Combined Bacterial Antigen Lipopolysaccharide and Lipoteichoic Acid Increase Cal 27 Oral Cancer Cell Proliferation

Oral biofilms harbour gram-negative bacterial antigen lipopolysaccharide (LPS) involved in oral cancer progression and gram-positive bacterial surface-associated adhesive, lipoteichoic acid (LTA). Thus, we hypothesised that LPS and LTA together would increase the proliferation of cancer cells compared to stimulation by LPS alone. Oral cancer cell lines SCC4, SCC9, SCC25, Cal 27 and the normal oral cell line, OKF6, were studied. The bacterial antigen stimulation indices were determined using the MT Glo assay. Cell proliferation after bacterial antigen stimulation was validated by clonogenic assays. Phosphokinase array, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western blot were employed to study proliferative and apoptotic pathways in bacterial antigen-stimulated cells. Bacterial antigens significantly stimulated Cal 27 (p ≤ 0.001) alone. SCC4 and SCC9 showed negligible stimulation with either antigen, while SCC25 results were comparable to OKF6. The combined antigen stimulation of Cal 27 led to a decrease in phosphorylated p53 and β-catenin and higher PI3K compared to LPS only stimulated cells (p ≤ 0.001). Combined bacterial antigen stimulation results in increased proliferation of Cal 27 cells due to lowering of tumor suppressor proteins and increased tumor proliferation-related proteins. © 2021 Shoba Ranganathan. Hosting by Science Repository. All rights reserved.


I Sample Preparation for Phosphokinase Array
CAL27 cells in 75-cm 2 T flasks were kept in a humidified incubator at 37°C, 5% CO2. After confluence of 80% was reached, the cells were treated with 5 μg/ml of bacterial antigen. Therefore, a total of 8 ml of DMEM (without 10% FBS and 1% P/S) was added to each flask, Specific assays required: 8 ml of 5μg/ml LPS for the LPS treatment; 8 ml of 5μg/ml LTA for LTA treatment; and 4 ml of each of 10 μg/ml LPS 10 μg/ml LTA, for the combined LPS+LTA treatment. The cells were incubated in a humidified incubator at 37°C, 5% CO2 for 72 hrs. After the treatment period, the cells were then detached using trypsin-EDTA for 4 mins. After the desired detachment of the cells, they were centrifuged at 1000 g for 5 mins at 4°C. After the final acquisition of cell pellets, the pellet from each condition was added to 100 μl of lysis solution, made up of 90 μl of RIPA (radioimmunoprecipitation assay buffer containing 20 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4) and 10 μl of protease inhibitor (10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin). The protease inhibitor was added to inhibit proteases from cleaving the total protein. The lysate (cell suspension + lysis buffer) was then kept on ice for 5 mins, sonicated at a speed of 40 kHz for 5-10 secs. The lysates were kept on the ice for a further 30 mins and then centrifuged at 14,000 g for 15 mins at 4°C. After centrifugation, the pellet (containing the cytoplasmic inclusions) was discarded and the supernatant (containing the protein desired for each condition) was transferred to -80°C until it was used for further protein estimation and proteome profiling.

II Bicinchoninic Acid Protein Assay
Pierce BCA Protein Assay kit (Thermo Fisher Scientific) was used to estimate the total amount of protein present in the samples. A series of dilutions of known concentration was prepared from bovine serum albumin (BSA; supplied) and assayed alongside the unknowns and the concentration of each unknown was determined based on a standard curve generated from the known-concentration BSA standards. For protein estimation for the samples of CAL27 (untreated), LPS (treated), LTA (treated) and LPS+LTA (treated), BSA standards were first prepared. Then, a BCA working solution was prepared by mixing Reagent A (sodium carbonate, sodium bicarbonate, Pierce Detection Reagent A, sodium tartrate in 0.1 N sodium hydroxide) and Reagent B (Pierce Detection Reagent B). For each well, 200 μl of Reagent A + Reagent B at a 1:50 ratio was added. Ten μl of standard sample and 10 μl of sample (1 μl of sample + 9 μl of diluent) of unknown concentration was added to the BCA working solution, in triplicate, in Greiner 96-well flat-bottomed (transparent) plates. The reaction was then kept in the incubator at 37°C for 30 mins. Absorbance was then recorded using a spectrophotometer at 562 nm with shaking frequency 300 g, double orbital shaking mode and a settling time of 0.2 secs: with a reading direction of bidirectional, horizontal left to right, top to bottom, at a constant temperature of 37°C. The readings from the standards were plotted and estimation of the unknown protein concentration of the samples was done based on the formula for the standard curve, y = Slope * x + offset. Here, y was the absorbance and x were the unknown protein concentration of the samples. After the average blank corrected absorbance (optical density) of the samples were determined, they were multiplied by a factor of 10. Dilutions were used in the assay to ensure that the unknown sample absorbance fell within the range of the standards. Following protein concentration analysis, the samples were kept on ice while reagents were prepared for the proteome profiling assay. All reagents were brought to room temperature prior to the start of the assay.

III Primer Design
The complete gene sequences of target genes were retrieved from Link 1. The Primer blast tool Link 2 from the National Center for Biotechnology Information was then used to design the primers. As the primers were to be used for downstream qPCR amplification, stringent criteria were employed in their design: PCR product size 100-200 bp, 18-22 bp (length of the primer), Tm (melting temperature) > 60°C, and GC content 50-60%. It was also considered that the primers should hybridise to around half the 3'end of one exon and the 5'end of the next exon; that is, one of the primers should be in the exon-exon junction to avoid gDNA co-amplification. Further screening and validation of the specificity of the primers was done using the multi-functional primer analysis software Oligo (version 6.67, Wojciech & Piotr Rychlik, Molecular Biology Insights, USA). Primers that formed 3' terminal dimers, hairpin loops or cross-dimers, and those with palindromes or repeats were excluded. Other primer characteristics such as upper primer and lower primer composition, upper primer and lower primer Tm difference (which should < 4°C for optimum annealing of both primers), negative free energy (∆G) (nucleic acid duplex stability) for all primers to maintain stability, internal stability of primers and primer efficiency were also evaluated to ensure maximum specificity of the primers to achieve the best PCR results. This is because using low-specificity primers may result in high background because of base pairing between a primer's 3' terminus and sites other than the intended target in the DNA (template). Characteristics of the primers used to determine the expression of proliferation protein are shown in (Supplementary Tables  5-7).

IV Detail procedure of Western Blot i Sample Preparation for Western Blot
The protocol for sample preparation was identical to Section S2. The amount of protein amount was estimated following the Pierce BCA protein estimation procedure Section S3. Using the formula, protein concentration = mass/volume, the volume of total protein extract of each sample was determined to ensure 10 μg of total protein was loaded in each well of the gel. NuPAGE LDS sample buffer (4X) was added to the sample at a ratio of 1:3. Each cell lysate in sample buffer was then boiled at 95°C for 5 mins to denature the protein and the tubes centrifuged at 16,000 g in a microcentrifuge for 1 min.

ii Protein Separation by Gel Electrophoresis
After the samples were prepared, they were placed on ice until the gel electrophoresis setup was completed. It was planned to run 10-well gels for samples CAL27 (untreated), LPS treated Cal27, and LPS+LTA treated Cal27. CAL27 (untreated) was the control in the experiment. A Novex Sharp Pre-Stained Protein Standard (Life Technologies) ladder was used for the entire experiment. First, 20X NuPAGE MOPS SDS Running Buffer (Invitrogen) was diluted to produce 1X running buffer compatible with the NuPAGE 10% Bis-Tris mini gels used in the project. Then the samples were loaded onto their respective gels and subjected to electrophoresis at 110 V, 5 W, 0.05 Amp for 2 hours, ensuring that the dye front had run off the bottom of the gel.

iii Electro-Transfer
Transfer of proteins from the gel to the membrane was achieved using the iBlot 2 dry blotting system (Invitrogen). The manufacturer's instructions were followed to configure the iBlot 2. For electro-transfer, the 'P0' program was selected.

iv Blocking and Antibody Incubation
The blots were rinsed with distilled water and then stained with Ponceau S solution (Sigma-Aldrich) to check the transfer quality. Then the membranes were washed three times with Tris-buffered saline-Tween 20 (TBST) buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 10 mins on a rocking platform. The membranes were blocked with 5% skim milk in TBST for 1 hour on a rocking platform and then washed three times with TBST buffer for 10 mins on a rocking platform. They were then incubated with primary antibodies in 5% skim milk overnight on a rocking platform at 4°C. On the following day the membranes were washed three times with TBST buffer for 10 mins on a rocking platform and further incubated with the respective secondary antibody in 5% skim milk for 2 hours on a rocking platform at room temperature. All primary and secondary antibodies were provided by R&D Systems. After incubation, membranes were washed like the previous steps and then the chemiluminescent substrate (ECL mix) (Bio-Rad) was prepared following the manufacturer's instructions and added to the membranes; the chemiluminescent signals were then captured.