Reversing adriamycin resistance of human breast cancer cells by hyperthermia combined with Interferon alpha and Verapamil

One of the major obstacles related to chemotherapy is resistance against anticancer drugs, including Adriamycin (ADM). The purpose of the present work is to investigate the reversal effects on ADM resistance by hyperthermia (42.5 degrees C) combined with two reversal agents (Interferon alpha and Verapamil) in MCF-7/ADR (ADM-resistant MCF-7 breast cancer cell line), and its relevant molecular mechanism of action. The cell survival rate and ADM IC50 of different experiment groups were measured by MTT test. The quantitative expression of MDR1 gene in cells was detected by Real-time PCR, and the expression of P-glycoprotein (P-gp) on the cells surface and the intracellular ADM accumulation was detected by flow cytometry (FCM). The ADM IC50 of the MCF-7/ADR cells decreased 830-fold after combined with Interferon alpha (IFN-alpha) and Verapamil (VRP). Although there was no distinction in the mRNA expression of MDR1, the P-gp on the MCF-7/ADR cell membrane was significantly reduced and the cellular ADM uptake increased markedly as compared to pretreatment. Our results suggeste that hyperthermia induces a considerably reversal activity against ADM resistance synergizing other reversal agents (IFN-alpha and VRP). The reversal mechanism needs further study. However, these features of hyperthermia may be exploited in clinical cancer chemotherapy.     

Combined effect of navelbine with medroxyprogesterone acetate against human breast carcinoma MCF-7 cells in vitro.

Navelbine (NVB, vinorelbine ditartrate, KW-2307), a new vinca alkaloid analogue, has been shown to be clinically effective against advanced breast cancer. In this report, the combined effect of NVB with medroxyprogesterone acetate (MPA), a synthetic progesterone derivative, was examined in vitro against human breast carcinoma MCF-7 cells. The combined effect was demonstrated to be synergistic using the isobologram and median-effect plot analyses. To elucidate the mechanism of action, we further examined effects of both drugs on cell cycle distribution of the cells in combination and/or alone. NVB at 2 nM induced apparent G1-phase accumulation as well as the induction of cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1) protein and the dephosphorylated form of retinoblastoma protein (pRb). In contrast, MPA at 0.1 microM also induced G1-phase accumulation as well as the reduced expression of cyclin D1 protein. In addition, the combination of both drugs induced augmented G1-phase accumulation, which occurred along with p21(WAF1/CIP1) protein induction, cyclin D1 protein reduction and pRb dephosphorylation. These results demonstrate that the synergistic combined effect of NVB with MPA was mediated through enhancement of G1-phase accumulation that resulted from the different action point(s) of each drug. Furthermore, the synergistic combined effect of NVB with MPA was also observed in other human breast carcinoma cell lines, such as T-47D and ZR-75-1. These results suggest that combination therapy of NVB with MPA in breast cancer might be effective in clinical studies.     

人参皂甙Rh2逆转P-gp介导的MCF-7/ADM多药耐药性的基础研究

目的：研究人参皂甙Rh：在逆转多药耐药（MDR）方面的作用及其机理。方法：Rh：和阿霉素单独或联合作用于MCF-7及MCF-7／ADM细胞，应用MTT法确定各组细胞的IC50。罗丹明123加入各组细胞，流式细胞仪分析Rh2和维拉帕米抑制罗丹明123外排的情况。RT—PCR检测各组mdrl mRNA表达量及流式细胞仪检测各组P—gP的表达情况。结果：Rh2可以显著降低MCF-7／ADM的ADM IC50（P〈0．05），对MCF-7细胞没有影响。Rh2和维拉帕米可以增加MCF-7／ADM细胞内罗丹明123荧光表达率（P〈0．05），Rh3比维拉帕米抑制罗丹明123外排作用更强。在MCF-7细胞内Rh2和维拉帕米无此作用。Rh2对MCF-7／ADM细胞mdrl mRNA表达及P—gP表达没有影响。结论：人参皂甙Rh2可以有效逆转P—gP介导的人乳腺癌细胞耐药细胞系MCF-7／ADM的耐药性。     

microRNA-21对乳腺癌MCF-7/ADR细胞株多柔比星耐药性影响的研究

目的:探讨microRNA-21(miR-21)基因表达改变对乳腺癌细胞多柔比星化疗耐药的影响.方法:人工合成miR-21模拟序列或干扰序列,以脂质体为载体,转染乳腺癌细胞MCF-7及其多柔比星耐药细胞株MCF-7/ADR;应用荧光定量RT-PCR检测miR-21的表达;应用MTT法检测细胞转染前后对多柔比星的耐药性;应用流式细胞仪检测细胞凋亡;应用蛋白质印迹法检测PTEN、BAX及Bcl-2蛋白的表达.结果:MTT检测结果示,MCF-7及其耐药株MCF-7/ADR细胞多柔比星半数抑制浓度(IC50)分别为(0.28±0.03)和(17.5±0.12) μmol/L.miR-21表达上调,MCF-7细胞对多柔比星的IC50明显增高为(3.65±0.12) μmol/L;miR-21表达下调,MCF-7/ADR细胞对多柔比星的IC50明显降低为(7.53±0.11) μmol/L.流式细胞分析显示,miR-21下调后MCF-7/ADR细胞凋亡率明显增加.同时,细胞内PTEN、BAX蛋白表达水平增加,Bcl-2表达降低.结论:miR-21在乳腺癌化疗耐药中具有重要作用,抑制miR-21的表达可以逆转乳腺癌多柔比星耐药细胞株对多柔比星的耐药性.     

Natural interferon-alpha activity in hormone-sensitive,hormone-resistant and autonomous human breast-cancer cell lines

This study explored the activity of natural interferon-alpha (nIFN-α) in regulating cell growth of 6 breast-cancer cell lines. The anti-proliferative effect of the combination nIFN-α and tamoxifen (TAM) or medroxyprogesterone acetate (MPA) in CG-5 estrogen-sensitive mammary cancer cells was investigated, and the ability of nIFN-α to restore hormone-sensitivity in the MPA-resistant MCF-7 SK sub-line was examined. nIFN-α, at concentrations ranging from 10 to 1000 IU/ml, inhibited cell proliferation of all cell lines tested after 3 and 6 days of treatment. In particular, the highest concentration of the drug used was equally effective in hormone-sensitive and in hormone-insensitive cells. A 6-day pre-treatment of CG-5 cells with nIFN-α, at the above-mentioned doses, sensitized them to the growth-inhibiting activity of subsequent exposure to 10⁻⁷ M TAM or MPA, which resulted in a synergistic effect, and could be explained on the basis of the observed enhancement of estrogen and progesterone receptors due to IFN activity. Conversely, the simultaneous drug combination did not modify the response to the hormone in CG-5 cells. Pre-treatment with nIFN-α (from 10 to 1000 IU/ml) restored MPA sensitivity in the MCF-7 SK sub-line, but no modulation of progesterone receptors was seen in this model. The hormone-sensitivity of the parental cell line was not substantially affected by pre-exposure to nIFN-α. These data indicate that nIFN-α may be potentially useful in enhancing the clinical effectiveness of TAM and MPA and in overcoming hormone resistance.Int. J. Cancer 71: 1103-1108, 1997. © 1997 Wiley-Liss Inc.     

Adriamycin action on human breast cancer cells: enhancement by medroxyprogesterone acetate

Exposure of cultured human breast cancer cells (MCF-7) to medroxyprogesterone acetate (MPA) prior to treatment with adriamycin (ADR) enhanced the effect of the cytotoxic drug on cell yield after further growth of viable cells. Pretreatment with oestradiol (E2) produced similar synergism, but no such effect was observed using dexamethasone. Exposure to ADR before MPA led to an additive effect, but not to synergism. When cells resistant to the growth-inhibitory action of MPA were examined, progestogen/drug synergism could still be demonstrated. We conclude that the effects of ADR, like those of methotrexate and vincristine, can be enhanced by pre-treatment with MPA and that further clinical trials of such a regimen are needed.     

Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells

The effect of medroxyprogesterone acetate (MPA) on breast cancer cell proliferation kinetics was investigated in ten human breast cell lines growing as monolayer cultures. Significant inhibition of growth occurred only in the estrogen receptor-positive, progesterone receptor-positive cell lines, T-47D, MCF-7, ZR 75-1, BT 474, and MDA-MB-361. Among these cell lines sensitivity to MPA varied widely; concentrations required for 20% inhibition of growth ranged from 0.04 nM for T-47D to greater than 100 nM for ZR 75-1 cells. Furthermore, although the most sensitive line, T-47D, had the highest level of PR, sensitivity to MPA was not correlated with PR levels among the responsive cell lines. More detailed studies were undertaken with the T-47D cell line. The growth-inhibitory response was confined to the progestins: MPA, ORG 2058, R5020, and progesterone, while androgens, estrogens, and glucocorticoids were without effect over the same concentration range (0.1-100 nM). MPA-induced growth inhibition was associated with a significant decrease in the proportion of S-phase cells with an accumulation of cells in the G0-G1 phase of the cell cycle. Cells began to accumulate in G0-G1 after 12 h of drug treatment and the effect was maximal by 24 h, i.e., maximal effects were observed during the first cell cycle following drug treatment. By contrast, significant accumulation in G0-G1 required exposure of MCF-7 cells to MPA for at least two cell cycle times, i.e., 48 h and the effect was still increasing at 96 h. Stathmokinetic studies revealed that in both cell lines accumulation in the G0-G1 phase was due to an MPA-induced increase in the G1 transit time. These data indicate that MPA and other progestins have direct growth inhibitory effects on estrogen receptor-positive and progesterone receptor-positive human breast cancer cells in vitro and these effects can be accounted for by a decrease in the rate at which cells traverse the G1 phase of the cell cycle.     

植物多酚类化合物抑制人乳腺癌MCF-7/ADM细胞的筛选

目的研究从儿茶素、槲皮素、大豆苷元、芝麻素、阿魏酸和白藜芦醇等六种植物多酚类化合物,筛选出对人乳腺癌MCF-7/ADM细胞具有抑制作用的药物。方法MTT法初筛对MCF-7/ADM细胞具有毒性作用的植物多酚类化合物;再通过均匀设计筛出无毒性剂量时联合多柔比星（阿霉素）对MCF-7/ADM细胞抑制作用最强的植物多酚类化合物;最后经单细胞凝胶电泳检测筛出后的植物多酚类化合物联合阿霉素对正常肝细胞的毒性作用。结果槲皮素、白藜芦醇、阿魏酸能抑制MCF-7/ADM细胞增殖;经均匀设计后认为白藜芦醇联合阿霉素对MCF-7/ADM细胞的抑制作用比其余两者明显;单细胞凝胶电泳试验证明白藜芦醇联合阿霉素对正常肝细胞无毒性作用。结论白藜芦醇联合阿霉素对人乳腺癌MCF-7/ADM细胞具有抑制作用,且基本无毒害作用。     

Reversal of multidrug resistance by a novel quinoline derivative,MS-209

MS-209, a novel quinoline derivative, was examined for its reversing effect on multidrug-resistant tumor cells. MS-209 at 1–10 M completely reversed resistance against vincristine (VCR) in vitro in multidrug-resistant variants of mouse leukemia P388 cells (VCR-resistant P388/VCR and Adriamycin (ADM)-resistant P388/ADM) and human leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM). MS-209 at 1–10 M also completely reversed resistance against ADM in vitro in P388/VCR cells, K562/VCR cells, and K562/ADM cells. In ADM-resistant P388 (P388/ADM) cells, however, ADM resistance was only partially reversed at the MS-209 concentrations tested. MS-209 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When MS-209 was given p.o. at 80 mg/kg twice a day (total dose, 160 mg/kg per day) with 100 g/kg VCR, a treated/control (T/C) value of 155% was obtained. MS-209 also enhanced the chemotherapeutic effect of ADM in P388/ADM-bearing mice. The most prominent effects were obtained when MS-209 was given with 2 mg/kg ADM, yielding T/C values of 150%–194% for the combined treatment at an MS-209 dose of 200–450 mg/kg. MS-209 inhibited [³H]-azidopine photolabeling of P-glycoprotein efficiently. Furthermore, the accumulation of ADM in K562/ADM cells was increased more eficiently by MS-209 than by verapamil. These results indicate that MS-209, like verapamil, directly interacts with P-glycoprotein and inhibits the active efflux of antitumor agents, thus overcoming multidrug resistance in vitro and in vivo.This work was supported by grants-in-aid from the Ministry of Education Science and Culture, Japan     

甲基莲心碱对耐阿霉素人乳腺癌细胞多药耐药性的逆转

Objective: To study the reversal effect of neferine on adriamycin (ADM) resistant human breast cancer cell line MCF-7/ADM. Methods: The cytotoxic effect of Nef or ADM was determined by 3-[4, 5-dimethylthiazol-2.-yl], 5-diphenyl tetraxolium bromid (MTT) assay. Apoptosis and the expression of P-glycoprotein (P-gp) were detected by flow cytometry (FCM). The intracellular ADM concentration was measured by HPLC. Results: Nef at 1, 5, 10 mol/L decreased the IC₅₀ of ADM to MCF-7/ADM from 11.63 g/mL to 4.59, 2.44, 0.27 g/mL respectively. MCF-7/ADM could resist the apoptosis induced by ADM while Nef (1-10 mol/L) could augment ADR-mediated apoptosis. Nef (10 mol/L) increased the accumulation of ADM up to 2.88 fold in MCF-7/ADM but not in sensitive cells MCF-7/S and reduced the expression of P-gp in MCF-7/ADM cells. Conclusion: Nef can circumvent multidrug resistance (MDR) of MCF-7/ADM cells and the mechanism was associated with the increase of intracellular accumulation of ADM and the reduced expression of P-gp in MCF-7/ADM cells.     

Acute Induction of Adriamycin-resistance in Human Colon Carcinoma HT-29 Cells Exposed to a Sublethal Dose of Adriamycin

To study the mechanisms of the acute induction of drug resistance in cancer cells, we have established a model system in which adriamycin (ADM) induces immediate drug resistance. In this system, human colon carcinoma HT-29 cells were pretreated for 1 h with a subtoxic dose of ADM (0.3 μg/ml) and incubated for 24 h in drug-free medium. Then the cells were treated for 1 h with ADM, and the cell survival was determined in terms of colony-forming ability. The survival of the pretreated cells was increased up to 100-fold, as compared with that of untreated cells. Such increased survival, however, was observed only after high doses of ADM (2 to 8 μg/ml); more than 99% of the cells were killed. These results indicate that only a small fraction of ADM-pretreated cells acquire the ADM-resistant phenotype. Similar induced resistance was observed in five of seven subclones isolated from HT-29 cells by limiting dilution, suggesting that the majority of cells in the parental HT-29 population could acquire the ADM-resistant phenotype. In the subclone HT-29T9, the ADM pretreatment induced concomitant resistance to daunomycin, VP-16, and VM-26 but not to agents other than topoisomerase II inhibitors. The ADM-induced drug resistance did not accompany MDR1 gene expression and could not be overcome by verapamil, a P-glycoprotein inhibitor. The present system could be useful to study the acute induction mechanism(s) of ADM-resistance, which could be relevant to clinical resistance in patients.     

乳腺癌细胞耐药过程中p38MAPK活性与细胞凋亡的关系

目的研究乳腺癌细胞耐药过程中p38MAPK活性与细胞凋亡的关系,探讨p38MAPK信号转导途径在其中的作用。方法 以p38MAPK特异性抑制剂SB203580处理乳腺癌耐药细胞MCF-7／ADM,采用流式细胞技术分析对细胞凋亡的影响;MTT检测MCF-7／ADM细胞对阿霉素的半数药物抑制浓度（IC50）;Western blot检测SB203580处理MCF-7／ADM和MCF-7两株细胞后p38MAPK蛋白表达水平;RT—PCR检测细胞内MDR-1 mRNA水平。结果SB203580（10μmol／L）干预24h后MCF-7／ADM细胞的凋亡率为（26．73±4．90）％,与未干预组和对照组凋亡率相比差异有显著统计学意义（F=143．80,P〈0．001）;MCF-7／ADM细胞对阿霉素的敏感性明显提高（F=148927．10,P〈0．001）,相对逆转率达68．45％;与对照组和未干预组相比,干预组的MDR1 mRNA（F=9139．24,P〈0．001）及p38MAPK（F=685．42,P〈0．001）蛋白表达水平明显降低。结论p38MAPK信号转导途径与乳腺癌耐药密切相关,其可能机制为p38MAPK保护人乳腺癌耐药细胞（MCF-7／ADM）逃避凋亡,阻断该通路可增强乳腺癌耐药细胞发生凋亡。     

Apoptosis inhibition mediated by medroxyprogesterone acetate treatment of breast cancer cell lines

Several reports suggested that steroidogenic hormones could be directly involved in the regulation of apoptosis in vitro, but whether this is due to blocking or promoting mechanism of these hormones remains controversial. However, it was shown that progesterone exhibited a protective effect against the apoptotic process during mouse mammary gland involution in vivo. In this study, we analyzed the effect of medroxyprogesterone acetate (MPA) treatment, an agonist of progesterone, on serum starvation induced apoptosis on breast cancer cell lines. Positive and negative progesterone receptor (PgR+ and PgR–) breast cancer cell lines were treated with MPA (10nM), either in standard culture conditions or in serum-free medium to induce apoptosis. Cell survival, proliferation and apoptosis were simultaneously analyzed with the expression of apoptosis-related genes measured by a real time quantitative RT-PCR. At non cytotoxic doses, MPA protected PgR+ T47-D, MCF-7 and H466-B cell lines against serum depletion-induced apoptosis, while MPA did not protect PgR-MDA-MB-231 cells against serum depletion induced apoptosis. In PgR+ cell lines and in concordance with the protective effect, the pro-apoptotic HRK and BAK1 mRNAs were up-regulated after apoptosis induction, while they were no more induced in condition of protection against apoptosis after MPA treatment. We also observed, specifically in PgR+ cells, an up-regulation of BCLX-L and BCLX-S and a down-regulation of BCL2 mRNAs, which are specific to the MPA response and unrelated to apoptotic process. Involvement of these genes with regard to the MPA-mediated protection against apoptosis is discussed.     

Effect of a chimeric anti-ganglioside GM2 antibody on ganglioside GM2-expressing human solid tumors in vivo.

Ganglioside GM2 is expressed on the surface of neuroblastoma and glioblastoma cells, and may also be detected on lung cancer cells. We reported previously that anti-ganglioside GM2 antibody exhibited strong in vitro anti-tumor activity against adriamycin-resistant cancer cells, which overexpressed ganglioside GM2. In the present study, we examined the in vivo anti-tumor effect of the chimeric anti-ganglioside GM2 antibody, KM966, against human lung and breast carcinoma cells, SBC-3 and MCF-7, and respective adriamycin-resistant clones, SBC-3/ADM and AdrR MCF-7 in BALB/c nu/nu mice. Ratios of tumor volume (T/C) between KM966-treated group and control group were 0.01 for SBC-3, 0.00 for SBC-3/ADM, 0.85 for MCF-7 and 0.34 for AdrR MCF-7 cells, respectively. Nude mice, which were pretreated with anti-asialo GM1 antibody to remove natural killer cells, were transplanted with 4 x 10(7) of SBC-3 and SBC-3/ADM subcutaneously. Seven days later, when tumors had grown to a diameter of over 8 mm, mice began to receive intravenous treatment of 120 microgram/mouse KM966 daily. Fourteen daily treatments induced regression to less than 4-mm diameter in 4/5 SBC-3 tumors and 5/5 of SBC-3/ADM tumors. All SBC-3/ADM tumors disappeared completely, suggesting that KM966 exerts a strong in vivo anti-tumor effect on ganglioside GM2-expressing cancer cells. In KM966-treated mice, the surface of the tumor cells stained positive with anti-human IgG. In addition, numerous leukocytes had infiltrated into the tumor mass. Antibody-dependent cell-mediated cytotoxicity (ADCC) of KM966 against tumor cells was examined in vitro by (51)Cr-release assay and revealed that KM966 induces ADCC activity against ganglioside GM2-expressing tumors. Our results suggest that immunotherapy using KM966 may be useful for the treatment of ganglioside GM2-expressing solid tumors.     

新型光敏剂联合阿霉素对乳腺癌细胞增殖及周期的影响

目的:研究新型光敏剂叶绿素衍生物(Chlorophyl derivative4,CPD4),联合阿霉素对人乳腺癌MCF-7细胞增殖及周期的影响,初步探讨联合用药的作用机制,为临床开辟新的治疗方法提供实验依据.方法:以人乳腺癌MCF-7细胞系为研究对象,新型光敏剂和乳腺癌传统化疗药物阿霉素(ADM)联合给药.采用流式细胞仪检测ADM组(2个浓度组分别预处理24小时、48小时)、光动力效应组、联合用药组细胞凋亡率和周期分布:流式细胞仪分析ADM(20ng/mL)预处理24小时、48小时对细胞平均荧光强度的影响以及ADM预处理24小时、48小时后加入CPD4 1.5μg/mL孵育不同时间细胞平均荧光强度的变化.结果:联合用药组细胞凋亡率明显高于单药组,差异有统计学意义(P＜0.01);光动力效应可造成MCF-7细胞G_0/G_1期阻滞,低浓度ADM预处理后GdM期细胞增加,联合用药时G_2/M期细胞升高.ADM预处理MCF-7细胞24小时、48小时细胞平均荧光强度与对照组荧光强度比较差异无统计学意义(P＞0.05);ADM预处理MCF-7后能增加光敏剂CPD4进入细胞的量,在CPD4孵育2小时细胞平均荧光强度最强,且ADM预处理48小时组＞预处理24小时组＞对照组.结论:ADM预处理MCF-7细胞后能够增加光敏剂CPD4进入细胞的量;光动力效应联合ADM具有协同作用.     

Telomerase enzyme activity and RNA expression in adriamycin-resistant human breast carcinoma MCF-7 cells.

Telomerase activity has been reported in cancer cells after treatment with antineoplastic agents. Assessment of telomerase activity could be a valuable tool to measure the reduction of aggression caused by chemotherapy. This study was designed to investigate the significance of telomerase for chemotherapy with respect to Adriamycin (ADM)-resistance. MCF-7 and its ADM-resistant line (AdrR) were treated with ADM, 5-fluorouracil (5FU) or taxotere (TAXO). Telomerase activity and human telomerase RNA component (hTR) were quantitatively measured by the telomeric repeat amplification protocol assay and RT-PCR, respectively. Cell counting and MTT assay were also performed. In MCF-7, enzyme activity was significantly reduced by ADM and 5FU treatments. In AdrR, 5FU and TAXO reduced enzyme activity, while ADM significantly increased the activity. No significant changes in hTR were seen in these two cell lines after treatment with any of these drugs. When Bcl-2 expression was examined after drug treatments, ADM increased Bcl-2 expression in AdrR cells, while not changing it in MCF-7 cells. We conclude that an unusual reaction of telomerase activity in AdrR may explain, at least in part, one of the mechanisms of the malignant biological behavior related with the drug-resistance to ADM.