Anti-Epidermal Growth Factor Receptor (EGFR) Antibodies Overcome Resistance of Ovarian Cancer Cells to Targeted Therapy and Natural Cytotoxicity

The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.     

Radiation-Sensitising Effects of Antennapedia Proteins (ANTP)-SmacN7 on Tumour Cells

The objective of this study was to investigate the underlying mechanisms behind the radiation-sensitising effects of the antennapedia proteins (ANTP)-smacN7 fusion protein on tumour cells. ANTP-SmacN7 fusion proteins were synthesised, and the ability of this fusion protein to penetrate cells was observed. Effects of radiation on the expression of X-linked inhibitor of apoptosis protein (XIAP) were detected by western blotting. The radiation-sensitising effects of ANTP-SmacN7 fusion proteins were observed by a clonogenic assay. The effects of drugs and radiation on tumour cell apoptosis were determined using Annexin V/FITC double staining. Changes in caspase-8, caspase-9 and caspase-3 were detected by western blot before and after ANTP-SmacN7 inhibition of XIAP. The ANTP-SmacN7 fusion protein could enter and accumulate in cells; in vitro XIAP expression of radiation-induced tumour cells was negatively correlated with tumour radiosensitivity. The ANTP-SmacN7 fusion protein promoted tumour cell apoptosis through the activation of caspase3. ANTP-SmacN7 fusion protein may reduce tumour cell radioresistance by inducing caspase3 activation.     

Multiple Mechanisms Mediate Resistance to Sorafenib in Urothelial Cancer

Genetic and epigenetic changes in the mitogen activated protein kinase (MAPK) signaling render urothelial cancer a potential target for tyrosine kinase inhibitor (TKI) treatment. However, clinical trials of several TKIs failed to prove efficacy. In this context, we investigated changes in MAPK signaling activity, downstream apoptotic regulators and changes in cell cycle distribution in different urothelial cancer cell lines (UCCs) upon treatment with the multikinase inhibitor sorafenib. None of the classical sorafenib targets (vascular endothelial growth factor receptor 1/-receptor 2, VEGFR1/-R2; platelet-derived growth factor receptor α/-receptor β, PDGFR-α/-β; c-KIT) was expressed at significant levels leaving RAF proteins as its likely molecular target. Low sorafenib concentrations paradoxically increased cell viability, whereas higher concentrations induced G1 arrest and eventually apoptosis. MAPK signaling remained partly active after sorafenib treatment, especially in T24 cells with an oncogenic HRAS mutation. AKT phosphorylation was increased, suggesting compensatory activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Sorafenib regularly down regulated the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) protein, but combinatorial treatment with ABT-737 targeting other B-cell lymphoma 2 (Bcl-2) family proteins did not result in synergistic effects. In summary, efficacy of sorafenib in urothelial cancer cell lines appears hampered by limited effects on MAPK signaling, crosstalk with further cancer pathways and an anti-apoptotic state of UCCs. These observations may account for the lack of efficacy of sorafenib in clinical trials and should be considered more broadly in the development of signaling pathway inhibitors for drug therapy in urothelial carcinoma.     

Cetuximab-Induced MET Activation Acts as a Novel Resistance Mechanism in Colon Cancer Cells

Aberrant MET expression and hepatocyte growth factor (HGF) signaling are implicated in promoting resistance to targeted agents; however, the induced MET activation by epidermal growth factor receptor (EGFR) inhibitors mediating resistance to targeted therapy remains elusive. In this study, we identified that cetuximab-induced MET activation contributed to cetuximab resistance in Caco-2 colon cancer cells. MET inhibition or knockdown sensitized Caco-2 cells to cetuximab-mediated growth inhibition. Additionally, SRC activation promoted cetuximab resistance by interacting with MET. Pretreatment with SRC inhibitors abolished cetuximab-mediated MET activation and rendered Caco-2 cells sensitive to cetuximab. Notably, cetuximab induced MET/SRC/EGFR complex formation. MET inhibitor or SRC inhibitor suppressed phosphorylation of MET and SRC in the complex, and MET inhibitor singly led to disruption of complex formation. These results implicate alternative targeting of MET or SRC as rational strategies for reversing cetuximab resistance in colon cancer.     

Cancer-Associated Fibroblasts Modify the Response of Prostate Cancer Cells to Androgen and Anti-Androgens in Three-Dimensional Spheroid Culture

Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool.     

Clinical Application of Circulating Tumour Cells in Prostate Cancer: From Bench to Bedside and Back

Prostate cancer is the most common cancer in men worldwide. To improve future drug development and patient management, surrogate biomarkers associated with relevant outcomes are required. Circulating tumour cells (CTCs) are tumour cells that can enter the circulatory system, and are principally responsible for the development of metastasis at distant sites. In recent years, interest in detecting CTCs as a surrogate biomarker has ghiiukjrown. Clinical studies have revealed that high levels of CTCs in the blood correlate with disease progression in patients with prostate cancer; however, their predictive value for monitoring therapeutic response is less clear. Despite the important progress in CTC clinical development, there are critical requirements for the implementation of their analysis as a routine oncology tool. The goal of the present review is to provide an update on the advances in the clinical validation of CTCs as a surrogate biomarker and to discuss the principal obstacles and main challenges to their inclusion in clinical practice.     

Modulation of MDR1 and MRP3 Gene Expression in Lung Cancer Cells after Paclitaxel and Carboplatin Exposure

Carboplatin-paclitaxel is a reference regimen in the treatment of locally advanced or disseminated non-small cell lung cancer (NSCLC). This paper discusses the multidrug resistance developed with this drug combination, which is one of the major obstacles to successful treatment. In order to understand and overcome the drug resistance pattern of NSCLC after carboplatin plus paclitaxel exposure, levels of mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 3 (MRP3) were investigated in primary NSCLC cell lines (A-549 and A-427) and a metastasis-derived NSCLC cell line (NODO). Our results showed that exposure of the three NSCLC lines to plasma concentrations of paclitaxel (5 μM) produced an increase in MDR1 expression, while MRP3 showed no alteration in expression. By contrast, the same cells exposed to carboplatin plasma concentrations (30 μM) showed overexpression of MRP3. In these cells, MDR1 showed no expression changes. Interestingly, the combination of both paclitaxel and carboplatin caused increased expression of the MDR1 drug resistance gene rather than the individual treatments. These results suggest that carboplatin and paclitaxel may induce drug resistance mediated by MDR1 and MRP3, which may be enhanced by the simultaneous use of both drugs.     

Apoptotic Death of Cancer Stem Cells for Cancer Therapy

Cancer stem cells (CSCs) play crucial roles in tumor progression, chemo- and radiotherapy resistance, and recurrence. Recent studies on CSCs have advanced understanding of molecular oncology and development of novel therapeutic strategies. This review article updates the hypothesis and paradigm of CSCs with a focus on major signaling pathways and effectors that regulate CSC apoptosis. Selective CSC apoptotic inducers are introduced and their therapeutic potentials are discussed. These include synthetic and natural compounds, antibodies and recombinant proteins, and oligonucleotides.     

镀锌及低铬钝化锌镀层的电化学行为

本文在３％ＮａＣｌ溶液中采用线性极化及交流阻抗技术测定了镀锌层和钝化后的锌镀层的极化电阻,实验表明：低铬钝化后腐蚀电阻明显增大,是纯锌的２－６倍,同时利用Ｓｔｅｒｎ－Ｇｅａｒｙ方程,根据极化电阻Ｒｐ和Ｔａｆｅｌ斜率计算腐蚀电流,同时探讨了纯化后的镀锌层在静止电位下的交流阻抗行为。     

抗坏血酸与乳腺癌MDA-MB-231细胞增殖和凋亡的相关性

目的 探讨抗坏血酸与乳腺癌MDA-MB-231细胞增殖和凋亡的相关性。方法分别用0.5、1.0、2.0和4.0mmol/L的抗坏血酸处理MDA-MB-231细胞,MTT法检测细胞增殖水平;RT-PCR检测细胞p53和bcl-2基因mRNA的转录水平;West-ern blot检测细胞P53和bcl-2蛋白的表达;流式细胞术检测细胞的凋亡率。结果不同浓度的抗坏血酸均可明显抑制MDA-MB-231细胞增殖,促进其凋亡,并使细胞p53基因mRNA转录水平及P53蛋白的表达水平明显升高,而bcl-2基因mRNA转录水平及bcl-2蛋白表达水平明显降低,且均呈剂量依赖性。结论抗坏血酸能抑制MDA-MB-231细胞增殖,诱导其凋亡,其机制可能是通过上调P53,下调bcl-2介导的。     

肿瘤细胞多药耐药机制的研究进展

随着抗肿瘤药物在临床上被广泛使用,使越来越多不同种类的肿瘤细胞出现耐药现象,大大降低了肿瘤药物的治疗效果。因此,弄清肿瘤细胞的耐药机制,寻找体内治疗指数高、细胞选择性强、稳定且具有新型作用机制的抗肿瘤药物迫在眉睫。     

Ipr1和GFP基因真核共表达穿梭质粒的构建及其在人肺癌细胞中的表达

目的构建胞内病原体抗性基因1(Ipr1)和绿色荧光蛋白(GFP)基因真核共表达穿梭质粒,并在人肺腺癌细胞A549中表达。方法采用PCR方法,分别从质粒pEGFP-C1-Ipr1和pEGFP-C1中扩增Ipr1和GFP基因,将GFP基因、分枝杆菌复制子OriM和Ipr1基因同时克隆入多启动子真核共表达载体pBudCE4.1中,构建pBud-GFP-OriM-Ipr1穿梭质粒,脂质体法转染A549细胞,荧光显微镜观察GFP的表达,免疫组化方法检测Ipr1蛋白的表达。结果酶切和测序分析表明,pBud-GFP-OriM-Ipr1真核共表达穿梭质粒构建正确。转染A549细胞后,荧光显微镜下可观察到转染细胞中有GFP表达,免疫组化法可检测到Ipr1蛋白的表达,且定位于细胞核内。结论已成功构建Ipr1和GFP基因真核共表达穿梭质粒,为进一步研究Ipr1抗结核的功能奠定了基础。     

Proteomic Analysis Identified DJ-1 as a Cisplatin Resistant Marker in Non-Small Cell Lung Cancer

The aim of study is to identify cisplatin-resistance associated biomarkers for non-small cell lung cancers (NSCLC). We use two-dimensional electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to compare the proteome between lung cancer cell line A549 and its cisplatin-resistant subline A549/DDP. Nine cisplatin resistance-related proteins were identified, and DJ-1, one of the differently expressed proteins, was selected for further validation and evaluation. Immunohistochemical results demonstrated that high expression level of DJ-1 was associated with cisplatin resistance and a predictor for poor prognosis in 67 locally advanced NSCLC patients. Furthermore, in vitro results showed that silencing DJ-1 increased the proliferation inhibitory effect of cisplatin to A549/DDP cells. In conclusion, DJ-1 might play an important role in the resistibility to cisplatin, and it could also act as a novel candidate biomarker for predicting the response of NSCLC patients to cisplatin-based chemotherapy.     

镀锌层高耐蚀三价铬黑色钝化工艺

采用正交试验优化镀锌层三价铬黑色钝化液的各个组分,在此基础上考察各种工艺条件对钝化膜的外观、耐蚀性和附着力的影响,确定了三价铬黑色钝化工艺的配方及工艺参数。三价铬黑色钝化工艺为:7.5 g/L Cr~(3+),21 g/L络合剂,22 g/L磷酸二氢钠,15 g/L过渡金属盐,0.4 mL/L有机硫化物,0.4 mL/L纳米硅溶胶,pH为2.0,θ为50℃,钝化t为45 s。该钝化工艺得到的钝化膜均匀黑亮,附着力良好,在未加封闭的情况下,耐蚀性达到中性盐雾60 h以上。     

Efficient pH Dependent Drug Delivery to Target Cancer Cells by Gold Nanoparticles Capped with Carboxymethyl Chitosan

Doxorubicin (DOX) was immobilized on gold nanoparticles (AuNPs) capped with carboxymethyl chitosan (CMC) for effective delivery to cancer cells. The carboxylic group of carboxymethyl chitosan interacts with the amino group of the doxorubicin (DOX) forming stable, non-covalent interactions on the surface of AuNPs. The carboxylic group ionizes at acidic pH, thereby releasing the drug effectively at acidic pH suitable to target cancer cells. The DOX loaded gold nanoparticles were effectively absorbed by cervical cancer cells compared to free DOX and their uptake was further increased at acidic conditions induced by nigericin, an ionophore that causes intracellular acidification. These results suggest that DOX loaded AuNPs with pH-triggered drug releasing properties is a novel nanotheraputic approach to overcome drug resistance in cancer.     

电解磷化工艺对锌系磷化膜耐蚀性影响的研究

为提高碳钢的耐蚀性与冷加工性能,采用电解磷化法制备了锌系电解磷化膜,通过盐雾试验、Tafel曲线及交流阻抗等方法研究了电解磷化工艺对锌系电解磷化膜耐蚀性的影响,并通过X-射线衍射仪分析了电解磷化膜的成分。结果表明,磷化膜成分为Zn_3(PO_4)_2、Fe_3(PO_4)_2,在Jκ为45 A/dm~2,磷化t为10 s,θ为60℃的条件下,电解磷化膜盐雾试验35 h不锈蚀。     

镀锌层钼酸盐转化膜及其耐蚀机理

应用极化曲线和交流阻抗的方法研究了镀锌层钼酸盐转化膜在5％NaCl的溶液中的耐蚀性,结果表明,钼酸盐转化膜处理能显著地提高镀锌层的耐腐蚀能力,分析了转化膜的成膜机理,并从转化膜对镀锌层在5％ NaCl溶液中的阴、阳极反应历程的影响及交流阻抗行为的变化,分析了钼酸盐转化膜的耐蚀机理。     

铝元素对锌镀层耐蚀性的影响

研究了用复合电沉积法制备的锌铝镀层在微酸性腐蚀介质中的耐蚀性。试验结果证明,氢氧化铝也能改善镀层的耐蚀性,但不如铝粉的作用显著。EPMA和ICP显示含氢氧化铝和含铝粉镀层中的铝呈均匀分布状态,但前者铝的含量为50ppm,后者为0.15wt％。x-射线衍射法揭示溶液中的氢氧化铝能使镀层的结晶细致,晶面择优取向程度增加。当镀液中含有铝粉时,所得镀层的结晶晶面的择优程度更大,结构则成为层状的。因此,氢氧化铝和铝粉是通过提高镀层的晶面择优取向程度,从而改善了其耐蚀性能。