Stimulatory effect of estrogen on the growth of endometrial cancer cells is regulated by cell-cycle regulators

Estrogen is known as a major risk factor in tumorigenesis of the endometrium. The aim of this study is to establish stable estrogen-responsive endometrial cancer cell lines and to investigate the mechanism of estrogen action, focusing on cell-cycle regulation. Human wild-type estrogen receptor cDNA was transfected into endometrial cancer cells (Ishikawa) and estrogen-responsive cell lines were cloned. Their estrogen responsiveness was evaluated by the effect of estrogen on cellular growth and progesterone receptor expression. It was quantitatively estimated by immunocytochemistry or immunoblotting how the expression of cell-cycle regulators such as cyclin D1, cyclin E, Cyclin A, p53, p21 and p27 was regulated by estrogen. A cell line stably responsive to estrogen was established, and cells proliferated and the glandular structure was formed by estrogen stimulation. Cyclin D1 expression increased at 6–24 h and cyclin A gradually increased until 48 h of estrogen treatment compared with untreated cells. On the other hand, p53 and p21 expressions decreased at 6–24 h, and p27 gradually decreased until 24 h by estrogen. Our results show that the stimulatory effect of estrogen on cell proliferation may be regulated by the up-regulation of cyclin D1 and cyclin A, and down-regulation of p53, p21 and p27. This cell line is useful to clarify the molecular mechanism of estrogen action on endometrial cancer.     

Gene expression signature analysis identifies vorinostat as a candidate therapy for gastric cancer

Background

Gastric cancer continues to be one of the deadliest cancers in the world and therefore identification of new drugs targeting this type of cancer is thus of significant importance. The purpose of this study was to identify and validate a therapeutic agent which might improve the outcomes for gastric cancer patients in the future.

Methodology/Principal Findings

Using microarray technology, we generated a gene expression profile of human gastric cancer–specific genes from human gastric cancer tissue samples. We used this profile in the Broad Institute''s Connectivity Map analysis to identify candidate therapeutic compounds for gastric cancer. We found the histone deacetylase inhibitor vorinostat as the lead compound and thus a potential therapeutic drug for gastric cancer. Vorinostat induced both apoptosis and autophagy in gastric cancer cell lines. Pharmacological and genetic inhibition of autophagy however, increased the therapeutic efficacy of vorinostat, indicating that a combination of vorinostat with autophagy inhibitors may therapeutically be more beneficial. Moreover, gene expression analysis of gastric cancer identified a collection of genes (ITGB5, TYMS, MYB, APOC1, CBX5, PLA2G2A, and KIF20A) whose expression was elevated in gastric tumor tissue and downregulated more than 2-fold by vorinostat treatment in gastric cancer cell lines. In contrast, SCGB2A1, TCN1, CFD, APLP1, and NQO1 manifested a reversed pattern.

Conclusions/Significance

We showed that analysis of gene expression signature may represent an emerging approach to discover therapeutic agents for gastric cancer, such as vorinostat. The observation of altered gene expression after vorinostat treatment may provide the clue to identify the molecular mechanism of vorinostat and those patients likely to benefit from vorinostat treatment.     

Protein kinase C alpha (PKCα) regulates growth and invasion of endometrial cancer cells

The etiology of endometrial cancers remains poorly understood, particularly with respect to signal transduction pathways underlying the development and progression of the more aggressive, type II steroid‐independent tumors. Protein kinase C alpha (PKCα) regulates cellular processes critical to malignancy and has been implicated in the pathogenesis of endometrial cancers. The objective of these studies was to determine the functional role of PKCα in endometrial cancer cell proliferation, anchorage‐independent growth, and invasion. PKCα expression in endometrial cancer cell lines was examined by Western blotting. PKCα levels were increased in type II HEC‐50, HEC‐1‐A and HEC‐1‐B cell lines relative to the type I Ishikawa and RL‐95‐2 lines. Retroviral constructs were used to either overexpress PKCα or selectively knockdown levels by shRNA in Ishikawa and HEC 50 cells, respectively. Knockdown of PKCα expression in HEC‐50 cells resulted in a diminished growth rate and attenuation of anchorage‐independent growth. Correspondingly, Ishikawa cells overexpressing PKCα protein exhibited increased proliferation, resistance to growth factor deprivation and enhanced anchorage‐independent growth. Consistent with the observed changes in cell proliferation, PKCα also modulated cyclin D1 promoter activity in both cell lines. A reduction in PKCα levels rendered HEC‐50 cells significantly less invasive, whereas PKCα overexpression enhanced invasion of Ishikawa cells. These data indicate that PKCα promotes growth and invasion of endometrial cancer cells, suggesting that PKCα dependent signaling pathways could provide novel prognostic indicators or therapeutic targets, particularly in clinically aggressive type II endometrial tumors. J. Cell. Physiol. 220: 112–118, 2009. © 2009 Wiley‐Liss, Inc.     

Megestrol acetate drives endometrial carcinoma cell senescence via interacting with progesterone receptor B/FOXO1 axis

Megestrol acetate is a common and efficient anticancer progesterone. To explore the activity and the therapeutic mechanisms of megestrol acetate in endometrial cancer, human endometrial cancer cell lines Ishikawa and HHUA overexpressing progesterone receptor A (PR-A) and progesterone receptor B (PR-B) were treated with megestrol acetate. Cell viability, apoptosis, cycle arrest, and senescence, as well as the expressions of p21 and p16, two hallmarks of cellular senescence, were evaluated. Compared with the control, >10 nmol/L megestrol acetate treatment could significantly reduce endometrial cancer cell growth, and induce the irreversible G1 arrest and cell senescence. The expression of cyclin D1 in megestrol acetate treated cells was downregulated, while the expressions of p21 and p16 were upregulated via PR-B isoform. FOXO1 inhibitor AS1842856 could significantly abrogate megestrol acetate-induced cell senescence, suggesting that FOXO1 was involved in megestrol acetate/PR-B axis. These findings may provide a new understanding for the treatment of human endometrial cancer.     

Decorin antagonizes IGF receptor I (IGF-IR) function by interfering with IGF-IR activity and attenuating downstream signaling

We have recently discovered that the insulin-like growth factor receptor I (IGF-IR) is up-regulated in human invasive bladder cancer and promotes migration and invasion of transformed urothelial cells. The proteoglycan decorin, a key component of the tumor stroma, can positively regulate the IGF-IR system in normal cells. However, there are no available data on the role of decorin in modulating IGF-IR activity in transformed cells or in tumor models. Here we show that the expression of decorin inversely correlated with IGF-IR expression in low and high grade bladder cancers (n = 20 each). Decorin bound with high affinity IGF-IR and IGF-I at distinct sites and negatively regulated IGF-IR activity in urothelial cancer cells. Nanomolar concentrations of decorin promoted down-regulation of IRS-1, one of the critical proteins of the IGF-IR pathway, and attenuated IGF-I-dependent activation of Akt and MAPK. This led to decorin-evoked inhibition of migration and invasion upon IGF-I stimulation. Notably, decorin did not cause down-regulation of the IGF-IR in bladder, breast, and squamous carcinoma cells. This indicates that decorin action on the IGF-IR differs from its known activity on other receptor tyrosine kinases such as the EGF receptor and Met. Our results provide a novel mechanism for decorin in negatively modulating both IGF-I and its receptor. Thus, decorin loss may contribute to increased IGF-IR activity in the progression of bladder cancer and perhaps other forms of cancer where IGF-IR plays a role.     

Regulatory role of osteopontin in malignant transformation of endometrial cancer

Osteopontin (OPN) involves in the tumor-promoting or metastasis in human endometrial cancer. Depletion of OPN gene expression in endometrial cancer cells was significantly decreased in cell viability and the cells undergo apoptotic cell death. The status of OPN in THESC, RL95, Hec1A and Ishikawa cell lines were analyzed by RT-PCR and western blot. After OPN-siRNA transfection, mRNA and protein expression levels of OPN were determined in Hec1A and Ishikawa cells. Cell proliferation and cell cycle distribution were observed by MTT and flow cytometry analysis. DNA fragmentation assay was used to measure cell apoptosis. Cell migration was assessed by wound healing assay. Depletion of OPN gene expression in endometrial cancer cell lines (Hec1A and Ishikawa cells) reproducibly changed their ability of proliferation. Concomitant changes were seen in the expression of OPN binding cell surface receptors, cell cycle-regulatory genes, cell invasion and colony formation nature of the tumor cells. Decreased colonizing potential in the absence of OPN was reversed in the presence of recombinant OPN. Inhibition of anchorage-independent growth was observed in the presence of metabolic inhibitors of the PI3K, Src and integrin signaling cascades, which was ameliorated in the presence of exogenously added OPN. Our result showed the role of OPN in endometrial cancer, in particular on the malignancy-promoting aspects of OPN that may pave way for new approaches to the clinical management of endometrial cancer.     

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

Background

Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

Methods

HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity^® Pathway Analysis.

Results

Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

Conclusion

This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.     

Regulation of transforming growth factor gene expression in human endometrial adenocarcinoma cells

We have examined the effects of medroxyprogesterone acetate (MPA) and 4-hydroxytamoxifen (OH-TAM) on the cell proliferation and the expression of TGF- and TGF-β genes in Ishikawa cells and HEC-50 human endometrial adenocarcinoma cells. The effects of exogenous TGF-, TGF-β and anti-EGF receptor monoclonal antibody on cell proliferation were also determined. Antisense oligonucleotides were used to determine the effects of endogenous expression of TGF- and TGF-β. In both cell lines, MPA resulted in a time and dose-dependent inhibition of cell proliferation whereas OH-TAM had no effect on HEC-50 cell proliferation. The relative abundance of TGF- mRNA was significantly reduced by MPA in Ishikawa cells but not in HEC-50 cells. In Ishikawa cells, a reduction in TGF- mRNA abundance was observed with OH-TAM under conditions where both inhibition and stimulation of cell proliferation were demonstrated. Anti-EGF receptor monoclonal antibody inhibited Ishikawa cell growth but had little effect on HEC-50 cell proliferation. Exogenous TGF- stimulated proliferation of both cell lines whereas exogenous TGF-β inhibited proliferation of Ishikawa cells but stimulated proliferation of HEC-50 cells. Antisense oligonucleotides to TGF-β inhibited proliferation of HEC-50 cells. From these data we conclude that the antiproliferative effects of progestins and OH-TAM on endometrial cancer cells appear to be mediated by different mechanisms.     

Terpenoids from Zingiber officinale (Ginger) Induce Apoptosis in Endometrial Cancer Cells through the Activation of p53

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC₅₀ of 1.25 µg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC₅₀ 10 µM (2.3 µg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53^neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer.     

Effects of N-[N-(3, 5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) on cell proliferation and apoptosis in Ishikawa endometrial cancer cells

Endometrial cancer is one of the most common gynecological malignancies in Japan, where the disease shows an increasing morbidity. However, surgical therapy remains the treatment of choice for endometrial cancers that tend to be insensitive to radiation therapy and chemotherapy. Therefore, novel therapeutic strategies are required. The Notch signaling pathway regulates embryogenesis and cellular development, but deregulated Notch signaling may contribute to tumorigenesis in several cancers. Moreover, γ-secretase inhibitors have been shown to be potent inhibitors of the Notch signaling pathway; they suppress cellular proliferation and induce apoptosis in several cancer cells. In the present study, we investigated the effect of N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, γ-secretase inhibitor) on the cell proliferation and apoptosis in Ishikawa endometrial cancer cells. Real-time PCR detected mRNA derived from NOTCH1 and HES1, which are target genes of the Notch signaling pathway, in Ishikawa endometrial cancer cells. After blocking Notch signaling, cellular proliferation decreased, accompanied by increased expression of p21 mRNA and decreased expression of the cyclin A protein. Furthermore, blockade of Notch signaling induced apoptosis. These results suggest that the Notch signaling pathway may be involved in cell proliferation through cell cycle regulation and apoptosis in Ishikawa endometrial cancer cells. Inhibition of the Notch signaling pathway by γ-secretase inhibitors is expected to be a potential target of novel therapeutic strategies for endometrial cancer.     

The Reciprocal Regulation of γ-Synuclein and IGF-I Receptor Expression Creates a Circuit That Modulates IGF-I Signaling

Insulin-like growth factor (IGF) system plays important roles in carcinogenesis and maintenance of the malignant phenotype. Signaling through the IGF-I receptor (IGF-IR) has been shown to stimulate the growth and motility of a wide range of cancer cells. γ-Synuclein (SNCG) is primarily expressed in peripheral neurons but also overexpressed in various cancer cells. Overexpression of SNCG correlates with tumor progression. In the present study we demonstrated a reciprocal regulation of IGF-I signaling and SNCG expression. IGF-I induced SNCG expression in various cancer cells. IGF-IR knockdown or IGF-IR inhibitor repressed SNCG expression. Both phosphatidylinositol 3-kinase and mitogen-activated protein kinase were involved in IGF-I induction of SNCG expression. Interestingly, SNCG knockdown led to proteasomal degradation of IGF-IR, thereby decreasing the steady-state levels of IGF-IR. Silencing of SNCG resulted in a decrease in ligand-induced phosphorylation of IGF-IR and its downstream signaling components, including insulin receptor substrate (IRS), Akt, and ERK1/2. Strikingly, SNCG physically interacted with IGF-IR and IRS-2. Silencing of IRS-2 impaired the interaction between SNCG and IGF-IR. Finally, SNCG knockdown suppressed IGF-I-induced cell proliferation and migration. These data reveal that SNCG and IGF-IR are mutually regulated by each other. SNCG blockade may suppress IGF-I-induced cell proliferation and migration. Conversely, IGF-IR inhibitors may be of utility in suppressing the aberrant expression of SNCG in cancer cells and thereby block its pro-tumor effects.     

Valproic acid inhibits the growth of cervical cancer both in vitro and in vivo

Valproic acid (VPA), a well-known anti-convulsant, is currently under extensive evaluation as an anti-cancer agent. It is known to exert its anti-cancer effect mainly by inhibiting the enzyme histone deacetylase I. In our study, we investigated the effects of VPA on cervical cancer both in vitro and in vivo cancer models. We examined the effects of acute VPA (0, 1.2, 2.4, 5.0 mM) treatment on cell proliferation in cervical cancer cell lines HeLa, SiHa and Ca Ski and histone acetylation, p21 and p53 gene expression in HeLa cell line. We also investigated the effect of chronic VPA administration in tumour xenograft growth studies. Our results show that with acute treatment, VPA can increase the expression of net histone H3 acetylation and up-regulate p21 expression with no effect on p53 expression. Chronic administration of VPA had a net cytostatic effect that resulted in a statistically significant reduction of tumour growth and improved survival advantages in tumour xenografts studies. Furthermore, we also demonstrated that VPA has a direct anti-angiogenic effect in tumour studies and could potentially be a promising candidate for further cervical cancer trails.     

Curcumin exhibits anti-tumor effect and attenuates cellular migration via Slit-2 mediated down-regulation of SDF-1 and CXCR4 in endometrial adenocarcinoma cells

Although curcumin shows anti-proliferative and anti-inflammatory activities in various cancers, the effect of curcumin on cellular migration in endometrial adenocarcinoma cells remains to be understood. The current investigation was aimed to explore the anti-proliferative and anti-migratory effects of curcumin and its mechanism of action in endometrial cancer cells. Our in-vitro and in-vivo experimental studies showed that curcumin inhibited the proliferation of endometrial cancer cells and suppressed the tumor growth in Ishikawa xenograft mouse model. Curcumin induced ROS-mediated apoptosis in endometrial cancer cells. Curcumin suppressed the migration rate of Ishikawa and Hec-1B cells as analyzed by scratch wound assay. In transwell migration studies, knock down of Slit-2 reversed the anti-migratory effect of curcumin in these cell lines. Curcumin significantly up-regulated the expression of Slit-2 in Ishikawa, Hec-1B and primary endometrial cancer cells while it down-regulated the expression of stromal cell-derived factor-1 (SDF-1) and CXCR4 which in turn, suppressed the expression of matrix metallopeptidases (MMP) 2 and 9, thus attenuating the migration of endometrial cancer cells. In summary, we have demonstrated that curcumin has inhibitory effect on cellular migration via Slit-2 mediated down-regulation of CXCR4, SDF-1, and MMP2/MMP9 in endometrial carcinoma cells. These findings helped explore the role of Slit-2 in endometrial cancer cells.     

Insulin-like growth factor I receptor involvement in proliferation of NOR-P1 cells in serum-free media

Insulin-like growth factor (IGF)-I is up-regulated in pancreatic cancer tissues. Pancreatic cancer cell lines were analyzed in serum-free media as a model of the fibrous tissues that these cells often invade. Pancreatic cancer surgical specimens were immunostained with anti-IGF-I receptor (IGF-IR)β antibody. The growth of pancreatic cancer cells in serum-free media was also analyzed. Cell lysates were analyzed for protein by western blot analysis. Cells cultured in the presence of picropodophyllin (PPP), LY294002, or PD98059, were subjected to cell proliferation and scratch assays. In addition, BrdU uptake and apoptosis were analyzed in these cells. IGF-IRβ was detected in pancreatic cancer cells invading fibrous tissues. NOR-P1 grew most rapidly in serum-free media. The concentrations of IGF-I and IGF-II in the media were higher in NOR-P1 than the other cell lines. Cell proliferation in NOR-P1 cells was enhanced by IGF-I or IGF-II treatment more than in MIA-Paca2 or PK-1 cells. PPP, LY294002, and PD98059 suppressed proliferation and motility of NOR-P1 cells and inhibited BrdU uptake, while PPP induced apoptosis. IGF-IRβ may be a potential therapeutic target to inhibit invasion of pancreatic cancer.