Fig leaf polysaccharides inhibit the proliferation and promote apoptosis of gastric cancer cells
Ficus carica L. belongs to the Ficus genus of the Moraceae family and is a common deciduous fruit tree in warm temperate regions. It is also one of the earliest domesticated and cultivated fruit trees by humans. In 1990, a collaborative study between Nanjing Agricultural University and Jiangsu Cancer Prevention and Control Research Institute proved that figs have significant anti-cancer effects, and the national fig industry began to flourish. Nowadays, in addition to being processed into food such as wine, dried fruit, jam, etc., figs still attract people’s attention as a mystery in terms of their medicinal and health benefits, especially their anti-cancer properties. Only by developing and utilizing this value can this ancient fruit tree truly benefit humanity.

Fig is rich in various medicinal active ingredients, such as flavonoids, benzaldehyde, polysaccharides, etc. Its roots, stems, leaves, fruits, and milk can all be included in traditional medicinal sources. Many researchers are dedicated to extracting natural polysaccharides from plants, believing that they have medical benefits such as enhancing immune activity, lowering blood sugar, and anti-tumor effects. On figs, some scholars attribute their anti-cancer activity to polysaccharide components. In Guo et al.’s report, fig fruit polysaccharides have antioxidant activity and can significantly inhibit the growth of human liver cancer HepG2 and gastric cancer SGC-7901 cells. Jiang confirmed that polysaccharides from fig leaves have varying degrees of inhibitory effects on human lung cancer cells A549, cervical cancer cells Hela, and liver cancer cells HepG2. Therefore, fig polysaccharides are expected to be used for the prevention and treatment of human tumors. However, there are many varieties of figs with different characteristics. It is necessary to conduct experiments to determine whether polysaccharides from all varieties have anti-cancer activity. In addition, fig leaves are large and abundant, which can be used to make fig tea. However, the production and consumption process of fig tea mainly focuses on taste, and rarely involves the role of polysaccharides in human health. In fact, fig tea soup soaked in high-temperature hot water contains a certain amount of polysaccharides. This is an issue that needs to be explored in the development of fig products. Previous studies on fig polysaccharides have mainly focused on the fruit, with limited research on leaf polysaccharides. There have been no reports on the anticancer activity of leaf polysaccharides among different varieties. To this end, this experiment first isolated, extracted, and purified polysaccharides from three different varieties and months of fig leaves. Then, the MTT method was used to compare the inhibitory activity of polysaccharides from different sources on the proliferation of human gastric cancer SGC-7901 cells. Based on this, the effect and mechanism of fig leaf polysaccharides on cancer cell apoptosis were further studied, in order to provide theoretical basis for the comprehensive development and utilization of fig leaf varieties.

It has been proven that fig polysaccharides have in vitro antioxidant activity, can lower blood sugar, enhance immunity, and have medicinal effects such as anti-tumor effects. Jiang proposed that fig polysaccharides have significant inhibitory activity on cervical cancer Hela and liver cancer HepG2 cells, but have weaker inhibitory activity on lung cancer A549 cells, indicating that fig polysaccharides have different inhibitory effects on different tumor cells. Guo et al. demonstrated that different components of fig polysaccharides have significant inhibitory effects on human liver cancer HepG2, gastric cancer SGC-7901, and colon cancer SW1116 cells. The inhibitory rate of 2mg/mL polysaccharide component-3 on gastric cancer 7901 cells was 54.49%. The results of this article are similar. When the concentration of polysaccharides in the leaves of the ‘Braunschweig’ variety was 2mg/mL, the inhibition rate of SGC-7901 cell proliferation was 46.67% (see Figure 1); At a concentration of 4mg/mL, the inhibition rate reached 52.92% (see Figure 2). Once again, it has been proven that fig polysaccharides have significant anti-cancer activity. Treatment with appropriate concentrations of polysaccharides can significantly inhibit cancer cell proliferation and lead to cell death (see Figure 3).

Previous studies on fig polysaccharides and their anticancer activity have rarely involved variety and sampling time. However, the activity of plant contents may vary depending on the variety and time. Zhang et al. found that extracts from four varieties of fig leaves have hypoglycemic effects, but there are differences in the effects of different varieties on liver glycogen. This experiment also obtained similar results. We found that there were significant differences in the inhibitory activity of leaf polysaccharides on SGC-7901 cells among different fig varieties and collection times (see Figure 1). In the 2mg/mL experimental system, the average inhibition rate of ‘Braunschweig’ was the highest, followed by ‘Bojihong’, and finally ‘Masyitaofen’, indicating that the leaves of the ‘Braunschweig’ variety are more suitable for polysaccharide development. In addition, the anticancer activity of polysaccharides in the leaves of this variety is highest in September, followed by July and August, and the lowest in October and November. These results provide a theoretical basis for the development and utilization of the anticancer effect of fig leaf polysaccharides, as well as the selection of raw materials.

Previous studies on the anti-tumor mechanism of fig polysaccharides have focused on enhancing antioxidant activity or immune function. However, this article observed that treatment with fig polysaccharides would cause an increase in intracellular ROS levels in SGC-7901 cells. This effect was significantly observed after treatment with 0.5mg/mL fig polysaccharide, and increased with the increase of polysaccharide concentration. When the concentration of polysaccharides was 4mg/mL, the ROS content of treated cells was 5.5 times higher than that of the control (see Figure 6), indicating that the anticancer activity of fig polysaccharides may promote cancer cell apoptosis by inducing an increase in ROS in cancer cells. This viewpoint also conforms to the current popular view. For example, Halliwell proposed that excessive ROS can lead to oxidative damage to cell membranes, lipids, and DNA. Brosche et al. believe that oxidative stress is closely related to cell apoptosis. Ding et al. demonstrated using exogenous H2O2 that reactive oxygen species can induce cell apoptosis. Recently, it has been demonstrated that astragalus polysaccharides can induce an increase in cellular ROS during the apoptosis process of human gastric cancer MGC-803. From this perspective, the observed increase in ROS induced by fig polysaccharides in this article is an important aspect of its anti-cancer effect.

This experiment observed for the first time that fig polysaccharides induce apoptosis in SGC-7901 gastric cancer cells (see Figure 3). After treatment, the number of live cells decreased with increasing polysaccharide concentration, while the number of late apoptotic and dead cells significantly increased (see Figure 5). Further observation suggests that the anticancer effect of fig leaf polysaccharides may be related to their inhibition of cell cycle. From Figure 4, it can be seen that the inhibitory effect of fig polysaccharides on the cell cycle of SGC-7901 is mainly manifested in the S phase. As the concentration of polysaccharides increases, the number of cells stuck in the S phase significantly increases. It has been reported that the inhibitory effect of citrus peel polysaccharides on the proliferation of H22 cells with transplantable ascites tumor is also manifested in the S phase of the cell cycle, while goji berry polysaccharides inhibit the cell cycle of Hela cells with cervical cancer in the S and G2/M phases. The above results indicate that cell cycle arrest, especially the inhibition of S phase DNA synthesis, may be an important mechanism by which fig polysaccharides inhibit cancer cell proliferation.

It has been established that cyclins and cyclin dependent kinases (CDKs) play a central regulatory role in the cell cycle. There are dozens of Cyclins in different organisms that can form Cyclin CDK complexes with different types of CDKs, acting as kinases at different stages of the cell cycle, inducing protein phosphorylation, and regulating cell cycle progression. The CDK2/Cyclin E complex is highly expressed in the late G1 and S phases. If the expression of CDK2 weakens, the initiation of the S phase is blocked. The Cyclin D1/CDK4/CDK6 protein complex is located upstream of the CDK2/Cyclin E complex and also regulates G1/S phase cell division. Zhong et al. found that when mulberry yellow polysaccharides inhibit the proliferation of human colon tumor HT-29 cells, a large number of cells stagnate in the S phase, and the expression levels of CDK2 and Cyclin E proteins decrease. Ma et al. found that hawthorn polysaccharides can also induce downregulation of CDK1, CDK2, Cyclin A1, Cyclin D1, and Cyclin E1 gene expression when inhibiting the proliferation of human colon cancer HCT116 cells. The results of this experiment are similar to this. We observed that fig polysaccharides inhibited the cell cycle arrest of human bone cancer SGC-7901 cells in the S phase, while significantly downregulating the expression of cell cycle protein genes (see Figure 7B). The gene expression of CDK1, CDK2, CDK6, Cyclin B, Cyclin D1, and Cyclin E in SGC-7901 cells treated with fig polysaccharides was significantly reduced (see Figure 7B). Perhaps it is the suppression of gene expression encoding cell cycle proteins that causes fig polysaccharides to block the cell cycle, thereby inhibiting cancer cell proliferation.

In addition to the aforementioned Cyclins and CDKs, polysaccharide treatment of fig leaves also affected the expression of tumor suppressor genes p53 and Bax, as well as the oncogene Bcl-2 (see Figure 7A). Among these three genes, Bax and Bcl-2 belong to the same gene family. Bcl-2 promotes cell cycle, while Bax forms a dimer with Bcl-2. When Bax is highly expressed, it forms a homodimer on its own to promote cell apoptosis. It has been reported that treatment of HepG2 cells with polysaccharides from Hedyotis diffusa resulted in a decrease in Bcl-2 gene expression; Treatment of HepG2 cells with ginger polysaccharides resulted in upregulation of gene expression such as Bax and p53, as well as downregulation of Bcl-2 gene expression, inducing cell apoptosis. After conducting pairwise correlation analysis between the concentration of fig polysaccharides, SGC-7901 cell proliferation inhibition rate, and the relative expression levels of Bax, Bcl-2, and p53, we found that the correlation coefficient between fig polysaccharide concentration and proliferation inhibition rate was r=0.938, and the correlation coefficient between cell proliferation inhibition rate and Bcl-2 relative expression level was r=-0.932. However, the correlation between other indicators did not reach a significant level of P=0.05. This suggests that although fig polysaccharides can induce upregulation of tumor suppressor genes Bax and p53 expression, in terms of concentration effect, downregulation of oncogene Bcl-2 expression may be more important.

In summary, this article analyzed the inhibitory effect of fig leaf polysaccharides from different varieties and months on the proliferation of human gastric cancer SGC-7901 cells, and found that the ‘Braunschweig’ fig variety had the highest inhibition rate of leaf polysaccharides in September. As the concentration of polysaccharides increases, cancer cell proliferation is inhibited, density and adhesion decrease, cell morphology changes from spindle to round, live cells decrease, and the proportion of dead cells and late apoptotic cells increases. Research on the mechanism of tumor inhibition has shown that polysaccharides from fig leaves block the cell cycle in the S phase and a small amount in the G2 phase. The expression of oncogenes Bcl-2 and cell cycle related genes (Cyclins and CDKs) is downregulated, while the expression of tumor suppressor genes (Bax and p53) is upregulated, leading to an increase in intracellular reactive oxygen species accumulation. These are important reasons why fig polysaccharides promote cancer cell apoptosis. These results provide a theoretical basis for the collection of fig leaves and the development of anticancer drugs. In addition, the developed fig tea mainly uses leaves as raw materials, and during the hot water brewing process, fig polysaccharides should be able to be extracted. However, whether they have anti-cancer activity still needs further research.