Donatella Vizza

Omega-3 PUFA ethanolamides DHEA and EPEA induce autophagy through PPARγ activation in MCF-7 breast cancer cells†‡§

The omega‐3 long chain polyunsaturated fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), elicit anti‐proliferative effects in cancer cell lines and in animal models. Dietary DHA and EPA can be converted to their ethanolamide derivatives, docosahexaenoyl ethanolamine (DHEA), and eicosapentaenoyl ethanolamine (EPEA), respectively; however, few studies are reported on their anti‐cancer activities. Here, we demonstrated that DHEA and EPEA were able to reduce cell viability in MCF‐7 breast cancer cells whereas they did not elicit any effects in MCF‐10A non‐tumorigenic breast epithelial cells. Since DHA and EPA are ligands of Peroxisome Proliferator‐Activated Receptor gamma (PPARγ), we sought to determine whether PPARγ may also mediate DHEA and EPEA actions. In MCF‐7 cells, both compounds enhanced PPARγ expression, stimulated a PPAR response element‐dependent transcription as confirmed by the increased expression of its target gene PTEN, resulting in the inhibition of AKT‐mTOR pathways. Besides, DHEA and EPEA treatment induced phosphorylation of Bcl‐2 promoting its dissociation from beclin‐1 which resulted in autophagy induction. We also observed an increase of beclin‐1 and microtubule‐associated protein 1 light chain 3 expression along with an enhanced autophagosomes formation as revealed by mono‐dansyl‐cadaverine staining. Finally, we demonstrated the involvement of PPARγ in DHEA‐ and EPEA‐induced autophagy by using siRNA technology and a selective inhibitor. In summary, our data show that the two omega‐3 ethanolamides exert anti‐proliferative effects by inducing autophagy in breast cancer cells highlighting their potential use as breast cancer preventive and/or therapeutic agents. J. Cell. Physiol. 228: 1314–1322, 2013.

Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells

ETHNOPHARMACOLOGICAL RELEVANCE Ziziphus extracts have been used in Traditional Chinese Medicine for the treatment of cancer. AIM OF THE STUDY In the present study we have investigated the effects of Ziziphus jujube extracts (ZEs) on breast cancer. MATERIALS AND METHODS We evaluated the effects of increasing concentrations of ZEs on ERα positive MCF-7 and ERα negative SKBR3 breast cancer cell proliferation using MTT assays. Apoptosis was analyzed by evaluating the involvement of some pro-apoptotic proteins, including Bax, Bad, Bid and PARP cleavage by immunoblotting analysis. Moreover, the effects of ZEs treatment on apoptosis were tested by both DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. By using chromatographic techniques, we identified the constituents of the effective extracts. RESULTS ZE1, ZE2, and ZE4 exerted significant antiproliferative effects on estrogen receptor alpha (ERα) positive MCF-7 (IC(50) values of 14.42, 7.64, 1.69μg/mL) and ERα negative SKBR3 (IC(50) values of 14.06, 6.21, 3.70μg/mL) human breast cancer cells. Remarkably, ZEs did not affect cell viability of both normal human fibroblasts BJ1-hTERT and nonmalignant breast epithelial MCF-10A cells. Treatment with ZEs induced cell death by apoptosis in both malignant breast cells. We found that the most effective extracts ZE2 and ZE4 shared a number of triterpenic acids, already known for their anticancer activities. CONCLUSIONS Our data provide a rational base for the use of Ziziphus extracts in the treatment of breast cancer in Traditional Chinese Medicine.

Combined Low Doses of PPARγ and RXR Ligands Trigger an Intrinsic Apoptotic Pathway in Human Breast Cancer Cells

Ligand activation of peroxisome proliferator-activated receptor (PPAR)gamma and retinoid X receptor (RXR) induces antitumor effects in cancer. We evaluated the ability of combined treatment with nanomolar levels of the PPARgamma ligand rosiglitazone (BRL) and the RXR ligand 9-cis-retinoic acid (9RA) to promote antiproliferative effects in breast cancer cells. BRL and 9RA in combination strongly inhibit of cell viability in MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer cells, whereas MCF-10 normal breast epithelial cells are unaffected. In MCF-7 cells, combined treatment with BRL and 9RA up-regulated mRNA and protein levels of both the tumor suppressor p53 and its effector p21(WAF1/Cip1). Functional experiments indicate that the nuclear factor-kappaB site in the p53 promoter is required for the transcriptional response to BRL plus 9RA. We observed that the intrinsic apoptotic pathway in MCF-7 cells displays an ordinated sequence of events, including disruption of mitochondrial membrane potential, release of cytochrome c, strong caspase 9 activation, and, finally, DNA fragmentation. An expression vector for p53 antisense abrogated the biological effect of both ligands, which implicates involvement of p53 in PPARgamma/RXR-dependent activity in all of the human breast malignant cell lines tested. Taken together, our results suggest that multidrug regimens including a combination of PPARgamma and RXR ligands may provide a therapeutic advantage in breast cancer treatment.

Leptin increases HER2 protein levels through a STAT3-mediated up-regulation of Hsp90 in breast cancer cells

Obesity condition confers risks to breast cancer development and progression, and several reports indicate that the adipokine leptin, whose synthesis and plasma levels increase with obesity, might play an important role in modulating breast cancer cell phenotype. Functional crosstalk occurring between leptin and different signaling molecules contribute to breast carcinogenesis.

Farnesoid X receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through downregulation of HER2 expression

Tamoxifen (Tam) treatment is a first-line endocrine therapy for estrogen receptor-α-positive breast cancer patients. Unfortunately, resistance frequently occurs and is often related with overexpression of the membrane tyrosine kinase receptor HER2. This is the rationale behind combined treatments with endocrine therapy and novel inhibitors that reduce HER2 expression and signaling and thus inhibit Tam-resistant breast cancer cell growth. In this study, we show that activation of farnesoid X receptor (FXR), by the primary bile acid chenodeoxycholic acid (CDCA) or the synthetic agonist GW4064, inhibited growth of Tam-resistant breast cancer cells (termed MCF-7 TR1), which was used as an in vitro model of acquired Tam resistance. Our results demonstrate that CDCA treatment significantly reduced both anchorage-dependent and anchorage-independent epidermal growth factor (EGF)-induced growth in MCF-7 TR1 cells. Furthermore, results from western blot analysis and real-time reverse transcription–PCR revealed that CDCA treatment reduced HER2 expression and inhibited EGF-mediated HER2 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation in these Tam-resistant breast cancer cells. Transient transfection experiments, using a vector containing the human HER2 promoter region, showed that CDCA treatment downregulated basal HER2 promoter activity. This occurred through an inhibition of nuclear factor-κB transcription factor binding to its specific responsive element located in the HER2 promoter region as revealed by mutagenesis studies, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Collectively, these data suggest that FXR ligand-dependent activity, blocking HER2/MAPK signaling, may overcome anti-estrogen resistance in human breast cancer cells and could represent a new therapeutic tool to treat breast cancer patients that develop resistance.

Epigallocatechin gallate inhibits growth and epithelial-to-mesenchymal transition in human thyroid carcinoma cell lines

Well‐differentiated papillary and follicular thyroid carcinoma are the most frequent types of thyroid cancer and the prognosis is generally favorable however, a number of patients develops recurrences. Epigallocatechin‐3‐gallate (EGCG), a major catechin in green tea, was shown to possess remarkable therapeutic potential against various types of human cancers, although data on thyroid cancer cells are still lacking. The aim of this study was to investigate the effect of EGCG on the proliferation and motility of human thyroid papillary (FB‐2) and follicular (WRO) carcinoma cell lines. Our results demonstrate that EGCG (10, 40, 60 μM) treatment inhibited the growth of FB‐2 and WRO cells in a dose‐dependent manner. These changes were associated with reduced cyclin D1, increased p21 and p53 expression. Furthermore, EGCG suppressed phosphorylation of AKT and ERK1/2. In addition EGCG treatment results in reduction of cell motility and migration. Changes in motility and migration in FB‐2 were associated with modulation in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. After 24 h EGCG caused an increase of the E‐cadherin expression and a concomitant decrease of SNAIL, ZEB and the basic helix–loop–helix transcription factor TWIST. Besides expression of Vimentin, N‐cadherin and α5‐integrin was down‐regulated. These data well correlate with a reduction of MMP9 activity as evidenced by gelatin zymography. Our findings support the inhibitory role of EGCG on thyroid cancer cell proliferation and motility with concomitant loss of epithelial‐to‐mesenchymal cell transition markers. J. Cell. Physiol. 228: 2054–2062, 2013.

Bid as a potential target of apoptotic effects exerted by low doses of PPARγ and RXR ligands in breast cancer cells.

The combined treatment with nanomolar doses of the PPARγ ligand Rosiglitazone (BRL) and the RXR ligand 9-cis‑retinoic acid (9RA) induces a p53-dependent apoptosis in MCF7, SKBR3 and T47D human breast cancer cells. Since MCF7 cells express a wild-type p53 protein, while SKBR3 and T47D cells harbor endogenous mutant p53, we elucidated the mechanism through which PPARγ and RXR ligands triggered apoptotic processes independently of p53 transcriptional activity. We showed an upregulation of Bid expression enhancing the association between Bid/p53 in both cytosol and mitochondria after the ligand treatment. Particularly in the mitochondria, the complex involves the truncated Bid that plays a key role in the apoptotic process induced by BRL and 9RA, since the disruption of mitochondrial membrane potential, the induction of PARP cleavage and the percentage of TUNEL-positive cells were reversed after knocking down Bid. Moreover, PPARγ and RXR ligands were able to reduce mitochondrial GST activity, which was no longer noticeable silencing Bid expression, suggesting the potential of Bid in the regulation of mitochondrial intracellular reactive oxygen species scavenger activity. Our data, providing new insight into the role of p53/Bid complex at the mitochondria in promoting breast cancer cell apoptosis upon low doses of PPARγ and RXR ligands, address Bid as a potential target in the novel therapeutical strategies for breast cancer.

High doses of hydroxytyrosol induce apoptosis in papillary and follicular thyroid cancer cells

PurposeRecent evidences indicates that hydroxytyrosol, one of the main olive oil phenols, possess antitumor effects because of its pro-oxidant properties and the capacity to inhibit proliferation and to promote apoptosis in several tumor cell lines, although most of the results were obtained for breast and digestive systems cancers.MethodsIn this study, we evaluated the activities of hydroxytyrosol against papillary (TPC-1, FB-2) and follicular (WRO) thyroid cancer cell lines.ResultsCellular viability revealed that high doses of hydroxytyrosol reduced cancer cells viability concomitantly with a reduction of cyclin D1 expression and an up-regulation of cell cycle key modulator p21 levels. In the same experimental conditions, Annexin V–PI staining and DNA laddering revealed that hydroxytyrosol exerts proapoptotic effects on papillary and follicular cancer cells. Furthermore, by Western blot analysis, we observed that hydroxytyrosol treatment reduced thyroid cancer cells viability by promoting apoptotic cell death via intrinsic pathway.ConclusionsCollectively, our results demonstrated for the first time that in thyroid cancer cells hydroxytyrosol promoted apoptosis at higher doses with respect to other cancer cells lines. Therefore, further studies will reveal the mechanisms by which thyroid cancer cells are more resistant to the proapoptotic effect exerted by hydroxytyrosol as well as the potential application as novel target therapeutic in thyroid cancer.

T3 enhances thyroid cancer cell proliferation through TRβ1/Oct-1-mediated cyclin D1 activation

Several studies have demonstrated that thyroid hormone T3 promotes cancer cell growth, even though the molecular mechanism involved in such processes still needs to be elucidated. In this study we demonstrated that T3 induced proliferation in papillary thyroid carcinoma cell lines concomitantly with an up-regulation of cyclin D1 expression, that is a critical mitogen-regulated cell-cycle control element. Our data revealed that T3 enhanced the recruitment of the TRβ1/Oct-1 complex on Octamer-transcription factor-1 site within cyclin D1 promoter, leading to its transactivation. In addition, silencing of TRβ1 or Oct-1 expression by RNA interference reversed both increased cell proliferation and up-regulation of cyclin D1, underlying the important role of both transcriptional factors in mediating these effects. Finally, T3-induced increase in cell growth was abrogated after knocking down cyclin D1 expression. All these findings highlight a new molecular mechanism by which T3 promotes thyroid cancer cell growth.

Sperm metabolism in pigs: a role for peroxisome proliferator-activated receptor gamma (PPARγ)

SUMMARY Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor expressed predominantly in adipose tissue, also implicated in energy homeostasis. In this study, we used western blotting and immunofluorescence techniques to demonstrate for the first time that pig spermatozoa express PPARγ. To investigate the functional role of PPARγ in pig sperm, we evaluated its action on different events that characterize the biology of sperm cells, i.e. motility, capacitation, viability and acrosome reaction, using the PPARγ-agonist 15-deoxy-12,14-prostaglandin J2 (PGJ2). In responses to PGJ2 treatment, motility, cholesterol efflux and tyrosine phosphorylation were increased, which broadens the role of PPARγ from that previously described in the literature, as it also acts to improve sperm functionality. To further our understanding of the significance of PPARγ in pig sperm, we focused its effects on lipid and glucose metabolism. Evaluation of triglyceride content and lipase, acyl-CoA dehydrogenase and G6PDH activities suggests that PPARγ induces energy expenditure in pig spermatozoa. These data represent a meaningful advance in the field of sperm energy metabolism. Taken together, our results demonstrate for the first time that PPARγ is expressed by pig sperm, thus improving its functionalities in terms of motility, capacitation, acrosome reaction, survival and metabolism.