Background: Colorectal cancer (CRC) is one of the diseases with high prevalence and mortality worldwide. Conclusion: Our results demonstrate the anti-metastatic effect and therapeutic potential of betulin in metastatic CRC treatment. < 0.05. 2. Materials and Methods 2.1. Reagents We purchased betulin from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), cell counting kit (CCK)-8 from DoGen (Daejeon, Korea), compound C (CC) from MedChemExpress (Monmouth Junction, NJ, USA), and crystal violet solution from SigmaCAldrich (St Louis, MO, USA). 2.2. Cell Culture The murine CRC cell line colon 26 (CT26) and human CRC cell lines HCT116 and SW620 were purchased from Korean Cell Line Lender (Seoul, Republic of Korea). CT26 cells were maintained in Dulbeccos modified Eagles medium. HCT116 and SW620 cells were cultured in RPMI 1640. The mediums contained 10% fetal bovine serum and 100 U/mL Cysteine Protease inhibitor Penicillin-Streptomycin (Thermo Fisher Scientific, MA, USA). 2.3. Cell Viability Measurement The viability of cells after betulin (0C8 M) treatment was measured using the CCK-8 reagent. Cells were seeded in a 96-well plate (3 103 cells/well/200 L) and treated with betulin for 72 h. New medium made up of CCK-8 was added to the plate, and the absorbance was measured using a microplate reader. 2.4. Colony Formation Cells were seeded into a 12-well culture plate (5 102 cells/well) and incubated with betulin for 7 days. The colonies Cysteine Protease inhibitor were fixed with 3.7% formaldehyde for 30 min and washed using phosphate buffered saline (PBS). Colonies were stained using crystal violet solution (0.1%) for 20 min. The stained colonies were photographed after PBS washing. 2.5. Cell Cycle Distribution Cell cycle analysis was conducted using the Muse Cell Cycle Kit and Muse Cell Analyzer (MUSE, Millipore, Bedford, MA, USA). CT26 and HCT116 cells (5 105 cells/well) in 6-well plates were treated with betulin (0C8 M) for 24 h. Manufacturer protocols were followed for staining and analysis of propidium iodide (PI)-positive cells. 2.6. Real-Time Cysteine Protease inhibitor Reverse Transcription Polymerase Chain Reaction (RT-PCR) Total RNA was extracted using an RNA-spinTM Total RNA Extraction Kit (iNtRon Biotech, Seoul, Republic of Korea) and reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Expression of target Cysteine Protease inhibitor genes was quantified using the Power SYBR? Green PCR Grasp Mix and Step-one PlusTM Real-Time PCR Systems (Applied Biosystems, Foster Town, CA, USA). The mouse primers for real-time RT-PCR had been the following: cyclin D1, 5-TAGGCCCTCAGCCTCACTC-3 (forwards) and 5-CCACCCCTGGGATAAAGCAC-3 (invert); cdk4, 5-AGAGCTCTTAGCCGAGCGTA-3 (forwards) and 5-TTCAGCCACGGGTTCATATC-3 (invert); and gapdh, 5-GACATGCCGCCTGGAGAAAC-3 (forwards) and 5-AGCCCAGGATGCCCTTTAGT-3 (change). The individual primers for real-time RT-PCR had been the following: Cyclin D1, 5-ATGCCAACCTCCTCAACGAC-3 (forwards) and 5-GGCTCTTTTTCACGGGCTCC-3 (invert); CDK4, 5-GTGCAGTCGGTGGTACCTG-3 (forwards) and 5-TTCGCTTGTGTGGGTTAAAA-3 (invert); GAPDH, 5-TGCACCACCACCTGCTTAGC-3 (forwards) and 5-GGCATGGACTGTGGTCATGAG-3 (invert). 2.7. Recognition of Autophagy Muse TM Autophagy LC3-antibody structured package (MUSE, Millipore, Bedford, MA, USA) was utilized to identify autophagy of tumor cells after incubation with betulin for 24 h. Based on the producers protocol, cells had been permeabilized and incubated using the anti-LC3 Alexa Fluor 555-conjugated antibody for 30 min. Intracellular LC3 fluorescence was analyzed and detected using the Muse Cell Analyzer. 2.8. Traditional western Blot Evaluation PRO-PREP TM Proteins Extraction Option (iNtRon Biotech, Seoul, Korea) was utilized to remove total proteins from cells and tissue. Lysates had been blended with 5 test buffer quantity and total protein had been separated using gel electrophoresis. Focus on proteins had been detected with the next antibodies: Anti-phopho-AMPK, LC3-II, beclin-1, phospho-PI3K, phospho-Akt, phospho-mTOR, phospho-p38, phospho-ERK, phospho-JNK, AMPK, PI3K, PARP, caspase-3, caspase-9, Bcl-xL, and Bax (Cell Signaling, Danvers, MA, USA). Rabbit Polyclonal to iNOS Anti-Akt, p38, ERK, JNK, Bcl-2, GAPDH, cyclin D1, CDK4, and -tubulin antibodies had been purchased from.