Based polymer nanoparticles from bee pollen attenuate non-small lung cancer through enhancement of apoptosis and cell cycle arrest in vivo

doi:10.21203/rs.3.rs-2871939/v1

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Based polymer nanoparticles from bee pollen attenuate non-small lung cancer through enhancement of apoptosis and cell cycle arrest in vivo

https://doi.org/10.21203/rs.3.rs-2871939/v1

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published 06 Oct, 2023

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Bee pollen extract (BPE)-based polymer nanoparticles (BPENP) were fabricated by using bovine serum albumin (BSA) and targeted with folic acid and were further characterized. Mice groups are : Group 1 received saline, whereas Groups 2, 3, 4, 5, and 6 received a single dose of urethane, followed by weekly injections of butylated hydroxy-toluene (BHT). After the BHT injection, the mice in Groups 3, 4, 5, and 6 received BPE, Avastin,BPENP, and BNENP + Avastin, respectively. The number and size of tumors decreased in Group 6 compared to those in the other groups. The ratios of early and late apoptotic cells in Groups 3, 4, 5, and 6 (42.8%, 41.4%, 26.2%, and 45.4%, respectively) were lower than that in the untreated group. The PCNA-labeling indices (LI)% in tissues and lesions from Group 6 were lower than those in the other groups; on the other hand, the caspase 3 LI (%) was higher than those in the other groups. No significant differences in HRAS and MAPK levels were observed between Group 6 and the other groups. However, the level of Baxwas significantly increased, whereas those of Bcl2 and P21 were decreased in Group 6 compared to those in Groups 4 and 5.

Bee pollen

nanoparticles

lung cancer

Avastin

apoptosis

in vivo

Lung cancers account for the majority (11.4%) of malignant tumors in the world after breast cancer (11.7%) [1]. The heterogeneity of the disease may be attributed to the pathologically and clinically significant subtypes. Small-cell lung carcinoma (SCLC; 12.9% of cases) and non-small-cell lung carcinoma (NSCLC; 83.9% of cases) are the two main types of lung malignancies, which can be distinguished based on their primary histotypes, prognoses, and therapeutic effects [2]. Smoking is a known primary risk factor for SCLC, the most aggressive and rapidly developing type of lung cancer [3]. The three primary subtypes of NSCLC include squamous cell lung carcinoma (SqCC), non-SqCC (often known as adenocarcinoma; ADC), and large-cell (undifferentiated) lung carcinoma (LCLC). Sarcoma-like tumors and tumors of the salivary glands account for a relatively minor portion of all cases of NSCLC [4].

ADC, the most common form of NSCLC, accounts for 47.9% of all instances of lung cancer [5]. SqCC normally develops in the center and arises from the main or lobar bronchus [6]. LCLCs, the least common NSCLC, account for 1.5% of all lung cancers; they are squamous cell, glandular, or small-cell non-differentiated NSCLCs that lack histological or immunohistochemical (IHC) evidence of any differentiation [7].

Bee pollen (BP) is a round, seed-like particle ranging from 2.5 to 250 m in size. It is encased in a protective shell and loaded with nutrients such as proteins, lecithin, active enzymes, folic acid, vitamins (particularly vitamin B), and other minerals. The physical forms of BP vary depending on the plant species. Numerous studies have shown that BP can cure obesity, allergies, and infertility [8]. It is a potential source of flavonoids, such as hesperidin, rutin, kaempferol, apigenin, luteolin, quercetin, and isorhamnetin, and natural antioxidants, such as vanillic, protocatechuic, gallic, and p-coumaric acid [9]. These naturally-occurring polyphenols have a range of actions, including anti-inflammatory, anti-carcinogenic, anti-fungal, and antibacterial activities [10]. In addition, the cytotoxic effects of these polyphenols on several tumor cell lines via interruption of the cell cycle or induction of apoptosis have been demonstrated previously [11].

A submicron colloidal dispersion system called drug nanocrystal boosts the saturation solubility of the drug and increases its bioavailability by adding active ingredients, which are stabilized by polymers or surfactants. Polymeric nanoparticles are created when amphiphilic copolymers self-assemble into nanoparticles in water. They often have better stability, tighter size distribution, and more regulated drug release characteristics [12].

Recently, several nano-systems were to enhance the biological activity and stability of drugs when stored and consumed [13]. The purpose of this study was to demonstrate the effects of BP extract (BPE) and BPE-based polymer nanoparticles (BPENP) on lung cancer in mice chemically induced with urethane and butylated hydroxy-toluene (BHT).

2.1 Chemicals for BPE encapsulation

Folic acid was purchased from Fluka-Sigma-Aldrich, St. Louis, MO, USA; 7; and ethanol was obtained from Baker Analyzed, Fisher Scientific, the Netherlands. BSA, protamine (PRM), formaldehyde, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, St. Louis, MO, USA.

2.2 BPE for the encapsulation process

The bee pollen of Trifolium alexandrinum (Egyptian clover; also known as Berseem), was used for extraction. In brief, 3g of dried BP powder and a flask containing 5 ml of DMSO and 45 ml of 96% ethanol were used. The mixture was stirred using a magnetic stirrer at 200 rpm for 1 h at 55°C. The supernatant was spun for 10 min at a speed of 6000 rpm, collected, and stored at 4°C until use for the creation of the nanoparticles.

2.3 BPENP preparation

The BPE (20 mL) and BSA (50 mg/50 mL) were combined and mixed with a magnetic stirrer for 5 min at room temperature. PRM-conjugated folic acid (50 mg/100 ml) was added to the liquid and stirred with the magnetic stirrer for 5 min. The BP was dialyzed against distilled water and stored at − 20°C for lyophilization [14].

2.4 Characterization of BPE and BPENP

A scanning electron microscope (SEM; JEOL JSMIT100) was used at 15 kV to characterize the BPE and BNEP. Images from a transmission electron microscope (TEM) were captured using an EOL 2100 at an accelerating voltage of 200 kV. X-ray diffraction (XRD) tests were carried out using a Shimadzu 6000-XRD X-ray diffractometer at a copper K (Cu K) radiation wavelength of ʎ = 1.54 18. The potassium bromide (KBr) pellet technique and a JASCO Fourier transform infrared (FTIR)-6800 Spectrometer were used to detect the active functional groups of the produced nanoparticles in the 500–4000 cm1 range.

2.5 Chemicals for the animal model

Urethane (97%) and BHT were obtained from Acros-Organics (New Jersey, USA), Avastin (Bevacizumab; 100 mg/4 ml) was purchased from Hoffmann-La Roche Ltd., and BP was obtained from a bee product placed in Egypt.

2.6 Animals protocol

Five-week-old, male Swiss mice were obtained from the Holding Company for Biological Products and Vaccines (VACSERA), Helwan, Egypt. The weights of the mice ranged from 18 to 20 g. The animals were housed in plastic cages (5 per cage) in a conventional animal house, with wood chips for bedding and a natural day/ night cycle at a room temperature of 21°C ± 30°C for two weeks. Diet and water were available ad libitium. Approval for the study design was obtained from The Institutional Animal Care and Use Committee (IACUC) of the Faculty of Science at Tanta University (IACUC:SCI:TU:0129).

2.7 Experimental design

At the age of 7 weeks, 120 male Swiss albino mice were separated into six groups based on their body weights: Group 1 received saline (0.9%) intraperitoneally (i.p), whereas Groups 2, 3, 4, 5, and 6 received a single dose of urethane (1 mg/g, i.p), followed by weekly injections of BHT after every 7 days (first dose, 150 mg/kg, and the remaining five doses, 200 mg/kg) [15]. After the last BHT injection, the mice in Group 3 received an intragastric injection of concentrated BPE (0.5 ml/mouse) [16]. Avastin (5 mg/kg, i.p) was administered to the mice in Group 4 [17], an intragastric injection of BPENP (0.5 ml/mice) was provided to those in Group 5 [18], and Avastin (5 mg/kg, i.p) along with an intragastric injection of BPENP (0.5 ml/mice) was administered to the mice in Group 6. The animals were sacrificed 14 weeks after the start of the experiment.

2.8 Preparation of biological samples

Diethyl ether was used to anesthetize each animal. The abdomen of each mouse was opened, and the lung was taken out. After thoroughly cleaning with 0.09% normal saline, the lungs were injected with buffered formalin from the tracheal opening. Subsequently, the lungs were processed for histological analysis after fixing in 10% phosphate-buffered formalin for at least 48 h, and the diagnosis was made using the standards outlined by two animal pathologists [19].

2.9 Histopathological investigations

The lung samples were fixed in 10% neutral buffer formalin and stored in 70% ethyl alcohol until further processing. After embedding in paraffin, the fixed tissues were dehydrated using a graduated series of ethanol. The slides were routinely stained with Hematoxylin and Eosin [20]. The incidence and multiplicity (total number of nodules and cancer nodules) of the tumor and the average square areas of the nodules within an area of 1 cm² in the section were measured using the stage and ocular micrometer method, wherein the ocular micrometer was calibrated with the standard stage micrometer. The high-power objective of the ocular micrometer in a light microscope was calibrated using a stage micrometer, and the readings were consistent as long as the specific eyepiece and objective were used [21].

2.10. IHC analysis for PCNA and Caspase 3

The avidin-biotin complex method was used to visualize PCNA and caspase 3 in 4-µm-thick sections of the lung tissues. The sections were incubated with anti-PCNA rabbit polyclonal antibody IgG (Catalog No. NBP1-89434, Novus Biologicals, USA; dilution, 1:50) and anti-caspase 3 rabbit polyclonal antibody IgG (Product. No. NB100-56113, Novus Biologicals, USA; dilution, 1:5000) overnight at 4°C. Subsequently, the sections were counterstained with Mayer's hematoxylin and visualized under a light microscope [22]. Briefly, sections were deparaffinized with xylene and hydrated through a graded ethanol series, rinsed in sodium citrate buffer (pH 6.0), and then were heated till boiling in an autoclave and then cooled for antigen retrieval (repeated more than once). The sections were then treated with 0.3% hydrogen peroxide, and normal horse serum, The sections were then progressively treated with 0.3% hydrogen peroxide after being rinsed with TBS-T and submitted to ABC-peroxidase operations (using an ABC kit from Vector Laboratories). Normal serum was utilised as a negative control in place of main antibodies. All IHC procedures used the chromogen 3,3-diaminobenzidine tetrahydrochloride (DAB) to visualise immune complexes [22].

2.10.1. IHC evaluation and generation of labeling index (LI)

The positively-stained cells for the two IHC antibodies displayed brown nuclear staining, whereas the negative cells were stained blue. Sections with no discernible brown stain were deemed as immune-negative. The number of positively-stained nuclei or cells from 15–20 high-power fields in each section was counted and divided by the total number of nuclei in each crypt X100 to generate the labeling indices (LI) for the specific antibodies (PCNA and caspase 3). Two animal pathologists evaluated the sections in a blinded manner without being aware of the experimental groups [21].

2.11 Flow cytometry analysis

Aliquots of homogenate cells (about one gram in weight) from the lungs of mice from different groups were suspended in phosphate-buffered saline, stored on ice, and transported to the flow cytometer (Fluorescence-activated cell sorting caliber flow cytometer; Becton Dickinson, Sunnyvale, CA, USA) located at the Flow Cytometry Unit, Mansoura University Children's Hospital; the cytometer was equipped with a compact air-cooled low power 15 mW argon ion laser beam (488 nm) [23].

2.11.1 Determination of apoptosis using flow cytometry

Apoptotic and necroptotic cell death were identified by dual labeling with FITC-labeled annexin V and propidium iodide (PI). The cells in the lungs treated with BPE, Avastin, BPENP, and BPENP + Avastin were digested, washed, and resuspended in a binding solution. Then, the cells were stained with PI and annexin V-FITC and subjected to flow cytometry following a 15-min dark incubation period. The BD FACSDiva software was used to examine each sample (Becton Dickinson, USA).

2.11.2 Cell cycle analysis by flow cytometry

The labeling of mammalian DNA for flow cytometry was performed to synchronize the cell cycle as described previously [24]. Cells from the lung tissues of mice from the different groups were collected, fixed in ice-cold 70% ethanol, and left at 4°C overnight. The next day, the cells were stained with PI (eBioscience), according to the manufacturer's recommendations, in order to examine the cell cycle.

2.12 RNA isolation and real-time quantitative PCR

Total RNA was isolated from the specimens using (QIAGEN, Analytik Jena Biometra AG, Berlin, Germany) and quantified using a Nanodrop 2000 (Thermo Scientific, San Jose, CA, USA). The RNA was then reverse transcribed into cDNA using the Sensiscript Reverse Transcriptase kit (QIAGEN, Germany). The following primers were used for RT-PCR: HRAS: forward, 5-CTCGCAGCTATGGCATCC-3; and reverse, 5-CAACGTGTGCCCTCACAG-3 MAPK: forward, 5-TCAAGCCTTCCAACCTC − 3 and reverse, 5- GCAGCCCACAGACCAAA-3; Bcl2: Forward, 5-TTGGCCCCCGTTGCTT-3 Reverse, 5-CGGTTATCGTACCCCGTTCTC-3, Bax: Forward, 5-TCCCCCCGAGAGGTCTTTT-3 Reverse, 5-CGGCCCCAGTTGAAGTTG-3; P21: Forward, 5-AGGTGGACCTGGAGACTCTCAG-3, Reverse, 5-TCCTCTTGGAGAAGATCAGCCG-3 B-actin: forward, 5-CTGTCCCTGTATGCCTCTG-3; and reverse, 5-ATGTCACGCACGATTTCC-3. RT-PCR was carried out in two-step using the SYBR GREEN PCR master mix (QuantiTect® SYBR® Green PCR for quantitative, real-time PCR and two-step RT-PCR using SYBR Green I kit) and the Applied Biosystems Steps One™ instrument. The amount of mRNA was calculated using the comparative CT method, which depends on the ratio of the amount of RNA from the target genes to that of the reference gene (B-actin).

2.13 Statistical analysis

Group data expressed as means ± S.D. were analyzed using the two-tailed t-test or ANOVA analyses while the data expressed as percentages were analyzed using the Chi-squared (X²) analysis using the GraphPad Prism version 8.2.0 USA. P ≤ 0.05 was considered significant.

3.1 Characterization of nanoparticles

The BSA nanoparticles typically exhibited favorable properties, such as biocompatibility, biodegradability, longer plasma residence, and improved tumor dispersion [25]. BSA functions as a protective colloid and remains in the bloodstream for a long time [26]. Protamine, protamine-based RNA delivery systems in vaccines against infectious disease and their use in cancer treatment [27]. Folic acid was used to maintain the stability of nanoparticles and boost their adherence to cells, particularly cancer cells [28]. TEM images of BPENP in the form of a 3D assembled structure are shown in Figs. 1A and B. C. The average size of the nanoparticle was 25–35 nm. BP nanoparticles in the SEM image demonstrated a smooth surface and a nearly spherical form (Fig. 1D). This outcome is consistent with that reported by KESKN, (2022) [29], wherein the particle sizes of the BP-based silver nanoparticles ranged between 40 and 60 nm.

FTIR was used to detect the modifications in the chemical bands. As seen in the FTIR spectrum of BSA alone (Fig. 2A), the 3421 cm^− 1 band was assigned to the stretching vibration of the hydroxyl group, whereas the 1650 cm^− 1 band was related to amide I (mainly the C–O stretching vibrations) with a high proportion of α-helix. The band at 1512 cm^− 1 was assigned to amide II [30]. The FTIR of BPE showed a broad band at 3283 cm^− 1, corresponding to the stretching vibration of O–H -polyphenolic compounds, and two strong peaks at 2913 and 2851 cm^− 1 that were assigned to the stretching vibration of C–H. Furthermore, the peak at 1589 cm^− 1 was associated with the C = O groups, wherein a 1034 cm^− 1 band corresponding to the stretching vibration of C–O, C–N, and C–C was detected [31]. The FTIR of the PRM spectrum showed bands located at 1660 and 1540 cm^− 1 that were attributed to the stretching vibrations of C = O and NH–, respectively. An intense band located at 1096 cm^− 1 was assigned to the arginine of protamine [32].

The folic acid spectrum showed a strong peak at 3360 cm^− 1 associated with O–H and N–H (Fig. 2A). Additionally, strong peaks at 1650cm^− 1 were observed for the carboxylic acid groups. Likewise, a strong peak at 1096 cm^− 1 was seen due to the aromatic ring structure [33]. The FTIR of BPENP showed a broad band at 3283 cm^− 1 corresponding to the O–H stretching vibration, and two strong peaks at 2913 and 2851 cm^− 1, the peaks of which were assigned to the C–H stretching vibrations. Furthermore, a peak at 1650 cm^− 1 due to the presence of amide 1 in BSA and one at 1512 cm^− 1 due to folic acid, which is expressed on the phenyl and pterin ring, was observed. The 1200–500 cm^− 1 range is the so-called fingerprint region, which consists of a strong peak at 1034 cm^− 1, corresponding to the C–O, C–N, and C–C stretching vibrations of sugar and proteins during adding PRM shifted from 1096 cm^− 1 to 1034 cm^− 1 [34].

The crystallinity of a formulation is a critical parameter that can be used to assess its physical characteristics. Powder XRD was performed to evaluate the crystallinity of BPENP and FA by measuring the intensity in units of degree 2θ and a range of 5°–80°, as shown in Fig. 2B. The loaded folic acid nanoparticles presented with peaks at 38°, 45°, 65°, and 80° It established folic acid's crystalline form and demonstrated its effective fusion with the other components of the BPENP. XRD patterns for the pollen-coated AuNPs samples showed four distinctive diffraction peaks at 2θ (38.14°, 44.41°, 64.75°, and 77.78°), as described by Kumar et al., 2022) [35].

3.4 Histopathology, incidence (%), multiplicity, and average area of the tumor

Treatment with urethane + BHT resulted in the development of several lesions (nodules) on the outer surface of the lungs, which were later identified as bronchioloalveolar adenocarcinomas, alveolar epithelial adenomas, or alveolar epithelial hyperplasia (Fig. 3, 4). After the lungs were removed, the number of lung nodules on the surface of the lungs was counted with the naked eye. Table 1 displays the tumor incidence, number of nodules, number of tumors (multiplicity), and mean square areas of lesions within a 1 cm² area in the lung sections in the urethane + BHT-treated groups. The lungs in Group 1 did not present with any morphological or histological nodules. The number of nodules was marginally reduced in Group 3 after treatment with BPE and dramatically reduced in Groups 4, 5, and 6 after treatment with Avastin, BPENP, and BPENP + Avastin, respectively. On the other hand, 6.8 nodules in Group 2, which received only urethane + BHT, were identified as adenomas or adenocarcinomas. Furthermore, the average area of the lung lesions within the 1 cm² area was 12.9 mm² in Group 2. The average nodule counts after treatment with urethane + BHT were 4.2 in Group 3 and 2.2 in Group 4 (diagnosed as adenoma or adenocarcinoma). The relative tumor areas were marginally lower in Group 3 and dramatically decreased in Group 4 when compared to that in Group 2 (Table 1) (Supplementary Table 1 and Supplementary Fig. 1).

Additionally, the incidence and number of tumors in Groups 5 and 6 were considerably reduced compared to those in Group 2. Likewise, the average tumor areas in Groups 5 and 6 (6.7 mm² and 3.2 mm², respectively) were significantly lower than that in Group 2. Group 6 demonstrated a markedly reduced inhibitory response to combination therapy compared to the other treatment groups (Table 1). To the best of our knowledge, this is the first study to demonstrate the effect of BPE, BPENP, and BPENP + Avastin in lung cancer in vivo. However, other research combine NP and flavonoid, BPENP + Avastin was consistent with that reported by Parashar et al (2018) [36], which suggested that co-therapy is superior to individual therapy for improving therapeutic efficacy. Flavonoid also supports the development of a safe, effective, and targeted delivery system for the treatment of lung cancer using gefitinib (Gnb) and naringenin (Nar), both of which have been studied for their potential therapeutic and synergistic effects against lung cancer; co-administration of Gnb and Nar nanoparticles showed a substantial decrease in tumor volume [36].

Table 1

Histological incidence (%), multiplicity, and average tumor area per 1 cm² of the lung tissue.
Groups	Treatment	Mice (n)	No. of lung nodules ^a	Tumor incidence (%)	Tumor Multiplicity (Number of tumors / 1 cm²)^b	Average tumor areas / 1 mm^2c
G1	Control -ve	10	0	0	0	0
G2	Ure + BHT	10	8.40 ± 0.12	9/10 (92%)	6.88 ± 4.2	12.99 ± 13.8
G3	Ure + BHT + BPE	8	6.25 ± 0.17 ^*	6/8 (75%)	4.28 ± 2.2 ^*	7.6 ± 3.1 ^*
G4	Ure + BHT + Avastin	12	4.58 ± 0.17 ^*	7/12 (58.3%)	2.25 ± 2.22 ^*	4.98 ± 6.4 *
G5	Ure + BHT + BPENP	10	5.20 ± 0.12 ^*	6/10 (60%)	3.85 ± 3.7 ^*	6.78 ± 5.8 ^*
G6	Ure + BHT + BPENP + Avastin	8	3.75 ± 0.08 ^*	4/8 (50%)	1.80 ± 1.0 , ,**	3.25 ± 4.3 ^{, ,**}

n, number; Ure, urethane; G1: normal control; G2: Ure + BHT; G3: Ure + BHT + BPE; G4: Ure + BHT + Avastin; G5: Ure + BHT + BPENP; G6: Ure + BHT + BPENP + Avastin. a: Lesions: Total microscopical (histological) lesions in lung tissues in tumor-bearing mice regardless of histological type; b: Number of lesions histologically diagnosed as adenomas and adenocarcinomas in 1cm2 of lung tissue; c: Areas (mm²) (tumor size) in 1cm² of lung tissues; Values are means ± standard deviation (S.D.). *: Significant vs. G2 at P < 0.05; **: Significant vs. G4, ***: significant vs. G5 at P < 0.05.

3.5 Flow cytometry analysis data for Annexin

As shown in the dot plots of the log data from the flow cytometry analysis, the percentages of annexin V-FITC-positive cells in the urethane + BHT-treated groups were considerably higher than those in the treatment groups and negative group (Fig. 5). The cells in the BPENP + Avastin group mainly resulted in apoptosis with a significant increase in necrosis when compared to those in the untreated group. After that, when compared to the urethane + BHT (Group 2) versus treated groups dramatically decrease at in the early apoptosis.

The ratios of the percentages of early and late apoptotic cells in Groups 3, 4, 5, and 6 were significantly higher at 42.8%, 41.4%, 26.2%, and 45.4%, respectively, compared to that in the untreated group (Group 2), which displayed the regularly anticipated numbers (Fig. 5).

According to Saisavoey et al. (2021) [37], the observed underlying mechanisms, such as the stimulation of apoptosis, inhibition of cell proliferation in several cell lines, and reduction in tumor growth, account for the anticancer effects of BP.

3.6 Cell cycle analysis data by flow cytometry

The mean numbers of apoptotic cells in the urethane + BHT-treated groups (Groups 3, 4, 5, and 6) were considerably greater than those in Group 2 (Fig. 6). Subsequent treatment with BPENP and Avastin increased by 66.9% when compared to the similar Groups that had just received urethane + BHT treatment. The treated groups (G3,G4&G5) were significantly increased compared to untreated group 2. The initial gap phase (G0/1) in Group 2 was significantly lower than that in the control group (Group 1); alternatively, this phase was substantially longer in Groups 3, 4, 5, and 6 when compared to that in Group 2. However, the gap phase in Group 6 was significantly more than that in Groups 4 and 5. The S phase (G2/M) in Group 2 was significantly lower than that in Group 1; similarly, this phase was significantly lower in Groups 3, 4, 5, and 6 than in Group 2. However, the S phase in Group 6 was significantly longer than those in Groups 2 and 4. The mitotic phase (G2/M) in Group 2 was significantly longer than that in the control (Group 1) and the treated groups (3, 4, 5, and 6); however, the phase in Group 6 was longer than those in Groups 4 and 5. According to Khan et al., (2021) [38] ROS can cause apoptosis by controlling the transcription of a number of redox-sensitive signal proteins. However, some flavones may also cause apoptosis by stopping the cell cycle in a ROS-independent manner at the G2/M phase.

3.7 PCNA IHC levels in lung tissues and tumors

The lungs of mice in Group 2 showed a significant increase in the number of positively-stained nuclei when compared to those in Group 1 (Fig. 7). Additionally, the PCNA LI (%) in Groups 3, 4, 5, and 6 were considerably lower than that in Group 2. Furthermore, the PCNA LI (%) in the tissues and lesions of mice belonging to Group 6 were significantly lower than those in Groups 3, 4, and 5, Group 6. Reddish-brown nuclear staining was seen in the lung tissues after IHC labeling for PCNA. These findings are consistent with that reported by Granja et al. (2016) [39], wherein the anti-tumor effects of green tea and EGCG nanoparticles were demonstrated in vivo by assessing the levels of Ki-67, PCNA, and CDK4 in the tumor tissues. In the study by Salim et al. [21], decreases in the incidence and size of the lung tumor were followed by a decline in cell proliferation, as shown by the PCNA LI (%). After urethane + BHT treatment, the use of balanitoside resulted in a dose-dependent reduction in PCNA LI (%) in mouse lungs. Previous studies have reported a gradual increase in PCNA LI (%) during carcinogenesis [21].

3.8 Caspase IHC levels in lung tissues and tumors

A significant number of strongly positive caspase-stained nuclei were seen in the lungs of mice in Group 2 when compared to that in Group 1 (Fig. 8). Additionally, the lung parenchyma of mice in Groups 3, 4, 5, and 6 presented with significantly higher caspase LI (%) than those in Group 2. Additionally, lungs from mice belonging to Group 6 presented with the highest caspase LI (%) values compared to those from Groups 3, 4, and 5. Lung tissues stained with caspase showed reddish-brown nuclear staining. Ben Bacha et al. (2020) [40] reported that the anti-inflammatory properties of BPE decreased levels of IFN-γ, inhibited the apoptotic cascade, lowered the concentration of p53, and ultimately reduced the production of caspase-3.

3.9 qRT- PCR data analysis

Gene expression levels of HRAS, MAPK, Bcl2, Bax, andp21in the lung tissues

The normalized mRNA expression levels of HRAS and MAPK did not differ substantially among the groups (Figs. 9A, B).

Compared to the negative non-treated control group 1 and the positive control group 2, the normalized mRNA expression level of the Bax gene was significantly increased (P < 0.05) by 4.8-, 2.3-, 2.2-, and 3.3-fold in Groups 3, 4, 5, and 6, respectively (Fig. 10A). Treatment with BPENP (Group 5) considerably reduced the mRNA expression levels relative to that in Group 3 by around 2.2-fold; however, the level was statistically different from that in the normal control. Treatment with BPENP + Avastin (Group 6) significantly increased the mRNA expression levels of Bax by 3.3-fold when compared to those in Groups 4 and 5.

The normalized mRNA expression levels of the Bcl2 gene were significantly downregulated (P < 0.05) in Groups 2, 4, and 6 (by 0.73-, 0.71-, and 0.5-fold, respectively) compared to the control group (Fig. 10B). Treatment with BPE (Group 3) and the combination of BPE and urethane + BHT (Group 6) significantly reduced the mRNA expression levels relative to those in Group 2 by around 0.96- and 0.5-fold, respectively; however, the levels remained significantly different from those in Group 1. Treatment with BPENP + Avastin (Group 6) resulted in a 0.5-fold drop in the mRNA expression levels of Bcl2 when compared to those in Groups 4 and 5 (Fig. 10B).

The normalized mRNA expression levels of the P21gene were found to be significantly (P < 0.05) elevated in Groups 2, 3, and 6 by 2.2, 1.9, and 1.4-fold, respectively when compared to those in the negative non-treated control group 1(Fig. 10C). The levels of the gene in Groups 3, 4, 5, and 6 were significantly lower than that in Group 2, and remained significantly different from that in Group 1. BPENP treatment resulted in a significant decrease in the level of P21 compared to that in Group 3 (Supplementary Fig. 2 and Supplementary Fig. 3) .

The pro-apoptotic gene Bax is the primary apoptosis regulator that initiates apoptosis in cells. The anti-apoptotic gene Bcl2 can prevent apoptosis by inactivating Bax. Consequently, the Bax/Bcl2 transcription ratio can affect the growth or inhibition of cancer cells [41]. Naseri et al. (2021) [42] investigated the effect of BP on the expression of the pro-apoptotic (Bax and caspase-3) and anti-apoptotic (Bcl-2) genes; they demonstrated increased expression levels of Bax, caspase-3, -8, and − 9 and decreased levels of Bcl-2, Ki-67, CD31, and (VEGF). In another study by Zakhireh et al. (2022) [43], the mRNA expression level of Bax was dramatically increased (P < 0.05), whereas that of Bcl2 was significantly downregulated (P < 0.05) in cells seeded on PShs/Fe3O4NPs when compared to those in bare PShs. Cells cultivated on a substrate containing the highest concentration of Fe3O4NPs had the highest Bax/Bcl2 ratio. Increased expression of caspase-3 and an increase in the Bax/BCL2 ratio revealed that the intrinsic mitochondria-dependent pathway mediates apoptosis by Fe3O4NPs.

Similary our previous results confirmed the present finding that bioactive materials isolated from bee pollen have potential therapeutic effect on non small lung cancer celllines [44, 45].

The in vivo lung cancer mouse model in the current study revealed that treatment with adjuvant chemotherapies combined with polyphenolic compound isolated from bee pollen activates significantly apoptotic pathway as evaluated by flowcytometry, immunohistochemistry and apoptotic genes, in addition, decreased tumors volume and tumor growth as determined by histopathology. It can be concluded that hybrid polymeric protein nanoparticles involved natural bioactive materials, can be recommended strongly to be administrated with chemotherapy to potentiate their effect and minimize their required dose. Thus, dropping the maximum dose of adjuvant chemotherapies can be minimized to diminish their toxicity on healthy cells which support their chemo-preventive and protective agents and can be used as a dietary complement during the chemotherapeutic progression.

ANOVA: Analysis of variance; Avast., Avastin; BPE: Bee pollen extract; BPENP: Bee pollen extract nanoparticles; DMSO: Dimethylsulfoxide;; NPs: Nanoparticles; NSCLC: Non-small cell lung cancer; SCLC: small cell lung carcinoma; ADC: adenocarcinoma : SqCC: squamous cell lung carcinoma; LCLC : large cell lung cancer; TTF1: thyroid transcription factor 1; IHC: Immunohistochemistry; Ure: Urethane; BHT: Butylated hydroxytoluene (BHT); Ros: Reactive Oxygen species; PCNA: Proliferating cell nuclear antigen; PI: Propidium iodide; FACS: Flourescence activated cell sorting; RT-PCR: Real-time-polymerase chain reaction; SD: Standard deviation; XRD: X ray differaction; FTIR: Fourier Trans; CS:Cationic chitosan; PLGA: poly(lactic-co-glycolic acid); HRAS: Harvey Rat sarcoma virus; MAPK: mitogen-activated protein kinase; BSA: Bovine serum albumin; PRM: protamine; HPLC: High-performance liquid chromatography; FA: Folic acid; LI: labeling indexes.

Ethics approval and consent to participate: All experiments were conducted in accordance with US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and cell line experiments Guide for the Care and Use of Laboratory Animals after being approved by the relevant Ethical Committee and authorized by the Italian and German Ministry of Health. This study was also approved by the Research Ethics Committee of Tanat University (KFS.Ph.001/21).

Consent for publication: All authors have provided their agreement to publish the submitted manuscript.

Availability of data and materials: Data is available in a publicly accessible repository.

Competing interests: The authors declare no conflict of interest.

Funding: This research received no funds.

Authors' contributions: Elsayed I. Salim: Methodology, data curation, investigation, writing original draft, preparation, design the manuscript, format analysis and supervision. Magdy E. Mahfouz: Methodology, data curation, investigation, format analysis, write original draft. Eman A. Eltonouby: Methodology, data curation, investigation, design the manuscript, reviewing and editing original draft. Ezar H. Hamed: Supervision, review and editing, investigation, design the manuscript. Nemany A. N. Hanafy: Methodology, data curation, investigation, supervision, review and editing, investigation, design the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgments: . Eman A. Eltonouby would like to thank professor Dr. Elsayed I. Salim, and Magdy E. Mahfouz, Ezar H. Hamed and . Nemany A. N. Hanafy for their kind supervision, support, and their encouragement.

Authors' information: Data of authors will be available in proofreading manuscript after being accepted.

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No competing interests reported.

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Based polymer nanoparticles from bee pollen attenuate non-small lung cancer through enhancement of apoptosis and cell cycle arrest in vivo

Status:

Journal Publication

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Chemicals for BPE encapsulation

2.2 BPE for the encapsulation process

2.3 BPENP preparation

2.4 Characterization of BPE and BPENP

2.5 Chemicals for the animal model

2.6 Animals protocol

2.7 Experimental design

2.8 Preparation of biological samples

2.9 Histopathological investigations

2.10. IHC analysis for PCNA and Caspase 3

2.10.1. IHC evaluation and generation of labeling index (LI)

2.11 Flow cytometry analysis

2.11.1 Determination of apoptosis using flow cytometry

2.11.2 Cell cycle analysis by flow cytometry

2.12 RNA isolation and real-time quantitative PCR

2.13 Statistical analysis

3. Results and Discussion

3.1 Characterization of nanoparticles

3.4 Histopathology, incidence (%), multiplicity, and average area of the tumor

3.5 Flow cytometry analysis data for Annexin

3.6 Cell cycle analysis data by flow cytometry

3.7 PCNA IHC levels in lung tissues and tumors

3.8 Caspase IHC levels in lung tissues and tumors

3.9 qRT- PCR data analysis

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1