Background
Breast cancer, similar to other cancers, exhibits major morbidity and mortality. It is continued to be the most common female cancer and prevalence is still rising [
1]. Cancer-prevention is aimed at interfering with the basic processes of carcinogenesis through chemical agents or regimens that may block neoplastic induction [
2]. It results in prevention of the progression of transformed cells into malignant types [
2]. The other purpose is to prevent the development of second primary tumors that may arise from patients who had already been cured from the initial cancer [
2]. Potential cancer-preventive agents include vitamins, minerals, phytochemicals, synthetic compounds and natural products such as honey [
3,
4].
Honey has been used since ancient times as a traditional medicine. Honey has recently received attention as a complementary and alternative treatment in modern medicine [
4,
5]. It is mainly composed of various sugars, phenolic acids, flavonoids, enzymes, amino acids, proteins, phytochemicals and other miscellaneous compounds [
4]. It is also considered as a natural phytoestrogen [
6] with epigenetic modification [
7]. Honey has a potential to be preventive agent against cancer [
4]. Tualang honey (TH) is a multi-floral jungle honey. It is produced by
Apis dorsata bee specie which builds their hives high on Tualang trees (
Kompassia excelsa) in Malaysian tropical rainforests [
8,
9]. Published data has shown that TH exhibits antimicrobial [
10,
11], anti-inflammatory [
9], antioxidant [
12] and antidiabetic effects [
13]. It has been demonstrated that TH as a cancer-preventive agent ameliorates 7,12-dimethyl-benz-anthracene (DMBA) induced breast cancer in vivo by modulating tumor severity, histological grading and increased apoptosis [
14]. Another study has reported that TH reduces tamoxifen-induced cytotoxicity against breast cancer
in vitro, demonstrating its preventive effects [
15]. TH has been shown to have anticancer effects against oral squamous cell carcinoma [
16], human osteosarcoma cell lines [
16], human breast cancer cell lines [
17] and cervical cancer cell lines [
17]. The aim of our study was to evaluate the chemo-preventive effects of Tualang honey against experimental breast cancer in vivo.
Hematological parameters have been correlated with prognosis in different malignancies [
18]. Pre and post-treatment studies have shown that breast cancer patients have deranged or abnormal blood count pattern [
18]. The functioning of the immune system at hematological level has a direct influence on breast cancer [
19]. Prolonged exposure to estrogens has been associated with increased risk of breast cancer development [
20]. Elevated serum levels of endogenous estrogen (E2) and estrogen receptors (ESR1, ESR2) are associated with increased risks [
21]. Estrogen receptor positive breast cancer is the most common type and anti-estrogen therapy has been shown to be very effective in preventing recurrence [
22].
Apoptosis is recognized as the principal mechanism of drugs-induced regression in breast cancer [
23]. The expression of pro and anti-apoptotic proteins is considered a hallmark for prognosis of this disease [
23]. Apoptotic protease activating factor-1(Apaf-1) is a tumor suppressor gene [
24]. A reduction in Apaf-1 occurs during tumor progression from primary to systemic metastasis and can contribute to the ability of tumor cells to evade Caspase-9 apoptotic pathway [
24]. Increased levels of Bcl-xL expression are seen in primary high grade human breast carcinomas [
25].
The beneficial ‘preventive’ effects of TH on breast cancer are based on the premise that there is a reduction of tumor incidence, increased latency period and a slower tumor growth through modulation of hematologic, estrogenic and apoptotic activities. To our knowledge, this is the first study to report the modification of hematological parameters, E2, ESR1 and Apaf-1 by administering honey as a preventive measure in an in-vivo breast cancer model.
Discussion
The cancer-related death toll remains one of the highest among chronic diseases in humans [
32]. A major concern for anti-cancer drugs is their potential toxicity [
3]. Thus, alternative measures continue to be exerted to identify natural products with potential to complement existing preventive and therapeutic modalities [
4]. Studies have shown the potential effectiveness of honey against cancer [
4,
17]. Our study highlights some intriguing findings regarding the utility of TH as a potential cancer-preventive agent.
The findings of our study show that all the treatment groups that received TH had a lower tumor incidence and a higher latency than the non-treated positive control (Table
1). One of the primary purposes for cancer-preventive studies is to delay cancer appearance in healthy subjects or in subjects with an increased risk of cancer development who are otherwise healthy [
33]. Thus, our findings suggest that tumor incidence was reduced and tumor initiation was delayed by TH treatment. Honey exhibits strong anti-oxidant and anti-mutagenic activity [
9,
34], which could probably inhibit the carcinogenesis to transform the normal cells into malignant ones. Hence, may affect tumor latency and incidence.
The results of our study demonstrate that all varying strengths of TH appeared to slow down the progression of breast tumors development with lower multiplicity, size and weight compared to control (Fig.
1 and Table
1). It was also evident by the macroscopic evaluations of the tumors (Fig.
2). TH seems to be capable of reversing the tumorigenesis. It is shown by the reduced tumor size and weight in treated groups. Carcinogenesis is a multistep process and can be divided into three main stages; initiation, promotion and progression [
35]. Cancer-preventive agents may act as anti-promoting agents via intervening at initiation or promotion stages of carcinogenesis. Thus, we can assume that TH may intervene at the initiation or promotion stage to inhibit tumor growth. Honey, which exhibits strong anti-oxidant and anti-mutagenic activity [
9,
34], could potentially inhibit or disrupt critical steps in carcinogenesis, the initiation or promotion stage to inhibit tumor growth. The lower tumor multiplicity observed implies that TH may also acts as an anti-metastatic agent.
Some of the breast lesions in our study were found to be completely disappeared at the end of the study. It has been demonstrated that tumors can be eliminated or diminished by chronic administration of low doses of chemotherapeutic drugs [
36]. It is quite possible that TH treatments behaves similarly. Our findings are consistent with another previous in vivo study investigated by Kadir and colleagues that honey may modulate tumor latency, incidence, multiplicity and progression [
14]. In that study, a different carcinogen DMBA (7,12-dimethylbenzanthracene) was used. MNU has several advantages as it is more organ specific (breasts) and it induces tumors of breast ductal epithelium [
28].
Histological grading of cancers has pivotal importance for the prognosis of the cancer [
37]. The three criteria of grading breast cancer are based on the scores for mitotic activity, tubular formation by cancer cells and cellular pleomorphism. Our study shows that the breast cancers that developed in the TH treatment groups were of grades I and II, whereas tumors in the negative control group were mainly of grade III (Table
3 and Fig.
3). These findings support the antimutagenic activity of TH as it has been reported that honey exhibits antimutagenic activity [
34]. Polyphenols and flavonoids are reported to be solely responsible for the anticancer activity of honey [
38]. Thus, anti-neoplastic or anti-tumoral effects of TH may also be attributed to these compounds. Thus far, the specific compounds which are responsible for these protective effects are still unknown.
Full blood count is a prerequisite investigation in cancer patients before treatment and poor blood parameters affect the outcome of malignancies [
18]. It has been demonstrated that hematological parameters are correlated with prognosis of cancer [
18]. Breast cancer patients have been observed with abnormal or poor blood parameters [
18]. Our results showed the TH-treated groups had blood parameters closer to the normal control rats. The untreated negative control rats had a lower level of RBC, Hb, PCV, lymphocytes and platelets compared to the rats of TH treated groups. Treatments with varying strengths of TH had potentiating effect on the hematological parameters such as RBC, Hb, PCV, lymphocytes, eosinophils and platelets (Table
4). Lymphocytes and eosinophils are cells of the immune system and good level of Hb could aid in recovery from the injury due to cancer. Cancer patients are reported to have lower level of RBC, Hb, MCV, MCH, MCHC and lymphocytes and higher level of RDW, TWBC, polymorphs during pre and post-treatment [
18,
39]. Our findings show that TH may alter or tend to normalize the hematological parameters to ameliorate carcinogenesis in induced breast cancer. Exclusive honey feeding in the absence of any disease have been shown to significantly modify hematological parameters [
40‐
43].
We noted that rats in negative control group had higher serum levels of E2 and a lower Apaf-1 concentration than TH treated rats. When given various strengths of honey (groups 2, 3 and 4), the levels of E2 and Apaf-1 tend to almost near normal levels (Fig.
4). Higher serum estradiol (E2) levels are associated with an increased risk of breast cancer in postmenopausal women [
44]. The risk is higher in postmenopausal women and lower in premenopausal women [
44]. Higher levels of E2 have been reported in breast cancer patients [
21]. E2 promotes cell proliferation and suppresses apoptosis by directly modulating the genetic expression and thus is considered a crucial target in breast cancer treatment [
45]. The reduction of E2 in TH treated groups could be attributed to honey. Treatment with estrogen-lowering drugs shrinks breast cancer masses in patients [
46]. Thus, TH behaves as a natural estrogen-lowering agent.
At the tissue level, majority of tumors from the negative control group demonstrated a higher ESR1 expression than treated groups as detected by immunohistochemistry (Table
5 and Fig.
5). These tissue findings are validated when we found same results for E2 at serum level. Breast cancer patients had higher levels of estrogens and ER-mediated bioactivity [
21]. Estrogen receptors (ERs) bind to estrogens to dimerize and then translocate into the nuclei. These complexes then bind to specific DNA base sequences called estrogen-response elements (EREs) resulting in transcription and translation in the targeted tissue [
47]. This signaling cascade induced by estrogens may be modulated at any stage [
47]. TH possibly modulates E2 and ESR1, hindering this signaling pathway. Exogenous or synthetic estradiol (E2) can be used as a treatment in estrogen receptor (ER) positive breast cancer to stimulate the apoptotic pathway [
48]. Honey, which is a natural phytoestrogen [
6], may play a role in modulating endogenous estrogen and estrogen receptors and may stimulate the apoptotic pathway.
TH may bind to the ER in a similar manner as other drugs that disrupt receptor dimerization to block ER nuclear localization [
49]. Honeys from various floral sources exert estrogen agonist effects at high concentrations (20–100 μg/mL) and antagonistic effects at low concentrations (0.2–5 μg/mL) [
50]. These effects were attributed to polyphenols or flavonoids content [
50]. This study reported in vitro analysis of estrogen receptors only. Our study reports in vivo analysis of serum level estrogen and estrogen receptors at tumor tissues level. Our findings demonstrate agonist effect with all varying strengths of TH. The medium dose and low dose of TH seem more effective to modulate E2 and ESR1 respectively.
Loss of pro-apoptotic protein Apaf-1 can aid tumor cells in evading programmed cell death or apoptosis [
51]. Apaf-1 is an essential target in the intrinsic or Caspase-9 apoptotic pathway [
51]. Our results show TH has a potentiating effect on Apaf-1 and Caspase-9 at cancer tissues level (Fig.
4). This finding is further substantiated for Apaf-1 at serum level (Table
5 and Fig.
5). It could be concluded that TH enhances the expression of Caspase-9 and Apaf-1 resulting in slower tumor growth rate and better histological grading. We postulate that TH causes the up-regulation of Apaf-1 and Caspase-9 expression and may activate the intrinsic apoptotic pathway to modulate tumor growth. The possible mechanism demonstrates that TH akin to chemotherapeutic agents may induce apoptosis through multiple signaling pathways that converge on the mitochondria to cause the release of cytochrome c. Cytochrome c binds to Apaf-1 in the presence of dATP/ATP (deoxyadenosine triphosphate/adenosine triphosphate), which then binds to procaspase-9 to form a cytochrome c–Apaf-1–caspase-9 complex, called apoptosome. Apoptosome enables enzymatic self-activation of caspase-9 that subsequently activates procaspase-3. This ultimately results in cell death [
51]. Honey mediates cell death mainly through the intrinsic apoptotic pathway and by enhancing pro-apoptotic proteins expression [
4,
17].
Our findings showed no evidence of the expression of FASLG and FADD, hence no involvement of caspase-8 or the extrinsic apoptotic pathway in TH mediated apoptosis. Our results are in line with another study which demonstrated that Manuka honey induces intrinsic or caspase-9 apoptotic pathway in breast cancer [
52].
A study has reported that over-expression of Bcl-xL in breast cancer patients is associated with metastasis and worse prognosis [
53]. The decrease of Bcl-xL expression observed in TH treated tumors suggests that the administration of TH can lead to lower tumor cells proliferation and increased apoptosis by blocking mitochondrial swelling and membrane hyper polarization [
54]. Our findings suggest that Bcl-xL expression was hindered by TH at its intrinsic mitochondrial apoptotic pathway. This ultimately promotes apoptosis through increased expression of mitochondrial pathway proteins; Caspase-9 and Apaf-1, as observed in our study. This can be presumed that TH could be a viable option to mediate hematological parameter, and expression of Caspase-9, Apaf-1, E2, Bcl-xL and ESR1 against breast cancer. The variations in the dose dependent effect of TH need further research to elucidate the reasons and the underlying mechanisms.
Acknowledgments
We thank Federal Agricultural Marketing Authority (FAMA), Ministry of Agriculture and Agro-based Industry, Malaysia, for providing Tualang honey, and we thank TWAS (Third World Academy of Sciences) for providing USM-TWAS fellowship to author (SA). We would also like to thank Professor Siti Amrah Sulaiman (USM) for her advice on the animal experimentation and the technologists in pathology laboratory who helped the first author in tissue sectioning and Immunohistochemistry.