Cell cycle progression data on human skin cancer cells with anticancer synthetic peptide LTX-315 treatment

Skin cancer, including melanoma and non-melanoma (NMSC), represents the most common type of malignancy in the white population [1]. The incidence rate of melanoma is increasing worldwide, while the associated mortality remains stable. On the other hand, the incidence of NMSC varies widely [1,2]. Camilio and collaborators recently described the anticancer properties of LTX-315, a novel synthetic anticancer peptide, commercialized as Oncopore™ [3,4]. Despite various studies demonstrating the efficiency of LTX-315 therapy in inducing cancer cell death, the effects on cell cycle progression of this antitumoral peptide are poorly understood. In this research, we present data about the effect of LTX-315 on the cell cycle of two skin cancer cell lines: epidermoid carcinoma cells (A431) and melanoma cells (A375); as well as on an immortalized normal keratinocyte cell line, HaCaT. Additionally, its cytotoxicity on the cells was determined by measuring the uptake of propidium iodide, in order to establish its relationship with cell cycle progression. The analysed data obtained by flow cytometry show different cell cycle distributions in non-tumoral and skin cancer-derived cell lines in response to LTX-315 treatment. Non-tumoral cells showed a sub-G1 peak, while for tumoral cells there was a shift in the G1peak without producing an obvious distant and distinct sub-G1 peak. This data is in accordance with a major decrease in cell viability in non-cancer cells.


a b s t r a c t
Skin cancer, including melanoma and non-melanoma (NMSC), represents the most common type of malignancy in the white population [1] . The incidence rate of melanoma is increasing worldwide, while the associated mortality remains stable. On the other hand, the incidence of NMSC varies widely [1 , 2] . Camilio and collaborators recently described the anticancer properties of LTX-315, a novel synthetic anticancer peptide, commercialized as Oncopore TM [3 , 4] . Despite various studies demonstrating the efficiency of LTX-315 therapy in inducing cancer cell death, the effects on cell cycle progression of this antitumoral peptide are poorly understood. In this research, we present data about the effect of LTX-315 on the cell cycle of two skin cancer cell lines: epidermoid carcinoma cells (A431) and melanoma cells (A375); as well as on an immortalized normal keratinocyte cell line, HaCaT. Additionally, its cytotoxicity on the cells was determined by measuring the uptake of propidium iodide, in order to establish its relationship with cell cycle progression. The analysed data obtained by flow cytometry show different cell cycle distributions in non-tumoral and skin cancer-derived cell lines in response to LTX-315 treatment. Non-tumoral cells showed a sub-G1 peak, while for tumoral cells there was a shift in the G1peak without producing an obvious distant and distinct sub-G1 peak. This data is in accordance with a major decrease in cell viability in non-cancer cells.
©  • The data provide valuable information on the relationships between cell cycle distribution and cell viability in non-tumoral and skin cancer-derived cells in response to LTX-315 treatment. • Researchers can use the data as additional evidence to investigate the selectivity of the treatment with LTX-315.

Data
Histograms ( Fig. 1 ) and bar graphs ( Fig. 2 ) showed that treatment with peptide LTX-315, compared to non-treated cells, induced a sub-G1 peak in non-tumoral cells; this is a dosedependent effect, the data are statistically different at 75 μM. The distinct sub-G1 peak might represent condensed or fragmented DNA, which was stained with low level DNA fluorescence and further suggests an apoptotic death [5 , 6] . These data are in marked contrast to the effect of the peptide observed in melanoma and epidermoid carcinoma cells, in which no sub-G1 peak is evident. The analyses of the viability data after performing PI uptake assays ( Fig. 3 ) showed that HaCaT cells were more sensitive to LTX-315 treatment than the cancer cells.

LTX
LTX-315 (K-K-W-W-K-K-W-Dip-K-NH 2 , Lot.91799130 0 07/PE4961) was synthesized by the solid-phase method according to the sequence published by Camilio et al. [4] and purchased from GenScript (Piscataway Township, NJ, USA). The purity of the peptide was determined by analytical HPLC to be higher than 95%, and the molecular weight was confirmed with MALDI-TOF mass spectrometry.

Cell culture
Human skin cancer cell lines, A375 and A431, were purchased from American Type Cell Culture (ATCC). The human keratinocyte line HaCaT was employed as normal cell control. Cells were cultured at a final concentration of 2.5 × 10 5 cells/ml in Dulbecco's modified Eagle's medium (DMEM, Gibco). The medium was complemented with 5% fetal bovine serum (FCS, Gibco) and 1% penicillin/streptomycin (Gibco). Cell cultures were incubated at 37 °C with 5% CO 2 .

Treatment outline
Cells were disaggregated using trypsin-EDTA. Cell suspensions were centrifuged at 3500 rpm for 5 min, the supernatant was discarded and cell pellets were plated in 6-well plates at a final concentration of 2.5 × 10 5 cells/ml. After adhesion, cells were treated with different concentrations of LTX-315 (25-75 μM) for 24 h and were then processed for cell cycle and cell viability. Peptide concentrations were selected based on previous studies of LTX-315 on different tumoral cell lines [3 , 7-10] . In these studies, concentrations ranging from 8 to 400 μM were evaluated. However, a maximum concentration of 75 μM was considered appropriate to induce changes in the cell cycle.

Cell cycle analyses
Measuring the DNA content of cells is a well-established method to monitor cell cycle [11] . In order to analyze cell progression through the cycle, exponentially growing cells were exposed to 25 , 50 or 75 μM of LTX-315 for 24 h, washed with PBS, and then disaggregated using trypsin-EDTA. One million cells per milliliter were suspended in ice-cold 70% ethanol and fixed for 1 h at 4 °C. Next, cell suspensions were washed in PBS, treated with 100 μg/ml RNase, and incubated with 100 μg/ml propidium iodide (PI) for 30 min. PI fluorescence of 10,0 0 0 events was analyzed using a BD LSRFortessa flow cytometer. Percentages of cells in each phase were calculated using FlowJo. Each experiment was performed three times.

PI staining (cell viability analysis)
One of the best markers for cell death is membrane integrity. In flow cytometry, plasma membrane permeability is assessed using propidium iodide, a dye that is not permeant to live cells [12] . To assess cell viability after 24 h of 25 , 50 or 75 μM LTX-315 treatments, cells were washed with PBS and disaggregated using trypsin-EDTA. Cell pellets were resuspended, stained with 1 mg/ml PI, and incubated in the dark at room temperature for 20 min. Following this, 10,0 0 0 events were analyzed by flow cytometry in BD LSRFortessa. Fluorescence was quantified using FlowJo. Each experiment was performed three times.

Statistical analysis
Data were expressed as mean ± standard error of the mean. ANOVA analyses were performed using via GraphPad Prism, with post-hoc comparisons carried out by Fisher's protected least significant difference (FPLSD) tests. p ≤ 0.05 was considered statistically significant.