Introduction

Peptides have emerged as a potent therapeutic candidate for several diseases, including cancer, because of their small size, facile manufacturing, easy membrane permeability, and enhanced specificity. Excessive research has supported several small therapeutic peptides, among which many are undergoing clinical trials (Bose et al. 2022; Das et al. 2022, 2023). Small peptides (20–30 amino acids) are essentially lower immunogenic and stable at room temperature than full-length proteins and antibodies (Timur and Gürsoy 2021; Karami Fath et al. 2022; Das et al. 2023). Studying the anticancer role of small peptides in several forms of cancer (Conibear et al. 2020; Bose et al. 2022; Das et al. 2022; Karami Fath et al. 2022) has now gained much attention. Colon cancer (CC) is one of the most frequent malignant cancers around the globe, and in recent years, the incidence rate has risen globally (Xi and Xu 2021; Wu and Lui 2022; Saraiva et al. 2023). Epigenetic and genetic alterations within a cell are intrinsic in CC, activating oncogenes and proliferative signals from the abnormal microenvironment surrounding the cell (Hossain et al. 2022). Adenomatous polyposis coli (APC), a tumor suppressor protein, is deregulated at the somatic and germline levels (Jung et al. 2020; Kawazu et al. 2022). In CC, APC gene mutations cause the APC protein to stabilize and accumulate β-catenin in the cytoplasm, and cytoplasmic β-catenin translocates to the nucleus and upregulates genes that promote uncontrolled cell growth and division (Ayala‑Calvillo et al. 2017). Cellular activities, including cell adhesion, motility, differentiation, proliferation, and chromosome separation, are all influenced by the APC gene, which is found to be mutated in about 80% of colon tumors (Siraj et al. 2020; Liu et al. 2020; Wu et al. 2020; Ghasemi et al. 2021). For 80–85% of sporadic CC, changes in the APC gene are an early, if not an initiating, event, except those that display a CpG island methylator phenotype (CIMP) or hypermutable microsatellite instability (MSI) phenotype because of a lack of mismatch repair (MMR) (Fennell et al. 2020; Moshawih et al. 2022). Although there are several strategies for CC treatments, the main issues with most of these therapies include the relapse of the cancer (Noe et al. 2021; Graf et al. 2023).

The antimicrobial peptide BMAP-27 is a cathelicidin peptide characterized by an α-helical structure and a C-terminus of 27 amino acid residues amidated. The structural analysis of BMAP-27 revealed an elongated α-helix at the N-terminal region, which exhibited distinct cationic and aromatic patterns. Additionally, a central kink was seen, along with a tiny hydrophobic helix at the C-terminal end (McGwire et al. 2003; Haines et al. 2003; Lee et al. 2011; Yang et al. 2019). The peptide’s cationic NH2-terminal portion (residues 1 to 18) is predicted to create an amphipathic α-helix, followed by a hydrophobic tail (residues 19 to 27). The hydrophobic tail is discovered to be essential for the peptide to exert its cytotoxic effect. The N-terminal amphipathic helix mediates initial binding to the anionic membrane surface.

On the other hand, the C-terminal hydrophobic helix is a key structure part that allows the peptide to be rapidly and effectively inserted into the hydrophobic core of the lipid bilayer, which kills bacteria and cancer cells (Hoskin and Ramamoorthy 2008). When BMAP-27 peptide was added to different human leukemia cell lines, it was found that BMAP-27 was able to kill cancer cells, increasing the permeability of the cell membranes leading to Ca2+ penetration (Risso et al. 1998; Hoskin and Ramamoorthy 2008; Yang et al. 2019), subsequently causing fragmentation of the DNA, and resulting in induced apoptosis (Risso et al. 2002). The current study aimed to investigate the role of the BMAP-27 in suppressing proliferation and inducing apoptosis in colon cancer cell lines.

Materials and Methods

Chemicals

Primary (SW480) and metastatic (SW620) colon cancer cell lines were purchased from NCCS, Pune, India, where sixteen short tandem repeat (STR) profiling were done using AmpFISTR® Identifiler® Plus Amplification Kit and Applied Bio system® 3500 Genetic analyser at NCCS, Pune (Supplementary material 1). BMAP-27 peptide with the sequence (GRFKRFRKKFKKLFKKLSPVIPLLHL) was purchased from PRIVEEL PEPTIDES: CHENNAI, India, with > 90% purity.

Cell Culture Conditions

Primary (SW480) and metastatic (SW620) colon cancer cell lines were cultured in DMEM containing 10% FBS, 1% glutamine, and 1% penicillin-streptomycin at 37 °C in 5% CO2.

Dosage Selection and Cell Proliferation Analysis

MTT Assay

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) analysis was performed for dose fixation of BMAP-27 peptide for further experiments. 2 × 103 per well of SW480 and SW620 cells were seeded in a 96-well plate and were grown under culture conditions for 24 h in standard 10% FBS in DMEM medium. After 24 h incubation, the cells were treated with various concentrations of BMAP-27 peptide (1 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, 1 µg/mL) for 72 h. MTT solution (2 mg/mL) was added to the cells and kept for incubation at 37 °C for 4 h. Then 40 µl of DMSO was added to the wells, and the absorbance was taken at 546 nm using an ELISA reader (Robonik Touch Well Reader, India) (Rahimi et al. 2018). The IC50 value was determined through analysis of the optical density obtained from the MTT assay, using the linear equation (y = mx + c), where x represented the IC50 value of BMAP-27 peptide, and y represented the 50% viability of colon cancer cell line (Olaokun et al. 2020).

Cell Proliferation Assay

The Cell Counting Kit-8 (CCK-8, Abbkine) was used to determine cell proliferation. About 2 × 103 cells per well were seeded in a 96-well plate. After 24 h of seeding the cells, they were treated with the selected doses of BMAP-27 (500 ng/mL and 250 ng/mL) peptide and allowed to proliferate for different intervals (24 h, 48 h, and 72 h). Absorbance was measured at 450 nm with a microplate reader (Robonik Touch Well Reader, India).

Lactate Dehydrogenase (LDH) Activity

LDH assay was performed to measure the degree of damaged cells using the EZcount™ lactate dehydrogenase cell assay kit (Cat. No: CCK036, HiMedia) as per the manufacturer’s protocol.

Catalase Activity Analysis

Catalase activity was measured using a standard protocol with slight modification (Hadwan and Abed 2016).

Assessment of Cellular Senescence

β-Galactosidase Activity Assay

The β-galactosidase activity is a known biomarker of cell senescence which is measured using an EZdetect™ cell senescence detection kit (Cat. No-CCK063, HiMedia).

Gene Expression Analysis by qPCR

RNAzol (Cat. No. R4533, Sigma–Aldrich) was used to isolate RNA from the cells. NanoDrop was used to measure the concentration of total RNA in each sample at 260 and 280 nm. Eurogentec Reverse Transcriptase PCR kit (Cat. No. RT-RTCK-03, Eurogenetec) was used for the reverse transcription. Ct values were obtained for all the genes (CASPASE3, BAX, BCL-2, CDK-6, PCNA, TP53, WNT11, AXIN1, CTNNB1) with Takara Bio’s RR420A Syber Green qPCR master mix (Cat. No: RR420A, Takara) and normalized with housekeeping gene GAPDH to generate ∆Ct values. The fold change was determined by calculating the relative quantification (RQ) values (2−∆∆CT). Further, the pre-designed primers (Sigma–Aldrich) details are given below in Table 1.

Table 1 Table representing the primer details
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Assessment of Expression of IL-6, IFN-γ and CXCL-10, Human Cytochrome c

The evaluation of the inflammatory and apoptotic markers was performed with the help of the proteins that were isolated from the cell lysate. IL-6, IFN-γ, CXCL-10 and cytochrome c expression were studied using IL-6 ELISA kit (Cat No. KET6017, Abbkine), IFN-γ ELISA kit (Cat No. KET6011, Abbkine), CXCL-10 ELISA kit (Cat. No. KTE62958, Abbkine), and cytochrome c ELISA kit (Cat. No. KTE62179, Abbkine) according to the manufacturer’s protocol.

Statistical Analysis

The statistical significance was determined with the help of the unpaired student’s t-test and the GraphPad V8.4.2 software. The statistical significance of the results, (*) indicates a p value less than 0.05 (p < 0.05), (**) indicates a p value less than 0.01 (p < 0.01), (***) indicates p value less than 0.001 (p < 0.001) and ns = non-significant. The data are presented as mean ± standard error. All the experiments were performed in triplicates.

Methodology for Molecular Docking and Simulation Analysis

Using two repeating peptide sequences the structure of BMAP-27 was prepared from the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index), and then after generation of the structure, only one sequence is taken for molecular docking and Molecular Dynamics simulation. The PDB files of the crystal structure of adenomatous polyposis coli (APC) protein (PDB ID: 1LUJ) and β-catenin (PDB ID: 4YJL) were retrieved from the protein data bank (https://www.rcsb.org/). Both the structures of the proteins were cleaned using UCSF-chimers. The molecular docking study between BMAP-27, APC, and β-catenin protein was then performed using the patchdock web server (http://bioinfo3d.cs.tau.ac.il/PatchDock/php.php). Furthermore, to investigate the effect of BMAP-27 on APC-protein and β-catenin, we have performed MD-simulation studies using GROMACS (Version 2018.5), protein-peptide composite system using the amber99sb force field and TIP3P solvation model. A periodic boundary condition with a cubical box of 1 nm edge length was considered. Na+ or Cl− ions were added to neutralize the system. Energy minimization was carried out until the maximum force became < 10 kJmol−1 nm−1. Then isochoric-isothermal (NVT) followed by isobaric-isothermal (NPT) ensembles were used to equilibrate the system for 1000 ps at 300 K. For the NPT ensemble, a modified Berendsen thermostat was used. NVT and NPT equilibration 1 nm of electrostatic and van der Waals interaction cut-off was kept. Using the same electrostatic and van der Waals cut-off with the equilibrated ensembles, MD simulation for 10 ns was performed. A modified Berendsen thermostat and a Parinello–Rahman barostat were used with reference temperature and pressure at 300 K and 1 bar, respectively.

Molecular Dynamic Simulations

To realign the protein, peptide, and other molecules within the cubical box, the trjconv tool was used. Xmgrace tool was used to plot Root mean square deviation (RMSD), Radius of gyration (Rg), and solvent-accessible surface area (SASA) plots.

Results

MTT Assay

SW480 and SW620 cells were treated with various concentrations (1 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL, 1 µg/mL) of BMAP-27 peptide for 72 h. BMAP-27 was found to exhibit a strong dose as well as time-dependent reduction of cell viability. IC50 value of 556 ng/mL was calculated, and doses of 500 ng/mL and 250 ng/mL were selected for further study (Fig. 1).

Fig. 1
figure 1

Cell viability analysis of BMAP-27- treated control, primary (SW480), and metastatic (SW 620) colon cancer cells by MTT assay. a SW480 and BMAP-27 peptide-treated SW480 cells; b SW620 and BMAP-27 peptide-treated SW620 cells at 72 h. The statistical significance results represents p values *p < 0.05; **p < 0.01; ***p < 0.001 and ns = non significant

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CCK-8 Assay

The Cell Counting Kit-8 (CCK-8) was used to measure the proliferation of SW480 and SW620 cells. The BMAP-27 peptide (500 ng/mL and 250 ng/mL) showed a reduction in cell growth compared to the untreated SW480 and SW620 cells. It was found to decrease cell survival by inhibiting growth, and statistically significant data is shown in Fig. 2a, b.

Fig. 2
figure 2

Bar diagram representing the difference in proliferation between control and BMAP-27 peptide treated cells at 24, 48, and 72 h. a SW480 and BMAP-27 peptide-treated SW480 cells; b SW620 and BMAP-27 peptide-treated SW620 cells. The statistical significance results represents p values *p < 0.05; **p < 0.01; ***p < 0.001 and ns = non significant

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Lactate Dehydrogenase Assay

The lactate dehydrogenase (LDH) study was used to evaluate the cell-damaging potential of the BMAP-27 peptide. After 72 h of treatment with the peptide, cellular damage was seen as increased LDH release in peptide-treated SW480 cells compared to non-treated cells. However, it did not show any significance in metastatic colon cancer cell lines Fig. 3a, b.

Fig. 3
figure 3

Determination of cellular damage by BMAP-27 in colon cancer cells. Bar graph represents the LDH release percentage by a SW480 and BMAP-27 peptide-treated SW480 cells; b SW620 and BMAP-27 peptide-treated SW620 cells. The statistical significance results represents p values *p < 0.05; **p < 0.01; ***p < 0.001 and ns = non significant

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Catalase Activity

The activity of catalase was assessed in non-treated groups as well as in the peptide-treated groups. Our findings showed that BMAP-27 peptide, at concentrations of 500 ng/mL and 250 ng/mL, respectively, produced a substantial increase in the catalase activity of primary cells, thus restoring the initial levels of catalase. However, it showed no significant difference in metastatic colon cancer cell line Fig. 4a, b.

Fig. 4
figure 4

Catalase activity was assessed to evaluate the impact of BMAP-27 on colon cancer cells. The figure illustrates the catalase activity observed in two scenarios: a SW480 cells and BMAP-27 treated SW480 cells and b SW620 cells and BMAP-27 treated SW620 cells. The statistical significance represents values as *p < 0.05; **p < 0.01; ***p < 0.001 and ns = non significant

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Cell Senescence Assay

Senescence-Associated-β-Galactosidase (SA-β-gal) Assay

Senescence-associated-β-galactosidase enzyme expression was studied on SW480 and SW620 cells, revealing a low level of senescence. However, SW480 and SW620 cells supplemented with BMAP-27 (500 ng/mL and 250 ng/mL) peptide exhibited more significant number of blue-stained cells indicating significantly higher levels of senescence as shown in Fig. 5a, b.

Fig. 5
figure 5

β-Gal cell senescence analysis in control and BMAP-27 treated cells. a SW480 and BMAP-27 peptide-treated SW480 cells; b SW620 and BMAP-27 peptide-treated SW620 cells. The statistical significance represents values as *p < 0.05; **p < 0.01; ***p < 0.001 and ns = non significant

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Gene Expression Analysis Using qPCR

The expression of apoptotic markers CASPASE3, BAX, and BCL-2 was analyzed by real-time PCR. In the present study, the apoptotic proteins in primary and metastatic colon cancer cell lines showed that BMAP-27 treatment for 72 h significantly regulated apoptosis in the primary colon cancer cell line compared to the metastatic colon cancer cell line. Further, BMAP-27 decreased CDK6 and PCNA expression in colon cancer cell lines, indicating its cell-cycle regulatory effects in both primary and metastatic cell lines. Moreover, BMAP-27 treatment for 72 h increased p53 expression in the primary colon cancer cell line. Subsequently, the effect of BMAP-27 was also assessed in several cancer-associated genes, where it showed a decreased expression of WNT11 after the treatment with BMAP-27 peptide to the colon cancer cell lines. Similarly, BMAP-27 increases AXIN1 expression, which may control CTNNB1 and reduce the expression of β-catenin in peptide-treated colon cancer cell lines Fig. 6a, b.

Fig. 6
figure 6

Expression profile of pro-apoptotic marker CASPASE3 and BAX, anti-apoptotic marker BCL-2, cell proliferation markers CDK-6 and PCNA, tumour suppressor marker TP53, cancer-specific markers WNT11, AXIN1, and CTNNB1 in a SW480 and BMAP-27 peptide-treated SW480 cells; b SW620 and BMAP-27 peptide treated SW620 cells. The statistical significance represents values as *p < 0.05; **p < 0.01; ***p < 0.001 and ns = non significance

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Modulation of Inflammatory Markers After the Treatment of BMP 27

Our results suggested that the BMAP-27 peptide (500 ng/mL) reduced the IL-6 concentration significantly in the SW480 colon cancer cell line. However, it could not show any significant effect on metastatic colon cancer cell line. In the same line, BMAP-27 (500 ng/mL) was also able to reduce the IFN-γ concentration significantly in both SW480 and SW620 cells, indicating that BMAP-27 treatment might regulate the activation of immune response. Moreover, BMAP-27 significantly decreases the expression level of CXCL-10 in both primary and metastatic colon cancer cell lines, indicating the anticancer properties of BMAP-27. In the current investigation, we found that the cytochrome c level increased significantly in SW480 cells, suggesting that BMAP-27 might induce apoptosis in the colon cancer cell line. Figure 7a–h.

Fig. 7
figure 7

Effect of BMAP-27 on inflammatory and apoptotic markers expression in control, primary, and metastatic colon cancer cells. Bar diagram representing the concentration of a, b Expression of IL-6, c, d Expression of IFN-γ, e, f Expression of CXCL-10, g, h Expression of cytochrome c in SW480, SW620, and BMAP-27 peptide-treated SW480 and SW620 cells. The statistical significance represents values as *p < 0.05; **p < 0.01; ***p < 0.001 and ns = non significant

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Molecular Docking and Molecular Simulation Study

The molecular docking study of BMAP-27 with APC protein and β-catenin was performed using patchdock server, and the results obtained from the server were tabulated in Table 2. The docking score between BMAP-27 and β-catenin is higher compared to APC protein. Also, the higher confidence score and lower RMSD of BMAP-27 prove more selective docking against β-catenin (4YJL).

Table 2 Molecular docking results between BMAP-27 of APC-protein and β-catenin
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Figure 8 represents the docked structure of BMAP-27 with APC protein (1LUJ) and β-catenin (4YJL). Due to the smaller binding cavity of both proteins, BMAP-27 has docked at the surface of the proteins. The minimum distance between BMAP-27 and 1LUJ was found to be 2.73 Å in the docked structure, while that between BMAP-27 and 4YJL was found to be 2.68 Å, which suggests a good binding affinity between BMAP-27 and 4YJL.

Fig. 8
figure 8

Docked structure of a 1LUJ and BMAP-27 and b 4YJL and BMAP-27

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To further investigate the interaction of BMAP-27 with the proteins (β-catenin and APC), MD simulation was performed for a period of 10 ns with the docked and undocked structures. The RMSD plots obtained from the MD-simulation are shown in Fig. 9. It is clear from Fig. 9a that the deviation of the plot is lesser after 8 ns with the BMAP-27 bind protein indicating the stabilization of the protein due to the presence of BMAP-27. While Fig. 9b indicates the stabilization of the plot after 4 ns due to the presence of BMAP-27. Furthermore, the deviation is more in the docked 1LUJ compared to 4YJL, revealing the more effectiveness of BMAP-27 against β-catenin (4YJL) compared to APC protein (1LUJ).

Fig. 9
figure 9

RMSD plot of a 1LUJ and BMAP-27 and b 4YJL and BMAP-27

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Figure 10 represents the Rg plot of BMAP-27 with APC protein and β-catenin. Rg-plots measure the compactness of the protein during MD simulation. The more compact the proteins more they will bind with the BMAP-27. It is clear from Fig. 10 that the proteins are more compact in the docked structures. The compactness of BMAP-27 docked 1LUJ is achieved after 8 ns, while for 4YJL, it is after 4 ns, which is consistent with the RMSD plot. The compactness is more in BMAP-27 docked 4YJL compared to 1LUJ.

Fig. 10
figure 10

Radius of gyration plot of a 1LUJ and BMAP-27 and b 4YJL and BMAP-27

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To interpret the structural stability of the native structure, we have analyzed SASA. Figure 11 represents the SASA plot of BMAP-27 docked 1LUJ and 4YJL, which determines the stability and folding of proteins. SASA values for BMAP-27 docked 4YJL is lowered compared to that for 1LUJ.

Fig. 11
figure 11

Solvent-accessible surface area of a 1LUJ and BMAP-27 and b 4YJL and BMAP-27

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In both cases, BMAP-27 docked conformers show less fluctuation (for 1LUJ-BMAP within 250 nm2 and for 4YJL-BMAP within 190 nm2) compared to undocked conformation, suggesting the stability of the docked conformation.

Discussion

Conventional cancer therapies are associated with multiple adverse effects due to the non-selective nature of frequently administered anticancer agents, which impacts the growth of all rapidly dividing cells rather than only targeting malignant cells. On the other hand, certain anticancer peptides appear to exhibit selectivity towards malignant cells. It is evident that additional research is required to thoroughly investigate the precise mechanism through which anticancer peptides (ACPs) interact with cancer cells. BMAP-27, which belongs to the cathelicidin family of peptides, has been found to decrease cancer cell proliferation without toxic hemolytic activity (Zanetti et al. 2002; Cragg and Pezzuto 2016; Zhang and Shay 2017; Liang et al. 2020; Lath et al. 2023). The C-terminal hydrophobic helix of the BMAP-27 peptide may be inserted into the lipid bilayer, and this contact results in membrane permeabilization and depolarization, which may be involved in its anticancer actions (Li et al. 2012; Amerikova et al. 2019; Yang et al. 2019; Yadav and Misra 2021). We observed a significant reduction in cellular proliferation in a dose and time-dependent manner (24 h, 48 h, and 72 h) in which 500 ng/mL and 250 ng/mL concentrations of the peptides showed significant results in 72 h compared to 24 and 48 h. However, it is observed from the results that BMAP-27 showed a significant impact in the primary colon cancer cell line than in the metastatic cell line.

LDH levels are associated with the development of colon cancers (Tas et al. 2001; Bohers et al. 2018; Dall’Olio et al. 2022). Our results showed that BMAP-27 can induce LDH levels at a significant rate in primary colon cancer cells. However, BMAP-27 could not show a significant difference in the metastatic colon cancer cell line. An increase in LDH release can indicate cell death, particularly necrosis, in which cells often release their intracellular contents, including LDH, into the surrounding environment (Li et al. 2016).

Catalase enzymatic activity is found to be reduced in colon cancer cell lines. Furthermore, we have also observed a decreased level of catalase activity in the untreated group, which may indicate an increased susceptibility to redox imbalance and oxidative damage. It has been demonstrated to be crucial in several different cellular processes (Zińczuk et al. 2019). While hydrogen peroxide (H2O2) does not possess free radical properties, it has the ability to traverse the cell membrane. Hence, it is unsurprising that a reduction in the activity of catalase, the enzymes accountable for the detoxification of H2O2, has been found to be rational. However, our results showed a significant increase in the levels of catalase activity with the peptide-treated groups in SW480 cells. Still, they showed no substantial difference in the metastatic colon cancer cell line. Cancer cells reduce cell senescence by maintaining the telomere length, epigenetic regulation, a mutation in the tumor suppressor genes, and immune evasion (Özsoy et al. 2020; Dai et al. 2022). Senescence-associated secretory phenotype (SASP), a group of cytokines and chemokines generated by senescent cells, may cause inflammation. Thus, in SW480 and SW620 colon cancer cell lines, BMAP-27 peptide dosages significantly increased the number of blue-stained cells, indicating senescent cells compared to untreated SW480 and SW620 cells. Cells can potentially undergo senescence that may lead to apoptosis, serving as a secondary response towards cell death. Senescence can function as a defensive mechanism to inhibit the proliferation of impaired cells. However, when the extent of damage is beyond a certain threshold, cells may ultimately trigger apoptosis as a means of self-elimination.

The expression levels of CASPASE-3, BAX, and BCL-2 are the potential predictors of colon cancer progression. Elevated expression of BCL-2 or decreased expression of BAX is associated with a more unfavorable prognosis (Scopa et al. 2001; Khodapasand et al. 2015). The expression of pre and anti-apoptotic genes on primary and metastatic colon cancer cell lines indicates that the administration of BMAP-27 for 72 h induces apoptosis. The genes CDK6, PCNA, and WNT11 showed a decrease in expression. Previous research has demonstrated that TP53 mutations were consistently present in 80% of individuals with advanced cervical cancer and metastases (Li 2015; Zhou et al. 2018; Nakayama and Oshima 2019; Gorroño-Etxebarria et al. 2019; Yousuf et al. 2020). In the present study, we also found that colon cancer cell lines showed reduced levels of p53 expression. Still, after the administration of the BMAP-27 peptide, a significant upregulation of p53 expression in the primary colon cancer cell line was observed. The development of colon cancer is closely linked to the inactivation of the AXIN1 gene (Sanson et al. 2023).

In some cases of colon cancer, mutations in the AXIN1 gene have been found to enhance the different proteins involved in the Wnt/β-catenin signaling pathway. Dysfunction of AXIN1 leads to the nuclear accumulation and stability of β-catenin, resulting in the upregulation of target genes like c-myc and cyclin D1 that facilitates cell proliferation, survival, and development of cancer (Nakamura et al. 1998). Our findings demonstrate that the administration of BMAP-27 peptide treatment leads to a significant upregulation of AXIN1 expression and downregulating β-catenin expression.

IL-6 promotes various malignancies, including CC (Lin et al. 2013). In the same line with other studies, colon cancer cell lines had an increased level of IL-6. However, the primary colon cancer cell line treated with BMAP-27 peptide showed significantly lower levels of IL-6. The decrease in IL-6 expression may suggest that the peptide has an anti-inflammatory effect. The relationship between inflammation and cancer growth is highly related, with the pro-inflammatory cytokine IL-6 significantly promoting inflammation. The reduction of IL-6 levels may indicate the anti-inflammatory potential of BMAP-27.

Moreover, the BMAP-27 treatment decreased the levels of inflammatory cytokine IFN-γ in SW480 and SW620 cells, confirming its anti-inflammatory potential. CXCL10, which promotes tumor growth, migration, and invasion in colon cancer (Wang et al. 2021), was observed to be decreased after the peptide treatment in primary and metastatic colon cancer cell lines, suggesting that BMAP-27 may have anticancer activities.

Furthermore, reduced cytochrome c expression or subcellular localization has been linked to worse cancer outcomes, including advanced tumor stage, higher metastatic risk, and worse survival rates (Robichaux et al. 2023). In the present study, SW480 and SW620 cells treated with BMAP-27 peptide significantly increased the cytochrome c expression. An increase in cytochrome c expression in peptide-treated colon cancer cell lines may be correlated with the induction of apoptosis. The mitochondrial dysfunction may increase cytochrome c expression as the cell attempts to respond to the stress, causing cytochrome c to be released into the cytoplasm, which may trigger the apoptotic cascade.

The in-silico analyses on the effect of BMAP-27 peptide on APC and β-catenin proteins were studied through molecular docking studies followed by molecular dynamics simulation. The results indicated significant binding between BMAP-27 and the selected proteins with a high binding affinity, revealing strong binding interactions. The molecular dynamics simulation conducted throughout 10 ns in a water-based environment provided additional evidence regarding the impact of BMAP-27 on the structural arrangement of both β-catenin and APC proteins. The RMSD plot showed less fluctuation in the case of docked proteins during MD simulation, indicating their stability and compactness. The radius of the gyration plot also demonstrated increased compactness of both proteins after binding with BMAP-27. Additionally, the solvent-accessible surface area plot showed more scattered conformation in the docked state, suggesting increased stability and access to solvent molecules like water.

Conclusions

BMAP-27 peptide demonstrated the ability to induce cell death, inhibit cell proliferation, and regulate cancer-specific gene regulation. Moreover, the present and initial data found that BMAP-27 activity was more in the primary colon cancer cell line (SW480) than in the metastatic colon cancer cell line (SW620). The molecular docking and MD simulations further revealed the interactions between BMAP-27 and its target proteins, APC and β-catenin, providing valuable insights into the mechanisms underlying the anticancer properties of BMP 27. Further research on different colon cancer cell lines and in vivo studies, including zebrafish and colon cancer mice models, will be necessary to validate the anti-colon cancer potential of BMAP-27.