Dermaseptin B2’s Anti-Proliferative Activity and down Regulation of Anti-Proliferative, Angiogenic and Metastatic Genes in Rhabdomyosarcoma RD Cells in Vitro

Background: Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children, representing approximately 50% of pediatric sarcomas and can develop in any part of the body though more frequently at the extremities. Aim: Evaluating the in vitro anti-proliferative activity of Dermaseptin B2 on Rhabdomyosarcoma RD (CCL-136TM) cells and its effect on the expression of MYC, FGFR1, NOTCH1, and CXCR7 genes involve in processes including proliferation, angiogenesis and metastasis. Methods: RD cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% Fetal Bovine Serum. Exponentially growing cells were treated with Dermaseptin B2 and Antiproliferative activity was assayed using the resazurin and migration assays at three time-points. In order to determine the gene expression profiles of MYC, NOTCH1, FGFR1 FGFR1 (fc; 2.3515, 2.0809, 2.2543), NOTCH1 (fc; 2.4667, 4.6274, 4.3352) genes for the three-time points respectively. NOTCH1 and CXCR7 showed higher fold changes with respect to β-Actin than MYC and FGFR1. Conclusion: The results of this study indicate that Dermaseptin B2 is a target molecule for signaling pathways including PI3K/AKT, RTK and NOTCH pathways that could affect the transcription of these genes and overall inhibition of cancer progression. Further studies are needed to give a better understanding of the detailed mechanisms of action as well as the effects of the Dermaseptin B2 peptide in vivo.

MYC proto-oncogene participates in many cellular processes including cell proliferation, differentiation and apoptosis [17]. It is the most frequent proto-oncogene that is amplified in 40% of numerous human cancers [18] [19]. Missiaglia et al. (2009) have reported a high amplification and overexpression of the oncogene MYC in the ERMS (RD) cells and other primary tumors [20]. Targeting and inhibition of MYC have also been shown to reduce the progression and tumorigenicity of RMS and myogenic differentiation [21], indicating MYC as a potential target for rhabdomyosarcoma treatment. Though MYC is under tight regulation in normal cells through various signaling pathways like WNT, Hedgehog and receptor tyrosine kinases, constitutive activation of these pathways results MYC activation in cancer cells [22] [23]. PI3K drug resistance was also associated with MYC amplification illustrating this gene as downstream of PI3K for tumorigenesis [22]. Inhibition of MYC expression, interrupting Myc-Max dimerization and final alteration of Myc-Max binding to DNA is crucial to hinder target MYC genes [18].
The Notch signaling pathway involves regulation and determination of cell destiny and is dysregulated in human cancers [24]. This pathway can be targeted by inhibition of notch receptors using small molecule inhibitors [25]. The enzyme, gamma-secretase leads to the increase in the Notch intercellular domain and overactivation of the Notch pathway followed by transcriptional activation in the nucleus [26]. Belyea et al. (2011) showed using Notch1 RNAi or Ƴ-secretase small molecule inhibitors reduce ERMS growth in vitro [25]. A study by Roma et al. (2011) have shown upregulation of the Notch pathway in fusion positive (FP) and fusion negative (FN) RMS with notable correlation between the invasiveness and motility of RMS cells up on Notch pathways activation [27]. The motility and invasiveness of the cells also reduced with gamma-secretase inhibition in in vitro models [27]. Furthermore, NOTCH1 was shown to cause tumor propagation in embryonal RMS zebrafish models via upregulation of SNAIL1 transcription factor, ultimately leading to dedifferentiation of the embryonal RMS cells [28]. The study also indicated the possible role of NOTCH1/SNAIL1/MEF2C pathway in the metastatic capacity of embryonal RMS [28]. Novel compounds that can inhibit this pathway will form promising therapeutic agents to treat metastatic cancers [29].
Fibroblast growth factor receptor (FGFR) belongs to a signaling pathway that is involved in diverse biological processes including growth, survival, differentiation, angiogenesis, tumor growth and development [30] [31]. Understanding the different roles of the fibroblast growth factor receptor family in cancer development will elucidate the importance of this pathway as potential target for cancer treatment and the development of new target therapies [32]. Mouron et al. (2021) reported that 25% of hormone receptor positive (HR+) breast cancer patients have amplification or overexpression of FGFR1 [33]. Studies have reported the role of the Fibroblast growth factor receptor 1 (FGFR1) in the tumorigenesis of breast cancer and mammary development [34] [35]. Additionally, amplifications and overexpression of this factor (FGFR1) has been linked to poor prognosis of in breast cancer, functioning as a predictor of poor outcome [36] [37]. FGFR1 has also been reported to be overexpressed in RMS [32]. A study by Missiaglia et al. (2009) has confirmed the amplification and gain of FGFR1 and its overexpression in RMS cells with higher expressions in embryonal type than alveolar sub-type [20]. Goldstein et al. (2007) reported FGFR1 overexpression in RMS cells with hypomethylations at the promoter region [38]. These data suggest FGFR1 as a potential therapeutic target for RMS and search for therapeutic compounds that can target this gene and its pathway specifically [38]. Recent reports showed the correlation between resistance to chemotherapy and FGF/FGFR signaling and underscore drugs that target this signaling pathway as potential for cancer treatment [39].
The chemokine CXCR7 is a seven transmembrane receptor that was re-named ACKR3 as it is a member of the atypical chemokine receptor family (ACKR) [40]. Overexpression of CXCR7 was shown to be important in the angiogenic process in cancers, suggesting its role in tumor progression and its regulatory activity in the process of angiogenesis [40]. Sheng et al. (2010) showed the role of CXCR7 in endothelial proliferation, migration and production of vascular endothelial growth factors that enhance tumor propagation through mediation of angiogenesis [41]. Embryonal RMS cells have been shown to highly express the CXCR7 chemokine [42]. This chemokine was also reported to respond to stimulation by stromal derived factor (SDF)-1 also known as CXCL12, leading to ultimate activation of MAPK and AKT pathways. This activation was linked to the motility of RMS cells rather than proliferation and survival [43]. There is focus on small molecules as CXCR7 antagonists which show different affinities [40]. Simultaneous blocking of CXCR7 and CXCR4 also represents therapeutic approach as they involve both in cancer malignance [44].
Chemotherapy, surgery, radiation and their combination are the standard treatments for cancer. However, these are associated with limitations including lack of specificity, toxicity and development of drug resistance [45]. Therefore, the need to search for alternative therapeutic agents with less tendency for development of resistance and precisely target cancer cells [23]. Recently, there has been growing interest in antimicrobial peptides (AMPs) as a source of therapeutic agents with low development of resistance, higher efficacy and less toxicity to the cells [46] [47] [48]. Antimicrobial peptides (AMP) are short cationic peptides that are amphipathic in nature [49] [50], with diverse amino acids and secondary structures [51]. Recent studies suggest that antimicrobial peptides kill cancer cells by two mechanisms; membrane disruption leading to necrosis [52] [53], induction of apoptosis resulting from their binding to the mitochondrial membrane [54] [55]. Papo et al. (2004) showed that the D-K 6 L 9 peptide (15-mer D,L-amino acid peptide) inhibits proliferation of prostate adenocarcinoma cells after intratumoral injection with no activity on non-malignant cells [56]. Systematic administration of this peptide also inhibited growth of primary and metastatic tumors [57]. Dermaseptin B2 [58], an α-helical polypeptide, is the most potent within the family of Dermaseptins, with activities in micromolar ranges [59] [60]. In vitro studies reported the antitumor and antiangiogenic activities of the antimicrobial peptide Dermaseptin B2 (Drs B2) [61].  demonstrated that Drs B2 inhibits colony formation of PC3 and MDA-MB231 tumor cells, the proliferation and capillary formation of endothelial HUVEC cells [62]. This later study reported necrotic effects of Drs B2 rather than apoptotic activities with no activation of caspase-3 and no changes in mitochondrial potential [62]. In the current study, we investigated the antiproliferative activity of Drs B2 against RMS (RD) cells and evaluated the activity of this peptide at the molecular level by assessing the expression of genes involved in the proliferation of cells (MYC), angiogenesis (FGFR1, CXCR7) and metastasis (NOTCH1, CXCR7).

Determination of Cell Viability Using Resazurin Reduction Assay
The resazurin reduction assay was performed as previously described [64] [65]. At 65% -75% Cells were trypsinased, counted with hemocytometer and plated into 96-well cell culture plates at seeding density of 1 × 10 4 cells/well and incubated in an incubator at 37˚C, 5% CO 2 and 95% humidity. The previous media was gently removed from the cells and 100 µL of increasing concentrations (3, 6, 9, 12 and 15 µM) of Drs B2 in complete media was added to each well in triplicates. Doxorubicin used as standard control drug was prepared the same way. Plates were then incubated for 24, 48, 72-hours to determine the anti-proliferative activity of Drs B2 compared to Doxorubicin. After 24, 48, 72-hours of incubation, 20 µL (0.15 mg/mL) of resazurin dye was added and incubated for 4-hours.
The optical density of the cultures was then read with plate reader (Infinite M1000, Tecan), at an absorbance of 570 nm and 600 nm. The percentage cell viability was calculated from the net absorbance of 570 nm and 600 nm readings using the equation; percentage cell viability (% viability) = (Net absorbance of the treated cells/Net absorbance of the blank) × 100 [66]. The effect of the Drs B2 on the proliferation of the cells was then presented in graphs of % cell viability against the log concentration (µM) of the treatment. The 50% inhibitory concentration (IC 50 ) of the treatments was then calculated using non-linear regression analysis in GraphPad Prism software (v8.4.3).

Cell Migration Assay
The 2-dimensional wound healing assay was performed as previously described [67] [68] [69]. RD cells were grown in T-75 cell culture flasks using DMEM supplemented with 10% FBS. Cells were trypsinased, counted and plated in 12-well plates with a cell density of 5 × 10 4 cells/well. Cell growth was monitored until the cells attained 95% -100% confluence [68]. Plates were removed from the incubator and using sterile conditions in the biosafety cabinet, a 200 µL pipette tip was used to make a vertical wound on the cell monolayer. The cell debris together with the media was then removed and 2 ml of media containing either Drs B2 and Doxorubicin at their pre-determined IC 50

Primer Designing and Optimization
The primers used in this study were designed using the National Center for Bio- technology Information's (NCBI) Primer Blast tool https://www.ncbi.nlm.nih.gov/tools/primer-blast. Reference sequences of the genes were retrieved from GenBank in FASTA format and used to design the individual sequences for forward and reverse primers. A range of 70 -300 bps, 40% -60% GC content, and self-complementarity not exceeding 2 were targeted.
To confirm the PCR products sizes, primer sequences were checked using the Sequence Manipulation Suite (SMS). Primers sequences and optimization conditions are presented in Table 2.

qRTPCR Analysis
The expression levels of the target genes were assessed using qRTPCR real time thermal cycler qTOWER 3 84 GmbH (Analytik Jena). The qRTPCR reaction was performed using Luna ® Universal qRTPCR Master Mix (NEBM3003S, New England Biolabs) following manufacturer's instructions. A 20 µl reaction contained 10 µl of the Luna ® master mix, 0.5 µl each of the forward and reverse primers (10 pmol/µl), 2 µl of cDNA and 7 µl of nuclease-free water. All reactions were carried out in triplicate. The qRTPCR was done using the program; initial denaturation: 95˚C for 60 seconds, 40 cycles of; denaturation: 95˚C for 15 seconds, annealing/extension: 62˚C for 30 seconds, melting at 60˚C -95˚C. Data were analyzed using Microsoft excel software (version 2019). Expression levels of the target genes were determined by using the 2^− ddCt method [70], with reference to β-Actin housekeeping gene. Figure 1 shows the qPCR products of the target genes.

Relative Gene Expression
The relative expression levels of the target genes were calculated using the 2^− ddCt method. The house-keeping gene β-Actin was used as reference gene to normalize the expression of the target genes. The relative expression was then presented as fold change relative to the controls.

Data Analysis
All experiments were performed in triplicates of at least three replicates. Data were presented in Microsoft Excel software (version 2019) and expressed as mean ± SE. Analysis of variance was carried out using Graph-pad Prism software (v8.4.3) and used to compare the difference between the groups. P. value < 0.05 was considered as statistically significant.

Effect of Dermaseptin B2 on the Proliferation of RD Cells
The antiproliferative activity of Drs B2 and Doxorubicin was assessed against rhabdomyosarcoma (RD) cell line. Treatments were performed in time-points of 24, 48, and 72-hours. Doxorubicin was used as positive a control. Results indicate that treatments beyond 9 µM lead to complete inhibition of cell proliferation. The inhibitory concentrations (IC50) of Drs B2 and Doxorubicin for the time-points of 24-hours (Figure 2), 48-hours ( Figure 3) and 72-hours ( Figure  4) are presented in Table 3. The data reveals that, the activity of both Drs B2 (5.993 µM) and Doxorubicin (1.886 µM) increases with longer incubation (72-hours) of the cells with the treatments. Though Doxorubicin showed greater inhibition of the cell growth, when these results were compared using analysis of variance (ANOVA), they did not show a significant difference between Drs B2 and Doxorubicin in all of the treatment time-points.

Effect of Drs B2 on Cell Migration
The effect of Drs B2 and Doxorubicin on cell migration is depicted in Figure 5. The normal morphology of the RD cells before each treatment are shown (letters

Gene Expression Analysis
The effect of Drs B2 on the expression profiles of MYC, NOTCH1, FGFR1, and CXCR7 was investigated. Relative gene expression was carried out by comparing the expression profiles of the target genes between the treated and untreated RD cells and the results were normalized to the reference gene β-Actin. The Livak [70] method was used to determine the fold change expression of the target genes between the treated and untreated cells.  Figure 6. At 72-hour treatment (P. value = 0.01), MYC was significantly downregulated in the RD cells treated with Drs B2 (fold change = 2.4144) and Doxorubicin (fold change: 3.5067). Fold change expressions for all the genes are shown in Table 4. There were no significant differences in the 24-hour and 48-hour time-points between the treated and untreated cells (P. value = 0.4). Conversely, no significant differences were found between Drs B2 and Doxorubicin in all of the treatment time-points (P. value = 0.53).       Table 4. There was no significant difference between Drs B2

Effect of Drs B2 and Doxorubicin on the expression of MYC in RD cells at 24, 48 and 72-hours is shown in
and Doxorubicin in of the treatment time-points.

Discussion
The current study sought to investigate the antiproliferative activity of the antimicrobial peptide Drs B2 against rhabdomyosarcoma (RD) cells and its effect on expression of genes involved in the proliferation of cells (MYC), angiogenesis (FGFR1, CXCR7) and metastasis (NOTCH1, CXCR7), while using doxorubicin as a control anti-cancer drug. Results indicated that Drs B2 has strong antiproliferative activity against rhabdomyosarcoma (RD) cells in time-dependent manner and the inhibitory action of Drs B2 was deducted as time and concentration dependent. These findings were in line with previous studies which reported the inhibition of cell growth by Drs B2 against various cancer cells with micromolar concentrations [62]. In comparison, a Dermaseptin-L1, member of the Dermaseptin family, was reported to have selective inhibitory activity (GI 50 ) of 45µM [71], suggesting the higher activity of Drs B2 compared to other family members of Dermaseptins. These results notwithstanding, in comparison with Doxorubicin, Drs B2 showed less specificity given the lower selectivity index towards Vero Cells, however, the difference is not significant, thus Drs B2 can be a potential alternative for treatment. One of the main complications associated with cancer treatment is due to metastasis to distant organs leading to poor prognosis and survival [72]. Study of cell migration is an important aspect of cell's motility and their ability to metastasize. Drs B2 and Doxorubicin strongly inhibited the migration of the cells and prolonged exposure (48 and 72-hours) resulted in the failure of the cells to re-establish the wound/scratch. Inhibition of cell's communication and recovery through contact inhibition is an indication of their inability to move and hence interference of their metastatic potential [72]. This result shows that Drs B2 affects factors or genes that involve in cell migration and invasiveness. This result is also in further support of the downregulation of FGFR1 gene that involve metastatic process by Drs B2.
MYC plays many critical functions including proliferation, growth, and apoptotic process [21]. This gene has been shown to be dysregulated in different human cancers and its inactivation leads to tumor regression and can be targeted for cancer treatment [73]. The downregulation of MYC gene in RD by Drs B2 is an indication that it affected processes involving cell proliferation and growth. This result can be further supported by the cytotoxicity results of the treatments which showed greater inhibitory activity with longer incubation of the cells with the treatments. One of the main mechanisms of action of cancer therapeutic drugs is induction of apoptosis [74], Drs B2 could also play part the process, although a previous study reported that Drs B2 had more necrotic than apoptotic activity [62]. Ignatius et al. (2017) have shown the role of NOTCH1 in embryonal rhabdomyosarcoma differentiation and its effect on overall tumorigenesis [28]. A previous study reported that Ƴ-secretase inhibitors reduced the mobility of RMS cells but had no effect on cell cycle or apoptotic processes [27]. This indicates Notch pathway inhibition reduces the invasiveness of the cells, therefore, novel compounds that can inhibit this pathway could be of critical importance in RMS treatment and might have anti-metastatic potential. In this study, Drs B2 (fc: 4.6275) and Doxorubicin (fc: 5.7878), had significant effect on NOTCH1 expression in the treated cells. Though fold change expression of NOTCH1 slightly reduced for Drs B2 in the 72-hour (fc: 4.3352) interval, this gene was significantly downregulated all the treatment intervals (P. value 0.0001). These results can be further supported by the activity Drs B2 and Doxorubicin on cell migration which indicates their possible effect on factors or genes that involve in cell motility and invasiveness. The higher efficacy of Doxorubicin could be due to the fact that Doxorubicin is used in the management of later stages of RMS and metastatic solid tumors [8].
Fibroblast growth factor receptors (FGFRs) play important roles involving survival, motility, homeostasis and carcinogenesis processes [75]. Studies have confirmed the amplification and overexpression of FGFR1 in RMS [20] [32] [38]. Therefore, novel therapeutic agents that can target and inhibit processes including neovascularization of the cells would be crucial in the treatment of RMS. Exposure of RD cells to both Drs B2 and Doxorubicin resulted in significant downregulation of FGFR1 in all of the treatment intervals, though for both Drs B2 (fc: 2.3515, p. value; 0.01) and Doxorubicin (fc: 2.5943, p. value; 0.007), were more potent in the 24-hour intervals. Our results are in support of the previously reported angiostatic effects of Drs B2 [61]. As FGFR1 involves many processes including neovascularization and angiogenesis, its downregulation in the cells is an indication of the novel anti-angiogenic activity of Drs B2 and inhi-bition of the neovascularization in the RD cells.
CXCR7 was shown to be highly expressed RMS cells and was linked to increase in the adhesiveness of these cells [43]. It was also reported that CXCR7 has key roles in controlling angiogenic process [40]. The role CXCR7 in controlling the metastatic propagation, adhesion, and invasion of RMS cells was also reported [41] [43], indicating this gene axis as target for RMS treatment. The relative downregulation of CXCR7 in the treated cells compared to the untreated cells is further indication of the effect treatment of Drs B2 on neovascularization and the anti-angiogenic potential of Drs B2. Chemokines and their receptors regulate pro-metastatic propagation of RMS cells increasing motility, chemotaxis and expression of MMP as well as cell adhesion [76] [77] [78], but not directly involve in RMS cell growth, blocking of the newly identified SDF-1 binding receptor for CXCR7 [43], by Drs B2 might be target in these cells.

Conclusion
Drs B2 shows strong antiproliferative activity against RMS cells. Cell migration assay results showed that Drs B2 exhibits strong inhibitory activity on cell migration which could be due to its effect on factors that involve in cell migration and motility. Gene expression results showed that Drs B2 downregulates genes that involve in proliferation, angiogenesis and metastatic propagation of the cells. Further studies are needed to give a better understanding of the detailed mechanisms of action as well as the effects of the Dermaseptin B2 peptide in vivo.

Institutional Review Board Statement
Not applicable.

Informed Consent Statement
Not applicable.

Data Availability Statement
All the raw data for this study can be obtained from the corresponding author up on reasonable request.