Effect of Dioscorea extract on Bax and Bcl-2 gene expression in MCF-7 and HFF cell lines

Abstract

Background

In cancer cells, the balance between proliferation and apoptosis is disturbed. There is a direct relationship between gene expression and the process of apoptosis. The two genes involved in apoptosis are Bax and Bcl-2, and it is now well established that some plant compounds can alter the expression of genes. The aim of this study is to determine the rate of change in the expression of these genes in the cell line MCF-7 treated with Dioscorea extract for 24, 48 and 72 h. For this purpose, the plant extract was prepared by Soxhlet method and diluted in different concentrations. MCF-7 and HFF cell lines were treated in three replicates with different concentrations of the extract at intervals of 24, 48, and 72 h. To evaluate the toxicity of the extract, the MTT assay was performed and the IC₅₀ value was calculated. Both cell types were cultured at IC₅₀ concentration with three treatments and three replicates. RNA extraction, cDNA synthesis and real-time PCR were then performed. Flow cytometry was performed to further confirm apoptosis.

Results

MTT results showed that 72 h treatment with Dioscorea extract in IC₅₀ concentration had the greatest effect on the death of MCF-7 cancer cells, while the cells of the control cell line remained healthy. The results of the study of gene expression changes showed that when treated with the plant extract for 24 h, the increase in Bax gene expression and the decrease in Bcl-2 gene expression were not statistically significant. At 48-h treatment, the decrease in Bcl-2 expression was not statistically significant, whereas the increase in Bax expression, which was 2.1 times, was statistically significant. When treated with the plant extract for 72 h, Bax expression increased 2.72 times and Bcl-2 gene expression decreased 0.67 times. Flow cytometry showed that 72-h treatment with plant extract at a concentration of 438.35 µg/ml was the most effective treatment for MCF-7 cancer cell death.

Conclusions

The expression ratio of Bax gene to Bcl-2 is equal to 4.06, which indicates the induction of more apoptosis by treatment with plant extract.

Background

Breast cancer accounts for about one-third of all cancers that affect women. Worldwide statistics show that 2.3 million new cases of breast cancer are diagnosed each year. It is the leading cause of death after lung cancer [1] and that too in Iranian women [2, 3]. In breast cancer, cancer cells enter in the lymphatic vessels. They migrate to the lymph nodes and spread to other parts of the body through the lymphatic system [4]. Oncogenes were the first tumorigenic genes to be identified. In fact, proto-oncogenes regulate normal cell division, and mutation turns them into oncogenes [5]. Bcl-2 is located on chromosome 18 and encodes a 24-kDa protein found in the nuclear membrane, endoplasmic reticulum, and mitochondrial membrane. The Bcl-2 gene is involved in the inhibition of apoptosis and increases cancer cell survival. In contrast, expression of the Bax gene triggers apoptosis and death of cancer cells [6]. Studies have shown that plants are effective in treating gastrointestinal cancer, skin cancer, oral cancer, and breast cancer [7]. Plants play an important role in the prevention and treatment of cancer. In recent years, the use of natural plant products has increased due to the side effects of chemotherapeutic agents [8, 9]. Different species of Dioscorea are used as food and medicine. The substances contained in this plant such as flavonoids, diosgenin, dioscorin, tannins, saponins, and phenols are valuable foods with beneficial effects [10]. A study was conducted on the presence of diosgenin in white yam. This is because the tubers are the staple food of hundreds of thousands of indigenous people in Africa, especially in Nigeria. [11]. The results of another study conducted in Japan to evaluate the effect of Dioscorea extract on reducing cancer risk were satisfactory. The antioxidants in Dioscorea can scavenge free radicals [12]. A study shows that ethanolic extracts of D. rotundata can be used to effectively treat breast cancer tumors [13]. In 2021, a study was conducted on the hemolytic activity of the extract and the cytotoxic effect of D. bulbifera on MDA-MB-231 cell line. The results of the 24-, 48-, and 72-h treatments showed that an increase in estrogen levels increased the risk of breast cancer. It was also shown that MDA-MB-231 was reduced by lowering serum estrogen levels [14]. The results of a study on a variety of chemicals from D. bulbifera suggest that this plant may be preferred for contraceptive capsules and pills to cure diseases [15]. The results of another study showed that Dioscorea is converted into medicines and used as a valuable diet and prevention of diseases [16]. In 2019, Li showed the beneficial clinical effect of a modified Dioscorea tablet in the treatment of diseases associated with chronic cerebral hypoperfusion [17]. In 2018, a study was conducted on the effect of Dioscorea Opposit extract on MCF-7 cell line. This Chinese herb has no side effects and could become a new drug for the treatment of premenopausal syndrome [18]. In 2010, a study was conducted on the effect of dioscorealide B extract on MCF-7 cell line and the changes of Bcl-2 and Bax gene expression. The results showed that dioscorealide B treatment decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pre-apoptotic proteins Bax and Bak. As a result, caspase 9 and 7 were activated, respectively. Therefore, this study provided a molecular basis for the further development of dioscorealide B as a new chemotherapeutic agent for the treatment of breast cancer [19]. In 2010, another study was conducted on the effect of caspase-9 inhibitor on MCF-7 cell line. The results showed that treatment with dioscorealid B extract increased the gene expression of Bax and decreased the gene expression of Bcl-2 [20]. In 2016, a study was conducted on the effect of D. membranacea extract on apoptosis of L929 cancer cell line. The L929 cancer cell line increases Bax expression and decreases Bcl-2 gene expression after treatment with the extract. Thus, it is involved in the apoptosis of L929 cancer cells [21]. In 2019, a study was conducted on MDA-MB -231 and MCF-7 cells with two treatments of 24 and 48 h. In this study, DiosR and DiosB containing diosgenin were applied to cancer cells. Comparison of 24- and 48-h treatments with DiosB and DiosR showed that 48-h treatments with plant extracts were more effective than 24-h treatments in the MDA-MB-231 cell line. In addition, both extracts were more effective in the cancer cell line MCF-7 than in the cell line MDA-MB -231. The greatest effect on MCF-7 cells compared to MDA-MB -231 was seen with the 48-h treatment [22]. In 2021, Mainasara et al. showed that the methanolic extract of D. bulbifera exhibited ant proliferative activity compared with a standard drug. The antioxidant activity of the methanol, ethyl acetate, and hexane extracts of the plant was significant compared to ascorbic acid, indicating the potential of the leaves of this species as natural antioxidants. Thus, the ant proliferative activities shown by the leaf extracts confirm the ancient use of this plant against various breast cancers [14].

Since it is important to investigate the effect of phytochemicals on cancer cell growth, in the present study, Dioscorea extract was investigated on MCF-7 cell line as cancer cell line and HFF fibroblast cell line as control cell line.

Methods

Dioscorea was obtained from China and MCF-7 and HFF cell lines from the National Genetic Resources Center of Iran. For every 10 g of plant powder, 100 cm³ of 70% ethanol was used as solvent. The solution was boiled at 90 °C for 10 min and filtered through a sieve. The extract was then transferred to the micro tube and DMEM culture medium was added. MCF-7 Cells were cultured with DMEM and stored in a 37° CO₂ incubator. When the cell density reached 80%, cell passage was performed. MCF-7 cells at a density of 20,000 cells and HFF cells at a density of 20,000 cells were cultured in 96-house plates. The Dioscorea extract plate was then added to the first row, and dilution was performed in each row with each pipette. These steps were performed for 24, 48, and 72 h with three replicates. The concentrations used in this study are 2000, 1000, 500, 250, 125, 62.5, 31.25 µg/ml, respectively.

MTT assay technique

The MTT method is based on mitochondrial activity in living cells. This method is based on the reduction and disruption of yellow crystals (tetrazolium) by the enzyme mitochondrial succinate dehydrogenase and the formation of purple crystals.

After the treatment time, 20 μl of MTT dye was added to the wells containing the cells and extract, and the plate was incubated for 4 h. Then, 100 μl of isopropanol reagent was added to the wells and incubated for 15 min. The plate was placed in an ELISA instrument to determine the absorbance at a wavelength of 570 nm. Then, the IC₅₀ concentration was determined for each treatment in each cell line.

Evaluation of apoptosis induced by flow cytometry method

Flow cytometry was used to determine the degree of apoptosis and necrosis induced by 24-, 48-, and 72-h treatment with plant extracts in the MCF-7 and HFF cell lines. Cells were stained with Annexin-v and propidium iodide (PI). Data analysis was performed using instrument software in 4 areas of the curve Q1 to Q4. After staining cells together with PI and annexin-v, necrotic cells are stained with PI and young apoptotic cells are stained with annexin-v and old apoptotic cells are stained with PI and annexin-v.

Real-time PCR technique

RNA was extracted from cells treated with IC₅₀ extract for 24, 48, and 72 h. The fermentase kit was used for RNA extraction. A Nanodrop device (generator) was used before performing real-time PCR to ensure optimal quality of RNA. A mixture of 20 µl, including 10 µl of master mix green, 2 µl of primer for each gene (Table 1), and 6 µl of distilled water were added to the micro tube. Then, the real-time PCR reaction (Table 2) was performed using the instrument (Cycler96-roche). The changes in gene expression were measured at 2^−ΔΔCt. Changes in the expression of Bax and Bcl-2 genes compared with the reference gene β-actin were examined by real-time PCR.

Table 1 Sequence of primers used in this research

Table 2 PCR steps and repeat cycle

Result

Result of extraction

The extraction of the Dioscorea plant was carried out by the Soxhlet method. After determining the concentration of 667 mg/ml extract, the desired concentrations were prepared using DMEM extract and culture medium and by sequential dilution.

Evaluation of biological percentage of MCF-7 cell line under the influence of different concentrations of Dioscorea extract after 24, 48 and 72 h

After determining the optimal cell number, 20,000–30,000 cells were cultured in each well.

Figure 1 shows that the plant extract did not cause healthy cells to die in the HFF cell line. As the concentration of the extract increases, the survival rate of cancer cells decreases. This indicates that the plant extract can be used to kill cancer cells without harming healthy cells. This condition was also observed in the 48- and 72-h treatments. In the 24-h treatment of cancer cells with the plant extract, the IC₅₀ was reached at a concentration of 1178.95 µg/ml.

Fig. 1

Percentage of survival of MCF-7 cells (right) and normal cells (left) to different concentrations of Dioscorea extract after 24 h by colorimetric method (*p value < 0.05, **p value < 0.01, ***p value < 0.001)

When treated with the plant extract for 48 h, it was found that the plant extract had no lethal effect on the HFF cell line. As the concentration of the extract increases, the survival rate of the cancer cell lines decreases. In the 48-h treatment with the plant extract, the IC₅₀ was determined at a concentration of 568.25 µg/ml (Fig. 2).

Fig. 2

Percentage of survival of MCF-7 cells (right) and normal cells (left) to different concentrations of Dioscorea extract after 48 h by colorimetric method (*p value < 0.05, **p value < 0.01, ***p value < 0.001)

After 72 h of treatment with the plant extract, it was found that the plant extract had no lethal effect on the HFF cell line. During the 72-h treatment with the plant extract, the IC₅₀ value was 438.35 µg/ml (Fig. 3).

Fig. 3

Percentage of survival of MCF-7 cells (right) and normal cells (left) to different concentrations of Dioscorea extract after 72 h by colorimetric method (*p value < 0.05, **p value < 0.01, ***p value < 0.001)

Table 3 compares the IC₅₀ results obtained by treatment with the plant extract in the cancer cell line and the control. The results show that at the IC₅₀ concentrations obtained for the cancer cell line, the control cells remained healthy, but the cancer cells died.

Table 3 IC₅₀ value of MCF-7 cell line (right) and normal cell line (left)

The statistical studies on the 24-, 48-, and 72-h treatments with the plant extract with p value < 0.01 are shown in Tables 4 and 5. According to this table, the survival rate of MCF-7 cells is highest at a concentration of 31.25 µg/ml. The viability of MCF-7 cells decreases with increasing concentration.

Table 4 Mean bioavailability of MCF-7 cell line with different concentrations of Dioscorea extract in 24, 48 and 72 h by MTT assay

Table 5 Mean bioavailability of normal cells with different concentrations of Dioscorea extract in 24, 48 and 72 h by MTT assay

The result of the quantified RNA extract

After the amount of RNA extracted from the cells of the cell line was evaluated using the Nanodrop device at an absorbance ratio of 260 to 280 nm, the RNA concentration was 480.5 ng/µl (Fig. 4).

Fig. 4

The results of quantitative analysis of RNA concentrations extracted from the MCF-7 breast cancer cell line using Nanodrop

Real-time PCR results

The melting curve shows that specific amplification of the desired gene fragments occurred well, and the absence of priming and nonamplification of nonspecific fragments for each gene indicates that there is no contamination in the samples. β-actin was considered as a control. After cDNA synthesis, a real-time PCR reaction was performed using primers for the Bax and Bcl-2 genes. Ct values were determined based on the amplification plot. Subsequently, gene expression changes were calculated using the Livak method (Table 6).

Table 6 Ct values and fold changes in genes expression

Expression profile of the studied genes in normal and cancer cells treated with Dioscorea extract

Using the Ct values from the real-time PCR reaction of the treated cells and the β-actin gene, gene expression analysis was performed using the Livak method. In Table 7 and Fig. 5, the fold change and the changes in the expression of Bax and Bcl-2 genes were calculated, respectively, compared with the reference gene and the control cell. The greatest decrease and increase in gene expression was seen at the 72-h treatment.

Table 7 Comparison of Bax and Bcl-2 gene expression in MCF-7 cancer cell lines treated with Dioscorea extract at 24, 48 and 72 h

Fig. 5

Fold change of Bax (right) and Bcl-2 (left) genes in 72-h treatment

The expression of Bcl-2 and Bax genes in 72 h after treatment with the extract has a significant decrease of 0.67 (p value < 0.05) and a significant increase of 2.72 (p value < 0.001), respectively.

The expression of Bax and Bcl-2 genes at 24, 48 and 72 h and the expression of Bax and Bcl-2 genes in MCF-7 cell lines treated with Dioscorea extract are shown in Table 7 and Fig. 6.

Fig. 6

Expression rate of Bax and Bcl-2 genes in 24-, 48- and 72-h treatment (*p value < 0.05, **p value < 0.01, ***p value < 0.001)

Results of induced apoptosis at 24, 48 and 72 h

Based on the quadrant plots, it can be seen that the apoptosis induced by the studied plant extract increased in 48 h compared to the control group. After 48 h, the apoptosis induced by IC₅₀ dilution of the plant extract was statistically significant in the cell line compared to the control group (p value < 0.001) (Table 8). The difference with the control group was not significant. After 72 h, more primary apoptosis was observed than delayed apoptosis and necrosis (Table 9).

Table 8 Comparison of induced apoptosis with necrosis by plant extract in 24-, 48- and 72-h treatment on MCF-7 cell line

Table 9 Evaluation of apoptosis induced by plant extract in 24-, 48- and 72-h treatment on MCF-7 and HFF cell lines

Flow cytometry results in Fig. 7 show the rate of induced apoptosis in HFF cells. The results show that 99.8% of the control cells survived because they did not become apoptotic as a result of treatment with the plant extract. The number of cells showing necrosis during the collection and processing phase is very low, 0.032%. The percentage of cells with primary apoptosis is 0.074% and the percentage of cells with delayed apoptosis is 0.064% (Fig. 7). The results of induction of apoptosis with three replicates in MCF-7 cells treated with IC₅₀ dose for 24 h are shown in Fig. 7. The results show that at the IC₅₀ concentration of the plant extract (1178.95 µg/ml), 92.9% of the cancer cells remained healthy. In this study, we used two different assays on separate instruments to measure two different parameters. It is important to note that it is very unlikely to get identical results, especially when comparing the percentage of cell populations to the relative absorbance.

Fig. 7

Results from the study of induction of apoptosis in control cells (left side) and in MCF-7 cells treated with IC₅₀ concentration of Dioscorea extract in 24 h (right side)

The MTT assay was used to assess the metabolic activity of the cells, whereas the annexin V/PI (FACS) assay was used to measure the cell populations stained with a DNA antibody, which allowed the assessment of apoptosis induction and progression. The results do not have to match exactly; rather, it is critical that a similar trend be observed.

From the results of the MTT and FACS cytotoxicity assays, it can be concluded that the cytotoxicity of the extract increases with time. The tendency to cytotoxicity depends on duration, as the degree of cytotoxicity increases with time.

The results of induction of apoptosis with three replicates in MCF-7 cells treated with IC₅₀ dose for 48 h are shown in Fig. 8. The results show that at IC₅₀ concentration of the plant extract (568.25 µg/ml), 84.6% of cancer cells survived. The percentage of cells affected by necrosis in the collection and preparation phase was 1.12%, the percentage of cells with primary apoptosis was 4.51%, and the percentage of cells with delayed apoptosis was 9.77%. The results of apoptosis induction upon 72-h treatment with the plant extract are shown in Fig. 8. MCF-7 Cells were treated with an IC₅₀ concentration of 438.35 µg/ml for 72 h. The results show that 77.5% of the cancer cells survived. Comparison of the three treatments shows that the 72-h treatment has the greatest effect on cancer cell death. The percentage of cells affected by necrosis was 1.94%, and the percentage of cells with primary and delayed apoptosis was 11.2% and 9.29%, respectively.

Fig. 8

Results of apoptosis induction in MCF-7 cells treated with IC₅₀ concentration of Dioscorea extract in 48 h (right side) and 72 h (left side)

The amount of primary and delayed apoptosis in the 24-h treatment compared to the control group was 6.63% and the rate of necrosis was 0.477%, which is statistically significant with a p value < 0.001. In the 48-h treatment with the plant extract, the amount of primary and delayed apoptosis compared to the control group was 14.28% and the rate of necrosis was 1.12%, which is statistically significant (p value < 0.001). In the 72-h treatment with the plant extract, the percentage of primary and late apoptosis compared to the control group is 20.49% and the percentage of necrosis is 1.94%, which is statistically significant (p value < 0.001) (Table 8). Therefore, it can be concluded that 72 h treatment with a plant extract in IC₅₀ concentration has the greatest effect on killing MCF-7 cancer cells. The rate of primary apoptosis, delayed apoptosis, and necrosis for all three treatments can be seen in Fig. 9 and Table 9.

Fig. 9

Comparison of delayed apoptosis, primary apoptosis and necrosis in 24-, 48- and 72-h treatment with plant extract (*p value < 0.05, **p value < 0.01***, p value < 0.001)

As shown in Table 9, the amount of primary apoptosis and necrosis was not significant at 24- and 48-h treatment. Delayed apoptosis was significant at 24- and 48-h treatment with the plant extract (p value < 0.001). In 72-h treatment with the plant extract, the amount of primary and delayed apoptosis was significant with p value < 0.001, while the amount of necrosis was not statistically significant. Therefore, it can be said that the rate of necrosis is not statistically significant in any of the three treatments. Among the three treatments of 24, 48 and 72 h with the plant extract, the 72-h treatment with the plant extract at a concentration of (IC₅₀) 438.35 µg/ml has the best effect on inducing apoptosis in MCF-7 cell line cells.

Discussion

When treated with the plant extract for 72 h, Bax gene expression increased by 2.72, which is statistically significant (p value < 0.001). Also, Bcl-2 gene expression decreased 0.67-times, which is significant at a p value < 0.05. Therefore, it can be concluded that 72 h treatment with the plant extract is the most effective treatment for the death of MCF-7 cancer cells. The ratio of Bax gene expression to Bcl-2 is 4.06.

The flow cytometry results also show that the 72-h treatment with the plant extract is the most effective treatment. The amounts of apoptosis induced at 24, 48, and 72 h are 6.63, 14.28, and 20.49, respectively. The amounts of necrosis induced in these treatments are 0.477, 1.12, and 1.94, respectively. In the healthy cell line, the values of apoptosis induced and necrosis induced are 0.318 and 0.032, respectively. This means that the treatment with the plant extract induced apoptosis and necrosis in cancer cells but had no effect on healthy cells. Therefore, 72-h treatment with a plant extract at a concentration of 438.35 µg/ml is the most effective treatment for the death of MCF-7 cancer cells, and the amount of primary and delayed apoptosis induced was significant (p value < 0.001).

In the following, we compare the results of the present study with those of previous studies.

Our results are in agreement with those of Saekoo et al. [19] and Saekoo et al. [20]. In the present study, Bax gene expression increased 2.72-times and Bcl-2 gene expression decreased 0.67-times under the influence of 72-h treatment with the plant extract.

The results of this study are consistent with those of Jeonghun et al. [22]. In the present study, the expression of Bax gene increased and the expression of Bcl-2 gene decreased under the influence of 72-h treatment with the plant extract.

Our results are in agreement with those of Mainasara et al. [14]. In the present study, apoptosis was shown to be induced in MCF-7 cells treated with the extract. The results of this study are also in agreement with those of Nantana et al. [21]. In the present study, after 72 h of treatment with the plant extract, the expression of Bax gene increases and the expression of Bcl-2 gene decreases.

Conclusion

According to the obtained results, Dioscorea rhizoma extract can be proposed as a therapeutic agent in cancer treatment, which of course requires further studies.