Growth suppressive effect of pegylated arginase in malignant pleural mesothelioma xenografts

Abstract

Background: Malignant pleural mesothelioma (MPM) is a difficult-to-treat global disease. Pegylated arginase (BCT-100) has recently shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and melanoma. This study aims to investigate the effects of PEG-BCT-100 in MPM.

Methods: A panel of 5 mesothelioma cell lines (H28, 211H, H226, H2052 and H2452) was used to study the in vitro effects of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 were studied using 211H and H226 nude mice xenografts. Protein expression (argininosuccinate synthetase, ornithine transcarbamylase, cleaved PARP, cleaved caspase 3, cyclins (A2, D3, E1 and H), CDK4 and Ki67) and arginine concentration were evaluated by Western blot and ELISA respectively. Cellular localization of BCT-100 was detected by immunohistochemistry and immunoflorescence. TUNEL assay was used to identify cellular apoptotic events.

Results: Argininosuccinate synthetase was expressed in H28, H226, and H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 reduced in vitro cell viability (IC₅₀ values at 13-24 mU/ml, 72 h) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth (p < 0.01) with prolonged median survival (p < 0.01) in both xenograft models. Combining BCT-100 with pemetrexed or cisplatin conferred no additional benefits over single agents. Serum and intratumoral arginine levels were effectively decreased by BCT-100, associated with cytosolic accumulation of BCT-100 within tumor cells. Apoptosis (PARP cleavage in 211H xenografts; Bcl-2 downregulation, and cleavage of PARP and caspase 3 in H226 xenografts; positive TUNEL staining in both) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenografts; suppression of cyclin A2, E1, H and CDK4 in H226 xenografts) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms.

Conclusions: BCT-100 suppressed tumor growth with prolonged median survival partially mediated by intratumoral arginine depletion resulting in apoptosis and G1 arrest in mesothelioma xenograft models. The findings provide scientific evidence to support further clinical development of BCT-100 in treatment of MPM.

Keywords: Apoptosis; BCT-100; Cell cycle arrest; Mesothelioma; Pegylated arginase; Xenografts.

Fig. 1

Relative cell viability upon BCT-treatment, basal expression of ASS1 and combination effect of BCT-100 with cisplatin or pemetrexed in a panel of mesothelioma cell lines. a BCT-100 reduced cell viability in a dose-dependent fashion. b ASS1 was found in H28, H226 and 2452 cells but not in 211H and H2052 cells. c No synergistic effect in combination of BCT-100 with cisplatin or pemetrexed in different mesothelioma cell lines

Fig. 2

BCT-100 suppressed tumor growth and increased median survival in MPM xenograft models. BCT-100 inhibited tumor growth in a dose-dependent manner in (a) 211H and (b) H226 xenografts. BCT-100 (60 mg/kg) increased median survival in (c) 211H and (d) H226 xenograft models

Fig. 3

Combination effects of BCT-100 and pemetrexed or cisplatin in MPM xenograft models. No synergistic or additive effect was observed when combining BCT-100 with pemetrexed in (a) 211H and (b) H226 xenograft models. The tumor suppressive effect of BCT-100 was comparable to pemetrexed in 211H xenograft model. Pemetrexed exerted no tumor suppression in H266 xenografts. c There was no beneficial effect when combining BCT-100 with cisplatin in 211H xenograft model

Fig. 4

Basal expression of ASS1, BCT-100-induced arginine depletion and localization of BCT-100 in xenograft models. a ASS1 was highly expressed in H226 xenograft but relatively low in 211H xenograft. BCT-100 decreased serum arginine concentration in (b)(i) 211H and (b)(ii) H226 xenograft models. BCT-100 reduced intratumoral arginine level in a dose-dependent fashion in (b)(iii) 211H and (b)(iv) H226 xenografts. Intratumoral level of PEG-BCT-100 was increased in BCT-100 treatment arms with a dose-dependent manner in (c)(i) 211H and (c)(ii) H226 xenografts. BCT-100 was mostly located in the central part of tumor in (d)(i) 211H and (d)(ii) H226 xenografts, with cytosolic compartmentalization ((e)(i), (e)(ii)) as shown by immunoflorescence staining (pictures taken with 400X magnification)

Fig. 5

BCT-100 induced apoptosis and G1 arrest in MPM xenografts. Upon BCT-100 (60 mg/kg) treatment, (a) cleaved PARP (cPARP) was upregulated in 211H xenograft, (b) downregulation of Bcl-2 as well as cleavage of caspase 3 (CC3) and PARP (cPARP) in H226 xenograft and (c) CC3 was mainly found in the core part of tumor in H226 xenograft. (d) Nuclei were stained by DAPI (blue signal). There was no green TUNEL signal in control groups in both xenografts. The TUNEL signal was noted in both treatment arms in (d)(i) 211H and (d)(ii) H226 xenografts. Overlay of TUNEL and DAPI indicated DNA breaks were mainly located in nuclei. e(i) BCT-100 reduced the expression of cyclin A2, D3, E1 and CDK4 in 211H xenograft. e(ii) Downregulation of cyclin A2, E1, H and CDK4 by BCT-100 was observed in H226 xenograft. BCT-100 induced downregulation of proliferation factor Ki67 in (f)(i) 211H and (f)(ii) H226 xenografts