*3.9. Proteasome*

The proteasome is a protein complex that is dedicated to degrade damaged or useless proteins that are unusable to cell [153]. It is strictly dependent on ubiquitins, molecules that tag unwanted proteins and continues with the degradation process mediated by proteases [154]. The proteasome has been investigated as a key target for multiple diseases, given its importance in cell physiology. E ffectively, three approved drugs are available, bortezomib (for multiple myeloma and mantle cell lymphoma), carfilzomib and ixazomib (for relapsed or refractory multiple myeloma), and many others are in development [155]. Di fferent cell types were tested with EGCG: human Jurkat T, prostate cancer (LNCaP, PC-3), breast cancer (MCF-7), normal (WI-38), and SV40-transformed (VA-13) human fibroblast cells [156,157]. The works showed that EGCG specifically blocked proteasome activity in LNCaP, PC-3, and MCF-7 cell extracts with a range of IC50 from 86 to 194 nm. In addition, in cell models, EGCG 1–10 μM induced the accumulation of two natural proteasome substrates, p27(Kip1) and IkappaB-alpha, an inhibitor of transcription factor NF-κB, together with G1 cell cycle phase arrest. Moreover, EGCG derivatives were studied both to understand the biochemical role of such molecules in proteasome inhibition and to find novel potent or stabilized alternatives to EGCG [158]. For example, a work of Kazi and collaborators revealed that the A-ring and gallate ester/amide bond are necessary for the blockade of proteasome [159]. Similarly, EGCG modified with peracetate presented higher proteasome-inhibitory activity if compared to EGCG, with increased cell death mechanism in Jurkat T cells [160]. Again, other EGCG analogs were tested and showed that B-ring/D-ring peracetate-protected EGCG derivatives could induce cell death [161,162]. Additionally, peracetate-protected EGCG in breast carcinoma cell line (MDA-MB-231) and in MDA-MB-231 mouse xenografts (daily *sub cute* injection with 50 mg/kg) was tested, showing tumor growth decrement, with concomitant proteasome inhibition and apoptosis activation [163]. Later, di fferent fluoro-substituted EGCG analogs were generated and, among these, Pro-F-EGCG4 ((-)-(2R,3R)-5,7-Diacetoxy-2-(3,4,5-triacetoxyphenyl)chroman-3-yl3,4-difluorobenzoate) had potent e ffects than peracetate EGCG inducing apoptosis and decreasing cell proliferation (concentration-dependent e ffects from 10 to 50 μM) in the Jurkat and MDA-MB-231 cell lines, in addition to reducing tumor size in MDA-MB-231 mouse xenografts (daily *subcutaneous* injection with 50 mg/kg) [164,165]. In the same cell line (MDA-MB-231), EGCG was tested with catechol-σ-methyltransferase (COMT), an human enzyme that degrades catecholamines [166]. COMT can methylate EGCG, reducing its biological activity. The work reported that decreasing COMT activity could augmen<sup>t</sup> EGCG e fficacy through apoptosis induction and proteasome inhibition. In YT (human natural killer) and Jurkat cell lines, EGCG (and other proteasome inhibitors) produced cell cycle modulation (subG0/G1 phase increase in Jurkat cells and G2/M phase arrest in YT cells), apoptosis with caspase-3 activation, and with mitochondrial membrane potential decrement [167]. Moreover, in Caco-2 cells, EGCG inhibited 20S proteasomes activity with antioxidant and proteasome blocking properties in cell lysates [168]. In the human neuroblastoma cell line (SH-SY5Y), EGCG at low concentration (1 μM) impacted on BCL2 associated agonist of cell death (Bad) levels, while the protein kinase C (PKC) inhibitor GF109203X impeded Bad degradation [169]. The authors claimed that EGCG promoted neuronal survival through the rapid removal of Bad (PKC-mediated) by proteasome, while using low doses, whereas most of works showed general EGCG inhibitory e ffects at 50 μM. This di fference could probably reside in the specific cell model used. In a mouse model of experimental autoimmune encephalomyelitis (EAE), EGCG administered by gavage 300 μL per mouse twice daily was able to decrease clinical severity, as suggested by reduced brain inflammation and neuronal damage [170]. In addition, in the same work with human myelin-specific CD4+ T cells, EGCG impacted cyclin-dependent kinase 4 expression (with cell cycle arrest) and on 20S/26S proteasome complex activity (with subsequent NF-κB inhibition), demonstrating anti-inflammatory and neuronal protective capacities. In skin cancer cells (SCC-13 and A431), EGCG alone or associated to 3-deazaneplanocin A (a potent inhibitor of S-adenosylhomocysteine hydrolase, enzyme that is involved in homocysteine metabolism) suppressed cell cycle progression and increased apoptosis, through proteasome-dependent degradation of the polycomb group proteins, molecules that are involved in development, di fferentiation, and survival [171]. A study investigated the e ffects of extremely low frequency electromagnetic fields (ELFEF) on 20S proteasome while using EGCG in Caco-2 cells [172]. It is known that ELFEF can modulate intracellular ROS levels and the cell cycle progression, indeed EGCG was able to reduce the pro-oxidant e ffects, regulate cell cycle, and proteasome functionality. However, EGCG did not alter cell viability as measured by MTT assay.

Though numerous works demonstrated a positive interaction of EGCG in proteasome function, others showed a deleterious activity of EGCG. For example, in HepG2 cells, EGCG 50 μM exerted anti-cholesterolemic activity, reducing apolipoprotein B-100 (apoB) production and lipid assembly, thorough a proteasome-independent pathway, as demonstrated by a lack of response to N-acetyl-leucyl-leucyl-norleucinal, a proteasome inhibitor [173]. Moreover, the association of EGCG and bortezomib (proteasome inhibitor for multiple myeloma) should not be suggested, as their experimentation in human multiple myeloma cells (RPMI/8226 and U266) showed strong antagonism, with EGCG blocking proteasome inhibition being produced by bortezomib and, thus, stopping tumor cell death [174,175]. Similarly, in prostate cancer cells (PC3), bortezomib anti-tumor e ffects was counteracted by EGCG which induced autophagy while decreasing endoplasmic reticulum stress [176]. These data underline in a di fferent cell model, as EGCG can antagonize bortezomib, blocking prostate cancer cells death, and thus negatively supporting tumor pro-survival.
