Alma Boninsegna

Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, characterized by degeneration of the anterior horn cells of the spinal cord. SMA presents with a highly variable phenotype ranging from very severe to mild (type I–III). No cure for SMA is available at present. All forms of SMA are caused by homozygous loss of the functional survival motor neuron (SMN1) gene. However, all patients have one or more copies of the SMN2 gene, nearly identical to SMN1. Both genes encode the SMN protein but the level produced by SMN2 is insufficient to protect from disease. Increasing SMN2 gene expression could be of considerable therapeutic importance. The aim of this study was to assess whether SMN2 gene expression can be increased by 4-phenylbutyrate (PBA). Fibroblast cell cultures from 16 SMA patients affected by different clinical severities were treated with PBA, and full-length SMN2 transcripts were measured by real-time PCR. In all cell cultures, except one, PBA treatment caused an increase in full-length SMN2 transcripts, ranging from 50 to 160% in type I and from 80 to 400% in type II and III cultures. PBA was found also effective in enhancing SMN protein levels and the number of SMN-containing nuclear structures (gems). These data show that SMN expression is considerably increased by PBA, and suggest that the compound, owing also to its favorable pharmacological properties, could be a good candidate for the treatment of SMA.

Lycopene induces cell growth inhibition by altering mevalonate pathway and Ras signaling in cancer cell lines.

Several evidences suggest that cancer cells have abnormal cholesterol biosynthetic pathways and prenylation of small guanosine triphosphatase proteins. Tomato lycopene has been suggested to have beneficial effects against certain types of cancer, including that of prostate, although the exact molecular mechanism(s) is unknown. We tested the hypothesis that lycopene may exert its antitumor effects through changes in mevalonate pathway and in Ras activation. Incubation of the Ras-activated prostatic carcinoma LNCaP cells with a 24 h lycopene treatment (2.5-10 μM) dose dependently reduced intracellular total cholesterol by decreasing 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase expression and by inactivating Ras, as evidenced by its translocation from cell membranes to cytosol. Concomitantly, lycopene reduced the Ras-dependent activation of nuclear factor-kappaB (NF-κB). Such a reduction was parallel to an inhibition of reactive oxygen species production and to a decrease in the phosphorylation ofc-jun N-terminal kinase, extracellular signal-regulated kinase 1/2 and p38. These effects were also accompanied by an arrest of cell cycle progression and by apoptosis induction, as evidenced by a decrease in cyclin D1 and phospho-AKT levels and by an increase in p21, p27 and p53 levels and in Bax:Bcl-2 ratio. The addition of mevalonate prevented the growth-inhibitory effects of lycopene as well as its increase in Ras cytoplasmatic accumulation and the subsequent changes in NF-κB. The ability of lycopene in inhibiting HMG-CoA reductase expression and cell growth and in inactivating Ras was also found in prostate PC-3, colon HCT-116 and HT-29 and lung BEN cancer cells. These findings provide a novel mechanistic insight into the growth-inhibitory effects of lycopene in cancer.

Growth-inhibitory effects of the astaxanthin-rich alga Haematococcus pluvialis in human colon cancer cells.

The growth-inhibitory effects of the astaxanthin-rich Haematococcus pluvialis were studied in HCT-116 colon cancer cells. H. pluvialis extract (5-25 microg/ml) inhibited cell growth in a dose- and time-dependent manner, by arresting cell cycle progression and by promoting apoptosis. At 25 microg/ml of H. pluvialis extract, an increase of p53, p21(WAF-1/CIP-1) and p27 expression (220%, 160%, 250%, respectively) was observed, concomitantly with a decrease of cyclin D1 expression (58%) and AKT phosphorylation (21%). Moreover, the extract, at the same concentration, strongly up-regulated apoptosis by modifying the ratio of Bax/Bcl-2 and Bcl-XL, and increased the phosphorylation of p38, JNK, and ERK1/2 by 160%, 242%, 280%, respectively. Growth-inhibitory effects by H. pluvialis were also observed in HT-29, LS-174, WiDr, SW-480 cells. This study suggests that H. pluvialis may protect from colon cancer.

Lycopene prevents 7-ketocholesterol-induced oxidative stress, cell cycle arrest and apoptosis in human macrophages.

The present study was undertaken to examine whether lycopene is able to counteract 7-ketocholesterol (7-KC)-induced oxidative stress and apoptosis in human macrophages. Human THP-1 macrophages were exposed to 7-KC (10-25 microM) alone and in combination with lycopene (0.5-2 microM), and we monitored changes in cell oxidative status [reactive oxygen species (ROS) production, NOX-4, hsp70 and hsp90 expressions, 8-OHdG formation] and in cell proliferation and apoptosis. After 24 h of treatment, lycopene significantly reduced the increase in ROS production and in 8-OHdG formation induced by the oxysterol in a dose-dependent manner. Moreover, the carotenoid strongly prevented the increase of NOX-4, hsp70 and hsp90 expressions as well as the phosphorylation of the redox-sensitive p38, JNK and ERK1/2 induced by the oxysterol. The attenuation of 7-KC-induced oxidative stress by lycopene coincided with a normalization of cell growth in human macrophages. Lycopene prevented the arrest in G0/G1 phase of cell cycle induced by the oxysterol and counteracted the increased expression of p53 and p21. Concomitantly, it inhibited 7-KC-induced apoptosis, by limiting caspase-3 activation and the modulatory effects of 7-KC on AKT, Bcl-2, Bcl-xL and Bax. Comparing the effects of lycopene, beta-carotene and (5Z)-lycopene on ROS production, cell growth and apoptosis show that lycopene and its isomer were more effective than beta-carotene in counteracting the dangerous effects of 7-KC in human macrophages. Our study suggests that lycopene may act as a potential antiatherogenic agent by preventing 7-KC-induced oxidative stress and apoptosis in human macrophages.

Regulation of cell-cycle progression and apoptosis by beta carotene in undifferentiated and differentiated HL-60 leukemia cells: possible involvement of a redox mechanism.

Although epidemiologic studies have demonstrated that a high intake of vegetables containing β‐carotene lowers the risk of cancer, recent intervention studies have revealed that β‐carotene supplementation to smokers resulted in a high incidence of lung cancer. We hypothesized that β‐carotene may act as a pro‐ or anticancerogenic agent by modulating pathways involved in cell growth and that such a modulation may involve a redox mechanism. To test this hypothesis, cell proliferation, apoptosis and redox status were evaluated in undifferentiated and dimethylsulfoxide‐differentiated HL‐60 cells exposed to β‐carotene. The carotenoid modified cell cycle progression and induced apoptosis in a dose‐dependent manner. These effects were more remarkable in undifferentiated cells than in differentiated cells. In accord with these findings, in undifferentiated cells, β‐carotene was more effective in decreasing cyclin A and Bcl‐2 expression and in increasing p21 and p27 expression. Neither Bcl‐xL nor Bax expression were significantly modified by the carotenoid. From a mechanistic point of view, the delay in cell growth by β‐carotene was highly coincident with the increased intracellular reactive oxygen species production and oxidized glutathione content induced by the carotenoid. Moreover, α‐tocopherol minimized the effects of β‐carotene on cell growth. These data provide evidence that β‐carotene modulates molecular pathways involved in cell cycle progression and apoptosis and support the hypothesis that a redox mechanism may be implicated. They also suggest that differentiated cells may be less susceptible to the carotenoid than highly neoplastic undifferentiated cells.

Docosahexaenoic acid induces apoptosis in lung cancer cells by increasing MKP-1 and down-regulating p-ERK1/2 and p-p38 expression

Different agents able to modulate apoptosis have been shown to modify the expression of the MAP-kinase-phosphatase-1 (MKP-1). The expression of this phosphatase has been considered a potential positive prognostic factor in lung cancer, and smoke was shown to reduce the levels of MKP-1 in ferret lung. Our aim was to assess whether the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), known to inhibit the growth of several cancer cells mainly inducing apoptosis, may exert pro-apoptotic effect in lung cancer cells by modifying MKP-1 expression. We observed that DHA increased MKP-1 protein and mRNA expression and induced apoptosis in different lung cancer cell lines (mink Mv1Lu adenocarcinoma cells, human A549 adenocarcinoma and human BEN squamous carcinoma cells). We inhibited the pro-apoptotic effect of DHA by treating the cells with the phosphatase inhibitor Na3VO4 or by silencing the MKP-1 gene with the specific siRNA. This finding demonstrated that the induction of apoptosis by DHA involved a phosphatase activity, specifically that of MKP-1. DHA reduced also the levels of the phosphorylated MAP-kinases, especially ERK1/2 and p38. Such an effect was not observed when the MKP-1 gene was silenced. Altogether, the data provide evidence that the DHA-induced overexpression of MKP-1 and the resulting decrease of MAP-kinase phosphorylation by DHA may underlie the pro-apoptotic effect of this fatty acid in lung cancer cells. Moreover, they support the hypothesis that DHA may exert chemopreventive action in lung cancer.

Targeted inhibition of the epidermal growth factor receptor‐tyrosine kinase by ZD1839 (‘Iressa’) induces cell‐cycle arrest and inhibits proliferation in prostate cancer cells

The epidermal growth factor (EGF) plays a role in the development of prostate cancer, which becomes essential after androgen resistance has emerged. The EGF receptor (EGFR) is therefore a potential target for anticancer therapy. We evaluated the effects of ZD1839 (‘Iressa’), an orally active EGFR—tyrosine kinase inhibitor, on prostate cancer cell lines. The effects of ZD1839 were evaluated on the anchorage dependent and independent growth of androgen‐responsive (LNCaP) and androgen‐independent (DU145 and PC3) cells by a cell proliferation assay, cell counting, and soft agar analysis. Flow cytometric analysis and Western blotting were used to assess the effects on the cell‐cycle and on protein expression levels, respectively. ZD1839 caused a dose‐ and time‐dependent growth inhibition in all three cell lines. A dose‐dependent supra‐additive increase in growth inhibition was observed when ZD1839 was combined with the antiandrogen flutamide or ionizing radiation (IR). The antiproliferative effect of ZD1839 was mainly cytostatic and associated with a block in the G0/G1 phase of the cell‐cycle, evident after about 12 h of treatment. In the DU145 cells this block was associated with an increase in expression of the CDK inhibitor p27Kip1, both in the cytoplasmic and nuclear fractions. The increase in p27Kip1 was not evident in the LNCaP and PC3 cells. No changes were observed in the expression of cyclin D1 protein. These results demonstrate the antiproliferative effects of ZD1839 on the growth of prostate cancer cells and suggest that inhibition of EGFR‐associated signal transduction pathway might represent a promising novel therapeutic strategy for the treatment of prostate cancer. J. Cell. Physiol. 201: 97–105, 2004.

Dystroglycan expression is reduced during prostate tumorigenesis and is regulated by androgens in prostate cancer cells

Prostate cancer, the most frequently diagnosed cancer in Western men, can display a high variability in term of clinical aggressiveness and prognosis and none of the available markers is able to accurately predict its clinical course. Dystroglycan (DG), a non‐integrin adhesion molecule, is a complex formed by two subunits, α‐ and β‐DG, which bind to extracellular matrix molecules and cytoskeleton, respectively. DG expression is frequently reduced in human cancers and has been related to tumor grade and aggressiveness. This study investigated the role of DG in human prostate tumorigenesis and its suitability as a prognostic marker. The expression level of extracellular α‐DG subunit was frequently reduced in human prostate cancer cell lines and primary tumors and the percentage of positive tumor cells was significantly further decreased in vivo following androgen ablation therapy (median = 1%) compared to pre‐treatment samples (median = 28%). A significant relationship was observed between α‐DG staining on the post‐treatment samples and tumor recurrence. A dose‐ and time‐dependent decrease of DG expression also occurred in human prostate cancer cells following treatment with the anti‐androgen flutamide. Stable expression of an exogenous DG cDNA in the LNCaP human prostate carcinoma cell line resulted in a marked inhibition of both anchorage‐dependent and independent growth and of the in vivo tumorigenicity. These findings confirm and extend previous evidence that disturbances in the function of the DG complex might contribute to the definition of the malignant behavior of prostate cancer cells and suggest that androgens might regulate DG expression in these cells. J. Cell. Physiol. 213: 528–539, 2007.

Increased expression of geminin stimulates the growth of mammary epithelial cells and is a frequent event in human tumors

Geminin is a potent inhibitor of origin assembly and re‐replication in multicellular eukaryotes and is a negative regulator of DNA replication during the cell cycle. Thus, it was proposed as an inhibitor of cell proliferation and as a potential tumor suppressor gene. However, the protein was found specifically expressed in proliferating lymphocytes and epithelial cells and up‐regulated in several malignancies. Therefore, geminin is now regarded as an oncogene but its role in tumor development remains unknown. In this study, we evaluated by Western blot analysis the expression of geminin in a series of human cancer cell lines of various histogenetic origin and in a series of human primary colon, rectal, and breast cancers. Expression of geminin was variable in different cell lines and not related to the expression level of the corresponding mRNA. Moreover, geminin was expressed at higher level in 56% and 58% of colon and rectal cancers, respectively, compared with the corresponding adjacent normal mucosa. A high expression of geminin was also detected by immunohistochemistry in 60% of human primary breast cancers. We also transfected a full‐length geminin cDNA in a human non‐tumorigenic and a cancer breast cell lines and obtained derivatives expressing high levels of the protein. Geminin overexpression stimulated cell cycle progression and proliferation in both normal and cancer cells and increased the anchorage—independent growth of breast cancer cells. These results demonstrate that expression of geminin is frequently deregulated in tumor cells and might play an important role in the regulation of cell growth in both normal and malignant cells.

Post-translational modulation of CD133 expression during sodium butyrate-induced differentiation of HT29 human colon cancer cells: implications for its detection.

The CD133 molecule has been proposed as a surface marker of cancer stem cells in several human malignancies, including colon cancers. The function and the mechanisms regulating CD133 expression remain unknown. The HT29 human colon cancer cells undergo differentiation following treatment with various agents and represent a useful in vitro model of colon differentiation. This study evaluated the behavior of CD133 during sodium butyrate‐induced differentiation of HT29 cells. Treatment with sodium butyrate induced a progressive decrease of CD133 expression, as assessed by flow cytometry using the AC133 monoclonal antibody. Indeed, expression of CD133, which was about 47% in untreated control cells, gradually decreased down to about 3% after 72 h in a time‐ and dose‐dependent manner. No relationship was observed between CD133 protein evaluated by flow cytometry and mRNA expression level, and no changes were detected in the methylation status of the CD133 gene promoter during HT29 differentiation. Moreover, the expression of the CD133 protein, evaluated by Western blot analysis using a specific anti‐CD133 antibody directed against the C‐terminal intracytoplasmic region of human CD133 protein, did not correlate with flow cytometry results. Different results were also obtained using the two antibodies to analyze the expression of the CD133 molecule in human colon cancers. These findings demonstrate that membrane expression of the CD133 stem cell marker might undergo a complex regulation during differentiation of colon cells and suggest that HT29 cells are a useful in vitro model to study the mechanisms involved in this regulation which likely occurs at a post‐transcriptional level. J. Cell. Physiol. 224:234–241, 2010