E-钙黏蛋白在人非小细胞肺癌细胞多西他赛耐药中的作用

目的:探讨非小细胞肺癌(non-small cell lung cancer,NSCLC)A549、H1299、H358、H441多西他赛（docetaxel，DTX）耐药细胞株与上皮间质转化（epithelial-mesenchymal transition,EMT）的关系，并初步研究逆转E-cadherin的表达对NSCLC细胞多西他赛耐药性的影响。方法:应用Real-time PCR 和Western blot 方法检测上皮间质转化相关标志物E-cadherin、Vimentin、N-cadherin、Snail在亲本细胞和多西他赛耐药细胞之间的表达差异；应用慢病毒载体介导构建稳定表达E-cadherin的NSCLC多西他赛耐药细胞；MTS法检测NSCLC细胞多西他赛耐药特性。结果:与A549、H1299、H358、H441四株亲本细胞相比，多西他赛耐药细胞（A549DTX，H1299DTX，H358DTX，H441DTX）形态呈长梭形，呈上皮间质转化（EMT）样改变。多西他赛耐药细胞中E-cadherin表达下调，Vimentin、N-cadherin、Snail表达上调。成功构建了过表达E-cadherin的NSCLC多西他赛耐药细胞。过表达E-cadherin细胞株细胞形态与空载对照细胞株以及亲本耐药细胞株相比呈间质上皮转化(MET)样改变。MTS结果显示四种不同E-cadherin过表达的NSCLC多西他赛耐药细胞对多西他赛的敏感性均强于其亲本耐药细胞和空载对照细胞。结论:NSCLC多西他赛耐药细胞发生EMT样改变，上调E-cadherin能增加NSCLC多西他赛耐药细胞对多西他赛的敏感性。     

Stat1 negatively regulates angiogenesis, tumorigenicity and metastasis of tumor cells

Stat1 is deficient or inactive in many types of human tumors whereas some tumors have activated Stat1. Whether Stat1 affects tumor growth and metastasis is unclear. In the present study, we used Stat1 knockout tumor cells to determine (1) whether Stat1 can regulate angiogenesis, growth, and metastasis of tumor cells; and (2) whether Stat1 is required for the inhibitory effect of IFN-beta on the expression of angiogenic factor bFGF. Highly tumorigenic and metastatic RAD-105 tumor cells derived from a fibrosarcoma of a Stat1 knockout mouse were reconstituted with a Stat1 expression vector. The reconstitution of Stat1 suppressed the tumorigenicity and metastasis of RAD-105 cells in nude mice which correlated with a decreased microvessel density and decreased expression of proangiogenic molecules bFGF, MMP-2, and MMP-9 in vivo. Moreover, noncytotoxic concentrations of IFN-beta significantly inhibited the in vitro expression of bFGF in the Stat1-reconstituted cells but not in the Stat1-deficient cells, which was consistent with decreased bFGF expression of Stat1-reconstituted tumors in vivo. Therefore, Stat1 is essential for IFN-mediated inhibition of bFGF production, suggesting that tumor-intrinsic Stat1 is an important mediator for antiangiogenic signals, such as IFN. Collectively, these data demonstrate that Stat1 expressed by tumor cells is a negative regulator of tumor angiogenesis and, hence, tumor growth and metastasis.     

多西紫杉醇治疗前列腺癌的分子机制研究

  目的  探讨多西紫杉醇治疗男性雄激素抵抗前列腺癌(castration-resistant prostate cancer, CRPC)的分子机制。  方法  前列腺癌细胞株LNCaP、PC-3和CW22-rv1体外培养后, 通过蛋白质印迹、细胞转染、荧光素酶分析、细胞存活率分析等试验分析多西紫杉醇(Docetaxel, Doc)处理后细胞株的存活、AR及p-c-jun的表达情况及其与细胞生存的关系, 同时实时定量PCR检测相应mRNA的表达情况。  结果  多西紫杉醇对不同前列腺癌细胞株的敏感性不同, 其中PC-3细胞最敏感, CW22-rv1和LNCaP细胞中度敏感, 其敏感性与p-c-jun表达呈负相关。转染c-jun基因可降低细胞对多烯紫杉醇的敏感性, 而PC-3细胞转染c-jun和AR基因后则可以使细胞恢复到中等程度敏感性, 细胞存活率为30%。长期暴露于Bicalutmide(比卡鲁胺)后的LNCaP细胞经Doc处理后PSA蛋白表达增加, AR蛋白表达水平降低, AR的mRNA却增加。  结论  p-c-jun降低前列腺癌细胞株对多西紫杉醇的敏感性, 而AR可以增加前列腺癌细胞株对多西紫杉醇的反应, AR的上调降低c-jun/p-c-jun的转录活性, 从而增加前列腺癌细胞株对Doc的反应, 可能是Doc治疗前列腺癌的机制之一。      

Role of ras oncogene in adriamycin resistance in human prostate tumor cells

We have previously isolated a drug-resistant, [PC3(R)], variant of human prostate PC3 cell line, which showed significant resistance (>10-fold) to adriamycin. No known mechanisms of drug resistance were found; however, resistant cells expressed more bcl2, c-myc, and ms oncogenes compared to the sensitive cells. In this study, we found that buthionine sulfoximine (BSO), an inhibitor of gamma-glutamyl-cysteine synthetase, decreased glutathione levels by 80-90% in both cell lines. BSO treatment down-modulated Ras protein only in PC3(R) cells and caused a 4-fold sensitization of PC3(R) cells to adriamycin without affecting PC3(W) cells. Farnesol treatment also inhibited expression of Ras protein and concomitantly reversed adriamycin resistance in PC3(R) cells, indicating that altered levels of ras expression plays an important role in drug resistance in PC3(R) cells.     

Gene expression profiling of the androgen independent prostate cancer cells demonstrates complex mechanisms mediating resistance to docetaxel

The molecular mechanisms conferring resistance to docetaxel in prostate cancer patients remain partially understood. We generated docetaxel resistant derivatives of the androgen independent prostate cancer cell lines PC-3 and DU-145. Docetaxel rapidly induces DU-145 cell death via apoptosis and the drug resistant cells were produced by periodically exposing proliferating DU-145 cultures to small doses of docetaxel. In PC-3 cells docetaxel induces delayed cell death via mitotic catastrophe evident by profound multinucleation and formation of giant cells. Mononucleated progeny of the giant PC-3 cells shows significant resistance to docetaxel. Gene expression profiling of these docetaxel resistant PC-3 cells revealed sets of docetaxel inducible and constitutively expressed genes associated with major cancer pathways. A contradictory overlap with DU-145 docetaxel resistant cells was also found. Analyses suggested significant changes associated with apoptotic function, DNA repair, cell growth, survival and proliferation, metabolism, maintenance of cytoskeleton and extracellular matrix formation. These cellular processes often contribute to drug resistance and our study identified a set of genes managing this phenotype. Additional analyses of the drug resistant PC-3 cells using shRNA constructs determined direct relevance of Cyclin G2 to docetaxel resistance as well as prevention of multinucleation, whereas the knockdown of upregulated CYP1B1 showed no effect on either of these processes. Downregulated GBP1 was explored by ectopic overexpression and even though GBP1 has a potential to mediate resistance to docetaxel, it was not utilized in PC-3 cells. The results suggest complex combination of gene expression pattern changes that enables resistance to docetaxel while preventing death via multinucleation.     

NOTCH3 promotes docetaxel resistance of prostate cancer cells through regulating TUBB3 and MAPK signaling pathway

Docetaxel is the preferred chemotherapeutic agent in patients with castrate-resistant prostate cancer (CRPC). However, patients eventually develop docetaxel resistance and in the absence of effective treatment options. Consequently, it is essential to investigate the mechanisms generating docetaxel resistance and develop novel alternative therapeutic targets. RNA sequencing was undertaken on docetaxel-sensitive and docetaxel-resistant prostate cancer (PCa) cells. Subsequently, chemoresistance, cancer stemness, and lipid metabolism were investigated. To obtain insight into the precise activities and action mechanisms of NOTCH3 in docetaxel-resistant PCa, immunoprecipitation, mass spectrometry, ChIP, luciferase reporter assay, cell metabolism, and animal experiments were performed. Through RNA sequencing analysis, we found that NOTCH3 expression was markedly higher in docetaxel-resistant cells relative to parental cells, and that this trend was continued in docetaxel-resistant PCa tissues. Experiments in vitro and in vivo revealed that NOTCH3 enhanced stemness, lipid metabolism, and docetaxel resistance in PCa. Mechanistically, NOTCH3 is bound to TUBB3 and activates the MAPK signaling pathway. Moreover, NOTCH3 was directly regulated by MEF2A in docetaxel-resistant cells. Notably, targeting NOTCH3 and the MEF2A/TUBB3 signaling axis was related to docetaxel chemoresistance in PCa. Overall, these results demonstrated that NOTCH3 fostered stemness, lipid metabolism, and docetaxel resistance in PCa via the TUBB3 and MAPK signaling pathways. Therefore, NOTCH3 may be employed as a prognostic biomarker in PCa patients. NOTCH3 could be a therapeutic target for PCa patients, particularly those who have developed docetaxel resistance.     

Cyclin D1 expression during rat mammary tumor development and its potential role in the resistance of the Copenhagen rat

Background  

Resistance to mammary tumorigenesis in Copenhagen rats isassociated with loss of early preneoplastic lesions known as intraductalproliferations. The cause of this disappearance, however, is unknown.     

siRNA-mediated silencing of Notch-1 enhances docetaxel induced mitotic arrest and apoptosis in prostate cancer cells

Purpose: Notch is an important signaling pathway that regulates cell fate, stem cell maintenance and theinitiation of differentiation in many tissues. It has been reported that activation of Notch-1 contributes totumorigenesis. However, whether Notch signaling might have a role in chemoresistance of prostate cancer isunclear. This study aimed to investigate the effects of Notch-1 silencing on the sensitivity of prostate cancercells to docetaxel treatment. Methods: siRNA against Notch-1 was transfected into PC-3 prostate cancer cells.Proliferation, apoptosis and cell cycle distribution were examined in the presence or absence of docetaxel byMTT and flow cytometry. Expression of p21waf1/cip1 and Akt as well as activation of Akt in PC-3 cells were detectedby Western blot and Real-time PCR. Results: Silencing of Notch-1 promoted docetaxel induced cell growthinhibition, apoptosis and cell cycle arrest in PC-3 cells. In addition, these effects were associated with increasedp21waf1/cip1 expression and decreased Akt expression and activation in PC-3 cells. Conclusion: Notch-1 promoteschemoresistance of prostate cancer and could be a potential therapeutic target.     

Med1 plays a critical role in the development of tamoxifen resistance

Understanding the molecular pathways that contribute to the development of tamoxifen resistance is a critical research priority as acquired tamoxifen resistance is the principal cause of poor prognosis and death of patients with originally good prognosis hormone-responsive breast tumors. In this report, we provide evidence that Med1, an important subunit of mediator coactivator complex, is spontaneously upregulated during acquired tamoxifen-resistance development potentiating agonist activities of tamoxifen. Phosphorylated Med1 and estrogen receptor (ER) are abundant in tamoxifen-resistant breast cancer cells due to persistent activation of extracellular signal-regulated kinases. Mechanistically, phosphorylated Med1 exhibits nuclear accumulation, increased interaction with ER and higher tamoxifen-induced recruitment to ER-responsive promoters, which is abrogated by inhibition of Med1 phosphorylation. Stable knockdown of Med1 in tamoxifen-resistant cells not only reverses tamoxifen resistance in vitro but also in vivo. Finally, higher expression levels of Med1 in the tumor significantly correlated with tamoxifen resistance in ER-positive breast cancer patients on adjuvant tamoxifen monotherapy. In silico analysis of breast cancer, utilizing published profiling studies showed that Med1 is overexpressed in aggressive subsets. These findings provide what we believe is the first evidence for a critical role for Med1 in tamoxifen resistance and identify this coactivator protein as an essential effector of the tamoxifen-induced breast cancer growth.     

The tumor suppressive role of CAMK2N1 in castration-resistant prostate cancer

Prostate cancer at advanced stages including metastatic and castration-resistant cancer remains incurable due to the lack of effective therapies. The CAMK2N1 gene, cloned and characterized as an inhibitor of CaMKII (calcium/calmodulin-dependent protein kinase II), has been shown to affect tumorigenesis and tumor growth. However, it is still unknown whether CAMK2N1 plays a role in prostate cancer development. We first examined the protein and mRNA levels of CAMK2N1 and observed a significant decrease in human prostate cancers comparing to normal prostate tissues. Re-expression of CAMK2N1 in prostate cancer cells reduced cellular proliferation, arrested cells in G₀/G₁ phases, and induced apoptotic cell death accompanied by down-regulation of IGF-1, ErbB2, and VEGF downstream kinases PI₃K/AKT, as well as the MEK/ERK-mediated signaling pathways. Conversely, knockdown of CAMK2N1 had a significant opposite effects on these phenotypes. Our analyses suggest that CAMK2N1 plays a tumor suppressive role in prostate cancer cells. Reduced CAMK2N1 expression correlates to human prostate cancer progression and predicts poor clinical outcome, indicating that CAMK2N1 may serve as a biomarker. The inhibition of tumor growth by expressing CAMK2N1 established a role of CAMK2N1 as a therapeutic target.     

miR-221增强乳腺癌细胞对多西他赛的耐药性

目的:探究miR-221在人乳腺癌细胞T47D和MCF-7多西他赛(docetaxel)耐药性中的作用及机制。方法:qPCR检测多西他赛处理乳腺癌细胞T47D和MCF-7不同时间点,细胞中miR-221含量的变化;qPCR和Western blot检测miR-221 mimics的转染效率。用阴性对照（NC）或miR-221 mimics转染细胞,不同浓度的多西他赛刺激细胞72小时后,MTT法检测细胞对多西他赛的耐药性;PI和Annexin V双染法检测miR-221 过表达对T47D和MCF-7细胞凋亡的影响;qPCR和Western blot检测靶蛋白p27的表达。结果:在T47D和MCD-7中多西他赛刺激明显促进miR-221的表达量升高;miR-221在细胞内可以发挥生物学效应,降低靶蛋白p27的表达;且过表达miR-221明显增强T47D和MCF-7对多西他赛的耐药性,降低其凋亡率。结论:miR-221过表达可明显增强T47D和MCF-7细胞对多西他赛的耐药性。     

Novel stimulatory role for insulin-like growth factor binding protein-2 in prostate cancer cells

The insulin-like growth factor (IGF) axis is a complex system composed of 2 mitogenic ligands, IGF-I and -II, 2 receptors, IGF-1R and IGF-2R, and 6 binding proteins, IGFBP-1 to -6. The IGFBPs exert their actions through their regulation of IGF bioavailability for IGF receptors. In addition, some IGFBPs have also been found to have direct cellular actions independent of IGFs. IGFBP-2 is a major IGFBP in the prostate and in seminal fluid. IGFBP-2 levels, which are elevated in many malignancies, are markedly increased in prostate cancer, and show a positive correlation with prostate specific antigen (PSA) and prostate tumor aggressiveness. We investigated the potential role of IGFBP-2 in the pathogenesis of prostate cancer by evaluating its ability to stimulate growth and the expression and activity of the nuclear enzyme, telomerase. We found IGFBP-2 to have a modest suppressive effect on the growth of primary cultures of normal prostate epithelial cells and a potent IGF-antagonistic effect in these cells, similar to previous reports on the inhibitory nature of this molecule. Surprisingly, IGFBP-2 had a potent stimulatory effect on growth of LAPC-4 prostate cancer cells, an effect that was more pronounced in the absence of androgens. IGFBP-2 growth stimulation of LAPC-4 cells was completely blocked by MAP-kinase inhibitors and partially blocked by PI3-kinase inhibitors. IGFBP-2 stimulation of LAPC-4 cell growth seen in serum-free conditions was lost in the presence of 10% FBS. IGFBP-2 also had a growth stimulatory effect on DU 145 prostate cancer cells. IGFBP-2 significantly stimulated telomerase activity in LAPC-4 cells in the absence of androgens. In addition, IGFBP-2 significantly stimulated hTERT expression and telomerase activity in DU 145 cells. Thus, we demonstrated an inhibitory effect of IGFBP-2 on non-malignant prostate cells, but showed it to be stimulatory for prostate cancer cells in a MAP-kinase and androgen-modulated process. In conclusion, IGFBP-2 may play a role in the progression, but not in the initiation of the prostate cancer disease process, suggesting the existence of a molecular switch transitioning IGFBP-2 from a growth inhibitor to a pro-carcinogenic molecule.     

Prognostic role of circulating tumor cells and disseminated tumor cells in patients with prostate cancer: a systematic review and meta-analysis

Circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) displayed their roles in prognosis prediction in prostate cancer. The objective of the present study was to conduct a systematic review and meta-analysis of published literature while investigating the correlation between survival outcome and CTCs or DTCs counts in patients with prostate cancer. Relevant literature was searched in Pubmed and Embase. Survival data of included study were extracted. Forrest plots were used to estimate the effect of CTCs/DTCs on the survival of patients. Publication bias was evaluated using Begg’s test. The estimated HRs and 95 % confidence interval for the effect of CTCs/DTCs on overall survival (OS) and biochemical relapse-free survival (bRFS) or disease-free survival (DFS) were 2.43 [2.07, 2.86] (p?<?0.00001) and 2.15 [1.69, 2.73] (p?<?0.00001), respectively. Subgroup analysis revealed that CTCs were also relevant to poor prognosis (hazard ratio (HR) 2.43 [2.05, 2.89] for OS, HR 2.46 [2.08, 2.90] for bRFS/DFS). A similar result was yielded in DTCs (1.47 [1.21, 1.80] for DFS). CTCs/DTCs could also predict poor OS in metastatic prostate cancer (2.37 [1.99, 2.82], p?<?0.00001) and in localized stage (HR 1.84 [1.47, 2.28], p?<?0.00001). In addition, CTCs/DTCs detected by different methods, especially by CellSearch system (HR for OS 2.36 [1.95, 2.85] and HR for bRFS/DFS 2.53 [1.66, 3.85]), were relevant to poor prognosis. Available evidence supported the notion of the strong prognostic value of CTCs. CTCs are promising biomarkers that are clinically implemented in the therapeutic decision-making process in patients with prostate cancer.