Recent findings revealed in cancer cells novel stress response pathways, which in response to many chemotherapeutic drugs causing nucleolar stress, will function independently from tumor protein p53 ...(p53) and still lead to cell cycle arrest and/or apoptosis. Since it is known that most cancers lack functional p53, it is of great interest to explore these emerging molecular mechanisms. Here, we demonstrate that nucleolar stress induced by 5-fluorouracil (5-FU) in colon cancer cells devoid of p53 leads to the activation of ribosomal protein L3 (rpL3) as proapoptotic factor. rpL3, as ribosome-free form, is a negative regulator of cystathionine-β-synthase (CBS) expression at transcriptional level through a molecular mechanism involving Sp1. The rpL3-CBS association affects CBS stability and, in addition, can trigger CBS translocation into mitochondria. Consequently apoptosis will be induced through the mitochondrial apoptotic cell death pathway characterized by an increased ratio of Bax to Bcl-2, cytochrome c release and subsequent caspase activation. It is noteworthy that silencing of CBS is associated to a strong increase of 5-FU-mediated inhibition of cell migration and proliferation. These data reveal a novel mechanism to accomplish p53-independent apoptosis and suggest a potential therapeutic approach aimed at upregulating rpL3 for treating cancers lacking p53.
Hydrogen sulfide (H₂S) is synthesized by 2 enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). L-Cysteine (L-Cys) acts as a natural substrate for the synthesis of H₂S. Human ...penile tissue possesses both CBS and CSE, and tissue homogenates efficiently convert L-Cys to H₂S. CBS and CSE are localized in the muscular trabeculae and the smooth-muscle component of the penile artery, whereas CSE but not CBS is also expressed in peripheral nerves. Exogenous H₂S sodium hydrogen sulfide (NaHS) or L-Cys causes a concentration-dependent relaxation of strips of human corpus cavernosum. L-Cys relaxation is inhibited by the CBS inhibitor, aminoxyacetic acid (AOAA). Electrical field stimulation of human penile tissue, under resting conditions, causes an increase in tension that is significantly potentiated by either propargylglycine (PAG; CSE inhibitor) or AOAA. In rats, NaHS and L-Cys promote penile erection, and the response to L-Cys is blocked by PAG. Our data demonstrate that the L-Cys/H₂S pathway mediates human corpus cavernosum smooth-muscle relaxation.
Penile erection is a perfect example of microcirculation modulated by psychological factors and hormonal status. It is the result of a complex neurovascular process that involves the integrative ...synchronized action of vascular endothelium; smooth muscle; and psychological, neuronal, and hormonal systems. Therefore, the fine coordination of these events is essential to maintain penile flaccidity or allow erection; an alteration of these events leads to erectile dysfunction (ED). ED is defined as the consistent or recurrent inability of a man to attain and/or maintain a penile erection sufficient for sexual activity. A great boost to this research field was given by commercialization of phosphodiesterase-5 (PDE5) inhibitors.
Indeed, following the discovery of sildenafil, research on the mechanisms underlying penile erection has had an enormous boost, and many preclinical and clinical papers have been published in the last 10 years. This review is structured to provide an overview of the mediators and peripheral mechanism(s) involved in penile function in men, the drugs used in therapy, and the future prospective in the management of ED. Indeed, 30% of patients affected by ED are classified as “nonresponders,” and there is still an unmet need for therapeutic alternatives. A flowchart suggesting the guidelines for ED evaluation and the ED pharmacological treatment is also provided.
Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing ...several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Background Phosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the treatment of lower urinary tract symptom (LUTS), although their mechanism of action is still unclear. PDE5-Is ...cause bladder detrusor relaxation, and this effect is partially independent of nitric oxide. Hydrogen sulfide (H2 S) is a newly discovered transmitter with myorelaxant properties. It is predominantly formed from L-cysteine by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Objective To evaluate whether the L-cysteine/H2 S pathway contributes to the relaxing effect of sildenafil on the human detrusor dome. Design, setting, and participants Samples of bladders obtained from men undergoing open prostatectomy for benign prostatic hyperplasia (BPH) were used. The presence of CBS and CSE enzymes was assessed by western blot. H2 S production was measured by a colorimetric assay in basal and stimulated conditions with L-cysteine and in response to sildenafil (1, 3, 10, and 30 μM), 8-bromo–cyclic guanosine monophosphate (8-bromo–cGMP; 100 μM) or dibutyryl–cyclic adenosine monophosphate (dibutyryl-cAMP; 100 μM). A curve concentration effect of sodium hydrosulfide (NaHS), H2 S donor (0.1 μM to 10 mM), L-cysteine (0.1 μM to 10 mM), and sildenafil (0.1–10 μM) was performed on precontracted detrusor dome strips. To investigate H2 S signaling in a sildenafil effect, CBS and CSE inhibitors were used. Outcome measurements and statistical analysis Analysis of variance was used, followed by the Bonferroni post hoc test. Results and limitations CBS and CSE are present in the human bladder dome and efficiently convert L-cysteine into H2 S. Both NaHS and L-cysteine relaxed human strips. Sildenafil caused (1) a relaxation of bladder dome strips and (2) a concentration-dependent increase in H2 S production. Both effects were significantly reduced by CBS and CSE inhibitors. Similar to sildenafil, both 8-bromo-cGMP and dibutyryl-cAMP caused an increase in H2 S production. Conclusions The sildenafil relaxant effect on the human bladder involves the H2 S signaling pathway. This effect may account in part for the efficacy of PDE5-Is in LUTS. A better definition of the pathophysiologic role of the H2 S pathway in the human bladder may open new therapeutic approaches.
Breast cancer is the most frequent form of cancer occurring in women of any age. Among the different types, the triple-negative breast cancer (TNBC) subtype is recognized as the most severe form, ...being associated with the highest mortality rate. Currently, there are no effective treatments for TNBC. For this reason, the research of novel therapeutics is urgently needed. Natural products and their analogs have historically made a major contribution to pharmacotherapy and the treatment of various human diseases, including cancer. In this study, we explored the potential anti-cancer effects of erucin, the most abundant H2S-releasing isothiocyanate present in arugula (Eruca sativa) in MDA-MB-231 cells, a validated in vitro model of TNBC. We found that erucin, in a concentration-dependent manner, significantly inhibited MDA-MB-231 cell proliferation by inducing apoptosis and autophagy. Additionally, erucin prevented intracellular ROS generation promoting the expression of key antioxidant genes and halted MDA-MB-231 cell migration, invasion, and colony formation. In conclusion, using a cellular and molecular biology approach, we show that the consumption of erucin could represent a novel and promising strategy for intervention against TNBC.
The role of H2S in urothelial carcinoma (UC) is still unclear. Here we have evaluated the expression of H2S producing enzymes as well as the effect of endogenous and exogenous H2S on human bladder UC ...cells. In human UC cells the expression of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST); is significantly lower as compared to healthy cells. A modulatory role for the H2S pathway is supported by the finding that, the overexpression of CSE or CBS, but not 3-MST, inhibits cell proliferation and promotes apoptosis. A similar effect is obtained by using exogenous H2S. Diallyl trisulfide (DATS), which is a fully characterized H2S donor, inhibits the proliferation of UC cells in a time and concentration-dependent manner as well as promotes apoptosis. Moreover, DATS also induces autophagy, as determined by transcriptomic and western blot analysis. Finally, DATS inhibits mRNA expression levels of canonical markers of epithelial-mesenchymal transition by limiting migration and clonogenic ability of human UC cells in vitro. In conclusion, in urothelial carcinoma, there is an impairment of H2S pathway that involves CSE and CBS- derived hydrogen sulfide. Thus, targeting H2S signaling pathway in urothelial carcinoma could represent a novel therapeutic strategy.
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•The expression of H2S producing enzymes is significantly lower in urothelial carcinoma (UC) cells than healthy control.•Genetic and/or pharmacological replenishment of H2S impacts on UC cell proliferation, apoptosis, and autophagy.•DATS prevents epithelial-mesenchymal transition by limiting cell migration and colony formation in UC cells.
Sarcopenia is a gradual and generalized skeletal muscle (SKM) syndrome, characterized by the impairment of muscle components and functionality. Hydrogen sulfide (H2S), endogenously formed within the ...body from the activity of cystathionine-γ-lyase (CSE), cystathionine- β-synthase (CBS), and mercaptopyruvate sulfurtransferase, is involved in SKM function. Here, in an in vitro model of sarcopenia based on damage induced by dexamethasone (DEX, 1 μM, 48 h treatment) in C2C12-derived myotubes, we investigated the protective potential of exogenous and endogenous sources of H2S, i.e., glucoraphanin (30 μM), L-cysteine (150 μM), and 3-mercaptopyruvate (150 μM). DEX impaired the H2S signalling in terms of a reduction in CBS and CSE expression and H2S biosynthesis. Glucoraphanin and 3-mercaptopyruvate but not L-cysteine prevented the apoptotic process induced by DEX. In parallel, the H2S-releasing molecules reduced the oxidative unbalance evoked by DEX, reducing catalase activity, O2− levels, and protein carbonylation. Glucoraphanin, 3-mercaptopyruvate, and L-cysteine avoided the changes in myotubes morphology and morphometrics after DEX treatment. In conclusion, in an in vitro model of sarcopenia, an impairment in CBS/CSE/H2S signalling occurs, whereas glucoraphanin, a natural H2S-releasing molecule, appears more effective for preventing the SKM damage. Therefore, glucoraphanin supplementation could be an innovative therapeutic approach in the management of sarcopenia.
Endocrine-disrupting chemicals (EDCs) are different natural and synthetic chemicals that may interfere with several mechanisms of the endocrine system producing adverse developmental, metabolic, ...reproductive, and neurological effects in both human beings and wildlife. Among pesticides, numerous chemicals have been identified as EDCs. MicroRNAs (miRNAs) can regulate gene expression, making fine adjustments in mRNA abundance and regulating proteostasis. We hypothesized that exposure to low doses of atrazine, cypermethrin, and vinclozolin may lead to effects on miRNA expression in SH-SY5Y cells. In particular, the exposure of SH-SY5Y cells to subtoxic concentrations of vinclozolin is able to downregulate miR-29b-3p expression leading to the increase in the related gene expression of ADAM12 and CDK6, which may promote a pro-oncogenic response through the activation of the PI3K/Akt/mTOR pathway and counteracting p53 activity. A better understanding of the molecular mechanisms of EDCs could provide important insight into their role in human disease.
Inhaled antivirulence drugs are currently considered a promising therapeutic option to treat Pseudomonas aeruginosa lung infections in cystic fibrosis (CF). We have recently shown that the ...anthelmintic drug niclosamide (NCL) has strong quorum sensing (QS) inhibiting activity against P. aeruginosa and could be repurposed as an antivirulence drug. In this work, we developed dry powders containing NCL nanoparticles that can be reconstituted in saline solution to produce inhalable nanosuspensions. NCL nanoparticles were produced by high-pressure homogenization (HPH) using polysorbate 20 or polysorbate 80 as stabilizers. After 20 cycles of HPH, all formulations showed similar properties in the form of needle-shape nanocrystals with a hydrodynamic diameter of approximately 450 nm and a zeta potential of −20 mV. Nanosuspensions stabilized with polysorbate 80 at 10% w/w to NCL (T80_10) showed an optimal solubility profile in simulated interstitial lung fluid. T80_10 was successfully dried into mannitol-based dry powder by spray drying. Dry powder (T80_10 DP) was reconstituted in saline solution and showed optimal in vitro aerosol performance. Both T80_10 and T80_10 DP were able to inhibit P. aeruginosa QS at NCL concentrations of 2.5–10 μM. NCL, and these formulations did not significantly affect the viability of CF bronchial epithelial cells in vitro at microbiologically active concentrations (i.e., ≤10 μM). In vivo acute toxicity studies in rats confirmed no observable toxicity of the NCL T80_10 DP formulation upon intratracheal administration at a concentration 100-fold higher than the anti-QS activity concentration. These preliminary results suggest that NCL repurposed in the form of inhalable nanosuspensions has great potential for the local treatment of P. aeruginosa lung infections as in the case of CF patients.
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