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Beyond its canonical role in ribosome biogenesis, the nucleolus operates as a first-responder to stress stimuli that impair cell growth leading to a cellular condition, known as nucleolar stress, that is able to activate p53-dependent or p53-independent stress response signaling pathways. In this condition a subset of RPs translocates to the nucleoplasm where can exert their extra-ribosomal functions leading to cell cycle arrest and/or apoptosis. Among these RPs, we have indicated ribosomal protein uL3 (RPL3) as a key mediator of nucleolar stress pathway induced by chemotherapeutics as 5-Fluorouracile, Actinomycin D, and Oxaliplatin independently from p53 status. Here, we investigated more in depth the extra-ribosomal functions of uL3 in response to drug-induced nucleolar stress in colon cancer cells lacking p53 (HCT 116p53-/-). In addition, we have identified a strictly correlation between uL3 expression levels and drug sensitivity. Specifically, in our experimental model, the lower expression of uL3 is associated to enhancement of autophagic flux and epithelial-mesenchymal transition (EMT) phenotype resulting in Actinomycin D resistance. These observations imply a possibility that silencing of uL3 might increase the resistance of p53-deleted colon cancer cells to drug treatment through autophagy activation, enhancement in cell motility and EMT phenotype. In conclusion, our data might have a considerable value in the development of new targeted anticancer therapies for colorectal tumors lacking functional p53 and showing low expression levels of uL3 protein.

Our previous data demonstrated that nucleolar stress induced by anticancer drugs in colon cancer cells devoid of p53 leads to the activation of ribosomal protein L3 as inhibitor of cell cycle. In particular, nucleolar stress induces L3 expression and promote its nucleolar exit. To understand the molecular mechanism underlying this effect, HCT 116p53-/- cells were treated with Actinomycin D to induce nucleolar stress and with cycloheximide to block NMD pathway. Results showed that the delocalization of L3 during nucleolar stress was associated with an increase alternative L3 pre-mRNA, substrate of the NMD. Next, we quantified known cell cycle-associated E2F target genes as CycD1, CycE1 and CDK1 by quantitative RT-PCR in HCT 116p53-/- and L3ΔHCT 116p53-/- cells, in which L3 is stably silenced. We detected a significant up-regulation of E2F target genes associated with L3 knockdown. Results from reporter gene assays in presence and in absence of L3 upon drug- induced nucleolar stress demonstrated that L3 acts as positive regulator of E2F transcriptional activity. In conclusion, L3 regulates cell proliferation independently of p53 by targeting E2F and inhibiting its function.

Our previous data demonstrated that ribosomal stress induced by anticancer drugs in colon cancer cells devoid of p53 leads to the activation of ribosomal protein L3 as inhibitor of cell cycle. In particular, ribosomal stress induces L3 expression and promotes its nucleolar exit. To understand the molecular mechanism underlying this effect, HCT 116p53-/- cells were treated with Actinomycin D to induce ribosomal stress and with cycloheximide to block NMD pathway. Results showed that the delocalization of L3 during ribosomal stress was associated with an increase of alternative L3 pre-mRNA, substrate of the NMD. Next, we quantified known cell cycle-associated E2F target genes as CycD1, CycE1 and CDK1 by quantitative RT-PCR in HCT 116p53-/- and L3∆HCT 116p53-/- cells, in which L3 is stably silenced. We detected a significant up-regulation of E2F target genes associated with L3 knockdown. Results from reporter gene assays in presence and in absence of L3 upon drug-induced ribosomal stress demonstrated that L3 acts as negative regulator of E2F transcriptional activity. In conclusion, L3 regulates cell proliferation independently of p53 by targeting E2F and inhibiting its function.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Despite recent therapeutic advancements, resistance to 5-fluorouracil (5-FU) remains a major obstacle to the successful treatment of this disease. We have previously identified the ribosomal protein uL3 as a key player in the cell response to 5-FU, and loss of uL3 is associated with 5-FU chemoresistance. Natural products, like carotenoids, have shown the ability to enhance cancer cell response to drugs and may provide a safer choice to defeat chemoresistance in cancer. Transcriptome analysis of a cohort of 594 colorectal patients revealed a correlation between uL3 expression and both progression-free survival and response to treatment. RNA-Seq data from uL3-silenced CRC cells demonstrated that a low uL3 transcriptional state was associated with an increased expression of specific ATP-binding cassette (ABC) genes. Using two-dimensional (2D) and three-dimensional (3D) models of 5-FU-resistant CRC cells stably silenced for uL3, we investigated the effect of a novel therapeutic strategy by combining β-carotene and 5-FU using nanoparticles (NPs) as a drug delivery system. Our results indicated that the combined treatment might overcome 5-FU chemoresistance, inducing cell cycle arrest in the G2/M phase and apoptosis. Furthermore, the combined treatment significantly reduced the expression levels of analyzed ABC genes. In conclusion, our findings suggest that β-carotene combined with 5-FU may be a more effective therapeutic approach for treating CRC cells with low levels of uL3.

In this paper, we study the T30923 antiproliferative potential and the contribution of its loop residues in six different human cancer cell lines by preparing five T30923 variants using the single residue replacement approach of loop thymidine with an abasic site mimic (S). G-rich oligonucleotides (GRO) show interesting anticancer properties because of their capability to adopt G-quadruplex structures (G4s), such as the G4 HIV-1 integrase inhibitor T30923. Considering the multi-targeted effects of G4-aptamers and the limited number of cancer cell lines tested, particularly for T30923, it should be important to find a suitable tumor line, in addition to considering that the effects also strictly depend on G4s. CD, NMR and non-denaturating polyacrylamide gel electrophoresis data clearly show that all modified ODNs closely resemble the dimeric structure of parallel G4s’ parent aptamer, keeping the resistance in biological environments substantially unchanged, as shown by nuclease stability assay. The antiproliferative effects of T30923 and its variants are tried in vitro by MTT assays, showing interesting cytotoxic activity, depending on time and dose, for all G4s, especially in MDA-MB-231 cells with a reduction in cell viability approximately up to 30%. Among all derivatives, QS12 results are the most promising, showing more pronounced cytotoxic effects both in MDA-MB-231 and Hela cells, with a decrease in cell viability from 70% to 60%. In summary, the single loop residue S substitution approach may be useful for designing antiproliferative G4s, considering that most of them, characterized by single residue loops, may be able to bind different targets in several cancer cell pathways. Generally, this approach could be of benefit by revealing some minimal functional structures, stimulating further studies aimed at the development of novel anticancer drugs.

Purpose: In order to study novel therapeutic approaches taking advantage of natural compounds showing anticancer and anti-proliferative effects, we focused our interest on S-adenosyl-l-methionine, a naturally occurring sulfur-containing nucleoside synthesized from adenosine triphosphate and methionine by methionine adenosyltransferase, and its potential in overcoming drug resistance in colon cancer cells devoid of p53. Results: In the present study, we demonstrated that S-adenosyl-l-methionine overcomes uL3-mediated drug resistance in p53 deleted colon cancer cells. In particular, we demonstrated that S-adenosyl-l-methionine causes cell cycle arrest at the S phase, inhibits autophagy, augments reactive oxygen species, and induces apoptosis in these cancer cells. Conclusions: Results reported in this paper led us to propose S-adenosyl-l-methionine as a potential promising agent for cancer therapy by examining p53 and uL3 profiles in tumors to yield a better clinical outcomes.

It is now well established that the urothelium does not act as a passive barrier but contributes to bladder homeostasis by releasing several signaling molecules in response to physiological and chemical stimuli. Here, we investigated the potential contribution of the hydrogen sulfide (H2S) pathway in regulating human urothelium function in β3 adrenoceptor-mediated relaxation. The relaxant effect of BRL 37344 (0.1–300 µM), a selective β3 adrenoceptor agonist, was evaluated in isolated human bladder strips in the presence or absence of the urothelium. The relaxant effect of BRL 37344 was significantly reduced by urothelium removal. The inhibition of cystathionine-γ-lyase (CSE), but not cystathionine-β-synthase (CBS), significantly reduced the BRL 37344 relaxing effect to the same extent as that given by urothelium removal, suggesting a role for CSE-derived H2S. β3 adrenoceptor stimulation in the human urothelium or in T24 urothelial cells markedly increased H2S and cAMP levels that were reverted by a blockade of CSE and β3 adrenoceptor antagonism. These findings demonstrate a key role for urothelium CSE-derived H2S in the β3 effect on the human bladder through the modulation of cAMP levels. Therefore, the study establishes the relevance of urothelial β3 adrenoceptors in the regulation of bladder tone, supporting the use of β3 agonists in patients affected by an overactive bladder.

In this paper, we report our investigations on five T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties.

Eukaryotic cells are exposed to many internal and external stimuli that affect their fate. In particular, the exposure to some of these stimuli induces stress triggering a variety of stress responses aimed to re-establish cellular homeostasis. It is now established that the deregulation of stress response pathways plays a central role in cancer initiation and progression, allowing the adaptation of cells to an altered state in the new environment. Autophagy is a tightly regulated pathway which exerts “housekeeping” role in physiological processes. Recently, a growing amount of evidence highlighted the crucial role of autophagy in the regulation of integrated stress responses, including nucleolar and endoplasmic reticulum. In this review, we attempt to afford an overview of the complex role of nucleolar and endoplasmic reticulum stress-response mechanisms in the regulation of autophagy in cancer and cancer treatment.

The nucleolus is a distinct sub-cellular compartment structure in the nucleus. First observed more than 200 years ago, the nucleolus is detectable by microscopy in eukaryotic cells and visible during the interphase as a sub-nuclear structure immersed in the nucleoplasm, from which it is not separated from any membrane. A huge number of studies, spanning over a century, have identified ribosome biogenesis as the main function of the nucleolus. Recently, novel functions, independent from ribosome biogenesis, have been proposed by several proteomic, genomic, and functional studies. Several works have confirmed the non-canonical role for nucleoli in regulating important cellular processes including genome stability, cell-cycle control, the cellular senescence, stress responses, and biogenesis of ribonucleoprotein particles (RNPs). Many authors have shown that both canonical and non-canonical functions of the nucleolus are associated with several cancer-related processes. The association between the nucleolus and cancer, first proposed by cytological and histopathological studies showing that the number and shape of nucleoli are commonly altered in almost any type of cancer, has been confirmed at the molecular level by several authors who demonstrated that numerous mechanisms occurring in the nucleolus are altered in tumors. Recently, therapeutic approaches targeting the nucleolus in cancer have started to be considered as an emerging “hallmark” of cancer and several therapeutic interventions have been developed. This review proposes an up-to-date overview of available strategies targeting the nucleolus, focusing on novel targeted therapeutic approaches. Finally, a target-based classification of currently available treatment will be proposed.

Several experimental strategies in the treatment of cancer include drug alteration of cell cycle regulatory pathways as a useful strategy. Extra-ribosomal functions of human ribosomal protein L3 (uL3) may affect DNA repair, cell cycle arrest and apoptosis. In the present study, we demonstrated that uL3 is required for the activation of G1/S transition genes. Luciferase assays established that uL3 negatively regulates the activity of E2F1 promoter. Induced ribosome-free uL3 reduces Cyclin D1 mRNA and protein levels. Using protein/protein immunoprecipitation methods, we demonstrated that uL3 physically interacts with PARP-1 affecting E2F1 transcriptional activity. Our findings led to the identification of a new pathway mediated by uL3 involving E2F1 and Cyclin D1 in the regulation of cell cycle progression.

In the last years, a lot of evidences have been accumulated suggesting that translational reprogramming plays a key role in tumor initiation, invasion and multidrug resistance (MDR). Translational reprogramming is associated to alteration in eukaryotic translation initiation factors (eIFs) activity, in particular the overexpression of the eIF4F complex, is associated with chemoresistance in melanoma, colon and thyroid cancer cell lines. In our previously study, we have analyzed the role of human ribosomal protein rpL3 in MDR. Our studies revealed that rpL3 is a key determinant in cellular stress responce and in MDR in HCT 116p53-/- cells. Here we report results from GST pull-down and mass spectrometry demonstrating that eiF4A, a component of eIF4F complex, is able to interact with rpL3 in vitro. Immunoprecipitation experiments confirm the presence of a complex rpL3/eiF4A/eiF4G in HCT 116p53-/- cells treated with 5-FU. The translation status of known mRNAs regulated by eIF4A (i.e. BCL2-family, Cyclins and c-Myc mRNAs) has been analyzed by qPCR in 5-FU resistant HCT 116p53-/- cells and in L3DHCT 116p53-/- cells. Results from these experiments will be presented.