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. 2014 Aug 26:4:49.
doi: 10.1186/2045-3701-4-49. eCollection 2014.

Peptidylarginine deiminase IV promotes the development of chemoresistance through inducing autophagy in hepatocellular carcinoma

Tingting Fan  1 , Changsong Zhang  2 , Ming Zong  1 , Qiudong Zhao  3 , Xue Yang  3 , Chong Hao  3 , Hui Zhang  1 , Shanshan Yu  1 , Jinhu Guo  1 , Ruhan Gong  1 , Shasha Fan  1 , Lixin Wei  3 , Lieying Fan  1
Affiliations

Peptidylarginine deiminase IV promotes the development of chemoresistance through inducing autophagy in hepatocellular carcinoma

Tingting Fan et al. Cell Biosci. .

Abstract

Background: Peptidylarginine deiminase IV (PADI4) is widely distributed in several tissues and the expression is correlated with many pathological processes. Chemotherapy remains a major treatment alternatively to surgery for a large number of patients at the advanced stage of hepatocellular carcinoma (HCC). However, the role of PADI4 in the chemoresistance of HCC has not been identified.

Methods: MTT and PI/Annexin V assay were employed to examine the proliferation and apoptosis of HCC cell lines. The expression of MDR1 is detected by Realtime PCR. GFP tagged LC3 expression vector and electron microscopy are utilized to demonstrate the occurrence of autophagy.

Results: We observed that the elevated PADI4 expression is associated with chemoresistance in HCC patients with TACE after surgery. In addition, we found that overexpression of PADI4 in HCC cell lines lead to the resistance to chemotherapeutic agents in vitro and in vivo. Interestingly, the HCC cells that overexpressed PADI4 were observed to undergo autophagy which was known as a protective mechanism for cells to resist the cell tosicity from chemotherapy. Autophagy inhibitor could effectively restore the sensitivity of HCC cells to chemotherapy in vitro and in vivo.

Conclusions: These results indicate that PADI4 may induce chemoresistance in HCC cells by leading autophagy.

Keywords: Autophagy; Chemoresistance; Hepatocellular Carcinoma; PADI4.

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Figures

Figure 1
Figure 1
The elevated PADI4 mRNA expression in MDR1(+) HCC patients with TACE. (A) The MDR1 mRNA expression were detected in blood samples before and after TACE. - ΔΔCt, -(ΔCtbefore TACE – ΔCtafter TACE); MDR(+):- ΔΔCt > =0; MDR(-):- ΔΔCt < 0. (B) The progression-free survival (PFS) after transcatheter arterial chemoembolization (TACE) in partial hepatectomy for hepatocellular carcinoma determined by Kaplan-Meier analysis. (C) The PADI4 mRNA expression in HCC tumor tissues associated with chemoresistance. - ΔΔCt, -(ΔCtbefore TACE – ΔCtafter TACE); MDR(+):- ΔΔCt > =0; MDR(-):- ΔΔCt < 0.
Figure 2
Figure 2
PADI4 enhanced chemoresistance of hepatocellular carcinoma cells in vitro. (A) SMMC-7721 cells were treated with 5-Fu (120 μg/mL) with overexpression of PADI4 or not. The morphology of the cells was observed by microscope. (B) PADI4 was overexpressed in SMMC-7721 or HepG2 cells and the cells (1 × 104/well) were cultured in a 96-well plate with an existence of 5-Fu (120 μg/mL) or cisplatin (8 μg/mL) for 24 hours. MTT was employed to examine the viability of SMMC-7721 or Hep-G2 cells. (C) PADI4 was overexpressed in SMMC-7721 cells and the cells were cultured in a 6-well plate with an existence of 5-Fu (120 μg/mL) for 24 hours. Flow cytometry was used to measure apoptosis of the cells. (*Compared with the group that untreated with chemotherapy drugs, P < 0.05; #Compared with the group that treated with chemotherapy drugs, P < 0.05)
Figure 3
Figure 3
PADI4 enhanced chemoresistance of hepatocellular carcinoma cells in vivo. (A) PADI4 was overexpressed in SMMC-7721 cells and then the cells (5 × 106) to perform subcutaneous administration in the nude mice armpit area. After implantation, recipients were injected in tumor in situ with 5-Fu (30 mg/kg) every 3 days. After 27 days of implantation, the animals were sacrificed and tumors were dissected. (B) The tumor weight and volume of each groups were measured after been removed from the mice. (*P < 0.05)
Figure 4
Figure 4
PADI4 induced autophagy in hepatocellular carcinoma cells. (A) GFP-tagged LC3 plasmid was transfected into SMMC-7721 cells, after 24 hours transfection, the cells were incubated with Ad-PADI4. Fluorescence microscope was used to observe the punctate GFP-LC3 in the cytoplasm. (B) The number of punctate GFP-LC3 in each cell of SMMC-7721 and HepG2 was counted and at least 100 cells were included for each group. (C) Electron micrographs was exployed to examine the autophagic vacuoles ultrastructure in the cytoplasm of SMMC-7721 and HepG2 cells which PADI4 were overexpressed. Magnification, ×10,000. (D) Western blot was used to analysis the expression of LC3-I and LC3-II in SMMC7721 and HepG2 cells. a-d: SMMC-7721 in different treatment conditions; e: HepG2 treated with Ad-PADI4. GAPDH expression was used as control.
Figure 5
Figure 5
Inhibition of autophagy restored the sensitivity of HCC cells to chemotherapy. (A) SMMC-7721 and Hep-G2 cells (1 × 104/well) that overexpressed PADI4 were cultured in a 96-well plate with an existence of 5-Fu (120 μg/mL) for 24 hours. The occurrence of autophagy was inhibited by autophagy inhibitor-3-MA or sh-Atg7 and sh-Beclin1. MTT was used to detect the viability of the cells. (B) PADI4 was overexpressed in SMMC-7721 cells. The occurrence of autophagy was inhibited by sh-Atg7. Then the cells (5 × 106) were performed subcutaneous administration in the nude mice armpit area. After implantation, recipients were injected in tumor in situ with 5-Fu (30 mg/kg) every 3 days. After 27 days of implantation, the animals were sacrificed and tumors were dissected. The tumor weight and volume of each groups were measured after been removed from the mice. (*Compared with the group that untreated with chemotherapy drugs, P < 0.05; #Compared with the group that treated with chemotherapy drugs, P < 0.05)
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References

    1. Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays. 2003;25(11):1106–1118. doi: 10.1002/bies.10357. - DOI - PubMed
    1. Gyorgy B, Toth E, Tarcsa E, Falus A, Buzas EI. Citrullination: a posttranslational modification in health and disease. Int J Biochem Cell Biol. 2006;38(10):1662–1677. doi: 10.1016/j.biocel.2006.03.008. - DOI - PubMed
    1. Darrah E, Rosen A, Giles JT, Andrade F. Peptidylarginine deiminase 2, 3 and 4 have distinct specificities against cellular substrates: novel insights into autoantigen selection in rheumatoid arthritis. Ann Rheum Dis. 2012;71(1):92–98. doi: 10.1136/ard.2011.151712. - DOI - PMC - PubMed
    1. Chang X, Hou X, Pan J, Fang K, Wang L, Han J. Investigating the pathogenic role of PADI4 in oesophageal cancer. Int J Biol Sci. 2011;7(6):769–781. doi: 10.7150/ijbs.7.769. - DOI - PMC - PubMed
    1. Wang L, Chang X, Yuan G, Zhao Y, Wang P. Expression of peptidylarginine deiminase type 4 in ovarian tumors. Int J Biol Sci. 2010;6(5):454–464. doi: 10.7150/ijbs.6.454. - DOI - PMC - PubMed