Abstract
Background/Aim: γ-Glutamylcyclotransferase (GGCT) is up-regulated in a broad range of cancers, including breast cancer, and GGCT inhibition has been shown to be a promising strategy for therapy. Herein, we evaluated the efficacy and mechanism of action of pro-GA, a GGCT enzymatic inhibitor, in MCF7 breast cancer cells. Materials and Methods: Proliferation was evaluated by WST-8 and trypan blue dye exclusion assays. Western blot analysis was conducted to examine the expression of cyclin-dependent kinase inhibitors (CDKI), including p21, p27, and p16. Induction of senescence was assessed by senescence-associated β-galactosidase staining. Generation of mitochondrial superoxide reactive oxygen species (ROS) was assessed using flow cytometry. The effect of N-acetylcysteine (NAC) on pro-GA dependent inhibition of proliferation, ROS generation, and senescence was also studied. The efficacy of systemic administration of pro-GA was evaluated in a MCF7 xenograft mouse model. Results: Treatment with pro-GA inhibited proliferation of MCF7 cells, increased CDKI expression and mitochondrial ROS, and induced cellular senescence. We found that cotreatment with NAC restored proliferation in pro-GA treated cells. NAC similarly suppressed CDKI expression, mitochondrial ROS generation, and senescence induced by pro-GA. Furthermore, the systemic administration of pro-GA in an MCF7 xenograft model had significant antitumor effects without toxicity. Conclusion: Pro-GA may be a promising therapeutic agent for the treatment of breast cancer.
- γ-glutamylcyclotransferase
- N-acetylcysteine
- reactive oxygen species
- cyclin-dependent kinase inhibitors
- pro-GA
- breast cancer
γ-Glutamylcyclotransferase (GGCT; also known as C7orf24) was originally identified by proteomic analysis as a highly expressed protein in bladder cancer tissue (1). GGCT protein plays a role in glutathione homeostasis, catalyzing the generation of 5-oxoproline and free amino acids from gamma-glutamyl peptides (2). It has been reported that GGCT is highly expressed in various cancer tissues, including breast, cervical, lung, colon, ovary, stomach, osteosarcoma, esophageal, and brain (3-5).
Knockdown of GGCT has been shown to inhibit cell growth via the induction of autophagy, up-regulation of cyclin-dependent kinase inhibitors followed by cell-cycle arrest, and induction of senescence (6-9). It was shown that targeting GGCT using polyethylene glycol–hyaluronic acid-modified liposomal siRNA can inhibit the growth of MCF7 breast cancer xenografts in vivo (10). Thus, GGCT constitutes a promising molecular target for novel breast cancer therapeutics.
We recently developed a substrate-mimicking GGCT inhibitor, N-glutaryl alanine (11), using screening methods reported by Board et al. (12). We further synthesized a diester-type cell-permeable prodrug of this GGCT inhibitor, pro-GA (13), which significantly inhibited the growth of PC3 prostate cancer cells both in vitro and in vivo (14). These data demonstrated that the specific inhibition of GGCT enzymatic activity is an attractive strategy for the development of novel anticancer therapeutics. In this study, we further investigated pro-GA activity in the human breast cancer cell-line MCF7, demonstrating growth inhibition in vitro by GGCT blockade, and explored the in vivo anticancer activity of this compound in an MCF7 xenograft mouse model.
Materials and Methods
Cell culture. MCF7 cells (RIKEN BRC, Rockville, Tsukuba, Japan) were maintained in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution (Wako) at 37°C in humidified air containing 5% CO2.
Reagents. Pro-GA was purchased from Funakoshi Co., Ltd. (Tokyo, Japan) and dissolved in DMSO at 200 mM for in vitro study or 200 mg/ml for in vivo study. N-acetylcysteine (NAC) was purchased from Wako and dissolved in PBS at 500 mM.
Cell growth assay. A standard trypan blue dye exclusion test was performed using a Countess II (Invitrogen, Waltham, MA, USA). The WST-8 assay was performed using an SF kit (Nacalai Tesque, Kyoto, Japan). Absorbance at 450 nm was recorded and normalized against a DMSO-treated control at day 0.
Antibodies. The following antibodies were used for western blotting: GGCT (6-1E; Cosmo, Tokyo, Japan), GAPDH (Wako), p21WAF1/CIP1 (BD Biosciences, Franklin Lakes, NJ, USA), p16INK4A (Abcam, Cambridge, MA, USA), and p27 (Santa Cruz Biotechnology, Dallas, TX, USA). Horse anti-mouse IgG-horseradish peroxidase conjugates were purchased from Vector Laboratories (PI-2000; Burlingame, CA, USA). Horseradish peroxidase-linked goat anti-rabbit IgG was purchased from the Jackson Laboratory (#7074; Bar Harbor, ME, USA).
Western blot analysis. Proteins solubilized in 1% sodium dodecyl sulfate (SDS) lysis buffer were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% fat-free dried milk in Tris-buffered saline containing 0.05% Tween 20, membranes were incubated with the antibodies detailed above. Targets were visualized using Clarity ECL Substrate (Bio-Rad, Hercules, CA, USA).
Bromodeoxyuridine (BrdU) incorporation assay. At 72 h post-treatment with pro-GA, the percentage of cells undergoing active DNA synthesis was examined using an APC BrdU flow kit (BD Biosciences) according to the manufacturers’ instructions. At least 10,000 cells from each experiment were analyzed using a BD LSRFortessa X-20 cell analyzer (San Diego, CA, USA).
Detection of cellular senescence. Senescence-associated β-galactosidase (SA-β-gal) staining was performed using a Cellular Senescence Kit (OZ BioScience, San Diego, CA, USA) according to the manufacturers’ instructions.
Measurement of mitochondrial reactive oxygen species (ROS). At 72 h post-treatment with pro-GA, the levels of mitochondrial ROS were measured using a MitoROS 580 kit (#16052; AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturers’ instructions and a BD LSRFortessa X-20 cell analyzer.
Xenograft study. All experiments were conducted with the approval of the Institutional Ethics Committee for Animal Experiments of Kyoto Pharmaceutical University (Approval number: CLON-19-001). Female CB-17 severe combined immunodeficient mice (14 weeks old, n=8 per group) were purchased from Japan SLC (Shizuoka, Japan). MCF7 cells (1×107 cells) were inoculated into mammary fat pad. Mice were intraperitoneally injected with pro-GA at a dose of 25 mg/kg in a total volume of 100 μl saline twice a week for 4 weeks starting 1 day after cell-inoculation. Tumor size was measured with vernier calipers twice a week for 4 weeks, and the tumor volume was calculated as (length × width2) × 0.5. The tumor weight was measured after the 4-week treatment.
Statistical analyses. Statistical analysis was performed with Bell Curve for Excel (Social Survey Research Information Co., Ltd. Tokyo, Japan). Student’s t-test, two-way ANOVA, and Dunnet’s or Bonferroni’s multiple comparison test were used, and a value of p<0.05 was considered statistically significant. In vitro experimental results are expressed as the mean±S.D. In vivo experimental results are expressed as the mean±S.E.
Results
Pro-GA suppresses MCF7 proliferation. We examined whether pro-GA treatment inhibited proliferation of MCF7 cells using a cell viability assay. Treatment with pro-GA inhibited MCF7 cell growth in a dose-dependent manner (Figure 1A). BrdU incorporation assay showed that pro-GA significantly reduced the percentage of cells undergoing DNA synthesis (Figure 1B and C). These findings indicate that pro-GA suppresses MCF7 cell proliferation.
Pro-GA inhibits MCF7 cell growth via cell-cycle arrest. A) MCF7 cells were treated with (●) DMSO, (▲) 50 μM pro-GA, (◆) 75 μM pro-GA, or (■) 100 μM pro-GA for the indicated number of days, and WST-8 assay was performed (n=3). B) MCF7 cells were treated with DMSO and 50 or 75 μM pro-GA for 3 days, and the proportion of BrdU-incorporating cells was assessed by flow cytometry (n=5). **p<0.01 and ***p<0.001 by Dunnet’s multiple comparison test.
Restorative effects of NAC on pro-GA dependent growth suppression in MCF7 cells. Next, we investigated the mechanisms underlying this pro-GA activity. We found that cotreatment with NAC, an established antioxidant (15), restored proliferation in cells treated with pro-GA (Figure 2A). Western blot analyses revealed that pro-GA induced the protein expression of CDKI, such as p21, p27, and p16, which was similarly disrupted by NAC cotreatment (Figure 2B). We also found that pro-GA significantly increased the level of mitochondrial ROS in MCF7 cells (Figure 2C). Again, this was reduced following NAC cotreatment (Figure 2D). These results suggest that pro-GA inhibits the proliferation of MCF7 cells by inducing mitochondrial ROS generation, which further increases the expression of CDKI.
Pretreatment with NAC rescues the growth inhibitory effect of pro-GA in MCF7 cells. A) MCF7 cells were pretreated with 100 μM NAC for 10 min and then treated with DMSO or pro-GA (50 or 75 μM) for 5 days. Cell viability was assessed using a trypan blue dye exclusion test (n=3; ***p<0.001 by one-way ANOVA with Bonferroni’s multiple comparison test). B) Western blot analysis of p21, p27, p16, and GGCT expression in MCF7 cells treated with 50 μM pro-GA for 3 days. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as a loading control. C) Mitochondrial reactive oxygen species (ROS) levels in MCF7 cells pretreated with 100 μM NAC for 10 min and then treated with DMSO or 75 μM pro-GA for 3 days. Light gray: control; yellow: NAC; gray: NAC plus pro-GA; red: pro-GA. D: Mean fluorescence intensities (mitochondrial ROS levels) taken from 2C. (n=6, ***p<0.001 by two-way ANOVA with Bonferroni’s multiple comparison test).
Pro-GA induced senescence in MCF7 cells. Cellular senescence is induced by ROS generation (16); therefore, we next examined senescence using SA-β-gal staining in pro-GA-treated cells. Quantitative analysis of the SA-β-gal-positive senescent cells demonstrated that pro-GA treatment significantly induced cellular senescence (Figure 3A and B).
NAC inhibits cellular senescence induced by pro-GA in MCF7 cells. A) MCF7 cells were pretreated with 100 μM NAC for 10 min and then treated with DMSO or pro-GA (50 or 75 μM) for 4 days. Representative images following SA-β-gal staining are shown. Scale bar: 50 μm. B) Quantitative analysis of SA-β-gal-positive cells. Data are the mean±standard deviation (n=3 or 5). ***p<0.001 by two-way ANOVA with Bonferroni’s multiple comparison test.
Systemic administration of pro-GA suppressed the growth of MCF7 xenografts in vivo. Finally, we examined whether pro-GA treatment inhibited the growth of MCF7 xenografts in vivo. We found that intraperitoneal injection of pro-GA (25 mg/kg) significantly inhibited the growth of MCF7 xenograft tumors in immunodeficient mice (Figure 4A and B). Reduction of the tumor weight following this 4-week treatment with pro-GA was also confirmed (Figure 4C); however, no obvious change in body weight was observed during the treatment (Figure 4D). These results suggest that systemic treatment with pro-GA safely suppresses MCF7 tumor progression in vivo.
Pro-GA suppresses the growth of MCF7 xenografts in immunocompromised mice. A) Relative tumor volume normalized to the tumor volume on day 7; **p<0.01 by a two-tailed Student’s t-test. The graph represents the tumor volume expressed as the mean±standard error (n=8 per group). B) Representative images of tumors from control (n=3) and pro-GA-treated (n=3) mice. Scale bars, 1 cm. C) The tumor weight from the mice treated with vehicle (n=7) or pro-GA (n=5); *p<0.05 using the two-tailed Student’s t-test. D) Relative body weight of mice treated with vehicle or pro-GA normalized to the weight on day 0 (n=8 per group).
Discussion
GGCT generates cysteine from γ-glutamyl cysteine, which has an antioxidative effect in cancer cells (5). In this study, we showed that the GGCT inhibitor pro-GA disrupts the growth of MCF7 breast cancer cells by increasing mitochondrial ROS in MCF7 cells. Intracellular stress, including ROS production, has been shown to induce p21-mediated cell-cycle arrest and subsequent cellular senescence (16), and it has been reported that an acceleration of ROS generation and p21 induction is critical for senescence (17). For instance, increased ROS production induced by the naturally occurring compound cristacarpin up-regulates CDKI and stress-induced cellular senescence, which can be suppressed by NAC treatment (18). In addition, we have reported that the knockdown of GGCT in A549 lung cancer cells using specific antisense oligonucleotides increases mitochondrial ROS (19), while others have shown that GGCT knockout induces cellular senescence in mouse embryonic fibroblast cells (4). Consistent with these findings, the data presented here show that pro-GA treatment induces CDKI, which can be suppressed by NAC treatment. These data suggest that the mechanism by which pro-GA inhibits MCF7 cell proliferation occurs via CDKI-induced cellular senescence mediated by mitochondrial ROS production.
The data presented here demonstrate that systemic administration of pro-GA has an antitumor effect in MCF7 tumor-bearing mice, suggesting that the inhibition of GGCT activity by pro-GA may be useful as a novel breast cancer therapy.
In summary, this study reveals that pro-GA has inhibitory effects on cell proliferation by producing mitochondrial ROS in MCF7 cells, arresting the cell-cycle through the induction of CDKI, and inducing cellular senescence. In addition, NAC scavenges the mitochondrial ROS generated by pro-GA and suppresses these inhibitory effects, indicating that the antitumor effect of pro-GA requires the accumulation of mitochondrial ROS produced by GGCT inhibition.
Acknowledgements
The Authors would like to thank Kengo Kanatani, Rina Nakajima, Shino Hayakawa, and Saeko Shiomi for the experimental support.
Footnotes
Authors’ Contributions
HI performed the experiments and drafted the manuscript. KT and YN performed the experiments. SK, AK, and TY designed the experiments. SN designed and supervised the study, and wrote the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest pertaining to the present study.
- Received July 4, 2022.
- Revision received July 19, 2022.
- Accepted July 20, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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