3.1 Physicochemical, Allergenicity, Digestion Resistance and Anticancer Assessment of moricin peptide
The antibacterial peptide ‘moricin’ consists of 42 amino acid sequences. The physicochemical properties reveal that moricin is a highly basic entity, and greater the basicity, the more anti-microbial effects it possesses (Table 1). The electrostatic association(s) between positively charged moricin peptide and negatively charged bacterial membrane is mediated with an isoelectric point (pI) calculated to be at 11.3. The instability index value is 8.32. Furthermore, the peptide did not possess any toxic, allergenic and digestion resistance responses as predicted by the in silico study. Anticancer prediction servers AntiCP and iACP server with SVM scores 0.75 and 0.99 reveal their potent anticancer property prediction (Table 2). Additionally, the SVM score of 0.16 has been over served for the cell-penetrating property of the peptide (Table 2). The iDNA-Prot server reveals that the peptide interacts with the nucleic acids and the interacting amino acids predicted were Tyr27, Glu30, Val31, Asp33, Phe34 (Table 3). The C-score value ranges from 0-1 predicting for ligand binding where, a higher score indicates a more reliable prediction. The secondary structures of the peptide were predicted using NPS@: SOPMA secondary structure prediction tool (https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html) were the percentage of α-helices, coil and strand were determined (Table 2).
3.2 Docking Analysis
Protein-peptide interactions where moricin was docked with selected target proteins in the notch signaling pathway namely human DLL4, crystal structure notch 1, notch 3, and jagged-1 proteins. By analyzing the individual docked models generated shows that maximum binding of moricin peptide was observed with notch 3 NRR with a BFE of -72.13 kcal/mol and the interacting amino acids are depicted in table (Table 4). As evident from results, the binding affinity of moricin with the target proteins decreased in the order notch 3 (BFE: -72.13 kcal/mol)>DLL4 (BFE: -59.79 kcal/mol)>TACE (BFE: -51.14 kcal/mol)>notch 1 (BFE: -48.92 kcal/mol)>jagged-1 (BFE: -41.49kcal/mol). The DNA binding amino acids of the peptide have been writtenin italicized form in Table 5predicted by iDNA-Prot server, suggesting that Tyr27, Glu30, Val31, Asp33 and Phe34 are the active site residues of the peptide.
3.3 Moricin is a-helix predominant structure with a MW of 4543.5 Da revealed by CD and LC/MS
Peptides were analyzed and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with a linear gradient of water (0.1% TFA). The RP-HPLC chromatogram of purified peptide showed one major peak at approximate retention time 8.1 min (Fig. 1A). Most of the contaminants and unwanted side products eluted before 4 min. when the amount of organic solvent was very less (< 10%). The specific fraction was collected (peak of interest) and then freeze-dried before used for other applications. The purified peptide was further characterized by mass spectrometry and circular dichroism. The CD spectrum of synthesized moricin peptide showed negative band ~190 nm in water, indicating random coil conformation. However, when analysed in presence of 30% (v/v) trifluoroethanol-water mixture, two distinct minima were observed at 197 and 208 nm region. In addition, a positive band was observed at ~182 nm. This indicates a-helix as the predominant secondary structure. A closer look into the position and relative intensity of the bands indicate presence of 44.4 % a-helix and 13.5 % b-strand structure (Fig. 1B). For further confirmation, the molecular mass of the peptide was measured by ion spray mass spectrometry on a single quadrupole LC/MS spectrometer. The obtained value, 4544.0 ± 0.4 Da, was coincident with the literature-reported value, 4543.5 Da (Fig. 1C) considering that the C terminus of the peptide is unmodified.
3.4 Moricin induced growth inhibition of MDA-MB-231 cells at 6.25µg/ml concentration
To determine the effective dose of moricin peptide on MDA-MB-231 cells, we assessed the moricin peptide-induced cytotoxicity in MDA-MB-231cells by measuring cell viabilities. As shown (Fig. 2), cells were treated with various concentrations of moricin peptide (0-100μg/mlor0-22 μM) for 24 h. Moricin peptide exposure up to 3.125 μg/ml (0.688 μM) of did not cause any significant loss in cell viability. However, the cell viabilities were found to be decreased significantly at 6.25 (1.37μM) and 12.5 μg/ml (2.75μM) concentrations of peptide by24% and 50% respectively, while at above concentrations more that 75% of decrease in cell viabilities were observed compared to untreated cells. Additionally, the cytotoxic effects of moricin peptide were also confirmed by assessing LDH release (Fig. 2C) and by the trypan blue assay (Fig. 2D) Similar to the MTT assay, results of LDH release and trypan blue assays also showed significant decrease in cell viability at 6.25μg/ml and above concentrations of moricin peptide. Hence, based on these results, we selected 6.25(1.37μM) and 12μg/ml (2.75μM) concentrations of moricin peptide for our subsequent studies.
3.5 Moricin induces intracellular Reactive Oxygen Species (ROS) generations in MDA-MB-231 cells
There are several growing evidences that anticancer drugs exposure causes significant alteration in the cellular redox status of cancer cells. Therefore, we were also interested to know the effects of moricin peptide on cellular redox in MDA-MB-231 cells. As shown (Fig. 3A & 3B), moricin exposure causes significant rise of 1.5 and 2 fold in the levels of intracellular ROS levelsin dose 6.25and 12μg/ml respectively after 24 hour. Flow cytometry results revealed the intracellular ROS were 11.85 %, 26.85% and 46.80% in untreated and 6.25 and 12μg/ml moricin treated cells respectively.
3.6 Moricin induces Mitochondrial ROS mediated structural damage to mitochondria and lysosome
Several studies have reported that more than 90 percent of cellular ROS is generated through mitochondria, therefore we were also interested to know the effects of moricin exposure on mitochondrial ROS. It was observed that moricin exposure causes significant increase of mitochondrial superoxide anion by1 and 1.4 fold in 6.25 and 12μg/ml moricin treated cells respectively as compared to control (Fig. 4A & 4B). In this connection, we further checked the effects of superoxide anion at lysosome and mitochondrial morphology by utilizing fluorescence microscopy. There were significant morphological alteration were observed in the mitochondria and lysosomal structure at 12.5 µg/ml of moricin concentration. However at low doses (6.25µg/ml) no significant morphological alterations were observed in the mitochondria and lysosome (Fig. 4C).
3.7 Moricin reduces intracellular GSH level and enhances TBARS in MDA-MB-231 cells
In the previous result, we have shown that cytotoxic concentrations of moricin (6.25 and 12.5 μg/ml) cause a significant rise in the total intracellular and mitochondrial ROS, therefore we were further interested to know the effects of moricin induced oxidative stress on cellular glutathione and lipid peroxidation. Results shown that moricin exposure leads to the significant decrease in the total intracellular glutathione levels (Fig. 5A &5B) whereas, the TBARS assay results shows the significant rise in the level of lipid peroxidation in dose dependant manner. (Fig. 5C).
3.8 Moricin retards proliferation, migration and colony formation rate in MDA-MB-231 cells
As shown in previous results, moricin induces ROS mediated morphological alterations in the mitochondria, therefore, we were keen to investigate the effects of moricin exposure onMDA-MB-231 cells migration and proliferation activity. We observed, moricin causes a significant decline in the rate of migration after 12 and 24 hrs of exposure. However 12.5 μg/ml concentration of morcin shows relatively higher anti-migration effects after 24 hrs (Fig. 6A & 6B). Similarly, BrdU incorporation also shows the significantly decrease in the rate of cell proliferation after moricin exposure and causes 60 and 70% lower cell proliferation at 6.25 and 12.5 μg/ml respectively. (Fig. 6C &D). Soft agar assay (Fig. S1) also shows the significantly decrease in colony formation under moricin exposure.
3.9 Moricin induces caspase dependent cell death via down regulating the Notch-1/NFKB in MDA-MB-231 cells
As in our in silico study the protein peptide docking analysis indicates that moricin have relatively higher binding affinity with Notch-1 receptor, therefore to validate our in silico finding along with our in vitro study we performed immunoblotting in moricin exposed MDA-MB-231 cells to delineate the molecular pathways of morcin induced cell death. It was observed thatNotch-1 and NFƙB protein shows significantly low level of expression in moricin exposed cells (Fig. 7A &B), Next, we check for the expression of tumor suppressor gene P53, interestingly our results shows significantly high expression of P53 (Fig. 7C&D),, subsequently we also checked for the protein expression of apoptotic pathways (viz. Bad, Bcl-2, Caspase3 and Caspase9). We observed the level of Bcl-2 proteins were significantly downregulated (Fig. 7C&D), whereas the protein expression of Bad, Caspase3 and Caspase9 were found to be significantly upregulated (Fig. 7E &F),