Ubenimex enhances the radiosensitivity of renal cell carcinoma cells by inducing autophagic cell death

Liu,Shuai; Wang,Xiaoqing; Lu,Jiaju; Han,Liping; Zhang,Yongfei; Liu,Zheng; Ding,Sentai; Liu,Zhao; Bi,Dongbin; Niu,Zhihong

doi:10.3892/ol.2016.5036

Ubenimex enhances the radiosensitivity of renal cell carcinoma cells by inducing autophagic cell death

Authors:
- Shuai Liu
- Xiaoqing Wang
- Jiaju Lu
- Liping Han
- Yongfei Zhang
- Zheng Liu
- Sentai Ding
- Zhao Liu
- Dongbin Bi
- Zhihong Niu
View Affiliations

Affiliations: Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China, Department of Neurology, Shandong Police Hospital, Jinan, Shandong 250021, P.R. China, Department of Dermatology, Shandong University, Jinan, Shandong 250000, P.R. China
Published online on: August 22, 2016 https://doi.org/10.3892/ol.2016.5036
Pages: 3403-3410

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Renal cell carcinoma (RCC) is resistant to standard radiotherapy. Ubenimex, an aminopeptidase N inhibitor, is widely used as an adjunct therapy after surgery to enhance the function of immunocompetent cells and confer antitumor effects. Our previous study demonstrated that ubenimex induces autophagic cell death in RCC cells. Recently, the molecular mechanism of autophagy induction has been associated with radiosensitivity in RCC cells. In the present study, the ability of ubenimex to enhance RCC cell sensitivity to radiation via the induction of autophagic cell death was determined, and the mechanism of action of this effect was investigated. The 786‑O and OS‑RC‑2 human RCC cell lines were treated with 0.5 mg/ml ubenimex and different doses of irradiation (IR). The cell viability was measured using a colony‑formation assay and flow cytometry. Acridine orange (AO)‑ethidium bromide (EB) staining was assessed by fluorescence microscopy as an indicator of autophagic cell death. Protein expression was assessed by western blotting. Autophagosomes were evaluated using transmission electron microscopy. RCC cells were used to evaluate the sensitivity to radiation using clonogenic survival and lactate dehydrogenase assays. Furthermore, these parameters were also tested at physiological oxygen levels. The AO‑EB staining and flow cytometry of the OS‑RC‑2 cells indicated that the combined treatment significantly enhanced autophagic cell death compared with ubenimex or IR alone. Therefore, treatment with ubenimex did not significantly alter cell cycle progression but increased cell death when combined with radiation. An Akt agonist could significantly weaken this effect, indicating that ubenimex may act as an Akt inhibitor. Furthermore, the western blot analysis indicated that the combined treatment inhibited the Akt signaling pathway compared with ubenimex treatment or IR alone. Ubenimex may enhance RCC cell sensitivity to radiation by inducing cell autophagy. This induction changes the role of autophagy from protective to lethal in vitro, and this switch is associated with the inhibition of the Akt signaling pathway.

View Figures

View References

1	Siegel R, Ma J, Zou Z and Jemal A: Cancer statistics, 2014. CA Cancer J Clin. 64:9–29. 2014. View Article : Google Scholar : PubMed/NCBI
2	Hollingsworth JM, Miller DC, Dunn RL, Montgomery JS, Roberts WW, Hafez KS and Wolf JS Jr: Surgical management of low-stage renal cell carcinoma: Technology does not supersede biology. Urology. 67:1175–1180. 2006. View Article : Google Scholar : PubMed/NCBI
3	Hollingsworth JM, Miller DC, Daignault S and Hollenbeck BK: Rising incidence of small renal masses: A need to reassess treatment effect. J Natl Cancer Inst. 98:1331–1334. 2006. View Article : Google Scholar : PubMed/NCBI
4	Jin Z and El-Deiry WS: Overview of cell death signaling pathways. Cancer Biol Ther. 4:139–163. 2005. View Article : Google Scholar : PubMed/NCBI
5	Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM and Lowe SW: A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell. 109:335–1146. 2002. View Article : Google Scholar : PubMed/NCBI
6	Chu K, Teele N, Dewey MW, Albright N and Dewey WC: Computerized video time lapse study of cell cycle delay and arrest, mitotic catastrophe, apoptosis and clonogenic survival in irradiated 14-3-3sigma and CDKN1A (p21) knockout cell lines. Radiat Res. 162:270–286. 2004. View Article : Google Scholar : PubMed/NCBI
7	Ichinose Y, Genka K, Koike T, Kato H, Watanabe Y, Mori T, Iioka S, Sakuma A and Ohta M: NK421 Lung Cancer Surgery Group: Randomized double-blind placebo-controlled trial of bestatin in patients with resected stage I squamous-cell lung carcinoma. J Natl Cancer Inst. 95:605–610. 2003. View Article : Google Scholar : PubMed/NCBI
8	Wan J, Liu T, Mei L, Li J, Gong K, Yu C and Li W: Synergistic antitumour activity of sorafenib in combination with tetrandrine is mediated by reactive oxygen species (ROS)/Akt signaling. Br J Cancer. 109:342–350. 2013. View Article : Google Scholar : PubMed/NCBI
9	Nomiya T, Tsuji H, Hirasawa N, Kato H, Kamada T, Mizoe J, Kishi H, Kamura K, Wada H, Nemoto K and Tsujii H: Carbon ion radiation therapy for primary renal cell carcinoma: Initial clinical experience. Int J Radiat Oncol Biol Phys. 72:828–833. 2008. View Article : Google Scholar : PubMed/NCBI
10	Bursch W: The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 8:569–581. 2001. View Article : Google Scholar : PubMed/NCBI
11	Klionsky DJ and Emr SD: Autophagy as a regulated pathway of cellular degradation. Science. 290:1717–1721. 2000. View Article : Google Scholar : PubMed/NCBI
12	Liu Z, Antalek M, Nguyen L, Li X, Tian X, Le A and Zi X: The effect of gartanin, a naturally occurring xanthone in mangosteen juice, on the mTOR pathway, autophagy, apoptosis, and the growth of human urinary bladder cancer cell lines. Nutr Cancer. 65(Suppl 1): 68–77. 2013. View Article : Google Scholar : PubMed/NCBI
13	Mizushima N, Tsukamoto S and Kuma A: Autophagy in embryogenesis and cell differentiation. Tanpakushitsu Kakusan Koso. 53:(16 Suppl). S2170–S2174. 2008.(In Japanese).
14	Horita H, Frankel AE and Thorburn A: Acute myeloid leukemia-targeted toxin activates both apoptotic and necroptotic death mechanisms. PLoS One. 3:e39092008. View Article : Google Scholar : PubMed/NCBI
15	Ling YH, Aracil M, Zou Y, Yuan Z, Lu B, Jimeno J, Cuervo AM and Perez-Soler R: PM02734 (elisidepsin) induces caspase-independent cell death associated with features of autophagy, inhibition of the AKT/mTOR signaling pathway, and activation of death-associated protein kinase. Clin Cancer Res. 17:5353–5366. 2011. View Article : Google Scholar : PubMed/NCBI
16	Kim KW, Moretti L, Mitchell LR, Jung DK and Lu B: Combined Bcl-2/mammalian target of rapamycin inhibition leads to enhanced radiosensitization via induction of apoptosis and autophagy in non-small cell lung tumor xenograft model. Clin Cancer Res. 15:6096–6105. 2009. View Article : Google Scholar : PubMed/NCBI
17	Lin CI, Whang EE, Donner DB, Du J, Lorch J, He F, Jiang X, Price BD, Moore FD Jr and Ruan DT: Autophagy induction with RAD001 enhances chemosensitivity and radiosensitivity through Met inhibition in papillary thyroid cancer. Mol Cancer Res. 8:1217–1226. 2010. View Article : Google Scholar : PubMed/NCBI
18	Chiu HW, Lin JH, Chen YA, Ho SY and Wang YJ: Combination treatment with arsenic trioxide and irradiation enhances cell-killing effects in human fibrosarcoma cells in vitro and in vivo through induction of both autophagy and apoptosis. Autophagy. 6:353–365. 2010. View Article : Google Scholar : PubMed/NCBI
19	Rajecki M, af Hällström T, Hakkarainen T, Nokisalmi P, Hautaniemi S, Nieminen AI, Tenhunen M, Rantanen V, Desmond RA, Chen DT, et al: Mre11 inhibition by oncolytic adenovirus associates with autophagy and underlies synergy with ionizing radiation. Int J Cancer. 125:2441–2449. 2009. View Article : Google Scholar : PubMed/NCBI
20	Zhuang W, Li B, Long L, Chen L, Huang Q and Liang Z: Induction of autophagy promotes differentiation of glioma-initiating cells and their radiosensitivity. Int J Cancer. 129:2720–2731. 2011. View Article : Google Scholar : PubMed/NCBI
21	Altmeyer A, Jung AC, Ignat M, Benzina S, Denis JM, Gueulette J, Noël G, Mutter D and Bischoff P: Pharmacological enhancement of autophagy induced in ahepatocellular carcinoma cell line by high-LET radiation. Anticancer Res. 30:303–310. 2010.PubMed/NCBI
22	Gewirtz DA, Hilliker ML and Wilson EN: Promotion of autophagy as a mechanism for radiation sensitization of breast tumor cells. Radiother Oncol. 92:323–328. 2009. View Article : Google Scholar : PubMed/NCBI
23	Yang ZJ, Chee CE, Huang S and Sinicrope F: Autophagy modulation for cancer therapy. Cancer Biol Ther. 11:169–176. 2011. View Article : Google Scholar : PubMed/NCBI
24	Mathew R, Karantza-Wadsworth V and White E: Role of autophagy in cancer. Nat Rev Cancer. 7:961–967. 2007. View Article : Google Scholar : PubMed/NCBI
25	Rodriguez-Rocha H, Garcia-Garcia A, Panayiotidis MI and Franco R: DNA damage and autophagy. Mutat Res. 711:158–166. 2011. View Article : Google Scholar : PubMed/NCBI
26	Chen W, Wu J, Shi H, Wang Z, Zhang G, Cao Y, Jiang C and Ding Y: Hepatic stellate cell coculture enables sorafenib resistance in Huh7 cells through HGF/c-Met/Akt and Jak2/Stat3 pathways. Biomed Res Int. 2014:7649812014.PubMed/NCBI
27	Zhai B, Hu F, Jiang X, Xu J, Zhao D, Liu B, Pan S, Dong X, Tan G, Wei Z, et al: Inhibition of Akt reverses the acquired resistance to sorafenib by switching protective autophagy to autophagic cell death in hepatocellular carcinoma. Mol Cancer Ther. 13:1589–1598. 2014. View Article : Google Scholar : PubMed/NCBI
28	Ishii K, Usui S, Sugimura Y, Yoshida S, Hioki T, Tatematsu M, Yamamoto H and Hirano K: Aminopeptidase N regulated by zinc in human prostate participates in tumor cell invasion. Int J Cancer. 92:49–54. 2001. View Article : Google Scholar : PubMed/NCBI
29	Fontijn D, Duyndam MC, van Berkel MP, Yuana Y, Shapiro LH, Pinedo HM, Broxterman HJ and Boven E: CD13/Aminopeptidase N overexpression by basic fibroblast growth factor mediates enhanced invasiveness of 1F6 human melanoma cells. Br J Cancer. 94:1627–1636. 2006.PubMed/NCBI
30	Mina-Osorio P: The moonlighting enzyme CD13: Old and new functions to target. Trends Mol Med. 14:361–371. 2008. View Article : Google Scholar : PubMed/NCBI
31	Liu S, Xie F, Wang H, Liu Z, Liu X, Sun L and Niu Z: Ubenimex inhibits cell proliferation, migration and invasion in renal cell carcinoma: The effect is autophagy-associated. Oncol Rep. 33:1372–1380. 2015.PubMed/NCBI
32	Mujtaba SF, Dwivedi A, Yadav N, Ch R, Kushwaha HN, Mudiam MK, Singh G and Ray RS: Superoxide mediated photomodification and DNA damage induced apoptosis by Benz(a)anthracene via mitochondrial mediated pathway. J Photochem Photobiol B. 142:92–102. 2015. View Article : Google Scholar : PubMed/NCBI
33	Liu K, Liu PC, Liu R and Wu X: Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry. Med Sci Monit Basic Res. 21:15–20. 2015. View Article : Google Scholar : PubMed/NCBI
34	Zhang S, Qi J, Sun L, Cheng B, Pan S, Zhou M and Sun X: Matrine induces programmed cell death and regulates expression of relevant genes based on PCR array analysis in C6 glioma cells. Mol Biol Rep. 36:791–799. 2009. View Article : Google Scholar : PubMed/NCBI
35	Burdak-Rothkamm S and Prise KM: New molecular targets in radiotherapy: DNA damage signalling and repair in targeted and non-targeted cells. Eur J Pharmacol. 625:151–155. 2009. View Article : Google Scholar : PubMed/NCBI
36	Elmore S: Apoptosis: A review of programmed cell death. Toxicol Pathol. 35:495–516. 2007. View Article : Google Scholar : PubMed/NCBI
37	Anbalagan S, Pires IM, Blick C, Hill MA, Ferguson DJ, Chan DA and Hammond EM: Radiosensitization of renal cell carcinoma in vitro through the induction of autophagy. Radiother Oncol. 103:388–393. 2012. View Article : Google Scholar : PubMed/NCBI
38	Rosenfeldt MT and Ryan KM: The multiple roles of autophagy in cancer. Carcinogenesis. 32:955–963. 2011. View Article : Google Scholar : PubMed/NCBI
39	Morani F, Titone R, Pagano L, Galetto A, Alabiso O, Aimaretti G and Isidoro C: Autophagy and thyroid carcinogenesis: Genetic and epigenetic links. Endocr Relat Cancer. 21:R13–R29. 2013. View Article : Google Scholar : PubMed/NCBI
40	Takeuchi H, Kondo Y, Fujiwara K, Kanzawa T, Aoki H, Mills GB and Kondo S: Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer Res. 65:3336–3346. 2005.PubMed/NCBI
41	Viola G, Bortolozzi R, Hamel E, Moro S, Brun P, Castagliuolo I, Ferlin MG and Basso G: MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells. Biochem Pharmacol. 83:16–26. 2012. View Article : Google Scholar : PubMed/NCBI
42	Mujumdar N, Mackenzie TN, Dudeja V, Chugh R, Antonoff MB, Borja-Cacho D, Sangwan V, Dawra R, Vickers SM and Saluja AK: Triptolide induces cell death in pancreatic cancer cells by apoptotic and autophagic pathways. Gastroenterology. 139:598–608. 2010. View Article : Google Scholar : PubMed/NCBI