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In order to expand the class of diazocompounds able to act as photo-activable microtubule inhibitors the potential of azo-heteroarenes has been explored. In this paper we focus on the synthesis, physical properties and biological effects of methyl rac-2-(2-((E)-(4-((R)-2,3-dihydroxypropoxy)phenyl) diazenyl)-1H-pyrrol-1-yl)-3-hydroxypropanoate (1a) and methyl rac-2-(2-((E)-(4-((S)-2,3-dihydroxypropoxy)phenyl) diazenyl)-1H-pyrrol-1-yl)-3-hydroxypropanoate (1b). Preliminary biological studies on the HCT-116 p53-/- cancer cell line have shown that the weak antiproliferative action of the trans isomers of these molecules, especially of 1a, is enhanced upon LED light irradiation at 435 nm. On A375 cells the molecules have not shown any effect on cell viability either in the dark or under irradiation. Moreover, the two diastereomeric components of 1a as pure stereomers have been synthesized and characterized for their chemical-physical properties. Interestingly, upon irradiation, 1a has shown an antiproliferative activity on the HCT-116 p53-/- cells greater than that of the pure stereomers, 1RR and 1RS. Tubulin polymerization assay has also demonstrated that 1a, 1RR and 1RS inhibit tubulin aggregation mostly after exposure of the samples to LED light irradiation.

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.

Lung cancer is one of the most common causes of cancer-related death among adults. Chemotherapy is recognized as an important component of treatment for all stages of lung cancer and is crucial in determining patient survival and quality of life. However, intrinsic or acquired multidrug resistance (MDR) is the main reason for tumor recurrence continuing to pose a significant challenge in the management of cancer. We previously reported evidence of the role of uL3 (formerly rpL3) in mediating drug resistance showing that the resistance of A549 lung cancer cells to Cisplatin correlates to the loss of uL3 expression. In this study we identified a key role of uL3 in the control of the redox status conferring multidrug resistance to lung cancer cells lacking p53. We established and characterized a multidrug resistant Calu-6 lung cell line. We found that uL3 down-regulation correlates positively with multidrug resistance. Restoration of the uL3 protein level re-sensitized the resistant cells to the drug by regulating the reactive oxygen species (ROS) levels, glutathione content, glutamate release, and cystine uptake. Chromatin immunoprecipitation experiments and luciferase assays demonstrated that uL3 coordinated the expression of stress-response genes acting as transcriptional repressors of solute carrier family 7 member 11 (xCT) and glutathione S-transferase α1 (GST-α1), independently of Nuclear factor erythroid 2-related factor 2 (Nrf2). Altogether our results describe a new function of uL3 as a regulator of oxidative stress response genes and advance our understanding of the molecular mechanisms underlying multidrug resistance in cancers. These data suggest the possibility of targeting uL3 to modulate the redox status of cancer cells for future therapeutic purposes in order to overcome MDR.

5-FU is a chemotherapy drug commonly used for the treatment of human cancers; however drug resistance represents a major challenge for its clinical application. In the present study, we reporte that rpL3 induced by 5-FU treatment in Calu-6 cells represses CBS transcription and reduces CBS protein stability leading to a decrease of CBS protein levels. rpL3 also regulates negatively the activation of NFκB by preventing NFκB nuclear translocation through IκB-α up-regulation. Furthermore, we demonstrate that rpL3 significantly enhances the apoptosis of 5-FU treated Calu-6 cells promoting the overexpression of the pro-apoptotic proteins Bax and the inhibition of the anti-apoptotic protein Bcl-2. We finally demonstrate that rpL3 potentiates 5-FU efficacy inhibiting cell migration and invasion. Our results suggest that combination of rpL3 and 5-FU is a promising strategy for chemotherapy of lung cancers lacking functional p53 that are resistant to 5-FU.

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.