The Research On Inhibitory Effects Of Matrine To Human Salivary Adenoid Cystic Carcinoma ACC-M Cells

Salivary adenoid cystic carcinoma(SACC) is one of the most common malignant tumor of the salivary glands. It has higher rate of invasion and distant metastasis. Therefore, it usually needs a supplementary chemotherapy after original tumor had been resected. But traditional chemotherapy often fail because of drug-resistant. It is important for research workers to find a kind of effective medicine that can be applied to treat SACC. Matrine has a great effect on anti-inflammation, anti-arrhythima and anti-virus as an active component of Chinese traditional medicine. The recent evidences indicated that matrine also plays an important role in anti-tumor, such as inhibitingproliferation, reducing invasion and metastasis of tumor cell, inducing differentiation and apoptosis, down-regulating telomerase activity of tumor cell, improving ability of immune and easing harmful reaction of chemotherapy. But it was seldom been reported in paper about anti-tumor of Head and Neck. It could be a new way and thinking for research workers to study anti-SACC on matrine. Objective:To investigate the inhibitory effects of matrine on human SACC ACC-M cell lines and study the related mechanism about inhibitory effects. Materials and methods:ACC-M cells were provided by Shanghai Second Medical University. Matrine was produced by Guangzhou Mingxing Medicine Factory. ACC-M cells were cultured in common RPMI-1640 culture medium .The test groups were treated with the culture medium that has different concentration of matrine(0.25mg/ml, 0.50mg/ml, 0.75mg/ml, 1.00mg/ml). The control group had no matrine in culture medium. All indexes were detected after ACC-M cells have treated. Effect of inhibiting proliferation was survey on a way of MTT. Curve lines of proliferation was described according to results of MTT. The hTERT-mRNA expression was determined by RT-PCR assay. Cell cycle change was analyzed with the flow cytometry(FCM). Bcl-2, Bax andPCNA expression were detected by an immunocytochemical method, indirectly immunofluorescene and FCM. The statistical analysis was executed by SPSS. 10.0 software, using Chi-square, Fisher's exact test, analysis of variance(ANOVA) and t-test. Statistically significant level was considered as "alpha equals 0.05". Results:(1) Matrine can inhibit proliferation of ACC-M cell. Effect of inhibition is gradually increasing along with the increasing of matrine conentration and treated time. The statistical graph demonstrated that effect of inhibition is positive correlation with concentration and treated time.(2) Matrine can change distribution of ACC-M cell cycle. Change of cell cycle shows that ratio of Gi phase is gradually increasing and ratio of S phase is gradually decreasing along with the increasing of matrine concentration and treated time. The statistical graph show change of the cell cycle is positive correlation with matrine concentration and treated time. The statistical analysis has significant difference between the test groups and the control group(3) After ACC-M cells were treated by 0.75mg/ml matrine, PCNA and Bcl-2 expression of ACC-M cell decreased and cytoplasmic or nuclear staining weakened. Quantitative analysis shows that average fluorescene intensity of PCNA was respectively 613.9+11.99 and 380+8.41 between the control group and the test group, and average fluorescene intensity of Bcl-2 was 570.6+11.62 and 512.4 + 5.76 between the control group and the test group. Statistically analysis has significant difference(p<0.05).(4) After ACC-M cells were treated by 0.75mg/ml matrine, Bax expression increased and cytoplasmic staining strengthened. Quantitative analysis shows that average fluorescene intensity of Bax was 197.5+6.92 to 313.7+6.88 between the control group and the test group. Statistically analysis has significant difference(p<0.05).(5) Results of RT-PCR displayed that bands of hTERT-mRNA were gradually weakening with matrine concentration increased. Statistically analysis proved that 0.50mg/ml group, 0.75mg/ml group and l.OOmg/ml group have significant difference compared to the control group. On the contrary, 0.25mg/ml group has no significant difference compared to the control group.Conclusions:(1) Matrine can evidently inhibit proliferation of ACC-M cells. Effect of inhibiting proliferation is positive correlation to matrine concentration and treated time.(2) Matrine can change distribution of ACC-M cell cycle. It has evident effect of Gi phase arrested. This effect is positive correlation to matrine concentration and treated time.(3) Matrine may regulate expression Bcl-2 and Bax protein related apoptosis of ACC-M cells. It induced apoptosis of ACC-M cells by altering rate of Bcl-2/Bax.(4) Matrine can inhibit activity of telomerase of ACC-M cells. Mechanism of inhibitory effect is down-regulated expression of hTERT.(5) Cell cycle was arrested, and activity of telomerase was inhibited, and expression of Bcl-2 and Bax gene were regulated, all of them may be one of mechanism that ACC-M cells were inhibited by matrine.