Epigallocatechin‑3‑gallate inhibits self‑renewal ability of lung cancer stem‑like cells through inhibition of CLOCK

Abstract

Circadian rhythm plays an important role in diverse physiological processes. Abnormal expression of circadian rhythm genes is associated with increased risk of disease, including different types of cancer. The cancer stem cell (CSC) hypothesis suggests that there is a small subset of stem-like cells within tumors that are responsible for tumor initiation. However, the biological effect of circadian rhythm on CSCs remains largely unknown. Studies have highlighted that the circadian rhythm protein CLOCK controls key aspects of various diseases. In the present study, lung cancer stem-like cells were successfully enriched using a sphere formation assay. Next, it was observed that CLOCK mRNA and protein expression levels in the A549 and H1299 sphere cells were notably increased compared with those in the corresponding parental cells. In addition, flow cytometry was performed to isolate CD133 + cells and, consistently, CLOCK expression was also found to be markedly upregulated in CD133 + lung cancer cells. Subsequently, to determine the effect of CLOCK on lung cancer stem cells in detail, CLOCK was knocked down using targeted short inhibiting RNA and the results demonstrated that the sphere-forming ability of the A549 and H1299 cell lines was reduced. In addition, CSC-like properties, including the expression of CD133, CD44, sex determining region Y-box 2, Nanog and octamer-binding transcription factor 4, were markedly decreased in the A549 and H1299 sphere cells following knockdown of CLOCK. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, has been reported to be a potential anticancer phytochemical. EGCG was found to repress CLOCK expression in A549 and H1299 sphere cells. In addition, EGCG also decreased the ratio of CD133 + cells. The Wnt/β-catenin pathway was notably inactivated by the knockdown of CLOCK in A549 and H1299 sphere cells. Subsequently, using a xenograft model, it was demonstrated that EGCG suppressed the CSC-like characteristics of lung cancer cells by targeting CLOCK. In conclusion, the present study demonstrated that EGCG inhibited the self-renewal ability of lung cancer stem-like cells by targeting CLOCK.

New Insights Into Cancer Chronotherapies

“…However, in esophageal tumors, CLOCK , PER1 , PER2 , PER3 , CRY1 as well as CRY2 levels are down-regulated and their downstream proteins accordingly become disordered ( van der Watt et al, 2020 ). As for lung cancer cells, the primary circadian gene CLOCK is up-regulated in CD133+ cells, and this can also be observed in A549 and H1299 cells ( Jiang et al, 2020 ; Yoshida et al, 2013 ). A comparative analysis revealed that PER1 levels were nearly 30% those of normal tissues, PER2 was 70%, CRY1 was 66%, CRY2 was 30%, and BMAL1 was 80% in non-small cell lung cancer ( Chen et al, 2020 ).…”

Recent Advances in Chronotherapy Targeting Respiratory Diseases

“…Interestingly, the knockdown of CLOCK by siRNA resulted in a reduced ability for sphere formation and the inactivation of the Wnt/β-catenin signaling pathway, while treatment with EGCG repressed CLOCK expression in the spheroids. Moreover, in a xenograft model, EGCG suppressed the cancer stem-cell-like features (CD133, CD44, Sox2, Nanog and Oct4 protein) of A549 and H1299 cells by targeting CLOCK, suggesting EGCG is a compound with chronotherapeutic potential to inhibit the self-renewal capacity of lung cancer stem-like cells by modulating CLOCK [101]. In another study, it was observed that doxorubicin (DOX) altered the core clock genes and various cytokines in macrophages extracted from tumor (Lewis lung carcinoma)-bearing mice.…”

Circulating Tumour Cells (CTCs) in NSCLC: From Prognosis to Therapy Design

“…Numerous clinical trials have confirmed a strong correlation between PD-L1 expression and the OS and PFS of NSCLC patients [63,68,[81][82][83][84][85], which is deeply associated with tumour immunoresistance [86].…”