A review of the anticancer properties of bee products and their molecular mechanisms: An overview on lung cancer

Lung cancer is one of the most common types of cancer in the world, and it represents a significant percentage of all diagnosed cancers. Despite better clinical outcomes associated with current drugs, lung cancer has a worse survival rate than any of the other commonly occurring malignancies. Evidence suggests that natural products offer promising potential for improving the current treatment approaches and/or developing novel treatment strategies. Bee products such as honey, propolis, and venom have been reported to exert anti-cancer effects on a variety of human cancer cell lines, thereby paving the way for a wide range of treatment possibilities. The current review focuses on the cytotoxic effects of bee bioactive compounds on all types of cancer, and the molecular pathways by which they might reduce tumor cell growth or trigger apoptosis of cancer cells, with particular reference to lung cancer. This strategy could potentially improve the efficacy of current remedies, reduce the doses of chemotherapy drugs, and decrease adverse effects of chemotherapy drugs. Furthermore, in vitro studies on anti-cancer properties of bee products are carefully reviewed. The results reveal that bee products have the potential to become effective treatment agents against many forms of cancer in the future.


INTRODUCTION
Cancer is a life-threatening morbidity that arises primarily due to an abnormality in the cell cycle that induces either excessive cell proliferation, incomplete apoptosis, or both [1]. According to the World Health Organisation (WHO), cancer is the second leading cause of death globally; it caused an estimated 9.6 million deaths in 2018 alone [2]. Lung cancer is the most common kind of cancer, with high frequency and fatality equivalent to those of other malignancies [3].
There were 2 million new cancer cases diagnosed globally in 2018 [4]. Furthermore, the American Cancer Society predicted that 228,820 new cases of lung cancer would be diagnosed in the United States in the year 2020 [5]. Although smoking is regarded as a predominant cause of lung cancer, followed by genetic mutations, parameters such as co-morbid disease, environmental factors and lifestyle factors are also involved in the pathogenesis of lung cancer [6]. Among genetic mutations, the p53 gene which indues genetic instability, is the first gene that has been implicated in lung carcinogenesis [7].
Several other causes such as retinoblastoma/p16 abnormality and the chronic lung inflammation, also contribute to development of lung cancer [7]. Studies have shown that patients with advanced stage cancer have a low 5-year survival estimate of 4.7 %, but patients with surgically removed stage I tumours have a 5-year survival of 70 % [8]. It is true that lung cancer deaths decrease, and percentage survival have seen steady increases over the past 30 years, thanks to improvements in the care approaches involved [9]. Low-dose computed tomography screening of heavy smokers enhances early diagnosis of lung cancer, leading to timely care which is considered to reduce mortality [10]. Data from the International Early Lung Cancer Action Program indicates that patients with stage I lung cancer diagnosed via screening have an estimated survival of 88 % over 10 years [11]. The current therapeutic options for cancer are surgery, chemotherapy, radiation therapy, immunotherapy, and hormone therapy. However, these treatments appear to be ineffective in advanced stages [12]. On the other hand, high risk of recurrence along with late detection, lead to poor prognosis and general mortality of about 10 percent [7]. The present treatments barely alleviate cancer. Chemotherapy is toxic to normal surrounding cells, since it could lead to gene modification, DNA methylation, and histone changes, resulting in resistance to some chemotherapeutic agents [13].

Lung cancer management and current therapy
Despite significant advances in oncology, clinical outcomes of lung cancer remain inadequate, owing mostly to late diagnosis, patient age, coexisting disorders, and relatively limited treatment options. [14]. Lung cancer therapy should be assessed on the basis of the advanced stage of cancer, clinical and functional state, co-morbidities, nutritional status, and cognitive functions [15]. It should be noted that age is not an influencing factor for care initiation. Surgery is the treatment of choice for early stage of non-small-cell lung cancer [NSCLC]. Elderly patients who are disqualified from surgery should be considered for extreme radiotherapy. Singleagent chemotherapy has been observed to be beneficial for elderly people with advanced NSCLC who have general good health, essentially due to its low toxicity and good survival estimate. Polychemotherapy is the firstline therapy for patients with small-cell lung cancer (SCLC) who require prophylactic brain radiation [15]. However, its toxicity seems to be greater than the benefits of the therapy. Increased lung cancer cases and late diagnosis, high mortality, and poor efficiency of chemotherapy and radiation contribute to the need for more efficient screening measures as well as safer and more effective treatments for elderly persons with lung cancer. A significant number of medications used as chemotherapeutic agents may lose their effectiveness if patients develop resistance to drugs [16]. These drugs certainly have the potential to significantly improve survival of cancer patients, but they have also been found to significantly increase the probability of complications and adverse effects associated with therapy. Therefore, in order to minimize the number of lung cancer deaths, it is crucial to develop a treatment with improved efficacy and decreased adverse effects. Several studies have been conducted to achieve this in order to obtain potent sources of natural products which function as supplementary anti-cancer agents for suppressing and delaying carcinogenesis. Numerous studies have revealed the efficiency of several natural products in the treatment of lung cancer, including the use of propolis [17], honey [18], and bee venom [19].
A large number of studies have identified the biological properties of bee products, although other articles indicate their medicinal properties and uses as nutraceutical, pharmaceutical and cosmetic agents [20]. Bee products have been studied and tested in clinical settings, but pharmacological and therapeutic standardization remain complicated because of their high chemical heterogeneity due to wide differences in honeybees and their botanical origins. While several compounds or classes of compounds have been isolated, and only a few of them were pharmacologically identified, the findings from these studies highlight the importance of bee products in the introduction of medicines from natural sources. This review was aimed at providing research information on thorough screening of the bioactive compounds present in bee pollen, venom and propolis as remedies for lung cancer or prophylactic medication, taking into account the importance of this field in the quest for new treatments for cancer.

Bee products
Since ancient times, bee products have been used as medicines. For example, bee pollen has been shown to improve energy and stamina [20]. Propolis has been shown to be beneficial in restoring good health, while royal jelly has been shown to improve the immune system and increase energy [21]. Honey, which is primarily used as a natural sweetener, is also used as an antiseptic agent for treating wounds and sore throat. The therapeutic standardization of these drugs is hindered by variations in chemical properties because of the wide differences in the botanical origins of honeybee, although some molecules have been isolated and pharmacologically characterized [22]. Phenolics, methylglyoxal, major royal jelly proteins [MRJPs], and oligosaccharides are the chief bioactive compounds found in honey. The antimicrobial jelleins and royalisin peptides, MRJPs, and hydroxy-decanoic acid derivatives found in royal jelly, as well as 10-hydroxy-2-decenoic acid [10-HDA], have antimicrobial, anti-inflammatory, immunomodulatory, neuro-modulatory, metabolic syndrome-preventing, and anti-aging characteristics [22]. Propolis, on the other hand, contains antiviral, immunomodulatory, antiinflammatory caffeic acid phenethyl ester and artepillin C. These compounds, and their effects on cancer, are unique to Brazilian propolis. Bee venom consists of poisonous peptides such as pain-inducing melittin, channel K-blocking apamine, and allergic phospholipase A2 [22].

Honey
Honey, one of the main bee products, consists of more than 200 components, most of which are sugars (75 % monosaccharides and 10-15% disaccharides), water, enzymes, vitamins (vitamin B6, riboflavin, niacin and thiamine); minerals, phenolic compounds (flavonoids and phenolic acids), and volatile compounds [21]. Honey has long been used as a supplementary source of food and medicine. Reactive oxygen species (ROS) and inflammation play a significant role in carcinogenesis [23]. The phenolic compounds in honey, on the other hand, have anti-inflammatory and antioxidant properties [24]. To a large extent, the phenolic compounds are responsible for the antioxidant potential of honey. However, as described earlier, the composition of phenolic compounds varies significantly as a function of floral origin. Therefore, honey can be expected to display a wide range of antioxidant intensities. Moreover, honey suppresses the growth of several microbes. The antibacterial properties displayed by specific honey samples have been evaluated with sequential neutralization, keeping in mind that the mechanisms of action are complicated and vary with the source of honey. Evidence suggests that honey has an intrinsic regulatory effect on blood glucose level [25]. Moreover, honey intrinsically possesses wound-and burnhealing capacities [26].

Propolis
Propolis is a resin-like, pliable and compact substance formed by bees by combining bee wax and saliva with materials taken from tree buds, sap flows, and other plant components [27]. Raw propolis generally contains more than 300 components, majority of which are triterpenes (50 % w/w), wax (25-30 % w/w), and volatile mono-and sesquiterpenes [8-12 % w/w). Typically, propolis has odour of resin and phenolics which constitute 5-10 % w/w [27]. Since propolis contains mostly phenolic compounds, it has been extensively researched for its antioxidant and free radical-scavenging effects. Pinocembrin, pinobanksin, and chrysin are examples of such compounds with significant antioxidant and antiradical capabilities [28]. Propolis volatile components such as βeudesmol in Bulgarian propolis; delta-cadinene, α-pinene and trans terpineol in Greek propolis; terpineol, spatulenol, and ledol in Canary Islands; propolis insular, farnesol, dihydroeudesmol and guaiol in Democratic propolis, have shown significant anti-bacterial properties. Furthermore, propolis contains antifungal agents effective against disease-causing yeasts such as C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata [29]. The antifungal properties were due mostly to volatile compounds from Brazilian propolis viz. a-pinene, b-pinene and d-cadinene, as well as volatile compounds from Turkish propolis viz. phenyl-, ethyl-, and benzyl alcohols, and decanal [30]. Propolis also possesses potential antiviral properties, sometimes better than those of regular antiviral drugs. For example, it has been demonstrated that a topical ointment containing Canadian propolis outperformed Acyclovir ® or placebo in clinical trials for genital herpes simplex [31]. Propolis is also well-known for modulating immune responses, which largely accounts for its antimicrobial and antiviral properties [32]. Several studies have reported the antiinflammatory properties of propolis, which could be due to the production of phenolic acids. Caffeic acid phenethyl ester is the second most important anti-inflammatory constituent of propolis, with potential to regulate the complex NF-kB signal pathway [33]. Moreover, it has been reported that caffeic acid phenethyl ester modulated the ERK MAPK signal pathway in T cells and mastocytes [34] and controlled the PI3K/Akt signal route in different human cells lines [35].

Bee venom
Bee venom, also known as apitoxin, is an important chemical generated in the bee abdomen by the venomous gland cavity. It is injected into victims through a stinger, leading to local inflammation, anticoagulation effects and stimulation of immune responses. Bee venom consists of several amphipathic polycationic peptides, among which the most commonly found are melittin and apamine enzyme (phospholipase A2), and low-molecular weight compounds including bioactive amines such as histamine and catecholamines [36]. Since ancient times, bee venom has been used for acupuncture and apitherapy in cases of chronic pain, inflammation, Parkinson's disease and immunotherapy. It has also been shown to exhibit anticancer, antimutagenic, antinociceptive, and radioprotective properties. The efficiency of bee venom in relief and management of chronic pain has been studied, with a view to assessing its beneficial potential for therapeutic use [37]. Due to its potential interaction with biological membranes and strong antimicrobial properties, studies have been done on the effect of melittin on human pathogens such as methicillin-resistant S. aureus [38], as well as plant pathogens [39]. Moreover, several studies have revealed anticancer properties of melittin. In addition, in vitro investigations have been carried out to establish the molecular mechanisms involved [40]. Although it has enormous therapeutic potential, in vivo injection of melittin led to some serious adverse effects such as haemolysis and injury to the liver, indicating a need to study non-toxic hybrid derivatives of the venom [40].

Bee product and cancer
Chemotherapy medications and radiotherapy surely destroy cancerous cells. However, more often than not, they also harm healthy cells in the process of killing cancer cells, leading to undesirable as well as debilitating adverse effects, which in some cases, could possibly kill the patient faster than the cancer itself. Therefore, studies on alternative anticancer drugs, especially natural products, have become an important field of cancer research. Natural products are considered weak agents, but they are much safer and stronger. A few natural products are also extremely harmful to healthy cells. However, most of them do not act directly on cells. Rather, they stimulate the immune system, relying on the natural immunological distinction between healthy and infected or transformed cells. Still, natural products with the potential to fight a variety of diseases, remain a relatively unexplored area. Therefore, only a few isolated compounds with proven clinical effectiveness are currently available. These natural compounds can be utilized as bases for developing more effective analogues via full or combination synthesis [41]. Bee products, including raw materials, crude extracts, and distilled bioactive compounds, were employed in ancient medicine for antibacterial, antiinflammatory, and antioxidant purposes. In recent times too, bee products have been used for combating several immune-related disorders, as well as tumours. It has been observed that bee-derived peptides induce apoptotic cell death in vitro in several human cancer cell lines such as renal, lung, liver, prostate, thyroid, and lymphoid cancers [42]. New studies revealed that some specific natural bee compounds inhibit tumour cell growth and metastasis, and induce cancer cell apoptosis, indicating their potential benefits as alternative medical treatments for several human cancers [43].

Molecular mechanism involved in the anticancer effects of honey
The anti-cancer properties of honey have been widely hypothesized, and the underlying molecular mechanism have been investigated. Honey induces anticancer effect through cell cycle arrest, mitochondria pathway stimulation, activation mitochondrial outer membrane permeabilization [MOMP], activation of apoptosis, oxidative stress modulation, amelioration of inflammation, insulin signal modulation, and inhibition of angiogenesis [44].
Honey exerts its effect by inhibiting cancer growth at the cell cycle level. This inhibition was observed in the sub-G1 phase of bladder neoplastic cell lines [45], and at G0/G1 phase in non-small cell carcinoma cell [NCI-H460] [46]. Honey activates the mitochondrial pathway of apoptosis, leading to release of cytochrome C. Flavonoids from honey have been shown to activate the mitochondrial pathway of apoptosis [47]. Treatment of neoplastic cells with honey triggered apoptosis-induced death via activation of caspase-3, caspase-7 and caspase-9 [48]. Honey has also been shown to enhance tamoxifen-induced apoptosis by activating caspase-3, caspase-7, caspase-8 and caspase-9 [49]. The effects of honey on many apoptosisrelated enzymes, genes and transcription factors have also been demonstrated. Treatment of colorectal neoplastic cell lines HCT-15 and HT-29 with honey induced down-regulation of expression of poly-ADP-ribose polymerase (PARP), a vital enzyme involved in apoptosis and repair of DNA. The honey-induced inhibition of PARP activity blocked DNA repair, and thus caused increased cytotoxicity in cancer cells [50]. In addition, the expressions of caspase-3, Bax and p53 were triggered or activated by honey, while Bcl-2 expression was down-regulated. A study has demonstrated that honey exerted antimutagenic property by inhibiting error-prone repair process in DNA [51]. In diethyl nitrosamine [DEN]-induced carcinogenic rats; honey treatment was reported to produce antineoplastic effects owing to restoration of p53 expression. The apoptotic effect of honey is mediated by activation of caspase-9 and, caspase-3, and down-regulation of Bcl-2 expression [52]. Honey may also prevent cancer growth by regulating oxidative stress, that is, by oxidative stress enhancement or induction. The anti-cancer effects of honey which it exerts via antioxidants or pro-oxidants, appears to be entirely dependent on the level of oxidative stress in cancer cells. If cancer cell survival is dependent on low levels of ROS and oxidative stress, honey acts as a pro-oxidant, thereby increasing ROS and oxidative stress. Inflammation is a very important element in the pathophysiology of many other cancers/malignancies [53]. Two essential components of the inflammatory pathway activated in cancers are mitogen-activated protein kinase [MAPK] and nuclear factor kappa-B [NF-kB] [54]. Activation of MAPK and/or NF-kB stimulates the synthesis of inflammatory proteins and genes such as cyclooxygenase 2 [COX 2], C-reactive protein [CRP], lipoxygenase-2 [LOX2], and pro-inflammatory mediators or cytokines such as interleukin 1 [IL-1], IL-6, and tumor necrosis alpha (TNF-α). These pathways and pro-inflammatory mediators are known to have significant roles in cancer angiogenesis and pathogenesis of inflammation [55]. Honey therapy reduced the expressions of MAPK and NF-kB in HIT-T15 cells [56]. Chrysin, a normal component of honey, has also been shown to induce apoptosis in melanoma cells of B16-F1 and A375 by modulation of MAPK [57]. In addition, chrysin enhanced apoptosis of neoplastic cell lines mediated by TNF-related apoptosis-inducing ligand [TRAIL] [58]. Study reported that high levels of serine phosphorylation in MAPK, NF-kB, and insulin receptor substrate 1 [IRS-1] were characterized by insulin resistance assembly, while Akt expression and insulin content were significantly decreased [56]. Honey therapy increased the expression of Akt, while the expressions of phosphorylated IRS-1, MAPK and NF-κB were decreased. These findings demonstrated that honey also modulates insulin signaling, which may contribute to its anti-cancer effects [56]. In cancer cells, honey prevents angiogenesis. The potential anti-angiogenic impact of honey on cancer cells (hepatoma HePG2 cell lines) was based on evidence that honey decreased the viability, metastasis, and gelatinase and protease activities of cancer cells [59]. Caffeic acid phenethyl ester, chrysin and other cytotoxic honey constituents exert anti-angiogenic and anti-cancer effects [60].

Bee products and lung cancer
Bee venom possesses unique pharmacological effect due to its complex protein composition, which includes enzymes and peptides. Melittin is the key component of bee venom, and it accounts for 50 % of the total constituents of the venom. Recent studies showed that bee venom has tremendous benefits for tumor treatment due to its synergistic antitumor effects with melittin. Earlier, a study was conducted on the effect of propolis on the growth and apoptosis of human lung adenocarcinoma [A549 cells], and its mechanism of action [61]. The propolis cells were incubated for 72 h, and 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were employed for analysis of cell viability and inhibitory concentration [IC]. Findings from the study suggested that propolis produced antiproliferative and cytotoxic effects on A549 cells in dose-and time-dependent manner but failed to inhibit the growth of normal Vero cells. Compared to control A549 cells, increased apoptosis was observed in cells treated with propolis after 48 h. Propolis was shown to induce over-expressions of pro-apoptotic genes Bax and Noxa, which resulted in decreased mitochondrial membrane potential and decreased expression of anti-apoptotic gene Bcl-XL. The study also showed that the expression levels of other genes such as p53, Caspse-3, and Bax remained unaffected, except p21 which showed increased expression.
Propolis induced caspaseindependent apoptosis through a p53independent mitochondrial pathway, causing arrest of the cell cycle via p21 upregulation [62]. Another study was conducted on the cytotoxicity of Turkish propolis ethanolic extract [EEP] on human lung cancer [A549] cells, and the mechanism involved. Results from MTT assay revealed the cytotoxicity of EEP on A549 cells [63]. The mechanism of EEP cytotoxicity on A549 cells was evaluated in relation to apoptosis, mitochondrial membrane, and the cell cycle using flow cytometry; endoplasmic reticular tension using RT-PCR, and luminometric analysis of caspase activity. It was observed that EEP produced selective toxicity on A549 cells, when compared to normal fibroblast cells. The study revealed that EEP arrested the cell cycle of A549 in the G1 stage; increased endoplasmic reticulum stress, caspase activity, and apoptosis, and decreased mitochondrial membrane potential. These results indicate the potential of Turkish propolis to halt the proliferation of cancer cells, and its likely application in the development of new anticancer drugs [63].
A study on the antiproliferative effect of an ethanolic extract of Algerian propolis [EAP] on the human lung adenocarcinoma cell line [A549] in albino Wistar rats demonstrated its chemopreventive potential against benzo [a]pyreneinduced lung carcinogenesis. The cytotoxic effect of Algerian propolis on A549 cells was evaluated using MTT assay and cell adhesion [17]. A single intraperitoneal dose of benzo [a]pyrene [200 mg/kg] was injected into the rats, along with propolis (25 mg/kg) for 5 days before lung cancer was induced experimentally. Body weight, lung weight, lipid peroxidation, marker enzymes, and enzymatic and non-enzymatic antioxidants were measured prior to treatment. Pre-treatment evaluation revealed increases in enzymatic and non-enzymatic antioxidants and decreases in lipid peroxidation [17].
Another study was carried out to assess the antiproliferative potential of Acacia honey on NCI-H460 cells, and the associated molecular mechanism, using cell cycle, cell viability, cytokine, calcium ion, and gene expression analyses. Acacia honey dose-dependently inhibited aberrant cell division, arrested the cell cycle in G0/G1 stage, activated cytokines, released calcium ions, and reduced the expressions of p53 and Bcl-2 [64]. The antiproliferative effect of Acacia honey on the NCI-H460 cell line was attributed to cell cycle arrest, cytokine stimulation, calcium ion release, as well as downregulation of the genes Bcl-2 and p53 [64].
A study was conducted on the cytotoxic effect of Kelulut honey (Trigona itama), a Malaysian multiflora honey, as a probable natural anticancer agent for inducing apoptosis and cell cycle arrest on an epithelial cell line of human lung adenocarcinoma (A549) cells [65]. The cells were exposed consecutively to various concentrations of Trigona itama honey for 24, 48 and 72 h. Cytotoxicity and cell viability were analysed using trypan blue exclusion assay [TBEA] and flow cytometric analysis. Trigona itama honey produced maximum cytotoxic effect at 72 h at a concentration of 20 %, with 100 % inhibition. The moisture content of the honey was 14.3 ± 0.8 %, which was within the international standard range. The pH was 3.17 ± 0.02, and the electrical conductivity was 0.47 -0.55 mS/cm, while the proline contents were 19.1 and 20.2 mg/kg [65].
Chen et al conducted a study involving in vivo and in vitro investigations of the antitumor effect of melittin [66]. The study revealed that epidermal growth factor influenced inhibition of the invasion and migration of non-small cell lung cancer cells. In the study, subcutaneous injection of melittin at a dose of 1 mg/kg led to 27 % suppression of non-small cell lung cancer cells. However, a dose of 10 mg/kg resulted in 61% suppression. Melittin also suppressed the protein expressions of vascular endothelial growth factor [VEGF] and hypoxia-inducible factor 1 α. Therefore, it is reasonable to expect that the anticancer effect of melittin is related to its antiangiogenic effect as demonstrated via inhibition of VEGF and hypoxia-inducible factor signaling pathways [66].
For so many decades, honey was used for nutritional purposes and topical wound care. There is no evidence of any adverse effects from most of the available varieties of honey. Gastrointestinal distress and hepatitis were reported after ingestion of Rhododendronderived honey, colloquially known as crazy honey [67]. However, such incidents are uncommon and are limited to unique sources. Lately, a study performed by Fernandez-Cabezudo et al demonstrated the efficacy of honey as a medicinal product [68].

CONCLUSION
A plethora of evidence and data from cell culture and animal studies have proven that honey is a beneficial chemo-preventive agent and an important alternative to medications used for cancer therapy. Honey is very easily available, cheap, and easy to use. Moreover, it possesses minimal risk of detrimental side effects. The basic composition and therapeutic qualities, such as anticancer characteristics, vary according to bee species, nectar source, habitat, geographical location, extent of processing, packing, and storage. Bee products should be further studied in order to determine their prophylactic effects on invasive neoplastic lesions, and to determine the length of time that these effects last after surgery. Therefore, more research is required in order to evaluate the anticancer potential of honey before making any recommendations for its use in clinical settings.

Acknowledgement
The author would like to thank Dr Sirajudheen Anwar, Department of Pharmacology and Toxicology, University of Hail, Hail for editing and critically reviewing this manuscript.