Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in VSports app下载. gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2018 Oct 6;8(19):5200-5212.
doi: 10.7150/thno.27806. eCollection 2018.

VSports手机版 - Down-regulation of OGT promotes cisplatin resistance by inducing autophagy in ovarian cancer

Fuxing Zhou  1 , Xiaoshan Yang  2 , Hang Zhao  3 , Yu Liu  1 , Yang Feng  4 , Rui An  5 , Xiaohui Lv  1 , Jia Li  1 , Biliang Chen  1
Affiliations

Down-regulation of OGT promotes cisplatin resistance by inducing autophagy in ovarian cancer

Fuxing Zhou et al. Theranostics. .

Abstract

Cisplatin resistance significantly affects the survival rate of patients with ovarian cancer. However, the main mechanism underlying cisplatin resistance in ovarian cancer remains unclear. Methods: Immunohistochemistry was used to determine the expression of OGT, OGA and O-GlcNAc in chemoresistant and chemosensitive ovarian cancer tissues VSports手机版. Functional analyses (in vitro and in vivo) were performed to confirm the role of OGT in cisplatin resistance. Autophagy-related proteins were tested by western blot. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus reporter were used for autophagy flux analysis. Immunoprecipitation assay was utilized to detect protein-protein interactions. Results: We found that O-GlcNAc and O-GlcNAc transferase (OGT) levels were significantly lower in chemoresistant ovarian cancer tissues than in chemosensitive tissues, whereas O-GlcNAcase (OGA) levels did not differ. The down-regulation of OGT increased cisplatin resistance in ovarian cancer cells but had no effect on the efficacy of paclitaxel. The down-regulation of OGT improved tumor resistance to cisplatin in a mouse xenograft tumor model. OGT knockdown enhanced cisplatin-induced autophagy, which reduced apoptotic cell death induced by cisplatin, and promoted autolysosome formation. A reduction in O-GlcNAcylated SNAP-29 levels caused by the down-regulation of OGT promoted the formation of the SNARE complex and autophagic flux. Conclusion: Our findings suggest that down-regulation of OGT enhances cisplatin-induced autophagy via SNAP-29, resulting in cisplatin-resistant ovarian cancer. OGT may represent a novel target for overcoming cisplatin resistance in ovarian cancer. .

Keywords: OGT; SNARE V体育安卓版. ; autophagy; chemoresistance; ovarian cancer. .

PubMed Disclaimer

V体育平台登录 - Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
OGT and O-GlcNAc are decreased in chemoresistant ovarian cancer tissues. Immunohistochemical staining of OGT, O-GlcNAc and OGA were carried out on chemoresistant and chemosensitive ovarian cancer tissue sections. (A) Stained tissues are shown at 200× and 400× magnifications. Scale bar represent 50 μm. (B) H-SCORE of the two groups. **P < 0.01, *P < 0.05.
Figure 2
Figure 2
Down-regulation of OGT enhances cisplatin resistance in ovarian cancer cell lines. (A) A2780 and SKOV3 were transfected with control or OGT shRNA to establish stable OGT-deficient cell lines. Western blotting was used to test the expression of OGT in control and OGT-deficient cells. (B-C) Control and OGT-deficient cells were treated with different concentrations of cisplatin for 48 h. The cell viability of A2780 (B) and SKOV3 (C) were measured by CCK-8 after cisplatin treatment. (D) Control and OGT-deficient cells were treated with cisplatin (5 µg/mL) for 24 h. Apoptotic cells were measured by ANXA5 and PI staining. The numbers shown are the sum of ANXA5-positive and double-positive cells. (E-F) Control and OGT-deficient cells were treated with different concentrations of paclitaxel for 48 h. The cell viability of A2780 (E) and SKOV3 (F) were measured by CCK-8 after paclitaxel treatment. (G) Control and OGT-deficient cells were treated with paclitaxel (100 nM) for 24 h. Apoptotic cells were measured by ANXA5 and PI staining. The numbers shown are the sum of ANXA5-positive and double-positive cells. The values are presented as a mean ± SD (n = 3). **P < 0.01, *P < 0.05.
Figure 3
Figure 3
OGT deficiency leads to the development of cisplatin resistance in vivo. (A) SKOV3 control and OGT-deficient cells were injected in the flanks of BALB/c nude mice. Tumor volume was measured every other day. Data are shown as mean ± SEM (n = 16). (B) Twenty-three days after cell injection, mice were injected intraperitoneally with PBS or cisplatin (3 mg/kg) 3 times per week for 2 weeks. Tumor volume was measured every other day. Data are shown as mean ± SEM (n = 8). *P < 0.05 vs Con sh-PBS, OGT sh-PBS, or OGT sh-cis group. (C) Representative tumors. (D) Expression of OGT in tumors tested by IHC. Scale bar represent 50 μm.
Figure 4
Figure 4
Down-regulation of OGT enhances autophagy induced by cisplatin in ovarian cancer cells. (A-B) A2780 and SKOV3 cells were treated with or without cisplatin (5 µg/mL) for 24 h. The expression levels of LC3 and p62 were tested by western blotting. (C-D) Control and OGT-deficient ovarian cancer cells were treated with cisplatin (5 µg/mL) for 24 h. LC3 and p62 expression levels were determined by western blotting. (E) Control and OGT-deficient ovarian cancer cells were cultured with cisplatin (5 µg/mL) for 24 h. LC3 puncta were detected by anti-LC3 by fluorescence microscopy. Scale bar represent 50 μm. The values are presented as mean ± SD (n = 3). **P < 0.01, *P < 0.05.
Figure 5
Figure 5
Down-regulation of OGT promotes the formation of autolysosome induced by cisplatin. (A) OGT-deficient A2780 and SKOV3 cells were cultured in different concentrations of cisplatin with 3-MA (3 mM) or Baf (200 nM) for 48 h. Cell viability was measured by CCK-8. (B) OGT-deficient A2780 and SKOV3 cells were treated with 3-MA (3 mM) or Baf (200 nM) in the presence of cisplatin (5 µg/mL) for 24 h. Apoptotic cells were identified by ANXA5 and PI staining. (C-D) Control and OGT-deficient A2780 (C) and SKOV3 (D) cells were transfected with mRFP-GFP-LC3 vector for 24 h and then treated with cisplatin (5 µg/mL) for 24 h. Representative images of fluorescent LC3 puncta are shown. The mean number of yellow puncta representing autophagosomes and the mean number of red puncta representing autolysosomes are plotted. *P < 0.05 compared with Con sh+cis group. Scale bar represent 10 μm. (E) Control and OGT-deficient A2780 and SKOV3 cells were treated with cisplatin for 24 h and then analyzed by transmission electron microscopy. Scale bars represent 500 nm. (F) OGT-deficient SKOV3 cells were transfected with RFP-LC3 plasmids and then treated with cisplatin (5 µg/mL) for 24 h. Lamp1 puncta were detected by anti-Lamp1. Scale bar represent 10 μm. The values are presented as mean ± SD (n = 3). **P < 0.01, *P < 0.05.
Figure 6
Figure 6
The O-GlcNAcylation level of SNAP-29 is associated with autophagy activity induced by cisplatin. (A) Control and OGT-deficient SKOV3 cells were transfected with NC siRNA or SNAP-29 siRNA and then treated with cisplatin (5 µg/mL) for 24 h. The expression levels of LC3 and p62 were examined by western blotting. (B) OGT-deficient SKOV3 cells were transfected with mRFP-GFP-LC3 vector for 24 h and then transfected with NC siRNA or SNAP-29 siRNA. After culturing in cisplatin (5 µg/mL) for 24 h, cells were imaged with a confocal microscope. Representative images of fluorescent LC3 puncta are shown. *P < 0.05 compared with OGT sh group. Scale bar represent 10 μm. (C) SKOV3 cells stably expressing control shRNA or OGT shRNA were treated with cisplatin (5 µg/mL) for 24 h and then the expression of SNAP-29 was tested by western blotting. (D) After being treated with cisplatin (5 µg/mL) for 24 h, the control and OGT-deficient SKOV3 cell extracts were immunoprecipitated with anti-SNAP-29 and the resulting precipitants were immunoblotted against O-GlcNAc. Whole-cell lysates were tested for SNAP-29 and actin. (E) Control and OGT-deficient SKOV3 cells were transfected with SNAP-29-GFP and RFP-LC3 vector and then treated with cisplatin (5 µg/mL) for 24 h. Colocalization of SNAP-29 and LC3 was determined by confocal fluorescence microscopy. Scale bar represent 10 μm. The values are presented as mean ± SD (n = 3). **P < 0.01, *P < 0.05.
Figure 7
Figure 7
O-GlcNAcylation of SNAP-29 regulates the interaction of SNAP-29 with Stx17 and VAMP8. (A) SKOV3 cells were transfected with SNAP-29 and SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. The O-GlcNAcylation levels of SNAP-29 were tested by co-immunoprecipitation. (B) SKOV3 cells were transfected with SNAP-29-GFP, SNAP-29 (Mut)-GFP and RFP-LC3 vectors and then treated with cisplatin (5 µg/mL) for 24 h. Colocalization of SNAP-29 and LC3 was determined by confocal fluorescence microscopy. Scale bar represent 10 μm. (C) SKOV3 cells were transfected with control, SNAP-29 or SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. The expressions of LC3 and p62 were measured by western blotting. (D) The expressions of LC3 and p62 in SKOV3 cells that were transfected with si Stx17 or si VAMP8 and treated with cisplatin (5 µg/mL) for 24 h. (E) SKOV3 cell were transfected with SNAP-29 or SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. Then, cell extracts were immunoprecipitated with anti-SNAP-29 and the resulting precipitants were immunoblotted against Stx17 and VAMP8. Whole-cell lysates were tested for SNAP-29 and actin. The values are presented as mean ± SD (n = 3). **P < 0.01, *P < 0.05.
See this image and copyright information in PMC

References

    1. Chornokur G, Amankwah EK, Schildkraut JM. et al. Global ovarian cancer health disparities. J Gynecol Oncol. 2013;129:258–64. - PMC (VSports在线直播) - PubMed
    1. Krzystyniak J, Ceppi L, Dizon DS. et al. Epithelial ovarian cancer: the molecular genetics of epithelial ovarian cancer. Ann Oncol. 2016;27(Suppl 1):i4–i10. - PMC - PubMed
    1. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin. 2017;67:7–30. - PubMed
    1. Kobayashi H, Yamada Y, Sado T. et al. A randomized study of screening for ovarian cancer: a multicenter study in Japan. Int J Gynecol Cancer. 2008;18:414–20. - PubMed
    1. Buys SS, Partridge E, Black A. et al. Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial. JAMA. 2011;305:2295–303. - PubMed

Publication types

MeSH terms