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Pheochromocytoma/paragangliomas (PPGLs) are neuroendocrine tumours, often non-metastatic, but without available effective treatment for their metastatic form. Recent studies have shown that metformin exhibits antiproliferative activity in many human cancers, including PPGLs. Nevertheless, no data are available on the role of metformin on PPGL cells (two-dimension, 2D) and spheroids (three-dimension, 3D) migration/invasion. In this study, we observed that metformin exerts an antiproliferative effect on 2D and 3D cultures of pheochromocytoma mouse tumour tissue (MTT), either silenced or not for the SDHB subunit. However, metformin did not affect MTT migration. On the other hand, metformin did not have a short-term effect on the proliferation of mouse primary fibroblasts, but significantly decreased their ability to migrate. Although the metabolic changes induced by metformin were similar between MTT and fibroblasts (i.e., an overall decrease of ATP production and an increase in intracellular lactate concentration) the activated signalling pathways were different. Indeed, after metformin administration, MTT showed a reduced phosphorylation of Akt and Erk1/2, while fibroblasts exhibited a downregulation of N-Cadherin and an upregulation of E-Cadherin. Herein, we demonstrated that metformin has different effects on cell growth and spread depending on the cell type nature, underlining the importance of the tumour microenvironment in dictating the drug response.

Pheochromocytomas and Paragangliomas (Pheo/PGL) are rare catecholamine-producing tumours derived from adrenal medulla or from the extra-adrenal paraganglia respectively. Around 10–15% of Pheo/PGL develop metastatic forms and have a poor prognosis with a 37% of mortality rate at 5 years. These tumours have a strong genetic determinism, and the presence of succinate dehydrogenase B (SDHB) mutations are highly associated with metastatic forms. To date, no effective treatment is present for metastatic forms. In addition to cancer cells, the tumour microenvironment (TME) is also composed of non-neoplastic cells and non-cellular components, which are essential for tumour initiation and progression in multiple cancers, including Pheo/PGL. This review, for the first time, provides an overview of the roles of TME cells such as cancer-associated fibroblasts (CAFs) and tumour-associated macrophages (TAMs) on Pheo/PGL growth and progression. Moreover, the functions of the non-cellular components of the TME, among which the most representatives are growth factors, extracellular vesicles and extracellular matrix (ECM) are explored. The importance of succinate as an oncometabolite is emerging and since Pheo/PGL SDH mutated accumulate high levels of succinate, the role of succinate and of its receptor (SUCNR1) in the modulation of the carcinogenesis process is also analysed. Further understanding of the mechanism behind the complicated effects of TME on Pheo/PGL growth and spread could suggest novel therapeutic targets for further clinical treatments.

Pheochromocytomas/paragangliomas (PPGLs) are neuroendocrine tumours, mostly resulting from mutations in predisposing genes. Mutations of succinate dehydrogenase (SDH) subunit B (SDHB) are associated with high probability of metastatic disease. Since bioelectrical properties and signalling in cancer are an emerging field, we investigated the metabolic, functional and electrophysiological characteristics in human SDHB knockout pheochromocytoma cells. These cells exhibited reduced SDH function with elevated succinate-to-fumarate ratio and reduced intracellular ATP levels. The analysis of membrane passive properties revealed a more hyperpolarized membrane potential and a lower cell capacitance of SDHB deficient cells compared to parental ones. These bioelectrical changes were associated with reduced proliferation and adhesion capacity of SDHB deficient cells. In SDHB deficient cells only, we also observed an increased amplitude of potassium currents suggesting an activation of ATP-sensitive potassium channels (KATP). Indeed, exposure of the SDHB deficient cells to glibenclamide, a specific KATP inhibitor, or to ATP caused normalization of potassium current features. In this work, we show for the first time that reduced intracellular ATP levels in SDHB deficient chromaffin cells impaired cell bioelectrical properties, which in turn, increased cell aggressiveness. Moreover, we first ever demonstrated that glibenclamide, not only reduced the outward potassium currents in SDHB deficient cells, but increased their growth capacity, reduced their ability to migrate, and shifted their phenotype towards one more similar to that of parental one.

Introduction Female sexual response is a complex phenomenon that involves multiple psychophysiological factors. Sex steroids play a fundamental role in the mechanisms of regulation of sexual arousal, modulating the sexual response and preserving the integrity and functionality of the vaginal tissue. For this reason, imbalances in the sexual environment of steroids in women favor the onset of important pathophysiological conditions such as hypoactive sexual desire disorder (HSDD) and genitourinary syndrome of menopause (GSM). Previous research has focused primarily on the effects of estrogen supplementation on vaginal physiology, but only few studies evaluated the effects of androgen administration, particularly on the vaginal smooth muscle compartment. The calcium sensitizing pathway of RhoA/ROCK may play a role in regulating vaginal contractility but, at present, the regulatory mechanisms of RhoA/ROCK signaling in the female genitourinary tract have yet to be fully elucidated. In particular, no specific research has been conducted on the effects of testosterone (T) on the contractility mechanism of smooth muscle in the vagina. Objective The aim of this study was to evaluate the sex steroid regulation on the distal vagina contractility mechanism, mediated by the RhoA/ROCK pathway, using a validated animal model of female ovariectomized rats. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (OVX+E), testosterone (OVX+T), or testosterone and letrozole (OVX+T+L) and compared with an intact animals’ group (CTRL). In vitro contractility studies were performed on noradrenaline (NA) pre-contracted vaginal strips to investigate the effect of OVX and in vivo treatments on vaginal smooth muscle activity. The mRNA from vagina tissues isolated from each experimental group was analyzed by semi-quantitative RT-PCR, and membrane translocation of RhoA was evaluated by Western blot analysis. Results Y-27632 induced a dose-dependent relaxation on NA pre-contracted vaginal strips that was significantly decreased by OVX; T, with or without L, further decreased OVX-induced hyporesponsiveness to Y-27632, whilst it was fully restored up to control level by 17β-estradiol (E) supplementation. Accordingly, gene expression analysis indicated that OVX decreased the expression of RhoA/ROCK pathway markers, which was normalized by E. However, at variance with contractility results, androgen receptor (AR) activation upregulated, rather than downregulating, RhoA/ROCK expression. In Western blot analysis, OVX induced activation and membrane translocation of the active form of RhoA as compared to controls. Interestingly, T reverted this effect by reducing RhoA translocation to membrane to a level even significantly below the control one. This effect was not exerted by E administration. Considering that AR activation increases NO signaling (as measured by Prkg1 mRNA expression), we tested the effect of abolishing NO formation through the NO synthase inhibitor L-NAME. Pre-incubation with L-NAME substantially increased Y-27632 responsiveness in the OVX +T group and partially in the CTRL one and, but not in the OVX group. Conclusions This study confirms that in vagina estrogens increase the expression of the calcium-sensitizing pathway RhoA/ROCK, while androgen administration functionally decreases RhoA/ROCK activity, by hampering RhoA translocation to the membrane, a mechanism which is most probably driven by NO. Disclosure No

Introduction The RhoA/ROCK calcium-sensitizing pathway plays a role in clitoral contractility, and therefore we deemed interesting to investigate its involvement in the vagina. Objective The aim of this study was to investigate the sex steroid regulation of the vagina contractility through the RhoA/ROCK pathway, using a validated animal model. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (E2), testosterone (T), with or without letrozole (T+L), and compared with intact animals. Contractility studies were performed on vaginal strips to test the effect of ROCK inhibitor Y-27632 and NO synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated, mRNA expression was analyzed by semi-quantitative RT-PCR, and RhoA membrane translocation was evaluated by Western blot (Wb) analysis. Finally, rat smooth muscle cells (rvSMCs) were isolated from distal vagina of intact animals and the quantification of RhoA inhibitory protein RhoGDI was performed by Wb after stimulation with NO-donor SNP, with or without the soluble guanylate cyclase inhibitor ODQ or the PKGR1 inhibitor KT5823 administration. Results The results show that ROCK1 was immunolocalized in the smooth muscle bundles and in the blood vessel wall of vagina, while a weak positivity was detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline pre-contracted vaginal strips, decreased by OVX and completely restored by E2, while T and T+L further decreased it even below OVX level increased it. Accordingly, in Wb analysis, compared to controls, OVX significantly induced RhoA activation, as reveled by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing the NO formation via L-NAME increased Y-27632 responsiveness in the OVX+T group; L-NAME had only a partial effect in controls whilst it did not modulate Y-27632 responsiveness in OVX. Finally, stimulation of rvSMCs with SNP significantly increased the RhoGDI protein expression, an effect counteracted by ODQ and, partially, by the protein kinase inhibitor KT5823 incubation. Conclusions In conclusion, in vagina androgen administration in OVX functionally decreases RhoA/ROCK activity by hampering RhoA membrane translocation and plays a critical role in the inhibitory mechanism of the smooth muscle distal vagina contractility. Accordingly, androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring the sexual intercourse. Disclosure No

Background Sex steroids have been demonstrated as important modulators of vaginal function. The RhoA/ROCK calcium-sensitizing pathway plays a role in genital smooth muscle contractile mechanism, but its regulation has never been elucidated. Aim This study investigated the sex steroid regulation of the vaginal smooth muscle RhoA/ROCK pathway using a validated animal model. Methods Ovariectomized (OVX) Sprague-Dawley rats were treated with 17β-estradiol (E2), testosterone (T), and T with letrozole (T + L) and compared with intact animals. Contractility studies were performed to test the effect of the ROCK inhibitor Y-27632 and the nitric oxide (NO) synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated; mRNA expression was analyzed by semiquantitative reverse transcriptase–polymerase chain reaction; and RhoA membrane translocation was evaluated by Western blot. Finally, rat vaginal smooth muscle cells (rvSMCs) were isolated from the distal vagina of intact and OVX animals, and quantification of the RhoA inhibitory protein RhoGDI was performed after stimulation with NO donor sodium nitroprusside, with or without administration of the soluble guanylate cyclase inhibitor ODQ or PRKG1 inhibitor KT5823. Outcomes Androgens are critical in inhibiting the RhoA/ROCK pathway of the smooth muscle compartment in the distal vagina. Results ROCK1 was immunolocalized in the smooth muscle bundles and blood vessel wall of the vagina, with weak positivity detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline precontracted vaginal strips, decreased by OVX and restored by E2, while T and T + L decreased it below the OVX level. In Western blot analysis, when compared with control, OVX significantly induced RhoA activation, as revealed by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing NO formation via L-NAME increased Y-27632 responsiveness in the OVX + T group; L-NAME had partial effects in controls while not modulating Y-27632 responsiveness in the OVX and OVX + E2 groups. Finally, stimulation of rvSMCs from control animals with sodium nitroprusside significantly increased RhoGDI protein expression, counteracted by ODQ and partially by KT5823 incubation; no effect was observed in rvSMCs from OVX rats. Clinical Implications Androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring sexual intercourse. Strengths and Limitations This study describes the role of androgens in maintaining vaginal well-being. The absence of a sham-operated animal group and the use of the only intact animal as control represented a limitation to the study.