Morusin inhibits the growth of human colorectal cancer HCT116­derived sphere­forming cells via the inactivation of Akt pathway.

The existence of colorectal cancer stem­like cells (CSC) is responsible for the failure of current treatments against colorectal cancer. Therefore, novel therapies need be developed to target CSCs. Some natural agents, including morusin have been proposed as possible candidates for this purpose. Morusin has been shown to exert antitumor effects. In the present study, it is demonstrated that morusin exerts antitumor effects on colorectal CSCs (CCSCs). The viability of human CCSCs was enhanced when the CCSCs formed spheroids in a serum­free and non­adhesive floating culture system. HCT116 sphere cells exhibited an increased proliferative capacity and a higher expression of stemness markers [octamer­binding transcription factor 4 (Oct4), Sox2 and Nanog]. Morusin inhibited the development of cancer spheroids and suppressed the growth of sphere cells via the induction of cell cycle arrest. Similarly, morusin decreased the expression levels of the stemness markers, Nanog and Oct4. The data partially revealed the molecular mechanisms involved: ß­catenin signaling maintains the growth of CSCs and directly modulates the expression of Nanog and Oct4. Morusin suppressed the activity of ß­catenin signaling via the inactivation of Akt; the executive ß­catenin/TCF4 complex and the downstream targets, c­Myc, survivin and cyclin D1, were also downregulated. Moreover, the morusin­induced inactivation of Akt also increased the expression of p21Cip1/WAF1 and p27Kip, which can block the cell cycle by interacting with cyclin­dependent kinase (CDK) complexes. On the whole, the present study demonstrates that morusin inhibited the growth of colorectal cancer sphere cells, which were enriched with CCSCs via the inactivation of the Akt pathway.

Desialylation of Sonic-Hedgehog by Neu2 Inhibits Its Association with Patched1 Reducing Stemness-Like Properties in Pancreatic Cancer Sphere-forming Cells.

Cancer stem cells (CSCs) are crucial regulators of tumor recurrence/progression. The maintenance of CSCs is dependent on aberrant activation of various pathways, including Hedgehog. Prevalent sialylations contribute to aggressiveness in CSCs. Here, we have addressed the role of sialylation in regulating stemness-like properties of pancreatic cancer sphere-forming cells (PCS) through modulation of the Hedgehog (Hh) pathway. The status of CD133/CD44/surface-sialylation was checked by flow cytometry and effects of Neu2 overexpression in PCS were compared using qPCR, immunoblotting, co-immunoprecipitation and also by colony-formation assays. The work was also validated in a xenograft model after Neu2 overexpression. Neu2 and Shh status in patient tissues were examined by immunohistochemistry. PCS showed higher Hh-pathway activity and sialylation with reduced cytosolic-sialidase (Neu2). Neu2 overexpression caused desialylation of Shh, thereby reducing Shh-Patched1 binding thus causing decreased Hh-pathway activity with lower expression of Snail/Slug/CyclinD1 leading to reduction of stemness-like properties. Neu2-overexpression also induced apoptosis in PCS. Additionally, Neu2-overexpressed PCS demonstrated lower mTORC2 formation and inhibitory-phosphorylation of Gsk3ß, reflecting a close relationship with reduced Hh pathway. Moreover, both Neu2 and Rictor (a major component of mTORC2) co-transfection reduced stem cell markers and Hh-pathway activity in PCS. Neu2-overexpressed tumors showed reduction in tumor mass with downregulation of stem cell markers/Shh/mTOR and upregulation of Bax/Caspase8/Caspase3. Thus, we established that reduced sialylation by Neu2 overexpression leads to decreased stemness-like properties by desialylation of Shh, which impaired its association with Patched1 thereby inhibiting the Hh pathway. All these may be responsible for enhanced apoptosis in Neu2-overexpressed PCS.

High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

Epigallocatechin gallate hinders human hepatoma and colon cancer sphere formation.

BACKGROUND AND AIM: The long-term survival of patients with hepatocellular carcinoma remains unsatisfactory because of the presence of cancer stem cells (CSCs), which are responsible for tumor recurrence and chemoresistance after hepatectomy. Drugs that selectively target CSCs thus offer great promise for cancer treatment. Although the antitumor effects of epigallocatechin gallate (EGCG) have been reported in some cancer cells, its effects on CSCs remain poorly studied. In this study, we investigated the effects of EGCG on human hepatoma and colon CSCs. METHODS: HepG2 and HCT-116 cell lines were enriched by sphere formation, and their gene-expression profiles were analyzed by quantitative real-time polymerase chain reaction. EGCG-induced growth inhibition in the parental cells was determined by WST-8 assay, and protein expression was assessed by western blotting. Cell cycle profile and apoptosis analysis was performed using flow cytometer. RESULTS: Sphere-derived cells grown in serum-free, nonadherent cultures showed increased expression of stem cell markers, Nek2, and ATP-binding cassette transporter genes, compared with parental cells grown in conventional culture. EGCG induced growth inhibition in the parental cells in a dose-dependent manner. EGCG also inhibited self-renewal in hepatoma and colon CSCs, attenuated the expression of stem cell markers and ATP-binding cassette transporter genes, which are putative molecules associated with treatment resistance in CSCs, and decreased the transcription of Nek2 and p-Akt, resulting in the inhibition of Akt signaling. EGCG also altered cell cycle profile and apoptosis, which may in part play an important role in EGCG-induced cancer cell death. CONCLUSIONS: Overall, these results suggest that EGCG could be a useful chemopreventive agent for targeting hepatocellular carcinoma and colon CSCs, in combination with standard chemotherapies.

Cultivation and identification of colon cancer stem cell-derived spheres from the Colo205 cell line

Our group established a method to culture spheres under serum-free culture condition. However, the biological characteristics and the tumorigenicity of spheres are unknown. Here, we demonstrate that sphere cells expressed high levels of the putative colorectal cancer stem cell markers CD133 and CD44. The CD133-positive rates were 13.27 ± 5.62, 52.71 ± 16.97 and 16.47 ± 2.45 percent in sphere cells, regular Colo205 cells and differentiated sphere cells, respectively, while the CD44-positive rates were 62.92 ± 8.38, 79.06 ± 12.10 and 47.80 ± 2.5 percent, respectively, and the CD133/CD44-double-positive rates were 10.77 ± 4.96, 46.89 ± 19.17 and 12.41 ± 2.27 percent, respectively (P < 0.05). Cancer sphere cells formed crypt-like structures in 3-D culture. Moreover, cells from cancer spheres exhibited more tumorigenicity than regular Colo205 cells in a xenograft assay. The cancer sphere cells displayed much higher oncogenicity than regular Colo205 cells to initiate neoplasms, as assayed by H&E staining, Musashi-1 staining and electron microscopy. Our findings indicated that the sphere cells were enriched with cancer stem cells (CSCs), and exhibited more proliferation capacity, more differentiation potential and especially more tumorigenicity than regular Colo205 cells in vitro and in vivo. Further isolation and characterization of these CSCs may provide new insights for novel therapeutic targets and prognostic markers.