Mechanism of resistance of Yoshida sarcoma to nitrogen mustard

Uptake and binding of methyl-bis(2-chloroethyl)amine hydrochloride (HN₂) to the cells of Yoshida sarcoma during their acquisition of resistance to this drug was investigated. With the increase of resistance index, drug uptake by the cells decreased. A good parallelism was found between resistance index and binding rate of the drug with cellular constituents when the intact cells were kept contacted in vitro with the drug in an incubation medium. When the cells were homogenized before contact with the drug, there was no difference in binding rate of the drug between sensitive and resistant cell lines. Suppression of cell membrane transport seemed to play a role in the acquisition of resistance by the cells.     

Brain and plasma pharmacokinetics and anticancer activities of cyclophosphamide and phosphoramide mustard in the rat

Summary By a sensitive and quantitative fluorometric assay, brain and plasma time-dependent concentration profiles were generated for phosphoramide mustard (PM) and active alkylating metabolites derived from cyclophosphamide (CPA) administration to rats. Whereas PM rapidly disappeared from plasma, with a monophasic half-life of 15.1 min, equimolar administration of CPA generated active metabolites in plasma that disappeared monoexponentially, with a composite half-life of 63 min. As a consequence, the time-dependent concentration integral of active alkylating metabolites derived from CPA administration, calculated between 5 min and infinity, was 3-fold that of PM. Pharmacokinetic parameters were calculated for each compound. The brain/plasma concentration-integral ratios of PM and active alkylating metabolites derived from CPA were 0.18 and 0.20, respectively. The cerebrovascular permeability-surface area product of PM was 7.5×10^–5s^–1, which is similar to that of other watersoluble anticancer agents that are restricted from entering the brain. The activities of a range of daily doses of PM and CPA were assessed against subcutaneous and intracerebral implants of Walker 256 carcinosarcoma tumor in rats. Inhibition of subcutaneous tumor growth by 50% was caused by CPA and PM doses of 6.6 and 12.0 mg/kg (daily for 5 consecutive days, starting 36 h after tumor implantation), respectively. However, administration of daily doses of up to 40 mg/kg did not significantly increase the survival of animals with intracerebral tumor implants. These studies indicate that active metabolites of CPA are restricted from entering the brain and that only subtherapeutic concentrations are achieved in brain tissue after systemic administration of CPA or PM.Abbreviations CPA cyclophosphamide - PM phosphoramide mustard - 4-HC 4-hydroxycyclophosphamide - AP aldophosphamide - PA cerebrovascular permeability-surface area product     

Quantitation by gas chromatography-chemical ionization mass spectrometry of cyclophosphamide, phosphoramide mustard, and nornitrogen mustard in the plasma and urine of patients receiving cyclophosphamide therapy

Unambiguous and sensitive methods based on gas chromatography-chemical ionization mass spectrometry have been developed to quantitate cyclophosphamide and two alkylating and cytotoxic metabolites, phosphoramide mustard and nornitrogen mustard. The levels of these materials have been determined in the plasma and urine of five patients receiving cyclophosphamide, 60 or 75 mg/kg i.v. Peak plasma levels of phosphoramide mustard of 50 to 100 nmoles/ml were found at 3 hr after cyclophosphamide administration. Variable levels of nornitrogen mustard were found in the plasma. This product may be arising in part from the decomposition of other metabolites during sample storage and preparation.     

Validation of a novel procedure for quantification of the formation of phosphoramide mustard by individuals treated with cyclophosphamide

Purpose

Use of the patient’s body surface area (mg m^?2) as a basis for dosing does not take individual variation in metabolic capacity and rate of clearance into account. Here, we evaluated a novel approach for individual monitoring of short-lived cytotoxic agents formed from cytostatic drugs such as cyclophosphamide (CP).

Methods

The accumulated blood dose of the cytotoxic active agent phosphoramide mustard (PAM) formed from CP was measured as a reaction product with hemoglobin (Hb adduct). This adduct, N-[2-(2-oxazolidonyl)ethyl]-valyl Hb (OzVal-Hb), was detached from Hb with the adduct FIRE procedure?, and the formed analyte was quantified using LC-MS/MS. This dose biomarker for PAM and the analytical procedure was evaluated in accordance with the guidelines on bioanalytical method validation formulated by the European Medicine Agency. The evaluated method was applied to quantify blood dose levels of PAM in female breast cancer patients (n = 12) before and after three cycles of polychemotherapy regimes containing CP.

Results

OzVal-Hb, a specific and stable biomarker, could be measured with great sensitivity (lower limit of quantification = 33 pmol g^?1 Hb), high accuracy (within ±20 %) and good repeatability (CV < 20 %). The inter-individual variability in the blood level of this adduct in women with breast cancer (n = 12) who received three doses of CP in combination with one or two other cytostatic drugs was 250 % following the first dose and approximately 150 % after each subsequent dose.

Conclusions

Measurement of the biomarker OzVal-Hb can be used to quantify the short-lived cytotoxic agent PAM in a single blood sample drawn several days after therapy. This procedure may aid in individualizing doses of CP, thereby improving efficacy while both reducing the risk of and increasing the predictability of side-effects.     

Mechanism of resistance of Yoshida sarcoma to nitrogen mustard. 3. Mechanism of suppressed transport of nitrogen mustard

Mechanism of decreased transport of nitrogen mustard (HN₂) in HN₂-resistant Yoshida sarcoma cells was investigated. HN₂ uptake by Yoshida sarcoma cells proved to be temperature sensitive, saturable, affected by metabolic inhibitors, and also repressed competitively by choline chloride. In resistant cells, the uptake of choline was markedly depressed. Kinetics of choline transport in resistant cells was comparatively examined, and decreased V_max and increased K_m were observed in some resistant sublines. Accordingly, decrease in choline uptake may be due to decreased activity of choline transport-carrier, both qualitatively and quantitatively. Decreased HN₂ uptake by resistant cells seemed to be produced by the same mechanism.     

Mechanism of resistance of Yoshida sarcoma to nitrogen mustard. II. Further evidence for suppressed transport of nitrogen mustard

Sensitivity of Yoshida sarcoma lines resistant to nitrogen mustard (HN²) was not enhanced by pretreatment in vitro with SH-blocking agents such as 2,2′-dithiodi-pyridine. This fact seems to indicate that content of cellular sulfhydryl groups did not play a great role in acquisition of resistance to HN². No difference was observed between the sensitive and resistant lines in retention rate of HN² bound to DNA of the cells during incubation of the cells in an HN-free medium. These results suggested no considerable difference in the ability to repair DNA in both lines. Further evidence was added for suppressed transport through the cell membranes of the resistant lines. Damage to cell membranes was proved by vital staining when a concentration of SH-blocking agents in the pretreatment reached a certain level, and the binding rate of HN² to DNA of the resistant lines was recovered almost to the same level of the sensitive line by this pretreatment. However, the binding rate of the sensitive line was not changed by pretreatment with the blocking agents at any concentrations.     

Characterisation of a mouse tumour cell line with in vitro derived resistance to verapamil

We have established a subline (EMT6/VRP) of the mouse tumour cell line EMT6/P with acquired resistance to the calcium transport blocker verapamil (VRP). The subline was 4-fold resistant to the cytoxicity of VRP alone compared with the parent line but of similar sensitivity to adriamycin, vincristine or colchicine. EMT6/VRP cells growing in 75 micrograms ml-1 VRP were morphologically different from and larger in diameter than EMT6/P cells, but these two parameters reverted almost to normal within 3 days of VRP removal, although resistance was retained. Expression of an mRNA coding for P-glycoprotein was similar in EMT6/VRP and the parent cell line, although considerable hyperexpression was seen in a multidrug resistant subline, EMT6/AR1.0. Cellular accumulation of both 3H-daunorubicin and 3H-VRP were greater in EMT6/VRP than in the parent line. Sensitisation to adriamycin by 3.3 micrograms ml-1 VRP was, however, somewhat reduced in EMT6/VRP (i.e. to 6.1-fold) compared with the 11-fold sensitisation seen in the parent line. It is clear that resistance to VRP seen in this cell line occurs via a different mechanism from the resistance to drugs such as adriamycin, vincristine and colchicine seen in multidrug resistant cell lines.     

Comparative effects of cyclophosphamide, isophosphamide, 4-methylcyclophosphamide, and phosphoramide mustard on murine hematopoietic and immunocompetent cells.

The effects of equimolal doses of cyclophosphamide (CY), isophosphamide (IP), 4-methylcyclophosphamide (4-MCY), and phosphoramide mustard (PM) on murine hematopoietic spleen colonies and adoptively transferred antibody-forming cells in vivo were compared. Equimolal doses of the drugs produced significantly different effects. All the drugs exerted an increasing effect against the ability of adoptively transferred immunocompetent cells to produce a significant anti-sheep red blood cell titer as the length of time between cell transfer and drug administration was increased. The maximum effect was seen when a drug was given 48--72 hours after antigen and spleen cell transfer. CY and IP produced significantly greater immunosuppressive effects than did the other drugs at all times after cell transfer and at all doses administered. PM had the least immunosuppressive effect at each dose evaluated. Against hematopoietic spleen colonies, the cytotoxic effects of 4-MCY and PM were similar and, at most doses studied, significantly greater than the effect of either CY or IP. Inasmuch as PM is an active metabolite of CY, it appeared either that one of the prior metabolites of CY was responsible for this marked immunosuppressive effect or that due to differences in polarity, PM was differentially distributed within the two cell systems as compared to CY. The differences in hematopoietic effects among all drugs were much less than those seen against immunocompetent cells and were not dependent on time of drug administration.     

The mechanism of acquired resistance to cisplatin by a human ovarian cancer cell line

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G1-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 micrograms/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.     

回苏宝液对A549细胞抑制作用及其机制的初步探讨

目的:观察红藤脂酸钠中药制剂回苏宝体外对人肺腺癌细胞系A549的生长抑制作用,并对其作用机制作初步探讨.方法:MTT法测定不同时间点不同浓度紫杉醇、回苏宝对A549细胞生长的抑制百分率,计算IC50(半数抑制浓度) 值,用DIP染色法观察A549细胞核的改变.结果:对照组(紫杉醇)作用30min后对A549细胞抑制率可达93%,24h达到100%;实验组血浆药浓度(80.3mg/ml)或2倍稀释回苏宝(40.15mg/ml)在8h内对A549细胞抑制率迅速升高,12h后缓慢上升(抑制率超过90%),48h到达平台期,72h达到98%左右.回苏宝在24h、48h的IC50值均为22.1mg/ml.正常A549细胞8h、24h DAPI染色其细胞核染色质饱满;2倍稀释回苏宝液作用8h的A549细胞核染色质有边集、减少现象,作用24h后显微镜视野中完整的A549细胞极少,核有固缩、凋亡现象;血浆浓度回苏宝液作用8h、24h后细胞残存很少,细胞核染色质边集、固缩,有凋亡小体形成的现象.结论:回苏宝体外对A549细胞生长有明显的抑制作用,其作用机制可能主要是诱导肿瘤细胞发生凋亡.     

回苏宝液对A549细胞抑制作用及其机制的初步探讨

目的：观察红藤脂酸钠中药制剂回苏宝体外对人肺腺癌细胞系A549的生长抑制作用,并对其作用机制作初步探讨。方法：MTT法测定不同时间点不同浓度紫杉醇、回苏宝对A549细胞生长的抑制百分率,计算IC50（半数抑制浓度）值,用DIP染色法观察A549细胞核的改变。结果：对照组（紫杉醇）作用30min后对A549细胞抑制率可达93％,24h达到100％;实验组血浆药浓度（80．3mg/ml）或2倍稀释回苏宝（40．15mg／ml）在8h内对A549细胞抑制率迅速升高,12h后缓慢上升（抑制率超过90％）,48h到达平台期,72h达到98％左右。回苏宝在24h、48h的IC50值均为22．1mg／ml。正常A549细胞8h、24hDAPI染色其细胞核染色质饱满;2倍稀释回苏宝液作用8h的A549细胞核染色质有边集、减少现象,作用24h后显微镜视野中完整的A549细胞极少,核有固缩、凋亡现象;血浆浓度回苏宝液作用8h、24h后细胞残存很少,细胞核染色质边集、固缩,有凋亡小体形成的现象。结论：回苏宝体外对A549细胞生长有明显的抑制作用,其作用机制可能主要是诱导肿瘤细胞发生凋亡。     

卵巢癌细胞拓扑替康耐药机制的探讨

目的　探讨人卵巢癌细胞对拓扑替康 (TPT)的耐药机制。方法　流式细胞仪检测卵巢癌TPT耐药细胞与亲本细胞的胞内罗丹明 (Rh12 3)荧光强度 ,RT PCR法检测各膜转运蛋白 (P gp、MRP、BCRP)的基因表达。将包含BCRPmRNA翻译起始位点的反义寡核苷酸 (ASODN)片段转染进耐药细胞 ,分别检测耐药细胞经体外转染后 ,BCRP的基因表达及胞内Rh12 3荧光强度的改变。结果　耐药株的胞内Rh12 3荧光强度是亲本细胞的 31.19% (P <0 .0 1)。耐药株中无P 糖蛋白 (P gp)的基因表达 ;多药耐药相关蛋白 (MRP)基因有极微弱表达 ,相对表达值为 0 .0 5 7;而BCRP基因在耐药株中高表达 ,相对表达值为 0 .6 6 ,亲本细胞不表达BCRP基因。将ASODN转染进耐药细胞后 ,BCRP的基因表达显著下降了 5 9.4 2 % (P <0 .0 5 ) ,胞内Rh12 3荧光强度由 5 .4 2增加到 16 .6 3(P <0 .0 5 )。结论　BCRP的高表达致胞内化疗药物浓度减少 ,是卵巢癌细胞对TPT耐药的主要原因。     

A transferable "resistance factor" from in vitro cultured MDMS-resistant Yoshida sarcoma cells

Cells of the methylene dimethanesulphonate-(MDMS)-resistant Yoshida sarcoma cell line contain a low molecular weight “resistance factor” which is present in the culture medium of these cells and may be utilized by MDMS-sensitive Yoshida sarcoma cells either by co-culturing the two cell lines or by culturing the MDMS-sensitive Yoshida cells in a medium containing 20% used medium of MDMS-resistant Yoshida cells or in the presence of dialysed medium from resistant cells. The “resistance factor” does not inactivate the drug itself or its metabolites, and it has no influence on the sensitivity of the cells if added after MDMS treatment. Twenty-four hours seems to be enough time for the transfer of the resistance factor, but its effect on whole populations decreases within 24 hours of ceasing the supply. The relationship between these findings and the known phenomena of metabolic co-operation are discussed.     

Expression microarray analysis reveals genes associated with in vitro resistance to cisplatin in a cell line model

We aimed to investigate the mechanisms of cisplatin resistance using an in vitro cancer model. A derivative breast cancer cell line (MCF-7CR) was established which demonstrated significant resistance to cisplatin at clinically relevant low concentrations compared to the MCF-7 parental cell line. Expression microarray analysis was used to identify targets from a 3k cancer-related oligonucleotide platform which were differentially expressed between the derivative and parental cell lines. Real-time quantitative PCR was used to confirm the difference in expression of a subset of genes which demonstrated significant up- or down-regulation. Using expression microarray analysis a total of 28 genes were identified to be differentially expressed (by at least 2-fold) between the MCF-7 and MCF-7CR cells. Real-time quantitative PCR expression analysis confirmed the differential expression of a selection of these genes (ACTG2, ARHD, CTSL, GSTM3, GSTM4 and EHF) between the two cell lines. An in vitro model of cisplatin resistance has been established and expression microarray analysis revealed 28 genes which may be associated with cisplatin resistance.     

Characterization of a human small cell lung carcinoma cell line with acquired resistance to cis-diamminedichloroplatinum(II) in vitro

A 6.4-fold cis-diamminedichloroplatinum(II) (CDDP) resistant human small cell lung carcinoma cell line (GLC4-CDDP) was developed to study acquired CDDP resistance in vitro. Compared to the sensitive cell line (GLC4), the GLC4-CDDP showed an increase in doubling time and a decrease in cloning efficiency, cellular size, double minutes per cell, cellular protein, and nuclear protein content. While a complete cross-resistance for tetraplatin and a partial cross-resistance for doxorubicin, melphalan, cadmium chloride, carboplatin, and cis-dichloro-trans-dihydroxo-cis-bis(isoprolylamine)platinum (IV) (resistance factor, respectively,4.0,5.8,2.1,1.5,2.9) was found, no cross-resistance for vincristine was found. In the GLC4-CDDP line in comparison to the GLC4 line, glutathione and total amount of sulfhydryl compounds was significantly increased, while glutathione S-transferase and glutathione reductase was the same. The platinum content in cells and nuclei was lower in the resistant line, but after correction for cellular protein or volume no difference was found. The amount of platinum bound to DNA was significantly lower in the GLC4-CDDP line. After a 1-h incubation with CDDP, the amount of Pt-GG adducts was the same and the amount of interstrand cross-links was reduced in the GLC4-CDDP line as compared to GLC4. In conclusion, in the GLC4-CDDP line the phenotype and genotype are changed and various mechanisms, such as decreased Pt-DNA binding, elevated glutathione, and reduced interstrand cross-links, play a role in the development of the CDDP resistance.     

Multidrug resistance in Yoshida rat ascites hepatoma cell lines.

Rat ascites hepatoma (AH) cell lines that were induced by dimethylaminoazobenzene and established as transplantable tumors had different sensitivities to vinblastine (VBL). The most VBL-resistant cells, AH66, showed more cross-resistance to vincristine and anthracyclines than AH66F cells. The resistance of AH66 cells was significantly decreased by verapamil. VBL-resistance of AH66 cells was inhibited by other drugs reported as overcoming acquired multidrug resistance, while the sensitivity of AH66F cells was hardly influenced by these drugs. The lowered uptake and enhanced extrusion of the antitumor drug in AH66 cells were suppressed by verapamil. M(r) 160,000 protein in the plasma membrane from AH66 was labeled with a photoactive VBL analog and was immunopositive to a monoclonal antibody against P-glycoprotein, C219. The sensitive cells had barely detectable levels of the surface membrane components. Specific photo-labeling with a VBL analog of P-glycoprotein of AH66 cell membrane was inhibited by reserpine and verapamil which restored the VBL resistance. These results indicate that AH66 cells are a naturally acquired multidrug-resistant cell line overexpressing a P-glycoprotein, and AH cell lines are useful to study multidrug resistance of hepatic carcinomas and development of counteracting drugs.     

Multidrug resistance in a human leukemic cell line selected for resistance to trimetrexate

Trimetrexate (TMQ) is a lipophilic antifolate shown to have antitumor activity in humans. TMQ-resistant sublines of the MOLT-3 human acute lymphoblastic leukemia cell line were developed and were designated as MOLT-3/TMQ200, MOLT-3/TMQ800, and MOLT-3/TMQ2500 based on degrees of resistance to TMQ. The TMQ resistance was accompanied by 5- to 7-fold increases in dihydrofolate reductase activity and markedly reduced cellular TMQ accumulation. Methotrexate accumulation was not impaired in TMQ-resistant cells. TMQ retention (efflux) was unchanged in these TMQ-resistant cells. Verapamil enhanced the TMQ accumulation in the resistant cells to the level seen in the parent cells but had no effects on the TMQ retention. These sublines were cross-resistant not only to methotrexate but also to vincristine, doxorubicin, daunorubicin, and mitoxantrone. There was no cross-resistance to bleomycin or cisplatin. Resistance to vincristine, doxorubicin, daunorubicin, and mitoxantrone was reversed by verapamil. TMQ resistance was only minimally reversed by verapamil and methotrexate resistance not affected at all. Both cellular accumulation and retention of vincristine and daunorubicin in the TMQ-resistant cells were markedly decreased. Verapamil enhanced both accumulation and retention of the drug. Plasma membrane fractions of the TMQ-resistant cells analyzed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue revealed the presence of a distinct band with a molecular weight of 170,000. Immunoblot analysis with 125I-labeled monoclonal antibody raised against P-glycoprotein of multidrug-resistant Chinese hamster ovary cells (C219) cross-reacted with the Mr 170,000 protein of the TMQ-resistant cells. These results show that the TMQ-resistant cells displayed not only decreased TMQ uptake and increased dihydrofolate reductase but also characteristics associated with a classical multidrug-resistant phenotype. Multidrug resistance includes lipophilic antifolate.     

乳腺癌细胞株MDA-MB-231获得性放疗抵抗机制探讨

目的探讨乳腺癌细胞株MDA-MB-231产生获得性放疗抵抗的可能机制。方法采用CCK8及流式细胞术检测产生乳腺癌细胞株MDA-MB-231获得放疗抵抗能力后细胞增殖及细胞周期的变化,采用蛋白质印迹法检测放疗抵抗后相关信号通路蛋白的变化,初步探讨乳腺癌细胞产生放疗抵抗的可能机制。结果 CCK8法检测结果显示,第1天实验组吸光度值为0.305 7±0.013 1,明显高于对照组的0.277 8±0.011 8,差异无统计学意义,F=7.571,P=0.051;第2天实验组为0.401 7±0.048 0,明显高于对照组的0.289 0±0.020 9,差异有统计学意义,F=13.907,P=0.020;第4天实验组为0.635 7±0.026 1,明显高于对照组的0.434 2±0.080 6,差异有统计学意义,F=16.994,P=0.015;第6天实验组为1.5347±0.0391,显著高于对照组的1.075 7±0.036 8,差异有统计学意义,F=218.953,P〈0.001。提示231/RR10细胞株在获得放疗抵抗能力的同时,增殖能力也增强,差异有统计学意义,P〈0.05。流式细胞术检测结果显示,放疗抵抗细胞株中,G0-G1及S期的细胞比例减少,但差异无统计学意义,P值分别为0.083和0.165;G2-M期的细胞比例明显增加,差异有统计学意义,P值分别为0.002和〈0.01。蛋白质印迹法检测结果显示,抑癌基因PTEN的表达明显减少,表皮生长因子的活化状态pEGFR的表达明显增加,AKT蛋白的表达水平无变化,而AKT磷酸化蛋白的表达明显增加。结论乳腺癌细胞株MDA-MB-231中EGFR/AKT信号通路激活,促进细胞生长和生存,从而导致放疗抵抗的产生。这一信号通路可能由抑癌基因PTEN负性调控。     

结肠癌耐药细胞株LoVo/L-OHP的建立及其耐药机制

目的：建立获得性奥沙利铂（L-OHP）耐药的结肠癌细胞模型LoVo/L-OHP,并初步研究其耐药机制。方法：采用L-OHP浓度递增法建立人结肠癌细胞耐药模型LoVo/L-OHP,观察其生长规律并绘制细胞生长曲线;用MTT法鉴定耐药细胞株耐药性并计算耐药指数（RI）;用半定量RT-PCR方法对部分耐药相关基因在耐药细胞及其亲本细胞中的表达情况进行分析。结果：成功建立了耐药的结肠癌细胞模型LoVo/L-OHP,LoVo/L-OHP细胞与LoVo细胞相比,生长缓慢,触角增多。通过RT-PCR半定量分析,P-gp、bcl-2、ERCC-1在LoVo/L-OHP中的表达上调,而p53基因表达下调。结论：LoVo/L-OHP细胞株耐药性稳定,耐药机制可能与P-gp、bcl-2、ERCC-1基因上调、p53基因下调多因素有关。