Rossella Farra

Bortezomib arrests the proliferation of hepatocellular carcinoma cells HepG2 and JHH6 by differentially affecting E2F1, p21 and p27 levels.

Despite the broad anti-tumour potential of the proteasome inhibitor bortezomib, partial information is available with regard to its effects on hepatocellular carcinoma (HCC) cells. Here we studied the effects of bortezomib on two human HCC cell lines displaying a different phenotype, hepatocyte-like for HepG2 and undifferentiated for JHH6. Bortezomib induced a dose- and time-dependent increase in cell toxicity and decrease of cell viability, with JHH6 being less sensitive than HepG2. Moreover, a differential influence on major cell cycle regulatory genes was responsible for the observed decrease of S and increase of G(2)-M phase cells. In HepG2, bortezomib induced a post-transcriptional increase of cyclin E1 together with a transcriptional-mediated decrease of the transcription factor E2F1. This in turn resulted in the reduction of the hyper-phosphorylated form of pRB and in the transcriptional down-regulation of the E2F1 targets cyclin D1, cyclin A2 and CdK2 but not cyclin E1. Up-regulation of LRH1, a liver specific cyclin E1 transcription factor, accounted for the unvaried cyclin E1 mRNA levels. Additionally, bortezomib induced both transcriptional and post-translational increase of p21(waf1/cip1) and p27(kip1). In JHH6, an overall more contained variation in cell cycle mediators was observed with the reduction of E2F1, cyclin A2, LRH1 and the increase of p21(waf1/cip1) being the most evident. In conclusion, the presented data show the mechanisms regulating cell proliferation inhibition by bortezomib in two different HCC cell lines. Despite a certain phenotype-dependent effect, the potent action exerted by bortezomib makes this drug attractive for future experimentation in animal models, possibly leading to novel treatments for HCC.

Dissecting the expression of EEF1A1/2 genes in human prostate cancer cells: the potential of EEF1A2 as a hallmark for prostate transformation and progression

Background:In prostate adenocarcinoma, the dissection of the expression behaviour of the eukaryotic elongation factors (eEF1A1/2) has not yet fully elucidated.Methods:The EEF1A1/A2 expressions were investigated by real-time PCR, western blotting (cytoplasmic and cytoskeletal/nuclear-enriched fractions) and immunofluorescence in the androgen-responsive LNCaP and the non-responsive DU-145 and PC-3 cells, displaying a low, moderate and high aggressive phenotype, respectively. Targeted experiments were also conducted in the androgen-responsive 22Rv1, a cell line marking the progression towards androgen-refractory tumour. The non-tumourigenic prostate PZHPV-7 cell line was the control.Results:Compared with PZHPV-7, cancer cells showed no major variations in EEF1A1 mRNA; eEF1A1 protein increased only in cytoskeletal/nuclear fraction. On the contrary, a significant rise of EEF1A2 mRNA and protein were found, with the highest levels detected in LNCaP. Eukaryotic elongation factor 1A2 immunostaining confirmed the western blotting results. Pilot evaluation in archive prostate tissues showed the presence of EEF1A2 mRNA in near all neoplastic and perineoplastic but not in normal samples or in benign adenoma; in contrast, EEF1A1 mRNA was everywhere detectable.Conclusion:Eukaryotic elongation factor 1A2 switch-on, observed in cultured tumour prostate cells and in human prostate tumour samples, may represent a feature of prostate cancer; in contrast, a minor involvement is assigned to EEF1A1. These observations suggest to consider EEF1A2 as a marker for prostate cell transformation and/or possibly as a hallmark of cancer progression.

Proliferation of human primary vascular smooth muscle cells depends on serum response factor.

Smooth muscle cells (SMCs) can switch between a differentiated/contractile and an alternative proliferative phenotype. The transcription factor serum response factor (SRF) has been implicated in the regulation of gene expression profiles determining both phenotypes. Whereas strong evidence exists for a role of SRF in SMC differentiation, the contribution of SRF to SMC proliferation is less well defined. For primary human vascular SMCs in particular, existing data are non-conclusive. To study SRF functions in primary human vascular SMCs, we used an siRNA approach. siRNA-mediated SRF suppression affected the expression of established SRF target genes such as smooth muscle alpha-actin (ACTA2) or SM22alpha (TAGLN) and decreased both F-actin formation and cell migration. Furthermore, SRF knockdown caused a cell-cycle arrest in G1 associated with reduced hyperphosphorylated pRB, cyclin A and SKP2 levels, and increased p27(kip1) (CDKN1B) protein levels. SRF-depleted cells expressed senescence-associated beta-galactosidase indicating an irreversible G1 arrest. siRNA-mediated suppression of SKP2 triggered senescence to a similar extent as SRF depletion, indicating that SRF knockdown-induced senescence may be dependent on a decrease in SKP2. Thus, SRF is an essential regulator of primary human vascular SMC proliferation and senescence. Interfering with SRF function may therefore be a promising strategy for the treatment of hyperproliferative SMC disorders such as atherosclerosis and in-stent restenosis.

Role of E2F1-cyclin E1-cyclin E2 circuit in human coronary smooth muscle cell proliferation and therapeutic potential of its downregulation by siRNAs.

Aberrant coronary vascular smooth muscle cell (CSMC) proliferation is a pivotal event underlying intimal hyperplasia, a phenomenon impairing the long-term efficacy of bypass surgery and angioplasty procedures. Consequently research has become focused on efforts to identify molecules that are able to control CSMC proliferation. We investigated downregulation of CSMC growth by small interfering RNAs (siRNAs) targeted against E2F1, cyclin E1, and cyclin E2 genes, whose contribution to CSMC proliferation is only now being recognized. Chemically synthesized siRNAs were delivered by two different transfection reagents to asynchronous and synchronous growing human CSMCs cultivated either in normo- or hyperglycemic conditions. The depletion of each of the three target genes affected the expression of the other two genes, demonstrating a close regulatory control. The clearest effects associated with the inhibition of the E2F1-cyclin E1/E2 circuit were the reduction in the phosphorylation levels of the retinoblastoma protein pRB and a decrease in the amount of cyclin A2. At the phenotypic level the downmodulation of CSMC proliferation resulted in a decrease of S phase matched by an increase of G1-G0 phase cell amounts. The antiproliferative effect was cell-donor and transfectant independent, reversible, and effective in asynchronous and synchronous growing CSMCs. Importantly, it was also evident in hyperglycemia, a condition that underlies diabetes. No significant aspecific cytotoxicity was observed. Our data demonstrate the interrelation among E2F1-cyclin E1-cyclin E2 and the pivotal role this circuit exerts in CSMC proliferation. Additionally, our work validates the concept of utilizing anti-E2F1-cyclin E1-cyclin E2 siRNAs to develop a potential novel therapy to control intimal hyperplasia.

Temperature-sensitive hydrogels

Thermo-sensitive polymers are appealing materials for several therapeutic applications, such as in regenerative medicine and in situ drug release. These macromolecules are characterized by the ability to undergo swelling/deswelling processes during temperature change-induced phase transitions. Swelling and shrinking temperatures depend on the specific physicochemical properties, namely salt concentration or pH, of the thermo-sensitive gels as well as the incubation environment. An understanding of the mechanisms underlying the gel-swelling equilibrium and kinetics is necessary for the selection of an appropriate gel in relation to the specific pharmaceutical application. Thermo-sensitive polymers used in medicine include polyacrylamides, polyvinyls, polyethers, polysaccharides, and polyphosphazenes. A few of them have been successfully used as 3-dimentional supports for cell cultivation, allowing for the production of scaffolds with excellent biologic properties for application in regenerative medicine. Stem cells that can undergo specific differentiation under the appropriate stimulation have also been cultivated. The ability of drug/polymer solutions to turn into gels at physiologic temperature has been exploited for local drug delivery. The prolonged in situ presence and slow drug release enhances the therapeutic performance of antibiotics used in urogenital pathologies, anti-inflammatory agents, and anticancer drugs. The reduced toxicity as well as lower fluctuations in peak-to-trough drug concentrations make these systems superior to traditional gels. Thermo-sensitive hydrogels have also been demonstrated to be interesting formulations for the delivery of biotechnological drugs. Proteins and oligonucleotides can be loaded under mild conditions, stabilized, and released at a controlled rate. Finally, thermo-reversible polymers have been investigated for protein conjugation to enhance the physicochemical, biologic, immunologic, and pharmacokinetic properties of biotechnological products.

Polysaccharides for the Delivery of Antitumor Drugs

Among the several delivery materials available so far, polysaccharides represent very attractive molecules as they can undergo a wide range of chemical modifications, are biocompatible, biodegradable, and have low immunogenic properties. Thus, polysaccharides can contribute to significantly overcome the limitation in the use of many types of drugs, including anti-cancer drugs. The use of conventional anti-cancer drugs is hampered by their high toxicity, mostly depending on the indiscriminate targeting of both cancer and normal cells. Additionally, for nucleic acid based drugs (NABDs), an emerging class of drugs with potential anti-cancer value, the practical use is problematic. This mostly depends on their fast degradation in biological fluids and the difficulties to cross cell membranes. Thus, for both classes of drugs, the development of optimal delivery materials is crucial. Here we discuss the possibility of using different kinds of polysaccharides, such as chitosan, hyaluronic acid, dextran, and pullulan, as smart drug delivery materials. We first describe the main features of polysaccharides, then a general overview about the aspects ruling drug release mechanisms and the pharmacokinetic are reported. Finally, notable examples of polysaccharide-based delivery of conventional anti-cancer drugs and NABDs are reported. Whereas additional research is required, the promising results obtained so far, fully justify further efforts, both in terms of economic support and investigations in the field of polysaccharides as drug delivery materials.

Improving siRNA bio-distribution and minimizing side effects.

The RNA interference (RNAi) is a biological process by which a double stranded RNA (dsRNA also called small interfering RNA - siRNA) triggers the sequence-dependent degradation of a target RNA within the cellular environment. Thus siRNAs can be used to combat the expression of deleterious gene(s) causing disease or to destroy invading pathogen RNAs. Despite their enormous therapeutic potential, the use of siRNA as drugs presents two major problems: the difficulties to identify optimal delivery systems and the possible induction of different unwanted side effects. In this review, after presenting an overview about the mechanisms ruling the process of RNAi, we focus the attention on the description of the strategies developed to optimise systemic siRNA delivery; in this sense, considerations about the attempts to improve siRNA stability in the biological environment, the development of synthetic vectors for siRNA delivery, the siRNA bio-distribution and pharmacokinetics together with the selection of siRNA targeted delivery systems, are discussed. Since in the optimisation of the siRNA delivery systems the minimization of siRNA side effects should not be neglected, in the last part of the review we consider the problems related to the possible induction of siRNA mediated side effects focusing on the so called microRNA like off-targeting.

Effects of E2F1-cyclin E1-E2 circuit down regulation in hepatocellular carcinoma cells

BACKGROUND No effective therapy is available for hepatocellular carcinoma. To identify novel therapeutic strategies, we explored the effects of the depletion of E2F1, cyclin E1-E2 whose inter-relationships in hepatocellular carcinoma cell proliferation have never been defined. METHODS siRNA-mediated depletion of the targets was studied in the hepatocellular carcinoma cells HepG2, HuH7 and JHH6 characterized by high, medium and low hepatocyte differentiation grade, respectively; a model of normal human hepatocytes was also considered. RESULTS The depletion of each target mRNA reduced the levels of the other two mRNAs, thus demonstrating a close regulatory control, also confirmed by over-expression experiments. At the protein level, an exception to this trend was observed for cyclinE1 whose amount increased upon cyclin E2 (HepG2, HuH7, JHH6) and E2F1 (HepG2) depletion. In HepG2, reduced cyclinE1 proteolysis accounted for this observation. Additionally, cyclin E1-E2-E2F1 targeting decreased the levels of cyclin A2 mRNA and of the hyper-phosphorylated form of pRb thus leading to an S-phase cell decrease; migration was impaired as well. Finally, the model of human hepatocytes used was clearly less affected by target mRNAs depletion than hepatocellular carcinoma cells. CONCLUSION Our data provide novel mutual relationships amongst cyclin E1-E2-E2F1 and indicate their role in sustaining hepatocellular carcinoma cell proliferation/migration, validating the concept of an anti-cyclin E1-E2-E2F1 therapeutic approach for hepatocellular carcinoma.

Serum response factor depletion affects the proliferation of the hepatocellular carcinoma cells HepG2 and JHH6

For hepatocellular carcinoma (HCC), a leading cause of cancer death world-wide, there is no effective therapy especially for the advanced stage of the disease. Thus, we started the investigations about a novel anti HCC approach based on the depletion of the transcription factor serum response factor (SRF) in HCC cell lines; SRF choice was based on its recently proposed contribution to HCC tissue development and on its important role in cell proliferation. SRF depletion, obtained by a siRNA (siSRF797), was studied in two HCC cell lines, i.e. HepG2 and JHH6 assigned to high and low hepatocytic differentiation grade on the base of the capacity to synthesize albumin. In the HCC cell lines examined, siSRF797 reduced both the mRNA and protein levels of SRF without inducing unspecific interferon response or cytotoxicity. Moreover, SRF depletion induced the reduction of S-phase cells and a decrease in cell number and vitality. Particularly in HepG2, cell growth impairment was paralleled by the decrease of the levels of the transcription factor E2F1 together with some of its regulated genes. In HepG2 but not in JHH6, SRF depletion was associated with apoptosis. Finally, in both HepG2 and JHH6, the combined administration of siSRF797 and bortezomib, a proteasome inhibitor whose therapeutic potential for HCC is considered attractive, further reduced cell viability compared to either siSRF797 or bortezomib treatment alone. In conclusion, SRF depletion affects the expansion of the high and low differentiation grade HCC cells HepG2 and JHH6. These results can pave the way to understand the role of SRF in HCC development and possibly to identify novel anti HCC therapeutic strategies.

Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system

Small interfering RNAs (siRNAs) have the potential to be of therapeutic value for many human diseases. So far, however, a serious obstacle to their therapeutic use is represented by the absence of appropriate delivery systems able to protect them from degradation and to allow an efficient cellular uptake. In this work we developed a siRNA delivery system based on inulin (Inu), an abundant and natural polysaccharide. Inu was functionalized via the conjugation with diethylenetriamine (DETA) residues to form the complex Inu-DETA. We studied the size, surface charge and the shape of the Inu-DETA/siRNA complexes; additionally, the cytotoxicity, the silencing efficacy and the cell uptake-mechanisms were studied in the human bronchial epithelial cells (16HBE) and in the hepatocellular carcinoma derived cells (JHH6). The results presented here indicate that Inu-DETA copolymers can effectively bind siRNAs, are highly cytocompatible and, in JHH6, can effectively deliver functional siRNAs. Optimal delivery is observed using a weight ratio Inu-DETA/siRNA of 4 that corresponds to polyplexes with an average size of 600nm and a slightly negative surface charge. Moreover, the uptake and trafficking mechanisms, mainly based on micropinocytosis and clatrin mediated endocytosis, allow the homogeneous diffusion of siRNA within the cytoplasm of JHH6. Notably, in 16 HBE where the trafficking mechanism (caveolae mediated endocytosis) does not allow an even distribution of siRNA within the cell cytoplasm, no significant siRNA activity is observed. In conclusion, we developed a novel inulin-based siRNA delivery system able to efficiently release siRNA in JHH6 with negligible cytotoxicity thus opening the way for further testing in more complex in vivo models.