Androgen receptor status predicts response to chemotherapy, not risk of breast cancer in Indian women

Voltage gated potassium channels have been extensively studied in relation to cancer. In this review, we will focus on the role of two potassium channels, Ether à-go-go (Eag), Human ether à-go-go related gene (HERG), in cancer and their potential therapeutic utility in the treatment of cancer. Eag and HERG are expressed in cancers of various organs and have been implicated in cell cycle progression and proliferation of cancer cells. Inhibition of these channels has been shown to reduce proliferation both in vitro and vivo studies identifying potassium channel modulators as putative inhibitors of tumour progression. Eag channels in view of their restricted expression in normal tissue may emerge as novel tumour biomarkers.     

Calcitriol inhibits Ether-à go-go potassium channel expression and cell proliferation in human breast cancer cells

Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is known regarding the molecular basis of this process in breast carcinoma. Ether-à-go-go (Eag1) potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Since calcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factors antagonically regulate cell cycle progression, we investigated a possible regulatory effect of calcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferative activity of this hormone in human breast tumor-derived cells. RT real-time PCR and immunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor (VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation, which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistry and Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductal carcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferative effects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment and prevention.     

Resistance to cisplatin does not affect sensitivity of human ovarian cancer cell lines to mifepristone cytotoxicity

Background  

The prototypical antiprogestin mifepristone exhibits potent growth inhibition activity towards ovarian cancer cells in vitro and in vivo. The aim of this research was to establish whether mifepristone is capable of inhibiting cell proliferation and inducing apoptotic cell death regardless of the degree of sensitivity ovarian cancer cells exhibit to cisplatin.     

The potassium ion channel opener NS1619 inhibits proliferation and induces apoptosis in A2780 ovarian cancer cells

Diverse types of voltage-gated potassium (K⁺) channels have been shown to be involved in regulation of cell proliferation. The maxi-conductance Ca²⁺-activated K⁺ channels (BK channels) may play an important role in the progression of human cancer. To explore the role of BK channels in regulation of apoptosis in human ovarian cancer cells, the effects of the specific BK channel activator NS1619 on induction of apoptosis in A2780 cells were observed. Following treatment with NS1619, cell proliferation was measured by MTT assay. Apoptosis of A2780 cells pretreated with NS1619 was detected by agarose gel electrophoresis of cellular DNA and flow cytometry. Our data demonstrate that NS1619 inhibits the proliferation of A2780 cells in a dosage and time dependent manner IC₅₀ = 31.1 μM, for 48 h pretreatment and induces apoptosis. Western blot analyses showed that the anti-proliferation effect of NS1619 was associated with increased expression of p53, p21, and Bax. These results indicate that BK channels play an important role in regulating proliferation of human ovarian cancer cells and may induce apoptosis through induction of p21^Cip1 expression in a p53-dependent manner.     

Tumour suppressive function and modulation of programmed cell death 4 (PDCD4) in ovarian cancer

Background

Programmed cell death 4 (PDCD4), originally identified as the neoplastic transformation inhibitor, was attenuated in various cancer types. Our previous study demonstrated a continuous down-regulation of PDCD4 expression in the sequence of normal-borderline-malignant ovarian tissue samples and a significant correlation of PDCD4 expression with disease-free survival. The objective of the current study was to further investigate the function and modulation of PDCD4 in ovarian cancer cells.

Principal Findings

We demonstrated that ectopic PDCD4 expression significantly inhibited cell proliferation by inducing cell cycle arrest at G₁ stage and up-regulation of cell cycle inhibitors of p27 and p21. Cell migration and invasion were also inhibited by PDCD4. PDCD4 over-expressing cells exhibited elevated phosphatase and tensin homolog (PTEN) and inhibited protein kinase B (p-Akt). In addition, the expression of PDCD4 was up-regulated and it was exported to the cytoplasm upon serum withdrawal treatment, but it was rapidly depleted via proteasomal degradation upon serum re-administration. Treatment of a phosphoinositide 3-kinase (PI3K) inhibitor prevented the degradation of PDCD4, indicating the involvement of PI3K-Akt pathway in the modulation of PDCD4.

Conclusion

PDCD4 may play a critical function in arresting cell cycle progression at key checkpoint, thus inhibiting cell proliferation, as well as suppressing tumour metastasis. The PI3K-Akt pathway was implied to be involved in the regulation of PDCD4 degradation in ovarian cancer cells. In response to the stress condition, endogenous PDCD4 was able to shuttle between cell compartments to perform its diverted functions.     

ALDH1A1 Maintains Ovarian Cancer Stem Cell-Like Properties by Altered Regulation of Cell Cycle Checkpoint and DNA Repair Network Signaling

Objective

Aldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance.

Methods

Isogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators.

Results

ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDH^HIGH displayed significantly lower progression free survival than the patients with ALDH^LOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells.

Conclusion

This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.     

Whole ovary immunohistochemistry for monitoring cell proliferation and ovulatory wound repair in the mouse

Background  

Ovarian surface epithelial cells are thought to be a precursor cell type for ovarian carcinoma. It has been proposed that an increased rate of ovarian surface epithelial cell proliferation during ovulatory wound repair contributes to the accumulation of genetic changes and cell transformation. The proliferation of ovarian surface epithelial cells during ovulatory wound repair has been studied primarily using immunohistochemical staining of paraffin-embedded ovary sections. However, such analyses require complex reconstruction from serially-cut ovary sections for the visualization and quantification of the cells on the ovarian surface. In order to directly visualize the proliferation and organization of the ovarian surface epithelial cells, we developed a technique for immunohistochemical staining of whole mouse ovaries. Using this method, we analyzed cell proliferation and morphologic changes in mouse ovarian surface epithelial cells during follicle growth and ovulatory wound repair.     

Targeting cleavage and polyadenylation specific factor 1 via shRNA inhibits cell proliferation in human ovarian cancer

Cleavage and polyadenylation specificity factor 1 (CPSF1), a member of CPSF complex, has been reported to play a key role in pre-mRNA 3′-end formation, but its possible role in ovarian cancer remains unclear. In the present study, we found the mRNA level of CPSF1 was overexpressed in ovarian cancer tissues using Oncomine Cancer Microarray database. Then the loss-of-function assays, including CCK-8, colony formation and flow cytometry assays, were performed to determine the effects of CPSF1 on cell viability, proliferation, cell cycle and apoptosis of human ovarian cancer cell lines (SKOV-3 and OVCAR-3). The results indicated that depletion of CPSF1 suppressed cell viability, impaired colony formation ability, induced cell cycle arrest at G0/G1 phase and promoted cell apoptosis in ovarian cancer cells. Furthermore, knockdown of CPSF1 upregulated the expression of cleaved caspase-3 and PARP and downregulated CDK4/cyclin D1 expression. These data suggested that CPSF1 could promote ovarian cancer cell growth and proliferation in vitro and its depletion might serve as a potential therapeutic target for human ovarian cancer.     

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells

Background

Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays.

Principal Findings

LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis.

Conclusion

LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.     

Specific thiazolidinediones inhibit ovarian cancer cell line proliferation and cause cell cycle arrest in a PPARγ independent manner

Background

Peroxisome Proliferator Activated Receptor gamma (PPARγ) agonists, such as the thiazolinediones (TZDs), have been studied for their potential use as cancer therapeutic agents. We investigated the effect of four TZDs—Rosiglitazone (Rosi), Ciglitazone (CGZ), Troglitazone (TGZ), and Pioglitazone (Pio)—on ovarian cancer cell proliferation, PPARγ expression and PPAR luciferase reporter activity. We explored whether TZDs act in a PPARγ dependent or independent manner by utilizing molecular approaches to inhibit or overexpress PPARγ activity.

Principal Findings

Treatment with CGZ or TGZ for 24 hours decreased proliferation in three ovarian cancer cell lines, Ovcar3, CaOv3, and Skov3, whereas Rosi and Pio had no effect. This decrease in Ovcar3 cell proliferation was due to a higher fraction of cells in the G₀/G₁ stage of the cell cycle. CGZ and TGZ treatment increased apoptosis after 4 hours of treatment but not after 8 or 12 hours. Treatment with TGZ or CGZ increased PPARγ mRNA expression in Ovcar3 cells; however, protein levels were unchanged. Surprisingly, luciferase promoter assays revealed that none of the TZDs increased PPARγ activity. Overexpression of wild type PPARγ increased reporter activity. This was further augmented by TGZ, Rosi, and Pio indicating that these cells have the endogenous capacity to mediate PPARγ transactivation. To determine whether PPARγ mediates the TZD-induced decrease in proliferation, cells were treated with CGZ or TGZ in the absence or presence of a dominant negative (DN) or wild type overexpression PPARγ construct. Neither vector changed the TZD-mediated cell proliferation suggesting this effect of TZDs on ovarian cancer cells may be PPARγ independent.

Conclusions

CGZ and TGZ cause a decrease in ovarian cancer cell proliferation that is PPARγ independent. This concept is supported by the finding that a DN or overexpression of the wild type PPARγ did not affect the changes in cell proliferation and cell cycle.     

K+ Channel Expression in Human Breast Cancer Cells: Involvement in Cell Cycle Regulation and Carcinogenesis

K⁺ channels are a most diverse class of ion channels in the plasma membrane and are distributed widely throughout a variety of cells including cancer cells. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K⁺ channels and that these K⁺ channels play important roles in regulating tumor cell proliferation, cell cycle progression and apoptosis. Moreover, a significant increase in K⁺ channel expression has been correlated with tumorigenesis, suggesting the possibility of using these proteins as transformation markers and perhaps reducing the tumor growth rate by selectively inhibiting their functional activity. Significant progress has been made in defining the properties of breast K⁺ channels, including their biophysical and pharmacological properties and distribution throughout different phases of the cell cycle in breast cell line MCF-7. This review aims to provide a comprehensive overview of the current state of research into K⁺ channels/currents in breast cancer cells. The possible mechanisms by which K⁺ channels affect tumor cell proliferation and cell cycle progression are discussed.     

SPARC Is a Key Regulator of Proliferation, Apoptosis and Invasion in Human Ovarian Cancer

Background

Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, is implicated in the progression of many cancers. In this study, we investigated the expression and function of SPARC in ovarian cancer.

Methods

cDNA microarray analysis was performed to compare gene expression profiles of the highly invasive and the low invasive subclones derived from the SKOV3 human ovarian cancer cell line. Immunohistochemistry (IHC) staining was performed to investigate SPARC expression in a total of 140 ovarian tissue specimens. In functional assays, effects of SPARC knockdown on the biological behavior of ovarian cancer cells were investigated. The mechanisms of SPARC in ovarian cancer proliferation, apoptosis and invasion were also researched.

Results

SPARC was overexpressed in the highly invasive subclone compared with the low invasive subclone. High SPARC expression was associated with high stage, low differentiation, lymph node metastasis and poor prognosis of ovarian cancer. Knockdown of SPARC expression significantly suppressed ovarian cancer cell proliferation, induced cell apoptosis and inhibited cell invasion and metastasis.

Conclusion

SPARC is overexpressed in highly invasive subclone and ovarian cancer tissues and plays an important role in ovarian cancer growth, apoptosis and metastasis.     

Involvement of STIM1 and Orai1 in EGF-mediated cell growth in retinal pigment epithelial cells

Background

In non-excitable cells, one major route for calcium entry is through store-operated calcium (SOC) channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca²⁺ store. STIM1 and Orai1 are major regulators of SOC channels. In this study, we explored the functions of STIM1 and Orai1 in epidermal growth factor (EGF)-induced cell proliferation and migration in retinal pigment epithelial cells (ARPE-19 cell line).

Results

EGF triggers cell proliferation and migration in ARPE-19 cells. Cell proliferation and migration involve STIM1 and Orai1, as well as phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, and Akt. Pharmacological inhibitors of SOC channels and siRNA of Orai1 and STIM1 suppress cell proliferation and migration. Pre-treatment of mitogen-activated protein kinase kinase (MEK) inhibitors and a phosphatidylinositol 3 kinases (PI3K) inhibitor attenuated cell proliferation and migration. However, inhibition of the SOC channels failed to prevent EGF-mediated ERK 1/2 and Akt phosphorylation.

Conclusions

Our results showed that STIM1, Orai1, ERK 1/2, and Akt are key determinants of EGF-mediated cell growth in ARPE-19 cells. EGF is a potent growth molecule that has been linked to the development of PVR, and therefore, STIM1, Orai1, as well as the MEK/ERK 1/2 and PI3K/Akt pathways, might be potential therapeutic targets for drugs aimed at treating such disorders.     

Growth inhibition of androgen-responsive prostate cancer cells with brefeldin A targeting cell cycle and androgen receptor

Background  

Androgen ablation is one of the viable therapeutic options for patients with primary hormone (androgen)-dependent prostate cancer. However, an antibiotic brefeldin A (BFA) has been shown to exhibit the growth inhibitory effect on human cancer cells. We thus investigated if BFA might inhibit proliferation of androgen-responsive prostate cancer LNCaP cells and also explored how it would be carried out, focusing on cell cycle and androgen receptor (AR).     

Roflumilast enhances cisplatin‐sensitivity and reverses cisplatin‐resistance of ovarian cancer cells via cAMP/PKA/CREB‐FtMt signalling axis

Objective

We previously demonstrated the roflumilast inhibited cell proliferation and increased cell apoptosis in ovarian cancer. In this study, we aimed to investigate the roles of roflumilast in development of cisplatin (DDP)‐sensitive and ‐resistant ovarian cancer.

Methods

OVCAR3 and SKOV3 were selected and the corresponding DDP‐resistant cells were constructed. Cell viability, proliferation, apoptosis, cycle were performed. Expression cAMP, PKA, CREB, phosphorylation of CREB and FtMt were detected. The roles of roflumilast in development of DDP‐sensitive and ‐resistant ovarian cancer were confirmed by xenograft model.

Results

Roflumilast + DDP inhibited cell proliferation, and induced cell apoptosis and G0/G1 arrest in OVCAR3 and SKOV3 cells, roflumilast induced expression of FtMt, the activity of cAMP and PKA and phosphorylation of CREB in ovarian cancer cells and the above‐effect were inhibited by H89. Downregulation of CREB inhibited the roflumilast‐increased DDP sensitivity of ovarian cancer cells, and the roflumilast‐induced FtMt expression and phosphorylation of CREB. Also, roflumilast reversed cisplatin‐resistance, and induced expression of FtMt and activation of cAMP/PKA/CREB in DDP‐resistant ovarian cancer cells. Similarly, treated with H89 or downregulation of CREB inhibited the changes induced by roflumilast. In vivo, roflumilast inhibited the development of SKOV3 or SKOV3‐DDP‐R xenograft models.

Conclusions

Roflumilast enhanced DDP sensitivity and reversed the DDP resistance of ovarian cancer cells via activation of cAMP/PKA/CREB pathway and upregulation of the downstream FtMt expression, which has great promise in clinical treatment.

     

Functional characterization of Trip10 in cancer cell growth and survival

Background

The Cdc42-interacting protein-4, Trip10 (also known as CIP4), is a multi-domain adaptor protein involved in diverse cellular processes, which functions in a tissue-specific and cell lineage-specific manner. We previously found that Trip10 is highly expressed in estrogen receptor-expressing (ER⁺) breast cancer cells. Estrogen receptor depletion reduced Trip10 expression by progressively increasing DNA methylation. We hypothesized that Trip10 functions as a tumor suppressor and may be involved in the malignancy of ER-negative (ER^-) breast cancer. To test this hypothesis and evaluate whether Trip10 is epigenetically regulated by DNA methylation in other cancers, we evaluated DNA methylation of Trip10 in liver cancer, brain tumor, ovarian cancer, and breast cancer.

Methods

We applied methylation-specific polymerase chain reaction and bisulfite sequencing to determine the DNA methylation of Trip10 in various cancer cell lines and tumor specimens. We also overexpressed Trip10 to observe its effect on colony formation and in vivo tumorigenesis.

Results

We found that Trip10 is hypermethylated in brain tumor and breast cancer, but hypomethylated in liver cancer. Overexpressed Trip10 was associated with endogenous Cdc42 and huntingtin in IMR-32 brain tumor cells and CP70 ovarian cancer cells. However, overexpression of Trip10 promoted colony formation in IMR-32 cells and tumorigenesis in mice inoculated with IMR-32 cells, whereas overexpressed Trip10 substantially suppressed colony formation in CP70 cells and tumorigenesis in mice inoculated with CP70 cells.

Conclusions

Trip10 regulates cancer cell growth and death in a cancer type-specific manner. Differential DNA methylation of Trip10 can either promote cell survival or cell death in a cell type-dependent manner.     

Novel roles for hERG K+ channels in cell proliferation and apoptosis

The human ether-a-go-go-related gene potassium channel (hERG, Kv11.1, KCNH2) has an essential role in cardiac action potential repolarization. Electrical dysfunction of the voltage-sensitive ion channel is associated with potentially lethal ventricular arrhythmias in humans. hERG K⁺ channels are also expressed in a variety of cancer cells where they control cell proliferation and apoptosis. In this review, we discuss molecular mechanisms of hERG-associated cell cycle regulation and cell death. In addition, the significance of hERG K⁺ channels as future drug target in anticancer therapy is highlighted.