The highly malignant OSCC cell line, HSC-3 was used in xenograft tumourigenicity to investigate the anti-tumour growth ability of hinokitiol in vivo. No significant differences were observed in body weight between the control and feeding 10 mg/kg hinokitiol group during the experiment (Fig. 6A). The average tumour size exhibited by the mice in the hinokitiol group was smaller than that of the mice in the control group from Day 9, and the average tumour size of the hinokitiol group was smaller than the control group (1.348±1.029 cm3
and 3.167±1.175 cm3
, P=0.0586) on Day 33 (Fig. 6B). The mice were sacrificed, and the tumours were isolated from the flank of the mice on day 33. The tumour weight of the hinokitiol group were smaller than control group (0.332±0.213 g and 0.916±0.443 g, P<0.05) (Fig. 6C). The IHC pictures were taken in the same area on tissue section. The xenograft tumours showed that the total histone H3, total histone H4, cleaved caspase 3, and PCNA were decreased both
in cytoplasm and nucleus of hinokitiol group (Fig. 6D).
Cleaved caspase 3 activates the apoptotic cascade and has been characterized as direct link to cell apoptosis. It is present in cytosol and mitochondria in cells (Samali, Zhivotovsky, Jones, & Orrenius, 1998), and expressed higher in cytoplasm of less well-differentiated tumour (Hague et al., 2004). Caspase-3 has been identified in
numerous benign or malignant neoplasm, including breast cancer (O'Donovan et al., 2003), gastric cancer (Sun et al., 2006), and head and neck cancer, and expressed more in head and neck malignant tumour (Chrysomali, Nikitakis, Tosios, Sauk, &
Papanicolaou, 2003; Hague et al., 2004; Parenti et al., 2006), which is related to tumour progression and poor differentiation. It is conflicted with the idea that caspase 3 is reduced in malignancy. There are references of nonapoptotic roles of caspase 3 including stimulation of differentiation (Szymczyk, Freeman, Adams, Srinivas, &
Steinbeck, 2006), dedifferentiation (Li et al., 2010), T cell proliferation (Kennedy, Kataoka, Tschopp, & Budd, 1999), and is involved in tumour cell development and repopulation (Huang et al., 2011). Whether or not the caspase 3 play nonapoptotic role in head and neck malignant tumour is unknown. In the present study, hinokitiol reduced the xenograft tumour size, and the IHC data showed reduced cleaved caspase 3 expression in both cytoplasm and nucleus of hinokitiol group. It provides the evidence that hinokitiol effectively reduced the malignant phenotypes not only in vitro but also in vivo. We inferred that hinokitiol might be a potential compound of therapeutic approach for caspase 3 overexpression cancer.
Conclusions
Hinokitiol suppressed pan-histone expression by decreasing NPAT and FLASH
expression, then, the binding of NPAT or FLASH to the histone promoters were decreased. As a result, the histone promoter activities were down-regulated, and the histone mRNA and protein expressions were decreased in both hinokitiol-treated HSC-3 and SAS cells. A relative low dose hinokitiol effectively inhibited the malignancy of OSCC cells in vitro, and showed a positive result of suppressing xenograft tumour
growth in vivo.
Hinokitiol is used as a food additive for preservative purposes, and the suggested concentration is 125 ppm (762 µM) per day, which is higher than the dose used in the present study. No known toxic effect from chronic exposure to hinokitiol has been reported (Cho et al., 2011). In addition, the hinokitiol containing oral hygiene products have been used to prevent dental caries and periodontal inflammation. Except being a remedy for oral cancer, combining the anti-microbial and anti-cancer activities, hinokitiol might be applied to reduce SSI effects and cancer recurrence in postoperative wound care. Hinokitiol exhibited specific cytotoxicity to OSCC cells than normal oral cells. Direct role for hinokitiol suppressing pan-histone expression in OSCC cells has not been described before to our knowledge. Future work should investigate the potential effect as a chemoprevention compound against OSCC pre-cancerous lesion.
5. Acknowledgments
This study was supported by grants from China Medical University (CMU98-N1-30, CMU100-S-30), the National Science Council, Taiwan (NSC99-2314-B-039-023-MY3, and NSC102-2628-B-040-001-MY3), and Ministry of Science and Technology,
Taiwan (MOST103-2811-B-040-007).
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