Ewa Witort

Natural compounds for cancer treatment and prevention.

We describe here the main natural compounds used in cancer therapy and prevention, the historical aspects of their application and pharmacognosy. Two major applications of these compounds are described: as cancer therapeutics and as chemopreventive compounds. Both natural compounds, extracted from plants or animals or produced by microbes (antibiotics), and synthetic compounds, derived from natural prototype structures, are being used. We also focus on the molecular aspects of interactions with their recognized cellular targets, from DNA to microtubules. Some critical aspects of current cancer chemotherapy are also discussed, focusing on genetics and genomics, and the recent revolutionary theory of cancer: aneuploidy as the primum movens of cancer.

Autologous lipofilling: coenzyme Q10 can rescue adipocytes from stress-induced apoptotic death.

Background: Autologous fat transplantation (or lipofilling) is an excellent technique for correction of cosmetic defects. The success of the procedure relies strongly on the techniques of harvesting and transferring viable adipocytes. The purpose of this study was to evaluate effects of two harvesting methods and coenzyme Q10 on the viability and apoptotic death of adipocytes collected for autologous lipofilling. Methods: Human adipose tissue from six patients was collected by Luer-Lok syringe according to Coleman’s technique or by means of an aspirator with a 680-mmHg vacuum. Half of each sample collected using Coleman’s technique was treated with 10 &mgr;M Coenzyme Q10, and the other half served as untreated control. Viability and apoptosis were assessed by immunoenzymatic, biochemical, and morphological methods. Results: The harvesting of adipose tissue by aspirator reduced the viability and increased apoptotic death significantly more than harvesting tissue using Coleman’s technique. Biochemical and morphological analyses confirmed that treatment of adipose tissue with coenzyme Q10 reduced and even inhibited apoptotic death of harvested adipocytes. Conclusion: Coenzyme Q10 can rescue adipocytes from stress-induced apoptotic death.

Overexpression of the 18 kDa and 22/24 kDa FGF-2 isoforms results in differential drug resistance and amplification potential.

We investigated the role of low molecular weight (LMW) and high molecular weight (HMW) isoforms of basic fibroblast growth factor 2 (FGF‐2) in the expression of transformation‐related phenotypic alterations, drug sensitivity modulation, and gene amplification potential. For this purpose, we used NIH 3T3 and A31 cells transfected with different cDNA FGF‐2 constructs allowing expression of the different proteins. Both cell lines showed marked phenotypic alterations when expressing the LMW FGF‐2 or the four HMW FGF‐2 isoforms: they acquired a transformed morphology, grew at higher saturation densities in 10% serum, and exhibited anchorage‐independent growth and increased invasive potential. However, HMW FGF‐2‐expressing cells also grew in 1% serum and their invasive potential was lower than in cells expressing all FGF‐2 forms or LMW FGF‐2 alone. We have grown the different cell lines under a selective pressure of N‐(phosphonacetyl)‐l‐aspartate (PALA), a drug which specifically inhibits the aspartate transcarbamylase activity of the multifunctional carbamyl‐P‐synthetase/aspartate transcarbamylase/dihydro‐orotase genes (CAD) enzyme (and thus inhibits de novo pyrimidine biosynthesis) and selects for cells with amplified copies of the CAD gene. Our results demonstrate that aberrant expression of the LMW FGF‐2 and/or HMW FGF‐2 isoforms differently modulates drug resistance and gene amplification properties in the NIH 3T3 and A31 cell lines by differential amplification of the CAD gene. Coexpression of all isoforms appears to be necessary to obtain cumulative effects and nuclear‐targeted HMW FGF‐2 has a pivotal role in such a cooperation. J. Cell. Physiol. 193: 64–72, 2002.

Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified, synthetic ribozyme

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.

Coenzyme Q10 instilled as eye drops on the cornea reaches the retina and protects retinal layers from apoptosis in a mouse model of kainate-induced retinal damage.

PURPOSE To evaluate if coenzyme Q10 (CoQ10) can protect retinal ganglion cells (RGCs) from apoptosis and, when instilled as eye drops on the cornea, if it can reach the retina and exert its antiapoptotic activity in this area in a mouse model of kainate (KA)-induced retinal damage. METHODS Rat primary or cultured RGCs were subjected to glutamate (50 μM) or chemical hypoxia (Antimycin A, 200 μM) or serum withdrawal (FBS, 0.5%) in the presence or absence of CoQ10 (10 μM). Cell viability was evaluated by light microscopy and fluorescence-activated cell sorting analyses. Apoptosis was evaluated by caspase 3/7 activity and mitochondrion depolarization tetramethylrhodamine ethyl ester analysis. CoQ10 transfer to the retina following its instillation as eye drops on the cornea was quantified by HPLC. Retinal protection by CoQ10 (10 μM) eye drops instilled on the cornea was then evaluated in a mouse model of KA-induced excitotoxic retinal cell apoptosis by cleaved caspase 3 immunohistofluorescence, caspase 3/7 activity assays, and quantification of inhibition of RGC loss. RESULTS CoQ10 significantly increased viable cells by preventing RGC apoptosis. Furthermore, when topically applied as eye drops to the cornea, it reached the retina, thus substantially increasing local CoQ10 concentration and protecting retinal layers from apoptosis. CONCLUSIONS The ability of CoQ10 eye drops to protect retinal cells from apoptosis in the mouse model of KA-induced retinal damage suggests that topical CoQ10 may be evaluated in designing therapies for treating apoptosis-driven retinopathies.

Anticancer activity of an antisense oligonucleotide targeting TRADD combined with proteasome inhibitors in chemoresistant hepatocellular carcinoma cells

Abstract Chemoresistance is a major cause of mortality of patients with advanced and metastatic hepatocellular carcinoma (HCC), the fifth most common cancer in the world. We employed a molecular approach to inhibit cell proliferation and induce apoptosis in HepG2 cells, originated from human hepatocarcinoma. TRADD gene expression was knocked down by an antisense oligonucleotide (ASO TRADD), resulting in TRADD protein decrease by 60%, coinciding with increase of apoptotic cell death of up to 30%. Combination of the ASO TRADD with the cytotoxic drugs 5-fluorouracil or paclitaxel did not improve chemosensitivity of HepG2 cells, while the combined administration of the ASO TRADD with proteasome inhibitors MG132 or ALLN inhibited cell proliferation by 80% and 93%, respectively. Taken together, these findings reveal the importance to combine proteasome inhibitors with silencing of anti-apoptotic signalling components to target HCC cells effectively and provide useful data for developing potential treatments of HCC.

ζ-crystallin is a bcl-2 mRNA binding protein involved in bcl-2 overexpression in T-cell acute lymphocytic leukemia

The human antiapoptotic bcl‐2 gene has been discovered in t(14;18) B‐cell leukemias/lymphomas because of its overexpression caused at a transcriptional control level by the bcl‐2/IgH fusion gene. We were the first to disclose the post‐transcriptional control of bcl‐2 expression mediated by interactions of an adenine + uracil (AU)‐rich element (ARE) in the 3′‐UTR of bcl‐2 mRNA with AU‐binding proteins (AUBPs). Here, we identify and characterize ζ‐crystallin as a new bcl‐2 AUBP, whose silencing or overexpression has impact on bcl‐2 mRNA stability. An increased Bcl‐2 level observed in normal phytohemagglutinin (PHA)‐activated T lymphocytes, acute lymphatic leukemia (ALL) T‐cell lines, and T cells of patients with leukemia in comparison with normal non‐PHA‐activated T lymphocytes was concomitant with an increase in ζ‐crystallin level. The specific association of ζ‐crystallin with the bcl‐2 ARE was significantly enhanced in T cells of patients with ALL, which accounts for the higher stability of bcl‐2 mRNA and suggests a possible contribution of ζ‐crystallin to bcl‐2 overexpression occurring in this leukemia.—Lapucci, A., Lulli, M., Amedei, A, Papucci, L., Witort, E., Di Gesualdo, F., Bertolini, F., Brewer, G., Nicolin, A., Bevilacqua, A., Schiavone, N., Morello, D., Donnini, M., Capaccioli, S. ζ‐Crystallin is a bcl‐2 mRNA binding protein involved in bcl‐2 overexpression in T‐cell acute lymphocytic leukemia. FASEB J. 24, 1852–1865 (2010). www.fasebj.org

Coenzyme Q10 protects retinal cells from apoptosis induced by radiation in vitro and in vivo

The key pathogenetic event of many retinopathies is apoptosis of retinal cells. Our previous studies have demonstrated that Coenzyme Q10 (CoQ10) prevents apoptosis of corneal keratocytes both in vitro and in vivo, by virtue of its ability to inhibit mitochondrial depolarization, independently of its free radical scavenger role. The aim of this study was to evaluate whether CoQ10 can protect cultured retinal cells and the retinas of rats from radiation-induced apoptosis, if instilled as eye drops in the cornea. In vitro experiments were carried out on cultured ARPE-19 or RGC-5 cells pretreated with CoQ10 before eliciting apoptosis by UV- and γ-radiation, chemical hypoxia (Antimycin A) and serum starvation. Cell viability was evaluated by light microscopy and fluorescence activated cell sorting analysis. Apoptotic events were scored by time-lapse videomicroscopy. Mitochondrial permeability transition was evaluated by JC-1. The anti-apoptotic effectiveness of CoQ10 in retina was also evaluated by an in situ end-labeling assay in Wistar albino rats treated with CoQ10 eye drops prior to UV irradiation of the eye. CoQ10 substantially increased cell viability and lowered retinal cell apoptosis in response both to UV- and γ-radiation and to chemical hypoxia or serum starvation by inhibiting mitochondrion depolarization. In the rat, CoQ10, even when applied as eye drops on the cornea, protected all retina layers from UVR-induced apoptosis. The ability of CoQ10 to protect retinal cells from radiation-induced apoptosis following its instillation on the cornea suggests the possibility for CoQ10 eye drops to become a future therapeutic countermeasure for radiation-induced retinal lesions.

CD63 Tetraspanin Is a Negative Driver of Epithelial-to-Mesenchymal Transition in Human Melanoma Cells

The CD63 tetraspanin is highly expressed in the early stages of melanoma and decreases in advanced lesions, suggesting it as a possible suppressor of tumor progression. We employed loss- and gain-of-gene-function approaches to investigate the role of CD63 in melanoma progression and acquisition of the epithelial-to-mesenchymal transition (EMT) program. We used two human melanoma cell lines derived from primary tumors and one primary human melanoma cell line isolated from a cutaneous metastasis, differing by levels of CD63 expression. CD63-silenced melanoma cells showed enhanced motility and invasiveness with downregulation of E-cadherin and upregulation of N-cadherin and Snail. In parallel experiments, transient and stable ectopic expression of CD63 resulted in a robust reduction of cell motility, invasiveness, and protease activities, which was proportional to the increase in CD63 protein level. Transfected cells overexpressing the highest level of CD63 when transplanted into immunodeficient mice showed a reduced incidence and rate of tumor growth. Moreover, these cells showed a reduction of N-cadherin, Vimentin, Zeb1, and a-SMA, and a significant resistance to undergo an EMT program both in basal condition and in the following stimulation with TGFβ. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

Ocular Safety of Infliximab in Rabbit and Cell Culture Models

PURPOSE To undertake the safety testing of infliximab in animal and cell culture models. METHODS Sixteen New Zealand albino rabbits were divided into 4 groups of 4 animals. Each group received 2 mg of infliximab in the right eye and saline solution in the left eye. Clinical examination and retinal histology were performed at months 1, 2, and 3 for groups A, B, and C, respectively. Electroretinography (ERG) recordings were made before the injection, and at months 1 and 2 for group D. The assessment of the safety of infliximab in retinal pigment epithelial (RPE) and retinal ganglion (RGC-5) cells was performed by Western blot analysis of caspase-3 involved in apoptosis pathway, the analysis of cytotoxicity of infliximab by WST-1 proliferation assay, and time-lapse video-microscopy to register the morphology of treated cells in time-lapse. RESULTS Clinical examination of the eyes, histological study of the retina, and ERG recordings showed no sign of ocular toxicity after a single intravitreal injection of 2 mg of infliximab in the rabbit model. RPE and RGC viability was not reduced either in the range of concentration (up to 3 mg) or during the follow-up interval. CONCLUSION This study suggests that infliximab has no direct retinal toxicity using rabbit (2 mg) and cell culture (2 and 3 mg) models.