Abstract
Background/Aim: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family, is a target for ovarian cancer therapy. The present study investigated the administration schedule of BK-UM, an anticancer agent targeting HB-EGF. Materials and Methods: The ovarian cancer cell line, RMG-I, was injected subcutaneously into five-week-old female nude mice. The BK-UM was administered intraperitoneally, using three administration schedules with different doses. The tumor volume was calculated every week. Statistical significance was assessed using the Mann–Whitney U-test. Results: At doses >0.1 mg/kg, BK-UM displayed significant antitumor effects, although the antitumor effects and body weights of mice did not significantly differ by dose or by three different administration schedules. At a dose <0.1 mg/kg, however, BK-UM had little inhibitory effect on tumor growth. Conclusion: Daily administration of BK-UM, which has a potentially dose-dependent antitumor effect, may be the optimal schedule for clinical application.
Ovarian cancer is the most common cause of gynecological cancer-related death (1, 2). As ovarian cancer cells are widely spread through the peritoneal cavity by the peritoneal fluid, ovarian cancer is most often diagnosed in advanced stages, leading to a poor prognosis. Accumulating evidence indicates that this peritoneal fluid is a rich source of growth factors, termed ovarian cancer-activating factors (OCAFs), which promote the survival and proliferation for ovarian cancer cells (3). We have previously reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is the most widely expressed OCAF, and has a pivotal role in ovarian cancer progression (4, 5). We, therefore, considered HB-EGF to be a therapeutic target for ovarian cancer (6).
HB-EGF is a member of the epidermal growth factor superfamily, and can bind to the epidermal growth factor receptor (EGFR). Membrane-anchored HB-EGF (proHB-EGF) is synthesized similarly to other EGF proteins (7, 8). ProHB-EGF is cleaved by a member of the disintegrin and metalloproteinases family at the cell surface (a mechanism called ‘ectodomain shedding’), resulting in soluble HB-EGF (9, 10). As soluble HB-EGF is essential for the formation of some tumors, HB-EGF is a potential therapeutic target for many types of cancer, including ovarian (4, 11), gastric and breast cancer (5, 12, 13).
ProHB-EGF also functions as a diphtheria toxin receptor. Cross-reacting material 197 (CRM197), which has recently been made using a Good Manufacturing Process, designated BK-UM, is a non-toxic mutant of diphtheria toxin (14). BK-UM inhibits the mitogenic action of HB-EGF. BK-UM binds to both the pro- and soluble forms of human HB-EGF, thus inhibiting the binding of HB-EGF to the EGFR (15). However, BK-UM does not inhibit the mitogenic activity of other EGFR ligands, and is therefore recognized as a specific inhibitor of HB-EGF (16). Accordingly, BK-UM is a candidate agent for ovarian cancer treatment, and warrants further investigation. No pre-clinical studies have been performed to investigate the most effective schedule for BK-UM administration. Therefore, this study aimed to validate the optimal administration of BK-UM in preparation for its investigation in phase I clinical trials in patients with advanced or recurrent ovarian cancer.
Materials and Methods
Reagents. BK-UM, which is composed of CRM197 and is an anticancer agent (15), was obtained from the Research Institute for Microbial Diseases, Osaka University (Osaka, Japan).
Cells and cell culture. A human ovarian cancer cell line, RMG-I, was obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (ICN Biomedicals, Irvine, CA, USA), 100 U/ml of penicillin G and 100 μg/ml of streptomycin (Invitrogen Corp., Carlsbad, CA, USA) in a humidified atmosphere of 5% CO2 at 37°C.
Antitumor effects of BK-UM in a mouse xenograft model. Subconfluent cell cultures were detached from plates with trypsin-EDTA. A total volume of 250 μl containing 5×106 cells suspended in serum-free RPMI-1640 was injected subcutaneously into five-week-old female nude mice (Charles River Laboratories Japan Inc., Yokohama, Japan). To assess the inhibitory effects of BK-UM on tumor growth, different doses (5, 10, 25 or 50 mg/kg) of BK-UM were injected intraperitoneally into tumor-bearing mice by three different schedules after the tumor reached an estimated volume of more than 100 mm3. The BK-UM was administered 10 times during the course of the treatment. The first schedule was daily administration of BK-UM for 10 days. The second schedule was administration of BK-UM on alternate days for 21 days. The third schedule was weekly administration of BK-UM for 10 weeks. Tumor size and body weight of mice were measured every week. The tumor volume was estimated from two-dimensional tumor measurements as follows: tumor volume (mm3)=(length×width2)/2.
Statistical analysis. The statistical significance of differences among the groups was assessed using the Mann–Whitney U-test. A value of p<0.05 was considered statistically significant.
Results
To determine the optimal administration schedule of BK-UM for use in a future clinical study, we examined the antitumor effects on RMG-I xenograft tumor in mice. The tumors treated with 5, 10, 25 or 50 mg/kg of BK-UM using all three administration schedules were significantly smaller by week 11 compared to the initial tumors, and compared to those of control mice (p<0.05 for all; Figure 1A-C). However, the tumor sizes and mouse body weights did not significantly differ at week 11 according to doses or schedules of BK-UM administration (Figure 1D-F).
Using the same BK-UM administration schedules, we compared the tumor sizes at week 4 and week 11 for all BK-UM doses. In the daily and alternate-day BK-UM administration groups, the tumor size at week 11 did not significantly differ by dose, whereas the tumor size at week 4 in the group treated with 50 mg/kg of BK-UM tended to be lower compared to mice treated with 5 mg/kg of BK-UM (Figure 2A-D). In the weekly BK-UM administration group, the tumor size at week 4 and week 11 in the 50-mg/kg group showed a small reduction compared to that in the 5 mg/kg groups (Figure 2E and F). Taken together, these results suggest that BK-UM has a dose-dependent antitumor effect.
To assess the overall antitumor effects of BK-UM, we estimated the frequency of the disappearance of tumor at week 11. Daily administration of 5, 10, 25 or 50 mg/kg of BK-UM, and alternate-day administration of 25 or 50 mg/kg of BK-UM made some tumors disappear completely (Figure 3A and B). The frequency of tumor disappearance was highest for mice daily administered 50 mg/kg of BK-UM (Figure 3A). Taken together, these findings suggest that daily BK-UM administration is optimal for inducing antitumor effects.
To confirm that the antitumor effects of BK-UM occur in a dose-dependent manner, we examined the mouse tumor size and body weight after daily administration of 0.01, 0.1, 0.5 or 5 mg/kg of BK-UM. At the 0.1, 0.5 and 5 mg/kg doses of BK-UM, the tumor sizes at week 11 were significantly reduced compared to the control and group treated with 0.01 mg/kg of BK-UM (all p<0.05; Figure 4A). However, the body weight of the control mice did not differ significantly in mice treated daily with 0.01, 0.1, 0.5 or 5 mg/kg of BK-UM (Figure 4B).
Discussion
In the present study, BK-UM showed significant, dose-dependent antitumor effects on mice bearing RMG-I xenograft tumors. The daily administration of BK-UM for 10 days yielded the greatest antitumor activity, thus suggesting that daily administration is more effective than the other (alternate day and weekly) schedules.
In ovarian cancer, cytotoxic agents, including carboplatin and paclitaxel, are widely used therapeutic agents. However, most patients with advanced ovarian cancer experience disease relapse within 2 years of initial treatment and ultimately die due to drug resistance. Our previous study has shown that BK-UM effectively suppressed the growth of paclitaxel-resistant cells and had a markedly synergistic antitumor effect when used with paclitaxel (21). In the near future, BK-UM might be used with cisplatin or paclitaxel as a combination therapy for patients with ovarian cancer. In general, chemotherapy for patients with ovarian cancer is performed every three or four weeks as one course. When BK-UM is administered as a single-agent, it should be administered daily. The daily administration of BK-UM could be performed for two weeks, with the subsequent two weeks used as an observation/rest time for one course of chemotherapy.
The effects of BK-UM (CRM197) on the tumor size and body weight of mice bearing RMG-I xenograft tumors. A and D: Daily administration of BK-UM for 10 days. B and E: Administration of BK-UM on alternate days for 21 days. C and F: Weekly administration of BK-UM for 10 weeks. Mice were weighed each time the tumor sizes were measured. Black arrows indicate the days when BK-UM was administered. The number of the mice of all the groups is 6. The tumor volume is presented as the mean±SEM. *p<0.05 versus the control groups for all groups administered BK-UM.
BK-UM is a mutant form of diphtheria toxin. Although BK-UM is recognized as being non-toxic, its safety has not been reported, and non-mutant diphtheria toxic can induce fatal reactions, such as cardiac arrest. Accordingly, divided BK-UM administration would seem to be safer than using a single injection. Additionally, a previous clinical trial using CRM197 showed that it had a half-life of 16-20 h and a mean half-life of 18.1 h when given subcutaneously (22). Therefore, the daily administration of BK-UM might maintain the serum BK-UM level at a constant, safe and efficacious level.
A comparison of the mean tumor size at the fourth week and eleventh week. The tumor size at week 4 and week 11 after the initial administration of BK-UM was compared for all doses. A and B: Daily administration of BK-UM. C and D: Alternate-day administration of BK-UM. E and F: Weekly administration of BK-UM. The number of the mice of all the groups is 6. Error bars indicate the SEM.
The frequency of disappearance of the tumor. The number of mice whose tumors disappeared by week 11 after the initial BK-UM administration using daily administration of BK-UM for 10 days (A) and administration of BK-UM on alternate days for 21 days (B). The number of the mice of all the groups is 6.
The dose-dependency of BM-UK when administered daily at the indicated doses for 10 days. A: The tumor volume. B: The mouse body weight. The black arrows indicate the days when BK-UM was administered. The tumor volumes represent the means±SEM. *p<0.05 versus the control or 0.01 mg/kg BK-UM group. The number of the mice of all the groups is 8.
In conclusion, we have shown that the daily administration of BK-UM was most effective for ovarian cancer therapy in this pre-clinical study, performed as part of Good Laboratory Practice. Pending the approval of our Ethics Committee, a phase I study of BK-UM will be performed at the Fukuoka University for patients with advanced and recurrent ovarian cancer.
Footnotes
-
↵* These Authors contributed equally to this work.
- Received April 4, 2014.
- Revision received June 10, 2014.
- Accepted June 11, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- Different combinations of ErbB receptor dimers generate opposing signals that regulate cell proliferation in cardiac valve development
- Association of Serum HB-EGF Value and Response to Chemotherapy in Patients with Recurrent Ovarian Cancer
- Activated EphA2 Processing by MT1-MMP Is Involved in Malignant Transformation of Ovarian Tumours In Vivo
- Antitumour Effects of Intravenous Administration of BK-UM, a Novel Inhibitor of HB-EGF, in Ovarian Cancer Therapy
- ErbB1 and ErbB4 generate opposing signals regulating mesenchymal cell proliferation during valvulogenesis
- Anti-tumor Effect of Intravenous Administration of CRM197 for Triple-negative Breast Cancer Therapy