Tumor Biology

, Volume 34, Issue 3, pp 1531–1536

Effect of lentinan combined with docetaxel and cisplatin on the proliferation and apoptosis of BGC823 cells

Authors

    • Department of OncologyShengjing Hospital of China Medical University
  • Yuping Xiao
    • Gastrointestinal Tumor Pathology Laboratory of Cancer Institute, Department of Tumor Pathology of General Surgery InstituteFirst Hospital of China Medical University
  • Nan Xiao
    • China Medical University
Research Article

DOI: 10.1007/s13277-013-0680-8

Cite this article as:
Zhao, L., Xiao, Y. & Xiao, N. Tumor Biol. (2013) 34: 1531. doi:10.1007/s13277-013-0680-8

Abstract

We studied the inhibitory effects of lentinan alone or lentinan combined with docetaxel and cisplatin on growth of gastric cancer cell line BGC823. The cells were divided into lentinan group, docetaxel combined with cisplatin group, and lentinan combined with docetaxel and cisplatin group. Gastric cancer cell line BGC823 was treated with different concentrations of drugs in each group. Tetrazolium-based colorimetric assay (MTT), Annexin V/propidium iodide method and flow cytometry were used to determine the proliferation and apoptosis of the cells in each group. The inhibition ratio was positively related with the concentrations of drugs when BGC823 cells were treated with docetaxel combined with cisplatin from low to high dose. The inhibition ratio of each group further increased after lentinan was added into the medium. The apoptosis rate of 6.25 μg/ml lentinan on BGC823 cells was 19.84 %. The apoptosis rate of BGC823 cells was significantly increased from 50.22 % to 72.06 % after treatment with 6.25 μg/ml lentinan combined with 2.5 μg/ml docetaxel and 50 μg/ml cisplatin. Lentinan has an inhibitory effect on the proliferation of gastric cancer cell line BGC823. Lentinan combined with docetaxel and cisplatin increases the inhibitory effect on the proliferation of BGC823 cells mediated by docetaxel combined with cisplatin. Low concentration of lentinan combined with docetaxel and cisplatin has better therapeutic effects on the proliferation of BGC823 cells compared with high concentrations of docetaxel combined with cisplatin. Lentinan has the ability of inducing BGC823 cell apoptosis and this effect is enhanced when combined with docetaxel and cisplatin.

Keywords

LentinanDocetaxelCisplatinCell proliferationGastric cancer cell line BGC823

Introduction

Gastric cancer [1] is one of the malignant tumors and has a higher incidence and mortality in China. Surgical operation and chemotherapy are the regular treatments for gastric cancer. Some studies demonstrate that gastric cancer is sensitive to chemotherapy [2], and efficiency of combination chemotherapy is 30–50 %. The efficiency of chemotherapy has been improved dramatically due to new drugs being used in recent years. The commonly used schemes of combination chemotherapy include taxanes, anthracyclines, cisplatin and 5-Fu [3]. Docetaxel combined with cisplatin is one of the more frequently used schemes. However, long-term application of docetaxel and cisplatin leads to drug-resistance and reduced therapeutic effect. Furthermore, more side effects will appear as the dose increases.

Lentinan, extracted from the bodies of Lentinus edodes by Chihara et al. in 1969 [4], is a polysaccharide with antitumor activity. Lentinan has obvious inhibition effect on primary tumor, xenografted tumor and the allogeneic transplantation tumor [5], and has minimal side effects [6, 7]. Moreover, lentinan can reduce the toxicity and enhance the effectiveness of chemotherapy drugs [8]. Chemo-immunotherapy with lentinan offers a significant advantage over S-1-based chemotherapy alone in terms of survival in patients with advanced gastric cancer [9]. The addition of lentinan to the chemotherapy regimen could improve the general condition, symptoms and signs, and quality of life of patients with esophageal carcinoma [10].

The aim of this study was to observe the effect of lentinan combined with chemotherapy drugs docetaxel and cisplatin on the proliferation and apoptosis of human gastric cancer cell line BGC823 compared with docetaxel and cisplatin. This study may provide an experimental basis for exploring the molecular mechanism of lentinan on antitumor and chemotherapy sensitization.

Materials and methods

Cell culture

Human gastric cancer cell line BGC823 was a gift from Department of Cell Biology, China Medical University. BGC823 cells were cultured in RPMI-1640 medium (Gibco, CA) supplemented with 10 % fetal bovine serum (FBS; Hyclone, Utah, USA) at 37 °C in a humidified incubator with 5 % CO2. Cell growth was observed under an inverted microscope (TMS, Nikon, Japan). Cells were passaged by trypsinization at the ratio of 1:3 or 1:2 after the cell reached 70–80 % confluency.

Tetrazolium-based colorimetric assay (MTT test)

BGC823 cells in logarithmic growth phase were seeded in 96-well plates at a density of 1 × 104 per well in 100 μl medium and grown for 24 h at 37 °C in a humidified incubator with 5 % CO2. The cells were divided into three groups. For Group 1, the cells were treated with lentinan alone at the concentrations of 0 (control), 1.56, 3.125, 6.25, and 12.5 μg/ml. In Group 2, the cells were treated with docetaxel combined with cisplatin at the concentrations of 0 + 0 (control), 0.625 + 1, 1.25 + 10, 2.5 + 50, and 5 + 100 μg/ml, respectively. In Group 3, the cells were treated with lentinan combined with docetaxel and cisplatin at the concentrations of 0 + 0 + 0 (control), 1.56 + 0.625 + 1, 3.125 + 1.25 + 10, 6.25 + 2.5 + 50, and 12.5 + 5 + 100 μg/ml, respectively. Docetaxel solution was prepared by dissolving docetaxel in docetaxel solvents at 40 mg/ml (product NO. 1110282TA; Qilu Pharmaceutical Co. Ltd., China). Cisplatin was prepared by dissolving cisplatin in normal saline at 1 mg/ml (product NO. 2020062DB; Qilu Pharmaceutical Co. Ltd.), and lentinan was prepared by dissolving lentinan in lentinan solution at 0.5 mg/ml (product NO. 1110281; Zhendong Taisheng Pharmaceutical Co. Ltd., China). They were then diluted to the required concentrations. The supernatant of cultured cells were discarded and above concentrations of medicine were added into each well in 100 μl. Five replicated samples were assayed in each concentration at the same time and cultured for another 24 h, followed by the addition of 50ul MTT (Kaiji Bioengineering Co. Ltd., China) in each well. After a 4-h culture, the supernatant were discarded, 150 μl dimethyl sulfoxide (Sigma, St. Louis, MO) was added to each well, and the plate was shaken for 10 min to dissolve the MTT. Absorbance of each well was measured by an ELISA microplate reader (ELX808; Bio-TEK, USA) at 490 nm wavelength and recorded. Inhibition ratio of cell proliferation was determined according to the following formula:
$$ \mathrm{Inhibition}\,\mathrm{ratio}\,\mathrm{of}\,\mathrm{proliferation}\,\mathrm{in}\,\mathrm{cancer}\,\mathrm{cell}\left( \% \right)=\left( {1-{{{\mathrm{OD}\,\mathrm{values}\,\mathrm{of}\,\mathrm{treated}\,\mathrm{group}}} \left/ {{\mathrm{OD}\,\mathrm{values}\,\mathrm{of}\,\mathrm{control}\,\mathrm{group}}} \right.}} \right)\times 100\%. $$

The coefficient of drug interaction (CDI) was used to analyze the synergistically inhibitory effect of drug combinations. CDI is calculated as follows: \( \mathrm{CDI}={AB \left/ {{\left( {A \times B} \right)}} \right.} \). According to the absorbance of each group, AB is the ratio of the combination groups to the control group; A or B is the ratio of the single agent groups to the control group. Thus, CDI value less than, equal to or greater than 1 indicates that the drugs are synergistic, additive or antagonistic, respectively. A CDI value less than 0.7 indicates that the drugs are significantly synergistic.

Cell apoptosis assay

Cells in logarithmic growth phase were seeded in 6-well plates at a density of 1 × 105 per well for 24 h. The cells were divided into three treated groups: Group 1 (lentinan alone, 6.25 μg/ml), Group 2 (2.5 + 50 μg/ml), and Group 3 (lentinan combined with docetaxel and cisplatin, 6.25 + 2.5 +50 μg/ml). Another group without drug treatment was considered as control. The supernatant of cultured cells were discarded and above concentrations of medicine (2 ml) were added into each well. The cells were incubated for 24 h in 5 % CO2 at 37 °C and digested with trypsin (Hyclone). After washing with PBS twice (5 min each), the cells were resuspended gently with 195 μl Annexin V-FITC conjugated solution (Beyotime Institute of Biotechnology, China) followed by the addition of another 5 μl Annexin V-FITC solution. The cells were incubated in the dark at room temperature for 10 min. The cells were resuspended gently with 190 μl Annexin V-FITC conjugated solution and then 10 μl staining solution of propidium iodide (PI) was added. The cells were incubated on ice bath for 15 min in the dark and assayed by FACSCalibur flow cytometry (BD, New Jersey, USA).

Statistical analysis

SPSS 11.0 software was used for statistical analysis. Data are presented as mean ± standard deviation (SD) and were compared by t-test within groups. Differences with a P value <0.05 were considered statistically significant.

Results

Effect of lentinan alone or combined with docetaxel and cisplatin on the viability of BGC823 cells

The viability of BGC823 cells was determined by MTT method after treated with different concentrations of lentinan, docetaxel and cisplatin. The results indicated that lentinan alone significantly inhibited the proliferation of BGC823 cells in a dose-dependent manner compared with control (P < 0.05; Table 1 and Fig. 1). Different concentrations of docetaxel combined with cisplatin significantly inhibited the proliferation of BGC823 cells as well (P < 0.05; Table 1 and Fig. 2). Lentinan combined with docetaxel and cisplatin significantly increased the inhibition ratio of cell proliferation compared with docetaxel combined with cisplatin group at the same concentration. Interestingly, a lower concentration of lentinan (3.125 μg/ml) combined with docetaxel (1.25 μg/ml) and cisplatin (10 μg/ml) had an even more significant inhibition ratio of cell proliferation compared with a higher concentration of docetaxel (5 μg/ml) combined with cisplatin (100 μg/ml) (Table 1 and Fig. 2). In addition, interaction between lentinan (6.25 μg/ml), and docetaxel (2.5 μg/ml)–cisplatin (50 μg/ml) was additive, with the CDI was equal to (Table 1).
Table 1

Inhibitory effect of different drugs on BGC-823 cells (24 h)

Drug

Concentration (μg/ml)

OD490 nm

Inhibitory rate (%)

CDI

Control

0

0.922 ± 0.004

 

Lentinan

1.56

0.717 ± 0.026

23.2 ± 2.41*

 

3.125

0.550 ± 0.014

42.1 ± 2.49*

 

6.25

0.428 ± 0.029

56.0 ± 3.44*

 

12.5

0.324 ± 0.008

67.7 ± 3.36*

 

Docetaxel + cisplatin

0.625 + 1

0.721 ± 0.004

22.4 ± 1.21*

 

1.25 + 10

0.603 ± 0.011

34.2 ± 1.54*

 

2.5 + 50

0.514 ± 0.023

46.4 ± 3.26*

 

5 + 100

0.411 ± 0.019

57.8 ± 4.13*

 

Lentinan + docetaxel + cisplatin

1.56 + 0.625 + 1

0.521 ± 0.011

48.0 ± 2.06*

0.98

3.125 + 1.25 + 10

0.341 ± 0.024

64.7 ± 3.41*#

0.97

6.25 + 2.5 + 50

0.214 ± 0.026

76.2 ± 3.42*#

1.00

12.5 + 5 + 100

0.102 ± 0.005

88.1 ± 4.33*#

1.02

CDI coefficient of drug interaction

*Compared with control group, P < 0.05

#Compared with docetaxel + cisplatin group with the same concentration, P < 0.05

https://static-content.springer.com/image/art%3A10.1007%2Fs13277-013-0680-8/MediaObjects/13277_2013_680_Fig1_HTML.gif
Fig. 1

Correlation between lentinan concentrations and inhibitory rate. The viability of BGC823 cells was determined by MTT method after treated with different concentrations of lentinan. The results indicated that lentinan significantly inhibited the proliferation of BGC823 cells in a dose dependent manner compared with control (*P < 0.05)

https://static-content.springer.com/image/art%3A10.1007%2Fs13277-013-0680-8/MediaObjects/13277_2013_680_Fig2_HTML.gif
Fig. 2

Correlation between lentinan, docetaxel and concentrations and inhibitory rate. The viability of BGC823 cells was determined by MTT method after treated with different concentrations of lentinan, docetaxel and cisplatin. Different concentrations of docetaxel combined with cisplatin significantly inhibited the proliferation of BGC823 cells. Lentinan combined with docetaxel and cisplatin significantly increased the inhibition ratio of cell proliferation compared with docetaxel combined with cisplatin group at the same concentration (#P < 0.05)

Effect of lentinan alone or combined with docetaxel and cisplatin on the apoptosis rate of BGC823 cells

Based on the CDI assay, the apoptosis assay was performed as the following groups: Group 1 (lentinan alone, 6.25 μg/ml), Group 2 (2.5 + 50 μg/ml), and Group 3 (lentinan combined with docetaxel and cisplatin, 6.25 + 2.5 +50 μg/ml). The results of flow cytometry indicated that the apoptosis rate of BGC823 cells was 19.84 % when treated with lentinan alone. However, docetaxel combined with cisplatin increased the apoptosis rate of BGC823 cells up to 50.22 %. There was statistical difference compared with the control group (P < 0.05). Lentinan combined with docetaxel and cisplatin significantly increased the apoptosis rate of BGC823 cells from 50.22 % to 72.06 % (P < 0.05; Table 2 and Fig. 3).
Table 2

Effect of drugs on apoptosis rate of BGC823 cells (%)

Drug

Concentration (μg/ml)

Apoptosis rate (%)

Control

0

0.15 ± 0.01

Lentinan

6.25

19.84 ± 1.32*

Docetaxel + cisplatin

2.5 + 50

50.22 ± 2.91*

Lentinan + docetaxel + cisplatin

6.25 + 2.5 + 50

72.06 ± 4.53*#

*Compared with control group, P < 0.05

#Compared with docetaxel and cisplatin group, P < 0.05

https://static-content.springer.com/image/art%3A10.1007%2Fs13277-013-0680-8/MediaObjects/13277_2013_680_Fig3_HTML.gif
Fig. 3

The result of drugs on apoptosis rate of BGC823 cells. a Control group; b lentinan (6.25 μg/ml) group; c docetaxel (2.5 μg/ml) + cisplatin (50 μg/ml) group; d lentinan (6.25 μg/ml) + docetaxel (2.5 μg/ml) + cisplatin (50 μg/ml) group. The apoptosis rate of BGC823 cells was 19.84 % when treated with lentinan alone. Docetaxel combined with cisplatin increased the apoptosis rate of BGC823 cells up to 50.22 %. There was statistical difference compared with the control group (*P < 0.05). Lentinan combined with docetaxel and cisplatin significantly increased the apoptosis rate of BGC823 cells from 50.22 % to 72.06 % compared with docetaxel combined with cisplatin group (#P < 0.05)

Discussion

Docetaxel combined with cisplatin is a commonly used clinical chemotherapy scheme for gastric cancer. Docetaxel is an inhibitor of cell growth and possesses strong cytotoxicity. A side chain of ester at its C13 site can bind to the β site of free microtubules in eukaryotic cell to stabilize microtubes and inhibit depolymerization simultaneously. The cell cycle is thus blocked in G2/M phase and, normal mitosis and proliferation are shut off. Cisplatin is a common and effective chemotherapy drug in the treatment of solid tumors with cell cycle nonspecific. It blocks the translocation of methionine into the cell by changing the permeability of tumor cell membrane. The metabolism system of folic acid is enhanced, which promotes the synthesis of a plenty of methionine from homocysteine, thus inhibiting DNA replication [11]. In vivo experiments confirm that docetaxel and cisplatin have a synergic effect [12]. Further research finds that docetaxel can form a cytidine analog, which stabilizes the combination of cisplatin and DNA and represses the repair of DNA damaged by cisplatin.

Clinical application of lentinan is widespread in recent years. Some studies demonstrate that lentinan has many properties such as anti-virus, anti-tumor, regulation of immunity, antidiabetic and antioxidant [13, 14]. Good efficacy is gained in the treatment of gastric cancer, colon cancer, breast cancer and lung cancer. The survival time of patients with advanced cancer could be significantly prolonged by lentinan [15, 16]. Eight patients with inoperable advanced gastric cancer are treated with combination chemotherapy of S-1 (80 mg/m2), low-dose cisplatin (15 mg/m2) and lentinan (2 mg/body). One complete response and four partial responses are observed for an overall response rate of 63 % [17]. Murata et al. [18] reported that the antitumor activity of lentinan is significantly enhanced when combined with cisplatin in the treatment of colon cancer. The mechanism of antitumor activity of lentinan is complicated. The inhibition of tumor cell proliferation and promotion of apoptosis are thought to be involved. Interestingly, the spatial structure of lentinan was found to be related to its antitumor activity. Zhang et al. [19] reported that the inhibition rate of sarcoma 180 by lentinan with triple helix structure is 49.5 %, but this rate is only 12.3 % by single-stranded lentinan. This result indicates that the antitumor activity of lentinan with a triple helix structure is higher than that with a single chain. In addition, double-stranded RNA within lentinan can inhibit the proliferation of tumor cells [20]. Induction of cell apoptosis is an important way to kill tumor cells for many antitumor drugs. Cell apoptosis is a programmed cell death after receiving some kind of signal or stimulation by certain factors. Cell apoptosis is measured using the TUNEL method in the tumor of nude mice injected with oral squamous cell carcinoma KB cell, and the results show that the cell apoptosis is increased in lentinan combined with S-1 group [21]. Cells apoptosis is regulated by multiple signals inside and outside the cells such as Ca2+, cAMP and cGMP. These factors play an important role in the process of apoptosis. Chen et al. [22] observe that the concentration of free Ca2+ within cytoplasm increases rapidly after the spleen cells are treated with lentinan. The elevated levels of Ca2+ in cells may be due to lentinan-activated cell membrane Ca2+ channel, which stimulates the influx of extracellular Ca2+. The calcium pool in the endoplasmic reticulum and mitochondria can be triggered and Ca2+ released passively. Elevated Ca2+ activates Ca2+- and Mg2+-dependent endogenous endonuclease, leading to cellular DNA breakage, which in turn causes apoptosis. In this study, inhibition ratio of gastric cancer cell line BGC823 was positively related to the concentration of lentinan. Moreover, the proliferation of gastric cancer cell line BGC823 was significantly reduced after treated with lentinan combined with docetaxel and cisplatin. Meanwhile, cell apoptosis was induced by lentinan as well. Our results indicated that lentinan alone has the ability of inducing BGC823 cell apoptosis and this effect was enhanced when combined with docetaxel and cisplatin.

In conclusion, lentinan combined with docetaxel and cisplatin significantly inhibits the proliferation of BGC823 cell line. Particularly, a low concentration of lentinan combined with docetaxel and cisplatin has better therapeutic effects compared with high concentrations of docetaxel combined with cisplatin. Meanwhile, a low concentration of lentinan promotes apoptosis of BGC823 cells effectively. These data provide the theoretical basis for the strategy of low toxicity and high performance in gastric cancer treatment.

Conflicts of interest

None

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© International Society of Oncology and BioMarkers (ISOBM) 2013