The subunits of glutamate cysteine ligase enhance cisplatin resistance in human non-small cell lung cancer xenografts in vivo

Glutamate cysteine ligase (GCL) is a key enzyme in glutathione (GSH) synthesis, and is thought to play a significant role in the intracellular detoxification of anticancer drugs, especially of cisplatin (CDDP). GCL is composed of a modifier or light chain subunit (GCLM) and a catalytic or heavy chain subunit (GCLC). It was unclear whether the subunits are essential to CDDP-resistance. We examined the gene expression of GCLM and GCLC in 39 xenografts of human non-small cell lung cancer [NSCLC; 10 adenocarcinoma (Ad), 17 squamous cell carcinoma (Sq) and 12 large cell carcinoma (La)] by real-time polymerase chain reaction (PCR) with human-specific primers. Drug sensitivity to CDDP was evaluated in the 9 xenografts (4 Ad, 2 Sq and 3 La) using an in vivo drug sensitivity test. There was a significant association between the expression of GCLM and GCLC mRNA in each xenograft (Fisher's test, p<0.045). Squamous cell carcinoma xenografts significantly showed higher expression of GCLM gene than adenocarcinoma xenografts (p=0.023, t-test), while there was no significant difference in GCLC gene expression levels between each histopathological xenograft. Three of nine xenografts were sensitive to CDDP (Mann-Whitney U test, p<0.01, one-sided), while the other 6 xenografts were resistant. There was a significant relationship between drug sensitivity to CDDP and the co-overexpression of GCL subunits (chi2 test for independence, Yates' correction, p=0.014). These results suggested that the co-overexpression of GCL subunits correlated with CDDP-resistance in human NSCLC xenograft in vivo.     

The modifier subunit of glutamate cysteine ligase (GCLM) is a molecular target for amelioration of cisplatin resistance in lung cancer

It is unclear whether a subunit of glutamate cysteine ligase [a modifier subunit (GCLM) and a catalytic subunit (GCLC)] is an effective target for ameliorating cisplatin (CDDP)-resistance. We inhibited each subunit of GCL mRNA using a specific ribozyme (M-Rz and C-Rz) in the pulmonary adenocarcinoma cell line A549. GCL activity was suppressed by the ribozyme. CDDP-resistance was more effectively ameliorated when GCLM rather than GCLC was inhibited. GCLM is a potentially more effective pharmacologic target for ameliorating CDDP-resistance in non-small cell lung cancer than GCLC.     

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.     

Up-regulation of fas reverses cisplatin resistance of human small cell lung cancer cells

Background/Aim

Fas/FasL system is a major regulator of apoptosis. The mechanisms by which Fas mediates cisplatin resistance remain unclear. The aim of this study is to explore the effect of Fas over-expression on cisplatin resistance of small cell lung cancer cells and its possible mechanisms.

Materials and methods

Fas was over-expressed in H446/CDDP cells by infection with the adenoviruses containing Fas. Sensitivity of Fas-overexpressed H446/CDDP cells to cisplatin was evaluated using MTT assay. Expressions of Fas, GST-π and ERCC1 were detected by RT-PCR and Western blot analysis. Apoptosis rate was examined by FACS.

Results

Over-expression of Fas in H446/CDDP cells significantly decreased the expressions of GST-π and ERCC1 at mRNA and protein levels, and increased the cell apoptosis. Furthermore, up-regulation of Fas significantly decreased the tolerance of H446/CDDP cells to cisplatin.

Conclusion

Over-expression of Fas reverses drug resistance of H446/CDDP cells, possibly due to the increased cell sensitivity to apoptosis and the decreased expressions of GST-π and ERCC1.     

Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts.

Tumour cell adhesion, detachment and aggregation seem to play an important part in tumour invasion and metastasis, and numerous cell adhesion molecules are expressed by tumour cells. Several families of cell-cell adhesion molecules have been described, of which two groups are particularly well characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were examined. All tumours but one expressed both cadherin and NCAM. The tumours expressed one, two or rarely three cadherin bands, and different combinations of two major isoforms of NCAM with M(r)'s of approximately 190,000 and 135,000. Polysialylation of NCAM, a feature characteristic of NCAM during embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression.     

Influence of docosahexaenoic acid on cisplatin resistance in a human small cell lung carcinoma cell line

In a sensitive, human, small cell lung carcinoma cell line (GLC4) and a cisplatin (CP)-resistant subline (GLC4-CP), the effect of co-culturing with docosahexaenoic acid (DCHA) on CP cytotoxicity was studied. Cells were cultured for 4 days, with 32 microM of DCHA added on days 1 and 3. Incorporation of DCHA into the cellular phospholipids was demonstrated by fatty acid analysis. Supplementation with DCHA led to almost a threefold decrease of resistance in GLC4-CP and had no influence on CP cytotoxicity in GLC4. After culturing with DCHA, cellular platinum (Pt); total Pt bound to DNA; and Pt-GG, Pt-AG, G-Pt-G, and Pt-GMP adduct contents increased in both lines, whereas interstrand cross-link formation was elevated only in GLC4-CP. These experiments demonstrate that DCHA reduces CP resistance. Although an effect on cellular membranes resulting in an increased CP uptake apparently was present, this mechanism does not seem to be responsible for resistance modulation. Rather, an effect on nuclear, probably DNA-related, structures is likely and leads to an increased formation of interstrand cross-links in GLC4-CP.     

The in vitro effects of lonidamine combined with cisplatin in human small cell lung cancer cell lines.

Two human small cell lung cancer (SCLC) cell lines were used to evaluate the in vitro response to lonidamine and cisplatin exposure. The two cell lines both showed growth inhibition when exposed to lonidamine alone at concentrations greater than 50 micrograms/ml; however, one cell line (H69) was more sensitive. When cisplatin was combined with lonidamine a synergistic interaction was observed when cells were exposed to 10 microM cisplatin for 1 hour combined with lonidamine at concentrations of 50 micrograms/ml or greater. At a concentration of 25 micrograms/ml lonidamine combined exposure with cisplatin had no effect on cell growth or viability.     

Expression of copper-transporting P-type adenosine triphosphatase (ATP7B) correlates with cisplatin resistance in human non-small cell lung cancer xenografts

Copper-transporting P-type adenosine triphosphatase (ATP7B) is reportedly associated with platinum drug resistance in various solid carcinomas. However, the impact of ATP7B on platinum drug resistance in non-small cell lung cancer (NSCLC) remains unknown. We investigated ATP7B expression in nine human NSCLC xenografts using real-time polymerase chain reaction (PCR) and immunohistochemistry, and examined the relationship between the expression level of ATP7B and in vivo cisplatin (CDDP) sensitivity. ATP7B mRNA expression was significantly correlated with in vivo cisplatin sensitivity [coefficient of determination (R(2))=0.949, p=0.005]. ATP7B protein was detected in the nine xenografts. The ATP7B protein expression level was comparable to that of ATP7B mRNA. ATP7B mRNA and protein expression levels in the CDDP-resistant xenografts were significantly higher than those in the CDDP-sensitive xenografts (p=0.0389 and p=0.0357, respectively, Mann-Whitney U test). These results suggest that ATP7B is a CDDP-resistance marker in human NSCLC xenografts in vivo.     

Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin.

We have established cis-diamminedichloroplatinum(II) (cisplatin) resistant human small cell lung cancer cell lines, H69/CDDP0.2 and H69/CDDP, to investigate the mechanism of acquired resistance to cisplatin. H69/CDDP0.2 and H69/CDDP were 6- and 11-fold resistant to cisplatin compared with the H69 parental cell line. H69/CDDP was also resistant to cadmium chloride (2-fold), cis-diammine(glycolato)platinum (4-fold), 4-hydroperoxycyclophosphamide (3-fold) and 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosour ea (4-fold) if the drug concentrations that inhibit cell growth by 50% from growth inhibition assay were compared. There was no significant difference in the cisplatin accumulation among these cell lines. Although DNA interstrand cross-link formations, determined by filter elution assay in H69/CDDP0.2 and H69/CDDP, was decreased to 20 to 30% of that in H69 parental cells, the repair capacity of DNA interstrand cross-links was equivalent in all three cell lines. Intracellular glutathione content was equal in all cell lines. H69/CDDP had the highest glutathione S-transferase activity (H69, 11 nmol/min/mg protein, H69/CDDP0.2, 12 nmol/min/mg protein; H69/CDDP, 74 nmol/min/mg protein, respectively) and an overexpression of glutathione S-transferase pi mRNA. The drug concentrations that inhibit cell growth by 50% for cisplatin in all cell lines were decreased by treatment with ethacrynic acid, an inhibitor of glutathione S-transferase pi, but this did not alter the relative degree of resistance. Intracellular metallothionein content (H69, 14 pmol/mg protein, H69/CDDP0.2, 22 pmol/mg protein; H69/CDDP, 33 pmol/mg protein, respectively) and expression of metallothionein mRNA were correlated with the drug concentrations that inhibit cell growth by 50% of the three cell lines for cisplatin and cadmium chloride. The present study suggested the importance of metallothionein in the mechanisms of cisplatin resistance.     

Effect of lung resistance-related protein on the resistance to cisplatin in human ovarian cancer cell lines

The mechanisms of drug-resistance in human ovarian cancer cells have not been entirely clarified. The purpose of this study was to investigate whether LRP is involved in the resistance of ovarian cancer cell lines to cisplatin and its molecular mechanism. Human ovarian cisplatin-resistant cancer cell lines (A2780/DDP and COC1/DDP) and their parental cisplatin-sensitive cell lines (A2780 and COC1), alone or transfected with antisense LRP-specific oligonucleotides (ODN) or sense ODN, were treated with cisplatin to induce differentiation. Expression of LRP was examined by RT-PCR and Western blot analysis. The sensitivities of cells to cisplatin were assessed using sulforhodamine B (SRB) assay and flow cytometry, and the accumulation and efflux of cisplatin in the cells and isolated nuclei were examined by high performance liquid chromatographic (HPLC) assay. The expressions of LRP in A2780/DDP and COC1/DDP cells were higher than those in A2780 and COC1 cells and conferred resistance to cisplatin. Transfection of LRP AsODN into A2780/DDP and COC1/DDP cells down-regulated LRP expression and reversed the resistance phenotype. Levels of cisplatin accumulating in cells were increased by LRP-specific AsODN and anti-LRP monoclonal antibody. Isolated nuclei from A2780 and COC1 cells or A2780/DDP and COC1/DDP cells incubated with anti-LRP antibody contained more cisplatin than the nuclei of A2780/DDP and COC1/DDP cells not treated with anti-LRP antibody. Efflux of cisplatin was greater from the nuclei of A2780/DDP and COC1/DDP cells than those of A2780 and COC1 cells, and was inhibited by anti-LRP monoclonal antibody. Thus, LRP was involved in the resistance of ovarian cancer cells to cisplatin and has an important role in the transport of cisplatin both in exocytotic vesicles and between the nucleus and cytoplasm.     

Inhibition of glutamate cysteine ligase activity sensitizes human breast cancer cells to the toxicity of 2-deoxy-D-glucose

It has been hypothesized that cancer cells increase glucose metabolism to protect against metabolic fluxes of hydroperoxides via glutathione-dependent peroxidases. 2-Deoxy-D-glucose, inhibits glucose metabolism and has been shown to cause cytotoxicity in cancer cells that is partially mediated by disruptions in thiol metabolism. In the current study, human breast cancer cells were continuously treated (24 hours) with 2-deoxy-D-glucose, and total glutathione content as well as the expression of the first enzyme in the glutathione synthetic pathway [glutamate cysteine ligase (GCL)] were found to be induced 2.0-fold. Inhibiting GCL activity during 2-deoxy-D-glucose exposure using l-buthionine-[S,R]-sulfoximine (BSO) significantly enhanced the cytotoxic effects of 2-deoxy-D-glucose and caused increases in endpoints indicative of oxidative stress, including % oxidized glutathione and steady-state levels of pro-oxidants as assayed using an oxidation-sensitive fluorescent probe. These results show that treatment of human breast cancer cells with 2-deoxy-d-glucose causes metabolic oxidative stress that is accompanied by increases in steady-state levels of GCL mRNA, GCL activity, and glutathione content. Furthermore, inhibition of 2-deoxy-D-glucose-mediated induction of GCL activity with BSO increases endpoints indicative of oxidative stress and sensitizes cancer cells to 2-deoxy-D-glucose-induced cytotoxicity. These results support the hypothesis that drug combinations capable of inhibiting both glucose and hydroperoxide metabolism may provide an effective biochemical strategy for sensitizing human cancer cells to metabolic oxidative stress.     

Resistance to alkylating agents and tumour differentiation in xenografts of small cell lung cancer

Small cell bronchial carcinoma (SCC) xenografts with differing sensitivity to cyclophosphamide (CY) were investigated using a variety of techniques. Two xenografts (HX78 and HX88) were relatively sensitive to CY, one xenograft (HX72) was inherently resistant to CY and a fourth xenograft (HX78Cy) was a CY induced resistant subline of HX78 and was unstable when maintained without CY exposure. Conventional light microscopy, cytology and electron microscopy examination of the xenografts revealed appearances consistent with SCC. An antikeratin antibody demonstrated the presence of keratin in the inherent and the induced resistant xenografts (and in the unstable revertant) but not in the two sensitive xenografts; the presence of keratin suggested squamous differentiation. Monolayer culture morphology of the sensitive HX78 and the unstable revertant was anchorage independent with the cells forming floating aggregates; the induced resistant subline of this xenograft (HX78Cy) showed, by contrast, flattened, angular adherent cells. Beta HCG production was detected in the monolayer culture supernatant of sensitive HX78 cells; the level of production of beta HCG was increased in the induced resistant HX78Cy cells. Karyotype and flow cytometry studies were also performed. The morphological responses of small cell lung cancer to treatment are discussed.     

HA14-1联合顺铂诱导人小细胞肺癌细胞凋亡的研究

目的:观察HA14-1及其联合顺铂对人小细胞肺癌NCI-H446细胞凋亡诱导作用并探讨其可能的作用机制,探讨其对Bcl-2、Bax表达的的影响。方法:选用人小细胞肺癌NCI-H446细胞株为研究对象,分为空白组、DDP组、HA14-1组及DDP+HA14-1组,分别作用24、48 h后,采用MTT法检测细胞抑制率,流式细胞仪检测细胞凋亡,Real time PCR法比较细胞内Bcl-2、Bax表达变化。结果:随着HA14-1及DDP浓度的增加,细胞抑制率逐渐增加,细胞凋亡率增高,联合用药组较单独用药组细胞的抑制率,凋亡率均增高,HA14-1作用细胞后,Bcl-2表达水平下降,Bax表达水平增高。结论:HA14-1可以诱导人小细胞肺癌凋亡,并增加肺癌细胞对DDP的化疗作用。     

Multidrug resistance in a human small cell lung cancer cell line selected in adriamycin

A multidrug resistant variant (H69AR) of the human small cell lung cancer cell line NCI-H69 was obtained by culturing these cells in gradually increasing doses of Adriamycin up to 0.8 microM after a total of 14 months. H69AR expresses the multidrug resistant phenotype because it is cross-resistant to anthracycline analogues including daunomycin, epirubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vinblastine. H69AR is also similar to other multidrug resistant cell lines in that it displays little or no cross-resistance to bleomycin, 5-fluorouracil, and carboplatin. It has a slight collateral sensitivity to 1-dehydrotestosterone and lidocaine. H69AR has increased cell-cell adhesiveness compared to H69, but a similar growth rate in vitro and tumorigenicity in nude mice. When cultured in the absence of Adriamycin, there is a 40% decrease in resistance by 35 days of culture, compared to cells in continuous culture in drug, but no further decrease in resistance up to 181 days. Monoclonal antibodies to P-glycoprotein have no detectable reactivity with H69AR cells as determined by enzyme-linked immunosorbent assay and immunoblotting techniques. Thus, unlike most multidrug resistant cell lines, H69AR does not appear to express enhanced levels of P-glycoprotein. H69AR will provide a useful model for the study of multidrug resistance in human small cell lung cancer.     

Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells

Cisplatin produces good responses in solid tumours including small cell lung cancer (SCLC) but this is limited by the development of resistance. Oxaliplatin is reported to show activity against some cisplatin-resistant cancers but there is little known about oxaliplatin in SCLC and there are no reports of oxaliplatin resistant SCLC cell lines. Studies of drug resistance mainly focus on the cellular resistance mechanisms rather than how the cells develop resistance. This study examines the development of cisplatin and oxaliplatin resistance in H69 human SCLC cells in response to repeated treatment with clinically relevant doses of cisplatin or oxaliplatin for either 4 days or 2 h. Treatments with 200 ng/ml cisplatin or 400 ng/ml oxaliplatin for 4 days produced sublines (H69CIS200 and H69OX400, respectively) that showed low level (approximately two-fold) resistance after eight treatments. Treatments with 1,000 ng/ml cisplatin or 2,000 ng/ml oxaliplatin for 2 h also produced sublines, however, these were not stably resistant suggesting shorter treatment pulses of drug may be more effective. Cells survived the first five treatments without any increase in resistance, by arresting their growth for a period and then regrowing. The period of growth arrest was reduced after the sixth treatment and the H69CIS200 and H69OX400 sublines showed a reduced growth arrest in response to cisplatin and oxaliplatin treatment suggesting that ‘regrowth resistance’ initially protected against drug treatment and this was further upregulated and became part of the resistance phenotype of these sublines. Oxaliplatin dose escalation produced more surviving sublines than cisplatin dose escalation but neither set of sublines were associated with increased resistance as determined by 5-day cytotoxicity assays, also suggesting the involvement of regrowth resistance. The resistant sublines showed no change in platinum accumulation or glutathione levels even though the H69OX400 subline was more sensitive to buthionine sulphoximine treatment. The H69CIS200 cells were cross-resistant to oxaliplatin demonstrating that oxaliplatin does not have activity against low level cisplatin resistance. Relative to the H69 cells, the H69CIS200 and H69OX400 sublines were more sensitive to paclitaxel and taxotere suggesting that the taxanes may be useful in the treatment of platinum-resistant SCLC. These novel cellular models of cisplatin and oxaliplatin resistant SCLC will be useful in developing strategies to treat platinum-resistant SCLC.     

新城疫病毒对小细胞肺癌裸鼠移植瘤生长的抑制作用

目的:探讨新城疫病毒(Newcastle disease virus, NDV)7793株在体内对肿瘤生长的抑制作用及机制.方法:体外培养人小细胞肺癌NCI-H446细胞,通过皮下接种NCI-H446细胞建立人小细胞肺癌裸鼠移植瘤模型.NDV组裸鼠瘤内注射新城疫病毒,阳性对照组注射顺铂(cisplatin,DDP),阴性对照组注射PBS.观察移植瘤生长情况和裸鼠体质量变化,绘制肿瘤生长曲线.6周后处死裸鼠剥取肿瘤组织,HE染色观察细胞学变化,免疫组织化学法检测移植瘤细胞Bax和Bcl-2蛋白表达情况.结果:NDV注射对肿瘤生长有显著抑制作用,抑瘤率达34.1%;NDV对荷瘤鼠无不良反应;光学显微镜下观察发现,NDV治疗组肿瘤组织有许多散在分布的凋亡/坏死区域.NDV能明显上调Bax蛋白的表达量(P＜0.01),对Bcl-2蛋白表达则无影响.结论:新城疫病毒7793株在体内能够有效抑制移植瘤生长,其抗肿瘤机制可能与上调Bax蛋白的表达有关.     

Acquired resistance to cisplatin and doxorubicin in a small cell lung cancer cell line is correlated to elevated expression of glutathione-linked detoxification enzymes

A human small cell lung cancer cell line, U-1906, developedaltered functional properties upon continuous in vitro cultivation.Cells obtained at late (U-1906 L) and early (U-1906 E) passagesof cultivation differ in drug resistance to the cytostatic therapeuticagents cisplatin and doxorubicin. The U-1906 L cells are 1.6-foldand 1.3-fold more resistant to cisplatin and doxorubicin respectively,than are the U-1906 E cells. In the more resistant U-1906 Lcells, the total glutathione (GSH plus GSSG) level is 40% lower,whereas the activities of GSH-linked enzymes such as GSH peroxidaseand GSH transferases are significantly higher. Quantitativeanalysis with isoenzyme-specific ELISAs demonstrated increasedconcentrations of all three of the measurable GSTs, M1-1, M3-3and P1-1, in the more resistant cells. The Intracellular proteinexpression patterns of the U-1906 E and the U-1906 L cells arevery similar as revealed by two-dimensional denaturing electrophoresis,but show significant alterations in the concentrations of somecomponents. Two 35 kDa proteins of different pI values, theconcentrations of which are increased in the U-1906 L cells,were both identified as glyceraldehyde-3-phosphate dehydrogenase,either by N-terminal or by internal amino acid sequence analysis.The present study demonstrates that the increased resistanceof the U-1906 L cells may involve multiple detoxification mechanismsand that the contribution of the GSH-linked detoxification canbe ascribed to the elevation of cytosolic GST lsoenzymes, GSHperoxidase and glutathione reductase, rather than to the IntracellularGSH concentrations.