ABSTRACT
Objective To analyze the effect of trametes robiniophila on the apoptosis and the expressions of invasion and metastasis related matrix metalloproteinase 2 (MMP-2) and MMP-9 in human gastric carcinoma poorly differentiated cell line MKN-45 and medium differentiated cell line SGC-7901.Methods Human gastric carcinoma cell lines MKN-45 and SGC-7901 incubated with trametes robiniophila at the different concentrations [0 (the negative control group), 5, 10, and 20 mg/ml] for 24 h. When bifluorescently stained with acridine orange-ethidium bromide (AO-EB), morphology was observed by using microscopy. Flow cytometry was used to test the apoptosis ratio after 24, 48, and 72 h. Reverse transcription polymerase chain reaction (RT-PCR) was applied to analyze the expressions of mRNA of MMP-2 and MMP-9 after 24 h, and the absorbance values of the bands after gel electrophoresis were used as their expression levels. Results Under the fluorescence microscope, the cells of the negative control group were green (normal cells), and the membrane was even in size and integrity. The proportion of apoptotic and necrotic cells was increased with the concentration enrichment of trametes robiniophila. The apoptotic cells were yellow, and their cell membrane lost integrity. Apoptotic bodies were found in cancer cells, and cell membrane showed bud projection. The nuclei of the apoptotic cells showed brightly condensed chromatin or fragmented. Chromatin was strongly stained and located in karyotheca. The necrotic cells were dyed red with integrity of size. The apoptotic ratio of MKN-45 cell line induced by trametes robiniophila after 24 h was (6.5 ±0.8)％, (14.6±1.0)％, (18.0±1.1)％, and (23.1±1.2)％, respectively (F= 333.972, P< 0.01) in negative control group and 5, 10, and 20 mg/ml trametes robiniophila group. After 48 h, the apoptotic ratio was (7.3 ±1.2)％, (18.3 ± 1.6)％, (24.5±1.3)％, and (27.2±1.7)％, respectively (F= 528.432, P= 0.001); after 72 h, the apoptotic ratio was (7.5 ±0.9)％, (50.2 ±1.6)％, (58.0 ±1.9)％, and (69.0 ±1.4)％, respectively (F= 3814.238, P< 0.01). After SGC-7901 cell line induced by trametes robiniophila for 24 h, the apoptotic ratio was (12.9 ±1.0)％, (19.4 ± 1.2)％, (22.0±1.7)％, and (23.0±1.9)％, respectively (F= 120.190, P< 0.01) in negative control group and 5, 10, and 20 mg/ml trametes robiniophila group. For 48 h, the apoptotic ratio was (10.2 ±1.3)％, (40.9 ±1.4)％, (51.6 ±1.9)％, and (66.2 ±1.9)％, respectively (F= 1281.342, P< 0.01). For 72 h, the apoptotic ratio was (27.4 ±1.8)％, (49.7 ±1.4)％, (65.1 ±1.4)％, and (69.0 ±2.0)％, respectively (F= 1112.767, P< 0.01). The induction of apoptosis showed time and dose dependence (both P< 0.01). There was a trendency that the apoptotic ratio of SGC-7901 cell line was higher than that of the MKN-45 cell line at the same condition. RT-PCR showed that mRNA relative expression level of MMP-2 was 0.64±0.02, 0.49±0.01, 0.36±0.02, and 0.32±0.01, respectively (F= 274.321, P< 0.01) of negative control group and 5, 10, and 20 mg/ml trametes extract group after the effect of trametes extract on gastric cancer MKN-45 cell line. The relative expression level of MMP-9 in MKN-45 cell line was 0.71±0.01, 0.54±0.02, 0.47±0.02, and 0.39±0.02, respectively (F=203.948, P< 0.01). The expression level of MMP-2 in SGC-7901 cell line was 0.64±0.01, 0.42±0.02, 0.34± 0.20, and 0.29±0.01, respectively (F= 305.877, P< 0.01), while the mRNA expression level of MMP-9 was 0.65 ±0.15, 0.47 ±0.01, 0.44 ±0.01, and 0.39 ±0.02, respectively (F= 265.259, P< 0.01). Compared with the negative control group, the expression levels of MMP-2 and MMP-9 of the two cell lines were both decreased, after incubated with trametes robiniophila at the different concentrations (all P< 0.05), and with the addition of concentration, the expression levels of MMP-2 and MMP-9 were decreased. Conclusion Trametes robiniophila can induce the apoptosis of human gastric carcinoma cell lines MKN-45 and SGC-7901 in vitro and can inhibit the expressions of MMP-2 and MMP-9 and the effect of trametes robiniophila may be related with the differentiation degree.