Background Samsoeum (SSE), a traditional natural formula, has been widely used to treat cough, fever, congestion, and emesis for centuries. initially found that SSE caused dose- and time-dependent cell death in cancer cells but not in normal primary hepatocytes. In addition, during early SSE treatment (6C12?h), cells were arrested in G2/M phase concomitant with up-regulation of p21 and p27 and down-regulation of cyclin D1 and cyclin B1, followed by an increase in apoptotic YO-PRO-1 (+) cells. SSE also induced autophagy via up-regulation of Beclin-1 Rabbit Polyclonal to CRHR2 expression, conversion of microtubule-associated protein light chain 3 (LC3) I to LC3-II, and re-distribution of LC3, indicating autophagosome formation. Moreover, the level of B-cell lymphoma 2 (Bcl-2), Rolziracetam which is critical for Rolziracetam cross-talk between apoptosis and autophagy, was significantly reduced in SSE-treated cells. Phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was increased, followed by suppression of the protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathway, and phosphorylation of mitogen-activated protein kinases (MAPKs) in response to SSE treatment. In particular, among MAPKs inhibitors, only the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 nearly blocked SSE-induced increases in Beclin-1, LC3-II, and Bax expression and decreases in Bcl-2 expression, indicating that JNK activation plays critical role in cell death caused by SSE. Conclusions These findings suggest that SSE efficiently induces cancer cell death via apoptosis as well as autophagy through modification of the Akt/mTOR and JNK signaling pathways. SSE may be as a potent traditional herbal medicine for treating malignancies. in Chinese, in Japanese), a traditional herbal medicine, was first described during the Song Dynasty of China and has been widely used as a remedy for headache, cough, rhinorrhea, and fever. SSE also has been used to treat congestion with phlegm, tidal fever, and emesis. Recent studies have reported the pharmacological efficacy of SSE in allergic and asthma reactions and pulmonary damage from ozone [20]. SSE modulates allergic and inflammatory reactions via inhibition of the expression of cyclooxygenase 2 (COX-2) and inflammatory cytokines and suppression of nuclear factor-B (NF-B) activation [21]. However, the anti-cancer effect of SSE and its exact mechanism of action remain to be examined. Therefore, the present study aimed to elucidate the effect of SSE on the cell growth and cell death in cancer cells and investigate the detailed mechanism of its anti-cancer activity. Methods Cell lines The human gastric carcinoma AGS cell line, human fibrosarcoma HT1080 cell line, human epidermoid carcinoma A431 cell line, and murine melanoma B16F10 cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA). Each cell line was maintained as a monolayer culture in Roswell Park Memorial Institute (RPMI) 1640 or Dulbeccos Modified Eagle Medium (DMEM; Lonza, Walkersville, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; GIBCO/Invitrogen, Carlsbad, CA), 100 units/mL penicillin, and 100?g/mL streptomycin (Welgene) at 37C in a humidified 5% CO2 incubator. Murine hepatocytes were isolated from 6C8?weeks old female ICR mouse purchased from Nara Bio animal center (Nara Biotech, Korea). Mice were housed under standard conditions at a temperature of 24 1C and humidity of 55 5%, and experimental procedures were approved by Korea Institute of Oriental Medication Care and Make use of Committee having a research quantity 12C122. Mice had been cared for relative to the dictates from the Country wide Animal Welfare Regulation of Korea Rolziracetam and tests had been carried out relative to the Korea Institute of Oriental Medication Care Committee Recommendations. Murine hepatocytes had been isolated utilizing a perfusion program with some changes [22]. After suspending within the Williams E moderate including 10% FBS, 100?IU/mL insulin, 2?mM?L-glutamine, 15?mM HEPES, 100 devices/mL penicillin, and 100?g/mL streptomycin, hepatocytes were seeded for the tradition dish coated with 10% gelatin/phosphate buffered saline (PBS), and incubated at 37C inside a humidified 5% CO2 incubator. Antibodies and reagents Propidium iodide (PI), Ribonuclease A (RNase A) from bovine pancreas, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) had been bought from Sigma Chemical substance Co. (St Louis, MO, USA). Antibodies against Cyclin D1, Cyclin B1, Cdc25, and -tubulin Rolziracetam had been from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-p21Waf1/Cip1, anti-p27Kip1, anti-caspase-3, poly (ADP-ribose) polymerase (PARP), anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-extracellular signal-related kinase1/2 (ERK), anti-phospho-ERK (Thr202/Tyr204), Rolziracetam anti-c-Jun-N-terminal kinase (JNK), anti-phopsho-JNK (Thr183/Tyr185), anti-Akt, anti-phopho-Akt (Ser473), anti-mTOR, anti-phospho-mTOR (Ser2481), anti-adenosine monophosphate activated-activated proteins kinase (AMPK), anti-phospho-AMPK (Thr172), anti-Bcl-2, anti-Bax, and anti-Beclin-1 antibodies had been bought from Cell Signaling Technology (Danvers, MA, USA). Anti-microtubule-associated proteins light string 3 (LC3) and anti-cleaved caspase-3 antibodies had been from Sigma Chemical substance Co. and Abcam.
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