Effect of space flight on the behavior of human retinal pigment epithelial ARPE-19 cells and evaluation of coenzyme Q10 treatment.

Astronauts on board the International Space Station (ISS) are exposed to the damaging effects of microgravity and cosmic radiation. One of the most critical and sensitive districts of an organism is the eye, particularly the retina, and > 50% of astronauts develop a complex of alterations designated as spaceflight-associated neuro-ocular syndrome. However, the pathogenesis of this condition is not clearly understood. In the current study, we aimed to explore the cellular and molecular effects induced in the human retinal pigment ARPE-19 cell line by their transfer to and 3-day stay on board the ISS in the context of an experiment funded by the Agenzia Spaziale Italiana. Treatment of cells on board the ISS with the well-known bioenergetic, antioxidant, and antiapoptotic coenzyme Q10 was also evaluated. In the ground control experiment, the cells were exposed to the same conditions as on the ISS, with the exception of microgravity and radiation. The transfer of ARPE-19 retinal cells to the ISS and their living on board for 3 days did not affect cell viability or apoptosis but induced cytoskeleton remodeling consisting of vimentin redistribution from the cellular boundaries to the perinuclear area, underlining the collapse of the network of intermediate vimentin filaments under unloading conditions. The morphological changes endured by ARPE-19 cells grown on board the ISS were associated with changes in the transcriptomic profile related to the cellular response to the space environment and were consistent with cell dysfunction adaptations. In addition, the results obtained from ARPE-19 cells treated with coenzyme Q10 indicated its potential to increase cell resistance to damage.

In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression.

Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases.

zeta-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 zeta-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 zeta-crystallin level. The specific association of zeta-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 zeta-crystallin to bcl-2 overexpression occurring in this leukemia.

B cell lymphoma (Bcl)-2 protein is the major determinant in bcl-2 adenine-uridine-rich element turnover overcoming HuR activity.

In the 3'-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins.

Divergent pathways for TNF and C(2)-ceramide toxicity in HTC hepatoma cells.

We previously showed that, in the rat hepatoma cell line HTC, TNF brings about a non-caspase-dependent, apoptosis-like process requiring NADPH oxidase activity, an iron-mediated pro-oxidant status, and a functional acidic vacuolar compartment. This process may thus involve mechanisms such as autophagy or relocation of lysosomal enzymes, perhaps secondary to the formation of ceramide by acidic sphingomyelinase. Here we investigated whether ceramide formation contributes to the apoptogenic process. HTC cells were found to be sensitive to exogenous ceramide and significantly protected against TNF by desipramine, an inhibitor of lysosomal acid sphingomyelinase. However, Bcl-2 transfection and Bcl-x(L) upregulation by dexamethasone significantly diminished the apoptogenic effect of ceramide but not that of TNF, suggesting that ceramide is not directly involved in TNF toxicity. Moreover, Bcl-x(L) silencing precluded dexamethasone-induced protection against ceramide and, by itself, induced massive death, demonstrating the strict dependence of HTC cells on Bcl-x(L) for survival also under standard culture conditions.

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.

Cannabinoid receptor activation induces apoptosis through tumor necrosis factor alpha-mediated ceramide de novo synthesis in colon cancer cells.

PURPOSE: Cannabinoids have been recently proposed as a new family of potential antitumor agents. The present study was undertaken to investigate the expression of the two cannabinoid receptors, CB1 and CB2, in colorectal cancer and to provide new insight into the molecular pathways underlying the apoptotic activity induced by their activation. EXPERIMENTAL DESIGN: Cannabinoid receptor expression was investigated in both human cancer specimens and in the DLD-1 and HT29 colon cancer cell lines. The effects of the CB1 agonist arachinodyl-2'-chloroethylamide and the CB2 agonist N-cyclopentyl-7-methyl-1-(2-morpholin-4-ylethyl)-1,8-naphthyridin-4(1H)-on-3-carboxamide (CB13) on tumor cell apoptosis and ceramide and tumor necrosis factor (TNF)-alpha production were evaluated. The knockdown of TNF-alpha mRNA was obtained with the use of selective small interfering RNA. RESULTS: We show that the CB1 receptor was mainly expressed in human normal colonic epithelium whereas tumor tissue was strongly positive for the CB2 receptor. The activation of the CB1 and, more efficiently, of the CB2 receptors induced apoptosis and increased ceramide levels in the DLD-1 and HT29 cells. Apoptosis was prevented by the pharmacologic inhibition of ceramide de novo synthesis. The CB2 agonist CB13 also reduced the growth of DLD-1 cells in a mouse model of colon cancer. The knockdown of TNF-alpha mRNA abrogated the ceramide increase and, therefore, the apoptotic effect induced by cannabinoid receptor activation. CONCLUSIONS: The present study shows that either CB1 or CB2 receptor activation induces apoptosis through ceramide de novo synthesis in colon cancer cells. Our data unveiled, for the first time, that TNF-alpha acts as a link between cannabinoid receptor activation and ceramide production.

Impact of targeting the adenine- and uracil-rich element of bcl-2 mRNA with oligoribonucleotides on apoptosis, cell cycle, and neuronal differentiation in SHSY-5Y cells.

We have identified previously a destabilizing adenine- and uracil-rich element (ARE) in the 3'-UTR of bcl-2 mRNA that interacted with ARE-binding proteins to down-regulate bcl-2 gene expression in response to apoptotic stimuli. We have also described three contiguous 2'-O-methyl oligoribonucleotides (ORNs) in both sense and antisense orientation with respect to the bcl-2 ARE that are able to regulate the bcl-2 mRNA half-life and Bcl-2 protein level in two different cell lines. Here we show that treatment of neuronal cell line (SHSY-5Y) with antisense ORNs targeting the bcl-2 ARE (bcl-2 ARE asORNs) prevents bcl-2 down-regulation in response to apoptotic stimuli with glucose/growth factor starvation (Locke medium) or oxygen deprivation and enhances the apoptotic threshold as evaluated by time-lapse videomicroscopy, fluorescence-activated cell sorting analysis, and caspase-3 activation. Additional effects of bcl-2 ARE asORNs included inhibition of cell cycle entry and a marked increase of cellular neurite number and length, a hallmark of neuronal differentiation resulting from bcl-2 up-regulation. The ability of bcl-2 ARE asORNs to enhance the apoptotic threshold and to induce neuronal differentiation implies their potential application as a novel informational tool to protect cells from ischemic damage and to prevent neuronal degeneration.

Inhibition of 5-lipoxygenase by MK886 augments the antitumor activity of celecoxib in human colon cancer cells.

Cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) are key enzymes involved in arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in colorectal tumor development. We aimed at evaluating whether combined blocking of the COX-2 and 5-LOX pathways might have additive antitumor effects in colorectal cancer. The expression/activity of COX-2 and 5-LOX were assessed in 24 human colorectal cancer specimens. The effects of the COX-2 inhibitor celecoxib and the 5-LOX inhibitor MK886 on prostaglandin E(2) and cysteinyl leukotriene production, tumor cell proliferation, cell apoptosis, and Bcl-2/Bax expression were evaluated in the Caco-2 and HT29 colon cancer cells. We also investigated the effect of the enzymatic inhibition on mitochondrial membrane depolarization, one of the most important mechanisms involved in ceramide-induced apoptosis. Up-regulation of the COX-2 and 5-LOX pathways was found in the tumor tissue in comparison with normal colon mucosa. Inhibition of either COX-2 or 5-LOX alone resulted in activation of the other pathway in colon cancer cells. Combined treatment with 10 micromol/L celecoxib and MK886 could prevent this activation and had additive effects on inhibiting tumor cell proliferation, inducing cell apoptosis, decreasing Bcl-2 expression, increasing Bax expression, and determining mitochondrial depolarization in comparison with treatment with either inhibitor alone. The administration of the ceramide synthase inhibitor fumonisin B1 could prevent some of these antineoplastic effects. In conclusion, our study showed that inhibition of 5-LOX by MK886 could augment the antitumor activity of celecoxib in human colorectal cancer.

The role of cyclooxygenase-2 in mediating the effects of histamine on cell proliferation and vascular endothelial growth factor production in colorectal cancer.

PURPOSE: Activity of histidine decarboxylase, the key enzyme in the synthesis of histamine, has been shown to be increased in several types of human tumors. We attempted to establish whether the possible involvement of histidine decarboxylase and histamine in colorectal carcinogenesis might be mediated by the activation of the cyclooxygenase-2 (COX-2) pathway. EXPERIMENTAL DESIGN: Expression/activity of histidine decarboxylase, histamine content, and prostaglandin E2 (PGE2) production were analyzed in 33 colorectal cancer samples and in the HT29, Caco-2, and HCT116 colon cancer cell lines. The effects of histamine, celecoxib, and H1, H2, and H4 receptor antagonists on COX-2 expression/activity, cell proliferation, and vascular endothelial growth factor (VEGF) production were assessed in the three colon cancer lines that showed different constitutive COX-2 expression. RESULTS: We showed the up-regulation of histidine decarboxylase protein expression and activity in the tumor specimens when compared with normal colonic mucosa. Histidine decarboxylase activity and histamine content were also significantly higher in metastatic tumors than in nonmetastatic ones. These variables significantly correlated with tumor PGE(2) production. The administration of histamine increased COX-2 expression/activity, cell proliferation, and VEGF production in the COX-2-positive HT29 and Caco-2 cells. Treatment with either H2/H4 receptor antagonists or celecoxib prevented these effects. Histamine had no effect on both the COX-2 pathway and VEGF production in the COX-2-negative HCT116 cells. CONCLUSIONS: Our data showed that histamine exerts both a proproliferative and a proangiogenic effect via H2/H4 receptor activation. These effects are likely to be mediated by increasing COX-2-related PGE2 production in COX-2-expressing colon cancer cells.

Albumin Cys34 adducted by acrolein as a marker of oxidative stress in ischemia-reperfusion injury during hepatectomy.

The aim of this study was to measure and identify the reactive carbonyl species (RCSs) released in the blood of humans subjected to hepatic resection. Pre-anesthesia malondialdehyde (MDA) plasma content (0.36 ± 0.11 nmol/mg protein) remained almost unchanged immediately after anaesthesia, before clamping and at the 10th min after ischemia, while markedly increased (to 0.59 ± 0.07 nmol/mg; p < 0.01, Tukey's post test) at the 10th min of reperfusion. A similar trend was observed for the protein carbonyls (PCs), whose pre-anesthesia levels (0.17 ± 0.13 nmol/mg) did not significantly change during ischemia, while increased more than fourfold at the 10th min of reperfusion (0.75 ± 0.17 nmol/mg; p < 0.01, Tukey's post test). RCSs were then identified as covalent adducts to the albumin Cys34, which we previously found as the most reactive protein nucleophilic site in plasma. By using a mass spectrometry (MS) approach based on precursor ion scanning, we found that acrolein (ACR) is the main RCS adducted to albumin Cys34. In basal conditions, the adducted albumin was 0.6 ± 0.4% of the native form but it increased by almost fourfold at the 10th min of reperfusion (2.3 ± 0.7%; p < 0.01, t-test analysis). Since RCSs are damaging molecules, we propose that RCSs, and ACR in particular, are new targets for novel molecular treatments aimed at reducing the ischemia/reperfusion damage by the use of RCS sequestering agents.

LMW-PTP is a positive regulator of tumor onset and growth.

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are an enzyme family that plays a key role in cell proliferation control by dephosphorylating/inactivating both tyrosine kinase receptors (such as PDGF, insulin, and ephrin receptors) and docking proteins (such, as beta-catenin) endowed with both adhesion and transcriptional activity. Besides being a frequent event in human tumors, overexpression of LMW-PTP has been recently demonstrated to be sufficient to induce neoplastic transformation. We recently demonstrated that overexpression of LMW-PTP strongly potentiates the stability of cell-cell contacts at the adherens junction level, which powerfully suggests that LMW-PTP may also contribute to cancer invasivity. Focusing on mechanisms by which LMW-PTP is involved in cancer onset and progression, the emerging picture is that LMW-PTP strongly increases fibronectin-mediated cell adhesion and mobility but, paradoxically, decreases cell proliferation. Nevertheless, LMW-PTP-transfected NIH3T3 fibroblasts engrafted in nude mice induce the onset of larger fibrosarcomas, which are endowed with higher proliferation activity as compared to mock-transfected controls. Quite opposite effects have been obtained with engrafted fibroblasts transfected with a dominant-negative form of LMW-PTP. Notably, in sarcoma extracts, LMW-PTP overexpression greatly influences the ephrin A2 (EphA2) but not PDGF receptor or beta-catenin tyrosine phosphorylation. The high association of dephosphorylated EphA2 overexpression with most human cancers and our observation that cell growth stimulation by LMW-PTP overexpression is restricted to the in vivo model, strongly suggest that LMW-PTP oncogenic potential is mediated by its EphA2 tyrosine dephosphorylating activity.

Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt.

The serine/threonine kinase mTOR, the major sensor of cell growth along the PI3K/Akt pathway, can be activated by agents acting on microtubules. Damaged microtubules induce phosphorylation of the Bcl-2 protein and lower the threshold of programmed cell death, both of which are inhibited by rapamycin. In HEK293 cells expressing Akt mutants, the level of Bcl-2 phosphorylation and the threshold of apoptosis induced by taxol or by nocodazole are significantly modified. In cells expressing dominant-negative Akt (DN-Akt), Bcl-2 phosphorylation and p70S6KThr421/Ser424 phosphorylation induced by taxol or nocodazole were significantly enhanced as compared to cells expressing constitutively active Akt (CA-Akt) and inhibited by rapamycin. Moreover, DN-Akt cells were more sensitive to antitubule agents than CA-Akt cells. In nocodazole-treated HEK293 cells sorted according to cell cycle, the p70S6KThr421/Ser424 phosphorylation was associated to the G2/M fraction. More relevant, nocodazole inhibited, in a dose-response manner, mTOR phosphorylation at Ser2448. This activity, potentiated in DN-Akt cells, was not detectable in CA-Akt cells. Our results suggest that death signals originating from damaged microtubules in G2/M can compete with G1 survival pathways at the level of mTOR. These findings have implications for cancer therapy and drug resistance.

Molecular characterization of established human colon carcinoma cell lines (HCT-8) made resistant to 5-fluorouracil by different selection schedules.

To provide some insight into molecular mechanisms of 5 fluorouracil (5-FU) clinical resistance in colorectal cancer, we hypothesized that different in vitro exposure schedules of human colorectal cancer cell lines mimicking clinical infusion or bolus regimens could lead to differential gene expression. Resistant HCT-8 colon cancer cell lines (HCT-8/FUI/15R and HCT-8/FUB/2R) were selected from parental sensitive HCT-8 cells by long-term and short-term exposure schedules, respectively. Expression levels of the 437 genes evaluated by the Atlas Select cDNA Expression Human Tumor Array were not substantially different between HCT-8/FUB/2R and HCT-8 cell lines except for three genes downregulated in the resistant subline. Several genes were differentially expressed in HCT-8/FUI/15R cells compared to the parental cell line: 43 genes, including three chemoresistance-related genes, were upregulated, and three genes were downregulated. HCT-8/FUB/2R cells were substantially more resistant to 5-FU in comparison to HCT-8/FUI/15R cells after both 4- and 72-h exposures. No substantial differences were observed among resistant and parental cells in sensitivity to SN-38, the active metabolite of irinotecan, and oxaliplatin. Analysis of the mRNA levels of thymidylate synthase, thymidine phosphorylase, and bcl-2 genes evaluated by reverse transcription and real time PCR (RT-PCR) assay showed comparable results in resistant sublines and sensitive parental cells, whereas expression of the dihydropyrimidine dehydrogenase gene was markedly increased in both resistant cell lines compared to parental cells.

In vitro study on the safety of near infrared laser therapy in its potential application as postmastectomy lymphedema treatment.

Clinical studies demonstrated the effectiveness of laser therapy in the management of postmastectomy lymphedema, a discomforting disease that can arise after surgery/radiotherapy and gets progressively worse and chronic. However, safety issues restrict the possibility to treat cancer patients with laser therapy, since the effects of laser radiation on cancer cell behavior are not completely known and the possibility of activating postmastectomy residual cancer cells must be considered. This paper reports the results of an in vitro study aimed to investigate the effect of a class IV, dual-wavelength (808 nm and 905 nm), NIR laser system on the behavior of two human breast adenocarcinoma cell lines (namely, MCF7 and MDA-MB361 cell lines), using human dermal fibroblasts as normal control. Cell viability, proliferation, apoptosis, cell cycle and ability to form colonies were analyzed in order to perform a cell-based safety testing of the laser treatment in view of its potential application in the management of postmastectomy lymphedema. The results showed that, limited to the laser source, treatment conditions and experimental models used, laser radiation did not significantly affect the behavior of human breast adenocarcinoma cells, including their clonogenic efficiency. Although these results do not show any significant laser-induced modification of cancer cell behavior, further studies are needed to assess the possibility of safely applying NIR laser therapy for the management of postmastectomy lymphedema.

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.

Concomitant effect of topical ubiquinone Q10 and vitamin E to prevent keratocyte apoptosis after excimer laser photoablation in rabbits.

PURPOSE: To investigate in vivo whether ubiquinone Q10 together with vitamin E protects rabbit corneas from keratocyte apoptosis after excimer laser irradiation. METHODS: Photorefractive keratectomy (PRK) was performed in both eyes of three New Zealand white rabbits. During 3 days before surgery, each right eye received four-times-daily instillation of an eye-drop solution containing ubiquinone Q10 0.20% and vitamin E 0.04%; each left eye was treated with a solution that did not contain ubiquinone or vitamin E. The central cornea was analyzed after surgery using the in situ end labelling (ISEL) technique of nicked DNA to detect DNA fragmentation. To determine the number of ISEL positive nuclei, an average of 70 random microscopic fields (five for each de-epithelialized tissue section) of 138,000 mu2 were examined in the right and left cornea samples at 250X by two different observers. RESULTS: Light microscopic examination of the sections from corneas treated before PRK showed that cells committed to apoptosis by PRK were about 50% compared to those of untreated controls. CONCLUSION: Treatment of rabbit eyes before PRK with ubiquinone Q10 lowered the number of apoptotic events.

A pathophysiological view of the long non-coding RNA world.

Because cells are constantly exposed to micro-environmental changes, they require the ability to adapt to maintain a dynamic equilibrium. Proteins are considered critical for the regulation of gene expression, which is a fundamental process in determining the cellular responses to stimuli. Recently, revolutionary findings in RNA research and the advent of high-throughput genomic technologies have revealed a pervasive transcription of the human genome, which generates many long non-coding RNAs (lncRNAs) whose roles are largely undefined. However, there is evidence that lncRNAs are involved in several cellular physiological processes such as adaptation to stresses, cell differentiation, maintenance of pluripotency and apoptosis. The correct balance of lncRNA levels is crucial for the maintenance of cellular equilibrium, and the dysregulation of lncRNA expression is linked to many disorders; certain transcripts are useful prognostic markers for some of these pathologies. This review revisits the classic concept of cellular homeostasis from the perspective of lncRNAs specifically to understand how this novel class of molecules contributes to cellular balance and how its dysregulated expression can lead to the onset of pathologies such as cancer.

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 TGFß. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

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

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.