Chemosensitivity Of Breast Cancer Cells Research Articles

In vitro effects of imatinib mesylate on radiosensitivity and chemosensitivity of breast cancer cells

BackgroundBreast cancer treatment is based on a combination of adjuvant chemotherapy followed by radiotherapy effecting intracellular signal transduction. With the tyrosine kinase inhibitors new targeted drugs are available. Imatinib mesylate is a selective inhibitor of bcr-abl, PRGFR alpha, beta and c-kit. The purpose of this study was to determine whether Imatinib has an influence on the effectiveness of radiotherapy in breast cancer cell lines and if a combination of imatinib with standard chemotherapy could lead to increased cytoreduction.MethodsColony-forming tests of MCF 7 and MDA MB 231 were used to study differences in cell proliferation under incubation with imatinib and radiation. Changes in expression and phosphorylation of target receptors were detected using western blot. Cell proliferation, migration and apoptosis assays were performed combining imatinib with doxorubicin.ResultsThe combination of imatinib and radiotherapy showed a significantly stronger inhibition of cell proliferation compared to single radiotherapy. Differences in PDGFR expression could not be detected, but receptor phosphorylation was significantly inhibited when treated with imatinib. Combination of imatinib with standard chemotherapy lead to an additive effect on cell growth inhibition compared to single treatment.ConclusionsImatinib treatment combined with radiotherapy leads in breast cancer cell lines to a significant benefit which might be influenced through inhibition of PDGFR phosphorylation. Combining imatinib with chemotherapy enhances cytoreductive effects. Further in vivo studies are needed to evaluate the benefit of Imatinib in combination with radiotherapy and chemotherapy on the treatment of breast cancer.

Abstract 2183: High-throughput screening, identification and characterization of novel kinases involved in chemosensitization of breast cancer cells

Abstract Background - Development of drug resistance is considered to be one of the most challenging issues in chemotherapy for cancer patients. As one of the conventional chemotherapeutic agent, paclitaxel was originally extracted from Pacific yew tree and has been applied widely in ovarian cancer, breast cancer and lung cancer. Resistance to paclitaxel has posed severe problems in cancer treatment. We are therefore interested in identifying the genes that render paclitaxel-resistant breast cancer cells to become more sensitive with the drug treatment. The mechanisms and signaling pathways of these kinases in relations to paclitaxel resistance and their molecular targets have been investigated. Methods - We have screened a shRNA library containing 592 novel kinase genes with 1306 clones for the chemosensitizer loci. DNA was extracted in a high throughput manner, and cell transfection/transduction was carried out on 384 well plates using a unique solid-phase DNA delivery method. The positive hits were selected according to the alteration on their IC50 values for the drug using cell viability assays. Quantitative PCR, Western blot, and caspase assays have been employed to elucidate the signaling pathways of these kinases in response to paclitaxel. Protein purification and interaction studies have also been carried out in attempt to elucidate the putative substrates for the kinases and their interaction partners. Results and Conclusions - We have established a high throughput screening platform for identification of the chemosensitizer loci. High transfection/transduction efficiencies can be achieved using our solid-phase gene delivery method. Several novel unknown kinase genes that render drug-resistant cancer cells to become sensitive have been selected. The positive shRNA hits were further verified by quantitative real-time PCR. Among the 20 hits that showed diminished IC50 values of paclitaxel in breast cancer cells, we have selected two candidates for further studies. Knocking down of TAOK3 or VRK3 in breast cancer cells resulted in reduction of IC50 of paclitaxel to half of the original value. Furthermore, we have investigated the signaling pathways of these 2 kinases in relation to paclitaxel resistance. The different signaling pathways elucidated have revealed the complex nature of drug resistance development in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2183.

Abstract 690: Estrogen receptor beta (ERβ) enhances the chemosensitivity of breast cancer cells by altering DNA damage response signaling

Abstract Estrogen Receptor beta (ERβ) is a mediator of estrogen signaling that regulates growth in mammary gland and is involved in breast cancer. In response to DNA damage most tumor cells due to disruptions in the p53 pathway and consequent loss of G1 cell cycle arrest are entirely dependent in S and G2/M checkpoints to maintain genomic integrity. Inhibition of these checkpoints following DNA damage induces selective killing of p53-defective tumor cells. We show that expression of ERβ caused abrogation of Cdc25C-mediated G2/M checkpoint following cisplatin and doxorubicin exposure selectively in p53-deficient cells, resulting in mitotic catastrophe and decreased cancer cell survival. The loss of G2/M checkpoint observed was due to ERβ-mediated inhibition of the DNA damage-induced expression of brca1. ERβ by modulating DNA damage response signaling seems to selectively enhance the chemosensitivity of tumor cells. This suggests that co-operation of compounds that specifically induce the ERβ signaling with DNA damaging chemotherapeutic drugs may represent an attractive new cancer therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 690.

A systems analysis of the chemosensitivity of breast cancer cells to the polyamine analogue PG-11047

BackgroundPolyamines regulate important cellular functions and polyamine dysregulation frequently occurs in cancer. The objective of this study was to use a systems approach to study the relative effects of PG-11047, a polyamine analogue, across breast cancer cells derived from different patients and to identify genetic markers associated with differential cytotoxicity.MethodsA panel of 48 breast cell lines that mirror many transcriptional and genomic features present in primary human breast tumours were used to study the antiproliferative activity of PG-11047. Sensitive cell lines were further examined for cell cycle distribution and apoptotic response. Cell line responses, quantified by the GI50 (dose required for 50% relative growth inhibition) were correlated with the omic profiles of the cell lines to identify markers that predict response and cellular functions associated with drug sensitivity.ResultsThe concentrations of PG-11047 needed to inhibit growth of members of the panel of breast cell lines varied over a wide range, with basal-like cell lines being inhibited at lower concentrations than the luminal cell lines. Sensitive cell lines showed a significant decrease in S phase fraction at doses that produced little apoptosis. Correlation of the GI50 values with the omic profiles of the cell lines identified genomic, transcriptional and proteomic variables associated with response.ConclusionsA 13-gene transcriptional marker set was developed as a predictor of response to PG-11047 that warrants clinical evaluation. Analyses of the pathways, networks and genes associated with response to PG-11047 suggest that response may be influenced by interferon signalling and differential inhibition of aspects of motility and epithelial to mesenchymal transition.See the related commentary by Benes and Settleman: http://www.biomedcentral.com/1741-7015/7/78

Expression of the Breast Cancer Metastasis Suppressor 1 (BRMS1) maintains in vitro chemosensitivity of breast cancer cells

The Breast Cancer Metastasis Suppressor 1 (BRMS1) belongs to an expanding category of proteins called metastasis suppressors that demonstrate in vivo metastasis suppression while still allowing growth of the orthotopic tumor. Since BRMS1 decreases either the expression or function of multiple mediators implicated in resistance to chemotherapy (NF-κB, AKT, EGFR), we asked whether breast carcinoma cells expressing BRMS1 could be sensitized upon exposure to commonly used therapeutic agents that inhibit some of these same cellular mediators as BRMS1. In this report, we demonstrate that chemosensitivity of breast cancer cells is preserved in the presence of BRMS1. Further, BRMS1 does not change expression of AKT isoforms or PTEN, implicated in chemoresistance to common drug agents. Overall, our data with two different metastatic breast cancer cell lines indicates that BRMS1 expression status may not interfere with the response to commonly used chemotherapeutic agents in the management of solid tumors such as breast cancer. Since tumor protein expression analysis increasingly guides therapy decisions, our data may be of clinical benefit in disease management including profiling for BRMS1 expression before start of therapy.

Effects of folate and folylpolyglutamyl synthase modulation on chemosensitivity of breast cancer cells

Folylpolyglutamyl synthase (FPGS) converts intracellular folates and antifolates to polyglutamates. Polyglutamylated folates and antifolates are retained in cells longer and are better substrates than their monoglutamate counterparts for enzymes involved in one-carbon transfer. FPGS modulation affects the chemosensitivity of cancer cells to antifolates, such as methotrexate, and 5-fluorouracil (5FU) by altering polyglutamylation of antifolates and specific target intracellular folate cofactors. However, this effect may be counterbalanced by FPGS modulation-induced changes in polyglutamylation of other intracellular folate cofactors and total intracellular folate pools. We generated an in vitro model of FPGS overexpression and inhibition in breast cancer cells by stably transfecting human MDA-MB-435 breast cancer cells with the sense FPGS cDNA or FPGS-targeted small interfering RNA, respectively, and investigated the effects of FPGS modulation on chemosensitivity to 5FU and methotrexate. FPGS modulation-induced changes in polyglutamylation of both antifolates and folate cofactors and in intracellular folate pools affected chemosensitivity of breast cancer cells to pemetrexed and trimetrexate whose cytotoxic effects do or do not depend on polyglutamylation, respectively, in a predictable manner. However, the effects of FPGS modulation on the chemosensitivity of breast cancer cells to 5FU and methotrexate seem to be highly complex and depend not only on polyglutamylation of a specific target intracellular folate cofactor or methotrexate, respectively, but also on total intracellular folate pools and polyglutamylation of other intracellular folate cofactors. Whether or not FPGS modulation may be an important clinical determinant of chemosensitivity of breast cancer cells to 5FU and methotrexate-based chemotherapy needs further exploration.

Up-regulation of α Vβ 3 integrin expression is a novel molecular response to chemotherapy-induced cell damage in a heregulin-dependent manner

α vβ 3 integrin has a dual role in apoptosis. Whereas ligated α vβ 3 activates cell survival pathways and suppresses pro-apoptotic signals, unligated α vβ 3 or integrins bound to soluble ligands promote apoptosis. In this study, we assessed the role of α vβ 3 in chemosensitivity of breast cancer cells expressing different levels of heregulin (HRG). Expression levels of the RGD-binding integrins α vβ 3 were measured in MDA-MB-231 human breast cancer cells and its low HRG-expressing derivative (MDA-MB-231/AS31) treated with the microtubule-interfering agents (MIAs) paclitaxel and vincristine. Following treatment, only α vβ 3 levels were significantly increased in MDA-MB-231 cells. Interestingly, α vβ 3 expression was more significantly up-regulated in the MDA-MB-231/AS31 cells than in the parental cells. This MIA-induced increase of α vβ 3 expression was correlated with a decrease in cell viability and an increase in apoptosis in MDA-MB-231/AS31 cells, indicating that overexpression of α vβ 3 is linked to chemotherapy-induced cell death in low HRG-expressing breast cancer models. Moreover, a paclitaxel-induced increase of α vβ 3 was also observed in MCF-7 cells but not in an doxorubicin-resistant derivative that shows cross-resistance to paclitaxel, further providing evidence that the extent of α vβ 3 up-regulation is related to cell damage. These results indicate that α vβ 3 integrin is dramatically up-regulated in low HRG-expressing breast cancer models that are highly responsive to MIAs, thus providing a novel molecular marker of chemosensitivity influenced by HRG levels in breast cancer cells.

Inactivation of NF-κB by 3,3′-diindolylmethane contributes to increased apoptosis induced by chemotherapeutic agent in breast cancer cells

Constitutive activation of Akt or nuclear factor-kappaB (NF-kappaB) has been reported to play a role in de novo resistance of cancer cells to chemotherapeutic agents, which is a major cause of treatment failure in cancer chemotherapy. Previous studies have shown that 3,3'-diindolylmethane (DIM), a major in vivo acid-catalyzed condensation product of indole-3-carbinol, is a potent inducer of apoptosis, inhibitor of tumor angiogenesis, and inactivator of Akt/NF-kappaB signaling in breast cancer cells. However, little is known regarding the inactivation of Akt/NF-kappaB that leads to chemosensitization of breast cancer cells to chemotherapeutic agents, such as Taxotere. Therefore, we examined whether the inactivation Akt/NF-kappaB signaling caused by B-DIM could sensitize breast cancer cells to chemotherapeutic agents both in vitro and in vivo. MDA-MB-231 cells were simultaneously treated with 15 to 45 micromol/L B-DIM and 0.5 to 1.0 nmol/L Taxotere for 24 to 72 h. Cell growth inhibition assay, apoptosis assay, electrophoretic mobility shift assay, and Western blotting were done. The combination treatment of 30 micromol/L B-DIM with 1.0 nmol/L Taxotere elicited significantly greater inhibition of cell growth compared with either agent alone. The combination treatment induced greater apoptosis in MDA-MB-231 cells compared with single agents. Moreover, we found that NF-kappaB activity was significantly decreased in cells treated with B-DIM and Taxotere. We also have tested our hypothesis using transfection studies, followed by combination treatment with B-DIM/Taxotere, and found that combination treatment significantly inhibited cell growth and induced apoptosis in MDA-MB-231 breast cancer cells mediated by the inactivation of NF-kappaB, a specific target in vitro and in vivo. These results were also supported by animal experiments, which clearly showed that B-DIM sensitized the breast tumors to Taxotere, which resulted in greater antitumor activity mediated by the inhibition of Akt and NF-kappaB. Collectively, our results clearly suggest that inhibition of Akt/NF-kappaB signaling by B-DIM leads to chemosensitization of breast cancer cells to Taxotere, which may contribute to increased growth inhibition and apoptosis in breast cancer cells. The data obtained from our studies could be a novel breakthrough in cancer therapeutics by using nontoxic agents, such as B-DIM, in combination with other conventional therapeutic agents, such as Taxotere.

Inhibition of Fatty Acid Synthase (FASN) synergistically enhances the efficacy of 5-fluorouracil in breast carcinoma cells

The lipogenic enzyme fatty acid synthase (FASN) is differentially overexpressed and hyperactivated in a biologically aggressive subset of breast carcinomas and minimally in most normal adult tissues, rendering it an interesting target for anti-neoplastic therapy development. We previously reported that the FASN blockade can induce a synergistic chemosensitization of breast cancer cells to microtubule interfering agents (MIAs) such as docetaxel, paclitaxel and vinorelbine. Upon pharmacological inhibition of FASN activity using the natural antibiotic cerulenin [(2S,3R)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide], we evaluated the role of FASN-catalyzed endogenous fatty acid biogenesis on the sensitivity of SK-Br3, MCF-7 and MDA-MB-231 breast cancer cell lines to the anti-metabolite 5-fluorouracil (5-FU). Cells were exposed simultaneously to cerulenin and 5-FU, sequentially to 5-FU followed by cerulenin or cerulenin followed by 5-FU. Cell viability was determined by MTT assays and the increase in 5-FU-induced cell growth inhibition was measured by dividing 5-FU IC30 and IC50 values (i.e., 30% and 50% inhibitory concentrations, respectively) that were obtained in the absence of cerulenin by those in its presence. Co-exposure to cerulenin enhanced 5-FU efficacy up to 20-, 81-, and 58-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. Pre-treatment with cerulenin followed by the addition of 5-FU increased 5-FU efficacy up to 31-, 87-, and 126-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. Pre-treatment with 5-FU followed by the addition of cerulenin augmented 5-FU efficacy up to 107-, 20-, and 18-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. When isobologram transformations of multiple dose-response analyses were performed to detect in vitro synergy, we concluded that the nature of the interaction between cerulenin and 5-FU in individual breast cancer cells lines generally exhibited sequence-dependency. Thus, while synergism was mainly observed when breast cancer cells were exposed to 5-FU prior to cerulenin, moderate synergism or additive interactions was obtained either when the chemical FASN blocker preceded 5-FU or when both drugs were concurrently administered. Of note, no antagonist interactions occurred upon any schedule of combined treatment with cerulenin and 5-FU. Our current findings revealing a schedule-dependent synergistic interaction between 5-FU and cerulenin represents, to the best of our knowledge, the first evidence that FASN-catalyzed de novo FA biogenesis plays a key role in regulating breast cancer cell response to antimetabolite-based therapies.

Evidence that Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Induction by 5-Aza-2′-Deoxycytidine Sensitizes Human Breast Cancer Cells to Adriamycin

The DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) inhibits DNA methyltransferase activity and sensitizes cancer cells to chemotherapy, but the mechanisms of its sensitization are not fully understood. Here, we show that 5-aza-CdR induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the human breast cancer MDA-231 cells. Induction of TRAIL by 5-aza-CdR correlated with inactivation of Akt. Furthermore, we show that overexpression of the active form of Akt by adenovirus infection or inhibition of the Akt downstream target glycogen synthase kinase 3 by its pharmacologic inhibitors abolishes TRAIL induction by 5-aza-CdR. Importantly, we show that the combined treatment of breast cancer cells with 5-aza-CdR and Adriamycin significantly increases apoptotic cell death compared with the treatment with either agent alone. Moreover, the combined treatment activated both death receptor and mitochondrial apoptotic pathways, whereas Adriamycin alone activated only the mitochondrial pathway while 5-aza-CdR failed to activate either. More importantly, down-regulation of TRAIL by small interference RNA silencing decreased 5-aza-CdR-mediated Adriamycin-induced caspase activation and apoptosis, thus conferring Adriamycin resistance. Taken together, our results suggest that induction of TRAIL by 5-aza-CdR is critical for enhancing chemosensitivity of breast cancer cells to Adriamycin.

Targeted therapy against Bcl-2-related proteins in breast cancer cells

IntroductionBcl-2 and Bcl-xL confer resistance to apoptosis, thereby reducing the effectiveness of chemotherapy. We examined the relationship between the expression of Bcl-2 and Bcl-xL and chemosensitivity of breast cancer cells, with the aim of developing specific targeted therapy.MethodsFour human breast cancer cell lines were examined, and the effects of antisense (AS) Bcl-2 and AS Bcl-xL phosphorothioate oligodeoxynucleotides (ODNs) on chemosensitivity were tested in vitro and in vivo. Chemosensitivity was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, and the antitumor effect was assessed in vivo by the success of xenograft transplantation into athymic mice.ResultsTreatment with AS Bcl-2 and Bcl-xL ODNs resulted in a sequence-specific decrease in protein expression, compared with controls. Treatment of BT-474, ZR-75-1, and MDA-MB-231 cells with AS Bcl-2 increased chemosensitivity to doxorubicin (DOX), mitomycin C (MMC), paclitaxel (TXL), and docetaxel (TXT). Transfection of the Bcl-2 gene into MDA-MB-453 cells decreased sensitivity to DOX and MMC. Treatment of MDA-MB-231, BT-474, and ZR-75-1 cells with AS Bcl-xL increased chemosensitivity to DOX, MMC and taxanes to a smaller extent than AS Bcl-2. This occurred in the setting of increased Bax and cleaved poly(ADP-ribose) polymerase, as well as decreased Bcl-2 and pAkt. AS Bcl-2 ODNs induced splenomegaly in association with increased serum IL-12, which was attenuated by methylation of the CpG motifs of AS Bcl-2; however, methylated CpG failed to negate the increased antitumor effect of AS Bcl-2. Bcl-2 and Bcl-xL, to a smaller extent, are major determinants of chemosensitivity in breast cancer cells.ConclusionTargeted therapy against Bcl-2 protein with the use of AS ODNs might enhance the effects of chemotherapy in patients with breast cancer.

Blockade of Epidermal Growth Factor Receptors Chemosensitizes Breast Cancer Cells through Up-Regulation of Bnip3L

Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling pathways activated by EGFR and HER2 may help unravel novel therapeutic targets and molecular markers of response. Here, we show that treatment of breast cancer cell lines with blocking antibodies against EGFR (cetuximab) or HER2 (trastuzumab) promotes the specific induction of proapoptotic Bnip3L and chemosensitization. Moreover, we found that the Bnip3L gene is transcriptionally activated by FoxO3a. Trastuzumab-mediated induction of Bnip3L and nuclear translocation of FoxO3a was also shown in pleural effusion cells from a breast cancer patient. Transfection of breast cancer cells with constitutively active FoxO3a or with Bnip3L promotes sensitization to chemotherapy-induced apoptosis. On the contrary, blockade of Bnip3L expression by a small interfering RNA strategy significantly diminished the chemosensitizing effect of cetuximab. We found also an inverse correlation between EGFR and Bnip3L expression in surgical specimens from patients with breast cancer. Therefore, blockading EGFR or HER2 specifically up-regulates Bnip3L, which is required for chemosensitization of breast cancer cells. This novel pathway provides also the rationale for therapeutic strategies aimed to induce the expression of Bnip3L.

Study on relationship between chemosensitivity of breast cancer and expression of p73α and p53

Objective: To investigate the relationship between the expression of p73α and p53 versus the chemosensitivity in breast cancer cells. Methods: Twelve surgical samples of breast cancer diagnosed by pathology were used. The cancer sample cells were separately cultured in the incubator at 37°C, 5% CO2in vitro. The relative inhibition rate of cancer cells by 4 kinds of anticancer drugs, which were EPI, MMC, 5-Fu and DDP, were assayed by MTT method. Immunocytochemistry was used to detect the expression of p73α and p53 in the cancer cells. Results: The positive expression of p73α was found in 5/12 (41.67%), and p53 positive expression rate was 50.0% (6/12). The relative inhibition rate of MMC, EPI, 5-Fu and DDP were significantly higher in the p73α positive cancer cells than in the p73α negative cancer cells. A positive correlation was found between expression of p73α and chemosensitivity for all the four anticancer drugs. Conclusion: The expression of p73α is related with the chemosensitivity of the breast cancer cells, and it may become one of the markers for judging the effect of chemotherapy in clinic.

Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate.

Although single nucleotide polymorphisms may be potentially important pharmacogenetic determinants of cancer therapy, functional evidence regarding their relevance is currently lacking. The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with changes in cellular composition of folates. We hypothesized that this polymorphism may modulate the cytotoxic effect of 5-fluorouracil (5FU) and methotrexate (MTX), two commonly used chemotherapeutic agents for colon and breast cancers, because the modes of action of 5FU and MTX are critically dependent on cellular composition of folates. Human HCT116 colon and MDA-MB-435 breast cancer cells were stably transfected with wild-type or mutant 677T human MTHFR cDNA. MTHFR enzyme activity and thermolability, intracellular folate composition, growth rate, and catalytic thymidylate synthase activity were determined. In vitro chemosensitivity to 5FU and MTX was determined using the sulforhodamine B assay. In vivo chemosensitivity of HCT116 cells to 5FU was determined in nude mice. Compared with cells expressing the wild-type MTHFR, HCT116 and MDA-MB-435 cells expressing the mutant 677T MTHFR had decreased MTHFR activity, MTHFR thermolability, changed intracellular folate distribution, accelerated cellular growth rate, and increased thymidylate synthase activity. The MTHFR 677T mutation increased chemosensitivity of colon and breast cancers to 5FU, but decreased chemosensitivity of breast cancer cells to MTX. In nude mice, xenografts expressing the mutant 677T MTHFR grew faster, but were more sensitive to 5FU, than xenografts expressing the wild-type protein. Our data provide evidence that the MTHFR C677T polymorphism affects the concentration and intracellular distribution of folates and changes the growth and chemosensitivity of colon and breast cancer cells. The MTHFR C677T polymorphism may be a useful pharmacogenetic determinant for providing rational and effective tailored chemotherapy.

HER-2/neu and topoisomerase IIalpha in breast cancer.

In breast cancer, the predominant genetic mechanism for oncogene activation is through an amplification of a gene. The HER-2 (also known as ErbB2/c-erbB2/HER-2/neu) oncogene is the most frequently amplified oncogene in breast cancer, and its overexpression is associated with poor clinical outcome. In addition to its important role in breast cancer growth and progression, HER-2 is also a target for a new form of chemotherapy. Breast cancer patients have been treated with considerable success since 1998 with trastuzumab, a recombinant antibody designed to block signaling through HER-2 receptor. HER-2 has also been implicated in altering the chemosensitivity of breast cancer cells to different forms of conventional cytotoxic chemotherapy, particularly of topoII-inhibitors (e.g., anthracyclines). Topoisomerase IIalpha gene is located just by the HER-2 oncogene at the chromosome 17q12-q21 and is amplified or deleted in almost 90% of the HER-2 amplified primary breast tumors. Recent data suggests that amplification and deletion of topoisomerase IIalpha may account for both relative chemosensitivity and resistance to anthracycline therapy, depending on the specific genetic defect at the topoIIalpha locus. Expanding our understanding of HER-2 amplification also changes its role in the pathogenesis of breast cancer. HER-2 is an oncogene that clearly can drive tumor induction and growth and is also a target for a new kind of chemotherapy, but its function as a marker for chemoselection may be due to associated genetic changes, of which topoisomerase IIalpha is a good example. Moreover, despite potential evidence that genes other than HER-2, such as topoisomerase IIalpha, may be more important predictors of therapeutic response in breast cancer, HER-2 status still has a very significant role in therapeutic selection, mainly as the major criterion for administering trastuzumab in treating breast cancer. Thus, the clinical and therapeutic importance of the HER-2 and topoisomerase IIalpha status to breast cancer management should only increase in the next few years.

Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer.

The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents.

Overexpression of bax sensitizes breast cancer MCF-7 cells to cisplatin and etoposide.

Bax, one of the bcl-2 family genes, is expressed in a number of untransformed cell lines and various breast tissues, whereas only weak or no expression has been detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weak bax gene expression, were stably transfected with pCX2neo bax, encoding human bax; and two unique clones, MCF-7/bax-1 and MCF-7/ bax-2, that expressed different levels of bax were generated. Sensitivity to cisplatin (CDDP) and etoposide (VP-16) was examined and each stable transfectant was more sensitive to these agents than the parental MCF-7 cells. The degree of enhancement in sensitivity to these anticancer agents was dependent on the expression level of bax. The enzyme-linked immunosorbent assay (ELISA), which quantifies DNA damage, demonstrated that this sensitization was due to apoptosis. Thus, we suggest that exogenous bax-alpha overexpression may be one of the factors determining cellular chemosensitivity in MCF-7 breast cancer cells and that it could be applied therapeutically to enhance chemosensitivity in breast cancer cells.

Value of the adenosine triphosphate chemosensitivity assay for pre-therapeutic determination of the chemosensitivity of breast cancer cells in cell culture

This presentation gives a summary of the chemosensitivity test results in breast cancer using the ATP-Cell-Viability Assay (ATP-CVA). Other assays and test systems for chemosensitivity testing are also evaluated. Our results with the ATP-CVA in breast cancer cell lines showed that this test system is a reproducible and reliable tool in the screening of new drugs. Our results with drug combinations showed that with the ATP-CVA and the "median effect principle" synergistic and antagonistic effects can be reliably calculated. The application of the ATP- CVA in fresh human breast cancer made it possible to test tumor chemosensitivity in individual patients. We found an evaluability rate of the ATP-CVA of 95% in 116 patients. The chemosensitivity profiles of the tested tumors revealed a big heterogeneity of chemosensitivity results for single and drug combinations. The 22 in vitro/in vivo correlations from patients with metastatic breast cancer treated with at least three cycles of chemotherapy showed a sensitivity of the ATP-CVA of 92% and a specificity of 89%. These data are encouraging and further investigations on chemosensitivity testing of fresh tumours are currently ongoing in a prospective study at the University of Zurich.