The anti-tumor effect of cross-reacting material 197, an inhibitor of heparin-binding EGF-like growth factor, in human resistant ovarian cancer

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Abstract

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to represent possible chemotherapeutic agent for ovarian cancer. However, the effect of CRM197 on the resistant ovarian carcinoma cells has not been sufficiently elucidated. Here, we found that HB-EGF was over-expressed in a paclitaxel-resistant human ovarian carcinoma cell line (A2780/Taxol) and a cisplatin-resistant cell line (A2780/CDDP), as well as the xenograft mouse tissue samples with these cells. To investigate the possible significance of the HB-EGF over-expression in A2780/Taxol and A2780/CDDP cells, we inhibited HB-EGF expression by CRM197 to investigate the effect of CRM197 treatment on these cells. We observed that CRM197 significantly induced anti-proliferative activity in a dose-dependent manner with the cell-cycle arrest at the G0/G1 phase and enhanced apoptosis in A2780/Taxol and A2780/CDDP cells. The sensitive ovarian carcinoma parental cell line (A2780), A2780/Taxol and A2780/CDDP cells formed tumors in nude mice, and enhanced tumorigenicity was observed in drug-resistant tumors. Furthermore, we observed that CRM197 significantly suppressed the growth of drug-resistant ovarian cancer xenografts in vivo (p < 0.001). These results suggest that CRM197 as an HB-EGF-targeted agent has potent anti-tumor activity in paclitaxel- and cisplatin-resistant ovarian cancer which over-express HB-EGF.

Highlights

► HB-EGF over-expression in A2780/Taxol, A2780/CDDP cells and the matched xenografts. ► CRM197 induces enhanced apoptosis in A2780/Taxol and A2780/CDDP cells. ► CRM197 arrests A2780/Taxol and A2780/CDDP cells at G0/G1 phase. ► CRM197 suppressed the A2780/Taxol and A2780/CDDP growth of xenografts.

Introduction

Ovarian cancer is the leading lethal cause among all gynecological malignancies. More than 80% of patients respond to first-line chemotherapy with platinum and taxane. However, nearly all the patients relapse and become refractory to traditional chemotherapeutics. Patients who relapse, and those who do not initially respond to chemotherapy, are thought to carry hidden drug-resistant cells, leading to tumor relapse and lethality [1]. Therefore, a effective novel therapy for drug-resistant ovarian cancer at the molecular level is eagerly awaited.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), an epidermal growth factor receptor (EGFG) ligand, is synthesized as a membrane-anchored protein (pro-HB-EGF), which is cleaved at the cell surface by a protease to release a soluble N-terminal ectodomain (s-HB-EGF) via a mechanism referred to as ectodomain shedding [2]. S-HB-EGF, a potent mitogen and chemoattractant, drives signal-transduction cascades through activating EGFR or erythroblastic leukemia viral oncogene homolog 4 (ErbB4), which have critical roles in diverse pathophysiological processes [3], [4], [5]. Meanwhile, the C-terminal domain of HB-EGF (after ectodomain shedding) translocates to the nucleus and modulates the cell cycle and cell proliferation [6]. HB-EGF participates in a variety of pathophysiological processes and is over-expressed in many human malignancies, such as bladder carcinoma [7], gastric cancer [8], head and neck squamous cell carcinoma [9] and ovarian cancer.

So far, the role of HB-EGF over-expression in ovarian cancer cells has been increasingly reported [10]. The tumors and ascitic fluid obtained from patients with ovarian cancer express higher levels of HB-EGF than the normal ovaries and ovarian cysts. Meanwhile, there were large differences in expression between HB-EGF and the other six EGFR ligands [11]. HB-EGF has been proven to play a pivotal role in tumorigenicity [11], proliferation [12], metastasis and angiogenesis [13], making HB-EGF a promising therapeutic target for ovarian carcinoma. HB-EGF is over-expressed in 5-fluorouracil (5-FU)- and cisplatin (CDDP)-resistant cells and is regarded as a chemoresistance-related gene in gastric cancer, colon cancer and et al. [14], [15], [16]. However, the expression level of HB-EGF and its precise role in drug-resistant ovarian cancer remains unclear.

Cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin, is a strong specific HB-EGF inhibitor [17]. CRM197 binds to the sHB-EGF, as well as to proHB-EGF, and inhibits the mitogenic action of HB-EGF by inhibiting its binding to ErbB receptors [18]. Currently, a phase I study on CRM197 is being conducted for patients with advanced ovarian cancer at Fukuoka University, Japan [10]. CRM197 has been proven to represent possible chemotherapeutic agent for ovarian cancer [10], [19], [20]. However, its effect on resistant ovarian cancer cells has not been sufficiently elucidated.

The current study examined the inhibitory effects of CRM197 on resistant human ovarian cancer cells. We found that the levels of HB-EGF expression in paclitaxel- and cisplatin-resistant cells were higher than the levels in parental sensitive cells, and CRM197 treatment which inhibited HB-EGF expression could significantly suppress the tumorigenicity of resistant ovarian cancer in vitro and in vivo.

Section snippets

Cell culture and CRM197 treatment

The cisplatin-resistant human ovarian cancer cell line (A2780/CDDP; catalog No. 93112517) was purchased from the European Collection of Cell Cultures (ECACC, UK) and cultured according to the ECACC recommendations. The sensitive human ovarian cancer parental (A2780) and the paclitaxel-resistant (A2780/Taxol) cell lines were gifts of Dr. Lan Xiao (Department of Gynecology and Obstetrics, the Third Affiliated Hospital of Sun Yan-sen University, Guangzhou, China) and were cultured as previously

Enhanced expression of HB-EGF in drug-resistant human ovarian cancer cells and xenograft mice models

HB-EGF is the primary EGFR ligand altered in ovarian cancer. Its expression level in ovarian cancer is much higher than those of normal ovaries and ovarian cysts. We first determined HB-EGF expression in drug-resistant ovarian cancer cells by western blot analysis using drug-sensitive cells as a control (Fig. 1A, p < 0.001). The data showed that the A2780/Taxol and A2780/CDDP cells exhibited significant increases in HB-EGF expression compared with the A2780 cells. We also used immunohistochemical

Discussion

In ovarian cancer, the combination of paclitaxel with platinum is one of the most active drug combinations. However, most advanced-stage patients relapse and ultimately die of drug resistance. Elevated EGFR expression level has been proven to enhance chemoresistance in ovarian cancer [22]. HB-EGF, which is the only EGFR ligand with particularly enhanced expression in ovarian cancer, may be associated with drug resistance [14]. Heparin-binding epidermal growth factor-like growth factor (HB-EGF)

Competing interest

The authors have declared that no competing interests exist.

Funding

This study was supported by Heilongjiang Provincial Natural Science Foundation of China (No. ZD200906). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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