Pre-clinical studies of honey-related synthetic peptides in malignancy control

Backgound: The challenge with malignancy control approaches, although of all traditional methods of treatment like surgery, chemotherapy, radiotherapy, immunotherapy etc. makes more stress upon the scientific community to look for a solution for this challenge even far from the traditional ways. Methods: A growth inhibitory assay was performed. Samples of peptide A and peptide F, 5 mg each, were dissolved in 500 μL Dulbecco’s Phosphate-Buffered Saline to prepare the stock solution, which was added into the wells directly. A 1% trifluoroacetic acid (TFA) solution was used as the control. (a) Cells were placed into a 96-well assay plate at a density of 5000 and 10000 cells/well for adherent and suspension cell lines, respectively, with a 50 μL complete cell culture medium. (b) Stock solutions (10 μL) of the compounds were added into the wells. (c) For data analysis, 10 μL 1% TFA solution was used as control. (d) Complete medium of 40 μL was added into the wells to reach a total volume of 100 μL. (e) The plate was incubated at 37℃ for 72 hours. (f) CellTiter-Glo assay mix solution (50 μL) was added to each well and mixed gently at room temperature for 10 minutes; subsequently, the luminescence was read using PHERAstar Plus (Molecular Devices). Results: Apparent inhibitory effects of those peptides were noticed in 17 of 18 tumour cell lines. Similar effects were noted in an in vivo study using the U87MG tumour mouse model. Conclusion: This study has introduced new synthetic peptides derived from honey proteins, which may help in cancer control through direct apoptotic action not limited to only a certain tumour cell line.


Background
Bee genome was disclosed and published in 2006 [1], and many hypothetical proteins were postulated. Honey was known as a remedy thousands of years ago as it was mentioned in old books, either religious or others, emphasising its medical and nutritional value [2][3][4][5][6].
Royal jelly (RJ) is the most abundant protein found in honey, and some researchers have discussed its inhibitory effects on malignant cells [15].
This study focused on RJ and two hypothetical proteins (XP 001120220 of LOC724386 gene and XP 397512 of LOC408608 gene) for possible anticancer effects [16,17,18]. After a series of experiments, we postulated the following signal peptides in proteins that may present significant anticancer effects [19,20,21]:

3-MTKWLLLVVC LGIACQ
However, these peptides were hydrophobic; many biotechnology companies could manufacture crude products but failed to synthesise highly pure states (>95%) of these peptides to fit our laboratory requirements. Therefore, we modified the C-terminus of these peptides by adding lysine either after deleting some amino acids in two peptides or without any deletion in the third peptide [22] as follows: Because of the increased proportions of ageing populations and habits of smoking and related predisposing factors, the prevalence of cancer is expected to increase, particularly in developing countries. In 2008, approximately 12.7 million cancer cases and 7.6 million cancer deaths were estimated, and 56% of these cases and 64% of the deaths occurred in economically developing countries [23]. Additionally, more than 16 million new cancer cases every year are expected by 2020 [24].
Currently, primary approaches to cancer treatment include surgery, chemotherapy, radiotherapy, and biologic treatment.
The "biologics" option to treat cancer includes the use of proteins, monoclonal antibodies, and peptides. Because of the large size of monoclonal antibodies and protein, its usage was limited to haematological malignancies and suboptimal penetration in other malignancies [25][26][27][28][29][30]. By contrast, peptides with their small size and high penetration plus easy synthesis and modifications prove their superiority over proteins and monoclonal antibodies in biologic treatment of cancer [31].
Therefore, peptide use in clinical trials has increased from 1.2 per year in the 1970s to 16.8 per year in the 2000s [32].
Peptides may induce anticancer activity through one or more mechanisms, such as angiogenesis, protein-protein interactions, enzymes, proteins, signal transduction pathways, or gene expression [39][40][41][42][43][44][45]. Additionally, peptide antagonists, considering their binding ability to a known receptor, exhibit anticancer activity [46,47]. The peptides assessed in this study induced apoptosis and programmed cell death in tumours [48,49,50]. 5 RGD-(KLAKLAK)2 and NGR-(KLAKLAK)2 are examples of angiogenesis inhibitors, which may help prolong survival in some patients with cancer. By contrast, LyP-1 was limited to certain tumours of lymphatic vessels that inhibit breast cancer cell lines and not melanoma cell lines. Additionally, the inhibition of breast cancer cell lines was found only in vivo in the MDA-MB-435 breast cancer xenografts-mice-animal model [
(A+F) per well: Peptide A and peptide F were stored at −20°C. The samples of peptide A and peptide F, 5 mg each, were dissolved in 500 μL DPBS to prepare the stock solution, which was added to the wells directly. TFA solution of 0.1% was used as control. 2) Add 10 μL stock solutions of the compounds into the wells.
3) A 10 μL 1% TFA solution is used as control for data analysis. 4) Add 40 μL complete medium into the wells to adjust the total volume to 100 μL. 5) Incubate the plate at 37°C for 72 hours. 6) Add 50 μL CellTiter-Glo assay mix solution to each well and mix gently at room temperature for 10 minutes; subsequently, read the luminescence with PHERAstar Plus (Molecular Devices).

In Vivo Study
The study protocol and the animal use were approved by the IACUC of Genscript.

Dosage and Acute Toxicity:
Before the main experiment, we performed a pilot study to mimic the treatment and studied mouse tolerance to intravenous (IV) injections of the peptides. We tested IV injections of the mixed peptide A+F in nude mice at different doses: (1) 50 μg/g per peptide, which was a total of 100 μg peptide/g for the mixed peptide; (2) 25 μg/g per peptide, and (3) 12.5 μg/g per peptide. In group (1), both the mice died immediately after injection; in group (2), all three mice tolerated the dose. They showed weakness and reluctance to move after the injection but recovered in a few hours; in group (3), all three mice tolerated the dose well without significant stress. The combination of peptide A+F through IV route showed strong acute toxicity at a relatively high dosage, whereas injection through subcutaneous (SC) route showed tolerate for more than 100 μg/g per peptide, which was a total of 200 μg/g for the mixed peptide. These findings were consistent with the observation in rats with IV injection of peptide F for the PK study.

Groups and dosage:
When the tumour size reached a volume of 100-200 mm 3 , U87 tumour-bearing nude mice were randomly assigned to three groups (four mice/group) and started with dosing immediately: Group 1 was administered with a vehicle and served as control group.
Group 2 was administrated with peptide A+F (10 g/g of mouse weight with each peptide; IV, biweekly, three injections; then 20 μg/g intratumoral SC injection, every other day, two injections. Therefore, five injections in total). Group 3 was administrated with peptide A+F (10 μg/g of mouse weight with each peptide, IV, only a single dose at first week; then 20 μg/g intratumoral SC injection, every other day, two injections. Therefore, three injections in total).
The administration period lasted for 12 days. Terminal procedures and necropsy: Early death/unscheduled necropsy: If a mouse died during the study, the time of death would be estimated as closely as possible and recorded, and the mouse would be necropsied as soon as possible. If the necropsy could not be performed immediately, the mouse would be refrigerated (not frozen) to minimise tissue autolysis and would be necropsied no later than 12 hours following death. If a mouse appeared in poor condition or morbid, the mouse was euthanised after discussing with the client. The mouse might be euthanised (as described later) per the Testing Facility's policies on humane care of animals. If the tumour burden was larger than 2000 mm 3 or the body weight loss was greater than 20% of the baseline body weight, the mice would be euthanised. If the weight dropped significantly, the data would be viewed with a caveat. All unscheduled-necropsy mice would be necropsied immediately, or, if this could not be performed, the mouse would be refrigerated to minimise autolysis and necropsy would be performed no later than 12 hours after death.
Scheduled necropsy: At the end of the experiment, tumour-bearing mice were euthanised with CO 2 by the end of the study. The tumour samples were harvested, weighed, and photographed.

Endpoints:
Tumour volume: Tumour volume was measured twice a week in two dimensions using a calliper, and the volume was expressed in mm 3 using the formula V = 1/2*axb 2 where a and b were the long and short diameters of the tumour, respectively.
Body weight: The body weight of the tumour-bearing mice was measured and recorded twice a week after administration. If the body weight loss was greater than 20% of the baseline body weight, the dosage would be stopped or decreased.
Tumour weight: The tumour masses were weighed at the end of the experiment after being harvested. Three tumours, one from each group, were fixed in 10% formalin for paraffin block preparation.
Inhibition rate: Inhibition rate (%) = (average tumour weight of control group − average tumour weight of test group)/average tumour weight of control group × 100%

In Vivo Study (Pharmacokinetic Study):
The study protocol and all the animal use were approved by the IACUC of Genscript.

Study Purpose:
To evaluate the pharmacokinetic parameters of peptide F in Wistar rats following IV administration.

13
The study protocol and all the animal use were approved by the IACUC of Genscript.
The flow rate was maintained at 0.2 mL/min, and the following mobile phases were used: Table 1. The study demonstrated superior response for peptide F in 14 of 15 cell lines followed by peptide A and lastly peptide E; excellent response was observed when we added peptides F, E, and A in one group.
EOL-1 cell viability assay results are not presented in Figure 1; EOL-1 cells were sensitive to 0.1% TFA control. Approximately 13% cell viability was detected in 1% TFA-treated EOL-1 cells as compared with a medium without TFA-treated EOL-1 cells. Therefore, the data were presented as either normalised by 0.1% TFA control or medium control for EOL-1 cells. 15

In Vitro Study:
The peptides (combination A+F) were prepared and tested according to the aforementioned protocols.

Cell Growth Inhibition:
Results are shown in Figures 1 and 2. Apparent tumour cell inhibition for all tested lines increased with increased peptide doses.

Apoptosis Assay:
Apoptosis Effect of Compound A+F on Tumour Cell (TFA as Control).
As shown in Figure 3, high apoptosis was noted with peptides A+F in the U87MG and MDA-MB-468 cell lines, moderate apoptosis was noted in the K562 cell line, and mild apoptosis was noted in the A375 cell line.

Tumour Growth Curve:
The data showed that the tumour volumes of two treatment groups were reduced compared with the saline group from Day 9 to Day 12 post-treatment but not significantly as shown in Figure 4.

Body Weight Change Curve:
The body weights of the mice who received peptide A+F were not significantly reduced after dosing.

Photo of Tumour Mass and Tumour Weight:
The tumour masses were harvested and weighed.
Tumour weight: Tumour weights of the two treatment groups decreased compared with the saline group without any statistically significant difference.

Inhibition Rate:
16 The data demonstrated that a combination of peptide A and F moderately inhibited U87 tumours and xenograft growth but not significantly, as shown in Figure 5.

In Vivo Study (Pharmacokinetic Study):
3.4.1. Data Analysis: Peak areas of the analyte and internal standard were calculated using Analyst™ 1.5.2 software (Applied Biosystems). Regression analysis was performed in this software, and the standardise on linear curve fit will 1/x or 1/x2 in the first instance. Acceptance criteria should be ±30% of the target value for standards and quality control (QC).As shown in Fig   6. A minimum of five calibration points must be used in the quantification. At least three of the four QC standards must be within the ±30% target value. If the sample concentrations are significantly higher than the lowest QC point (100 ng/mL), that particular QC may be disregarded and the run accepted.
At least one of the high QC points must be within the target acceptance. Plasma versus time data were analysed using non-compartmental approaches with the WinNonlin version 5.2 software program.

Pharmacokinetic Parameters:
As shown in Table 2, pharmacokinetic parameters of peptide F after IV administration (10 μg/g) showed significantly short half-life.

Stability in Plasma and Stability after Sample Preparation: as shown
in Table 3 and Table 4.
Peptide F was not stable in plasma at room temperature; therefore, the standard samples must be prepared on ice.

Discussion
17 Flavonoids, phenolics, and many other components in honey are reported for inhibitory effects in cancer colon [52], glioma [53], and melanoma [54] cell lines.
Therefore, honey is a strong antimutagenic agent and presents anticarcinogenic properties. Honey on Escherichia coli exposed to radiation exhibits SOS response (SOS is an error-prone repair pathway contributing to mutagenicity) [56].
According to an in vivo study of CBA mice for breast carcinoma and fibrosarcoma, RJ administered intraperitoneally and subcutaneously exhibits no effect on the formation of metastases; however, simultaneous IV administration of RJ with tumour cells significantly inhibited metastases [58]. Anti-oestrogenic activities of RJ have been reported, which result in an inhibition of MCF-7 mammary cancer cell proliferation [59,60]. By contrast, if the lipid oestrogenic RJ component is added to MCF-7 cells, the proliferation increases [61].
The protein fraction is of RJP3O, which showed antitumor activities when it was tested in HeLa cells of cervical-uterine carcinoma [62].
Peptides related patents for cancer treatments are growing up and increase nowadays [63][64][65][66][67][68][69][70][71][72][73][74][75][76][77] As demonstrated in our studies, in vitro efficacy studies presented positive data; however, studies involving animal model presented only moderate inhibition, although the date were promising for a preliminary human test. These study outcomes present a scope for a debating whether a non-satisfactory effective data in an animal tumour model is sufficient to stop proceeding with the clinical trial before performing a preliminary human test.
Considering that several studies have presented positive effective data in animal tumour models while presenting negative outcomes in human models, a reverse possibility may also be contemplated.
The PK study of rats has demonstrated a significantly short half-life of peptide F in plasma. This may partially explain why IV injection of the peptide combination did not present significant tumour inhibition. These findings must be considered for future in vivo studies. Currently, an intratumoral injection, which minimises the loss of peptide during blood transportation, can be an ideal choice. Moreover, administration frequency and dosage might be adjusted accordingly. However, modification of the peptide or formulation to prolong its plasma half-life is necessary. mouse model. Moreover, pharmacokinetics, dosing, safety, and stability of peptide F were investigated, which presented significantly short half-life, instability of peptide at room temperature, and safety in certain doses. Therefore, modification and improvement of peptide F and other peptides was mandatory to yield more potent, stable, and safe peptides using D-form amino acids instead of L-form in both C and N termini [78]. We foresee introduction of new peptides to the scientific community to be tested and modified 19 for superior long-term malignancy management.  Tumour weights of the two groups were decreased compared with the saline group weight Figure 6 Typical chromatography of plasma sample (rate 1-5 min, PL, IV, 10 μg/g) 33 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download.