Phosphorylation of Akt (Ser473) predicts poor clinical outcome in oropharyngeal squamous cell cancer.

BACKGROUND: Several lines of laboratory evidence support a role of persistent activation of Akt pathway in oropharyngeal squamous cell carcinoma (OSCC) progression. Loss of phosphatase PTEN is one of the proposed mechanisms of Akt activation. We sought to determine the prognostic significance of Akt activation in a cohort of patients with OSCC as well as the association between phosphorylated (activated) Akt and PTEN levels. METHODS: Using a novel system of in situ quantitative protein expression analysis (AQUA), we studied the protein expression levels of phosphorylated Akt (p-Akt) and PTEN on a tissue microarray. The array included 79 OSCCs with a mean follow-up of 36 months. RESULTS: Patients with tumors expressing low tumor p-Akt levels had lower 5-year local recurrence rates (5% versus 38%). Additionally, these patients had improved 5-year overall survival rates (45% versus 27%). This survival effect was likely due to disease recurrence, as there was no difference in death without recurrence between low- and high-expressing groups. In adjusted analysis, tumor p-Akt expression was a strong predictor of local recurrence. A significant inverse relationship was found between nuclear p-Akt and nuclear PTEN: Tumors with high nuclear p-Akt had low nuclear PTEN and vice versa. CONCLUSIONS: Akt activation in OSCC is associated with adverse patient outcome, indicating that Akt is a promising molecular target in OSCC. PTEN loss may be one of the mechanisms of Akt activation in OSCC.     

Phosphorylated Akt overexpression and loss of PTEN expression in non-small cell lung cancer confers poor prognosis

Akt, a downstream mediator of phosphatidylinositol 3-kinase (PI3K), is a signal transduction protein that plays a central role in tumorigenesis. The tumor suppressor gene PTEN negatively regulates the PI3K/Akt signaling pathway. However, the roles of Akt and PTEN function in patients with non-small cell lung cancer (NSCLC) is not well established. To clarify roles of expression of phosphorylated Akt (p-Akt) and loss of PTEN expression in biological behavior and prognosis of NSCLC. Immunohistochemical staining was used to determine the expression of p-Akt and PTEN in 20 cases of normal lung tissues and 102 cases patients with NSCLC. All patients with NSCLC were followed from 3 to 60 months. The positive incidence of p-Akt expression and loss incidence of PTEN expression in NSCLC were 41.2% (42/102) and 46.1% (47/102), while negative of p-Akt expression (0%, 0/20) and positive of PTEN expression (100%, 20/20) in normal lung tissues. Overexpression of p-Akt and loss of PTEN expression were correlated to poor differentiation, lymph node involvement, distant metastasis and late stages. A significant negative correlation was observed between expression of p-Akt and PTEN (r = -0.425, P < 0.001). Patients with p-Akt positive expression (42/102) and loss of PTEN expression (47/102) showed significantly worse 5 years survival rate and median survival time than relevant those with p-Akt negative expression (14.29% versus 33.33%, 14 months versus 32 months, Log-rank test X(2) = 14.24, P < 0.001) and PTEN positive expression (10.64% versus 38.18%, 15 months versus 40 months, Log-rank test X(2) = 21.06, P < 0.001). A univariate analysis revealed that smoking, tumor size, lymph node involvement, distant metastasis, stage, p-Akt and loss of PTEN expression were significant correlative factors with prognosis. The result of multivariate Cox analysis showed that smoking, stage and loss of PTEN expression were independent prognosticators. p-Akt is overexpressed and accompanied by the loss of PTEN in clinical specimens of NSCLC. Both p-Akt and PTEN are concerned with invasion and metastasis of NSCLC. Loss of PTEN expression is an independent poor prognostic factor for patients with NSCLC.     

磷酸化Akt和PTEN在非小细胞肺癌组织中的表达及其相关性

目的:探讨磷酸化Akt(p-Akt)与PTEN表达在非小细胞肺癌(NSCLC)侵袭、转移中的作用及其相关关系。方法:应用免疫组织化学SP法检测20例正常肺组织和102例非小细胞肺癌癌组织中p-Akt和PTEN蛋白的表达。结果:正常肺组织p-Akt为阴性表达,而PTEN为阳性表达;非小细胞肺癌癌组织中p-Akt表达的阳性率为41.2%(42/102),PTEN失表达率为46.1%(47/102)。p-Akt阳性表达和PTEN的失表达与肿瘤组织分化程度、临床分期以及有无淋巴结和远处转移相关。p-Akt和PTEN表达呈显著负相关(Phi r=-0.425,P<0.001)。结论:p-Akt过表达伴随着PTEN表达缺失,两者与NSCLC侵袭、转移有关。     

miR-19通过靶向PTEN调节PI3K-Akt通路促进子宫内膜癌KLE细胞 的侵袭和迁移

目的：分析miR-19对PTEN及PI3K-Akt通路的调节作用,揭示miR-19和PTEN对子宫内膜癌KLE细胞侵袭、迁移的影响及其机制。方法：收集2017年5月至2020年8月河北医科大学第一医院收治的74例子宫内膜癌患者经手术切除（术前未接受放化疗等治疗）且病理确诊为子宫内膜癌的肿瘤组织及对应癌旁组织,采用qPCR和WB法检测PTEN在子宫内膜癌组织中的表达水平以及转染miR-19模拟物或抑制物对KLE细胞中PTEN表达的影响。通过TargetScan及双荧光素酶报告基因实验预测并验证PTEN是否为miR-19的靶基因,将KLE细胞随机分为对照（NC）组、miR-19模拟物组和miR-19+PTEN组,分别转染阴性对照片段（miR-NC）、miR-19 模拟物或共转染 miR-19 模拟物+PTEN 过表达载体,采用 Transwell 和细胞划痕实验检测 miR-19 和PTEN对KLE细胞迁移和侵袭能力的影响,WB法检测对PI3K-Akt通路相关蛋白表达的影响。结果：子宫内膜癌组织中PTEN的mRNA和蛋白表达水平均明显低于癌旁组织（均P<0.01）。miR-19能与PTEN mRNA的3’-UTR特异性结合,上调miR-19的表达能抑制PTEN的mRNA和蛋白的表达,下调miR-19的表达则出现相反的结果（均P<0.01）。与miR-NC组相比,miR-19 模拟物组的迁移、侵袭细胞数以及划痕愈合率均显著升高（均 P<0.01）,而 miR-19+PTEN 组的细胞迁移和侵袭能力以及划痕愈合率较miR-19摸拟物组则明显降低（均P<0.01）;与miR-NC 组相比,miR-19 模拟物组中 PTEN 蛋白的表达明显降低,而p-Akt 308和p-Akt 473蛋白的表达明显升高,Akt蛋白的表达无明显变化（均P<0.01）;而在miR-19+PTEN组中,p-Akt 308和p-Akt 473蛋白的表达均明显低于miR-19模拟物组（均P<0.01）。结论：miR-19可能通过抑制PTEN的表达从而活化PI3K-Akt通路,进而促进子宫内膜癌KLE细胞的迁移和侵袭。     

Correlation between loss of PTEN expression and Akt phosphorylation in endometrial carcinoma.

The tumor suppressor PTEN acts as a lipid phosphatase, regulates the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway, and modulates cell cycle progression and cell survival. Somatic mutations of PTEN have been reported in a variety of cancers, especially in endometrial carcinoma. To clarify whether and how PTEN and the PI3K/Akt pathway relates to endometrial carcinoma, we examined the expression of those pathway-related proteins in patients with endometrial carcinoma. Of 103 endometrial carcinomas, 37 (36%) showed negative immunohistochemical staining of PTEN. Western blotting revealed that the expression of PTEN in PTEN-negative cases was significantly lower compared with that in positive cases. In contrast, phospho-Akt level in negative cases was significantly higher. We found a significant inverse correlation between PTEN and phospho-Akt (r = -0.796). The expression of phospho-Bad was greater in negative cases, suggesting that Bad might be a target for AKT: The present study demonstrates the phosphorylation of Akt accompanied by the loss of PTEN in clinical specimens of endometrial carcinomas.     

RGS19 stimulates cell proliferation by deregulating cell cycle control and enhancing Akt signaling

RGS19 is a regulator of G protein signaling which is upregulated in ovarian cancers and its overexpression promotes cell proliferation in several mammalian cell types. Here we showed that cyclin D1/3 and Cdk6 were upregulated in HEK293 cells overexpressing RGS19, while INK4A and INK4B were reduced. Moreover, RGS19 augmented serum-stimulated PTEN/PDK/Akt and Rb phosphorylations in 293/RGS19 and Caco2/RGS19 cells. These changes were reversed upon the knockdown of RGS19. Consistent with an elevated Akt activity, increased levels of phosphorylated Bad and c-Raf and a diminished expression of TSC2 were detected, thus demonstrating that RGS19 can deregulate cell proliferation via multiple pathways.     

Phosphorylated Akt Protein at Ser473 Enables HeLa Cells to Tolerate Nutrient-Deprived Conditions

Background: Despite angiogenesis, many tumours remain hypovascular and starved of nutrients while continuing to grow rapidly. The specific biochemical mechanisms associated with starvation resistance, austerity, may be new biological characters of cancer that are critical for cancer progression. Objective: This study aim was to investigate the effect of nutrient starvation on HeLa cells and the possible mechanism by which the cells are able to tolerate nutrient-deprived conditions. Methods: Nutrient starvation was achieved by culturing HeLa cells in nutrient-deprived medium (NDM) and cell survival was estimated by using cell counting kit-8. The effect of starvation on cell cycle distribution and the quantitative analysis of apoptotic cells were investigated by flow cytometry using propidium iodide staining. Western blotting was used to detect the expression levels of Akt and phosphorylated Akt at Ser473 (Ser473p-Akt) proteins. Results: HeLa cells displayed extremely long survival when cultured in NDM. The percentage of apoptotic HeLa cells was significantly increased by starvation in a time-dependent manner. A significant increase in the expression of Ser473p-Akt protein after starvation was also observed. Furthermore, it was found that Akt inhibitor III molecule inhibited the cells proliferation in a concentration- and time-dependent manner. Conclusion: Results of the present study provide evidence that Akt activation may be implicated in the tolerance of HeLa cells for nutrient starvation and may help to suggest new therapeutic strategies designed to prevent austerity of cervical cancer cells through inhibition of Akt activation.     

Prognostic significance of activated Akt expression in pancreatic ductal adenocarcinoma.

PURPOSE: Akt is a serine/threonine kinase that plays a central role in tumorigenesis. Among the members of Akt family, Akt2 is associated with the development of human cancers. The present study was designed to clarify the prognostic significance of Akt2 and activated Akt expression in pancreatic ductal adenocarcinoma (PDAC). In addition, activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the proliferation activity of tumor cells detected by Ki-67 immunohistochemistry were examined. EXPERIMENTAL DESIGN: Immunohistochemical analysis was performed on paraffin-embedded specimens from 65 patients with PDAC; 36 males and 29 females with ages ranging from 48 to 79 years (median, 66 years) of age. Expression levels of Akt2, phosphorylated Akt (p-Akt), and phosphorylated ERK 1/2 (p-ERK 1/2) were categorized as either weaker (low intensity) or equal to stronger (high intensity) compared with those in the endothelial cells of the same specimens. For Ki-67 immunohistochemistry, cases were divided into two groups: level 1, Ki-67 labeling index (LI), <20%; level 2, Ki-67 LI, > or = 20%. RESULTS: Twenty-six (42.6%), 28 (45.9%), 39 (63.9%), and 46 (75.4%) of the tumors showed high intensity of Akt2, p-Akt, and p-ERK 1/2 expression, and Ki-67 LI level 2, respectively. A significant positive correlation was observed between Akt2 and p-Akt expression (P < 0.01). Multivariate analysis revealed that p-Akt expression, Ki-67 LI, and histological differentiation are independent prognosticators for PDAC. CONCLUSIONS: p-Akt expression is a significant prognostic indicator for PDAC. Inhibition of Akt is a possible molecular approach for treatment of PDAC.     

Loss of neutral endopeptidase and activation of protein kinase B (Akt) is associated with prostate cancer progression

BACKGROUND: Neutral endopeptidase (NEP) is a cell-surface peptidase that can regulate the activation of Akt kinase through catalytic-dependent and independent mechanisms. NEP expression is absent in approximately 50% of prostate cancers. The authors investigated whether NEP loss in vivo would result in Akt phosphorylation and potentially contribute to prostate cancer progression by examining the interaction of NEP, Akt, and phosphatase and tensin homolog (PTEN) in a prostate xenograft model and in clinical specimens from patients with prostate cancer. METHODS: Using a tetracycline-repressible expression system to express NEP in a tumor animal xenograft model, the effects of NEP were tested on tumor growth, Akt phosphorylation, and PTEN expression. The clinical relevance of NEP, phosphorylated Akt, and PTEN protein expression also was investigated in 204 patients who had undergone radical prostatectomy. RESULTS: The results indicated that the induction of NEP expression inhibited established xenograft tumor growth, diminished Akt phosphorylation, and increased PTEN protein levels. In humans, prostate cancers with complete loss of NEP expression were significantly more likely to express phosphorylated Akt (P = .02). Moreover, patients who had prostate cancers with concomitant loss of NEP and expression of phosphorylated Akt had an increased, independent risk of prostate-specific antigen (PSA) recurrence (P = .03). In the study cohort, loss of PTEN protein expression did not correlated significantly with phosphorylated Akt or with patients' clinical outcome. CONCLUSIONS: The findings from this investigation demonstrated that NEP loss leads to Akt activation and contributes to the clinical progression of prostate cancer.     

Survivin associates with cell proliferation in renal cancer cells: regulation of survivin expression by insulin-like growth factor-1, interferon-gamma and a novel NF-kappaB inhibitor

Although survivin has been widely recognized as an attractive target for cancer therapy, the exact mechanism regarding the regulation of survivin and its effect on cell proliferation have yet to be clearly defined in renal cell carcinoma (RCC). We investigated herein the association between survivin expression and cell proliferation in a RCC cell line, KU19-20. In KU19-20 cells, cell proliferation and survivin expression were significantly induced by IGF-1, whereas they were inhibited by a novel NF-kappaB inhibitor dehydroxymethyl-epoxyquinomicin (DHMEQ) and IFN-gamma. The combination of DHMEQ and IFN-gamma inhibited cell proliferation synergistically with a pronounced attenuation of survivin expression. Furthermore, treatment with survivin-specific siRNA reduced expression of survivin and significantly inhibited cell proliferation. Survivin expression was thus associated with cell proliferation in KU19-20 cells. The regulation of survivin by IFN-gamma and/or an NF-kappaB inhibitor may therefore be a potential treatment modality for RCC.     

Evidence for PTEN-independent Akt activation and Akt-independent p27(Kip1) expression in advanced bladder cancer

In the treatment of advanced bladder cancer (BC), attention has recently focused on small molecule therapy concerning EGFR and the downstream Akt signalling pathway. Cellular deregulation processes are poorly understood, and biological determinants for the selection of therapy and monitoring are currently not available. The proteins PTEN, p-Akt and p27(Kip1) are suggested to be potentially significant biomarkers of Akt signalling. In this study, we investigated the expression of these proteins in advanced BC. PTEN, p-Akt and p27(Kip1) expression was determined immunohistochemically in 86 T2-4 BC specimens using a tissue microarray technique. Staining was documented with regard to intensity, cellular frequency and a multiplied staining score. Staining characteristics of the three proteins were correlated by regression analysis with the parameters of tumour stage and grade. A positive correlation was observed in the expression scores of PTEN and p-Akt, p-Akt and p27(Kip1) as well as PTEN and p27(Kip1) (p<0.02 for all combinations). The positive correlation between PTEN and p-Akt resulted mainly due to the strong correlation of PTEN intensity with p-Akt (p=0.0003 and p=0.0006 to p-Akt frequency and intensity, respectively). A positive correlation between p-Akt and p27(Kip1) was noted for p-Akt frequency as well as intensity (p<0.05 for all combinations). The positive correlation between PTEN and p27(Kip1) resulted due to the correlation of PTEN intensity alone with p27(Kip1) (p<0.03 for p27(Kip1) frequency and intensity), whereas no significance was noted for PTEN frequency. No correlation was found between T or G and expression of the proteins. However, activation of Akt in BC is known to occur independently of PTEN protein loss and appears not to cause a decrease of p27(Kip1). However, a direct regulatory impact of PTEN on p27(Kip1) was found. PTEN intensity, rather than frequency, appears to be a superior biomarker. These results may provide information to support research into protein profiling-predicted targeted therapy for BC. Correlations to benign urothelium, superficial BC specimens and follow-up data remain to be investigated.     

PTEN/PI3K/Akt信号通路对K562细胞凋亡调控的研究

 目的 探讨PTEN/PI3K/Akt信号传导通路对人慢性粒细胞白血病细胞系K562的增殖、凋亡调控的研究及可能的分子作用机制。 方法 将携带有野生型PTEN及绿色荧光蛋白的腺病毒（Ad-PTEN-GFP）及空载体（Ad-GFP）腺病毒,转染人慢性粒细胞白血病细胞系K562。通过MTT检测细胞生长曲线,流式细胞术检测细胞凋亡率和细胞增殖指数,同时用细胞光镜、电镜形态等方法检测细胞凋亡,荧光定量PCR（FQ-PCR）检测PTEN及凋亡相关基因Bcl-2、Bcl-_xL、Bax mRNA水平变化,Western blot检测PTEN及Akt、p-Akt蛋白水平变化。 结果 与Ad-GFP组相比,Ad-PTEN-GFP 转染K562细胞后,细胞增殖受抑,增殖指数降低,凋亡率增加,p-Akt表达降低,抗凋亡相关基因Bcl-2、Bcl-_xL mRNA表达降低,促凋亡基因Bax mRNA表达增加。 结论 过表达PTEN可能通过抑制PI3K/Akt通路抑制K562细胞系增殖,促进细胞凋亡。     

抑制Notch和PI3K/Akt信号通路对食管腺癌细胞增殖、侵袭及迁移的影响

目的 探讨Notch和PI3K/Akt两条信号通路对人食管腺癌细胞OE33增殖、侵袭及迁移能力的影响及两条信号通路之间的联系。方法 应用Notch信号通路阻滞剂DAPT和PI3K/Akt信号通路阻滞剂LY294002分别单独和联合处理OE33细胞,设置对照组;Western blot和实时定量PCR法检测OE33细胞中NICD、Hes1、p-Akt、PTEN蛋白和mRNA的表达变化;CCK-8法检测细胞的增殖抑制率;划痕实验用于检测细胞迁移能力的变化;Transwell细胞侵袭实验用于检测细胞的侵袭能力变化。结果 Notch1通路阻滞剂DAPT能减低Notch1通路相关蛋白NICD和Hes1的表达,并能提高p-Akt蛋白的表达;PI3K/Akt路阻滞剂LY294002不仅使p-Akt的蛋白表达水平减低,还能降低Hes1的蛋白表达水平,同时使NICD的蛋白和Hes1的mRNA表达水平增高。DAPT和LY294002联合处理组的NICD、Hes1、p-Akt的蛋白表达均较空白对照组和单药处理组降低,同时细胞的增殖、迁移和转移能力亦明显减弱。各处理组中PTEN的蛋白和mRNA表达水平较对照组均有一定程度的升高。结论 Notch和PI3K/Akt两条信号通路在食管腺癌细胞OE33中可能存在串话, 同时抑制这两种信号通路能有效的抑制OE33的增殖、侵袭及迁移。     

2-Methoxyestradiol attenuates phosphatidylinositol 3-kinase/Akt pathway-mediated metastasis of gastric cancer

The major obstacle for the treatment of gastric cancer is recurrence and metastasis; yet, its molecular mechanism is largely unknown. 2-methoxyestradiol (2-ME), a metabolite of the estradiol-17beta, has recently been demonstrated to have multifactorial effects against tumor proliferation and angiogenesis; how these effects are interrelated and act cooperatively is the key question to be elucidated. Akt activation was shown to promote cancer cell invasiveness, and inhibition of Akt phosphorylation by 2-ME was also noted. We herein investigated the significance of PI3K/Akt activation in gastric cancer metastasis and the anti-metastatic effect of 2-ME through attenuation of Akt activity. Immunohistochemistry of PI3K, phosphorylated Akt (p-Akt) and phosphorylated Erk (p-Erk) was performed in tumors from 56 gastric cancer patients, and a significant correlation between PI3K/p-Akt and tumor stage/prognosis was demonstrated (p < 0.05). An in vitro study of 7 gastric cancer cell lines showed a remarkable correlation between PI3K and p-Akt. PI3K/p-Akt overexpression was associated with invasiveness/migration; in contrast, phosphorylation of Erk was not shown to be correlated with invasiveness. In addition, metastatic gastric cancer clones expressed a higher level of PI3K/p-Akt. The anti-metastatic effect of a low dose of 2-ME and inactivation of Akt was demonstrated. 2-ME also exhibited an ability to inhibit gastric cancer cell proliferation and induce G2/M cell cycle arrest at a higher concentration than that required for inhibition of migration. We conclude that the activation of PI3K/Akt pathway is involved in the late-stage progression and metastasis of gastric cancer, and attenuation of p-Akt by 2-ME suppresses metastasis.     

miR-142-5p对人上皮性卵巢癌细胞增殖和凋亡的影响

目的:探讨miR-142-5p靶向PTEN-PI3K/Akt信号调控人上皮性卵巢癌SKOV3细胞增殖和凋亡的具体机制。方法:Real-time PCR检测miR-142-5p在人卵巢癌组织及各卵巢癌细胞系中的表达情况。利用MTT、细胞克隆形成实验观察miR-142-5p对SKOV3细胞增殖活力的影响;Caspase-3活力检测miR-142-5p对SKOV3细胞凋亡情况;信号通路抑制剂处理及Western blot检测miR-142-5p、PTEN与PI3K/Akt信号通路的关系及PI3K/Akt信号下游基因Akt、FOXO1、cyclin D1等的表达情况;Luciferase实验验证miR-142-5p和PTEN 3' UTR的结合。结果:人上皮性卵巢癌组织及其细胞系中miR-142-5p表达显著增加（P<0.05)。与对照组相比,SKOV3细胞转染miR-142-5p mimics后活细胞数量显著增加,增殖能力显著上升,细胞凋亡下降（P<0.05);与之相反的,转染miR-142-5p inhibitor后SKOV3细胞增殖能力下调,凋亡增加。信号通路抑制剂处理显示miR-142-5p过表达激活PI3K/Akt信号通路。Luciferase实验显示miR-142-5p与PTEN 3' UTR直接结合。与对照组相比,miR-142-5p mimics组PTEN表达显著降低,p-Akt蛋白表达则显著上调（P<0.05）,同时过表达PTEN后p-Akt表达则显著降低（P<0.05）。结论:miR-142-5p通过抑制PTEN基因表达活化PI3K/Akt信号通路,促进人上皮性卵巢癌组织及SKOV3细胞增殖存活,抑制细胞凋亡。     

贝母素乙通过调控PI3K/Akt信号通路诱导乳腺癌MCF-7细胞凋亡

目的：探究贝母素乙（Peiminine）对乳腺癌细胞MCF-7细胞凋亡的影响及其可能作用机制。方法：采用不同浓度贝母素乙或联合PI3K抑制剂LY294002干预MCF-7细胞,MTT法检测细胞增殖能力;Hoechst33258染色和Annexin V-FITC/PI流式细胞术检测细胞凋亡情况;JC-1染色法检测细胞线粒体膜电位变化;Western blotting检测细胞中PI3K(p110α)、Akt、p-Akt(ser473)、Bad、Bax、Bcl-2、cleaved-Caspase-3以及线粒体和胞浆中细胞色素C(Cyt C)等蛋白表达水平。结果：贝母素乙可呈时间-浓度依赖性抑制MCF-7细胞增殖,诱导细胞出现凋亡形态改变,促进细胞凋亡,并降低线粒体膜电位,上调细胞中Bad、Bax、cleaved-Caspase-3及胞浆中Cyt C蛋白表达水平,下调PI3K(p110α)、p-Akt、Bcl-2和线粒体中Cyt C蛋白表达水平,而Akt蛋白表达水平无显著变化。然而,联合LY294002干预可增强贝母素乙对MCF-7细胞凋亡的促进作用。结论：贝母素乙可诱导乳腺癌MCF-7细胞凋...     

Effect of BRAF-mediated PI3K/Akt/mTOR pathway on biological characteristics and chemosensitivity of NSCLC A549/DDP cells

The present study aimed to explore the biological characteristics of non-small cell lung cancer (NSCLC) cells and the mechanism of chemosensitivity through the role of the PI3K/Akt/mTOR signaling pathway mediated by BRAF gene silencing. Following cell transfection and grouping, an MTT assay detected the activity of NSCLC cells, a scratch wound test assessed the migration ability, flow cytometry using PI staining detected the cell cycle phase, TUNEL and flow cytometry through Annexin V-PI staining assessed the apoptosis, and colony formation was used to detect the sensitivity of lung cancer cells to cisplatin chemotherapy. Furthermore, the relative expression levels of BRAF, PTEN, PI3K, mTOR mRNA were assessed by RT-qPCR, and the protein expression levels of BRAF, PTEN, PI3K, phosphorylated (p)-PI3K, Akt, p-Akt, mTOR, p-mTOR, cisplatin resistance-related enzymes ERCC1 and BRCA1, apoptotic proteins Bax and Bcl-2 were assessed by western blotting. Compared with the control group and NC group, there were differences in decreased BRAF mRNA expression levels in the small interfering (si)BRAF group and siBRAF + IGF-1 group (both P<0.05). In addition, compared with the control group, the siBRAF, NVP-BEZ235 and siBRAF + NVP-BEZ235 groups had significant decreased cell viability at 2–6 days, decreased migration ability, shortened proportion of S-phase cells, increased proportion of G₁/G₀-phase cells, increased apoptosis rate, decreased number of colony-forming cells, decreased mRNA expression of PI3K, Akt and mTOR, increased PTEN mRNA expression, decreased protein expression levels of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, ERCC1, BRCA1 and Bcl-2, and increased protein expression levels of PTEN and Bax (all P<0.05); and more obvious trends were revealed in the siBRAF + NVP-BEZ235 group (all P<0.05); whereas opposite results were detected in the siBRAF + IGF-1 group when compared with the siBRAF group and NVP-BEZ235 group (all P<0.05). Silencing of BRAF gene expression to inhibit the activation of the PI3K/Akt/mTOR signaling pathway exerted a synergistic effect decreasing cell viability, inhibiting the cell cycle and migration, increasing the apoptosis rate, decreasing the number of colony-forming cells and increasing chemosensitivity of NSCLC. Activation of the PI3K/Akt/mTOR signaling pathway may reverse the role of silencing of BRAF gene expression, providing a potential approach for improving the chemosensitivity of NSCLC. The present study for the first time, to the best of our knowledge, clarified the possible mechanism of NSCLC cell biological characteristic changes and chemosensitivity from the perspective of BRAF gene silencing and PI3K/Akt/mTOR signaling pathway activation, providing a potential reference for suppressing tumor aggravation and improving the therapeutic outcomes of NSCLC at the genetic level.     

Topotecan and small interfering RNA suppress survivin expression synergistically in Caki-1 renal cancer cells: Direct suppression of survivin and enhancement of transfection efficiency by topotecan

Survivin is an apoptosis inhibitor found in many tumors but not in most normal differentiated tissues, which makes it an exciting target for cancer therapy. Survivin expression has been shown to be associated with cell proliferation in renal cancer and we previously demonstrated the possibility of treating renal cancer with survivin-specific siRNA and topotecan, which increased the uptake of siRNA by KU19-20 renal cancer cells. We found in the present study that the combination of siRNA and topotecan inhibits survivin expression in Caki-1 renal cancer cells by another mechanism. Caki-1 renal cancer cells expressed higher level of survivin than KU19-20 cells did and survivin-specific siRNA alone suppressed survivin expression only slightly. The combination of topotecan and siRNA suppressed survivin expression completely and inhibited cell proliferation. Topotecan itself did not increase cellular uptake of siRNA but suppressed survivin expression. It also increased transfection efficiency without increasing cellular uptake of siRNA. Although the mechanism of action varies between cell lines, combination therapy using topotecan and siRNA should offer an attractive approach for treatment of advanced renal cancer.     

HK2通过Akt1/p-Akt1上调Cdc42表达促进宫颈癌细胞迁移和侵袭

目的:探究己糖激酶2（HK2）通过Akt1/p-Akt1/Cdc42促进宫颈癌细胞迁移和侵袭的作用机制。方法:G418压力筛选并获取稳定表达HK2蛋白的HeLa和SiHa细胞系;Western blot和细胞免疫化学鉴定HK2蛋白在HeLa和SiHa细胞系中的表达水平;细胞划痕实验检测HK2对HeLa和SiHa体外划痕愈合能力的影响;Transwell迁移、侵袭实验检测HK2对HeLa和SiHa细胞体外迁移和侵袭能力的影响;GEPIA数据库分别分析宫颈癌中HK2的表达与Akt1、Cdc42表达的相关性;Western blot检测Akt1、p-Akt1、Cdc42蛋白在HK2过表达的HeLa细胞和SiHa细胞中的表达情况;在HK2过表达的HeLa、SiHa细胞中应用Akt1/p-Akt1抑制剂MK2206观察Cdc42的表达及细胞迁移和侵袭能力的变化。结果:成功构建HK2稳定表达的HeLa、SiHa细胞系;过表达HK2促进了HeLa、SiHa细胞的划痕愈合能力,促进了HeLa、SiHa细胞的体外迁移和侵袭;GEPIA在线数据库结果显示在宫颈癌中HK2表达与Akt1、Cdc42表达均呈正相关;过表达HK2上调了Akt1、p-Akt1、Cdc42蛋白在HeLa、SiHa细胞中的表达;在HK2过表达HeLa、SiHa细胞中,MK2206的使用抑制了HK2对Cdc42表达上调及细胞迁移和侵袭的促进作用。结论:HK2可能通过Akt1/p-Akt1途径上调Cdc42蛋白的表达促进HeLa和SiHa细胞的迁移和侵袭。     

Regulation of carcinoma cell invasion by protein C inhibitor whose expression is decreased in renal cell carcinoma

Protein C inhibitor (PCI), a member of the serine protease inhibitor family, is produced in various human tissues, including the liver, kidney and testis. In addition to inhibiting the anticoagulant protein C pathway, PCI also inhibits urinary plasminogen activator (uPA), which is a well-known mediator of tumor cell invasion. In the present study, to clarify the biologic significance of PCI in the kidney, we compared the expression of PCI between human renal cell carcinoma (RCC) tissue and nontumor kidney tissue. The PCI antigen level in RCC tissue was found to be significantly lower than in nontumor kidney tissue, and expression of PCI mRNA was detected in normal renal proximal tubular epithelial cells (RPTEC), but not in RCC or in an RCC cell line (Caki-1 cells). No differences were detected between the nucleotide sequence of the major cis-elements in the promoter region of the PCI gene from nontumor kidney and RCC tissues, RPTEC and Caki-1 cells, an RPTEC-derived RCC cell line. The in vitro invasiveness of Caki-1 cells transfected with a PCI expression vector was significantly decreased compared to mock-transfected Caki-1 cells, and it was blocked in the presence of anti-PCI antibody. Since PCI itself did not affect the proliferation rate of Caki-1 cells or cell expression of uPA in vitro, the effect of uPA, PCI, heat-inactivated PCI and plasminogen activator inhibitor (PAI)-1 on the invasive potential of cultured RCC cells was evaluated. The in vitro invasiveness of Caki-1 cells, which express uPA, was significantly enhanced by the addition of uPA, and it was inhibited by anti-uPA antibody, PCI and PAI-1, but not by heat-inactivated PCI. In addition, uPA activity was significantly decreased and uPA-PCI complex level was significantly increased in the culture medium of PCI expression vector-transfected Caki-1 cells as compared to mock-transfected Caki-1 cells. These findings strongly suggest that PCI regulates the invasive potential of RCC cells by inhibiting uPA secreted by these cells. The results of our study suggest that PCI might be a potential therapeutic agent for inhibiting renal tumor invasion.