HCC1937 Breast Cancer Cell Lines Research Articles

Synthesis and bioassay of 3-Aryl -1-(pyridin-4-yl)benzo[4,5]imidazo[1,2-d][1,2,4]- triazin-4(3H)-ones as anti-cancer agents.

Four novel 3-Aryl -1-(pyridin-4-yl)benzo[4,5]imidazo[1,2-d][1,2,4]- triazin-4(3H)-ones derivatives (C1 to C4) have been designed, synthesized, and evaluated for their anticancer activity. The structure of compounds was characterized by IR,1H NMR, 13C NMR and high-resolution mass (HRMS). The crystal structures of C1, C2 and C4 were previously determined by single-crystal X-ray analysis.The results from docking experiments with EGFR suggested the binding of the compounds at the active site of EGFR. The new compounds exhibited different levels of cytotoxicity against HCC1937 and MCF7 breast cancer cells. Results of the MTT assay identified C3 as the most cytotoxic of the series against both MCF7 and HCC1937 breast cancer cell lines with IC50 values of 36.4 and 48.2 µM, respectively. In addition to its ability to inhibit cell growth and colony formation ability, C3 also inhibited breast cancer cell migration. Western blotting results showed that C3 treatment inhibited EGFR signaling and induced cell cycle arrest and apoptosis as indicated by the low level of p-EGFR and p-AKT and the increasing levels of p53, p21 and cleaved PARP. Our work represents a promising starting point for the development of a new series of compounds targeting cancer cells.

The Anticancer Effects of Novel Imidazo[1,2-a]Pyridine Compounds against HCC1937 Breast Cancer Cells.

Anticancer drugs confront clinical obstacles such as drug resistance and adverse effects. Imidazo[1,2-a]pyridines (IPs) compounds have lately gained considerable interest as possible anticancer therapeutics due to their potent inhibitory function against cancers cells. This study was to determine the anticancer activities of three novel IPs (IP-5, IP-6, and IP-7) against the HCC1937 breast cancer cell line in vitro. The cytotoxic and anti-proliferative effects of IPs compounds in HCC1937 cells were determined by cell viability (MTT) assay, trypan blue assay, and clonogenic survival assay. Scratch motility assay was used to test the antimigration ability of the IPs. Western blot analysis was carried out to detect the level of apoptosis and cell cycle protein markers and to understand the mechanism of action of IPs compounds. IP-5 and IP-6 have a strong cytotoxic impact against HCC1937 cells with IC50 values of 45µM and 47.7µM respectively. IP-7 possesses less cytotoxic effect against HCC1937 cells with IC50 of 79.6µM. Trypan blue assay showed that the three compounds induce significant cell death in the HCC1937 cells. Clonogenic and mammosphere assays demonstrated that IP-5 reduced the HCC1937 cells survival rate by more than 25.0% at 1000 cell concentrations. Western blotting analysis showed that IP-5 compound causes cell cycle arrest as noted by the increasing levels of p53 and p21 in treated cells. IP-5 induced an extrinsic apoptosis pathway as reveals from the increased activity of caspase 7, caspase 8, and the increasing level of PARP cleavage in treated cells. Also, IP-5 treated cells revealed segmented chromatin which is characteristic of apoptotic cells as shown by DAPI stain. Importantly, In comparison to control cells, IP-5-treated cells exhibited lower levels of pAKT. The novel three IPs compounds represent potential active anticancer compounds against HCC1937 breast cancer cells in vitro.

The mechanism of action of Fangji Huangqi Decoction on epithelial-mesenchymal transition in breast cancer using high-throughput next-generation sequencing and network pharmacology

Ethnopharmacological relevanceFangji Huangqi Decoction (FHD) is widely used in traditional Chinese medicine (TCM). FHD has been hypothesized to inhibit the epithelial-mesenchymal transition (EMT) process, which may positively impact breast cancer prevention and treatment. However, its exact mechanism of action is still unknown. Aim of the studyThis study aimed to screen potential targets of FHD for the treatment of EMT in breast cancer through network pharmacology, and to verify their therapeutic effects in vitro experiments and high-throughput second-generation sequencing. Materials and methodsThe data sets of effective components and targets of FHD were established through the Traditional Chinese Medicine Systems Pharmacology database. The GeneCards and OMIM databases were used to establish breast cancer-related target datasets, which were then matched with the TCM target data. The interaction between key target proteins was analyzed using the STRING database; the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to identify the associated biological processes and enriched signal pathways, respectively. The active ingredient disease target network was analyzed using Cytoscape. Finally, next generation sequencing was used to verify the related pathways of FHD intervention in EMT in breast cancer. High-content screening was used to identify the genes/pathways affected by FHD. MDA-MB-231 and HCC-1937 breast cancer cell lines were used to evaluate the impact of FHD on migration, invasion, and EMT. ResultsEighty possible significant targets were identified for the treatment of breast cancer EMT with FHD; GO and KEGG were used to identify 173 cell biological processes associated with breast cancer (P < 0.05), including the NF-κB and PI3K-Akt signaling pathways. The high-throughput sequencing and network pharmacology results were highly consistent. The migration and invasion ability of MDA-MB-231 cells was reduced and their EMT status could be reversed by DSHR2 knockdown. The results of morphology and scratch assays showed that FHD could improve the EMT status of HCC-1973. ConclusionsThis study provides more evidence to support the clinical application of FHD, which has reliable interventional effects on breast cancer EMT. Its therapeutic effects may involve a multi-target, multi-pathway, and multi-mechanism effect.

BRCA1 participates in the expression of noncoding XIST RNA

Introduction . Noncoding RNA of XIST gene (X inactivation-specific transcript) initiates inactivation of one of X chromosomes in cells of female organism. Further stages of this process include chromatin epigenetic modifications leading to the inhibition of the most genes on X chromosome. Recently the data were obtained that tumor suppressor BRCA1 is associated with inactive X chromosome (Xi) participating in XIST RNA localization on Xi and influencing XIST RNA expression.Objective: to reveal the role of BRCA1 in XIST RNA expression.Materials and methods . The objects of the study were mutant breast cancer cell lines (BRCA1–/–): HCC1395, HCC1937, SUM149PT, and, as controls – cell lines containing wild type of BRCA1 gene (BRCA1+/+): IMR90 и 293T. Method of reverse transcription coupled with polymerase chain reaction (RT-PCR) was used for the analysis of XIST RNA expression.Results . In the clone of doxycycline-inducible HCC1937 breast cancer cell line XIST RNA expression was observed upon BRCA1 induction. In HCC1395, HCC1937 and SUM149PT breast cancer cell lines containing mutant BRCA1 gene (BRCA1–/–) and nonfunctional BRCA1 protein the absence of XIST RNA expression was observed using RT-PCR. This observation indicates the indispensable role of functional BRCA1 protein in XIST RNA expression.Conclusion . Altogether, the data obtained in this study confirm the role of BRCA1 in the expression of noncoding inhibiting XIST RNA and suggest the involvement of BRCA1 in the inhibition of gene expression on Xi.

Abstract P5-04-04: Eribulin differentially disrupts TGF-β signaling pathway in BT-549 and HCC1937 breast cancer cell lines

Abstract Microtubule targeting agents (MTAs) continue to be valuable in treating breast cancer. While these drugs were classically identified as only antimitotic agents, recent evidence demonstrates that the ability of MTAs to disrupt key signaling components in interphase cells likely contributes to their anticancer actions. Yoshida and colleagues demonstrated that eribulin reverses TGF-β-induced epithelial-to-mesenchymal transition (EMT) in preclinical models of triple negative breast cancer (TNBC) within 7days. To gain a deeper understanding of the interphase effects of eribulin in comparison with other MTAs on the TGF-β signaling pathway, we tested the hypothesis that a short-term, 2 h treatment of TNBC cells with MTAs would disrupt TGF- β signaling. The effects of the microtubule destabilizers eribulin and vinorelbine and the microtubule stabilizers paclitaxel and ixabepilone on downstream targets of the TGF-β pathway were evaluated in two TNBC cell lines. In the mesenchymal type BT-549 cells, eribulin and vinorelbine significantly inhibited TGF-β induced expression of Snail and Slug, while the microtubule stabilizers had no effect on their expression. In the basal-like 1 type HCC1937 cells, the microtubule destabilizers inhibited the expression of Snail when compared to vehicle and stabilizer-treated cells, but caused increased expression of Slug. These data suggest that MTAs have different effects on the TGF-β signaling pathway in distinct molecular contexts. Our efforts are directed towards deciphering the effects of eribulin and other MTAs on the TGF- β pathway and how this contributes to differential sensitivity. Preliminary data suggest that eribulin initiated inhibition of Snail and Slug in BT-549 cells is mediated by the scaffold protein NEDD9 and we are testing whether the differential levels of expression of NEDD9 help explain the differences in the effects of eribulin in BT-549 and HCC1937 cells. These results will begin to decipher differences among MTAs in different molecular contexts and might help identify biomarkers for optimal therapies. Funding for this work was provided by Eisai Inc. Citation Format: Kaul R, Risinger AL, Mooberry SL. Eribulin differentially disrupts TGF-β signaling pathway in BT-549 and HCC1937 breast cancer cell lines [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-04-04.

Cytotoxicity Study of Gold Nanoparticles on the Basal-Like Triple-Negative HCC-1937 Breast Cancer Cell Line

The Triple Negative “Basal-like” breast cancer (TNBL) tumours have a high proliferative capacity and develop a resistance phenotype associated with metastases. However, the management of TNBL carcinomas is still not standardized. Among the promising trails, gold nanoparticles could be a relevant tool for the development of a targeted treatment for this breast cancer subtype in monotherapy, associated and/or conjugated with other drugs. In this work, we report the cytotoxicity impact of gold nanoparticles wrapped in Poly-Ethylene Glycol (PEG) on the TNBL HCC-1937 breast cancer cell line. PEG-coated gold nanoparticles (PEG-Au NPs) were synthesized by a two-step method using a reduction process followed by a post-functionalization called PEGylation. PEG-Au NPs were characterized using transmission electron microscopy and X-ray diffraction. The gold content of the samples was determined using atomic absorption spectrometer. The cytotoxicity tests were performed using Sulforhodamine B survival test and resazurin viability test. PEG-Au NPs impact analysis on HCC1937 TNBL cell line showed a clear toxic action of type dose dependent and at long term. These PEGylated gold nanoparticles present a promising tool for the development of tumor-specific radiosensitizing vectors, with or without the association of other treatment strategies.

Abstract 1015: Repurposing the FDA-approved drug carbidopa to treat human cancers

Abstract Carbidopa is used in combination with L-DOPA to treat Parkinson's disease; it does not have any therapeutic use by itself in Parkinson's disease, but when used along with L-DOPA prevents the conversion of the latter into dopamine in the periphery by inhibiting aromatic amino acid decarboxylase. Carbidopa however does not cross the blood-brain barrier; thus does not impact on the conversion of L-DOPA into dopamine in the brain. We hypothesized that carbidopa might have potential as an anticancer drug with the following rationale: (a) carbidopa is an amino acid derivative and therefore might block the entry of amino acids into cancer cells via certain amino acid transporters; (b) the stress hormones epinephrine and norepinephrine are known to promote cancer progression, and carbidopa as an inhibitor of aromatic amino acid decarboxylase might interfere with the generation of these tumor-promoting hormones; (c) carbidopa is also an analog of phenylhydrazine, which is an inhibitor of the immunosuppressive enzyme indoleamine-2,3-dioxygenase, a drug target for cancer treatment; carbidopa might inhibit this enzyme and thus enhance the ability of the immune system to recognize cancer cells as foreign and fight against them. Based on this rationale, we examined the efficacy of carbidopa to treat pancreatic and breast cancer. We found carbidopa to be effective in blocking the proliferation of pancreatic cancer cell in vitro. We then examined its efficacy in vivo using xenografts of pancreatic cancer cells in nude mice; again, the drug was effective in decreasing the growth of the xenografted tumor cells into tumors. We also tested its efficacy on proliferation of breast cancer cell lines; we used ZR-75.1, MB-231, and HCC-1937 breast cancer cell lines as models for ER-positive, ER-negative and BRCA-1 mutant breast cancers, respectively. Carbidopa decreased the proliferation of all three cell lines. We then examined its in vivo efficacy against breast cancer using the MMTV-PyMT-transgenic mouse as a model of spontaneous breast cancer. In this model, breast cancer develops initially as an ER-positive subtype but then turns into an ER-negative subtype. Carbidopa markedly decreased the growth of breast cancer in this mouse model. Based on these in vitro and in vivo data, we conclude that carbidopa has promise for use as an anticancer drug. As the drug potentially elicits its anticancer effects by targeting multiple pathways, the anticancer efficacy of the drug is likely to be broad against different types of human cancers. For in vivo studies, we used the drug intraperitoneally at a dose of 1 mg/mouse that approximately translates to a human dose of 300-400 mg/day. As the drug has been shown to have no detectable side effects in humans at doses as high as 400 mg/day, it can be taken to clinical trials readily to test its efficacy in humans as an anticancer drug. Citation Format: Vadivel Ganapathy, Ellappan Babu, Sabarish Ramachandran, Yangzom D. Bhutia. Repurposing the FDA-approved drug carbidopa to treat human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1015.

Abstract POSTER-BIOL-1346: Growth-suppressive effect of WRN RECQ helicase inactivation in breast and ovarian cancer cells

Abstract One important and high-yielding paradigm in the development of novel, targeted anticancer therapies is to identify genes that are not essential to normal cells but that have become critically important to particular cancer cells as they rely on these genes for survival. This concept, sometimes called addiction of cancer cells to specific genes or pathways can be exploited in the clinic because the function of the genes that cancer cells are addicted to can be disrupted using genetic or chemical manipulation, leading to selective killing of cancer cells while normal cells are minimally affected. For example, it was shown that homologous recombination-defective BRCA1-deficient cancer cells rely heavily on other repair pathways such as NHEJ, one of the pathways facilitated by PARP1. This finding has led to clinical trials designed to determine if PARP inhibitor drugs are an effective treatment for patients with BRCA1-deficient breast and ovarian cancer. We have previously discovered, via a high throughput screen, that simultaneous deficiency in BRCA1 and the WRN RECQ helicase resulted in additive growth-suppressive phenotype in human fibroblasts. WRN is a gene mutated in the Werner syndrome of premature aging, and it is a multifunctional exonuclease/helicase involved in maintenance of genomic stability. Congenital loss of WRN also predisposes to sarcomas and melanomas, though interestingly, not to the most common type of age-related cancer – carcinoma. Near-complete inactivation of WRN in human fibroblasts and common cell lines such as sarcoma-derived U2OS and HT1080, is typically non-lethal, though somewhat reduces cellular growth rate and fitness. Following our original observation, we were interested to determine whether loss of WRN is synthetically lethal with loss of BRCA1 in breast and ovarian cancer cell lines, and if loss of WRN restores sensitivity to platinum or impairs growth in platinum resistant clones derived from breast and ovarian cancer cell lines. Our results show that depletion of WRN in BRCA1-negative or positive breast or ovarian cancer cell lines dramatically suppresses their growth. Both in breast and ovarian cancer cells, at least some of this growth suppression could be attributed to the induction of RNAi-triggered cytotoxicity by WRN downregulation. In addition, WRN depletion also hypersensitized the HCC1937 breast cancer cell line to platinum, however this effect of WRN was attenuated in platinum-resistant clones derived in vitro from this line. Strikingly, non-cancerous mammary epithelial line MCF10a showed virtually no growth suppression by WRN depletion, suggesting that breast and ovarian cancer cells may be more reliant on WRN than normal cells of at least some of the relevant epithelial lineages — which is a therapeutically exploitable trait. We are currently following up on this observation using other non-cancerous epithelial cells, such as primary mammary and fallopian tube epithelial cells, as well as primary human keratinocytes. We will present these data as well as the update on reproducing WRN growth-suppressive phenotype using small molecule inhibitors of WRN. Significance: Our results highlight WRN as a potential novel target for development of breast and ovarian cancer therapies. Citation Format: Piri Welcsh, Keffy Kehrli, Paul Lazarchuk, Julia Sidorova. Growth-suppressive effect of WRN RECQ helicase inactivation in breast and ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1346.

Integration of BRCA1-mediated miRNA and mRNA profiles reveals microRNA regulation of TRAF2 and NFκB pathway

Microarray-based techniques are being useful to obtain miRNA and gene expression signatures associated with different tumors. BRCA1 deregulation is a frequent event in the pathogenesis of breast as well as other cancers. In addition to DNA repair functions of BRCA1, it is involved in a wide range of cellular processes such as cell cycle, chromatin remodeling or transcription. However, the molecular events underlying BRCA1-associated tumorigenesis are still largely unknown. In order to deepen our understanding of BRCA1-associated tumorigenesis, we integrated data from mRNA and miRNA microarray experiments on HCC1937 breast cancer cell line, and the isogenic HCC1937 stably expressing BRCA1, to obtain significant miRNA-mRNA relationships associated with the presence of BRCA1 gene. By using bioinformatic integration of gene and miRNA expression data, we found significant miRNA-gene relationships underlying the array signatures. We additionally evaluated the role of these statistically significant pairs at the biological pathways level and identified MAPK and NF-κB pathways associated with these expression changes. Furthermore, we experimentally validated miRNAs induced by BRCA1 that commonly regulate TRAF2, a key regulator of NF-κB and MAPK pathways. We demonstrate that miR-146a, miR-99b and miR-205, induced in HCC1937 BRCA1-expressing cells, bind and regulate TRAF2 gene. In addition, re-expression of miR-146a, miR-99b or miR-205 in HCC1937 BRCA1-null cells was sufficient to modulate NF-κB activity. Our results demonstrate that integration of mRNA and miRNA associated with BRCA1 expression was useful to discover new miRNA-gene interactions as molecular events underlying BRCA1-mediated tumorigenesis.

Abstract 1190: Integration of mRNA and miRNA signatures induced by BRCA1 gene

Abstract During the past years it has been well established that microRNAs (miRNAs) modulate the activity of mRNAs. Microarray-based techniques are being useful to obtain miRNA and gene expression signatures associated to different samples. The aim of this study is to integrate data from both types of microarray experiments to obtain significant relationships between miRNA profile and gene expression signature associated to the absence or presence of BRCA1 gene in a cellular system. Whole genome transcriptional mRNA profiling and global miRNA expression profiling was performed on a BRCA1 deficient, HCC1937 breast cancer cell line, and the isogenic HCC1937 expressing BRCA1. We have related each differentially expressed miRNA and their miRanda predicted transcripts targets obtained from miRBase and TargetScan, to find consistent pairs of downregulated targets for upregulated miRNAs and viceversa, which were non randomly associated (Fisher, FDR&amp;lt;0.05). In our study we found over 8000 differentially expressed genes and 9 differentially expressed miRNAs between HCC1937 and HCC1937/BRCA1 cells (FDR p&amp;lt;0.05). Integration of these data revealed significant interactions between three differentially upregulated microRNAs and their downregulated targets. Moreover, we have evaluated the role of these experimentally consistent pairs at the biological pathways level (Ingenuity Pathway Analysis). We identified potential signaling pathways significantly repressed associated with miRNA upregulation in BRCA1-expressing HCC1937 cells, which included MAPK or NFκB pathway. Down regulation of these pathways was experimentally validated by luciferase assays. Integration analysis between miRNA-mRNA data allows us to define genes and pathways controlled by differentially expressed miRNA upon BRCA1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1190. doi:10.1158/1538-7445.AM2011-1190

Abstract 2469: ATM inhibitor 55933 abolishes the radiation resistance of CD44+/CD24−or low subsets of breast cancer cells

Abstract CD44+/CD24− subsets of breast cancer cells are enriched in cancer stem-like cells (CSC) and are radiation resistant. We isolated ESA+/CD44+/CD24− or low subsets by flow cytometry from MCF7, MDA-MB231, and HCC1937 breast cancer cell lines and primary cultures of breast cancer cells from two patients. The isolated CD44+/CD24−orlow subsets showed a 50 to 70% increase of sphere formation by in suspension culture in serum-free medium compared to CD44−/CD24high subsets. CD44+/CD24−or low subsets of the cell lines and primary cultures showed a significantly increased radiation resistance compared to the CD44−/CD24high cell line subsets or the unsorted primary cells with &amp;gt; 40% increased survival at 2Gy. Post-radiation survival showed that the radiation resistance was dependent on the CD44+/CD24 −or low cell phenotype. The correlation of CD44/CD24 status with radiation resistance was demonstrated by culture of the subsets. After 2 weeks of culture the following transitions occurred: 1) CD44+/CD24−or low cells changed to CD44−/CD24high cells (MCF-7 and HCC1937) or to CD44+/CD24+ (MDA-MB-231); and 2) CD44−/CD24 high (MCF-7 and HCC1937) and CD44+/CD24+ (MDA-MB-231) cells became CD44+/CD24−or low cells. Cells that lost the CD44+/CD24 −or low phenotype were no longer radiation resistant. The mechanisms of increased radiation resistance in CD44+/CD24−orlow subsets was not a function of non-homologous end joining (NHEJ) as there was no in vivo and in vitro change in NHEJ activity in the resistant cells nor altered expression of the NHEJ related proteins Ku 80, Ku70, PARP-1, and DNA-PK. However, there were differences ATM activity between CD44+/CD24−or low and the CD44−/CD24 high (MCF-7 and primary breast cancer) or CD44+/CD24+ (MDA-MB-231) subsets. Depending on the cell type and radiation dose, ATM phsophorylation increased by 2-5 fold in radiated CD44+/CD24−or low subsets of breast cancer cell lines and primary cultured breast cancer cells compared to the control subsets. The ATM inhibitor 55933 in a dose-dependent manner increased radiation sensitivity of all subsets including significantly abolishing the radiation resistance of CD44+/CD24−or low subsets of cells. After 2Gy of radiation, the survival fractions of cells treated with10μM of 55933 were 8-10 fold lower than that of untreated cells and were now similar between CD44+/CD24−or low subsets and control subsets for the cell lines and primary cultures. In conclusion, ATM is a candidate target for eliminating the radiation resistance of CSC's of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2011-2469

Accumulation of Oxidatively Induced DNA Damage in Human Breast Cancer Cell Lines Following Treatment with Hydrogen Peroxide

Breast cancer is a leading cause of cancer deaths in women. Although the causes of this disease are largely unknown, inefficient repair of oxidatively induced DNA lesions has been thought to play a major role in the transformation of normal breast tissue to malignant breast tissue. Previous studies have revealed higher levels of 8-hydroxyguanine in malignant breast tissue compared to non-malignant breast tissue. Furthermore, some breast cancer cell lines have greatly reduced capacity to repair this lesion suggesting that oxidatively induced DNA lesions may be elevated in breast cancer cells. We used liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of 8-hydroxy-2’-deoxyadenosine, (5’S)-8,5’-cyclo-2’-deoxyadenosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine in MCF-7 and HCC1937 breast cancer cell lines before and after exposure to H2O2 followed by a DNA repair period. We show that H2O2-treated HCC1937 and MCF-7 cell lines accumulate significantly higher levels of these lesions than the untreated cells despite a 1 h repair period. In contrast, the four lesions did not accumulate to any significant level in H2O2-treated non-malignant cell lines, AG11134 and HCC1937BL. Furthermore, MCF-7 and HCC1937 cell lines were deficient in the excision repair of all the four lesions studied. These results suggest that oxidatively induced DNA damage and its repair may be critical in the etiology of breast cancer.

Reduced repair of 8-hydroxyguanine in the human breast cancer cell line, HCC1937

BackgroundBreast cancer is the second leading cause of cancer deaths in women in the United States. Although the causes of this disease are incompletely understood, oxidative DNA damage is presumed to play a critical role in breast carcinogenesis. A common oxidatively induced DNA lesion is 8-hydroxyguanine (8-OH-Gua), which has been implicated in carcinogenesis. The aim of this study was to investigate the ability of HCC1937 and MCF-7 breast cancer cell lines to repair 8-OH-Gua relative to a nonmalignant human mammary epithelial cell line, AG11134.MethodsWe used oligonucleotide incision assay to analyze the ability of the two breast cancer cell lines to incise 8-OH-Gua relative to the control cell line. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the levels of 8-OH-Gua as its nucleoside, 8-OH-dG in the cell lines after exposure to H2O2 followed by 30 min repair period. Protein expression levels were determined by Western blot analysis, while the hOGG1 mRNA levels were analyzed by RT-PCR. Complementation of hOGG1 activity in HCC1937 cells was assessed by addition of the purified protein in the incision assay, and in vivo by transfection of pFlagCMV-4-hOGG1. Clonogenic survival assay was used to determine sensitivity after H2O2-mediated oxidative stress.ResultsWe show that the HCC1937 breast cancer cells have diminished ability to incise 8-OH-Gua and they accumulate higher levels of 8-OH-dG in the nuclear genome after H2O2 treatment despite a 30 min repair period when compared to the nonmalignant mammary cells. The defective incision of 8-OH-Gua was consistent with expression of undetectable amounts of hOGG1 in HCC1937 cells. The reduced incision activity was significantly stimulated by addition of purified hOGG1. Furthermore, transfection of pFlagCMV-4-hOGG1 in HCC1937 cells resulted in enhanced incision of 8-OH-Gua. HCC1937 cells are more sensitive to high levels of H2O2 and have up-regulated SOD1 and SOD2.ConclusionThis study provides evidence for inefficient repair of 8-OH-Gua in HCC1937 breast cancer cell line and directly implicates hOGG1 in this defect.

BRCA1 expression modulates chemosensitivity of BRCA1-defective HCC1937 human breast cancer cells.

Germline mutations of the tumour suppressor gene BRCA1 are involved in the predisposition and development of breast cancer and account for 20–45% of all hereditary cases. There is an increasing evidence that these tumours are characterised by a specific phenotype and pattern of gene expression. We have hypothesised that differences in chemosensitivity might parallel molecular heterogeneity of hereditary and sporadic breast tumours. To this end, we have investigated the chemosensitivity of the BRCA1-defective HCC1937 breast cancer cell line, and the BRCA1-competent MCF-7 (hormone-sensitive) and MDA-MB231 (hormone-insensitive) breast cancer cell lines using the MTT assay. The 50% inhibitory concentration (IC50) for the individual compounds were derived by interpolate plot analysis of the logarithmic scalar concentration curve after a 48 h exposure. HCC1937 cells were significantly (P<0.005) more sensitive to cisplatin (CDDP) (IC50 : 30–40 μM) compared with MCF-7 (IC50 : 60–70 μM) and MDA-MB231 (IC50 : 90–100 μM) cells. On the other hand, BRCA1-defective breast cancer cells were significantly less sensitive to doxorubicin (Dox) (IC50 : 45–50 μM) compared with MCF-7 (IC50 : 1–5 μM) and MDA-MB231 (IC50 : 5–10 μM) (P<0.02), as well as to paclitaxel (Tax) (IC50 : >2 μM for HCC1937, 0.1–0.2 μM for MCF-7 and 0.01–0.02 μM for MDA-MB231) (P<0.001). Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/WTBRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar. BRCA1 reconstitution reverted the hypersensitivity to CDDP (P<0.02), and restored the sensitivity to Dox (P<0.05) and Tax (P<0.001), compared with parental HCC1937 cells. Taken together, our findings suggest a specific chemosensitivity profile of BRCA1-defective cells in vitro, which is dependent on BRCA1 protein expression, and suggest prospective preclinical and clinical investigation for the development of tailored therapeutical approaches in this setting.