| Objective: Salivary adenoid cystic carcinoma(SACC) is also called cylindroma or adeno-carcinoma of cylindroma type, one of the most common malignant epithelial tumors in human salivary glands, which can occur at any age without gender difference. SACC can occur in any salivary gland, but most common in parotid gland, submandibular gland and the minor salivary glands of the palate. As malignancy tumor, SACC is characterized by highly invasive growth, infiltrating along facial nerve and high metastasis rate. The lung is the most common site for distant metastasis. And the distant metastasis rate of SACC ranks the first place in oral and maxillofacial tumors. Currently, the clinical treatment of SACC is combined treatment that postoperative radiotherapy after surgical excision. But the curative effect and prognosis of SACC after surgery and radiotherapy is not satisfactory for local recurrence and distant metastasis. However, the mechanism of the tumor’s incidence, highly invasive and distant metastasis is still unclear now. It is necessary to explore new therapies for SACC which can improve the curative effect, decrease local recurrence and distant metastasis, and improve the quality of life of the patients. Rhizoma Paridis is the dry root of the general term for Liliaeeae Parisandis, the present studies showed that it has lots of effect such as anti-inflammatory, hemostasis, acesodyne, antalgic anti-tumor and so on. Polyphyllin I(PP I) is a kind of steroidal saponins of Rhiaoma Paridis. More and more studies showed that PP I has the effect of anti-tumor. Nevertheless, whether the PP I has an effect on the proliferation of SACC and its mechanism has not been reported yet. The aim of this study is to observe the effect of PP I on proliferation in salivary adenoid cystic carcinoma cell line ACC-M in vitro at different times and concentrations and investigate the potential mechanism, in order to provide the experimental evidence for screening new phytochemical compounds in Multimodal treatment of Salivary adenoid cystic carcinoma.Methods: 1 Materials 1.1Cell line ACC-M was purchased from the laboratory of oral and maxillofacial surgery of Shanghai Jiao Tong University, which was established in 1995; 1.2 Polyphyllin I(PP I) was obtained from Pufei De Biotech Co., Ltd, Chengdu, China. 2 Methods 2.1 Preparation of physic liquorPP I was dissolved with DMSO medium into 10mg/m L and Stored in RT, which was diluted with RPMI 1640 before each assay; 2.2 Cell cultureACC-M cells were cultured in RPMI 1640 medium containing 10 %( v/v) newborn bovine serum, penicillin(100U/ml) and streptomycin(100μg/ml), and then incubated at 37℃ in 5% CO2 gas incubator with saturation humidity. After 72-96 hours,a new generation was formed and the cells were harvested with the mixed liquid containing trypsin. 2.3 MTT assay5×104/ml cell suspension in logarithmic phase were seeded into 96-well plates at 100μl culture medium every well. After 24 h incubating, 200μl PP I solution was added into each well, the final concentrations were 1μg/m L, 2μg/m L, 4μg/m L, 6μg/m L, 8μg/m L, 10μg/m L respectively. And blank and control were set. Quintuplicate were done in each experimental group. After cultured for 24 h and 48 h, 20μl MTT solution(5mg/m L in PBS) was added to each well. Then additional 4h incubation was carried on, supernatant was sucked out and 200μl DMSO was added to solubilize crystallize with concussion sufficiently. The absorbance was measured with Microplate reader, the inhibition rate of growth was calculated. 2.4 Trypan blue exclusion assay5×104/ml cell suspension in logarithmic phase were seeded into 6-well plates at 3ml tissue culture medium every well. After 24 h incubating, 3ml PPⅠ solution was added into each well, the final concentrations were 2μg/m L, 4μg/m L, respectively. And negative control was set. After cultured for 24 and 48 h, all of the groups were stained with Trypan blue solution and the number of both stained cells(dead cells) and unstained cells(the living cells) would be recorded for calculating. 2.5 Observation under the inversion phase contrast microscope5×104/ml cells were seeded into 50 ml glass culture flask, the growth status of cells in control group and different concentration groups(2μg/m L, 4μg/m L) were observed at different times(24h, 48h). 2.6 Observation under the light microscopeCells were inoculated into 6-well plate, and grown on coverslips placed in every well. After the cells attached to the coverslips by overnight incubation the culture medium with and without PP Ⅰ(2μg/m L, 4μg/m L) were added and cultured for different time(24h, 48h). Then after Giemsa staining, cells were observed under the microscope. 2.7 Observation under the transmission electronmicroscopyCells were inoculated into 50 ml culture flask. After scheduled processing and preparation of electron microscope specimens, the growth status of cells in control group and 2μg/m L PPⅠ treatment group were observed with TEM at 24 h. 2.8 Flow cytometric analyses(FCM) of apoptotic ACC-MAfter cells were Treated with or without PP at different concentrations Ⅰ(0μg/m L 2μg/m L, 4μg/m L) for different time(24h 、 48h). Cells were determined with double staining of Annexin-V-FITC/PI as analyzed by FCM. 3 Statistical analysesAll the experiments were repeated three times and the experimental data were collected. One Way Variance analysis, correlation analysis were performed by SPSS13.0 software.Results:1 MTT assayPPⅠ significantly inhibited the proliferation of ACC-M cells, The rate of maximal growth inhibition could reached 100%.According to statistical analysis, among different concentration groups, the rate of growth inhibition were different at different times(P <0.05). The inhibitory effect of PPⅠ on ACC-M is positive correlated with the concentration and time.2 Trypan blue exclusion assayThe ratio of survival cell of the treated group showed a obvious decreased trend with the growth of drug concentration and time. There was significantly difference(P <0.05) between each time group and each concentration group.3 Observation under the inversion phase contrast microscopethe cells of control groups grew quickly and attached in the wall like paving stones, most of which were flat and polymorphic, full of cytoplasm, and the rounded nucleus was found at the center of cells, cells contact closely, and the intercellular space is small. In the contrary, the growth of PPⅠ-treated cells was clearly constrained.Also a few treated cells were found in the medium,which was desquamated from the glass wall,the shape of the cells became round, bigger than the control, the connections between cells were loose,and the intercellular space became larger.4 Observation under the light microscopeThe outline of cells was clear, multiple nucleoli were found in the cells of control group. The cytomembrane was damaged. Cytoplasm vacuolation or disintegration and nuclear pyknosis were seen in the treated group.5 Observation under the transmission electronmicroscopyCells were complete, nucleoli and organelles were clearly visible in the control group and hyperthyroidism of the treated cells showed obvious mitotic figures. The microvilli of the treated cells were decreased or disappeared, cytomembrane were damaged and cytoplasm vacuolation or even disintegration; mitochondrion pyknosis, and the nucleus was squeezed to one side by the vacuoles of cytoplasm.Part of the nuclei were disappeared.6 FCM assayIn the control groups, the rate of apoptosis was low, and there is no obvious growth trend with the change of time. The apoptosis rate of the treated groups went up with the increase of drug concentration and action time, but there was no statistical difference between the group of each time group and each concentration group(P>0.05)Conclusion: This study suggested that PPⅠ could inhibit the proliferation of the salivary adenoid cystic carcinoma ACC-M cells in vitra and the main mechanism of this inhibition of proliferation is due to cytotoxicity of PP I, and inhibition was dependent association of the drug concentration and treating time. The inhibition of cells is by inducing necrosis but not by inducing apoptosis of the tumor cells. |
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