Hypoxia Induced Multidrug Resistance of Laryngeal Cancer Cells via Hypoxia-inducible Factor-1α

Objectives: To investigate whether hypoxia has an effect on regulation of multidrug resistance (MDR) tochemotherapeutic drugs in laryngeal carcinoma cells and explore the role of hypoxia-inducible factor-1α (HIF-1α). Methods: Laryngeal cancer cells were cultured under normoxic and hypoxic conditions. The sensitivityof the cells to multiple drugs and levels of apoptosis induced by paclitaxel were determined by MTT assay andannexin-V/propidium iodide staining analysis, respectively. HIF-1α expression was blocked by RNA interference.The expression of HIF-1α gene was detected by real-time quantitative RT-PCR and Western blotting. The valueof fluorescence intensity of intracellular adriamycin accumulation and retention in cells was evaluated by flowcytometry. Results: The sensitivity to multiple chemotherapy agents and induction of apoptosis by paclitaxelcould be reduced by hypoxia (P<0.05). A the same time, the adriamycin releasing index of cells was increased(P<0.05). However, resistance acquisition subject to hypoxia in vitro was suppressed by down-regulating HIF-1αexpression. Conclusion: HIF-1α could be considered as a key regulator for mediating hypoxia-induced MDR inlaryngeal cancer cells via inhibition of drug-induced apoptosis and decrease in intracellular drug accumulation.     

Hypoxia and drug resistance

Biologically and therapeutically important hypoxia occurs in many solid tumor masses. Hypoxia can be a direct cause of therapeutic resistance because some drugs and radiation require oxygen to be maximally cytotoxic. Cellular metabolism is altered under hypoxic conditions. Hypoxia can result in drug resistance indirectly if under this condition cells more effectively detoxify the drug molecules. Finally, there is evidence that hypoxia can enhance genetic instability in tumor cells thus allowing more rapid development of drug resistance cells. The current review describes the effects of hypoxia on tumor response to a variety of anticancer agents and also describes progress toward therapeutically useful methods of delivering oxygen to tumors in an effort to overcome therapeutic resistance due to hypoxia. Finally, the use of hypoxic cell selective cytotoxic agents as a means of addressing hypoxic drug resistance is discussed.     

Mislocalization of death receptors correlates with cellular resistance to their cognate ligands in human breast cancer cells

Multiple clinical trials are ongoing to evaluate the potential antitumor activity of human TNF variants, Fas ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL) and its agonistic antibodies. These drug products act through the death receptors (DRs) TNF receptor 1 (TNFR1), Fas/CD95, DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2), respectively. Therefore, characterization of the level and localization of DR expression in cancer cells is important for DR-targeted therapy. In this study, we examined the subcellular distribution of the four DRs in a panel of 10 human breast cancer cell lines by western blots and flow cytometry and 50 human breast tumors by immunohistochemistry. Despite their total protein expressions, the DRs were found to be absent on the surface of some cell lines. Consistent with this result, all four DRs were found to be mostly expressed in the cytoplasm and/or the nucleus of primary breast tumors (n=50). We further determined the growth inhibition activity (GI50) of the death ligands, recombinant human TNFα, FasL and TRAIL, and found a correlation with the subcellular localization of the corresponding DRs. These results demonstrate an aberrant expression of the death receptors in breast cancer cells, and suggest that the lack of surface DRs appears to be predictive of tumor resistance to DR-targeted therapies.     

SRPX2 is overexpressed in gastric cancer and promotes cellular migration and adhesion

SRPX2 (Sushi repeat containing protein, X‐linked 2) was first identified as a downstream molecule of the E2A‐HLF fusion gene in t(17;19)‐positive leukemia cells and the biological function of this gene remains unknown. We found that SRPX2 is overexpressed in gastric cancer and the expression and clinical features showed that high mRNA expression levels were observed in patients with unfavorable outcomes using real‐time RT‐PCR. The cellular distribution of SRPX2 protein showed the secretion of SRPX2 into extracellular regions and its localization in the cytoplasm. The introduction of the SRPX2 gene into HEK293 cells did not modulate the cellular proliferative activity but did enhance the cellular migration activity, as shown using migration and scratch assays. The conditioned‐medium obtained from SRPX2‐overexpressing cells increased the cellular migration activity of a gastric cancer cell line, SNU‐16. In addition, SRPX2 protein remarkably enhanced the cellular adhesion of SNU‐16 and HSC‐39 and increased the phosphorylation levels of focal adhesion kinase (FAK), as shown using western blotting, suggesting that SRPX2 enhances cellular migration and adhesion through FAK signaling. In conclusion, the overexpression of SRPX2 enhances cellular migration and adhesion in gastric cancer cells. Here, we report that the biological functions of SRPX2 include cellular migration and adhesion to cancer cells. © 2008 Wiley‐Liss, Inc.     

缺氧调控端粒酶活性及抑制鼻咽癌细胞增殖实验研究

目的探讨缺氧及siRNA沉默缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)后对鼻咽癌细胞中端粒酶催化亚单位(telomerase catalytic subunit,hTERT)、细胞周期和化疗耐药的影响。方法采用三气培养箱对鼻咽癌细胞5-8F和CNE2进行缺氧处理(1%O2),蛋白质印迹法检测不同乏氧时相(0~72h)HIF-1α和hTERT蛋白的表达。将HIF-1α基因特异性siRNA分别转染鼻咽癌细胞株5-8F和CNE2,筛选出沉默效率最高的siRNA,实验分为未处理组(常氧)、未处理组(缺氧)、Negative-siRNA(缺氧)和HIF-1α-siRNA(缺氧),荧光定量PCR及蛋白质印迹检测瞬时转染后hTERT及HIF-1α的表达。流式细胞术(flow cytometry,FCM)分析缺氧或沉默HIF-1α后对细胞周期的影响。MTT法检测缺氧或沉默HIF-1α后,鼻咽癌细胞对顺铂(DDP)和5-氟尿嘧啶(5-FU)的化疗敏感性。结果缺氧处理0~72h后鼻咽癌5-8F细胞HIF-1α(F=37.147,P<0.001)和hTERT(F=70.069,P<0.001)蛋白的表达上调,差异有统计学意义。HIF-1α-siRNA对5-8F细胞瞬时转染率>98%。HIF-1α-siRNA组hTERT mRNA表达量为0.37±0.05,显著低于未处理组(缺氧)的1.00±0.00和Negative-siRNA(缺氧)的0.95±0.01,F=360.339,P<0.001;hTERT蛋白表达量为(0.27±0.05),显著低于未处理组(缺氧)0.54±0.00和Negative-siRNA组(缺氧)0.53±0.01,F=24.010,P<0.001。未处理组(缺氧)G0/G1期细胞比例明显增加(45.63±2.01)%,显著高于未处理组(常氧)的(26.75±1.28)%,P<0.001。5-8F细胞未处理组(常氧)对5-FU的IC50分别为(17.30±3.31)μg/mL,未处理组(缺氧)为(32.04±12.75)μg/mL,Negative-siRNA组为(33.90±0.87)μg/mL,HIF-1α-siRNA组为(13.72±2.36)μg/mL,F=3.704,P<0.001。5-8F细胞缺氧组对DDP的化疗敏感性也降低,沉默HIF-1α后,5-8F细胞对DDP的化疗敏感性明显提高。除细胞周期外,CNE2与5-8F的结果均一致。结论缺氧促使鼻咽癌细胞发生G1/S阻滞及化疗耐药,可能与上调hTERT表达,进而上调端粒酶活性有关;沉默HIF-1α显著逆转缺氧诱导的肿瘤耐药并下调端粒酶活性。     

食管癌组织上皮细胞黏附分子表达的临床意义

 【摘要】　目的　探讨上皮细胞黏附分子（Ep-CAM）在食管鳞状细胞癌组织中表达的临床意义。方法　应用免疫组织化学方法检测70例食管正常黏膜、癌组织和72枚区域淋巴结内Ep-CAM的表达情况。结果　在正常食管组织中，Ep-CAM未见阳性表达；但在食管癌组织中表达阳性率为94.3 %；其表达强度与食管癌的病变长度、浸润深度无明显相关，但与肿瘤分化程度、淋巴结是否转移明显相关（P＜0.001）；Ep-CAM表达强度与患者术后3年生存率呈明显负相关（P＜0.001）。结论　Ep-CAM在食管癌组织中特异性高表达，可作为食管癌诊断、预后判断及治疗的有用指标。     

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1.     

Efficacy of adjuvant chemotherapy according to Prion protein expression in patients with estrogen receptor-negative breast cancer.

BACKGROUND: Prion protein (PrPc) has been previously reported to be associated with resistance to proapoptotic stimuli. We evaluated whether the expression of PrPc was associated with the resistance to adjuvant chemotherapy in patients with estrogen receptor (ER) -negative breast cancer. PATIENTS AND METHODS: The expression of PrPc by primary tumors was assessed by immunohistochemistry in a series of 756 patients included in two randomized trials that compared anthracycline-based chemotherapy to no chemotherapy. The PrPc expression was correlated with ER expression and the benefit of adjuvant chemotherapy was assessed according to PrPc expression in patients with ER-negative tumors. RESULTS: Immunostaining analysis showed that PrPc was mainly expressed by myoepithelial cells in normal breast tissue. Tissue microarray analysis from 756 breast tumors showed that PrPc was associated with ER-negative breast cancer subsets (P < 0.001). Adjuvant chemotherapy was not associated with a significant risk reduction for death in patients with ER-negative/PrPc-positive disease [adjusted hazard ratio (HR) for death = 0.98, 95% confidence interval (CI) 0.45-2.1, P = 0.95], while it decreased the risk for death (HR = 0.39, 95% CI 0.2-0.74, P = 0.004) in patients with ER-negative/PrPc-negative tumors. CONCLUSION: These data indicate that ER-negative/PrPc-negative phenotype is associated with a high sensitivity to adjuvant chemotherapy.     

Drosophila homologue of Diaphanous 1 (DIAPH1) controls the metastatic potential of colon cancer cells by regulating microtubule-dependent adhesion

Drosophila homologue of Diaphanous 1 (DIAPH1) regulates actin polymerization and microtubule (MT) stabilization upon stimulation with lysophosphatidic acid (LPA). Recently, we showed strongly reduced lung metastasis of DIAPH1-depleted colon cancer cells but we found accumulations of DIAPH1-depleted cells in bone marrow. Here, we analyzed possible organ- or tissue-specific metastasis of DIAPH1-depleted HCT-116 cells. Our data confirmed that depletion of DIAPH1 strongly inhibited lung metastasis and revealed that, in contrast to control cells, DIAPH1-depleted cells did not form metastases in further organs. Detailed mechanistic analysis on cells that were not stimulated with LPA to activate the cytoskeleton-modulating activity of DIAPH1, revealed that even under basal conditions DIAPH1 was essential for cellular adhesion to collagen. In non-stimulated cells DIAPH1 did not control actin dynamics but, interestingly, was essential for stabilization of microtubules (MTs). Additionally, DIAPH1 controlled directed vesicle trafficking and with this, local clustering of the adhesion protein integrin-β1 at the plasma membrane. Therefore, we conclude that under non-stimulating conditions DIAPH1 controls cellular adhesion by stabilizing MTs required for local clustering of integrin-β1 at the plasma membrane. Thus, blockade of DIAPH1-tubulin interaction may be a promising approach to inhibit one of the earliest steps in the metastatic cascade of colon cancer.     

A new superinvasive in vitro phenotype induced by selection of human breast carcinoma cells with the chemotherapeutic drugs paclitaxel and doxorubicin

Doxorubicin- and paclitaxel-selected variants of an in vitro invasive clonal population of the human breast cancer cell line, MDA-MB-435S, were established by pulse selection, and exhibited a novel 'superinvasive' phenotype. This phenotype is characterised by an ability to relocate to another surface following invasion through matrigel and membrane pores, by decreased adhesion to extracellular matrix proteins and by increased motility. This may represent an in vitro model of a step in the metastatic process occurring subsequent to invasion. The paclitaxel-resistant variants, MDA-MB-435S-F/Taxol-10p and MDA-MB-435S-F/Taxol-10p4p were resistant to paclitaxel, vincristine and docetaxel, but not to doxorubicin, carboplatin, etoposide or 5-fluorouracil. The doxorubicin-selected variants MDA-MB-435S-F/Adr-10p and MDA-MB-435S-F/Adr-10p10p, in contrast, exhibited only small increases in resistance to doxorubicin, although they were slightly resistant to VP-16 and docetaxel, and exhibited increased sensitivity to paclitaxel, carboplatin and 5-fluorouracil.     

Androgen deprivation results in time‐dependent hypoxia in LNCaP prostate tumours: Informed scheduling of the bioreductive drug AQ4N improves treatment response

Androgen withdrawal induces hypoxia in androgen‐sensitive tissue; this is important as in the tumour microenvironment, hypoxia is known to drive malignant progression. Our study examined the time‐dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT‐induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti‐androgens and tumour oxygenation measured. Dorsal skin fold (DSF) chambers were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide‐treated and vehicle‐only‐treated tumours were re‐established in vitro, and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2 mg/kg/day) decreased tumour oxygenation by 45% within 24 hr, reaching a nadir of 0.09% oxygen (0.67 ± 0.06 mmHg) by Day 7; this persisted until Day 14 when it increased up to Day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at Days 7 and 14 with revascularisation occurring by Day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide‐treated tumours were more malignant than vehicle‐treated controls. Combining bicalutamide with AQ4N (50 mg/kg, single dose) caused greater tumour growth delay than bicalutamide alone. Our study shows that bicalutamide‐induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.     

肿瘤细胞耐药机制的研究进展

细胞耐药性的产生是导致肿瘤化疗失败的重要因素,尤其是多药耐药是目前研究的一个重点.肿瘤细胞耐药的分子机制涉及癌基因和抑癌基因的表达异常,DNA损伤修复能力改变及多药耐药相关蛋白异常表达等多个方面,全面了解肿瘤细胞耐药的分子机制将有助于指导临床用药及为新药的研发提供理论依据.本文综述了肿瘤细胞耐药机制的研究进展.     

缺氧微环境诱导胶质母细胞瘤耐药机制研究进展

胶质母细胞瘤（glioblastoma,GBM）的耐药性是导致其临床治疗失败的主要原因。目前,诸多研究认为肿瘤内部缺氧的微环境能够诱导GBM对放化疗的抵抗。GBM的生长极为迅速,贫瘠的氧供不能满足其对氧气的需求,缺氧的微环境由此形成。缺氧可通过不同机制影响GBM的耐药性。本文将根据缺氧微环境的形成与缺氧诱导GBM耐药的机制进行综述。      

耐阿霉素食管癌细胞株EC9706/ADM的EMT-MET现象及机制

目的:建立耐阿霉素食管癌细胞株EC9706/ADM,探讨耐药细胞的EMT-MET现象及机制。方法:采用中等浓度间歇法建立耐阿霉素食管癌细胞株EC9706/ADM,用MTT法测定细胞的耐药指数及多药耐药性,Transwell小室实验检测耐药细胞的体外侵袭能力,实时荧光定量PCR和Western blot 检测细胞在不同黏附时间EMT相关分子标志物E-cadherin、N-cadherin、Vimentin、Fibronectin和基质金属蛋白酶MMP2、MMP7、MMP9等的表达。结果:成功建立耐阿霉素食管癌细胞株EC9706/ADM,细胞的耐药指数为32.2,对顺铂、5-氟尿嘧啶、环磷酰胺产生交叉耐药性。与EC9706亲本细胞相比,EC9706/ADM细胞体外侵袭能力明显增强。黏附实验中,在黏附早期,耐药细胞 E-cadherin表达降低而N-cadherin表达显著增高,黏附后期,E-cadherin表达升高而N-cadherin表达显著降低,基质金属酶在细胞黏附过程中表达均升高。结论:食管癌耐阿霉素细胞EC9706/ADM侵袭能力增强,可能与基质金属酶表达量增高和细胞发生EMT-MET转换相关。