Bacterial Ribonuclease MazF-Mediated Apoptosis as Potential Cancer Therapy

: 1 Programmed cell death is a dynamic and critical mechanism of cell suicide in eukaryotes and 2 prokaryotes. MazF is a ribonuclease protein involved in bacterial intracellular programmed death. This 3 protein cleaves mRNAs at ACA sequences, leading to inhibition of protein synthesis and triggering cell 4 death. Given that cancer is heterogenic and has varied susceptibility to treatment, we examined the 5 impact of MazF proteins on the growth and viability of three cancer cell lines: MCF7, HT29, and AGS. These 6 cell lines were transfected with ACA-less mazF mRNAs and evaluated for MazF-mediated cell death. The 7 data illustrated that efficient MazF translation leads to a significant reduction in cell viability and is 8 modulated by structural elements of ACA-less mazF mRNAs. In the presence of MazF, the levels of 9 activated caspase-3 and -7 were significantly elevated in transfected cells, confirming the occurrence of 10 apoptosis. We also quantified mRNA translation on a single-cell basis in MCF7 and AGS cell lines to 11 examine MazF-mediated inhibition of protein synthesis. MazF expression significant decreases the levels 12 of protein translation in the examined cell lines. This is the first report of MazF as a potential anti-cancer 13 agent via induction of apoptosis in MCF7, AGS, and HT-29 cell lines.

As small proteins, ribonucleases have been a promising option in cancer therapy at the level of 1 gene expression 3 and function as anti-angiogenic, neurotoxic, antitumor, or immunosuppressive agents 2 against cancer 4,5 . These proteins occur naturally in both prokaryotic and eukaryotic cells. Bacteria 3 generate diverse groups of RNase proteins with various cytotoxic activities from anti-tumor effects to 4 involvement in bacterial programmed cell death (PCD) and population stability 6 . 5 Bacteria are routinely exposed to harsh conditions, such as amino-acid starvation, antibiotics, 6 temperature change, and DNA damage, that lead to the regulation of cell growth and cell death 6 . Toxin-7 antitoxin (TA) is an intracellular death mechanism present in almost all bacteria, comprising of bicistronic 8 operons containing a toxin and an antitoxin. In the TA system, toxins target processes such as DNA 9 replication, mRNA stability, protein synthesis, and ATP synthesis while their corresponding antitoxins 10 prevent toxin activities under normal conditions 7 . MazF is a well-studied toxin in bacteria that halts 11 protein translation but does not affect DNA or RNA synthesis. MazF protein targets specific sites in single-12 stranded mRNAs, 16S rRNA, and transfer-messenger RNAs (tmRNAs), tRNA-mRNA hybrids that bind to the 13 aminoacyl site of ribosomes 6,8-10 . MazF degrades RNAs at either the 5´or the 3´ ends of the first A residue 14 of ACA sequences. Similar to RNase A, the 2-OH group at the cleavage site is crucial for the function of 15 MazF 11 . 16 Shimazu et al. reported that MazF has the ability to impede protein synthesis and become 17 involved in Bak-dependent apoptosis in mammalian cells 12 . However, the injection of MazF DNA into solid 18 tumors, in spite of tumor shrinkages, did not prevent tumor regression. This failure stems from the 19 occurrence of mutations in the mazF gene, degradation of MazF or the lack of continued expression of 20 MazF in cells 13 . Therefore, DNA delivery may not be an optimal approach to deliver this RNase into cancer 21 cells or tumors. 22 In vitro transcribed mRNA (IVT mRNA)-based therapy, unlike other nucleic acid-based therapies, 23 offers exceptional advantages in the medical field. Translation in both dividing and non-dividing cells, 24 30-2004), while HT-29 (ATCC  HTB-38) cells were cultured in McCoys 5A medium (ATCC  30-2007). All 1 media were supplemented with 10% fetal bovine serum (Atlanta Biologicals, Inc., Flowery Branch, GA, 2 USA . cat. S11095) and 1% penicillin/streptomycin (Thermo Fisher, cat. 15140122). Cells were maintained 3 in fresh medium every 2 days at 37 ºC with 5% CO2. 4 Transfection 5 All cell lines were seeded in concentrations of 5x10 5 cells per well or 2.5x10 4 cells per well in 6-6 well or 96-well plates, respectively. Transfection was carried out using Lipofectamine® MessengerMAX 7 mRNA Transfection Reagent (Thermo Fisher, LMRNA003) following manufacturer's instructions. 8 The viability of transfected cells was analyzed via trypan blue exclusion. The cells were seeded in 20 6-well plates and transfected with mazF mRNA the following day. The transfected cells were trypsinized 21 and counted using a hemocytometer. 22

Western Blot Analysis
The toxicity of mazF mRNA on different cell lines was determined using the MTT assay following 1 the CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA cat. G4000) 2 instructions. 3

11
The transfected cells were prepared for analysis using the Click-iT Plus OPP Protein Synthesis 12 Assay Kit (Thermo Fisher, C10458) following the manufacturer's instructions, with one modification: cells 13 were fixed using 4% paraformaldehyde for 15 minutes instead of the recommended 3.7% formaldehyde. 14 Following staining, samples were imaged using a GE IN Cell Analyzer 2500HS then analyzed using GE 15 InCarta software (version 1.5). For analysis, we created a mask using HCS Nuclear Blue Stain. 16

17
All experiments were conducted in triplicates. Data are shown as the mean ± standard deviation. 18 The data were analyzed through t-tests. The difference was considered statistically significant when P< 19 0.05. 20

Results:
1 mazF mRNA Structure 2 mRNA therapy is an attractive approach for delivering genetic constructs into cells to treat a 3 disease. mRNA therapy owes its success to its superior transfection and expression efficiency, simplicity 4 and safety profile 18 . These advantages led us to examine whether ACA-less mazF mRNA can be effectively 5 transfected and translated in cancer cells. To this end, a T7-mazF construct (pT7-mazF) was designed. To 6 compare the expression efficacy of cap-dependent and IRES-dependent mRNAs for in vitro transcription 7 purposes, we developed a pIRES-mazF-1 plasmid. Additionally, several ACA-less mazF mRNAs lacking one 8 or more critical structural elements such as a cap, an IRES, poly-As, or Kozak sequences were constructed 9 and their effective translations in mammalian cells were measured. 10 Our results indicated that capped-mazF-polyadenylated or IRES-mazF-polyadenylated mRNA 11 could be translated in cells while the omission of any one of these mRNA structural elements, i.e., cap/IRES 12 or poly-As, was enough to block MazF protein expression. However, the lack of Kozak sequences did not The Caspase-3 and -7 detection assay contains aspartic acid-glutamic acid-valine-aspartic acid (DEVD), a 8 caspase substrate that has a high affinity for activated caspases. Our results showed that the expression 9 of MazF caused apoptosis reactions in MCF7, AGS, and HT29 cells (Figure 3). MCF7, AGS, and HT29 cells 10 exhibited MazF-mediated apoptosis 12, 18, and 36 hours post transfection, respectively. Additionally, the 11 activity of caspase was quantified in these cell lines (Figure 3). Further, we investigated whether cleaved 12 PARP, a marker of cells undergoing apoptosis, was present in the cell lines translating mazF mRNA. As 13 shown in Figure 4, the cleaved PARP is detectable in AGS cells transfected with mazF mRNA. Altogether, 14 these results demonstrate that MazF could induce apoptosis in cancer cells. Even with recent advancements in the medical field, cancer remains a significant concern. 9 Introduction of novel therapies can bestow hope to many patients worldwide. Ribonucleases provide 10 promising new treatment options that could be effective at transcription and translation levels. However, 11 it quickly became evident that certain ribonucleases, due to their instability, the lack of cytotoxic or 12 cytostatic activity, and the presence of ribonuclease inhibitors (RI) within cells making them poor 13 candidates for cancer therapy 21 . 14 MazF is a small and stable ribonuclease in bacterial cells that cleaves mRNAs, tRNA, and rRNA at 15 In this study, we demonstrated that the delivery of mazF mRNA into cancer cells enhances 1 translation of the encoded protein. mRNA is an attractive candidate in non-viral gene therapy due to its 2 ability to be active in the cytosol without entering the nucleus. However, mRNA lability has always 3 overshadowed the advantages of mRNA-based therapy. In order to address this concern, we studied the 4 necessity of the main structural elements of mRNA to boost the translation efficiency of MazF in in vitro 5 cell line conditions. Our results demonstrated that the presence of Cap/IRES and poly-A elements enables 6 the translation of the encoded protein within cells. We also synthesized and delivered GFP and mazF 7 mRNA into HEK293, MCF, AGS and HT29 cell lines. mazF mRNA deficient for caps or poly-As resulted in no 8 induction of death in transfected cells. These results may suggest synergy between cap structure and poly-9 A tail in translation efficiency, a finding reported by various researchers 23-26 . This cooperation stems from 10 mRNA circulation, where the cap-eIF4E-eIF4G-PABP-poly-A complex hampers the function of 11 exonucleolytic nucleases in order to degrade mRNA 16 . 12 The 5′ ends of eukaryotic mRNAs contain a methylated m 7 GpppN cap structure that is crucial for 13 mRNA splicing, stabilization, transport, and translation. In IVT reactions, the cap structure is linked to the 14 synthesized mRNA through an enzymatic reaction. However, this capping process caps either the 5′ side 15 or both ends of the mRNA, rendering half of the synthesized mRNAs nonfunctional 16 . Recently, several 16 cap analogues have been designed to optimize the capping process in in vitro conditions. One example of 17 these cap analogs is the anti-reverse-cap analogue (ARCA), which contains a methylated 3'-OH group that 18 enforces ARCA to localize in the proper orientation 16 . 19 Although the cap is a fundamental element in mRNA stability and translation, some viral and 20 cellular mRNAs possess internal ribosome entry sites (IRES) that carry out their translation in a cap-21 independent manner. IRES sequences directly recruit ribosomes and start translation at either a non-AUG 22 codon or an overlapping +1 frame gene 27 . The discovery of IRES has led to an exciting path in cancer 23 therapy. This element enables researchers to design bicistronic, tricistronic or tetracistronic operons 24 encoding two or more genes under the control of a single promoter. Here, we recruited the IRES sequence 1 from encephalomyocarditis virus (EMCV) RNA to synthesize uncapped mRNAs. We then compared the 2 expression of capped and cap-independent mRNAs in cell-culture systems. As mentioned previously, both 3 IRES-mazF-A and C-mazF-A were expressed in the examined cell lines in a time-dependent manner. As a part of their defense responses, eukaryotic and prokaryotic cells have the capability to 1 degrade mRNA and subsequently suppress protein synthesis. For example, when bacteria are exposed to 2 harsh conditions (e.g., extreme amino acid starvation or antibiotic-blocked transcription or translation), 3 they use MazF proteins to cleave mRNAs and therefore block protein synthesis 34