Use of Stingless Bee Propolis and Geopropolis against Cancer—A Literature Review of Preclinical Studies

Cancer is one of the major maladies affecting humankind and remains one of the leading causes of death worldwide. The investigation of the biological activities of stingless bee products, especially propolis and geopropolis, has revealed promising therapeutic properties, especially in the research on new antineoplastic agents. This literature review of preclinical trials, involving biological assays of antitumor activity and identification of the chemical composition of propolis and geopropolis of stingless bee species, describes the cytotoxicity in tumor lineages (breast, lung, ovarian, liver, mouth, pharynx, larynx, colon, stomach, colorectal, cervix, kidney, prostate, melanoma, human glioblastoma, canine osteosarcoma, erythroleukemia, human chronic myelocytic leukemia, and human promyelocytic leukemia) of propolis and geopropolis of 33 species of stingless bees. The chemical composition of propolis and geopropolis was identified, indicating that these belong to the chemical classes of phenolic acids, flavonoids, coumarins, benzophenones, anthraquinones, alkaloids, terpenes, steroids, saponins, fatty acids, and carbohydrates and are possibly responsible for the cytotoxicity in tumor cells. Apoptosis was one of the main mechanisms of cytotoxicity of extracts and substances isolated from stingless bee products. Although the results found are encouraging, other preclinical studies and clinical trials are essential for the discovery of new anticancer agents.


Introduction
Stingless bees, also known as meliponines, live in colonies and are characterized by having atrophied stingers [1]. They are social insects of great diversity and wide geographic distribution, occupying almost all of Latin America and Africa, besides southeast Asia and northern Australia [2][3][4]. There are more than 600 described species, and they are spread across all tropical and subtropical areas of the globe [5]. Among the genera with the largest number of known species are Plebeia, Trigona, Melipona, Scaptotrigona, and Trigonisca [6].
Meliponines make great contribution to environmental conservation, as they perform pollination of native plant species and contribute to a reduction in deforestation and environmental damage [4,7]. In addition, they are commercially known for their role in the production of natural products, such as honey, wax, royal jelly, propolis, and geopropolis and accumulation of pollen [3,4,8,9].
Propolis is a mixture of salivary secretions and plant resins collected by bees and is produced to seal the hive and prevent the entry of air and invading insects, besides having antimicrobial activity, protecting the colony from diseases [10,11].
Some meliponin species mix propolis with clay or soil. The result of this mixture is a resinous material more rigid than propolis. Despite the differences in the formation of both products, geopropolis has similar functions to propolis regarding the protection of the hive [12].  [30] Human epidermoid laryngeal carcinoma (HEp-2)

Ehrlich Tumor
Ehrlich tumor is a model of mammary adenocarcinoma of female mice that is used in the evaluation of antitumor drugs [45]. According to Araújo et al. [28], animals inoculated by Ehrlich tumor cells in the paws treated with the hydroethanolic propolis extract produced by Scaptotrigona aff. postica at doses of 0.5 and 5 mg/kg showed significant inhibition in tumor development from the 6th day after inoculation. There was a significant increase in the number of cells in the spleen and bone marrow of animals treated with the extract in relation to the control, showing that these doses induce an increase in the production of peripheral immune cells and precursors.

Glioblastoma
Glioblastomas are the most frequent and aggressive primary brain tumors, classified, according to the World Health Organization (WHO), as grade IV due to their malignancy [46]. Borges et al. [22] showed an in vitro antiproliferative effect of the ethanolic propolis extract produced by Scaptotrigona sp. against human adult glioblastoma cell lines (U251 and U343), with a decrease in cell proliferation by 48% to 59% in 72 h at a dose of 2 mg/mL, as well as a reduction in colony formation. They also observed that the combination of the propolis extract (2 mg/mL) with temozolomide (50 µM) had a synergistic antiproliferative effect, reducing cell proliferation to less than 20%. This association showed superior results related to the extract and drug when evaluated separately.

Erythroleukemia
Erythroleukemia is a rare form of acute myeloid leukemia characterized by the proliferation of erythropoietic elements in the bone marrow; erythroblasts with foreign, lobulated nuclei; and pathological myeloblasts in the peripheral blood [47].
The mechanism of in vitro cytotoxicity of the ethanolic propolis extract produced by Melipona orbignyi against erythroleukemia cells (K562) was elucidated by Campos et al., [33], demonstrating that the cell viability decreased to less than 25% at the concentration of 500 µg/mL and that necrosis was the predominant form of cell death of cells treated with propolis. The data are interesting with regard to therapy against tumor cells resistant to cell death by apoptosis, which usually occurs with the use of conventional chemotherapy.
Bonamigo et al. [31] observed the in vitro cytotoxic activity of ethanolic propolis extracts of Scaptotrigona depilis and Melipona quadrifasciata anthidioides against erythroleukemia cells (K562) as the concentration increased with cell growth of 32.6% ± 3.2% and 21.2% ± 4.1%, respectively. At the concentration of 500 µg/mL, by flow cytometry using the annexin and propidium iodide markers, after 24 h of treatment, the ethanolic propolis extract produced by Scaptotrigona depilis promoted death by necrosis in 52.9% ± 4.1% of the cells and death by late apoptosis in 12.1% ± 0.6% of the cells. The ethanolic propolis extract produced by Melipona quadrifasciata anthidioides promoted, after 24 h of treatment, death by necrosis in 57.5% ± 3.8% of the cells and death by late apoptosis in 19.4% ± 1.6% of the cells.

Melanoma
Melanoma skin cancer originates from normal pigment cells called melanocytes. These melanocytes produce melanin, the pigment responsible for giving color to the skin and which protects the body from damage by the sun's ultraviolet rays. Similar to other cells in the body, melanocytes can transform into cancer cells and when this transformation occurs, the result is the development of melanoma [48].
According to Cisilotto et al. [32], in vitro cytotoxicity of the hydroethanolic propolis extract produced by Scaptotrigona bipunctata against melanoma cells (SK-MEL-28) occurs by cell death by apoptosis, also evidenced in the accumulation of reactive oxygen species (ROS), reduction of mitochondrial membrane potential (∆ψm), and induction of decreased levels of Bcl-2 proteins (antiapoptotic proteins) and AKT-3 (cell-growth-related protein). The extract also causes a decrease in migration and invasion of melanoma cells.
The combination of the extract (30 µg/mL) with the antineoplastic vemurafenib (15 µM) against melanoma cells demonstrated a synergistic effect, showing a cytotoxic effect, suggesting reduced resistance and increased cell death in cells with BRAF (protooncogene regulator of cell function) mutation.

Osteosarcoma Cells
Osteosarcoma is a primary malignant bone tumor that can occur in any age group but mainly affects children, adolescents, and young adults and can also occur in animals [49]. Cinegaglia et al. [23] demonstrated that the hydroethanolic geopropolis extract produced by Melipona fasciculata exerts an in vitro cytotoxic effect against canine osteosarcoma cells in a dose-and time-dependent manner (24, 48, and 72 h). This was also evidenced by morphological analysis, showing the sensitivity of these cells to the extract.

Laryngeal Carcinoma
Laryngeal carcinoma is among the most common head and neck cancers, accounting for about 2.4% of all newly diagnosed cases and 0.7% of all cancer-related deaths occurring worldwide/year [50].
Studies by Bartolomeu et al. [25] demonstrated in vitro cytotoxic activity of the hydroethanolic geopropolis extract produced by Melipona fasciculata against the growth of HEp-2 cells (larynx epidermoid carcinoma) and significant reduction in cell migration after 24 h of treatment with the extract. In the same study, the combination of the extract (25 mg/mL) with doxorubicin (1 mM) significantly affected the sensitivity of HEp-2 cells after 72 h, promoting apoptosis of this tumor lineage, presenting morphological changes, such as cytoplasmic membrane fragmentations (apoptotic bodies), loss of membrane, and integrity. Araújo et al. [30] also verified a significant decrease in cell viability observed after 6 h of incubation with 50 and 100 µg/mL of extract, and after 24, 48, and 72 h of incubation, there was a significant decrease in cell viability from 25 to 100 µg/mL.

Ovarian Adenocarcinoma
Ovarian cancer is the second-most-common gynecological neoplasm, second only to cervical cancer. Almost all ovarian neoplasms (95%) are derived from the epithelial cells lining the ovary [51].
Da Cunha et al. [24] demonstrated the in vitro antiproliferative activity of the ethanolic geopropolis extract produced by Melipona scutellaris against ovarian adenocarcinoma with a multidrug-resistant phenotype (NCI/ADR-RES) and ovarian adenocarcinoma (OVCAR-03), with an IC 50 range from 11.93 to 23.92 µg/mL. The total growth of these strains was inhibited at low concentrations when compared to normal strains (keratinocytes and normal murine fibroblast with IC 50 of 43.20 and 52.73 µg/mL, respectively), thus demonstrating the selectivity of the ethanolic extract geopropolis for tumor cells.
The in vitro cytotoxicity of the hydroethanolic geopropolis extract produced by Melipona fasciculata (specifically at the concentration of 62.5 µg/mL, the highest exposed) against ovarian cancer lineage (A2780) was demonstrated through the visualization of several alterations in the morphology of these cells, such as cell rounding and shrinkage, presenting decreased density, standing out from the substrate. These characteristics are suggestive of cell death induced by the hydroethanolic extract mediated by apoptosis. This fact was confirmed with the increased expression of cleaved caspase-3 and of PARP, which is poly(ADP-ribose) polymerase cleaved by the Western blotting technique [8].

Colorectal Adenocarcinoma
Colorectal cancer (CRC) accounts for 9% of all cancers worldwide, making it the second-most-common cancer in women and the third-most-common cancer in men. Adenocarcinoma arising from the rectal and colonic epithelium accounts for 90% of the CRC cases [52][53][54].
Umthong et al. [41] observed decreased cell viability for 23% of the colon cancer cells (SW620) treated with the aqueous propolis extract produced by Trigona laeviceps, and morphological changes were visualized in these cells, such as chromatin condensation, DNA fragmentation, internucleosomal DNA degradation, cell shrinkage, membrane blebbing, nuclear pyknosis, and apoptotic body formation, events typical of the apoptosis process.
Choudhari et al. [26] suggested that the hydroethanolic propolis extract produced by Trigona spp. exhibits in vitro cytotoxicity against human colon adenocarcinoma tumor cell lines (HT-29) as the incubation time and concentration were increased. The IC 50 value found was 250 µg/mL. Morphological changes in cells demonstrated typical changes of apoptosis, i.e., apoptotic membrane blebbing and detachment of cells.

Carcinoma of the Pharynx
Pharynx cancer represents about 25% of malignant tumors affecting this area and 2% of all malignant diseases. The most prevalent histological type, in more than 90% of the patients, is squamous cell carcinoma [51].
Utispan et al. [36] evaluated the in vitro cytotoxicity of three fractions derived from the dichloromethane propolis extract produced by Trigona sirindhornae against cell lines derived from primary pharyngeal lesions (HN30) and lymph node metastases (HN31), both from the same patient. The fractions significantly decreased the viability of both cell lines at concentrations of 50 to 200 µg/mL.

Gastric Adenocarcinoma
Stomach cancer is also called gastric cancer. The adenocarcinoma type accounts for about 95% of the stomach tumor cases and mostly affects men around 60-70 years of age. About 65% of the patients are over 50 years old [51].
Gastric cancer tumor lines (AGS, MKN-45, NUGC-4, and MKN-74) were treated in vitro with the ethanolic propolis extract produced by Tetragonula biroi by Desamero et al. [29], which revealed a proportional reduction in cancer cell proliferation as a function of higher concentration and longer incubation times, showing an IC 50 range from 39 to 925 µg/mL after 72 h. Data confirmed in an in vivo assay using an animal model mimicking a gastric adenocarcinoma of a differentiated type indicated that after treatment with an ethanolic extract, there was a remarkable regression of macroscopic mucosal elevation, corresponding histologically to a substantial reduction in the pyloric mucosal thickness and infiltration of lymphocyte T.
In view of this, the evaluation of the antitumor activity of stingless bee products showed encouraging results. The predominance of in vitro studies to the detriment of animal assays was observed. In addition, the mechanism of cell death was not so explored and detailed in some studies. Sparse data from anticancer trials of stingless bees propolis and geopropolis demonstrate the need for these trials to prove the efficacy and safety of these products.

Chemical Identification of Antitumor Extracts from Propolis and Geopropolis
The chemical composition of propolis and geopropolis from stingless bees is shown in Table 2. The main classes of compounds identified are phenolics compounds (comprising phenolic acids, flavonoids, coumarins, and benzophenones) terpenes, steroids, alkaloids, fatty acids, and sugars. Qualitative approaches were used to define the classes of compounds; and analytical techniques, such as high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS), liquid chromatography coupled to mass spectrometry (LC/MS), and gas chromatography coupled to spectrometry (GC/MS), were employed to identify the compounds.
β-amyrin, a compound identified in samples from stingless bees Tetrigona apicalis, Scaptotrigona bipunctata, Melipona quadrifasciata anthidioides, and Melipona fasciculata, was the subject of a study by Wen et al. [55] that highlighted the significant cytotoxic activity of this substance against HepG2 (hepatocellular carcinoma) cells. The cytotoxic effects were justified by the induction of apoptosis and the arrest of the G2/M cycle in a dose-dependent manner.
Cinnamic acid, a chemical compound identified in Melipona orbignyi and Tetragonisca fiebrigi samples, was explored by [56], who observed a reduced cell proliferation rate and a significant change in nuclear cytoplasmic ratio of nasopharyngeal carcinoma (NPC) after treatment with cinnamic acid. In addition, the treatment partially restored normal cell morphology and drove cell differentiation toward a benign phenotype and revealed cell death by apoptosis.
Ma et al. [57] showed that the compound taraxerone (identified in Tetrigona apicalis and Melipona fasciculata samples) exerts potent antiproliferative effects against A-549 (lung adenocarcinoma) in a strong dose-dependent and time-dependent manner. Furthermore, fluorescence microscopy revealed that taraxerone is able to induce cell shrinkage and chromatin condensation, recorded features of apoptosis.
p-Coumaric acid, identified in samples of species Scaptotrigona bipunctata, Melipona quadrifasciata anthidioides, and Tetragonisca fiebrigi, was studied by Sharma et al. [58], who observed significant inhibition of the proliferation of A375 (human melanoma) and B16 (mouse melanoma) cells after treatment with p-coumaric acid, as well as morphological changes in these cells after 48 h of treatment with different concentrations of the compound. They found increased levels of cleaved caspase-3 and cleaved caspase-9 in A375 and B16 cells, indicating that apoptosis is regulated by the family of caspases.
Artepillin C, present in the ethanolic extract produced by Scaptotrigona bipunctata, exhibited dose-and time-dependent cytotoxic effects on prostate cancer (HSC-3) cell lines. Flow cytometry analysis showed that 22% of the HSC-3 cells untreated with the compound suffered spontaneous cell death, while 77.32% of the cells were killed in response to the highest dose of artepillin C at 72 h. The antitumor activity of artepillin C is mediated by one of the following mechanisms: induction of cell cycle arrest in cancer cells, inhibition of angiogenesis, and inhibition of the oncogenic PAK1 signaling cascade [59].
Gallic acid, identified in the hydroethanolic extract geopropolis of Melipona mondury and in the ethanolic extract propolis of Melipona quadrifasciata quadrifasciata, inhibited the progression of prostate cancer cells (PC-3), was a mitochondrial potential enhancer (∆Ψm), and increased the number of apoptotic cells and DNA fragmentation. A Western blot analysis revealed negatively regulated expression of histone deacetylases (HDAC) 1 and 2, reported in various cancers, leading to the positive regulation of acetyl-p53 expression at the protein level, subsequent to the negative regulation of cell-cycle-related gene expression, i.e., proliferating cell nuclear antigen (PCNA) and cyclin D1 and E1; positively regulating the expression of the cell cycle arrest gene p21; and regulating the expression of genes related to the intrinsic apoptosis pathway, such as Bax, Bcl-2, cleaved caspase-3, and poly(ADP-ribose) polymerase [60].
Apigenin, presented in the ethanolic extract produced by Melipona quadrifasciata anthidioides propolis, inhibited proliferation, prevented cell cycle progression, and promoted apoptosis in both ovarian cancer cells (SKOV3) and cisplatin-resistant cells (SKOV3/DDP). In addition, apigenin reduced mitochondrial transmembrane potential and elevated caspase-3/cleaved caspase-3 and Bax/Bcl-2 ratios in both cell types. Quantitative reverse transcription PCR and Western blotting results demonstrated that apigenin significantly downregulates Mcl-1 transcription and translation levels in SKOV3 and SKOV3/DDP cells, which is responsible for its cytotoxic functions and chemosensitizing effects [61].

Isolation of Compounds
Compounds isolated from propolis and geopropolis of stingless bee species that were tested against tumor cell lines are shown in Figure 2.

Isolation of Compounds
Compounds isolated from propolis and geopropolis of stingless bee species that were tested against tumor cell lines are shown in Figure 2.
Eight compounds were isolated from the hydroethanolic geopropolis extract produced by Melipona scutellaris by [62] and tested in vitro against two colon cancer cell lines (COLO205 and KM12). The coumarins mammeisin (2) and mammein (3) (Figure 2) showed a higher average percentage of growth inhibition, of 56% and 83%, respectively. The mechanism of cytotoxicity of the extract against tumor cells was not investigated by the authors.
Vongsak et al. [27] tested the in vitro cytotoxicity of the methanolic propolis extract produced by Tetragonula pagdeni against oral squamous cell carcinoma (KB), hepatocellular carcinoma (HepG2), colon adenocarcinoma (CaCo-2), and melanoma (SK-MEL-28) cell lines and observed variation in IC50 values from 33. 38 to 80.81 μg/mL. In contrast, the IC50 value of normal human fibroblast cells was 228.75 μg/mL, demonstrating greater selectivity of propolis metabolites toward tumor cells. From the said extract, γmangostin (4) and α-mangostin (5) were isolated by preparative thin layer chromatography and their structures identified by spectrometric methods (NMR 1 H and 13 C) (Figure 2). These substances expressed comparable cytotoxicity to the positive control, Kustiawan et al. [38][39][40] submitted the methanolic propolis extract produced by Trigona incisa to chromatographic fractionation, isolating, among other compounds, cardol (1), which was identified by NMR spectrometric analysis. Biological cytotoxicity tests indicated that compound 1 induces cell death by apoptosis in the initial incubation period (≤6 h) and modulates cell cycle arrest in the G1 subphase in SW620 cells (colon cancer cells). Kustiawan et al. [40] observed that compound 1 promotes changes in cell morphology in SW620 cells; a significant increase in caspase-3 and caspase-9 activities; and cleavage of pro-caspase-3, pro-caspase-9, and PARP.
Eight compounds were isolated from the hydroethanolic geopropolis extract produced by Melipona scutellaris by [62] and tested in vitro against two colon cancer cell lines (COLO205 and KM12). The coumarins mammeisin (2) and mammein (3) (Figure 2) showed a higher average percentage of growth inhibition, of 56% and 83%, respectively. The mechanism of cytotoxicity of the extract against tumor cells was not investigated by the authors.
Vongsak et al. [27] tested the in vitro cytotoxicity of the methanolic propolis extract produced by Tetragonula pagdeni against oral squamous cell carcinoma (KB), hepatocellular carcinoma (HepG2), colon adenocarcinoma (CaCo-2), and melanoma (SK-MEL-28) cell lines and observed variation in IC 50 values from 33. 38 to 80.81 µg/mL. In contrast, the IC 50 value of normal human fibroblast cells was 228.75 µg/mL, demonstrating greater selectivity of propolis metabolites toward tumor cells. From the said extract, γ-mangostin (4) and α-mangostin (5) were isolated by preparative thin layer chromatography and their structures identified by spectrometric methods (NMR 1 H and 13 C) (Figure 2). These substances expressed comparable cytotoxicity to the positive control, doxorubicin, against these tumor cell lines, with IC 50 values from 2.84 to 15.12 µg/mL and 1.63 to 7.07 µg/mL for (4) and (5), respectively.
The ethanolic propolis extract produced by Trigona minor was subjected to partionation with solvents of different polarities. The n-hexane extract showed the most potent preferential cytotoxicity against human pancreatic cancer cells (PANC-1), with an IC 50 value of 3.6 µg/mL. Further separation and purification of this fraction led to the identification of 16 triterpenoids, most notably 23-hydroxyisomangiferolic acid B (10) and 27-hydroxyisomangiferolic acid (11), which showed stronger preferential cytotoxicity, with IC 50 values of 4.3 and 3.7 µM, respectively. Subsequently, compound (10) was evaluated for its effect on the cell morphology of PANC-1 cells. When these cells were treated with 5 µM of compound (10) for 24 h, the PANC-1 cells changed morphologically and gave a unique red fluorescence, indicating the apoptosis process. Furthermore, in the colony formation assay in PANC-1 cells, compound (10) significantly inhibited colony formation in a concentration-dependent manner [64].
Three more substances were isolated from the ethanolic propolis extract produced by Trigona minor, with emphasis on 2 hydroxyl-6-(14 Z-nonadecenyl) benzoic acid (12), which showed preferential cytotoxicity against the human pancreatic cell line PANC-1, with an IC 50 value of 2.4 µM. The cytotoxicity of this compound is related to the substituents on the alkenylphenol ring. The presence of the carboxylic acid group or one more hydroxyl group appears to increase the activity [65].
Chromatographic separation of the ethyl acetate propolis extract produced by Lisotrigona furva led to the isolation of five cycloartane-type triterpenes, which were tested on lung cancer cell lines (LU-1) and breast cancer cell lines (MCF-7), most notably mangiferolic acid (14), which showed an IC 50 value of 13.33 and 62.85 µg/mL, respectively [66].

Materials and Methods
This review covered ScienceDirect, Scopus, Pubmed, and Scielo databases, as updated on October 2021. The references obtained in the review were consulted and analyzed in detail. The key words employed alone or in combination in the literature review were propolis, geopropolis, stingless bee, cancer, cytotoxicity, and antiproliferative. Articles on propolis from sting bee species (i.e., Apis mellifera) were excluded from the search.

Conclusions
The propolis and geopropolis extracts from stingless bees analyzed in this revision had diverse and complex chemical compositions, and their constituents belong to the chemical classes of phenolic acids, flavonoids, coumarins, benzophenones, terpenes, steroids, alkaloids, fatty acids, and sugars. The extracts and isolated substances showed selective cytotoxicity against different tumor cell lines, suggesting an antineoplastic potential and synergism with standard chemotherapeutics. Although the preliminary results of propolis and geopropolis from stingless bees are encouraging, further preclinical studies and clinical trials are essential to validate the safety, efficacy, and effectiveness of the products from these species in cancer therapy.