MicroRNA-302a Suppresses Tumor Cell Proliferation by Inhibiting AKT in Prostate Cancer

Micro (mi) RNAs are important regulators involved in various physical and pathological processes, including cancer. The miRNA-302 family has been documented as playing a critical role in carcinogenesis. In this study, we investigated the role of miRNA-302a in prostate cancer (PCa). MiRNA-302a expression was detected in 44 PCa tissues and 10 normal prostate tissues, and their clinicopathological significance was analyzed. Cell proliferation and cell cycle analysis were performed on PCa cells that stably expressed miRNA-302a. The target gene of miRNA-302a and the downstream pathway were further investigated. Compared with normal prostate tissues, miRNA-302a expression was downregulated in PCa tissues, and was even lower in PCa tissues with a Gleason score ≥8. Overexpression of miRNA-302a induced G1/S cell cycle arrest in PCa cells, and suppressed PCa cell proliferation both in vitro and in vivo. Furthermore, miRNA-302a inhibits AKT expression by directly binding to its 3΄ untranslated region, resulting in subsequent alterations of the AKT-GSK3β-cyclin D1 and AKT-p27^Kip1 pathway. These results reveal miRNA-302a as a tumor suppressor in PCa, suggesting that miRNA-302a may be used as a potential target for therapeutic intervention in PCa.     

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2^YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER⁺ and R26-SmoM2^YFP+ mice in this protocol); 2) inducing SmoM2^YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.     

A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P⁺ cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G₁ phase and inhibited the G₁/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P⁺ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.     

Proliferation of Toxoplasma gondii Suppresses Host Cell Autophagy

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.     

Truncated SSX Protein Suppresses Synovial Sarcoma Cell Proliferation by Inhibiting the Localization of SS18-SSX Fusion Protein

Synovial sarcoma is a relatively rare high-grade soft tissue sarcoma that often develops in the limbs of young people and induces the lung and the lymph node metastasis resulting in poor prognosis. In patients with synovial sarcoma, specific chromosomal translocation of t(X; 18) (p11.2;q11.2) is observed, and SS18-SSX fusion protein expressed by this translocation is reported to be associated with pathogenesis. However, role of the fusion protein in the pathogenesis of synovial sarcoma has not yet been completely clarified. In this study, we focused on the localization patterns of SS18-SSX fusion protein. We constructed expression plasmids coding for the full length SS18-SSX, the truncated SS18 moiety (tSS18) and the truncated SSX moiety (tSSX) of SS18-SSX, tagged with fluorescent proteins. These plasmids were transfected in synovial sarcoma SYO-1 cells and we observed the expression of these proteins using a fluorescence microscope. The SS18-SSX fusion protein showed a characteristic speckle pattern in the nucleus. However, when SS18-SSX was co-expressed with tSSX, localization of SS18-SSX changed from speckle patterns to the diffused pattern similar to the localization pattern of tSSX and SSX. Furthermore, cell proliferation and colony formation of synovial sarcoma SYO-1 and YaFuSS cells were suppressed by exogenous tSSX expression. Our results suggest that the characteristic speckle localization pattern of SS18-SSX is strongly involved in the tumorigenesis through the SSX moiety of the SS18-SSX fusion protein. These findings could be applied to further understand the pathogenic mechanisms, and towards the development of molecular targeting approach for synovial sarcoma.     

紫草素对人脑胶质瘤U251 细胞增殖和凋亡的影响

目的:观察紫草素对体外培养的人脑胶质瘤U251细胞的增殖和凋亡的影响。方法:应用MTT法和Annexin V-FITC/PI双染流式细胞术分别检测2.5、5、10μM/L的紫草素对U251细胞的体外抑杀作用以及凋亡诱导作用,进一步应用Western blot方法检测紫草素对凋亡相关蛋白Bcl-2及Bax表达水平的影响。结果:紫草素对人U251胶质瘤细胞具有明显的增殖抑制作用,且呈一定的剂量依赖性和时间依赖性。紫草素可明显上调U251细胞Bax的表达,下调Bcl-2的表达,与对照组相比存在显著性差异(P0.05)。结论:紫草素对人胶质瘤U251细胞具有明显的抑制增殖和促进凋亡作用。     

Luteolin Suppresses Cancer Cell Proliferation by Targeting Vaccinia-Related Kinase 1

Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.     

Mitofusin 2-Deficiency Suppresses Cell Proliferation through Disturbance of Autophagy

Mitofusin2 (Mfn2), a mitochondrial outer membrane protein serving primarily as a mitochondrial fusion protein, has multiple functions in regulating cell biological processes. Defects of Mfn2 were found in diabetes, obesity, and neurodegenerative diseases. In the present study, we found that knockdown of Mfn2 by shRNA led to impaired autophagic degradation, inhibited mitochondrial oxygen consumption rate and cell glycolysis, reduced ATP production, and suppressed cell proliferation. Inhibition of autophagic degradation mimicked Mfn2-deficiency mediated cell proliferation suppression, while enhancement of autophagosome maturation restored the suppressed cell proliferation by Mfn2-deficiency. Thus, our findings revealed the role of Mfn2 in regulating cell proliferation and mitochondrial metabolism, and shed new light on understanding the mechanisms of Mfn2 deficiency related diseases.     

AG490上调BATF2表达抑制淋巴瘤细胞增殖

目的：AG490 作为JAK2/STAT3 通路的抑制剂,在对肿瘤细胞的抑制作用上所展现出的高效低毒性,使其有望成为临床上治疗肿瘤的一种可能的药物。然而,AG490 的抗瘤机制尚未明确。因此,本文拟对AG490 抑制淋巴瘤细胞增殖的效应及其作用机制进行进一步探讨,为AG490 应用于临床提供实验依据。方法：用不同剂量的AG490 处理淋巴瘤细胞（Namalwa 和JeKo-1）、Jurkat T 淋巴细胞性白血病细胞和THP-1 单核细胞性白血病细胞24小时,CCK-8 法检测AG490 （0 滋M、2 滋M、20 滋M、50 μM、200滋M）对上述细胞的增殖抑制作用,实时定量PCR 法检测BATF2 mRNA 的变化,Western blot 法检测其蛋白水平的变化,细胞转染siRNA 法抑制BATF2 表达后CCK8 法检测AG490 对Namalwa 细胞的增殖抑制效应。结果：AG490 呈剂量依赖性地抑制Namalwa、JeKo-1、Jurkat 细胞的增殖（P〈0.05）,同时上调其BATF2 mRNA 水平和蛋白水平的表达（P〈0.05）。对于无显著抑制作用的THP-1 细胞,BATF2 的表达亦未见升高（P〉0.05）。siRNA 法抑制BATF2 基因表达后,AG490 对Namalwa 细胞的增殖抑制效果明显降低（P〈0.05）。结论：AG490 杀肿瘤细胞的效率与其诱导的BATF2 的表达呈正相关,抑制BATF2 的表达后AG490 抑制肿瘤细胞增殖的效率明显降低。因此,AG490可能是通过上调BATF2表达的方式抑制淋巴瘤细胞增殖。这意味着BATF2 是AG490 杀伤淋巴瘤细胞的作用靶点,可能为新药的开发做出一定的贡献。     

人乳头瘤病毒诱导细胞增殖和抗凋亡的信号转导途径

人乳头瘤病毒(human papillomavirus,HPV)通过激活Ras—MAP激酶通路可以诱导细胞增殖,而HPV编码3种蛋白质彼此独立地发挥作用,但又相互联系,形成一个大的信号网络：E5蛋白通过调节EGF信号通路抑制细胞凋亡;E6蛋白和Tyk2结合后减弱Jak—STAT通路;E7可直接作用于Smad蛋白,并且调节TGF—β信号转导。通过了解HPV及其编码的3种蛋白质的信号转导途径以及诱导细胞增殖和抑制细胞凋亡机制,对于进一步探明HPV的致病机制及防治HPV所引起的疾病具有重要的意义。     

CDC42 抑制剂ML141对喉癌Hep-2 细胞增殖的抑制作用

目的:探讨CDC42抑制剂ML141对喉癌细胞增殖的抑制作用,为喉癌的分子治疗提供新的靶点。方法:体外培养人喉癌Hep-2细胞。实时定量聚合酶链反应(Real-time PCR)检测CDC42在Hep-2细胞中的表达。利用GLISA法检测ML141对CDC42活性的抑制效果。利用CCK8法检测ML141对Hep-2细胞增殖能力的抑制效果。结果:1Real-time PCR结果显示在人喉癌Hep-2细胞中CDC42显著高表达(P0.001),证明全基因组的结果准确。2GLISA结果显示表皮生长因子作用的Hep-2细胞中CDC42的活性明显高表达,但加入ML141的Hep-2细胞中CDC42的活性受到明显抑制。(P0.001)。3CCK8结果显示24 h,48h和72 h时,ML141处理的Hep-2细胞的增殖能力与对照组相比均受到明显抑制。(P0.001)。结论:促癌基因CDC42抑制剂ML141能够抑制人喉癌Hep-2细胞增殖,具有成为抗喉癌新药的潜力,为喉癌的分子治疗提供新的切入点。     

Neisseria gonorrhoeae Suppresses Dendritic Cell-Induced, Antigen-Dependent CD4 T Cell Proliferation

Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.     

AG490 上调 BATF2 表达抑制淋巴瘤细胞增殖 *

摘要 目的： AG490 作为 JAK2/STAT3 通路的抑制剂, 在对肿瘤细胞的抑制作用上所展现出的高效低毒性, 使其有望成为临床上 治疗肿瘤的一种可能的药物。 然而, AG490 的抗瘤机制尚未明确。因此, 本文拟对 AG490 抑制淋巴瘤细胞增殖的效应及其作用机 制进行进一步探讨,为 AG490 应用于临床提供实验依据。方法： 用不同剂量的 AG490 处理淋巴瘤细胞 （Namalwa 和 JeKo-1 ） 、 JurkatT 淋巴细胞性白血病细胞和 THP-1 单核细胞性白血病细胞 24 小时, CCK-8 法检测 AG490 (0 μM、 2 μM、 20μM、 50μM、 200μM)对上述细胞的增殖抑制作用, 实时定量 PCR 法检测 BATF2 mRNA 的变化, Western blot 法检测其蛋白水平的变化, 细胞转染 siRNA 法抑制 BATF2 表达后 CCK8 法检测 AG490 对 Namalwa 细胞的增殖抑制效应。结果： AG490 呈剂量依赖性地抑制 Namalwa、 JeKo-1、 Jurkat 细胞的增殖(P<0.05), 同时上调其 BATF2 mRNA 水平和蛋白水平的表达(P<0.05)。对于无显著抑制作用的 THP-1 细胞, BATF2 的表达亦未见升高(P>0.05)。siRNA 法抑制 BATF2 基因表达后, AG490 对 Namalwa 细胞的增殖抑制效果明 显降低(P<0.05)。 结论： AG490 杀肿瘤细胞的效率与其诱导的 BATF2 的表达呈正相关, 抑制 BATF2 的表达后 AG490 抑制肿瘤细 胞增殖的效率明显降低。因此, AG490 可能是通过上调 BATF2 表达的方式抑制淋巴瘤细胞增殖。这意味着 BATF2 是 AG490 杀 伤淋巴瘤细胞的作用靶点, 可能为新药的开发做出一定的贡献。     

Down-Regulating Overexpressed Human Lon in Cervical Cancer Suppresses Cell Proliferation and Bioenergetics

The human mitochondrial ATP-dependent Lon protease functions in regulating the metabolism and quality control of proteins and mitochondrial DNA (mtDNA). However, the role of Lon in cancer is not well understood. Therefore, this study was undertaken to investigate the importance of Lon in cervical cancer cells from patients and in established cell lines. Microarray analysis from 30 cancer and 10 normal cervical tissues were analyzed by immunohistochemistry for Lon protein levels. The expression of Lon was also examined by immunoblotting 16 fresh cervical cancer tissues and their respective non-tumor cervical tissues. In all cases, Lon expression was significantly elevated in cervical carcinomas as compared to normal tissues. Augmented Lon expression in tissue microarrays did not vary between age, tumor-node-metastasis grades, or lymph node metastasis. Knocking down Lon in HeLa cervical cancer cells by lentivrial transduction resulted in a substantial decrease in both mRNA and protein levels. Such down-regulation of Lon expression significantly blocked HeLa cell proliferation. In addition, knocking down Lon resulted in decreased cellular bioenergetics as determined by measuring aerobic respiration and glycolysis using the Seahorse XF24 extracellular flux analyzer. Together, these data demonstrate that Lon plays a potential role in the oncogenesis of cervical cancer, and may be a useful biomarker and target in the treatment of cervical cancer. Lon; immunohistochemistry; cervical cancer; cell proliferation; cellular bioenergetics.