硫氧还蛋白系统蛋白在舌癌中的表达及作用
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摘要
研究背景：
口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)是最常见的十大恶性肿瘤之一,而在我国口腔鳞状细胞癌中,舌鳞癌(tongue squamous cell carcinoma, TSCC)的发病率居第一位。舌癌生长快,侵袭性较强,易发生淋巴结转移,预后相对较差。其治疗方法常是手术、放疗、化疗等的综合治疗,但患者的五年生存率仍然很低,因此寻找积极有效的预防途径及有效的治疗药物显得尤为迫切和必要。
硫氧还蛋白系统是生物体内主要的抗氧化系统之一,由硫氧还蛋白(Thioredoxin, Trx)、硫氧还蛋白还原酶(Thioredoxin reductase, TrxR)和NADPH三部分组成,是一个广泛分布的NADPH依赖性二硫化物还原酶系统。Trx通过直接清除过氧化氢以及还原某些特异性酶而发挥其抗氧化作用,而TrxR是唯一可以还原Trx的还原剂。学者证实,Trx与TrxR在多种肿瘤组织中呈高表达状态,且高表达的Trx与细胞增殖活性的增高呈正相关关系,因此硫氧还蛋白系统在肿瘤细胞的增殖和分化中起着重要的作用。抑制硫氧还蛋白系统的功能可能会抑制肿瘤细胞的生长。因此,本研究旨在探讨Trx系统蛋白在舌癌中表达情况,以及其在舌癌发生发展中的作用。研究内容共分为以下三部分：
第一部分硫氧还蛋白系统蛋白在人舌癌组织中的表达及其与临床病理因素、预后的关系
目的检测硫氧还蛋白(Trx, Thioredoxin)和硫氧还蛋白还原酶(Thioredoxin reductase, TrxR)在人舌鳞癌(tongue squamous cell carcinoma, TSCC)组织中的蛋白表达水平,及与临床病理因素、预后的关系。材料和方法选取武汉大学口腔医院2003年至2005年经手术切除的舌癌组织共65例作为研究对象,并以正常口腔黏膜上皮做对照。采用免疫组化SP法分别检测Trx和TrxR-1的表达水平,应用SPSS16.0进行统计分析：采用卡方检验,四格表确切概率法以及相关性分析等方法检测Trx和TrxR-1在舌癌组织和正常口腔黏膜上皮的表达差异及相关性,并对全部病例进行随访。结果Trx与TrxR-1主要表达在肿瘤细胞的细胞质,少数表达于细胞核。65例肿瘤组织中,Trx与TrxR-1的表达率分别为100%(65／65),95.4%(62／65)；而在10例正常口腔黏膜中,Trx在9例、TrxR-1在10例中均为低表达。舌癌组织中Trx与TrxR-1的表达显著高于正常对照(P=0.000,0.000)。在与临床病理学因素的比较中发现,Trx与肿瘤分化程度明显相关(P=0.001)；而TrxR-1与肿瘤分化程度,性别,年龄,TNM分期,淋巴转移,复发等均无显著相关性(P>0.05)。相关分析表明,Trx与TrxR-1呈高度正相关关系,随着TrxR-1的表达增高,Trx也随之增高(P=0.001)。生存分析发现,Trx低表达组与Trx中,高度表达组间存在显著的差异性,Trx中度及高度表达的患者预后较差,生存时间较短(P=0.033)。结论舌癌组织中Trx与TrxR-1过表达,并且二者呈显著相关性,高表达的Trx可以预测舌癌的不良预后,还需要进一步的实验证明Trx与TrxR-1在舌癌发生中的具体作用机制。
第二部分硫氧还蛋白还原酶抑制剂杨梅黄酮对人舌癌CAL-27细胞增殖、凋亡的影响
目的采用体外培养人舌癌CAL-27细胞的方法,观察不同浓度的杨梅黄酮对其生长产生的作用。材料和方法光学显微镜观察未经和经杨梅黄酮作用的CAL-27细胞的形态学变化；应用透射电镜观察经120μmol/L杨梅黄酮作用12h的细胞超微结构变化；浓度为120μmol/L的杨梅黄酮作用12h后,Hoechst33258荧光染色观察细胞形态；浓度为40、80、120、160、200μmol／L杨梅黄酮作用于CAL-27细胞,分别培养6,12,24h后,采用MTT比色法,检测CAL-27细胞增殖活性的改变,观察不同浓度杨梅黄酮作用不同时间后对人舌癌CAL-27的毒性效应；经浓度为40、80、120、160、200μmol／L杨梅黄酮作用于CAL-27细胞12h后,用Annexin V/PI双染色流式细胞术法检测细胞凋亡率；采用统计学软件SPSS16.0对数据进行统计学处理,数据用均数标准差(X±S)表示。结果经杨梅黄酮作用后,CAL-27细胞的形态以及超微结构发生了明显变化。透射电镜及荧光染色观察到肿瘤细胞发生了明显的形态学改变,可见细胞骨架破坏,染色体固缩,细胞核碎裂以及凋亡小体形成等。MTT结果显示杨梅黄酮对CAL-27细胞具有明显的生长抑制作用,并呈现明显的浓度和时间依赖性；不同浓度组间、不同时间组间生长抑制率均有显著性差异(P<0.05)。200μmol/L杨梅黄酮作用24h后细胞生长抑制率达94.21%。流式细胞仪检测发现肿瘤细胞的凋亡率随着杨梅黄酮浓度的增高而升高,由(14.60%±2.44%)增加到(62.91%±3.54%),各实验组与对照组相比具有统计学意义(P<0.05)。结论杨梅黄酮对人舌癌细胞CAL-27具有明显的生长抑制及促进凋亡作用,并且作用强度随着杨梅黄酮浓度的升高以及时间的延长而增强。
第三部分杨梅黄酮促进人舌癌CAL-27细胞凋亡的作用机制
目的分别研究在不同时间、不同浓度的杨梅黄酮作用下,人舌癌细胞CAL-27中TrxR的活性变化,重点研究Trx下游调控分子及caspase3在杨梅黄酮促进舌癌细胞凋亡过程中的参与机制。材料和方法浓度为40、80、120、160、200μmol/L杨梅黄酮作用于CAL-27细胞,分别培养6、12、24h后,用TrxR活性试剂盒检测TrxR的活性；浓度为40、80、120、160、200μmol/L杨梅黄酮作用于CAL-27细胞培养12h后,或浓度为120μmol/L杨梅黄酮作用于肿瘤细胞分别培养1、3、6及12h后,采用免疫蛋白印记法(Western Blotting)检测药物作用后的CAL-27细胞中p-ASK1, ASK1, p-p38, p38, p-JNK和JNK的蛋白表达水平,并对检测结果进行半定量分析；应用caspase3活性检测试剂盒测定经浓度为40、80、120、160、200μmol/L杨梅黄酮分别作用6、12、24h后肿瘤细胞caspase3的活性；采用统计学软件SPSS16.0对数据进行统计学处理,数据用均数±标准差(x±s)表示。结果杨梅黄酮作用后的CAL-27细胞的TrxR活性被显著抑制,并具有浓度及时间依赖性；120μmol/L作用12h,TrxR活性抑制率达70%以上,200μmol/L作用24h以上,TrxR活性几乎被完全抑制。Western Blotting检测结果显示经杨梅黄酮作用后,CAL-27细胞的ASKl,p38和JNK表达减弱,相反p-ASK1, p-p38和p-JNK表达逐渐升高且具有时间浓度依赖性。Caspase3活性检测结果显示,经杨梅黄酮作用后的CAL-27细胞caspase3活性增强,并呈显著的浓度及时间依赖性,结果具有统计学意义(P<0.05)。结论杨梅黄酮诱导人舌鳞癌CAL-27细胞的凋亡作用部分通过调控TrxR/Trx/ASK1/p38/JNK信号通路来实现。
Background
Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the top ten at present, while the incidence of tongue squamous cell carcinoma (TSCC) ranks the first among the oral cancer in China. TSCC is known for its invasive nature, propensity for lymph node metastasis, and poor prognosis. Comprehensive treatment usually involves surgery, radiotherapy, chemotherapy etc., but the five-year survival of TSCC patients is still low. To explore positively effective preventive measures and drugs becomes very urgent and necessary.
The thioredoxin (Trx) system, comprising Trx and Trx reductase (TrxR) plus a reduced form of NADPH, is a major antioxidant system integral to maintenance of the intracellular redox state. Trx protein is one of the most important regulators of redox balance and, thus, of redox-controlled cell functions. The oxidized form of Trx can only be reduced by TrxR within the cell. Several studies have investigated Trx system function or expression in a variety of human tumors and overexpression of Trx was related to poor prognosis and increased proliferation of tumor cells. Therefore, the Trx system protein may play an important role in proliferation and differentiation of tumor cells. Inhibition of the Trx system protein function may induce growth-inhibition of tumor cells. Therefore, the purpose of the present study was to investigate the expression levels of Trx and TrxR-1in human TSCC tissues and the relationships with clinicopathological features and clinical outcome, as well as its role in development of TSCC. The study was divided into three parts as follows:
Part One:Overexpression of thioredoxin system proteins predicts poor prognosis in patients with squamous cell carcinoma of the tongue
Objective To evaluate the expression of Trx and TrxR-1and the relationships with clinicopathological features and clinical outcome in TSCC. Materials and Methods Immunohistochemistry was employed to analyze the protein expression levels of Trx and TrxR-1in65TSCC tissue samples and10normal oral mucosa samples. The results were then evaluated semiquantitatively and compared to other clinicopathological variables. Statistical analysis was performed with the SPSS16.0. P <0.05was considered statistically significant. Results The expression of Trx and TrxR-1was predominantly manifested in the cytoplasm of tumour cells, with diffuse distribution in nuclei. The percentages of TSCC specimens with Trx and TrxR-1expression were100%(65/65) and95.4%(62/65), respectively, whereas the corresponding rates of expression in normal tissues were almost low, with Trx (9/10) and TrxR-1(10/10). Both Trx and TrxR-1expression levels were significantly higher in TSCC tissues as compared with the10normal oral mucous samples (P<0.01). A highly significant association between Trx and TrxR-1expression in TSCCs was revealed (P=0.001), and the expression of Trx was correlated with tumour cell differentiation (P=0.001). Moreover, Kaplan-Meier analysis revealed that Trx expression and TNM stage were significantly related with5-year survival rate (P=0.033,0.000), while TrxR-1expression was not associated with survival (P=0.092).
Conclusion The results indicated that high expression of Trx and TrxR-1was associated with tumourigenesis in TSCC, and overexpression of Trx might predict poor prognosis. Much additional studies into these areas are required to fully elucidate the role of the Trx system in tumourigenesis and as a possible therapeutic target.
Part Two:Induction of apoptosis of human tongue squamous cell carcinoma cells by myricetin targeting thioredoxin reductase
Objective To detect the proliferative and apoptotic effect on CAL-27cells when treated with myricetin. Materials and Methods Morphology changes of CAL-27cells treated or without treated by myricetin were observed by optical microscope; the changes of ultramicrostructure in the cells treated with myricetin at120μmol/L for12h were observed by transmission electron microscope; tumor cells were treated with different concentrations of myricetin for6,12, and24h and detected the growth inhibition by MTT assay; cancer cells were treated with Hoechst33258for12h and fluorescent microscopy was used to observe the morphological feature; cells were treated with0,40,80,120,160, and200μmol/L of myricetin for12h and stained with Annexin V and PI, and florescence intensity was measured by flow cytometry; SPSS16.0was used for all analysis and data was indicated by mean±standard deviation (χ±S). Results The morphology changes of CAL-27cells treated with myricetin was observed. When treated with certain myricetin, we observed the cancer cells developed morphological changes, including chromatin condensation, nuclear fragmentation, and apoptotic bodies by transmission electron microscope and fluorescent microscopy. To determine the proliferative activity of HOSCC cells, we found that cell viability was inhibited by myricetin in a time-, and dose-dependent manner. There were significant differences between different concentration-groups and time-groups (P<0.05). The percentage of growth inhibition of the cancer cells treated with200μmol/L myricetin for24h reached to94.21%. Flow cytometry showed that the apoptosis rate of CAL-27cells increased from (14.60%±2.44%) to (62.91%±3.54%) gradually, with the prolonging of myricetin role. There were significant differences (P<0.05). Conclusion Being treated with myricetin induced growth-inhibition and apoptosis of human tongue squamous cell carcinoma (HTSCC) CAL-27cells.
Part Three:The apoptotic mechanisms of human tongue squamous cell carcinoma CAL-27cells treated with myricetin
Objective To investigate the TrxR activity in CAL-27cells treated with certain myricetin and explain the possible molecular mechanism with special emphasis on the downstream regulator of Trx and caspase for induction of apoptosis of human tongue squamous cell carcinoma (HTSCC) CAL-27cells by myricetin. Materials and Methods CAL-27cell were treated with different concentrations of myricetin in serum-free medium for6,12, and24hours and the cells lysates were subjected to TrxR activity assay; the protein expression status of p-ASK1, ASK1, p-p38, p38, p-JNK and JNK in the CAL-27cells treated with myricetin at40,80,120,160, and200μmol/L for12h, or120μmol/L for30min,1,6, and12h was respectively detected by western blotting assay and the test results were investigated through semi-quantitative analysis; CAL-27cell were treated with different concentrations of myricetin for6,12, and24hours and the cells lysates were subjected to caspase3activity assay; SPSS16.0was used for all analysis and data was indicated by mean±standard deviation (χ±S).
Results TrxR activity of CAL-27was decreased when treated with myricetin in a time-, and dose-dependent manner. When treated with120μmol/L myricetin for12h, the rate of inhibition exceeded90%;200μmol/L myricetin for24h, the TrxR activity was almost absent. Western blotting analysis demonstrated that myricetin treatment increased protein phosphorylation of ASK1, p38, and JNK in CAL-27cells with a time-, and dose-dependent manner. Caspase3activity was also increased when treated with myricetin in CAL-27cells with a time-, and dose-dependent manner. There was also statistical difference (P<0.05). Conclusion Myricetin-exerted apoptotic effects on human tongue squamous cell carcinoma CAL-27cells involve TrxR/Trx/ASK1/p38/JNK signaling pathway.
口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)是最常见的十大恶性肿瘤之一,而在我国口腔鳞状细胞癌中,舌鳞癌(tongue squamous cell carcinoma, TSCC)的发病率居第一位。舌癌生长快,侵袭性较强,易发生淋巴结转移,预后相对较差。其治疗方法常是手术、放疗、化疗等的综合治疗,但患者的五年生存率仍然很低,因此寻找积极有效的预防途径及有效的治疗药物显得尤为迫切和必要。
硫氧还蛋白系统是生物体内主要的抗氧化系统之一,由硫氧还蛋白(Thioredoxin, Trx)、硫氧还蛋白还原酶(Thioredoxin reductase, TrxR)和NADPH三部分组成,是一个广泛分布的NADPH依赖性二硫化物还原酶系统。Trx通过直接清除过氧化氢以及还原某些特异性酶而发挥其抗氧化作用,而TrxR是唯一可以还原Trx的还原剂。学者证实,Trx与TrxR在多种肿瘤组织中呈高表达状态,且高表达的Trx与细胞增殖活性的增高呈正相关关系,因此硫氧还蛋白系统在肿瘤细胞的增殖和分化中起着重要的作用。抑制硫氧还蛋白系统的功能可能会抑制肿瘤细胞的生长。因此,本研究旨在探讨Trx系统蛋白在舌癌中表达情况,以及其在舌癌发生发展中的作用。研究内容共分为以下三部分：
第一部分硫氧还蛋白系统蛋白在人舌癌组织中的表达及其与临床病理因素、预后的关系
目的检测硫氧还蛋白(Trx, Thioredoxin)和硫氧还蛋白还原酶(Thioredoxin reductase, TrxR)在人舌鳞癌(tongue squamous cell carcinoma, TSCC)组织中的蛋白表达水平,及与临床病理因素、预后的关系。材料和方法选取武汉大学口腔医院2003年至2005年经手术切除的舌癌组织共65例作为研究对象,并以正常口腔黏膜上皮做对照。采用免疫组化SP法分别检测Trx和TrxR-1的表达水平,应用SPSS16.0进行统计分析：采用卡方检验,四格表确切概率法以及相关性分析等方法检测Trx和TrxR-1在舌癌组织和正常口腔黏膜上皮的表达差异及相关性,并对全部病例进行随访。结果Trx与TrxR-1主要表达在肿瘤细胞的细胞质,少数表达于细胞核。65例肿瘤组织中,Trx与TrxR-1的表达率分别为100%(65／65),95.4%(62／65)；而在10例正常口腔黏膜中,Trx在9例、TrxR-1在10例中均为低表达。舌癌组织中Trx与TrxR-1的表达显著高于正常对照(P=0.000,0.000)。在与临床病理学因素的比较中发现,Trx与肿瘤分化程度明显相关(P=0.001)；而TrxR-1与肿瘤分化程度,性别,年龄,TNM分期,淋巴转移,复发等均无显著相关性(P>0.05)。相关分析表明,Trx与TrxR-1呈高度正相关关系,随着TrxR-1的表达增高,Trx也随之增高(P=0.001)。生存分析发现,Trx低表达组与Trx中,高度表达组间存在显著的差异性,Trx中度及高度表达的患者预后较差,生存时间较短(P=0.033)。结论舌癌组织中Trx与TrxR-1过表达,并且二者呈显著相关性,高表达的Trx可以预测舌癌的不良预后,还需要进一步的实验证明Trx与TrxR-1在舌癌发生中的具体作用机制。
第二部分硫氧还蛋白还原酶抑制剂杨梅黄酮对人舌癌CAL-27细胞增殖、凋亡的影响
目的采用体外培养人舌癌CAL-27细胞的方法,观察不同浓度的杨梅黄酮对其生长产生的作用。材料和方法光学显微镜观察未经和经杨梅黄酮作用的CAL-27细胞的形态学变化；应用透射电镜观察经120μmol/L杨梅黄酮作用12h的细胞超微结构变化；浓度为120μmol/L的杨梅黄酮作用12h后,Hoechst33258荧光染色观察细胞形态；浓度为40、80、120、160、200μmol／L杨梅黄酮作用于CAL-27细胞,分别培养6,12,24h后,采用MTT比色法,检测CAL-27细胞增殖活性的改变,观察不同浓度杨梅黄酮作用不同时间后对人舌癌CAL-27的毒性效应；经浓度为40、80、120、160、200μmol／L杨梅黄酮作用于CAL-27细胞12h后,用Annexin V/PI双染色流式细胞术法检测细胞凋亡率；采用统计学软件SPSS16.0对数据进行统计学处理,数据用均数标准差(X±S)表示。结果经杨梅黄酮作用后,CAL-27细胞的形态以及超微结构发生了明显变化。透射电镜及荧光染色观察到肿瘤细胞发生了明显的形态学改变,可见细胞骨架破坏,染色体固缩,细胞核碎裂以及凋亡小体形成等。MTT结果显示杨梅黄酮对CAL-27细胞具有明显的生长抑制作用,并呈现明显的浓度和时间依赖性；不同浓度组间、不同时间组间生长抑制率均有显著性差异(P<0.05)。200μmol/L杨梅黄酮作用24h后细胞生长抑制率达94.21%。流式细胞仪检测发现肿瘤细胞的凋亡率随着杨梅黄酮浓度的增高而升高,由(14.60%±2.44%)增加到(62.91%±3.54%),各实验组与对照组相比具有统计学意义(P<0.05)。结论杨梅黄酮对人舌癌细胞CAL-27具有明显的生长抑制及促进凋亡作用,并且作用强度随着杨梅黄酮浓度的升高以及时间的延长而增强。
第三部分杨梅黄酮促进人舌癌CAL-27细胞凋亡的作用机制
目的分别研究在不同时间、不同浓度的杨梅黄酮作用下,人舌癌细胞CAL-27中TrxR的活性变化,重点研究Trx下游调控分子及caspase3在杨梅黄酮促进舌癌细胞凋亡过程中的参与机制。材料和方法浓度为40、80、120、160、200μmol/L杨梅黄酮作用于CAL-27细胞,分别培养6、12、24h后,用TrxR活性试剂盒检测TrxR的活性；浓度为40、80、120、160、200μmol/L杨梅黄酮作用于CAL-27细胞培养12h后,或浓度为120μmol/L杨梅黄酮作用于肿瘤细胞分别培养1、3、6及12h后,采用免疫蛋白印记法(Western Blotting)检测药物作用后的CAL-27细胞中p-ASK1, ASK1, p-p38, p38, p-JNK和JNK的蛋白表达水平,并对检测结果进行半定量分析；应用caspase3活性检测试剂盒测定经浓度为40、80、120、160、200μmol/L杨梅黄酮分别作用6、12、24h后肿瘤细胞caspase3的活性；采用统计学软件SPSS16.0对数据进行统计学处理,数据用均数±标准差(x±s)表示。结果杨梅黄酮作用后的CAL-27细胞的TrxR活性被显著抑制,并具有浓度及时间依赖性；120μmol/L作用12h,TrxR活性抑制率达70%以上,200μmol/L作用24h以上,TrxR活性几乎被完全抑制。Western Blotting检测结果显示经杨梅黄酮作用后,CAL-27细胞的ASKl,p38和JNK表达减弱,相反p-ASK1, p-p38和p-JNK表达逐渐升高且具有时间浓度依赖性。Caspase3活性检测结果显示,经杨梅黄酮作用后的CAL-27细胞caspase3活性增强,并呈显著的浓度及时间依赖性,结果具有统计学意义(P<0.05)。结论杨梅黄酮诱导人舌鳞癌CAL-27细胞的凋亡作用部分通过调控TrxR/Trx/ASK1/p38/JNK信号通路来实现。
Background
Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the top ten at present, while the incidence of tongue squamous cell carcinoma (TSCC) ranks the first among the oral cancer in China. TSCC is known for its invasive nature, propensity for lymph node metastasis, and poor prognosis. Comprehensive treatment usually involves surgery, radiotherapy, chemotherapy etc., but the five-year survival of TSCC patients is still low. To explore positively effective preventive measures and drugs becomes very urgent and necessary.
The thioredoxin (Trx) system, comprising Trx and Trx reductase (TrxR) plus a reduced form of NADPH, is a major antioxidant system integral to maintenance of the intracellular redox state. Trx protein is one of the most important regulators of redox balance and, thus, of redox-controlled cell functions. The oxidized form of Trx can only be reduced by TrxR within the cell. Several studies have investigated Trx system function or expression in a variety of human tumors and overexpression of Trx was related to poor prognosis and increased proliferation of tumor cells. Therefore, the Trx system protein may play an important role in proliferation and differentiation of tumor cells. Inhibition of the Trx system protein function may induce growth-inhibition of tumor cells. Therefore, the purpose of the present study was to investigate the expression levels of Trx and TrxR-1in human TSCC tissues and the relationships with clinicopathological features and clinical outcome, as well as its role in development of TSCC. The study was divided into three parts as follows:
Part One:Overexpression of thioredoxin system proteins predicts poor prognosis in patients with squamous cell carcinoma of the tongue
Objective To evaluate the expression of Trx and TrxR-1and the relationships with clinicopathological features and clinical outcome in TSCC. Materials and Methods Immunohistochemistry was employed to analyze the protein expression levels of Trx and TrxR-1in65TSCC tissue samples and10normal oral mucosa samples. The results were then evaluated semiquantitatively and compared to other clinicopathological variables. Statistical analysis was performed with the SPSS16.0. P <0.05was considered statistically significant. Results The expression of Trx and TrxR-1was predominantly manifested in the cytoplasm of tumour cells, with diffuse distribution in nuclei. The percentages of TSCC specimens with Trx and TrxR-1expression were100%(65/65) and95.4%(62/65), respectively, whereas the corresponding rates of expression in normal tissues were almost low, with Trx (9/10) and TrxR-1(10/10). Both Trx and TrxR-1expression levels were significantly higher in TSCC tissues as compared with the10normal oral mucous samples (P<0.01). A highly significant association between Trx and TrxR-1expression in TSCCs was revealed (P=0.001), and the expression of Trx was correlated with tumour cell differentiation (P=0.001). Moreover, Kaplan-Meier analysis revealed that Trx expression and TNM stage were significantly related with5-year survival rate (P=0.033,0.000), while TrxR-1expression was not associated with survival (P=0.092).
Conclusion The results indicated that high expression of Trx and TrxR-1was associated with tumourigenesis in TSCC, and overexpression of Trx might predict poor prognosis. Much additional studies into these areas are required to fully elucidate the role of the Trx system in tumourigenesis and as a possible therapeutic target.
Part Two:Induction of apoptosis of human tongue squamous cell carcinoma cells by myricetin targeting thioredoxin reductase
Objective To detect the proliferative and apoptotic effect on CAL-27cells when treated with myricetin. Materials and Methods Morphology changes of CAL-27cells treated or without treated by myricetin were observed by optical microscope; the changes of ultramicrostructure in the cells treated with myricetin at120μmol/L for12h were observed by transmission electron microscope; tumor cells were treated with different concentrations of myricetin for6,12, and24h and detected the growth inhibition by MTT assay; cancer cells were treated with Hoechst33258for12h and fluorescent microscopy was used to observe the morphological feature; cells were treated with0,40,80,120,160, and200μmol/L of myricetin for12h and stained with Annexin V and PI, and florescence intensity was measured by flow cytometry; SPSS16.0was used for all analysis and data was indicated by mean±standard deviation (χ±S). Results The morphology changes of CAL-27cells treated with myricetin was observed. When treated with certain myricetin, we observed the cancer cells developed morphological changes, including chromatin condensation, nuclear fragmentation, and apoptotic bodies by transmission electron microscope and fluorescent microscopy. To determine the proliferative activity of HOSCC cells, we found that cell viability was inhibited by myricetin in a time-, and dose-dependent manner. There were significant differences between different concentration-groups and time-groups (P<0.05). The percentage of growth inhibition of the cancer cells treated with200μmol/L myricetin for24h reached to94.21%. Flow cytometry showed that the apoptosis rate of CAL-27cells increased from (14.60%±2.44%) to (62.91%±3.54%) gradually, with the prolonging of myricetin role. There were significant differences (P<0.05). Conclusion Being treated with myricetin induced growth-inhibition and apoptosis of human tongue squamous cell carcinoma (HTSCC) CAL-27cells.
Part Three:The apoptotic mechanisms of human tongue squamous cell carcinoma CAL-27cells treated with myricetin
Objective To investigate the TrxR activity in CAL-27cells treated with certain myricetin and explain the possible molecular mechanism with special emphasis on the downstream regulator of Trx and caspase for induction of apoptosis of human tongue squamous cell carcinoma (HTSCC) CAL-27cells by myricetin. Materials and Methods CAL-27cell were treated with different concentrations of myricetin in serum-free medium for6,12, and24hours and the cells lysates were subjected to TrxR activity assay; the protein expression status of p-ASK1, ASK1, p-p38, p38, p-JNK and JNK in the CAL-27cells treated with myricetin at40,80,120,160, and200μmol/L for12h, or120μmol/L for30min,1,6, and12h was respectively detected by western blotting assay and the test results were investigated through semi-quantitative analysis; CAL-27cell were treated with different concentrations of myricetin for6,12, and24hours and the cells lysates were subjected to caspase3activity assay; SPSS16.0was used for all analysis and data was indicated by mean±standard deviation (χ±S).
Results TrxR activity of CAL-27was decreased when treated with myricetin in a time-, and dose-dependent manner. When treated with120μmol/L myricetin for12h, the rate of inhibition exceeded90%;200μmol/L myricetin for24h, the TrxR activity was almost absent. Western blotting analysis demonstrated that myricetin treatment increased protein phosphorylation of ASK1, p38, and JNK in CAL-27cells with a time-, and dose-dependent manner. Caspase3activity was also increased when treated with myricetin in CAL-27cells with a time-, and dose-dependent manner. There was also statistical difference (P<0.05). Conclusion Myricetin-exerted apoptotic effects on human tongue squamous cell carcinoma CAL-27cells involve TrxR/Trx/ASK1/p38/JNK signaling pathway.
引文
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