Littérature scientifique sur le sujet « NCI-H295R »

The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3β-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3β-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the ‘androgenic’ steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.

We investigated the effects of epidermal growth factor (EGF) and prostaglandins (PG) on the expression of aromatase (CYP19) in human adrenocortical carcinoma cell line NCI-H295R cells. EGF significantly increased aromatase activity and CYP19 gene transcript in NCI-H295R cells. Exon PII was selected from among several tissue-specific exon I regions. Promoter II that abuts on exon PII was activated by EGF. PGE2 also significantly increased aromatase activity, CYP19 gene transcript, and promoter II activity. The results of experiments using protein kinase (PK) inhibitors suggest that the cAMP–PKA signaling pathway is involved in the up-regulation of aromatase expression by EGF. PGE2 activated promoter II activity in 4 h, while12 h was required for its activation by EGF. In addition, PGE2 was secreted from NCI-H295R cells in response to EGF. Selective agonists for prostaglandin receptors EP1 and EP2 significantly increased aromatase activity, which was decreased by the corresponding antagonists. Finally, antagonists for EP1 and EP2 inhibited the up-regulation of aromatase expression following EGF. These results suggest that PGE2 secondarily acts as an autocrine signal in the up-regulation of aromatase expression by EGF in NCI-H295R cells.

Hypocretins/orexins act through two receptor subtypes: OX1 and OX2. Outside the brain, orexin receptors are expressed in adrenal glands, where orexins stimulate the release of glucocorticoids. To further address the regulation of steroidogenesis, we analyzed the effect of orexins on the expression of steroidogenic enzymes in human adrenocortical National Cancer Institute (NCI) H295R cells by qPCR. In NCI H295R cells, OX2 receptors were highly expressed, as they were in human adrenal glands. After treatment of NCI H295R cells with orexin A for 12–24 h, the cortisol synthesis rate was significantly increased, whereas 30 min of treatment showed no effect. While CYP11B1 and CYP11B2 mRNA levels were increased already at earlier time points, the expression of HSD3B2 and CYP21 mRNA was significantly up-regulated after treatment with orexin A for 12 h. Likewise, orexin B increased CYP21 and HSD3B2 mRNA levels showing, however, a lower potency compared with orexin A. The mRNA levels of CYP11A and CYP17 were unaffected by orexin A. OX2 receptor mRNA levels were down-regulated after 12 and 24 h of orexin A treatment. Orexin A increased intracellular Ca2+ but not cAMP concentrations in NCI H295R cells. Furthermore, inhibition of PKC and MAPK kinase/ERK kinase (MEK1/2) prevented the increase of HSD3B2 expression by orexin A. Accordingly, orexin A treatment of NCI H295R cells markedly enhanced ERK1/2 phosphorylation that was prevented by PKC and, in part, PKA inhibition. In conclusion, orexins may influence adrenal steroidogenesis by differential regulation of the expression of steroidogenic enzymes involving Ca2+, as well as PKC-ERK1/2 signaling.

Adrenocortical carcinomas are rare tumors with poor prognosis and limited treatment options. Although widely used as in vitro models to test novel therapeutic strategies, the adrenocortical carcinoma-derived cell lines NCI-H295R and SW-13 have only partially been described genetically. Our aim was to characterize the mutational landscape of these cells to improve their experimental utility and map them to clinical subtypes of adrenocortical carcinoma. Genomic DNA from NCI-H295R and SW-13 cells was subjected to whole-exome sequencing. Variants were filtered for non-synonymous mutations and curated for validated adrenocortical and pan-cancer driver gene mutations. Genes mutated in the cell lines were mapped using gene ontology and protein pathway tools to determine signaling effects and compared to mutational and clinical characteristics of 92 adrenocortical carcinoma cases from The Cancer Genome Atlas. NCI-H295R and SW-13 cells carried 1325 and 1836 non-synonymous variants, respectively. Of these, 61 and 76 were known cancer driver genes, of which 32 were shared between cell lines. Variant interaction analyses demonstrated dominant TP53 dysregulation in both cell lines complemented by distinct WNT (NCI-H295R) and chromatin remodeling (SW-13) pathway perturbations. Both cell lines genetically resemble more aggressive adrenocortical carcinomas with worse prognosis, for which development of targeted therapies is most critical. Careful incorporation of the genetic landscapes outlined in this study will further the in vitro utility of these cell lines in testing for novel therapeutic approaches for adrenocortical malignancy.

Several important physiological functions are regulated by cortisol. Previously, we demonstrated the involvement of human organic anion transporter 3 (hOAT3) in cortisol release. In the present study, we investigated the influence of dehydroepiandrosterone sulfate (DHEA-S) and estrone sulfate on cortisol release in a human adrenocortical cell line (NCI-H295R) compared with forskolin stimulation. Additionally, we examined the impact of forskolin and DHEA-S on the expression of key enzymes in steroid biosynthesis and expression of hOAT3 and -4 in NCI-H295R cells. The cortisol release was increased 10-fold after 24-h incubation with DHEA-S, but incubation with estrone sulfate did not show any significant change in cortisol release. When cells were incubated with DHEA-S in the presence of forskolin, an additive influence of DHEA-S stimulation of cortisol was recorded over forskolin alone. The 24-h stimulation of NCI-H295R cells with forskolin increased the expression of steroidogenic acute regulatory protein (StAR), CYP17, CYP21A2, and CYP11A1, whereas only StAR mRNA expression was increased significantly by incubation with DHEA-S. Immunofluorescence analyses revealed strongly elevated expression of hOAT3 by forskolin as well as by DHEA-S stimulation. We conclude that the increased cortisol release of adrenocortical cells by DHEA-S and forskolin stimulation is probably due to high expression of the key enzymes of steroid biosynthesis and hOAT3.

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

Adrenocorticotropin is the major regulator of adrenocortical development and function. It acts mainly through the cAMP-dependent protein kinase A (PKA) pathway. Our aim was to study the interaction of tumor necrosis factor-α (TNFα) and the PKA pathway in adrenocortical cell proliferation and apoptosis. The PKA activator Dibutyryl cAMP ((Bu)2cAMP) strongly induced differentiation and inhibited proliferation in the human adrenocortical cell line NCI-H295R (H295R). TNFα induced apoptosis of H295R cells. Interestingly, (Bu)2cAMP treatment clearly enhanced TNFα-induced apoptosis in H295R cells, but not in another human adrenocortical cell line SW-13, the mouse adrenocortical Y-1 cell line or the human HeLa cell line. This synergistic effect was not due to the (Bu)2cAMP-induced glucocorticoid secretion since dexamethasone had no significant effect on the TNFα-induced apoptosis. (Bu)2cAMP treatment rapidly increased the expression of the proto-oncogene c-myc in H295R cells, but not in SW-13, Y-1 or HeLa cells. In transient c-myc transfection assay, c-myc expression associated with decreased expression of the proliferation marker Ki-67 in H295R cells. In conclusion, cAMP-dependent protein kinase activation reduced proliferation and augmented TNFα-induced apoptosis in adrenocortical H295R cells, and these effects were associated with increased c-myc expression.

High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) can be effectively reversed if the herb Scutellaria baicalensis is included in traditional Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum concentration and intervention time for baicalin treatment of NCI-H295R cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and ELISA. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, baicalin decreased the testosterone concentrations in a dose- and time-dependent manner in NCI-H295R cells. GEP revealed that 3β-hydroxysteroid dehydrogenase type II (HSD3B2) was the key enzyme of androgen biosynthesis, and baicalin inhibited the expression of HSD3B2 by regulating the binding of transcription factor GATA-binding factor 1 (GATA1) to the HSD3B2 promoter. Hyperandrogenic PCOS model rats treated with baicalin significantly reversed the high androgen levels of serum and the abnormal ovarian status, restored the estrous cyclicity and decreased the expression of HSD3B2 in ovarian. In summary, our data revealed that GATA1 is an important transcription factor activating the HSD3B2 promoter in steroidogenesis, and baicalin will potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue.

Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy with no effective therapy for patients with unresectable disease. The aim of the current study was i) to evaluate TOP2A expression and function in human adrenocortical neoplasm and ACC cells and ii) to determine the anticancer activity of agents that target TOP2A. TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 ACCs). In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth, and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cells. TOP2A mRNA and protein expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (P<0.05). Knockdown of TOP2A gene expression in ACC cell lines significantly decreased cell proliferation, anchorage-independent growth, and invasion (P<0.05). A screening assay in NCI-H295R cells showed that 11 of 14 TOP2A inhibitors had antiproliferative activity, 5 of the 14 TOP2A inhibitors had a higher antiproliferative activity than mitotane, and aclarubicin was the agent with the highest activity. Aclarubicin was validated to significantly decrease proliferation and tumor spheroid size in both NCI-H295R and SW13 ACC cell lines (P<0.05). Our results suggest that TOP2A is overexpressed in ACC, regulates cellular proliferation and invasion in ACC cells, and is an attractive target for ACC therapy. Of the TOP2A inhibitors screened, aclarubicin is a good candidate agent to test in future clinical trials for patients with locally advanced and metastatic ACC.

Background: Angiotensin II (Ang-II) regulates aldosterone and cortisol secretion via type 1 angiotensin II receptor (AT1R) in the adrenal cortex. Although the type 2 angiotensin II receptor (AT2R) and Mas receptor (MasR), form an important part of the so-called protective renin-angiotensin system (RAS), and counter regulate AT1R-mediated effects in various tissues, their function in the adrenal cortex remains largely unknown. Furthermore, the angiotensin-converting enzyme 2 (ACE2), another component of the protective RAS, cleaves Ang II and is also known to regulate effects of AT1R in several tissues. Aim: Therefore, our aim was to investigate: - the presence of AT2R, MasR, and ACE2 in human adrenocortical tissue and in aldosterone-producing adenoma (APA); - the role of Ang-(1-7) and compound 21 (C21), an AT2R agonist, and diminazene aceturate (DIZE) in modulation of aldosterone and cortisol production in humans. Methods: We detected AT2R, MasR, and ACE2 by immunoblotting and RT-PCR. Furthermore, using immunohistochemistry we localized AT2R and MasR in aldosterone-producing adenoma (APA) and in normal adrenal glang (NAG). We used Ang-(1-7) to stimulate MasR, C21 to stimulate AT2R, Ang II to stimulate AT1R and AT2R, and DIZE to activate ACE2. Irbesartan and A779 were used as blockers for AT1R and MasR respectively. RT-PCR was used to quantify CYP11B1/CYP11B2 genes expression at baseline and after stimulation. Results: AT2R and MasR were found to be heterogeneously express in human adrenal cortex and in APA. At low concentrations [nM], Ang-(1-7) had no effect on CYP11B1 and CYP11B2 gene expression, but at high concentrations [μM] it increased them. A779 did not significantly decrease the Ang-(1-7) effects, whereas irbesartan blunted them. In human adrenocortical cell lines (HAC15 and NCI-H295R), the AT2R-agonist C21 had no effect at low concentrations [nM], but at high concentrations [μM] it increased CYP11B1 and CYP11B2 expression. Irbesartan blunted these effects Finally, DIZE effect was no potent in CYP11B1 and CYP11B2 gene expression, but it may be involved in ACE2 gene expression increase. Conclusions: The expression of AT2R and MasR in the human adrenal cortex is, respectively 10-fold and 150-fold lower than AT1R. Moreover, whereas AT1R and MasR genes are expressed at the same level in the healthy adrenal cortex and in APA, AT2R gene expression is significantly lower in APA. The secretagogue effect of Ang-(1-7) and C21 on aldosterone and cortisol production seen with high concentrations likely occurs via AT1R. Thus, Ang-(1-7) and C21 may not be used as therapeutically tool to decrease aldosterone and cortisol production in human.
Background: L'angiotensina II (Ang-II) regola aldosterone e secrezione di cortisolo via di tipo 1 del recettore dell'angiotensina II (AT1R) nella corteccia surrenale. Anche se il tipo 2 del recettore dell'angiotensina II (AT2R) e del recettore Mas (Masr), costituiscono parte importante del cosiddetto sistema renina-angiotensina protettivo (RAS), e il contatore di regolare effetti AT1R mediata in vari tessuti, la loro funzione nel corteccia surrenale rimane in gran parte sconosciuta. Inoltre, l'enzima di conversione dell'angiotensina 2 (ACE2), un altro componente della RAS di protezione, si unirà Ang II ed è noto anche per regolare gli effetti di AT1R in diversi tessuti. Obiettivo: Pertanto, il nostro obiettivo era quello di indagare: - La presenza di AT2R, Masr e ACE2 nel tessuto adrenocorticale umana e aldosterone adenoma (APA); - Il ruolo di Ang (1-7) e composto di 21 (C21), un agonista AT2R, e Diminazene aceturato (Dize) nella modulazione di aldosterone e cortisolo produzione negli esseri umani. Metodi: Abbiamo rilevato AT2R, Masr, e ACE2 da immunoblotting e RT-PCR. Inoltre, utilizzando l'immunoistochimica abbiamo localizzato AT2R e Masr in aldosterone producono adenoma (APA) e in normale gLang surrene (NAG). Abbiamo usato Ang (1-7) per stimolare Masr, C21 per stimolare AT2R, Ang II per stimolare AT1R e AT2R, e Dize di attivare ACE2. Irbesartan e A779 sono state usate come bloccanti rispettivamente per AT1R e Masr. RT-PCR è stato utilizzato per quantificare l'espressione dei geni CYP11B1 / CYP11B2 al basale e dopo stimolazione. Risultati: AT2R e Masr sono risultati essere eterogeneo espressa in corteccia surrenale umana e in APA. A basse concentrazioni [Nm], Ang (1-7) non ha avuto effetti sulla espressione genica e CYP11B1/CYP11B2, ma ad alte concentrazioni [mM] li aumenta. A779 non è diminuito in modo significativo il Ang (1-7) gli effetti, mentre irbesartan loro smussati. In linee cellulari umane surrenalici (HAC15 e NCI-H295R), il AT2R-agonisti C21 non ha avuto effetto a basse concentrazioni [Nm], ma ad alte concentrazioni [micron] è aumentata CYP11B1 e CYP11B2 espressione. Irbesartan smussati questi effetti Infine, effetto Dize non era potente nel CYP11B1 e l'espressione genica CYP11B2, ma può essere coinvolto in aumento dell'espressione genica ACE2. Conclusioni: L'espressione di AT2R e Masr nella corteccia surrenale umano è, rispettivamente di 10 volte e 150 volte inferiori rispetto AT1R. Inoltre, mentre AT1R e Masr geni sono espressi allo stesso livello nel sano corteccia surrenale e in APA, AT2R espressione genica è significativamente più bassa in APA. L'effetto secretagogo di Ang (1-7) e C21 sulla aldosterone e cortisolo produzione visto con alte concentrazioni probabilmente avviene attraverso AT1R. Così, Ang (1-7) e C21 non possono essere utilizzate come strumento terapeutico per diminuire aldosterone e cortisolo.

Il carcinoma corticosurrenalico (ACC) è un tumore raro e altamente aggressivo che origina nella corteccia surrenalica, con una prognosi infausta dovuta al suo fenotipo maligno e alla mancanza di opzioni terapeutiche efficaci. Il fatto che a tutt’oggi non siano ancora state sviluppate terapie specifiche si deve attribuire alla scarsa conoscenza dei meccanismi patogenetici di questo tipo di tumore. L’unico farmaco attualmente in uso per il trattamento dei pazienti con ACC ad uno stadio avanzato è il mitotane, di cui tuttavia non sono ancora stati studiati i possibili effetti tossici a livello delle cellule surrenali tumorali e i meccanismi coinvolti. Il primo obiettivo di questo lavoro di tesi si è pertanto incentrato sullo studio dei profili di espressione proteica del tumore e della ghiandola surrenale sana, allo scopo di individuare proteine differenzialmente espresse e di conseguenza in grado di discriminare i due gruppi sperimentali. Lo studio è stato condotto mediante l’utilizzo della tecnica di 2D-DIGE (Differential In-gel Electrophoresis), utilizzata per la prima volta per questo tipo di tumore, che ci ha permesso di confrontare e quantificare i profili proteici di 19 campioni bioptici (11 tessuti tumorali e 8 tessuti surrenalici sani) corsi su gel differenti, grazie alla presenza di uno standard interno costituito da un pool proteico di tutti i campioni analizzati. La spettrometria di massa associata all’analisi DIGE ha identificato 22 proteine differenzialmente espresse (con un’ average ratio ≤-2 o ≥2 e una significatività statistica p<0.05) tra la condizione patologica e quella normale. La maggior parte delle proteine sono risultate overespresse negli ACC, ad eccezione di una downregolata, la tiosolfato sulfotransferasi. Per confermare la modulazione delle proteine identificate, abbiamo utilizzato due diverse metodiche: Western Blotting e immunoistochimica; la scelta delle proteine da validare si è basata sul ruolo che alcune di esse rivestono in alcune vie di segnalazione coinvolte nella tumorigenesi e nella progressione tumorale, ed in particolare abbiamo confermato l’espressione differenziale di ALDH6A1, TRANSFERRINA, FASCINA1, LAMINA A/C, CAP1 e ADX REDUTTASI (fold icrease+SE di 7.5±1.4, 3.6±1.2, 2.9±0.2, 2.6±2.1, 1.9±1.4, 1.6±0.8, p<0.05, rispettivamente) negli ACC rispetto alle surreni sane. In conclusione, i nostri risultati preliminari hanno identificato un profilo proteomico specifico per gli ACC, che si differenzia da quello dei tessuti surrenalici sani, e, all’interno di esso, abbiamo osservato il coinvolgimento di numerosi enzimi mitocondriali che potrebbero rappresentare dei validi biomarcatori per la diagnosi e lo sviluppo di terapie targetspecifiche, se ulteriormente validati in una coorte più estesa di pazienti. Se da un lato sono ancora in gran parte sconosciuti i meccanismi molecolari alla base dello sviluppo e della progressione del tumore corticosurrenalico, e diventa quindi di fondamentale importanza l’utilizzo di differenti approcci per l’individuazione di nuovi possibili markers molecolari che permettano lo sviluppo di terapie più efficaci, dall’altro lato è altrettanto importante uno studio approfondito del meccanismo di azione del mitotane, che ad oggi rimane l’unico farmaco efficacie utilizzato per il trattamento dei pazienti con ACC allo stadio avanzato. Nonostante venga utilizzato da lungo tempo, infatti, non se ne conoscono i target intracellulari e molecolari, e uno studio approfondito potrebbe essere di aiuto per la scelta di eventuali molecole da somministrare in combinazione con esso per ridurne le dosi e ottenere degli effetti citotossici maggiori e maggiormente specifici. Nella seconda parte di questo lavoro, pertanto, siamo andati ad indagare gli effetti del mitotane a livello della linea cellulare di carcinoma corticosurrenalico, H295R, focalizzandoci sullo studio dei meccanismi intracellulari alla base del suo effetto tossico: in particolare, siamo andati ad analizzare le alterazioni nella morfologia e nella funzionalità dei mitocondri. Dopo aver osservato che il mitotane (DDD) viene metabolizzato e si accumula insieme al metabolita DDE all’interno delle cellule H295R in maniera dose-dipendente, abbiamo effettuato dei saggi di proliferazione che hanno confermato il suo effetto citotossico a livello delle cellule tumorali della corteccia surrenalica; in particolare, l’effetto del farmaco è evidente già a partire dalla dose di 10 micromolare dopo 48h di somministrazione, e raggiunge il suo effetto massimo dopo 7 giorni ad alte dosi (con un’inibizione della crescita tumorale del 92+1.12 % alla dose di 30 micromolare, corrispondente alla dose più bassa della finestra terapeutica del farmaco, dopo 7 giorni e significatività statistica p<0,0001). Successivamente siamo andati a valutare l’interessamento delle strutture intracellulari mediante analisi in microscopia elettronica: abbiamo osservato un’alterazione nella morfologia di questi organelli indotta dal mitotane in maniera dose- e tempo-dipendente; i mitocondri hanno mostrato un progressivo rigonfiamento, in concomitanza con una significativa diminuzione del numero delle creste interne. Dal momento che la riduzione nell’estensione del numero delle creste interne può interferire con la funzionalità mitocondriale, siamo andati a valutare se le alterazioni strutturali si riflettessero anche in un’alterazione della funzionalità di questi organelli. A questo scopo abbiamo valutato il potenziale di membrana in cellule vive in adesione, e abbiamo osservato una depolarizzazione dose-dipendente, che esita in una completa disgregazione degli organelli. Infine, per valutare se la depolarizzazione del potenziale di membrana influenzasse anche la respirazione, siamo andati a valutare il consumo di ossigeno in mitocondri vivi isolati da cellule H295R e abbiamo osservato una riduzione del consumo di ossigeno che sembra principalmente dovuta alla presenza di un danno di membrana (come confermato mediante esperimenti in Western Blotting) piuttosto che ad una specifica alterazione degli enzimi della catena respiratoria. Questi risultati contribuiscono a chiarire il meccanismo di azione del mitotane, mostrando che l’alterazione mitocondriale rappresenta uno dei principali bersagli dell’azione citotossica del farmaco. In conclusione, con questo lavoro sperimentale di tesi abbiamo individuato, mediante la tecnica 2D-DIGE, la presenza di un pattern proteico overespresso, specifico dell’ACC, e, all’interno di esso, abbiamo osservato il coinvolgimento di numerosi enzimi mitocondriali, che potrebbero rappresentare dei validi biomarcatori per la diagnosi dell’ACC. Con i dati di questo lavoro di tesi abbiamo quindi evidenziato che il mitocondrio rappresenta uno dei candidati più promettenti per lo sviluppo di nuove terapie target-specifiche, dal momento che anche il mitotane ha dimostrato di svolgere il suo effetto citotossico a livello di questo organello.