NDUFAF4激活Wnt/β-catenin通路增加肝癌辐射抵抗性机制研究

目的 研究泛醌氧化还原酶复合物组装因子4（NDUFAF4）的表达与肝癌患者临床预后的相关性,探究NDUFAF4对人肝癌细胞系的辐射敏感性的影响及其机制。方法 通过数据库及肝癌组织样本探究NDUFAF4在肝癌中的表达及其表达水平与临床预后的相关性。通过脂质体将敲减NDUFAF4基因的质粒si-NDUFAF4转入HepG2和Huh7细胞,克隆形成实验检测辐射敏感性。同时通过裸鼠开展荷瘤实验,免疫荧光法检测β联蛋白（β-catenin）,蛋白质印迹法检测上皮钙黏素（E-cadherin）和神经钙黏素（N-cadherin）的蛋白表达。结果 生物信息学分析结果证实,NDUFAF4在肝癌组织中显著高表达,其表达水平越高,患者预后越差（P<0.05）。NDUFAF4在肝癌组织中的表达水平显著高于癌旁组织;克隆形成实验证实,敲减NDUFAF4显著降低肝癌细胞的生存率（P<0.01）;体内实验证实,敲减NDUFAF4可以减慢癌细胞增殖,减少β-catenin及Ki-67的表达。敲减NDUFAF4显著减少肝癌细胞系核内β-catenin的蛋白水平,提示NDUFAF4可以激活Wnt/β-catenin通路。敲减NDUFAF4显著上调E-cadherin和下调N-cadherin的蛋白表达水平。结论 敲减NDUFAF4可以通过抑制Wnt/β-catenin通路增强人肝癌细胞系的辐射敏感性,并且与临床预后紧密相关,或可为克服肝癌的辐射抗性提供新的靶点。     

miR-148b在肺腺癌中的表达及其功能分析

目的 探讨微小RNA-148b（miR-148b）在肺腺癌组织和细胞株中的表达及临床意义。方法 采用实时定量PCR检测13例肺腺癌及癌旁组织中miR-148b的表达,并检测其在肺腺癌细胞株H1437、H1975、A549、PC14/B和正常人肺上皮细胞株HBE中的表达,选取肺腺癌细胞株miR-148b表达水平最低者分别转染miR-148b模拟物（mimics）和miR-148b control。通过MTT检测、低密度细胞集落形成实验、Transwell实验检测miR-148b对细胞增殖、集落形成和侵袭能力的影响。结果 miR-148b在肺腺癌和癌旁组织中的相对表达量分别为0.61±0.42和0.91±0.32（P＜0.05）;与正常肺上皮细胞株HBE（相对表达量设为1.00）比较,4种肺腺癌细胞株H1437、H1975、A549、PC14/B中miR-148b的表达量分别为0.42±0.08、0.38±0.02、0.29±0.03和0.21±0.04（P＜0.05）,选取PC14/B细胞株进行后续实验。MTT检测显示,实验第3天,转染miR-148b mimics的PC14/B细胞吸光值明显低于转染miR-148b control组（P＜0.05）;低密度细胞集落形成实验中,与转染了miR-148b control的PC14/B细胞（相对克隆数设为100）相比,转染miR-148b mimics的PC14/B细胞的相对克隆数为47±8,差异有统计学意义（P＜0.05）;Transwell实验显示,转染miR-148b mimics后穿过基底膜的相对PC14/B细胞数为82±22,与miR-148b control组（200±34）相比,差异有统计学意义（P＜0.05）。结论 miR-148b在肺腺癌组织和细胞中表达下调,且可抑制肺腺癌PC14/B细胞株的增殖、集落形成和侵袭能力。     

放射性核素骨显像在建立人肺腺癌骨转移细胞株中的应用

目的:探讨采用放射性核素骨显像技术建立人肺腺癌骨转移细胞株SIC-A-1BM以及在免疫缺陷BNX小鼠的转移动物模型活体成像中的作用.方法:将人肺腺癌细胞株SIC-A-1注射入小鼠左心室,形成骨转移,在放射性核素骨显像剂示踪下寻找到骨转移灶,然后切除病变组织进行体外培养以获得转移性肺腺癌细胞.利用获得的第1代肺腺癌骨转移细胞,经血液(动、静脉系统)和肺原位途径进行种植,按上述步骤重复多个循环.结果:染色体分析结果确定亲代SPC-A-1细胞经小鼠体内-体外连续筛选,通过放射性核素骨显像后显示,获得的肺腺癌骨转移细胞株(SIC-A-1BM)未改变人源细胞属性.99mTcMDP和X射线的放射剂量对SPC-A-1BM细胞生长影响的结果表明,111 MBq的99mTc-MDP对人肺腺癌骨转移细胞生长的影响类似于且略小于40 kV、2 mA、4 s的X射线.放射性核素骨显像与X线摄片检测出的小鼠骨转移的敏感度、特异度和准确度分别为97.6%和31.3%、73.3%和100%以及94%和43%.结论:放射性核素骨显像技术为建立人肺腺癌骨转移细胞株SIC-A-1BM及其免疫缺陷BNX小鼠转移动物模型活体成像提供了一种实用且便捷的实验手段.     

肺腺癌恶性胸水中外泌体来源双链DNA的鉴定及其在EGFR突变检测中的意义

目的 探索肺腺癌恶性胸水中外泌体来源DNA(exoDNA)用于表皮生长因子受体(EGFR)突变检测的可行性。方法 采用沉淀法收集胸水外泌体,透射电镜和Western blot对外泌体进行鉴定;同时对exoDNA的稳定性和物理特征进行鉴定。收集28例初诊肺腺癌患者的胸水及对应癌组织,采用扩增阻滞突变系统方法对胸水脱落癌细胞、exoDNA及对应癌组织的EGFR基因突变进行检测,比较外泌体用于EGFR基因突变检测的敏感性、特异性和一致性。结果 电镜证实提取的外泌体符合外泌体的大小特征;Western blot印证了外泌体富含的特征蛋白CD63。exoDNA含有大片段DNA且纯度符合要求、适合EGFR突变分析;在肺腺癌初治患者的恶性胸水exoDNA中含有EGFR突变,与配对的组织和胸水脱落癌细胞相比,敏感性和特异性为100%和92.9%,符合率为96.4%(Kappa=0.929,P<0.001)。结论 肺腺癌胸水exoDNA质量好,稳定性高,是EGFR基因突变检测的另一全新的、重要的DNA来源。     

Bcl-2和整合素α3蛋白在非小细胞肺癌中的表达及临床意义

目的：研究Bcl-2和整合素α3蛋白在非小细胞肺瘟组织中的阳性及其对肿瘤生物学行为和患者预后的影响。方法：应用免疫组化方法检测269例根治术后非小细胞肺癌组织中Bcl-2和整合素α蛋白表达水平，SPSS10．0统计软件包进行数据分析。结果：在269例非小细胞肺癌中，Bcl-2蛋白阳性153制．占56.9％；整合素α3蛋白阳性127例．占472％．Bcl-2和整合索α3均阳性94例．占34.9％。Bcl-2蛋白在女性非小细胞肺癌组织内阳性率显著高于男眭患者．而且与肿瘤的大小呈负相关。整合素α3蛋白在女性和非鳞癌组织内阳性率显著高于男性和肺鳞癌、COX模型多因素分析显示．Bcl-2蛋白阳性是Ⅱ期非小细胞肺癌独立的预后不良因素(五年生存率39．3％对该蛋白阴性者为52．1%,P=0.004)；Bcl-2和整合素α3蛋白均阳性是阳期非小细胞肺癌独立的预后良好因素(五年生存率34.5％对两蛋白其他结果组合为23．8％P=0．02)。结论：Bcl-2和整合素α3蛋白的阳性影响非小细胞肺癌的生物学行为．检测两种蛋白的阳性情况对患者预后有一定预测价值。     

αvβ 3整合素在小细胞肺癌骨转移细胞株SBC-5中的表达及意义

目的:分析αvβ3整合素在特异性小细胞肺癌骨转移细胞株中的表达,探讨肺癌骨转移的机制.方法:RT-PCR、Western-blot和免疫荧光染色检测小细胞肺癌特异性骨转移细胞株SBC-5和非骨转移细胞株SBC-3中αvβ3整合素的表达差异.结果:αvβ3整合素在SBC-5细胞株中的表达显著高于SBC-3细胞株.结论:αvβ3整合素可能与小细胞肺癌骨转移的发生相关,可能促进了小细胞肺癌骨转移的发生.     

驱动基因阳性肺腺癌合并骨转移患者的临床特征及预后分析

目的探讨驱动基因阳性肺腺癌合并骨转移患者的临床特征以及抗肿瘤治疗后骨转移病灶变化情况对预后的影响。方法根据纳入、排除标准获得56例于2017年8月至2023年3月在上海交通大学医学院附属第六人民医院就诊的驱动基因阳性肺腺癌合并骨转移患者的资料, 分析其临床、病理及影像学特征, 并采用Kaplan-Meier法分析出现成骨型病灶增大或新发成骨型病灶对预后的影响。结果 56例患者中, 11例(20%)出现病理性骨折, 3例(5%)出现脊髓压迫, 10例(18%)接受骨转移放疗, 9例(16%)接受骨转移手术治疗, 上述患者均为溶骨型或混合型骨转移患者。所有人群的中位无进展生存(PFS)期为13.6个月(95%CI为10.3～16.9个月)。20%(11/56)患者出现成骨型病灶增大或新发成骨型病灶, 上述患者PFS为37.7个月(95%CI为5.4～70.0个月), 无成骨型病灶增大或新发成骨型病灶的患者中位PFS为13.1个月(95%CI为9.3～16.9个月)(χ2=4.141, P=0.0418)。结论驱动基因阳性肺腺癌靶向治疗后出现成骨型病灶增大或新发成骨型病灶患者较未出现上述变化...     

αvβ_3整合素在小细胞肺癌骨转移细胞株SBC-5中的表达及意义

目的：分析αvβ3整合素在特异性小细胞肺癌骨转移细胞株中的表达,探讨肺癌骨转移的机制。方法：RT-PCR、Western-blot和免疫荧光染色检测小细胞肺癌特异性骨转移细胞株SBC-5和非骨转移细胞株SBC-3中αvβ3整合素的表达差异。结果：αvβ3整合素在SBC-5细胞株中的表达显著高于SBC-3细胞株。结论：αvβ3整合素可能与小细胞肺癌骨转移的发生相关,可能促进了小细胞肺癌骨转移的发生。     

肺腺癌新分类在预测Ⅰb期非小细胞肺癌预后中的价值

[目的]探讨新的肺腺癌分类在判断早期可手术的Ⅰb期非小细胞肺腺癌患者中的预后价值。[方法]回顾性分析168例非小细胞肺癌患者,根据第7版TNM分期明确诊断为Ⅰb期非小细胞肺腺癌。采用Kaplan-Meier法比较不同病理亚型患者的无复发生存期和总生存期。Cox回归分析探讨影响患者无复发生存期和总生存期的影响因素。[结果]168例患者中,无原位腺癌患者,2例患者为微浸润腺癌,51例患者为乳头状为主型,55例患者为腺泡样为主型,24例为微乳头为主型,20例为鳞屑状为主型,12例为实性为主型腺癌,4例为变异性腺癌（其中2例为实性腺癌,2例为胎儿型腺癌）。单因素分析显示,是否淋巴管,血管侵犯（P=0．042）和不同病理类型（P=0．004）是影响患者5年无复发生存的因素。多因素分析显示。病理类型是同时影响患者5年无复发生存和总生存期的惟一因素（P分别为0．002和0．035）。[结论]新肺腺癌分类在预测可手术的Ⅰb期非小细胞肺腺癌的无复发生存和总生存方面有一定的价值。     

hsa-miR-23a-3p在肺腺癌组织中的表达及意义

[目的]探讨hsa-miR-23a-3p在肺腺癌织中的表达水平及临床意义。[方法]收集我院呼吸科就诊的42例肺腺癌患者的癌组织及癌旁组织样本,同时选取37例非肺腺癌患者的肺穿刺样本为对照组。采用荧光定量PCR法检测各组患者肺组织中hsa-miR-23a-3p相对表达水平,分析hsa-miR-23a-3p在肺腺癌组织中的表达及与患者临床特征的关系;以患者血清癌胚抗原(CEA)指标为参考,利用受试者工作特征曲线(ROC)评估肺腺癌患者癌组织中hsa-miR-23a-3的表达临床应用价值。[结果]对照组与癌旁组织中hsa-miR-23a-3p的表达水平差异无统计学意义(4.32±0.34 vs 4.16±0.41,P>0.05),与对照组与癌旁组织比,肺腺癌组织hsa-miR-23a-3p表达较高(6.08±0.47 vs 4.32±0.34,6.08±0.47 vs 4.16±0.41,P<0.01)。不同性别(6.16±0.82 vs 5.89±0.73)、分化程度(6.20±0.84 vs 5.97±0.67)癌组织的hsa-miR-23a-3p表达差异无统计学意义(P>0.05),TNM分期越高(5.13±0.49 vs 6.26±0.51)、肿瘤越大(6.47±0.65 vs 5.94±0.71)、淋巴结转移(6.32±0.53 vs 5.83±0.46)及CEA阳性(6.47±0.44 vs 5.79±0.41)癌组织的hsa-miR-23a-3p表达显著性增高(P<0.001)。ROC曲线分析显示,与CEA比,hsa-miR-23a-3p的曲线下面积高(0.779vs 0.683)。[结论]肺腺癌组织hsa-miR-23a-3p呈高表达,与淋巴结转移、肿瘤大小及TNM分期相关,可用于辅助诊断肺腺癌。     

Chromobox 4 facilitates tumorigenesis of lung adenocarcinoma through the Wnt/β-catenin pathway

Chromobox 4 (CBX4) is a core component of polycomb-repressive complex 1 with important roles in cancer biology and tissue homeostasis. Aberrant expression of CBX4 has been implicated in several human malignancies. However, its role and underlying mechanisms in the tumorigenesis of lung adenocarcinoma (LUAD) have not been defined in vivo. Here, we found that expression of CBX4 was frequently up-regulated in human LUAD samples and correlated with poor patient survival. Importantly, genetic ablation of CBX4 greatly dampened lung tumor formation and improved survival in the Kras^G12D/P53^L/L (KP) autochthonous mouse model of LUAD. In addition, CBX4 depletion significantly inhibited proliferation and anchorage-independent growth of KP mouse embryonic fibroblasts. Moreover, ectopic CBX4 expression clearly promoted proliferation and anchorage-independent growth in both human and mouse LUAD cells, whereas silencing of CBX4 exerted opposite effects. Mechanistically, CBX4 promoted growth of LUAD cells through activation of the Wnt/β-catenin pathway. Furthermore, expression levels of CBX4 were positively correlated with β-catenin in human LUAD samples. In conclusion, our data suggest that CBX4 plays an oncogenic role via the Wnt/β-catenin pathway and could serve as a potential therapeutic target in LUAD.     

CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

Cadherin 6 (CDH6) is significantly overexpressed in advanced ovarian and renal cancers. However, the role of CDH6 in cancer metastasis is largely unclear. Here, we investigated the impact of CDH6 expression on integrin‐mediated metastatic progression. CDH6 preferentially bound to αIIbβ3 integrin, a platelet receptor scarcely expressed in cancer cells, and this interaction was mediated through the cadherin Arginine–glycine–aspartic acid (RGD) motif. Furthermore, CDH6 and CDH17 were found to interact with α2β1 in αIIbβ3^low cells. Transient silencing of CDH6, ITGA2B, or ITGB3 genes caused a significant loss of proliferation, adhesion, invasion, and lung colonization through the downregulation of SRC, FAK, AKT, and ERK signaling. In ovarian and renal cancer cells, integrin αIIbβ3 activation appears to be a prerequisite for proper α2β1 activation. Interaction of αIIbβ3 with CDH6, and subsequent αIIbβ3 activation, promoted activation of α2β1 and cell adhesion in ovarian and renal cancer cells. Additionally, monoclonal antibodies specific to the cadherin RGD motif and clinically approved αIIbβ3 inhibitors could block pro‐metastatic activity in ovarian and renal tumors. In summary, the interaction between CDH6 and αIIbβ3 regulates α2β1‐mediated adhesion and invasion of ovarian and renal cancer metastatic cells and constitutes a therapeutic target of broad potential for treating metastatic progression.     

LncRNA MEG3 inhibits retinoblastoma invasion and metastasis by inducing β-catenin degradation

In our previous study, we found that low expression of LncRNA-MEG3 was closely associated with the invasion and metastasis of retinoblastomas. The molecular mechanism by which MEG3 inactivation induces the invasion and metastasis of retinoblastoma cell lines remains unclear. We used the GEO database to analyze the expression of MEG3 in retinoblastoma tissues and MEG3-related pathways. The scratch, transwell migration, mouse tumor metastasis, and mouse fluorescence live imaging assays were performed to detect migration and invasion of retinoblastoma cell lines. The RNA pull down, electrophoretic mobility shift, RIP, co-immunoprecipitation, and ubiquitination assays were performed to analyze the molecular mechanisms. The GEO database showed that the expression of MEG3 was low in retinoblastoma tissues and was closely associated with the invasion of retinoblastoma cells and activity of the Wnt pathway. Both in vivo and in vitro experiments confirmed that MEG3 inhibited the migration and invasion of retinoblastoma cells. Cell experiments confirmed that MEG3 could promote the binding of β-catenin and GSK-3β and induce phosphorylation, ubiquitination and degradation of β-catenin indirectly. In conclusion, MEG3 can promote the degradation of β-catenin via GSK-3β, which in turn inactivates the Wnt pathway and ultimately inhibits the invasion and metastasis of retinoblastoma cells.     

Constitutive activation of integrin αvβ3 contributes to anoikis resistance of ovarian cancer cells

Epithelial ovarian cancer involves the shedding of single tumor cells or spheroids from the primary tumor into ascites, followed by their survival, and transit to the sites of metastatic colonization within the peritoneal cavity. During their flotation, anchorage‐dependent epithelial‐type tumor cells gain anoikis resistance, implicating integrins, including αvß3. In this study, we explored anoikis escape, cisplatin resistance, and prosurvival signaling as a function of the αvß3 transmembrane conformational activation state in cells suspended in ascites. A high‐affinity and constitutively signaling‐competent αvß3 variant, which harbored unclasped transmembrane domains, was found to confer delayed anoikis onset, enhanced cisplatin resistance, and reduced cell proliferation in ascites or 3D‐hydrogels, involving p27^kip upregulation. Moreover, it promoted EGF‐R expression and activation, prosurvival signaling, implicating FAK, src, and PKB/Akt. This led to the induction of the anti‐apoptotic factors Bcl‐2 and survivin suppressing caspase activation, compared to a signaling‐incapable αvß3 variant displaying firmly associated transmembrane domains. Dissecting the mechanistic players for αvß3‐dependent survival and peritoneal metastasis of ascitic ovarian cancer spheroids is of paramount importance to target their anchorage independence by reversing anoikis resistance and blocking αvß3‐triggered prosurvival signaling.

Abbreviations

CLSM

confocal laser scanning microscopy

ECM

extracellular matrix

EGF‐R

epidermal growth factor receptor

EOC

epithelial ovarian cancer

FAK

focal adhesion kinase

FIGO

Fédération Internationale de Gynécologie et d''Obstétrique

GAPDH

glyceraldehyde 3‐phosphate dehydrogenase)

GpA

glycophorin A

IMD

integrin‐mediated death

MAPK

mitogen‐activated protein kinases

PI

propidium iodide

RGD

Arg‐Gly‐Asp

TMD

transmembrane domain

     

Bone sialoprotein‐αvβ3 integrin axis promotes breast cancer metastasis to the bone

The underlying mechanisms of breast cancer cells metastasizing to distant sites are complex and multifactorial. Bone sialoprotein (BSP) and αvβ3 integrin were reported to promote the metastatic progress of breast cancer cells, particularly metastasis to bone. Most theories presume that BSP promotes breast cancer metastasis by binding to αvβ3 integrin. Interestingly, we found the αvβ3 integrin decreased in BSP silenced cells (BSPi), which have weak ability to form bone metastases. However, the relevance of their expression in primary tumor and the way they participate in metastasis are not clear. In this study, we evaluated the relationship between BSP, αvβ3 integrin levels, and the bone metastatic ability of breast cancer cells in patient tissues, and the data indicated that the αvβ3 integrin level is closely correlated to BSP level and metastatic potential. Overexpression of αvβ3 integrin in cancer cells could reverse the effect of BSPi in vitro and promote bone metastasis in a mouse model, whereas knockdown of αvβ3 integrin have effects just like BSPi. Moreover, The Cancer Genome Atlas data and RT‐PCR analysis have also shown that SPP1, KCNK2, and PTK2B might be involved in this process. Thus, we propose that αvβ3 integrin is one of the downstream factors regulated by BSP in the breast cancer‐bone metastatic cascade.     

Modified heparins inhibit integrin αIIbβ3 mediated adhesion of melanoma cells to platelets in vitro and in vivo

The adhesion of tumor cells with platelets is important in the process of tumor metastasis. A huge work has indicated that anti‐adhesion is an effective strategy for metastasis inhibition. In this article, we assess the role of platelet integrin α_IIbβ₃ in adhesion of melanoma cells to platelets and the effects of heparin and modified heparins on the adhesion in vitro and in vivo. We show that platelet integrin α_IIbβ₃ is involved in the interaction of human melanoma A375 cells with platelets, and the high affinity epitope resides on the α_IIb subunit rather than β₃ subunit. Heparin sulfate‐like proteoglycans on tumor cell surface are implicated in the adhesion of A375 cells to integrin α_IIbβ₃. We also show that RO‐heparin, CR‐heparin, N‐2,3‐DS‐heparin and 2,3‐O‐DS‐heparin can significantly inhibit A375 cells binding to the CHO cells expressing integrin α_IIbβ₃ under static and flow conditions, and remarkably inhibit the adhesion of A375 cells to the immobilized platelet layers under flow conditions. We find that A375 cells and B16F10 cells are arrested in the pulmonary vessels and adhered to platelets, and the initial interaction of tumor cells with platelets in lung vessel and long‐term establishment of metastatic foci can be inhibited by heparin as well as CR‐heparin and N‐2,3‐DS‐heparin. These data suggest that modified heparins can inhibit tumor cell‐platelet interaction mediated by platelet integrin α_IIbβ₃ and modified heparins may be a potential substitute for heparin in inhibiting tumor metastasis. © 2009 UICC     

Targeting cancer stem cells via integrin β4

Integrins mediate cell-cell interactions and communication with the extracellular matrix (ECM). These transmembrane protein receptors allow binding between a cell and its surroundings, initiating a breadth of intracellular signaling resulting in proliferation, differentiation, survival, or migration. Such responses have made integrins an attractive target for cancer therapy. Self-renewing and highly tumorigenic cancer stem cells (CSCs) are most resistant to traditional radiation treatment and chemotherapy, and therefore may contribute directly to the metastasis and relapse of the disease. In both the 4T1 mouse metastatic mammary tumor model and SCC7 head and neck squamous cell carcinoma model, integrin β4 (ITGB4) was expressed on ALDH^high 4T1 and SCC7 CSCs. Using two immunological approaches, we targeted ITGB4 through 1) ITGB4 protein-pulsed dendritic cell (ITGB4-DC) vaccination or 2) via anti-CD3/anit-ITGB4 bispecific antibody (ITGB4 BiAb)-armed T cell adoptive transfer. These two therapies reduced ITGB4-expressing CSCs and inhibited local tumor growth and lung metastasis through ITGB4 specific cellular and humoral immune responses. Additionally, the combination of anti-PD-L1 immunotherapy with our two ITGB4-targeted approaches significantly improved treatment efficacy. We also found increased concentrations of serum IFN-γ and IL-6 in the 4T1 and SCC7 models which may help define future directions of this ITGB4-targeted study. Together, these results emphasize ITGB4 as a practical CSC immunological target with possible therapeutic benefits across tumor types with high ITGB4 expression.     

Perphenazine and prochlorperazine decrease glioblastoma U-87 MG cell migration and invasion: Analysis of the ABCB1 and ABCG2 transporters,E-cadherin, α-tubulin and integrins (α3, α5, and β1) levels

Glioblastoma multiforme is the most frequent type of malignant brain tumor, and is one of the most lethal and untreatable human tumors with a very poor survival rate. Therefore, novel and effective strategies of treatment are required. Integrins play a crucial role in the regulation of cellular adhesion and invasion. Integrins and α-tubulin are very important in cell migration, whereas E-cadherin plays a main role in tumor metastasis. Notably, drugs serve a crucial role in glioblastoma treatment; however, they have to penetrate the blood-brain barrier (BBB) to be effective. ABC transporters, including ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily G member 2 (ABCG2), are localized in the brain endothelial capillaries of the BBB, have a crucial role in the development of multidrug resistance and are modulated by phenothiazine derivatives. The impact of perphenazine and prochlorperazine on the motility of human Uppsala 87 malignant glioma (U87-MG) cells was evaluated using a wound-healing assay, cellular migration and invasion were assessed by Transwell assay, and the protein expression levels of ABCB1, ABCG2, E-cadherin, α-tubulin and integrins were determined by western blotting. The present study explored the effects of perphenazine and prochlorperazine on the levels of ABCB1, ABCG2, E-cadherin, α-tubulin and integrins (α3, α5, and β1), as well as on the migratory and invasive ability of U87-MG cells. The results suggested that perphenazine and prochlorperazine may modulate the expression levels of multidrug resistance proteins (they decreased ABCB1 and increased ABCG2 expression), E-cadherin, α-tubulin and integrins, and could impair the migration and invasion of U-87 MG cells. In conclusion, the decrease in migratory and invasive ability following treatment with phenothiazine derivatives due to the increase in ABCG2 and E-cadherin expression, and decrease in α-tubulin and integrins expression, may suggest that research on perphenazine and prochlorperazine in the treatment of glioblastoma is worth continuing.     

Mitochondrial malic enzyme 2 promotes breast cancer metastasis via stabilizing HIF-1α under hypoxia

Objectiveα-ketoglutarate (α-KG) is the substrate to hydroxylate collagen and hypoxia-inducible factor-1α (HIF-1α), which are important for cancer metastasis. Previous studies have shown that the upregulation of collagen prolyl 4-hydroxylase in breast cancer cells stabilizes the expression of HIF-1α by depleting α-KG levels. We hypothesized that mitochondrial malic enzyme 2 (ME2) might also affect HIF-1α expression via modulating α-KG levels in breast cancer cells.MethodsWe evaluated ME2 protein expression in 100 breast cancer patients using immunohistochemistry and correlated with clinicopathological indicators. The effect of ME2 knockout on cancer metastasis was evaluated using an orthotopic breast cancer model. The effect of ME2 knockout or knockdown on the levels of α-KG and HIF-1α proteins in breast cancer cell lines was determined both in vitro and in vivo. ResultsME2 was found to be upregulated in the human breast cancerous tissues compared with the matched precancerous tissues (P<0.001). The elevated expression of ME2 was associated with a poor prognosis (P=0.019). ME2 upregulation was also related to lymph node metastasis (P=0.016), pathological staging (P=0.033), and vascular cancer embolus (P=0.014). Also, ME2 knockout significantly inhibited lung metastasisin vivo. In the tumors formed by ME2 knockout cells, the levels of α-KG were significantly increased and collagen hydroxylation level did not change significantly but HIF-1α protein expression was significantly decreased, compared to the control samples. In cell culture, cells with ME2 knockout or knockdown demonstrated significantly higher α-KG levels but significantly lower HIF-1α protein expression than control cells under hypoxia. Exogenous malate and α-KG exerted similar effect on HIF-1α in breast cancer cells to ME2 knockout or knockdown. Additionally, treatment with malate significantly decreased 4T1 breast cancer lung metastasis. ME2 expression was associated with HIF-1α levels in human breast cancer samples (P=0.008). ConclusionsOur results provide evidence that upregulation of ME2 is associated with a poor prognosis of breast cancer patients and propose a mechanistic understanding of a link between ME2 and breast cancer metastasis.