Mono-PEGylated thermostable Bacillus caldovelox arginase mutant (BCA-M-PEG20) induces apoptosis, autophagy, cell cycle arrest and growth inhibition in gastric cancer cells

Abstract

Gastric cancer is one of the most common malignant solid tumors in the world, especially in Asia with high mortality due to a lack of effective treatment. The potential usage of the newly constructed arginine-depleting enzyme-mono-PEGylated Bacillus caldovelox arginase mutant (BCA-M-PEG20), an effective drug against multiple cancer cell lines such as cervical and lung cancers, for the treatment of gastric cancer was demonstrated. Our results indicated that BCA-M-PEG20 significantly inhibited argininosuccinate synthetase (ASS)-positive gastric cancer cells, MKN-45 and BGC-823, while another arginine-depleting enzyme, arginine deiminase (ADI, currently under Phase III clinical trial), failed to suppress the growth of gastric cancer cells. In vitro studies demonstrated that BCA-M-PEG20 inhibited MKN-45 cells by inducing autophagy and cell cycle arrest at the S phase under 0.58 U/mL (IC₅₀ values). Significant caspase-dependent apoptosis was induced in MKN-45 after the treatment with 2.32 U/mL of BCA-M-PEG20. In vivo studies showed that administrations of BCA-M-PEG20 at 250 U/mouse twice per week significantly suppressed about 50% of tumor growth in the MKN-45 gastric cancer xenograft model. Taken together, BCA-M-PEG20 demonstrated a superior potential to be an anti-gastric cancer drug.

Keywords: Apoptosis; Autophagy; BCA-M-PEG20; Cell cycle arrest Cytostatic.

Fig. 1

Growth inhibitory effects on gastric cancer cell lines after the treatment of BCA-M-PEG20. a Protein expressions of ASS and OTC were measured by western blot analysis in MKN-45 and BGC-823. Mouse liver served as a positive control. b Cell proliferation assay for BCA-M-PEG20 and ADI on BGC-823 and MKN-45 cell lines were determined by MTT assay. Three independent experiments were performed in triplicate (each n = 3)

Fig. 2

BCA-M-PEG20 induced apoptosis in MKN-45 cells in a dose-dependent manner after 72 h of incubation as shown by a flow cytometry with Annexin V-FITC and propidium iodide staining, and b western blot analysis of cleaved-PARP level. The percentages of apoptotic cells are presented in the bar charts and band intensity was quantified using the ImageJ software. c BCA-M-PEG20 at a dosage 2.32 U/mL induced apoptosis in MKN-45 cells in a time-dependent manner. d MKN-45 cells were treated with 2.32 U/mL BCA-M-PEG20 and 0.1 mM 5-FU (positive control) with or without the caspase inhibitor z-VAD-FMK for 72 h. The results are shown as mean ± S.D and analyzed by Student’s t-test (n = 3, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001)

Fig. 3

BCA-M-PEG20 induced cell cycle arrest and autophagy in MKN-45 cells. a BCA-M-PEG20 induced S phase arrest in MKN-45 cells after 72 h of incubation as shown by flow cytometry with propidium iodide staining. The percentages of cell distribution are presented in the bar charts. b BCA-M-PEG20 with 0.58 U/mL resulted in S phase arrest in MKN-45 cells in a time-dependent manner. c Green punctate signals represented autophagosome formation in MKN-45 after 24–72 h of BCA-M-PEG20 (0.58 U/mL) treatment. d An increase in the ratio of LC3-II/LC3-I was detected after the treatment of 0.58 U/mL of BCA-M-PEG20. The results are shown as mean ± S.D and analyzed by Student’s t-test (n = 3, ^*p < 0.05, ^**p < 0.01, ***p<0.001)

Fig. 4

BCA-M-PEG20 and 5-fluorouracil (positive control) showed notable tumor inhibition in term of a Relative tumor volume and b % final tumor-to-body weight. c Macroscopic appearance of tumors dissected from PBS, BCA-M-PEG20 and 5-fluorouracil treated mice after the experiment (n = 10). d The body weight of each mouse in the three groups was measured weekly. The results are shown as mean ± SEM. through Two-way ANOVA with Bonferroni correction. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001