Antitumorigenic effect of insect-derived peptide poecilocorisin-1 in human skin cancer cells through regulation of Sp1 transcription factor

Malignant melanoma is highly resistant to conventional treatments and is one of the most aggressive types of skin cancers. Conventional cancer treatments are limited due to drug resistance, tumor selectivity, and solubility. Therefore, new treatments with fewer side effects and excellent effects should be developed. In previous studies, we have analyzed antimicrobial peptides (AMPs), which showed antibacterial and anti-inflammatory effects in insects, and some AMPs also exhibited anticancer efficacy. Anticancer peptides (ACPs) are known to have fewer side effects and high anticancer efficacy. In this study, the insect-derived peptide poecilocorisin-1 (PCC-1) did not induce toxicity in the human epithelial cell line HaCaT, but its potential as an anticancer agent was confirmed through specific effects of antiproliferation, apoptosis, and cell cycle arrest in two melanoma cell lines, SK-MEL-28 and G361. Additionally, we discovered a novel anticancer mechanism of insect-derived peptides in melanoma through the regulation of transcription factor Sp1 protein, which is overexpressed in cancer, apoptosis, and cell cycle-related proteins. Taken together, this study aims to clarify the anticancer efficacy and safety of insect-derived peptides and to present their potential as future therapeutic agents.


PCC-1 inhibits the viability of malignant melanoma cells.
To analyze the cancer cell-specific cell viability of the peptide PCC-1 selected through the transcriptome analysis of Poecilocoris lewisi, SK-MEL-28, G361, and HaCaT cells were treated with various concentrations of PCC-1. The MTS assay was used to compare cell viability of untreated (control) skin cancer cells and normal cells at two time points (24 and 48 h), as shown in Fig. 1A,B. Both skin cancer cell lines showed decreased viability with increasing treatment concentration, which did not change significantly between the two time points (24 and 48 h). However, the decrease became significant as the treatment concentration increased over time. In addition, the IC50 values of SK-MEL-28 and G361 cells were 50.8 and 57.8 μM, respectively. However, the HaCaT cell line did not show a significant change when exposed to 80 μM PCC-1, showing the greatest decrease in cell viability. These results were verified by analyzing the morphological changes (Fig. 1C). SK-MEL-28 and G361 cell lines were treated with PCC-1 at 20, 40, and 80 μM for 48 h. The number of cells decreased, and the cells became more rounded. In contrast, HaCaT cells showed no change in morphology when PCC-1 was treated at 80 μM for 48 h.

PCC-1 increases apoptosis in malignant melanoma cells.
In SK-MEL-28 and G361 cell lines, the apoptosis effect was increased by PCC-1 treatment, and this result was verified in the image of morphological changes. It was confirmed that apoptosis was induced by PCC-1 in SK-MEL-28 and G361 cell lines using DAPI staining analysis, which specifically stains the cell nucleus. Compared to the control nuclei, the cells cultured for 48 h after treatment with PCC-1 showed fragmented and condensed nuclei as the concentration of PCC-1 increased (20,40, and 80 μM) On the other hand, morphological and nuclear changes were not observed in HaCaT cell lines when PCC-1 was treated at a concentration of 80 μM for 48 h ( Fig. 2A,B). In addition, the apoptotic effect of PCC-1 on SK-MEL-28 and G361 cells was analyzed after 48 h using Annexin V staining (Fig. 2C,D). In Fig. 2C, the average percentage of early apoptotic cells in the SK-MEL-28 group was 0 ± 0.1, 15.7 ± 1.1, 23.9 ± 1.2, and 16.2 ± 2.1%, respectively, as the concentration of PCC-1 increased, and the ratio of dead cells and late apoptotic cells was, on average, 0.1 ± 0.06, 20.6 ± 0.15, 33.4 ± 0.19, and 59.5 ± 1.26%, respectively. The proportion of live cells in the control group was 98.4%, which decreased to 63.1, 42.24, and 24.28% as the treatment concentration increased. Figure 2D shows the proportions of the G361 group. The average percentage of cells in the initial apoptosis phase was 1.1 ± 0.3, 35.5 ± 1.6, 31 ± 1.9, 19.6 ± 1.6%, respectively, as the concentration of PCC-1 increased. The average percentage of dead cells and late apoptotic cells was 0.5 ± 0.03, 10.8 ± 0.64, 23.4 ± 2.61, and 52.6 ± 1.39% as the treatment concentration increased. The proportion of live cells in the control group was 96.9%, which decreased to 53.41, 45.4, and 27.25% as the treatment concentration increased. As the treatment concentration increased, the total apoptotic cell population increased, including dead, early and late apoptosis.

PCC-1 regulates Sp1 protein levels in melanoma cells.
The transcription factor Sp1 has been studied that it is involved in cell cycle progression and apoptotic cell death [32][33][34][35] . The level of reduction in the expression of Sp1 according to the treatment concentration of PCC-1 analyzed through Western blot was evident. As the treatment dose of PCC-1 increased, the level of Sp1 protein (related to β-actin levels) decreased in both SK-MEL-28 and G361 cell lines. On the other hand, in the HaCaT cell line, when PCC-1 was treated at a concentration of 80 μM for 48 h, the protein level of Sp1 did not change (Fig. 3A, Supplementary Fig. 1). The comparison in the same amount of protein was ensured through a constant β-actin level, in particular the level of Sp1 expression decreased significantly when the treatment concentration of PCC-1 was 80 μM. In addition, it was confirmed through real-time PCR analysis that the level of Sp1 mRNA was decreased compared to the level of GAPDH mRNA. On the other hand, in the HaCaT cell line, when PCC-1 was treated at a concentration of 80 μM for 48 h, the mRNA level of Sp1 did not change (Fig. 3B). Consequently, in both Western blot and real-time PCR analysis, the levels of Sp1 protein and genes decreased as the treatment concentration of PCC-1 increased. Furthermore, it was confirmed that various apoptosis-related proteins such as caspase 3, PARP, and each truncated form, including Sp1, were time-dependently regulated when the concentration of PCC-1 was 80 μM (Fig. 3C, Supplementary  Fig. 1). In addition, in order to analyze in more detail the involvement of PCC-1 in apoptosis, two cancer cells were treated with z-DEVD-fmk, which inhibits caspase3, and PCC-1. As a result, it was confirmed that cleaved caspase3 was not expressed after caspase3 was inhibited (Fig. 3D, Supplementary Fig. 1). We also inhibited translation using cycloheximide (CHX) to analyze the pathway by which PCC-1 induces apoptosis in skin cancer cell lines. As a result, it was confirmed that PCC-1 was involved in extrinsic apoptosis pathway through Sp1 protein regulation after translation was inhibited (Fig. 3E, Supplementary Fig. 1). Based on the above results, immuno- www.nature.com/scientificreports/ cytochemical analysis in SK-MEL-28 and G361 cell lines confirmed a decrease in the Sp1 level and an increased level of cleaved caspase3 in a concentration-dependent manner of PCC-1 (Fig. 3F). This study concludes that PCC-1 treatment in skin cancer cells leads to apoptosis through inhibition of Sp1.

PCC-1 regulates cell cycle arrest and migration in melanoma cells.
In SK-MEL-28 and G361 cell lines, an analysis was performed on how the treatment of PCC-1 affects the cell cycle. During cell proliferation, G1/G0, S, and G2/M each represent different steps. Through analysis of the DNA content of control cells and PCC-1 treated (20, 40, and 80 μM) cells for 24 h after PCC-1 treatment, it was possible to determine the minimum time required for sufficient cell replication (Fig. 4A,B). Two typical G1/G0 and S peaks were observed in the control cells of both cell lines (Fig. 4A). As the treatment concentration of PCC-1 increased, a sub-G1 phase was appear indicating the formation of debris due to apoptosis. As the concentration of PCC-1 increased from 20 to 80 μM, the percentage of cells in the sub-G1 phase gradually increased with the change in the total percentage of cell cycling (such as G1/G0, S, and G2/M). In addition, when treated with 20 μM of PCC-1 compared to the control cells, the percentage of cells in the G1/G0 phase increased to 68.45% and 58.64%, respectively, for SK-MEL-28 and G361. This result means that PCC-1 is treated so that cells in the G1/G0 phase cannot proceed to www.nature.com/scientificreports/ the subsequent S phase, which induces cell cycle arrest. Then, the proteins p53, p21, p27, and cyclin D1 involved in advancing the cell cycle to the next phase in G1/G0 were analyzed (Fig. 4C, Supplementary Fig. 1). Expression of negative cell cycle regulatory proteins such as p53, p21 and p27 increased with time from 0 to 48 h in both cell lines, SK-MEL-28 and G361, respectively, after treatment with 80 μM PCC-1. In contrast, the levels of cyclin D1, a positive cell-cycle regulation protein, decreased as time elapsed. The time-dependent regulation of protein expression in cell cycle-related proteins indicates that cell cycle arrest and apoptosis are caused by PCC-1 treatment. Figure 4D shows the proliferation rate of cell migration when PCC-1 was treated with 30 μM, which is similar to that observed in the in vitro metastasis. The two control cell lines SK-MEL-28 and G361 almost recovered 72 h after scratching the center of the monolayer, but the PCC-1 treated group did not recover for the same time. In contrast, the group treated with PCC-1 increased by about 20% after 24 h, and the recovery area gradually increased after 48 h and 72 h, but compared with the control group, the group treated with PCC-1 for 72 h Only about 45% of the rate was recovered (Fig. 4D,E). Based on these results, it was confirmed that when PCC-1 was treated with SK-MEL-28 and G361 cell lines, a certain range of proliferation was inhibited, it can be strongly assumed that this result is directly related to the expression of the various proteins analyzed above.

Discussion
Peptides have various therapeutic effects, and among them, they have been attracting attention for their excellent safety and anticancer efficacy. In particular, discovering therapeutic peptides for the development of anticancer drugs is a novel and promising approach 8,9 . ACPs are divided into antimicrobial/pore-forming peptides, cellpermeable peptides, and tumor-targeting peptides groups according to their main mechanisms of action 28 . AMPs (antimicrobial/pore-forming peptides) has specific biological activity and occurs naturally in all living organisms 10 . They have the potential as antimicrobial agents such as defensins and cathelicidins, and are part of an innate immune defense mechanism 36,37 . These AMPs form an amphiphilic structure in a non-polar solvent, and most of them are short and have a cationic charge 38 . They disrupt function through electrostatic interactions that bind to negatively charged bacterial cell membranes, leading to cell death 38,39 . Pore-forming peptides that induce cell death through such necrosis or apoptosis target cancer cell membranes. In necrosis, AMP destroys the mitochondrial membrane to induce apoptosis and causes cell lysis by targeting negatively charged molecules in the cancer cell membrane 28 . One of the AMPs with these properties, the insect-derived peptide CopA3, inhibits the growth of pancreatic and liver cancer cells and has antibacterial activity 16,29 . This study is a representative study of synthetic peptides based on bioactive substances occurring in nature with potential as novel anticancer agents of AMPs that exhibit inhibitory activity against microorganisms.    30 . PCC-1 is a peptide derived from Poecilocoris lewisi, which has excellent antibacterial and antifungal effects. Moreover, PCC-1 inhibits NO secretion and the expression of IL-6, iNOS, and COX-2Therefore, PCC-1 has potential as a therapeutic agent for various inflammatory diseases (Fig. 1A) 50 . As the first step to elucidate the anticancer efficacy of PCC-1, melanoma cells (SK-MEL-28 and G361 cell lines) were treated with the peptide, and the cytotoxic and cancer cell proliferation inhibitory effects were confirmed at this time. However, the human epithelial cell line HaCaT did not show toxicity, and through MTS analysis, it was confirmed that PCC-1 has anticancer efficacy specifically for skin cancer cells (Fig. 1B,C). Sp1 represents an essential promoter in cancer cells and plays an important role as a basal transcription factor. Numerous studies on Sp1 in various cancer cells have been reported 3,31,[40][41][42][43] . In addition, Sp1 expression levels are higher in cancer cells than in normal cells, and the growth and metastasis of cancer cells are weakened in nude mice in which Sp1 is knocked out 44,45 . Therefore, lowering Sp1 levels is a good strategy to prevent tumor cell growth. However, the anticancer effects of Sp1 using the insect-derived peptide PCC-1 have not been reported yet. To the best of our knowledge, this is the first study to demonstrate the anticancer effects of PCC-1.
To analyze the anticancer effect of PCC-1 on the melanoma cell lines, the results of the untreated cells and cells treated with various concentrations of PCC-1 were compared. Furthermore, Sp1 protein is closely related to apoptosis and cell cycle, and changes in the expression levels of related regulatory proteins were observed. For this, DAPI staining and Annexin V analyses were performed to classify the different stages of apoptosis based on cell morphology (early apoptosis, late apoptosis, and dead cells). As the concentration of PCC-1 increased, abnormal DNA fragmentation was observed with DAPI staining in both SK-MEL-28 and G361 cell lines ( Fig. 2A,B). In addition, total apoptosis increased, and the percentage of living cells decreased with increasing treatment The results of flow cytometry were quantified. Data represent the mean ± standard deviation (SD) (n = 3; *p < 0.05). (C) SK-MEL-28 and G361 cells were treated with PCC-1 (80 μM) and examined at 12, 24, and 48 h, and whole-cell extracts were prepared, separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and subjected to western blotting using p53, p21, p27, and cyclin D1 antibodies. Actin was employed as a loading control. The results represent three independent experiments. Western blot analysis data complied with the digital image and integrity policy, and blots exposed from the same gel were used. (D) SK-MEL-28 and G361 cells were treated with PCC-1 (30 μM), and confluent cells were carefully scratched using sterile pipette tips and then re-cultured with or without PCC-1. At 24, 48, and 72 h, the cells were photographed under a microscope. (E) The results of the migration assay were quantified. Data represent the mean ± SD (n = 3; *p < 0.05). www.nature.com/scientificreports/ concentrations (Fig. 2C,D). These apoptotic effects were analyzed by western blotting and real-time PCR analysis (Fig. 3A,B), and the expression levels of Sp1, caspase 3, PARP, cleaved caspase 3, and cleaved PARP were consistent with and provided detailed insights into the cell viability test results (Fig. 3C,F). Additionally, we inhibited caspase3 through z-DEVD-fmk to determine how PCC-1 affects the apoptosis pathway. In skin cancer cell lines, cleaved caspase3 was not expressed when PCC-1 was treated in a state where caspase3 was inhibited, and from this result, it was confirmed that PCC-1 was involved in the apoptosis pathway of cancer cells (Fig. 3D). In addition, in order to check which pathway (intrinsic or extrinsic) PCC-1 regulates Sp1 in skin cancer cell lines, translation was inhibited through CHX and then PCC-1 was treated. As a result, it was confirmed that PCC-1 is involved in the extrinsic apoptosis pathway through the regulation of Sp1 protein after translation is inhibited. However, considering that PCC-1 regulates the mRNA level of Sp1, it is presumed that PCC-1 acts directly or indirectly in the intrinsic or extrinsic pathway (Fig. 3E).
Based on the result that increasing PCC-1 concentration induced apoptosis in melanoma cells, the expression of Sp1 target proteins, including p53, p21, p27, and cyclin D1 46 , were investigated in terms of cell cycle. Cell cycle arrest was analyzed based on measurements of DNA content at specific stages of the cell cycle, and the number of cells in the sub-G1 phase increased with increasing dosing time (Fig.4A,B). The p53, p21, and p27 proteins are negative regulators of the cell cycle 47,48 , and the kinase of the protein is upregulated, resulting in cell cycle arrest that blocks progression to the next phase (Fig. 4C). In contrast, cyclin D1, which is involved in tumor formation, is a positive modulator 49 . Thus, positive and negative modulators exhibited cell cycle arrest and increased the duration of the lower G1 phase. Lastly, through wound healing analysis, a direct wound was caused to the cancer cells, and when PCC-1 was administered to the melanoma cells, the recovery ability of the cancer cells was suppressed compared to the control group (Fig. 4D,E).
In conclusion, our study demonstrated that the insect-derived peptide PCC-1 induces cell proliferation inhibition, apoptosis, and cell cycle arrest by downregulating Sp1 expression in the melanoma cell lines SK-MEL-28 and G361. The results of this study support the anticancer efficacy of PCC-1, which regulates Sp1 in human cancer cells, for clinical application. Furthermore, we are currently researching on the possibility of a safe and effective peptide that can be applied clinically by studying the interaction between the peptide and an immune checkpoint inhibitor based on the results of this study.
SK-MEL-28 and G361 cells treated with PCC-1 were harvested by trypsinization and fixed in 100% methanol at room temperature (RT) for 20 min. The cells were seeded onto slides, stained with DAPI (2 μg/mL), and monitored using an inverted fluorescence microscope (Korea Lab Tech, KI-3000F, Gyeonggi-do, Korea). DAPI staining analysis was performed as previously described 50 . Annexin V staining. SK-MEL-28 (3 × 10 5 cells/well) and G361 (3 × 10 5 cells/well) cells were seeded into sixwell plates and cultured overnight. After cells were treated with different concentrations (20,40, and 80 µM) of PCC-1 for 48 h, they were stained with Muse™ Annexin V and Dead Cell reagents (Muse™ Apoptosis Assay kit, EMD Millipore). Stained cells were analyzed using the Muse™ Cell Analyzer. Annexin V staining analysis was performed as previously described 50 . Cell-cycle analysis. Cells were seeded into six-well plates and exposed to PCC-1 at different concentrations (20,40, and 80 µM) for 24 h. Cells were harvested and washed three times with phosphate-buffered saline (PBS). The harvested cells were fixed in 70% ethanol at 20 °C overnight. Cells were washed with PBS, mixed with 200 µL of Muse™ cell-cycle reagent (EMD Millipore, Billerica, MA, USA), and incubated at RT for 30 min in the dark.