the Akt/mTOR pathway in colorectal cancer cells. (A). Immunoblotting of phosphorylation of Akt (S473), mTOR (S2448), p70S6K (S424/T421), and 4EBP1 (S65/T70) in cells indicated with the designed concentrations of Ziyu II for 24 h. (B). The empty vector (pECE) or with a constitutively active CA-Akt transfected into the DLD-1 and HCT116 cells for 48 h and then indicated with Ziyu II for another 24 h. The Western blot detected the Akt phosphorylation and LC3 lipidation (C). The formation of endogenous LC3 puncta was evaluated in cells treated as in (D, F), the total number of endogenous LC3 puncta per cell (E, G). Scale bars, 10 μm. All data are means ± SD. ***p < 0.001. Akt expression in orthotopic xenografts was examined by IHC. The charecteristic images were provided as shown (H), and relative immunohistochemical scores were shown (I). Scale bar, 50 μm. ***p < 0.001.
Figure 6
Figure 6
The antiproliferation of the Ziyuglycoside II is mediated through the inhibition of autophagy in the colorectal cancer cells. (A). MTT assay of DLD-1 and HCT116 cells were indicated with or without CQ in the presence or absence of Ziyu II. The characteristic images (B) and the quantification of colonies (C) were shown. (D) LDH release assay in cells treated as in (A). (E) The Western blot detected the Akt phosphorylation. (F) After transfecting an empty vector (pECE) or with a constitutively active CA-Akt for 48 h, DLD-1 and HCT116 cells were used for MTT analysis and the colony formation assay in indicated with Ziyu II. Characteristic images (G) and quantification of colonies (H) were shown. All data are means ± SD. *p < 0.05 **p < 0.01.

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