In Vitro and In Silico Screening and Characterization of Antimicrobial Napin Bioactive Protein in Brassica juncea and Moringa oleifera.

The study aimed to investigate the antibacterial activity of Mustard (Brassica juncea) and Moringa (Moringa oleifera) leaf extracts and coagulant protein for their potential application in water treatment. Bacterial cell aggregation and growth kinetics studies were employed for thirteen bacterial strains with different concentrations of leaf extracts and coagulant protein. Moringa oleifera leaf extract (MOS) and coagulant protein showed cell aggregation against ten bacterial strains, whereas leaf extract alone showed growth inhibition of five bacterial strains for up to 6 h and five bacterial strains for up to 3 h. Brassica juncea leaf extract (BJS) showed growth inhibition for up to 6 h, and three bacterial strains showed inhibition for up to 3 h. The highest inhibition concentration with 2.5 mg/mL was 19 mm, and furthermore, the minimum inhibitory concentration (MIC) (0.5 mg/mL) and MBC (1.5 mg/mL) were determined to have a higher antibacterial effect for <3 KDa peptides. Based on LCMS analysis, napin was identified in both MOS and BJS; furthermore, the mode of action of napin peptide was determined on lipoprotein X complex (LpxC) and four-chained structured binding protein of bacterial type II topoisomerase (4PLB). The docking analysis has exhibited moderate to potent inhibition with a range of dock score -912.9 Kcal/mol. Thus, it possesses antibacterial-coagulant potential bioactive peptides present in the Moringa oleifera purified protein (MOP) and Brassica juncea purified protein (BJP) that could act as an effective antimicrobial agent to replace currently available antibiotics. The result implies that MOP and Brassica juncea purified coagulant (BJP) proteins may perform a wide degree of antibacterial functions against different pathogens.

Natural Products as Anti-Cancerous Therapeutic Molecules Targeted towards Topoisomerases.

Topoisomerases are reported to resolve the topological problems of DNA during several cellular processes, such as DNA replication, transcription, recombination, and chromatin remodeling. Two types of topoisomerases (Topo I and II) accomplish their designated tasks by introducing single- or double-strand breaks within the duplex DNA molecules, and thus maintain the proper structural conditions of DNA to release the topological torsions, which is generated by unwinding of DNA to access coded information, in the course of replication, transcription, and other processes. Both the topoisomerases have been looked at as crucial targets against various types of cancers such as lung, melanoma, breast, and prostate cancers. Conceptually, targeting topoisomerases will disrupt both DNA replication and transcription, thereby leading to inhibition of cell division and consequently stopping the growth of actively dividing cancerous cells. Since the discovery of camptothecin (an alkaloid) as an inhibitor of Topo I in 1958, a number of derivatives of camptothecin were developed as potent inhibitors of Topo I. Two such derivatives of camptothecin, namely, topotecan and irinotecan, have been commonly used as US Food and Drug Administration (FDA) approved drugs against Topo I. Similarly, the first Topo II inhibitor, namely, etoposide, an analogue of podophyllotoxin, was developed in 1966 and got FDA approval as an anti-cancer drug in 1983. Subsequently, several other inhibitors of Topo II, such as doxorubicin, mitoxantrone, and teniposide, were developed. These drugs have been reported to cause accumulation of cytotoxic non-reversible DNA double-strand breaks (cleavable complex). Thus, the present review describes the anticancer potential of plant-derived secondary metabolites belonging to alkaloids, flavonoids and terpenoids directed against topoisomerases. Furthermore, in view of the recent advances made in the field of computer-aided drug design, the present review also discusses the use of computational approaches such as ADMET, molecular docking, molecular dynamics simulation and QSAR to assess and predict the safety, efficacy, potency and identification of these potent anti-cancerous therapeutic molecules.

Spica Prunellae Extract Enhances Fluorouracil Sensitivity of 5-Fluorouracil-Resistant Human Colon Carcinoma HCT-8/5-FU Cells via TOP2α and miR-494.

The use of 5-fluorouracil (5-FU) has been proven benefits, but it also has adverse events in colorectal cancer (CRC) chemotherapy. In this study, we explored the mechanism of 5-FU resistance by bioinformatics analysis of the NCBI public dataset series GSE81005. Fifteen hub genes were screened out of 582 different expressed genes. Modules of the hub genes in protein-protein interaction networks gathered to TOP2α showed a decrease in HCT-8 cells but an increase in 5-FU-resistant HCT-8/5-FU cells with 5-FU exposure. Downregulation of TOP2α with siRNA or miR-494 transfection resulted in an increase of cytotoxicity and decrease of cell colonies to 5-FU for HCT-8/5-FU cells. Moreover, we found that an ethanol extract of Spica Prunellae (EESP), which is a traditional Chinese medicine with clinically beneficial effects in various cancers, was able to enhance the sensitivity of 5-FU in HCT-8/5-FU cells and partly reverse the 5-FU resistance effect. It significantly helped suppress cell growth and induced cell apoptosis in HCT-8/5-FU cells with the expression of TOP2α being significantly suppressed, which increased by 5-FU. Consistently, miR-494, which reportedly regulates TOP2α, exhibited reverse trends in EESP/5-FU combination treatment. These results suggested that Spica Prunellae may be beneficial in the treatment of 5-FU-resistant CRC patients.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy.

PURPOSE: High activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC. METHODS: Patients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed. RESULTS: We found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39+CCL18- or YKL-39-CCL18+) and only one patient had M2- macrophage phenotype (YKL-39-CCL18-). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2- phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2- phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39-CCL18-). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2- phenotypes between pathologically responding and non-responding patients within DOX-Sense and DOX-Res groups were statistically significant. CONCLUSIONS: Thus, we showed that in patients with breast cancer who received anthracycline-containing NAC the absence of clinical response is associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18 + or YKL-39 + CCL18-) based on TOP2a overexpression data.

Suppression of PTEN/AKT signaling decreases the expression of TUBB3 and TOP2A with subsequent inhibition of cell growth and induction of apoptosis in human breast cancer MCF-7 cells via ATP and caspase-3 signaling pathways.

The aim of the present study was to evaluate the effects of PTEN/AKT signaling on TUBB3 and TOP2A expression and on the subsequent cell growth of human breast cancer MCF-7 cells. We found that the disease-free survival (DFS) and overall survival (OS) of breast cancer patients with TUBB3­positive tumors were lower than these rates in the patients with TUBB3-negative tumors. Meanwhile, DFS and OS of breast cancer patients with TOP2A-positive tumors were also lower than these rates in patients with TOP2A-negative tumors. Suppression of PTEN reduced the protein expression of TUBB3 and TOP2A in MCF-7 cells. Suppression of PTEN also reduced cell proliferation and induced apoptosis and caspase-3 activity in MCF-7 cells. Moreover, an increase in ATP also reduced TUBB3 and TOP2A protein expression, reduced cell proliferation and induced apoptosis and caspase-3 activity in the MCF-7 cells following suppression of PTEN. Suppression of phosphorylation-AKT (p-AKT) reduced the protein expression of TUBB3 and TOP2A in the MCF-7 cells. Suppression of p-AKT also reduced cell proliferation and induced apoptosis and caspase-3 activity in the MCF-7 cells. Then, ATP also reduced TUBB3 and TOP2A protein expression, reduced cell proliferation and induced apoptosis and caspase-3 activity in MCF-7 cells following suppression of p-AKT. These results suggest that PTEN/AKT signaling affects the expression of TUBB3 and TOP2A reducing cell growth and inducing apoptosis of human breast cancer MCF-7 cells through ATP and caspase-3 signaling pathways. TUBB3 and TOP2A may be promising prognostic markers for the efficacy of adjuvant cisplatin-based chemotherapy.

Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts.

Leishmaniasis is a disease caused by pathogenic Leishmania parasites; current treatments are toxic and expensive, and drug resistance has emerged. While pentamidine, a diamidine-type compound, is one of the treatments, its antileishmanial mechanism of action has not been investigated in depth. Here we tested several diamidines, including pentamidine and its analog DB75, against Leishmania donovani and elucidated their antileishmanial mechanisms. We identified three promising new antileishmanial diamidine compounds with 50% effective concentrations (EC50s) of 3.2, 3.4, and 4.5 µM, while pentamidine and DB75 exhibited EC50s of 1.46 and 20 µM, respectively. The most potent antileishmanial inhibitor, compound 1, showed strong DNA binding properties, with a shift in the melting temperature (ΔTm) of 24.2°C, whereas pentamidine had a ΔTm value of 2.1°C, and DB75 had a ΔTm value of 7.7°C. Additionally, DB75 localized in L. donovani kinetoplast DNA (kDNA) and mitochondria but not in nuclear DNA (nDNA). For 2 new diamidines, strong localization signals were observed in kDNA at 1 µM, and at higher concentrations, the signals also appeared in nuclei. All tested diamidines showed selective and dose-dependent inhibition of kDNA, but not nDNA, replication, likely by inhibiting L. donovani topoisomerase IB. Overall, these results suggest that diamidine antileishmanial compounds exert activity by accumulating toward and blocking replication of parasite kDNA.

Bioassay-Guided Isolation of Two Flavonoids from Derris scandens with Topoisomerase II Poison Activity.

Derris scandens (ROXB.) BENTH. (Fabaceae) is used as an alternative treatment for cancer in Thai traditional medicine. Investigation of the topoisomerase II (Top2) poison of compounds isolated from this plant may reveal new drug leads for the treatment of cancer. Bioassay-guided isolation was performed on an extract of D. scandens stems using a yeast cell-based assay. A yeast strain expressing the top2-1 temperature-sensitive mutant was used to assay Top2 activity. At the permissive temperature of 25°C, yeast cells were highly sensitive to Top2 poison agents. At the semi-permissive temperature of 30°C, where enzyme activity was present but greatly diminished, cells displayed only marginal sensitivity. The bioassay-guided fractionation of the extract led to the isolation of two known isoflavones: 5,7,4'-trihydroxy-6,8-diprenylisoflavone (1) and lupalbigenin (2). These two compounds also displayed cytotoxicity against three different cancer cell lines, KB, MCF-7 and NCI-H187. In conclusion, Top2 poison agents from D. scandens are reported for the first time, substantiating the use of D. scandens in Thai traditional medicine for cancer treatment.

Tetramethylpyrazine induces SH-SY5Y cell differentiation toward the neuronal phenotype through activation of the PI3K/Akt/Sp1/TopoIIß pathway.

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Previously, we have shown that TMP induces human SH-SY5Y neuroblastoma cell differentiation toward the neuronal phenotype by targeting topoisomeraseIIß (TopoIIß), a protein implicated in neural development. In the present study, we aimed to elucidate whether the transcriptional factors specificity protein 1 (Sp1) and nuclear factor Y (NF-Y), in addition to the upstream signaling pathways ERK1/2 and PI3K/Akt, are involved in modulating TopoIIß expression in the neuronal differentiation process. We demonstrated that SH-SY5Y cells treated with TMP (80µM) terminally differentiated into neurons, characterized by increased neuronal markers, tubulin ßIII and microtubule associated protein 2 (MAP2), and increased neurite outgrowth, with no negative effect on cell survival. TMP also increased the expression of TopoIIß, which was accompanied by increased expression of Sp1 in the differentiated neuron-like cells, whereas NF-Y protein levels remained unchanged following the differentiation progression. We also found that the phosphorylation level of Akt, but not ERK1/2, was significantly increased as a result of TMP stimulation. Furthermore, as established by chromatin immunoprecipitation (ChIP) assay, activation of the PI3K/Akt pathway increased Sp1 binding to the promoter of the TopoIIß gene. Blockage of PI3K/Akt was shown to lead to subsequent inhibition of TopoIIß expression and neuronal differentiation. Collectively, the results indicate that the PI3K/Akt/Sp1/TopoIIß signaling pathway is necessary for TMP-induced neuronal differentiation. Our findings offer mechanistic insights into understanding the upstream regulation of TopoIIß in neuronal differentiation, and suggest potential applications of TMP both in neuroscience research and clinical practice to treat relevant diseases of the nervous system.

Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

Predicting Anthracycline Benefit: TOP2A and CEP17-Not Only but Also.

PURPOSE: Evidence supporting the clinical utility of predictive biomarkers of anthracycline activity is weak, with a recent meta-analysis failing to provide strong evidence for either HER2 or TOP2A. Having previously shown that duplication of chromosome 17 pericentromeric alpha satellite as measured with a centromere enumeration probe (CEP17) predicted sensitivity to anthracyclines, we report here an individual patient-level pooled analysis of data from five trials comparing anthracycline-based chemotherapy with CMF (cyclophosphamide, methotrexate, and fluorouracil) as adjuvant chemotherapy for early breast cancer. PATIENTS AND METHODS: Fluorescent in situ hybridization for CEP17, HER2, and TOP2A was performed in three laboratories on samples from 3,846 of 4,864 eligible patients from five trials evaluating anthracycline-containing chemotherapy versus CMF. Methodologic differences did not affect HER2-to-CEP17 ratios but necessitated different definitions for CEP17 duplication: > 1.86 observed copies per cell for BR9601, NEAT, Belgian, and DBCG89D trials and > 2.25 for the MA.5 trial. RESULTS: Fluorescent in situ hybridization data were available in 89.3% (HER2), 83.9% (CEP17), and 80.6% (TOP2A) of 3,846 patient cases with available tissue. Both CEP17and TOP2A treatment-by-marker interactions remained significant in adjusted analyses for recurrence-free and overall survival, whereas HER2 did not. A combined CEP17 and TOP2A-adjusted model predicted anthracycline benefit across all five trials for both recurrence-free (hazard ratio, 0.64; 95% CI, 0.51 to 0.82; P = .001) and overall survival (hazard ratio, 0.66; 95% CI, 0.51 to 0.85; P = .005). CONCLUSION: This prospectively planned individual-patient pooled analysis of patient cases from five adjuvant trials confirms that patients whose tumors harbor either CEP17 duplication or TOP2A aberrations, but not HER2 amplification, benefit from adjuvant anthracycline chemotherapy.

Tetramethylpyrazine promotes SH-SY5Y cell differentiation into neurons through epigenetic regulation of Topoisomerase IIß.

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Recently, it has been reported that TMP enhances neurogenesis, and promotes neural stem cell differentiation toward neurons. However, its molecular basis remains unknown. Topoisomerase IIß (TopoIIß) is a nuclear enzyme with an essential role in neuronal development. This study aimed to investigate whether TopoIIß is involved in TMP-induced neuronal differentiation. We examined the effect of TMP on neuronal differentiation of SH-SY5Y cells. It was found that TMP inhibited cell proliferation and induced G0/G1 cell cycle arrest. TMP promoted SH-SY5Y cells to differentiate toward post-mitotic neurons characterized by long, out-branched neurites and up-regulated neuronal markers, microtubule-associated protein 2 (MAP2) and tau. Meanwhile, we demonstrated that TopoIIß was highly expressed following TMP treatment. To unravel how TMP affects TopoIIß expression, two chromatin active markers, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4) were examined in this study. Our data showed that the levels of Ac-H3 and Ac-H4 were positively correlated with TopoIIß expression in the processes of neuronal differentiation. We further performed chromatin immunoprecipitation (ChIP) analysis and identified that TMP enhanced the recruitment of Ac-H3 and Ac-H4 to the TopoIIß gene promoter region. Therefore, we concluded that TMP may stimulate neuronal differentiation of SH-SY5Y cells through epigenetic regulation of TopoIIß. These results suggest a novel molecular mechanism underlying TMP-promoted neuronal differentiation.

Shikonin shortens the circadian period: possible involvement of Top2 inhibition.

The naphthoquinone pigment, shikonin, is a natural product derived from Lithospermum erythrorhizon and an active component of a Chinese traditional herbal therapeutic. We identified shikonin as a candidate for shortening the circadian period using real-time reporter gene assays based on NIH3T3-derived stable reporter cells. Period length that became shortened in cells incubated with shikonin or etoposide reverted to that of control cells after continued incubation without these compounds. These findings indicated that shikonin and etoposide shorten the circadian period reversibly and through similar mechanisms. Topoisomerase II (Top2)-specific decatenation assays confirmed that shikonin, liker etoposide, is a Top2 inhibitor. Shikonin was incorporated into the nucleus and Top2 was located in the Bmal1 promoter, suggesting the relationship between Bmal1 transcription and Top2 inhibition. Top2a siRNA also shortened period length, suggesting that Top2 is involved in this process. Promoter assays showed that Top2a siRNA, etoposide and shikonin reduce Bmal1 promoter activity. These findings indicated that Top2 is involved in Bmal1 transcription and influences the circadian period, and that shikonin is a novel contributor to the control of period length in mammalian cells.

Mechanism study of goldenseal-associated DNA damage.

Goldenseal has been used for the treatment of a wide variety of ailments including gastrointestinal disturbances, urinary tract disorders, and inflammation. The five major alkaloid constituents in goldenseal are berberine, palmatine, hydrastine, hydrastinine, and canadine. When goldenseal was evaluated by the National Toxicology Program (NTP) in the standard 2-year bioassay, goldenseal induced an increase in liver tumors in rats and mice; however, the mechanism of goldenseal-associated liver carcinogenicity remains unknown. In this study, the toxicity of the five goldenseal alkaloid constituents was characterized, and their toxic potencies were compared. As measured by the Comet assay and the expression of γ-H2A.X, berberine, followed by palmatine, appeared to be the most potent DNA damage inducer in human hepatoma HepG2 cells. Berberine and palmatine suppressed the activities of both topoisomerase (Topo) I and II. In berberine-treated cells, DNA damage was shown to be directly associated with the inhibitory effect of Topo II, but not Topo I by silencing gene of Topo I or Topo II. In addition, DNA damage was also observed when cells were treated with commercially available goldenseal extracts and the extent of DNA damage was positively correlated to the berberine content. Our findings suggest that the Topo II inhibitory effect may contribute to berberine- and goldenseal-induced genotoxicity and tumorigenicity.

Is TIMP-1 immunoreactivity alone or in combination with other markers a predictor of benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial?

INTRODUCTION: Predictive cancer biomarkers to guide the right treatment to the right patient at the right time are strongly needed. The purpose of the present study was to validate prior results that tissue inhibitor of metalloproteinase 1 (TIMP-1) alone or in combination with either HER2 or TOP2A copy number can be used to predict benefit from epirubicin (E) containing chemotherapy compared with cyclophosphamide, methotrexate and fluorouracil (CMF) treatment. METHODS: For the purpose of this study, formalin fixed paraffin embedded tumor tissue from women recruited into the BR9601 clinical trial, which randomized patients to E-CMF versus CMF, were analyzed for TIMP-1 immunoreactivity. Using previously collected data for HER2 amplification and TOP2A gene aberrations, we defined patients as "anthracycline non-responsive", that is, 2T (TIMP-1 immunoreactive and TOP2A normal) and HT (TIMP-1 immunoreactive and HER2 negative) and anthracycline responsive (all other cases). RESULTS: In total, 288 tumors were available for TIMP-1 analysis with (183/274) 66.8%, and (181/274) 66.0% being classed as 2T and HT responsive, respectively. TIMP-1 was neither associated with patient prognosis (relapse free survival or overall survival) nor with a differential effect of E-CMF and CMF. Also, TIMP-1 did not add to the predictive value of HER2, TOP2A gene aberrations, or to Ki67 immunoreactivity. CONCLUSION: This study could not confirm the predictive value of TIMP-1 immunoreactivity in patients randomized to receive E-CMF versus CMF as adjuvant treatment for primary breast cancer.

Epimerization of green tea catechins during brewing does not affect the ability to poison human type II topoisomerases.

(-)-Epigallocatechin gallate (EGCG) is the most abundant and biologically active polyphenol in green tea (Camellia sinensis) leaves, and many of its cellular effects are consistent with its actions as a topoisomerase II poison. In contrast to genistein and several related bioflavonoids that act as interfacial poisons, EGCG was the first bioflavonoid shown to act as a covalent topoisomerase II poison. Although studies routinely examine the effects of dietary phytochemicals on enzyme and cellular systems, they often fail to consider that many compounds are altered during cooking or cellular metabolism. To this point, the majority of EGCG and related catechins in green tea leaves are epimerized during the brewing process. Epimerization inverts the stereochemistry of the bond that bridges the B- and C-rings and converts EGCG to (-)-gallocatechin gallate (GCG). Consequently, a significant proportion of EGCG that is ingested during the consumption of green tea is actually GCG. Therefore, the effects of GCG and related epimerized green tea catechins on human topoisomerase IIα and IIß were characterized. GCG increased levels of DNA cleavage mediated by both enzyme isoforms with an activity that was similar to that of EGCG. GCG acted primarily by inhibiting the ability of topoisomerase IIα and IIß to ligate cleaved DNA. Several lines of evidence indicate that GCG functions as a covalent topoisomerase II poison that adducts the enzyme. Finally, epimerization did not affect the reactivity of the chemical substituents (the three hydroxyl groups on the B-ring) that were required for enzyme poisoning. Thus, the activity of covalent topoisomerase II poisons appears to be less sensitive to stereochemical changes than interfacial poisons.

TIMP-1 in combination with HER2 and TOP2A for prediction of benefit from adjuvant anthracyclines in high-risk breast cancer patients.

HER2 amplification, TOP2A aberrations, and absence of tissue inhibitor of metalloproteinase (TIMP-1) expression in breast carcinomas have been shown to be associated with incremental benefit from anthracycline-containing adjuvant chemotherapy, and this study was undertaken to validate these findings in a similar, but independent, randomized clinical trial. TIMP-1 was examined by immunohistochemistry in archival tumor tissue from 403 of 716 premenopausal high-risk patients with known HER2 and TOP2A status who were randomized to cyclophosphamide, epirubicin, and fluorouracil (CEF) or cyclophosphamide, methotrexate, and fluorouracil (CMF) in the MA.5 trial. Ninety-eight (24%) patients had no TIMP-1 staining of tumor cells, 27% were HER2 amplified, and 18% were TOP2A aberrant. Forty-four percentage was classified as HT responsive (HER2 amplified and/or TIMP-1 negative) and 37% as 2T responsive (TOP2A aberrant and/or TIMP-1 negative). There was no heterogeneity in treatment effect of CEF versus CMF according to TIMP-1. In HT-responsive patients, CEF was superior to CMF with an improved RFS (adjusted HR, 0.64; 95% CI, 0.42-0.97), but this was not significant for OS (adjusted HR, 0.66; 95% CI, 0.42-1.04). A significant HT profile versus treatment interaction was detected for OS (P = 0.03). In 2T-responsive patients, CEF seemed to improve RFS compared to CMF (adjusted HR, 0.67; 95% CI, 0.43-1.03) and improved OS (adjusted HR, 0.58; 95% CI, 0.36-0.93). A significant 2T profile versus treatment interaction was detected for OS (P = 0.01). With this study, we validate a more substantial reduction in mortality by CEF compared to CMF in patients with an HT- or 2T-responsive profile; however, we could not show a similarly significant reduction in RFS events, where a benefit of CEF over CMF was found irrespective of TIMP-1 status. Further studies are necessary before the HT and 2T profiles may be used to direct the use of anthracyclines.

Utilization of fluorescence in situ hybridization with cytokeratin discriminators in TOP2A assessment of chemotherapy-treated patients with breast cancer.

Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P ≤ .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue.

Tumor topoisomerase II alpha status and response to anthracycline-based neoadjuvant chemotherapy in breast cancer.

OBJECTIVES: Individualized chemotherapy for breast cancer improves the outcome. Anthracyclines target the enzyme topoisomerase IIα (TOP2A). We set out to perform a retrospective study of the presence of gene abnormalities and the expression of TOP2A in a cohort of breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy. METHODS: Forty-three patients with 45 breast cancers were treated with neoadjuvant docetaxel-epirubicin with/without capecitabine chemotherapy. The TOP2A status of the cancers, determined retrospectively by fluorescent in situ hybridization and immunohistochemistry, was analyzed in relation to the standard clinical and pathological data. RESULTS: Clinically and pathologically complete remission (pCR) was achieved in 15 (33.3%) and 9 (20%) cases, respectively. The TOP2A gene was amplified in 2 human epidermal growth factor receptor 2 (HER2)-positive cancers (8%), and 32 (84.2%) overall exhibited TOP2A expression in >15% of the cells. The expression of TOP2A exhibited a strong correlation with the expression of Ki67 (R = 0.743, p < 0.001), and was negatively correlated with estrogen receptors (ER; R = 0.404, p = 0.012) and progesterone receptors (R = 0.430, p = 0.007). The expression of TOP2A was not related to the amplification of the TOP2A gene or the HER2 status of the tumor. The proportions of Ki67- and TOP2A-positive tumor cells were significantly reduced after chemotherapy (56.1 ± 23.6 vs. 19.0 ± 27.7%, p = 0.004, and 41.0 ± 27.9 vs. 12.7 ± 24.8%, p < 0.001, respectively). The development of pCR was related to a high grade (p = 0.054), ER negativity (p = 0.027) and high TOP2A expression (p = 0.037). The expression of TOP2A was an independent predictor of pCR (OR = 1.460, for every 10% increase, 95% CI: 1.016-2.096, p = 0.041). After a median follow-up time of 31.0 months, neither relapse-free survival nor overall survival was related to the tumor response. CONCLUSIONS: TOP2A expression is a marker of the tumor's proliferation rate and sensitivity to anthracycline-based chemotherapy, and does not depend on the amplification of its gene.

Topoisomerase II alpha protein and responsiveness of breast cancer to adjuvant chemotherapy with CEF compared to CMF in the NCIC CTG randomized MA.5 adjuvant trial.

Overexpression of topoisomerase II protein (topo 2α) is postulated to be more closely associated with responsiveness to anthracycline-containing chemotherapy than human epidermal growth factor receptor type 2 (HER2) gene amplification or alterations in the topoisomerase II alpha gene (TOP2A). The authors used tissue microarrays from 477 of 710 premenopausal women with node-positive breast cancer randomized to CEF or CMF adjuvant chemotherapy in the NCIC Clinical Trials Group Mammary 5 (MA.5) trial. No significant interaction was found between treatment and continuous topo 2α level in either relapse-free (RFS) or overall survival (OS). In 136 patients (28.5%) whose tumors showed topo 2α overexpression by immunohistochemistry based on a cut-off of 13%, CEF was superior to CMF for RFS (adjusted HR 0.45; 95% CI 0.25-0.82; P = 0.009) and OS (adjusted HR 0.50; 95% CI 0.26-0.96; P = 0.04). When tumors lacked topo 2α overexpression, CEF was not superior for RFS (adjusted HR 0.88; 95% CI 0.64-1.22; P = 0.46) or OS (adjusted HR 0.95; 95% CI 0.66-1.38; P = 0.80). Interaction between topo 2α and treatment was borderline significant for RFS (P = 0.04) and OS (P = 0.05) and not substantially more significant than between TOP2A gene alteration (P (interaction) = 0.09 for RFS and 0.02 for OS) or HER2 overexpression (P (interaction) = 0.002 for RFS and 0.009 for OS). Topo 2α protein overexpression based on the cut-off identified in this study, TOP2A gene alterations and HER2 protein overexpression were each associated with responsiveness to anthracycline-containing chemotherapy. The topo 2α protein analysis was exploratory and will require further validation.

Prediction of epigenetically regulated genes in breast cancer cell lines.

BACKGROUND: Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profiles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profiles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fixed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. RESULTS: Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically significant negative correlation between methylation profiles and gene expression in the panel of breast cancer cell lines. Subnetwork enrichment of these genes has identified 35 common regulators with 6 or more predicted markers. In addition to identifying epigenetically regulated genes, we show evidence of differentially expressed methylation patterns between the basal and luminal subtypes. CONCLUSIONS: Our results indicate that the proposed computational protocol is a viable platform for identifying epigenetically regulated genes. Our protocol has generated a list of predictors including COL1A2, TOP2A, TFF1, and VAV3, genes whose key roles in epigenetic regulation is documented in the literature. Subnetwork enrichment of these predicted markers further suggests that epigenetic regulation of individual genes occurs in a coordinated fashion and through common regulators.