Functional targeting of the TGF-βR1 kinase domain and downstream signaling: A role for the galloyl moiety of green tea-derived catechins in ES-2 ovarian clear cell carcinoma

 The galloyl moiety is a specific structural feature which dictates, in part, the chemopreventive properties of diet-derived catechins.

• In ovarian cancer cells, galloylated Catechins were recently demonstrated to target the transforming growth factor (TGF)-β-mediated control of the epithelial-mesenchymal transition process, but their impact on signaling remains poorly understood. Here, we questioned whether the sole Galloyl Moiety interacted with TGF-β-receptors to alter signal transduction and chemotactic migratory response in an ES-2 serous carcinoma-derived ovarian cancer cell model.

Cancer chemoprevention with green tea

• Green tea intake, decreased ovarian cancer occurrence, and better prognosis have been observed from observational studies
• Evidence points to the actions of a group of molecules termed polyphenols, which in combination with other phytochemicals or micronutrients, can target multiple molecular processes involved in cancer development and progression
• Metastasis, which combines complex molecular processes involving, in part, cancer cell migration away from the primary site of tumor [10], has been thought to be targeted by green tea components
• The exact relationship between the structure and biological actions of several green tea catechins against the early upstream signaling events that trigger EMT remains unaddressed
• Catechins presenting a galloyl moiety, namely (-)-epigallocatechin-3-gallate (EGCG), epicatechin gallate (ECG), catechin gallates (CG), and gallocatechin Gallate (GCG), inhibited TGF-β-induced phosphorylation of Smad-3 more potently than their ungalloylated counterparts
• Directophysical interaction of RTKs and EMTs and the interaction of the transforming growth factor-β receptor (IGF-β) and the epidermal growth factor receptor (RTKs) is still investigated

Materials

• Sodium dodecylsulfate (SDS), all catechins, galloylated compounds, and bovine serum albumin were purchased from Sigma-Aldrich Canada.
• Galunisertib (LY2157299) was from MedChemExpress (Monmouth Junction, NJ, USA).
• Electrophoresis reagents and enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech (Baie d’Urfé, QC, Canada).

Cell culture

• Serous carcinoma-derived ES-2 ovarian cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
• They were grown as a monolayer with McCoy's 5a Modified Medium (Wisent, 317-010-CL) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin.
• Cells were cultured at 37°C under a humidified 95%-5% (v/v) mixture of air and CO2.

Immunoblotting procedures

• ES-2 ovarian cancer cells were lysed in a buffer containing 1 mM each of NaF and Na3VO4, and proteins were separated by SDS-polyacrylamide gel electrophoresis (30 µg)
• Proteins were electrotransferred to polyvinylidene difluoride membranes, which were then blocked for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline
• Membranes were washed in TBST, incubated with the appropriate primary antibodies (1/1,000 dilution), and immunoreactive material was visualized by enhanced chemiluminescence

TGF-βR1 kinase assay

• This assay measures measures of the ADP formed from a kinase reaction.
• The amount of adenosine triphosphate (ATP) remaining after completion of the kinase is depleted prior to an ADP-to-ATP conversion step and quantitation of the newly synthesized ATP converted into light by Ultra-Glo Luciferase.

Cell migration assay

• Adherent ES-2 ovarian cancer cells were trypsinized and seeded (20,000 cells/well) onto CIM-Plates 16 (Roche Diagnostics, Basel, Switzerland).
• These migration plates are similar to conventional Transwells (8-µm pore size) but have gold electrode arrays on the bottom side of the membrane to provide real-time measurement of cell migration.
• Prior to cell seeding, the underside of the wells from the upper chamber were coated with 25 µL of 0.15% gelatin in phosphate buffered saline (PBS) and incubated for 1 h at 37°C.

Surface plasmon resonance analysis

• SPR analyses were performed using a Biacore T200 instrument
• The recombinant kinase domain of TGF-βR1 protein was immobilized on a carboxymethylated dextran CM7 sensor chip (GE Healthcare, Chicago, IL, USA) following the amine coupling kit protocol
• Steady-state bindings of catechins or galloylated molecules over the immobilized recombinant protein sensor chip were evaluated at increasing concentrations (5 nM-5 µM) at a flow rate of 30 µL/min.

CMDA

• Molecular docking analysis of catechins and galloylated molecules to the TGF-βR1 kinase domain (PDB ID: 5e8s) was performed using AutoDock Vina (Scripps Research Institute, San Diego, CA, USA)
• The box size was chosen to allow the entire surface of the protein to be used for docking, and the exhaustiveness parameter was increased from 8 to 100 to account for the large size of the chosen box.
• Docking was performed with ATP molecule aligned to the structure of the kinase of MLK4 (pdb 4uya) that contains ATPγS, and visualized using PyMOL and Discovery Studio Visualizer.

Statistical data analysis

• Data and error bars were expressed as means ± SEM of three or more independent experiments.

The galloyl moiety of green tea-derived catechins alter TGF-β-mediated Smad-3 phosphorylation

• EGCG virtually inhibited completely such signalling.
• Galloyl requirement for Smad 3 pathway inhibition was investigated with other members from the catechin family, and found to be non-trivial.

The galloyl moiety contributes to the inhibition of the Smad-3 phosphorylation pathway.

• When ES-2 cells were pretreated with the indicated galloylated molecules, a pharmacological inhibition of TGF-β-induced SmAD-3 was observed for all gallates (Fig. 2A).

The galloyl moiety antagonizes TGF-β-induced cell migration

• ES-2 ovarian cancer cell chemotactic response to TGF was next assessed.
• In cells treated with Butyl-, Octyl-, and Dodecyl-gallate, the effective COVID-19 was found to be as effectively decreased as in cells treated only with Galunisertib.

The galloyl moiety likely competes with ATP for the binding of the TGF-βR1 kinase domain.

• CG, GCG, and EGCG were significantly more potent than their respective catechin, gallocatechin, and epigallocatechin ungalloylated counterparts to inhibit the kinase activity, whereas no significant difference could be observed between ECG and ECG.

Galloylated catechins directly interact with TGF-βR1

• they have the highest affinity for the kinase domain.
• They also exhibited higher capacity to cross the plasma membrane than their ungallated parent compound, suggesting that cellular uptake may be higher for poorly hydrophobic compounds with low LogP and high LogS features.

CMDA illustrate how the galloyl moiety could inhibit kinase activity by competing with ATP for the binding of TGF-βR1

• To determine in silico whether galloylated compounds could effectively localize at the ATP binding site and compete with ATP
• Galunisertib was effectively localized to the ATP-binding cleft, and gallate moieties were accommodated in a sub-pocket (sub-pocket A) through interactions with the side chains of Lys-232, Glu-245, Tyr-249, Leu-260, Ser-280 as well as the main chain at positions 278 and 351.
• In the case of octyl gallate, all of the highest scoring molecules adopted very similar orientations within the ATP Binding pocket (Fig. 6D).
• The ability of gallate compounds to inhibit TGFβ-β-mediated signaling could be achieved through binding to and acting as an ATP competitive inhibitor

Discussion EMT is a latent developmental process that is re-activated during cancer progression and, as such, plays a crucial role in the aggressiveness of EOC through increased invasion ability and resistance to therapy.

• The discovery of novel therapeutic modalities that target EMT in EOC should improve the clinical outcome of patients suffering from such lethal gynecological malignancies.

Conclusion

• Our study further reinforces other recent structure-function studies where a role for the galloyl moiety in both direct and indirect interactions of green tea catechins was demonstrated with fatty acid synthase and MT1-MMP interactors-mediated oncogenic processes, respectively.
• The gallate moiety would thus be a good candidate to include in the future design of pharmacological molecules targeting metastatic molecular processes.

Dietary sugar is an important determinant of the development and progression of nonalcoholic fatty liver disease (NAFLD).

• However, the molecular mechanisms underlying the deleterious effects of sugar intake on NAFLD under energy-balanced conditions are still poorly understood.
• Here, we provide a comprehensive analysis of the liver lipidome and mechanistic insights into the pathogenesis of the chronic consumption of high-sugar diet (HSD).

Abbreviations

• Acadl acyl-coenzyme
• A dehydrogenase long-chain
• Acar acylcarnitine
• ARACHIDonic acid
• CE cholesteryl ester
• Cer ceramide
• Cholesterol
• CHREBP carbohydrate response element binding protein
• DAG diacylglycerol
• DHA docosahexaenoic acid
• DNL de novo lipogenesis
• EPA eicosapentaenoinic acid
• FFA free fatty acids
• MUFAs monounsaturated fatty acids
• NAFL nonalcoholic fatty liver, NAFLD nonalcoholics fatty liver disease, NASH non-alcoholic steatohepatitis
• oPC plasmanyl-phosphatidylcholine, oPE plasmid, PC, PCA principal component analysis
• Ppara peroxisome proliferator activated receptor alpha - activated receptor gamma, coactivator 1 alpha - active receptor, gamma

Reference

• 10.1016/j.jnutbio.2020.108518