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The Cytotoxicity and Anticancer Effects of Propolis against the Oral Squamous Cell Carcinoma: In Vitro Study

BACKGROUND: Propolis has a wide range of therapeutic properties, including anti-cancer properties. METHOD AND MATERIALS: Two types of propolis were collected from two Iranian Provinces (Ardabil (KhalKhal) and Gilan (Qaleh Rudkhan)). The study was performed using ethanolic extract of propolis on Human dermal fibroblast cells and oral squamous cell carcinoma (OSCC) cell lines. The cell viability was evaluated by MTT assay. Cancer cell lines were assessed for the expression of matrix metalloproteinases (mmp) 2 and 9, caspase 3 and 9, BCL2-associated X protein ( bax ), and B-cell lymphoma 2 ( bcl-2 ). RESULTS: Increased concentrations of two types of propolis reduced cell viability but did not cause significant cytotoxicity. Real-time polymerase chain reaction indicated that the Khalkhal propolis sample produced more effects as the level of mRNA gene expression of bax in the Khalkhal propolis sample was increased in concentration dependent manner, and mRNA levels of bcl-2 , mmp-2, and mmp-9 were reduced. The activities of caspase 3 and 9 were increased in concentration dependent manner. CONCLUSION: Two types of propolis had cytotoxic effects on OSCC cell line, but the Khalkhal propolis was more effective than Gilan propolis.


Introduction
The most common type of cancer in southern Asia is the squamous cell cancer of the head and neck squamous cell cancer (HNSCC). This group of malignancies is considered the most prevalent cancer worldwide, and more than 550,000 new cases are diagnosed annually [1]. There are currently three main methods of treating HNSCC, namely, surgery, chemotherapy, and radiation therapy. Similar to other tumors, proliferating, invading, and metastasizing are key determining factors. An extreme microenvironment allows cancer cells to evade apoptosis [2].
The matrix metalloproteinase (mmp) is involved in invasion and metastasis of HNSCC. Basement membrane destruction and extracellular matrix degradation by mmp-2 and mmp-9 lead to tumor invasion [2]. As a result of developmental and genetic processes, cancer arises when cells proliferate excessively and evade apoptosis [3]. Therefore, they may invade and metastasize into adjacent tissues and organs [4].
Even though billions of dollars are spent on cancer research annually, there is still much to be learned about its development. There are different advancements in cancer treatment methods, but no desired strategies exist for curing these cancers. Since the side effects of drugs have become evident, people have resorted to alternative therapies, such as herbs and natural medicines. Natural compounds have been considered potential resources for discovering new drugs [5], [6], [7].
There has been intensive research demonstrating that the proteins in propolis can be toxic against numerous types of cancer cells, such as DU145 prostate cancer cells, PC-3 human prostrate cancer cells, HeLa cervical cancer cells, HT-29 colorectal cancer cells, MCF-7 breast cancer cells, Caco-2 colorectal cancer cells, and B16F1 melanoma cells [1]. Researchers have discovered that the phenolic compounds in propolis induce apoptosis [20], [25], [26], mitochondrial stress [27], and prevent cancer growth [20], [27], [28]. In this study, the various aspects of in vitro anticancer effects of two Iranian propolis were assessed by evaluating cell viability and apoptosis pathways on oral cancer cells.

Propolis extract preparation
At first, the samples were frozen at −20°C then were grounded in the cooled grinder. Approximately 10 g of raw propolis were extracted through diluting with a 10:1 volume of 70% ethanol solution in sealed tubes at room temperature for 3 days in the dark condition. After freezing the suspensions at −20°C for 24 h to eliminate wax and less soluble compounds, the samples were filtered using Whatman filter papers (No.1). Three freezing-filtration cycles were performed. The final filtrates were called ethanolic extract of propolis as they represented the balsam (Tincture) of propolis. In the next step, under reduced pressure at 64°C, rotary evaporators (IRE 3001) (MedZist company, Iran) were used to evaporate the solutions to near-dryness, and then they were freeze-dried.

Cell culture
The OSCC cell line was grown in monolayer method RPMI-1640 medium (Gibco, Germany) containing 10% FBS (Gibco, Germany), supplemented with an antibiotic solution of penicillin/streptomycin 1% (100 mg/L streptomycins and 100 U/mL penicillin, Gibco, Germany) (37 ˚C, 5% CO 2 , 72 h). HDF were cultured at 37°C in DMEM with 10 % FBS and 1% penicillin/streptomycin antibiotics. A DMEM containing 0.1% FBS was added to the cultures after confluence. A culture of HDF cells were washed twice with PBS and then dissociated with 0.25% Trypsin-EDTA at 37°C for 5 min. By adding 15% FBS to DMEM, the enzyme was neutralized. A Neubauer chamber was used to count HDF cells using microscopy. The HDF cells were ready to be used for the next step after 3-4 passages.

Cell viability assay (MTT assay)
MTT assay kit was used for cell toxicity. In first step, 1×10 4 cells/mL were cultured in 96-well plates (with two types of propolis at 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, and 500 µg/ml for 24, 48, and 72 h). Untreated cells of OSCC and HDF were considered as control groups in MTT test. Then, MTT solution was added to the cells (37°C, 5% CO 2 , 4 h). Finally, the DMSO (100 µL) was replaced with the culture medium, and absorbance was determined (570 nm, the ELISA reader (BMG Labtech, Germany). For further assays and according MTT results, we continued our research work using the propolis that had maximum cytotoxicity on cancer cells and/or the highest survival rate on normal cells.

Evaluation of caspase 3 and 9 activations
An assay kit of caspase-3 and -9 colorimetric measurements was applied on untreated cells of OSCC (control group) and treated cells (5×10 5 cells/mL) with 31.2, 62.5, and 125 µg/ml propolis for 72 h to measure the activity of caspase 3 and 9 as described in manufacturer instructions. As a final step, we measured the absorbance at 405 nm (ELISA Reader (BMG Labtech, Germany)) to determine the caspase activity in the sample.

Statistical analysis
The statistical analysis was done by SPSS software, Ver 26.0 (I.B.M. Corp. Released 2021. Armonk, NY), GraphPad Prism 9 (GraphPad Software, La Jolla California, USA). Routinely, an ANOVA test was performed on distributed data. In this study, p < 0.05 was considered statistically significant.

Cell viability of propolis on HDF
The cell viability of Gilan and Khalkhal propolis was measured on fibroblast cells (normal cells) by MTT assay. As shown in Figure 1a, the percentage of cell viability was related to the extract concentrations. The results showed that the highest cytotoxicity and the lowest cell viability were at 500 and 250 µg/ml (p < 0.001). In addition, the cell viability was higher than 99% at concentrations of ≤ 0.15.6 µg/ml. Cell viability at three concentrations of 125, 62.5, and 31.2 µg/ml (p < 0.001 and p < 0.05) was detected at more than 70% in 24 h of treatment. This cell survival rate was increased by more than 80% after 48 h (Figure 1b). These three concentrations indicated lower cytotoxicity compared to the 24 h duration. Figure 1c shows 72 h of treatment, the rate of cell viability was notably increased with cells adaptation during this time.

Cell viability of propolis on oral cancer cell line
The cytotoxicity of propolis extracts against OSCC cells was significantly high in 24 h (500 and 250 µg/ml) (p < 0.001) and led the survival rate of cancer cells to <10 and 20%, respectively. The survival rate of cancer cells was approximately between 50 and 85% in three concentrations of 125, 62.5, and 31.2 µg/ ml, and the cytotoxicity of Khalkhal extract was higher than Gilan extract (Figure 2a). The extract ≤15.6 µg/ ml had little effect on the cytotoxicity of cancer cells. However, by increasing the concentration and extending the time to 48 and 72 h, the cytotoxicity of the extracts was increased. The survival rate of cancer cells in 48 h was lower than 24 h (Figure 2b). In addition, this rate dropped after 72 h (Figure 2c). The concentrations of 500 and 250 µg/ml had the highest level of cytotoxicity at 3 times intervals (24, 48, and 72 h), and the cytotoxicity of the three concentrations (125, 62.5, and 31.2 µg/ml) (p < 0.001 and p < 0.05) was measured approximately between 50 and 85%. According to the obtained results, Khalkhal extract was selected as the desirable one with maximum cytotoxicity on cancer cells.

Expression level of genes that regulate apoptosis (bcl-2 and bax) and metastasis genes (mmp-2 and mmp-9)
Using RT-PCR, we measured the level of gene expression in oral cancer cells treated with propolis for the gene expression of mmp-2, mmp-9, bcl-2, and bax. A study showed that if the concentration of propolis was increased, there would be an increase in the amount of bax (proapoptotic mRNA) expressed. This is consistent with the fact that bax was a dosedependent factor, with the highest level of expression being detected at 125 µg/ml (p < 0.001). As a contrast, a higher concentration of propolis decreased mRNA expression levels of bcl-2 (antiapoptotic mRNA) in response to a higher concentration of propolis. As shown in Figure 3, the lowest expression was observed at 125 µg/ml (p < 0.001). The level of expression of the bcl-2 gene occurred at concentrations of 125, 62.5, and 31.2 µg/ml, respectively, and was around 0.85, 0.79, and 0.75 fold of that of bcl-2. As shown in Figure 3, mmp-2 and mmp-9 expression levels were reduced while concentrations increased, indicating that the expression levels were dose-dependent in response to the concentration increase. According to the results of these experiments, the expression levels of these genes were reduced from 31.2 µg/ml  (p < 0.05) to 62.5 µg/ml (p < 0.01) compared to the control, and the lowest expression level was found at 125 µg/ml (p < 0.01).

Caspase 3 and 9 activities
The activities of caspase 3 and 9 were measured in OSCC to determine the apoptosis. As regards Figure 4a, the caspase -3 activity was remarkably increased after the treatment at concentrations of 62.5 and 125 µg/ml (p < 0.01) compared to the control group. In addition, caspase 9 was similar to caspase 3 in these two concentrations (Figure 4b). The results indicated that the activities of caspase 3 and 9 were increased by increasing the concentration of the extract. The highest level of caspase activity was at 125 µg/ml (p < 0.01), and the lowest level of caspase 3 and 9 activities was at the concentration of 31.2 µg/ml.

Discussion
Traditional medicine has been supplanted by the use of natural products to treat diseases rather a a https://oamjms.eu/index.php/mjms/index than conventional treatments. Today's biomedical applications of natural products cover a broad spectrum of diseases that can be treated with them [31]. The ninth most prevalent type of malignancy is head and neck cancer worldwide, with a high mortality rate in developing countries [32], [33], [34], [35]. These tumors behave aggressively and can cause harmful diseases and form early local lymphoma and distant metastasis in the future [32], [36].
Propolis is a natural material that bees use to build and maintain their colonies [31]. There are a number of biological activities associated with the use of propolis, including anti-tumor properties, which make it a widely used bee product [20]. It has been found that several types of propolis have been shown to be effective in inhibiting breast cancer cell proliferation [37], lung cancer [38], and melanoma [39].
Jiang et al. in 2020 studied the anticancer properties of Chinese propolis in a similar manner to the observations we made here that was altered by the expression of proteins involved in apoptosis [40]. It has also been reported that the ethanol extract of Chilean propolis has an antiproliferative activity against human mouth epidermoid carcinoma cells in another similar study was demonstrated by Russo et al. [41].
To assess the effect of propolis on proliferation and apoptotic processes in vitro in the first instance, a highgrade squamous cell carcinoma cell line, one of the most commonly used to model tumors, was used in this study to evaluate the efficacy of propolis on cell proliferation and apoptosis. Specifically, it has been found that Gilan and Khalkhal propolis both displayed antiproliferative activity against OSCC. This discovery was a result of measuring the cytotoxic activity of Iranian propolis in vitro. Human fibroblast cell lines were observed to have the lowest level of cytotoxic effects. Both propolis extracts showed great potential as anti-proliferative agents to inhibit the proliferation of oral cancer cells, according to the results of the MTT test. In spite of the fact that Khalkhal propolis was found to inhibit oral cancer cells more effectively than Gilan, it was selected for further research based on its superior results.
The uncontrolled proliferation of cancerous cells is accompanied by a loss of resistance to apoptosis. Consequently, in the future, propolis ability to induce apoptosis may be considered as a potential characteristic for developing chemotherapeutics agent against cancer [42]. According to the results, the activities of caspases 3 and 9 increased in a concentration-dependent manner when Khalkhal propolis were used indicating that caspase pathways that control apoptosis are influenced by propolis.
Furthermore, the results of this study showed that by increasing the concentration of propolis the level of bax gene expression increased and the level of bcl-2 gene expression decreased in OSCC cells, which are the genes that are responsible for the internal process of apoptosis, by a dose-dependent mechanism [43]. A high level of expression was observed in the bax gene when the concentration was 125 µg/ml. As a result of the bax/bcl-2 pathway, several pharmaceutical agents can have a detrimental effect on antitumor proliferation and activity [44]. It was evident that bcl-2 levels were decreasing and bax levels were increasing, indicating the dysregulation of two key molecules (bcl-2 and bax) and the activation of caspases 3 and 9 in the cells [45].
As the concentration of propolis was increased, it was found that there was a decrease in the expression levels of mmp-2 and mmp-9 genes. We found that 62.5 µg/ml of mmp-2 and 31.2 µg/ml of mmp-9 gene expression were decreased, respectively. After treatment at concentrations of 125 and 62.5 µg/ml, the caspase 3 and 9 activities in oral cancer cells were significantly increased, when compared to the caspase 3 and 9 activities of normal cells.

Conclusion
These results showed two types of propolis had cytotoxic effects on OSCC cell line, but the Khalkhal propolis was more effective than Gilan propolis and they can be considered as an appropriate candidate for preventing oral cancer cells.