All-trans Retinoic Acid-induced Differentiation Study In Hepatic Cancer Stem Cells

Part 1Detection, isolation, culture and identification of hepatic cancer stem cellsObjective:Testing cancer stem cell markers expression in different hepatic cell lines, culturing isolated hepatic cancer stem cells and identifying their sternness.Methods:Flowcytometry analyzes expressions of cancer stem cells marker, CD 133, in various hepatic cancer cell lines; According to flowcytometry analysis, one cell line high expressing CD 133 was applied to magnetic isolation. Isolated CD133+ cells were cultured in stem cell medium containing high growth factors to maintain their undifferentiation conditions. For further confirming sternness of CD133+ cells, expressions of embryo stem markers, NANOG, OCT4 and SOX2 were analysis by western blot in CD 133" and CD133+ hepatic cancer cells.Results:Flowcytometry analysis indicated that CD 133 positive cells of HepG2, Huh-7 and PLC-PRF-5 are 10.8|%,0.176% and 0.07% respectively. Isolated CD133+ HepG2 cells were maintained in stem cell medium with high growth factors. In western blot analysis, CD133+ HepG2 cells showed high expressions of embryo stem markers, NANOQ OCT4 and SOX2 compared with CD 133" cells.Conclusion:HepG2 cells express CD133 antigens. CD133 positive cells showed a great expression of embryo stem cell markers, that indicating it features cancer stem cells.Part 2All-trans retinoic acid differentiates hepatic cancer stem cellsObjective:Isolated CD133+hepatic cancer stem cells were cultured in stem cell medium. All-trans retinoic acid treats CD133+ hepatic cancer stem cells for differentiation.Methods:Normal culture HepG2 cells were used to process magnetic isolation for CD133+ cells. To maintain their sternness, the cells were cultured in serum-free DMEM/F12 medium supplemented with 20 ng/mL EGF,10 ng/mL FGF2 and B27. CD133+ hepatic cancer stem cells were exposed on various dose of all-trans retinoic acid (10-9～10-5M) for 5 days, then cell lysate was analyzed by western blot to detect the expression of embryonic stem cell markers including NANOG, OCT4 and SOX2.Results:Flowcytometry analysis results indicated decreased expression of cellular membrane CD 133 after 12 or 24 hours ATRA exposure. Also western blot assay detected attenuated CD 133 level. After 5 days treatment of ATRA, CD133+ HepG2 cells significantly decreased their embryonic stem cell markers protein level, including NANOG, OCT4 and SOX2.Conclusions:All-trans retinoic acid attenuates stemness of CD133+ HepG2 cells significantly.Part 3Retinoic acid metabolism in hepatic carcinoma tissuesObjective:Studies reported that leukemia patient cure after ATRA administration was related to ATRA-induced differentiation and apoptosis of cancer cells. Liver is endogenous resource of retinoic acid, its cancerization and retinoic acid metabolism study will contribute to explore hepatic tumorigenesis, metastasis and undifferentiation maintaining.Methods:Oncomine database was applied to analyze the enzyme activities related to retinoic acid synthesis, including alcohol dehydrogenases, retinol dehydrogenases, retinaldehyde dehydrogenases. The enzyme activities of hepatic cancer patient compared with normal liver. Immunohistochemistry measured the expression of three enzymes.Results:Alcohol dehydrogenases, dehydrogenases and retinaldehyde dehydrogenases are involved in the biosynthesis of retinoic acid from retinol. Gene expression profiling of RALDH1, ADH1 and RDH10 was analyzed using the Oncomine database (Bittner Multi-cancer data set, including 1,911 clinical patients, pathologically classified by 16 different cancer types). (B) RALDH1; (C) ADH1; (D) RDH10. Liver cancer (HCC) group is highlighted by dark blue. (1) Bladder cancer (n= 32); (2) Brain and central nervous system cancer (n= 4); (3) Breast cancer (n= 328); (4) Cervical cancer (n= 35); (5) Colorectal cancer (n= 330); (6) Esophageal cancer (n= 7); (7) Gastric cancer (n= 7); (8) Head and neck cancer (n= 41); (9) Kidney cancer (n = 254); (10) Liver cancer (n= 11); (11) Lung cancer (n= 107); (12) Lymphoma (n= 19); (13) Ovarian cancer (n= 166); (14) Pancreatic cancer (n= 19); (15) Prostate cancer (n= 59); (16) Sarcoma (n= 49). Data show most increased expression of RALDH1, ADH1 and RDH10 in hepatic carcinoma than other type of cancer. Immunohistochemistry analysis also confirmed expression of three enzymesConclusions:mRNA levels of three enzymes related to the biosynthesis of retinoic acid are highest in 16 types of cancer. Hepatic carcinoma tissue expresses ADH1, RDH10, RALDH1 in immunohistochemistry analysis.Part 4Increased sensitivities of All-trans retinoic acid -exposed hepatic carcinoma stem cells to anticancer drugObjective:Recurrences and drug resistance are most common circumstances in cancer management, including hepatic carcinoma. Increasing studies elaborate there are a small population with high level of embryonic stem cell markers in cancer cells, which greatly contribute to tumorigenesis and metastasis. Procedures differentiating cancer cells and increasing sensitivities of cancer cells to tumor drugs will give more rise to cancer therapy.Methods:Hepatic cancer stem cells HepG2/CD133+ were exposed on various dose of ATRA (10-7、10-6、10-5M) for differentiation. Anti-cancer drug docetaxel was employed to harm ATRA-exposed HepG2/CD133+ cells. Cellular apoptosis and proliferative activity were compared among docetaxel group and combination group of ATRA with docetaxel. Subcutaneous injection of hepatic cancer stem cells to nude mice were carried out for investigating its tumorigenesis.Results:HepG2/CD133+ cells were exposed on all-trans retinoic acid (10-7,10-6, 10-5M) for 5 days. Unexpectedly, high concentration induced slight increase of cellular proliferation activity (from 100% to 120%). Significant impairing (from 100% to 85%,70%,55%) of cellular proliferation activity was observed in docetaxel-exposed HepG2/CD133 cells (10-10,10-9,10-8M). Finally, Combining of DOC(10-9M) with ATRA (10-7, 10-6,10-5M) significantly attenuated cellular proliferation activity compared with DOC only. Apoptosis assay (PI and Annexin V staining) also showed increased apoptotic cells in combination group.55 days after subcutaneous injection of hepatic cancer stem cells, the volume of tumor in DMSO group is 400.00 mm3,321.73 mm3 and 150.59 mm3 for ATRA and DOCgroup separatively. However, the combination of ATRA with DOC induced dramatic decrease of tumor volume to 22.93 mm3.Conclusions:All-trans retinoic acid-induced differentiation of HepG2/CD 133+cancer stem cells may be beneficial to cancer therapy. Combination of ATRA with DOC significantly impaired cellular activities of HepG2/CD133+ cancer stem cells, induced obvious apoptosis and decreased tumorigenesis.Part 5Involving of β-catenin signaling in All-trans retinoic acid-induced differentiation of hepatic cancer stem cellsObjective:Studies focusing on the mechanism of all-trans retinoic acid-induced differentiation of hepatic cancer stem cells may contribute to find out more targets for clinic application of hepatic cancer therapies.Methods:A large number of studies showed PI3K/AKT and β-catenin signaling regulated cellular proliferation, activity, tumorigenesis, tumor development and differentiation of embryonic stem cells. This study will molecular signaling inducing differentiation of hepatic cancer stem cells. To achieve that, RNA interference of b-catenin was applied. The sternness of hepatic cancer stem cells and involving of PI3K/AKT signals was evaluated under the interference condition.Results:CD133+ hepatic cancer stem cells exposed on various dose of all-trans retinoic acid (10-9～10-5M) for 5 days showed significant decrease of PI3K protein and phosphorylation of AKT. The protein level of P-catenin also decreased accompanying risen phosphorylation. Meanwhile, mRNA level of (3-catenin did not change obviously. This results indicated that impaired β-catenin was induced via phosphorylation degradation. In detail, ATRA decreased PI3K/AKT, leading to signals released GSK3β that responsible for further protein degradation of β-catenin.Conclusions:Attenuated PI3K/AKT signals by ATRA exposure free GSK3β, leading to GSK3β-induced degradation of β-catenin, that finally differentiates HepG2/CD133+hepatic cancer stem cells.