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Introduction
Hochu-ekki-to (HET) (bu-zhong-yi-qi-tang in Chinese) is a Kampō herbal medicine. Kampō medicines are traditional remedies composed of several herbs, and have been used for several hundred years in Japan (1). To date, the Japanese Ministry of Health, Labour and Welfare has approved >120 Kampō prescriptions for clinical use (2). These Kampō medicines have been considered to be clinically effective since antiquity in Japan. However, studies on the biochemical and pharmacological mechanisms of action of Kampō medicines are not extensive.
HET is mainly used for the treatment of appetite loss (3), general fatigue (4) and loss of vigor (1). It is particularly used for treating elderly people and, more recently, patients with infectious and malignant diseases (5,6), or after chemotherapy or radiation therapy of malignant tumors (7,8). It has been reported that HET activates macrophages (9) and natural killer cells (10) and restores impaired immune function, which is beneficial for the prevention of cancer (11,12). In studies on mice, HET suppressed the development and metastasis of several malignant tumor types, including biliary carcinoma and uterine cancer (3,13). In an in vitro study, inhibition of viability measured by MTT assay was observed when Hep3B hepatocellular carcinoma cells were incubated with HET (14). In addition, flow cytometric analysis showed that HET induced cell cycle arrest and apoptosis in Hep3B cells (14). These results suggested that the underlying mechanisms of the anti-tumor activity of HET involve the suppression of cell viability and induction of apoptosis.
However, high concentrations of HET in culture medium may have non-specific cytotoxic effects owing to components including saponins (e.g. saikosaponin in HET) and other detergent-like compounds. When cells are incubated with >5,000 µg/ml, necrosis has been identified to be markedly increased in ovarian cancer cells by flow cytometric analysis (15). To rule out the non-specific cytotoxic effects, the present study used a low dose of HET (50 µg/ml) that did not suppress cell viability, and assessed the synergistic effect of HET pre-treatment on the potency of cisplatin against HeLa cells. Cell survival was measured by colony survival and crystal violet assays, and the apoptotic rate was analyzed by flow cytometry. Proteins associated with cell viability and apoptosis, including phosphorylated Akt (p-Akt), p53, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and...