乌骨藤中C21甾体苷诱导SACC-83和SACC-LM细胞凋亡的作用及其机制研究

Apoptosis Induced by the C21 Sterols in Marsdeniae Tenacissimae Caulis and Its Molecule Mechanism of Action in SACC-83 and SACC-LM Cells

关键词:  乌骨藤  C₂₁ 甾体苷  SACC-83细胞  SACC-LM细胞  细胞增殖  细胞凋亡

Key words:  Marsdeniae Tenacissimae Caulis  C₂₁ sterols  SACC-83 cell  SACC-LM cell  cell proliferation  cell apoptosis

摘要:

目的 研究乌骨藤提取物C₂₁甾体苷对唾液腺腺样囊性癌（salivary adenoid cystic carcinomacv，SACC）低侵袭细胞株（salivary adenoid cystic carcinoma-83，SACC-83）和肺高转移细胞株（SACC-LM）增殖抑制和诱导凋亡的作用及其机制。方法 用不同浓度（5，10，20，40，60，80，100 μmol·L^-1） C₂₁甾体苷处理SACC-83和SACC-LM细胞48 h后，MTT法检测细胞活力，并计算药物的IC₂₀和IC₅₀；细胞克隆形成实验检测细胞增殖能力；流式细胞术检测SACC-83及SACC-LM细胞凋亡情况；实时荧光定量PCR和Western blot检测SACC-83和SACC-LM细胞Bcl-2、Bax、caspase 3的mRNA和蛋白的表达。结果 不同浓度的C₂₁甾体苷降低SACC-83和SACC-LM细胞活力，抑制细胞增殖，并且作用于SACC-83细胞C₂₁甾体苷的IC₂₀浓度为7.49 μmol·L^-1，IC₅₀浓度为38.34 μmol·L^-1；作用于SACC-LM细胞的C₂₁甾体苷IC₂₀浓度为9.30 μmol·L^-1，IC₅₀浓度为46.04 μmol·L^-1；细胞克隆集落形成明显减少。C₂₁甾体苷IC₂₀浓度分别促进SACC-83及SACC-LM细胞凋亡，且随着给药时间延长，凋亡率增加，具有显著性差异（P<0.05，P<0.01）。经7.49，9.30 μmol·L^-1 C₂₁甾体苷分别处理SACC-83及SACC-LM细胞后，Bcl-2的mRNA及蛋白水平显著降低（P<0.01），而Bax、Caspase 3的mRNA和蛋白水平显著升高（P<0.05）。结论 乌骨藤C₂₁甾体苷抑制SACC-83及SACC-LM细胞增殖、促进凋亡，其作用机制可能与调控Bcl-2、Bax和Caspase 3表达有关。

Abstract:

OBJECTIVE To evaluate the effects and mechanism of C₂₁ sterols in Marsdeniae Tenacissimae Caulis extract on inhibition of proliferation and induction of apoptosis of low invasion and lung hypermetastasis in salivary adenoid cystic carcinomacv cell line SACC-83 and SACC-LM, respectively. METHODS SACC-83 and SACC-LM cells were treated with C₂₁ sterols at different doses (5, 10, 20, 40, 60, 80, 100 μmol·L^-1) for 48 h, then the cell viability was measured by MTT assay, and calculated the value of IC₂₀ and IC₅₀ for drug. The capacity of clone formation of cells were detected by clone formation assay. The apoptotic of SACC-83 and SACC-LM cells were determined by flow cytometry. The expression levels of Bcl-2, Bax and caspase-3 mRNA and proteins in SACC-83 and SACC-LM cells were examined by q-PCR and Western blot method. RESULTS C₂₁ sterols at different doses decreased the viability and inhibited proliferation of SACC-83 and SACC-LM cells, and the concentration of IC₂₀ was 7.49 μmol·L^-1, and IC₅₀ was 38.34 μmol·L^-1 for SACC-83 cells. In addition, the concentration of IC₂₀ was 9.30 μmol·L^-1, and IC₅₀ was 46.04 μmol·L^-1 for SACC-LM cells. The apoptotic rate of SACC-83 and SACC-LM cells under concentration of IC₂₀ with C₂₁ sterols was significantly increased(P<0.05, P<0.01). The q-PCR and Western blot results showed that the expression level of Bcl-2 in the SACC-83 and SACC-LM cells was decreased significantly(P<0.01), and the expression level of Bax and Caspase-3 were increased significantly (P<0.05). CONCLUSION C₂₁ sterols in Marsdeniae Tenacissimae Caulis extract can depress the proliferation and promote apoptosis of SACC-83 and SACC-LM cells, which mechanism may be related to decreasing the Bcl-2 level, and increasing the Bax and Caspase-3 level.