Gemcitabine sensitizes lung cancer cells to Fas/FasL system‐mediated killing

Gemcitabine is a chemotherapy agent commonly used in the treatment of non‐small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine‐induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real‐time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL‐expressing cells was evaluated by flow cytometry, and caspase‐8 and caspase‐3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine‐activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti‐Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane‐bound FasL (mFasL) and of mFasL‐positive apoptotic H292 cells, as well as caspase‐8 and caspase‐3 cleavage. Moreover, gemcitabine increased CH11‐induced caspase‐8 and caspase‐3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine‐treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up‐regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas‐dependent apoptosis mediated by caspase‐8 and caspase‐3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine‐induced lung cancer cell killing.     

CD44和Fas在人乳腺癌中的表达及其相互关系

目的:通过观察人乳腺癌组织中CD44和凋亡调节蛋白Fas的表达,探讨CD44和Fas与乳腺癌的发生、转移和免疫逃逸的关系。方法:收集手术切除的人乳腺癌组织和癌旁相对正常乳腺组织,对36例乳腺癌标本进行检测。结果:CD44在相对正常乳腺组织中的表达率为2.8%,在有淋巴结转移和无淋巴结转移的阳性表达率分别为88.2%和42.1%,差异有统计学意义;Fas在相对正常乳腺组织中的表达率77.8%,在有淋巴结转移和无淋巴结转移的阳性表达率分别为58.9%和31.6%,差异有统计学意义;CD44与Fas之间存在相关关系。结论:CD44、Fas在乳腺癌的发生、转移和免疫逃逸方面可能起着重要的协同作用,为临床开展乳腺癌的生物治疗提供实验依据。     

Prognostic value of CD44 expression in non-small cell lung cancer: a systematic review

Background: CD44 is a potentially interesting prognostic marker and therapeutic target in non-small cell lung cancer (NSCLC). Although the expression of CD44 has been reported to correlate with poor prognosis of NSCLC in most literatures, some controversies still exist. Since the limited patient numbers within independent studies, here we performed a meta-analysis to clarify the correlations between CD44 expression and prognosis and clinicopathological features in NSCLC. Methods: Relevant literatures were identified using PubMed, EMBASE and CNKI (China National Knowledge Infrastructure) databases (up to February 2014). Data from eligible studies were extracted and included into meta-analysis using a random effects model. Studies were pooled. Summary hazard ratios (HR) and clinical parameters were calculated. Results: We performed a final analysis of 1772 patients from 23 evaluable studies for Prognostic Value and 2167 patients from 28 evaluable studies for clinicopathological features. Our study shows that the pooled hazard ratio (HR) of overexpression CD44-V6 for overall survival in NSCLC was 1.63 [95% confidence interval (CI): 1.20-2.21] by univariate analysis and 1.29 (95% CI: 0.71-2.37) by multivariate analysis.The pooled HR of overexprssion panCD44 for overall survival in NSCLC was 1.53 (95% CI: 0.58-4.04) by univariate analysis and 3.00 (95% CI: 1.53-5.87) by multivariate analysis. Overexpression of CD44-V6 is associated with tumor differentiation (poor differentiation, OR = 1.66, 95% CI: 1.12-2.45), tumor histological type [squamous cell carcinomas (SCC), OR = 2.6, 95% CI: 1.63-5.02], clinical TMN stage (TMN stage III, OR = 2.22, 95% CI: 1.44-3.43) and lymph node metastasis (N1-3, 3.52, 95% CI: 2.08-5.93) in patients with NSCLC. However, there was no significant association between CD44-V6 and tumor size [T category, OR = 1.42, 95% CI: 0.73-2.78]. Conclusion: Our meta-analysis showed that CD44-V6 is an efficient prognostic factor for NSCLC. Overexpression of CD44-V6 was significantly associated with tumor differentiation, tumor histological type, clinical TMN stage and lymph node metastasis. However, there was no significant association between CD44-V6 and tumor size. Large prospective studies are now needed to confirm the clinical utility of CD44 as an independent prognostic marker.     

Motility of colon cancer cells: modulation by CD44 isoform expression

The invasion of cancer cells at primary tumor sites and their migration during metastatic spread require the expression of cell adhesion and motility proteins. Whether accelerated cell motility is necessary in these two processes is not universally accepted. In this study we took advantage that CD44, a cell adhesion protein, has different metastatic potentials depending on its splicing isoforms to examine how they affect cell motility. We established stable transfectants of standard and variant isoforms of CD44 in SW620 cells, a human colon carcinoma cell line that does not express CD44. The morphology of the cells varied according to the CD44 isoform expressed, but actin filament distribution remained unchanged. Using the wound assay in a two-dimensional in vitro cell motility system, we found that the expression of standard CD44 increases cell motility, whereas CD44 isoforms containing an exon sequence associated with metastatic dissemination has a slowing effect. Cell proliferation was also decreased by the expression of variant CD44 isoforms. Overall, colon cancer cells expressing variant CD44 isoforms had slower cell motility, possibly due to alterations in their cell adhesion properties. In conclusion, this study suggests that, contrary to common models, the metastatic phenotype is associated with a slow rate of cell migration when tested in vitro.     

Loss of CD28 expression on CD8(+) T cells is induced by IL-2 receptor gamma chain signalling cytokines and type I IFN, and increases susceptibility to activation-induced apoptosis

CD8(+)CD28(-) T cells are selectively expanded during viral infections, indicating their importance in anti-viral immune responses. Since little is known about the differentiation of CD8(+)CD28(-) cells, we investigated the generation, function and survival characteristics of this subset. In healthy individuals CD8(+)CD28(-) T cells contained more elevated levels of perforin and IFN-gamma than the CD8(+)CD28(+) subset, indicating that they can have an effector function. CD8(+)CD28(-) cells were selectively expanded when activated CD8(+)CD28(+) T cells were cultured in IL-2, IL-7 or IL-15. Moreover, the generation of CD8(+)CD28(-) cells was accelerated by type I IFN suggesting that these cytokines which are released during viral infections influence CD8(+) T cell differentiation. We did not observe re-expression of CD28 by CD8(+)CD28(-) T cells in any of the experiments performed. Activated T cells are susceptible to activation-induced cell death (AICD) if re-stimulated in the absence of co-stimuli. AICD was induced in both CD28(+) and CD28(-) subsets of activated T cells when stimulated with anti-CD3 antibody in the absence of co-stimuli but the magnitude of death was greater in the CD28(-) subset. While co-stimulation through LFA-1 (CD11a and CD18) significantly reduced AICD in the CD8(+)CD28(+) subset, death was not prevented in CD8(+)CD28(-) cells. These results suggest that CD8(+)CD28(-) T cells are more functionally differentiated than the CD8(+)CD28(+) subset and indicate they may represent a terminally differentiated effector population which is destined for clearance by apoptosis at the end of the immune response.     

Increased expression of FLIP,an inhibitor of Fas-mediated apoptosis,in stomach cancer

Despite the cell surface expression of Fas (Apo-1/CD95), many types of tumor cells, including stomach cancer cells, are resistant to Fas-mediated apoptosis, indicating the presence of inactivating mechanisms of Fas signaling. Expression of FLICE-like inhibitory protein (FLIP), one of the inhibitory proteins of Fas-mediated apoptosis, has been reported in several cancer types, but not in stomach cancer. In the present study, we analyzed the expression of Fas and FLIP in 60 advanced gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. Immunopositivity (defined as >/=30% of the neoplastic cells) was observed for Fas in 58 (97%) and FLIP in 54 (90%) of the 60 cancers. All of the tumors with FLIP immunostaining also showed Fas immunostaining. Loss of cell surface Fas immunostaining, another mechanism of Fas resistance, was observed in 45 tumors (75%). By contrast, normal gastric mucosal cells showed no or weak expression of both Fas and FLIP. Taken together, these results indicate that increased expression of FLIP is a frequent event in stomach carcinomas, and suggest that for evading apoptosis stomach carcinoma cells in vivo may need FLIP expression, which might contribute to tumor development.     

CD44v6 expression in patients with stage II or stage III sporadic colorectal cancer is superior to CD44 expression for predicting progression

Background: Currently, it is difficult to predict the prognosis of patients exhibiting stage II or stage III colorectal cancer (CRC) and to identify those patients most likely to benefit from aggressive treatment. The current study was performed to examine the clinicopathological significance of CD44 and CD44v6 protein expression in these patients. Study design: We retrospectively investigated 187 consecutive patients who underwent surgery with curative intent for stage II to III CRC from 2007 to 2013 in the Beijing Civil Aviation Hospital. CD44 and CD44v6 protein expression levels were determined using immunohistochemistry and compared to the clinicopathological data. Results: Using immunohistochemical detection, CD44 expression was observed in 108 (57.75%) of the CRC patients; and its detection was significantly associated with greater invasion depth, lymph node metastasis, angiolymphatic invasion, and a more advanced pathological tumor-lymph node-metastasis (TNM) stage. CD44v6 expression was observed in 135 (72.19%) of the CRC patients; and its expression was significantly associated with a poorly differentiated histology, greater invasion depth, lymph node metastasis, angiolymphatic invasion, and a more advanced pathological TNM stage. Expression of CD44v6 was higher than that of CD44 in stage II and stage III sporadic CRC. Conclusion: CD44v6 is a more useful marker for predicting a poor prognosis in stage II and stage III sporadic CRC as compared to CD44.     

Peripheral T lymphocyte depletion by apoptosis after CD4 ligation in vivo: selective loss of CD44- and 'activating' memory T cells.

We have demonstrated that a single intravenous bolus of rat anti-CD4 MoAb caused a small but prolonged increase in apoptosis in murine lymph nodes. We have quantified this process using the novel Highly Optimized Microscope Environment (HOME) interactive images analysis system and shown that the increase in apoptosis was sufficient to account for the observed depletion of the peripheral CD4+ T cell subset. This occurred in the absence of any other exogenous signal. Furthermore, there was no evidence of an inflammatory or necrotic response in the tissues, indicating that this was unlikely to be Fc or complement-mediated antibody killing. The anti-CD4-induced depletion selectively removed CD44- T cells. Using mice previously immunized with yeast-derived HIV-1 p24 recombinant protein there was sparing of memory T cell function after in vivo anti-CD4 treatment, except during a window of less than 24 h duration, when simultaneous exposure to antigen and anti-CD4 antibody resulted in the depletion of specific memory T lymphocyte function. This indicated that a very minor alteration in the frequency of apoptosis had a marked effect on cell number over time, and suggested that opportunistic infection associated with CD4+ T cell depletion may be explained by loss of memory cells when there is antigenic stimulation at the same time as CD4 ligation. These results have implications for the pathology of HIV-associated disease which is associated with ligation of CD4 molecules in vivo.     

小细胞和非小细胞肺癌晚期患者CD3~+ CD4~+及CD3~+ CD8~+T淋巴细胞亚群的差异

目的:探索小细胞和非小细胞肺癌晚期患者CD3+CD4+及CD3+CD8+T淋巴细胞亚群是否存在差异,并为治疗提供参考。方法:选取肺癌晚期患者共65例,其中包括小细胞肺癌14例,非小细胞肺癌51例以及20例健康对照。用流式细胞仪检测研究对象外周血淋巴细胞表面CD3+CD4+及CD3+CD8+的表达情况。结果:CD3+CD4+T细胞所占比例无论是小细胞还是非小细胞肺癌晚期的患者都较健康对照显著降低;CD3+CD8+T细胞所占比例在肺癌晚期的患者较健康对照并无显著变化;CD4+/CD8+比值在小细胞肺癌晚期患者较健康对照显著下降。结论:无论是小细胞还是非小细胞肺癌晚期的患者CD3+CD4+T细胞的水平较健康人都显著降低,说明肺癌晚期患者细胞免疫功能严重受损。     

CD44反义寡核苷酸对肿瘤细胞表面抗原和对CD3AK杀伤敏感性的调节作用

目的：观察CD44反义寡核苷酸调节人低分化黏液腺胃癌MCC80-3细胞Fas分子和凋亡抵抗基因bcl-2的表达水平，提高免疫效应细胞杀伤敏感性的作用和机制。方法：RT-PCR法检测CD44、bcl-2mRNA的表达水平。MTT法检测CD3AK杀伤活性。流式细胞术检测细胞表面CD44及Fas、FasL蛋白表达水平。结果：CD44反义寡核苷酸（1.6μmol／L）处理后，明显地抑制MGC80-3细胞CD44mRNA和蛋白表达水平，在CD44配体低分子量透明质酸（HA）存在下，使MCC80-3表面下调的Fas分子显著增高，表达率从6．69％提高到16．81％（P〈0．01）。CD3AK对反义寡核苷酸处理的MGC80-3细胞杀伤活性显著增高，并呈效靶比依赖效应（P〈0．01）。MGC80-3细胞bcl-2mRNA的相对表达定量值由1．06降至0．32。结论：CD44反义寡核苷酸可通过抑制MGC80-3细胞CD44mRNA和蛋白表达，上调Fas分子的表达，下调凋亡抵抗基因bcl-2的表达，并提高其对免疫效应细胞的杀伤敏感性。     

硅对动脉硬化家兔外周血白细胞凋亡及CD44表达的影响

为探讨Na2SiO3对动脉粥样硬化模型动物外周血白细胞凋亡和CD44表达的影响,将家兔分四组正常对照组、动脉硬化模型组、硅剂Ⅰ组(造模9周后以饮水给予Na2SiO3(2mg/kg)溶液)、硅剂Ⅱ组(造模同时即给予Na2SiO3溶液).17周后测血脂和血中白细胞凋亡率及CD44表达量,观察主动脉形态变化.结果发现,①模型组白细胞凋亡率和CD44表达量均显著高于正常组( P＜0.01),②服用Na2SiO3溶液可显著降低白细胞凋亡率和CD44表达量(P＜0.01).提示动脉粥样硬化发生时,伴有白细胞凋亡率和CD44表达量增加,Na2SiO3能降低这两个指标,可能是Na2SiO3抗动脉粥样硬化的机理之一.     

CD44 promotes resistance to apoptosis in human colon cancer cells

Overexpression of CD44, especially its variant isoforms, occurs consistently in colon cancer, as compared to autologous normal colon, and this change occurs also in most other types of cancer. One of the basic features of malignant transformation is the acquisition of resistance to apoptosis. In this study, we asked whether the expression of CD44 and some of its variant isoforms commonly found in colon cancer participate in resistance to apoptosis and what are the mechanisms involved. A human colon cancer cell line, SW620, which does not express CD44 was stably transfected with standard, v3-10, and v8-10 containing isoforms of CD44. Mock-transfected and CD44-transfected cells were exposed to etoposide to induce apoptosis. Apoptotic and concomitant changes relevant to the mechanisms of apoptosis were monitored by flow cytometry, DNA fragmentation, and immunoblot analyses. It was observed that resistance to apoptosis induced by etoposide is promoted by CD44 expression in SW620, and this resistance is better sustained by the full variant isoform, v3-10. Concomitant alterations in caspase 9, caspase 3, Bcl-xl, and Bak indicated that the resistance to apoptosis in this model involved the mitochondrial pathway. The differential response of CD44 transfectants was associated with a downregulation of pRb and phosphorylated AKT. The results of this study are consistent with the conclusion that expression of variant CD44 isoforms which is characteristic of colon cancer, and most other types of cancer, confers a selective advantage to resist apoptosis, thereby promoting cell transformation into a malignant phenotype, in conjunction with other anti-apoptotic factors.     

非小细胞肺癌中CD44v6的表达

目的　探讨CD4 4v6表达与非小细胞肺癌临床病理特征之间的关系。方法　采用免疫组化S P法检测 132例非小细胞肺癌、6 6例淋巴结转移癌和 115例正常肺组织CD4 4v6表达的情况。结果　非小细胞肺癌CD4 4v6阳性表达率为4 8 4 8% ,高于正常肺组织 17 39%的阳性表达率。CD4 4v6阳性表达与淋巴结转移状况、TNM分期、肿瘤大小和组织学类型有相关性。非小细胞肺癌淋巴结转移组、TNMⅢ期患者、直径 >3cm的肿瘤和鳞癌CD4 4v6阳性表达率分别高于无淋巴结转移组、TNMⅠ期和Ⅱ期患者、直径≤ 3cm的肿瘤和腺癌。淋巴结转移癌CD4 4v6阳性表达率高于肺原发癌。CD4 4v6阳性表达与患者的性别、年龄和组织学分级无相关性。结论　CD4 4v6阳性表达预示非小细胞肺癌具有较强的侵袭和转移能力 ,可作为一项预测非小细胞肺癌转移潜能生物学指标。     

肿瘤干细胞相关标记物CD44及CD24在乳腺癌中的表达及其意义

目的探讨肿瘤干细胞相关标记物CD44及CD24在乳腺癌组织中的表达特点、CD44+/CD24-细胞与HER-2、ER、PR、CK5/6表达的相互关系及其与临床病理因素的关系。方法采用免疫组化SP单染及双染法检测42例乳腺导管原位癌及126例乳腺浸润性导管癌组织中CD44及CD24的表达情况,检测126例乳腺浸润性导管癌组织中HER-2、ER、PR、CK5/6表达状况以进行免疫分型。结果 (1)CD44阳性定位于癌细胞膜。在浸润癌中阳性率为56.3%,在导管原位癌中阳性率为85.7%。两者差异有显著性意义(P<0.05)。在不同分化程度的乳腺浸润性癌中,CD44阳性率分别为69.2%、58.1%及44.7%,差异有显著性意义(P<0.05)。(2)CD24阳性表达于非癌性乳腺组织中小管的腔缘;在癌组织中除腔缘阳性外,可出现膜质阳性。在浸润癌中阳性率为32.5%,在导管原位癌中阳性率为64.3%。两者差异有显著性意义(P<0.05)。(3)126例浸润性导管癌中CD44+/CD24-者65例,占51.6%;CD44+/CD24-阳性细胞在Luminal A型为47.5%、Luminal B型为42.9%、HER-...     

CD4 engagement induces Fas antigen-dependent apoptosis of T cells in vivo

CD4 is a T lymphocyte receptor for major histocompatibility complex class II antigens. It is referred to as coreceptor because it synergizes with the T cell receptor for antigen when both receptors become engaged simultaneously. We show here in mice that when engaged by antibody independently of the T cell antigen receptor, CD4 induces T cells to undergo apoptosis. Several features of this process were identified. The expression of an intact Fas protein is a requirement for CD4-mediated T cell death. Mice homozygous for the lpr mutation which are defective in the expression of Fas and in their ability to delete lymphocytes apoptotically fail to delete anti-CD4-reactive T cells. Sessile anti-CD4-reactive T cells leave their homing environment in lymphoid organs and modulate their cell surface molecules, e.g. CD2, CD3, CD4. A massive influx of lymphoid cells with null-cell phenotype occurs in the blood where they begin to reexpress cell surface markers. With their arrival in the circulation, anti-CD4-reactive T cells develop features of DNA degradation typical of apoptosis. More than one third of the circulating lymphoid cells show apoptotic features 7–8 h after anti-CD4 injection. Their frequency declines subsequently presumably due to their physical disintegration via shedding of apoptotic bodies and phagocytosis. Our data show that when not obliged to the activation process by the antigen receptor, CD4 can mediate deletion signals. Thus, besides functioning as coreceptor with the antigen receptor, CD4 has a function of its own in facilitating the induction of apoptosis.     

甲状腺髓样癌中CD133和CD44的表达及意义

目的 探讨肿瘤干细胞(cancer stem cells,CSC)标志物CD133及CD44在甲状腺髓样癌(medullary thyroid cancer,MTC)组织中的表达及临床病理意义.方法 采用免疫组化SP法检测51例MTC组织中CD133和CD44的表达.结果 (1)CD133蛋白在MTC伴有包膜浸润组的阳性率(76.2％,16/21)明显高于无包膜浸润组(43.3％,13/30);CD133蛋白在MTC病灶直径≥1 cm组的阳性率(64.4％,29/45)显著高于病灶直径＜1 cm组(0,0/6),差异均有统计学意义(P均＜0.05).(2)CD44蛋白在MTC伴有包膜浸润组的阳性率(66.7％,14/21)明显高于无包膜浸润组(20％,6/30),差异有统计学意义(P＜0.01).(3) MTC中CD133和CD44蛋白的表达与患者年龄、性别、是否散发、甲状腺受累范围、淋巴结转移、远处脏器转移、TNM分期均无关(P＞0.05).(4)CD133与CD44共表达与MTC包膜浸润密切相关(P＜0.01).结论 MTC中存在CSC,其与肿瘤的浸润生长及恶性增殖相关,可考虑将CD133及CD44作为MTC临床评价肿瘤生物学行为的指标.二者可能存在互相调节的机制,共表达在MTC的恶性进展中发挥作用.