ZNF488对电离辐射诱发的鼻咽癌细胞CNE1侵袭迁移能力的影响

[目的]研究ZNF488 siRNA对电离辐射诱发的鼻咽癌CNE1侵袭迁移能力的影响.[方法]采用qRT-PCR和Western blot检测ZNF488 siRNA转染效率.采用划痕实验和Transwell侵袭实验检测电离辐射对鼻咽癌CNE1细胞侵袭迁移能力的影响.采用划痕实验和Transwell侵袭实验检测沉默ZNF488后,电离辐射诱发的CNE1细胞侵袭迁移能力是否改变.[结果]在mRNA和蛋白水平,实验组(siZNF488组)表达量均明显低于对照组(siRNA-Ctrl 组),其中实验组ZNF488 mRNA水平为对照组的(0.54±0.12)倍(P=0.023).划痕实验证明电离辐射明显增强鼻咽癌CNE1细胞迁移能力;Traoswell侵袭实验检测到0Gy组侵袭细胞数为302.67± 18.77,4Gy组侵袭细胞数为371.67± 15.63,4Gy组侵袭细胞数为0Gy组(1.23±0.03)倍(P=0.006).沉默ZNF488后,电离辐射诱发的CNE1细胞侵袭迁移能力明显减弱,这一功能的实现与上皮间质转化(EMT)进程的逆转息息相关.[结论]ZNF488 siRNA通过逆转EMT进程抑制鼻咽癌细胞CNE1电离辐射诱发的侵袭迁移能力.     

Comparison of Amino Acid Metabolisms in Normal Prostate (PNT-1A) and Cancer Cells (PC-3)

Prostate cancer is the second most common cancer in men. Prostate-specific antigen (PSA) levels, commonly used in the diagnosis of prostate cancer, are increased in both malign and benign conditions, such as prostate hyperplasia (BPH) and prostatitis. Thus, more specific markers are urgently needed to discriminate between prostate cancer and benign diseases of the prostate. The purpose of this study is to examine both the intracellular and extracellular free amino acid profiles of metastatic prostate cancer cells (PC-3), normal prostate cells (PNT-1A), and metabolic changes (e.g., pH). In this study, cancer and normal cells were incubated in the appropriate medium. Then, the cells were collected and lysed in a cold medium. Finally, intracellular and extracellular free amino acid profiles were analyzed using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Extracellular metabolites were analyzed via blood gas measurements, including pH, CO₂, and O₂. We determined that intracellular prostate cancer cells include high amounts of glutamic acid, aspartic acid, glutamine, alpha aminobutyric acid, glycine, and proline, while cancer cells mostly use hydroxyproline, serine, alpha aminobutyric acid, ethanolamine, and valine from the extracellular medium. It was also determined that the extracellular pH of cancer cells is more acidic than that of normal cells, and normal cells have higher levels of Ca⁺, Cl^-, and Glu. In this study, we found that group amino acids have significant potential in prostate cancer pathogenesis; therefore, they have potential usefulness as a biomarker in the diagnosis of prostate cancer.     

Dual Targeting of Sorafenib-Resistant HCC-Derived Cancer Stem Cells

Sorafenib, an oral multi-tyrosine kinase inhibitor, has been the first-line therapy for the treatment of patients with advanced HCC, providing a survival benefit of only three months in approximately 30% of patients. Cancer stem cells (CSCs) are a rare tumour subpopulation with self-renewal and differentiation capabilities, and have been implicated in tumour growth, recurrence and drug resistance. The process of epithelial-to-mesenchymal transition (EMT) contributes to the generation and maintenance of the CSC population, resulting in immune evasion and therapy resistance in several cancers, including HCC. The aim of this study is to target the chemoresistant CSC population in HCC by assessing the effectiveness of a combination treatment approach with Sorafenib, an EMT inhibitor and an immune checkpoint inhibitor (ICI). A stem-cell-conditioned serum-free medium was utilised to enrich the CSC population from the human HCC cell lines Hep3B, PLC/PRF/5 and HepG2. The anchorage independent spheres were characterised for CSC features. The human HCC-derived spheres were assessed for EMT status and expression of immune checkpoint molecules. The effect of combination treatment with SB431542, an EMT inhibitor, and siRNA-mediated knockdown of programmed cell death protein ligand-1 (PD-L1) or CD73 along with Sorafenib on human HCC-derived CSCs was examined with cell viability and apoptosis assays. The three-dimensional spheres enriched from human HCC cell lines demonstrated CSC-like features. The human HCC-derived CSCs also exhibited the EMT phenotype along with the upregulation of immune checkpoint molecules. The combined treatment with SB431542 and siRNA-mediated PD-L1 or CD73 knockdown effectively enhanced the cytotoxicity of Sorafenib against the CSC population compared to Sorafenib alone, as evidenced by the reduced size and proliferation of spheres. Furthermore, the combination treatment of Sorafenib with SB431542 and PD-L1 or CD73 siRNA resulted in an increased proportion of an apoptotic population, as evidenced by flow cytometry analysis. In conclusion, the combined targeting of EMT and immune checkpoint molecules with Sorafenib can effectively target the CSC tumour subpopulation.     

Dihydrotanshinone-I modulates Epithelial Mesenchymal Transition (EMT) Thereby Impairing Migration and Clonogenicity of Triple Negative Breast Cancer Cells

Background: Salvia miltiorrhiza Bunge (Danshen), has been used for its therapeutic value in Traditional Chinese Medicine (TCM), for almost a thousand years. Dihydrotanshinone-I (DHTS) is a lipophilic compound isolated from the plant Salvia miltiorrhiza that has been shown to induce anti-proliferative and apoptotic effects on breast cancer cells. In the present study, we investigated the anti-migratory effect of DHTS on TNBC cell lines by studying the Epithelial Mesenchymal Transition (EMT) changes. Methods: IC50 values for DHTS in TNBC breast cancer cells were either discovered by literature search or by performing MTT assay. DHTS effect on EMT markers (viz. CD44, E-cadherin, Vimentin, N-cadherin, and active β-catenin) was studied using western blotting. Association between EMT and migration was further carried out in DHTS treated TNBC cells by wound healing assay. Cancer stemness and proliferation potential were further accessed using colony formation assay. Results: MTT assay revealed IC50 of MDA-MB-468 cells at 2 µM for 24 h. Subsequently, DHTS treatment in TNBC cell lines (MDA-MB-468 and MDA-MB-231) led to decrease in mesenchymal markers i.e. vimentin, N-cadherin and, active β-catenin. DHTS treated MDA-MB-468 cells showed a decrease in adhesion protein CD44 and an increase in epithelial protein E-cadherin. Additionally, a decrease in EMT potential was positively associated with the inhibition of migration and clonogenic potential in DHTS treated TNBC cells. Conclusion: In this study, we have demonstrated for the first time that DHTS has the potential to inhibit the migration and clonogenicity of highly aggressive TNBC cells by obstructing Epithelial to Mesenchymal Transition.     

Apogossypolone诱导前列腺癌PC-3细胞在体外的自噬

目的研究ApoG2对前列腺癌PC-3细胞在体外的作用,了解其杀伤肿瘤细胞的机制。方法采用MTT法、吖啶橙染色、透射电镜、流式细胞技术、Western blot、免疫组织化学等方法观察了ApoG2对PC-3细胞的自噬与凋亡的诱导作用。结果ApoG2可明显抑制PC-3细胞增殖;ApoG2作用于PC-3细胞72小时可诱导细胞自噬;加入自噬抑制剂3-MA可增强ApoG2诱导凋亡作用;ApoG2可以增强细胞内LC-3Ⅱ及Beclin-Ⅰ的表达,降低Bcl-2的表达水平。结论ApoG2主要以诱导PC-3细胞发生自噬为主,抑制自噬可以促进凋亡的发生。     

Anticancer Properties of Teucrium persicum in PC-3 Prostate Cancer Cells

Crude extracts or phytochemicals obtained from some plants have potential anti-cancer properties. Teucriumpersicum is an Iranian endemic plant belonging to the Lamiaceae family which has traditionally been used torelieve abdominal pains. However, the anti-cancer properties of this species of the Teucrium genus have notbeen investigated previously. In this study, we have used a highly invasive prostate cancer cell line, PC-3, whichis an appropriate cell system to study anti-tumor properties of plants. A methanolic extract obtained from Tpersicum potently inhibited viability of PC-3 cells. The viability of SW480 colon and T47D breast cancer cellswas also significantly decreased in the presence of the T persicum extract. Flow cytometry suggested that thereduction of cell viability was due to induction of apoptosis. In addition, the results of wound healing andgelatin zymography experiments supported anti-cell invasion activity of T persicum. Interestingly, sublethalconcentrations of T persicum extract induced an epithelial-like morphology in a subpopulation of cells with anincrease in E-Cadherin and β-Catenin protein levels at the cell membrane. These results strongly suggest thatT persicum is a plant with very potent anti-tumor activity     

Menadione (Vitamin K3) Induces Apoptosis of Human Oral Cancer Cells and Reduces their Metastatic Potential by Modulating the Expression of Epithelial to Mesenchymal Transition Markers and Inhibiting Migration

Oral cancer is one of the most commonly occurring cancers worldwide, decreasing the patient’s survival ratedue to tumor recurrence and metastasis. Menadione (Vitamin K3) is known to exhibit cytotoxicity in variouscancer cells but the present study focused on its effects on viability, apoptosis, epithelial to mesenchymal transition(EMT), anchorage independent growth and migration of oral cancer cells. The results show that menadioneis more cytotoxic to SAS (oral squamous carcinoma) cells but not to non-tumorigenic HEK293 and HaCaTcells. Menadione treatment increased the expression of pro-apoptotic proteins, Bax and p53, with a concurrentdecrease in anti-apoptotic proteins, Bcl-2 and p65. Menadione induced the expression of E-cadherin but reducedthe expression of EMT markers, vimentin and fibronectin. Menadione also inhibited anchorage independentgrowth and migration in SAS cells. These findings reveal and confirm that menadione is a potential candidate inoral cancer therapy as it exhibits cytotoxic, antineoplastic and antimigratory effects besides effectively blockingEMT in oral cancer cells.     

低氧对前列腺癌PC3细胞上皮间质转化的影响

 目的 探讨低氧对前列腺癌细胞上皮间质转化的影响。方法 分别在常氧（常氧组）和低氧（低氧组）条件下培养PC3细胞24 h,细胞增殖MTT实验检测低氧对前列腺癌PC3细胞增殖力的影响,Transwell侵袭实验评估低氧对前列腺癌PC3细胞侵袭力的影响,Western blot检测HIF-1α、E-cadherin、N-cadherin和Vimentin的表达水平。另外,在常氧条件下用siRNA抑制HIF-1α表达（干扰组）,用Western blot检测E-cadherin、N-cadherin和Vimentin的表达水平变化。结果 与常氧组比较,低氧组前列腺癌PC3细胞的增殖力和侵袭力增加,HIF-1α表达水平增加（P=0.0004）,HIF-1α向核内转位;N-cadherin（P<0.0001）和Vimentin（P<0.0001）的表达水平显著增加,E-cadherin的表达水平显著下降（P<0.0001）。同时,干扰组跟常氧组比较,抑制HIF-1α表达使N-cadherin（P=0.0002）和Vimentin（P=0.0002）的表达水平显著下降,而使E-cadherin的表达水平显著增加（P<0.0001）。结论 低氧可能通过调节HIF-1α表达促进前列腺癌PC3细胞的上皮间质转化。     

三氧化二砷重新激活PC-3前列腺癌细胞雄激素受体基因的表达

目的探讨三氧化二砷（As2O3）重新激活PC-3前列腺癌细胞雄激素受体（AR）基因的表达。方法用免疫组织化学的方法，对用浓度为2mg／L的As2O3处理前后的PC-3前列腺癌细胞和阳性对照的LNCaP前列腺癌细胞（AR）基因的表达情况进行检测分析。结果As2O3处理过的PC-3前列腺癌细胞AR的阳性表达率明显增高（P〈0．05）。结论As2O3可以重新激活PC-3前列腺癌细胞（AR）基因的表达。     

MicroRNA 495 Inhibits Proliferation and Metastasis and Promotes Apoptosis by Targeting Twist1 in Gastric Cancer Cells

Recently, microRNAs (miRNAs) have been reported to participate in multiple biological processes. However, the effects of miR-495 on gastric cancer (GC) remain unclear. The purpose of this study was to explore the functions of miR-495 in GC cell proliferation, metastasis, and apoptosis. SGC-7901 and BGC-823 cell lines were transfected with miR-495 mimic, miR-495 inhibitor, and negative controls (mimic control and inhibitor control). The expressions of miR-495, cell viability, migration, apoptosis, and apoptosis-related factors were examined by qRT-PCR, trypan blue staining, Transwell, flow cytometry, and Western blot, respectively. Simultaneously, key factor expression levels of EMT were detected by qRT-PCR and Western blot. The direct target of miR-495 was confirmed by dual-luciferase assay. Additionally, sh-Twist1, pc-Twist1, and corresponding controls were transfected into SGC-7901 and BGC-823 cells, and the protein levels of EMTassociated factors were detected by Western blot. miR-495 was downregulated in GC cells. miR-495 expression level was effectively overexpressed or suppressed in SGC-7901 and BGC-823 cells. Overexpression of miR-495 significantly decreased cell viability and migration, increased apoptosis, and inhibited the EMT process. Suppression of miR-495 showed contrary results. Twist1 was clarified as a target gene of miR-495, and Twist1 silencing obviously reduced the promoting effect of miR-495 suppression on these biological processes. Twist1 silencing significantly blocked the EMT process in both SGC-7901 and BGC-823 cells. miR-495 inhibited proliferation and metastasis and promoted apoptosis by targeting Twist1 in GC cells. These data indicated that miR-495 might be a novel antitumor factor of GC and provide a new method for the treatment of GC.     

Molecular Mechanism Underlying Hesperetin-induced Apoptosis by in silico Analysis and in Prostate Cancer PC-3 Cells

Aim: To investigate the molecular mechanisms underlying triggering of apoptosis by hesperetin using in silicoand in vitro methods. Methods: The mechanism of binding of hesperetin with NF-kB and other apoptotic proteinslike BAX, BAD, BCL2 and BCLXL was analysed in silico using Schrodinger suite 2009. In vitro studies were alsocarried out to evaluate the potency of hesperetin in inducing apoptosis using the human prostate cancer PC-3cell line. Results: Hesperetin was found to exhibit high-affinity binding resulting from greater intermolecularforces between the ligand and its receptor NF-kB (-7.48 Glide score). In vitro analysis using MTT assay confirmedthat hesperetin reduced cell proliferation (IC50 values of 90 and 40μM at 24 and 48h respectively) in PC-3 cells.Hesperetin also downregulated expression of the anti-apoptotic gene BCLXL at both mRNA and protein levels andincreased the expression of pro-apoptotic genes like BAD at mRNA level and BAX at mRNA as well as proteinlevels. Conclusion: The results suggest that hesperetin can induce apoptosis by inhibiting NF-kB.     

PC-1基因表达对前列腺癌细胞迁移能力的影响

背景与目的:PC-1基因在雄激素非依赖和高转移能力的前列腺癌C4-2细胞中高表达,在雄激素依赖及不转移的前列腺癌LNCaP细胞中低表达。本实验旨在研究PC-1对前列腺癌细胞迁移能力的影响。方法:构建PC-1稳定高表达的LNCaP细胞株和反义核酸调低内源性PC-1表达的C4-2细胞株。利用体外迁移系统检测PC-1表达对LNCaP和C4-2细胞迁移运动能力的影响。结果:体外迁移实验表明稳定转染提高PC-1的表达水平并未使LNCaP迁移细胞数增多(P>0.05),而反义核酸降低PC-1表达则使C4-2迁移细胞数降低(P<0.05)。PC-1蛋白水平升高不能提高LNCaP细胞迁移能力,但降低内源性PC-1表达则使C4-2细胞迁移能力明显降低。结论:PC-1可能在前列腺癌细胞侵袭过程中起一定作用。     

IGF-1 from Adipose-Derived Mesenchymal Stem Cells Promotes Radioresistance of Breast Cancer Cells

Purpose: The aim of this study was to investigate effects of adipose-derived mesenchymal stem cells (AMSCs)on radioresistance of breast cancer cells. Materials and Methods: MTT assays were used to detect any influenceof AMSC supernatants on proliferation of breast cancer cells; cell migration assays were used to determine theeffect of breast cancer cells on the recruitment of AMSCs; the cell survival fraction post-irradiation was assessedby clonogenic survival assay; γ-H2AX foci number post-irradiation was determined via fluorescence microscopy;and expression of IGF-1R was detected by Western blotting. Results: AMSC supernatants promoted proliferationand radioresistance of breast cancer cells. Breast cancer cells could recruit AMSCs, especially after irradiation.IGF-1 derived from AMSCs might be responsible for the radioresistance of breast cancer cells. Conclusions:Our results suggest that AMSCs in the tumor microenvironment may affect the outcome of radiotherapy forbreast cancer in vitro.     

Quantification of Mesenchymal Stem Cells (MSCs) at Sites of Human Prostate Cancer

Circulating bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. The prostate is bombarded by numerous infectious & inflammatory insults over a lifetime. Chronic inflammation is associated with CXCL12, CCL5, and CCL2, which are highly overexpressed in prostate cancer. Among other cell types, these chemoattractant stimuli recruit BM-MSCs to the tumor. MSCs are minimally defined as plastic-adhering cells characterized by the expression of CD90, CD73, and CD105 in the absence of hematopoietic markers, which can differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs are immunoprivileged and have been implicated in tumorigenesis through multiple mechanisms, including promoting proliferation, angiogenesis, and metastasis, in addition to the generation of an immunosuppressive microenvironment. We have demonstrated that MSCs represent 0.01-1.1% of the total cells present in core biopsies from primary human prostatectomies. Importantly, these analyses were performed on samples prior to expansion in tissue culture. MSCs in these prostatectomy samples are FAP-, CD90-, CD73-, and CD105-positive, and CD14-, CD20-, CD34-, CD45-, and HLA-DR-negative. Additionally, like BM-MSCs, these prostate cancer-derived stromal cells (PrCSCs) were shown to differentiate into osteoblasts, adipocytes, & chondrocytes. In contrast to primary prostate cancer-derived epithelial cells, fluorescently-labeled PrCSCs & BM-MSCs were both shown to home to CWR22RH prostate cancer xenografts following IV injection. These studies demonstrate that not only are MSCs present in sites of prostate cancer where they may contribute to carcinogenesis, but these cells may also potentially be used to deliver cytotoxic or imaging agents for therapeutic and/or diagnostic purposes.     

双氢青蒿素诱导前列腺癌PC-3细胞凋亡

目的研究双氢青蒿素（DHA）诱导前列腺癌PC-3细胞凋亡作用,并观察其形态学变化。方法采用人前列腺癌PC-3细胞建立裸鼠种植瘤模型,20只模型鼠随机分为对照组、溶剂组、DHA大剂量组及小剂量组,经13天干预后,计算抑瘤率;HE染色观察肿瘤组织形态学表现;TUNEL法及Hoechst 33258荧光染色检测凋亡。结果DHA大小剂量组抑瘤率分别为69.221%和63.186%;肿瘤组织内凋亡细胞明显增多;肿瘤细胞凋亡率及凋亡细胞数密度均明显升高（P＜0.05）。结论DHA具有较强的抗肿瘤作用,其作用机制可能与诱导肿瘤细胞凋亡有关。     

丁酸钠对前列腺癌LNCaP细胞HER-2信号通路的影响

 目的 研究组蛋白去乙酰化酶抑制剂丁酸钠对前列腺癌LNCaP细胞HER 2信号通路的影响，探讨其抗肿瘤作用的分子机制。方法 四甲基偶氮唑蓝(MTT)检测药物对肿瘤细胞增殖的影响；hoechst 33342染色观察细胞凋亡的形态学变化，Western blot检测凋亡标志蛋白、HER2/ neu、Phos Akt、Phos Erk等信号蛋白的表达。结果 丁酸钠能够有效抑制LNCaP细胞的增殖并诱导细胞凋亡，半效杀伤剂量（EC50）为5.6mmol/L；药物能够抑制HER 2基因的转录和蛋白的表达，并抑制下游信号通路中MAPK和AKT的活化。结论 丁酸钠能够阻断对前列腺癌细胞生长具有重要作用的HER 2信号通路，从而对肿瘤细胞发挥抑制作用。     

miR-203 Inhibits the Invasion and EMT of Gastric Cancer Cells by Directly Targeting Annexin A4

Many studies have shown that downregulated miR-203 level is in a variety of cancers including gastric cancer (GC). However, the precise molecule mechanisms of miR-203 in GC have not been well clarified. In the current study, we investigated the biological functions and molecular mechanisms of miR-203 in GC cell lines. We found that miR-203 is downregulated in GC tissues and cell lines. Moreover, the low level of miR-203 was associated with increased expression of annexin A4 in GC tissues and cell lines. The invasion and EMT of GC cells were suppressed by overexpression of miR-203. However, downregulation of miR-203 promoted invasion and EMT of GC cells. Bioinformatics analysis predicted that annexin A4 was a potential target gene of miR-203. Next, luciferase reporter assay confirmed that miR-203 could directly target annexin A4. Consistent with the effect of miR-203, downregulation of annexin A4 by siRNA inhibited the invasion and EMT of GC cells. Introduction of annexin A4 in GC cells partially blocked the effects of miR-203 mimic. Introduction of miR-203 directly targeted annexin A4 to inhibit the invasion and EMT of GC cells. Overall, reactivation of the miR-203/annexin A4 axis may represent a new strategy for overcoming metastasis of GC.