Molecular-Scale Investigations Reveal the Effect of Natural Polyphenols on BAX/Bcl-2 Interactions.

Apoptosis signaling controls the cell cycle through the protein-protein interactions (PPIs) of its major B-cell lymphoma 2-associated x protein (BAX) and B-cell lymphoma 2 protein (Bcl-2). Due to the antagonistic function of both proteins, apoptosis depends on a properly tuned balance of the kinetics of BAX and Bcl-2 activities. The utilization of natural polyphenols to regulate the binding process of PPIs is feasible. However, the mechanism of this modulation has not been studied in detail. Here, we utilized atomic force microscopy (AFM) to evaluate the effects of polyphenols (kaempferol, quercetin, dihydromyricetin, baicalin, curcumin, rutin, epigallocatechin gallate, and gossypol) on the BAX/Bcl-2 binding mechanism. We demonstrated at the molecular scale that polyphenols quantitatively affect the interaction forces, kinetics, thermodynamics, and structural properties of BAX/Bcl-2 complex formation. We observed that rutin, epigallocatechin gallate, and baicalin reduced the binding affinity of BAX/Bcl-2 by an order of magnitude. Combined with surface free energy and molecular docking, the results revealed that polyphenols are driven by multiple forces that affect the orientation freedom of PPIs, with hydrogen bonding, hydrophobic interactions, and van der Waals forces being the major contributors. Overall, our work provides valuable insights into how molecules tune PPIs to modulate their function.

Single-molecule scale quantification reveals interactions underlying protein-protein interface: from forces to non-covalent bonds.

Protein-protein interactions (PPIs) between the B-cell lymphoma 2 (Bcl-2) family are considered a major driving force in cell cycle regulation and signaling. However, how this interfacial noncovalent interaction is achieved molecularly remains poorly understood. Herein, anti-apoptotic protein (Bcl-2) and pro-apoptotic protein (BAX) were used as models and their PPIs were explored for the first time using atomic force microscopy-based single-molecule force spectroscopy (SMFS) and in silico approaches. In addition, we used advanced analytical models, including multiple kinetic models, thermodynamic models, Poisson distributions, and contact angle molecular recognition to fully reveal the complexity of the BAX/Bcl-2 interaction interfaces. We propose that the binding kinetics between BAX/Bcl-2 are mainly mediated by specific (hydrogen bonding) and non-specific forces (hydrophobic interactions and electrostatic interactions) and show that the complicated multivalent binding interaction induces stable BAX/Bcl-2 complexes. This study enriches our understanding of the molecular mechanisms by which BAX interacts with Bcl-2. It provides valuable insights into the physical factors that need to be considered when designing PPI inhibitors.

Effect of various hepatectomy procedures on circulating tumor cells in postoperative patients: a case-matched comparative study.

Background: The objective of this study is to elucidate the prevalence of systemic circulating tumor cells (CTCs) prior to and following resection of hepatocellular carcinoma (HCC), and to compare the disparities in postoperative CTCs in terms of quantity and classifications between the open liver resection (OPEN) and laparoscopic liver resection (LAP) cohorts. Patients materials and methods: From September 2015 to May 2022, 32 consecutive HCC patients who underwent laparoscopic liver resection at Southwest Hospital were retrospectively enrolled in this study. The clinicopathological data were retrieved from a prospectively collected computer database. Patients in the OPEN group matched at a 1:1 ratio with patients who underwent open liver resection during the study period on age, gender, tumor size, number of tumors, tumor location, hepatitis B surface antigen (HBsAg) positivity, alpha-fetoprotein (AFP) level, TNM and Child-Pugh staging from the database of patients to form the control group. The Can-Patrol CTC enrichment technique was used to enrich and classify CTCS based on epithelial-mesenchymal transformation phenotypes. The endpoint was disease-free survival (DFS), and the Kaplan-Meier method and multiple Cox proportional risk model were used to analyze the influence of clinicopathological factors such as total CTCs and CTC phenotype on prognosis. Results: The mean age of the 64 patients with primary liver cancer was 52.92 years (23-71), and 89.1% were male. The postoperative CTC clearance rate was more significant in the OPEN group. The total residual CTC and phenotypic CTC of the LAP group were significantly higher than those of the OPEN group (p = 0.017, 0.012, 0.049, and 0.030, respectively), which may increase the possibility of metastasis (p = 0.042). In Kaplan-Meier analysis, DFS was associated with several clinicopathological risk factors, including Barcelona Clinical Liver Cancer (BCLC) stage, tumor size, and vascular invasion. Of these analyses, BCLC Stage [p = 0.043, HR (95% CI) =2.03(1.022-4.034)], AFP [p = 0.007, HR (95% CI) =1.947 (1.238-3.062)], the number of positive CTCs [p = 0.004, HR (95% CI) =9.607 (2.085-44.269)] and vascular invasion [p = 0.046, HR (95% CI) =0.475 (0.22-1.023)] were significantly associated with DFS. Conclusion: In comparison to conventional OPEN technology, LAP technology has the capacity to augment the quantity of epithelial, mixed, and mesenchymal circulating tumor cells (CTCs). Following the surgical procedure, there was a notable increase in the total CTCs, epithelial CTCs, and mixed CTCs within the LAP group, indicating a potential drawback of LAP in facilitating the release of CTCs.

Investigating the Effect of Tyrosine Kinase Inhibitors on the Interaction between Human Serum Albumin by Atomic Force Microscopy.

It is important for elucidating the regulation mechanism of life activities, as well as for the prevention, diagnosis, and drug design of diseases, to study protein-protein interactions (PPIs). Here, we investigated the interactions of human serum albumin (HSA) in the presence of tyrosine kinase inhibitors (TKIs: imatinib, nilotinib, dasatinib, bosutinib, and ponatinib) using atomic force microscopy (AFM). The distribution of rupture events including the specific interaction force Fi and the non-specific interaction force F0 between HSA pairs was analyzed. Based on the force measurements, Fi and F0 between HSA pairs in the control experiment were calculated to be 47 ± 1.5 and 116.1 ± 1.3 pN. However, Fi was significantly decreased in TKIs, while F0 was slightly decreased. By measuring the rupture forces at various loading rates and according to the Bell equation, the kinetic parameters of the complexes were investigated in greater detail. Molecular docking was used as a complementary means by which to explore the force of this effect. The whole measurements indicated that TKIs influenced PPIs in a variety of ways, among which hydrogen bonding and hydrophobic interactions were the most important. In conclusion, these outcomes give us a better insight into the mechanisms of PPIs when there are exogenous compounds present as well as in different liquid environments.

Molecular Mechanisms of Resistance to Tyrosine Kinase Inhibitors Associated with Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death, which can be attributed to the high incidence and first diagnosis at an advanced stage. Tyrosine kinase inhibitors (TKIs), a class of small-molecule targeting drugs, are primarily used for the clinical treatment of HCC after chemotherapy because they show significant clinical efficacy and low incidence of clinical adverse reactions. However, resistance to sorafenib and other TKIs, which can be used to treat advanced HCC, poses a significant challenge. Recent mechanistic studies have shown that epithelial-mesenchymal transition or transformation (EMT), ATP binding cassette (ABC) transporters, hypoxia, autophagy, and angiogenesis are involved in apoptosis, angiogenesis, HCC cell proliferation, and TKI resistance in patients with HCC. Exploring and overcoming such resistance mechanisms is essential to extend the therapeutic benefits of TKIs to patients with TKI-resistant HCC. This review aims to summarize the potential resistance mechanism proposed in recent years and methods to reverse TKI resistance in the context of HCC.

Evaluating effect of metallic ions on aggregation behavior of ß-amyloid peptides by atomic force microscope and surface-enhanced Raman Scattering.

BACKGROUND: Excessive aggregation of ß-amyloid peptides (Aß) is regarded as the hallmark of Alzheimer's disease. Exploring the underlying mechanism regulating Aß aggregation remains challenging and investigating aggregation events of Aß in the presence and absence of metallic ions at molecular level would be meaningful in elucidating the role of metal cations on interactions between Aß molecules. In this study, chemical self-assembled monolayer (SAM) method was employed to fabricate monolayer of ß-amyloid peptides Aß42 on gold substrate with a bolaamphiphile named 16-Mercaptohexadecanoic acid (MHA). Firstly, the samples of gold substrate (blank control), the MHA-modified substrate, and the Aß42-modified substrate were detected by X-ray photoelectron spectroscopy (XPS) to track the self-assembly process. Aggregation behaviors of Aß42 before and after metallic ions (Zn2+, Ca2+, Al3+) treatment were monitored by atomic force microscopy (AFM) and the interaction between Aß42 and metallic ions (Zn2+, Ca2+, Al3+) was investigated by surface-enhanced Raman Scattering (SERS). RESULTS: The XPS spectra of binding energy of gold substrate (blank control), the MHA-modified substrate, and the Aß42-modified substrate are well fitted with the corresponding monolayer's composition, which indicates that Aß42 monolayer is well formed. The recorded surface morphology of different experimental groups obtained by AFM showed markedly different nanostructures, indicating occurrence of aggregation events between Aß42 molecules after adding metal ions to the solution. Compared to the control group, the presence of metallic ions resulted in the increased size of surface structures on the observed 3D topography. Besides, the intermolecular rupture force of Aß42 increased with the addition of metallic ions. Further study by SERS showed that the Raman strength of Aß42 changes significantly after the metal cation treatment. A considerable part of the amide bonds interacts with metal cations, leading to a structural change, which is characterized by the weakened ß-fold Raman peak. CONCLUSION: The AFM imaging results suggest that aggregation events occurred between Aß42 molecules with the addition of metal cations. In addition, the results of force tests indicate that the presence of metallic ions could promote adhesion between Aß42 molecules, which is likely to be the trigger for aggregation behavior of Aß42. Furthermore, the effect of metallic cations on the conformational change of Aß42 studied by SERS supported the results obtained by AFM. Taken together, the results showed that the presence of substoichiometric metal cations promotes aggregation behavior between Aß42 molecules on the substrate at pH 7.4.

Structural design of tetravalent T-cell engaging bispecific antibodies: improve developability by engineering disulfide bonds.

Since the advances in protein engineering and manufacture, over the last 30 years, antibody-based immunotherapeutic has become a powerful strategy to treat diseases. The T-cell engaging bispecific antibody (BsAb) by combining the Fab binding domain of tumor antigens and Fab or single-chain variable fragments (scFvs) binding domain of CD3 molecules, could redirect cytotoxic T cells to kill tumor cells. The IgG-scFv format of BsAb is a dual bivalent and asymmetrical design, which adds the benefit of potent cytotoxicity and less complicated for manufacture but limits the stability and production. Here, we engineered a series of interchain disulfide bonds in the Fab region of IgG-svFv BsAbs and evaluated its biophysical and biological properties. We found that simultaneously replaced the position of VH44-VL100 and CH1126-CL121 residues with cysteine, to form two additional disulfide bonds, could markedly increase monomeric BsAb formation and yield. The thermostability and stability against aggregation and degradation also performed better than BsAbs without extra disulfide bonds introduction. Besides, the affinity of engineered BsAbs was maintained, and the h8B-BsAb antibody had a slight enhancement in an inhibitory effect on target cells.

Association of Preoperative NANOG-Positive Circulating Tumor Cell Levels With Recurrence of Hepatocellular Carcinoma.

BACKGROUND: Cancer stem cells (CSCs) and Circulating tumor cells (CTCs) have been proposed as fundamental causes for the recurrence of hepatocellular carcinoma (HCC). CTCs isolated from patients with HCC illustrate a unique Nanog expression profile analysis. The aim of this study was to enhance the prediction of recurrence and prognosis of the CTC phenotype in patients with HCC by combining Nanog expression into a combined forecasting model. SUBJECTS MATERIALS AND METHODS: We collected 320 blood samples from 160 patients with HCC cancer before surgery and used CanPatrol™ CTC enrichment technology and in situ hybridization (ISH) to enrich and detect CTCs and CSCs. Nanog expression in all CTCs was also determined. In addition, RT-PCR and immunohistochemistry were used to study the expression of Nanog, E-Cadherin, and N-Cadherin in liver cancer tissues and to conduct clinical correlation studies. RESULTS: The numbers of EpCAM mRNA+ CTCs and Nanog mRNA+ CTCs were strongly correlated with postoperative HCC recurrence (CTC number (P = 0.03), the total number of mixed CTCS (P = 0.02), and Nanog> 6.7 (P = 0.001), with Nanog > 6.7 (P = 0.0003, HR = 2.33) being the most crucial marker. There are significant differences in the expression of Nanog on different types of CTC: most Epithelial CTCs do not express Nanog, while most of Mixed CTC and Mesenchymal CTC express Nanog, and their positive rates are 38.7%, 66.7%, and 88.7%, respectively, (P=0.0001). Moreover, both CTC (≤/> 13.3) and Nanog (≤/>6.7) expression were significantly correlated with BCLC stage, vascular invasion, tumor size, and Hbv-DNA (all P < 0.05). In the young group and the old group, patients with higher Nanog expression had a higher recurrence rate. (P < 0.001). CONCLUSIONS: The number of Nanog-positive cells showed positive correlation with the poor prognosis of HCC patients. The detection and analysis of CTC markers (EpCAM and CK8, 18, CD45 Vimentin,Twist and 19) and CSCs markers (NANOG) are of great value in the evaluation of tumor progression.

Development of a Tetravalent T-Cell Engaging Bispecific Antibody Against Glypican-3 for Hepatocellular Carcinoma.

Cancer therapies benefit from accelerated development of biotechnology, and many immunotherapeutic strategies spring up including vaccines, the immune checkpoint blockade, chimeric antigen receptor T cells, and bispecific antibodies (BsAbs). Glypican-3 (GPC3) is a member of the heparan sulfate proteoglycan family of proteins and is highly expressed in hepatocellular carcinoma (HCC) cell membranes. Here, the authors describe a new tetravalent BsAb h8B-BsAb targeting GPC3 and CD3 antigens and studied its antitumor activities against HCC. h8B-BsAb was designed based on immunoglobulin G with a fragment variable fused to the light chain, whose biophysical stabilities including degradation resistance and thermostability were improved by introducing disulfide bonds. In vitro activity of h8B-BsAb showed potent T-cell recruitment and activation for HCC cell lysis by the presence of peripheral blood mononuclear cells, but no specific killing in GPC3-negative cells. In HCC xenograft mouse studies, h8B-BsAb induced robust regression of tumors. In summary, we engineered a highly stable and efficacious BsAb as a potential candidate for HCC treatment.

Probing Changes in Ca2+-Induced Interaction Forces between Calmodulin and Melittin by Atomic Force Microscopy.

Mechanobiology studies the means by which physical forces and mechanical properties change intra- or inter- biological macromolecules. Calmodulin (CaM) is involved in physiological activities and various metabolic processes in eukaryotic cells. Although the configuration changes in the interaction between calmodulin and melittin have been studied, the biomechanical relationship of their interaction has rarely been explored. Here, we measured the adhesion forces between calmodulin and melittin in solutions of gradient concentration of calcium ions using atomic force microscopy (AFM). We found that the specific (Fi) and nonspecific (F0) adhesion forces between single melittin and calmodulin in a PBS solution were 69.4 ± 5.0 and 29.3 ± 8.9 pN, respectively. In the presence of 10-7 to 10-3 M Ca2+ PBS solution, the Fi increased significantly to 93.8 ± 5.0, 139.9 ± 9.0, 140.4 ± 9.7, 171.5 ± 9.0, and 213.3 ± 17.8 pN, indicating that the unbinding force between melittin and calmodulin increased in the presence of Ca2+ in a concentration-dependent manner. These findings demonstrated that biomechanical studies based on AFM could help us better understand the melittin/calmodulin-binding processes in the presence of calcium and help us design and screen peptide drugs based on calmodulin.

Evaluating the effect of aminoglycosides on the interaction between bovine serum albumins by atomic force microscopy.

Characterization and determination of protein-protein interactions (PPIs) plays an important role in molecular biological science. In this study, the effect of aminoglycosides (AGs: streptomycin, gentamycin, lincomycin and clindamycin) on interactions between bovine serum albumin (BSA) was evaluated employing imaging and probing adhesion event by AFM. Multi-spectroscopy and molecular docking were supplementary to investigate the acting forces of the effect. AFM measurements revealed the aggregation of BSA grains and changes of adhesion forces at single molecule level. With adhesion forces between BSA pairs decomposed by Poisson method, specific forces in streptomycin, gentamycin, lincomycin and climdamycin were obviously decreased with the rate of 33.1%, 26.4%, 32.3% and 31.3% while non-specific forces slightly decreased with 5.5%, 3.3%, 4.0% and 7.7%. Combined with results of multi-spectroscopy as well as molecular docking, the whole determination showed AGs affected PPIs by multiple forces, where the hydrogen bonding and hydration effect were the main reasons. The binding of drugs and proteins acted by hydrogen bonding affected the interaction forces between BSA. Consequently, AFM was proposed to be an effective and precise tool in application including evaluating the effects of exogenous compounds on biomacromolecular interactions and rapid screening of drug candidates to avoid potential damages in disease treatment.