Beta Defensin 1 human ≥98% (HPLC), 10% acetonitrile, 0.1% TFA in H₂O, cationic peptides from Sigma product no. SRP3011, lot no. 090202, molecular mass 7.8 kDa and cathelicidin LL37 human ≥95% (HPLC), 10% acetonitrile, 0.1% TFA in H₂O, cationic peptides from GenScript product no. RP-20332, lot no. PE1081804, molecular mass 4495.1 g.mol⁻¹. Defensin and cathelicidin LL37 are positively charged, amphiphatic molecules and preferentially bind to anionic phospholipids from cell membranes with the formation of dynamic peptide-lipid supramolecular pore and cell permeabilization.

The peptides under survey (Defensin and Cathelicidin LL37) were freeze-dried in a RPMI-1640 (Sigma-Aldrich) medium (pH 7.35), in a sterile environment. Consecutive peptide dilutions with RPMI-1640 (Sigma-Aldrich) were used for the two peptides, whereas the working concentrations were 20 µM, 15 µM, and from 10 to 1 µM, ten concentrations for 1 to 1 µM in 100 µl/well with an optimal and constant number of cells by 10⁵ tumor cells/well.

We used two adherent tumor cell lines: HT-29 is a colorectal carcinoma cell line (ATCC^® HTB-38™) and A549 is a human alveolar carcinoma adherent cell line (ATCC^® CCL-185™).

All the tumor cell lines were cultivated in RPMI-1640 medium (Sigma-Aldrich) with 10% FBS (fetal bovine serum; Gibco) (1). Triplicate cell lines (with viability >97%) were prepared for each peptide concentration. The negative control was represented by target cells incubated without peptide. In order to prevent the edge effect and preserve humidity in the plate, only culture medium was pipetted in the edge columns and lines. Their viability was determined after 48 h of incubation at 37°C, 5% CO₂, using the vital dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by MTT Cell Proliferation Assay kit from Cayman, product no. 10009365, lot no. 0518531. Absorbance was measured at 595 nm by reference at wavelength of 620 nm with the FilterMax F5 Mode Microplate Reader spectrophotometer.

The second method for viability was flow cytometry using Annexin V and propidium iodide (PI) from EXBIO by ApoFlowEx^® FITC kit product no. ED7044, lot no. 526846. FACSCanto II cytometer was used for data acquisition and processing using BD FACSDiva 6.12 program (both from Becton Dickinson Biosciences) (8).

Molecular biology techniques were used to detect metabolic changes in tumor cells, by assessing gene expression for particular molecular targets.

RNA extraction from tumor cell cultures was performed by automated methods with the RiboZol™ RNA Extraction Reagent from VWR AMRESCO LLC, lot no. 1587C464. The RNA solution was stored at −20°C until reverse transcription in cDNA was performed. This process was accomplished by using the SuperScript IV RT Enzymes kit from Promega, lot no. 0000361199 with Agilent SureCycler 8800 equipment. The complementary DNA thus obtained may be used immediately or stored for a short time at −20°C or for a longer term at −80°C. The purity of the DNA introduced as matrix is an important parameter for the success of a PCR reaction.

An in-house method of qRT-PCR with SYBR-Green (Promega product no. A6001, lot no. 0000322091) was used. This method requires a melting curve at the end of the amplification stage, which translates into a slowly and incrementally growing temperature gradient (1°C approximately every 2–3 sec) starting from the hybridization temperature of the primers (Table I) up to the temperature of denaturation of the entire reaction mixture.

The fluorescence obtained by melting the reaction products were read in each denaturation stage. Each product resulting in the amplification reaction thus produces a melting peak. Depending on the melting temperature of the product(s) and the number of peaks obtained, the specificity of the reaction may be interpreted (Fig. 1).

The desired amplicons of different concentrations were obtained through RT-PCR amplification, which were compared to the concentration of GAPDH extracted from each cell line used in the experiment. Results were reported as percentages of increase or decrease of the gene expression compared to the control, as compared to the reference gene (GAPDH): % gene = number of amplicon genes/µl/reference gene (GAPDH) ×100.

For the study we used GraphPad Prism 5, t-test and Excel method of calculation for gene expression percent. Data analysis results are presented as the mean ± S.E.M. Means of 2 continuous normally distributed variables were compared by independent samples Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

For the HT-29 tumor line, when the defensin concentration was 4 µM, the MTT colorimetric technique revealed intense cytotoxicity (P<0.001), which was also demonstrated by flow cytometry, when a 33.4% apoptosis was obtained after 48 h incubation (Fig. 1). Cytotoxicity was also significant when incubation occurred at the high 15 µM and 20 µM concentrations. This was revealed by both the MTT method (P<0.001) and by the flow cytometry method, with 57% apoptosis at a defensin concentration of 15 µM, and only 10% for the control (cells that were not incubated with the peptide).

For the A549 tumor line, when the defensin concentration was 4 µM, the MTT colorimetric technique revealed intensive statistically significant cytotoxicity (P<0.001), which was also demonstrated by flow cytometry, when a significant 52% apoptosis was obtained after 48 h incubation (Fig. 2). Cytotoxicity was also intensive when incubation occurred at the high 15 µM and 20 µM concentrations. This was revealed by both the MTT method (P<0.001) and by the flow cytometry method, with 32% apoptosis and 10% cell mortality rate.

Addition of cathelicidin LL37, after 48 h incubation, by flow cytometry revealed significant apoptosis only for the high peptide concentrations (15 µM). Of these cells 83.67% were in late apoptosis compared to the incubation of these cells at 4 µM concentrations, for which the behavior of the cells was similar to that of the negative control.

The experimental findings achieved for the HT-29 and A549 tumor cell lines, used molecular biology techniques to determine the gene expression of some molecular targets involved in the metabolic reprogramming of the tumor cell both in the presence and in the absence of peptides with tumoricidal potential (defensin and cathelicidin LL37). These molecular targets refer to genes involved in the pro-apoptotic pathway (CHOP, XBP1, IRE1α, PERK) and anti-apoptotic pathway (BCL2), as well as to genes that stimulate cell proliferation (NRF2) and survival (AKT, HIFα, PIK3).

The analysis of the findings revealed a significant increase (80%) in the CHOP gene expression in the HT29 line incubated with 4 µM defensin for 48 h (HT4), compared to the control (HT0), and 2% increase for the A549 line incubated with 4 µM defensin for 48 h (A4), compared to control (A0). The increase in CHOP gene expression was 16% in non-peptide cells 24 h after incubation, and there was no change in the cells incubated with 15 µM peptide for 24 h.

The analysis of these findings revealed a significant increase (302.67%) in the AKT gene expression in the HT29 line incubated with 15 µM defensin for 48 h (HT15), compared to the control (HT0), for which the increase reached 92.04% compared to the ABL reference gene. The increase for the A549 line incubated with defensin was 76.48% (A15) compared to A0.

For the NRF gene expression in the HT29 line, a 2-fold decrease in gene expression in the 15 µM defensin-treated tumor over the untreated line was observed. Also, approximately fourfold decrease was observed in the A549 line treated with 4 µM defensin incubated for 48 h (A4), compared to the control (A0).

The XBP gene expression presented a highly significant 45-fold increase in the gene expression in the 4 µM defensin-treated HT29 line and a 15-fold increase for the 15 µM defensin concentration compared to the untreated line. Furthermore, the increase was 248.32% in the A549 line incubated with 15 µM for 48 h (A15), compared to the control (A0, for which the increase was 94.2% compared to the ABL reference gene), showing, a 3-fold increase in the gene expression in XBP.

The findings for the PERK gene expression revealed a 2-fold decrease in the gene expression only in the A549 line incubated with 4 µM concentrated peptide, and showed no expression whatsoever at higher peptide concentrations. Gene expression is absent for the HT29 defensin-incubated line compared to the control that expressed this gene.

CHOP gene expression for the HT29 cell line decreased 1.5-fold over the control and 300-fold as a percentage relative to the reference gene ABL at 4 µM cathelicidin LL37 concentration. It also decreased significantly against the control at 15 µM concentration. This indicates that under toxic conditions, the CHOP gene that is involved in the pro-apoptotic pathway of the cell decreases its expression and thus has an anti-apoptotic effect. Compared to line A549, for which CHOP gene expression is 3-fold higher at 4 µM and only 2-fold higher at 15 µM. This indicates that, at lower concentrations of cytotoxic peptide, the neoplastic cell evolves towards apoptosis faster than at high concentrations (Fig. 3).

The BCL2 gene expression is involved in the anti-apoptotic pathway of the cell, i.e., in supporting its survival and therefore, in toxic conditions, its gene expression should decrease (Fig. 3).

When cathelicidin LL37 occurred in its living environment, the HT29 line showed a 4.5-fold increase at 4 µM concentrations and a 16-fold increase at 15 µM concentration of the gene expression compared to the control, which means improved cell defense. As for the A549 line, a significant 13-fold decrease was noted in gene expression at 4 µM and a 19-fold decrease at 15 µM, showing poorer cell defense. This proves that cathelicidin LL37 has a tumoricidal effect on the A549 cell line.

The manner in which cathelicidin LL37 influenced cell proliferation was determined by assessing the expression of the NRF2 gene, which presented a 274-fold decrease at 4 µM concentration and total suppression at 15 µM concentration, in the HT29 line. Therefore, although it triggers an improvement in cell defense by modifying pro- and anti-apoptotic gene expression, this peptide significantly reduces or blocks tumor cell proliferation. For the A549 line, a similar evolution was noted, as NRF2 gene expression decreased 58-fold at 4 µM concentration and 46-fold at 15 µM concentration (Fig. 3).

AKT gene expression decreased dramatically in both cell lines, namely over 80 times regardless of the peptide concentration (Fig. 4). This meant that cell survival and proliferation was intensely inhibited regardless of the peptide concentration, for both cell lines.

Our findings also revealed the presence of RE stress in the tumor cells. Expression of IRE1α and PI3K genes was not high for control tumor cells, only for peptide-incubated cathelicidin LL37, which indicates the presence of stress in the peptide-incubated tumor cells (Fig. 4).