Lung response to propolis treatment during experimentally induced lung adenocarcinoma

Abstract

Propolis is a natural agent produced by honeybees from several botanical sources. The purpose of our study is to evaluate the antitumor effect of propolis extract (PE) against urethane-induced lung cancer compared to Cisplatin (CIS) in rats. Some biochemical parameters of oxidative stress, gene expression and histopathology and transmission electron microscopy were estimated and performed. PE improved the induced changes in glutathione, malondialdehyde and superoxide dismutase concentration generated by urethane. Moreover, PE increased the expression of EGFR-mRNA. Examination of the light and electron microscopic sections manifested that the URT group displayed the excessive abnormal distribution of collagen fibers and the growth of papillary adenocarcinoma in the terminal bronchial lumen with thickening in the bronchial wall, inflammation and alveolar collapse. PE was able to decrease the above induced changes. In conclusion, a recent study concluded that PE could be an excellent source of natural chemicals that fight lung cancer.

KEYWORDS:

• Propolis extract
• urethane
• lung cancer
• Clara cells
• cisplatin
• rats
• mucin

1. Introduction

Lung cancer is a major cause of human cancer mortality in both sexes worldwide [1]. Non-small cell lung cancer “NSCLC” and small cell lung cancer “SCLC” are the two most prevalent forms. Non-small cell lung cancer is responsible for approximately 85% of all instances of lung cancer and is further categorized into adenocarcinoma, large cell carcinoma and squamous cell carcinoma [2,3]. Nearly 40% of all lung malignancies are adenocarcinomas [4].

Genetic changes are the critical cause of the malignant transformation of epithelial cells. Also, the microenvironments in which the cells live govern carcinogenesis. Most cancers arise from a cellular milieu characterized by decreased host immunity, dysregulated inflammation and elevated production of cellular growth and survival factors that promote angiogenesis and block apoptosis [5].

Lung cancer prevention has received large attention and the decrease in smoking in recent decades has helped, but smoking is not the only problem. Lung cancer in people who have never smoked is currently the fifth leading reason for cancer death in the United States [4]. Several factors contribute to the lethality of lung cancer, involving the rapidity of tumour growth, advanced stage at diagnosis, early development of metastases and resistance to therapy [6].

The environmental carcinogen urethane (IARC category 2A) largely promotes lung cancers in mice [7]. Lung tumours in mice are believed to derive from non-ciliated airway epithelial (Clara) or type II alveolar epithelial cells, and gene mutations described in mice lung tumour cells are analogous to those recognized in human lung cancers [8]. The currently used drug, urethane, is known to influence the progress of benign or malignant lung cancers “adenocarcinomas” [9]. It produces malignant lesions in a range of sites owing to the manufacture of the active metabolite vinyl carbamate. Repeated exposure to the urethane found in fermented foods and beverages increases the risk of lung cancer by producing persistent mitochondrial dysfunction and increasing cellular mitotic activity [10].

The treatment of NSCLC mainly depends on the surgery, radiation therapy and chemotherapy for the early stages and chemotherapeutic treatment, immunotherapy and targeted therapy for the advanced stages of the disease [2,11]. The first platinum-containing coordination complex, applied for the treatment of cancer including, SCLC and NSCLC, is Cisplatin “cis-dichlorodiamineplatinum II”. It is a potent chemotherapeutic agent utilized alone or in combination therapies. Its capability to inhibit cellular division was discovered by Burnett Rosenburg in 1965. Cisplatin was approved by US Food and Drug Administration “FDA” in 1978 [12–14]. Cisplatin’s anti-tumour actions stem from its ability to limit cell proliferation by intercalating into the DNA of replicating cells [15]. Nonetheless, the survival rate of patients with advanced NSCLC is low, highlighting the need for additional therapeutic alternatives [2,11].

Phytochemicals are effective, useful and non-toxic in treatments for various diseases, involving cancer [16]. Honey bees gather the resinous substance known as propolis from a variety of plants and use it to seal openings in their hives, soften the interior walls and protect the entrance from intruders. Honey bee propolis and its components are among the most effective anti-tumour agents [17]. A wide variety of biological effects, including antioxidant capacity, are produced by various forms of propolis. The medicinal benefits of propolis depend on polyphenols, particularly flavonoids [18]. The most efficient ingredients are thought to be terpenoids and polyphenols [19]. Flavonoid compounds encompass chrysin, quercetin, pinocembrin, kaempferol, galangin, apigenin, tectochrysin and pinostrobin. Aromatic acids are another important group of propolis compounds, with the most common being caffeic, cinnamic, ferulic, benzoic, salicylic and p-cumaric acids [20]. Propolis has a high medicinal value and is used for a wide range of purposes. In addition to cytotoxic activity, it has anti-tumour and anti-cancer properties [21]. The aim of this study is to compare the antitumour effect of propolis extract to cisplatin in adult male albino rats with urethane-induced lung cancer.

2. Materials and methods

2.1. Drugs and chemicals

Urethane: 99.0% (GC), synonym; Ethyl Carbamate, Sigma-Aldrich product, linear formula; NH2COOC2H5. Cisplatin: 50 MG/50 ML (1 mg/mL) 1 VIAL (MYLAN). Oxidative stress Kits were obtained from the Bio-diagnostic Company, Egypt. QPCR kit: Qiagen RNA extraction/BioRad syber green PCR MMX. Other chemicals of analytical grade were purchased from El-Gomhoria Co. Cairo, Egypt.

2.2. Biopropolis

Each capsule contains bee Propolis extract 400 mg standardized as 1% flavonoids, purchased from Sigma Pharmaceutical Industries, Egypt. According to Pakkirisamy et al. [22] an FTIR (Fourier transform infrared spectrophotometric) analysis was performed for PE. The range of the transmittance scanning was between 4000 and 400 cm⁻¹ (the mid-IR region).

2.3. Animals

A total of 40 adult male albino rats (Rattus norvigicus) weighing 150 ± 20 g (six to eight weeks old) at the beginning of the experiment were purchased from the New Veterinary Office in Giza, Egypt. Rats were housed in wired cages in a thermally controlled environment with 12-hour light/dark cycles. During the experiment, water and food water were available freely.

2.4. Experimental design and sample collection

Four groups were used in this study, each group has 10 rats, as follows:

The Control negative (CON) group: Rats were injected intra-peritoneally with phosphate-buffered solution for seven months. The Urethane (URT) group: Animals were presented as the carcinogenic non-treated group. For the induction of lung cancer, 30 rats were intra-peritoneally injected with urethane solution (1 g/kg/b.wt.) once weekly per each rat for a month [23]. Lung cancer developed within a five-month dormancy period from the first urethane injection. Ten rats were left without treatment for two months representing this group (the positive control group). The URT + propolis extract (PE) treated group: 10 lung cancer rats were given PE (500 mg/kg/b.wt.) daily via gastric tubes [24]. The treatment started after the fifth month and continued till the experiment was completed. The URT + Cisplatin (CIS)-treated group: 10 rats with lung cancer were intraperitoneally injected with cisplatin (2.5 mg/kg/b.wt.) once weekly [25]. The treatment started after the fifth month and continued till the completion of the experiment.

At the end of the experiment, all rats were sacrificed via 6% isoflurane anaesthesia, and their lungs were instantly excised, rinsed in saline, blotted dry and then weighed. Each resected lung was dissected into three parts: The first one was rapidly stored at −20°C in a deep freezer for biochemical analyses, while the second part was fixed in 10% neutral buffered formalin for histopathology examination and the third part was put in glutaraldehyde for transmission electron microscopy examination of bronchial Clara cells.

2.5. Lung index measurement

A digital balance was used to weigh five rats from each group after scarification to calculate the lung index (weight of lung per mg to the weight of rat per g × 100).

3. Histopathological studies

The lung was fixed in 10% neutral-buffered formalin [26]. The fixed lungs were set for paraffin sectioning. Sections (5 microns) were prepared [27] and stained for histological, histochemical and immunohistochemical examinations with a light microscope (LEICA DM4 P, Wetzlar, Germany) and photographed using a Zeiss camera.

For the general histo-pathological study of the tissue and cells, we used haematoxylin and Eosin (H&E) stain [28]. Haematoxylin and Eosin staining of lung tissues is used to determine the bronchial wall thickness, alveolar lumen diameter and inflammatory score.

A quantifiable inflammatory score has been used, with grades 0 representing no inflammation, 1 representing minimal inflammatory cells in some microscope fields, 2 representing mild inflammation with an influx of a ring of cells 1 cell layer deep, 3 representing moderate inflammation with a ring of cells 2–4 cells deep and 4 representing severe inflammation with a ring of cells >4 cells deep [29]. The bronchiolar wall thickness (µm) and alveolar lumen diameter (µm) were quantified and analyzed using ImageJ analysis software (ImageJ version 1.46, NIH, USA) and analyzed statistically. Mean bronchial wall thickness and alveolar lumen diameter and inflammatory scores were calculated from six random microscopic fields at ×40 magnification per each group.

The Masson trichrome stain was used to demonstrate collagen fibres [30]. At high magnification, paraffin sections of lungs from each group were stained with the Masson trichrome stain and photographed at power [31]. Collagen fibre is coloured blue by Masson’s trichrome staining, cytoplasm, muscle and erythrocytes are pink and nuclei are dark brown to black [32]. The optical density of the Masson trichrome-stained area was outlined and quantified using ImageJ software [33].

Periodic acid Schiff is a technique for detecting neutral mucins in tissue, but it also detects other tissue components such as glycogen [34]. The Alcian blue-PAS staining method is used to distinguish between acid and neutral mucins [35]. Paraffin sections of lungs from each of the main groups were stained with Alcian blue-PAS stain and photographed at a high magnification power [36]. Neutral mucin was stained magenta, while acid mucin was stained blue. The quantitative analysis of mucin content in the lungs was performed using image analysis software (ImageJ, 1.46, NIH, USA) across ten different fields for each section.

3.1. PCNA immunohistochemistry

The proliferating cell nuclear antigen (PCNA) is a cell proliferation marker [37]. Lung sections from each group were stained with anti-PCNA immunostain and photographed at high magnification. Streptavidin–biotin immunoperoxidase complex staining was used for immunohistochemical staining [38]; the positive reaction appears as a brown nuclear reaction for PCNA.

3.2. Histomorphometric analysis

The mean area per cent of collagen fibre, mucin and PCNA positive reactions were measured in ten non-overlapping high-power fields of paraffin sections of the lungs in each group using image analysis software (ImageJ version 1.46, NIH, USA) and analyzed statistically.

3.3. Transmission electron microscope studies

Transmission electron microscope (TEM) processing was carried out for observing ultra-structural features of bronchial Clara cells in the lungs of different groups [39]. A part of the lung was promptly fixed in cold glutaraldehyde (5%) for 24 h, rinsed in 0.1 ml phosphate buffer (pH 7.2) for 20 min (three changes) and post-fixed for 1.5 h with 1% osmium tetroxide. The specimens were then rinsed in phosphate buffer again before being dehydrated in varying degrees of alcohol and embedded in epoxy resin to determine the area that was subjected to ultrathin cutting; semi-thin slices (1 micron thick) were cut on an LKB ultratome, stained with toluidine blue and viewed under a light microscope. The ultra-thin sections were examined under an electron microscope (JEOL1010 EX II, Japan) at Regional Mycology and Biotechnology Center at Al-Azhar University, Cairo, Egypt.

3.4. Biochemical analysis

Homogenization of the lungs was done in 5–10 ml cold buffer (50 mM potassium phosphate at pH 7.5) per gram of the tissue. Centrifugation was done out at 4°C for 15 min at 2000 × g and the generated supernatant was kept at −80°C for further analyses. Malondialdehyde, superoxide dismutase and reduced glutathione concentrations in lung homogenate were determined using the techniques described by Beutler et al. [40], Nishikimi et al. [41] and Ohkawa et al. [42], respectively.

3.5. Quantitative RT-PCR

Total RNA was separated from the lungs of the main groups of rats using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA) and kept at −80°C according to the manufacturer’s instructions. A Beckman dual spectrophotometer was utilized to determine the concentration of extracted RNA (Beckman Instruments, Ramsey, MN, USA). QPCR was used to examine the expression level of the wild-type EGFR gene. The wild-type EGFR primer sequence (F: 5′-GAGAGGAGAACTGCCAGAA-3′) & (R: 5′-GTAGCATTTATGGAGAGTG-3′) was described by Shahin et al. [43] while the primer sequence of the housekeeping gene, B-actin, (F: 5′-GGCACCACACCTTCTACAATG-3′) & (5′-GGGGTGTTGAAGGTCT CA AAC-3′) was described by Hussein et al. [44]. Complementary DNA (cDNA) synthesis: Using a high-capacity cDNA reverse transcriptase kit, 10 ng of total RNA was extracted from each sample and used to synthesize cDNA (Applied Biosystems, Thermo Fischer Scientific, USA). Real-Time Quantitative polymerase chain reaction: The cDNA was then amplified using the Sybr Green I PCR master kit (Thermo Fisher Scientific Inc., Lithuania) and the Step One apparatus (Applied Biosystems, Thermo Fischer Scientific) was as follows: The enzyme was activated for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C for denaturation, 20 s at 55°C for annealing and 30 s at 72°C for extension. The ΔCT method was used to normalize changes in target gene expression relative to the mean critical threshold (CT) values of b-actin, the housekeeping gene. Fold change was calculated as a relative quantitation using the 2−ΔΔCt method [45] for each sample, and the Ct value of the target gene (EGFR) mRNA was normalized against the B-actin endogenous control as ΔCT (ΔCt = Ct target gene – Ct B-actin). The fold change of the target gene mRNA in the experimental sample relative to the control sample was determined by 2−ΔΔCt, where ΔΔCT = ΔCt Experimental – ΔCt Control.

3.6. Statistical analysis

The whole numerical data were statistically analyzed and expressed as mean standard deviation. To compare the significant difference between groups, one-way analysis of variance (ANOVA) was used to perform multi-group comparisons of means. When the p-value was less than 0.05, the result was considered statistically significant. Further group comparisons were made using the Statistical Package for the Social Sciences (SPSS) version 25 and the post-hoc Tukey’s test.

3.7. Ethical statement

All studies were authorized by the Committee of the Institutional Animal Ethics for Laboratory Animal Care at Helwan University’s Zoology Department, Faculty of Science (Approval number: HU-IACUC/Z/MI1101-23).

4. Results

4.1. FTIR analysis of PE

IR spectrum analysis of PE is shown in Figure 1 and Table 1. However, there are only a few reports on the analysis of PE by using FTIR. Most of the frequencies are group frequencies which tell us the presence or absence of specific functional groups in a sample. The characterization of PE reveals the presence of OH, CH, C–N, C–O, C–F, C–Cl, C=O, C=C stretching. It may, therefore, be inferred aliphatic or aromatic alcohols, esters, amine, fluro-compound and halo-compound are some of the constituents of extract.

Figure 1. IR spectrum analysis of Propolis extract.

Table 1. FTIR spectrum analysis of Propolis extract.

Absorption (cm^−1)	Appearance	Transmittance (%)	Group	Compound class
3266.51	Strong broad	14.97712	O–H stretching	Alcohol intermolecular bonded
2920.94	Medium	13.39266	C–H stretching	Alkane
2851.81	Medium	13.0757	C–H stretching	Alkane
1742.48	Strong	7.989363	C=O stretching	Vinyl/phenyl ester
1597.56	Medium	7.324897	C=C stretching	Conjugated alkene
1415.72	Medium	6.491151	O–H bending	Alcohol
1337.20	Medium	6.131133	O–H bending	Phenol
1241.47	Medium	5.692205	C–N stretching	Amine
1148.68	Medium	5.266758	C–N stretching	Amine
1075.21	Strong	4.929895	C–O stretching	Primary alcohol
1017.22	Strong	4.664007	C–F stretching	Fluoro compound
865.44	Strong	3.968088	C–H bending	1,3-Di-substitute
757.78	Strong	3.474461	C–H bending	1,3-Di-substitute
571.98	Strong	2.622558	C–Cl stretching	Halo compound

4.2. Lung index data

Lung index or the relative lung weight was considerably increased in the urethane group as matched to the control negative group. URT + PE and URT + CIS-treated groups showed a significant decrease in the lung index compared to the URT group (Figure 2).

Figure 2. Mean ± SD data of lung index between different study groups. *: Significance against the CON group, ^#: significance against the URT group. P < 0.05.

4.3. Histopathological findings in the lung

The control group showed normal histo-architecture of the lung of terminal bronchiole, alveolar sac and regular-sized alveolar spaces separated by a thin inter-alveolar septum and thick interstitial tissue (Figure 3(A)). The URT group displayed atypical lung histo-architecture with infiltrative papillary bronchial adenocarcinoma raised as a tumour mass, invading and obliterating the lumen of terminal bronchiole showed well-defined tumour mass made of irregular papillae lined by hyperplastic columnar epithelial cells besides basal vesicular nuclei and some pleomorphic different in cells in their shape and size (Figure 3(B)). The URT + PE-treated group exhibited the absence of tumour mass, restoring normal lung structure with some macrophages (Figure 3(C)). The URT + CIS-treated group showed no tumour mass and terminal bronchiole with peri-bronchial inflammation, thick blood vessels and extravasation of RBCs observed in air spaces (Figure 3(D)).

Figure 3. The transverse section in the lungs of the study groups (H&E stain). (A) The CON group shows normal lung histo-architecture; terminal bronchiole (TB), alveolar sac (AS), alveoli (A), thin inter-alveolar septum (black arrow) and thick interstitial tissue (red arrow). (B) The URT group showing papillary adenocarcinoma in the lumen of a terminal bronchiole (TB) formed of tumour mass (TM) of irregular papillae lined by hyperplastic columnar epithelial cells with basal vesicular nuclei (black arrow) with some pleomorphic cells differ in their shape and size (brown arrows). (C) The URT + PE-treated group shows terminal bronchiole (TB), alveolar sac (AS), variable-sized alveoli (A), thin inter-alveolar septum (black arrow), moderate thickened interstitial tissue (red arrow), some macrophages (MK) in the lung parenchyma. (D) The URT + CIS-treated group shows terminal bronchiole (TB), alveolar sac (AS), alveoli (A), thickened BV, peri-bronchial inflammation (PBI), RBCs extravasation (EV). Bar = 50 µm.

Figure 4 show semi-quantitative determinations of thickness of bronchial wall, alveolar wall and inflammation score of lungs of control (CON), Urethane (URT), Propolis-treated (URT + PE) and cisplatin-treated (URT + CIS) groups.

Figure 4. Semi-quantitative determinations of (A) the bronchiolar wall thickness, (B) alveolar lumen diameter and (C) inflammatory score in lungs of different study groups. Data represents Mean ± S.D.*: Significance against CON group, ^#: significance against URT group. P < 0.05.

The data revealed that the mean bronchial wall thickness in the URT group (96.20 ± 6.37) was significantly enhanced compared to CON (32.00 ± 3.08). Treatment with PE (31.73 ± 5.12) and CIS (34.05 ± 2.38) significantly lowered the mean bronchial wall thickness compared to the URT group (Figure 4(A)). Lung histo-scoring also revealed that the alveolar lumen diameter in the URT group (18.30 ± 1.15) was significantly lowered than that in the CON group (39.14 ± 3.41). Treatment with PE (38.10 ± 1.43) and CIS (40.24 ± 5.14) significantly enhanced the alveolar lumen diameter compared with the URT group (Figure 4(B)). Moreover, lung histo-scoring revealed that the infiltration of inflammatory cells in the URT group (4.00 ± 0.38) was significantly enhanced compared to the CON group (1.04 ± 0.27). Treatment with PE (1.82 ± 0.23) and CIS (2.42 ± 0.19) significantly lowered the inflammatory cell infiltration compared to the urethane group (Figure 4(C)).

Figure 5 show the morphometric area per cent of collage fibres, mucin content and PCNA positive reaction in different study groups. There was a significant increase (P < 0.05) in the area per cent of collagen fibres, AB-PAS positive reaction and PCNA positive immune reaction in the URT group compared to the CON group. Rats of the URT + PE group and URT + CIS group showed a significant decrease in these measurements compared to the URT group (Figure 5).

Figure 5. Mean ± SD quantitative analysis of area % of collagen fibres, AB-PAS positive Rx. and anti-PCNA positive immune Rx. in the lungs of different study groups. *Significance against the CON group, ^#significance against the URT group. P < 0.05.

According to Masson trichrome stain, the Control group showed normal distribution of a moderate amount of blue collagen fibres within interstitial tissues and scattered around the terminal bronchiole and blood vessels (Figure 6(A)). The URT group showed an abnormal distribution of an excessive amount of blue collagen fibres within and around the terminal bronchiole (Figure 6(B)). The URT + PE-treated group showed moderate collagen fibre deposition distributed around the terminal bronchiole and scattered in between alveoli (Figure 6(C)). The URT + CIS-treated group showed moderate collagen fibre deposition distributed around the terminal bronchiole and in between alveoli (Figure 6(D)).

Figure 6. A transverse section in the lungs of different study groups (Masson trichrome stain). (A) The CON group shows a moderate amount of collagen fibres between lung alveoli (arrowhead) and around the terminal bronchiole, blood vessel (arrow), terminal bronchiole (TB), the bronchiolar epithelium (BE), smooth muscle fibres (MF), alveolar sac (AS), alveoli (A) and alveolar epithelium (AE). (B) The URT group shows an excessive amount of collagen fibres around and within the terminal bronchiole (TB) (arrows), terminal bronchiole (TB), the bronchial epithelium (BE) and disrupted layer of smooth muscle fibres (MF). (C) The URT + PE-treated rats group shows moderate collagen fibre deposited around terminal bronchiole (TB) (arrow) and in between alveoli (A) (arrowhead) (D) The URT + CIS-treated rats group shows moderate collagen fibre deposited around terminal bronchiole (TB) (arrow) and in between alveoli (A) (arrowhead), alveolar sac (AS), alveoli (A) and peribronchial inflammation (PBI). Bar = 50 µm.

According to the Alcian blue-PAS reaction, the Control group showed moderate positive AB-PAS of total mucin content (Figure 7(A)). The URT group showed a strong positive AB-PAS reaction of staining of total mucin content. The strong positive reaction of neutral mucin and acidic mucin (Figure 7(B)) elevated mucin content pointed to the induction of mucinous adenocarcinoma. The URT + PE-treated group showed moderate positive AB-PAS reaction of total mucin (Figure 7(C)). The URT + CIS-treated group showed moderate positive AB-PAS reaction of total mucin (Figure 7(D)).

Figure 7. A transverse sections in the lungs of the study groups shows AB-PAS positive reactions. (A) The CON group shows moderate positive AB-PAS Rx. with moderate mucinous content, a black arrow (intense reaction area), a blue arrow (moderate reaction area). (B) The URT group shows a strong positive AB-PAS Rx. with excessive neutral mucins (black arrow) and acidic mucins (arrowhead), terminal bronchiole (TB), muscle fibre (MF) and bronchial epithelium (BE). (C) The URT + PE-treated group shows moderate positive AB-PAS reaction with moderate mucinous content, a black arrow (intense reaction area) and a blue arrow (moderate reaction area). (D) The URT + CIS-treated group shows moderate positive AB-PAS Rx. with moderate mucinous content. Bar = 50 µm.

Regarding PCNA Immunohistochemistry, the CON group showed net mild-to-moderate positive immunoreaction for staining of PCNA in the lungs (Figure 8(A)). The URT group showed totally strong positive PCNA immune reactivity in the lungs (Figure 8(B)), highlighting the cellular proliferation feature characteristic of cancer development in the lungs. The URT + PE-treated group showed moderate positive PCNA immunoreaction in the lungs (Figure 8(C)). The URT + CIS-treated group showed moderate positive PCNA immunoreaction in the lungs (Figure 8(D)).

Figure 8. Transverse sections in the lungs of different study groups showing anti-PCNA positive Rx. in lung nuclei (A) The CON group shows general moderate PCNA positive immunoreaction where strong positive Rx. area (a black arrow), moderate positive Rx. area reaction (a blue arrow) and mild reaction positive Rx. area (a red arrow). (B) The URT group shows strong positive PCNA immunoreaction where a strong positive reaction area (black arrow) in bronchial epithelial cells (BE) of terminal bronchiole (TB), moderate positive Rx. the area around the terminal bronchiole (TB) and mild reaction positive Rx. area in the muscle fibre layer (MF) (a red arrow). (C) The URT + PE-treated group shows moderate positive PCNA immunoreaction where a strong reaction area (a black arrow) in alveolar cells, a moderate reaction in lining cells of a terminal bronchiole (TB) (a blue arrow) while a weak reaction in interstitial cells (a red arrow). (D) The URT + CIS-treated group shows a moderate positive PCNA immune reactivity where a strong reaction area around the terminal bronchiole (TB) (a black arrow), appeared in some interstitial cells, a moderate reaction in some alveolar cells (a blue arrow), while a weak reaction appeared in some bronchial lining cells (a red arrow). Bar = 50 µm.

4.4. TEM findings

The CON group demonstrated normal epithelial cell lining of the terminal bronchiole as represented by low columnar epithelium composed of ciliated cells and Clara cells that project into the lumen rather than ciliated cells. The Clara cell formed and bulging apex protruded towards the bronchial lumen and the main body. The apex contains smooth endoplasmic reticulum, pale elongated mitochondria and dense secretory granules, while the main body contains a notched vesicular nucleus. Ciliated cells have cilia directed towards the airway lumen (Figure 9(A)). The URT group demonstrated abnormal epithelial lining of the terminal bronchiole showing tumour mass of a closely packed group of pleomorphic cells with characteristic dense secretory granules, pleomorphic nuclei and distorted cell boundaries. Ciliated cells have cilia directed towards the airway lumen (Figure 9(B)). A magnified part of Clara cells in the URT group showed cells with pleomorphic nuclei surrounded by pale cytoplasm, a few dense secretory granules and many ruptured smooth endoplasmic reticulum. Cancer cells do not rest on the basement membrane with underlying collagen fibres. Ciliated cells have cilia directed towards the airway lumen (Figure 9(C)). The URT + PE-treated group showed almost restoration of Clara cell ultra-structure (Figure 9(D)). While the URT + CIS-treated group showed some restoration ultra-structure of Clara cell with dilated smooth endoplasmic reticulum and cytoplasmic vacuoles (Figure 9(E)).

Figure 9. Transmission electron-micrograph of terminal bronchial Clara cells in the lungs of different study groups (Uranyl acetate & Lead citrate stain). (A) The CON group shows the bronchial epithelial lining of the terminal bronchiole comprising ciliated cells (Ci) and Clara cells (Cl). Clara cells formed and bulging apex protrude towards the bronchial lumen (L) and the main body. The apex contains smooth endoplasmic reticulum (sER), pale elongated mitochondria (M) and dense secretory granules (SG), while the main body contains a notched vesicular nucleus (N) and some (SG). Ciliated cells have cilia directed towards the airway lumen (a red arrow). (B) The URT group showing tumour mass (TM) of a closely packed group of pleomorphic cells with characteristic dense secretory granules (SG), pleomorphic nuclei and distorted cell boundaries. Ciliated cells have cilia directed towards the airway lumen (a red arrow). (C) The URT group shows a magnified part of Clara cells with pleomorphic nuclei (N) surrounded by pale cytoplasms, a few dense secretory granules (SG) and many ruptured smooth endoplasmic reticulums (sER). Cells do not rest on the basement membrane (a blue arrow) with underlying collagen fibres (an arrowhead). Ciliated cells have cilia directed towards the airway lumen (a red arrow). (D) The URT + PE-treated group shows almost restoration of the bronchial epithelial lining of the terminal bronchiole comprising ciliated cells (Ci) and Clara cells (Cl). Clara cells formed and bulging apex protrude towards the bronchial lumen (L) and the main body. The apex of Clara’s cell contains electron-dense secretory granules (SG) and mitochondria (M). The main body of Clara cell contains a vesicular basal central nucleus (N) surrounded by smooth endoplasmic reticulum (sER). The ciliated cell has cilia directed towards the lumen (a red arrow). (E) The URT + CIS-treated group shows more or less restoration of Clara cells (Cl) that protrude larger into the lumen (L). The apex of Clara cell contains some spherical mitochondria (M), electron-dense secretory granules (SG), dilated smooth endoplasmic reticulum (sER), while the main body contains a central round nucleus (N), vacuoles (V) and many (SG). Scale bar = 500 nm, except for Fig. C = 2 µm.

4.5. Effect of PE on biochemical markers in the lungs of male rats

Table 2 shows GSH concentration, SOD activity and MDA content in the study groups. Glutathione concentration showed a significant decrease (P < 0.05) in the urethane group compared to the CON group. Rats of the URT + PE group-treated groups showed a significant increase (P < 0.05) compared with the URT group. Super-oxide dismutase activity showed a significant decrease (P < 0.05) in the URT group compared to the CON group. Rats of the URT + PE group and URT + CIS-treated groups showed a significant increase (P < 0.05) compared with the URT group. Malondialdehyde content showed a significant increase (P < 0.05) in the URT group compared to the CON group. Rats of the URT + PE group and URT + CIS-treated groups showed a decreased malondialdehyde content (P < 0.05) compared with the URT group.

Table 2. Mean ± SD of GSH concentration, SOD activity, MDA content in the lungs of control (CON), Urethane (URT), Propolis-treated (URT + PE) and cisplatin-treated (URT + CIS) groups.

Parameters Groups	GSH Conc. (mg/gram tissue)	SOD activity (U/gram tissue)	MDA content (nmol./gram tissue)
CON	3.7833 ± 0.86558	921.00 ± 61.098	97.67 ± 14.012
URT	1.8933 ± 0.208*	487.67 ± 26.312*	450.00 ± 66.566*
URT + PE	3.9367 ± 0.926^#	969.00 ± 83.594^#	128.33 ± 15.503^#
URT + CIS	3.6633 ± 0.675	898.00 ± 25.060^#	179.00 ± 15.111^#

*Significance at P < 0.05 against the CON group, ^#Significance at P < 0.05 against the URT group.

4.6. Effect of PE on EGFR mRNA expression in the lungs of male rats

Gene expression (Relative fold change) of wild-type EGFR estimated by quantitative real-time-PCR revealed a significant decrease (P < 0.05) in the URT group compared to the CON group. Rats of the URT + PE and URT + CIS-treated groups showed a considerable increase (P < 0.05) compared to the URT group (Figure 10).

Figure 10. Quantitative RT-PCR analysis of relative fold change of EGFR m-RNA expression in the lungs of different study groups. Data expressed as Mean ± SD. *Significance against CON group, ^#: significance against URT group. P < 0.05.

5. Discussion

The current study found that the URT group had a significantly higher lung index than the CON group, which could be attributed to inflammatory cell accumulation and increased proliferation of cancerous cells in the lungs [46]. There was a significant decrease in lung weight and relative lung weight in the URT + PE and URT + CIS-treated groups compared to the URT group in the current study.

Malignant lung carcinoma is the most common type of cancer, and it is caused by a series of genetic changes in cells that are triggered by chemical and environmental stimuli [47]. We successfully induced lung cancer using multidose urethane injection according to the method of Janker et al. [23]. Urethane can be metabolized into N-hydroxylamine epoxides and vinyl carbamate, and cause oxidative stress in the environment of lung cells by increasing the amounts of ROS, which causes oxidation and DNA damage [48]. In the present study, the URT group’s GSH concentration and SOD activity significantly decreased compared to the CON group. Compared to CON rats, the MDA content in the URT group significantly increased. By interfering with the integrity of proteins and lipids, oxidative stress has been shown to play a substantial influence on the incidence of lung injury [49]. Furthermore, oxidative stress can harm many cellular components such as DNA, protein and lipids, which cause carcinogenesis [50]. Repeated exposure to urethane significantly altered lipid peroxidation and oxidative stress compared to the CON group, as seen by a large rise in MDA and a decline in GSH in lung homogenate. This could be related to lung cell mitochondrial malfunction and an increase in intrinsic ROS produced in the mitochondria as a result of the production of electrophilic species [51].

When compared to the URT group in the current investigation, the GSH and SOD levels in the rats treated with the URT + PE group were significantly higher. The malondialdehyde (MDA) level in the URT + PE group was significantly lower than in the URT group. Our findings regarding the anti-oxidative properties of propolis were consistent with those of Brihoum et al. [52] who realized that propolis ethanol extract therapy increased levels of antioxidants, including enzymatic antioxidants such as SOD, CAT and GST and non-enzymatic antioxidants like GSH, compared to lung cancer caused by carcinogens, which was associated with a decrease in MDA level. Bee propolis’ antioxidant properties, which include inhibiting radical formation, scavenging free radicals, preventing membrane lipid peroxidation (thereby controlling membrane permeability) and increasing intracellular scavenger content, could be responsible for the ameliorative effect of bee propolis on the lungs as observed in this study [53,54].

The current study found that m-RNA expression of wild-type EGFR was significantly lower in the URT group compared to the CON group. This finding is in agreement with Meseure et al. [55] who discovered that wild-type EGFR was significantly under-expressed at both the mRNA and protein levels in invasive breast carcinomas compared to normal breast tissues. In contrast to our findings, Janku et al. [56] discovered EGFR overexpression and mutations in premalignant lung epithelium, squamous cell carcinoma and metastatic non-small cell lung carcinoma. Yarden and Pines [57] clarified EGFR low expression by confirming the presence of oncogenic EGFR mutations and significant genomic rearrangements in (lung, breast and ovarian malignancies), which frequently induce altered receptor endocytosis, which contributes to increased signalling qualities. In cancer, incorrect EGFR activation is caused by impaired receptor endocytosis and trafficking [58]. In the present study, wild-type EGFR m-RNA expression was significantly higher in rats treated with PE compared to the URT group; these findings could be attributed to propolis’ anti-proliferative properties [52].

In the current study, we used four injections of urethane at a dosage of (1 g/kg/b.wt.) once weekly to generate bronchial adenocarcinoma in the lungs of male albino rats. Smoke from cigarettes also contains urethane, a component of tobacco that naturally develops malignant lung cancers [59]. Our model of urethane-induced lung cancer was in line with the findings of Janker et al. [23] who found that mice exposed to urethane three times (1 mg/g Urethane in one week) developed significantly more tumour nodules than mice exposed just once after 20 weeks although there was no mortality or morbidity and that the incidence of tumours was 100%. The length of time the animals were exposed to urethane determines the induction of adenocarcinoma in the experimental model [60]. The carcinogenic activity of URT in our model could be attributed to the formation of an active metabolite, vinyl carbamate, which is a powerful mutagen and promotes the formation of electrophilic species that interact with DNA to form 2-oxoethyl adducts as vinyl chloride [61].

The infiltrative bronchial adenocarcinoma, which appeared in the present work as a tumour mass invading and obliterating the terminal bronchiole lumen, formed in the urethane group of our study. A mixture of pleomorphic cells with various cell sizes and shapes and hyperplastic columnar epithelial cells with basal vesicular nuclei make up the tumour mass. Stakisaitis et al. [62] noted that the nuclei of bronchial adenocarcinoma cells were pleomorphic cells with frequent mitosis, tumour cell infiltration into the bronchial lumen and wall push on the surrounding tissues. These features were among many characteristics of the induced adenocarcinoma in the present study that was documented.

In the current investigation, rats treated with PE displayed anticancer and amelioration of almost pathologic alterations connected with lung cancer, along with some inflammatory macrophages and a minor thickening of interstitial tissue. The anticancer effect of Algerian propolis was supported by Brihoum et al. [52] who also pointed out that the anti-proliferative effect of an ethanolic extract of Algerian propolis in A549 cells and its chemo-preventive role against benzo(a)pyrene-induced lung carcinogenesis in albino Wistar rats were due to caffeic acid and its derivatives, CAPE and (+)-chicoric acid. Furthermore, Frión-Herrera et al. [63] showed that Brazilian propolis is selective for tumour cells as opposed to healthy cells and that it inhibits the growth of A549 cells in a dose-dependent manner. Apoptosis activation and cell cycle arrest are thought to be the main mechanisms behind propolis’ anticancer effects [64]. The current study suggests that the occurrence of visible lesions following cisplatin therapy, such as inflammation, haemorrhagic RBCs and RBC extravasation in airway spaces, may be caused by the fact that cisplatin is not only harmful to cancer cells but also affects healthy cells [65].

The area per cent of collagen fibres increased significantly in the URT group compared to the CON in the current investigation, which was similar to the findings of Anandakumar et al. [66] discovered enhanced collagen deposition in lung cancer-induced mice. The significant increase in mean surface area % collagen fibre in the URT group might be attributable to immunological and inflammatory mechanisms that cause fibroblast activation and collagen deposition [67]. In the current study, the improvement in area % of collagen fibres following PE treatment might be attributed to propolis’ anti-inflammatory characteristics [68].

In the current study, the area per cent of total mucins increased significantly in the URT group compared to the CON group, indicating a strong positive reaction for total mucins. Several studies have indicated that mucins are overexpressed in non-small cell lung cancer (NSCLC) and can be used as biomarkers for detecting and tracking the progression of lung cancer [69,70]. Chronic mucus hypersecretion, in particular, is a significant predictor of lung cancer mortality [71]. In this study, lung cancer-bearing rats treated with PE had normal mucin content restored.

Cell proliferation was verified by PCNA immunohistochemistry. PCNA is a nuclear peptide with a molecular weight of 36 kDa that has been identified as a DNA polymerase delta auxiliary protein [72]. The area per cent of PCNA in the URT group increased dramatically compared to the control negative group, indicating a robust PCNA positive response in the nucleus of terminal bronchial lining cells and peri-bronchial inflammatory cells. Wang et al. [73] confirmed this finding by showing that PCNA expression was elevated in NSCLC tissues and cells and that overexpression of PCNA in lung cancer cells resulted in cell proliferation, clonal formation, tumour genesis and inhibited cell apoptosis, implying that PCNA acts as an oncogene in the progression of NSCLC. In the current investigation, rats treated with PE and CIS showed a moderate PCNA-positive reaction in the lungs. This finding might be due to the anti-proliferative properties of propolis [52].

The typical Clara cell ultrastructure seen in the CON group in this study was consistent with Singh and Katyal’s [74] description of Clara cells as cubical or low columnar with a bulging surface towards the bronchial lumen; they lack cilia and do not secrete mucus. It has mitochondria in the cell's apical area and smooth and rough endoplasmic reticulum. In the cytoplasm’s apical area, small amounts of secretory granules are seen. The peri-nuclear Golgi apparatus has undergone significant development. The cell nucleus is wrinkled and has several nucleoli at the centre of the cell. Clara cells can secrete both apocrine and merocrine secretions and a variety of other chemicals.

In the URT group, the bronchial Clara cells hyper-proliferated to form a tumour mass that partially obliterated the lumen. The Clara cell tumour was made up of densely packed pleomorphic Clara cells with imperceptible cell boundaries that varied in size and shape, and a few distinctive dense secretory granules the neoplastic Clara cells that cause the lung cancer in this study are caused by URT. This result was in line with Reznik-Schuller [75] who found that giving nitrosamine derivatives to hamster rats over an extended period of time causes the development of bronchogenic carcinomas made up of neoplastic Clara cells. Clara cells of the URT group have pleomorphic nuclei, peri-nuclear rough ER, vacuolated cytoplasm and dilated smooth ER containing flocculent material. Phimister et al. [76] justified our data on the changed ultrastructure of Clara cells in urethane-induced adenocarcinoma by revealing that significant GSH depletion in Clara cells induces significant swelling, actin cytoskeleton disturbances and plasma membrane blebbing. These cellular alterations have been seen in cells exposed to a variety of toxins and may represent a general cell response to GSH depletion and/or oxidative stress found in the early stages of cell death [77]. Blundell [78] supported our data as he noted several structural changes occur in Clara cells after exposure to toxic substances that enter the lungs with inhaled air including nuclear chromatin margination and clumping, mitochondrial swelling, dilation of the endoplasmic reticulum Clara cells become enlarged and many vacuoles form adjacent to the cellular membranes. Clara cells are thought to play a vital role in protecting the airways from the negative effects of a hazardous external environment [79].

The current study found that the URT + PE group appeared without the tumour mass and almost restored normal Clara cell ultra-structures. The ameliorating impact of PE on bronchial Clara cell ultra-structures observed in this study might be due to PE’s anti-proliferative action [52].

We effectively made bronchial adenocarcinoma derived from proliferated Clara cells by four intra-peritoneal injections of urethane at a dose of (1 g/kg b.wt.) once weekly for a month. Because of their high antioxidant effects in countering oxidative stress-derived-urethane carcinogenesis and improving histo-pathological and ultra-structural alterations accompanied by lung cancer, propolis extract may be used as anticancer agent in NSCLC.

Propolis extract may be an excellent source of natural chemicals that fight lung cancer, but more research is needed to understand the mechanism of propolis action and its effect on other organs, as this study is limited to histopathological and molecular effects.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R96), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia and also was supported by Researchers Supporting Project (RSPD2023R655), King Saud University, Riyadh, Saudi Arabia.