Background: The tumor suppressor genes p15INK4b and p16INK4a as well as the estrogen receptor-α (ESR1) gene are abnormally methylated and expressed in colon cancer. The cancer-preventative abilities of several bioactive food components have been linked to their estrogenic and epigenetic activities. Methods: The effect of folic acid, zebularine, resveratrol, genistein and epigallocatechin-3-gallate (EGCG) on tumor cell growth, promoter methylation of ESR1, p15INK4b and p16INK4a and gene expression of ESR1 and ESR2 was analyzed in Caco-2 cells. Gene expression was measured using real-time PCR, and promoter CpG methylation was assessed using bisulfite conversion and methylation-specific PCR. Results: After exposure to a high concentration of folic acid (20 µmol/l), enhanced cancer cell growth and concomitant increased methylation of the ESR1 (3.6-fold), p16INK4a and p15INK4b promoters was observed. A lower concentration of folic acid (2 µmol/l) decreased cell growth. The phytoestrogens genistein and resveratrol enhanced expression of ESR1 (genistein 200 µmol/l: 2.1-fold; resveratrol 50 µmol/l: 6.3-fold) and ESR2 (2.6- and 3.6-fold, respectively). Genistein and resveratrol treatment increased promoter methylation of ESR1 (genistein 200 µmol/l: 2.9-fold; resveratrol 50 µmol/l: 1.4-fold). For p16INK4a, increased methylation was found after exposure to 10 µmol/l resveratrol, but for p15INK4b, decreased methylation was found. Both components showed growth-inhibitory activities. For EGCG, growth inhibition at 100 µmol/l and suppressed promoter methylation of tumor suppressor genes (p16INK4a: 0.9-fold; p15INK4b: 0.6-fold) was seen. Conclusions: Our results show that these food compounds regulate ESR and tumor suppressor gene expression by multiple mechanisms including epigenetic processes. An improved understanding of these epigenetic effects could therefore support specific dietary concepts of epigenetic cancer prevention and intervention.
The antibody Fv module which binds antigen consists of the variable domains VL and VH. These exhibit a conserved ß-sheet structure and comprise highly variable loops (CDRs). Little is known about the contributions of the framework residues and CDRs to their association. We exchanged conserved interface residues as well as CDR loops and tested the effects on two Fvs interacting with moderate affinities (KDs of ~2.5 µM and ~6 µM). While for the rather instable domains, almost all mutations had a negative effect, the more stable domains tolerated a number of mutations of conserved interface residues. Of particular importance for Fv association are VLP44 and VHL45. In general, the exchange of conserved residues in the VL/VH interface did not have uniform effects on domain stability. Furthermore, the effects on association and antigen binding do not strictly correlate. In addition to the interface, the CDRs modulate the variable domain framework to a significant extent as shown by swap experiments. Our study reveals a complex interplay of domain stability, association and antigen binding including an unexpected strong mutual influence of the domain framework and the CDRs on stability/association on the one side and antigen binding on the other side.
Coformulations containing two therapeutic monoclonal antibodies (mAbs) could offer various benefits like enhanced therapeutic efficacy and better patient compliance. However, there are very few published studies on coformulations and binary mixtures of mAbs. It remains unclear to what extent mAbs with different physicochemical properties can be combined in solution without detrimental effects on protein stability. Here, we present a study including six model mAbs of the IgG1 subclass that are commercially available. In silico and biophysical characterization shows that the proteins have different physicochemical properties. Thus, their combinations represent various scenarios for coformulation development. We prepared all possible binary mixtures of the six mAbs and determined several biophysical parameters that are assessed during early-stage protein drug product development. The measured biophysical parameters are indicative of the conformational protein stability (inflection points of the thermal protein unfolding transitions) and the colloidal protein stability (aggregation onset temperatures and interaction parameter k D from dynamic light scattering). Remarkably, all 15 binary mAb mixtures do not exhibit biophysical parameters that indicate inferior conformational or colloidal stability compared to the least stable mAb in the mixture. Our findings suggest that the coformulation of some therapeutic monoclonal antibodies of the IgG1 subclass could be possible in a straightforward way as severe detrimental effects on the stability of these proteins in binary mixtures were not observed.
Despite their importance for antibody architecture and design, the principles governing antibody domain stability are still not understood in sufficient detail. Here, to address this question, we chose a domain from the invariant part of IgG, the C2 domain. We found that compared with other Ig domains, the isolated C2 domain is a surprisingly unstable monomer, exhibiting a melting temperature of ∼44 °C. We further show that the presence of an additional C-terminal lysine in a C2 variant substantially increases the melting temperature by ∼14 °C relative to C2 WT. To explore the molecular mechanism of this effect, we employed biophysical approaches to probe structural features of C2. The results revealed that Lys is key for the formation of three secondary structure elements: the very C-terminal β-strand and two adjacent α-helices. We also noted that a dipole interaction between Lys and the nearby α-helix, is important for stabilizing the C2 architecture by protecting the hydrophobic core. Interestingly, this interaction between the α-helix and C-terminal charged residues is highly conserved in antibody domains, suggesting that it represents a general mechanism for maintaining their integrity. We conclude that the observed interactions involving terminal residues have practical applications for defining domain boundaries in the development of antibody therapeutics and diagnostics.
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