Adrenocortical Cancer Cell Research Articles

Gene silencing of beta-catenin in adrenocortical cancer cells causes growth inhibition and reversal of epithelial-to-mesenchymal transition

Adrenal carcinoma (ACC) is a rare endocrine neoplasm, notorious for its aggressive behavior. Aberrant expressions of β-catenin have been found in a third of ACC. Here, we studied its effects on the growth of the human ACC cell line H295R. The cells were infected with short hairpin RNA (shRNA)-mediated silencing β-catenin. Two shRNAs used induced down-regulation of β-catenin protein levels. The expression of these shRNAs decreased cell growth and increased H295R cells in S and G 2 /M phases. This cytostatic effect is due in part to a decrease of phosphorylated MAPK and to an up-regulation expression of the cyclin-dependent kinase inhibitors p57 KIP2 , p21 WAF1/CIP1 and p27 KIP1 . In addition, the knockdown of β-catenin decreased phosphorylated Akt level and increased apoptosis. Finally, loss of β-catenin was sufficient to induce the reversal of the epithelial-to-mesenchymal transition. We then transplanted these genetically modified H295R cells in Scid mice. The animals were sacrificed 46 days following transplantation. Tumor growth suppression was achieved by the two shRNAs showing in vitro efficacy. Proliferation was not significantly reduced in silenced tumors compared to control ones. In contrast, p57, p27 and p21 proteins were found expressed at high levels in silenced tumors, which might modulate cell cycle progression. Moreover, the depletion of β-catenin induced an increase in apoptotic cells. Taken together, these findings indicate that the blockade of β-catenin inhibits tumor growth of β-catenin-activated tumor cells and finally, shRNA technology may have potential therapeutic use in ACC.

Estrogen related receptor α (ERRα) a promising target for the therapy of adrenocortical carcinoma (ACC).

The pathogenesis of the adrenocortical cancer (ACC) involves integration of molecular signals and the interplay of different downstream pathways (i.e. IGFII/IGF1R, β-catenin, Wnt, ESR1). This tumor is characterized by limited therapeutic options and unsuccessful treatments. A useful strategy to develop an effective therapy for ACC is to identify a common downstream target of these multiple pathways. A good candidate could be the transcription factor estrogen-related receptor alpha (ERRα) because of its ability to regulate energy metabolism, mitochondrial biogenesis and signalings related to cancer progression. In this study we tested the effect of ERRα inverse agonist, XCT790, on the proliferation of H295R adrenocortical cancer cell line. Results from in vitro and in vivo experiments showed that XCT790 reduced H295R cell growth. The inhibitory effect was associated with impaired cell cycle progression which was not followed by any apoptotic event. Instead, incomplete autophagy and cell death by a necrotic processes, as a consequence of the cell energy failure, induced by pharmacological reduction of ERRα was evidenced. Our results indicate that therapeutic strategies targeting key factors such as ERRα that control the activity and signaling of bioenergetics processes in high-energy demanding tumors could represent an innovative/alternative therapy for the treatment of ACC.

Loss of β-catenin in adrenocortical cancer cells causes growth inhibition and reversal of epithelial-to-mesenchymal transition.

Adrenal carcinoma (ACC) is a rare neoplasm with a poor outcome. Aberrant expression of β-catenin has been found in approximatively 30% of ACC. We herein studied its effects on the growth of the human ACC cell line H295R. The cells were infected with short hairpin RNA (shRNA)-mediated silencing β-catenin. Two shRNAs used induced down-regulation of β-catenin protein levels. The expression of these shRNAs decreased cell growth and increased H295R cells in S and G2/M phases. This cytostatic effect is due to a decrease of phosphorylated MAPK and to an up-regulation expression of the cyclin-dependent kinase inhibitors p57(KIP2), p21(CIP) and p27(KIP1). In addition, the knockdown of β-catenin decreased phosphorylated Akt and increased apoptosis. Finally, loss of β-catenin was sufficient to induce the reversal of the epithelial-to-mesenchymal transition. We then transplanted these genetically modified H295R cells in Scid mice. Tumor growth suppression was achieved by the two shRNAs showing in vitro efficacy. Proliferation was not reduced in silenced tumors. In contrast, p57, p27 and p21 proteins were found expressed at high levels in silenced tumors along with an increase in apoptotic cells. These findings indicate that β-catenin loss in H295R cells inhibits tumor growth by inducing transcriptional and functional changes.

Focus on vitamin D and the adrenal gland.

The main role of vitamin D is to maintain calcium and phosphorus homeostasis, thus preserving bone health. However, recent evidences have demonstrated that vitamin D may also play a role in a variety of nonskeletal disorders such as endocrine diseases and in particular type 1 diabetes, type 2 diabetes, adrenal diseases, and the polycystic ovary syndrome. Despite controversial results on an association of low vitamin D levels with cortisol and aldosterone overproduction, encouraging in vitro findings have been reported on vitamin D effects in adrenocortical cancer cells. The focus of this review is the role of vitamin D in adrenal diseases and the results of vitamin D supplementation studies in patients. Although many studies support a beneficial role of vitamin D in adrenal disease, randomized controlled trials and mechanistic studies are required to provide more insight into the efficacy and safety of vitamin D as a therapeutic tool.

C-MYC modulation induces responsiveness to paclitaxel in adrenocortical cancer cell lines.

C-MYC is overexpressed in many types of cancer linked to poor prognosis. We examined the c-Myc protein expression in adrenocortical cancer (ACC) cells to investigate the role of this protein in the neoplasm, its involvement in chemotherapy and finally to determine whether c-Myc could be considered a prognostic factor in patients with ACC. H295R and SW13 cell lines were treated with paclitaxel. c-Myc overexpressing cell clones were achieved by transfecting the H295R cell line with the pcDNA3-hMYC plasmid expressing the full-lengh C-MYC coding sequence. The SW13 cell line was transfected with siRNA oligonucleotides for C-MYC. Cell cycle analysis was evaluated by flow cytometry. c-Myc, cyclin B1 and pro caspase expression levels were evaluated by western blot analysis. We found that expression of c-Myc was highly expressed in the SW13 cells, whereas the protein was undetectable in the H295R cells. Different doses of paclitaxel were required in the two ACC cell line to induce a block in the G2 phase, characterized by increased cyclin B1 levels and to induce apoptosis by pro-caspase-3 activation. Interestingly, the silencing of C-MYC mRNA prevented paclitaxel induced apoptosis in SW13 cells, whereas in the H295R cells the overexpression of C-MYC rendered the cells more prone to growth inhibition after paclitaxel exposure. The present study directly demonstrates that C-MYC plays a central role in controlling proliferation in ACC cells after paclitaxel treatment and that c-Myc could be considered as a marker for predicting response to chemotherapeutic agents in ACC cell lines.

RRM1 modulates mitotane activity in adrenal cancer cells interfering with its metabolization

The anti-proliferative activity of mitotane (o,p′DDD) in adrenocortical cancer is mediated by its metabolites o,p′DDE and o,p′DDA. We previously demonstrated a functional link between ribonucleotide reductase M1(RRM1) expression and o,p′DDD activity, but the mechanism is unknown. In this study we assessed the impact of RRM1 on the bioavailability and cytotoxic activity of o,p′DDD, o,p′DDE and o,p′DDA in SW13 and H295R cells. In H295R cells, mitotane and its metabolites showed a similar cytotoxicity and RRM1 expression was not influenced by any drug. In SW13 cells, o,p′DDA only showed a cytotoxic activity and did not modify RRM1 expression, whereas the lack of sensitivity to o,p′DDE was associated to RRM1 gene up-modulation, as already demonstrated for o,p′DDD. RRM1 silencing in SW13 cells increased the intracellular transformation of mitotane into o,p′DDE and o,p′DDA. These data demonstrate that RRM1 gene interferes with mitotane metabolism in adrenocortical cancer cells, as a possible mechanisms of drug resistance.

Adhesion to type V collagen enhances staurosporine-induced apoptosis of adrenocortical cancer cells.

Adrenocortical carcinoma (ACC) is a rare and aggressive tumor characterized by poor prognosis and resistance to conventional chemotherapy. Many chemotherapy agents act determining apoptosis, therefore, studying the responsiveness of ACC to apoptosis inducing molecules, can help to identify possible conditions to promote cancer cell death. Tumor progression is strictly related to the interaction between cancer cells and stroma; yet, extracellular matrix remodeling regulates tumor cell proliferation and apoptosis. At this purpose, we have studied staurosporine-induced apoptosis of ACC cell line H295R adherent to different extracellular matrix molecules. H295R cells grown on plastic showed a low responsiveness to staurosporine, with an apoptotic rate of 24 %, as compared to breast cancer MCF7 cells, with an apoptotic rate of 60 %. The adhesion of H295R cells to type V collagen induced a significant increase of apoptosis up to 52 %; this effect was inhibited by anti-integrin alpha2 antibody. At the same time, the adhesion of H295R cells on polylysine, matrigel, lamimin, fibronectin, and type I-III collagens didn't modify staurosporine-induced apoptosis. Staurosporine-treated H295R cells showed an increase of PARP cleavage and of annexin-V expression, when adherent to type V collagen. Yet, staurosporine induced Akt and Erk activation on H295R cells: the adhesion on type V collagen didn't modify Akt activation, while determined a dramatic inhibition of Erk activation. The described data demonstrate that the adhesion to type V collagen specifically increases the responsiveness of ACC cells to staurosporine-induced apoptosis and that this is probably obtained through the inhibition of Erk activation.

The anti-proliferative effect of anti-EGFR tyrosine kinase inhibitor in combination with mitotane on H295R adrenocortical cancer cells

The anti-proliferative effect of anti-EGFR tyrosine kinase inhibitor in combination with mitotane on H295R adrenocortical cancer cells

Pre-clinical assessment of the impact of Erlotinib on adrenocortical cancer cells proliferation

Pre-clinical assessment of the impact of Erlotinib on adrenocortical cancer cells proliferation

Morphofunctional effects of mitotane on mitochondria in human adrenocortical cancer cells

At present, mitotane (MTT) represents the first-line pharmacological approach for the treatment of advanced adrenocortical carcinoma (ACC). Despite clear evidence that the drug can reduce the clinical signs of steroid excess in secreting ACC, the mechanism mediating the possible toxic effect of MTT on tumor cells still remains obscure. This study investigated the intracellular events underlying the toxic effect of MTT by studying qualitative and quantitative alterations in mitochondrial morphology and functions in human adrenocortical cancer cell lines, H295R and SW13. Increasing concentrations of MTT resulted in rapid intracellular accumulation and conversion of the drug. Cytostatic and cytotoxic effects were evident at doses corresponding to the therapeutic window (30-50 μM) through an apoptotic mechanism involving caspase 3/7. Electron microscopic analysis of cell mitochondria displayed MTT-induced dose- and time-dependent alterations in the morphology of the organelle. These alterations were characterized by a marked swelling and a decrease in the number of respiratory cristae, accompanied by a significant depolarization of the mitochondrial membrane potential, finally leading to the disruption of the organelle. A drastic reduction of oxygen consumption was observed due to mitochondrial membrane damage, which was accompanied by a decrease in the levels of VDAC1 integral membrane channel. These findings contribute to better understand the intracellular mechanism of action of MTT in ACC cells, showing that its cytotoxic effect seems to be mainly mediated by an apoptotic process activated by the disruption of mitochondria.

Griseofulvin increases steroid hormone levels in the supernatant of adrenocortical cells in vitro

It has been shown that griseofulvin, which is an antifungal drug, inhibits growth of adrenocortical cancer cells in vitro. The inhibition of growth is caused by interactions with the microtubule apparatus. Interestingly, the microtubule apparatus is also involved in the regulation of steroidogenesis. Therefore we posed the question if griseofulvin also affects steroid hormone secretion. We have applied the NCI-H295R adrenocortical cancer cell line for the analysis of the effects of griseofulvin on adrenocortical steroid hormone secretion. Cells were incubated with or without griseofulvin at concentrations of 0.1µM, 1µM, 10µM, 40µM and 100µM for 24 hours. Concentrations of aldosterone, cortisol and dehydroepiandrostenedione (DHEA) were analyzed in the supernatants using Enzyme Linked Immunosorbent Assays (ELISAs). In addition, we measured concentrations of potassium levels to rule out that increased steroid hormone levels were a consequence of changed electrolyte concentrations. The incubation with griseofulvin leads to a dose dependent increase of Aldosterone-(approximately 2.3-fold), Cortisol-(approximately 1.3-fold) and DHEA-(approximately 4.3 fold) concentrations. The effects were, at least in part, independent from the concentration of potassium. Griseofulvin interfere with the microtubule apparatus and may disrupt regular trafficking of intracellular steroidal compounds. Apoptosis of adrenocortical cells may contribute to the release of steroid hormones. However, the exact mechanism how griseofulvin leads to increased concentrations of steroid hormones in the supernatant of adrenocortical cells is still unclear.

BMP5 treatment of adrenocortical carcinoma delays tumor growth via activation of endogenous anti-tumoral macrophages

Bone morphogenetic proteins (BMPs) are known to affect a variety of biological processes and also have been reported to modulate tumor growth. Former investigations revealed loss or downregulation of BMP5 in adrenocortical cancer (ACC) samples compared to normal adrenal glands and a deficit of downstream signalling in the human ACC cell line NCI h295R. Nude mice were subcutaneously xenografted with NCI h295R cells to establish solid tumors and treated with intra-tumoral administration of human recombinant (hr) BMP5 protein. Upon BMP5 delivery we observed a significant reduction in tumor size in comparison to PBS treated animals and a reduced proliferation rate (Ki-67 index) was evident. The canonical SMAD1/5/8 signalling pathway was not relevant in total tumor tissue with no detectable changes in human ID1, ID3, SMAD 6/7 and SMURF gene expression and SMAD1/5/8 phosphorylation. As BMP6 is reported to activate macrophages towards a pro-inflammatory anti-tumoral phenotype we were interested in a possible activating effect of BMP5. Expression of murine IL-12p40 was upregulated and a slight increase in the anti-angiogenic chemokines Mig (CXCL-9), IP-10 (CXCL-10) and murine macrophage marker F4/80 was detectable in BMP5 treated total tumor tissue. We also detected an increase in human CCL-2 (MCP1) expression, a monocyte attractant protein. Immunohistochemistry for galectin-3 showed influx of inflammatory macrophage-like cells into tumor tissue following BMP5 delivery. Murine macrophage cells (RAW 264.7) were treated with hrBMP5 and an increase in murine TNF-α expression and in the expression of the differentiation-related macrophage transcription factors PU.1 and IRF-5 was detected as well as upregulation of murine ID1 and ID3. We assume that the BMP5 mediated reduction in tumor growth might be achieved not via intracellular BMP signalling but rather by activation of anti-tumoral macrophages exerting anti-angiogenic, cytostatic or cytotoxic function on ACC tumors.

Silencing Mutated β-Catenin Inhibits Cell Proliferation and Stimulates Apoptosis in the Adrenocortical Cancer Cell Line H295R

ContextAdrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating β-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/β-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target.ObjectiveSimilar to patient tumor specimen the H295 cell line derived from an ACC harbors a natural activating β-catenin mutation. We herein assess the in vitro and in vivo effect of β-catenin inactivation using a doxycyclin (dox) inducible shRNA plasmid in H295R adrenocortical cancer cells line (clone named shβ).ResultsFollowing dox treatment a profound reduction in β-catenin expression was detectable in shβ clones in comparison to control clones (Ctr). Accordingly, we observed a decrease in Wnt/βcatenin-dependent luciferase reporter activity as well as a decreased expression of AXIN2 representing an endogenous β-catenin target gene. Concomitantly, β-catenin silencing resulted in a decreased cell proliferation, cell cycle alterations with cell accumulation in the G1 phase and increased apoptosis in vitro. In vivo, on established tumor xenografts in athymic nude mice, 9 days of β-catenin silencing resulted in a significant reduction of CTNNB1 and AXIN2 expression. Moreover, continous β-catenin silencing, starting 3 days after tumor cell inoculation, was associated with a complete absence of tumor growth in the shβ group while tumors were present in all animals of the control group.ConclusionIn summary, these experiments provide evidences that Wnt/β-catenin pathway inhibition in ACC is a promising therapeutic target.

P53 Stabilization Induces Cell Growth Inhibition and Affects IGF2 Pathway in Response to Radiotherapy in Adrenocortical Cancer Cells

Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.wtp53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases.

Effects of Mitotane on Gene Expression in the Adrenocortical Cell Line NCI-H295R: A Microarray Study

The adrenolytic agent mitotane is widely used in the treatment of adrenocortical cancer; however, its mechanism of action is poorly elucidated. We have studied mitotane-induced mRNA expression changes in the NCI-H295R adrenocortical cancer cell line. Cell viability and hormone assays were used to select the optimal mitotane concentration effectively inhibiting hormone secretion without affecting cell viability. RNA isolated from cultures treated for 48 and 72 h was subjected to Agilent 4×44K microarray platforms. Microarray results were validated by quantitative reverse-transcription PCR. Altogether, 117 significantly differentially expressed genes were detected at 48 h and 72 h (p < 0.05) in mitotane-treated samples relative to controls. Three significantly underexpressed genes involved in steroid hormone biosynthesis (HSD3B1, HSD3B2 and CYP21A2) and four significantly overexpressed genes (GDF15, ALDH1L2, TRIB3 and SERPINE2) have been validated. Gene-expression changes might be involved in the adrenal action of mitotane and in the inhibition of hormone secretion.

Efficacy of the novel dual PI3-kinase/mTOR inhibitor NVP-BEZ235 in a preclinical model of adrenocortical carcinoma

Adrenocortical cancer is a rare malignancy for which current pharmacological therapies are still insufficient. We tested the effect of a novel PI3 kinase – mammalian target of rapamycin dual inhibitor (NVP-BEZ235) on proliferation of the H295R adrenocortical cancer cell line in vitro and grown as xenografts in immunodeficient mice. NVP-BEZ235 was able to significantly inhibit phosporylation of Akt kinase and S6 ribosomal protein in H295R cells and to significantly reduce their proliferation in vitro and xenograft growth in vivo. The drug also induced activation of Erk phosphorylation, which could be inhibited by simultaneous treatment with the Erk inhibitor FR180204. This latter drug synergized with NVP-BEZ235 in the inhibition of H295R proliferation in vitro. Our data suggest that dual PI3K/mTOR inhibitors may represent a useful pharmacological tool in the therapy of advanced adrenocortical cancer and that simultaneous inhibition of both Erk and PI3K – mTOR pathways may be required to obtain a higher antiproliferative effect in this type of tumor.

MiR-484 regulates mitochondrial network through targeting Fis1

Mitochondria constantly undergo fusion and fission, two necessary processes for the maintenance of organelle fidelity. The abnormal mitochondrial fission participates in the pathogenesis of many diseases, but its regulation remains poorly understood. Here we show that miR-484 can suppress translation of mitochondrial fission protein Fis1, and inhibit Fis1-mediated fission and apoptosis in cardiomyocytes and in the adrenocortical cancer cells. We demonstrate that Fis1 is necessary for mitochondrial fission and apoptosis, and is upregulated during anoxia, whereas miR-484 is downregulated. miR-484 is able to attenuate Fis1 upregulation and mitochondrial fission, by binding to the amino acid coding sequence of Fis1 and inhibiting its translation. In exploring the underlying mechanism of miR-484 downregulation upon apoptosis, we observe that Foxo3a transactivates miR-484 expression. Foxo3a transgenic or knockout mice exhibit, respectively, a high or low level of miR-484 and a reduced or enhanced mitochondrial fission, apoptosis and myocardial infarction. Our data reveal a model of mitochondrial fission regulation by a microRNA.

Bitter Melon (Momordica charantia) Extract Suppresses Adrenocortical Cancer Cell Proliferation Through Modulation of the Apoptotic Pathway, Steroidogenesis, and Insulin-Like Growth Factor Type 1 Receptor/RAC-α Serine/Threonine-Protein Kinase Signaling

Adrenocortical carcinomas are rare but present with extremely poor prognosis. One of the approaches to control cancer progression and reduce cancer risk is prevention through diet. Bitter melon is widely consumed as a vegetable and especially as a traditional medicine in many countries. In this study, we have used human and mouse adrenocortical cancer cells as an in vitro model to assess the efficacy of bitter melon extract (BME) as an anticancer agent. The protein concentrations of BME and other extracts were measured before use. First, BME treatment of adrenocortical cancer cells resulted in a significantly dose-dependent decrease in cell proliferation. However, we did not observe an antiproliferative effect in adrenocortical cancer cells treated with extracts from blueberry, zucchini, and acorn squash. Second, apoptosis of adrenocortical cancer cells was accompanied by increased caspase-3 activation and poly(ADP-ribose) polymerase cleavage. BME treatment enhanced cellular tumor antigen p53, cyclin-dependent kinase inhibitor 1A (also called p21), and cyclic AMP-dependent transcription factor-3 levels and inhibited G1/S-specific cyclin D1, D2, and D3, and mitogen-activated protein kinase 8 (also called Janus kinase) expression, suggesting an additional mechanism involving cell cycle regulation and cell survival. Third, BME treatment decreased the key proteins involved in steroidogenesis in adrenocortical cancer cells. BME treatment decreased the level of phosphorylation of cyclin-dependent kinase 7, which is required, at least in part, for steroidogenic factor 1 activation. Finally, we observed that BME treatment significantly reduced the level of insulin-like growth factor 1 receptor and its downstream signaling pathway as evidenced by lower levels of phosphorylated RAC-α serine/threonine-protein kinase. Taken together, these data illustrate the inhibitory effect of bitter melon on cell proliferation of adrenocortical cancer through modulation of diverse mechanisms.

Abstract 5587: Bitter melon (Momordica charantia) extract suppresses adrenocortical cancer cell proliferation and growth through modulation of the cell cycle pathway and steroidogenic activity

Abstract Malignant adrenal tumors or adrenocortical carcinomas are relatively rare; however, this form of cancer is highly malignant and presents with extremely poor prognosis as a consequence of metastasis or local invasion at the time of diagnosis. One of the approaches to control cancer progression and reduce cancer risk is prevention through diet. Bitter melon is widely consumed as a vegetable and especially as a traditional medicine in China, India, Japan, and Taiwan. In this study, we have used human and mouse adrenocortical cancer cells, H295R and Y1, respectively, as an in vitro model to assess the efficacy of bitter melon extract (BME) as an anticancer agent. First, we find that BME treatment of adrenocortical cancer cells results in a significant dose-dependent decrease in cell proliferation and growth. We also find that bitter melon extract has stronger anti-proliferation effect than blueberry extract on adrenocortical cancer cells. As expected, extracts from Acorn squash and Zucchini, two vegetables from the same Family of Cucurbitaceae as bitter melon, do not decrease cell proliferation in adrenocortical cancer cells. Second, apoptosis of adrenocortical cancer cells is accompanied by increased caspase activation and poly(ADP-ribose) polymerase cleavage. Further studies reveal that BME treatment enhances p53, p21, Mdm2, and ATF3 and inhibits cyclin D1, cyclin D2, cycline D3, mTOR, and JNK expression, suggesting an additional mechanism involving cell cycle regulation, cell motility, cell survival, and cell differentiation. Third, BME treatment also decreases steroidogenic activities in adrenocortical cancer cells by Western blotting and reporter gene assay. BME treatment of adrenocortical cancer cells results in a significant dose-dependent decrease in levels of NR5A1/SF1, MC2R, StAR proteins. Interestingly, we find that BME treatment decreases the level of phospho-CDK7, which is required, at least in part, for NR5A1/SF1 activation. Taken together, these data illustrate that inhibitory effect of bitter melon on the growth and proliferation of adrenocortical cancer cells through modulation of the cell cycle regulation and steroidogenic pathways. Therefore, BME can be used as a dietary supplement for prevention of adrenocortical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5587. doi:10.1158/1538-7445.AM2011-5587

Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

Thiazolidinediones, specific peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-γ-inhibitor, showed that rosiglitazone acts through both PPAR-γ-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-γ. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPKα and beclin-1. The autophagy seems to be independent of PPAR-γ activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.