Perifosine inhibits growth of human experimental endometrial cancers by blockade of AKT phosphorylation

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Abstract

Objective

Perifosine is an orally active alkylphospholipid analog, which has shown anti-tumor activity in a variety of cancers by inhibition of AKT phosphorylation. The objective of the current study was to evaluate its efficacy in in vitro models of human endometrial cancer.

Study design

The effect of 10 μM and 40 μM perifosine on AKT phophorylation in human endometrial cancer cell lines Ishikawa and HEC 1A was determined by Western blotting. To screen for a putative anti-tumor effect, HEC 1A and Ishikawa cells were incubated with increasing concentrations of perifosine for 24 h, 48 h and 72 h and the number of viable cells was determined by crystal violet staining. Also the effect of a combined treatment with cisplatin and perifosine was investigated in Ishikawa cells. Flow cytometric analysis of DNA content was used to determine the effect of perifosine on the cell cycle distribution of HEC 1A and Ishikawa cells and to assess potential toxic side effects of perifosine on peripheral blood lymphocytes (PBL).

Results

AKT phosphorylation was dose-dependently inhibited by perifosine. Concomitantly, perifosine displayed anti-tumor activity in both cell lines at concentrations that showed no effect on peripheral blood lymphocytes. Growth inhibitory effects became more pronounced with increasing treatment time. While IC 50 values at 24 h were >40 μM, IC 50 values after 48 h were approximately 7 μM in Ishikawa and 25 μM in HEC 1A cells. After 72 h, the IC 50 was below 1.25 μM for Ishikawa and about 6 μM for HEC 1A cells. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in human Ishikawa endometrial cancer cells. Of note, the anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle.

Conclusions

The small molecule AKT inhibitor perifosine showed substantial anti-tumor activity in human endometrial cancer cell lines. Since these effects were increased with cisplatin, perifosine seems to be a good candidate for treatment combinations with classical cytostatic compounds. Thus, perifosine should be further evaluated in clinical studies in endometrial cancer.

Introduction

Endometrial carcinoma is the fourth most common cancer and the eighth leading cause of death from malignancy in women. With 40,000 new cases resulting in 6800 deaths every year in the US alone, it is the most common neoplasm of the female genital tract and accounts for almost half of all gynecologic cancers. Lifetime risk of developing endometrial cancer is about 2–3% [1]. Primary surgery followed by radiation has become the most widely accepted treatment for endometrial cancer. In the case of late-stage or recurrent disease systemic, hormonal or chemotherapy are added. Early-stage endometrial cancer carries a good prognosis, with 5-year survival rates of 87% and 76% for FIGO stages I and II, respectively [2]. However, 5-year survival rate drops to 59% (FIGO III) and 18% (FIGO IV) in patients with late-stage disease [2]. Overall survival is poorest in patients with recurrent endometrial cancer (7.7%) [3]. Accordingly, new therapeutic modalities are needed for the treatment of late-stage and recurrent endometrial cancer.

The serine/threonine kinase AKT/PKB pathway presents a promising new target for molecular therapeutics, as it integrates both extracellular and intracellular oncogenic signals. Alterations of the AKT pathway have been detected in a variety of human malignancies including endometrial cancer [4]. AKT has a wide range of downstream targets that regulate tumor-associated cell processes such as cell growth, cell cycle progression, survival, migration, epithelial–mesenchymal transition and angiogenesis [5]. Blockage of AKT signaling results in programmed cell death (PCD) and growth inhibition of tumor cells with elevated AKT. For this reason, AKT has become a promising drug target for cancer therapy.

The PTEN tumor suppressor gene is a central negative regulator of the AKT signaling cascade and the most commonly altered component of the AKT pathway in human malignancies [6]. Mutations in PTEN result in significantly increased AKT activity [7]. Accordingly PTEN mutations occur in 50% of endometrial carcinomas [4] and patients with loss of PTEN expression followed by AKT phosphorylation are burdened with a poor prognosis [8]. Therefore targeting the AKT-pathway may be a favourable strategy in endometrial cancer.

Perifosine is a synthetic alkylphospholipid which is orally active and inhibits AKT activation [9]. AKT is activated by phosphorylation, therefore AKT phosphorylation is a surrogate marker for the activation of this enzyme. In addition to inhibition of AKT in some cell types perifosine downregulates the anti-apoptotic mitogene activated protein kinase extracellular signal regulated kinase (MEK-ERK 1/2) pathway and activates the proapoptotic c-jun-N-kinase (JNK) network, thus modulating the balance between the survival and death signaling cascades, thereby inducing PCD [10], [11]. Perifosine has shown anti-tumor activity in a variety of cancers in vitro[12] and in vivo[13]. So far perifosine was tested in clinical phase II studies in breast [14], head and neck [15], prostate [16], pancreatic [17] cancers and soft tissue sarcoma [18] patients.

The current study investigates a potential anti-tumor activity of perifosine in in vitro models of human endometrial cancers using perifosine either on its own or in combination with an established chemotherapeutic drug.

Section snippets

Reagents and cells

The alkylphospholipid perifosine was kindly provided by AeternaZentarisGmbH (Frankfurt, Germany) and dissolved in DMSO (concentration of the stock solution = 10 mM). HEC 1A and Ishikawa human endometrial cancer cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). HEC 1A cells were grown in RPMI-1640 medium, Ishikawa cells in Dulbecco's modified eagle medium (DMEM) (both from PAA, Cölbe, Germany). Both media were supplemented with 10% fetal calf serum (FCS) (Biochrom,

Perifosine inhibits AKT phosphporylation in Ishikawa and HEC 1A human endometrial cancer cells

The presence of phosphorylated and total AKT was assessed by Western blotting in Ishikawa and HEC 1A cells that had been treated with 0 μM, 10 μM and 40 μM perifosine. Specific antibody reactivity was detected at Mr = 60 kDa for both phosphorylated and total AKT. Phosphorylated AKT was dose-dependently decreased by treatment with perifosine (Fig. 1).

Effects of perifosine on the growth of HEC 1A and Ishikawa human endometrial cancer cells

To investigate whether impaired AKT phosphorylation would lead to reduced growth or to cell death in human endometrial cancer cells, Ishikawa and HEC 1A

Discussion

Treatment of advanced or recurrent endometrial cancer remains a major problem. Radiation therapy is frequently applied, even though the resulting survival benefit is modest at most. Combination chemotherapy shows objective responses in 40–60% of patients, but the survival is less than 1 year. The most commonly used therapeutic regimen combines cisplatin and doxorubicin. Improved response rates, longer progression-free survival and increased overall survival may be achieved by adding paclitaxel

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