Laura Naldi’s research while affiliated with Marche Polytechnic University and other places
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The apelinergic system exerts multiple biological activities in human pathologies, including cancer. Overactivation of apelin/APJ, which has been detected in many malignant tumors, and the strong correlation with progression-free and overall survival, suggested the role of an oncogene for the apelin gene. Emerging evidence sheds new light on the effects of apelin on cellular functions and homeostasis in cancer cells and supports a direct role for this pathway on different hallmarks of cancer: “sustaining proliferative signaling”, “resisting cell death”, “activating invasion and metastasis”, “inducing/accessing vasculature”, “reprogramming cellular metabolism”, “avoiding immune destruction” and “tumor-promoting inflammation”, and “enabling replicative immortality”. This article reviews the currently available literature on the intracellular processes regulated by apelin/APJ, focusing on those pathways correlated with tumor development and progression. Furthermore, the association between the activity of the apelinergic axis and the resistance of cancer cells to oncologic treatments (chemotherapy, immunotherapy, radiation) suggests apelin/APJ as a possible target to potentiate traditional therapies, as well as to develop diagnostic and prognostic applications. This issue will be also covered in the review.
Hyponatremia is the most common electrolyte disorder in cancer patients and is associated with a worse outcome. This finding has also been reported in patients with metastatic renal cell carcinoma (mRCC). We have previously demonstrated that low extracellular sodium concentrations ([Na⁺]) increase cell proliferation and invasion in several human cancer cell lines. The aim of the present study was to evaluate in vitro the effects of mild [Na⁺] alterations on two mRCC cell lines, ACHN and Caki-1. After growth in reduced extracellular [Na⁺], we observed that even mild reductions of [Na⁺] significantly enhanced different key cancer cell features, including proliferation, invasion and migration. Furthermore, we observed a reduction in reactive oxygen species (ROS) levels in low [Na⁺], with a significant increase of the antioxidant Nrf2/HMOX-1 pathway. Since an excess of ROS causes cell death, this finding clarifies the role of Nrf2/HMOX-1 in maintaining the balance between oxidant and antioxidant species in the tumor environment, promoting cell survival. Although further clinical studies are needed, aiming for instance to determine whether serum [Na⁺] correction improves the outcome of patients with mRCC, our findings suggest that attention should be deserved to serum [Na⁺] in this setting.
Sommario
Nei pazienti oncologici è frequente il riscontro di iponatriemia, spesso secondaria alla Sindrome da Inappropriata Antidiuresi. Vi sono evidenze, sia cliniche che dalla ricerca di base, che l’iponatriemia si associa a una peggiore prognosi nei pazienti affetti da cancro. Viceversa, è stato documentato un miglioramento della sopravvivenza in seguito a correzione della natriemia. La terapia dell’iponatriemia nei pazienti oncologici non si discosta dalle raccomandazioni pubblicate, e include restrizione dell’apporto di liquidi, infusione di soluzioni saline, urea, tolvaptan.
Hyponatremia is the most frequent electrolyte alteration among hospitalized patients and it has been reported in 20–40% of patients with SARS-CoV-2 (COVID-19) infection. Multiple causes of hyponatremia have been hypothesized in these patients. The syndrome of inappropriate antidiuresis (SIAD) has been considered one of the main reasons leading to hyponatremia in this condition. SIAD can be secondary to cytokines release, in particular IL-6. Positive pressure ventilation can be another cause of hyponatremia due to SIAD. Other possible etiologies of hyponatremia in COVID-19 patients can be related to secondary hypocortisolism, nausea, vomiting, heart and kidney damage. Similar to many other clinical conditions, there is strong evidence that hyponatremia is associated with a worse prognosis also in patients with COVID-19 infection. In particular, hyponatremia has been identified as an independent risk of ICU transfer, need of non-invasive ventilation and death. Hyponatremia in COVID-19 patients is in principle acute and symptomatic and should be treated as such, according to the published guidelines. Therefore, patients should be initially treated with i.v. hypertonic saline (3% NaCl) infusion and serum [Na⁺] should be frequently monitored, in order to remain within a safe rate of correction. There is evidence showing that serum [Na⁺] correction is associated with a better outcome in different pathologies, including COVID-19 infection.
We have previously demonstrated that the vasopressin type 2 receptor (AVPR2) antagonist tolvaptan reduces cell proliferation and invasion and triggers apoptosis in different human cancer cell lines. To study this effect in vivo, a xenograft model of small cell lung cancer was developed in Fox1nu/nu nude mice through the subcutaneous inoculation of H69 cells, which express AVPR2. One group of mice (n = 5) was treated with tolvaptan for 60 days, whereas one group (n = 5) served as the control. A reduced growth was observed in the tolvaptan group in which the mean tumor volume was significantly smaller on day 60 compared to the control group. In the latter group, a significantly lower survival was observed. The analysis of excised tumors revealed that tolvaptan effectively inhibited the cAMP/PKA and PI3K/AKT signaling pathways. The expression of the proliferative marker proliferating cell nuclear antigen (PCNA) was significantly lower in tumors excised from tolvaptan-treated mice, whereas the expression levels of the apoptotic marker caspase-3 were higher than those in control animals. Furthermore, tumor vascularization was significantly lower in the tolvaptan group. Overall, these findings suggest that tolvaptan counteracts tumor progression in vivo and, if confirmed, might indicate a possible role of this molecule as an adjuvant in anticancer strategies.
The functions of the gut are closely related to those of many other organs in the human body. Indeed, the gut microbiota (GM) metabolize several nutrients and compounds that, once released in the bloodstream, can reach distant organs, thus influencing the metabolic and inflammatory tone of the host. The main microbiota-derived metabolites responsible for the modulation of endocrine responses are short-chain fatty acids (SCFAs), bile acids and glucagon-like peptide 1 (GLP-1). These molecules can (i) regulate the pancreatic hormones (insulin and glucagon), (ii) increase glycogen synthesis in the liver, and (iii) boost energy expenditure, especially in skeletal muscles and brown adipose tissue. In other words, they are critical in maintaining glucose and lipid homeostasis. In GM dysbiosis, the imbalance of microbiota-related products can affect the proper endocrine and metabolic functions, including those related to the gut–liver–pancreas axis (GLPA). In addition, the dysbiosis can contribute to the onset of some diseases such as non-alcoholic steatohepatitis (NASH)/non-alcoholic fatty liver disease (NAFLD), hepatocellular carcinoma (HCC), and type 2 diabetes (T2D). In this review, we explored the roles of the gut microbiota-derived metabolites and their involvement in onset and progression of these diseases. In addition, we detailed the main microbiota-modulating strategies that could improve the diseases’ development by restoring the healthy balance of the GLPA.
The functions of the gut are closely related to those of many other organs in the human body. Indeed, the gut microbiota (GM) metabolize several nutrients and compounds that, once released in the bloodstream, can reach distant organs, thus influencing the metabolic and inflammatory tone of the host. The main microbiota-derived metabolites responsible for the modulation of endocrine responses are short-chain fatty acids (SCFAs), bile acids and glucagon-like peptide 1 (GLP-1). These molecules can (i) regulate the pancreatic hormones (insulin and glucagon), (ii) increase glycogen synthesis in the liver, and (iii) boost energy expenditure, especially in skeletal muscles and brown adipose tissue. In other words, they are critical in maintaining glucose and lipid homeostasis. In GM dysbiosis, the imbalance of microbiota-related products can affect the proper endocrine and metabolic functions, including those related to the gut–liver–pancreas axis (GLPA). In addition, the dysbiosis can contribute to the onset of some diseases such as non-alcoholic steatohepatitis (NASH)/non-alcoholic fatty liver disease (NAFLD), hepatocellular carcinoma (HCC), and type 2 diabetes (T2D). In this review, we explored the roles of the gut microbiota-derived metabolites and their involvement in onset and progression of these diseases. In addition, we detailed the main microbiota-modulating strategies that could improve the diseases’ development by restoring the healthy balance of the GLPA.
Hyponatremia is the prevalent electrolyte imbalance in cancer patients, and it is associated with a worse outcome. Notably, emerging clinical evidence suggests that hyponatremia adversely influences the response to anticancer treatments. Therefore, this study aims to investigate how reduced extracellular [Na⁺] affects the responsiveness of different cancer cell lines (from human colon adenocarcinoma, neuroblastoma, and small cell lung cancer) to cisplatin and the underlying potential mechanisms. Cisplatin dose–response curves revealed higher IC50 in low [Na⁺] than normal [Na⁺]. Accordingly, cisplatin treatment was less effective in counteracting the proliferation and migration of tumor cells when cultured in low [Na⁺], as demonstrated by colony formation and invasion assays. In addition, the expression analysis of proteins involved in autophagosome–lysosome formation and the visualization of lysosomal areas by electron microscopy revealed that one of the main mechanisms involved in chemoresistance to cisplatin is the promotion of autophagy. In conclusion, our data first demonstrate that the antitumoral effect of cisplatin is markedly reduced in low [Na⁺] and that autophagy is an important mechanism of drug escape. This study indicates the role of hyponatremia in cisplatin chemoresistance and reinforces the recommendation to correct this electrolyte alteration in cancer patients.
In cancer patients, hyponatremia is detected in about 40% of cases at hospital admission and has been associated to a worse outcome. We have previously observed that cancer cells from different tissues show a significantly increased proliferation rate and invasion potential, when cultured in low extracellular [Na⁺]. We have recently developed an animal model of hyponatremia using Foxn1nu/nu mice. The aim of the present study was to compare tumor growth and invasivity of the neuroblastoma cell line SK-N-AS in hyponatremic vs. normonatremic mice. Animals were subcutaneously implanted with luciferase-expressing SK-N-AS cells. When masses reached about 100 mm³, hyponatremia was induced in a subgroup of animals via desmopressin infusion. Tumor masses were significantly greater in hyponatremic mice, starting from day 14 and until the day of sacrifice (day 28). Immunohistochemical analysis showed a more intense vascularization and higher levels of expression of the proliferating cell nuclear antigen, chromogranin A and heme oxigenase-1 gene in hyponatremic mice. Finally, metalloproteases were also more abundantly expressed in hyponatremic animals compared to control ones. To our knowledge, this is the first demonstration in an experimental animal model that hyponatremia is associated to increased cancer growth by activating molecular mechanisms that promote proliferation, angiogenesis and invasivity.
In cancer patients, hyponatremia is detected in about 40% of cases at hospital admission and has been associated to a worse outcome. We have previously observed that cancer cells from differ-ent tissues show a significantly increased proliferation rate and invasion potential, when cul-tured in low extracellular [Na+]. We have recently developed an animal model of hyponatremia using Foxn1nu/nu mice. The aim of the present study was to compare tumor growth and inva-sivity of the neuroblastoma cell line SK-N-AS in hyponatremic vs. normonatremic mice. Ani-mals were subcutaneously implanted with luciferase-expressing SK-N-AS cells. When masses reached about 100 mm3, hyponatremia was induced in a subgroup of animals via desmopressin infusion. Tumor masses were significantly greater in hyponatremic mice, starting from day 14 and until the day of sacrifice (day 28). Immunohistochemical analysis showed a more intense vascularization and higher levels of expression of the proliferating cell nuclear antigen, chro-mogranin A and heme oxigenase-1 gene in hyponatremic mice. Finally, metalloproteases were also more abundantly expressed in hyponatremic animals compared to control ones. To our knowledge, this is the first in vivo demonstration that hyponatremia is associated to increased cancer growth by activating molecular mechanisms that promote proliferation, angiogenesis and invasivity.
Apelin is an endogenous ligand for the G protein-coupled receptor APJ and has multiple biological activities in human tissues and organs, including the heart, blood vessels, adipose tissue, central nervous system, lungs, kidneys, and liver. This article reviews the crucial role of apelin in regulating oxidative stress-related processes by promoting prooxidant or antioxidant mechanisms. Following the binding of APJ to different active apelin isoforms and the interaction with several G proteins according to cell types, the apelin/APJ system is able to modulate different intracellular signaling pathways and biological functions, such as vascular tone, platelet aggregation and leukocytes adhesion, myocardial activity, ischemia/reperfusion injury, insulin resistance, inflammation, and cell proliferation and invasion. As a consequence of these multifaceted properties, the role of the apelinergic axis in the pathogenesis of degenerative and proliferative conditions (e.g., Alzheimer’s and Parkinson’s diseases, osteoporosis, and cancer) is currently investigated. In this view, the dual effect of the apelin/APJ system in the regulation of oxidative stress needs to be more extensively clarified, in order to identify new potential strategies and tools able to selectively modulate this axis according to the tissue-specific profile.
... In particolare, gli autori hanno dimostrato che tolvaptan e mozavaptan, un altro AVRP2 antagonista, sono in grado di ridurre la crescita del tumore, diminuendo la proliferazione cellulare e l'angiogenesi tramite l'inibizione del pathway di ERK e aumentando l'apoptosi cellulare [22]. Uno studio in vivo recentemente pubblicato ha inoltre dimostrato che tolvaptan è risultato efficace nel ridurre la crescita delle masse tumorali e nel migliorare la sopravvivenza in un modello murino xenograft di microcitoma (Fig. 2) [23]. ...
... Exposure to P. gingivalis has been shown to induce activation of hepatic macrophages, particularly Kupffer cells, which release TNF-α and IL-6. The progression of MASLD may be triggered by these cytokines that mediate the inflammatory process [120]. The mechanism underlying this deterioration involves bacteria that impact liver function, resulting in steatosis and inflammation. ...
... impact cancer progression and immune response modulation, potentially leading to new combined therapeutic strategies in the near future 19-21 . In vitro and in vivo studies have suggested that alterations in extracellular [Na + ] can significantly affect cancer cell proliferation, invasion, and migration [22][23][24] , and promote resistance to anticancer treatment 25 . However, the specific effects of low [Na + ] on renal cancer cells, particularly mRCC, remain poorly understood. ...
... impact cancer progression and immune response modulation, potentially leading to new combined therapeutic strategies in the near future 19-21 . In vitro and in vivo studies have suggested that alterations in extracellular [Na + ] can significantly affect cancer cell proliferation, invasion, and migration [22][23][24] , and promote resistance to anticancer treatment 25 . However, the specific effects of low [Na + ] on renal cancer cells, particularly mRCC, remain poorly understood. ...
... Апелин способствует «побурению» БЖТ, стимулирует усвоение глюкозы, повышает чувствительность к инсулину, регулирует липолиз и окисление жирных кислот [60,63,64]. Апелин усиливает синтез антиоксидантных и подавляет экспрессию прооксидантных ферментов, а также увеличивает окислительную способность митохондрий [65]. Апелин уменьшает уровень глюкозы у мышей с СД 2-го типа и/или ожирением [63]. ...
... Circa il 50% dei pazienti oncologici presenta iponatriemia cronica, la cui prevalenza in questa coorte è perfino superiore rispetto ai soggetti ospedalizzati per qualsiasi causa [5]. È quindi facile intuire come l'impatto di questo disturbo elettrolitico sulle spese sanitarie (soprattutto in termini di ospedalizzazione e aumento dei tempi di degenza) e sulla qualità della vita dei pazienti affetti da cancro possa essere molto pesante [6]. ...
... It has also been shown that alterations in different tissues, such as bone loss, skeletal muscle sarcopenia, cardiomyopathy, hypogonadism, and liver steatosis, can be induced by hyponatremia in rats [9,10], although these findings have not been investigated systematically in humans, so far. ...
... Tolvaptan, a vasopressin V2 receptor antagonist, has been shown to reduce neuroblastoma growth in vitro 35 . Decitabine, a DNA methyltransferase inhibitor, has been evaluated in combination with chemotherapy, radiotherapy, and immunotherapy 36,37 . ...
... From a mechanistic perspective, both hypo-and hypernatremia, even within the clinically normal range, can trigger osmoregulatory stress at the cellular level. Hypohydration-induced hypertonicity elevates intracellular sodium concentrations, leading to increased vasopressin release and cellular shrinkage, which can stimulate pro-aging pathways such as oxidative stress and mitochondrial dysfunction (33,34). Conversely, low-normal sodium levels may reflect underlying conditions like chronic inflammation, subclinical illness, or dilutional hyponatremia, each of which have been independently associated with frailty and biological aging (35,36). ...
... impact cancer progression and immune response modulation, potentially leading to new combined therapeutic strategies in the near future 19-21 . In vitro and in vivo studies have suggested that alterations in extracellular [Na + ] can significantly affect cancer cell proliferation, invasion, and migration [22][23][24] , and promote resistance to anticancer treatment 25 . However, the specific effects of low [Na + ] on renal cancer cells, particularly mRCC, remain poorly understood. ...
![Analysis of cell proliferation and viability in low [Na⁺] (a) Cell proliferation of ACHN and Caki-1 was assessed by MTS assay. The reported images are representative of cell growth at different [Na⁺] (magnification 100×). Results are expressed as mean ± SE of the optical density (OD) at 490 nm/well normalized versus [Na⁺] 153 mM (*p ≤ 0.05 and **p ≤ 0.01). (b) Flow cytometry cell cycle analysis of ACHN and Caki-1. Results are expressed as percentage (%) of cells in G0/G1, S and G2/M phases (mean ± SE). (c) Western blot analysis of pAkt/Akt, pERK/ERK, cyclin D1 and PCNA protein expression at different [Na⁺]. Results are expressed as the number of positive pixels normalized versus [Na⁺] 153 mM (*p ≤ 0.05 and **p ≤ 0.01). Original blots are presented in Supplementary Fig. 1c.](https://www.researchgate.net/publication/389677189/figure/fig1/AS:11431281314574054@1741453221070/Analysis-of-cell-proliferation-and-viability-in-low-Na-a-Cell-proliferation-of-ACHN_Q320.jpg)
![Analysis of cell invasion in low [Na⁺] Cell invasion of ACHN and Caki-1 was assessed by Transwell invasion assay. The reported images are representative of cell invasion at different [Na⁺] (magnification 100×). Results are expressed as the optical density (OD) at 550 nm/well normalized versus [Na⁺] 153 mM (*p ≤ 0.05).](https://www.researchgate.net/publication/389677189/figure/fig2/AS:11431281314546041@1741453221544/Analysis-of-cell-invasion-in-low-Na-Cell-invasion-of-ACHN-and-Caki-1-was-assessed-by_Q320.jpg)
![Analysis of cell migration in low [Na⁺] (a, b) Cell migration of ACHN and Caki-1 was assessed by wound healing assay (a) and by Transwell migration assay (b). The reported images are representative of cell migration at the time of the creation of the wound (T0) and after 24 h (T24), and of cell migration using transwell assay at different [Na⁺] (magnification 100×). Results are expressed as the % area of wound closed and as the optical density (OD) at 550 nm/well normalized versus [Na⁺] 153 mM (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001).](https://www.researchgate.net/publication/389677189/figure/fig3/AS:11431281314499728@1741453222185/Analysis-of-cell-migration-in-low-Na-a-b-Cell-migration-of-ACHN-and-Caki-1-was_Q320.jpg)
![Expression analysis of MMP2, MMP9, RhoA, ROCK1 and pERM/ERM proteins in low [Na⁺] (a) MMP2 and MMP9 proteins expression was assessed by Zymography assay. The reported images are representative of MMP2 and MMP9 expression at different [Na⁺]. Results are expressed as the number of positive pixels normalized versus [Na⁺] 153 mM (*p ≤ 0.5 and **p ≤ 0.01). Original gels are presented in Supplementary Fig. 4a. (b) Western blot analysis of RhoA, ROCK1 and pERM/ERM protein expression at different [Na⁺]. Results are expressed as the number of positive pixels normalized versus [Na⁺] 153 mM (*p ≤ 0.05). Original blots are presented in Supplementary Fig. 4b.](https://www.researchgate.net/publication/389677189/figure/fig4/AS:11431281314507845@1741453222615/Expression-analysis-of-MMP2-MMP9-RhoA-ROCK1-and-pERM-ERM-proteins-in-low-Na-a_Q320.jpg)
![Analysis of oxidative stress in low [Na⁺] (a, b) Reactive oxidative species (ROS) expression levels were assessed by CellROX Deep Red Flow Cytometry Assay Kit by immunofluorescence (a) and cytometric analysis (b). (a) The reported images showed the double stain of nuclei with DAPI (blue) and ROS (red) and are representative of ROS expression at different [Na⁺] (magnification 200×). Results are expressed as CellROX integrated density normalized versus [Na⁺] 153 mM (*p ≤ 0.5 and **p ≤ 0.01). (b) Representative gating strategy of CellROX Deep Red FACS staining for assessment of ROS quantification at different [Na⁺]. Results are expressed as CellROX mean signal intensity normalized versus [Na⁺] 153 mM (*p ≤ 0.5, **p ≤ 0.01 and ***p ≤ 0.001). (c) Relative mRNA expression of HIF-1α, NQO1, HMOX-1 and MDM2 at different [Na⁺]. Results are expressed as the relative mRNA level normalized versus [Na⁺] 153 mM (*p ≤ 0.5). (d) Western blot analysis of Nrf2, HMOX-1 and Bcl-2 protein expression at different [Na⁺]. Results are expressed as the number of positive pixels normalized versus [Na⁺] 153 mM (*p ≤ 0.5). Original blots are presented in Supplementary Fig. 5d.](https://www.researchgate.net/publication/389677189/figure/fig5/AS:11431281314564912@1741453223027/Analysis-of-oxidative-stress-in-low-Na-a-b-Reactive-oxidative-species-ROS_Q320.jpg)
![Incremento del volume delle masse tumorali di topi di controllo e di topi trattati con tolvaptan. *, p≤0,05\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$p \leq 0{,}05$\end{document}; **, p≤0,02\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$p \leq 0{,}02$\end{document}; ***, p≤0,002\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$p \leq 0{,}002$\end{document}vs T0; #, p≤0,05\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$p \leq 0{,}05$\end{document}vs controlli (da [23], modificata)](https://www.researchgate.net/publication/388964632/figure/fig2/AS:11431281312389794@1740713009259/Incremento-del-volume-delle-masse-tumorali-di-topi-di-controllo-e-di-topi-trattati-con_Q320.jpg)
![DDP effects on human cancer cell proliferation. (a) WST-8 dose–response curves at extracellular [Na⁺] of 153, 127, 115, and 90 mM after DDP treatment (0–100 µM). Graphs are plotted based on three independent experiments and tables summarize the IC50 values and standard errors resulting from all [Na⁺]. (b) Colony formation assays of HCT-8 and SK-N-AS adherent cells cultured at a [Na⁺] of 153 mM or 115 mM without (indicated with DDP−) or with DDP (indicated with DDP+) (30 µM and 11 µM, respectively, in the two cell lines). Microscopic images: representative examples from a single experiment for each cell line. Bars: colony numbers, expressed as mean ± SEM from three different experiments at 153 and 115 mM [Na⁺] (**, *** = p ≤ 0.02 and p ≤ 0.002 vs. 153 mM DDP−; #, ### = p ≤ 0.05 and p ≤ 0.002, 153 mM DDP+ vs. 153 mM DDP−; $$$ = p ≤ 0.002 vs. 153 mM DDP+).](https://www.researchgate.net/publication/379865441/figure/fig1/AS:11431281387251716@1745116967244/DDP-effects-on-human-cancer-cell-proliferation-a-WST-8-dose-response-curves-at_Q320.jpg)
![DDP effects on HCT-8 (a), SK-N-AS (b), and H69 (c) cell invasiveness. (a–c) The 560 nm OD/well of invading cells at [Na⁺] of 153, 127, 115, and 90 mM was expressed as the ratio between DDP treated/untreated cells, normalized vs. the same ratio at 153 mM [Na⁺]. Microscopic images from three representative experiments are reported in the lower panels (magnification 100×) (* = p ≤ 0.05 vs. 153 mM).](https://www.researchgate.net/publication/379865441/figure/fig2/AS:11431281387290502@1745116968015/DDP-effects-on-HCT-8-a-SK-N-AS-b-and-H69-c-cell-invasiveness-a-c-The-560-nm_Q320.jpg)
![LC3A/B I-II protein expression. Western blot analysis of the LC3A/B II/I ratio at [Na⁺] of 153, 127, 115, and 90 mM in HCT-8 (a), SK-N-AS (b), and H69 (c). LC3A/B I and LC3A/B II protein expressions were normalized vs. stain-free blots. Data in the plots show the mean ± SEM (n = 3) (* = p ≤ 0.05 vs. 153 mM DDP−; #, ##, ### = p ≤ 0.05, p ≤ 0.02, and p ≤ 0.002 vs. 153 mM DDP+; $, $$$ = p ≤ 0.05 and p ≤ 0.002 DDP+ vs. DDP− for the same [Na⁺]).](https://www.researchgate.net/publication/379865441/figure/fig3/AS:11431281387403117@1745116969759/LC3A-B-I-II-protein-expression-Western-blot-analysis-of-the-LC3A-B-II-I-ratio-at-Na_Q320.jpg)
![Confocal imaging of LC3A/B. Images showed the double stain of nuclei with DAPI (blue) and LC3A/B (green) at 153 and 115 mM [Na⁺] in HCT-8 (a) and SK-N-AS (b) cells. The LC3A/B positive pixels are quantified, and the bars show the mean ± SEM (n = 3) (*, ** = p ≤ 0.05 and p ≤ 0.02 vs. 153 mM DDP−; #, ### = p ≤ 0.05 and p ≤ 0.002 vs. 153 mM DDP+; $, $$, $$$ = p ≤ 0.05, p ≤ 0.02, and p ≤ 0.002 DDP+ vs. DDP− for the same [Na⁺]).](https://www.researchgate.net/publication/379865441/figure/fig4/AS:11431281387309457@1745116970496/Confocal-imaging-of-LC3A-B-Images-showed-the-double-stain-of-nuclei-with-DAPI-blue-and_Q320.jpg)
![Transmission electron microscopy (TEM) analysis of autophagic vesicles. Images are indicative of cell samples grown at 153 mM or 115 mM [Na⁺] with or without DDP treatment (magnification 25,000×) in HCT-8 (a), SK-N-AS (b), and H69 (c) cells. Representative electron micrographs (left) and quantification (right) of lysosomal area per cell (n = 10 micrographs per each condition); one-way ANOVA followed by Bonferroni’s post hoc test, * = p ≤ 0.05 vs. 153 mM DDP−; #, ##, ### = p ≤ 0.05, p ≤ 0.02, and p ≤ 0.002 vs. 153 mM DDP+.](https://www.researchgate.net/publication/379865441/figure/fig5/AS:11431281387383101@1745116971996/Transmission-electron-microscopy-TEM-analysis-of-autophagic-vesicles-Images-are_Q320.jpg)
![Body weight and serum [Na⁺]. (a) Body weight fluctuations in the two experimental groups. Results are expressed as mean ± SE. * p ≤ 0.05, ** p ≤ 0.02 vs. T0 and # p ≤ 0.05 vs. control group. (b) Serum [Na⁺] (mEq/L) in control group and hyponatremic group. Results are expressed as mean ± SE. *** p ≤ 0.002 vs. control group.](https://www.researchgate.net/publication/375881911/figure/fig1/AS:11431281388586841@1745181769392/Body-weight-and-serum-Na-a-Body-weight-fluctuations-in-the-two-experimental-groups_Q320.jpg)
