Effect of Sodium Butyrate on p16INK4a, p14ARF, p15INK4b, Class I HDACs (HDACs 1, 2, 3) Class II HDACs (HDACs 4, 5, 6), Cell Growth Inhibition and Apoptosis Induction in Pancreatic Cancer AsPC-1 and Colon Cancer HCT-116 Cell Lines

Background: In higher eukaryotes, cell-cycle transitions are regulated by different cyclin-dependent kinases (Cdks) and Cdk inhibitors (CKIs). CKIs include two groups, the Ink4 (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the Cip/Kip (p21Cip1, p27Kip1, and p57Kip2) families. The hyperactivity of histone deacetylases (HDACs) is associated with cancer induction. Histone deacetylase inhibitors (HDACIs) such as sodium butyrate (NaBT) can inhibit HDAC activity resulting in apoptosis induction. The present study was designed to investigate the effect of sodium butyrate on p16INK4a, p14ARF, p15INK4b, class I HDACs (HDACs 1, 2, 3), and class II HDACs (HDACs 4, 5, 6), cell growth inhibition, and apoptosis induction in pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines. In fact, we want to know whether sodium butyrate can reactivate Ink4 and Cip/Kip families by HDACs inhibition. Materials and Methods: The AsPC-1 and HCT-116 cells were treated with sodium butyrate at different periods. Then, the MTT assay, cell apoptosis assay, and qRT-PCR were done to determine viability, apoptosis, and the relative expression level of the genes respectively. Results: The sodium butyrate increased p16INK4a, p14ARF, and p15INK4b and decreased class I and II HDACs significantly. Besides, HCT-116 cell was more sensitive to sodium butyrate in comparison to AsPC-1 cell. Conclusion: The sodium butyrate can reactivate the p16INK4a, p14ARF, and p15INK4b through inhibition of HDACs in AsPC-1 and HCT-116 cell lines.


Introduction
In higher eukaryotes, cell-cycle transitions are regulated by different cyclin-dependent kinases (Cdks), the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases, and their activating cyclin subunits and Cdk inhibitors (CKIs) (Hochegger et al., 2008). Cyclins are accumulated gradually during interphase and are abruptly destroyed during mitosis. Based on the timing of expression in the cell cycle, cyclins are divided into four classes three of which are the G1/S cyclins, S cyclins, and M cyclins, these three groups are directly involved in the cell cycle control. The fourth class, the G1 cyclins, controls the entry into the cell cycle in response to extracellular growth factors or mitogens (Yang et al., 2018). Cdks act as the engine of the cell cycle that drives cell cycle progression whereas cyclins play as the gears. Whereas, CKIs serve as brakes to halt cell cycle progression under abnormal conditions. CKIs include two groups based on their structure and Cdk specificity, the Ink4 and the Cip/Kip families. The Ink4 family members including p16INK4a, p15INK4b, p18INK4c, and p19INK4d which primarily target Cdk4 and Cdk6. Conversely, the Cip/Kip family members including p21Cip1, p27Kip1, and p57Kip2 which interfere with the activities of cyclin D-, E-, A-and B-dependent kinase complexes (Lim et al., 2013). Cellular neoplastic transformation is accompanied with aberrant expression of CDKs and/or cyclins and also the negative regulators, the CDKIs. Of all epigenetic modifications, DNA hypermethylation, which represses tumor suppressor genes (TSGs) such as the Ink4 and the Cip/Kip families leading to gene silencing, has been recognized as a cause of oncogenesis. DNA methylation is achieved by a group of enzymes known as the DNA methyltransferases (DNMTs) which are classified into DNMT1, DNMT1b, DNMT1o, DNMT1p, DNMT2, DNMT3A, DNMT3b with its isoforms, and DNMT3L (Sanaei et al., 2020). In addition to DNA methylation, histone deacetylation is associated with transcriptional

Materials
Human pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines were provided from the National Cell Bank of Iran-Pasteur Institute. Dulbecco's modified Eagle's medium (DMEM) and sodium butyrate were obtained from Sigma (St. Louis, MO, USA). Both compounds were dissolved in dimethyl sulfoxide (DMSO) to make a master stock solution. Further concentration was obtained by diluting the provided solution. Several compounds including materials and kits were purchased as provided for previous works Kavoosi et al., 2018). All other compounds including fetal bovine serum (FBS) (product number f2442), penicillin (CAS number 69 57 8), and streptomycin (CAS number 3810 74 0) obtained from sigma too. Total RNA extraction kit (TRIZOL reagent) and real-time polymerase chain reaction (PCR) kits (qPCR MasterMix Plus for SYBR Green I dNTP) were obtained from Applied Biosystems Inc. (Foster, CA, USA). The cells were maintained in DMEM supplemented with fetal bovine serum 10% and antibiotics in a humidified atmosphere of 5% CO 2 in air at 37 o C. This is a lab-trial study was approved by the Ethics Committee of Jahrom University of Medical science with a code number of IR.JUMS.REC. 1399.010.

Cell culture and cell viability
Human pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines were cultured in DMEM supplemented with 10% FBS and antibiotics at 37°C in 5% CO 2 overnight. After 24 h of culture, the cells were seeded into 96-well plates (3 × 10 5 cells per well). After one day, the growth medium was changed with the experimental medium containing various concentrations of sodium butyrate (0, 1, 5, 10, 25, and 50 μM). The control groups received DMSO only, at a concentration of 0.05 %. After 24 and 48h of treatment, the cells were evaluated by MTT assay according to Standard protocols to determine cell viability. In this regard, the MTT solution was added to each well for 4 h at 37 o C, the MTT solution was replaced by DMSO and shaken for 10 min to dissolve all of the crystals. Finally, the optical density was detected by a microplate reader at a wavelength of 570 nM. Each experiment was repeated three times (triplicates).

Cell apoptosis assay
To determine apoptotic cells, the pancreatic cancer AsPC-1 and colon cancer HCT-116 cells were cultured at a density of 3 × 10 5 cells/well and incubated overnight, and then the cells were treated with sodium butyrate (10 μM) for 24 and 48 h. Subsequently, the cells were harvested by trypsinization, washed with cold PBS, and resuspended in Binding buffer (1x). Finally, Annexin-V-(FITC) and PI were used according to the protocol to determine the apoptotic cells by FACScan flow cytometry (Becton Dickinson, Heidelberg, Germany).

Result of cell viability by the MTT assay
The viability of pancreatic cancer AsPC-1 and colon cancer HCT-116 cells treated with sodium butyrate (0, 1, 5, 10, 25, and 50 μM) was investigated by MTT assay. In this technique, the activity of cellular enzymes produced a dark-blue formazan which is dissolvable in DMSO by which the number of viable AsPC-1 and HCT-116 cells can be demonstrated. As shown in Figure 1, sodium butyrate induced significant cell growth inhibition with a dose-and time-dependent manner (P< 0.004), figure  24 and 48 h and then stained using annexin-V-(FITC) and PI to determine apoptotic cells in the early and late apoptosis stage. As indicated in Figures 2 and 3, sodium butyrate induced cell apoptosis significantly in a timedependent manner (P<0.001). The HCT-116 cells were more sensitive to sodium butyrate in comparison to 1. The IC 50 value of sodium butyrate was obtained with approximately 10 μM.

Result of cell apoptosis assay
To determine AsPC-1 and HCT-116 cells apoptosis, the cells were treated with sodium butyrate (10 μM) for   (24 and 48h). The cells were treated with sodium butyrate for 24 and 48h and then the apoptotic effect of sodium butyrate was evaluated by flow cytometric analysis. Results were obtained from three independent experiments and were expressed as mean ± standard error of the mean.

Discussion
Histone deacetylases catalyze the removal of acetate from modified lysine residues. The activity of these enzymes can induce cancer by deacetylation of TSGs, such as p16INK4a, p14ARF, and p15INK4b, resulting in silenced genes and tumorigenesis. Fortunately, reversible acetylation occurs on specific lysines by histone deacetylase inhibitors such as sodium butyrate leads to reactivation of silenced TSGs and apoptosis induction (Sanaei et al., 2021). In this study, we reported that sodium butyrate down-regulated class I histone deacetylases 1, 2, and 3 gene expression and class II HDACs (HDACs 4,5,6) genes, up-regulated p16INK4a, p14ARF, and p15INK4b, inhibited cell growth and induced apoptosis in pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines. Similarly, it has been shown that sodium butyrate induces cell growth arrest and apoptosis in human HCC cell lines HCC-M and HCC-T (Roberts et al., 2005), and pancreatic ductal adenocarcinoma (PDAC) cell lines IMIM-PC1, IMIM-PC2, and RWP-1 Schneider et al., (2010). As we reported in this study, other researchers have demonstrated that HDACIs activate a member of the INK4 family causing G1 phase arrest in the Jurkat human T cell leukemia cells (Yokota et al., 2004). In vitro studies have demonstrated that HDACIs trichostatin A (TSA) and sodium butyrate activate the p15INK4b gene in human immortalized keratinocyte HaCaT cells and human colorectal carcinoma cell line HCT116 (Hitomi et al., 2003). On the other hand, the analysis of p16INK4a and p19ARF mRNA levels has demonstrated that sodium butyrate and TSA modify the levels of these mRNAs in   primary fibroblasts (Matheu et al., 2005). Experimental studies have indicated that sodium butyrate increases p14ARF expression in human lung carcinoma cell line H460, colon carcinoma cell line HCT116, and breast cancer cell line MCF7 (Joseph et al., 2005). In mice, it has been shown that depsipeptide (FK228), a histone deacetylase (HDA) inhibitor, induces the expression of p16INK4a and up-regulated the expression of p21WAF1/Cip1 (Nishida et al., 2004). We observed that butyrate up-regulated p16INK4a, p14ARF, and p15INK4b genes expression. This result encouraged us to evaluate the further molecular mechanism of this agent. Therefore, we investigated the effect of sodium butyrate on class I histone deacetylases (HDAC 1, 2, and 3) and class II HDACs (HDACs 4, 5, 6) gene expression. Our finding demonstrated that sodium butyrate down-regulated class I histone deacetylases (HDAC 1, 2, and 3) and class II HDACs (HDACs 4, 5, 6) gene expression significantly.
Our results are consistent with previously published ones reporting that sodium butyrate inhibits HDAC1, HDAC2, and HDAC3 expression in HCT116 and Caco-2 colon cancer cells (Wilson et al., 2006). In addition to the molecular mechanisms mentioned by our groups, other investigators have reported several pathways for sodium butyrate in various cancers. They indicated that this agent decreases anti-apoptosis gene Bcl-xl and Bcl-2 and increases pro-apoptosis gene Bax and Bak in prostate cancer DU145 and PC3 cell lines (Mu et al., 2013). In colon cancer, this compound can increase p21waf1/cip1 expression, block the activity of cdk-cyclin complexes, and cause cell cycle arrest (Coradini et al., 2000). Further, it increases Fas and Fas ligand levels by which induces apoptosis in breast cancer MCF-7, MCF-7ras, T47-D, and BT-20 cells, as well as an arrest in G2/M in MDA-MB-231 cell lines (Chopin et al., 2002). According to our results, sodium butyrate can upregulate p16INK4a, p14ARF, p15INK4b gene expression by inhibition of HDACs activity resulting in apoptosis induction.
In conclusion, our results indicated that sodium butyrate can reactivate tumor suppressor gene p16INK4a, p14ARF, and p15INK4b through down-regulation of class I histone deacetylases 1, 2, and 3 genes expression and class II HDACs (HDACs 4, 5, 6) gene expression in pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines. The colon cancer HCT-116 cell is more sensitive to sodium butyrate in comparison to the pancreatic cancer AsPC-1 cell. We did not evaluate the protein level of the genes of Ink4 and Cip/Kip families in this study because of budget limitations. Therefore, this evaluation is recommended.