Browsing by Author "Mammano, Fabio"
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Item Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing(2021) Nardin, Chiara; Peres, Chiara; Putti, Sabrina; Orsini, Tiziana; Colussi, Claudia; Mazzarda, Flavia; Raspa, Marcello; Scavizzi, Ferdinando; Salvatore, Anna María; Chiani, Francesco; Tettey-Matey, Abraham; Kuang, Yuanyuan; Yang, Guang; Retamal, Mauricio; Mammano, FabioIn this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.Publication Connexin46 in the nucleus of cancer cells: a possible role as transcription modulator(2025) Fernández, Ainoa; Orellana, Viviana; Llanquinao, Jesús; Nuñez, Gonzalo; Perez Moreno, Pablo; Contreras, Sebastián; Martin, Alberto; Mammano, Fabio; Alfaro, Ivan; Calderón, Juan; Stehberg, Jimmy; Sáez, Mauricio; Retamal, Mauricio A.Background: Oncogenes drive cancer progression, but few are active exclusively in tumor cells. Connexins (Cxs), traditionally recognized as ion channel proteins, can localize to the nucleus and regulate gene expression, playing key roles in both physiological and pathological processes. Cx46, once thought to be restricted to the eye lens, has been implicated in tumor growth, though its underlying mechanisms remain unclear. This study investigates the nuclear presence of Cx46 in cancer cells and its potential role as a transcriptional modulator. Methods: We employed ChIP-Seq, confocal immunofluorescence, and nuclear protein purification to assess Cx46 localization and DNA interactions. Functional assays were conducted to evaluate its effects on invasion, division, spheroid formation, and mesenchymal marker expression. Single-point mutations and molecular dynamics simulations were used to explore potential Cx46-DNA interactions. Results: Cx46 mRNA upregulation was found in a variety of tumors compared to adjacent healthy tissue. In HeLa cells, which do not express Cx46, its transfection promoted proliferation, invasion and self-renewal capacity, cancer stem cell traits and mesenchymal features. Consistently, in Sk-Mel-2, which naturally express Cx46, reduced Cx46 expression led to a decrease in the similar parameters. In HeLa cells, nuclear Cx46 was detected in two forms, full length 46 kDa and a 30 kDa fragment (GJA3-30 k), ChIP-Seq experiments revealed that Cx46 binds to the DNA at intergenic and promoter regions, leading to the activation of oncogenic pathways. Molecular dynamics simulations suggest that GJA3-30 k dimerizes in a RAD50-like structure, forming stable DNA complexes. Cx46 and in some cases GJA3-30 k were detected in the nuclei of multiple cancer cell lines, including prostate, breast and skin cancers. Conclusions: Our findings reveal a novel nuclear role for Cx46 in cancer, demonstrating its function as a transcriptional regulator and its potential as a therapeutic target.