Effect of Resveratrol on Metastasis of Nasopharyngeal Carcinoma and Its Related Mechanism
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摘要:
目的 研究白藜芦醇对鼻咽癌细胞侵袭、迁移能力的影响及其分子机制。 方法 （1）不同浓度白藜芦醇处理鼻咽癌CNE2细胞24、36、48 h，CCK8实验检测白藜芦醇对CNE2细胞增殖的抑制作用，确定适宜的浓度和时间以进行后续实验；（2）不同浓度白藜芦处理鼻咽癌CNE2细胞24 h：Transwell实验检测鼻咽癌细胞的侵袭、迁移能力；Western Blot实验检测与鼻咽癌转移相关的蛋白的表达水平；免疫荧光实验检测鼻咽癌细胞中NF-κB/p65的核转位情况；（3）裸鼠足掌皮下注射鼻咽癌CNE2细胞建立鼻咽癌原发灶模型，不同剂量白藜芦醇灌胃，观察腘窝淋巴结肿大情况，HE染色和免疫组织化学染色检测裸鼠腘窝淋巴结的转移情况。 结果 （1）CCK8实验发现，白藜芦醇对鼻咽癌CNE2细胞的增殖具有抑制作用，且作用呈浓度和时间依赖性（24，40 μmol/L，48 h μmol/L，P < 0.01；24，80 μmol/L及以上，36，40 μmol/L及以上，P < 0.001；36 h，20 μmol/L，P < 0.05，36，40 μmol/L及以上P < 0.001）；（2）Transwell实验发现：白藜芦醇能抑制鼻咽癌CNE-2细胞的侵袭迁移能力，随着白藜芦醇浓度的增高，对鼻咽癌CNE2细胞侵袭迁移能力的抑制作用增强（24，40 μmol/L，48，10 μmol/L，P < 0.01，24，80 μmol/L及以上，P < 0.001；36，20 μmol/L：P < 0.05，36，40 μmol/L及以上，48，20 μmol/L及以上，P < 0.001）；（3）Western Blot检测发现，随着白藜芦醇浓度增高：EMT相关蛋白E-cadherin的表达水平上升（20、40 μmol/L，P < 0.01，80 μmol/L，P < 0.001）；基质金属蛋白酶MMP9、MMP2的表达水平明显下调（MMP2，20 μmol/L，P < 0.05，40、80 mol/L，P < 0.001；MMP9，10、20 mol/L，P < 0.05，40、80 mol/L，P < 0.01）；转移相关蛋白Hsp27、Ezrin的磷酸化水平下调（Hsp27，20、40 μmol/L，P < 0.05；80 μmol/L，P < 0.01；Ezrin 40 μmol/L，P < 0.05； 80 μmol/L，P < 0.01）；随着白藜芦醇浓度增高：基质金属蛋白酶MMP9、MMP2的表达水平明显下调；EMT相关蛋白E-cadherin的表达水平上升；转移相关蛋白Ezrin、Hsp27的磷酸化水平下调；免疫荧光实验发现：白藜芦醇能抑制NF-κB/p65的核转位，且抑制作用呈浓度依赖性；（4）动物实验发现，白藜芦醇灌胃对荷瘤小鼠的体重无明显影响，但明显减轻瘤侧腘窝淋巴结的肿大程度（体积，50、100 mg/kg，P < 0.05；重量50 mg/kg，P < 0.05；100 mg/kg，P < 0.01）。 结论 组织学检测显示，白藜芦醇灌胃明显抑制了鼻咽癌细胞由足掌皮下成瘤部位向腘窝淋巴结的转移且抑制作用呈浓度依赖性。白藜芦醇可抑制鼻咽癌CNE2细胞的转移能力，其对鼻咽癌转移能力的抑制作用可能与降低鼻咽癌转移相关蛋白Ezrin和Hsp27的磷酸化水平、抑制NF-κB/p65的核转位，进而抑制EMT、降低MMP2和MMP9的表达水平有关。 Abstract:Objective To observe the effect of resveratrol on metastasis of nasopharyngeal carcinoma and explore its molecular mechanism. Methods （1）The inhibiting ability of resveratrol on nasopharyngeal carcinoma cells CNE2 was evaluated by CCK8 assay at different concentrations and times （24 h/40 μmol/L, P < 0.01; 24 h/80 μmol/L and above, P < 0.001; 36 h/20 μmol/L, P < 0.05; 36 h/40 μmol/L and above, P < 0.001; 48 h/10 μmol/L, P < 0.01, 48 h/20 μmol/L and above, P < 0.001）. （2） Resveratrol of different concentrations treated CNE2 cells for 24 hours: the migration and invasion ability of CNE2 cells were detected by Transwell experiment; Western Blot was used to explore the expression of E-cadherin, Vimentin, Hsp27, Ezrin and MMP; the nuclear translocation of NF-κB/p65 in CNE2 cells were detected by immunofluorescence assay increased the expression of E-cadherin （20, 40 μmol/L, P < 0.01; 80 μmol/L, P < 0.001）, inhibited the expression of MMP9 and MMP2 （MMP2, 20 μmol/L, P < 0.05; 40, 80 μmol/L, P < 0.001; MMP9, 10, 20 μmol/L, P < 0.05; 40, 80 μmol/L, P < 0.01）. （3）The LN metastasis experiment was constructed by injecting CNE2 cells into the left hind footpad of BALB/c nude mice. HE staining and immunohistochemistry staining was used to observe the effect of different doses of resveratrol gavage on the metastasis of nasopharyngeal carcinoma cells to popliteal lymph nodes Hsp27 and Ezrin （Hsp27, 20, 40 μmol/L, P < 0.05; 80 μmol/L, P < 0.01; Ezrin, 40 μmol/L, P < 0.05; 80 μmol/L, P < 0.01）. Results （1） CCK8 experiments showed that resveratrol had a toxic effect on nasopharyngeal carcinoma cell CNE2, and its toxicity was dose and time-dependent. （2） Transwell test found that resveratrol could inhibit the migration and invasion of nasopharyngeal carcinoma cell CNE2 in a dose and time-dependent manner. Western Blot assay showed that resveratrol significantly inhibited the expression of MMP9 and MMP2, and increased the expression of E-cadherin. Resveratrol inhibited the phosphorylation levels of Ezrin and Hsp27. Immunofluorescent experiments showed that resveratrol dose-dependently inhibited nuclear translocation of NF-κB/p65. （3） Animal experiments showed that resveratrol gavage treatment had no effect on the body weight of tumor-bearing nude mice, but significantly reduced the degree of swelling of the popliteal lymph nodes. Histological examination showed that resveratrol gavage significantly inhibited the metastasis of nasopharyngeal carcinoma cells from the subcutaneous tumor formation site to the popliteal lymph nodes in a dose-dependent manner. Conclusions Resveratrol can inhibit the metastasis of CNE2 cells in a time and dose-dependent manner. The mechanisms of resveratrol inhibiting the invasion and migration of CNE2 cells may relate to inhibiting the nuclear translocation of NF-κB/p65 by decreasing the phosphorylation value of Ezrin and Hsp27, and up-regulating the level of E-cadherin and down-regulating the expression of MMP2 and MMP9. -
Key words:
- Resveratrol /
- Nasopharyngeal carcinoma /
- Metastasis /
- Mechanism
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图 1 不同浓度白藜芦醇对鼻咽癌CNE2细胞增殖的抑制作用
A：白藜芦醇处理24 h；B：白藜芦醇处理36 h；C：白藜芦醇处理48 h。
Figure 1. The effect of different concentrations of Resveratrol on the proliferation of CNE2 cells
图 2 Transwell 实验检测白藜芦醇对鼻咽癌细胞CNE2侵袭和迁移能力的影响（结晶紫染色，×400）
A：结晶柴波色显示迁移细胞和侵袭细胞；B：不同浓度白藜芦醇处理后迁移细胞占比；C：不同浓度白藜芦醇处理后侵袭细胞占比。*P < 0.05，**P < 0.01，***P < 0.001。
Figure 2. Effect of Resveratrol treatment on migration and invasion of CNE2 cell by transwell assay （crystal violet staining，×400）
图 3 免疫荧光实验检测NF-κB/p65在鼻咽癌CNE2细胞中的定位（×400）
Figure 3. Immunofluorescence experiments showed the location of NF-κB/p65 in CNE2 cells （×400）
图 4 Western blot 检测白藜芦醇对转移相关蛋白表达水平的影响
*P < 0.05，**P < 0.01，***P < 0.001。
Figure 4. Effect of Resveratrol on expression of metastasis related proteins by western blot
图 5 白藜芦醇灌胃治疗对瘤侧腘窝淋巴结的肿大程度的影响
A：不同浓度白藜芦醇灌胃治疗后，瘤侧腘窝淋巴结大小；B：鼻咽癌足掌皮下移植及肿大腘窝淋巴结；C、D：不同浓度白藜芦醇灌胃治疗后瘤侧腘窝淋巴结重量及体积比较。与未给药组相比，*P < 0.05，**P < 0.01。
Figure 5. Effect of Resveratrol gavage treatment on the degree of swelling of the popliteal lymph nodes
图 6 白藜芦醇灌胃明显抑制了鼻咽癌细胞由足底向腘窝淋巴结的转移
A：HE 染色；B：免疫组化检测 。
Figure 6. Resveratrol gavage significantly inhibited the metastasis of nasopharyngeal carcinoma cells from the subcutaneous tumor formation site to the popliteal lymph nodes
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