hMS H2重组质粒对人卵巢癌细胞顺铂敏感性的影响

目的：研究hMSH2重组质粒对卵巢癌顺铂敏感性方面的影响。方法：构建重组真核表达质粒pCAN－hMSH2,通过酶切及测序法对重组质粒进行鉴定。采用脂质体转染技术对卵巢癌耐药细胞SKOV3／DDP进行瞬时转染,对照组为未转染细胞和SKOV3敏感细胞;hMSH2在细胞内的表达变化由Western blotting和RT－PCR进行检测;四甲基偶氮唑蓝（MTT）法对细胞的顺铂敏感性进行检测;耐药细胞的凋亡情况通过Hoechst染色法检测。结果：重组质粒pCAN－hMSH2转染进SKOV3／DDP后,经RT－PCR和Western blotting检测证实转染成功,hMSH2的表达得到增强;MTT结果提示转染后的卵巢癌耐药细胞对顺铂的敏感性明显增强;Ho-echst染色发现,转染后耐药细胞的凋亡明显增强。结论：hMSH2基因转染后能提高其在SKOV3／DDP细胞中的表达,增强SKOV3／DDP细胞对顺铂的敏感性,促进在顺铂作用下的凋亡。     

槲皮素逆转白血病细胞株HL-60/ADM多药耐药的研究

目的探讨槲皮素(Que)逆转白血病细胞多药耐药在膜转运蛋白方面的机制。方法通过四唑蓝体外药敏法,检测Que对柔红霉素(DNR)的增敏作用,并以不同浓度作用于HL-60／ADM耐药株及相应敏感株HL-60;运用逆转录多聚酶链式反应和流式细胞术,检测HL-60／ADM和HL-60细胞株多药耐药相关基因1(MRP1)及其膜蛋白产物MRP1蛋白的表达情况;借助激光共聚焦显微镜,观察DNR在亚细胞水平的分布变化。结果20～40μmol／L终浓度的Que在体外能明显提高DNR对HL-60／ADM耐药株的敏感性,并能下凋MRP1基因及其膜蛋白产物MRP1的表达,使DNR回归于细胞核内,从而逆转多药耐药,而且对细胞本身无明显毒性作用。结论Que有可能成为蒽环类药物治疗白血病的有效且低毒的化疗增敏剂。     

E1A基因对人肺腺癌细胞化疗敏感性的影响 *

目的：探讨Ｅ１Ａ基因对人肺腺癌细胞的化疗增敏作用。方法：通过脂质体介导将Ｅ１Ａ基因导入人肺腺癌细胞系Ａ－ｎｉｐ９７３,经Ｇ４１８筛选获得稳定表达Ｅ１Ａ的转染细胞（Ａｎｉｐ９７３－Ｅ１Ａ）。用不同浓度顺铂、泰素及ＶＰ１６等化疗药物处理细胞,观察Ｅ１Ａ基因对人肺腺癌细胞的化疗增敏作用。结果：Ａｎｉｐ９７３－Ｅ１Ａ细胞对顺铂、泰素的敏感性显著增加,顺铂的ＩＣ５０值减少了７倍,并且敏感性随时间的延长而增加。但对ＶＰ１６,Ｅ１Ａ基因的稳定表达在该细胞系未显示增敏作用。免疫细胞化学染色显示,Ｅ１Ａ基因抑制了ＨＥＲ－２／ｎｅｕ的表达。结论：Ｅ１Ａ基因能增加人肺腺癌细胞对顺铂、泰素等化疗药物的敏感性。该作用可能与Ｅ１Ａ基因抑制ＨＥＲ－２／ｎｅｕ的表达有关。为在恶性肿瘤治疗上化疗和基因治疗联合应用的可能性提供了实验依据。     

Nrf2及其靶基因在人肺腺癌A549顺铂耐药细胞株中的表达和意义

背景与目的 NF-E2相关因子2(NF-E2-related factor 2,Nrf2)是细胞调节抗氧化应激反应的重要转录因子,已有研究表明Nrf2及其信号传导通路与卵巢癌顺铂耐药密切相关,但Nrf2与肺腺癌耐药相关性的研究罕见报道,本研究的目的是测定转录因子Nrf2及其靶基因在人肺腺癌A549顺铂耐药细胞株(A549/DDP)及其亲本细胞(A549)中的定量表达,探讨肺腺癌顺铂耐约机制.方法 采用MTT法测定A549/DDP及A549细胞对DDP的敏感性;实时荧光定量PCR检测两株细胞中转录因子Nrf2及其靶基因定量表达.结果 A549/DDP对DDP的耐药指数为12.12,属于中度耐药;与亲本细胞比较,A549/DDP细胞Keap1、Nrf2、NQO1、GSTP1、GCL、HO-1、MRP4的mRNA表达增加(P＜0.01),MRP1、MRP2及MRP3的表达降低(P＜0.01).结论 Nrf2信号传导通路可能参与了人肺腺癌A549顺铂耐药现象的产生,这一发现对从基因水平开发新的耐药拮抗剂用于肿瘤耐药的辅助治疗,避免和克服耐药具有重要意义.     

MRP1介导肿瘤的多药耐药

目的:探讨多药耐药相关蛋白基因MRP1介导肿瘤耐药机制,寻找逆转耐药的方法.方法:建立肝细胞癌单基因耐药细胞株HepG2/MRP1,体外转录合成目的siRNA片断,脂质体介导转染单基因肝细胞癌耐药细胞株,MTT法检测细胞对化疗药的敏感性,流式细胞仪检测药物浓度及细胞表面MRP1蛋白表达.结果:成功建立多药耐药细胞株,并筛选出靶向MRP1基因高效的siRNA分子.转染HepG2/MRP1细胞后,MRP1 mRNA及MRP1表达水平明显下调,细胞内DNR蓄积量增加,对ADM的IC50下调,相对逆转率达90％.转染前后细胞对化疗药敏感性、细胞内药物浓度、细胞表面MRP1蛋白质表达均有显著差异.结论:肿瘤多药耐药与MRP1高表达密切相关,RNAi技术能有效逆转由MRP1介导的肿瘤细胞的多药耐药.     

Neu RNAi对卵巢癌细胞生长、凋亡及药物敏感性的作用

目的探讨nenRNAi对卵巢癌耐药细胞株SKOV3／DDP细胞生长、凋亡及药物敏感性的作用,寻找下调基因表达并逆转化疗耐药的方法,为卵巢癌的基因治疗探索新途径。方法应用含有u6启动子的pSilencer 1．0-U6表达载体构建neu基因的小干扰RNA（siRNA）表达载体（pSilencer—neu）,用脂质体方法将pSilencer—neu转染卵巢癌顺铂耐药细胞株SKOV3／DDP,另设未转染组及转染pSilencer—control组为对照。显微镜下观察转染前后细胞的变化;用RT—PCR及Western Blot检测neu基因mRNA及蛋白的表达;MTT法检测细胞生长情况;流式细胞仪检测细胞凋亡及周期的变化;加入顺铂后检测RNAi对SKOV3／DDP顺铂药物敏感性的影响。结果neuRNAi可明显下调卵巢癌耐药细胞株SKOV3／DDP中neu的基因表达;使其细胞生长受到抑制,生长速度减慢,生长曲线低平;细胞凋亡增加且细胞周期发生变化,G1／G0期细胞增多,S期细胞则减少;顺铂耐药实验显示,转染RNAi的SKOV3／DDP细胞IC50明显下调,细胞凋亡率显著增加。结论应用RNAi可以部分阻抑neu基因在卵巢癌顺铂耐药细胞株SKOV3／DDP中的表达,使SKOV3／DDP细胞生长减慢、凋亡增加,而且对顺铂的敏感性增强。     

Sox2对膀胱癌细胞化疗耐药的影响及其分子机制的探讨

摘 要：［目的］ 探讨Sox2对膀胱癌细胞化疗耐药的影响及其分子机制。［方法］ 选用T24人膀胱癌细胞系,采用浓度递增方式建立顺铂耐药细胞系T24/DDP,慢病毒载体转染对应细胞,进行Sox2基因过表达或敲除,得到T24-Sox2细胞和T24/DDP-shSox2细胞系。qRT-PCR和Western blot检测各组细胞中Sox2与多药耐药相关蛋白（MRP）表达水平。MTT实验检测各组细胞在不同浓度顺铂作用下的吸光度值并计算IC50,绘制耐药曲线。流式细胞术检测各组细胞凋亡率,并使用qRT-PCR和Western blot检测凋亡相关蛋白的表达水平。［结果］ qRT-PCR和Western blot结果证实Sox2和MRP在膀胱癌顺铂耐药细胞系中高表达,耐药曲线提示Sox2与膀胱癌的顺铂耐药存在一定关联,耐药细胞系的IC50为(15.24±1.12)μg/ml,而非耐药细胞系的IC50为(4.88±0.72) μg/ml,P<0.05。Sox2表达上调能够抑制膀胱癌细胞凋亡,凋亡率从74.62%下降到25.84%,P<0.05。Bax和Caspase-3在Sox2高表达的细胞中呈低表达,而Bcl-2则呈高表达。［结论］ Sox2基因在顺铂耐药膀胱癌细胞系中表达水平显著高于正常非耐药膀胱癌细胞,且Sox2可能是通过抑制细胞凋亡来实现对化疗敏感性的调控。     

转化生长因子诱导的基因蛋白与肿瘤耐药的相关性

目的：探讨转化生长因子诱导的基因蛋白与肿瘤耐药的相关性。方法：分别用载有 TGFBI 基因质粒以及空质粒转染人卵巢癌细胞株 SKOV3,采用 Western blot 技术检测转染后两组细胞 TGFBI 蛋白的表达情况,用 MTT 法检测不同浓度紫杉醇、顺铂对上述两组细胞的抑制率,采用流式细胞技术检测紫杉醇对上述两组细胞凋亡的影响。结果：用载有 TGFBI 基因质粒转染细胞后其 TGFBI 蛋白表达情况显著高于用空质粒转染细胞后 TGFBI 蛋白的表达。在紫杉醇、顺铂同种药物浓度下,TGFBI 转染后细胞抑制率显著高于空质粒转染细胞（P <0.05）。TGFBI 质粒转染细胞凋亡率为（67.35±12.36）%,而空质粒转染细胞凋亡率为（50.21±14.85）%,TGFBI 质粒转染细胞凋亡率显著高于空质粒转染细胞凋亡率（P <0.05）。结论：TGFBI 高表达能够降低人卵巢癌细胞 SKOV3的耐药性,提高肿瘤细胞对化疗药物的敏感性,有助于促进肿瘤细胞的凋亡。     

小分子干扰RNA对非小细胞肺癌MRP基因和药物敏感性的影响

目的：观察小分子干扰RNA（siRNA）对非小细胞肺癌（NSCLC）MRP基因、药物敏感性和细胞凋亡的影响，探讨体外肺癌耐药的高效和特异逆转耐药策略。方法：采用MRP siRNA转染，RT-PCR检测多药耐药相关蛋白MRP mRNA表达水平，流式细胞仪检测MRP蛋白表达，MTT法检测MRP siRNA的细胞毒作用，碘化丙啶（PI）染色检测MRP siRNA的细胞凋亡作用。结果：SW1573／2R120细胞转染MRP siRNA后，MRP mRNA表达明显降低，转染24h、48h和72h后MRP siRNA组的MRP蛋白表达阳性率较对照组明显减低，MRP siRNA与对照组相比对细胞生长抑制并无统计学差异，未见明显影响细胞增殖；MRP siRNA明显增加10μM阿霉素对SW1573／2R120细胞生长的抑制，并随作用时间的延长而增强，MRP siRNA＋ADM组细胞生长抑制作用与对照组相比有统计学差异，MRP siRNA协同阿霉素增强对化疗耐药肺癌细胞的凋亡作用。结论：MRP siRNA可以明显逆转体外NSCLC MRP基因编码MRP蛋白的多药耐药。     

人膀胱癌非经典型多药耐受性研究

目的：为了建立人膀胱癌耐阿霉素细胞系并研究它们的生物学特性及药物耐受性的机理。方法：采用人膀胱癌细胞系EJ,经递增阿霉素剂量的方法,历时1年,建立一株耐药亚株EJ／DOR,对其生物学特性及耐药机理进行了研究,并应用反转录PCR检测了MDR1、MRP和DNA TopoⅡ基因的表达。结果：EJ／DOR对阿霉素的相对耐受度较亲本细胞提高了14.3倍;对蒽环类、长春花属生物碱及DNA TopoⅡ靶刺剂足叶乙甙有明显的交叉耐药性,但对顺铂、丝裂霉素无明显的交叉耐受性;耐药细胞对柔红霉素的细胞内聚集量显著减少;EJ／DOR并不表达MDR1基因,而MRP基因过表达,但细胞内DNA TopoⅡ基因表达低于亲本细胞。结论：细胞内DNA TopoⅡ基因表达下降及MRP基因过表达是EJ／DOR表现为多药耐受性亚型的主要原因,这种非P-gp介导的非经典型多药耐受性细胞为寻求包括阿霉素在内的化疗方案提供了良好的实验模型。     

5—氟脲嘧啶对耐顺铂人肺腺癌细胞A549^DDP的程序依赖性逆转作用

用耐顺铂（ＣＤＤＰ）人肺腺癌细胞系Ａ５４９ＤＤＰ作模型，研究了５－ＦＵ对ＣＤＤＰ耐药的程序依赖性逆转作用。５－氟脲嘧啶（５－ＦＵ）预处理Ａ５４９ＤＤＰ２４ｈ后立即给予ＣＤＤＰ，ＣＤＤＰ细胞毒性增加３．９倍；５－ＦＵ预处理Ａ５４９ＤＤＰ后间隔２４或４８ｈ再给予ＣＤＤＰ，其细胞毒性分别增加２０倍和２５０倍，甚至较亲代细胞Ａ５４９更为敏感；如先给ＣＤＤＰ后给５－ＦＵ则细胞毒性仅增加１．８倍。５－ＦＵ对亲代细胞亦有类似效应但细胞毒性增加程度明显低于耐药细胞。５－ＦＵ预处理后间隔２４，４８ｈ，细胞内ＧＳＨ含量逐渐降低，与细胞毒性逐渐增加相一致。如用ＢＳＯ耗竭Ａ５４９ＤＤＰ细胞内ＧＳＨ，ＣＤＤＰ细胞毒性增加６．４倍，仅能部分逆转ＣＤＤＰ耐药。５－ＦＵ明显抑制ＭＲＰ的表达，但对ＧＳＴπ的表达无影响。在５－ＦＵ预处理后的无药间隔时间内，给予无毒浓度的三苯氧胺则有明显的协同效应。结论：程序性给予５－ＦＵ通过降低细胞内ＧＳＨ含量和抑制ＭＲＰ的表达能完全逆转ＣＤＤＰ耐药     

Lack of a role for MRP1 in platinum drug resistance in human ovarian cancer cell lines.

The level of expression of the multidrug resistance-associated protein (MRP1) in a panel of human ovarian carcinoma cell lines and their variants with acquired cisplatin resistance was determined using Western blotting. No overexpression of MRP1 was detected in any of the cell lines. In addition, we have transfected the MRP1 gene into an intrinsically cisplatin-resistant cell line SKOV3, previously shown to have elevated levels of glutathione (GSH). The MRP1-transfected line SKOV3-S2 was shown to be cross-resistant to doxorubicin, vincristine and etoposide but not to paclitaxel, vinblastine and platinum agents, such as cisplatin, JM216 [bis-acetato-ammine-dichloro-cyclohexylamine platinum (IV)] and AMD473 [cis-ammine dichloro (2-methyl-pyridine) platinum (II)]. No cross-resistance to any of the platinum agents was observed in a MRP1-overexpressing human lung cancer cell line with acquired doxorubicin resistance. Reduction of GSH levels (80-90%) by buthionine sulphoximine (BSO) produced significant potentiation in cisplatin sensitivity in the parental SKOV3, the vector-alone control SKOV3-puro and the MRP1-transfected line SKOV3-S2. The degree of sensitization was similar in all cell lines (1.6-fold). However, selective sensitization by BSO to vincristine was observed in the MRP1-transfected line (4.1-fold) but not in the vector control. No significant differences were observed in cisplatin accumulation in the SKOV3-puro and the SKOV3-S2 cells, although both these transfected lines accumulated significantly more than the parental line. Our results suggest that MRP1 does not play a significant role in platinum resistance in the human tumour cell lines investigated in this study.     

Determinants of Drug Response in a Cisplatin-resistant Human Lung Cancer Cell Line

To elucidate the mechanism(s) of cisplatin resistance, we have characterized a human non-small cell lung cancer cell line (PC-9/CDDP) selected from the wild type (PC-9) for acquired resistance to cisplatin. PC-9/CDDP demonstrated 28-fold resistance to cisplatin, with cross resistance to other chemotherapeutic drugs including chlorambucil (× 6.3), melphalan (× 3.7) and 3-[(4-amino-2-methyl-5-pyrimidinyl)]methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU) (× 3.9). There was no expression of mdr-1 mRNA in either wild-type or resistant cells. The mRNA and protein levels of glutathione S -transferase (GST) × were similar in the two lines. A GST-μ isozyme was present in equal amounts and the activities of selenium-dependent and independent glutathione peroxidase and glutathione reductase were unchanged. The mRNA level of human metallothionein IIA and the total intracellular metallothionein levels were reduced in the resistant cells. Significantly increased intracellular glutathione (GSH) levels were found in the resistant cells (20.0 vs. 63.5 nmol/mg protein) and manipulation of these levels with buthionine sulfoximine produced a partial sensitization to either cisplatin or chlorambucil. Increased GSH probably also played a role in determining cadmium chloride resistance of the PC-9/CDDP, even though this cell line had a reduced metallothionein level. Also contributing to the cisplatin resistance phenotype was a reduced intracellular level of platinum in the PC-9/CDDP. Thus, at least two distinct mechanisms have been selected in the resistant cells which confer the phenotype and allow degrees of cross resistance to other electrophilic drugs.     

Determinants of drug response in a cisplatin-resistant human lung cancer cell line

To elucidate the mechanism(s) of cisplatin resistance, we have characterized a human non-small cell lung cancer cell line (PC-9/CDDP) selected from the wild type (PC-9) for acquired resistance to cisplatin. PC-9/CDDP demonstrated 28-fold resistance to cisplatin, with cross resistance to other chemotherapeutic drugs including chlorambucil (X 6.3), melphalan (X 3.7) and 3-[(4-amino-2-methyl-5-pyrimidinyl)]methyl-1-(2-chloroethyl)-1-nitros our ea (ACNU) (x 3.9). There was no expression of mdr-1 mRNA in either wild-type or resistant cells. The mRNA and protein levels of glutathione S-transferase (GST) pi were similar in the two lines. A GST-mu isozyme was present in equal amounts and the activities of selenium-dependent and independent glutathione peroxidase and glutathione reductase were unchanged. The mRNA level of human metallothionein IIA and the total intracellular metallothionein levels were reduced in the resistant cells. Significantly increased intracellular glutathione (GSH) levels were found in the resistant cells (20.0 vs 63.5 nmol/mg protein) and manipulation of these levels with buthionine sulfoximine produced a partial sensitization to either cisplatin or chlorambucil. Increased GSH probably also played a role in determining cadmium chloride resistance of the PC-9/CDDP, even though this cell line had a reduced metallothionein level. Also contributing to the cisplatin resistance phenotype was a reduced intracellular level of platinum in the PC-9/CDDP. Thus, at least two distinct mechanisms have been selected in the resistant cells which confer the phenotype and allow degrees of cross resistance to other electrophilic drugs.     

Relationship of cellular glutathione to the cytotoxicity and resistance of seven platinum compounds.

The role of glutathione (GSH) in the effectiveness of and resistance to 7 platinum compounds [5 Pt(II) and 2 Pt(IV) drugs] was evaluated in a 8.6-fold cisplatin (CDDP)-resistant human small cell lung cancer cell line (GLC4/CDDP), the parent GLC4 line, a 3.7-fold CDDP-resistant human embryonal carcinoma cell line (Tera-CP), and the parent Tera line (NTera2/D1). Resistance factors for both CDDP-resistant cell lines were determined after continuous incubation (4 days) with CDDP. Continuous incubation with the other studied platinum drugs revealed complete cross-resistance for carboplatin (CBDCA) and zeniplatin but less for enloplatin (ENLO) and iproplatin in both models. Tetraplatin and lobaplatin showed, respectively, partial and complete cross-resistance in GLC4/CDDP but no cross-resistance in Tera-CP. GSH level, but not glutathione S-transferase activity, of the 4 cell lines correlated with platinum drug concentrations inhibiting cell survival by 50% after continuous incubation (r = 0.86, P < 0.05). GSH depletion by DL-buthionine-S,R-sulfoximine (BSO) increased sensitivity, as measured after a 4-h exposure to the drugs, of GLC4/CDDP for CDDP 2.0-fold, for CBDCA 1.7-fold, for zeniplatin 1.7-fold, and almost to the level of the sensitive GLC4 for ENLO, whereas no effect was observed for lobaplatin and the Pt(IV) compounds iproplatin and tetraplatin. BSO-modulating effect was higher in the sensitive GLC4 line for most compounds; therefore reduction of resistance could be achieved only for CDDP and ENLO. In contrast to GLC4, no modulation occurred in Tera. In Tera-CP BSO increased sensitivity for CDDP 1.5-fold, for CBDCA 1.9-fold, and for zeniplatin 1.2-fold; no effect was observed for ENLO, lobaplatin, and the Pt(IV) compounds. Reduction of CDDP resistance by BSO was known to occur with identical cellular platinum levels and higher Pt-DNA binding in GLC4/CDDP. However, pretreatment with BSO followed by 4 h ENLO incubation increased cellular platinum levels in both GLC4 and GLC4/CDDP while Pt-DNA binding remained unchanged. In conclusion, GSH reflected sensitivity to platinum-containing drugs. However, since the involvement of GSH differed between the models and the various platinum drugs, the effect of modulation with BSO was unpredictable.     

Retroviral transfer of MRP1 and gamma-glutamyl cysteine synthetase modulates cell sensitivity to L-buthionine-S,R-sulphoximine (BSO): new rationale for the use of BSO in cancer therapy

MRP1 (multidrug resistance protein 1) co-exports glutathione (GSH) and drug(s) and exports GSH, glucuronide, and sulphate-conjugated drugs. Human Fly-eco fibrosarcoma cells producing the MRP1-expressing retrovirus SF91MRP (Fly-eco MRP1), as well as 3T3 cells transduced with SF91MRP (3T3/MRP1), presented a decrease in intracellular GSH levels, as measured by two different methods. The enhanced export of GSH caused by the overexpression of MRP1 was partially counterbalanced by an increased rate of GSH synthesis. Fly-eco MRP1 and 3T3/MRP1 were hypersensitive to the GSH-depleting and cytotoxic activities of L-buthionine-S,R-sulphoximine (BSO), compared with their parental counterparts. In addition, the potentiation by BSO of the cytotoxic activity of chlorambucil and doxorubicin in Fly-eco MRP1 cells was greater than in parental Fly-eco cells. Although the turnover time of GSH, i.e. the theoretical time in which the entire GSH pool is resynthesised, was approximately 50% faster in Fly-eco MRP1 cells than in parental cells, this was not sufficient to fully restore the intracellular GSH level. In addition, mrp1 (-/-) mice were resistant to the GSH-depleting activity of intraperitoneally (i.p.) injected BSO, compared with mrp1 (+/+) mice. Co-transfer of the cDNAs for MRP1 and the heavy subunit of gamma-glutamyl cysteine synthetase (GCS) resulted in increased intracellular GSH levels and in high-level resistance to the GSH-depleting and cytotoxic activities of BSO. These data, and in particular the elevated single-agent cytotoxicity of BSO, provide a new rationale for the use of BSO in the treatment of MRP1-overexpressing tumours.     

Mechanism of cross-resistance to cisplatin in a mitomycin C-resistant human bladder cancer cell line

This study was undertaken to elucidate the mechanism(s) of cross-resistance to cisplatin (CDDP) in a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC. The J82/MMC celi line displayed 2- to 3-fold cross-resistance to CDDP and carboplatin when compared to the parental J82/WT cells. Drug uptake studies revealed that cross-resistance to CDDP in the J82/MMC cell line was independent of reduced platinum accumulation. The J82/MMC cetl line exhibited approximately a 1.5-fold resistance to cadmium chloride, an indicator for increased metallothionein (MT) content, when compared to the J82/WT cells. Northern blot analysis showed a 2.7-fold higher level of MT-II_A mRNA in the J82/MMC ceil line compared with J82/WT. We have reported previously that, whereas glutathi-one (GSH) level is comparable in these cells, GSH transferase (GST) activity is significantly higher in the J82/MMC eell line compared with J82/WT. Results of the present study showed that the elevated GST activity in the J82/MMC cell line was due to an over-expression of π-type GST protein. Although buthio-nine-S,R-sulfoximine (BSO)-induced GSH depletion significantly enhanced CDDP cytotoxicrty in both cell lines, the magnitude of potentiation was markedly higher in J82/MMC cells (about 2.1-fold) relative to J82/WT (about 1.6-fold). Our results suggest that cross-resistance to CDDP in the J82/MMC cell tine may be due to alterations in cellular thiols. © 1995 Wiley-Liss, Inc.     

The role of glutathione in resistance to cisplatin in a human small cell lung cancer cell line

The role of glutathione (GSH) in resistance to cisplatin (CDDP) was studied in a human small cell lung carcinoma cell line (GLC4) and a CDDP-resistant subline (GLC4-CDDP). In addition to studying the steady state of GSH, the kinetics of this defence system were also studied via the monitoring of the GSH status of the cells under continuous pressure of CDDP. GLC4-CDDP maintained its elevated GSH level whereas GLC4 (under pressure of CDDP) quickly synthesised GSH to about twice its initial level, corresponding with 80% of the GSH level of GLC4-CDDP. D,L-buthionine-S,R-sulphoximine (BSO) was used to analyse the role of GSH in resistance to CDDP. Pretreatment with BSO (48 h, 50 microM, GSH not detectable) increased the CDDP-induced cytotoxicity 2.8-fold in GLC4-CDDP and 1.7-fold in GLC4. In GLC4 no changes in the amount of platinum (Pt) bound to DNA could be observed after GSH depletion. Changes in formation of interstrand cross-links or the main Pt-containing intrastrand cross-link in digested DNA, the Pt-GG adduct, were also not observed. In GSH depleted GLC4-CDDP cells, an increase in the amount of Pt bound to DNA and in the Pt-GG adduct was observed. Pretreatment with BSO substantially reduced the repair of Pt bound to DNA in both cell lines. We conclude that an increased GSH level and GSH synthesis capacity were demonstrated in CDDP resistant cells. The observations after BSO treatment suggest two roles for GSH in CDDP resistance, namely that of a cytosolic elimination resulting in less DNA platination and a nuclear effect on the formation and repair of DNA platinum adducts.     

Overcoming tnf-alpha and cddp resistance of a human ovarian-cancer cell-line (c30) by treatment with buthionine sulfoximine in combination with tnf-alpha and or cddp

Previous studies have demonstrated that glutathione (GSH) plays an important role in a wide range of cellular functions including protection, detoxification, transport and metabolism. GSH has been implicated in tumor cell resistance to drugs and/or cytotoxic factors. Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, depletes intracellular GSH and thus could reverse resistance. The present study investigated the effect of BSO used in combination with tumor necrosis factor-alpha (TNF-alpha) or cisdiamminedichloroplatinum (II) (CDDP) on cytotoxicity of a TNF-alpha and CDDP resistant human ovarian cancer cell line (C30). Cytotoxicity was monitored by the MTT assay. Treatment of C30 cells with BSO and CDDP or BSO and TNF-alpha resulted in overcoming resistance and a synergistic cytotoxic effect was obtained. Pretreatment of the tumor cells by either agent for 4 h and wash and followed by the addition of the second agent for 20 h resulted in the same cytotoxicity as observed in the presence of the two agents. Furthermore, combination treatment with BSO, CDDP and TNF-alpha further augmented the synergistic cytotoxic activity achieved by two agents against C30 cells. The protective effect of GSH was shown for TNF-alpha but not for CDDP as treatment of C30 cells with TNF-alpha in combination with GSH or N-acetyl-cysteine (NAC) reduced the cytotoxic effect of TNF-alpha. One mechanism of resistance to TNF-alpha in tumor cells is through the induction of TNF-alpha mRNA and/or protein. The C30 cells did not constitutively express TNF-alpha mRNA, however, treatment of C30 cells with TNF-alpha upregulated the expression of TNF-alpha mRNA. When BSO was used in combination with TNF-alpha, the level of TNF-alpha mRNA induced by TNF-alpha was markedly reduced. Further, incubation of C30 cells with TNF-alpha in conjunction with GSH or NAC also downregulated the expression of TNF-alpha mRNA induced by TNF-alpha. These findings demonstrate that treatment with BSO in combination with TNF-alpha or CDDP can overcome the resistance of C30 tumor cells to TNF-alpha and CDDP. The depletion of intracellular GSH and downregulation of TNF-alpha mRNA by BSO may play a role in the enhanced cytotoxicity seen with the combination of BSO and TNF-alpha. The synergistic effect obtained with a CDDP selected resistant ovarian cancer cell line suggests that treatment with BSO in conjunction with either TNF-alpha or CDDP, or TNF-alpha and CDDP may have a clinical application in the therapy of TNF-alpha and/or CDDP resistant ovarian tumors'     

Effect of DNA-repair-enzyme modulators on cytotoxicity ofl-phenylalanine mustard andcis-diamminedichloroplatinum (II) in mammary carcinoma cells resistant to alkylating drugs

We investigated the effect of DNA-repair-enzyme inhibitors onl-phenylalanine mustard (L-PAM) andcis-diamminedichloroplatinum (II) (CDDP) cytotoxicity in rat mammary-carcinoma MatB cells sensitive (WT) and resistant (MLNr) to bifunctional alkylating drugs. Among the modulators tested, the combination of arabinofuranosylcytosine (Ara-C) and hydroxyurea (HU) significantly increased the sensitivity of the cells to CDDP and, to a lesser extent, L-PAM as compared with cells treated with drug alone. The modulation effect of HU+Ara-C on CDDP and L-PAM cytotoxicity was more effective when intracellular glutathione (GSH) was depleted byl-buthionine-(S,R)-sulfoximine (BSO). This was also associated with a significant increase in DNA-DNA interstrand crosslinks. Caffeine also sensitized both WT and MLNr cells to the cytotoxic effect of L-PAM and CDDP, and this effect was potentiated in GSH-depleted cells. No significant effect was observed with other repair modulators such as aphidicolin, 3-aminobenzamide, novobiocin, or etoposide. These results show (a) that inhibition of DNA repair by HU+Ara-C or caffeine could be a target for modulation of bifunctional alkylating-drug resistance and (b) that GSH depletion renders resistant cells more susceptible to the repair-enzyme modulators, suggesting that intracellular GSH may be involved in the regulation of some of these enzymes. Our results also indicate that a combination of a number of modulators may offer an advantage over the use of a single modulator in tumor resistance that may be associated with multifactorial mechanisms.Abbreviations 3-AB 3-Aminobenzamide - ADP adenosine diphosphate - APD aphidicolin - Ara-C arabinofuranosylcytosine - BSO 1--d-l-buthionine-(S,R)-sulfoximine - CDDP cis-diamminedichloroplatinum (II) (cisplatin) - GSH glutathione - GST glutathione-S-transferase - HU hydroxyurea - L-PAM l-phenylalanine mustard (melphalan) This work was supported in part by the Quebec Lung Association and the National Cancer Institute of Canada