Boschniakia Rossica Polysaccharide Triggers Laryngeal Carcinoma Cell Apoptosis by Regulating Expression of Bcl-2, Caspase-3, and P53.

BACKGROUND Laryngeal cancer is a malignant head and neck tumor with high morbidity and high mortality in humans. Recently, treatments with Chinese medicines and their extracts have gradually received great attention, and studies suggest that Boschniakia rossica polysaccharide (BRP) has potential anti-tumor activity. Therefore, this study investigating the role of BRP in inducing apoptosis in human laryngeal carcinoma cells. MATERIAL AND METHODS The BRP was extracted with organic solvent and HR column. We treated Hep2 laryngeal carcinoma cells with different concentrations of BRP, then assessed cell growth inhibition rate by flow cytometry and apoptosis index by TUNEL staining. The protein expression of p53, Bcl-2, Bax, and caspase-3 were analyzed by Western blot. RESULTS Flow cytometry results showed that BRP inhibited Hep2 cell proliferation in a dose-dependent manner (p<0.05), and TUNEL staining indicated that BRP significantly increased Hep2 apoptosis index (p<0.05). Western blot results showed that the expression levels of p53 and activation of caspase-3 in Hep2 cells were significantly up-regulated (p<0.05), while the expression of Bcl-2 was significantly down-regulated (p<0.05). CONCLUSIONS BRP might induce cell apoptosis by regulating the expression level of cell apoptosis-associated proteins, suggesting strong anti-laryngeal cancer activity.

Background

Recently, natural products have received increasing research attention in China and other countries because of the unique mechanism of action and low toxic adverse effects. As of 2013, more than a third of new molecular entities approved by the FDA were directly or indirectly derived from natural products [1]. Currently, a large number of studies have reported that a variety of plant extracts (e.g., resveratrol and epigallocatechin) have anti-tumor activity, [2, 3], some of which (e.g., Taxol and vincristine) have been used with significant effect as anticancer drugs in clinical practice. There are also some anti-cancer drugs that are derivatives of natural products such as etoposide, derived from the leaves of a grass from which podophylloxin is derived [4,5].

Boschniakia rossica is a protected plant in China. It belongs to the Orobanche family of parasitic plants, mainly growing in the mountains of northern Daxing’an. As a traditional Chinese herbal medicine, Boschniakia rossica has great medicinal value as a laxative and is good for the kidneys [6]. In recent years, based on the analysis of its biological activity from the components of Boschniakia rossica, researchers found that Boschniakia rossica polysaccharide (BRP) is the main active ingredient [7]. Studies showed that BRP has a variety of biological activities, including strong anti-tumor activity [8].

Laryngeal cancer (LC) is a head and neck cancer with a high mortality rate. Due to changes in lifestyle and other factors, the incidence of laryngeal cancer is gradually increasing in recent years. Traditional treatment for laryngeal cancer chemotherapy is trying to inhibit the progression of the disease, but this treatment also kills normal cells [9]. Therefore, the development of highly selective anticancer drugs is urgent. Recent studies suggest that BRP might have the potential to inhibit cancer cell division and induce apoptosis in malignant cancer [10]. The present study explored the role of BRP in triggering human laryngeal carcinoma cell apoptosis.

Material and Methods

BRP extraction

Boschniakia rossica was bought from a Chinese medicine store. We chopped 1.0 g of Boschniakia rossica and put it in a flask, then washed it with petroleum ether and used ethanol ultrasonic extraction. The solvent was removed by rotary evaporation, ultrasonic extraction with pure water, and low-temperature vacuum distillation, and then concentrated it. After filtration, ethanol was allowed to stand, then the ethanol was removed by rotary evaporation, and the residue was placed in a 5-mL distilled water bath at 60°C to be dissolved. Thus, the crude extract was converted into a polysaccharide solution. Crude extracts were processed by polyacrylamide dextran 200HR column purification, and the purified components were polysaccharides (BRP). For polysaccharide proteins, we detected protein residue extracts by using the Bradford method protein quantification kit (Regen Biotech). The concentration of polysaccharides in BRP glucose was measured by UV spectrophotometry as a control standard curve [11].

Cell culture

Hep2 human laryngeal carcinoma cell line cells were purchased from the Shanghai Institute of Life Science Cell Resource Center. We used RPMI 1640 culture medium supplemented with 10% inactivated fetal calf serum, 50 U/mL penicillin, and 50 U/mL streptomycin, with culture conditions of 37°C and 5% CO2. After they entered logarithmic growth phase, the Hep2 cells were seeded in 96-well plates. After the cells adhered, we added different concentrations of BRP extract in the experimental groups, and added the same volume of saline to the control group. Then the cells were cultured for another 24 h [12].

Flow cytometry detection of the inhibitory effect of BRP on Hep2 cell proliferation

The cells were fixed in 90% ethanol at 4°C overnight and then treated with RNase at 37°C for 30 min. Next, the cells were stained by PI and tested using flow cytometry (Becton Dickinson, USA). The excitation wavelength was 488 nm and the emission wavelength was 630 nm. FL-2 area and DNA histogram were analyzed using Modifit software. The cell percentage elevation in S phase shows cell proliferation enhancement.

We used the MTT assay to determine the cell proliferation inhibitory rate.

TUNEL detection of apoptotic cells in situ

Hep2 cells at the logarithmic growth phase were digested with trypsin, then placed on coverslips and seeded into 24-well plates. The experimental groups received 200 mg/L of BRP extract or saline as a control group. Each group has 6 parallel experiments, then the coverslips were taken out at 1, 2, 3, and 4 days, then we used a TUNEL detection kit (Roche, 11684795910) to analyze the apoptotic cells. Apoptotic cell nuclei were stained brown under the microscope, and 5 horizons of apoptotic cells were randomly selected to count the number of apoptotic cells and the total cell number, using the ratio of these 2 numbers as the apoptotic index [13].

Western blot

Hep2 cells at the logarithmic growth phase were digested with trypsin, placed on coverslips and 24-well plates. After being treated with different concentrations of BRP, cells were collected for protein extraction. Proteins were separated by SDS-PAGE and transferred onto membrane, then blocked with 5% fat-free milk. Membranes were incubated with primary antibody (rabbit anti-human Bcl-2 antibody (Proteintech, 25850-1-AP), rabbit anti-P53 antibody (Proteintech, 10442-1-AP), rabbit anti-human Bax antibody (Proteintech, 50599-2-Ig), rabbit anti-human Caspase-3 antibody (Proteintech, 19677-1-AP), and rabbit anti-human β-actin antibody (Proteintech, 20536–1-AP) at 4°C overnight. HRP-labeled secondary antibody (1: 1000) was used to incubate the membranes for 1 h after bring washed with TBST. Images were imported into the computer, then we used the gel imaging analysis system to detect the bands for calculation of the relative expression level [14].

Statistical analysis

All data are presented as mean ± standard deviation. The comparison among groups was analyzed by one-way ANOVA), and the inhibition rate and apoptosis index were calculated using the t test. p <0.05 was considered statistically significant.

Results

HPLC detection of BRP

Extracted BRP was hydrolyzed by TFA and tested HPLC (Figure 1), showing that the monosaccharide in the BRP was mainly composed of Fuc (peak 7), Xyl (peak 6), Man (peak 3), and Glc (peak 5). Peak 1 and 2 were unknown substances.

Figure 1

The growth inhibition rate changes at 48 h after adding various concentrations of BRP.

The effect of BRP on cell growth

BRP (50, 100, 200, and 400 mg/L) were added into the Hep2 cell lines, and an equal volume of water was added as a control. Then, to evaluate the effect of different concentrations of BRP on cell growth of Hep2 cell lines, the rate of cell growth inhibition was calculated by performing MTT assay (Table 1). As shown in Table 1, BRP significantly inhibited cell growth (p<0.01). In certain concentration ranges, cell growth inhibition rate gradually increased in a concentration-dependent manner. Based on 48-h changes in cell culture, the inhibition rates were plotted in Figure 1, showing that, at BRP concentrations less than 200 mg/L, the inhibition rate increased in a concentration-dependent manner, but when the concentration of BRP was more than 200 mg/L, cell growth inhibition showed no significant increase (p>0.05).

Table 1

Cell growth inhibition of Hep 2 from different concentrations of BRP.

Groups	Dose (mg/L)	Inhibition rate (%)
		12 h	24 h	48 h
Control	0	1.3±0.3	1.9±0.4	2.3±0.6
A	50	9.3±1.3*	13.2±1.5*	17.5±1.6*
B	100	18.8±1.7*	20.3±1.8*	24.2±1.8*
C	200	19.3±1.6*	27.9±1.5*	29.5±2.1*
D	400	22.4±2.3*	29.1±1.8*	31.3±1.9*

Compared with the control group, p<0.01.

BRP enhances the apoptosis index of Hep 2 cells

As shown in Figure 2A, apoptotic cells turned brown after TUNEL staining. Compared with the control group, the number of apoptotic cells in the BRP (200 mg/L) group increased significantly (Table 2). AI-incubation time curves of both groups are shown in Figure 2B based on the apoptotic index (AI). The results showed that the apoptosis index of the BRP group was significantly higher than in the control group (P<0.05). As the culture time lengthened, the apoptotic index increased gradually.

Figure 2

The apoptosis index of Hep 2 cells after treatment with BRP (200 mg/L). (A) TUNEL staining on day 4; arrows indicate nuclei stained brown, representing the apoptotic cells. (B) The apoptosis index of Hep 2 cells; each group underwent 6 parallel experiments. TUNEL staining was performed on days 1, 2, 3, and 4. Under an optical microscope, cells were photographed randomly, and 5 high-power fields were selected to count the apoptotic cells and total cell number; the apoptosis index is the ratio between these 2 values. * Compared with the control group, p<0.05.

Table 2

Apoptosis rate of Hep 2 cell after BRP treatment.

Time (d)	Apoptosis rate (%)
	Control	Treatment
0	2.3±0.5	2.4±0.6
1	4.0±0.6	13.3±0.9*
2	4.4±0.9	21.5±1.1*
3	5.1±0.9	30.9±1.4*

Compared with the control group, p<0.05.

Expression of apoptosis-related proteins P53, Bcl-2, Bax, and Caspase-3

To explore the mechanism of BRP affecting the Hep 2 cell apoptosis, we detected cell apoptosis-related proteins, including P53, Bcl-2, Bax, and Caspase-3 expression, by Western blot method at different concentrations of BRP. Each group was assessed 3 times, and the results are shown in Figure 3A. Figure 3B shows results of analysis by gel imaging system using the relative expression of proteins P53, Bcl-2, Bax, and Caspase-3. Compared with the control group, the expression of apoptosis-related proteins in the BRP-treated groups changed in varying degrees. The ratio of Bcl-2/Bax had the most significant decline at the concentration of 200 mg/L (p<0.05), and the 50 and 100 mg/L groups also showed significant decrease. The expression levels of p53 and cleavage of Caspase-3 were increased significantly (p<0.05) in a dose-dependent manner (Figure 3). The most significant increase was also at the concentration of 200 mg/L (p<0.05).

Figure 3

Expression of P53, Bcl-2, and Caspase-3 in different concentrations of BRP-treated Hep2 cells. (A) Immunoblot results of P53, Bcl-2, and Caspase-3 proteins; (B) The statistical results of expression of Bcl-2 protein; (C) The statistical results of expression level of p53 protein; (D) The statistical results of expression of Caspase-3 protein. Independent experiments were repeated 3 times. * Compared with the control group, p<0.05.

Discussion

Boschniakia rossica is used as a traditional Chinese medicine. It has complex components, but the polysaccharide component has been considered as the important part, showing biological activity without toxic adverse effects [4]. Early studies found that the biological activity of BRP was broad, including modulation of immune function, as well as anti-tumor, anti-oxidation, and protection effect on blood vessels and red blood cells [15]. The present study aimed to explore the role of BRP in triggering cell apoptosis in human laryngeal carcinoma cells. First, after treating Hep2 laryngeal cancer cell lines with different concentrations of BRP, we found that cell growth inhibition was significantly increased in a concentration-dependent manner, and TUNEL staining showed that the apoptotic index significantly increased, indicating that BRP can inhibit the proliferation of Hep2 cells.

Plant polysaccharides have complex structures, and the structure-activity relationship is not clear. So, the mechanism for inducing apoptosis of cancer cells is a multi-level anti-cancer effect [16]. We found that BRP can promote the expression of p53 and the further activation of Caspase-3, and down-regulate Bcl-2/Bax ratio as shown by Western blot analysis. It is now widely recognized that plant polysaccharides can induce apoptosis in cancer cells through multiple pathways, including regulating the expression of apoptosis genes such as Bcl-2 and p53 [16]. Bcl-2 is a proto-oncogene, which can inhibit apoptosis, mainly because Bcl-2 regulates a variety of cell proliferation- and apoptosis-related protein activity, such as by Caspase-3 [17,18]. The p53 is a tumor suppressor gene, which can block the cell cycle, and induce cell differentiation and apoptosis. BRP can increase the expression level of p53, thereby promoting apoptosis [19].

Another study showed that a variety of plant polysaccharides such as LBP and seaweed, have significant anti-cancer effects [16,20]. Wang et al. found that BRP showed synergistic anti-tumor effects with 5-fluorouracil. When BRP interacts synergistically with 5-fluorouracil, the tumor inhibition rate increased significantly. The level of expression of cell interleukin-2 (IL-2 cytokines) and TNF were also shown to increase significantly [21]. In vitro experiments showed that BRP can promote nitric oxide (NO) production and increase the secretion of TNF. As a tumor necrosis factor, TNF has an important effect on cancer progression and apoptosis [22]. Thus, BRP may also exert further inhibition on tumor grow by stimulating cells to produce TNF.

Human laryngeal cancer is a head and neck cancer with relatively high incidence and mortality. Traditional chemotherapy treatment is often unsatisfactory due to adverse effects, drug resistance, and other reasons [4]. Because herbal extracts have the advantage of less drug resistance and lower toxic adverse effects, it has received widespread attention in the medical research [15]. BRP not only induces apoptosis in cancer cells, but also regulates the immune system and has anti-oxidant and other effects, and therefore it has the potential to become a better cancer drug. However, because their structure is much more complex, there is a need to perform further structure-activity mechanism studies [23], which may be able to eventually promote proper and clinical treatment. In this study, a series of experiments confirmed the effect of BRP on inducing laryngeal carcinoma cell apoptosis, as well analyzing the expression of p53, Caspase-3, and Bcl-2 protein. However, due to the specific mechanism of apoptosis and complex regulation, the role of BRP in apoptosis mechanisms needs further research to provide a theoretical and practical foundation for the future use of BRP in cancer treatment.

Conclusions

Boschniakia rossica polysaccharide significantly decreases proliferation and promotes apoptosis of Hep2 cells in a dose-dependent manner. Boschniakia rossica polysaccharide promotes Hep2 apoptosis through regulating expression of p53, Caspase-3, and Bcl-2.