Antiproliferative Effect of Royal Jelly on Human Cancer Cell Lines: Potential Involvement of Bioactive Compounds

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Novel self-micellizing anticancer lipid nanoparticles induce cell death of colorectal cancer cells

Probiotics are live microorganisms, habituated in the human intestine, which have a beneficial effect on our health. In spite of many reports about the anticancer effect of these bacteria in in-vivo and in-vitro, their mechanisms of action are not completely understood. The goal of this study was to compare the extracellular fractions of Lactobacillus casei and L. paracasei on the anti proliferation and apoptosis induction in K562 cell line. L. casei and L. paracasei were cultured in MRS broth medium. Then extracellular secretions were collected and after enrichment, analyzed by electrophoresis. Fractionation were determined by gel filtration chromatography using sephadex G100 column, and the anticancer properties were evaluated. The results of SDS-PAGE showed various molecular weight of fractionated proteins of L. casei and L. paracasei. Bioactivity assessment illustrated that anti proliferative effects on K562 cells is dose and time dependent and the cytotoxic effects was parallel with protein concentration and the increase of time from 36 to 72 hours. Regarding the cell cytotoxicity results, the fractionated extracellular proteins of L. casei and L. paracasei have significant effects in inhibition of cancer cell proliferation. However, more study is needed to better elucidate the mechanisms of extracted proteins, and its effect on other human cancer cell lines.

Selective pro-apoptotic and antimigratory effects of polyphenol complex catechin:lysine 1:2 in breast, pancreatic and colorectal cancer cell lines

Several nutraceuticals, food, and cosmetic products can be developed using royal jelly. It is known for its potential health benefits, including its ability to boost the immune system and reduce inflammation. It is rich in vitamins, minerals, and antioxidants, which can improve general health. Royal jelly (RJ) is also being studied as a potential therapeutic agent for cancer and other chronic diseases. It is effective in reducing tumor growth and stimulating immunity. In this study, we investigated the effects of royal jelly on cancerous A549 cells and healthy MRC-5 cells at various doses ranging from 1.25 to 10 mg/mL. Royal jelly's anti-proliferative effect was evaluated by MTT and SRB assay for 48 h. The induction of necrosis and apoptosis was assessed by flow cytometry as well. The relative amounts of major molecules in Royal jelly were determined by FTIR spectroscopy to identify key functional groups and molecular structures. In addition, this technique was used for the first time to detect changes in the macromolecular composition of lung cells treated with royal jelly. Thus, it provided insights into the relative abundance of proteins, lipids, and carbohydrates, which could correlate with their bioactive properties. The antiproliferative effect of Royal jelly was found to be selective on A549 cells in a dose-dependent manner with an IC50 of 9.26 mg/mL, with no cytotoxic effects on normal MRC-5 cells. Moreover, Royal jelly induced predominantly necrotic cell death in A549 cells, %39.10 at 4 mg/mL and %57.88 at 10 mg/mL concentrations. However, the necrosis rate in MRC-5 cells was quite low, at 9.16% and 20.44% at the same doses. Royal jelly showed dose-dependent selective cytotoxicity toward A549 cells, whereas it exhibited no apparent cytotoxicity in MRC-5 cells. In order to identify the biomolecular changes induced by royal jelly, we used two unsupervised chemometric pattern recognition algorithms (PCA and HCA) on the preprocessed sample FTIR spectra to determine the effects of royal jelly on cell biochemistry. According to PCA and HCA results, RJ treatments especially affected biomolecules in A549 cells. The total spectral band variances in the PCA loading spectra were calculated for understanding biomolecular alterations. These plots revealed profound changes in the lipid, protein, and nucleic acid content of RJ-applied lung cells, primarily identifying RJ and H2O2 treated groups for A549 cells. Ultimately, the selective cytotoxicity of royal jelly toward A549 cancerous cells suggests that royal jelly may be a promising therapeutic agent for identifying innovative lung cancer treatment strategies. Additionally, understanding the molecular alterations induced by royal jelly could guide the development of novel cancer treatments that exploit its bioactive properties. This could lead to more effective and safer therapies.

In vitro study on anticancer effect of Dodder grown on fennel (Foeniculum vulgare) and camelthorn (Alhagi maorurum) against human cancer cells lines

Background: Signaling by the insulin-like growth factor 1 receptor (IGF-1R) promotes cell growth, migration and survival in several human tumor types. IGF-1R is overexpressed in CRC and is associated with a poor prognosis and resistance to chemotherapy. Our prior transcriptional profiling analysis of CRC cell lines treated with the small molecule IGF-1R/IR TKI, OSI-906, indicated that overexpression of the mitogen-activated protein kinase (MAPK) pathway conferred resistance to OSI-906. The purpose of this study was to evaluate the rational combination of the MEK1/2 inhibitor, U0126, and OSI-906 against human CRC cell lines. Methods and Results: The antiproliferative effects of OSI-906 and U0126 were assessed as single agents and in combination using the Sulforhodamine B (SRB) cell viability assay. Twenty-eight CRC cell lines were exposed to either OSI-906 (0–5µM), or U0126 (0–20µM). In reference to OSI-906, cell lines with IC50 ≤ 1µM were considered sensitive (S) and cell lines with IC50 ≥ 5µM were deemed resistant (R). Likewise, cell lines with IC50 < 5µM were regarded as S to U0126 and cell lines with IC50 > 10µM were considered R. We selected 12 cell lines for combination studies according to the following conditions: 1) S to both drugs, 2) R to both drugs, 3) S to OSI-906 but R to U0126, and 4) R to OSI-906 but S to U0126, and treated them with varying doses of the two drugs as single agents and in combination. Combination effects between OSI-906 and U0126 were evaluated using the Chou and Talalay method. Robust synergy was observed in most CRC cell lines, including those that were resistant to OSI-906 or U0126. Apoptosis was then analyzed using bioluminescent caspase 3/7 detection and validated through analysis of PARP cleavage by immunoblotting. Surprisingly, some of the cell lines demonstrated induction of apoptosis when exposed to the combination but not with either agent alone. The CRC cell lines with the greatest apoptotic induction were inherently sensitive to OSI-906 in the SRB assay. Conclusion: Our prior transcriptional profiling data revealed elevated expression of genes in the MAPK pathway in cell lines that were less sensitive to OSI-906, providing the basis for rational combination therapy with U0126. Interestingly, the combination of OSI-906 and U0126 displayed synergy in nearly all cell lines tested, regardless of sensitivity to either compound. Similarly, the apoptosis exhibited by some CRC cell lines could not be predicted by the sensitivity profile, or RAS/RAF mutational status. However, CRC cell lines with the greatest synergistic induction of apoptosis were sensitive to OSI-906. Further pathway and transcriptional profiling analysis is ongoing to reveal the underlying mechanism(s) of the observed synergy. These results, if further validated in vivo, will support the rational combination of OSI-906 and a MEK inhibitor in patients with CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A256.

The food and pharmaceutical industries have utilized royal jelly, an alternative medicinal food, as a natural pharmaceutical product since ancient times. Royal jelly has a unique remarkable composition containing lipids, proteins, carbohydrates, vitamins, minerals, hormones, and phenolic compounds. The rapidly expanding functional food market has coincided with the increasing consumer demand for royal jelly. Over the past two decades, royal jelly, a rich source of certain bioactive components, has been used by humans as a functional and nutritious food due to recent studies of the effect of royal jelly in underlying pathogenic processes in a variety of animal models. Scientific evidence has accumulated supporting a wide variety of health-promoting effects from the intake of royal jelly that supports cardiovascular health, immune and antioxidant function, wound healing, blood lipid, and glucose control in addition to antibacterial and antihypertensive effects. The main bioactive ingredients are Major Royal Jelly Proteins (MRJPs), essential oils, fatty acids, peptides, and phenolics, which are thought to have a significant role in the development of honeybee queens. The health-endorsing qualities of royal jelly make it a significant functional ingredient in the food, and cosmetic industry. Apisin is one of the main proteins in royal jelly that has antibacterial properties. Other bioactive ingredients of royal jelly that have multifunctional health-promoting properties include defensin-1, royalisin, apisimin, apidaecin, jelleins, royalactin and 10-hydroxy-2-decenoic acid (10HDA) in epigenetic diseases. This review highlights the important role that royal jelly plays as an agent in various fields of medicine, paying special attention to its biological features. Additionally, we discuss royal jelly’s composition as a possible therapeutic for vital natural sources of bioactive substances.

This dissertation investigated the role of the miR-200 family in normal colon epithelial (CCD 841) and Dukes’ C (HT-29) colorectal cancer (CRC) cell lines. Our aim was to characterize expression of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) in colorectal cell lines, study their effect on the tumor suppressor Ras Associated Domain-Containing Protein (RASSF) 2 and on subsequent activity within the mitogen-activated protein kinase (MAPK) signaling pathway. We wanted to determine whether regulation of miR-200 family members could change cell behavior towards more “cancer-like” in a normal colon epithelium (CCD 841) cell line, or less “cancer-like” in a Dukes’ C (HT-29) CRC cell line. We found the following: 1. All miR-200 family members were highly expressed in colorectal cancer cell lines compared to a normal colon epithelial cell line. 2. RASSF2 mRNA and protein expression was downregulated in all CRC cell lines compared to the normal colon epithelial (CCD 841) cell line. 3. Overexpression of miR-200 family members in a normal colon epithelial (CCD 841) cell line decreased expression of both RASSF2 mRNA and protein. 4. Inhibition of miR-200 family members in a Dukes’ C (HT-29) CRC cell line increased expression of both RASSF2 mRNA and protein. 5. Total K-Ras expression and phosphorylation of ERK 1/2 increased following overexpression of miR-200 family members in a normal colon epithelial (CCD 841) cell line, indicating increased activity within the MAPK pathway resulting in increased cell proliferation. 6. MAPK pathway activity decreased, as measured by reduced ERK 1/2 phosphorylation and reduced cell proliferation in a Dukes’ C (HT-29) CRC cell line following inhibition of miR-200 family members. These findings demonstrate a novel association of the miR-200 family, the tumor suppressor RASSF2, and the MAPK signaling pathway in CRC. In contrast to the previous understanding that miR-200 family dysregulation is considered to exhibit tumor suppressive behavior by blocking epithelial to mesenchymal transition, we refute this in the case of CRC and propose the miR-200 family contribute to CRC tumorigenesis. This improved understanding of the miR-200 family may have the potential to be developed as a therapeutic intervention in CRC.

Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells.

Simple SummarySpermatogenesis and hormones secretions are serious life-threating and complicated process, which can be improve through science-based approaches. Royal jelly is a thick white milky fluid secreted by the hypopharyngeal and mandibular glands of young nurse worker bees (Apis mellifera) and used to feed their queen to expand their life. The results of the study revealed that, the growth performance of testis in exposed mice to freeze-dried Royal Jelly for 35 consecutive days were significantly enhanced in moderate dose among other treated doses. However, at Post Natal Days (PNDs 14 and PNDs 21), obviously changes were observed in histological examination of the testis while at PNDs-07 no major changes were observed. The Tunnel assay showed that, less apoptotic cells were detected in the testis of mice in high dose of freeze-dried RJ and oral administration of freeze-dried royal jelly can aggravate adverse effects via tempestuous on sexual hormone secretion at both PNDs 21 and PNDs 35 respectively.Spermatogenesis and hormones secretions are crucial endocrine and physiological process for maintaining the life. Royal Jelly (RJ) bioactive components are Major Royal Jelly Proteins (MRJPs), owing exceptional biological properties. However, the effects of RJ on pup’s testicular development during neonatal and pubertal period exposure hasn’t been adequately studied. The aim of the study was to detect neonatal sexual hormones concentration and histopathological changes on testicular development of the male progeny after oral exposure to freeze-dried RJ for 35 consecutive days. After mice give birth, male pups were collected together, separated by sex, and randomly standardized to seven (7) male pups per dam. Male pups were assigned to control diet (CON group), low dose RJ (L-RJ group) diet (control diet + 125 mg/kg/day RJ), moderate dose RJ (M-RJ group) diet (control diet + 250 mg/kg/day RJ) and high dose of RJ (H-RJ group) diet (control diet + 500 mg/kg/day RJ). After weaning, male pups were continuously fed with freeze-dried RJ until the age of PNDs 35. The results revealed that, oral M-RJ (250 mg/kg/day) administration significantly (p < 0.05) increased the testis weight, the diameter of seminiferous tubule and the height of seminiferous epithelium of offspring mice at PNDs 14. However, high-dose RJ (500 mg/kg/day) decreased the diameter of seminiferous tubule but increased the height of seminiferous epithelium of male offspring (p < 0.05) at the same time point. Furthermore, oral M-RJ treatment significantly (p < 0.05) increased the testis weight and spermatogenesis at PNDs 21. Whereas, oral H-RJ treatment significantly (p < 0.05) reduced the diameter of seminiferous tubule and the height of seminiferous epithelium at PNDs 21. At PNDs 35, oral M-RJ treatment increased the testis weight, the diameter of seminiferous tubule and the level of FSH. While, high-dose of RJ reduced testis weight and size (diameter of seminiferous tubule and height of seminiferous epithelium), ratio of apoptotic germ cells and incomplete spermatogenesis collectively. In addition, sexual hormone secretions (FSH, LH, E2) were decreased after RJs treatment (L-RJ, M-RJ, H-RJ) at PNDs 21 respectively. In conclusion, the results concluded that oral administration of low and moderate doses of RJ could enhance the development of testis at neonate period until pubescent, whereas unfavorable adverse effects induced by high dose of RJ might remain.

ABSTRACT: Fruits and vegetables contain a variety of phytochemicals, including flavonoids, which have antioxidant and anticancer properties. The purpose of this study was to evaluate the antiproliferative effects and synergistic interactions of a variety of flavonoids in Hepa‐1c1c7, a mouse liver cancer cell line, and LNCaP, a human prostate cancer cell line. Aglycone flavonoids, such as quercetin, kaempferol, and naringenin (at 12.5 to 50 μM), inhibited cancer cell proliferation in both cell lines in a dose dependent manner without cytotoxicity. In contrast, glycone flavonoids (rutin, quercetrin, and naringin) did not inhibit cell growth. Significant synergistic antiproliferative effects were demonstrated in both cancer cell lines when flavonoids were provided in combination treatments. These results suggest that combinations of flavonoids, which are naturally present in whole fruits and vegetables, are more effective in cancer cell growth inhibition than the individual flavonoids.

BackgroundAs a prodrug of 5-fluorouracil (5-FU), orally administrated capecitabine (CAP) undergoes preliminary conversion into active metabolites in the liver and then releases 5-FU in the gut to exert the anti-tumor activity. Since metabolic changes of CAP play a key role in its activation, a single kind of intestinal or hepatic cell can never be used in vitro to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) nature. Hence, we aimed to establish a novel in vitro system to effectively assess the PK and PD of these kinds of prodrugs.MethodsCo-culture cellular models were established by simultaneously using colorectal cancer (CRC) and hepatocarcinoma cell lines in one system. Cell Counting Kit-8 (CCK-8) and flow cytometric analysis were used to evaluate cell viability and apoptosis, respectively. Apoptosis-related protein expression levels were measured using western blot analysis. A selective liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for cellular PK in co-culture models.ResultsCAP had little anti-proliferative effect on the five monolayer CRC cell lines (SW480, LoVo, HCT-8, HCT-116 and SW620) or the hepatocarcinoma cell line (HepG2). However, CAP exerted marked anti-tumor activities on each of the CRC cell lines in the co-culture models containing both CRC and hepatocarcinoma cell lines, although its effect on the five CRC cell lines varied. Moreover, after pre-incubation of CAP with HepG2 cells, the culture media containing the active metabolites of CAP also showed an anti-tumor effect on the five CRC cell lines, indicating the crucial role of hepatic cells in the activation of CAP.ConclusionThe simple and cost‑effective co-culture models with both CRC and hepatocarcinoma cells could mimic the in vivo process of a prodrug dependent on metabolic conversion to active metabolites in the liver, providing a valuable strategy for evaluating the PK and PD characteristics of CAP-like prodrugs in vitro at the early stage of drug development.

BackgroundNon-healing wounds have been a severe issue in the global healthcare system. Regrettably, royal jelly, a traditional remedy for various skin injuries, has not been widely applied in cutaneous wounds in clinical practice nowadays, which may be due to the confusion and the lack of knowledge about the efficacies of different types of royal jelly, the bioactive constituents, and the precise mechanisms underlying the wound repairing activity. Since the compositions and bioactivities of royal jelly are predominantly influenced by nectar plants, this study aims to explore the differences in the wound-healing properties of royal jelly produced by Apis mellifera L. during the blossom seasons of different floral sources, to provide guidelines for the future rational application of royal jelly in cutaneous wounds, and to promote the further discovery of wound repair-promoting substances.MethodsRoyal jelly samples were harvested during flowering seasons of Castanea mollissima Bl. (chestnut) and Brassica napus L. (rapeseed) in South China, from which hydrophilic and lipophilic fractions were extracted. The in vivo wound-healing potential was preliminarily assessed in Wistar rats’ excisional full-thickness wounds, followed by investigating the mechanisms of action through in vitro assays with human epidermal keratinocytes and LPS-stimulated inflammation in macrophages.ResultsThe results indicated that different royal jelly samples exhibited distinct wound-healing potential, in which Castanea mollissima Bl. royal jelly was more potent. It sped up wound closure between day 2 and day 4 to 0.25 cm2/day (p < 0.05), and could accelerate wound repair by enhancing the proliferative and migratory capabilities of keratinocytes by 50.9% (p < 0.001) and 14.9% (p < 0.001), modulating inflammation through inhibiting nitric oxide production by 46.2% (p < 0.001), and promoting cell growth through increasing the secretion of transforming growth factor-β by 44.7% (p < 0.001). In contrast, Brassica napus L. royal jelly could regulate inflammation by reducing the amount of tumour necrosis factor-α by 21.3% (p < 0.001).ConclusionsThe present study improves the application of royal jelly for curing difficult-to-heal wounds, in which the hydrosoluble extract of Castanea mollissima Bl. royal jelly promises the greatest potential. It also provides clues which may lead towards the identification of substances derived from royal jelly to treat wounds.

Royal jelly (RJ) is a natural product that reduces toxic effects and has anti-proliferative effects. The aim of the study is to increase the anticancer effect of Paclitaxel (PAX), which is used in cancer treatment, and to reduce its toxic effect with RJ in oral squamous carcinoma cells. Cytotoxicity tests of RJ and PAX substances were tested on healthy gingival HGF cells and their anti-proliferative effects on UPCI-SCC-131 cells with real-time cell analyzer (xCELLigence RTCA). Their anti-migratory properties were observed with wound healing assay. Glycolysis stress test was performed with Seahorse XFe24 to measure the glycolytic capacity. Total RNA-seq libraries were created and sequenced with NovaSeq 6000. Transcriptome profiles were created with bioinformatic analyses and functional enrichment analyses were performed. Results demonstrate that both RJ and PAX exhibit significant anti-proliferative effects against oral squamous cell carcinoma cells, as quantified by real-time cell analysis. Notably, RJ co-treatment mitigated PAX-induced cytotoxicity in healthy human gingival fibroblasts, suggesting a protective role against chemotherapy-associated toxicity. While both compounds inhibited cancer cell proliferation, PAX particularly displayed potent anti-migratory properties in wound healing assays, significantly impairing OSCC cell motility. Metabolic profiling revealed that the RJ-PAX combination therapy substantially reduced glycolytic capacity in OSCC cells, indicating disruption of their energy metabolism. Transcriptomic analysis identified downregulation of critical cell cycle regulators (MCM2, CDC25A, CCNE2) and DNA replication factors (RFC2, PCNA), along with modulation of MYC and E2F pathways, providing insights into the observed anti-cancer effects.