Jorge Sastre-Serra

Estrogen down-regulates uncoupling proteins and increases oxidative stress in breast cancer.

Oxidative stress has been postulated as one of the mechanisms underlying the estrogen carcinogenic effect in breast cancer. Estrogens are known to increase mitochondrial-derived reactive oxygen species (ROS) by an unknown mechanism. Given that uncoupling proteins (UCPs) are key regulators of mitochondrial energy efficiency and ROS production, our aim was to check the presence and activity of UCPs in estrogen receptor (ER)-positive and ER-negative breast cancer cells and tumors, as well as their relation to oxidative stress. Estrogen (1 nM) induced higher oxidative stress in the ER-positive MCF-7 cell line, showing increased mitochondrial membrane potential, H(2)O(2) levels, and DNA and protein damage compared to ER-negative MDA-MB-231 cells. All isoforms of uncoupling proteins were highly expressed in ER-positive breast cancer cells and tumors compared to negative ones. ROS production in mitochondria isolated from MCF-7 was increased by inhibition of UCPs with GDP, but not in mitochondria from MDA-MB-231. Estrogen treatment decreased uncoupling protein and catalase levels in MCF-7 and decreased GDP-dependent ROS production in isolated mitochondria. On the whole, these results suggest that estrogens, through an ER-dependent mechanism, may increase mitochondrial ROS production by repressing uncoupling proteins, which offers a new perspective on the understanding of why estrogens are a risk factor for breast cancer.

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 17β‐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 (ERβ) ratio were used: MCF‐7 (high ERα/ERβ ratio), T47D (low ERα/ERβ 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 ERβ 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/ERβ ratio in breast cells. Therefore, genistein treatment produces cell cycle arrest and an improvement of mitochondrial functionality in T47D cells with a low ERα/ERβ ratio, but not in MCF‐7 (high ERα/ERβ ratio) and MDA‐MB‐231 (ER‐negative) ones. J. Cell. Biochem. 115: 949–958, 2014.

Genistein modulates oxidative stress in breast cancer cell lines according to ERα/ERβ 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 (ERβ), although it has a higher affinity for the ERβ. The ERα/ERβ ratio is a prognostic marker for breast tumors, and ERβ 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/ERβ ratios. The lower ERα/ERβ 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/ERβ ratio, reported no changes for the control situation. On the whole, our results show different genistein effects depending on ERα/ERβ 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 ERβ presence, but could also be due to up-regulation of ERβ caused by the genistein treatment.

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. J. Cell. Biochem. 113: 3178–3185, 2012.

17β-Estradiol regulates oxidative stress in prostate cancer cell lines according to ERalpha/ERbeta ratio.

Estrogen action is mediated by the two receptor isoforms: estrogen receptor alpha and beta. Both receptors are expressed in human prostate tissue and have different action profiles. ERalpha is positively correlated with the malignancy of prostate cancer, while ERbeta may protect against abnormal prostate cell growth. 17β-Estradiol (E2), at least in part, induces cancerous transformations by causing deleterious mutations through the formation of reactive oxygen species (ROS). The aim was to study the effect of E2 on oxidative stress and the expression of uncoupling proteins (UCPs) and antioxidant enzymes in several prostate cancer cell lines with different ERalpha/ERbeta ratios. The cell prostate lines with a lower ERalpha/ERbeta ratio had lower oxidative stress, which could be partially explained by the increased expression of antioxidant enzymes and UCPs. Moreover, the action of E2 on the expression of antioxidant enzymes and UCPs was dual and dependent on the ERalpha/ERbeta ratio. Treatments with 0.1 nM E2 in cell lines with high ERalpha/ERbeta ratio produced a decrease in antioxidant enzymes and UCPs levels, with an increase in ROS production. These effects disappeared when the treatment was done in the presence of an ERalpha antagonist (MPP). In the cell lines with greatest levels of ERbeta and the lowest ERalpha/ERbeta ratio, E2 treatment caused the up-regulation of antioxidant enzymes and UCPs with a look-up decrease in ROS production. These effects were reversed when the cells were treated with E2 in the presence of an ERbeta antagonist (R,R-THC). On the whole, our results suggest a dual E2 effect; increasing or decreasing oxidative stress in part by modulation of UCPs and antioxidant enzymes according to the abundance ERbeta and ERalpha/ERbeta ratio in prostate cancer cell lines.

The effects of 17β-estradiol on mitochondrial biogenesis and function in breast cancer cell lines are dependent on the ERα/ERβ ratio.

Background/Aims: 17β-estradiol (E2) is a risk factor for the development of breast cancer, and cause tumorigenesis in epithelial breast cells. Moreover, E2 has distinct effects on different tissues that are attributed to the presence of two estrogen receptor isoforms, ERα and ERβ. Methods: The effect of E2 on mitochondrial biogenesis and function was investigated in two breast cancer cell lines with different estrogen receptor ratios, MCF-7 (high ERα/ERβ ratio) and T47D (low ERα/ERβ ratio) cell lines treated with physiological concentrations of E2 (1 nM). Results: Mitochondria of the MCF-7 cell line showed an increase in proliferation but a decrease in functionality, while the T47D cell line, with low ERα/ERβ ratio, maintained functionality with fewer mitochondria. Conclusion: Our results suggest that ERs endowment and its subtypes relation have an effect on treatment response and could contribute new ideas about mitochondria and ERs in breast cancer, as well as new indicators to the disease progression.

Mitochondrial dynamics is affected by 17β-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 17β-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 Dulbeccos modified Eagle medium charcoal-stripped fetal bovine serum and treated with 1nM of 17β-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 17β-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 17β-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 17β-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 17β-estradiol-estrogen receptor action, which can affect to mitochondrial biogenesis, concretely in mitochondria proliferation. According to this information, 17β-estradiol would modify mitochondrial dynamics, biogenesis and metabolism, and thus compromise the normal development and function of mitochondria in cancer affected tissues.

Chronic leptin treatment sensitizes MCF-7 breast cancer cells to estrogen.

Background/Aims: Obesity is associated with an increased risk of estrogen-dependent breast cancer. The adipokine leptin, whose levels are chronically increased in obese people, has been shown to stimulate ER positive cancer cell growth. Considering previous evidence of a crosstalk between leptin and estrogen signaling, the objective of this study was to establish the influence of chronic leptin treatment on estrogen-dependent cell growth. Methods: To this aim, we use the estrogen receptor (ER) positive MCF-7 breast cancer cell line treated chronically with leptin and analyzed estrogen-dependent cell growth, ERs (ERα and ERβ) expression, ER-dependent transcriptional activity as well as cell survival to the antiestrogenic agents tamoxifen and ICI 182,780. Results: Leptin signaling pathway kept activated after chronic stimulation (7 days) with leptin showing significant phosphorylation of JAK2 and STAT3 and higher cell proliferation rate. Chronic leptin at 100 ng/mL dose increased ERα to ERβ ratio and consistently enhanced estrogen-dependent transcriptional activity, increasing E2-dependent cell growth and resistance to antiestrogen agents. Conclusion: This study supports the existence of a crosstalk between leptin and estrogen, in which leptin might play an important role potentiating the mitogenic action of estrogen, probably by alteration of ERα to ERβ ratio.

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 17β-estradiol, which acts through two distinct estrogen receptors (alpha or beta), and the estrogen receptors ratio can determine the final effect of 17β-estradiol on mitochondria. Our aim was to study the effects of 17β-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 17β-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 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.