Topoisomerase IIα Expression Research Articles

Treatment with Gefitinib or Lapatinib Induces Drug Resistance through Downregulation of Topoisomerase IIα Expression

The EGF receptor (EGFR) is therapeutically targeted by antibodies and small molecules in solid tumors including lung, colorectal, and breast cancer. However, chemotherapy remains important, and efforts to improve efficacy through combination with targeted agents is challenging. This study examined the effects of short and long durations of exposure to the EGFR- and HER2-targeted tyrosine kinase inhibitors (TKI) gefitinib and lapatinib, on induction of cell death and DNA damage by topoisomerase IIα (Topo IIα) poisons, in the SK-Br-3 HER2-amplified breast cancer cell line. Short exposure to either gefitinib or lapatinib for 1 hour did not affect the induction of apoptosis by the Topo IIα poisons doxorubicin, etoposide, and m-AMSA. In contrast, cells treated for 48 hours were resistant to all three drugs. Short exposure (1 hour) to TKI did not alter the number of DNA single- or double-strand breaks (DSB) induced, whereas longer exposure (48 hours) reduced the number of DNA DSBs and the formation of γ-H2AX foci. Both gefitinib and lapatinib reduced the expression and activity of Topo IIα at 48 hours. Studies using a cell line with inducible downregulation of Topo IIα showed that expression of Topo IIα, and not Topo IIβ, determined the number of DNA strand breaks induced by these chemotherapeutic agents. These results indicate that prolonged exposure to TKIs targeting EGFR and HER2 induce resistance to doxorubicin, etoposide, and m-AMSA through downregulation of Topo IIα. This may explain why their addition to chemotherapy regimens have not increased efficacy.

Utility of ProEx C in the histologic evaluation of the neoplastic and nonneoplastic urothelial lesions

ProEx C is an antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein 2. ProEx C staining is being used mainly to assist in diagnoses of dysplasia in gynecological specimens. This study was designed to determine the utility of ProEx C in assessing the urothelial lesions. Sixty-four patient specimens were divided into 5 groups: group I, 22 benign; group II, 13 low-grade noninvasive papillary urothelial carcinoma; group III, 10 high-grade urothelial carcinoma in situ (flat lesion); and group IV, 19 high-grade noninvasive papillary and invasive urothelial carcinomas. ProEx C reactions were scored in basal, parabasal, intermediate, and superficial cell layers. A sample was recorded as positive when nuclear staining was seen in the cells of the intermediate and/or superficial layers. Of 22 cases in group I, 21 were negative for ProEx C with a specificity of 95%. Of 13 cases in group II, 11 had positive results with sensitivity of 85%. All cases in groups III and IV had positive staining pattern with ProEx C with a sensitivity of 100%. In this study, ProEx C stain had an overall 95% sensitivity and specificity with positive and negative predictive values of 98% and 91%, respectively, for detection of urothelial carcinoma. We conclude that ProEx C is effective in differentiating carcinoma in situ and benign urothelial lesions. It also resolves issues in low- versus high-grade papillary urothelial carcinomas. To our knowledge, this is the first publication on the diagnostic application of ProEx C in histopathology of the urothelial lesions.

A first-in-human phase I study of the CDK4/6 inhibitor, LY2835219, for patients with advanced cancer.

2500 Background: Cyclin dependent kinases 4 and 6 (CDK4/6) act with D-type cyclins to inactivate the retinoblastoma (Rb) tumor suppressor protein and enable cell cycle progression from G1 to S phase. LY2835219 is a selective inhibitor of CDK4/6 that shows antitumor activity in preclinical models of human cancer and also distributes efficiently to the brain. We performed a phase 1 study to evaluate safety, pharmacokinetics, pharmacodynamics, and antitumor activity of LY2835219. Methods: 3+3 dose escalation was followed by expansions in 5 tumor types (brain metastases permitted): non-small cell lung cancer (NSCLC), glioblastoma, breast cancer, melanoma, and colorectal cancer. LY2835219 was taken orally every 12 or 24 hours (in escalation) and every 12 hours (in expansions) on days 1-28 of a 28-day cycle. Results: 55 patients (pts) received LY2835219. In escalation, 33 pts received LY2835219 on 1 of 2 schedules: 50, 100, 150, 225 mg every 24 hours (Q24H) or 75, 100, 150, 200, 275 mg every 12 hours (Q12H). On the Q24H schedule, the maximum tolerated dose (MTD) was not identified. On the Q12H schedule, the MTD was 200mg Q12H with dose limiting toxicity of G3 fatigue at 200 mg (1/6 evaluable pts) and 275 mg (2/3 evaluable pts). At 200mg Q12H, the mean Cmax and AUC0-24hr at steady state were 285 ng/mL and 5502 ng-hr/ml, respectively. In skin, LY2835219 induced pharmacodynamic inhibition of both Rb phosphorylation and topoisomerase IIα expression. In the ongoing expansions, 22 pts have received LY2835219. Across the study, the most common related adverse events were diarrhea (52%, including 5% G3), nausea (30%, 4% G3), fatigue (21%, 7% G3), vomiting (18%, 2% G3), and neutropenia (16%, 7% G3). 15 pts have reached ≥4 cycles for stable disease or better with 3 pts achieving 8, 16, and 26 cycles. One pt with ovarian cancer had a durable CA-125 response with >50% decrease for 16 cycles. One pt with KRAS mutant NSCLC had a 27% decrease by RECIST. One pt with CDKN2A-/- NRAS mutant melanoma had a confirmed partial response. Early clinical activity has been observed in ovarian cancer, NSCLC, breast cancer, and melanoma. Conclusions: LY2835219 shows acceptable safety and early clinical activity as a single agent for patients with advanced cancer. Clinical trial information: NCT01394016.

Abstract 3112: The homeoprotein DLX4 induces topoisomerase IIα expression but reduces sensitivity of tumor cells to topoisomerase II poisons.

Abstract Chemotherapeutic drugs such as anthracyclines and etoposide that target topoisomerase II (TOP2) induce tumor cell death by causing accumulation of DNA double-strand breaks (DSBs). Experimental studies have shown that overexpression of topoisomerase IIα (TOP2A) increases sensitivity to TOP2 poisons. However, the clinical value of TOP2A in predicting responsiveness to TOP2 poisons has been controversial and it is not known why some TOP2A-overexpressing tumors respond poorly to these drugs. We identified that expression of TOP2A, the gene encoding TOP2, is induced by DLX4, a homeoprotein that is overexpressed in breast and ovarian cancers. Analysis of breast cancer datasets revealed that TOP2A-High cases that expressed DLX4 at low levels responded more favorably to anthracycline-based chemotherapy than TOP2A-High cases that also highly expressed DLX4. Overexpression of TOP2A alone in tumor cells increased sensitivity to TOP2 poisons. However, DLX4 decreased sensitivity to these drugs irrespective of its induction of TOP2A. We identified that DLX4 stimulates repair of DSBs and interacts with Ku proteins that are components of the non-homologous end-joining machinery. DLX4 did not reduce levels of TOP2 poison-induced DSBs in Ku-deficient cells, but reduced the level of DSBs when Ku was reconstituted in these cells. Taken together, our findings indicate that DLX4 induces TOP2A expression, but reduces the sensitivity of tumor cells to TOP2 poisons by stimulating Ku-dependent repair of DSBs. These dual and opposing effects of DLX4 could explain why some tumors with elevated TOP2A levels are not highly sensitive to TOP2 poisons. Citation Format: Bon Q. Trinh, Song Yi Ko, Nicolas Barengo, Shiaw-Yih Lin, Honami Naora. The homeoprotein DLX4 induces topoisomerase IIα expression but reduces sensitivity of tumor cells to topoisomerase II poisons. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3112. doi:10.1158/1538-7445.AM2013-3112

The predictive and prognostic significance of pre- and post-treatment topoisomerase IIα in anthracycline-based neoadjuvant chemotherapy for local advanced breast cancer

To investigate the predictive and prognostic value of topoisomerase IIα (Topo IIα, Topo II) expression in the primary tumors and residual tumors of local advanced breast cancer (LABC) patients being treated with anthracycline-based neoadjuvant chemotherapy (NCT). The data from 283 LABC patients who had been treated with anthracycline-based neoadjuvant chemotherapy were collected. The expression of Topo IIα, HER-2 and other biomarkers was determined via immunohistochemical analysis in pre- and post-chemotherapy specimens. The status of pre-treatment biomarkers was correlated with the clinical response determined by the RECIST 1.1 criteria, whereas the post-treatment biomarkers were studied for prognostic value using the Cox model. By analyzing the complete data from 99 patients, the co-expression of HER-2/Topo IIα was found to be significantly correlated with the clinical response to chemotherapy (Logistic regression P=0.042). Notably, a 20% alteration in the Topo IIα status during neoadjuvant chemotherapy was found, which could also influence the sensitivity to treatment. With a survival analysis performed in 245 patients with residual tumors after NCT, node metastasis, HER-2 and Ki-67 were independent predictors of patient outcome. However, post-treatment Topo IIα expression demonstrated significant prognostic value in HER-2+ patients (P=0.002). A relatively lower disease-free survival and overall survival was observed in HER-2+/Topo- patients (log rank P=0.010 for DFS and P<0.001 for OS). Topo IIα, together with HER-2, might help to select for patients who could benefit from anthracycline-based neoadjuvant chemotherapy and identify non-complete responders at a higher risk of disease recurrence or death.

ProExC as an adjunct marker to improve cytological detection of urothelial carcinoma in urinary specimens

ProEx C is an antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein-2. ProEx C staining is being used to assist in diagnoses of the gynecological specimens. This study was designed to determine the utility of ProEx C in urine cytology samples for improving the detection of urothelial carcinomas where a significant number of urine cytology specimens are diagnosed as "atypia." Sixty urinary specimens (12 negative, 13 positive, and 35 atypical cases) were stained with ProEx C, and ProEx C results were recorded as positive when nuclear staining was only seen in at least one morphologically atypical urothelial cell. All 12 benign cytology samples showed negative staining with ProEx C. Twelve of 13 cases that had a malignant cytologic diagnosis showed a positive nuclear staining of the malignant cells. Eighteen of 35 cases with atypical cytologic diagnoses showed positive nuclear staining. Of the 35 cases with "atypia," 17 had a malignant histopathologic follow-up. In this study, ProEx C stain had an overall sensitivity of 78.4%, specificity of 95.7%%, positive predictive value of 96.7%, and negative predictive value of 73.3% for the detection of urothelial carcinoma. ProEx C stain is a useful adjunct test to urine cytologic analysis, even in specimens with limited cellularity. In urinary smears, this test is most useful in stratification of the "atypical" diagnoses into benign and malignant subsets. To the authors' knowledge, this is the first study of ProEx C application in urine cytology as an adjunct marker for detection of urothelial carcinoma.

Promoter Methylation, BRAF Mutation Analysis and Topoisomerase IIa Expression for the Detection of Endometrial Carcinoma in Liquid Based Cytology Samples

Cancer of the corpus uteri remains the most common gynecological related cancer in developed countries. Cytology, after the induction of liquid based cytology, has reemerged as a possible first line non-interventional diagnostic procedure with promising results. Apart from slide preparation for cytology diagnosis, LBC allows the application of elaborate molecular tests on the residual material. Samples from 74 symptomatic women were collected in ThinPrep PreservCyt medium, from witch immunocytochemical and molecular tests were performed. Final diagnosis of 39 endometrioid carcinomas, 20 non-endometrioid carcinomas and 15 non-malignant was set after hysterectomy. Topoisomerase IIa expression was common (42%) in both types of cancer. Promoter methylation analysis revealed that hMLH1 is commonly methylated in cancers (52.7%), CDKN2A and MGMT less often (27.1%) and RARB rarely methylated (8.4%). BRAF activating mutation V600E was a rare event (8.4%) only found in low grade endometrioid carcinomas. Topoisomerase IIa expression correlated with BRAF mutations, hMLH1 and to lesser extent with CDKN2A methylation. Almost none of the biomarkers were positive in cytological negative or hyperplastic without atypia samples. Detection of methylation in any gene displayed sensitivity, specificity, PPV and NPV similar to cytology of cancer. However, inclusion of cytology diagnosis of hyperlasias with atypia increased sensitivity and NPV of cytology outperforming methylation of any gene. Further evaluation of the panel of promoter methylation, especially in cytology diagnoses of hyperplasia with or without atypia should be evaluated since initial results are promising. Even though methylation of MGMT and RARB are rare events, some patients could be benefit from specific chemotherapeutics that target either of them or the more frequently expressed topoisomerase IIa.

Gene Expression Profiles in the Fetal Mouse Brain after Etoposide (VP-16) Administration

The aim of this study was to analyze the response of gene expression caused by etoposide (VP-16) in the fetal mouse brain. Four miligrams/kilogram of VP-16 was intraperitoneally injected into pregnant mice on day 12 of gestation (GD 12). Gene expression profiling of the VP-16-treated fetal mouse brain by DNA microarray was performed. The expression changes of the target genes of p53 were also examined by real-time RT-PCR. VP-16 induced S-phase accumulation, G2/M arrest, and eventually apoptosis of neuroepithelial cells in the fetal brain. DNA microarray analysis revealed that 8 of cell cycle control- and apoptosis-related genes were upregulated and that 5 of DNA damage, repair, replication, and transcription genes were also upregulated in the fetal telencephalons at 4 h after VP-16 treatment (HAT). The results of real-time RT-PCR demonstrated that the expression of topoisomerase IIα was increased at 4 and 8 HAT. The expression of pro-apoptotic factors such as puma, noxa, bax, and cyclin G was also increased from 4 to 12 HAT. These results suggest that VP-16 induces DNA damage, DNA repair, cell cycle alternation, and apoptosis in the fetal mouse brain. In addition, VP-16-induced apoptosis is mediated through the mitochondrial pathway in a p53-related manner. The present study will provide a better understanding of the mechanisms of VP-16-induced fetal brain injury.

Free Circulating Tumor DNA as a Diagnostic Marker for Breast Cancer

Cell-free DNA (cfDNA) in the plasma of patients with both malignant and benign breast lesions was analyzed to determine whether the findings may have diagnostic and prognostic implications and to analyze the association between the levels of cfDNA and prognostic parameters. Plasma samples were obtained from 99 subjects; 42 with breast cancer (BC), 30 with benign breast lesions, and 27 healthy women as normal controls. Circulatory cfDNA was extracted from the plasma samples and quantified using a real-time quantitative PCR method. Immunohistochemistry was done on formalin-fixed paraffin-embedded sections to evaluate the status of hormonal receptors (estrogen receptor [ER] and progesterone receptor [PR]), and the protein expression of both Her2/neu and Topoisomerase IIα. The level of cfDNA in the BC group was significantly higher than in the benign lesions and control groups. cfDNA level was associated with malignant tumor size, lymph node involvement, stage, and grade as well as Her2/neu and Topoisomerase IIα expression, while it was not associated with ER or PR status. The present study suggests that the level of cfDNA can be easily quantified using plasma samples. Thus, level of plasma cfDNA might constitute an important noninvasive diagnostic and prognostic valuable tool in cancer breast patients' management.

DNA repair and replication proteins as prognostic markers in melanoma

Elevated expression of DNA repair and replication genes has been reported in thick, non-fixed primary melanomas that subsequently went on to metastasize, when compared to non-recurrent primary tumours. This increased expression could contribute to the extreme resistance shown by melanoma to DNA-damaging chemotherapeutics. We have investigated the hypothesis that levels of key DNA repair and replication proteins are prognostic biomarkers in melanoma. We used a tissue microarray containing samples from all stages of melanomagenesis to investigate the hypothesis that levels of key DNA repair and replication proteins are prognostic biomarkers in a larger, more representative and readily available set of fixed primary melanomas. High expression of topoisomerase IIα (TOP2A), that relieves torsional stress during DNA replication, and XRCC5 (Ku80), required for DNA double-strand break repair, were associated with significantly worse survival. Two (XRCC5 and TOP2A) of seven DNA repair and replication proteins studied were prognostic for melanoma.

Glucose-regulated protein 75 overexpression attenuates ionizing radiation-mediated injury in PC12 cells by inducing the expression of topoisomerase IIα

Glucose-regulated protein 75 (Grp75), also known as mortalin/mthsp70/PBP74, is a member of the heat-shock protein 70 (HSP70) family. Grp75 is known to protect cells from stress-induced injury. Previous studies have shown that the expression of Grp75 is upregulated by a low dose of ionizing radiation (IR). To evaluate the protective role of Grp75 on cell proliferation in response to IR, Grp75 was overexpressed in a rat adrenal pheochromocytoma cell line (PC12). It was revealed that Grp75 overexpression desensitized PC12 cells to IR-mediated cell injury. In addition, the expression of topoisomerase IIα (Topo IIα) was downregulated in PC12 cells following γ-ray IR. The effect of Grp75 overexpression on Topo IIα expression was examined. It was revealed that Grp75 overexpression reversed the inhibitory effect of IR on Topo IIα expression. In conclusion, the data indicated that Grp75 overexpression attenuates IR-induced injury in PC12 cells by maintaining the expression of Topo IIα.

DNA topoisomerase II alpha: a favorable prognostic factor in colorectal caner

There is a lack of study concerning expression of Topoisomerase IIα (Topo IIα) and long-term results in colorectal cancer patients. We aimed to investigate the relationship between expression of Topo IIα and clinicopathological parameters including overall survival in colorectal cancer. Paraffin-fixed specimens from a large prospective cohort of colorectal cancer patients who had been followed up for 4 years were assayed immunohistochemically. Of 490 colorectal cancer patients accessible for Topo IIα expression, expression of Topo IIα was scored as (-) in 4 (0.8%) patients, (+) in 41 (8.4%) patients, (++) in 396 (80.8%) patients, and (+++) in 49 (10.0%) patients. Overexpression of Topo IIα was found to be related with lower T stage (p = 0.042), lower N stage (p = 0.038), and a lower incidence of recurrence with nearly significance (p = 0.053). Kaplan-Meier analyses showed that overexpression of Topo IIα was related with prolonged overall survival (p = 0.022) and disease-free survival (p = 0.036). Multivariate analyses showed that elevated serum CEA (p < 0.001), elevated serum CA199 (p = 0.002), poor differentiation (p = 0.001), advanced Dukes stage (p < 0.001), and lower expression of Topo IIα (p = 0.017) were independent predictive factors for poor prognosis. Topo IIα expression is a valuable prognostic indicator for colorectal cancer and would be useful in treatment selection for early colorectal cancer and malignant colorectal polyps resected under endoscopy, especially when it is used in combination with serum CEA, CA199, and differentiation.

Expressions of Topoisomerase IIα and BCRP in Metastatic Cells are Associated with Overall Survival in Small Cell Lung Cancer Patients

The aim of this study was to investigate the mRNA expression levels of multidrug resistance-associated proteins in chemo-naïve metastatic lung cancer cells and to determine the correlation with response to chemotherapy and overall survival. Metastatic cells were obtained by transbronchial fine needle aspiration biopsy of enlarged mediastinal lymph nodes in 14 patients with small cell lung cancer (SCLC) and 7 patients with non-small cell lung cancer (NSCLC). After cytological confirmation of lung cancer type, total RNA was extracted from biopsy samples and reverse transcribed to cDNA, and real-time PCR for the genes of interest [P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), breast cancer resistance protein (BCRP), lung resistance protein (LRP) and topoisomerase IIα (TOPIIα)], was performed. We observed significantly decreased expression of BCRP and significantly increased expression of TOPIIα in metastatic SCLC cells compared to NSCLC. Furthermore, in SCLC high topoisomerase IIα and low BCRP expression levels positively correlated with longer overall survival. Our results showed higher expression levels of BCRP as well as lower levels of topoisomerase IIα in chemo-naïve metastatic cells in NSCLC than in SCLC. These results correlate with previous observations that metastatic SCLC cells at the beginning of chemotherapy are potentially more sensitive to chemotherapeutic agents while in metastatic NSCLC cells resistance is usually inherent. We also showed that altered levels of topoisomerase IIα and BCRP in SCLC are important factors that contribute to resistance to chemotherapeutics that interfere with the enzyme and/or DNA and are highly associated with overall survival.

Decreased topoisomerase IIα expression and altered histone and regulatory factors of topoisomerase IIα promoter in patients with chronic benzene poisoning

Topoisomerase IIα (Topo IIα) has been implicated in the benzene-induced hemotoxicity in vitro. This study was to examine the effect of in vivo chronic benzene exposure on Topo IIα in human bone marrow mononuclear cells, and to further explore the mechanism underlying decreased Topo IIα expression in patients with chronic benzene exposure. Topo IIα activity, expression, and mRNA level assessed by DNA cleavage/relaxation assay, Western blot, and reverse transcriptase-PCR, decreased in patients with benzene exposure. These changes were accompanied by reduced histone H4 and H3 acetylation and H3K4 methylation, and increased H3K9 methylation in the Topo IIα promoter, which were evaluated by chromatin immunoprecipitation (ChIP) assay. In addition, there were alterations in mRNA levels of Topo IIα promoter regulatory factors such as SP1, ATF-2, SP3, NF-YA, NF-M, P53, C-MYB, C-JUN, and ICBP90. Our results demonstrate that Topo IIα expression was reduced in patients with chronic benzene exposure, which was accompanied by alterations in histone acetylation and methylation and regulatory factor mRNA levels of Topo IIα promoter.

Novel Regulation of Nuclear Factor-YB by miR-485-3p Affects the Expression of DNA Topoisomerase IIα and Drug Responsiveness

Nuclear factor (NF)-YB, a subunit of the transcription factor nuclear factor Y (NF-Y) complex, binds and activates CCAAT-containing promoters. Our previous work suggested that NF-YB may be a mediator of topoisomerase IIα (Top2α), working through the Top2α promoter. DNA topoisomerase II (Top2) is an essential nuclear enzyme and the primary target for several clinically important anticancer drugs. Our teniposide-resistant human lymphoblastic leukemia CEM cells (CEM/VM-1-5) express reduced Top2α protein compared with parental CEM cells. To study the regulation of Top2α during the development of drug resistance, we found that NF-YB protein expression is increased in CEM/VM-1-5 cells compared with parental CEM cells. This further suggests that increased NF-YB may be a negative regulator of Top2α in CEM/VM-1-5 cells. We asked what causes the up-regulation of NF-YB in CEM/VM-1-5 cells. We found by microRNA profiling that hsa-miR-485-3p is lower in CEM/VM-1-5 cells compared with CEM cells. MicroRNA target prediction programs revealed that the 3'-untranslated region (3'-UTR) of NF-YB harbors a putative hsa-miR-485-3p binding site. We thus hypothesized that hsa-miR-485-3p mediates drug responsiveness by decreasing NF-YB expression, which in turn negatively regulates Top2α expression. To test this, we overexpressed miR-485-3p in CEM/VM-1-5 cells and found that this led to reduced expression of NF-YB, a corresponding up-regulation of Top2α, and increased sensitivity to the Top2 inhibitors. Results in CEM cells were replicated in drug-sensitive and -resistant human rhabdomyosarcoma Rh30 cells, suggesting that our findings represent a general phenomenon. Ours is the first study to show that miR-485-3p mediates Top2α down-regulation in part by altered regulation of NF-YB.

527 siRNA targeting of thymidylate synthase and thymidine kinase for anti-cancer therapy

Expression of DNA topoisomerase (topo) IIα is cell-cycle-regulated, with its peak in G2/M and its lowest level in G0/G1. In agreement with this expression pattern, we have shown that the topo IIα gene promoter shows cell-cycle-dependent activity, which is repressed in G0/G1 and activated exclusively in G2/M. However, the promoter sequence reveals no canonical CDE/CHR motifs, repressor elements commonly found in promoters of late S/G2-activated genes. Here, we show that at least two of the three proximal inverted CCAAT boxes (ICBs) are responsible for the G2/M-specific activation of the topo IIα promoter. Using antibody supershift experiments, we identify NF-Y as the ICB-binding transcription factor. However, the expression profile and binding capacity of NF-Y were constant during the cell cycle, suggesting a more global mechanism in topo IIα promoter regulation. Interestingly, we find that trichostatin A (TSA), a specific histone deacetylase inhibitor, greatly enhances topo IIα promoter activity in an ICB-dependent manner. In addition, the effect of TSA is predominant in G0/G1 and less obvious in G2/M. Our data, along with the recent findings that NF-Y associates in vivo with histone acetyltransferases (HATs), strongly suggest a mechanism, in which histone deacetylation plays a crucial role in the G0/G1-specific repression of the topo IIα promoter, and NF-Y recruits HATs to the promoter region, thereby stimulating histone acetylation and activating transcription in G2/M.

Phenolic-Containing Organic Extracts of Mulberry (Morus alba L.) Leaves Inhibit HepG2 Hepatoma Cells Through G2/M Phase Arrest, Induction of Apoptosis, and Inhibition of Topoisomerase IIα Activity

The entire plant of Morus alba L. (Family Moraceae), or mulberry, possesses medical benefits, including anticancer properties. In this study, we investigated the effect of mulberry leaf extracts on the human hepatoma HepG2 cell line, which is related to hepatocellular carcinoma. Mulberry leaf extracts were prepared using four solvents, each with different polarities: 100% methanol (MeOH), 50% aqueous MeOH, 1-butanol (BuOH), and hot water (W). The phenolic profile, total polyphenol content, antioxidant capacity, and effect on human hepatoma HepG2 cells of the leaf extracts were analyzed by examining cytotoxicity, cell cycle progression, apoptosis, expression of topoisomerase IIα, and proteins involved in cell cycle progression. High-performance liquid chromatography-mass spectrometry analysis revealed that 100% MeOH, 50% MeOH, and BuOH extracts contained rutin, isoquercetin, and various derivatives of kaempferol and quercetin glycosides as their major constituents; the W extract contained primarily chlorogenic acid and caffeoylquinic acid derivatives. Total phenolic content based on rutin equivalents was 17.1%, 9.6%, 8.3%, and 6.5% of dry 100% MeOH, 50% MeOH, BuOH, and W extracts, respectively. 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activities were 70.0%, 45.8%, 41.0%, and 33.6%, and 50% inhibitory concentration values were 33.1, 79.4, 35.6, and 204.2 μg/mL for HepG2 cell proliferation inhibition for 100% MeOH, 50% MeOH, BuOH, and W extracts, respectively. MeOH extracts caused cell cycle G2/M arrest and induced the caspase cascade and apoptosis, but the W extract had very little effect on cell cycle progression. MeOH extracts reduced the level of topoisomerase IIα but increased the level of p27(Kip1), with no significant effect on p21(Cip1/waf1). Therefore, we concluded that phenolic-containing organic extracts of mulberry leaves inhibit the growth of HepG2 hepatoma cells through coordinated actions of inducing cell cycle arrest in the G2/M phase (with p27(Kip1) protein expression), inhibiting topoisomerase IIα activity, and inducing cell apoptosis by activation of caspases.

ER, PgR, HER-2, Ki-67, topoisomerase IIα, and nm23-H1 proteins expression as predictors of pathological complete response to neoadjuvant chemotherapy for locally advanced breast cancer

The purpose of this study was to evaluate the importance of biological markers to predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) in patients with locally advanced breast cancer (LABC) One hundred and twelve consecutive patients with clinical stage III LABC who had received NCT with docetaxel and epirubicin from March 2006 to March 2009 were included in this retrospective study. The pre-NCT treatment expression levels of Ki-67 proliferation index, estrogen receptor (ER), progesterone receptor (PgR), epidermal growth factor receptor 2 (HER-2), Topoisomerase II alpha (Topo-II), and nm23-H1 were detected by immunohistochemistry (IHC). A total of 361 cycles were administered with the median number of three cycles per patient (range, 2-6). The pCR rate was 9.8% (95% CI, 4.3-15.3%). In univariate analysis, poor tumor differentiation, both negative of ER/PgR, negative Topo-II, and positive nm23-H1 were found to be significantly predictive of a pCR. ER/PgR status and nm23-H1 were significant for pCR on multivariate analysis (P = 0.006 and 0.025, respectively). ER/PgR status and nm23-H1 are independent predictive factors of pCR to neoadjuvant docetaxel plus epirubicin combination chemotherapy in patients with LABC.

Effect of hydroquinone on expression of topoisomerase enzyme IIα in human bone marrow mononuclear cells

To investigate the effects of hydroquinone (HQ) on expression of topoisomerase IIα (TOPOIIα) in human bone marrow mononuclear cells, and to explore the role and possible regulatory mechanism of TOPOIIα involved in toxicity of HQ to hematopoietic cells. After human bone marrow mononuclear cells were exposed to 50 µmol/L HQ (used the cells which were exposed to sterile distilled water as control); the activity of TOPOII was measured by TOPOII assay kit; the expression levels of TOPOIIα mRNA and protein were detected by RT-PCR technique and Western blotting method respectively; the chromatin immunoprecipitation (ChIP) assay was carried out to study the possible mechanism of TOPOIIα expression changes. (1) The activity of TOPOII was inhibited obviously; the protein and mRNA expression of TOPOIIα were 0.017 ± 0.029 and 0.610 ± 0.128, significantly lower than that in the control with the significant difference (P < 0.01) after treated with HQ for 10 h; (2) The decreased content of TOPOIIα was associated with descended level of histone H4 acetylation than in the control, from 1.198 ± 0.056 to 0.324 ± 0.229, with the significant difference (P < 0.01), without accompanied descended level of histone H3 acetylation, from 1.253 ± 0.045 to 1.177 ± 0.025 (P > 0.05); (3) TOPOIIα mRNA expression decreased gradually after HQ processing, and the chemical modification (histone H4 acetylation) of TOPOIIα promoter happened prior to the mRNA expression. HQ could repress the expression of TOPOIIα in human bone marrow mononuclear cells; the change of histone chemical modification plays an important role in the benzene's hematopoietic toxicity.

Topoisomerase II-α as a predictive factor of response to therapy with anthracyclines in locally advanced breast cancer

BackgroundTopoisomerase II-α is a molecular target of anthracyclines; several studies have suggested that topoisomerase II-α expression is related to response to anthracycline treatment. The objective of this study was to evaluate if topoisomerase II-α overexpression predicts response to anthracycline treatment in locally advanced breast cancer patients. Material and methodsTopoisomerase II-α, HER2, estrogen receptor (ER) and progesterone receptor (PR) expression were evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded breast tumors from 111 patients presenting with locally advanced breast cancer between 1995 and 2002. The prognostic value of these markers was analyzed using a multivariate proportional hazards regression model and an interaction analysis between topoisomerase II-α status and dose intensity. ResultsTumors from 40 patients (36%) showed topoisomerase II-α overexpression, 62 patients (56%) for ER, 39 (35%) for PR and 26 (23%) for HER2. There were no significant correlations between topoisomerase II-α expression and response to therapy, progression-free survival (PFS) or overall survival (OS). Anthracycline dose intensity had a significant impact on PFS and OS in patients overexpressing topoisomerase II-α (P=0.010 and 0.027, respectively). Negative PR (P=0.041), positive HER2 (P=0.013) were identified as risk factors in the multivariate model. The multivariate analysis in patients topoisomerase II-α negative shown no significance (HR=0.92, IC 95% 0.39–2.15, P=0.839) while the multivariate analysis in topoisomerase II-α positive, dose intensity shown to be statistically significant (HR=2.725, IC 95% 1.07–6.95, P=0.036). ConclusionsOur data do not support a correlation between topoisomerase II-α expression in breast cancer patients and improved clinical benefit with anthracycline therapy. However, they do suggest that tumors overexpressing topoisomerase II-α may experience better clinical benefit with higher anthracycline dose intensity.