Department of Zoology, NKR Government of Arts College for Women, Namakkal- 637 001, Tamil Nadu, India
Breast cancer (BC) is the leading cause of cancer-related deaths among females, and the development of resistance to chemotherapeutic drugs poses a significant challenge in its treatment. Royal Jelly (RJ) and Selenium are known sources of bioactive molecules with demonstrated anticancer properties. RJ has been shown to enhance cocoon production and silk yield in silkworms. This study aimed to evaluate the anti-cancer potential of silkworm extracts supplemented with RJ, Selenium, and a combination of RJ and Selenium on the MCF-7 BC cell line. MTT cytotoxicity assay was employed to assess the anti-cancer activity of the silkworm extracts. Different groups of extracts, including RJ, Selenium, and a mixture of RJ and Selenium, were tested for their cytotoxic effects on MCF-7 cells. The MTT assays demonstrated dose-dependent cytotoxic effects, with Group IV extract showing the highest potency, with a CC50 value of 130.07 µg/ml after 24 hours of incubation. The findings indicated that methanol extracts of RJ with Selenium exhibited significant potential as medicinal agents in BC treatment. The results of this study suggest that the combination of RJ and Selenium in silkworm extracts may hold promise as a therapeutic intervention for BC. The observed cytotoxic effects on MCF-7 cells highlight the potential of these extracts as valuable resources for the development of novel anticancer drugs. Further research is warranted to explore the mechanisms underlying the anticancer activity of RJ and Selenium in silkworm extracts and their potential clinical applications in BC therapy.
Breast cancer (BC) is a disease in which breast cells grow abnormally and form tumors. It typically begins inside the milk ducts or the milk-producing lobules of the breast and can potentially spread throughout the body, becoming fatal (1). When diagnosed in its early stages, it is not usually life-threatening. Cancer cells may invade nearby breast tissue, causing lumps or thickening. According to the Global Cancer Observatory, IARC-WHO in 2022, there were 2.3 million women diagnosed with BC and 670,000 deaths globally (2). The WHO Global BC Initiative (GBCI) is working to reduce global BC mortality by 2.5% per year between 2020 and 2040, aiming to prevent 2.5 million BC deaths (3). This initiative aims to avert 25% of BC deaths by 2030 and 40% by 2040 among women under the age of 70. In 2022, India had the highest number of estimated BC deaths among females, with 98,337 reported cases (4). Despite recent advances in medicine, the treatment of cancer remains a significant challenge. The side effects associated with conventional cancer treatments such as chemotherapy and radiation therapy have led researchers to seek new sources of drugs that are more targeted and have fewer side effects (5). Exploring novel approaches to combat cancer has become an exciting area of biomedical research (6). The silkworm (Bombyx mori), which has been domesticated for silk production for nearly 5000 years, is considered one of the best models for genetic and biochemical studies due to its complex metabolism, large body size, and the availability of mutants (7-9). In the future, silkworms could potentially be used in drug development as an alternative to mammals. Studies have shown that the silkworm infection model and the therapeutic efficacy of antiviral, antifungal, and antimicrobial agents (ED50) align well with mammalian models (7, 10, 11). Over the past decade, silkworms have been utilized as a model organism to investigate human microbial toxicology and pathology. Research has demonstrated that silkworms are highly sensitive to various human pathogenic microorganisms, pathogenic fungi, antibiotics, and pesticides (12, 13). Studies have been conducted on bacterial infections, fungal infections, viral infections, and natural immune stimulation using silkworms as models (14-16). Currently, the use of silkworms as a model organism for studying human tumors, degenerative diseases, and metabolic disorders has emerged as a prominent focus of research (16, 17). Royal Jelly (RJ) is a yellowish-white, acidic fluid produced by the hypopharyngeal and mandibular glands of nursing bees. It is used to feed young worker larvae for the first three days of their life and for the entire lifespan of the queen bee (18, 19). RJ is a highly esteemed natural product that has been traditionally used in medicines, health foods, and cosmetics around the world. It is the most extensively studied bee product, with researchers investigating its antibacterial, antioxidant, anti-aging, immunomodulatory, and general tonic properties in laboratory animals, microbiological species, farm animals, and clinical trials (20-23). RJ is commonly utilized in the treatment of various conditions such as cancer, diabetes, heart disease, and Alzheimer's disease (24, 25). The primary bioactive components of RJ are well-known for their medicinal and health-promoting properties (19). Recent studies have demonstrated that RJ exhibits anticancer, antioxidant, antimicrobial, anti-aging, and anti-inflammatory pharmacological effects, positioning it as a dietary supplement with functional health-enhancing benefits (18-24). Selenium is a crucial microelement in insect physiology that is naturally found in many foods and is also available as a dietary supplement. It is a component of 25 selenoproteins, including thioredoxin reductases, glutathione peroxidases, and selenoprotein P (26, 27). By integrating itself into selenoproteins in the form of selenocysteine (Sec), selenium plays a vital biological role in organisms (28). Selenoproteins are potent regulators of carcinogenesis and tumor progression, as they help inhibit tumor development by counteracting oxidative stress induced by inflammatory mediators (29). Selenium demonstrates cancer-inhibitory effects by disrupting antioxidant and pro-survival systems such as thioredoxin (Trx) and glutaredoxin (Grx)-coupled glutathione (GSH) to generate reactive oxygen species (ROS). Sodium selenite is identified as a potent compound against cancer cells due to its ability to induce ROS production (26, 30, 31). The present study aims to analyze the in vitro anti-cancer activity of the MCF-7 cell line using silkworms supplemented with a mixture of RJ and Selenium.
MATERIALS AND METHODS
Experimental design
The study were conducted in accordance of Dandin et al. (32), third instar silkworm larvae were divided into four groups, each comprising 50 larvae. The larvae were fed on mulberry leaves treated from the third to fifth instar and reared under standard recommended conditions at a temperature of 26 ± 2°C, relative humidity of 75 ± 5%, and a 12:12 light-dark photoperiod.
After reaching the fifth instar stage, the larvae from each group were collected, dried, and powdered. The samples were then extracted using a Soxhlet extractor, where a 500 ml flask containing 100 ml of methanol was used at its boiling temperature. The solvent was evaporated at its boiling point until complete removal of ethanol to determine the extract weight accurately (33).
In Vitro Anti-cancer activity
MTT-Assay-Chemicals
3-(4,5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide (MTT), Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and Trypsin were procured from Sigma Aldrich Co, St. Louis, USA. EDTA, Glucose, and antibiotics were sourced from Hi-Media Laboratories Ltd., Mumbai. Dimethyl Sulfoxide (DMSO) and Propanol were acquired from E. Merck Ltd., Mumbai, India.
Cell Lines and Culture Medium
The MCF-7 (Human BC) cell culture was obtained from the National Centre for Cell Sciences (NCCS) in Pune, India. The stock cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin B (5µg/ml) in a humidified atmosphere with 5% CO2 at 37°C until confluent. Cell dissociation was carried out using TPVG solution (0.2% trypsin, 0.02?TA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks, and all experiments were conducted in 96-well microtiter plates from Tarsons India Pvt. Ltd. in Kolkata, India.
Preparation of Test Solutions
For cytotoxicity studies, each weighed test drug was individually dissolved in distilled DMSO. The volume was adjusted with Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2% inactivated Fetal Bovine Serum (FBS) to achieve a stock solution concentration of 1 mg/ml. This solution was then sterilized by filtration. Subsequently, serial two-fold dilutions were prepared from this stock solution to conduct the cytotoxicity studies.
Determination of Cell Viability by MTT Assays
The monolayer cell culture was trypsinized, and the cell count was adjusted to 1.0 x 10^5 cells/ml using medium containing 10?tal Bovine Serum (FBS). These cells were then utilized for determining cell viability through MTT assays, following the protocol (34). The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using a specific formula, and the concentration of the test drug required to inhibit cell growth by 50% (CTC50) values was derived from the dose-response curves obtained for each cell line.
Mean OD of individual test group
%Growth in hibition =100 × 100
Mean OD control group
Statistical Analysis
The CTC50 value was extrapolated from the dose-response graph. By plotting data points across a range of concentrations and applying linear regression analysis using the PRISM program, the concentrations of the samples that reduced cell viability by 50% (CTC50) were determined. The dose-response curve analysis was conducted using GraphPad Prism 5.0 software.
RESULTS
In the current study, the in vitro anti-cancer activity of RJ and Selenium supplemented silkworms was assessed against the MCF-7 (Human breast carcinoma) cell line. The cytotoxic effects of the methanolic extracts from four different groups of silkworms were evaluated at doses of 50, 100, 150, 200, and 250 µg/ml using the MTT assay. The concentration required to inhibit cell growth by 50% (CTC50) was calculated using dose-response curves generated with GraphPad Prism software version 5.0. The results of the study are presented in Table 1 and Figure 1, illustrating the cytotoxic activity of the extracts, while Figure 2 shows the cell viability activity. The study findings indicated that all extracts were capable of inhibiting the proliferation of cancer cells. The methanolic extract from group IV exhibited a potent cytotoxic effect on MCF-7 cells in a concentration-dependent manner, with an excellent CTC50 value of 130.07 µg/ml. Group II extracts also demonstrated cytotoxic activity against MCF-7 cells with a CTC50 value of 160.88 µg/ml. Conversely, Group III and Group I displayed lower inhibition activity against the tested cell line compared to Group IV and Group II.
Microscopic observations revealed that the RJ and Selenium supplemented silkworm extract exhibited the highest reduction in MCF-7 cell population and cell volume compared to other groups. The cell viability decreased in the order of Group IV > Group II > Group III > Group I, with the cell viability of MCF-7 dropping to 38.65% at a high concentration of 250 µg/ml. Overall, Group IV (RJ and Selenium) demonstrated potent cytotoxic activity against MCF-7 BC cell lines compared to the other groups of supplemented silkworms
Table 1 shows the cytotoxicity effect of different groups of supplemented silkworms.
|
Concentration |
Groups |
|||
|
I |
II |
III |
IV |
|
|
(µg/ml) |
% CTC50 |
% CTC50 |
% CTC50 |
% CTC50 |
|
50 |
29.45 |
50 |
29.45 |
50 |
|
100 |
37.01 |
100 |
37.01 |
100 |
|
150 |
41.31 |
150 |
41.31 |
150 |
|
200 |
48.67 |
200 |
48.67 |
200 |
|
250 |
53.78 |
250 |
53.78 |
250 |
Figure 1: The inhibitory activity of various groups of supplemented silkworms at different concentrations.
Figure 2: The decrease in cell viability observed in the different groups of silkworm extracts is dependent on concentration.
DISCUSSION
Cancer is a significant health concern that can impact any part of the body worldwide. BC is one of the most prevalent cancers in women and remains a leading cause of death globally. The incidence of BC has decreased in developed countries due to advanced diagnostic techniques and increased awareness of mammographic screening (35). However, the tumor heterogeneity of BC often leads to drug resistance in chemotherapy, presenting a challenging obstacle in treatment (36, 37). Natural products have a long history of use in preventing and treating various diseases, including cancer, making them promising candidates for the development of anticancer drugs (38-40). There has been a growing interest in exploring the pharmacological effects of bioactive compounds for cancer treatment and prevention. In the current study, the anticancer activity of silkworms supplemented with RJ and Selenium, either individually or in combination, was investigated. RJ and Selenium are considered promising candidates for anticancer agents, as they have demonstrated various anticancer activities in different types of cancer cells by inducing cytotoxic effects while showing minimal harm to normal cells (41, 42). RJ has demonstrated potential anticancer properties by inhibiting tumorigenesis, cancer cell proliferation, and metastasis. This effect is believed to be achieved through the inhibition of tumor-induced angiogenesis and/or the activation of immune function (18-20). RJ has the ability to suppress the multiplication of MCF-7 human BC cells induced by bisphenol A (43). Estradiol, a key player in BC tumor development, is known to have crucial roles. RJ containing flavonoids such as naringenin, acacetin, apigenin, chrysin, genistein, coumestrol, luteolin, hesperetin, formononetin, kaempferol, glycoside, and isosakuranetin may contribute to its antitumor activity (44, 45). Flavonoids exert their anticancer effects by inhibiting malignant cell proliferation, promoting apoptosis, regulating enzyme antioxidant activities, inducing autophagy, and modulating the cell cycle (46-48). Moreover, polyphenols present in RJ have been shown to exhibit anticancer effects by influencing signaling pathways leading to cell death, inhibiting cell cycle progression, and promoting apoptosis (49). The presence of 10-HAD in RJ exerts its antitumor activity by modulating oxidative stress and inflammation in cells (50). Additionally, RJ reduced tumor weight in mice with 4T1 breast tumors at specific doses (51). In the current study, Group II exhibited a significant increase in anticancer activity against MCF-7 cells, attributed to the presence of flavonoids, polyphenols, and 10-HAD in Royal Jelly. Selenium is an essential trace element that plays a crucial role in maintaining health and is involved in various biological functions. It acts as a cofactor for several antioxidant enzymes and has been recognized as an effective agent in cancer prevention (27). Selenium supplementation has been linked to anti-cancer properties, including reducing cancer cell survival and stimulating the immune response by activating antibody formation, helper T-cells, cytotoxic T-cells, and natural killer cells (52). Sodium selenite (Na2SeO3) is an important inorganic selenium salt found in dietary supplements and has shown promising effects in cancer treatment (53). Studies have demonstrated that treatment with sodium selenite at specific concentrations can inhibit cell proliferation, induce apoptosis, and increase the expression of Se-binding protein 1 (SBP1) in gastric cancer cells. Selenium-containing compounds are valued for their ability to induce prooxidative stress, making them effective in cancer therapy (54, 55). Research has shown that selenium and vitamin C supplementation can reduce the incidence and mortality of gastric and lung cancer (56). Selenoproteins, which are proteins containing selenium, play critical roles in various biological processes such as thyroid hormone metabolism, DNA synthesis, reproduction, and protection against oxidative damage and infection (57). Furthermore, studies have reported synergistic effects when combining selenium compounds with other cancer treatments. For example a reduction in tumor growth in MCF-7 BC xenograft models when treated with a combination of tamoxifen and methylselenocysteine (58). This highlights the potential benefits of selenium supplementation in cancer therapy and its synergistic effects when combined with other anti-cancer agents. Based on the results of your study, it appears that the combined treatment of RJ with Selenium significantly enhances anticancer activity in MCF-7 cells in a dose-dependent manner. This suggests that the combination of RJ and Selenium may hold potential as a chemotherapeutic or chemopreventive agent, capable of inducing apoptosis in cancer cells while exhibiting relatively low toxicity to normal cells. The synergistic effects of RJ and Selenium in increasing anticancer activity highlight the promise of this combination as a potential strategy for cancer treatment or prevention.
CONCLUSION
The results of this study demonstrate the significant anti-cancer potential of methanol extracts of RJ with Selenium in BC treatment. The combination of RJ and Selenium showed dose-dependent cytotoxic effects on MCF-7 cells, with the Group IV extract exhibiting the highest potency. These findings suggest that the synergistic effects of RJ and Selenium in silkworm extracts hold promise as a potential medicinal intervention for BC. Further research is needed to elucidate the underlying mechanisms of action and to explore the clinical applications of these extracts in cancer therapy. The study underscores the importance of natural bioactive compounds in the development of novel anticancer drugs and highlights the potential of RJ and Selenium as valuable resources in the fight against BC.
REFERENCES
Poomani Vimala Manoharan, Sharmila Banu Gani*, Evaluation of Anti-Cancer Activity of Royal Jelly and Selenium Mixture Supplemented to Silkworms- An In-Vitro Study, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 1, 467-476. https://doi.org/10.5281/zenodo.14614203
10.5281/zenodo.14614203
