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Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

ABSTRACT: With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.

SUBMITTER: Chung SF

PROVIDER: S-EPMC7589785 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Recombinant <i>Bacillus caldovelox</i> Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

Chung Sai-Fung SF Kim Chi-Fai CF Chow Ho-Yin HY Chong Hiu-Chi HC Tam Suet-Ying SY Leung Yun-Chung YC Lo Wai-Hung WH

International journal of molecular sciences 20201009 20

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant <i>Bacillus caldovelox</i> arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with th ...[more]

PMID: 33050217

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Project description:BackgroundThe Securinega-type alkaloids occur in plants belonging to Euphorbiaceae family. One of the most widely distributed alkaloid of this group is securinine, which was identified next to allosecurinine in Phyllanthus glaucus (leafflower). Recently, some Securinega-type alkaloids have paid attention to its antiproliferative potency towards different cancer cells. However, the cytotoxic properties of allosecurinine have not yet been evaluated.MethodsThe cytotoxicity of the extract, alkaloid fraction obtained from P. glaucus, isolated securinine and allosecurinine against HeLa cells was evaluated by real-time xCELLigence system and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and 7-amino-actinomycin (7-AAD) staining and confirmed with fluorescent Hoechst 33342 dye. The assessment of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, the level of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), caspase-3/7 activity and cell cycle analysis were measured by flow cytometry. The enzymatic activity of caspase-9 was assessed by a luminometric assay. The expression of apoptosis associated genes was analyzed by real-time PCR.ResultsThe experimental data revealed that securinine and the alkaloid fraction were significantly potent on HeLa cells growth inhibition with IC50 values of 7.02 ± 0.52 μg/ml (32.3 μM) and 25.46 ± 1.79 μg/ml, respectively. The activity of allosecurinine and Phyllanthus extract were much lower. Furthermore, our study showed that the most active securinine induced apoptosis in a dose-dependent manner in the tested cells, increased the percentage of ROS positive cells and depolarized cells as well as stimulated the activity of ERK1/2, caspase-9 and -3/7. Securinine also induced cell cycle arrest in S phase. Real-time PCR analysis showed high expression of TNFRSF genes in the cells stimulated with securinine.ConclusionsSecurinine induces apoptosis and activates cell cycle checkpoints in HeLa cells which is associated with oxidative stress. The results indicate that the mitochondrial pathway is involved in the programmed cell death.

Dataset Information

Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

Publications

Recombinant <i>Bacillus caldovelox</i> Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

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