Skip to main content
PMC home page
Journal of Southern Medical University logoLink to Journal of Southern Medical University
. 2020 Sep 20;40(9):1288–1294. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2020.09.10

紫草素通过抑制人睾丸癌I-10和精原细胞瘤TCAM-2糖酵解诱导细胞死亡

Shikonin induces cell death by inhibiting glycolysis in human testicular cancer I-10 and seminoma TCAM-2 cells

姚 越 1, 张 冲 1, 韩 兵 1, 汤 玉锐 1, 熊 言骏 1, 汪 盛 1,*
PMCID: PMC7544591  PMID: 32990238

Abstract

目的

探究紫草素对睾丸癌细胞I-10和精原细胞瘤TCAM-2死亡形式的影响，并从线粒体功能和糖酵解方面探讨其可能机制。

方法

I-10细胞组加入浓度分别为0 μmol/L（对照组）、1.2、1.4、1.6 μmol/L的紫草素，TCAM-2细胞组加入浓度分别为0 μmol/L（对照组）、0.5、1、1.5 μmol/L的紫草素，JC-1试剂盒和ROS试剂盒分别用来检测线粒体膜电位和活性氧的变化；乳酸试剂盒检测胞内乳酸变化；MTT法检测细胞增殖抑制；透射电镜观察细胞死亡形式；Annexin V-FITC/PI双染法检测细胞凋亡；Western blot法检测线粒体通路相关凋亡蛋白Bax、Bcl-2、cleaved caspase 3和自噬相关蛋白LC3B以及糖酵解相关蛋白PKM2、GLUT1、HK2的相对表达水平。

结果

MTT结果表明，紫草素能够随时间和剂量依赖性的抑制I-10和TCAM-2细胞的增殖（P < 0.05），I-10组24、48、72 h的IC50值分别为1.8、1.36和1.16 μmol/L，TCAM-2组24、48、72 h的IC50值分别为2.37、0.8和0.41 μmol/ L；紫草素能通过下调I-10和TCAM-2细胞线粒体膜电位和增加细胞活性氧水平而影响其线粒体功能（P < 0.05），抑制乳酸水平影响细胞糖酵解能力（P < 0.05）；透射电镜和Annexin V-FITC/PI双染法检测出紫草素能够诱导I-10和TCAM-2细胞凋亡和过度自噬现象（P < 0.05）；Western blot证明，紫草素可以通过下调线粒体通路相关凋亡蛋白Bax、Bcl-2、cleaved caspase 3的表达而影响I-10和TCAM-2细胞线粒体功能，下调PKM2、GLUT1、HK2影响其糖酵功能，通过上调LC3B增加细胞自噬（P < 0.05）。

结论

紫草素对睾丸癌细胞I-10和TCAM-2具有增殖抑制及诱导凋亡和增加自噬作用，其机制可能为紫草素影响I-10和TCAM-2细胞能量代谢功能。

Keywords: 睾丸癌, 精原细胞瘤, 紫草素, 糖酵解, 死亡形式

睾丸癌是最常见的恶性肿瘤之一，在20~35岁的成年男性中[1-2]，睾丸癌的存活率很高[3]。顺铂（DDP）被认为是睾丸癌重要而有效的化学治疗剂治疗[4-5]，不幸的是，耐药性随着治疗的继续[6-12]，严重限制了DDP的临床使用。因此，寻找可以抑制睾丸癌新药物至关重要。

紫草素是一种天然染料和食品添加剂，具有抗病毒[13]，抗氧化[14]，抗炎[15]，加速伤口愈合和增强免疫力的特性[16]。研究表明，紫草素可以有效抑制PKM2，但对PKM1或丙酮酸激酶L（PKL）没有抑制作用，可限制糖酵解和ATP的产生[16]。紫草素已显示出诱导多种癌细胞凋亡的作用，包括HL60人早幼粒细胞白血病细胞系，肝癌，前列腺癌，结直肠癌，口腔鳞状细胞癌，基底细胞和骨肉瘤，宫颈癌HeLa，膀胱癌细胞系T24[18-27]。目前尚未有紫草素对人睾丸癌I-10和精原细胞瘤TCAM-2细胞的实验研究，本实验主要探讨紫草素对I-10和TCAM-2细胞的死亡形式，并从能量代谢（线粒体和糖酵解）方面探讨其可能机制，为临床治疗下睾丸癌提供新的策略。

1. 材料和方法

1.1. 实验材料、试剂

人睾丸癌I-10和精原细胞瘤TCAM-2细胞购自上海细胞库，DMEM培养基、胰蛋白酶（Hyclone），胎牛血清（fetal bovine serum, FBS）（浙江天杭生物科技），MTT试剂、JC-1、ROS、乳酸、Annexin V-FITC/PI双染试剂盒（上海碧云天），Bax、Bcl-2、cleaved caspase3、LC3B、PKM2、GLUT1、HK2蛋白抗体（CST）。

1.2. 细胞培养

人睾丸癌I-10精原细胞瘤TCAM-2细胞培养在含10%胎牛血清、100 mg/L链霉素、100 U/mL青霉素的DMEM培养基中，放置于37 ℃、5% CO2饱和湿度的细胞恒温培养箱中日常培养。

1.3. JC-1染色检测细胞线粒体膜电位差

将I-10和TCAM-2细胞制成分别单细胞悬液，每孔4×105个细胞接种于六孔板中，I-10细胞组加入浓度分别为0、1.2、1.4、1.6 µmol/L的紫草素，TCAM-2细胞组加入浓度分别为0、0.5、1、1.5 µmol/L的紫草素，给药12 h时，收集细胞，每孔加入1 mL JC-1染色溶液，充分混合，然后在细胞培养箱中于37 ℃孵育20 min（避光）。1200 r/min离心5 min，弃去上清液，并用JC-1染色缓冲液（1x）洗涤2次。最后用流式细胞仪上机检测，并将实验重复3次。

1.4. 细胞内ATP水平检测

将I-10和TCAM-2细胞分别制成单细胞悬液，每孔4×105个细胞接种于六孔板中，I-10细胞组加入浓度分别为0、1.2、1.4、1.6 µmol/L的紫草素，TCAM-2细胞组加入浓度分别为0、0.5、1、1.5 µmol/L的紫草素，作用6 h，离心收集细胞，弃去上清液。每个孔加入200 μL裂解液裂解20 min，剧烈涡旋以完全裂解细胞。使用4 ℃离心机以12 000 r/min将混合物离心5 min，并将上清液（保留在冰上）用于后续分析。裂解液中的蛋白质浓度通过BCA蛋白测定试剂盒进行定量。每个样中加入100 μL ATP工作液（1:9），并在室温下放置5 min（使反应在黑暗中进行），使背景ATP全部消耗，立即向每孔添加20 μL样品，用酶标仪测量吸光度，并将实验重复3次。

1.5. 活性氧（ROS）检测

将I-10和TCAM-2细胞分别制成单细胞悬液，每孔4×105细胞接种于六孔板中，I-10细胞组加入浓度分别为0、1.2、1.4、1.6 µmol/L的紫草素，TCAM-2细胞组加入浓度分别为0、0.5、1、1.5 µmol/L的紫草素，给药6 h，收集细胞，首先用无血清培养基将DCFH-DA以1:1000比例稀释至最终浓度10 μmol/ L。每个样加入1 mL稀释的DCFH-DA，并在37 ℃的细胞培养箱中培养20 min，用PBS洗涤2次后，用流式细胞仪上机检测，并将实验重复3次。

1.6. 乳酸的测定

将I-10和TCAM-2细胞分别制成单细胞悬液，每孔4×105细胞接种于六孔板中，I-10细胞组加入浓度分别为0、1.2、1.4、1.6 µmol/L的紫草素，TCAM-2细胞组加入浓度分别为0、0.5、1、1.5 µmol/L的紫草素，给药24 h时，然后收集细胞，重悬于适量的裂解物中，并转移至1.5 mL EP管中。将细胞在-20 ℃下裂解3次，并在4 ℃下以12 000 r/min离心30 min，上清液用于结果分析。参照乳酸测定试剂盒步骤（BioVision）进行测试，并将实验重复3次。

1.7. MTT测定

将I-10和TCAM-2细胞分别制成单细胞悬液，每孔1×104细胞接种于六孔板中，I-10细胞组加入浓度分别为0、1.2、1.4、1.6 µmol/L的紫草素，TCAM-2细胞组加入浓度分别为0、0.5、1、1.5 µmol/L的紫草素，于24、48、72 h时每孔加入15 µL MTT溶液，放入培养箱中孵育4 h，小心吸去培养液，每孔加入150 µL DMSO，置于37 ℃培养箱继续孵育30 min待甲臜完全溶解，在490 nm波长处用酶联免疫检测仪测吸光度值（A490 nm）。计算细胞存活率：细胞存活率（%）=实验组A490 nm/对照组A490 nm×100%。取平均值绘制药物剂量效应曲线，计算药物的半数抑制浓度（IC50）。

1.8. 透射电子显微镜观察细胞死亡的方式

将I-10和TCAM-2细胞分别制成单细胞悬液，接种于大皿中，待细胞长到70%左右时，I-10组加入浓度分别为0 µmol/L、1.4 µmol/L的紫草素，TCAM-2组加入浓度分别为0 µmol/L、1 µmol/L的紫草素，给药24 h，通过离心收集细胞，加入戊二醛固定细胞，低温保存，送至Saville公司进行后续处理，并通过透射电子显微镜观察，获取对照组和紫草素组的亚显微结构图像。

1.9. Western blot法检测蛋白表达水平

将I-10和TCAM-2细胞分别制成单细胞悬液，每孔4×105细胞接种于六孔板中，I-10细胞组加入浓度分别为0、1.2 L、1.4 L、1.6 µmol/L的紫草素，TCAM-2细胞组加入浓度分别为0、0.5、1、1.5 µmol/L的紫草素，给药48 h时收集细胞，冰上裂解30 mim，12000 r/min离心30 min，吸取上清液，每组样取60 μg蛋白进行SDS-PAGE电泳；蛋白转至聚偏氟乙烯（PVDF）膜；快速封闭液封闭15 min；4 ℃孵育一抗过夜；二抗室温下孵育2 h；ECL试剂盒暗室发光显影，凝胶成像系统获取图像。

1.10. Annexin V-FITC/PI双染法检测细胞凋亡

将I-10和TCAM-2细胞分别制成单细胞悬液，每孔4×105细胞接种于六孔板中，I-10细胞组加入浓度分别为0、1.2、1.4、1.6 µmol/L的紫草素，TCAM-2细胞组加入浓度分别为0、0.5、1、1.5 µmol/L的紫草素，给药24 h时收集细胞，每孔加入500 μL缓冲液重悬细胞，加入10 μL Annexin-V-FITC混匀，冰上15 min，之后每孔加15 μL PI染色剂，1 h内检测细胞凋亡情况。

1.11. 统计分析方法

实验数据采用SPSS21.0统计软件进行统计分析，实验数据均以均数±标准差表示，多组比较采用单因素方差分析及两因素方差分析，组间两两比较采用LSDt检验，P < 0.05认为差异具有统计学意义。

2. 结果

2.1. 紫草素对I-10和TCAM-2细胞线粒体功能的影响

I-10和TCAM-2细胞能够通过糖酵解和线粒体氧化磷酸化提供能量，首先通过评估活性氧、线粒体膜电位和胞内ATP含量，研究了紫草素对I-10和TCAM-2细胞线粒体功能的影响（图 1A、B），ROS测定的结果表明，随着紫草素浓度的增加，I-10和TCAM-2细胞活性氧水平逐渐增加。紫草素（持续24 h）显著降低了I-10和TCAM-2细胞的线粒体膜电位，也表明细胞处于凋亡的早期阶段（图 1E、F）。将紫草素作用于I-10和TCAM-2细胞6 h测量细胞内ATP的变化，与对照组相比，I-10细胞组1.2、1.4、1.6 μmol/ L的紫草素可以诱导I-10细胞中的ATP水平分别从基线的100%降至（80.1 ± 2.1）%，（60.2 ± 1.9）%和（48.7±1.34）%（图 1C）。TCAM-2细胞组0.5、1、1.5 μmol/ L的紫草素可以诱导TCAM-2细胞中的ATP水平分别从基线的100%降至（76.9± 2.6）%，（60±1.7）%和（48±1.3）%（图 1D）。

1.

1

Open in a new tab

紫草素对I-10和TCAM-2细胞线粒体功能的影响

Effect of shikonin on mitochondrial function in I-10 and TCAM-2 cells. A, B: Mitochondrial function of shikonin-treated I-10 cells and TCAM-2 cells, respectively; C, D: Changes of ATP level in I-10 and TCAM-2 cells after 6 h of shikonin treatment, respectively; E, F: Effect of shikonin on the mitochondrial membrane potential of I-10 and TCAM-2 cells, respectively. n=3; *P < 0.05, **P < 0.01 vs control group.

2.2. 紫草素对I-10和TCAM-2细胞糖酵解的影响

评估紫草素对I-10和TCAM-2细胞糖酵解的影响，如图 2C、G所示，糖原蛋白PKM2，GLUT1，HK2的表达随着紫草素浓度的增加而降低，糖酵解的最终产物乳酸受到抑制（图 2A、B）。

2.

2

Open in a new tab

紫草素对I-10和TCAM-2细胞糖酵解的影响

Effect of shikonin on glycolysis of I-10 and TCAM-2 cells. A: Effect of shikonin on lactic acid in I-10 cell line; B: Effect of shikonin on lactic acid in TCAM-2 cell; C-F: Effect of shikonin on the expression of glycolysis-related proteins PKM2, GLUT1, HK2 in I-10 cell; G-J: The effect of shikonin on the expression of glycolysis-related proteins PKM2, GLUT1 and HK2 in TCAM-2 cells (n=3, *P < 0.05, **P < 0.01 vs control group).

2.3. 紫草素对I-10和TCAM-2细胞增殖和凋亡的抑制作用

MTT分析的结果表明，随着紫草素浓度的增加和作用时间的延长，紫草素抑制I-10和TCAM-2细胞的增殖效果增强（图 2A、B）。I-10组24、48、72 h的IC50值分别为1.8、1.36和1.16 μmol/ L，TCAM-2组24、48、72 h的IC50值分别为2.37、0.8和0.41 μmol/ L，与对照组相比，差异具有统计学意义（*P < 0.05，**P < 0.01）。通过透射电子显微镜观察到I-10和TCAM-2对照组的细胞体正常，核仁清晰、核膜边缘完整，染色质均匀地分布在细胞核中（图 3C-a和3D-a箭头所指）。紫草素组中，核中的染色质横向移动，浓缩并聚集在核膜边缘，没有核仁，即显示出经典的凋亡特征（图 3C-b和3D-b箭头所指）。对照组线粒体呈现出椭圆形或棒状，隔膜排列整齐（图 3C-c和3D-c箭头所指）。紫草素组线粒体数量的减少，肿胀，缩短和空泡化（图 3C-d和3D-d箭头所指）。Annexin V-FITC / PI双染法验证的透射电镜凋亡结果（图 3E、F），I-10对照组的总凋亡率为（8.26 ± 0.39）%，1.2、1.4、1.6 μmol/L紫草素组的总凋亡率分别为（28.26±1.8）%，（36.6±1.5）%，（65.6±1.3）%，TCAM-2对照组的总凋亡率为（6.42±0.8）%，0.5、1、1.5 μmol/L紫草素组的总凋亡率分别为（12.16 ± 2.0）%，（20.14 ± 1.6）%，（65±1.2）%差异具有统计学意义（P < 0.05）。为了进一步研究紫草素诱导的I-10和TCAM-2细胞凋亡的可能机制，我们通过蛋白质印迹评估了线粒体凋亡相关蛋白的表达，如图 3G、K所示，随着紫草素浓度的增加，线粒体凋亡相关蛋白cleaved caspase 3（caspase-3的裂解产物）和促凋亡蛋白Bax的蛋白水平增加，而抑凋亡蛋白Bcl-2减少。。

3.

3

Open in a new tab

紫草素对I-10和TCAM-2细胞增殖和凋亡的抑制作用

Shikonin inhibits proliferation and apoptosis of I-10 and TCAM-2 cells. A: The effect of shikonin on the proliferation inhibition of I-10 cells; B: The effect of shikonin on the proliferation inhibition of TCAM-2 cells; C: Transmission electron microscopy was used to detect the effect of shikonin on apoptosis of I- 10 cells (The black arrow indicates the nucleus; the red arrow indicates the mitochondria); D: Transmission electron microscopey was used to detect the effect of shikonin on apoptosis of TCAM-2 cells(The black arrow indicates the nucleus; the red arrow indicates the mitochondria); E: Annexin V-FITC/PI double staining method was used to detect the effect of shikonin on apoptosis of I-10 cells; F: Annexin V-FITC/PI double staining was used to detect the effect of shikonin on apoptosis of I-10 cells; G-J: Western blot evaluation of the expression of mitochondrial apoptosis- related proteins Bax, Bcl- 2, cleaved caspase- 3 in I- 10 cells; K-N: Western blot evaluation of the expression of Bax, Bcl-2 and cleaved caspase-3 in TCAM-2 cells (n=3, *P < 0.05, **P < 0.01 vs control group.)

2.4. 紫草素诱导I-10和TCAM-2细胞增加自噬

透射电子显微镜显示，与对照组相比（图 4A-a、C-a箭头所指），紫草素组的I-10和TCAM-2细胞胞质中存在大量自噬体（图 4A-b、C-b箭头所指），并阻塞了线粒体和内质网。其特征在于细胞形成包含细胞器和胞质成分的双层或多层膜，大部分被液泡占据。为了确认自噬，进行了蛋白质印迹（图 4B、D），随着紫草素浓度的增加，自噬标志蛋白LC3B的表达增加。

4.

4

Open in a new tab

紫草素诱导I-10和TCAM-2细胞增加自噬

Shikonin enhances autophagy in I-10 and TCAM-2 cells. A, C: Transmission electron microscopy showing enhanced autophagy in shikonin-treated I-10 and TCAM-2 cells, respectively (The black arrow indicates the nucleus; the red arrow indicates the autophagosome); B, D: Effect of shikonin on the expression of LC3B in I-10 and TCAM-2 cells, respectively. n=3, *P < 0.05, **P < 0.01 vs control group.

3. 讨论

在癌变和肿瘤发展过程中，癌细胞将能量代谢重新规划成糖酵解表型，这是癌症的一个新的特征。肿瘤发生的最初原因是线粒体呼吸功能障碍，为了维持细胞生存和满足大分子合成的需要，细胞选择激活另一种能量代谢方式-有氧糖酵解[28]。已有文献表明睾丸癌细胞的糖酵解活性增加[29]，在本课题的研究中，我们从紫草素抑制I-10和TCAM-2细胞糖酵解和线粒体功能进而诱导其凋亡方面入手，并探寻其凋亡的可能机制，实验结果表明紫草素可以抑制I-10和TCAM-2细胞中胞内乳酸和糖酵解相关酶（PKM2，GLUT1和HK2）的表达。同时，紫草素会导致I-10和TCAM-2细胞内ATP和线粒体膜电位下降，而ROS的水平增加。这些早期的变化随后是线粒体结构破坏，其中包括释放小分子，例如细胞色素c，细胞凋亡诱导因子以及caspase-3和caspase-9反应[30]。线粒体是细胞ROS的主要来源[31]，ROS的异常积累可能引起氧化应激并导致DNA修饰和破坏[32]。与此同时，紫草素组的I-10和TCAM-2细胞透射电子显微镜观察更明显地显示了线粒体的肿胀，缩短和空泡化。这些数据表明，紫草素可对I-10和TCAM-2细胞的线粒体造成重大损害。以上结果表明，紫草素可通过影响I-10和TCAM-2细胞的线粒体功能和抑制其糖酵解来抑制I-10和TCAM-2细胞的能量代谢。

MTT分析的结果表明，随着紫草素浓度的增加和作用时间的延长，紫草素抑制I-10和TCAM-2细胞的增殖效果增强，为了检测紫草素抑制I-10和TCAM-2细胞糖酵解后是否会细胞凋亡，通过透射电镜观察到紫草素处理后I-10和TCAM-2细胞亚显微结构显示出细胞凋亡，这也被Annexin V-FITC双染法检测细胞凋亡所证实，与对照组相比，紫草素组的I-10和TCAM-2细胞凋亡明显增加。凋亡的机制似乎是通过下调线粒体通路相关凋亡蛋白Bax、Bcl-2、cleaved caspase 3的相对表达。自噬是一种进化上保守的机制适应微环境条件，例如代谢抑制，它确保了在缺氧和能量饥饿的情况下癌细胞的存活和增殖[33]。糖酵解抑制会产生能量剥夺并进一步触发自噬，最终导致自噬细胞死亡[34-36]，这有可能是抑制了肿瘤细胞中Akt/mTOR信号通路启动了自噬细胞的死亡[37]。通过透射电子显微镜我们发现紫草素组的I-10和TCAM-2细胞出现大量自噬溶酶体，自噬标记物LC3B蛋白的表达高于对照组，提示紫草素可促进I-10和TCAM-2细胞自噬的增加。以上结果表明，紫草素对睾丸癌细胞I-10和TCAM-2具有增殖抑制及诱导凋亡和增加自噬作用，其机制可能为紫草素影响I-10和TCAM-2细胞糖酵解和线粒体功能。

Biography

姚越，硕士，E-mail: 568243114@qq.com

Funding Statement

国家自然科学基金（81903142）；安徽省自然科学基金（1808085MH293）；安徽省高校自然科学研究重点项目（KJ2019A0293, KJ2019A0400）；蚌埠医学院科技发展基金项目（BYKF1763）

Supported by National Natural Science Foundation of China (81903142)

Contributor Information

姚 越 (Yue YAO), Email: 568243114@qq.com.

汪 盛 (Sheng WANG), Email: bydoctorw@163.com.

References

  • 1.Kamel MH, Elfaramawi M, Jadhav S, et al. Insurance status and differences in treatment and survival of testicular cancer patients. Urology. 2016;87:140–5. doi: 10.1016/j.urology.2015.06.059. [Kamel MH, Elfaramawi M, Jadhav S, et al. Insurance status and differences in treatment and survival of testicular cancer patients[J]. Urology, 2016, 87: 140-5.] [DOI] [PubMed] [Google Scholar]
  • 2.Kurobe M, Kawai K, Suetomi T, et al. High prevalence of hypogonadism determined by serum free testosterone level in Japanese testicular cancer survivors. Int J Urol. 2018;25:457–62. doi: 10.1111/iju.13537. [Kurobe M, Kawai K, Suetomi T, et al. High prevalence of hypogonadism determined by serum free testosterone level in Japanese testicular cancer survivors[J]. Int J Urol, 2018, 25: 457-62.] [DOI] [PubMed] [Google Scholar]
  • 3.Leveridge MJ, Siemens DR, Brennan K, et al. Temporal trends in management and outcomes of testicular cancer: A population-based study. Cancer. 2018;124:2724–32. doi: 10.1002/cncr.31390. [Leveridge MJ, Siemens DR, Brennan K, et al. Temporal trends in management and outcomes of testicular cancer: A population-based study[J]. Cancer, 2018, 124: 2724-32.] [DOI] [PubMed] [Google Scholar]
  • 4.Apps MG, Choi EH, Wheate NJ. The state-of-play and future of platinum drugs. Endocr Relat Cancer. 2015;22(4):R219–33. doi: 10.1530/ERC-15-0237. [Apps MG, Choi EH, Wheate NJ. The state-of-play and future of platinum drugs[J]. Endocr Relat Cancer, 2015, 22(4): R219-33.] [DOI] [PubMed] [Google Scholar]
  • 5.Matsuzaki S, Serada S, Morimoto A, et al. Annexin A4 is a promising therapeutic target for the treatment of platinum-resistant cancers. Expert Opin Ther Targets. 2014;18(4):403–14. doi: 10.1517/14728222.2014.882323. [Matsuzaki S, Serada S, Morimoto A, et al. Annexin A4 is a promising therapeutic target for the treatment of platinum-resistant cancers[J]. Expert Opin Ther Targets, 2014, 18(4): 403-14.] [DOI] [PubMed] [Google Scholar]
  • 6.Ghosh S. Cisplatin: The first metal based anticancer drug. Bioorg Chem. 2019;88:102925. doi: 10.1016/j.bioorg.2019.102925. [Ghosh S. Cisplatin: The first metal based anticancer drug[J]. Bioorg Chem, 2019, 88: 102925.] [DOI] [PubMed] [Google Scholar]
  • 7.Wu D, Li B, Liu H, et al. In vitro inhibited effect of gap junction composed of Cx43 in the invasion and metastasis of testicular cancer resistanced to cisplatin. Biomed Pharmacother. 2018;98:826–33. doi: 10.1016/j.biopha.2018.01.016. [Wu D, Li B, Liu H, et al. In vitro inhibited effect of gap junction composed of Cx43 in the invasion and metastasis of testicular cancer resistanced to cisplatin[J]. Biomed Pharmacother, 2018, 98: 826-33.] [DOI] [PubMed] [Google Scholar]
  • 8.Oechsle K, Honecker F, Cheng T, et al. Preclinical and clinical activity of sunitinib in patients with cisplatin-refractory or multiply relapsed germ cell tumors: a canadian urologic oncology group/ german testicular cancer study group cooperative study. Ann Oncol. 2011;22:2654–60. doi: 10.1093/annonc/mdr026. [Oechsle K, Honecker F, Cheng T, et al. Preclinical and clinical activity of sunitinib in patients with cisplatin-refractory or multiply relapsed germ cell tumors: a canadian urologic oncology group/ german testicular cancer study group cooperative study[J]. Ann Oncol, 2011, 22: 2654-60.] [DOI] [PubMed] [Google Scholar]
  • 9.Koster R, van Vugt MA, Timmer-Bosscha H, et al. Unravelling mechanisms of cisplatin sensitivity and resistance in testicular cancer. Expert Rev Mol Med. 2013;15:e12. doi: 10.1017/erm.2013.13. [Koster R, van Vugt MA, Timmer-Bosscha H, et al. Unravelling mechanisms of cisplatin sensitivity and resistance in testicular cancer[J]. Expert Rev Mol Med, 2013, 15: e12.] [DOI] [PubMed] [Google Scholar]
  • 10.Cheng L, Albers P, Berney DM, et al. Testicular cancer. Nat Rev Dis Primers. 2018;4:29. doi: 10.1038/s41572-018-0029-0. [Cheng L, Albers P, Berney DM, et al. Testicular cancer[J]. Nat Rev Dis Primers, 2018, 4: 29.] [DOI] [PubMed] [Google Scholar]
  • 11.Mao P, Hever MP, Niemaszyk L, et al. Serine/threonine kinase 17A is a novel p53 target gene and modulator of cisplatin toxicity and reactive oxygen species in testicular cancer cells. J Biol Chem. 2011;286:19381–91. doi: 10.1074/jbc.M111.218040. [Mao P, Hever MP, Niemaszyk L, et al. Serine/threonine kinase 17A is a novel p53 target gene and modulator of cisplatin toxicity and reactive oxygen species in testicular cancer cells[J]. J Biol Chem, 2011, 286: 19381-91.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Christensen JF, Andersen JL, Adamsen L, et al. Progressive resistance training and cancer testis (PROTRACT)-efficacy of resistance training on muscle function, morphology and inflammatory profile in testicular cancer patients undergoing chemotherapy: design of a randomized controlled trial. BMC Cancer. 2011;11:326. doi: 10.1186/1471-2407-11-326. [Christensen JF, Andersen JL, Adamsen L, et al. Progressive resistance training and cancer testis (PROTRACT)-efficacy of resistance training on muscle function, morphology and inflammatory profile in testicular cancer patients undergoing chemotherapy: design of a randomized controlled trial[J]. BMC Cancer, 2011, 11: 326.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Gao H, Liu L, Qu Z, et al. Anti-adenovirus activities of shikonin, a component of Chinese herbal medicine in vitro. Biol Pharm Bull. 2011;34:197–202. doi: 10.1248/bpb.34.197. [Gao H, Liu L, Qu Z, et al. Anti-adenovirus activities of shikonin, a component of Chinese herbal medicine in vitro[J]. Biol Pharm Bull, 2011, 34: 197-202.] [DOI] [PubMed] [Google Scholar]
  • 14.Tong Y, Bai L, Gong R, et al. Shikonin protects PC12 cells against β- amyloid peptide-induced cell injury through antioxidant and antiapoptotic activities. Sci Rep. 2018;8:26. doi: 10.1038/s41598-017-18058-7. [Tong Y, Bai L, Gong R, et al. Shikonin protects PC12 cells against β- amyloid peptide-induced cell injury through antioxidant and antiapoptotic activities[J]. Sci Rep, 2018, 8: 26.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Liao P, Lin C, Li C, et al. Anti-inflammatory properties of shikonin contribute to improved early-stage diabetic retinopathy. Sci Rep. 2017;7:44985. doi: 10.1038/srep44985. [Liao P, Lin C, Li C, et al. Anti-inflammatory properties of shikonin contribute to improved early-stage diabetic retinopathy[J]. Sci Rep, 2017, 7: 44985.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Li Y, Lu HL, Gu YC, et al. Enhancement of NK cells proliferation and function by Shikonin. Immunopharmacol Immunotoxicol. 2017;39(3):124–30. doi: 10.1080/08923973.2017.1299174. [Li Y, Lu HL, Gu YC, et al. Enhancement of NK cells proliferation and function by Shikonin[J]. Immunopharmacol Immunotoxicol, 2017, 39(3): 124-30.] [DOI] [PubMed] [Google Scholar]
  • 17.Lu B, Wang Z, Ding Y, et al. RIP1 and RIP3 contribute to shikonininduced glycolysis suppression in glioma cells via increase of intracellular hydrogen peroxide. Cancer Lett. 2018;425:31–42. doi: 10.1016/j.canlet.2018.03.046. [Lu B, Wang Z, Ding Y, et al. RIP1 and RIP3 contribute to shikonininduced glycolysis suppression in glioma cells via increase of intracellular hydrogen peroxide[J]. Cancer Lett, 2018, 425: 31-42.] [DOI] [PubMed] [Google Scholar]
  • 18.Trivedi R, Müller G, Rathore M S, et al. Anti-leukemic activity of shikonin: Role of ERP57 in shikonin induced apoptosis in acute myeloid leukemia. Cell Physiol Biochem. 2016;39:604–16. doi: 10.1159/000445652. [Trivedi R, Müller G, Rathore M S, et al. Anti-leukemic activity of shikonin: Role of ERP57 in shikonin induced apoptosis in acute myeloid leukemia[J]. Cell Physiol Biochem, 2016, 39: 604-16.] [DOI] [PubMed] [Google Scholar]
  • 19.Hashimoto S, Xu M, Masuda Y, et al. Beta-hydroxyisovalerylshikonin inhibits the cell growth of various cancer cell lines and induces apoptosis in leukemia HL-60 cells through a mechanism different from those of Fas and etoposide. J Biochem. 1999;125(1):17–23. doi: 10.1093/oxfordjournals.jbchem.a022255. [Hashimoto S, Xu M, Masuda Y, et al. Beta-hydroxyisovalerylshikonin inhibits the cell growth of various cancer cell lines and induces apoptosis in leukemia HL-60 cells through a mechanism different from those of Fas and etoposide[J]. J Biochem, 1999, 125 (1): 17-23.] [DOI] [PubMed] [Google Scholar]
  • 20.Gara R, Srivastava V, Duggal S, et al. Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway. J Biomed Sci. 2015;22:26. doi: 10.1186/s12929-015-0127-1. [Gara R, Srivastava V, Duggal S, et al. Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway[J]. J Biomed Sci, 2015, 22: 26.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Gara RK, Srivastava VK, Duggal S, et al. Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway. J Biomed Sci. 2015;22:26. doi: 10.1186/s12929-015-0127-1. [Gara RK, Srivastava VK, Duggal S, et al. Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway[J]. J Biomed Sci, 2015, 22: 26.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ruan M, Yan M, Yang WJ, et al. Role of NF-kappaB pathway in shikonin induced apoptosis in oral squamous cell carcinoma Tca- 8113 cells. Shanghai J Stomatol. 2010;19(1):66–71. [Ruan M, Yan M, Yang WJ, et al. Role of NF-kappaB pathway in shikonin induced apoptosis in oral squamous cell carcinoma Tca- 8113 cells[J]. Shanghai J Stomatol, 2010, 19(1): 66-71.] [PubMed] [Google Scholar]
  • 23.Andújar I, Martí-Rodrigo A, Giner RM, et al. Shikonin prevents early phase inflammation associated with azoxymethane/dextran sulfate sodium-induced colon cancer and induces apoptosis in human colon cancer cells. Planta Med. 2018;84(9/10):674–83. doi: 10.1055/a-0599-1145. [Andújar I, Martí-RodrigoA, Giner RM, et al. Shikonin prevents early phase inflammation associated with azoxymethane/dextran sulfate sodium-induced colon cancer and induces apoptosis in human colon cancer cells[J]. Planta Med, 2018, 84(9/10): 674-83.] [DOI] [PubMed] [Google Scholar]
  • 24.Li S, Zhang T, Xu W, et al. Sarcoma-targeting peptide-decorated polypeptide nanogel intracellularly delivers shikonin for upregulated osteosarcoma necroptosis and diminished pulmonary metastasis. Theranostics. 2018;8:1361–75. doi: 10.7150/thno.18299. [Li S, Zhang T, Xu W, et al. Sarcoma-targeting peptide-decorated polypeptide nanogel intracellularly delivers shikonin for upregulated osteosarcoma necroptosis and diminished pulmonary metastasis[J]. Theranostics, 2018, 8: 1361-75.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lu D, Qian J, Li Wei, et al. β-hydroxyisovaleryl-shikonin induces human cervical cancer cell apoptosis via PI3K/AKT/mTOR signaling. Oncol Lett. 2015;10:3434–42. doi: 10.3892/ol.2015.3769. [Lu D, Qian J, Li Wei, et al. β-hydroxyisovaleryl-shikonin induces human cervical cancer cell apoptosis via PI3K/AKT/mTOR signaling[J]. Oncol Lett, 2015, 10: 3434-42.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Wang X, Zhang FL, Wu XR. Inhibition of pyruvate kinase M2 markedly reduces chemoresistance of advanced bladder cancer to cisplatin. Sci Rep. 2017;7:45983. doi: 10.1038/srep45983. [Wang X, Zhang FL, Wu XR. Inhibition of pyruvate kinase M2 markedly reduces chemoresistance of advanced bladder cancer to cisplatin[J]. Sci Rep, 2017, 7: 45983.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Oronsky BT, Oronsky N, Fanger GR, et al. Follow the ATP: tumor energy production: a perspective. Anticancer Agents Med Chem. 2014;14(9):1187–98. doi: 10.2174/1871520614666140804224637. [Oronsky BT, Oronsky N, Fanger GR, et al. Follow the ATP: tumor energy production: a perspective[J]. Anticancer Agents Med Chem, 2014, 14(9): 1187-98.] [DOI] [PubMed] [Google Scholar]
  • 28.Zhou S, Min Z, Sun K, et al. MiR-199a-3p/Sp1/LDHA axis controls aerobic glycolysis in testicular tumor cells. Int J Mol Med. 2018;42:2163–74. doi: 10.3892/ijmm.2018.3771. [Zhou S, Min Z, Sun K, et al. MiR-199a-3p/Sp1/LDHA axis controls aerobic glycolysis in testicular tumor cells[J]. Int J Mol Med, 2018, 42: 2163-74.] [DOI] [PubMed] [Google Scholar]
  • 29.Bonatelli M, Silva E, Cárcano F, et al. The warburg effect is associated with tumor aggressiveness in testicular germ cell tumors. Front Endocrinol (Lausanne) 2019;10:417. doi: 10.3389/fendo.2019.00417. [Bonatelli M, Silva E, Cárcano F, et al. The warburg effect is associated with tumor aggressiveness in testicular germ cell tumors[J]. Front Endocrinol (Lausanne), 2019, 10: 417.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Duan D, Zhang B, Yao J, et al. Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL- 60 cells. Free Radic Biol Med. 2014;70:182–93. doi: 10.1016/j.freeradbiomed.2014.02.016. [Duan D, Zhang B, Yao J, et al. Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL- 60 cells[J]. Free Radic Biol Med, 2014, 70: 182-93.] [DOI] [PubMed] [Google Scholar]
  • 31.Holzerová E, Prokisch H. Mitochondria: Much ado about nothing? How dangerous is reactive oxygen species production? Int J Biochem. Cell Biol. 2015;63:16–20. doi: 10.1016/j.biocel.2015.01.021. [Holzerová E, Prokisch H, Mitochondria: Much ado about nothing? How dangerous is reactive oxygen species production[J]? Int J Biochem. Cell Biol, 2015, 63: 16-20.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Choi YH. Schisandrin A prevents oxidative stress-induced DNA damage and apoptosis by attenuating ROS generation in C2C12 cells. Biomedecine Pharmacother. 2018;106:902–9. doi: 10.1016/j.biopha.2018.07.035. [Choi YH. Schisandrin A prevents oxidative stress-induced DNA damage and apoptosis by attenuating ROS generation in C2C12 cells[J]. Biomedecine Pharmacother, 2018, 106: 902-9.] [DOI] [PubMed] [Google Scholar]
  • 33.Holzerová E, Prokisch H. Mitochondria: Much ado about nothing? How dangerous is reactive oxygen species production? Int J Biochem Cell Biol. 2015;63:16–20. doi: 10.1016/j.biocel.2015.01.021. [Holzerová E, Prokisch H. Mitochondria: Much ado about nothing? How dangerous is reactive oxygen species production[J]? Int J Biochem Cell Biol, 2015, 63: 16-20.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Kim J, Nam B, Choi Y, et al. Enhanced glycolysis supports cell survival in EGFR-mutant lung adenocarcinoma by inhibiting autophagy-mediated EGFR degradation. Cancer Res. 2018;78:4482–96. doi: 10.1158/0008-5472.CAN-18-0117. [Kim J, Nam B, Choi Y, et al. Enhanced glycolysis supports cell survival in EGFR-mutant lung adenocarcinoma by inhibiting autophagy-mediated EGFR degradation[J]. Cancer Res, 2018, 78: 4482-96.] [DOI] [PubMed] [Google Scholar]
  • 35.Shun C, Lin S, Hong C, et al. Sirtuin 6 modulates hypoxia-induced autophagy in nasal polyp fibroblasts via inhibition of glycolysis. Am J RhinolAllergy. 2016;30:179–85. doi: 10.2500/ajra.2016.30.4282. [Shun C, Lin S, Hong C, et al. Sirtuin 6 modulates hypoxia-induced autophagy in nasal polyp fibroblasts via inhibition of glycolysis[J]. Am J RhinolAllergy, 2016, 30: 179-85.] [DOI] [PubMed] [Google Scholar]
  • 36.Shi D, Zhao D, Niu P, et al. Glycolysis inhibition via mTOR suppression is a key step in cardamonin-induced autophagy in SKOV3 cells. BMC ComplementAltern Med. 2018;18:317. doi: 10.1186/s12906-018-2380-9. [Shi D, Zhao D, Niu P, et al. Glycolysis inhibition via mTOR suppression is a key step in cardamonin-induced autophagy in SKOV3 cells[J]. BMC ComplementAltern Med, 2018, 18: 317.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Dey P, Kundu A, Sachan R, et al. PKM2 knockdown induces autophagic cell death via AKT/mTOR pathway in human prostate cancer cells. Cell Physiol Biochem. 2019;52:1535–52. doi: 10.33594/000000107. [Dey P, Kundu A, Sachan R, et al. PKM2 knockdown induces autophagic cell death via AKT/mTOR pathway in human prostate cancer cells[J]. Cell Physiol Biochem, 2019, 52: 1535-52.] [DOI] [PubMed] [Google Scholar]

Articles from Journal of Southern Medical University are provided here courtesy of Editorial Department of Journal of Southern Medical University

RESOURCES