Diet-based cancer prevention and therapy have received considerable attention in recent years. Green tea, a popular beverage consumed worldwide, has been reported to have inhibitory effects against various types of cancer, such as breast, lung, prostate, and colon cancer.
Most of the chemo-preventive effects of green tea on cancer are attributed to polyphenol compounds, among which epigallocatechin-3-gallate (EGCG) is the most important1. EGCG accounts for 50–80% of the catechin in green tea. There is 200–300 mg of EGCG in a brewed cup (240 mL) of green tea2. By drinking cups of green tea or taking an EGCG tablet, a serum concentration of 0.1–1 μM EGCG can be achieved3,4.
The anti-cancer effect of EGCG has been demonstrated in epidemiological, cell culture, and animal studies, and in clinical trials5. A 10-year prospective study by Nakachi and Imai reported a decreased risk of cancer for those consuming over 10 cups of green tea a day, compared with those consuming below three cups6,7. Recently, Shin et al. found that green tea extract reduced the recurrence rate of colorectal adenomas by 44.2% in a randomized clinical trial in Korea8.
In vitro, EGCG was shown to promote cell growth arrest and induce apoptosis in a variety of human cancer cell lines, including prostate carcinoma cells9,10, epidermoid carcinoma cells11, bladder cancer cells12, and colon cancer cells13. In vivo, oral or intravenous administration of green tea or purified EGCG in mice inhibited angiogenesis and restrained solid tumor growth14,15.
At the molecular level, EGCG has been demonstrated to interact with cancer-related proteins, such as glucose-regulated protein 78 (GRP78)16 and Ras–GTPase-activating protein SH3 domain-binding protein 1 (G3BP1)17, with approximately μΜ affinities.
In EGCG-induced apoptosis and cell growth arrest, p53 was found to play an important role18,19. p53, often referred to as “the guardian of the genome”, is a crucial tumor suppressor mutated in over 50% of human cancer. p53 promotes cell-cycle arrest or apoptosis as a response to cellular stress stimuli, such as oxidative stress, oncogene activation, and DNA damage20,21.
As a transcription factor, p53 is tightly regulated with a short half-life. p53 protein is normally maintained at low levels in healthy mammalian cells by continuous ubiquitylation and subsequent degradation, mediated by murine double minute 2 (MDM2) E3 ligase. Under cellular stress, ubiquitylation of p53 is suppressed and p53 is stabilized. p53 then accumulates in the nucleus and turns on expression of target genes, triggering cell-cycle arrest, apoptosis, and DNA-repair processes20.
Besides acting as a transcription factor, p53 can also translocate to the cytoplasm or mitochondria. p53 interacts directly with anti-apoptotic proteins such as Bax and Bcl2 to induce apoptosis21,22 and is also involved in the anti‑senescent effect of EGCG23,24.
Full length p53 is composed of an N-terminal domain (NTD), a DNA-binding domain (DBD), a tetramerization domain (TET), and a C-terminal regulatory domain (REG) (Fig. 1). The NTD is further divided into two transcriptional activation domains (TAD1 and TAD2) and a proline-rich domain (PRD). NTD is an intrinsically disordered protein (IDP) and interacts with many proteins, acting as a hub for cellular signaling25,26.
NTD is not only required for transactivation, but also binds MDM2 to mediate the ubiquitylation and degradation of p53. Independent of ubiquitylation, MDM2 also inhibits transcription by preventing general transcription factors from binding to NTD27. The apoptosis effect of EGCG on human cancer cells was associated with its interference of MDM2-mediated p53 ubiquitylation28. EGCG is also reported to stabilize p53, with increased phosphorylation on critical serine residues29.
In a recent study, EGCG was identified from a library of 2295 phytochemicals as an inhibitor of p53–MDM2 interaction30. However, the molecular mechanism of how EGCG disrupts MDM2–p53 interaction is not yet understood.
In this work, we demonstrate the direct binding between EGCG and p53, mediated by NTD of p53. We show that the EGCG–p53 interaction disrupts p53 interaction with MDM2 and inhibits ubiquitination of p53, likely stabilizing p53 for anti-tumor activity, providing a structural mechanism for the anti-cancer effect of EGCG.
Results
EGCG–p53 interaction was studied using a sensor chip immobilized with full-length p53 or p53 NTD. As shown in Fig. 1a, b, specific binding of EGCG to full-length p53 and p53 NTD were detected by SPR. The dissociation constant (KD = 4 ± 2 μM) for p53 NTD and EGCG interaction was similar to that (KD = 1.6 ± 1.4 μM) for full-length p53 and EGCG interaction.
The two dissociation constants are within measurement error, indicating that NTD is the major mediator of the p53–EGCG interaction. NTD is also one of the primary binding sites for MDM231, suggesting this EGCG–NTD interaction may impact MDM2-mediated p53 degradation and inhibit NTD interactions with general transcription factors.
Full article: https://www.nature.com/articles/s41467-021-21258-5