ANTI-LEUKEMIC, ANTI-HIV, AND SIALIDASE ACTIVITIES OF ROYAL-JELLY PROTEINS

Abstract

This invention discloses isolated protein fractions from Apis mellifera royal jelly (RJ) have proven potent efficacy in inhibiting leukemia cell growth and HIV-1 reverse transcriptase (RT) as well as releasing the cellular sialic acid (sialidase catalytic activity). Methods for RJ fractionation, the investigation against leukemia cell lines (NFS-60 and Jurkat cells), HIV-1 RT, and cellular SA are disclosed.

Claims

1. A method of inhibiting myeloid and lymphoid leukemia cell growth and HIV-1 reverse transcriptase, RT, activity, the method comprising administering to the cells different protein fractions isolated from Apis mellifera royal jelly, RJ, named as protein fraction 25, PF.sub.25, 30, PF.sub.30, 40, PF.sub.40, 50, PF.sub.50, 60, PF.sub.60, major royal jelly protein 2, MRJP2, and MRJP2 isoform XI, resulting in inhibitory effects of different potency for myeloid and lymphoid leukemia cell growth and HIV-1 reverse transcriptase, RT, activity, and where some of the protein fractions having sialidase catalytic activity.

2. A method of inhibiting the myeloid and lymphoid leukemia cell growth according to claim 1, comprising the use of PF.sub.30 or one of its proteins.

3. A method of inhibiting the myeloid and lymphoid leukemia cell growth according to claim 1, comprising the use of PF.sub.40 or one of its proteins.

4. A method of inhibiting the myeloid and lymphoid leukemia cell growth according to claim 1, comprising the use of PF.sub.50

5. A method of inhibiting the myeloid and lymphoid leukemia cell growth according to claim 1, comprising the use of MRJP2.

6. A method of inhibiting the myeloid and lymphoid leukemia cell growth according to claim 1, comprising the use of MRJP2 isoform XI.

7. A method of inhibiting the HIV-1 RT activity according to claim 1, comprising the use of PF.sub.25 or one of its proteins.

8. A method of inhibiting the HIV-1 RT activity according to claim 1, comprising the use of PF.sub.30 or one of its proteins.

9. A method of inhibiting the HIV-1 RT activity according to claim 1, comprising the use of PF.sub.40 or one of its proteins.

10. A method of inhibiting the HIV-1 RT activity according to claim 1, comprising the use of PF.sub.60 or one of its proteins.

11. A method of inhibiting the HIV-1 RT activity according to claim 1, comprising the use of MRJP2.

12. Based on claims 7-11, PF.sub.25, PF.sub.30, PF.sub.40, PF.sub.60, and MRJP2 can prohibit the HIV replication and similarly other retroviruses such as Oncoviruses, Spumavirus, and many others in addition to HBV.

13. According to claim 1, the PF.sub.3o or one of its proteins able to release sialic acids (SAs) from the cellular surface i.e. they have sialidase activity.

14. According to claim 1, the PF.sub.40 or one of its proteins able to release sialic acids (SAs) from the cellular surface i.e. they have sialidase activity.

15. According to claim 1, the PF.sub.50 able to release sialic acids (SAs) from the cellular surface i.e. it has sialidase activity.

16. According to claim 1, the MRJP2 able to release sialic acids (SAs) from the cellular surface i.e. it has sialidase activity.

17. According to claim 1, the MRJP2 isoform XI able to release sialic acids (SAs) from the cellular surface i.e. it has sialidase activity.

18. Based on claims 13-17, PF.sub.30, PF.sub.40, and PF.sub.50 or one of their proteins in addition to MRJP2 and MRJP2 isoform XI able to prevent the infection with viruses that their entry to the host cells depends on the SA receptor such as Influenza virus, Isavirus Coronaviruses [including severe acute respiratory syndrome coronavirus 2], Adenoviruses, Rotaviruses, and many others.

19. Based on claims 13-17, PF.sub.30, PF.sub.40, and PF.sub.50 or one of their proteins in addition to MRJP2 and MRJP2 isoform XI able to prevent the infection with some types of bacteria and bacterial toxins that depend on the presence of SA receptor on the host cells to begin their infection. These include Helicobacter pylori, Streptococcus pneumoniae, Vibrio cholera toxin, Clostridium tetani toxin, and others.

Description

DETAILED DESCRIPTION

[0020] This invention provides certain protein fractions from RJ (obtained from the local market, Egypt) having high cytotoxic potency against leukemia cell growth, inhibitors for HIV-1 replication, and able to release the cellular SA (sialidase catalytic activity).

RJ Fractionation

[0021] RJ was fractionated following the method that used in our recently published PCT (EG2017/000022) into carbohydrate, lipid, and protein fractions.

[0022] Carbohydrate fraction preparation, 2 g of RJ was dissolved in water/methanol mixture (3:1) and deproteinized using Carrez I (potassium hexacyanoferrate II) and Carrez II (zinc acetate) reagents. Then, lipids were removed by washing the deproteinized RJ two times with dichloromethane. The aqueous layer (sugar fraction) was filtered through 0.2 m disposable syringe filter, lyophilized (Telstar, Terrassa, Spain) and kept at 80 C. until used.

[0023] Lipid fraction preparation, lipids were isolated from RJ with petroleum ether using Soxhlet apparatus for 30 min. The organic solvent was evaporated, and then the lipid fraction was stored at 80 C.

[0024] Crude protein fraction (CPF) preparation, the water-soluble proteins were extracted from RJ using ammonium sulfate crystals (Brixworth, Northants, UK). In brief, 1.5 g of RJ was dissolved in phosphate buffer saline (PBS, 0.1 M, pH 7) containing 1 protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo., USA) and the solution was centrifuged at 3800 g and 4 C. for 30 min. Then the water-soluble proteins in the supernatant were precipitated by adding crystals of ammonium sulfate until the saturation reach 60%. Pellet (CPF) was dissolved in PBS, dialyzed for 24 h against the same buffer and finally freeze-dried to obtain the powdered fraction.

[0025] The RJ CPF fractionation, CPF was further fractionated into five fractions (PF.sub.25, PF.sub.30, PF.sub.40, PF.sub.50, and PF.sub.60) using different ammonium sulfate saturation (20-25%, 25-30%, 30-40%, 40-50%, and 50-60%, respectively). The precipitated proteins were obtained by centrifugation at 3800 g (4 C.) for 30 min, dialyzed for 24 h against PBS, and lyophilized.

[0026] The major RJ protein 2 (MRJP2) and its isoform X1 purification, The PF.sub.50 was further fractionated by carboxymethyl (CM)-Sephadex ion-exchange column chromatography into two purified proteins, major RJ protein 2 (MRJP2) and its isoform X1. In brief, the amount of PF.sub.50 that obtained from 10 g of RJ was dissolved in 20 mL of the binding buffer (20 mM phosphate buffer containing 1 protease inhibitor cocktail, pH 6.7). The protein solution then applied to the CM-Sephadex column (162.5 cm) and left for 1 h at 4 C. The unbound protein (MRJP2 isoform X1, fraction 1) was obtained by washing the column with about 100 mL of the binding buffer. Elution of the bound protein (MRJP2, fraction 2) was achieved by a one-step gradient of about 50 mL of 0.5 M NaCl in the binding buffer. The protein content was determined in the purified fractions by UV measurement at 280 nm after dialysis for 24 h against PBS (pH 7) then freeze-dried.

Anti-Leukemic Activities of RI Fractions

[0027] The present study evaluated the anti-leukemic effect of RJ and its isolated fractions, including carbohydrates, lipids, CPF, PF.sub.25, PF.sub.30, PF.sub.40, PF.sub.50, PF.sub.60, MRJP2 and MRJP2 isoform X1 in comparison with Doxorubicin (DOX). This evaluation was done using two types of leukemia cell lines, murine myeloid (NFS-60) and human T lymphocyte (Jurkat).

[0028] Isolation of white blood cells (WBCs), The human WBCs were isolated from the blood of ten healthy volunteers (collected in heparin tubes). In brief, the blood was mixed gradually with a fresh cold lysing solution (80.2 mg % ammonium chloride, 8.4 mg % NaHCO.sub.3, and 3.7 mg % EDTA) then centrifuged at 1650 rpm for 5 min. The pellet (WBCs) was washed twice with RPMI-1640 medium and cells were stained with trypan blue for checking the viability and counting using a phase-contrast microscope (Olympus, Tokyo, Japan). Finally, cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and seeding as 10.sup.5 cells/well in 96 well cell culture plate.

[0029] Determination of the safe doses of RJ and its fractions on normal WBCs, About 100 L of serial dilutions of each of RJ fractions and standard chemotherapy (DOX) were incubated with WBCs in a CO.sub.2 incubator (New Brunswick Scientific, Netherlands) at 37 C., 5% CO.sub.2, and 90% relative humidity. After 72 h, 20 L of MTT (5 mg/mL in PBS) was added to each well and incubated for further 3 h, and then centrifuged for 10 min at 2000 rpm. One hundred microliters of DMSO was added to each well after supernatant aspiration and the absorbance was read at 570 nm using ELISA reader (BMG LabTech, Germany). Cell viability was determined and a relation between the cell viability and the studied fractions or DOX concentrations was plotted for calculating the safe concentrations (EC.sub.100, 100% cell viability) using GraphPadInstat program.

[0030] Anti-leukemic activity of RJ and its fractions, Both leukemia cells (NFS-60 and Jurkat) were seeded in RPMI containing 10% FBS as 3000 cells/well in 96 well cell culture plate. Then serial concentrations from the safe dose of each of the tested RJ and its fractions was added and incubated for 72 h in a 5% CO.sub.2 incubator at 37 C. The cytotoxic effect of RJ and its fractions in comparison with DOX against both leukemia cell lines were investigated using the MTT assay as described above. Then the concentration that inhibits leukemia cell growth by 50% (IG.sub.50 value) was determined for the RJ and each of its fractions and used to select the effective fraction (the lowest IG.sub.50 value). The morphological changes of untreated and treated leukemia cells were examined using the phase-contrast microscope.

[0031] Flow cytometric analysis of apoptosis, RJ, its effective fractions (CPF, PF.sub.50, MRJP2, and MRJP2 X1), and DOX at their IG.sub.50 was incubated for 72 h with each of the leukemia cell lines. After trypsinization, the untreated and treated cells were incubated with annexin V/propidium iodide (PI) for 15 min. Then cells were fixed and incubated with streptavidin-fluorescein (5 g/mL) for 15 min. The apoptosis-dependent anti-leukemic effect was determined by quantification of annexin-stained apoptotic cells using the Fluorescein isothiocyanate (FITC) signal detector (FL1) in the flow cytometer (Partec, Germany).

[0032] Fluorescence microscope investigation of apoptotic cells, NFS-60 and Jurkat cell lines were incubated separately with the most effective RJ fractions (PF.sub.50, MRJP2, and MRJP2 X1) and standard drugs (DOX) for 72 h in the CO.sub.2 incubator. Then leukemia cell apoptosis was investigated by ethidium bromide (EB)/acridine orange (AO) double staining (100 g/mL for each) and then visualized under the fluorescent phase contrast microscope (Olympus, Japan).

[0033] Cell cycle distribution by flow cytometry, The change in leukemia cell cycle distribution before and after treatment with IG.sub.50 of the most effective anti-leukemic RJ fractions (PF.sub.50, MRJP2, and MRJP2 X1) was determined by flow cytometry as described previously. Briefly, the untreated and treated leukemia cells were incubated with 5 g/mL RNase A (Sigma, USA) then mixed with 10 l of 1 mg/mL PI (Sigma, USA) for flow cytometry analysis at 488 nm using Cell Quist and Mod Fit softwares.

[0034] Table (1) represents the EC.sub.100 values of RJ and its isolated fractions. Results revealed the higher values (safer) for the RJ and its isolated factions more than DOX. In addition, results elucidated the higher safety of the RJ-PFs followed by the carbohydrate fraction, RJ, then the lowest safety was the lipid fraction. The Table also showed the IG.sub.50 values of RJ and its fractions against the two studied leukemia cells in comparison with DOX. Data revealed that DOX was significantly more potent than the tested fractions against the two studied leukemia cells and from these fractions, PF.sub.50 was the most potent (the lowest IG.sub.50).

[0035] FIG. 2 shows the morphology of the two leukemia cell lines under the phase contrast microscope after treatment with the most effective RJ-PFs and the standard chemotherapy. After 72 h incubation of the cancer cells with the different treatments, cells appeared as oval or irregular-shaped and shrinkage with condensed cytoplasm and apoptotic bodies. All of these features are the hallmarks of the apoptosis, which observed obviously with cancer cells-treated with the MRJP2 more than other treatments.

[0036] Apoptosis in the leukemia cell lines was clearly observed by annexin/PI flow cytometric analysis (FIG. 3) and the EB/AO double fluorescent staining (FIG. 4). The flow cytometric analysis showed that the highest percentage of the apoptotic cell populations was induced by MRJP2 followed by PF.sub.50 then MRJP2 X1 and this apoptotic effect was nearly equipotent to DOX. While the results of EB/AO double staining clarified that the leukemia viable cell number was depleted tremendously and no necrotic cells (red enlarged nuclei) were observed with all treatments. The treatment with PF.sub.50, MRJP2, and DOX increased the number of the late apoptotic cells (orange-red nuclei). However, the treatment with MRJP2 X1 elevated the number of early apoptotic cells (greenish-yellow nuclei) beside a few late apoptotic cells. In harmony with the flow cytometric results, MRJP2 showed the equipotent apoptotic effect to DOX and higher efficiency than PF.sub.50 and MRJP2 X1.

[0037] FIG. 5 showed the cell cycle regulatory effect of RJ-PFs. The FIG. clarified that both types of leukemia cells were arrested and accumulated at G1 phase. After 72 h incubation of each of these cancer cells with the RJ-PFs, the arrested cell populations were significantly decreased. Interestingly, these treatments significantly delay the G0 phase and blocked the cancer cell populations in this phase. This was accompanied by a decrease the cancer cell populations in both S and G2/M phases. For all of these effects, MRJP2 was the most effective fraction with the same or higher potency than DOX. These results concomitant with the apoptotic effect of these PFs due to the strong correlation between the G0 arrest and induction of apoptosis as confirmed by many previous studies. Therefore, the anti-leukemic effect of the RJ-PFs especially, MRJP2 mediated by significant induction of G0 phase arrest followed by induction of apoptosis.

Effect of RJ and its Fractions on the HIV-1 Reverse Transcriptase Activity

[0038] The current study evaluated the inhibitory effect of RJ and its isolated fractions (lipids, carbohydrates, CPF, PF.sub.25, PF.sub.30, PF.sub.40, PF.sub.50, PF.sub.60, MRJP2, and MRJP2 isoform X1) on the HIV-1 reverse transcriptase (RT) activity. The RT assay colorimetric kit (Roche Diagnostics GmbH, Mannheim, Germany) was used. The kit principle based on the use of the template/primer hybrid poly (A)oligo (dt).sub.15 and labeled nucleotides with digoxigenin and biotinin an optimized ratio for the synthesis of a freshly DNA molecule by RT transcriptase. The detection and quantification of the synthesized DNA follow a sandwich ELISA protocol.

[0039] For the RT inhibitory assay, the recombinant HIV-1 RT contained in the kit was prepared using autoclaved redistilled water into 10 mU/L (2 ng/L) final concentration. Then 20 L (containing 1, 0.5, 0.25, 0.125, 0.0625 mg) of RJ or each isolated fractions was incubated with the same volume of the prepared enzyme (4 ng/20 L) for 1 h at 37 C. Two controls were included, the negative control (without the enzyme) and the positive control (without the tested compounds). The enzymatic reaction was staid by adding 20 l of the substrate mixture [template/primer hybrid (750 mA.sub.260 nm/ml) and triphosphate (10 M, dUTP/dTTP)] and the reaction was continued for 1 h at 37 C. Then 60 L of the mixture was transferred into microplate (MP) modules precoated with streptavidin and post-coated with blocking reagent and incubated for another 1 h at 37 C. The MP wells were washed 5 times with the washing buffer provided by the kit, then the anti-digoxigenin-peroxidase working solution was added and followed by 1 h incubation at 37 C. The MP wells were washed again 5 times, after which the peroxidase substrate solution was added into each well and the absorbance of the produced color was measured at 405 nm using an ELISA reader (BMG LabTech, Germany). The inhibitory activity of the RJ and its fractions were calculated as percent inhibition compared to a control. Then the IC.sub.50 (the concentration that inhibits 50% of the enzyme activity) was calculated for each fraction.

[0040] The results in Table 1 showed that the IC.sub.50 values of all the RJ fractions are almost the same, except for the lipid fraction, which had a higher value (lower potency). When we look at the HIV-1 RT % inhibition of each of these fractions at the higher concentration used (1 mg), we can notice the most effective fractions. The PF.sub.25, PF.sub.30, PF.sub.40, PF.sub.60, and MRJP2 revealed inhibitory effect of more than 90% and they were considered the most effective RJ fractions against this enzyme. While lipid fraction was the lowest effective fraction with inhibitory effect less than 40%. Other fractions exhibited different inhibitory percentages between these two values.

Sialidase Activity of RJ Protein Fractions

[0041] The sialidase catalytic activity of the RJ isolated PFs was evaluated by incubating different concentrations (500, 250, 125, 62.5, 31.25) of each of the RJ-PFs (CPF, PF.sub.25, PF.sub.30, PF.sub.40, PF.sub.50, PF.sub.60, MRJP2 and MRJP2 isoform X1) with PBMCs or HepG2 cells at 37 C. for 2 h and 72 h. Two controls were included, each PF alone without cells and each cell alone without PF. At the end of the incubation period, the released SA concentration was quantified followed the previously published method.

[0042] Preparation of SA-attached cells, PBMCs were obtained by Ficoll-Hypaque density gradient centrifugation method as described previously. In brief, the blood samples from healthy volunteers were diluted with an equal volume of PBS, carefully layered on Ficoll-Hypaque, and centrifuged at 2000 rpm, 25 C. for 30 min. Then the undisturbed PBMCs layer (interface) was carefully transferred out, washed twice with 40 ml RPMI-1640 medium, and centrifuged at 1650 rpm for 10 min. Finally, the supernatant was removed and the cells were suspended in 5 ml of RPMI-1640 medium containing 10% FBS and counted using trypan blue stain. HepG2 cells were grown in RPMI-1640 medium (HyClone) supplemented with 10% heat-inactivated FBS.

[0043] The SA assay, SA concentration was measured by the alkali-Ehrlich method using 0.2 M borate buffer at pH 8.5. After the incubation period (2 h or 72 h), cell culture was centrifuged at 2000 rpm for 15 min and the SA content was quantified in the supernatant. To 0.5 mL of the supernatant, water (blank), or different standard concentrations (1-10 nmol/mL), 0.5 mL of the borate buffer solution was added. Then the mixture was heated at 100 C. for 45 min, cooled, treated with 3 ml of ethanol followed by 1 ml of the Ehrlich reagent and heated at 70 C. for a further 20 min. The developed violet color was read at 560 nm. The SA concentration (nmol/mL) was calculated from the standard SA calibration curve and used to calculate the sialidase activity of the RJPF as nmol/ml/min (IU). The specific activity (IU/mg protein) was calculated after determination of the protein content (mg/mL) in the supernatant using the Bradford method.

[0044] The results in FIG. 6 revealed the ability of all the studied RJ-PFs except PF.sub.25 and PF.sub.60 to release SA from the surface of PBMCs and HepG2 cells (i.e having sialidase activity) and this ability was time (FIG. 6E) and concentration (FIG. 6A-D)-dependent. The most potent enzymatic activity was observed for the PF.sub.50 more than other RJ-PFs and its purified proteins (MRJP2 and MRJP2 X1) separately. This effect clarified the synergistic catalytic activity of MRJPs in a combined form (PF.sub.50). The sialidase activity of the RJ-PFs, particularly PF.sub.50 has a crucial role in the prevention of many viruses, bacteria, and toxins entry into their host cells. Therefore, by cleaving SAs from the surface of the host cells, their receptors will be inactivated and thereby potentially renders the host cells resistant to this target infection.

Statistics

[0045] Data were expressed as meanSE and were analyzed by SPSS version 16. The mean values were compared using one-way analysis of variance (ANOVA) by Duncan's test and significance was determined at P<0.05. IC.sub.50 and EC.sub.100 values were calculated by the GraphPadInstat software version 3.

A BRIEF DESCRIPTION OF THE DRAWING

[0046] FIG. 1: Novel activities of Apis mellifera royal jelly proteins.

[0047] FIG. 2: Morphological changes in the murine myeloid (NFS-60) and human T lymphocyte (Jurkat) leukemia cell lines after the treatment with royal jelly (RJ) and its protein fractions (PFs) in comparison with the doxorubicin (DOX) chemotherapeutic drug as observed under the inverted microscope. CPF; crude protein fraction, MRJP; major royal jelly protein.

[0048] FIG. 3: Flow cytometric analysis using annexin V/propidium iodide (PI) double staining for detection of the apoptotic leukemia cells before and after the treatment with royal jelly (RJ) and its protein fractions (PFs) in comparison with the doxorubicin (DOX) chemotherapeutic drug. (A) Annexin V/PI flow charts for the control and treated-murine myeloid (NFS-60) and human T lymphocyte (Jurkat) leukemia cell lines. (B) Quantification of the % apoptotic cells in the control and treated-leukemia cells. CPF; crude protein fraction, MRJP; major royal jelly protein. Values are presented as meanSE (n=3) and different letters specify the significance at P<0.05.

[0049] FIG. 4: Acridine orange/ethidium bromide nuclear double staining of the apoptotic cell populations in the murine myeloid (NFS-60) and human T lymphocyte (Jurkat) leukemia cells before and after the treatment with the effective royal jelly (RJ) protein factions (PFs) in comparison with the doxorubicin (DOX) chemotherapeutic drug. MRJP; major royal jelly protein, VC; viable cells, EA, LA; early and late apoptotic cells, respectively.

[0050] FIG. 5: Cell cycle distribution of murine myeloid (NFS-60) and human T lymphocyte (Jurkat) leukemia cells before and after the treatment with the effective royal jelly (RJ) protein factions (PFs) in comparison with the doxorubicin (DOX) chemotherapeutic drug. (A) Flow cytometric images showed G2/M phase arrest (B, C) Quantification of the percentage of cells in the cell cycle phases (G0, G1, S, and G2/M). MRJP; major royal jelly protein. Values are presented as meanSE (n=3) and different letters specify the significance at P<0.05.

[0051] FIG. 6: Sialidase activity of royal jelly (RJ) protein fractions (PFs). (A, C) After 2 h incubation with peripheral blood mononuclear cells (PBMCs) and HepG2 cell, respectively (B, D) After 72 h incubation with PBMCs and HepG2 cell, respectively. (E) Time-dependent sialidase activity of RJ-PFs at the concentration of 500 g/mL. CPF; crude protein fraction, MRJP; major royal jelly protein.