紫檀芪通过抑制JAK2/STAT3信号通路抗人骨肉瘤细胞活性研究
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摘要
研究背景
骨肉瘤是一种儿童及青少年最常见的恶性骨肿瘤，高发于四肢长骨的干骺端。目前常规的化疗药物均为细胞毒性药物，副作用大。几乎一半的儿童期肿瘤幸存者因为抗肿瘤治疗至少承受一个不良后果。化疗药物早期毒性包括血液系统毒性，急性肝脏毒性和肾毒性。生存期的延长导致了晚期化疗毒性的增加。主要的晚期毒性有：心脏毒性、继发肿瘤、不育、慢性肾衰竭和神经系统毒性。因此寻找低毒的抗骨肉瘤药物具有重要的临床意义。
紫檀芪（PTE）是一种来源于我们食物的天然化合物，毒性极低，对人体是安全的。PTE是白藜芦醇的二甲基化类似物，具有多种生物学活性，可以抗炎、抗氧化、抗增殖、抗肿瘤及止痛。因甲氧基团替代了羟基增加了亲脂性，增加了生物利用度，从而增加了活性。研究表明PTE可以抑制多种肿瘤细胞的增殖并诱导其凋亡，包括肝癌、乳腺癌、前列腺癌、胰腺癌及白血病等。PTE是否具有抗骨肉瘤作用目前尚无人研究。
PTE抗肿瘤作用涉及多种细胞分子和信号通路，如单磷酸腺苷活化激酶(AMPK)、细胞基质钙超载、活性氧（ROS）、自噬、Wnt信号通路及溶酶体膜通透作用增强等，但具体的抗肿瘤机制目前尚不清楚。JAK/STAT通路对体内多种信号的转导都非常的关键，对多种哺乳动物发育及内环境的稳定都异常的重要。JAK家族包括JAK1、JAK2、JAK3及酪氨酸激酶2，这4个蛋白结构及功能相似。JAK的活化在细胞增殖、分化、迁移及凋亡中均有重要的作用。结构活化的JAKs会使细胞内许多重要的物质磷酸化，其中包括STAT家族，STAT家族中的STAT3与许多肿瘤的信号通路相关。结构活化后的STAT3和人实体肿瘤细胞的存活与生长关系密切。STAT3还可上调人肿瘤细胞抗凋亡基因编码的Mcl-1、Bcl-xL蛋白。PTE可以抑制STST3的磷酸化，磷酸化的STST3是一种肿瘤快速生长的标志。更为重要的是活化的JAK2/STAT3信号作为治疗实体肿瘤一个重要的目标分子已经被广泛的确认其有效性。PTE抗骨肉瘤的作用是否与抑制JAK2/STAT3通路的磷酸化程度有关目前尚不清楚。
第一部分PTE抗人骨肉瘤细胞活性研究
研究目的
研究PTE对人骨肉瘤细胞生长及凋亡的影响；研究PTE对人骨肉瘤细胞迁移及粘附能力的影响。
研究方法
1.细胞培养及处理
人骨肉瘤细胞系SOSP-9607(9607)，复苏后在10%的胎牛血清DMEM液中培养，加入L-谷酰胺、青霉素、链霉素及HEPES液。孵箱内温度维持在37°C，CO2浓度为5%。收集细胞备用。
PTE原液中加入二甲基亚砜，以培养液稀释，即配即用。对照组中加入0.01%的二甲基亚砜。实验组加入PTE，浓度梯度为1、2及4μM。
2.细胞活力检测
细胞预处理后以PBS液洗涤三次，加入MTT液，37°C的孵箱中孵育4h，加入二甲基甲酰胺，震荡，在酶联免疫监测仪上检测细胞活力。细胞活力以各孔吸光值（OD）的形式记录，测定各孔光吸收值，记录结果，绘制细胞生长曲线。并在倒置相差显微镜下观察细胞形态、照相。
3.细胞周期分析
细胞预处理后胰酶消化，70%的乙醇固定后PBS液洗涤，加入核糖核苷酸及碘化丙啶，避光室温下孵育30min。以配备数据采集及分析系统的流式细胞仪分析并记录数据。结果以荧光密度分布图的形式表示，荧光密度代表细胞数目。
4.线粒体跨膜电位（MMP）分析
将细胞放入六孔板中加入不同浓度的PTE，孵育24h。以PBS液洗涤，加入JC-1工作液，避光37°C孵育。细胞洗涤后悬浮于PBS液中，将染色的细胞用上述流式细胞仪分析，结果以较低MMP的细胞比例表示。
5.细胞凋亡分析
将细胞预处理后，冰PBS液冲洗，加入荧光标记的共轭膜联蛋白及碘化丙啶，使用上述流式细胞仪检测细胞凋亡情况并用相关软件进行分析。碘化丙啶阴性且共轭膜联蛋白阳性的细胞被认为是早期凋亡细胞，双阴性细胞被认为是正常细胞。
6.划痕试验
培养细胞至铺满瓶底后，微量管尖端在单层细胞处制造划痕。将分离的细胞以PBS洗涤干净，加入PTE24h后，使用显微镜观察并记录划痕边缘距离变化情况。结果以每组划痕之间的距离数值表示。
7.细胞粘附实验
PTE处理骨肉瘤细胞24h后消化、离心，悬浮在加入10%胎牛血清的DMEM液中。37°C下孵育30min，PBS液洗涤。将贴壁细胞用法MTT染色，在倒置相差显微镜下观察细胞粘附情况并照相。加入二甲基甲酰胺，37°C下震荡孵育15min，在490nm条件下使用分光光度计测量，结果以OD值表示，将对照组OD值设为100%。
8.统计学分析
所有的样本一式两份，重复至少3次，数据已平均值±标注差的形式表示。实验组间比较使用SPSS12.0，采用方差分析的方法(ANOVA)。差异P<0.05被认为统计学上有意义。
研究结果
1. PTE对骨肉瘤细胞生长及凋亡的影响
骨肉瘤细胞培养液中加入1,2及4μM浓度梯度的PTE后，分别培养12、24及36h，细胞生长均出现抑制。PTE对骨肉瘤细胞的生长抑制作用呈剂量和时间依赖。培养至24h时PTE的IC50(50%抑制浓度)约为1.81μM。与对照组比较，PTE导致细胞粘附率下降。加入1、2及4μM PTE后，与对照组相比较，凋亡指数分别增加16.75±3.91%、21.55±3.26%及35.87±4.23%(P<0.01)。我们发现凋亡指数呈剂量依赖性。这个结果说明PTE对人骨肉瘤细胞有生长抑制及诱导凋亡的作用。
2. PTE对骨肉瘤细胞迁移及粘附能力的影响
加入1、2及4μM的PTE，孵育24h后，随着浓度的增加，划痕边缘的距离显著增加，分别增加至对照组的123.07±9.05%、161.53±8.28%及223.08±11.30%(P<0.01)。粘附细胞显著减少，粘附率分别为对照组的62.29±9.43%、34.38±6.70%及13.64±3.35%(P<0.01)。这些结果表明PTE可以降低骨肉瘤细胞的迁移能力及粘附能力。
3. PTE对骨肉瘤细胞MMP的影响
加入1、2及4μM的PTE，孵育24h后，低MMP细胞的比例显著增加。各组低MMP的细胞比例分别增加至8.19±2.36%、16.17±2.68%及29.45±3.10%(与对照组比较，P<0.01)。结果表明PTE可以增加骨肉瘤细胞的MMP。
4. PTE对骨肉瘤细胞周期的影响
与对照组相比较，加入不同浓度的PTE后均会导致细胞G0-G1阶段的聚积。G0-G1阶段的细胞由对照组的33.27%增加至4μM PTE组的55.22%，可见PTE会导致细胞周期G0-G1期的阻滞。
结论
PTE具有很强的抗人骨肉瘤细胞活性的作用。PTE导致人骨肉瘤细胞G0-G1期阻滞，使细胞生长出现抑制；同时PTE降低了MMP，诱导了细胞的凋亡。PTE还可以减少骨肉瘤细胞的粘附能力及迁移能力。
第二部分PTE抗人骨肉瘤细胞活性的机制研究
研究目的：
研究PTE抗人骨肉瘤细胞活性的相关机制。
研究方法
1.细胞培养及处理
同第一部分最后一步实验中加入AG490，PTE的浓度为4μM
2.细胞内活性氧（ROS）及谷胱甘肽水平（GSH）检测
细胞预处理后胰酶裂解，加入DCFH-DA液，37oC下孵育2h。FLX800微量盘荧光光谱仪测量每孔二氯荧光素的强度。无细胞孔作为背景，将对照组荧光强度定为100%。
细胞加入培养皿中过夜后加入PTE24h后。PBS洗涤细胞。根据探针的操作指南，将细胞裂解后测量GSH的水平。在测量前1h用2-乙烯基吡啶遮蔽GSH。GSH和GSSG的含量与正常标准的蛋白含量比较。结果以GSH的表达（%对照含量）或GSH/GSSG比例的形式表达，使用还原形式的GSH或氧化形式的GSH（GSSG）作为标准。
3. Westen Blotting
将细胞样本在样本缓冲液中裂解，降噪处理，煮沸，跑凝胶，转膜，封闭。加入p-JAK2、JAK2、p-STAT3、STAT3、Mcl-1、Cyclin D1、p21、p27、Bcl-xL、Bax、Bak及GAPDH的抗体4oC过夜。洗膜，加入对应的二抗，室温下放置90min，洗涤。使用BioRad成像系统观察记录荧光强度，并以Image Lab软件定量分析。
4.细胞质中细胞色素C的萃取
处理离心细胞，PBS液洗涤2次后悬浮于冰的细胞萃取缓冲液中，4oC下孵育40min，离心，上清液用来提取细胞色素C，加入5倍上清的缓冲液，100oC下煮沸7min，将蛋白溶液用Western blotting的方法测定细胞色素C的含量。
5.统计学分析
同第一部分
研究结果
1. PTE对细胞周期相关蛋白的影响
为了进一步验证细胞G0–G1阻滞，我们利用Westernblotting的方法检测周期蛋白D1、p21及p27的表达。与细胞周期阻滞一致，Cyclin D1的表达水平降低，而p21和p27的表达水平升高。这个发现表明PTE可以导致细胞周期相关蛋白表达的变化。
2. PTE对骨肉瘤细胞活性氧生成及谷胱甘肽水平的影响
加入1、2及4μM的PTE，孵育24h后骨肉瘤细胞内产生的活性氧呈剂量依赖关系，分别增加至对照组的255.32±12.97%、411.59±16.24%及503.61±17.05%(与对照组比较，P<0.01)。
加入1、2及4μM的PTE，孵育24h后骨肉瘤细胞谷胱甘肽水平呈剂量依赖性降低，分别降低了80.51±3.06%、66.43±3.82%及52.30±3.49%。(与对照组比较，P<0.01)。PTE会导致骨肉瘤细胞内GSH/GSSG的比例呈剂量依赖性降低。
3.对人骨肉瘤细胞JAK2/STAT3信号通路及线粒体凋亡通路相关蛋白的影响
加入PTE后磷酸化的JAK2及STAT3的表达水平降低。与磷酸化的JAK2及STAT3的表达下调相一致的是PTE使Mcl-1和Bcl-xL的表达水平也降低，且呈剂量依赖关系。除此之外PTE使线粒体凋亡通路相关蛋白(Bax、Bak、细胞色素C)的表达上调。
4. PTE联合AG490对人骨肉瘤细胞活力及JAK2/STAT3信号的影响
联合应用PTE（4μM）和AG490(一种已知的JAK2/STAT3抑制剂，20μM)进一步抑制了骨肉瘤细胞的活力(与单独的PTE组或AG490组比较，P<0.01)。并且联合应用PTE和AG490也进一步抑制了JAK2及STAT3的磷酸化程度。
结论
PTE通过抑制JAK2/STAT3信号通路影响CDK/Cyclin复合物和CKIs的平衡，导致了细胞G0-G1期阻滞，使人骨肉瘤细胞出现了生长抑制；同时活化内源性线粒体凋亡途径，诱导了人骨肉瘤细胞的凋亡。
Background
Osteosarcoma is a most common high-grade malignant bone tumor occurringfrequently in children and adolescents. At the moment anti-osteosarcoma drugs havecytotoxicity and severe side effects which are harmful to our health. Nearly a half ofyoung adult survivors of childhood cancer have at least one major adverse outcome oftheir health status as a result of their cancer therapy. Early toxicity consists ofhematological toxicity, acute liver (MTX) and renal toxicity (CDP and IFO). Longersurvival has led to the increase in late chemotherapy toxicity. The main late toxicities are:cardiac, second tumor, sterility, chronic renal failure and neurologic toxicities (mainlyototoxicity due to CDP). It is urgent clinically to develop safe and efficient agents withhypotoxicity.
Pterostilbene(trans-3,5-dimethoxy-4’-hydroxystilbene, PTE), a natural compound occurring from our daily diet appears to be a safe and efficient agent. Pterostilbene,dimethylated analog of resveratrol from blueberries, is known to have diversepharmacologic activities, including anticancer, anti-inflammation, antioxidant,anti-proliferative and analgesic activities PTE has been shown to have potent antitumoractivity with low toxicity in various cancer types, including hepatoma, breast cancer,prostate cancer, pancreatic cancer and chronic myelogenous leukemia, among others. Inmost circumstances, PTE was as potent or significantly more potent than resveratrol,indicating that PTE might show better biological activity due to its increasedbioavailability, as the substitution of a hydroxy group with a methoxy group increases itslipophilicity. However, the effects of PTE in human osteosarcoma have not been clarified.
In addition, various molecules and signaling pathways are involved in the anti-tumoreffects of PTE, including adenosine monophosphate-activated protein kinase (AMPK),cytosolic Ca2+overload, ROS, autophagy, Wnt signaling, and lysosomal membranepermeabilization, among others. The molecular mechanisms underlying the effects of PTEremain largely unknown. The JAK/STAT pathway is pivotal for the transduction of amultitude of signals that are critical for development and homeostasis in mammals. TheJAK family of proteins consists of JAK1, JAK2, JAK3and tyrosine kinase2, all of whichshare similar structures and functions. JAK activation plays an important role in cellproliferation, differentiation, migration and apoptosis. Constitutively activated JAKsphosphorylate critical cellular substrates, such as the STAT family, which includesSTAT3, that are associated with oncogenic signaling pathways. Constitutive activation ofSTAT3plays a critical role in cell growth and survival in human solid tumor malignanciesand in the up-regulation of the anti-apoptotic genes encoding Mcl-1and Bcl-xL proteins inhuman cancer cells. PTE was also shown to inhibit STAT3phosphorylation, a marker ofaccelerated tumorigenesis, and decrease pancreatic tumor growth in vivo. Importantly,activated JAK2/STAT3signaling has been extensively validated as a new molecular targetfor the treatment of human solid tumors. The mechanisms of PTE in human osteosarcomahave not been clarified
Part I Antitumor activity of PTE against humanosteosarcoma cells
Objective
To investigate the effects of PTE on proliferation and apoptosis in humanosteosarcoma cells; to investigate the effects of PTE on migration and adhesion in humanosteosarcoma cells; to investigate the effects of PTE on cell cycle.
Methods
1. Cell culture and treatment
The human osteosarcoma cell line, SOSP-9607(9607), were grown in Dulbecco'smodified Eagle's medium, supplemented with10%fetal bovine serum, L-glutamine (2mM), penicillin (100units/ml), streptomycin (100units/ml), and HEPES (25mM). Thecells were maintained in the presence of5%CO2at37°C.
The PTE stock solution was prepared in DMSO and diluted with culture mediumimmediately prior to the experiment. The control group was treated with DMSO (0.01%).First, the cells were treated with PTE (1,2, and4μM).
2. Analysis of cell viability
After the cells were treated and washed with PBS,100μL of0.5mg/mL MTTsolution in phenol red-free DMEM was added to the cells, and the samples were incubatedfor4h at37°C. Finally,100μL ofN, N-dimethylformamide was added to each well, andthe samples were incubated for15min at37°C with shaking. The wells were measured at490nm using a microplatereader and cell viability was expressed as an optical density(OD) value. In addition, cell morphology was observed under an inverted/phase contrastmicroscope, and pictures were taken with an Olympus BX61camera.
3. Analysis of cell cycle
After treatment with PTE for24h, cells were collected by trypsinization, fixed in70%ethanol, washed in PBS, resuspended in1mL of PBS co ntaining0.02mg/mL RNaseand0.02mg/mL PI, and incubated in the dark for30min at room temperature. The cellcycle distribution was analyzed using a FACScan flow cytometer equipped with theFACStation data management system, running the Cell Quest software. The results areexpressed in a plot of fluorescence intensity vs. cell number.
4. Analysis of cell mitochondrial transme mbrane potential
MMP was estimated by flow cytometry after staining with JC-1fluorescent dye.When the cell is in a normal state, MMP is high, and JC-1predominantly appears as redfluorescence. When the cell is in an apoptotic or necrotic state, the MMP is reduced, andJC-1appears as a monomer that shows green fluorescence. A change in florescence fromred to green indicates a decrease in MMP. The cells were plated in6-well plates andtreated with PTE for24h. Then, the cells were washed with PBS and incubated with JC-1working solution for20min at37°C in the dark. The cells were washed with PBS andresuspended in500μl PBS. The stained cells were analyzed by the same flow cytometerand software used for the cell cycle analysis. The results are expressed as the proportion ofcells with low MMP.
5. Analysis of cell apoptosis
The apoptosis of osteosarcoma cells was detected using the fluoresceinisothiocyanate FITC-Annexin V/PI staining Kit. After being treated with PTE for24h, thecells were harvested, washed in ice-cold PBS, incubated for15min withfluorescein-conjugated Annexin V and PI, and analyzed using the same flow cytometerand software used for the cell cycle analysis. PI-negative and Annexin V-positive cellswere considered early apoptotic (lower right quadrant), while cells that were both PI-andAnnexin V-negative were considered normal (lower left quadrant).
6. Analysis of wound-healing
The cells were grown to confluence, and a linear wound was created in the confluentmonolayer using a200μl micropipette tip. The cells were then washed with PBS to eliminate detached cells. After being treated with PTE for24h, wound edge movementwas monitored with a microscope. The results are expressed as the distance between thecells on each side of the scratch.
7. Analysis of cell adhesion
After being treated with PTE for24h, the cells were centrifuged and resuspended inbasal medium with10%fetal bovine serum. The treated cells were placed on a96-wellplate and incubated for30min at37°C. After the cells were allowed to adhere for30min,they were gently washed3times with PBS. The adherent cells were stained with MTT andobserved under an inverted/phase contrast microscope, and pictures were taken with anOlympus BX61camera (Japan). Finally,100μL of N, N-dimethylformamide was addedto each well, and the samples were incubated for15min at37°C with shaking. The wellswere measured at490nm using a spectrophotometer (SpectraMax190, Molecular Device,USA), and the OD value in the control group was set as100%.
8. Statistical Analyses
All experiments were performed in duplicate and repeated at least three times. Thedata are expressed as the means±the standard deviation (SD). The treatment groups werecompared by one-way variance (ANOVA) with SPSS12.0. Differences were consideredstatistically significant at P <0.05.
Results
1. Effects of PTE on viability and apoptosis in osteosarcoma cells
Osteosarcoma cells were treated with PTE for12,24, and36h with1,2and4μM ofPTE, and cell growth was inhibited in a dose-and time-dependent manner. The IC50(50%inhibitory concentration) of PTE at24h was approximately1.81μM. As observed underthe microscope, PTE treatment resulted in a decrease in the rate of cellular attachmentcompared to the control group.
After treatment with1,2and4μM of PTE for24h, the apoptotic index increased by16.75±3.91%,21.55±3.26%and35.87±4.23%, respectively (P<0.01, compared with the control group). The induction of apoptosis was found to be dose-dependent. These resultsindicate that PTE induces apoptosis in osteosarcoma cells.
2. Effects of PTE on osteosarcoma cell migration and adhesion
After incubation with PTE (1,2and4μM) for24h, the distance between thescratches significantly increased to123.07±9.05%,161.53±8.28%, and223.08±11.30%, respectively (P<0.01, compared with the control group), and the cell adhesionratio decreased significantly to62.29±9.43%,34.38±6.70%, and13.64±3.35%,respectively (P<0.01, compared with the control group). These results indicate that PTEreduces the adhesive and migratory abilities of osteosarcoma cells.
3. Effects of PTE on mitochondrial me mbrane potential in osteosarcoma cells
After treatment with PTE (1,2and4μM) for24h, the proportion of cells with lowMMP increased significantly to8.19±2.36%,16.17±2.68%,29.45±3.10%, respectively(P<0.01, compared with the control group). These results indicate that PTE enhanced theMMP of osteosarcoma cells.
4. Effects of PTE on the cell cycle in osteosarcoma cells
To further investigate the effect of PTE on cell growth, we analyzed the effect of PTEon the cell cycle distribution of osteosarcoma cells. Compared with untreated control cells,PTE (1,2and4μM) induced an accumulation of cells in the G0–G1phase fractions. TheG0–G1phase fraction increased from33.27%in control cells to55.22%in PTE (4μM)induced cells.
Conclusion
PTE treatment resulted in a dose-and time-dependent inhibition of osteosarcoma cellviability. Additionally, PTE exhibited strong antitumor activity, as evidenced byreductions in tumor cell adhesion, migration and mitochondrial membrane potential.
Part Ⅱ Pterostilbene exerts antitumor activity byinhibiting the JAK2/STAT3signaling pathway
Objective
To investigate the role of the JAK2/STAT3signaling pathway on activity of PTEagainst human osteosarcoma cells; to investigate effects of PTE on the mitochondrialapoptotic pathway and cell cycle-related proteins in osteosarcoma cells.Methods
1. Cell culture and treatment
See part I. Then, the cells were treated withPTE (4μM) in the absence or presence ofAG490(a known JAK2/STAT3inhibitor,20μM). After the treatments, the cells wereharvested for further analysis.
2. Analysis of intracellular ROS generation and GSH levels
After being treated with PTE for24h, the cells were trypsinized and subsequentlyincubated with DCFH-DA (20μM) in PBS at37℃for2h. After incubation, the DCFHfluorescence of the cells in each well was measured using an FLX800microplatefluorescence reader at530nm as the emission wavelength and485nm as the excitationwavelength. A cell-free condition was used to determine the background, and thefluorescence intensity in the control group was defined as100%.
Briefly, cells were plated at a density of1×106in100-mm culture dishes, allowed toattach overnight, and treated with PTE on the second day. The cells were collected byscraping and washed with PBS. The resulting lysates were used to determine the GSHlevels using the previously mentioned kit, according to the manufacturer's instructions. Todetermine the GSSG levels, GSH was masked by2-vinylpyridine for1h before the assay.The samples were read at405nm at5min intervals for30min. The GSH and GSSG were evaluated by comparison with standards and normalized with protein content. The resultswere expressed as total GSH (%of control) or GSH/GSSG ratio, using the reduced formGSH or an oxidized form of GSH (GSSG) as the standard.
3. Western Blotting
Cell samples were lysed in sample buffer, sonicated, boiled, run through an8-12%Bis/Tris gel using5×MES buffer, transferred to Immobilon NC membrane and blocked in5%nonfat milk in TBST. The membranes were probed with p-JAK2, JAK2, p-STAT3,STAT3, Mcl-1, Cyclin D1, p21, p27(1:500), Bcl-xL, Bax, Bak and GAPDH antibodies(1:1000) overnight at4oC in blocking buffer. The membranes were then washed in TBSTand probed with the appropriate secondary antibodies (1:5000) in blocking buffer at roomtemperature for90min, followed by washing. Fluorescence was detected using a BioRadimaging system. The signals were quantified using the Image Lab Software.
4. Extract of cytosolic Cytochrome c
After being treated, the cells were harvested by centrifugation at1,000rpm for5min.The pellets were washed twice with ice-cold PBS, suspended with5-fold volume ice-coldcell extract buffer, and incubated for40min at4oC. Then, the cells were centrifuged at1,200rpm for10min at4oC and the final supernatant was used as cytosolic fraction ofCytochrome c. Then,5×loading buffer was added to the above obtained supernatant andthe mixture was boiled at100oC for7min. Thus, the protein solution was used foridentification of cytosolic Cytochrome c by Western blotting. The Cytochrome c proteinwas detected by using anti-Cytochrome c antibody in the ratio of1:500.
5. Statistical Analyses
See part I
Results
1. Effects of PTE on the expression of cell cycle-regulated proteins in osteosarcomacells
To further characterize the observed G0–G1phase arrest, we assayed the expressionof Cyclin D1, p21, and p27by Western blotting. Consistent with cell cycle arrest, theexpression level of Cyclin D1decreased, while the expression levels of p21and p27increased. This finding suggests that G0–G1phase arrest by PTE is, at least in part, due toprofound alterations in the expression of regulatory cell cycle-related factors.
2. Effects of PTE on ROS generation and GSH levels in osteosarcoma cells
Treatment with PTE (1,2and4μM) for24h induced a dose-dependent increase inROS generation in osteosarcoma cells (Fig.4A), with increases of255.32±12.97%,411.59±16.24%, and503.61±17.05%, respectively (P<0.01, compared to the controlgroup).
After treatment with PTE for24h, we observed a dose-dependent decrease (80.51±3.06%,66.43±3.82%,52.30±3.49%, respectively) in intracellular GSH levels inosteosarcoma cells,(P<0.01, compared with the control group). PTE induced adose-dependent decrease in the ratio of GSH/GSSG in osteosarcoma cells. These resultssupport the notion that PTE treatment affects cellular redox status.
3. Effects of PTE on the JAK2/STAT3signaling pathway and mitochondrialapoptotic pathway-related proteins in osteosarcoma cells
The phosphorylated forms ofJAK2and STAT3were assessed by Western blotanalysis in osteosarcoma cells treated with PTE for24h. We found that thephosphorylation of these factors was decreased by PTE in osteosarcoma cells. Consistentwith the down-regulation of p-JAK2and p-STAT3, the expression of Mcl-1and Bcl-xLwas reduced by PTE in a dose-dependent manner. In addition, mitochondrial apoptoticpathway-related proteins (Bax, Bak, cytosolic Cytochrome c and cleaved Caspase3) wereup-regulated by PTE treatment, suggesting that this apoptotic pathway was activated.
4. Effects of the combination of PTE and AG490on cell viability and theJAK2/STAT3signaling in osteosarcoma cells
Combined treatment with PTE (4μM) andAG490(a known JAK2/STAT3inhibitor,20μM) further inhibited the viability of osteosarcoma cells (P<0.01, compared with thePTE or AG490group). Thus, treatment with PTE and AG490further inhibits the phosphorylation of JAK2and STAT3.
Conclusions
In conclusion, these studies provide mechanistic evidence that PTE treatment inhibitsosteosarcoma cell growth via down-regulation of the JAK2/STAT3signaling pathway.PTE seems to regulate multiple molecular targets to produce its anti-osteosarcoma effect,including the activation of the mitochondrial apoptotic pathway and the regulation of cellcycle related proteins.
骨肉瘤是一种儿童及青少年最常见的恶性骨肿瘤，高发于四肢长骨的干骺端。目前常规的化疗药物均为细胞毒性药物，副作用大。几乎一半的儿童期肿瘤幸存者因为抗肿瘤治疗至少承受一个不良后果。化疗药物早期毒性包括血液系统毒性，急性肝脏毒性和肾毒性。生存期的延长导致了晚期化疗毒性的增加。主要的晚期毒性有：心脏毒性、继发肿瘤、不育、慢性肾衰竭和神经系统毒性。因此寻找低毒的抗骨肉瘤药物具有重要的临床意义。
紫檀芪（PTE）是一种来源于我们食物的天然化合物，毒性极低，对人体是安全的。PTE是白藜芦醇的二甲基化类似物，具有多种生物学活性，可以抗炎、抗氧化、抗增殖、抗肿瘤及止痛。因甲氧基团替代了羟基增加了亲脂性，增加了生物利用度，从而增加了活性。研究表明PTE可以抑制多种肿瘤细胞的增殖并诱导其凋亡，包括肝癌、乳腺癌、前列腺癌、胰腺癌及白血病等。PTE是否具有抗骨肉瘤作用目前尚无人研究。
PTE抗肿瘤作用涉及多种细胞分子和信号通路，如单磷酸腺苷活化激酶(AMPK)、细胞基质钙超载、活性氧（ROS）、自噬、Wnt信号通路及溶酶体膜通透作用增强等，但具体的抗肿瘤机制目前尚不清楚。JAK/STAT通路对体内多种信号的转导都非常的关键，对多种哺乳动物发育及内环境的稳定都异常的重要。JAK家族包括JAK1、JAK2、JAK3及酪氨酸激酶2，这4个蛋白结构及功能相似。JAK的活化在细胞增殖、分化、迁移及凋亡中均有重要的作用。结构活化的JAKs会使细胞内许多重要的物质磷酸化，其中包括STAT家族，STAT家族中的STAT3与许多肿瘤的信号通路相关。结构活化后的STAT3和人实体肿瘤细胞的存活与生长关系密切。STAT3还可上调人肿瘤细胞抗凋亡基因编码的Mcl-1、Bcl-xL蛋白。PTE可以抑制STST3的磷酸化，磷酸化的STST3是一种肿瘤快速生长的标志。更为重要的是活化的JAK2/STAT3信号作为治疗实体肿瘤一个重要的目标分子已经被广泛的确认其有效性。PTE抗骨肉瘤的作用是否与抑制JAK2/STAT3通路的磷酸化程度有关目前尚不清楚。
第一部分PTE抗人骨肉瘤细胞活性研究
研究目的
研究PTE对人骨肉瘤细胞生长及凋亡的影响；研究PTE对人骨肉瘤细胞迁移及粘附能力的影响。
研究方法
1.细胞培养及处理
人骨肉瘤细胞系SOSP-9607(9607)，复苏后在10%的胎牛血清DMEM液中培养，加入L-谷酰胺、青霉素、链霉素及HEPES液。孵箱内温度维持在37°C，CO2浓度为5%。收集细胞备用。
PTE原液中加入二甲基亚砜，以培养液稀释，即配即用。对照组中加入0.01%的二甲基亚砜。实验组加入PTE，浓度梯度为1、2及4μM。
2.细胞活力检测
细胞预处理后以PBS液洗涤三次，加入MTT液，37°C的孵箱中孵育4h，加入二甲基甲酰胺，震荡，在酶联免疫监测仪上检测细胞活力。细胞活力以各孔吸光值（OD）的形式记录，测定各孔光吸收值，记录结果，绘制细胞生长曲线。并在倒置相差显微镜下观察细胞形态、照相。
3.细胞周期分析
细胞预处理后胰酶消化，70%的乙醇固定后PBS液洗涤，加入核糖核苷酸及碘化丙啶，避光室温下孵育30min。以配备数据采集及分析系统的流式细胞仪分析并记录数据。结果以荧光密度分布图的形式表示，荧光密度代表细胞数目。
4.线粒体跨膜电位（MMP）分析
将细胞放入六孔板中加入不同浓度的PTE，孵育24h。以PBS液洗涤，加入JC-1工作液，避光37°C孵育。细胞洗涤后悬浮于PBS液中，将染色的细胞用上述流式细胞仪分析，结果以较低MMP的细胞比例表示。
5.细胞凋亡分析
将细胞预处理后，冰PBS液冲洗，加入荧光标记的共轭膜联蛋白及碘化丙啶，使用上述流式细胞仪检测细胞凋亡情况并用相关软件进行分析。碘化丙啶阴性且共轭膜联蛋白阳性的细胞被认为是早期凋亡细胞，双阴性细胞被认为是正常细胞。
6.划痕试验
培养细胞至铺满瓶底后，微量管尖端在单层细胞处制造划痕。将分离的细胞以PBS洗涤干净，加入PTE24h后，使用显微镜观察并记录划痕边缘距离变化情况。结果以每组划痕之间的距离数值表示。
7.细胞粘附实验
PTE处理骨肉瘤细胞24h后消化、离心，悬浮在加入10%胎牛血清的DMEM液中。37°C下孵育30min，PBS液洗涤。将贴壁细胞用法MTT染色，在倒置相差显微镜下观察细胞粘附情况并照相。加入二甲基甲酰胺，37°C下震荡孵育15min，在490nm条件下使用分光光度计测量，结果以OD值表示，将对照组OD值设为100%。
8.统计学分析
所有的样本一式两份，重复至少3次，数据已平均值±标注差的形式表示。实验组间比较使用SPSS12.0，采用方差分析的方法(ANOVA)。差异P<0.05被认为统计学上有意义。
研究结果
1. PTE对骨肉瘤细胞生长及凋亡的影响
骨肉瘤细胞培养液中加入1,2及4μM浓度梯度的PTE后，分别培养12、24及36h，细胞生长均出现抑制。PTE对骨肉瘤细胞的生长抑制作用呈剂量和时间依赖。培养至24h时PTE的IC50(50%抑制浓度)约为1.81μM。与对照组比较，PTE导致细胞粘附率下降。加入1、2及4μM PTE后，与对照组相比较，凋亡指数分别增加16.75±3.91%、21.55±3.26%及35.87±4.23%(P<0.01)。我们发现凋亡指数呈剂量依赖性。这个结果说明PTE对人骨肉瘤细胞有生长抑制及诱导凋亡的作用。
2. PTE对骨肉瘤细胞迁移及粘附能力的影响
加入1、2及4μM的PTE，孵育24h后，随着浓度的增加，划痕边缘的距离显著增加，分别增加至对照组的123.07±9.05%、161.53±8.28%及223.08±11.30%(P<0.01)。粘附细胞显著减少，粘附率分别为对照组的62.29±9.43%、34.38±6.70%及13.64±3.35%(P<0.01)。这些结果表明PTE可以降低骨肉瘤细胞的迁移能力及粘附能力。
3. PTE对骨肉瘤细胞MMP的影响
加入1、2及4μM的PTE，孵育24h后，低MMP细胞的比例显著增加。各组低MMP的细胞比例分别增加至8.19±2.36%、16.17±2.68%及29.45±3.10%(与对照组比较，P<0.01)。结果表明PTE可以增加骨肉瘤细胞的MMP。
4. PTE对骨肉瘤细胞周期的影响
与对照组相比较，加入不同浓度的PTE后均会导致细胞G0-G1阶段的聚积。G0-G1阶段的细胞由对照组的33.27%增加至4μM PTE组的55.22%，可见PTE会导致细胞周期G0-G1期的阻滞。
结论
PTE具有很强的抗人骨肉瘤细胞活性的作用。PTE导致人骨肉瘤细胞G0-G1期阻滞，使细胞生长出现抑制；同时PTE降低了MMP，诱导了细胞的凋亡。PTE还可以减少骨肉瘤细胞的粘附能力及迁移能力。
第二部分PTE抗人骨肉瘤细胞活性的机制研究
研究目的：
研究PTE抗人骨肉瘤细胞活性的相关机制。
研究方法
1.细胞培养及处理
同第一部分最后一步实验中加入AG490，PTE的浓度为4μM
2.细胞内活性氧（ROS）及谷胱甘肽水平（GSH）检测
细胞预处理后胰酶裂解，加入DCFH-DA液，37oC下孵育2h。FLX800微量盘荧光光谱仪测量每孔二氯荧光素的强度。无细胞孔作为背景，将对照组荧光强度定为100%。
细胞加入培养皿中过夜后加入PTE24h后。PBS洗涤细胞。根据探针的操作指南，将细胞裂解后测量GSH的水平。在测量前1h用2-乙烯基吡啶遮蔽GSH。GSH和GSSG的含量与正常标准的蛋白含量比较。结果以GSH的表达（%对照含量）或GSH/GSSG比例的形式表达，使用还原形式的GSH或氧化形式的GSH（GSSG）作为标准。
3. Westen Blotting
将细胞样本在样本缓冲液中裂解，降噪处理，煮沸，跑凝胶，转膜，封闭。加入p-JAK2、JAK2、p-STAT3、STAT3、Mcl-1、Cyclin D1、p21、p27、Bcl-xL、Bax、Bak及GAPDH的抗体4oC过夜。洗膜，加入对应的二抗，室温下放置90min，洗涤。使用BioRad成像系统观察记录荧光强度，并以Image Lab软件定量分析。
4.细胞质中细胞色素C的萃取
处理离心细胞，PBS液洗涤2次后悬浮于冰的细胞萃取缓冲液中，4oC下孵育40min，离心，上清液用来提取细胞色素C，加入5倍上清的缓冲液，100oC下煮沸7min，将蛋白溶液用Western blotting的方法测定细胞色素C的含量。
5.统计学分析
同第一部分
研究结果
1. PTE对细胞周期相关蛋白的影响
为了进一步验证细胞G0–G1阻滞，我们利用Westernblotting的方法检测周期蛋白D1、p21及p27的表达。与细胞周期阻滞一致，Cyclin D1的表达水平降低，而p21和p27的表达水平升高。这个发现表明PTE可以导致细胞周期相关蛋白表达的变化。
2. PTE对骨肉瘤细胞活性氧生成及谷胱甘肽水平的影响
加入1、2及4μM的PTE，孵育24h后骨肉瘤细胞内产生的活性氧呈剂量依赖关系，分别增加至对照组的255.32±12.97%、411.59±16.24%及503.61±17.05%(与对照组比较，P<0.01)。
加入1、2及4μM的PTE，孵育24h后骨肉瘤细胞谷胱甘肽水平呈剂量依赖性降低，分别降低了80.51±3.06%、66.43±3.82%及52.30±3.49%。(与对照组比较，P<0.01)。PTE会导致骨肉瘤细胞内GSH/GSSG的比例呈剂量依赖性降低。
3.对人骨肉瘤细胞JAK2/STAT3信号通路及线粒体凋亡通路相关蛋白的影响
加入PTE后磷酸化的JAK2及STAT3的表达水平降低。与磷酸化的JAK2及STAT3的表达下调相一致的是PTE使Mcl-1和Bcl-xL的表达水平也降低，且呈剂量依赖关系。除此之外PTE使线粒体凋亡通路相关蛋白(Bax、Bak、细胞色素C)的表达上调。
4. PTE联合AG490对人骨肉瘤细胞活力及JAK2/STAT3信号的影响
联合应用PTE（4μM）和AG490(一种已知的JAK2/STAT3抑制剂，20μM)进一步抑制了骨肉瘤细胞的活力(与单独的PTE组或AG490组比较，P<0.01)。并且联合应用PTE和AG490也进一步抑制了JAK2及STAT3的磷酸化程度。
结论
PTE通过抑制JAK2/STAT3信号通路影响CDK/Cyclin复合物和CKIs的平衡，导致了细胞G0-G1期阻滞，使人骨肉瘤细胞出现了生长抑制；同时活化内源性线粒体凋亡途径，诱导了人骨肉瘤细胞的凋亡。
Background
Osteosarcoma is a most common high-grade malignant bone tumor occurringfrequently in children and adolescents. At the moment anti-osteosarcoma drugs havecytotoxicity and severe side effects which are harmful to our health. Nearly a half ofyoung adult survivors of childhood cancer have at least one major adverse outcome oftheir health status as a result of their cancer therapy. Early toxicity consists ofhematological toxicity, acute liver (MTX) and renal toxicity (CDP and IFO). Longersurvival has led to the increase in late chemotherapy toxicity. The main late toxicities are:cardiac, second tumor, sterility, chronic renal failure and neurologic toxicities (mainlyototoxicity due to CDP). It is urgent clinically to develop safe and efficient agents withhypotoxicity.
Pterostilbene(trans-3,5-dimethoxy-4’-hydroxystilbene, PTE), a natural compound occurring from our daily diet appears to be a safe and efficient agent. Pterostilbene,dimethylated analog of resveratrol from blueberries, is known to have diversepharmacologic activities, including anticancer, anti-inflammation, antioxidant,anti-proliferative and analgesic activities PTE has been shown to have potent antitumoractivity with low toxicity in various cancer types, including hepatoma, breast cancer,prostate cancer, pancreatic cancer and chronic myelogenous leukemia, among others. Inmost circumstances, PTE was as potent or significantly more potent than resveratrol,indicating that PTE might show better biological activity due to its increasedbioavailability, as the substitution of a hydroxy group with a methoxy group increases itslipophilicity. However, the effects of PTE in human osteosarcoma have not been clarified.
In addition, various molecules and signaling pathways are involved in the anti-tumoreffects of PTE, including adenosine monophosphate-activated protein kinase (AMPK),cytosolic Ca2+overload, ROS, autophagy, Wnt signaling, and lysosomal membranepermeabilization, among others. The molecular mechanisms underlying the effects of PTEremain largely unknown. The JAK/STAT pathway is pivotal for the transduction of amultitude of signals that are critical for development and homeostasis in mammals. TheJAK family of proteins consists of JAK1, JAK2, JAK3and tyrosine kinase2, all of whichshare similar structures and functions. JAK activation plays an important role in cellproliferation, differentiation, migration and apoptosis. Constitutively activated JAKsphosphorylate critical cellular substrates, such as the STAT family, which includesSTAT3, that are associated with oncogenic signaling pathways. Constitutive activation ofSTAT3plays a critical role in cell growth and survival in human solid tumor malignanciesand in the up-regulation of the anti-apoptotic genes encoding Mcl-1and Bcl-xL proteins inhuman cancer cells. PTE was also shown to inhibit STAT3phosphorylation, a marker ofaccelerated tumorigenesis, and decrease pancreatic tumor growth in vivo. Importantly,activated JAK2/STAT3signaling has been extensively validated as a new molecular targetfor the treatment of human solid tumors. The mechanisms of PTE in human osteosarcomahave not been clarified
Part I Antitumor activity of PTE against humanosteosarcoma cells
Objective
To investigate the effects of PTE on proliferation and apoptosis in humanosteosarcoma cells; to investigate the effects of PTE on migration and adhesion in humanosteosarcoma cells; to investigate the effects of PTE on cell cycle.
Methods
1. Cell culture and treatment
The human osteosarcoma cell line, SOSP-9607(9607), were grown in Dulbecco'smodified Eagle's medium, supplemented with10%fetal bovine serum, L-glutamine (2mM), penicillin (100units/ml), streptomycin (100units/ml), and HEPES (25mM). Thecells were maintained in the presence of5%CO2at37°C.
The PTE stock solution was prepared in DMSO and diluted with culture mediumimmediately prior to the experiment. The control group was treated with DMSO (0.01%).First, the cells were treated with PTE (1,2, and4μM).
2. Analysis of cell viability
After the cells were treated and washed with PBS,100μL of0.5mg/mL MTTsolution in phenol red-free DMEM was added to the cells, and the samples were incubatedfor4h at37°C. Finally,100μL ofN, N-dimethylformamide was added to each well, andthe samples were incubated for15min at37°C with shaking. The wells were measured at490nm using a microplatereader and cell viability was expressed as an optical density(OD) value. In addition, cell morphology was observed under an inverted/phase contrastmicroscope, and pictures were taken with an Olympus BX61camera.
3. Analysis of cell cycle
After treatment with PTE for24h, cells were collected by trypsinization, fixed in70%ethanol, washed in PBS, resuspended in1mL of PBS co ntaining0.02mg/mL RNaseand0.02mg/mL PI, and incubated in the dark for30min at room temperature. The cellcycle distribution was analyzed using a FACScan flow cytometer equipped with theFACStation data management system, running the Cell Quest software. The results areexpressed in a plot of fluorescence intensity vs. cell number.
4. Analysis of cell mitochondrial transme mbrane potential
MMP was estimated by flow cytometry after staining with JC-1fluorescent dye.When the cell is in a normal state, MMP is high, and JC-1predominantly appears as redfluorescence. When the cell is in an apoptotic or necrotic state, the MMP is reduced, andJC-1appears as a monomer that shows green fluorescence. A change in florescence fromred to green indicates a decrease in MMP. The cells were plated in6-well plates andtreated with PTE for24h. Then, the cells were washed with PBS and incubated with JC-1working solution for20min at37°C in the dark. The cells were washed with PBS andresuspended in500μl PBS. The stained cells were analyzed by the same flow cytometerand software used for the cell cycle analysis. The results are expressed as the proportion ofcells with low MMP.
5. Analysis of cell apoptosis
The apoptosis of osteosarcoma cells was detected using the fluoresceinisothiocyanate FITC-Annexin V/PI staining Kit. After being treated with PTE for24h, thecells were harvested, washed in ice-cold PBS, incubated for15min withfluorescein-conjugated Annexin V and PI, and analyzed using the same flow cytometerand software used for the cell cycle analysis. PI-negative and Annexin V-positive cellswere considered early apoptotic (lower right quadrant), while cells that were both PI-andAnnexin V-negative were considered normal (lower left quadrant).
6. Analysis of wound-healing
The cells were grown to confluence, and a linear wound was created in the confluentmonolayer using a200μl micropipette tip. The cells were then washed with PBS to eliminate detached cells. After being treated with PTE for24h, wound edge movementwas monitored with a microscope. The results are expressed as the distance between thecells on each side of the scratch.
7. Analysis of cell adhesion
After being treated with PTE for24h, the cells were centrifuged and resuspended inbasal medium with10%fetal bovine serum. The treated cells were placed on a96-wellplate and incubated for30min at37°C. After the cells were allowed to adhere for30min,they were gently washed3times with PBS. The adherent cells were stained with MTT andobserved under an inverted/phase contrast microscope, and pictures were taken with anOlympus BX61camera (Japan). Finally,100μL of N, N-dimethylformamide was addedto each well, and the samples were incubated for15min at37°C with shaking. The wellswere measured at490nm using a spectrophotometer (SpectraMax190, Molecular Device,USA), and the OD value in the control group was set as100%.
8. Statistical Analyses
All experiments were performed in duplicate and repeated at least three times. Thedata are expressed as the means±the standard deviation (SD). The treatment groups werecompared by one-way variance (ANOVA) with SPSS12.0. Differences were consideredstatistically significant at P <0.05.
Results
1. Effects of PTE on viability and apoptosis in osteosarcoma cells
Osteosarcoma cells were treated with PTE for12,24, and36h with1,2and4μM ofPTE, and cell growth was inhibited in a dose-and time-dependent manner. The IC50(50%inhibitory concentration) of PTE at24h was approximately1.81μM. As observed underthe microscope, PTE treatment resulted in a decrease in the rate of cellular attachmentcompared to the control group.
After treatment with1,2and4μM of PTE for24h, the apoptotic index increased by16.75±3.91%,21.55±3.26%and35.87±4.23%, respectively (P<0.01, compared with the control group). The induction of apoptosis was found to be dose-dependent. These resultsindicate that PTE induces apoptosis in osteosarcoma cells.
2. Effects of PTE on osteosarcoma cell migration and adhesion
After incubation with PTE (1,2and4μM) for24h, the distance between thescratches significantly increased to123.07±9.05%,161.53±8.28%, and223.08±11.30%, respectively (P<0.01, compared with the control group), and the cell adhesionratio decreased significantly to62.29±9.43%,34.38±6.70%, and13.64±3.35%,respectively (P<0.01, compared with the control group). These results indicate that PTEreduces the adhesive and migratory abilities of osteosarcoma cells.
3. Effects of PTE on mitochondrial me mbrane potential in osteosarcoma cells
After treatment with PTE (1,2and4μM) for24h, the proportion of cells with lowMMP increased significantly to8.19±2.36%,16.17±2.68%,29.45±3.10%, respectively(P<0.01, compared with the control group). These results indicate that PTE enhanced theMMP of osteosarcoma cells.
4. Effects of PTE on the cell cycle in osteosarcoma cells
To further investigate the effect of PTE on cell growth, we analyzed the effect of PTEon the cell cycle distribution of osteosarcoma cells. Compared with untreated control cells,PTE (1,2and4μM) induced an accumulation of cells in the G0–G1phase fractions. TheG0–G1phase fraction increased from33.27%in control cells to55.22%in PTE (4μM)induced cells.
Conclusion
PTE treatment resulted in a dose-and time-dependent inhibition of osteosarcoma cellviability. Additionally, PTE exhibited strong antitumor activity, as evidenced byreductions in tumor cell adhesion, migration and mitochondrial membrane potential.
Part Ⅱ Pterostilbene exerts antitumor activity byinhibiting the JAK2/STAT3signaling pathway
Objective
To investigate the role of the JAK2/STAT3signaling pathway on activity of PTEagainst human osteosarcoma cells; to investigate effects of PTE on the mitochondrialapoptotic pathway and cell cycle-related proteins in osteosarcoma cells.Methods
1. Cell culture and treatment
See part I. Then, the cells were treated withPTE (4μM) in the absence or presence ofAG490(a known JAK2/STAT3inhibitor,20μM). After the treatments, the cells wereharvested for further analysis.
2. Analysis of intracellular ROS generation and GSH levels
After being treated with PTE for24h, the cells were trypsinized and subsequentlyincubated with DCFH-DA (20μM) in PBS at37℃for2h. After incubation, the DCFHfluorescence of the cells in each well was measured using an FLX800microplatefluorescence reader at530nm as the emission wavelength and485nm as the excitationwavelength. A cell-free condition was used to determine the background, and thefluorescence intensity in the control group was defined as100%.
Briefly, cells were plated at a density of1×106in100-mm culture dishes, allowed toattach overnight, and treated with PTE on the second day. The cells were collected byscraping and washed with PBS. The resulting lysates were used to determine the GSHlevels using the previously mentioned kit, according to the manufacturer's instructions. Todetermine the GSSG levels, GSH was masked by2-vinylpyridine for1h before the assay.The samples were read at405nm at5min intervals for30min. The GSH and GSSG were evaluated by comparison with standards and normalized with protein content. The resultswere expressed as total GSH (%of control) or GSH/GSSG ratio, using the reduced formGSH or an oxidized form of GSH (GSSG) as the standard.
3. Western Blotting
Cell samples were lysed in sample buffer, sonicated, boiled, run through an8-12%Bis/Tris gel using5×MES buffer, transferred to Immobilon NC membrane and blocked in5%nonfat milk in TBST. The membranes were probed with p-JAK2, JAK2, p-STAT3,STAT3, Mcl-1, Cyclin D1, p21, p27(1:500), Bcl-xL, Bax, Bak and GAPDH antibodies(1:1000) overnight at4oC in blocking buffer. The membranes were then washed in TBSTand probed with the appropriate secondary antibodies (1:5000) in blocking buffer at roomtemperature for90min, followed by washing. Fluorescence was detected using a BioRadimaging system. The signals were quantified using the Image Lab Software.
4. Extract of cytosolic Cytochrome c
After being treated, the cells were harvested by centrifugation at1,000rpm for5min.The pellets were washed twice with ice-cold PBS, suspended with5-fold volume ice-coldcell extract buffer, and incubated for40min at4oC. Then, the cells were centrifuged at1,200rpm for10min at4oC and the final supernatant was used as cytosolic fraction ofCytochrome c. Then,5×loading buffer was added to the above obtained supernatant andthe mixture was boiled at100oC for7min. Thus, the protein solution was used foridentification of cytosolic Cytochrome c by Western blotting. The Cytochrome c proteinwas detected by using anti-Cytochrome c antibody in the ratio of1:500.
5. Statistical Analyses
See part I
Results
1. Effects of PTE on the expression of cell cycle-regulated proteins in osteosarcomacells
To further characterize the observed G0–G1phase arrest, we assayed the expressionof Cyclin D1, p21, and p27by Western blotting. Consistent with cell cycle arrest, theexpression level of Cyclin D1decreased, while the expression levels of p21and p27increased. This finding suggests that G0–G1phase arrest by PTE is, at least in part, due toprofound alterations in the expression of regulatory cell cycle-related factors.
2. Effects of PTE on ROS generation and GSH levels in osteosarcoma cells
Treatment with PTE (1,2and4μM) for24h induced a dose-dependent increase inROS generation in osteosarcoma cells (Fig.4A), with increases of255.32±12.97%,411.59±16.24%, and503.61±17.05%, respectively (P<0.01, compared to the controlgroup).
After treatment with PTE for24h, we observed a dose-dependent decrease (80.51±3.06%,66.43±3.82%,52.30±3.49%, respectively) in intracellular GSH levels inosteosarcoma cells,(P<0.01, compared with the control group). PTE induced adose-dependent decrease in the ratio of GSH/GSSG in osteosarcoma cells. These resultssupport the notion that PTE treatment affects cellular redox status.
3. Effects of PTE on the JAK2/STAT3signaling pathway and mitochondrialapoptotic pathway-related proteins in osteosarcoma cells
The phosphorylated forms ofJAK2and STAT3were assessed by Western blotanalysis in osteosarcoma cells treated with PTE for24h. We found that thephosphorylation of these factors was decreased by PTE in osteosarcoma cells. Consistentwith the down-regulation of p-JAK2and p-STAT3, the expression of Mcl-1and Bcl-xLwas reduced by PTE in a dose-dependent manner. In addition, mitochondrial apoptoticpathway-related proteins (Bax, Bak, cytosolic Cytochrome c and cleaved Caspase3) wereup-regulated by PTE treatment, suggesting that this apoptotic pathway was activated.
4. Effects of the combination of PTE and AG490on cell viability and theJAK2/STAT3signaling in osteosarcoma cells
Combined treatment with PTE (4μM) andAG490(a known JAK2/STAT3inhibitor,20μM) further inhibited the viability of osteosarcoma cells (P<0.01, compared with thePTE or AG490group). Thus, treatment with PTE and AG490further inhibits the phosphorylation of JAK2and STAT3.
Conclusions
In conclusion, these studies provide mechanistic evidence that PTE treatment inhibitsosteosarcoma cell growth via down-regulation of the JAK2/STAT3signaling pathway.PTE seems to regulate multiple molecular targets to produce its anti-osteosarcoma effect,including the activation of the mitochondrial apoptotic pathway and the regulation of cellcycle related proteins.
引文
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