The Bcl-2 silencing with an Antisense Oligonucleotide: Increase early Apoptosis

Breast cancer is a heterogenic disease and hormone dependence. Estrogen receptor is positive in more than seventy percent of breast cancer patients. Tamoxifen is an estrogen receptor (ER) antagonist and used as the rst line of treatment. Drug resistance is a main reason in failure of cancer treatment and progression of the disease. Combination drug therapy is a method to treatment but is not sucient. New approaches like molecular therapy reveal new insight to cancer therapy. Studies shown, Bcl-2 gene family inhibitors and ER blockers enhance recovery. Interfering molecules such as antisense can inhibit the expression of Bcl-2 and push the cancer cells to apoptosis. Nevertheless, their effectiveness is low, mostly due to their direct use. increase of and We also the and effects of naked ASO and Nano-packed ASO.


Introduction
The Breast cancer (BC) is the most common type of cancer among women. It is second cause of death after lung cancer in females. According to last statistics estimations that published by International Agency for Research on Cancer (IARS); lung cancer, female breast cancer and colorectal cancer are most common types of cancer that are responsible for one third of the cancer incidence and mortality in worldwide. The estimations indicate breast cancer with approximately 2.1 million diagnoses has burden region with nearly 17000 new cases (1). However, breast cancer therapy divided to local and systemic.
Local therapy such as surgery or radiation that sometimes is not e cient. Systemic therapies including; chemotherapy, hormone therapy, targeted therapy and immunotherapy (2). Nevertheless, mortality rate is high. New approaches are essential because of high heterogeneity nature of breast cancer and failure of traditionally treatment (3). It seems that molecular therapies open a way to reduce mortality rate in early stages of cancer. Breast cancer can divided to subgroups based on the presence of estrogen receptor (ER), progesterone receptor (PR), HER2, and luminal (A and B), HER2-enriched, basal like and normal breast-like, respectively (4). There are a correlation between these subgroups and survival rate, cancer relapse, place of metastasis and chemotherapy response (5).
About 70% of breast cancers are Estrogen Receptor positive (ER + ) and usually treated with Tamoxifen which is an estrogen receptor antagonist rather than aromatize inhibitors(6). Long-time Tamoxifen usage can cause drug resistance (7). In addition, it also could led to some various side-effects in normal tissues that increases susceptibility to endometrial and liver cancers or thrombosis(8). Studies showed, Antiapoptotic molecules could lead to survival of cancer cells (9). They cause cancer cells avoid from apoptosis and promote tumor progression. They also act as a barrier to chemical cancer therapies. One of them is Bcl-2 family. Increases level of anti-apoptotic Bcl-2 family proteins such as BCL-2; MCL-1 or BCL-XL proteins prevent cancer cells apoptosis. Hence, anti-apoptotic Bcl-2 protein promotes cancer cells to survive and allows them to resist. Therefore, the anti-apoptotic Bcl-2 protein (Bcl-2) silencing could enhance apoptosis and increased sensitivity of cancer cells to treatment (10)(11)(12). Antisense technique is a way to silence anti-apoptotic Bcl-2 protein. Antisense Oligonucleotides (ASO) are short length (18-24 bp), synthetics molecules. They are single-strand, which complementary to the targeted mRNA gene, via Watson-Crick base paring. They can inhibit mRNA processing. Eventually, they lead to suppressing of gene expression through RNase H-mediated mechanism or by modulations of splicing to stop their translations to protein (13). However, they can reduce cancer gene expression but they have some limitations. For example, it degrades rapidly by endo and exo-nucleases, misleading off-target effects and weak cellular uptake. To solve these problems, chemical modi cation of nucleotides, various modi cation on backbone or sugar rings of Deoxyribonucleotides with sulfur ion to creating phosphorothionate (PS) linkage and 2'-deoxy residues modi cations have been con rmed (14). Nanopackaging is a good way to increase cellular uptake of ASO. Nano-packages Lipofectamine and micelles/niosome can increase cellular uptake and lead to decrease ASO dose (15)(16)(17). In this study, we used bioinformatics to predict the second structures of target's mRNA according to minimal delta G energy. It can predict a nity of ASO with target's mRNA. Our team designed an Antisense Oligonucleotides for Bcl-2 mRNA. A biodegradable cationic micelles/niosome and Lipofectamine utilized to evaluate cellular delivery of ASO and Tamoxifen. The Bcl-2 expression as an anti-apoptotic protein evaluated. The MCF-7 and the MDA-MB-231 cell lines selected as Estrogen Receptor-positive and ERnegative cells respectively. Finally, naked ASO, nano-pack ASO and Tamoxifen effects on Bcl-2 expression and cell apoptosis compared in several groups.

Methods And Materials
Cell Culture The Breast cancer cell lines (MCF-7 and MDA-MB-231) were purchased from the Pasteur Institute of Iran (NCBI codes: C135, and C578 respectively).They were cultured at RPMI 1640 with 10% FBS and were incubated at 5% CO2 and 37°C. The cells were passage when their con uences reached about 95% and cell medium was alter every 36h regularly according to the duplicate time of the cells.

RNA isolation and cDNA synthesis
Total RNA was isolated from MDA-MB-231 and MCF-7 cell lines using the TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. In addition, the isolated RNAs treated with DNase1 (YT8054, Iran) and then the integrity and quality of total mRNA was ascertained by gel electrophoresis and the 260/280 ratio spectrophotometer respectively. The rst strand cDNA synthesized by using the cDNA synthesis Kit Yektatajhiz Company (YTA; Iran) according to the manufacturer's instructions.

RT-PCR analysis
The Real-time RT-PCR was use to investigate the expression of Bcl-2 relative to a housekeeping gene (GAPDH) in different conditions. It was carrying out by using the ABI Step One Real Time Instrument (Applied Bio-systems, USA). All the reactions were performed in triplicate and in a total volume about 12 μl containing 1-2 µl cDNA, 0.5 μl each primer (5 pmol), 5 µl Master-mix (RealQ plus SYBR Green PCR Master Mix, Ampliqon; Denmark) and 4-5 µl water. The RT-PCR machine conditions were, 10 min at 95°C; 45 cycles at 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec.

Tamoxifen preparation
The Tamoxifen citrate salt is an estrogen receptor antagonist. Due to the low solubility of Tamoxifen in aqueous medium, the methanol used to prepare the Tamoxifen (see Additional le 1).

Lipofectamine preparation
The Lipofectamine 2000 (Invitrogen) used in the form of complex with the ASO based on 1:1 (w/w).The Lipofectamine (100 µg/ml) was allowed to complex with antisense oligonucleotides (ASO) (25,125,250,500 and 1000 nM) in serum and antibiotic free medium before dilution and addition to cells.

Nano-complex (Micelles/Niosome) preparation
The nano-complex (micelles/niosome) was prepared based on our previous research with some modi cations (25). The cationic nano-complex comprising polyethylenimine (PEI), tween80, squalene was synthesizing. Tween 80 & squalene was in molecular ratio 1:1 and PEI concentration was according to ratio of PEI to ASO (1). Electron microscopy were use to examine the morphology of the micelles/niosome. The zeta potential, size, and Polydispersity index (PDI) of the vesicles in PBS (pH 7.4) analyzed by DLS (Zetasizer Nano ZS; Malvern Instruments, Malvern, UK) using an argon laser beam at 633 nm and a 90° scattering angle (Table 1).

ASO and Nano-complex (micelles/Niosome) Preparation
The tween80 and squalene were dissolving in chloroform and methanol (ratio 3:2). This emulsion was rotate in vacuum rotary evaporator for 1h at 45°C until, thin lm formed. Thin lm was incubated in 37°C shaker for overnight and then it was dissolved in PBS and was sonicated for 30min. Afterward, to purify lipid particles, it was ltered by 22 micron lters. Finally, ASO was adding to the lipid Nano size particles with molar ratio of 1:1. To enhance electrostatic interaction, the nal emulsion was rotate for 30 seconds and incubated for 30 minutes at 37°C.

ASO, Nano-complex (Micelles/Niosome) and Tamoxifen Preparation
It was same as ASO and micelles/niosome preparation, but had difference in Tamoxifen loading process. Tamoxifen loading process was formulating using by nano-precipitate technique; 3ml of chloroform and 2 ml of methanol (in which 2 mg Tamoxifen was dissolve) were introduce to 2ml aqueous phase drop containing surfactant (Tween 80) and helper lipid (Squalene). The gel retardation assay has shown the ability of ASO packaging by PEI to form PEI/ASO complexes. PEI/ASO at various ratios was prepared. The resulting PEI/ASO various products subjected to 2.5 % agarose gel electrophoresis and were visualize by ethidium bromide staining (Data not shown).

Transfection of the cell lines
Approximately 5×10 5 cells of two breast cancer cell lines (MDA-MB-231 & MCF-7) were incubated in RPMI-1640 with 10% FBS for 24h. Then, the media was replace and the cells were transfected with 125nM, 250nM, 500nM and1000nM of Tamoxifen / ASO / Lipofectamine / ASO and Lipofectamine /ASO, Lipofectamine and Tamoxifen / Niosome/ ASO and Niosome /ASO, Niosome and Tamoxifen respectively. They were incubates again for 4h at 37°C. Each sample had a normal control.

Cellular analysis
The cells were culture in a 96-well plate. The labeled scrambled sequence with 5'-Cy3 was formulating in ASO -PEI -Lipid complex. The internalization observed by confocal uorescent Microscopy (Nikon, Japan).

MTT assay
The MTT {3-[4, 5-dimethylthiazol-2-yl]-2, 5-dipheyl tetrazolium bromide} powder (Sigma Aldrich Company, Germany) was use to evaluate cells viability based on NADPH-dependent cellular Oxidoreductase enzyme. NADPH-dependent cellular Oxidoreductase enzyme revives the Tetrazolium dye (MTT) to its insoluble Formazan, which has a purple color. After an incubation period, it was added 100 µl DMSO (Dimethyl sulfoxide, Sigma Aldrich Company, Germany) to solve insoluble Formazan and its absorbance was measure at 570 nm. The MCF7 and the MDA-MB-231 cell lines were seed at a density of

Statistical analysis
Expression data analysis was carried out by using the LinReg software version 11.0 (http://LinRegPCR.nl) and the Relative expression software tool (REST©) according to the manufacturer's instructions respectively. The graphs designed by Graph pad Prism 8 software. All data expressed as the means ± SEM (standard error of the mean). A one-way ANOVA and t-test performed to determine the statistically signi cant differences among groups. reduced about 30 to 40% respectively. At 500 nM of ASO, decrease expression was up to 50%. At 1000 nM of ASO, decrease expression was quite obvious and close to 70%. The lower concentrations did not show signi cant results (P-value < 0.05) (Fig. 2). The ASO 500 nM selected for further study. The Reverse microscopy was used to examine cell morphology indicating the onset of apoptotic activity, while to show modes of cell death stage, we used Annexin V-FITC/PI staining. At 500 nM of ASO, about 60% of the cells entered into initial phase of apoptosis compared to the control sample (Fig. 3).

Results
The Nano-complex (Micelles/Niosome) cytotoxicity  (Fig. 4). At 500 nM of ASO, Micelles/Niosome and PEI, about 85.2% of the cells entered into initial phase of apoptosis compared to the control sample (Fig. 5).

Discussion
A determined inactivated gene is a good way to understand how and why the gene works. The knockouts methods are time-consuming and costly process. An alternative method is gene silencing. It may do at different levels (transcript, splice, translate and post translate). For example, at the mRNA level, the RNA transcript of a desire gene targeted instead of the gene itself. However, it prevents expression of the target gene, or leads to transcript cleavage and resulting in mRNA degradation. Therefore, expression of the target gene stops. Sometimes, it may create a therapeutic effect. Two main strategies may do it, siRNA and Antisense technology. Although, siRNA is a potential therapeutic strategy, it has entered a lower therapeutic phase than antisense due to its cross-sectional structure and low e ciency in penetrating tissues and entering the cells. Second strategy is the Antisense technology. It used as a method to reduce or suppress gene expression at the mRNA level in the target genes. In this study, the Bcl-2 selected. The Bcl-2 mRNA expression increases in more than 70% of breast cancers. It can lead to further growth of cancer cells. An oligo antisense used to intervene at the mRNA level. An important problem in oligoantisense-based therapies is the stability of oligonucleotides at the target site in the cells. To solve this problem, some investigators used somatic changes of oligonucleotides-based structures such as replacement of phosphate bond with Phosphorothionate, addition of a sulfur atom, base changes, terminal changes, and changes in ribose sugar in carbon No. 2 by methyl group. In this research, we designed an oligo-antisense and involving Phosphorothionate as two wings. Another important problem in oligo-antisense-based therapies is the transfer of oligonucleotides to the target site. We used Lipofectamine 2000 for transport of Bcl-2 mRNA oligo-antisense into the cells. We also applied cationic micelles/niosome as a carrier. Tamoxifen selected as a therapeutic drug to evaluate the effect of our ASO. The designed oligo-antisense functional potency to assess apoptosis and Bcl-2 mRNA reducing expression compared in different groups as Tamoxifen, Naked Antisense, Antisense and Tamoxifen, Antisense and Lipofectamine, Antisense and Tamoxifen and Lipofectamine, Antisense and micelles/niosome, Antisense and Tamoxifen and micelles/niosome. The Real-Time RT PCR and Flow cytometry used to evaluate their effectiveness. On average, in two cell lines of breast cancer, Tamoxifen entered about 27% of the cells into the initial phase of apoptosis compared to the control sample. While, for the naked Antisense was 65%, the Antisense and Tamoxifen was 74%, the Antisense and Lipofectamine was 81%, the Antisense, Tamoxifen and Lipofectamine was 84%, the Antisense and micelles/niosome was 84%, the Antisense, Tamoxifen and micelles/niosome was 90%.

Conclusions
The designed ASO reduced the expression of Bcl-2. It was greater than the effect of the anticancer drug alone (Tamoxifen). It also had the synergistic effect with it. Tamoxifen entered about 27% the cells into the initial phase of apoptosis while ASO and Tamoxifen was 74%. The effectiveness of designed ASO would be different with various carriers. ASO and Lipofectamine entered about 81% the cells into the initial phase of apoptosis while ASO and micelles/niosome was 84%. The micelles/niosome e ciency was better than the Lipofectamine for transport of ASO into the cancer cells. It seems combination therapeutic approaches based on drug and gene therapy reveal a new vision in treatment of cancer.
-Consent for publication All authors agree to publish this study.
-Availability of supporting data All data and supporting data are available.

-Competing interests
There is no con ict of interest between the authors. -Funding The Vice Chancellor for Research and Technology of Golestan University of Medical Sciences provided the fund of this study.
Contract number is 120320.
Dr. Ali Akbar Saffar Moghadam received the grant.
-Authors' contributions The Mohsen Sedaghat Janaghard conducted the experiments. He also wrote the initial draft of the article.
The Vahid Erfani-Moghadam was adviser in the eld of nano-carriers. He also helped in the article.
The Ali Akbar Saffar Moghadam was supervisor. He designed the study and wrote the article. He also is corresponding author.
-Acknowledgements Tables   Table 1:  The Tamoxifen apoptosis in the MCF-7 cell line.  The Bcl-2 expression level in the MCF-7 cell line after 48 hr with different ASO, Niosome and PEI complex (Mean± Standard error, N=three, Statistical signi cance: P -value * less than 0.05, ** less than 0.01, *** less than 0.001).

Figure 5
The ASO, Niosome and PEI apoptosis in the MCF-7 cell line.