Rhodospirillum rubrum l-asparaginase targets tumor growth by a dual mechanism involving telomerase inhibition

https://doi.org/10.1016/j.bbrc.2017.08.078Get rights and content

Highlights

  • R. rubrum l-asparaginase mutant E149R, V150P, F151T (RrA) down-regulates telomerase.

  • Telomerase activity in treated cancer cells does not exceed 30% of control cells.

  • Continuous RrA exposure results in shortening of telomeres followed by cell death.

  • RrA reduces hTERT expression by 50% in SCOV-3, SkBr-3 and A549 xenografts.

Abstract

Rhodospirillum rubrum l-asparaginase mutant RrA E149R, V150P, F151T (RrA) was previously identified to down-regulate telomerase activity along with catalyzing the hydrolysis of l-asparagine. The aim of this study was to define the effect of prolonged RrA exposure on telomerase activity, maintenance of telomeres and proliferation of cancer cells in vitro and in vivo. RrA could inhibit telomerase activity in SCOV-3, SkBr-3 and A549 human cancer cell lines due to its ability to down-regulate the expression of telomerase catalytic subunit hTERT. Telomerase activity in treated cells did not exceeded 29.63 ± 12.3% of control cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell death in vitro. Using real time PCR we showed that length of telomeres in SCOV-3 cells has been gradually decreasing from 10105 ± 2530 b.p. to 1233 ± 636 b.p. after 35 days of cultivation. RrA treatment of xenograft models in vivo showed slight inhibition of tumor growth accompanied with 49.5–53.3% of decrease in hTERT expression in the all tumors. However down-regulation of hTERT expression, inhibition of telomerase activity and the loss of telomeres was significant in response to RrA administration in xenograft models. These results should facilitate further investigations of RrA as a potent therapeutic protein.

Introduction

Amino-acid cleaving enzymes are considered as promising agents for cancer therapy [1], [2], [3], [4]. l-Asparaginase (l-asparaginase amidohydrolase, l-ASNase) (EC 3.5.1.1) is an enzyme catalyzing the deamination of l-asparagine with the formation of aspartic acid and ammonia [5]. L-asparaginases are widely used for induction therapy of acute lymphoblastic leukemia (ALL). Recently l-asparaginase-containing regimens were introduced as standards of treatment for NK/T-cell lymphomas [6]. After treatment with l-asparaginase, malignant cells experience a significant lack of l-asparagine due to depletion of this amino acid in serum and extracellular fluid. Leukemia cells need an exogenous supply of l-asparagine for protein synthesis and are not able to synthesize asparagine due to low expression of asparagine synthetase (ASNS). The reduction of l-asparagine leads to death of the cancer cell [7], [8].

A number of new l-asparaginases from several sources, predominantly bacterial, have been isolated recently. l-asparaginases obtained from Thermococcus kodakaraensis, Synechococcus elongatus PCC6803, Nocardiopsis alba NIOT-VKMA08, Wollinella succinogenes showed high asparaginase activity and no glutaminase activity [9], [10], [11], [12]. l-asparaginases from Aspergillus oryzae CCT 3940 also showed no glutaminase activity and significant decrease in cell growth compared to the commercial l-asparaginases from Escherichia coli [13]. l-asparaginases obtained from actinomycetes Streptomyces fradiae NEAE-82, Enterobacter aerogenes KCTC2190/MTCC111, Erwinia carotovora and Yersinia pseudotuberculosis could also be considered as a potent anticancer drug [14], [15], [16], [17].

Our previous finding suggests that E149R, V150P, F151T mutant of l-asparaginase type I, derived from Rhodospirillum rubrum (RrA), suppress telomerase activity in leukemic cells, making a considerable contribution to anticancer activity of the enzyme [18], [19].

Telomerase synthesizes telomere repeats TTAGGG on human chromosomes' ends, which allows cells to maintain their telomere length. Most cancer cells have telomerase activity [20] which support their unlimited proliferation [21], [22]. The main components of human telomerase are hTR (human Telomerase RNA), which includes a template for synthesis of telomeric DNA and hTERT (human Telomerase Reversу transcriptase) that has reverse transcriptase activity and can synthesize telomere repeats on the hTR template [23]. It is known that the telomerase activity is strongly regulated by the level of hTERT synthesis [24]. Inhibition of telomerase in cancer cells leads to telomere attrition, limitation the lifespan of these cells, senescence and apoptosis [25], [26], [27]. Telomerase inhibitors have been expected to become promising agents for cancer treatment [28], [29].

The aim of the research is to evaluate RrA anti-telomerase activity and its effect on cell proliferation on various cancer models in vitro and in vivo.

Section snippets

Studied l-asparaginase

For all studies, RrA E149R, V150P, F151T mutant (RrA) was used. The upstream, downstream and enzymatic properties of the studied enzyme were described in Refs. [19], [30].

Cell cultures and cytotoxicity assay

Human breast adenocarcinoma SkBr-3, ovarian cancer SKOV-3 and lung carcinoma A549 (ATCC, Manassas, VA) were used as the in vitro model in this study. Cancer cells were routinely grown in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) containing 10% heat-inactivated (56 °C, 30 min) fetal bovine serum (Hyclone Laboratories, Logan,

RrA cytotoxicity for cancer cells

It is known that l-asparaginase activity induces direct cytotoxic effect on cells due to asparagine deprivation [36], [37], while indirect effect of RrA-mediated telomerase inhibition may develop for longer period of time [38], [39]. Direct cytotoxic activity of RrA was tested in 72 h of incubation. RrA showed dose-dependent cytotoxic effect for all cell lines (Fig. 1A). SkBr-3 cells were the most sensitive, while SCOV-3 cells showed the highest resistance for RrA. The values of determined RrA

Discussion

Apparently, the detailed mechanism of cell killing after l-asparaginase exposure is linked to protein synthesis depression. However, there are data indicating possible l-asparaginase involvement in different pro-apoptotic pathways. Apart from l-asparagine cleavage, l-asparaginase could inhibit mTORC1 and induce autophagy in acute myeloid leukemia cells [40]. In chronic myeloid leukemia cells l-asparaginase activates autophagy by inducing the conversion of light chain 3-I to light chain 3-II.

Conflict of interest

The authors declare no competing interests.

Acknowledgments

The work was performed in the framework of the Program for Basic Research of State Academies of Sciences for 2013–2020. This work was supported by 15-34-70020 mol_а_mos grant 2015–2016 from the Russian Fund of Fundamental Research.

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