Ejaculation sparing of classic and minimally invasive surgical treatments of LUTS/BPH.

BACKGROUND: The surgical landscape for Lower Urinary Tract Symptoms (LUTS) and Benign Prostatic Hyperplasia (BPH) has evolved with the introduction of Minimally Invasive Surgical Therapies (MISTs), recognizing the impact of sexual function on patients' well-being, and prioritizing ejaculation-sparing approaches. METHODS: This systematic review explored ejaculation sparing after classic endoscopic procedures and MISTs (iTind, Rezum, Urolift, Aquablation, and TPLA) and a literature search yielded 41 studies. RESULTS: While all procedures demonstrated efficacy in improving LUTS/BPH symptoms (IPSS, QoL, Qmax), a subset of studies evaluated ejaculatory function. Positive outcomes were noted, challenging the historical association of BPH surgeries with ejaculatory dysfunction. Variations in study design, patient cohorts, and limited long-term data present challenges. Notably, the lack of baseline specificity, use of alpha-blockers, and non-specific sexual function assessments underscore potential biases. CONCLUSIONS: Despite limitations, the review tentatively concluded that MISTs, including iTind, Rezum, Urolift, Aquablation, and TPLA, appear comparable in sparing ejaculation. Long-term studies are essential to validate sustainability, and comparative research should assess trade-offs between MISTs and traditional surgeries. Incorporating patient-reported outcomes and quality of life assessments will enhance future investigations, refining MISTs as standard therapeutic options for LUTS/BPH.

The Protective Role of Oleuropein Aglycone against Pesticide-Induced Toxicity in a Human Keratinocytes Cell Model.

The extensive use of agricultural pesticides to improve crop quality and yield significantly increased the risk to the public of exposure to small but repeated doses of pesticides over time through various routes, including skin, by increasing the risk of disease outbreaks. Although much work was conducted to reduce the use of pesticides in agriculture, little attention was paid to prevention, which could reduce the toxicity of pesticide exposure by reducing its impact on human health. Extra virgin olive oil (EVOO), a major component of the Mediterranean diet, exerts numerous health-promoting properties, many of which are attributed to oleuropein aglycone (OleA), the deglycosylated form of oleuropein, which is the main polyphenolic component of EVOO. In this work, three pesticides with different physicochemical and biological properties, namely oxadiazon (OXA), imidacloprid (IMID), and glyphosate (GLYPHO), were compared in terms of metabolic activity, mitochondrial function and epigenetic modulation in an in vitro cellular model of human HaCaT keratinocytes to mimic the pathway of dermal exposure. The potential protective effect of OleA against pesticide-induced cellular toxicity was then evaluated in a cell pre-treatment condition. This study showed that sub-lethal doses of OXA and IMID reduced the metabolic activity and mitochondrial functionality of HaCaT cells by inducing oxidative stress and altering intracellular calcium flux and caused epigenetic modification by reducing histone acetylation H3 and H4. GLYPHO, on the other hand, showed no evidence of cellular toxicity at the doses tested. Pretreatment of cells with OleA was able to protect cells from the damaging effects of the pesticides OXA and IMID by maintaining metabolic activity and mitochondrial function at a controlled level and preventing acetylation reduction, particularly of histone H3. In conclusion, the bioactive properties of OleA reported here could be of great pharmaceutical and health interest, as they could be further studied to design new formulations for the prevention of toxicity from exposure to pesticide use.

The CAIX inhibitor SLC-0111 exerts anti-cancer activity on gastric cancer cell lines and resensitizes resistant cells to 5-Fluorouracil, taxane-derived, and platinum-based drugs.

Gastric cancer (GC) is the fifth most frequent malignancy and the fourth leading cause of worldwide cancer-related death. Despite the usage of multimodal perioperative chemotherapy (pCT), GC progressively gains chemoresistance, thereby, the identification of suitable targets to overcome drug resistance is fundamental. Amongst the potential biomarkers, carbonic anhydrase IX (CAIX) - associated with a poor prognosis of several solid cancers - has gained the most attention. In a cohort of GC patients who received perioperative FLOT (i.e., Leucovorin, 5-Fluouracil, Docetaxel, and Oxaliplatin) or FOLFOX (i.e., Leucovorin, 5-Fluouracil, and Oxaliplatin), non-responder patients showed an increased expression of tumor CAIX compared to responder group. Moreover, GC cell lines induced to be resistant to 5-Fluouracil, Paclitaxel, Cisplatin, or the combination of 5-Fluorouracil, Oxaliplatin, and Docetaxel, overexpressed CAIX compared to the control. Accordingly, CAIX-high-expressing GC cells showed increased therapy resistance compared to low-expressing cells. Notably, SLC0111 significantly improved the therapy response of both wild-type and resistant GC cells. Overall, these data suggest a correlation between CAIX and GC drug resistance highlighting the potential of SLC-0111 in re-sensitizing GC cells to pCT.

Enhanced Vasculogenic Capacity Induced by 5-Fluorouracil Chemoresistance in a Gastric Cancer Cell Line.

Chemotherapy is still widely used as a coadjutant in gastric cancer when surgery is not possible or in presence of metastasis. During tumor evolution, gatekeeper mutations provide a selective growth advantage to a subpopulation of cancer cells that become resistant to chemotherapy. When this phenomenon happens, patients experience tumor recurrence and treatment failure. Even if many chemoresistance mechanisms are known, such as expression of ATP-binding cassette (ABC) transporters, aldehyde dehydrogenase (ALDH1) activity and activation of peculiar intracellular signaling pathways, a common and universal marker for chemoresistant cancer cells has not been identified yet. In this study we subjected the gastric cancer cell line AGS to chronic exposure of 5-fluorouracil, cisplatin or paclitaxel, thus selecting cell subpopulations showing resistance to the different drugs. Such cells showed biological changes; among them, we observed that the acquired chemoresistance to 5-fluorouracil induced an endothelial-like phenotype and increased the capacity to form vessel-like structures. We identified the upregulation of thymidine phosphorylase (TYMP), which is one of the most commonly reported mutated genes leading to 5-fluorouracil resistance, as the cause of such enhanced vasculogenic ability.

Streptomycin sulphate loaded solid lipid nanoparticles show enhanced uptake in macrophage, lower MIC in Mycobacterium and improved oral bioavailability.

Present study addresses the challenge of incorporating hydrophilic streptomycin sulphate (STRS; log P -6.4) with high dose (1 g/day) into a lipid matrix of SLNs. Cold high-pressure homogenization technique used for SLN preparation achieved 30% drug loading and 51.17 ± 0.95% entrapment efficiency. Polyethylene glycol 600 as a supporting-surfactant assigned small size (218.1 ± 15.46 nm) and mucus-penetrating property. It was conceived to administer STRS-SLNs orally rather than intramuscularly. STRS-SLNs remained stable on incubation for varying times in SGF or SIF. STRS-SLNs were extensively characterised for microscopic (TEM and AFM), thermal (DSC), diffraction (XRD) and spectroscopic (NMR and FTIR) properties and showed zero-order controlled release. Enhanced (60 times) intracellular uptake was observed in THP-1 and Pgp expressing LoVo and DLD-1 cell lines, using fluorescein-SLNs. Presence of SLNs in LoVo cells was also revealed by TEM studies. STRS-SLNs showed 3 times reduction in MIC against Mycobacterium tuberculosis H37RV (256182) in comparison to free STRS. It also showed better activity against both M. bovis BCG and Mycobacterium tuberculosis H37RV (272994) in comparison to free STRS. Cytotoxicity and acute toxicity studies (OECD 425 guidelines) confirmed in vitro and in vivo safety of STRS-SLNs. Single-dose oral pharmacokinetic studies in rat plasma using validated LCMS/MS technique or the microbioassay showed significant oral absorption and bioavailability (160% - 710% increase than free drug).

5-Fluorouracil Conversion Pathway Mutations in Gastric Cancer.

To date, 5-Fluorouracil (5FU) is a major component of several chemotherapy regimens, thus its study is of fundamental importance to better understand all the causes that may lead to chemoresistance and treatment failure. Given the evident differences between prognosis in Asian and Caucasian populations, triggered by clear genetic discordances and given the extreme genetic heterogeneity of gastric cancer (GC), the evaluation of the most frequent mutations in every single member of the 5FU conversion and activation pathway might reveal several important results. Here, we exploited the cBioPortal analysis software to query a large databank of clinical and wide-genome studies to evaluate the components of the three major 5FU transformation pathways. We demonstrated that mutations in such ways were associated with a poor prognosis and reduced overall survival, often caused by a deletion in the TYMP gene and amplification in TYMS. The use of prodrugs and dihydropyrimidine dehydrogenase (DPD) inhibitors, which normally catabolizes 5FU into inactive metabolites, improved such chemotherapies, but several steps forward still need to be taken to select better therapies to target the chemoresistant pools of cells with high anaplastic features and genomic instability.

Atorvastatin-loaded solid lipid nanoparticles as eye drops: proposed treatment option for age-related macular degeneration (AMD).

Statins, widely prescribed for cardiovascular diseases, are also being eyed for management of age-related macular degeneration (AMD). Poor bioavailability and blood-aqueous barrier may however limit significant ocular concentration of statins following oral administration. We for the first time propose and investigate local application of atorvastatin (ATS; representative statin) loaded into solid lipid nanoparticles (SLNs), as self-administrable eye drops. Insolubility, instability, and high molecular weight > 500 of ATS, and ensuring that SLNs reach posterior eye were the challenges to be met. ATS-SLNs, developed (2339/DEL/2014) using suitable components, quality-by-design (QBD) approach, and scalable hot high-pressure homogenization, were characterized and evaluated comprehensively for ocular suitability. ATS-SLNs were 8 and 12 times more bioavailable (AUC) in aqueous and vitreous humor, respectively, than free ATS. Three-tier (in vitro, ex vivo, and in vivo) ocular safety, higher corneal flux (2.5-fold), and improved stability (13.62 times) including photostability of ATS on incorporation in ATS-SLNs were established. Autoclavability and aqueous nature are the other highlights of ATS-SLNs. Presence of intact fluorescein-labeled SLNs (F-SLNs) in internal eye tissues post-in vivo application as eye drops provides direct evidence of successful delivery. Perinuclear fluorescence in ARPE-19 cells confirms the effective uptake of F-SLNs. Prolonged residence, up to 7 h, was attributed to the mucus-penetrating nature of ATS-SLNs. Graphical abstract.

MAP Kinases Pathways in Gastric Cancer.

Gastric cancer (GC) is turning out today to be one of the most important welfare issues for both Asian and European countries. Indeed, while the vast majority of the disease burden is located in China and in Pacific and East Asia, GC in European countries still account for about 100,000 deaths per year. With this review article, we aim to focus the attention on one of the most complex cellular pathways involved in GC proliferation, invasion, migration, and metastasis: the MAP kinases. Such large kinases family is to date constantly studied, since their discovery more than 30 years ago, due to the important role that it plays in the regulation of physiological and pathological processes. Interactions with other cellular proteins as well as miRNAs and lncRNAs may modulate their expression influencing the cellular biological features. Here, we summarize the most important and recent studies involving MAPK in GC. At the same time, we need to underly that, differently from cancers arising from other tissues, where MAPK pathways seems to be a gold target for anticancer therapies, GC seems to be unique in any aspect. Our aim is to review the current knowledge in MAPK pathways alterations leading to GC, including H. pylori MAPK-triggering to derail from gastric normal epithelium to GC and to encourage researches involved in MAPK signal transduction, that seems to definitely sustain GC development.

Zeta-crystallin: a moonlighting player in cancer.

Crystallins were firstly found as structural proteins of the eye lens. To this family belong proteins, such as ζ-crystallin, expressed ubiquitously, and endowed with enzyme activity. ζ-crystallin is a moonlighting protein endowed with two main different functions: (1) mRNA binding with stabilizing activity; (2) NADPH:quinone oxidoreductase. ζ-crystallin has been clearly demonstrated to stabilize mRNAs encoding proteins involved in renal glutamine catabolism during metabolic acidosis resulting in ammoniagenesis and bicarbonate ion production that concur to compensate such condition. ζ-crystallin binds also mRNAs encoding for antiapoptotic proteins, such as Bcl-2 in leukemia cells. On the other hand, the physiological role of its enzymatic activity is still elusive. Gathering research evidences and data mined from public databases, we provide a framework where all the known ζ-crystallin properties are called into question, making it a hypothetical pivotal player in cancer, allowing cells to hijack or subjugate the acidity response mechanism to increase their ability to resist oxidative stress and apoptosis, while fueling their glutamine addicted metabolism.

Update on gastric cancer treatments and gene therapies.

Gastric cancer is an active topic of clinical and basic research due to high morbidity and mortality. To date, gastrectomy and chemotherapy are the only therapeutic options for gastric cancer patients, but drug resistance, either acquired or primary, is the main cause for treatment failure. Differences in development and response to cancer treatments have been observed among ethnically diverse GC patient populations. In spite of major incidence, GC Asian patients have a significantly better prognosis and response to treatments than Caucasian ones due to genetic discordances between the two populations. Gene therapy could be an alternative strategy to overcome such issues and especially CRISPR/Cas9 represents one of the most intriguing gene-editing system. Thus, in this review article, we want to provide an update on the currently used therapies for the treatment of advanced GC. Graphical abstract.

Differential Responses of Colorectal Cancer Cell Lines to Enterococcus faecalis' Strains Isolated from Healthy Donors and Colorectal Cancer Patients.

The metabolites produced by the host's gut microbiota have an important role in the maintenance of intestinal homeostasis, but can also act as toxins and induce DNA damage in colorectal epithelial cells increasing the colorectal cancer (CRC) chance. In this scenario, the impact of some of the components of the natural human gastrointestinal microbiota, such as Enterococcus faecalis (E. faecalis), at the onset of CRC progression remains controversial. Since under dysbiotic conditions it could turn into a pathogen, the aim of this study was to compare the effect of E. faecalis' strains (isolated from CRC patients and healthy subjects' stools) on the proliferation of different colorectal cells lines. First, we isolated and genotyping characterized the Enterococcus faecalis' strains. Then, we analyzed the proliferation index (by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay) of three tumor and one normal intestinal cell lines, previously exposed to E. faecalis strains pre-cultured medium. Stool samples of CRC patients demonstrated a reduced frequency of E. faecalis compared to healthy subjects. In addition, the secreted metabolites of E. faecalis' strains, isolated from healthy donors, decreased the human ileocecal adenocarcinoma cell line HCT-8 and human colon carcinoma cell line HCT-116 cell proliferation without effects on human colorectal adenocarcinoma cell line SW620 and on normal human diploid cell line CLR-1790. Notably, the metabolites of the strains isolated from CRC patients did not influence the cell growth of CRC cell lines. Our results demonstrated a new point of view in the investigation of E. faecalis' role in CRC development, which raises awareness of the importance of not only associating the presence/absence of a unique microorganism, but also in defining the specific characteristics of the different investigated strains.

Plasmatic carbonic anhydrase IX as a diagnostic marker for clear cell renal cell carcinoma.

Carbonic anhydrase (CA, EC 4.2.1.1) IX is regarded as a tumour hypoxia marker and CA inhibitors have been proposed as a new class of antitumor agents, with one such agent in Phase II clinical trials. The expression of some CAs, in particular the isoforms CA IX and CA XII, has been correlated with tumour aggressiveness and progression in several cancers. The aim of this study was to evaluate the possibility that CA IX could represent a marker related to clear cell Renal Cell Carcinoma (ccRCC). Bcl-2 and Bax, and the activity of caspase-3, evaluated in tissue biopsies from patients, were congruent with resistance to apoptosis in ccRCCs with respect to healthy controls, respectively. In the same samples, the CA IX and pro-angiogenic factor VEGF expressions revealed that both these hypoxia responsive proteins were strongly increased in ccRCC with respect to controls. CA IX plasma concentration and CA activity were assessed in healthy volunteers and patients with benign kidney tumours and ccRCCs. CA IX expression levels were found strongly increased only in plasma from ccRCC subjects, whereas, CA activity was found similarly increased both in plasma from ccRCC and benign tumour patients, compared to healthy volunteers. These results show that the plasmatic level of CA IX, but not the CA total activity, can be considered a diagnostic marker of ccRCCs. Furthermore, as many reports exist relating CA IX inhibition to a better outcome to anticancer therapy in ccRCC, plasma levels of CA IX could be also predictive for response to therapy.

Roles of different IRES-dependent FGF2 isoforms in the acquisition of the major aggressive features of human metastatic melanoma.

Fibroblast growth factor 2 (FGF2) is involved in many physiological and pathological processes. Fgf2 deregulation contributes to the acquisition of malignant features of melanoma and other cancers. FGF2 is an alternative translation product expressed as five isoforms, a low-molecular-weight (18 KDa) and four high-molecular-weight (22, 22.5, 24, 34 KDa) isoforms, with different subcellular distributions. An internal ribosomal entry site (IRES) in its mRNA controls the translation of all the isoforms with the exception for the cap-dependent 34 KDa. The 18-KDa isoform has been extensively studied, while very few is known about the roles of high molecular weight isoforms. FGF2 is known to promote melanoma development and progression. To disclose the differential contribution of FGF2 isoforms in melanoma, we forced the expression of IRES-dependent low-molecular-weight (LMW, 18 KDa) and high-molecular-weight (HMW, 22, 22.5, 24 KDa) isoforms in a human metastatic melanoma cell line. This comparative study highlights that, while LMW isoform confers stem-like features to melanoma cells and promotes angiogenesis, HMW isoforms induce higher migratory ability and contribute to tumor perfusion by promoting vasculogenic mimicry (VM) when endothelial cell-driven angiogenesis is lacking. To conclude, FGF2 isoforms mainly behave in specific, antithetical manners, but can cooperate in different steps of tumor progression, providing melanoma cells with major malignant features. KEY MESSAGE: FGF2 is an alternative translation product expressed as different isoforms termed LMW and HMW. FGF2 is involved in melanoma development and progression. HMW FGF2 isoforms enhance in vitro motility of melanoma cells. LMW FGF2 confers stem-like features and increases in vivo metastasization. LMW FGF2 promotes angiogenesis while HMW FGF2 induces vasculogenic mimicry.

GDF5 regulates TGFß-dependent angiogenesis in breast carcinoma MCF-7 cells: in vitro and in vivo control by anti-TGFß peptides.

BACKGROUND: TGFß overproduction in cancer cells is one of the main characteristics of late tumor progression being implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGFß peptides in the control of angiogenesis elicited by conditional over-expression of TGFß. METHODS: We have inserted in human MCF7 mammary-cancer cells a mutated TGFß gene in a tetracycline-repressible vector to obtain conditional expression of mature TGFß upon transient transfection, evaluated the signaling pathways involved in TGFß-dependent endothelial cells activation and the efficacy of anti-TGFß peptides in the control of MCF7-TGFß-dependent angiogenesis. RESULTS: TGFß over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGFß-transfected MCF7 stimulated angiogenesis in vivo and in vitro by subsequent activation of SMAD2/3 and SMAD1/5 signaling in endothelial cells, as well as SMAD4 nuclear translocation, resulting in over-expression of the pro-angiogenic growth and differentiation factor-5 (GDF5). Inhibition or silencing of GDF5 in TGFß-stimulated EC resulted in impairment of GDF5 expression and of TGFß-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGFß antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGFß in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, GDF5 and uPAR overexpression. Antagonist peptides and anti-GDF5 antibodies efficiently inhibited in vitro and in vivo angiogenesis. CONCLUSIONS: TGFß produced by breast cancer cells induces in endothelial cells expression of GDF5, which in turn stimulates angiogenesis both in vitro and in vivo. Angiogenesis activation is rapid and the involved mechanism is totally opposed to the old and controversial dogma about the AKL5/ALK1 balance. The GDF-dependent pro-angiogenic effects of TGFß are controlled by anti-TGFß peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies.

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.

Differentiating and apoptotic dose-dependent effects in (-)-alpha-bisabolol-treated human endothelial cells.

The effect on angiogenesis of (-)-alpha-bisabolol [(-)-6-methyl-2-(4-methyl-3-cyclohexen-1-yl)-5-hepten-2-ol] (1), a widely distributed plant sesquiterpene alcohol, was investigated for the first time. Human endothelial cells treated with 1 were analyzed for their ability to differentiate and organize in microvessels and for their sensitivity to this compound in terms of cytotoxicity and cell growth inhibition. Within 24 h of the treatment with 5 microM 1, cells underwent massive death. Apoptosis induction was responsible for cytotoxicity triggered by 1 as revealed by the release of cytochrome c from the mitochondria, reduction of the Bcl-2/Bax ratio, and caspase 3 activation. At a lower, non-apoptotic concentration (0.25 microM), 1 showed a differentiating effect resulting in growth inhibition, invasiveness reduction, and tubule stabilization.

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.

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.