Cancer stem cells (CSCs) are a sub-population of cells within cancer tissues with tumor initiation, drug resistance and metastasis properties. CSCs also have been considered as the main cause of cancer recurrence. Targeting CSCs have been suggested as the key for successful treatment against cancer. Tumorsphere cultivation is based on culturing cancer cells onto ultralow attachment surface in serum-free media under the supplementation with growth factors such as epidermal growth factor and basic fibroblast growth factor. Tumorsphere cultivation is widely used to analyze the self-renewal capability of CSCs and to enrich these cells from bulk cancer cells. This method also provides a reliable platform for screening potential anti-CSC agents. The in vitro anti-proliferation activity of potential agents selected from tumorsphere assay is more translatable into in vivo anti-tumorigenic activity compared with general monolayer culture. Tumorsphere assay can also measure the outcome of clinical trials for potential anti-cancer agents. In addition, tumorsphere assay may be a promising strategy in the innovation of future cancer therapeutica and may help in the screening of anti-cancer small-molecule chemicals.

Oral submucous fibrosis (OSF) is considered as a pre-cancerous condition of the oral mucosa and is highly associated with habitual areca quid chewing. Arecoline is the major alkaloid in areca quid and is thought to be involved in the pathogenesis of OSF. Our previous studies have demonstrated that arecoline could induce epithelial–mesenchymal transition (EMT)-related factors in primary human buccal mucosal fibroblasts (BMFs). Therefore, we investigated the expression of zinc finger E-box binding homeobox 1 (ZEB1), which is a well-known transcriptional factor in EMT, in OSF tissues and its role in arecoline-induced myofibroblast transdifferentiation from BMFs. The expression of ZEB1, as well as the myofibroblast marker α-smooth muscle actin (α-SMA), was significantly increased in OSF tissues, respectively. With immunofluorescence analysis, arecoline induced the formation of α-SMA-positive stress fibres in BMFs expressing nuclear ZEB1. Arecoline also induced collagen contraction of BMFs in vitro. By chromatin immunoprecipitation, the binding of ZEB1 to the α-SMA promoter in BMFs was increased by arecoline. The promoter activity of α-SMA in BMFs was also induced by arecoline, while knockdown of ZEB1 abolished arecoline-induced α-SMA promoter activity and collagen contraction of BMFs. Long-term exposure of BMFs to arecoline induced the expression of fibrogenic genes and ZEB1. Silencing of ZEB1 in fibrotic BMFs from an OSF patient also suppressed the expression of α-SMA and myofibroblast activity. Inhibition of insulin-like growth factor receptor-1 could suppress arecoline-induced ZEB1 activation in BMFs. Our data suggest that ZEB1 may participate in the pathogenesis of areca quid–associated OSF by activating the α-SMA promoter and inducing myofibroblast transdifferentiation from BMFs.

Recent studies suggest that ALDH1 is a putative marker for HNSCC-derived cancer stem cells. However, the regulation mechanisms that maintain the stemness and metastatic capability of HNSCC-ALDH1+ cells remain unclear. Initially, HNSCC-ALDH1+ cells from HNSCC patient showed cancer stemness properties, and high expression of Bmi1 and Snail. Functionally, tumorigenic properties of HNSCC-ALDH1+ cells could be downregulated by knockdown of Bmi-1. Overexpression of Bmi-1 altered in expression property ALDH1− cells to that of ALDH1+ cells. Furthermore, knockdown of Bmi-1 enhanced the radiosensitivity of radiation-treated HNSCC-ALDH1+ cells. Moreover, overexpression of Bmi-1 in HNSCC-ALDH1− cells increased tumor volume and number of pulmonary metastatic lesions by xenotransplant assay. Importantly, knock-down of Bmi1 in HNSCC-ALDH1+ cells significantly decreased distant metastases in the lungs. Clinically, coexpression of Bmi-1/Snail/ALDH1 predicted the worst prognosis in HNSCC patients. Collectively, our data suggested that Bmi-1 plays a key role in regulating Snail expression and cancer stemness properties of HNSCC-ALDH1+ cells.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Patients diagnosed with GBM have a poor prognosis, and it has been reported that tumor malignancy and GBM recurrence are promoted by STAT3 signaling. As resveratrol (RV), a polyphenol in grapes, is reported to be a potent and non-toxic cancer-preventive compound, the aim of this study was to investigate the therapeutic effect and molecular mechanisms of RV on GBM-derived radioresistant tumor initiating cells (TIC). Firstly, our results showed that primary GBM-CD133(+) TIC presented high tumorigenic and radiochemoresistant properties as well as increased protein levels of phosphorylated STAT3. We consistently observed that treatment with shRNA-STAT3 (sh-STAT3) or AG490, a STAT3 inhibitor, significantly inhibited the cancer stem-like cell properties and radioresistance of GBM-CD133(+) in vitro and in vivo. Furthermore, treatment of GBM-CD133(+) with 100 µM RV induced apoptosis and enhanced radiosensitivity by suppressing STAT3 signaling. Microarray results suggested that RV or AG490 inhibited the stemness gene signatures of GBM-CD133(+) and facilitated the differentiation of GBM-CD133(+) into GBM-CD133(-) or astrocytoma cells. Finally, xenotransplant experiments indicated that RV or sh-STAT3 therapy could significantly improve the survival rate and synergistically enhance the radiosensitivity of radiation-treated GBM-TIC. In summary, RV can reduce in vivo tumorigenicity and enhance the sensitivity of GBM-TIC to radiotherapies through the STAT3 pathway.

Metastasis is the most common cause of cancer-related death in patients, and epithelial-to-mesenchymal transition (EMT) is essential for cancer metastasis, which is a multistep complicated process that includes local invasion, intravasation, extravasation, and proliferation at distant sites. When cancer cells metastasize, angiogenesis is also required for metastatic dissemination, given that an increase in vascular density will allow easier access of tumor cells to circulation, and represents a rational target for therapeutic intervention. Berberine has several anti-inflammation and anticancer biologic effects. In this study, we provided molecular evidence that is associated with the antimetastatic effect of berberine by showing a nearly complete inhibition on invasion (P , 0.001) of highly metastatic SiHa cells via reduced transcriptional activities of matrix metalloproteinase-2 and urokinase-type plasminogen activator. Berberine reversed transforming growth factor-b1-induced EMT and caused upregulation of epithelial markers such as E-cadherin and inhibited mesenchymal markers such as N-cadherin and snail-1. Selective snail-1 inhibition by snail-1-specific small interfering RNA also showed increased E-cadherin expression in SiHa cells. Berberine also reduced tumor-induced angiogenesis in vitro and in vivo. Importantly, an in vivo BALB/c nude mice xenograft model and tail vein injection model showed that berberine treatment reduced tumor growth and lung metastasis by oral gavage, respectively. Taken together, these findings suggested that berberine could reduce metastasis and angiogenesis of cervical cancer cells, thereby constituting an adjuvant treatment of metastasis control.

BackgroundThymoquinone (TQ), an active component of Nigella sativa or black cumin, elicits cytotoxic effects on various cancer cell lines. However, the anti-cancer effects of TQ on head and neck squamous cell carcinoma (HNSCC) remain unclear.Methodology/Principal FindingsIn this study, TQ elicited a strong cytotoxic effect on SASVO3, a highly malignant HNSCC cell line. The mechanisms of this cytotoxic effect were concentration dependent. TQ also induced apoptotic cell death in SASVO3 cells as indicated by an increase in Bax expression and caspase-9 activation. Apoptosis was possibly caspase-9 dependent because the exposure of cells to a caspase-9 inhibitor partially prevented cell death. The exposed cells also showed increased levels of autophagic vacuoles and LC3-II proteins, which are specific autophagy markers. Cell viability assay results further revealed that bafilomycin-A1, an autophagy inhibitor, enhanced TQ cytotoxicity; by comparison, Annexin V and propidium-iodide staining assay results showed that this inhibitor did not promote apoptosis. TQ treatment also increased the accumulation of autophagosomes. Using a lentivirus-shRNA system for LC3 silencing, we found that cell viability was eradicated in autophagy-defective cells. An in vivo BALB/c nude mouse xenograft model further showed that TQ administered by oral gavage reduced tumor growth via induced autophagy and apoptosis.ConclusionsThese findings indicated that TQ induced cell death in oral cancer cells via two distinct anti-neoplastic activities that can induce apoptosis and autophagy. Therefore, TQ is a promising candidate in phytochemical-based, mechanistic, and pathway-targeted cancer prevention strategies.

The feature of oral squamous cell carcinomas (OSCC) is commonly metastasizing to locoreginal lymph nodes, and the involvement of lymph nodes metastasis represents the one of important prognostic factors of poor clinical outcome. MicroRNAs (miRNAs) have been shown to be key players of cancer-related hallmarks including cancer stemness, EMT (epithelial-mesenchymal transition), and metastaisis. Herein we showed that OSCC-derived ALDH1+ cancer stem cells (OSCC-CSCs) express lower level of miR-204, and miR-204 over-expression suppresses cancer stemness and in vivo tumor-growth of OSCC-CSCs. miR-204 binds on their 3′UTR-regions of Slug and Sox4 and suppressing their expression in OSCC-CSCs. On the contrary, down-regulation of miR-204 significantly increased cancer stemness and the lymph nodes incidence of orthotopic animal models. Furthermore, co-knockdown with sh-Slug and sh-Sox4 synergistically rescued miR-204-supressing cancer stemness and EMT properties. Clinical results further revealed that a miR-204lowSlughighSox4high signature predicted the worse survival prognosis of OSCC patients by Kaplan-Meier survival analyses. Up-regulated miR-204-targeting Slug and Sox4 by epigallocatechin-3-gallate (EGCG) treatment significantly inhibited the proliferation rate, self-renewal capacity, and the percentage of ALDH1+ and CD44+ cells in OSCC-CSCs Oral-feeding of EGCG effectively alleviated tumor-progression in OSCC-CSCs-xenotransplanted immunocompromised mice through miR-204 activation. In conclusion, miR-204-mediated suppression of cancer stemness and EMT properties could be partially augmented by the anti-CSCs effect of EGCG.

Tumor initiating cells (TICs) possessing cancer stemness were shown to be enriched after therapy, resulting in the relapse and metastasis of head and neck squamous cell carcinomas (HNC). An effective therapeutic approach suppressing the HNC-TICs would be a potential method to improve the treatments for HNC. We observed that the treatment of silibinin (SB) dose dependently down-regulated the ALDH1 activity, CD133 positivity, stemness signatures expression, self-renewal property, and chemoresistance in ALDH1+CD44+ HNC-TICs. Using miRNA-microarray and mechanistic studies, SB increased the expression of microRNA-494 (miR-494) and both Bmi1 and ADAM10 were identified as the novel targets of miR-494. Moreover, overexpression of miR-494 results in a reduction in cancer stemness. However, knockdown of miR-494 in CD44−ALDH1−non-HNC-TICs enhanced cancer stemness and oncogenicity, while co-knockdown of Bmi1 and ADAM10 effectively reversed these phenomena. Mice model showed that SB treatment by oral gavage to xenograft tumors reduced tumor growth and prolonged the survival time of tumor-bearing mice by activation of miR-494-inhibiting Bmi1/ADAM10 expression. Survival analysis indicated that a miR494highBmi1lowADAM10low phenotype predicted a favourable clinical outcome. We conclude that the inhibition of tumor aggressiveness in HNC-TICs by SB was mediated by up-regulation miR-494, suggesting that SB would be a valuable anti-cancer drug for treatment of HNC.