Sirtuins are NAD+-dependent enzymes and are important for resistance to cellular stress. In this study, the expression of SIRT3 and 6 in Saos-2 cells was increased by 4HR administration (1–100 μM) (Fig. 1). The administration of 4HR increased pan-SIRT activity (Fig. 2). Additionally, the NAD+ level and NAD+ to NADH ratio were also in-creased by 4HR administration (Fig. 3). In conclusion, the administration of 4HR on Saos-2 cells showed increased SIRT3 and 6 expressions and pan-SIRT activity via an in-creased NAD+ to NADH ratio.
Prototype SIRTs are found in yeast, and their expression is associated with extension of the lifespan . When the cellular metabolism is restricted, the expression level of SIRT1 is increased in the muscles . SIRT3 is mainly expressed in mitochondria and associated with energy metabolism . SIRT6 inhibits glucose uptake and its metabolism . In this study, the administration of 4HR increased SIRT3 and SIRT6 in Saos-2 cells (Fig. 1). According to our recent study, 4HR administration reduced mitochondrial respiratory activity and ATP synthesis in human umbilical vein endothelial cells (HUVECs) . Therefore, 4HR-mediated inhibition of mitochondrial activity might be associated with increased SIRT3 and SIRT6 expression.
Reduced mitochondrial activity by 4HR administration might be fatal to cancer cells. Indeed, 4HR administration has been shown to have anti-cancer activity [18, 19]. Though the administration of 4HR would inhibit the growth of tumor, elevated SIRT3 and SIRT6 could play a protective role for cancer cells that survived after 4HR treatment, and these cancer cells might be more resistant to other cytotoxic cancer therapy. Superoxide dismutase (SOD) is activated by 4HR administration in RAW264.7 cells . SOD is also activated by SIRT3 activation .
Interestingly, 4HR administration increased the SIRT3 expression level (Fig. 1a). SIRT6 reduces pancreatic inflammation and fatty liver formation induced by a high-fat diet . SIRT6 decreases aging-associated inflammatory reactions via inhibiting the nuclear factor kappa B (NF-κB) pathway . 4HR administration increased the SIRT6 expression level in this study (Fig. 1b), and 4HR is strong inhibitor of the NF-κB path-way [23, 24]. The administration of 4HR decreases the expression of NF-κB, while increases ikappaB kinase in HUVECs .
The level of NAD+ is associated with cellular stress. When the available nutrients are restricted, AMP-activated protein kinase (AMPK) is activated, and the NAD+ level is in-creased as a consequence . Fibroblast growth factor 21 administration activates SIRT1 and reduces body weight . The activity of SIRTs can be regulated by the NAD+ level . In addition to SIRTs, poly(ADP-ribose) polymerases (PARPs) and cyclic ADP-ribose synthases also use NAD+ as an enzyme substrate [9, 11]. As PARPs are DNA repair enzymes and highly expressed in cancer, their inhibitor can be used in cancer treatment . PARPs are the main consumers of NAD+, and their inhibition may result in SIRT activation via flooding NAD+. However, the relation between PARPs and 4HR administration is yet to be clarified. In this study, 4HR administration increased the NAD+ level and NAD+/NADH ratio (Fig. 3).
4HR has been developed as an antiseptic . As the derivatives of resorcinolic lipid suppress microbial proliferation, the application of 4HR can inhibit microbial growth . When 4HR is prescribed for oral intake, its absorption rate from the gastro-intestinal tract is poor . When people receive 4HR per os, only 18% of the initial dosage is found in the urine within the first 12 h . Due to the poor absorption rate, it has been pre-scribed for killing intestinal pathogens . Russian scientists found that micro-organism surviving 4HR administration undergo dormancy and are more resistant to outer environmental stress . Therefore, 4HR has been considered as a chemical chaperone. A 4HR-mediated increase in SIRT activity and NAD+ level might be associated with its chaperone-like activity.
The possible anti-oxidant activity of 4HR might suggest that its applications can be useful in various conditions from orthodontic and surgical treatments. These conditions include gingivitis , root resorption , and dental pulp necrosis  during orthodontic treatment and several studies have reported that anti-oxidant activity can affect the expression of their manifestation [4, 5]. The application of 4HR ointment accelerates diabetic burn wound healing via improved capillary regeneration . Especially, the fact that 4HR has been proven to be safe as food additive for a long period  may make its utilization more promising.
A limitation of current study was that there are seven analogues of mammalian SIRTs. However, only SIRT1, 3, and 6 expression levels were evaluated after 4HR administration. The expression levels of SIRT2, 4, 5, and 7 should also be evaluated. According to our recent study on HUVECs, SIRT2, 4, 5, and 7 mRNA levels were increased by 4HR administration in HUVECs . In this study, pan-SIRT activity was increased by 4HR administration (Fig. 2). As individual SIRTs’ activity might be different after 4HR administration, individual enzyme activity after 4HR administration should also be studied in following projects. In this study, Saos-2 cell was used for evaluation. Saos-2 cell is originated from osteosarcoma cell and has been used for human osteoblastic cell . As it was cancer cell, additional cellular experiment using primary cultured human cell would be required for the confirmation of current finding.