Showing papers by "Luca Vanella published in 2013"

Increased heme-oxygenase 1 expression in mesenchymal stem cell-derived adipocytes decreases differentiation and lipid accumulation via upregulation of the canonical Wnt signaling cascade

Abstract: Heme oxygenase (HO), a major cytoprotective enzyme, attenuates oxidative stress and obesity. The canonical Wnt signaling cascade plays a pivotal role in the regulation of adipogenesis. The present study examined the interplay between HO-1and the Wnt canonical pathway in the modulation of adipogenesis in mesenchymal stem cell (MSC)-derived adipocytes. To verify the role of HO-1 in generating small healthy adipocytes, cobalt protoporphyrin (CoPP), inducer of HO-1, was used during adipocyte differentiation. Lipid accumulation was measured by Oil red O staining and lipid droplet size was measured by BODIPY staining. During adipogenesis in vitro, differentiating pre-adipocytes display transient increases in the expression of genes involved in canonical Wnt signaling cascade. Increased levels of HO-1 expression and HO activity resulted in elevated levels of β-catenin, pGSK3β, Wnt10b, Pref-1, and shh along with increased levels of adiponectin (P < 0.05). In addition, induction of HO-1 resulted in a reduction in C/EBPα, PPARγ, Peg-1/Mest, aP2, CD36 expression and lipid accumulation (P < 0.05). Suppression of HO-1 gene by siRNA decreased Wnt10b, pGSK3β and β-catenin expression, and increased lipid accumulation. The canonical Wnt responsive genes, IL-8 and SFRP1, were upregulated by CoPP and their expression was decreased by the concurrent administration of tin mesoporphyrin (SnMP), an inhibitor of HO activity. Furthermore, knockdown of Wnt10b gene expression by using siRNA showed increased lipid accumulation, and this effect was not decreased by concurrent treatment with CoPP. Also our results show that blocking the Wnt 10b antagonist, Dickkopf 1 (Dkk-1), by siRNA decreased lipid accumulation and this effect was further enhanced by concurrent administration of CoPP. This is the first study to demonstrate that HO-1 acts upstream of canonical Wnt signaling cascade and decreases lipogenesis and adipocyte differentiation suggesting that the HO-1 mediated increase in Wnt10b can modulate the adipocyte phenotype by regulating the transcriptional factors that play a role in adipogenesis. This is evidenced by a decrease in lipid accumulation and inflammatory cytokine levels, increased adiponectin levels and elevation of the expression of genes of the canonical Wnt signaling cascade.

Nuclear translocation of heme oxygenase-1 confers resistance to imatinib in chronic myeloid leukemia cells.

Abstract: Identification of imatinib mesylate as a potent inhibitor of the Abl kinase and the subsequent findings that this compound displays growth inhibitory and pro-apoptotic effects in Bcr-Abl+ cells, has deeply conditioned CML treatment. Unfortunately the initial striking efficacy of this drug has been overshadowed by the development of clinical resistance. A wide variety of molecular mechanisms can underlie such resistance mechanisms. In the recent years, heme oxygenase-1 (HO-1) expression has been reported as an important protective endogenous mechanism against physical, chemical and biological stress and this cytoprotective role has already been demonstrated for several solid tumors and acute leukemias. The aim of the present study was to investigate the effect of HO-1 expression on cell proliferation and apoptosis in chronic myeloid leukemia cells, K562 and LAMA-84 cell lines following imatinib treatment. Cells were incubated for 24h with Imatinib (1 μM) alone or in combination with Hemin (10μM), an inducer of HO-1. In addition, cells were also treated with HO byproducts, bilirubin and carbon monoxide (CO), or with a protease inhibitor (Ed64) to inhibit HO-1 nuclear translocation. Pharmacological induction of HO-1 was able to overcome the effect of imatinib. The cytoprotective effect of HO-1 was further confirmed after silencing HO-1 by siRNA. Interestingly, neither bilirubin nor CO was able to protect cells from Imatinib-induced toxicity. By contrast, the protective effect of HO-1 was mitigated by the addition of E64d, preventing HO-1 nuclear translocation. Finally, imatinib was able to increase the formation of cellular reactive oxygen species (ROS) and this effect was reversed by HO-1 induction or the addition of N-acetylcisteine (NAC). In conclusion, the protective effect of HO-1 on imatinib-induced cytotoxicity does not involve its enzymatic byproducts, but rather the nuclear translocation of HO-1 following proteolytic cleavage.

Effects of Ellagic Acid on Angiogenic Factors in Prostate Cancer Cells

Abstract: Background: Several natural antioxidants, including ellagic acid (EA), have been reported to have chemotherapeutic activity in vivo and in vitro settings. Cytochrome P450 (CYP) activity and synthesis of both epoxyeicosatrienoic acids (EETs) and 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), together with vascular endothelial growth factor (VEGF) and heme oxygenase system (HO) have emerged as important modulators of tumor growth and metastasis. Methods: The anti-angiogenic effects of EA were investigated in the human prostatic cancer cell line LnCap. HO-1, HO-2, CYP2J2 and soluble epoxyde hydrolase (sEH) expressions were evaluated by western blotting. Levels of VEGF and osteoprotegerin (OPG) were determined in the culture supernatant using an ELISA assay, while CYP mRNAs were determined by qRT-PCR. Results: EA treatment induced a significant decrease (p < 0.05) in HO-1, HO-2 and CYP2J2 expression, and in VEGF and OPG levels. Similarly CYP2J2, CYP4F2 and CYPA22 mRNAs were significantly (p < 0.05) down-regulated by EA treatment. The decrease in CYP2J2 mRNA was associated with an increase in sEH expression. Conclusions: Results reported in the present study highlighted the ability of EA to modulate a new pathway, in addition to anti-proliferative and pro-differentiation properties, via a mechanism that involves a decrease in eicosanoid synthesis and a down-regulation of the HO system in prostate cancer.

Apoptotic markers in a prostate cancer cell line: effect of ellagic acid.

Abstract: Ellagic acid (EA) inhibits cell growth and induces apoptosis in cultured cells; however, the precise molecular mechanism involved in EA-induced apoptosis in prostate cancer cells is unknown. The aim of the present study was to delineate possible apoptotic pathway(s) involved in the EA-mediated chemotherapeutic effects in the LNCaP human prostatic cancer cell line. EA produced anti-proliferative effects through inhibition of rapamycin (mTOR) activation and a reduction in intracellular levels of β-catenin. Moreover, we demonstrated that EA induced apoptosis via downregulation of the anti-apoptotic proteins, silent information regulator 1 (SIRT1), human antigen R (HuR) and heme oxygenase-1 (HO-1). EA modulated the expression of apoptosis-inducing factor (AIF) resulting in a significant increase in reactive oxygen species (ROS) levels and the activation of caspase-3. Finally, we demonstrated that EA reduced both transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) levels. EA treatment resulted in the increased expression of the tumor suppressor protein p21 and increased the percentage of apoptotic cells. In conclusion, the results suggest that EA treatment represents a new and highly effective strategy in reducing prostate cancer carcinogenesis.

Ellagic acid: cytodifferentiating and antiproliferative effects in human prostatic cancer cell lines.

Abstract: Background: Recently, increasing attention has been given to neuroendocrine differentiation (NED) of Prostate Cancer and its diagnostic, prognostic and therapeutic potential. During multistep carcinogenesis, cytodifferentiation of malignant/premalignant cells into more mature or normal-like cells, has become an attractive modality of treatment and promises to be a less toxic and a more specific targeting strategy than conventional chemotherapy. In this study we investigated the capacity of a polyphenol, ellagic acid (EA), to induce differentiation of two prostate cancer cell lines: LnCap and DU145. Methods: NED markers, Chromogranin A (CgA) and p75NGFR levels were evaluated by immunocytochemistry. DNA methyltransferase- 1 (DNMT-1) and phospho-Rb (p-Rb) expression were evaluated by western blotting. Akt activation was evaluated by ELISA. Finally the ability of EA to induce DNA damage in cancer cells was examined using the COMET assay. Results: Treatment with EA significantly reduced CgA levels and increased p75NGFR expression. Moreover p-Rb, DNMT-1 levels and Akt activation/phosphorylation were decreased. EA treatment induced, in a dose-dependent manner, a marked increase in DNA damage, both in LnCap and DU145 cell lines. Conclusions: The results of this study demonstrate that EA treatment represents a new approach and highly effective strategy in reducing carcinogenesis. Therefore, EA may be considered in a promising new class of cancer therapeutic agent, with both antiproliferative and pro-differentiation properties.

Antioxidant Activity of Extracts of Momordica Foetida Schumach. et Thonn.

Abstract: Momordica foetida Schumach. et Thonn. (Cucurbitaceae) is a perennial climbing herb with tendrils, found in swampy areas in Central Uganda. Antidiabetic and antilipogenic activities were reported for some Momordica species, however the mechanism of action is still unknown. Oxidative stress may represent an important pathogenic mechanism in obesity-associated metabolic syndrome. The present study evaluated free radical scavenging capacity of different concentrations of aqueous, methanolic and dichloromethane leaf extracts of Momordica foetida Schumach. et Thonn. and the ability of these extracts to inhibit in vitro plasma lipid peroxidation; in addition, healthy human adipose mesenchymal stem cell cultures were used in order to test the hypothesis that these extracts may affect adipocyte differentiation. Results obtained in this study suggested that aqueous extract might be useful in preventing metabolic syndrome.