N. Takada1,2, H. Sugano1,2, Y. Shirai1, N. Saito1, R. Hamura1,2, T. Taniai1,2, H. Shiba1, K. Eto1, T. Uwagawa1,3, T. Ohashi2, K. Yanaga1 1The Jikei University School of Medicine,Department Of Surgery,Minato-ku, Tokyo-to, JP, Japan 2The Jikei University School of Medicine,Division Of Gene Therapy, Research Center For Medical Science,Minato-ku, Tokyo-to, JP, Japan 3The Jikei University School of Medicine,Division Of Clinical Oncology And Hematology, Department Of Internal Medicine,Minato-ku, Tokyo-to, JP, Japan
Introduction: For gallbladder cancer (GBC), surgical resection is the only curative treatment, but it is difficult to detect early and the prognosis of advanced GBC remains very poor. Radiotherapy alone or in combination with chemotherapy has been reported as a neoadjuvant therapy for patients with locally advanced unresectable GBC, but its effect is limited. Nuclear factor kappa B (NF-κB) is activated in various cancer cells and involved in tumor growth, invasion and metastasis. In addition, NF-κB is activated by chemotherapy and radiotherapy. This radiation-induced NF-κB activation plays an important role in the regulation of cell apoptosis, inflammation and oncogenesis including invasion and angiogenesis, which result in treatment tolerance. We previously reported that nafamostat mesilate (NM), a synthetic serine protease inhibitor commonly used for pancreatitis and disseminated intravascular coagulation (DIC) in Japan inhibited NF-κB activation and induced antitumor effects for pancreatic cancer. We hypothesized that NM may inhibit radiation-induced NF-κB activation and enhance the antitumor effect of radiotherapy for GBC cells.
Methods: In vitro, we assessed NF-κB activity, cell viability, induction of caspase cascade and quantification of apoptosis of human GBC cells (NOZ) in the following four groups: 1) radiation (5Gy) alone, 2) NM alone, 3) combination (radiation?5Gy?and NM), or 4) vehicle as control. In combination group, the cells were incubated with NM for 3 hours before radiation therapy. In vivo, xenograft GBC mice model were established by injecting subcutaneously with 5×106 NOZ cells into the right flank. At one week after injection, the animals were treated with intraperitoneal (i.p.) injection of NM (30 mg/kg) 5 times a week as NM group, i.p. injection of the equal amount of distilled water 5 times a week and radiation (5Gy) once as radiation group, or i.p. injection of NM (30 mg/kg) 5 times a week and radiation (5Gy) once as combination group for 2 weeks. For control group, the equal amount of distilled water was injected (i.p.) 5 times a week.
Results:In vitro, as compared with radiation group, combination group had lower NF-κB activity (2026.12 ± 84.87 ng/mg protein vs. 1315.12 ± 88.98 ng/mg protein, p<0.01), and reduced cell viability (62.99 ± 4.02% vs. 31.07 ± 6.47%, p<0.01). The levels of apoptotic proteins (Cleaved caspase-3, -8, -9 and cleaved PARP) in combination group were greater than those in other groups. The proportion of apoptotic cells in combination group was significantly higher than that in radiation group (53.80 ± 2.83% vs. 15.14 ± 1.70%, p<0.01). In vivo, tumor growth in combination group was significant slower than that in radiation group (9.50 ± 3.27mm vs. 6.65 ± 1.77mm, p<0.05).
Conclusion:NM suppressed radiation-induced NF-κB activation and enhanced the antitumor effect of gallbladder cancer.