榄香烯乳逆转人肺癌细胞的实验研究

体外培养人肺癌细胞经３０μｇ／ｍｌ榄香烯乳作用后，生长受到抑制，＾３Ｈ－ＴｄＲ、＾３Ｈ－ｕＲ掺入率明显下降；流式细胞分析发现该药作用７２小时后Ｇ０／Ｇ１期细胞比例上升，Ｓ期比例下降；光镜、电镜观察可见药物作用后癌细胞增殖减慢，缩小变圆，微绒毛减少，核浆比例下降，异染色质增多。由此提示，榄香烯乳可在细胞生物学及形态学水平逆转人肺癌细胞的表型，诱导这些细胞趋于分化。     

β-榄香烯乳对舌鳞癌细胞株Tca-8113体外放射增敏观察

目的 探讨β-榄香烯乳对体外舌鳞癌细胞株的放射增敏作用。方法 MTT比色法测定不同浓度β-榄香烯乳对细胞生长抑制率，MTT法初步判断β-榄香烯乳对舌鳞癌细胞的放射敏感性：结果 β-榄香烯乳对Tca-8113细胞株有明显的抑制作用，40μg/mL、60μg／mL、80μg／mL、120μg／mL β-榄香烯乳作用细胞24h后生长抑制率分别为11％、19％、32％、48％。在相同放射剂量作用下，无细胞毒浓度（40μg/mL）作用后的OD值显著低于对照组。结论 β-榄香烯乳对Tca-8113细胞有放射增敏作用。     

榄香烯乳加联合化疗治疗急性非淋巴细胞白血病疗效观察

报道榄香烯乳加联合化疗治疗急性非淋巴细胞白血病的疗效。方法：将４３例急性非淋巴细胞白血病患者随机分为治疗组,用榄香烯乳２００～４００ｍｇ加生理盐水或５％葡萄糖５００ｍｌ,静脉滴注,持续１２～１５天,同时用ＨＡ方案：三尖杉酯碱４～６ｍｇ／ｄ１－７,阿糖胞苷１００－２００ｍｇ／ｄ１～７;对照组：单用ＨＡ方案,用量用法同治疗组。结果：治疗组ＣＲ率８０％,对照组ＣＲ率６０．９％;治疗组有效率９５％,对照组有效率７３．９％。结论：榄香烯乳对急性非淋巴细胞白血病有肯定的疗效,此药不良反应轻,无骨髓抑制,在化疗后的骨髓抑制期仍能继续使用榄香烯乳治疗,故榄香烯乳可成为急性非淋巴细胞白血病的有效药物之一。     

β－榄香烯诱发肿瘤细胞凋亡的研究

目的：探讨β－榄香烯的抗癌疗效及其作用机制。方法：采用ＭＴＴ方法、Ｈｏｅｃｈｓｔ３３３４２和ＰＩ荧光染色法、电镜及流式细胞术分析法,发现β－榄香烯乳对Ｋ５６２细胞生长的影响。结果：β－榄香烯明显抑制Ｋ５６２细胞的生长,对Ｋ５６２细胞的半数生长摇抑制剂量（ＩＣ５０）为１７．１４μｇ／ｍｌ。其抑制细胞生长能力主要是诱导细胞凋亡,并且呈浓度和时间依赖性。在影响细胞周期方面,主要使Ｇ、期细胞数目增多,Ｓ期     

榄香烯抗癌作用与诱发肿瘤细胞凋亡

榄香烯是从中药莪术中提取的抗癌有效成分，实验药理学和临床试验都证实该制剂对肿瘤有确切疗效。作者应用ＭＴＴ方法，分析了榄香烯对白血病细胞生长的影响。结果显示：榄香烯明显抑制白血病ＨＬ－６０和Ｋ５６２细胞的生长，对ＨＬ－６０细胞的半数生长的抑制剂量（ＩＣ５０）为２７．５μｇ／ｍｌ，对Ｋ５６２细胞的ＩＣ５０为８１μｇ／ｍｌ，而对正常人外周血白细胞（ＰＢＬ）的ＩＣ５０为２５４．３μｇ／ｍｌ。流式细胞术证实，榄香烯能阻滞肿瘤细胞从Ｓ期进入Ｇ２Ｍ期，并诱发其细胞凋亡。ＤＮＡ凝胶电泳及透射电镜超微结构观察都发现了榄香烯诱发肿瘤细胞凋亡所导致的生化与形态变化。结果表明，榄香烯诱导肿瘤细胞凋亡是其抗肿瘤作用机制的重要方面之一。     

榄香烯乳胸腔灌注对恶性胸水渗出液相关淋巴细胞(EAL)增殖及其LAK活性的增强作用实验研究

作者研究了榄香烯乳胸腔灌注对恶性胸水中渗出液相关淋巴细胞（EAL）的增殖和LAK活性的影响。结果显示，榄香烯乳能够明显促进EAL细胞增殖共产生显著的LAK活性，榄香烯乳还具有增强IL－2诱导的EAL细胞增殖效应及其LAK活性的作用，提示榄香烯乳与IL－2联合应用有可能产生增效作用。榄香烯乳的抗肿瘤免疫机制可能是通过作用于IL－2R／IL－2系统激活EAL细胞而发挥其抗肿瘤作用。结果提示，榄香烯乳有希望成为一种新型免疫增强剂而扩大应用于恶性肿瘤的免疫治疗。     

榄香烯对人肺腺癌细胞A549作用机理的初步研究

目的：研究榄香烯对体外培养的人肺腺癌细胞株A549的作用及其机制。方法：采用MTTI地检测药物对细胞的抑制作用。流式细胞术检测细胞周期变化以及电镜观察细胞的形态变化，结果：榄香烯能明显抑制肺腺癌细胞A549的生长，其半数生长抑制剂量为124．61μg/ml，流式细胞术证实榄香烯能阻滞肺腺癌细胞从S期进入G2／M期；透射电镜超微结构可见细胞浆内脂滴增多和坏死细胞明显增多的形态变化。结论：榄香烯能抑制肺腺癌细胞的生长，并阻滞肺腺癌细胞从S期进入G2／M期。     

人肺腺癌多药抗药细胞系LC—3／CDDP细胞周期及DNA含量变化

应用流式细胞仪测定人肺腺癌多药抗药细胞系ＬＣ－３／ＣＤＤＰ之细胞周期，ＤＮＡ指数，用＾３Ｈ－胸腺嘧啶核苷掺入法测定ＤＮＡ合成动态，结果显示，ＬＣ－３／ＣＤＤＰ细胞系Ｃ２＋Ｍ峰较亲代细胞明显增高，Ｇ期细胞比例降低，Ｓ期Ｇ２＋Ｍ期比例升高，ＤＮＡ指数增高，ＤＮＡ合成速率较亲代细胞显著增快。提示人肺腺癌多药抗药细胞纱ＬＣ－３／ＣＤＤＰ对ＤＮＡ损伤的修复功能增强。     

榄香烯乳抗肺癌细胞的实验研究

 作者观察了榄香烯乳在体外对人体肺癌细胞株LAX、Anip－93.7、Spc－A1、A549、H128、SPC和在体内对小鼠Lewis肺癌、大鼠WALKER－256肉瘤的抗癌作用。结果表明，榄香烯乳对上述肿瘤细胞均有明显的抑制作用。对各株体外癌细胞的IC50在20～45μg/ml，且与阳性对照组呈现不同的形态改变。腹腔内给药能明显抑制实验动物体内瘤结节的生长。药物作用后的肿瘤细胞DNA、RNA含量明显下降，脂滴增多，糖原颗粒减少，并出现细胞凋亡现象。     

榄香烯乳联合顺铂抑制胃癌AGS细胞生长的实验研究

目的：研究榄香烯乳（elemene）单独或联合顺铂（cisplatin）对人胃癌AGS细胞增殖和周期的影响,并探讨两药是否有协同作用及其可能的分子机制。方法：榄香烯乳单独或联合顺铂作用人胃癌AGS细胞。MTT法检测细胞增殖;流式细胞术检测细胞周期;BrdU掺入标记增殖的AGS细胞,免疫荧光细胞染色法测定增殖细胞数;Western印迹检测细胞周期蛋白D1（cyclinD1）的表达。结果：榄香烯乳（10-160μg/ml）对AGS细胞生长的抑制作用呈现浓度和时间依赖性,与对照组比较,除10μg/ml作用12h外,其它各组差异均有统计学意义（P〈0.05）。细胞周期中DNA合成前期（G0/G1期）细胞比例增加,DNA合成期（S期）细胞比例下降。榄香烯乳作用AGS细胞后,降低BrdU的渗入率,减少cyclinD1的表达。榄香烯乳（80μg/ml）联合顺铂（4μg/ml）对细胞增殖和周期的抑制明显强于单独用药（P〈0.05）。结论：榄香烯乳可抑制人胃癌AGS细胞的增殖并阻滞细胞于G0/G1期,与顺铂联合具有协同作用,其机理可能与降低cyclinD1的表达有关。     

体外观察中华眼镜蛇毒组分对KG1a 细胞增殖抑制作用及对其细胞周期的影响*

目的:探讨中华眼镜蛇毒组分（Naja Naja Actra Venom Component,NNAVC）对KG1a 细胞的增殖抑制作用及对其细胞周期的影响。方法:以人急性髓系白血病细胞株KG1a 细胞为研究对象进行实验。应用不同剂量的中华眼镜蛇毒组分作用于KG1a 细胞后,采用MTT 法测定其对KG1a 细胞的生长抑制作用,倒置显微镜镜下观察药物作用前后细胞形态学变化,流式细胞仪检测中华眼镜蛇毒组分对KG1a 细胞周期的影响。结果:中华眼镜蛇毒组分可以明显的抑制KG1a 细胞的增殖。在0.7 μ g/mL 至1.2 μ g/mL 的浓度范围内,中华眼镜蛇毒组分作用具有时间依赖性及剂量依赖性。显微镜镜下观察KG1a 细胞经中华眼镜蛇毒组分作用6h 后状态开始发生变化,随着药物作用时间的延长,细胞逐渐出现体积变小、变形、胞内空泡、细胞固缩等形态学变化。流式细胞仪检测证实中华眼镜蛇毒组分作用后,能够将KG1a 细胞阻滞于细胞周期的G0/G1 期,而处于G2/M期的细胞含量减少。结论:中华眼镜蛇毒组分对KG1a 细胞的增殖具有明显抑制作用,调节细胞周期停滞于G0/G1 期,减少G2/M期的细胞含量,提示影响细胞周期进程可能是中华眼镜蛇毒组分对KG1a 细胞的发挥增殖抑制作用的机制之一。      

Biomodulation by Hyperthermia of Topoisomerase II-Targeting Drugs in Human Colorectal Cancer Cells

We examined whether heat stress could enhance the sensitivity of human colon cancer WiDr cells to topoisomerase II-targeting anticancer agents, etoposide (VP-16) and teniposide (VM-26), and also determined the most effective timing for the drug administration after exposure to hyperthermia. Both topoisomerase II contents and topoisomerase II activity were significantly increased in WiDr cells 3 to 12 h after heat stress at 43°C for 1 h, in comparison with those immediately after the heat stress. Cytotoxicity by VP-16 was most significantly enhanced 3 to 12 h after exposure to 43°C for 1 h, but no synergistic effect was observed when the drug was administered immediately after the heat stress. A combination of VM-26 with heat stress, but not that of a topoisomerase I-targeting camptothecin derivative (CPT-11), or vincristine, showed a synergistic cytotoxic effect on WiDr cells. VP-16 alone induced cellular accumulation at the G2+M phase, whereas the combination of VP-16 and heat stress further increased the cell population at the G2+M phase, and decreased S-phase cells. A possible application of the combination of VP-16 and hyperthermia in clinical use is discussed.     

Survival and phase sensitivity of HeLa cells treated with dianhydrogalactitol (NSC-132313).

The effect of dianhydrogalactitol on survival and phase sensitivity of the cell cycle of HeLa cells was studied. In survival experiments no cells survived when the drug was added and left in contact with the cells at or above a dose of 0.7 mug/ml. Cells synchronized by the mitotic-selection method showed changing sensitivity to the drug during the cell cycle. G, late-S, and M phases were more sensitive to dianhydrogalactitol than the early-S and G phases. The DNA synthetic phase was divided into an early low sensitive and a late highly sensitive stage. The different sensitivity of mid-G and early-S phases was further established in separate survival experiments. Cells treated in G phase entered M phase in a manner similar to untreated control cells, while the progress of cells in S or G phase during treatment was delayed significantly. Lack of phase specificity of this drug is consistent with that of other alkylating agents. Changing sensitivity during some phases of the cell cycle proves that some metabolic events are especially sensitive to this particular drug.     

Cox-2抑制剂对骨肉瘤细胞抑制作用的实验研究

目的研究环氧化酶-2抑制剂Celecoxib对人骨肉瘤MG-63细胞的抑制作用及作用机制。方法用四唑盐（MTT）比色法检测不同浓度和不同作用时间对骨肉瘤细胞增殖的影响,用流式细胞仪检测药物对细胞周期和凋亡率的影响,透射电镜观察细胞的形态学改变。结果Celecoxlb能明显抑制细胞增殖,并呈剂量和时间依赖性。细胞凋亡率随药物浓度的增加逐渐增高,并引起细胞周期比例的改变。透射电镜显示细胞呈凋亡形态。结论Celecoxib对人骨肉瘤MG-63细胞有细胞毒作用,其机制与抑制肿瘤细胞增殖,使细胞阻滞于G0／G1期,并能诱导细胞凋亡有关。     

1,25（OH）2D3联合顺铂对原代肺腺癌细胞周期及调控因子的影响

目的:探讨1,25（OH）2D3联合顺铂对原代肺腺癌细胞周期及周期相关调控因子的影响。方法:体外培养手术切除的肺腺癌组织细胞,经药物作用后CCK-8法测定细胞抑制率,流式细胞仪检测细胞周期,RT-PCR检测细胞周期调控因子Cyclin D1、CDK4转录水平。结果:不同浓度的1,25(OH)2D3、顺铂单药作用于肺腺癌细胞均有明显的抑制作用,两药联合表现为协同作用,与单药相比,差异有统计学意义(P＜0.05)。细胞周期分析显示,经药物联合处理后的肺癌细胞,G0/G1期细胞数增多,Ｓ期和G2/M期细胞减少。细胞周期调控因子Cyclin D1、CDK4转录水平均有所降低,差异有统计学意义(P＜0.05)。结论:1,25（OH）2D3联合铂类抗癌药物抑制原代肺腺癌细胞的增殖能力、诱导细胞周期阻滞,与下调Cyclin D1和CDK4 的表达有关。     

Methyl protodioscin induces G2/M arrest and apoptosis in K562 cells with the hyperpolarization of mitochondria

Methyl protodioscin is a furostanol bisglycoside with antitumor properties. The present study investigated its effects on human chronic myelogenous leukemia K562 cells. Cell cycle analysis showed that methyl protodioscin caused distinct G2/M arrest, with the appearance of polyploidy population. The levels of cyclin B1 decreased, whereas Cdc2 kept at a steady level. Subsequent apoptosis after G2/M blockage was demonstrated through DNA fragmentation and the annexin V staining assay. Methyl protodioscin induced a biphasic alteration (i.e. an early hyperpolarization, followed by depolarization) in mitochondrial membrane potential of K562 cells. The transient decline of intracellular Ca2+ concentration was observed at early stage. The generation of reactive oxygen species was also detected. The anti-apoptotic Bcl-x(L) transiently increased and then decreased. And the pro-apoptotic Bax was markedly up-regulated. Taken together, these data demonstrated that methyl protodioscin inhibits K562 cell proliferation via G2/M arrest and apoptosis, with mitochondrial hyperpolarization and the disruption of Ca2+ homeostasis playing important roles.     

紫杉醇脂质体对卵巢癌细胞生长抑制作用的实验研究

目的分析紫杉醇脂质体对卵巢癌SKOV-3细胞的生长抑制作用,观察药物作用的时间-浓度关系,探究药物作用机制。方法应用不同剂量紫杉醇脂质体对SKOV-3细胞进行处理,通过MTT检测细胞存活率,倒置显微镜观察细胞形态,流式细胞术检测细胞凋亡作用及其对细胞周期的阻滞状态,Western blot分析Bcl-2基因蛋白表达情况。利用SPSS 13.0对数据进行统计学分析。结果紫杉醇脂质体对SKOV-3细胞有明显的剂量-时间依赖效应。紫杉醇与紫杉醇脂质体均可将SKOV-3细胞阻滞在G2/M期,且随药物浓度增加,凋亡细胞所占比例逐渐增加。通过Western blot方法发现,紫杉醇脂质体作用后SKOV-3细胞Bcl-2蛋白表达明显下降,Bax蛋白表达明显上调,Bax/Bal-2比例明显上调。结论紫杉醇以脂质体为转运载体后,并未改变其对卵巢癌SKOV-3细胞的抑制作用及作用周期,具有较好的临床应用前景。     

Cell cycle effects of CC-1065

CC-1065 is the most potent antitumor agent tested in our laboratory. It is lethal to B16 and CHO cells and to a variety of human tumors in the clonogenic assay at 1 ng/ml and is effective against L1210 leukemia and B16 melanoma in vivo at 1 to 50 micrograms/kg. CC-1065 inhibits DNA synthesis and binds to DNA in a nonintercalative manner in the minor groove. We report here the kinetics of inhibition of DNA synthesis and of cell progression and the phase-specific toxicity of the drug. To determine phase-specific toxicity, we started synchronous CHO cultures from mitotic cells harvested after Colcemid pretreatment. These cultures showed that mitotic cells were the most sensitive, and sensitivity decreased as the cells progressed through G1 to S and G2. Experiments with B16 and CHO mitotic cells harvested without Colcemid pretreatment also showed that mitotic cells were more sensitive than G1/S-phase cells. Cell progression studies showed that CC-1065 did not affect progression from mitosis to G1 or from G1 to S. Cells progressed slowly through S at low levels (1 ng/ml) of the drug but were blocked in S at 5 ng/ml. Cell progression from G2 to M was blocked by CC-1065. DNA synthesis in B16 cells was measured at different times after 2-hr exposure to CC-1065. The percentage of inhibition of DNA synthesis was minimum at 4 hr and maximum at 19 hr after drug exposure. Since B16 cell progression studies showed a marked change in percentage of S-phase cells during this time, the DNA synthesis rate was recalculated as cpm/S-phase cell. After this correction (i.e., expressing DNA synthesis as cpm/S-phase cell), the percentage of inhibition of DNA synthesis was minimum at 0 hr and gradually increased to maximum inhibition at 19 hr without the decrease seen previously at 4 hr.     

Arsenite induces prominent mitotic arrest via inhibition of G2 checkpoint activation in CGL-2 cells

Arsenic compounds, which are well-documented human carcinogens, are now used in cancer therapy. Knowledge of the mechanism by which arsenic exerts its toxicity may help in designing a more effective regimen for therapy. In this study, we showed that arsenite could induce prominent mitotic arrest in CGL-2 cells and demonstrated the presence of damaged DNA in arsenite-arrested mitotic cells. We then explored why these cells with arsenite-induced DNA damage were arrested at mitosis instead of G2 stage. When synchronized CGL-2 cells were treated with arsenite at stage G1, S or G2, all progressed into, and arrested at, the mitotic stage and contained damaged DNA, as demonstrated by the appearance of the DNA double-strand break marker, phosphorylated histone H2A.X (gamma-H2AX). Since X-irradiation induced G2 arrest in CGL-2 cells, these cells clearly have a functional G2 DNA damage checkpoint. However, treatment of X-irradiated CGL-2 cells with arsenite resulted in a decrease in G2 cells and an increase in mitotic cells, suggesting that arsenite may inhibit activation of the G2 DNA damage checkpoint and thus allow cells with damaged DNA to proceed from G2 into mitosis. Immunoblot analysis confirmed that arsenite treatment reduced the X-irradiation-induced phosphorylation of both ataxia-telangiectasia, mutated at serine 1981 and Cdc25C at serine 216, events which are crucial for G2 checkpoint activation and G2 arrest. Moreover, a higher frequency of apoptotic cells is observed in mitotic CGL-2 cells arrested by arsenite than those arrested by nocodazole or taxol. Our results show that the combined effects of arsenite in inducing DNA damages, inhibiting the activation of G2 checkpoint, and arresting cells with damaged DNA in the mitotic stage may subsequently enhance the induction of apoptosis in arsenite-arrested mitotic CGL-2 cells.     

Elevated phosphorylation of Chk1 and decreased phosphorylation of Chk2 are associated with abrogation of G2/M checkpoint control during transformation of Syrian hamster embryo (SHE) cells by Malachite green

Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the ability of MG to cause DNA damage, cell cycle arrest in mimosine synchronised and the possible roles of Chk1, Chk2, Cdc2, Cdc25C, 14-3-3 and Cyclin B1 in control and MG transformed SHE cells in order to understand the differential mechanisms associated with G2/M checkpoint control. Exposure of MG to control and transformed cells causes DNA damage. Flow cytometric analysis of mimosine synchronised cells when exposed to MG showed an increase of G2/M phase in control cells whereas no such accumulation of cells at the G2/M phase was observed in response to MG in transformed cells. Western blots of phosphoactive forms of Chk1 and Chk2 cells showed opposing levels. Control cells treated with MG showed a decrease in Chk1 and increase in Chk2, whereas the transformed cells treated with MG showed an increase in Chk1 and decrease in Chk2. Also a decrease in Cdc25C, 14-3-3 and Cyclin B1 was observed in MG treated transformed cells, whereas MG treated control cells showed elevated levels. Stabilization of the proteins seems to be the possible mechanism. The present study indicates elevated phosphorylation of Chk1 and decreased phosphorylation of Chk2 and decreased levels of Cyclin B1 are the critical changes associated with abrogation of G2/M checkpoint control during transformation of SHE cells by MG.