Margalida Torrens-Mas

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.

The presence of Estrogen Receptor β 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 ERβ increases mitochondrial functionality, decreasing ROS production. Our aim was to determine the interaction between the ERα/ERβ 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/ERβ ratio) and T47D (low ERα/ERβ 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 ERβ in MCF-7 caused a reduction in apoptosis, autophagy and ROS production, leading to higher cell survival; and the silencing of ERβ in T47D cells promoted the opposite effects. In TAM-treated cells, ERβ-overexpression led to less cell viability by an increment in autophagy; and the partial knockdown of ERβ in T47D triggered an increase in ROS production and apoptosis, leading to cell death. In conclusion, ERβ expression plays an important role in the response of cancer cells to cytotoxic agents, especially for cisplatin treatment.

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

Genistein (GEN) is a phytoestrogen found in soybeans. GEN exerts its functions through its interaction with the estrogen receptors (ER), ERα and ERβ, and we previously reported that the ERα/ERβ 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/ERβ ratio: MCF‐7 (high ratio), T47D (low ratio), and MCF‐7 overexpressing ERβ (MCF7 + ERβ) 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 + ERβ 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/ERβ ratio diagnosed breast cancer and it could be harmless or even beneficial in those patients with a lower ERα/ERβ ratio breast cancer cells. J. Cell. Biochem. 117: 218–229, 2016.

SIRT3 Silencing Sensitizes Breast Cancer Cells to Cytotoxic Treatments Through an Increment in ROS Production

SIRT3, the major deacetylase in mitochondria, plays a crucial role modulating ROS production and scavenging by regulating key proteins implicated in mitochondrial turnover and in antioxidant defenses. Therefore, SIRT3 could confer resistance to chemotherapy‐induced oxidative stress, leading to a lower ROS production and a higher cell survival. Our aim was to analyze whether SIRT3 silencing in breast cancer cells through a specific siRNA could increase oxidative stress and thus compromise the antioxidant response, resulting in a sensitization of the cells to cisplatin (CDDP) or tamoxifen (TAM). For this purpose, we studied cell viability, ROS production, apoptosis and autophagy in MCF‐7 and T47D cell lines treated with these cytotoxic compounds, these either alone, or in combination with SIRT3 silencing. Moreover, protein levels regulated by SIRT3 were also examined and survival curves were analyzed to study the importance of SIRT3 expression for the overall survival of breast cancer patients. When SIRT3 was silenced and combined with cytotoxic treatments, cell viability was highly decreased, and was accompanied by a significant increase in ROS production. While TAM treatment increased autophagic cell death, CDDP significantly triggered apoptosis, whereas SIRT3 silencing produced an enhancement of these two action mechanisms. SIRT3 knockdown also affected PGC‐1α and TFAM (mitochondrial biogenesis), and MnSOD and IDH2 (antioxidant defenses) protein levels. Finally, survival curves showed that higher SIRT3 expression is correlated to a poorer prognosis for patients with grade 3 breast cancer. In conclusion, SIRT3 could be a therapeutic target for breast cancer, improving the effectiveness of CDDP and TAM treatments. J. Cell. Biochem. 118: 397–406, 2017.

Sirtuin 3 silencing improves oxaliplatin efficacy through acetylation of MnSOD in colon cancer

Sirtuin 3 (SIRT3) is the major mitochondria deacetylase and regulates ROS levels by targeting several key proteins, such as those involved in mitochondrial function and antioxidant defenses. This way, SIRT3 balances ROS production and scavenging and promotes cell survival. The aim of this study was to analyze the effect of SIRT3 silencing on the antioxidant response in SW620 colon cancer cell line, and whether this intervention could improve efficacy of oxaliplatin, a common drug used to treat colon cancer. For this purpose, we obtained stable clones of SW620 with SIRT3 knockdown and determined parameters such as ROS levels and ROS production, levels of several antioxidant enzymes, cell viability, and apoptosis. Results showed that after SIRT3 silencing, both ROS levels and production were increased, and antioxidant enzymes gene expression was significantly reduced. Furthermore, manganese superoxide dismutase levels and enzymatic activity were reduced. Combination of SIRT3 knockdown with oxaliplatin treatment further increased ROS production and apoptosis, reducing cell viability. Finally, survival curves on colon cancer patients suggested that SIRT3 expression is related to a poorer prognosis. In conclusion, SIRT3 could be a target for colon cancer, since it regulates the antioxidant response and its knockdown improves the efficacy of oxaliplatin treatment.

PGC-1α in Melanoma: A Key Factor for Antioxidant Response and Mitochondrial Function

Melanocortin 1 receptor (MC1R) and BRAF are common mutations in melanoma. Through different pathways, they each regulate the expression of PGC‐1α, which is a key factor in the regulation of mitochondrial biogenesis and the antioxidant response. Our aim was to study the importance of the different regulatory characteristics of MC1R and BRAF on the pathways they regulate in melanoma. For this purpose, ROS production, levels of gene expression and enzymatic activities were analyzed in HBL and MeWo, with wild‐type MC1R and BRAF, and A375 cells with mutant MC1R and BRAF. HBL cells showed a functional MC1R‐PGC‐1α pathway and exhibited the lowest ROS production, probably because of a better mitochondrial pool and the presence of UCP2. On the other hand, MeWo cells showed elevated levels of PGC‐1α but also high ROS production, similar to the A375 cells, along with an activated antioxidant response and significantly low levels of UCP2. Finally, A375 cells are mutant for BRAF, and thus showed low levels of PGC‐1α. Consequently, A375 cells exhibited poor mitochondrial biogenesis and function, and no antioxidant response. These results show the importance of the activation of the MC1R‐PGC‐1α pathway for mitochondrial biogenesis and function in melanoma development, as well as BRAF for the antioxidant response regulated by PGC‐1α. J. Cell. Biochem. 118: 4404–4413, 2017.

Non-tumor adjacent tissue of advanced stage from CRC shows activated antioxidant response

Abstract Colorectal cancer (CRC) is a leading cause of malignant cancer‐related morbidity and mortality, with a higher incidence in developed countries and a high mortality rate mainly attributable to metastases. The aim of the present study was to determine the metabolic adaptations related to oxidative stress in tumor tissue from advanced stages (III and IV) of CRC and whether they could be used as potential biomarkers for clinical applications. To tackle this aim, we have analyzed the protein expression levels related to oxidative stress and the enzymatic activities of MnSOD and catalase, comparing samples of non‐tumor adjacent tissue and tumor tissue of CRC patients in stages III and IV. The results showed no differences between stage III and IV in tumor tissues for any of the proteins studied. However, some differences were found between samples of non‐tumor adjacent tissue and tumor tissue for some of the antioxidant enzymes. Overwhelmingly, the greatest differences were detected when comparing samples of non‐tumor adjacent tissue from stage III and stage IV. To the best of our knowledge, this is the first study where differences between the non‐tumor adjacent tissues of CRC patients from different cancer stages were determined. This study suggests that the parameters analyzed should be evaluated as biomarkers for the evolution of CRC. Furthermore, tumor tissue status should not be of sole importance for the prognosis of CRC, as the non‐tumor adjacent tissues could also merit consideration. Graphical abstract Figure. No caption available. HighlightsDifferences between non‐tumor adjacent tissues of CRC in stages III vs IV are shown.CRC stage IV non‐tumor adjacent tissue shows higher activated antioxidant response than in stage III.Changes in non‐tumor adjacent tissue could be evaluated as a CRC biomarkers source.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O 2ˉ · production in cancer cells

BackgroundThe TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 gain-of-function isoforms actively promote cancer malignancy.MethodsA 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.ResultsWe 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 O2ˉ· 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.ConclusionsThe 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.