野生型p53基因促进长春新碱诱导的细胞凋亡

为探讨VCR与p53介导的细胞凋亡的关系,将野生型p53基因导入一个内源性p53基因重组的多药耐药细胞系 KB_V200,经抗性筛选和杂交鉴定获得外源性p53基因稳定表达的转染细胞系KB_V200-p53。VCR处理细胞后,经电镜及荧光显微镜观察表明KB_V200-p53细胞呈现凋亡的典型形态学特征;而流式细胞术结果显示经600nmol·L^-1的VCR作用24h,KB_V200-p53细胞和KB_V200细胞的凋亡百分数分别为42.4%和8.4%。结果提示野生型p53基因能促进VCR诱导的细胞凋亡,肿瘤细胞对VCR的耐药性可能与细胞内p53基因失活有关。     

赛赓啶对 KBV200细胞多药抗性的逆转作用

研究赛赓啶对KB_V200细胞多药抗性的逆转作用及逆转机制。在KB_V200细胞,采用MTT法,测出赛赓啶对长春新碱、阿霉素和鬼臼乙叉甙耐药的逆转系数分别为5.5,2.0和1.9,而对5-氟尿嘧啶、美法仑的细胞毒性作用无明显影响,表明赛赓啶为多药抗性逆转剂。荧光分光光度法测定表明,赛赓啶可使KB_V200细胞内阿霉素蓄积量增加。流式细胞荧光测定显示赛赓啶可增加罗丹明123的蓄积并减慢其外排。免疫细胞化学及狭缝杂交表明赛赓啶不影响KB_V200细胞的P-糖蛋白染色深度和 mdr1 RNA 表达水平。以上结果提示赛赓啶的多药抗性逆转机制是抑制P-糖蛋白泵的功能。     

基因mdr1高表达多药耐药肿瘤细胞对力达霉素的药物敏感性

目的利用经药物诱导获得的mdr1基因高表达细胞株以及通过mdr1基因转染建立的稳定高表达细胞株，研究多药耐药肿瘤细胞对力达霉素(C-1027)的药物敏感性。方法构建mdr1重组真核表达质粒pcDNA3.1/mdr1，利用脂质体转染技术，获得mdr1高表达HepG2肝癌细胞。经RT-PCR、细胞荧光免疫化学及罗丹明外排实验，鉴定了细胞的mdr1表达水平和药物外排活性。MTT方法测定敏感细胞及相对应的多药耐药细胞对力达霉素等多种抗肿瘤药物的药物敏感性。结果mdr1稳定转染细胞株HepG2/mdr1、多药耐药KBv200细胞和MCF-7/ADR细胞对力达霉素的IC₅₀值分别为(0.020±0.011) nmol·L^-1，(0.24±0.20) nmol·L^-1和(0.028±0.011) nmol·L^-1。相对于各自的敏感细胞，多药耐药细胞HepG2/mdr1，KBv200和MCF-7/ADR对力达霉素的抗药倍数分别是1.3，6.8和1.6倍，对阿霉素的抗药倍数分别是8.8， 37.2和181.3倍，对紫杉醇的抗药倍数分别是40.3， 336.8和49.2倍。结论 mdr1高表达的多药耐药肿瘤细胞对力达霉素仍高度敏感，未表现出抗药性。     

柔红霉素-白蛋白交联物逆转多药耐药KB/VCR细胞株对柔红霉素的耐药性

将柔红霉素(Dau)与牛血清白蛋白(BSA)交联, 以克服耐长春新碱(VCR)的KB细胞株(KB/VCR)对Dau的耐药性. Dau抑制KB和KB/VCR细胞生长的IC₅₀分别为3.3 μg·L^-1和0.26 mg·L^-1. KB/VCR细胞对Dau的耐药性是KB细胞的79倍. 反转录聚合酶链式反应结果显示, KB/VCR细胞有很高水平的mdr-1基因表达. Dau-BSA交联物和Dau杀伤KB/VCR细胞的EC₅₀别是12 μg·L^-1(指其中Dau含量)和0.24 mg·L^-1, 即Dau BSA对KB/VCR细胞的细胞毒作用是Dau的20倍. 药物蓄积实验显示，使用Dau-BSA时，KB/VCR细胞内Dau蓄积量较使用Dau者增加1.4倍(24 h时)；药物外排实验显示，加入Dau时, Dau迅速从KB/VCR细胞排出, 而使用Dau-BSA时Dau能在KB/VCR细胞内长时间维持较高浓度. 结果提示，Dau与BSA交联后，能克服多药耐药细胞对Dau的耐药性.     

三氧化二砷对胃癌细胞SGC7901多药耐药的逆转作用及其机制

研究三氧化二砷(arsenic trioxide，As₂O₃)对胃癌细胞多药耐药的逆转作用及其机制。逐渐递增长春新碱(VCR)的浓度诱导胃癌细胞株SGC7901产生多药耐药性(SGC7901/VCR)。MTT法测定药物对肿瘤细胞的杀伤作用；Western blotting检测肿瘤细胞内P-糖蛋白(P-gp)、谷胱甘肽S-转移酶(GST-s)表达。结果表明，胃癌SGC7901/VCR细胞对长春新碱(VCR)、5-氟尿嘧啶(5-Fu)及表阿霉素的耐药倍数分别为16.56倍、2.69倍及13.05倍。经As₂O₃预处理24 h后，长春新碱、5-氟尿嘧啶及表阿霉素对SGC7901/VCR的耐药倍数显著下降(P<0.05)。SGC7901/VCR在静息时细胞内P-gp、GST-s蛋白表达显著高于SGC7901。而As₂O₃可使SGC7901/VCR细胞内P-gp、GST-s蛋白表达显著下降，但是对SGC7901无明显作用。从而证实As₂O₃部分逆转SGC7901/VCR的耐药性，其机制可能与P-gp、GST-s蛋白表达降低有关。     

姜黄素逆转人结肠癌细胞株HCT-8/VCR多药耐药的研究

目的探讨姜黄素对人结肠癌耐药细胞株HCT-8/VCR多药耐药性的逆转作用及可能机制。方法运用MTT法进行药敏试验,流式细胞仪检测细胞内罗丹明123(Rh123)的表达,RT-PCR法检测多药耐药基因mdr1 mR-NA水平的变化,Western blot法检测mdr1基因蛋白产物P糖蛋白(P-gp)表达水平的变化。结果经25μmol/L姜黄素处理后,增强了HCT-8/VCR细胞对VCR的敏感性,其逆转倍数为4.58倍,增加了细胞内Rh123的蓄积(P<0.05),明显抑制了多药耐药基因mdr1 mRNA及其蛋白的表达(P<0.05)。结论本研究结果提示姜黄素能抑制耐药基因的表达及功能,提高细胞对化疗药物的敏感性,从而逆转结肠癌细胞的多药耐药性。     

紫杉烷类多药耐药逆转剂的合成及其逆转耐药活性紫杉烷类多药耐药逆转剂的合成及其逆转耐药活性

目的设计合成一系列新型紫杉烷类化合物并进行逆转多药耐药肿瘤细胞耐药活性的筛选。方法以本所生物合成的紫杉烷，Sinenxan A为原料，合成了10个(I_1～7, II_1,2, III)新的紫杉烷类化合物，其结构经FABMS，2DNMR确证，并对多药耐药癌细胞进行了逆转耐药性的试验。结果9个(I_2～7,II_1,2,III)化合物对多药耐药的人口腔上皮癌细胞KB/V200，能够显著逆转其耐药性，增强抗癌药的活性，其中化合物I₂, I₃, I₄逆转活性明显好于已知对照药Verapamil。结论紫杉烷类化合物作为多药耐药逆转剂有较好的逆转耐药活性，值得进一步研究。     

下咽癌Hep—2v多药耐药细胞株的筛选

目的 建立Hep—2v多药耐药(MDR)细胞株，以进一步研究探讨咽喉部肿瘤MDR特征及其逆转方法。方法 以下咽癌Hep—2细胞为亲本细胞，用长春新碱(VCR)进行耐药细胞筛选，选择出Hep—2v耐药细胞株。用流式细胞仪测定筛选前后两组表面糖蛋白P-170的阳性率及细胞内罗丹明蓄积率。以MTT法测定筛选前后VCR对两组细胞的细胞生长半数抑制浓度(IC50)比较其细胞毒作用。结果 亲本细胞Hep—2经VCR筛选后检删其耐药性状，MTT法测得其VCR的IC50由筛选前的60nmol／L上升到约1200nmol／L，流式细胞仪检测细胞表面糖蛋白P—170阳性率由亲本细胞的20％左右增加到90％以上。流式细胞仪检测细胞内罗丹明蓄积率从筛选前的平均76．2％降至26.3％。结论 本研究建立的耐药细胞株Hep—2v主要表现为mdr1基因介导的MDR性状，其对选择药物VCR的敏感性明显降低，因此Hep—2v细胞株可作为MDR研究的良好模型。     

从海南哥纳香中分离的一种新化合物──海南哥纳香醇甲的抗肿瘤作用

体外用细胞生长曲线测定法、MTT试验、软琼脂集落形成分析法,及体内对移植性肿瘤实验,研究了海南哥纳香醇甲(GHM-10)的抗肿瘤作用,结果表明:GHM-10对肿瘤细胞有较强的抑制作用,IC₅₀在2μg·ml^-1左右;对正常细胞影响较小,骨髓祖细胞的敏感性则更低;耐药的KB/VCR200细胞及其亲本KB细胞具有相似的敏感性。GHM-10对HL-60细胞无分化诱导作用。GHM-10对实体型肝癌H22小鼠、Lewis肺癌小鼠及腹水型S180小鼠均有明显的治疗作用。     

人卵巢癌细胞阿霉素耐药株的建立及其耐药机制的探讨

人卵巢癌阿霉素耐药细胞株是用A2780人卵巢癌细胞株作亲本,获得的能耐受0.8μg.ml^-1 ADM的细胞株A2780/ADM。此细胞株对ADM的耐受程度约为A2780细胞的17倍。对其他抗肿瘤药如长春新碱、鬼臼乙叉甙有明显的交叉耐药,对5-氟脲嘧啶、顺铂、米尔法兰则无明显交叉耐药性。进一步研究表明耐药株对ADM蓄积明显减少;免疫细胞化学的研究显示:A2780/ADM细胞中P-170膜蛋白和谷胱甘肽S-转移酶(GsT-π)均较A2780表达高。对耐药的逆转也进行了初步研究,发现维拉帕米与ADM合用能提高A2780/ADM细胞对ADM的药物敏感性,细胞的耐受性得到部分逆转。     

Reversal of multidrug resistance of KBV200 cells by triterpenoids isolated from Poria cocos

Multidrug resistance (MDR) of tumor cells is one of the major problems encountered during cancer chemotherapy. In this paper, we isolated eight triterpenoids from Poria cocos and evaluated their effects on reversing MDR of KBV200 cells. Eight triterpenoids increase significantly vincristine-induced cytotoxicity in drug-resistant KBV200 cells at the concentrations of 12.5 μg/mL and 25 μg/mL. Dehydrotumulosic acid showed the best reversal effect: it increased KBV200 apoptosis induced by vincristine and inhibited P-gp function through enhancing the accumulation and retention of fluorescent P-gp substrate rhodamine 123 in KBV200 cells but had no effect on P-gp expression.     

粉防己碱与阿霉素或长春新碱联合用药的体外抗癌增效作用

AIM: To study the interaction between tetrandrine (Tet) and doxorubicin (Dox) or vincristine (Vin) against human breast cancer cell lines MCF-7 and MCF-7/Dox or human nasopharyngeal cancer cell lines KB and KBV200 in vitro. METHODS: Anticancer activities of a drug alone and a drug combination of Tet and Dox or Vin were determined by tetrazolium (MTT) method. The interaction between Tet and Dox or Vin was evaluated by both a value of a sum of fractional inhibitory concentration (SFIC) and an isobologram method. RESULTS: The SFIC values of the three-different-ratio combinations between Tet and Dox ranged from 0.14 to 0.38 for MCF-7, and 0.10 to 0.29 for MCF-7/Dox; those of Tet-Vin combinations ranged from 0.21 to 0.37 for KB, and 0.32 to 0.63 for KBV200. All the SFIC values of the combination between Tet and Dox or Vin were less than 1.0 when the 3 ratios of the 2 drugs in combination were used, and the shapes of isobolic curves obtained from the combination were concave. CONCLUSION: The interaction between Tet and Dox or Vin against the human cancer cells was markedly synergistic.     

苦参碱与3种抗肿瘤药物联合作用对KBV200耐药细胞株细胞周期的影响

目的:探讨苦参碱分别与3种常用抗肿瘤药物长春新碱(VCR)、阿霉素(ADM)、顺铂(DDP)合用对KBV200耐药细胞株细胞周期的影响。方法:将培养后的KBV耐药细胞分为对照组、抗肿瘤药物组及其与苦参碱合用组,采用流式细胞仪检测、分析各组细胞周期的变化及凋亡情况。结果:与抗肿瘤药物单用组比较,苦参碱与VCR、ADM合用组细胞处于不同周期的细胞比例及凋亡数均无明显变化;苦参碱与DDP合用组G0~G1期细胞数下降、G2~M期细胞数升高。结论:推测苦参碱逆转KBV200细胞对VCR和ADM的耐药作用可能与细胞周期及凋亡无关;苦参碱能轻度增强DDP的细胞毒作用。     

Reversal of P-gp mediated multidrug resistance in-vitro and in-vivo by FG020318

Overexpression of P-glycoprotein (P-gp) by tumours results in multidrug resistance (MDR) to structurally and functionally unrelated chemotherapeutic drugs. Combined therapy with MDR-related cytotoxins and MDR modulators is a promising strategy to overcome clinical MDR. This study was performed to explore the MDR reversal activity of a novel compound 2-[4-(2-pyridin-2-yl-vinyl) phenyl]-4,5-bis-(4-N,N-diethylaminophenyl)-1(H)-imidazole (FG020318) in-vitro and in-vivo. Tetrazolium (MTT) assay was used to evaluate the ability of FG020318 to reverse drug resistance in two P-gp-expressing tumour cell lines, KBv200 and MCF-7/adr. Intracellular doxorubicin accumulation was determined by fluorescence spectrophotometry in MCF-7/adr cell line. The effect of FG020318 on P-gp function was demonstrated by rhodamine 123 (Rh123) accumulation in KBv200 cells. KBv200 cell xenograft models were established to study the in-vivo effect of FG020318 on reversing MDR. FG020318 was not cytotoxic by itself against P-gp expressing KBv200 cells and MCF-7/adr cells and their parental drug-sensitive KB cells and MCF-7 cells. FG020318 could significantly increase the sensitivity of MDR cells to antitumour drugs including doxorubicin and vincristine in MCF-7/adr cells and KBv200 cells, respectively. It was much stronger than the positive control verapamil in reversal of MDR. FG020318 also increased the intracellular accumulation of doxorubicin in a concentration-dependent manner in MCF-7/adr cells, but did not affect the accumulation of doxorubicin in drug-sensitive MCF-7 cells. The Rh123 accumulation in resistant KBv200 cells was also increased by the addition of FG020318, but Rh123 accumulation was not affected by FG020318 in drug-sensitive KB cells. FG020318 potentiated the antitumour activity of vincristine to KBv200 xenografts and was an efficacious modulator in-vivo. Our results suggested that FG020318 was a highly potent, efficacious MDR modulator not only in-vitro but also in-vivo. The reversal of drug resistance by FG020318 was probably related to the increased anticancer drug accumulation and its inhibition of P-gp function of MDR tumour cells.     

High cell density-dependent resistance and P-glycoprotein-mediated multidrug resistance in mitoxantrone-selected Chinese hamster cells.

Mitoxantrone (MIT) resistance has been studied in a colony selected from the CHO AA8 parental line in one step under a low degree of selective pressure (9 nM). The cells of the clonal isolate AA8/MIT C1(0) were sensitive to 9 nM MIT at low cell density but able to grow at high density. Parental AA8 cells were not able to grow under the latter condition. Decreased MIT accumulation (-20%) was observed at this step (step 0) in the absence of overexpression of mdr RNA coding for the drug efflux pump P-glycoprotein. Furthermore, AA8/MIT C1(0) did not exhibit cross resistance to vincristine, Adriamycin and etoposide at low cell density. During subsequent controlled growth for 2 months at high cell density in the presence of 9 nM drug, an additional selection occurred leading to a 4-fold MIT-resistant subline AA8/MIT C1(+). This subline was characterized at this step (step I) and after an additional 4 months of culture in the presence of 9 nM MIT (step II). Analysis of mdr gene expression and gene copy number showed an increase in mdr RNA and a pattern of mdr gene amplification which changed between step I and II. AA8/MIT C1(+)II exhibited a classical multidrug resistance phenotype with decreased accumulation of [14C]MIT and cross-resistance to vincristine, Adriamycin and etoposide. The ability to form the cleavable complex in the presence of etoposide in DNA topoisomerase II-containing nuclear extracts was identical in AA8/MIT C1(+)II and AA8 cell lines. These results demonstrate a new sequence of events in MIT resistance: low level of drug resistance at high cell density followed by mdr gene amplification.     

Spontaneous Reversal of P‐Glycoprotein Expression in Multidrug Resistant Cell Lines*

Abstract: Increased expression of P‐glycoprotein encoded by the mdr‐1 gene is a well‐characterised mechanism for resistance to cancer chemotherapeutic drugs in cell lines. However, the P‐glycoprotein expression after removal of the selection pressure has not fully been elucidated. The stability of P‐glycoprotein expression in the presence (+) and absence (?) of vincristine (30 or 150 nM) was studied in multidrug resistant K562 cell lines (VCR30+, VCR150+, VCR30? and VCR150?) for 11 months. The P‐glycoprotein protein and mdr‐1 mRNA levels were determined at regular intervals using flow cytometry and real‐time PCR, respectively. Chemosensitivity to a panel of antineoplastic drugs was measured using an MTT assay. The presence of vincristine (VCR30+ and VCR150+) resulted in high and stable levels of P‐glycoprotein and mdr‐1 mRNA during the whole period compared to wild type. As for the VCR30? and VCR150? subcultures, the expressions of P‐glycoprotein and mdr‐1 mRNA were stable for five months, and then the levels decreased rapidly. Concomitantly, the sensitivity to drugs known as P‐glycoprotein substrates was restored. In conclusion, resistant cells growing in the presence of the inducing drug have a stable P‐glycoprotein expression and resistance level, but removing the inducing drug may result in a sudden and rapid lowering of P‐glycoprotein and mdr‐1 mRNA levels as long as five months after drug withdrawal.     

鬼臼毒素衍生物CIP-36诱导KBV200细胞凋亡

目的：研究鬼臼毒素衍生物CIP-36对多药耐药人口腔鳞状上皮癌细胞KBV200的抗肿瘤活性及其作用机制。方法：MTT法考察CIP-36对KBV200体外增殖的抑制作用;Giemsa染色、DNA ladder和流式细胞仪等方法进行细胞凋亡检测;免疫荧光法观察CIP-36对细胞骨架的作用;western-blot法检测CIP-36对KBV200细胞P-gp表达的影响。结果：CIP-36对KBV200细胞有明显的抑制作用,IC50值为（2.06±0.38）μmol/L,能够诱导细胞产生凋亡小体和DNAladder。流式细胞检测到了细胞凋亡峰,并观察到细胞周期出现S/G2＋M期阻滞。Western-blot结果显示P-gp表达降低,并且观察到CIP-36可破坏KBV200细胞的细胞骨架。结论：CIP-36可能通过降低P-gp的表达,破坏细胞骨架等多靶点克服KBV200细胞株的多药耐药性。