Unlimited growth, recurrence, and metastasis of cancer have been related to the behavior of CSC that can be identified by in vitro assay. Cancer stem cells undergoing metastasis that have been demonstrated to have EMT marker expressions associated with invasive and migratory properties easily invade the surrounding tissues. This invasive type of cancer cell acquired mesenchymal, fibroblast-like morphology and shows reduced intercellular adhesion and increased motility . Tumor progression is frequently associated with the down-regulation of E-cadherin, and up-regulation of vimentin, and several transcription factors including Snail, Twist and Slug [12, 25]. A major clinical feature of NPC is frequent involvement of regional lymph nodes and distant organ metastasis. Even after treatment, increased invasiveness and migration of NPC during metastasis leads to poor outcomes and increased patient mortality . Previous studies showed EMT molecular events associated with NPC metastasis were obvious in the absence of the Epstein-Barr virus genome .
The isolation of CSC from cancer cells was achieved successfully using several techniques. CSC can be enriched in spheres cultured in serum-free mediums supplemented with mitogens, such as the basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) [13, 28, 29]. Such cancer cells grown in a suspended condition exhibit resistance to anoikis, resulting in acquisition of ability to survive and proliferate .
We quantified and enriched CSC within NPC cell lines and subsequently characterized their phenotypical and functional properties, such as invasion capacity and epithelial-mesenchymal transition (EMT). These spheres contain putative CSC and can provide niches for the maintenance and growth of cancer cells. The EMT-type cells share many biological characteristics of CSC, which are linked closely with tumor recurrence and metastasis.
Previous studies exploited EGCG as a potential agent for prevention and treatment of cancers. EGCG suppresses the expression of HSP70 and HSP90, and exhibits anti-tumor activity in vitro and in vivo . Moreover, in addition to EGCG-induced apoptosis effects, previous researches have recognized that EGCG-induced p21, p53, p16, and p27 expression, which is associated with negative regulations of cell cycle progression [32–34]. However, there were limited reports about EGCG regulation effects on CSC. Our study proposes phenotype changes and functional consequences, by which the inhibitory effects of EGCG on CSC properties of NPC are seen.
Our designed experiments recognized the effects of EGCG on isolated sphere cells with anchorage-independent growth, and obtained novel findings. We discovered EGCG inhibits growth and induces apoptosis in parental NPC cells, but is inefficient in sphere-derived cells. Relative chemo-resistant NPC cells in sphere-derived cells, in accordance to Chen’s study on non-adhesive culture systems exhibiting CSC characteristics, can be used in cancer research .
On the other hand, EGCG can inhibit sphere formation and regulate invasive-like phenotypes characterized by EMT of cells grown in serum free non-adherent cultures. In addition, it inhibited colony formation of sphere-derived cells grown in agar in a dose-dependent manner. This implicated that EGCG can be effective in suppressing the self-renewal capacity of NPC CSC. Furthermore, at the mRNA level, the expression of stemness maintaining transcription factors Klf-4 and Oct-4 were inhibited by EGCG. Several studies suggested Klf-4 and Oct-4 play important roles in maintaining stem cell identity, and are responsible for increased cell migration and invasion [36, 37]. Our study further showed that EGCG inhibits the factors required for maintaining the self-renewal capacity in NPC CSC.
Because CSCs appear to have a significant role in invasion and metastasis, we measured the effects of EGCG on invasion and migration of NPC sphere-derived cells. The results showed that invasive and migratory behaviors were significantly reduced in the EGCG-treated group in a dose-dependent manner. This implied EGCG efficiently suppressed the metastasis ability in NPC CSC. Recent studies have also described the inhibitory activities of EGCG targeting cancer stem cells, which can suppress CSC self-renewal properties and block their migration and invasion [38, 39].
Enhanced EMT characteristics are associated with poor outcomes and declined survival in patients with NPC . We examined EGCG regulating translational levels of EMT expression by TW01 sphere-derived cells. The western blot result showed EGCG inhibits the expression of vimentin, Snail, and is accompanied by the upregulation of E-cadherin. Immunofluorescence staining showed that vimentin expression in sphere-derived cells was decreased in the EGCG group compared to the control group. These results showed that EGCG may block NPC early metastasis signaling. Dysregulation of E-cadherin and β-catenin functions in cell-cell adhesion is common in NPC, and correlates with advanced stage disease and lymph node metastasis . Our results further showed that EGCG inhibited NPC invasion and migration, correlating with the elevation of E-cadherin levels in NPC sphere-derived cells. E-cadherin is an important cell adhesion molecule, and has a key role in the early stage of tumor metastasis. Further investigation is required to study how EGCG modulates the expression of E-cadherin proteins.
Research regarding the use of EGCG against cancer has been proposed, however, most concentrations of EGCG used in cell lines are higher than the plasma concentrations observed in people after consuming tea because of its limited bioavailability in vivo. To increase bioavailability and stability of EGCG, particular studies have elucidated structure-modified compounds with higher efficacy, which also possess anticancer activities [39, 43]. Previous research has shown evidence of EGCG-induced apoptosis and cytotoxicity specifically on cancer cells, but not on normal cells under the same EGCG treatment [44, 45]. This suggests that EGCG serves as an acceptable safety agent.
Overall, our findings further clarify the anti-cancer effects of EGCG, which can eliminate the stem-cell characteristics of NPC, and inhibit epithelial-mesenchymal transition (EMT), partially because of regulated EMT-related proteins and lessen the migratory and invasive abilities of NPC. EGCG may be potentially effective in preventing the recurrence and metastasis of NPC in combination with standard treatment.