| Background In the globe, colorectal cancer causes 690 thousands deaths per year worldwide.Although the patients’ chance of survivial was greatly improved because of the advances in the therapies including surgery and chemoradiotherapy, the response rate to current systemic therapies is nearly 50%, and patients with advanced tumors have poor prognosis,the overall mortality of the disease is approximately 40%The initiation and advance of tumor is the result of multiple factors, cancer stem cell(CSC) is thought to be one of the primary causes, that was proved by an increasing number of evidences. Cancer stem cell is defined as “cells within a tumor that possess the capacity for self-renewal and that can cause the heterogeneous lineages of cancer cells that constitute the tumor”, it has been proven to exist in a variety of solid tumors, including colon cancer, gastric cancer, breast cancer and so on. Cancer stem cells have several biological characterization including self-renewal, unlimited proliferation, high tumorigenicity and strong resistance to chemoradiotherapy. CSCs might be the primary cause of cancer’s several malignant biological behaviors such as tumor initiation, advance,recurrence and metastasis. Therefore, it is proposed that CSCs should be a new target for anti-tumor strategy.Wnt/β-catenin signaling pathway plays an important role in colon cancer, the activation of the pathway is indispensable to maintain stemness of CSCs and promote tumorigenicity. The mechanism may involve the activation of its downstream target genes,such as cyclin D1, c-Myc and survivin which are related with cell cycle progression, cell proliferation and apoptosis; In addition, the pathway’s key protein β-catenin is accumulated in the cytoplasm, translocates to nucleus and binds to members of the T-cell factor(TCF)/lymphoid enhancer factor(LEF) family to directly modulate the expression of pluripotent stem cell transcription factors——Oct4 and Nanog, that may partially explain why Wnt/β-catenin signaling plays a role in sustaining stemness. When Wnt/β-catenin signaling pathway is activated, the cloning and tumorigenic ability of CSCs is enhanced; In contrast, the suppression of signaling results in the decrease of cloning and tumorigenic ability. Therefore, the inactivation of Wnt/β-catenin signaling pathway may be very important to anti-CSCs.Until now, no specific drug has definite effect to target CSCs. CSCs are resistant to chemoradiotherapy, conventional antitumor therapies which can eliminate bulk of tumor cells but cannot target CSCs may lead to a reduction of the tumor mass but not the regression of the tumor. To work through the challenges, researchers have targeted natural products.In China, traditional Chinese medicine are widely used to anti-tumor in clinical practice. Some natural agents such as curcumin, resveratrol and morusin have been proposed as candidates to target CSCs. However, there needs a lot of work to make it practicable to use these natural agents in clinical. Xiao Tan San Jie(XTSJ) decoction is used in clinical to treat gastrointestinal cancer for decades, it was demonstrated that it can improve patients’ quality of life and prolong the survival period. Base on our existing research results, XTSJ decoction can inhibit the proliferation and metastasis of colon cancer cells in vitro and in vivo, additionally, it can also inhibit the growth of gastric cancer stem cells which marked by CD44. Therefore, we hypothesized that targeting colon cancer stem cells may be involved in the mechanisms of XTSJ decoction’s effect on colon cancer. In the study, we will observe the inhibitory effect of XTST decoction on CCSCs’ proliferation and tumorigenicity and investigate the mechanism.Objective To determine whether the sphere cells which were cultured in serum-free and non-adhesive floating culture system are enriched with CCSCs, to observe cell proliferation in vitro and tumorigenicity in vivo, to detect the expression of stemness markers and the activity of Wnt/β-catenin signaling pathway in sphere cells; To observe XTSJ decoction’s inhibitory effect on CCSCs cell proliferation and tumorigenicity in vitro and in vivo, to investigate the role of XTSJ decoction in regulating the expression of tumor stem cell markers and the activity of Wnt/β-catenin signaling pathway.Methods Experiment 1: The cultivation of CCSCs and the identification of biological characterization.Human colon cancer HCT116 cells were cultured in serum-free and non-adhesive floating culture system to enrich CCSCs(sphere cells). CCK-8 assay and tumor transplantation were performed to detect the proliferation and tumorigenicity of sphere cells and parental cells. The immumofluorescence and PCR assays were performed to dectect the expression of pluripotent stem cell transcription factors(Oct4, Sox2, Nanog) and other CCSCs surface markers in sphere cells and parental cells.Experiment 2: The expression of stemness marker and Wnt/β-catenin signaling pathway.WB and PCR assays were performed to dectect the activity of Wnt/β-catenin signaling pathway.Experiment 3: The effect of XTSJ decoction on CCSCs in vitro and the investigation of the underlying mechanism.The water extract of XTSJ decoction was lyophilized、weighted、dissolved in cell culture and stored for using. CCK-8 and 5-Ethynyl-2’-deoxyuridine(Ed U) assay were performed to detect the proliferative ability of parental and sphere cells. After being pre-treated with XTSJ decoction for 72 h, observe the spheroid formation of HCT116 cells.Cell cycle distribution and cell apoptosis were detected by flow cytometry. WB and PCR assays were performed to dectect the expression of stemness marker and the activity of Wnt/β-catenin signaling pathway after the treatment of XTSJ decoction.Experiment 4: The effect of XTSJ decoction on CCSCs in vivo and the investigation of the underlying mechanism.Sphere cells were transplanted subcutaneous in the back of nude mice, different doses of XTSJ decoction were administrated everyday. Measure the size of subcutaneous xenografts and the body weight of mice everyday. After 4 weeks’ feeding,the mice were sacrificed and the tumors were collected and weighted. WB and PCR assays were performed to detect the expression of stemness marker and the activity of Wnt/β-catenin signaling pathway. After sphere cells were transplanted subcutaneous in the back of nude mice, high dose of XTSJ decoction was administrated everyday, then observe the survival time.Results1. Parental HTC116 cells which cultured in serum-free floating culture system can stability form form sphere cells. In vitro, sphere cells have stronger proliferative ability than parental cells,the difference was statistically significant(P<0.01). In vivo,causing the minimum number of cells required for sphere cells(50 000) was significantly less than the parental cells(500 000); to reach the minimum number of cells required for 100% tumor formation rate,the sphere cells(500 000) was significantly less than the parental cells(1000 000); after continuous feeding of animals for 4 weeks,the weight and volume of the transplanted tumor which induced by sphere cells is also significantly greater than the parental cells, the difference was statistically significant(p < 0.01). In parental cells,pluripotent stem cell transcription factors Nanog was expressed, while Oct4 and Sox2 were low expressed or undetectable; in sphere cells, the expression of Nanog, Oct4 and Sox2 were significantly up-regulated compared to the parental cell, the difference was statistically significant(p<0.01). Real-Time PCR was performed to detect m RNA expression levels of other stemness markers of CCSCs: In sphere cells, the m RNA expression of Lgr5, CD44 and CD133 were significantly stronger than the parental cells,the difference was statistically significant(p<0.01).2. In spheres cells, the key proteins of Wnt/β-catenin pathway were higher expressed and the m RNA expression levels of pathway ligands(Wnt1, Wnt3 a, Wnt10b), receptors(Frizzled1、Frizzled2、Frizzled3、Frizzled7) and co-receptors(Lrp5、Lrp6) were also higher than parental cells(p<0.05).3. In vitro, XTSJ decoction inhibited the proliferation of parental cells, and the IC50 value at 72 h was 0.9mg/ml; XTSJ decoction also inhibited the proliferation of CCSCs in a dose-time dependent manner, and the IC50 value at 72 h was 0.25 mg/ml; HCT116 parental cells which were pretreated with XTSJ decoction for 72 h could hardly form the sphere;Edu incorporation assay further confirmed that XTSJ decoction could inhibit the proliferative ability of CCSCs(p<0.01). The expression of Nanog and Oct4 was decreased in medium and high concentrations group, and the expression of Sox2 was decreased in the high concentration group; the m RNA expression levels of CD133 and Lgr5 were also decreased(p<0.01). XTSJ decoction significantly inhibited the activity of Wnt/β-catenin signaling pathway, the key protein β-catenin, TCF4 and downstream target gene protein c-Myc, Survivin was significantly down-regulated. The upstream mechanism involves two aspects: XTSJ decoction inhibited the expression of phosphorylated Akt and up-regulated the expression of GSK-3β;Additionally XTSJ decoction decreased the m RNA expression levels of the ligands, receptors and co-receptors at different levels; XTSJ decoction induced cell cycle arrest at S and G2/M phase, and the cell cycle regulatory proteins including Cyclin A2, Cyclin B1, CDK, CDK2 were down-regulated. In the meantime, XTSJ decoction activated Caspase3, Caspase7, up-regulated Bax, down-regulated Bcl-2, and induced cell apoptosis of CCSCs.4. In vivo, the low-dose group did not show a significant inhibition of transplanted tumors’ growth; medium and high doses group can inhibit transplanted tumors’ growth, tumor weight was significantly reduced compared with the control group, the difference was statistically significant(p<0.01). High-dose XTSJ decotion can prolong the survival time and improve the survival rate. In the medium and high dose group, the expression of β-catenin, TCF4 and c-Myc were decreased; the stemness marker Oct4 and Nanog were down-regulated, the m RNA expression levels of CD44 and CD133 were also reduced(p<0.01).Conclusion1. The sphere cells which cultured in serum-free and non-adhesive floating culture system are enriched with colon cancer stem cells. Their proliferation and tumorigenicity were stronger than parental cells, the expression of CCSCs markers were higher expressed and Wnt/β-catenin signaling pathway was significantly activated.2. XTSJ decoction suppressed CCSCs proliferation, induced cell cycle arrest and cell apoptosis in vitro, meanwhile inhibited tumorigenicity and prolonged survival in vivo; it also downregulated the expression of CCSCs markers in vitro and in vivo.3. XTSJ decoction induced the inactivation of Wnt/β-catening signaling pathway. The upstream mechanism involves two aspects: XTSJ decoction inhibited the expression of phosphorylated Akt and up-regulated the expression of GSK-3β;Additionally XTSJ decoction decreased the m RNA expression levels of the ligands, receptors and co-receptors at different levels... |
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