Daniela Rovito

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.

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.

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.

Mechanisms of divergent effects of activated peroxisome proliferator-activated receptor-γ on mitochondrial citrate carrier expression in 3T3-L1 fibroblasts and mature adipocytes☆

The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be essential for fibroblast differentiation into fat cells. Because peroxisome proliferator-activated receptor-gamma (PPARγ) is known to be one of the master regulators of adipogenesis, we aimed to study the regulation of CIC by the PPARγ ligand rosiglitazone (BRL) in 3T3-L1 fibroblasts and in adipocytes. We demonstrated that BRL up-regulated CIC mRNA and protein levels in fibroblasts, while it did not elicit any effects in mature adipocytes. The enhancement of CIC levels upon BRL treatment was reversed using the PPARγ antagonist GW9662, addressing how this effect was mediated by PPARγ. Functional experiments using a reporter gene containing rat CIC promoter showed that BRL enhanced CIC promoter activity. Mutagenesis studies, electrophoretic-mobility-shift assay and chromatin-immunoprecipitation analysis revealed that upon BRL treatment, PPARγ and Sp1 are recruited on the Sp1-containing region within the CIC promoter, leading to an increase in CIC expression. In addition, mithramycin, a specific inhibitor for Sp1-DNA binding activity, abolished the PPARγ-mediated up-regulation of CIC in fibroblasts. The stimulatory effects of BRL disappeared in mature adipocytes in which PPARγ/Sp1 complex recruited SMRT corepressor to the Sp1 site of the CIC promoter. Taken together, our results contribute to clarify the molecular mechanisms by which PPARγ regulates CIC expression during the differentiation stages of fibroblasts into mature adipocytes.

Omega-3 DHA- and EPA-dopamine conjugates induce PPARγ-dependent breast cancer cell death through autophagy and apoptosis.

BACKGROUND The omega-3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may form conjugates with amines that have potential health benefits against common diseases including cancers. Here we synthesized DHA-dopamine (DHADA) and EPA-dopamine (EPADA) conjugates and studied their biological effects on different breast cancer cell lines. METHODS AND RESULTS MTT assays indicated that increasing concentrations of DHADA and EPADA significantly affected viability in MCF-7, SKBR3 and MDA-MB-231 breast cancer cells, whereas no effect was observed in MCF-10A non-tumorigenic epithelial breast cells. DHADA and EPADA enhanced Beclin-1 expression, as evidenced by immunoblotting, real-time-PCR and functional analyses. Chromatin Immunoprecipitation (ChIP) and Re-ChIP assays revealed that both compounds induced recruitment of Peroxisome-Proliferator-Activated-Receptor gamma (PPARγ) and RNA Polymerase-II at the Retinoic-X-Receptor binding region on Beclin-1 promoter. Moreover, both compounds enhanced autophagosome formation, evaluated by LC-3 and monodansylcadaverine labeling, that was prevented by the PPARγ antagonist GW9662, addressing the direct involvement of PPARγ. Noteworthy, long-term treatment with DHADA and EPADA caused the blockade of autophagic flux followed by apoptotic cell death as evidenced by PARP cleavage and DNA fragmentation in all breast cancer cells. CONCLUSIONS We have provided new insights into the molecular mechanism through which PPARγ, as a central molecule in the cross talk between autophagy and apoptosis, mediates DHADA- and EPADA-induced cell death in breast cancer cells. GENERAL SIGNIFICANCE Our findings suggest that omega-3 DHADA- and EPADA activation of PPARγ may assume biological relevance in setting novel adjuvant therapeutic interventions in breast carcinoma.

Ligand-activated PPARγ downregulates CXCR4 gene expression through a novel identified PPAR response element and inhibits breast cancer progression

Stromal Derived Factor-1α (SDF-1α) and its cognate receptor CXCR4 play a key role in mediating breast cancer cell invasion and metastasis. Therefore, drugs able to inhibit CXCR4 activation may add critical tools to reduce tumor progression, especially in the most aggressive form of the breast cancer disease. Peroxisome Proliferator-Activated Receptor (PPAR) γ, a member of the nuclear receptor superfamily, has been found to downregulate CXCR4 gene expression in different cancer cells, however the molecular mechanism underlying this effect is not fully understood. Here, we identified a novel PPARγ-mediated mechanism that negatively regulates CXCR4 expression in both epithelial and stromal breast cancer cells. We found that ligand-activated PPARγ downregulated CXCR4 transcriptional activity through the recruitment of the silencing mediator of retinoid and thyroid hormone receptor (SMRT) corepressor onto a newly identified PPAR response element (PPRE) within the CXCR4 promoter in breast cancer cell lines. As a consequence, the PPARγ agonist rosiglitazone (BRL) significantly inhibited cell migration and invasion and this effect was PPARγ-mediated, since it was reversed in the presence of the PPARγ antagonist GW9662. According to the ability of cancer-associated fibroblasts (CAFs), the most abundant component of breast cancer stroma, to secrete high levels of SDF-1α, BRL reduced migratory promoting activities induced by conditioned media (CM) derived from CAFs and affected CXCR4 downstream signaling pathways activated by CAF-CM. In addition, CAFs exposed to BRL showed a decreased expression of CXCR4, a reduced motility and invasion along with a phenotype characterized by an altered morphology. Collectively, our findings provide novel insights into the role of PPARγ in inhibiting breast cancer progression and further highlight the utility of PPARγ ligands for future therapies aimed at targeting both cancer and surrounding stromal cells in breast cancer patients.

Benzofuran-2-acetic ester derivatives induce apoptosis in breast cancer cells by upregulating p21 Cip/WAF1 gene expression in p53-independent manner

Breast cancer is the most common malignancy and the leading cause of cancer-related death in women worldwide. High toxicity of used chemotherapeutics and resistance of cancer cells to treatments are a driving force for searching the new drug candidates for breast cancer therapy. In this study, we tested the antiproliferative effects of a series of benzofuran-2-acetic methyl ester derivatives, synthesized by a palladium-catalyzed carbonylative heterocyclization approach, on breast cancer cells. We observed that benzofuran compounds bearing a phenyl or tert-butyl substituent α to the methoxycarbonyl group significantly inhibited anchorage-dependent and -independent cell growth, and induced G0/G1 cell cycle arrest in human estrogen receptor alpha positive (MCF-7 and T47D) and in triple negative MDA-MB-231 breast cancer cells, without affecting growth of MCF-10A normal breast epithelial cells. Mechanistically, benzofuran derivatives enhanced the cyclin-dependent kinase inhibitor p21Cip/WAF1 expression at both mRNA and protein levels and this occurs transcriptionally in an Sp1-dependent manner. Moreover, benzofuran derivatives induced apoptosis, increased poly (ADP-ribose) polymerase cleavage and Bax/Bcl-2 ratio along with a marked DNA fragmentation along with a marked DNA fragmentation and a strong increase in TUNEL-positive breast cancer cells. Overall, we provide evidence that the newly tested benzofuran derivatives showed antiproliferative and pro-apoptotic activities against breast cancer cells regardless estrogen receptor status, suggesting their possible clinical development as anticancer agents.