Roberta Piva

In vitro antiproliferative effects on human tumor cell lines of extracts from the Bangladeshi medicinal plant Aegle marmelos Correa

In the present paper we show that extracts from Aegle marmelos Correa are able to inhibit the in vitro proliferation of human tumor cell lines, including the leukemic K562, T-lymphoid Jurkat, B-lymphoid Raji, erythroleukemic HEL, melanoma Colo38, and breast cancer MCF7 and MDA-MB-231 cell lines. Molecules present within the studied Aegle marmelos C. extracts were identified by gas-chromatography/mass-spectrometry analysis; three derivatives (butyl p-tolyl sulfide, 6-methyl-4-chromanone and butylated hydroxyanisole) were found to exhibit strong activity in inhibiting in vitro cell growth of human K562 cells. The antiproliferative activity of these compounds was found to be comparable to that of known antitumor agents, including cisplatin, chromomycin, cytosine arabinoside and 5-fluorouracil. In addition, the antiproliferative activity of butyl-p-tolyl sulfide, 6-methyl-4-chromanone and 5-methoxypsolaren was associated to activation of the differentiation pattern of K562 cells.

From microRNA functions to microRNA therapeutics: Novel targets and novel drugs in breast cancer research and treatment (Review)

MicroRNAs (miRNAs or miRs) are a family of small non-coding RNAs that regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation, depending on the degree of complementarity with target mRNA sequences. miRNAs play a crucial role in cancer. In the case of breast tumors, several studies have demonstrated a correlation between: i) the expression profile of oncogenic miRNAs (oncomiRs) or tumor suppressor miRNAs and ii) the tumorigenic potential of triple-negative [estrogen receptor (ER), progesterone receptor (PR) and Her2/neu] primary breast cancers. Among the miRNAs involved in breast cancer, miR-221 plays a crucial role for the following reasons: i) miR-221 is significantly overexpressed in triple-negative primary breast cancers; ii) the oncosuppressor p27 Kip1 , a validated miR-221 target, is downregulated in aggressive cancer cell lines; and iii) the upregulation of a key transcription factor, Slug, appears to be crucial, since it binds to the miR-221/miR-222 promoter and is responsible for the high expression of the miR-221/miR-222 cluster in breast cancer cells. A Slug/miR-221 network has been suggested, linking miR-221 activity with the downregulation of a Slug repressor, leading to Slug/miR-221 upregulation and p27 Kip1 downregulation. Interference with this process can be achieved using antisense miRNA (antagomiR) molecules targeting miR-221, inducing the down-regulation of Slug and the upregulation of p27 Kip1 .

Deficiency of polycystin-2 reduces Ca2+ channel activity and cell proliferation in ADPKD lymphoblastoid cells

Polycystin‐2 (PC2), encoded by the PKD2 gene, mutated in 10‐15% of autosomal‐dominant polycystic kidney disease (ADPKD) patients, is a Ca2+‐permeable cation channel present in kidney epithelia and other tissues. As PC2 was found expressed in B‐lymphoblastoid cells (LCLs) and Ca2+ signaling pathways are important regulators of B cell function activities, we investigated whether PC2 plays some role in B‐LCLs. In LCLs, PC2 was found mainly in ER membranes but ~8 times less than in kidney HEK293 cells. The same reductions were found in PKD2 and PKD1 RNA; thus, PKD genes maintained, in LCLs, the same reciprocal proportion as they do in kidney cells. In LCLs obtained from subjects carrying PKD2 mutations (PKD2‐LCLs) and showing reduced PC2 levels, intracellular Ca2+ concentrations evoked by platelet‐activating factor (PAF), were significantly lower than in non‐PKD‐LCLs. This reduction was also found in PKD1‐LCLs but without PC2 reductions. Likewise, cell proliferation, which is controlled by Ca2+, was reduced in PKD2‐ and PKD1‐LCLs. Moreover, in LCLs with PKD2 nonsense mutations, aminoglycoside antibiotics reduced the PC2 defect by promoting readthrough of stop codons. Therefore, PC2 and PC1 are functionally expressed in LCLs, which provide a model, easily obtainable from ADPKD patients, to study PKD gene expression and function.

Decoy oligodeoxynucleotides targeting NF-kappaB transcription factors: induction of apoptosis in human primary osteoclasts

Proteins belonging to the nuclear factor kappaB (NF-kappaB) superfamily are involved in osteoclast formation, playing a very important role for both differentiation of osteoclast precursors and survival of mature osteoclasts. Several drugs used to fight bone loss in a variety of human pathologies, including osteoporosis, act by increasing the frequency of osteoclast apoptosis, since it was demonstrated that small changes in osteoclast apoptosis can result in large changes in bone formation. In this respect, targeting of NF-kappaB transcription factor could be of great interest. Among nonviral gene therapy strategies recently proposed to inhibit or even block NF-kappaB activity, the transcription factor decoy (TFD) should be taken in great consideration. The main issue of the present study was to examine the effects of decoy DNA/DNA molecules targeting NF-kappaB on apoptosis of human osteoclasts (OCs), with the aim to interfere with the pathway regulating osteoclast differentiation and programmed cell death. To this aim, we used a mixture of receptor activator of NF-kappaB ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and parathyroid hormone (PTH) to prepare human OCs from peripheral blood cells. Then, transfection with the decoy molecules targeting NF-kappaB was performed. The results obtained demonstrate that in primary cells expressing typical osteoclast markers such as TRAP and MMP9, NF-kappaB decoy significantly stimulated apoptosis. Inhibition of IL-6 expression and induction of Caspase 3 were found in OCs treated with NF-kappaB DNA/DNA decoys. We consider these data as the basis for setting up experimental conditions allowing nonviral gene therapy of several bone disorders.

Induction of apoptosis of human primary osteoclasts treated with extracts from the medicinal plant Emblica officinalis

BackgroundOsteoclasts (OCs) are involved in rheumatoid arthritis and in several pathologies associated with bone loss. Recent results support the concept that some medicinal plants and derived natural products are of great interest for developing therapeutic strategies against bone disorders, including rheumatoid arthritis and osteoporosis. In this study we determined whether extracts of Emblica officinalis fruits display activity of possible interest for the treatment of rheumatoid arthritis and osteoporosis by activating programmed cell death of human primary osteoclasts.MethodsThe effects of extracts from Emblica officinalis on differentiation and survival of human primary OCs cultures obtained from peripheral blood were determined by tartrate-acid resistant acid phosphatase (TRAP)-positivity and colorimetric MTT assay. The effects of Emblica officinalis extracts on induction of OCs apoptosis were studied using TUNEL and immunocytochemical analysis of FAS receptor expression. Finally, in vitro effects of Emblica officinalis extracts on NF-kB transcription factor activity were determined by gel shift experiments.ResultsExtracts of Emblica officinalis were able to induce programmed cell death of mature OCs, without altering, at the concentrations employed in our study, the process of osteoclastogenesis. Emblica officinalis increased the expression levels of Fas, a critical member of the apoptotic pathway. Gel shift experiments demonstrated that Emblica officinalis extracts act by interfering with NF-kB activity, a transcription factor involved in osteoclast biology. The data obtained demonstrate that Emblica officinalis extracts selectively compete with the binding of transcription factor NF-kB to its specific target DNA sequences. This effect might explain the observed effects of Emblica officinalis on the expression levels of interleukin-6, a NF-kB specific target gene.ConclusionInduction of apoptosis of osteoclasts could be an important strategy both in interfering with rheumatoid arthritis complications of the bone skeleton leading to joint destruction, and preventing and reducing osteoporosis. Accordingly, we suggest the application of Emblica officinalis extracts as an alternative tool for therapy applied to bone diseases.

Correlation between Slug transcription factor and miR-221 in MDA-MB-231 breast cancer cells

BackgroundBreast cancer and its metastatic progression is mainly directed by epithelial to mesenchymal transition (EMT), a phenomenon supported by specific transcription factors and miRNAs.MethodsIn order to investigate a possible correlation between Slug transcription factor and miR-221, we performed Slug gene silencing in MDA-MB-231 breast cancer cells and evaluated the expression of genes involved in supporting the breast cancer phenotype, using qRT-PCR and Western blot analysis. Chromatin immunoprecipitation and wound healing assays were employed to determine a functional link between these two molecules.ResultsWe showed that Slug silencing significantly decreased the level of miR-221 and vimentin, reactivated Estrogen Receptor α and increased E-cadherin and TRPS1 expression. We demonstrated that miR-221 is a Slug target gene, and identified a specific region of miR-221 promoter that is transcriptionally active and binds the transcription factor Slug “in vivo”. In addition, we showed that in Slug-silenced cells, wich retained residual miR-221 (about 38%), cell migration was strongly inhibited. Cell migration was inhibited, but to a less degree, following complete knockdown of miR-221 expression by transfection with antagomiR-221.ConclusionsWe report for the first time evidence of a correlation between Slug transcription factor and miR-221 in breast cancer cells. These studies suggest that miR-221 expression is, in part, dependent on Slug in breast cancer cells, and that Slug plays a more important role than miR-221 in cell migration and invasion.

Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO‐PLEX technology

We have used cytokine protein array to analyze the secretion of cytokines from an osteoblastic clone derived from human umbilical cord blood mesenchymal stem cells (MSCs) cultured in an osteogenic differentiation medium. The analysis demonstrated the unexpected ability of osteoblast committed cells and their early progenitors to produce significant amounts of a range of soluble immune mediators without in vitro exposure to clinically relevant bacterial pathogens. The cells were expanded and their osteogenic potential analyzed over 45 days of culture was revealed by the expression of osteoblast‐specific markers (alkaline phosphatase and Runx2), and by matrix mineralization. Over this culture period, the cells secreted particularly high levels of IL‐8, MCP‐1 and VEGF, but did not express IL‐2, IL‐7, IL‐17, eotaxin, G‐CSF and IFN‐γ. These findings should encourage the use of human umbilical cord blood as a potential stem cells source for bone regeneration.

Estrogen induced increase of estrogen receptor RNA in human breast cancer cells

Estrogen Receptor (ER) RNA has been studied in ER positive and ER negative cells, grown in alpha medium and fetal calf serum (FCS), with and without estrogen addition. ER mRNA was detected only in ER positive cells. When cells were grown without estrogen addition, a progressive and transient increase of ER RNA levels was observed. When cells were cultured with estradiol, the ER RNA increase took place early and ER RNA levels remained 2 times higher than in untreated cells. Therefore, a down-regulation of ER RNA is not apparent in neoplastic breast cells. We hypothesize that the increase of ER expression is caused by stabilization of ER transcripts, due to the estrogen induced ER cytoplasmic depletion.

Slug gene expression supports human osteoblast maturation

This study aims to define the function of Slug transcription factor in human normal osteoblasts (hOBs). To date, Slug is considered exclusively a marker of malignancy in bone tissue. Here, we identified, for the first time, a role for Slug in hOBs using a knockdown approach. We demonstrated that Slug is positively correlated with osteoblast markers, including Runx2, osteopontin, osteocalcin, Collagen type 1, Wnt/β-catenin signaling mediators, and mineral deposition. At the same time, Slug silencing potentiates the expression of Sox-9, a factor indispensable for chondrogenic development. These data, with the finding that Slug is in vivo recruited by the promoters of Runx2 and Sox-9 genes, suggest that, in hOBs, Slug may act both as positive and negative transcriptional regulator of Runx2 and Sox-9 genes, respectively. In summary, our results support the hypothesis that Slug functions as a novel regulator of osteoblast activity and may be considered a new factor required for osteoblast maturation.

Induction of apoptosis of human primary osteoclasts treated with a transcription factor decoy mimicking a promoter region of estrogen receptor α

In this paper we investigated how the increase of human estrogen receptor alfa (ERα) gene expression may affect breast, osteoblast and osteoclast cells. Increase of ERα expression was obtained by interfering with the activity of a negative transcription factor and by removing it with a short and powerful decoy oligonucleotide (RA4-3′) mimicking a region of distal promoter C of ERα gene. We provide evidence that this decoy was able to induce apoptosis in osteoclasts, but not in osteoblasts and in breast cancer cells, in an estrogen dependent manner. This effect was associated with increase of the levels of Caspase 3 and Fas receptor. Since ERα is important in the transcription of different genes and is involved in several pathological processes, including neoplastic and osteopenic diseases, our findings may be of relevance for a possible new therapeutical approach of such diseases.