Unraveling malignant phenotype of peritumoral tissue: transcriptomic insights into early-stage breast cancer.

BACKGROUND: Early-stage invasive ductal carcinoma displays high survival rates due to early detection and treatments. However, there is still a chance of relapse of 3-15% after treatment. The aim of this study was to uncover the distinctive transcriptomic characteristics and monitoring prognosis potential of peritumoral tissue in early-stage cases. METHODS: RNA was isolated from tumoral, peritumoral, and non-tumoral breast tissue from surgical resection of 10 luminal early-stage invasive ductal carcinoma patients. Transcriptome expression profiling for differentially expressed genes (DEGs) identification was carried out through microarray analysis. Gene Ontology and KEGG pathways enrichment analysis were explored for functional characterization of identified DEGs. Protein-Protein Interactions (PPI) networks analysis was performed to identify hub nodes of peritumoral tissue alterations and correlated with Overall Survival and Relapse Free Survival. RESULTS: DEGs closely related with cell migration, extracellular matrix organization, and cell cycle were upregulated in peritumoral tissue compared to non-tumoral. Analyzing PPI networks, we observed that the proximity to tumor leads to the alteration of gene modules involved in cell proliferation and differentiation signaling pathways. In fact, in the peritumoral area were identified the top ten upregulated hub nodes including CDK1, ESR1, NOP58, PCNA, EZH2, PPP1CA, BUB1, TGFBR1, CXCR4, and CCND1. A signature performed by four of these hub nodes (CDK1, PCNA, EZH2, and BUB1) was associated with relapse events in untreated luminal breast cancer patients. CONCLUSIONS: In conclusion, our study characterizes in depth breast peritumoral tissue providing clues on the changes that tumor signaling could cause in patients with early-stage breast cancer. We propose that the use of a four gene signature could help to predict local relapse. Overall, our results highlight the value of peritumoral tissue as a potential source of new biomarkers for early detection of relapse and improvement in invasive ductal carcinoma patient's prognosis.

Metformin: From Diabetes to Cancer-Unveiling Molecular Mechanisms and Therapeutic Strategies.

Metformin, a widely used anti-diabetic drug, has garnered attention for its potential in cancer management, particularly in breast and colorectal cancer. It is established that metformin reduces mitochondrial respiration, but its specific molecular targets within mitochondria vary. Proposed mechanisms include inhibiting mitochondrial respiratory chain Complex I and/or Complex IV, and mitochondrial glycerophosphate dehydrogenase, among others. These actions lead to cellular energy deficits, redox state changes, and several molecular changes that reduce hyperglycemia in type 2 diabetic patients. Clinical evidence supports metformin's role in cancer prevention in type 2 diabetes mellitus patients. Moreover, in these patients with breast and colorectal cancer, metformin consumption leads to an improvement in survival outcomes and prognosis. The synergistic effects of metformin with chemotherapy and immunotherapy highlights its potential as an adjunctive therapy for breast and colorectal cancer. However, nuanced findings underscore the need for further research and stratification by molecular subtype, particularly for breast cancer. This comprehensive review integrates metformin-related findings from epidemiological, clinical, and preclinical studies in breast and colorectal cancer. Here, we discuss current research addressed to define metformin's bioavailability and efficacy, exploring novel metformin-based compounds and drug delivery systems, including derivatives targeting mitochondria, combination therapies, and novel nanoformulations, showing enhanced anticancer effects.

Therapy-Induced Senescence Enhances the Efficacy of HER2-Targeted Antibody-Drug Conjugates in Breast Cancer.

Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression. SIGNIFICANCE: Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.

Vitamin D analogues exhibit antineoplastic activity in breast cancer patient-derived xenograft cells.

Despite advances in breast cancer (BC) treatment, its mortality remains high due to intrinsic or acquired resistance to therapy. Several ongoing efforts are being made to develop novel drugs to treat this pathology with the aim to overcome resistance, prolong patient survival and improve their quality of life. We have previously shown that the non-hypercalcemic vitamin D analogues EM1 and UVB1 display antitumor effects in preclinical studies employing conventional cell lines and animal models developed from these cells. In this work, we explored the antitumor effects of EM1 and UVB1 employing BC cells derived from patient-derived xenografts (PDXs), which are a powerful preclinical tool for testing new drugs. We demonstrated that the analogues reduced the viability of HER2-positive and Triple Negative BC-PDXs. Moreover, using an in vitro model of acquired resistance to Trastuzumab-emtansine, UVB1 displayed anti-proliferative actions under 2D and 3D culture conditions. It inhibited both formation and growth of established organoids. In addition, a direct correlation between UVB1 antitumor effects and VDR expression in PDXs was found. In conclusion, all the results reinforce the potential use of these vitamin D analogues as antitumor agents to treat HER2-positive and Triple Negative BC.

The Second Generation Antibody-Drug Conjugate SYD985 Overcomes Resistances to T-DM1.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer. T-DM1 consists of trastuzumab covalently linked to the cytotoxic maytansinoid DM1 via a non-cleavable linker. Despite its efficacy, primary or acquired resistance frequently develops, particularly in advanced stages of the disease. Second generation ADCs targeting HER2 are meant to supersede T-DM1 by using a cleavable linker and a more potent payload with a different mechanism of action. To determine the effect of one of these novel ADCs, SYD985, on tumors resistant to T-DM1, we developed several patient-derived models of resistance to T-DM1. Characterization of these models showed that previously described mechanisms-HER2 downmodulation, impairment of lysosomal function and upregulation of drug efflux pumps-account for the resistances observed, arguing that mechanisms of resistance to T-DM1 are limited, and most of them have already been described. Importantly, SYD985 was effective in these models, showing that the resistance to first generation ADCs can be overcome with an improved design.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O2-· production in cancer cells.

BACKGROUND: The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 gain-of-function isoforms actively promote cancer malignancy. METHODS: A panel of wild-type and mutant p53 cancer cell lines of different tissues, including pancreas, breast, skin, and lung were used, as well as chronic lymphocytic leukemia (CLL) patients with different TP53 gene status. The effects of mutant p53 were evaluated by confocal microscopy, reactive oxygen species production assay, immunoblotting, and quantitative reverse transcription polymerase chain reaction after cellular transfection. RESULTS: We demonstrate that oncogenic mutant p53 isoforms are able to inhibit SESN1 expression and consequently the amount of SESN1/AMPK complex, resulting in the downregulation of the AMPK/PGC-1α/UCP2 axis and mitochondrial O2-· production. We also show a correlation between the decrease of reduced thiols with a poorer clinical outcome of CLL patients bearing mutant TP53 gene. The restoration of the mitochondrial uncoupling protein 2 (UCP2) expression, as well as the addition of the radical scavenger N-acetyl-L-cysteine, reversed the oncogenic effects of mutant p53 as cellular hyper-proliferation, antiapoptotic effect, and resistance to drugs. CONCLUSIONS: The inhibition of the SESN1/AMPK/PGC-1α/UCP2 axis contributes to the pro-oxidant and oncogenic effects of mutant p53, suggesting pro-oxidant drugs as a therapeutic approach for cancer patients bearing mutant TP53 gene.

Resistance to the Antibody-Drug Conjugate T-DM1 Is Based in a Reduction in Lysosomal Proteolytic Activity.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that was approved recently to treat HER2+ breast cancers. Despite its impressive clinical efficacy in many patients, intrinsic and acquired resistance to T-DM1 has emerged as a challenge. To identify mechanisms of T-DM1 resistance, we isolated several resistant HER2+ clones exhibiting stable drug refractoriness in vitro and in vivo Genomic comparisons showed substantial differences among three of the isolated clones, indicating several potential mechanisms of resistance to T-DM1. However, we observed no differences in HER2 levels and signaling among the resistant models and parental HER2+ cells. Bioinformatics studies suggested that intracellular trafficking of T-DM1 could underlie resistance to T-DM1, and systematic analysis of the path followed by T-DM1 showed that the early steps in the internalization of the drug were unaltered. However, in some of the resistant clones, T-DM1 accumulated in lysosomes. In these clones, lysosomal pH was increased and the proteolytic activity of these organelles was deranged. These results were confirmed in T-DM1-resistant cells from patient-derived HER2+ samples. We postulate that resistance to T-DM1 occurs through multiple mechanisms, one of which is impaired lysosomal proteolytic activity. Because other ADC may use the same internalization-degradation pathway to deliver active payloads, strategies aimed at restoring lysosomal functionality might overcome resistance to ADC-based therapies and improve their effectiveness. Cancer Res; 77(17); 4639-51. ©2017 AACR.

Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition.

Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.

The Phytoestrogen Genistein Affects Breast Cancer Cells Treatment Depending on the ERα/ERß Ratio.

Genistein (GEN) is a phytoestrogen found in soybeans. GEN exerts its functions through its interaction with the estrogen receptors (ER), ERα and ERß, and we previously reported that the ERα/ERß ratio is an important factor to consider in GEN-treated breast cancer cells. The aim of this study was to investigate the effects of GEN in breast cancer cells with different ERα/ERß ratio: MCF-7 (high ratio), T47D (low ratio), and MCF-7 overexpressing ERß (MCF7 + ERß) treated with cisplatin (CDDP), paclitaxel (PTX) or tamoxifen (TAM). Cell viability, ROS production, autophagy, apoptosis, antioxidant enzymes protein levels, and cell cycle were analyzed. GEN treatment provoked an increase in cell viability in MCF-7 cells and in the antioxidant enzymes protein levels in combination with the cytotoxic agents, decreasing ROS production (CDDP + GEN and TAM+GEN) and autophagy (TAM + GEN) or apoptosis (CDDP + GEN and TAM + GEN). Moreover GEN treatment enhanced the cell cycle S phase entry in CDDP+GEN- and TAM + GEN-treated MCF-7 cells and, in the case of CDDP + GEN, increased the proportion of cells in the G2/M phase and decreased it in the subG0 /G1 phase. Otherwise, in the T47D and MCF7 + ERß cells the combination of GEN with cytotoxic treatments did not cause significant changes in these parameters, even TAM + GEN-treated T47D cells showed less cell viability due to an increment in the autophagy. In conclusion, GEN consumption may be counterproductive in those patients receiving anticancer treatment with a high ERα/ERß ratio diagnosed breast cancer and it could be harmless or even beneficial in those patients with a lower ERα/ERß ratio breast cancer cells.

The presence of Estrogen Receptor ß modulates the response of breast cancer cells to therapeutic agents.

Breast cancer is a leading cause of death for women. The estrogen receptors (ERs) ratio is important in the maintenance of mitochondrial redox status, and higher levels of ERß increases mitochondrial functionality, decreasing ROS production. Our aim was to determine the interaction between the ERα/ERß ratio and the response to cytotoxic treatments such as cisplatin (CDDP), paclitaxel (PTX) and tamoxifen (TAM). Cell viability, apoptosis, autophagy, ROS production, mitochondrial membrane potential, mitochondrial mass and mitochondrial functionality were analyzed in MCF-7 (high ERα/ERß ratio) and T47D (low ERα/ERß ratio) breast cancer cell lines. Cell viability decreased more in MCF-7 when treated with CDDP and PTX. Apoptosis was less activated after cytotoxic treatments in T47D than in MCF-7 cells. Nevertheless, autophagy was increased more in CDDP-treated MCF-7, but less in TAM-treated cells than in T47D. CDDP treatment produced a raise in mitochondrial mass in MCF-7, as well as the citochrome c oxidase (COX) and ATP synthase protein levels, however significantly reduced COX activity. In CDDP-treated cells, the overexpression of ERß in MCF-7 caused a reduction in apoptosis, autophagy and ROS production, leading to higher cell survival; and the silencing of ERß in T47D cells promoted the opposite effects. In TAM-treated cells, ERß-overexpression led to less cell viability by an increment in autophagy; and the partial knockdown of ERß in T47D triggered an increase in ROS production and apoptosis, leading to cell death. In conclusion, ERß expression plays an important role in the response of cancer cells to cytotoxic agents, especially for cisplatin treatment.

UCP2 inhibition sensitizes breast cancer cells to therapeutic agents by increasing oxidative stress.

Modulation of oxidative stress in cancer cells plays an important role in the study of the resistance to anticancer therapies. Uncoupling protein 2 (UCP2) may play a dual role in cancer, acting as a protective mechanism in normal cells, while its overexpression in cancer cells could confer resistance to chemotherapy and a higher survival through downregulation of ROS production. Thus, our aim was to check whether the inhibition of UCP2 expression and function increases oxidative stress and could render breast cancer cells more sensitive to cisplatin (CDDP) or tamoxifen (TAM). For this purpose, we studied clonogenicity, mitochondrial membrane potential (ΔΨm), cell viability, ROS production, apoptosis, and autophagy in MCF-7 and T47D (only the last four determinations) breast cancer cells treated with CDDP or TAM, in combination or without a UCP2 knockdown (siRNA or genipin). Furthermore, survival curves were performed in order to check the impact of UCP2 expression in breast cancer patients. UCP2 inhibition and cytotoxic treatments produced a decrease in cell viability and clonogenicity, in addition to an increase in ΔΨm, ROS production, apoptosis, and autophagy. It is important to note that CDDP decreased UCP2 protein levels, so that the greatest effects produced by the UCP2 inhibition in combination with a cytotoxic treatment, with regard to treatment alone, were observed in TAM+UCP2siRNA-treated cells. Moreover, this UCP2 inhibition caused autophagic cell death, since apoptosis parameters barely increased after UCP2 knockdown. Finally, survival curves revealed that higher UCP2 expression corresponded with a poorer prognosis. In conclusion, UCP2 could be a therapeutic target in breast cancer, especially in those patients treated with tamoxifen.

Chronic-leptin attenuates Cisplatin cytotoxicity in MCF-7 breast cancer cell line.

BACKGROUND/AIMS: Large-scale epidemiological studies support a correlation between obesity and breast cancer in postmenopausal women. Circulating leptin levels are increased in obese and it has been suggested to play a significant role in mammary tumor formation and progression. Moreover, regulation of oxidative stress is another important factor in both tumor development and responses to anticancer therapies. The aim of this study was to examine the relationship between oxidative stress and chronic leptin exposure. METHODS: We treated MCF-7 breast cancer cells with 100 ng/mL leptin for 10 days and analyzed cell growth, ROS production and oxidative damage, as well as, some of the main antioxidant systems. Furthermore, since the hyperleptinemia has been associated with a worse pathology prognosis, we decided to test the influence of leptin in response to cisplatin anticancer treatment. RESULTS: Leptin signalling increased cell proliferation but reduced ROS production, as well as, oxidative damage. We observed an upregulation of SIRT1 after leptin exposure, a key regulator of stress response and metabolism. Additionally, leptin counteracted cisplatin-induced cytotoxicity in tumor cells, showing a decrease in cell death. CONCLUSION: Chronic leptin could contribute to the effective regulation of endogenous and treatment-induced oxidative stress, and it contributes to explain in part its proliferative effects.

Phytoestrogens and mitochondrial biogenesis in breast cancer. Influence of estrogen receptors ratio.

Phytoestrogens were originally identified as compounds having a close similarity in structure to estrogens and harboring weak estrogen activity. The interest in phytoestrogens as potential therapeutic agents has recently risen in the field of oncology, since population based studies have linked phytoestrogens consumption with a decreased risk of mortality due to several types of cancer. This review departs from the main focus of these articles by describing recent advances in our understanding of phytoestrogen potential action on mitochondria, specifically on mitochondrial biogenesis, dynamics and functionality, as well as mitoptosis in breast cancer. Further studies are necessary to explain the effects of individual phytoestrogens on mitochondrial biogenesis and dynamics and for designing of new therapy targets for cancer treatment, nevertheless area promising therapeutic approach.

Genistein modulates proliferation and mitochondrial functionality in breast cancer cells depending on ERalpha/ERbeta ratio.

Breast cancer is the most common malignancy in women of developed countries. The aim of this study was to investigate whether genistein, a soy phytoestrogen, and 17ß-estradiol (E2) could have effects on the cell cycle and mitochondrial function and dynamics. Three human breast cancer cell lines with different estrogen receptor alpha (ERα) and estrogen receptor beta (ERß) ratio were used: MCF-7 (high ERα/ERß ratio), T47D (low ERα/ERß ratio) and MDA-MB-231 (ER-negative). Cell proliferation, cell cycle, mitochondrial functionality, and mitochondrial dynamics parameters were analyzed. E2 and genistein treatment induced cell proliferation and apoptosis inhibition in MCF-7, but not in T47D and MDA-MB-231. Moreover, genistein treatment produced an up-regulation of ERß and a rise in cytochrome c oxidase activity in T47D cells, decreasing the ATP synthase/cytochrome c oxidase ratio. Finally, genistein treatment produced a drop in mitochondrial dynamics only in MCF-7 cells. In summary, the beneficial effects of genistein consumption depend on the ERα/ERß ratio in breast cells. Therefore, genistein treatment produces cell cycle arrest and an improvement of mitochondrial functionality in T47D cells with a low ERα/ERß ratio, but not in MCF-7 (high ERα/ERß ratio) and MDA-MB-231 (ER-negative) ones.

Genistein modulates oxidative stress in breast cancer cell lines according to ERα/ERß ratio: effects on mitochondrial functionality, sirtuins, uncoupling protein 2 and antioxidant enzymes.

Genistein is a biologically active isoflavone with estrogenic activity and can be found in a variety of soy products. This natural compound displays a wide array of biological activities, but it is best known for its ability to inhibit cancer progression, especially for hormone-related ones such as breast cancer. Genistein has been shown to bind both the estrogen receptor alpha (ERα) and the estrogen receptor beta (ERß), although it has a higher affinity for the ERß. The ERα/ERß ratio is a prognostic marker for breast tumors, and ERß expression could indicate the presence of tumors more benign in state, whereas ERα indicates malignant tumors. The objective of the present study was to investigate the effects of genistein on oxidative stress and mitochondrial functionality through its interaction with the estrogen receptor in breast cancer cell lines with different ERα/ERß ratios. The lower ERα/ERß ratio T47D cell line showed lower oxidative stress and greater mitochondrial functionality, along with an up-regulation of uncoupling protein 2 and sirtuins. On the other hand, genistein-treated MCF-7 cell line, with the highest ERα/ERß ratio, reported no changes for the control situation. On the whole, our results show different genistein effects depending on ERα/ERß ratio for oxidative stress regulation, mitochondrial functionality, and modulation of UCPs, antioxidant enzymes and sirtuins in breast cancer cell lines. Effects of genistein on oxidative stress and mitochondria could be due at least in part, to a higher ERß presence, but could also be due to up-regulation of ERß caused by the genistein treatment.

The over-expression of ERbeta modifies estradiol effects on mitochondrial dynamics in breast cancer cell line.

Mitochondrial biogenesis and function are under the control of 17ß-estradiol, which acts through two distinct estrogen receptors (alpha or beta), and the estrogen receptors ratio can determine the final effect of 17ß-estradiol on mitochondria. Our aim was to study the effects of 17ß-estradiol on mitochondrial biogenesis, dynamics and function in breast cancer cell lines with different estrogen receptors ratios. Mitochondrial biogenesis was increased in MDA-MB-231 (with only estrogen receptor beta expression), T47D (normal estrogen receptors ratio) and MCF-7 (highest estrogen receptors ratio) breast cancer cell lines, in response to different mitochondrial and cellular status. In fact, mitochondria of the MDA-MB-231 and T47D cell lines maintained their functionality, although, the MCF-7 cell line did suffer an important decrease in mitochondrial function. Thus, mitochondrial biogenesis increased in MCF-7 with the aim of mitigating these defective mitochondria. In normal conditions, mitophagic processes remove defective mitochondria to refresh the mitochondrial pool. Mitochondrial dynamics were also under control by 17ß-estradiol, and showed modifications in the fusion/fission processes and the modulation of mitochondrial removal. In fact, cells with only estrogen receptor beta or with a low estrogen receptors ratio, such as MDA-MB-231 and T47D, showed an increase in fusion processes. However, the MCF-7 cell line, with more estrogen receptor alpha, also showed an increase in fusion processes, even though the fission processes were diminished and led to an accumulation of unfunctional mitochondria. Finally, the importance of estrogen receptor beta in mitochondrial biogenesis, function, as well as in mitochondrial dynamics was examined. Using the T47D-estrogen receptor beta tetracycline-inducible cell line, the results confirmed that when the overexpression of estrogen receptor beta was inhibited, there was an increase in mitochondrial biogenesis, although these mitochondria were less functional, and with fewer fission events, although there was an increase in fusion processes.

The oxidative stress in breast tumors of postmenopausal women is ERα/ERß ratio dependent.

Estrogen receptor status is a diagnostic parameter in breast cancer treatment. Estrogen receptor presence is related to a better prognosis because the principal treatments attacking breast cancer tumors have their action site directed at the estrogen receptor. However, the two different subtypes of estrogen receptor, ERα and ERß, have different functions. In this work an alternative point of view focusing on oxidative stress is shown, given that estrogen receptors regulate several proteins related to this oxidative stress, such as antioxidant enzymes, sirtuins, and uncoupling proteins. Postmenopausal human breast tumors with different ERα/ERß ratios were analyzed to characterize the amount of oxidative stress, mitochondrial function, and proliferation-related and oxidative stress-activated signaling pathways. Results showed that tumors with a low ERα/ERß ratio have greater oxidative damage and higher antioxidant enzyme protein levels, as well as uncoupling protein (UCP) and sirtuin 3 (SIRT3), and have high studied signaling pathway activation. Glutathione peroxidase, Complex V, Complex III, Complex II, Complex IV, AKT, SAPK, and ERα were significantly and positively correlated with ERα/ERß ratio. However, carbonyl groups, catalase, CuZn-superoxide dismutase, UCP5, SIRT3, and ERß were significantly and negatively correlated with ERα/ERß ratio. From the independent variables included in the step-by-step stepwise multiple linear regression analysis, only the ERα/ERß ratio was independently associated with carbonyl groups. Surprisingly, these low ERα/ERß ratio tumors have poor prognosis for the patient, and these results and those of other authors suggest that these tumors are adapted to conditions of increased oxidative stress.

Initial activation status of the antioxidant response determines sensitivity to carboplatin/paclitaxel treatment of ovarian cancer.

BACKGROUND/AIM: Ovarian carcinoma is the main cause of gynecological cancer related deaths. The aim of this study was to determine the activation status of the antioxidant response in samples of ovarian serous carcinoma from paraffin-embedded biopsies and compare them with the response of patients to carboplatin-paclitaxel treatment. MATERIALS AND METHODS: Estrogen receptor alpha (ERα), antioxidant enzymes, and uncoupling protein (UCP) levels were analyzed by western blotting and the presence of estrogen receptor beta (ERß) was investigated by immunohistochemistry (IHC). RESULTS: Lower levels of ERα, antioxidant enzymes and UCPs were found in patients resistant to treatment in comparison to the carboplatin/paclitaxel-sensitive ones; IHC revealed a greater presence of ERß in sensitive patients. CONCLUSION: These results indicate that patients resistant to treatment have a lower level of antioxidant response activation compared to sensitive patients, fact which may be related to the efficacy of this treatment.

Mitochondrial dynamics is affected by 17ß-estradiol in the MCF-7 breast cancer cell line. Effects on fusion and fission related genes.

Mitochondrial dynamics, specifically fusion and fission processes, maintain mitochondria integrity and function, yet at this time, effect of estrogens on fusion and fission in breast cancer cell lines has not been studied. The aim of this study was to characterize the effect of 17ß-estradiol on fusion and fission-related genes, as well as on mitochondria proliferation and function. We used MCF-7 breast cancer cell line, which is estrogen sensitive (estrogen receptor positive). Cells were grown in Dulbecco's modified Eagle medium charcoal-stripped fetal bovine serum and treated with 1nM of 17ß-estradiol and with/without 100nM of ICI 182,780, a drug that caused rapid degradation of estrogen receptor. mRNA levels of fusion (mfn1, mfn2, opa1) and fission-related genes (drp1 and fis1) were examined by RT-PCR, cardiolipin content by N-acridyl-orange fluorescence and oxidative phosphorylation protein levels, as well as, the major fusion and fission related protein levels, by Western blot. mRNA expression of fusion-related genes increased after 17ß-estradiol-treatment for 4h; however fis1 fission-related gene expression decreased. All these effects were not found in cells pre-treated with ICI 182,780, save for the changes in mfn-1, conferring them the effects of 17ß-estradiol to estrogen receptor. The changes in protein levels were less prominent, but in the same way, than in mRNA levels, showing an increase in Mfn1 and Mfn2, as well as in Drp1, but there was no change in Fis1 protein levels. Mitochondrial biogenesis was also affected by 17ß-estradiol, showing an increase in mtDNA but with no change in N-acridyl-orange fluorescence. On the whole, our results suggest an imbalance in the fusion/fission ratio, with a high fusion by 17ß-estradiol-estrogen receptor action, which can affect to mitochondrial biogenesis, concretely in mitochondria proliferation. According to this information, 17ß-estradiol would modify mitochondrial dynamics, biogenesis and metabolism, and thus compromise the normal development and function of mitochondria in cancer affected tissues.

The ERalpha/ERbeta ratio determines oxidative stress in breast cancer cell lines in response to 17beta-estradiol.

The effects of 17beta-estradiol (E2) are mediated through activation of estrogen receptors (ER): ERalpha and ERbeta. It is known that ERalpha/ERbeta ratio is higher in breast tumors than in normal tissue. Since antioxidant enzymes and uncoupling proteins (UCPs) are reactive oxygen species (ROS) production and mitochondrial biogenesis regulators, our aim was to study the E2-effect on oxidative stress, antioxidant enzyme expression, and UCPs in breast cancer cell lines with different ERalpha/ERbeta ratios. The lower ERalpha/ERbeta ratio T47D cell line showed low ROS production and high UCP5 levels. However, the higher ERalpha/ERbeta ratio MCF-7 cell line showed an up-regulation of antioxidant enzymes and UCPs, yet exhibited high oxidative stress. As a result, a decrease in antioxidant enzyme activities and UCP2 protein levels, coupled with an increase in oxidative damage was found. On the whole, these results show different E2-effects on oxidative stress regulation, modulating UCPs, and antioxidant enzymes, which were ERalpha/ERbeta ratio dependent in breast cancer cell lines.