小剂量阿糖胞苷诱导神经母细胞瘤细胞分化的研究

[目的]通过人神经母细胞瘤(NB)SMS-KCNR细胞的体外实验,观察小剂量阿糖胞苷(Ara-C)对NB细胞的诱导分化作用.[方法]MTS法检测小剂量Ara-C对SMS-KCNR细胞增殖的影响;观察小剂量Ara-C处理后SMS-KCNR细胞的形态变化;流式细胞仪检测小剂量Ara-C处理后SMS-KCNR细胞的细胞周期分布;Western blot检测TrkA、N-myc蛋白的表达,RT-PCR检测TrkA及MYCN mRNA水平的变化.[结果]小剂量Ara-C对SMS-KCNR细胞的增殖具有抑制作用,诱导SMS-KCNR细胞周期停滞于S期,G2/M期细胞比例下降(P＜0.05).经小剂量Ara-C处理后SMS-KCNR细胞形态与正常神经节细胞类似,并使TrkA的表达上调(P＜0.05),MYCN的表达下调(P＜0.05).[结论]小剂量Ara-C对SMS-KCNR细胞具有诱导分化作用,该作用可能与TrkA表达上调,MYCN表达下调有关.     

全反式维甲酸体外诱导神经母细胞瘤细胞分化研究

[目的]观察全反式维甲酸（all trans retinoic acid,ATRA)体外对人神经母细胞瘤细胞系SK-N-SH细胞生长的影响并对其作用机制进行初步探讨。[方法]SK-N-SH细胞经ATRA处理后，采用相关显微镜，神经纤维银染法，透射电镜观察，逆转录PCRmRNA检测等手段，观察ATRA对SK-N-SH细胞生长的影响。[结果]ATRA作用SK-N-SH细胞12天后，细胞形态发生显著变化。表现为明显分化；出现神经原性表型，多个细胞形成神经节，节间形成粗而长的类神经纤维；尼氏小体和银染法亦证明SK-N-SH细胞产生显著分化，透射电镜观察结果表明，ATRA对该细胞无明显凋亡诱导作用，ATRA对SH细胞的维甲酸受体-β（RARβ）表达水平有上调作用。[结论]ATRA对神经母细胞瘤细胞体外能产生明显诱导分化作用，此作用与ATRA上调细胞的RARβmRNA水平有关。     

Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.     

抗CD44抗体HI44a对白血病细胞分化与凋亡作用的体外研究

背景与目的探讨抗CD44抗体HI44a对新鲜白血病细胞分化及凋亡的作用。材料与方法从细胞形态学、四氮唑蓝(NBT)还原反应和细胞分化特异性抗原CD11b,CD14和CD15的变化,体外研究HI44a对31例急性髓系白血病患者白血病细胞的诱导分化作用。并利用Annexin-Ⅴ试剂盒检测HI44a对白血病细胞的凋亡诱导,RT-PCR方法检测其对细胞分化相关因子G-CSF,M-CSF及原癌基因c-myc表达的影响。结果经HI44a作用后,白血病细胞形态向成熟方向转变;M2~M5各亚型的NBT还原反应阳性率分别升高到31%(对照为9%)、55%(对照为10%)、25%(对照为12%)和32%(对照为11%),与对照组相比,差异均有统计学意义(P<0.01)。CD11b,CD14和CD15表达分别由对照组的9.65%、27.40%、57.38%升高到19.29%、40.60%和66.82%(P均<0.01)。细胞的早期凋亡率由对照组的26.21%升高到41.18%。RT-PCR检测发现HI44a作用后,M-CSF表达增强,而原癌基因c-myc表达降低。结论HI44a能够有效的诱导白血病细胞分化及凋亡,为治疗急性髓性白血病提供了一条新思路。     

Growth Inhibition and Apoptosis Induction of HumanUmbilical Vein Endothelial Cells by Apogossypolone

Aims and Background: Prostate cancer is one of the most common malignant tumors in the male reproductivesystem, which causes the second most cancer deaths of males, and control of angiogenesis in prostate lesions is ofobvious importance. This study assessed the effect of apogossypolone (ApoG2) on proliferation and apoptosis ofhuman umbilical vein endothelial cells (HUVECs). Subjects and Methods: HUVECs were treated with differentconcentrations of ApoG2. The survival rate of HUVECs were determined by MTT assay. Utrastructural changesof HUVECs were assessed with transmission electron microscopy. Apoptosis in HUVECs was analyzed by flowcytometry and cell migration by Boyden chamber assay. Matrigel assays were used to quantify the development oftube-like networks. Results: ApoG2 significantly inhibited HUVEC growth even at 24 h (P<0.05). The inhibitoryeffect of ApoG2 is more obvious as the concentration and the culture time increased (P<0.05). These resultsindicate that ApoG2 inhibits the proliferation of HUVECs in a time- and concentration-dependent manner withincrease of the apoptosis rate. Besides, ApoG2 reduced the formation of total pseudotubule length and networkbranches of HUVECs. Conclusions: The results suggest that ApoG2 inhibits angiogenesis of HUVECs by growthinhibition and apoptosis induction.     

In Vitro and in Vivo Inhibition of SK-N-MC Neuroblastoma Growth Using Cyclic nucleotide Phosphodiesterase Inhibitors

The effect of cyclic nucleotide phosphodiesterase (PDE) inhibitors Zaprinast and DC-TA-46 has been tested on SK-N-MC neuroblastoma growth. Antiproliferative activity of the tested drugs was assayed both in vitro and in the xenograft model of nude mice. In clonal density experiments, the IC₅₀ value was 3.3M for Zaprinast and 1.9M for DC-TA-46, while 7.5M BCNU alkylating agent was required to obtain the same effect. SK-N-MC cells xenografted in the nude mouse showed that the administration of Zaprinast and DC-TA-46 caused a significant 50% decrease of the tumour weight. These data demonstrate that PDE inhibitors may be useful for at least reducing tumour growth; they may be of interest for further evaluation as alternative molecules in the design of multiple agent protocols for neuroblastoma treatment.     

细胞周期对肿瘤坏死因子诱导Hela 细胞凋亡的影响

 目的　研究细胞周期对肿瘤坏死因子 ( TNF)诱导 Hela细胞凋亡的影响。方法　通过胸腺嘧啶核苷酸 ( Td R)阻断法阻滞 Hela细胞的细胞周期 ,以 MTT法、流式细胞术和荧光染色分析 Td R阻滞细胞和周期化的 Hela细胞对 TNF诱导凋亡的敏感性。结果　Td R阻滞细胞周期较周期化的 Hela细胞对 TNF诱导的凋亡的敏感性降低。结论　揭示 TNF诱导 Hela细胞凋亡与细胞周期有关.     

电离辐射对血管内皮细胞和PC-3细胞增殖和凋亡的影响

目的 探讨不同剂量X射线照射对原代人脐静脉内皮细胞(HUVEC)和PC-3细胞周期和凋亡的影响,为临床放疗提供理论依据。方法 采用形态学观察和Annexin V-FITC联合PI双染法和ApO2.7单抗法流式细胞仪(FCM)定量检测原代HUVEC和PC-3细胞在0-10 Gy的6MV-X射线照射后细胞周期和凋亡的变化。结果 照射后24 h和48 h两种细胞均出现明显G0/G1期减少和G2/M期增加,但2 Gy照射时仅对PC-3细胞周期有影响;两种细胞照射后均未出现辐射相关的凋亡。结论 电离辐射可诱导两种细胞出现G2/M期阻滞但不引起凋亡发生,两种细胞均有一定的辐射抗拒性。     

神经母细胞瘤血管形成兔角膜模型观察与抑制实验

[目的]建立兔角膜神经母细胞瘤种植模型 ,观察血管形成过程并用TNP 470和环磷酰胺 (CTX)干扰肿瘤血管形成 ,探讨通过抗血管形成治疗神经母细胞瘤的新途径。[方法]15只新西兰白兔角膜接种一人神经母细胞瘤 ,观察肿瘤生长情况及血管密度计数。用TNP 470和CTX干预 ,观察血管形成和肿瘤生长抑制情况并检测血管内皮生长因子的表达情况。[结果]术后2天就有血管向肿瘤方向出芽生长 ,1周时达高峰形成血管丛。肿瘤生长明显分成两个期 ,即血管前期和血管期 ,在血管前期中 ,肿瘤生长缓慢 ,血管期 ,肿瘤开始明显生长。TNP 470、CTX药物干预血管形成 ,血管生长曲线明显被压低。用药后一周出现抑制肿瘤疗效 ,两药之间无明显差别(P>0.05)。VEGF在神经母细胞瘤和用TNP 470后均有高表达(P>0.05) ,而CTX可抑制其表达(P<0.05) ,图像分析值分别为31.27±5.81、25.17±7.61和16.76±4.38。[结论]神经母细胞瘤有较高的血管依赖性。TNP 470和CTX对抑制肿瘤血管形成、限制肿瘤的生长 ,有明显的作用。     

Apoptosis induction through proteasome inhibitory activity of cucurbitacin D in human T-cell leukemia

BACKGROUND:

Human T‐cell leukemia is an aggressive malignancy of T lymphocytes. T‐cell leukemia has a very poor prognosis, even with intensive chemotherapy, indicating the need for development of new drugs to treat the disease. Triterpenoid cucurbitacins have been shown to have antitumor activity, but the mechanism of this activity is not fully understood.

METHODS:

The effects of cucurbitacin D on the proliferation and apoptotic induction of T‐cell leukemia cells using the Cell viability assay and Annexin V staining were evaluated. To investigate the mechanisms of apoptosis, antiapoptotic protein, NF‐κB, and the proteasome activity of leukemia cells treated with cucurbitacin D were evaluated by Western blotting both in vitro and in vivo.

RESULTS:

In this study, cucurbitacin D was found to inhibit proliferation and to induce apoptosis of T‐cell leukemia cells. Constitutively activated NF‐κB was inhibited by cucurbitacin D in the nucleus, which resulted in accumulation of NF‐κB in the cytoplasm, leading to down‐regulation of the expression of antiapoptotic proteins Bcl‐xL and Bcl‐2. Furthermore, cucurbitacin D induced the accumulation of inhibitor of NF‐κB (IκB)α by inhibition of proteasome activity. Low doses of cucurbitacin D synergistically potentiated the antiproliferative effects of the histone deacetylase inhibitor VPA. Finally, the proapoptotic and proteasome inhibitory activities of cucurbitacin D also were demonstrated using SCID mice in an in vivo study.

CONCLUSIONS:

Cucurbitacin D induced apoptosis through suppression of proteasome activity both in vitro and in vivo, making cucurbitacin D a promising candidate for clinical applications in the treatment of T‐cell leukemia. Cancer 2011;. © 2010 American Cancer Society.     

Epigenetic siRNA and Chemical Screens Identify SETD8 Inhibition as a Therapeutic Strategy for p53 Activation in High-Risk Neuroblastoma

1. Download high-res image (233KB)
2. Download full-size image

     

Protein Kinase C Inhibition by UCN-01 Induces Apoptosis in Human Glioma Cells in a Time-dependent Fashion

Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly. we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3–6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2–4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.     

Luteolin Induced-growth Inhibition and Apoptosis of Human Esophageal Squamous Carcinoma Cell Line Eca109 Cells in vitro

Luteolin is a plant flavonoid which exhibits anti-oxidative, anti-inflammatory and anti-tumor effects. However,the antiproliferative potential of luteolin is not fully understood. In this study, we investigated the effect of luteolinon cell cycling and apoptosis in human esophageal squamous carcinoma cell line Eca109 cells. MTT assays showedthat luteolin had obvious cytotoxicity on Eca109 with an IC50 of 70.7±1.72μM at 24h. Luteolin arrested cell cycleprogression in the G0/G1 phase and prevented entry into S phase in a dose- and time-dependent manner. asassessed by FCM. Luteolin induced apoptosis of Eca109 cells was demonstrated by AO/EB staining assay andannexin V-FITC/PI staining. Moreover, luteolin downregulated the expression of cyclin D1, survivin and c-myc,and it also upregulated the expression of p53, in line with the fact that luteolin was able to inhibit Eca109 cellproliferation.     

刺五加叶皂苷联合冬凌草甲素对人食管癌Eca-109细胞的生长抑制作用

目的 研究刺五加皂苷联合冬凌草甲素对体外培养的Eca-109细胞的增殖、细胞生长周期和诱导凋亡的影响.方法 以浓度0.75 g/L的刺五加皂苷与不同浓度的冬凌草甲素作用于体外培养的Eca-109细胞,MTT法检测细胞生长抑制率,观察细胞生长状态的变化,应用流式细胞仪检测细胞周期分布及凋亡率.结果 两药联用时可明显的抑制Eca-109细胞的生长,流式细胞仪结果显示,各实验组S期细胞百分率明显升高,G0～G1期细胞百分比明显下降,细胞被阻滞于S期,细胞凋亡率升高.结论 刺五加皂苷与冬凌草甲素联合应用对Eca-109细胞的增殖具有明显抑制作用,对其细胞周期的影响及诱导细胞凋亡可能是其重要机制之一.     

Targeting the proteasome in mantle cell lymphoma: a promising therapeutic approach

Mantle cell lymphoma (MCL) is a distinctive non-Hodgkin's lymphoma sub-type, characterized by over-expression of cyclin D1 as a consequence of chromosomal translocation t(11;14)(q13;q32). MCL remains an incurable disease, combining the unfavorable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL, which is often associated with additional cytogenetic alterations, has an even worse prognosis and new treatment options are clearly needed. The 26S proteasome is a large multi-catalytic multi-protein complex, present in all eukaryotic cells. It is responsible for the degradation of a variety of short-lived proteins and exhibits a key position in cellular processes including apoptosis and cell cycle progression. Targeting the ubiquitin - proteasome pathway has only recently been identified as a promising new therapeutic option for cancer patients. Interestingly, an increased activity of the proteasome pathway has been described in MCL cells and the inhibition of the proteasome seems to be a promising therapeutic approach for this incurable disease.     

放射线诱导肺腺癌细胞增殖凋亡

 目的 观察人肺腺癌细胞和肺纤维母细胞照射后死亡的特征。方法 HE染色观察照射后死亡细胞的形态学改变,Comet检测凋亡细胞内DNA双链断裂,流式细胞仪检测照射后细胞周期的改变及凋亡发生时机。结果 肺腺癌细胞照射后出现凋亡,凋亡发生于细胞渡过照射诱导的C2停滞后的G1停滞期;肺纤维母细胞照射后仅见到坏死改变。结论 凋亡是肺腺癌细胞照射后增殖死亡的表现形式之一,增殖凋亡的发生具有剂量和时间依赖关系。而增殖坏死可能是肺纤维母细胞照射死亡的主要形式,揭示了不同种类细胞增殖死亡的复杂性和多样性。     

去甲斑蝥素诱导人胃癌SGC-7901细胞凋亡的机制研究

[目的]探讨去甲斑蝥素对人胃癌SGC-7901细胞抑制和诱导凋亡作用.[方法]采用不同浓度的去甲斑蝥素(noncantharidin,NCTD)作用于胃癌SGC-7901细胞,在扫描电镜下观察其形态特点,采用流式细胞术测定经NCTD作用后的细胞生长周期及凋亡率,Western-blot 检测Bcl-2、Mcl-1、Bax的表达.[结果]扫描电镜下可见,SGC-7901细胞出现凋亡形态学改变.经去甲斑蝥素处理SGC-7901细胞后,实验组G2/M细胞明显多于对照组(P＜0.05).10μg//m1和20μg/ml浓度去甲斑蝥素均能诱导胃癌SGC-7901细胞凋亡(P＜0.05),其抑制率与NCTD浓度及作用时间相关.Western-blot检测Bcl-2、Mcl-1蛋白表达减少,Bax蛋白表达增加,呈明显的剂量关系.[结论]去甲斑蝥素对人胃癌SGC-7901细胞有抑制作用,可能与其阻滞细胞周期诱导细胞凋亡有关.     

丹酚酸乙对乳腺癌MCF-7细胞的生长抑制及诱导凋亡研究

[目的]观察丹酚酸乙对乳腺癌MCF-7细胞的生长抑制及诱导凋亡作用。[方法]MTT法观察丹酚酸乙对体外培养MCF-7细胞的增殖抑制作用;Hoechst33258荧光染色法观察细胞凋亡的形态学变化;流式细胞仪检测丹酚酸乙作用于MCF-7细胞48h后细胞凋亡率和细胞周期的变化;Western Blot检测caspase-3蛋白表达的变化。[结果]丹酚酸乙能明显抑制MCF-7细胞的生长,呈剂量和时间依赖性;荧光染色结果显示,丹酚酸乙与MCF-7细胞共培养24、48和72h后,细胞呈现明显的核固缩、碎裂以及凋亡小体形成等细胞凋亡现象;流式细胞仪检测结果显示,不同浓度的丹酚酸乙处理MCF-7细胞48h后,细胞凋亡率和S期细胞的比例逐渐升高;Western Blot显示,随着丹酚酸乙浓度的增加,MCF-7细胞caspase-3蛋白表达水平逐渐升高。[结论]丹酚酸乙对MCF-7细胞具有明显的生长抑制和促凋亡作用,这种作用可能与上调caspase-3表达以及阻滞细胞于S期有关。