Blocking autophagy enhanced cytotoxicity induced by recombinant human arginase in triple-negative breast cancer cells - PubMed (original) (raw)

. 2014 Dec 11;5(12):e1563.

doi: 10.1038/cddis.2014.503.

X Shi 2, Y Li 2, J Fan 2, X Zeng 2, Z Xian 2, Z Wang 3, Y Sun 2, S Wang 2, P Song 2, S Zhao 4, H Hu 5, D Ju 2

Affiliations

Blocking autophagy enhanced cytotoxicity induced by recombinant human arginase in triple-negative breast cancer cells

Z Wang et al. Cell Death Dis. 2014.

Abstract

Depletion of arginine by recombinant human arginase (rhArg) has proven to be an effective cancer therapeutic approach for a variety of malignant tumors. Triple-negative breast cancers (TNBCs) lack of specific therapeutic targets, resulting in poor prognosis and limited therapeutic efficacy. To explore new therapeutic approaches for TNBC we studied the cytotoxicity of rhArg in five TNBC cells. We found that rhArg could inhibit cell growth in these five TNBC cells. Intriguingly, accumulation of autophagosomes and autophagic flux was observed in rhArg-treated MDA-MB-231 cells. Inhibition of autophagy by chloroquine (CQ), 3-methyladenine (3-MA) and siRNA targeting Beclin1 significantly enhanced rhArg-induced cytotoxic effect, indicating the cytoprotective role of autophagy in rhArg-induced cell death. In addition, N-acetyl-l-cysteine (NAC), a common antioxidant, blocked autophagy induced by rhArg, suggesting that reactive oxygen species (ROS) had an essential role in the cytotoxicity of rhArg. This study provides new insights into the molecular mechanism of autophagy involved in rhArg-induced cytotoxicity in TNBC cells. Meanwhile, our results revealed that rhArg, either alone or in combination with autophagic inhibitors, might be a potential novel therapy for the treatment of TNBC.Cell Death and Disease (2014) 5, e1563; doi:10.1038/cddis.2014.503; published online 11 December 2014.

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Figures

Figure 1

Figure 1

Five TNBC cell lines profiled for ASS, OTC expression and rhArg sensitivity. (a) Western blot analysis showed the protein expression of ASS and OTC in A549, MDA-MB-231, HCC-1806, HCC-1937, HS-578T and BT-549 cells. A549 cells were used as positive control. (b) MDA-MB-231, HCC-1806, HCC-1937, HS-578T and BT-549 cells were treated with rhArg at indicated concentrations for 3 days. The cell viability was determined by MTT assay at the wavelength of 570 nm (_n_=3, means±S.D.)

Figure 2

Figure 2

The role of caspase 3 in apoptosis induced by rhArg in MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with 1 U/ml of rhArg for 48 h. The collected cells were incubated with Annexin V/PI, and then the samples were analyzed by flow cytometry. (b) MDA-MB-231 cells were incubated with 1 U/ml of rhArg for 24, 48 and 72 h. The protein levels of cleaved caspase 3, cleaved caspase 9 and PARP were detected by western blot analysis. Paclitaxel was used as positive control. (c) MDA-MB-231 cells were treated with vehicle (untreated), 50 nM paclitaxel for 24 h or 1 U/ml of rhArg for 48 h and 20 mM Z-VAD-fmk was used to pretreat these cells. After that, caspase 3 activity was measured by Fluorescence Assay Kit (Nanjing, Jiangsu Province, China) and values were normalized to vehicle. (d) MDA-MB-231 cells were incubated with 1 U/ml rhArg in the presence or absence of pretreatment of 20 mM Z-VAD-fmk for 48 h. The collected cells were incubated with Annexin V/PI, and then the samples were analyzed by flow cytometry. (e) MDA-MB-231 cells were transfected with siRNA twice targeting caspase 3. After 48 h transfection, cells were treated with or without arginase for 3 days. And then the samples were analyzed by flow cytometry. Paclitaxel was used as positive control

Figure 3

Figure 3

RhArg-induced autophagy in MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with 0.5, 1 and 2 U/ml of rhArg for 24 h and the expression of LC3-I/II was measured by immunoblot analysis. (b) MDA-MB-231 cells were treated with 1 U/ml of rhArg for 6, 12 and 24 h and the expression of LC3-I/II was measured by immunoblot analysis. (c) Representative electron micrographs of MDA-MB-231 cells treated with vehicle control (50 nM rapamycin) or rhArg for 24 h were taken at × 5000 (left and middle) or × 20 000 (right). (d) MDA-MB-231 cells were treated with 1 U/ml of rhArg for 24 h. Rapamycin (50 nM) was regarded as a positive control. The green fluoresce was detected by confocal microscopy. Cyto-ID positive cells were counted manually by ImageJ (NIH, Bethesda, MD, USA) cell counter (_n_=3, means±S.D.)

Figure 4

Figure 4

Autophagic flux measurements in MDA-MB-231 cells after rhArg treatment. (a and b) After exposure to rhArg for different time, MDA-MB-231 cells were treated with 2 U/ml rhArg in the presence Baf A1 (20 nM) and CQ (20 _μ_M) for another 4 h. Cell lysates were analyzed by immunoblot analysis. (c) Representative immunofluorescence images of MDA-MB-231 cells costained with Cyto-ID and LysoTracker Red after exposed to rhArg for the indicated times. green (Cyto-ID) and red (LysoTracker Red) dots in cells were counted using the ImageJ (_n_=3, means±S.D.). (d) MDA-MB-231 cells were treated with 1 U/ml rhArg for indicated times. Cell lysates were analyzed by immunoblot analysis

Figure 5

Figure 5

Suppressing autophagy accelerated rhArg-induced apoptosis and growth inhibition of MDA-MB-231 cells. (a and c) MDA-MB-231 cells were incubated with 1 U/ml rhArg for 24 h, in the presence or absence of CQ (20 _μ_M) and 3-MA (2 mM) and the expression of LC3-I/II was measured by western blot analysis. (b and d) MDA-MB-231 cells were incubated with rhArg for 72 h in the presence or absence of CQ (20 _μ_M) and 3-MA (2 mM), the cell viability was determined by MTT (_n_=3, means±S.D., *P<0.05, **P<0.01 versus each respective rhArg group). (e) MDA-MB-231 cells were incubated with 1 U/ml rhArg in the presence or absence of CQ (20 _μ_M) before flow cytometry analysis. (f and g) MDA-MB-231 cells were transiently transfected with Beclin1 siRNA before treatment of 1 U/ml of rhArg. (f) The levels of Beclin1 were detected by western blot analysis. (g) The viability after treatment was measured by MTT (_n_=3, means±S.D., *P<0.05 versus each respective rhArg group)

Figure 6

Figure 6

The role of ROS in rhArg-induced autophagy and apoptosis. (a and b) MDA-MB-231 cells were treated with 0.5 U/ml and 2 U/ml of rhArg with or without NAC (20 mM) for 3 days. Then, the treated cells were subjected to flow cytometry to measure ROS level. Data were processed by Flowjo (Tree Star, Inc., Ashland, KY, USA). (c) MDA-MB-231 cells were incubated with 1 U/ml of rhArg for 24 h in the presence or absence of NAC (20 mM) and the expression of LC3-I/II were measured by western blot analysis. (d) The green fluoresce was detected by confocal microscopy before rhArg in the presence or absence of NAC in MDA-MB-231 cells. Cyto-ID positive cells were counted manually by ImageJ cell counter (_n_=3, means±S.D.). (e) MDA-MB-231 cells were treated with rhArg in the presence or absence of NAC (20 mM) for 3 days. Then, the treated cells were subjected to MTT assay. (f) MDA-MB-231 cells were treated with 4 U/ml of rhArg in the presence or absence of NAC (20 mM) for 3 days and flow cytometry was used to detect Annexin/V apoptosis

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References

    1. 1Li X, Kong X, Wang Y, Yang Q. BRCC2 inhibits breast cancer cell growth and metastasis in vitro and in vivo via downregulating AKT pathway. Cell Death Dis 2013; 4: e757. - PMC - PubMed
    1. 2Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90. - PubMed
    1. 3Tseng LM, Liu CY, Chang KC, Chu PY, Shiau CW, Chen KF. CIP2A is a target of bortezomib in human triple negative breast cancer cells. Breast Cancer Res 2012; 14: R68. - PMC - PubMed
    1. 4Bowles TL, Kim R, Galante J, Parsons CM, Virudachalam S, Kung HJ, Bold RJ. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase. Int J Cancer 2008; 123: 1950–1955. - PMC - PubMed
    1. 5Morrow K, Hernandez CP, Raber P, Del Valle L, Wilk AM, Majumdar S et al. Anti-leukemic mechanisms of pegylated arginase I in acute lymphoblastic T-cell leukemia. Leukemia 2013; 27: 569–577. - PMC - PubMed

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