Four human HCC cell lines, HepG2, SK-Hep1, SNU475 and Huh7, were treated with HsA at different concentrations (0, 5, 10, 30 and 50 M) for 24 h. arrest of the cell cycle, in which several regulatory proteins are involved. It has been reported that p53 did not function properly in over 50% of the human cancer cases examined . p53 activates the DNA Ropivacaine repair proteins when the DNA is damaged, arrests the cell cycle at Ropivacaine the G1/S point upon DNA damage recognition and initiates apoptosis if the DNA damage is not recoverable . For these reasons, p53 is recognized as a so-called anti-tumor protein. Another well-known tumor suppression protein, p21, is expressed with the help of p53 and Miz-1 to inhibit cyclin-dependent kinases and stop the cell cycle . Recent articles have demonstrated the functions of various proteins which induce p53-dependent metabolic checkpoints, typically AMP-activated protein kinase (AMPK) [19,20]. Activation of the AMPK pathway results in cross-talks with oxidative or genotoxic stresses regulating cell proliferation, which appears to be mediated via the Ras/PI3K/mTOR pathway and up-regulation of p53 and p21 [21,22]. The AMPK activation can thus be considered as a logical therapeutic target for the suppression of tumor cell proliferation . In this study, we performed a series of tests to determine whether HsA contains a therapeutic effect against HCC and how it works in cellular and molecular signaling systems. In our results, HsA clearly induced G0/G1 cell cycle arrest and apoptosis, which were mediated by the regulation of the p53/p21 axis and the AMPK pathway. Our data also showed that HsA effectively suppressed the growth of Huh7 cells xenografted in nude mice. Our findings provide mechanistic insights into the therapeutic application of HsA in HCC-targeted clinics. 2. Materials and Methods 2.1. Chemicals and Reagents Anti-PARP, anti-caspase 3, anti-ACC, anti-p-AMPK, anti-AMPK, anti-p-mTOR, anti-mTOR, anti-p-P70S6K1, anti-P70S6K1, anti-cyclin D1, anti-cyclin E1, anti-p53 and ant- actin antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-CDK2, anti-CDK6, anti-Bcl2, anti-BAX, and p21 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgGs were purchased from Zymed Laboratories (San Francisco, CA, USA). The compound C was purchased from Calbiochem (San Diego, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), propidium iodide (PI), JC-1 and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HsA was kindly provided by Prof. Jong Rok, Lee (Daegu Haany University, Gyeongsan, Korea), and isolated as previously described . The pure HsA (1.31 g) was isolated from the chloroform extract (150 g) of and was stored at ?20 C. The purity ( 97%) was Ropivacaine verified by comparing retention time with standard compounds. 2.2. Cell Culture Huh-7 cells, a human HCC cell line, were purchased from the Health Science Research Resources Bank (Osaka, Japan). The HepG2 and SK-Hep1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and SNU475 was purchased from the Korean Cell Line Bank (Seoul, Korea). The Huh7 cells were cultured in Roswell Park Memorial Institute Media 1640 (RPMI-1640), whereas HepG2, SK-Hep1 and SNU475 were cultured in Dulbeccos modified Eagles medium (DMEM) with 10% fetal bovine serum (FBS). Dimethyl sulfoxide (DMSO) (the dilution rate = 1:1000) was used as vehicle. All of the control was treated with DMSO alone. 2.3. Cell Proliferation Assay Cell proliferation and viability were assessed by MTT assay . In metabolically active cells, MTT salt is cleaved by the action mitochondrial dehydrogenases and is reduced to an insoluble formazan crystal displaying a purple color that is detectable in spectrophotometer . Cells were plated at a density of 1 1 105 cells per well in 48-well plates and incubated for 24 h, which was followed by the incubation in medium containing various concentrations (from 5 to 10 M) of HsA for 24 h. 2.4. Cell Cycle Analysis by Flow Cytometry Huh7 cells were seeded in six-well plates at a density of 5 105 cells . On the following day, cells were treated with HsA at different concentrations (from 1 to 10 M) for 24 h. After treatment and incubation, the cells were harvested and washing with phosphate-buffered saline (PBS). Cells were stained with BD CycletestTM Plus DNA kit (BD Biosciences, San Jose, CA, USA) according to the manufacturers instruction, and analyzed on the Accuri C6 (BD Biosciences, Mountain View, CA, USA). All experiments were performed RHOC in triplicate. 2.5. Senescence-Associated -Galactosidase (SA–gal) Staining The Senescence -Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA, USA) was used to Ropivacaine evaluate -galactosidase activity, a known characteristic of senescence, in Huh7 cells according to the manufacturers Ropivacaine instructions. Images were taken using phase-contrast microscopy (Olympus, Tokyo, Japan). 2.6. Apoptosis Analysis by Flow Cytometry Cells were seeded in six-well plates at a density of 5 105 and incubated for 24 h. On the following day, HsA was treated at different concentrations, ranging from 5 to 25 M and incubated for.