Novel actions of inhibitors of DNA topoisomerase II in drug-resistant tumor cells

We review herein current work on the cytotoxic and cellular actions of two classes of inhibitors of DNA topoisomerase II: one represented by etoposide and teniposide, which stabilize DNA-protein complexes, and another represented by merbarone and aclarubicin, which do not stabilize such complexes. We discuss current concepts of protooncogene activation and cell cycle perturbations by some of these inhibitors and summarize recent findings of novel actions of the latter compounds in tumor cells that express a mutant topoisomerase II.Paper presented at the Topoisomerase Inhibitors Conference, University of Maryland Cancer Center, 27–30 October 1993. Supported in part by Bristol-Myers Oncology Division.     

Presence of classical multidrug resistance and P-glycoprotein expression in human neuroblastoma cells

Background: The role of P-glycoprotein (Pgp) associated multidrug resistance for neuroblastoma patients is controversial. Therefore we asked whether at all the typical functional features of the multidrug resistance phenotype could be found in neuroblastoma cells and studied the prognostic relevance of Pgp expression.Patients and methods: Tumor touch preparations and tumor cell infiltrated bone marrow smears of 62 neuroblastoma patients were investigated. The expression of Pgp was determined by a highly sensitive immunosandwich technique. Drug resistance studies were performed by exposing cells to Pgp-dependent cytostatic drugs in tissue cultures. Intracellular drug accumulation was examined by rhodamine-123 fluorescence microscopy.Results: Pgp expression was demonstrable for the SK-N-SH cell line, but not detectable in CHP-100 and ten other neuroblastoma cell lines by immunocytochemistry. In tissue cultures, SK-N-SH cells showed a relative resistance to vincristine and adriamycin (45.1 and 12.7-fold resp.) and reduced intracellular accumulation of rhodamine-123 which could be normalized by the Pgp blocker verapamil.Pgp expression was detected by immunocytochemistry in 14 out of 62 tumors (22.6%). No correlation was found to the stage of the disease (P = 0.33), histopathological grading (P = 0.82), N-myc oncoprotein expression (P = 0.76) or N-myc oncogene amplification (P = 0.20). Kaplan–Meier analysis of event free survival for stage 4 tumors revealed a weak trend of inferior survival for patients with Pgp positive tumors (log-rank analysis: P = 0.069).Conclusions: Though Pgp expression is detectable and functional in neuroblastoma cells, but its presence does not provide much information to the complex phenomenon of chemotherapy resistance in patients.     

In vivo etoposide-resistant C6 glioma cell line: Significance of altered DNA topoisomerase II activity in multi-drug resistance

We have established an in vivo etoposide-resistant glioma cell line (C6/VP) from C6 rat glioma cells by stepwise exposure to increasing doses of etoposide. The C6/VP cells were 10 times more resistant to etoposide than the parental C6 cells. In addition C6/VP cells demonstrated cross-resistance to vincristine and vinblastine, but not to ADM or m-AMSA. Interestingly, the cells had collateral sensitivity to ACNU, cisDDP and Ara-C. The C6/VP cells did not express the MDR gene or p-glycoprotein, while they showed 16 times less topoisomerase II catalytic activity compared to the C6 cells. Although there was no significant difference between C6 and C6/VP cells in amounts of topoisomerase II in nuclear extracts, the C6/VP cells had 2.9 times higher amounts of the enzyme than C6 cells in nuclear scaffold prepared from a relatively low-salt buffer (0.5 M NaCl). Northern blot analysis demonstrated that mRNAs of topoisomerase II isoforms were expressed both in C6 and C6/VP cells, and that the amounts of topoisomerase II in C6/VP cells were 14 times greater than in C6 cells. The total uptake of etoposide in tumor tissues derived from C6/VP cells was 3 times less than those derived from parental C6 cells. These results indicate that the C6/VP acquired a multi-drug resistance phenotype by a reduction of the catalytic activity of topoisomerase II and/or diminished accumulation of drugs. This phenotype did not involve the p-glycoprotein. Alterations of topoisomerase II in the C6/VP cells also were accompanied by an increased amount of the topoisomerase II isoform, most of which was localized in the nuclear scaffold (matrix). This suggests that altered binding of topoisomerase II to topologically organized DNAs in the nuclear scaffold may be the molecular basis of this multi-drug resistance phenotype.     

Modulation of DNA topoisomerase II activity and expression in melanoma cells with acquired drug resistance

The role of DNA topoisomerases (Topo) IIalpha and IIbeta was investigated in various drug-resistant melanoma cells. Melanoma cells resistant to etoposide, exhibited an up to tenfold reduced Topo II activity corresponding to an increasing degree of drug resistance indicating that modulation of Topo II activity contribute to the drug-resistant phenotype. The reduction of Topo II activity was reflected by decreased nuclear amounts of both Topo II isoforms.     

Analysis of gene expression profiles in melanoma cells with acquired resistance against antineoplastic drugs

Resistance to various antineoplastic agents is common in the clinical management of malignant melanoma. The biological mechanisms conferring these different drug-resistant phenotypes are still unclear. To identify potential factors mediating drug resistance to melanoma cells, the mRNA expression profiles of the parental drug-sensitive human melanoma cell line MeWo and four derived drug-resistant sublines with acquired resistance against four commonly used drugs for melanoma treatment (cisplatin, etoposide, fotemustine and vindesine) were analysed. We investigated cDNA arrays with 43,000 cDNA clones ( approximately 30,000 unique genes) to study the expression patterns of these cell lines. We were able to simultaneously extract new candidate genes associated with drug resistance in malignant melanoma and to correlate the present findings with previously described resistance-associated genes. Using hierarchical clustering and analysing the overlap of genes with altered expression, we detected similarities between the expression signatures related to cisplatin and fotemustine resistance. The resistance against vindesine required a minimal set of changes in gene expression relative to the parental MeWo cell line. Our study provides new data that may be used to obtain further insight into the resistance characteristics of malignant melanoma.     

DNA拓扑异构酶抑制剂在逆转肿瘤多药耐药性中的研究进展

DNA拓扑异构酶（DNA topoisomerase,Topo）是细胞凋亡过程中关键的核内酶,Topo抑制剂通过作用于Topo抑制肿瘤的活性。然而在临床中,单独使用一种Topo抑制剂常常引发多药耐药性。研究证明,两种Topo抑制剂联合用药存在协同作用可以逆转这种多药耐药性,但这种联合与药物组配、肿瘤特点和给药顺序密切相关,本文就这种联合用药在逆转肿瘤多药耐药中的研究进展做一综述。     

Increased sensitivity to platinum drugs of cancer cells with acquired resistance to trabectedin

Background:

In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo.

Methods:

Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts.

Results:

402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G₂/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks.

Conclusions:

Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.     

Expression of MRP and mdr1 in human gastrointestinal cancer cell lines: A correlation with resistance against doxorubicin

The mRNA expression of mdr1 and MRP, each of which codes for a transport protein belonging to ATP-binding cassette superfamily and are reported to be responsible for multidrug resistance phenotype, were semiquantified by RT-PCR in a panel of gastrointestinal cancer cell lines. Although the expression of MRP was predominant in esophageal cancer cell lines, expression of either or both of the genes was detected in all the cell lines tested. Expression of these two genes added together correlated significantly with chemosensitivity against doxorubicin, implicating that expression of both genes should be evaluated in the future analysis of multidrug resistance phenotype. The ID₅₀ values for pirarubicin, although generally lower than the values for doxorubicin, correlated well with the latter, suggesting that the similar phenotype as that for doxorubicin might be responsible for drug resistance against this semisynthetic anthracycline glycoside. © 1996 Wiley-Liss, Inc.     

Reversal effects of Raloxifene on paclitaxel resistance in 2 MDR breast cancer cells

Raloxifene hydrochloride (RAL), one of second generation of selective estrogen receptor modulators (SERMs), is usually used in preventing osteoporosis and breast cancer. The present study evaluated whether Raloxifene might sensitize multidrug resistant (MDR) breast cancers to chemotherapies, especially in estrogen receptor negative (ER−) breast cancer. The results showed that RAL could significantly sensitize ER- MDR breast tumors to paclitaxel both in vitro and in vivo. Combination of Raloxifene could significantly enhance paclitaxel-induced cell apoptosis, G2-M arrest as well as inhibition of cell proliferation in MDR tumors. Further studies showed that the combined treatment did not alter P-glycoprotein expression but increased P-gp ATPase activity. These results suggested that raloxifene might be a valuable chemosensitizer agent for breast cancer therapy.     

Quinacrine has anticancer activity in breast cancer cells through inhibition of topoisomerase activity

The small molecule Quinacrine (QC, a derivative of 9-aminoacridine), an anti-malaria drug, displays activity against cancer cell lines and can simultaneously suppress nuclear factor-κB (NF-κB) and activate p53 signaling. In this study, we investigated the anticancer mechanism underlying these drug activities in breast cancer cell lines. QC caused a dose-dependent decrease of both anchorage dependent and independent growth of breast cancer cells (MCF-7 and MDA-MB-231) without affecting normal breast epithelial cells (MCF-10A), as evident from clonogenic cell survival, [3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide] viability, wound healing and soft agar growth. QC activated the proapoptotic marker Bax, PARP cleavage, p53 and its downstream target, p21 (Cip1/Waf1) and downregulated the antiapoptotic marker Bcl-xL and relative luciferase activity of NF-κB in MCF-7 cells. Results of DAPI nuclear staining and FACS analysis show that QC increased apoptosis in a dose-dependent manner. QC caused apoptosis by increasing the cell population in S-phase and simultaneously decreasing the G1 and G2/M populations. A dose-dependent increase of DNA damage as measured by the comet assay was seen in MCF-7 cells after exposure to QC. With regards to the mechanism of DNA damage, we found that QC inhibited topoisomerase activity in MCF-7 cells by increasing the unwinding of supercoiled DNA. Collectively, the results demonstrate that QC has efficient anticancer potential against breast cancer cells via not only an induction of p53 and p21 but also an induction of S phase arrest, DNA damage and inhibition of topoisomerase activity.     

A leukotriene B4 receptor-2 is associated with paclitaxel resistance in MCF-7/DOX breast cancer cells

Background:

Breast cancer is the most common malignancy in women. Although chemotherapeutic agents, such as paclitaxel, are effective treatments for the majority of breast cancer patients, recurrence is frequent and often leads to death. Thus, there is an urgent need to identify novel therapeutic targets that sensitise tumour cells to existing chemotherapy agents.

Methods:

The levels of leukotriene B₄ receptor-2 (BLT2) in multidrug-resistant MCF-7/DOX cells were determined using quantitative PCR and FACS analysis. The potential role of BLT2 in the paclitaxel resistance of MCF-7/DOX cells was assessed using a pharmacological inhibitor and small interfering RNA knockdown, and the BLT2-associated resistance mechanism was assessed.

Results:

The expression levels of BLT2 were markedly upregulated in MCF-7/DOX cells. The inhibition of BLT2 by pre-treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY255283","term_id":"1257961172","term_text":"LY255283"}}LY255283 or siBLT2 knockdown significantly sensitised MCF-7/DOX cells to paclitaxel and induced significant levels of apoptotic death, suggesting that BLT2 mediates paclitaxel resistance. We also demonstrated that BLT2-induced paclitaxel resistance was associated with the upregulation of P-glycoprotein. Finally, co-treatment with a BLT2 inhibitor and paclitaxel markedly reduced tumour growth in an MCF-7/DOX in vivo model.

Conclusion:

Together, our results demonstrate that BLT2 is a novel therapeutic target that sensitises drug-resistant breast cancer cells to paclitaxel.     

浸润性乳腺癌患者谷胱苷肽S-转移酶-π和肺耐药蛋白表达及其与DNA倍体的关系

目的:探讨谷胱苷肽S-转移酶-π(GST-π)、肺耐药蛋白(LRP)及DNA倍体在乳腺癌患者中的表达意义。方法:应用流式细胞术(FCM)定量分析56例原发浸润性乳腺癌GST-π、LRP蛋白表达及DNA倍体的状况。结果:乳腺癌组织GST-π和LRP表达的相对荧光强度(RFI)以中位数(M)表示分别为1·18和0·56,而相应癌旁组织分别为0·80和0·23,癌组织GST-π和LRP蛋白表达与相应癌旁组织比较差异有统计学意义,P<0·05。癌组织DNA指数(DI)、S期比例(SPF)和增殖指数(PI)显著高于相应癌旁组织,P<0·05。GST-π和LRP蛋白表达及DI、SPF、PI在不同年龄、不同肿瘤大小、不同病理类型、不同临床分期和雌、孕激素受体阴性与阳性患者间的表达差异均无统计学意义,P>0·05。有淋巴结转移组患者GST-π和LRP表达水平及DI、SPF、PI值显著高于无淋巴结转移组患者,P<0·05。异倍体与二倍体乳腺癌GST-π和LRP表达差异无统计学意义,P>0·05。结论:GST-π和LRP蛋白表达及DNA含量在乳腺癌发生、发展中起重要作用。GST-π和LRP蛋白表达及DI、SPF和PI与淋巴结转移有关。GST-π和LRP蛋白表达与DNA含量可作为预测乳腺癌预后的指标。     

New insights of extrachromosomal DNA in tumorigenesis and therapeutic resistance of cancer

In the past few decades, the studies of extrachromosomal DNA (ecDNA), which existed independently of chromosomes, were tepid. However, recent studies on ecDNA rekindled the enthusiasm of oncologists for further studying ecDNA. In this review, we summarized the recent advances of ecDNA in oncogenesis and oncotherapy. ecDNA consists of highly open chromatin, and its circular structure enables ultra-long-range chromatin contacts. ecDNA is not inherited in accordance with Mendel’s laws. Furthermore, ecDNA is widely existed in cancer cells, but almost never found in normal cells. It has been found that ecDNA played important roles in tumorigenesis and tumor progression, including oncogene amplification, tumor heterogeneity, enhancer hijacking and genomic rearrangement. More importantly, ecDNA is closely related to cancer treatment resistance. In hence, further understanding of ecDNA would contribute to developing innovative targeting ecDNA therapies.     

上皮性卵巢癌组织中肺耐药蛋白的表达及意义

目的探讨上皮性卵巢癌中肺耐药蛋白(LRP)的表达及其临床意义。方法采用免疫组化SP法及半定量分析方法,检测上皮性卵巢癌64例,复发后二次手术癌组织17例,卵巢良性上皮性肿瘤16例及正常卵巢组织14例中LRP的表达,并进行相关临床病理因素分析。结果在上皮性卵巢癌中LRP的阳性率为78.1%,与卵巢良性肿瘤及正常卵巢有显著性差异(P<0.01);化疗后癌组织中LRP的强阳性表达率与初治卵巢癌组织中相应的表达率差异有显著性。结论LRP在上皮性卵巢癌中较恒定表达,化疗可以增加癌组织中LRP的表达。检测LRP表达水平可以预测卵巢癌对铂类联合化疗疗效,也是判断其预后的可靠指标之一。     

Estrogen-induced potentiation of DNA damage and cytotoxicity in human breast cancer cells treated with topoisomerase II-interactive antitumor drugs

Hormone stimulation of responsive neoplasms is a potential strategy for improving the target selectivity of cancer chemotherapy. Using an alkaline DNA-unwinding technique to detect drug-induced DNA strand breakage, we have shown that estrogen stimulation of T-47D human breast cancer cells enhances induction of DNA cleavage by etoposide (VP-16), 4',9-acridinylaminomethanesulfon-m-anisidide (m-AMSA), mitoxantrone, and doxorubicin, drugs known to interact with the DNA-modifying enzyme topoisomerase II. No enhancement of DNA cleavage or cytotoxicity was seen in estrogen-treated cells exposed to X-rays or bleomycin. Novobiocin (an inhibitor of topoisomerase II) markedly antagonized the enhancing effect of estrogen on VP-16-induced DNA cleavage, while neutral nucleoid sedimentation detected less than 10% of such strand breaks revealed in estrogen-treated cells by alkaline unwinding. Estrogen did not affect DNA repair of lesions induced by X-rays, VP-16, or ultraviolet radiation. Enhancement of DNA cleavage was accompanied by a corresponding enhancement of cytotoxicity in cells treated with VP-16 or m-AMSA, but only minimal enhancement of cytotoxicity was seen following treatment with mitoxantrone or doxorubicin. Estrogen-treated and control cells treated with VP-16 and m-AMSA sustained similar levels of DNA cleavage for equivalent levels of cytotoxicity. These findings suggest that estrogen potentiates the cytotoxicity of VP-16 and m-AMSA by enhancing topoisomerase II-mediated DNA damage but that such "damage" does not contribute significantly to cytotoxicity induced by mitoxantrone or doxorubicin. Estrogen stimulation of receptor-positive breast cancer may prove to be a clinically relevant strategy for improving the selectivity and cytotoxicity of some, but not all, topoisomerase II-interactive drugs.     

No evidence of significant activity of the multidrug resistance gene product in primary human breast cancer

Background: The discovery of the multidrug resistance (MDR1) gene product P-glycoprotein (P-gp) has been widely seen as an important milestone in our understanding of the mechanisms underlying the clinical phenomenon of the emergence of resistant cells. MDR1 expression has been shown for numerous solid tumors and for virtually all hematologic malignancies. Nevertheless, results regarding MDR1/P-gp expression in human breast cancer have been controversial and the results of clinical trials on modulation of P-gp activity have not been encouraging.Patients and methods: MDR1/P-gp expression and the function of the P-gp pump were investigated in 61 tumor samples from patients with primary breast cancers by multiparameter analysis using MDR1-RT-PCR, immunohistochemistry with two MAbs (UIC2 and MRK 16) and the rhodamine 123 (Rh123) efflux assay. The cellular composition of the tumor cell suspension was analyzed by using specific MAbs against the P-gp expressing lymphocyte subsets CD4, CD8 and CD56, as well as against the HER-2/neu gene product, which was used to identify breast carcinoma cells.Results: UIC2 and MRK16 revealed a staining positivity in 72% and 75% of samples, respectively. A positive MDR1-RT-PCR signal was detected in 62% of the samples. Nevertheless, no correlation between immunohistochemistry and RT-PCR could be established. Furthermore, there was no correlation between HER-2/neu expression and MDR1-RT-PCR or P-gp immunohistochemical assays. A contamination by CD8+ and CD4+ lymphocytes was established in 100% and 84% of tumor cell suspensions, respectively. As assessed by the Rh123 efflux assay CD8+ and the CD4+ lymphocytes exhibited marked P-glycoprotein activity, whereas such activity was not detectable in a single instance for the breast carcinoma cells. In MDR1-RT-PCR positive samples, contamination by CD8 lymphocytes averaged 4.3%, while the contamination of CD8 cells in the MDR1 mRNA-negative samples was only 2.4% (P = 0.007). This signal vanished after elimination of the lymphocyte subpopulations by T-cell rosetting.Conclusions: In primary breast cancer detection of MDR1 gene expression by means of RT-PCR or immunohistochemical assays is not indicative for the MDR phenotype, since there is no evidence of significant activity of the P-gp pump.