The Inhibiting Effect of GB-2, (+)-Catechin, Theaflavin, and Theaflavin 3-Gallate on Interaction between ACE2 and SARS-CoV-2 EG.5.1 and HV.1 Variants.

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, continues to pose significant global health challenges. The results demonstrated that GB-2 at 200 µg/mL effectively increased the population of 293T-ACE2 cells with low RBD binding for both SARS-CoV-2 Omicron EG.5.1 and HV.1 variants by dual-color flow cytometry, indicating its ability to inhibit virus attachment. Further investigation revealed that (+)-catechin at 25 and 50 µg/mL did not significantly alter the ACE2-RBD interaction for the EG.5.1 variant. In contrast, theaflavin showed inhibitory effects at both 25 and 50 µg/mL for EG.5.1, while only the higher concentration was effective for HV.1. Notably, theaflavin 3-gallate exhibited a potent inhibition of ACE2-RBD binding for both variants at both concentrations tested. Molecular docking studies provided insight into the binding mechanisms of theaflavin and theaflavin 3-gallate with the RBD of EG.5.1 and HV.1 variants. Both compounds showed favorable docking scores, with theaflavin 3-gallate demonstrating slightly lower scores (-8 kcal/mol) compared to theaflavin (-7 kcal/mol) for both variants. These results suggest stable interactions between the compounds and key residues in the RBD, potentially explaining their inhibitory effects on virus attachment. In conclusion, GB-2, theaflavin, and theaflavin 3-gallate demonstrate significant potential as inhibitors of the ACE2-RBD interaction in Omicron variants, highlighting their therapeutic promise against COVID-19. However, these findings are primarily based on computational and in vitro studies, necessitating further in vivo research and clinical trials to confirm their efficacy and safety in humans.

The Immunosuppressive Receptor CD32b Regulation of Macrophage Polarization and Its Implications in Tumor Progression.

Macrophages, pivotal components of the immune system, orchestrate host defense mechanisms in humans and mammals. Their polarization into classically activated macrophages (CAMs or M1) and alternatively activated macrophages (AAMs or M2) dictates distinct functional roles in immunity and tissue homeostasis. While the negative regulatory role of CD32b within the FC gamma receptor (FCγR) family is recognized across various immune cell types, its influence on macrophage polarization remains elusive. This study aimed to elucidate the regulatory role of CD32b in macrophage polarization and discern the differential expression markers between the M1 and M2 phenotypes following CD32b siRNA transfection. The results revealed a decrease in the CD32b levels in lipopolysaccharide (LPS)-treated M1 and an increase in interleukin-4 (IL-4)-treated M2 macrophages, as observed in macrophage Raw264.7 cells. Furthermore, CD32b siRNA transfection significantly downregulated the M2 markers (IL-10, VEGF, Arg-1, and STAT6), while upregulating the M1 markers (IL-6, NF-κB, NOS2, and STAT1) in the Raw264.7 cells. Similar findings were recapitulated in macrophage-rich adherent cells isolated from mouse spleens. Additionally, the cytopathological analysis of pleural effusions and ascitic fluids from patients with cancer revealed a positive correlation between advanced tumor stages, metastasis, and elevated CD32b levels. In conclusion, this study highlights the regulatory influence of CD32b in suppressing M1 expression and promoting M2 polarization. Moreover, heightened M2 activation and CD32b levels appear to correlate with tumor progression. A targeted CD32b blockade may serve as a novel therapeutic strategy to inhibit M2 macrophage polarization and is promising for anti-tumor intervention.

Prognostic factors of early recurrence after complete cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

BACKGROUND: Although cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) offer the potential for long-term survival in peritoneal carcinomatosis, outcomes following CRS/HIPEC vary significantly. AIM: To identify the clinical factors associated with progression-free survival (PFS) after complete CRS/HIPEC in patients with colorectal/high-grade appendiceal, ovarian, and gastric cancers. METHODS: We retrospectively evaluated the risk of recurrence within 1 year after CRS/HIPEC and its impact on overall survival (OS) in patients recruited between 2015 and 2020. Logistic regression models were used to assess the prognostic factors for the risk of recurrence within 1 year. Kaplan-Meier survival curves and Cox proportional hazards models were used to evaluate the association between recurrence and OS. RESULTS: Of the 80 enrolled patients, 39 had an unfavorable PFS (< 1 year) and 41 had a favorable PFS (≥ 1 year). Simple logistic models revealed that the patients with a completeness of cytoreduction score of 0 (CC-0) or length of CRS ≤ 6 h had a favorable PFS [odds ratio (OR) = 0.141, P = 0.004; and OR = 0.361, P = 0.027, respectively]. In multiple logistic regression, achieving CC-0 was the strongest prognostic factor for a favorable PFS (OR = 0.131, P = 0.005). A peritoneal cancer index score > 12 was associated with a lower rate of achieving CC-0 (P = 0.027). The favorable PFS group had a significantly longer OS (median 81.7 mo vs 17.0 mo, P < 0.001). CONCLUSION: Achieving CC-0 was associated with a lower early recurrence rate and improved long-term survival. This study underscores the importance of selecting appropriate candidates for CRS/HIPEC to manage peritoneal carcinomatosis.

An optimized ligation-mediated PCR method for chromosome walking and fusion gene chromosomal breakpoints identification.

Molecular techniques that recover unknown sequences next to a known sequence region have been widely applied in various molecular studies, such as chromosome walking, identification of the insertion site of transposon mutagenesis, fusion gene partner, and chromosomal breakpoints, as well as targeted sequencing library preparation. Although various techniques have been introduced for efficiency enhancement, searching for relevant single molecular event present in a large-sized genome remains challenging. Here, the optimized ligation-mediated polymerase chain reaction (PCR) method was developed and successfully identified chromosomal breakpoints far away from the exon of the new exon junction without the need for nested PCR. In addition to recovering unknown sequences next to a known sequence region, the high efficiency of the method could also improve the performance of targeted  next-generation sequencing (NGS).

Synthesis and Structure-Activity Relationship of Salvinal Derivatives as Potent Microtubule Inhibitors.

Salvinal is a natural lignan isolated from the roots of Salvia mitorrhiza Bunge (Danshen). Previous studies have demonstrated its anti-proliferative activity in both drug-sensitive and -resistant cancer cell lines, with IC50 values ranging from 4-17 µM. In this study, a series of salvinal derivatives was synthesized and evaluated for the structure-activity relationship. Among the twenty-four salvinal derivatives, six compounds showed better anticancer activity than salvinal. Compound 25 displayed excellent anticancer activity, with IC50 values of 0.13-0.14 µM against KB, KB-Vin10 (overexpress MDR/Pgp), and KB-7D (overexpress MRP) human carcinoma cell lines. Based on our in vitro microtubule depolymerization assay, compound 25 showed depolymerization activity in a dose-dependent manner. Our findings indicate that compound 25 is a promising anticancer agent with depolymerization activity that has potential for the management of malignance.

Lipid Droplets in Lung Cancers Are Crucial for the Cell Growth and Starvation Survival.

For rapid and unlimited cell growth and proliferation, cancer cells require large quantities of nutrients. Many metabolic pathways and nutrient uptake systems are frequently reprogrammed and upregulated to meet the demand from cancer cells, including the demand for lipids. The lipids for most adult normal cells are mainly acquired from the circulatory system. Whether different cancer cells adopt identical mechanisms to ensure sufficient lipid supply, and whether the lipid demand and supply meet each other, remains unclear, and was investigated in lung cancer cells. Results showed that, despite frequent upregulation in de novo lipogenesis and the lipid transporter system, different lung cancer cells adopt different proteins to acquire sufficient lipids, and the lipid supply frequently exceeds the demand, as significant amounts of lipids stored in the lipid droplets could be found within lung cancer cells. Lipid droplet surface protein, PLIN3, was found frequently overexpressed since the early stage in lung cancer tissues. Although the expression is not significantly associated with a specific gender, age, histology type, disease stage, and smoking habit, the frequently elevated expression of PLIN3 protein indicates the importance of lipid droplets for lung cancer. These lipid droplets are not only for nutrient storage, but are also crucial for tumor growth and proliferation, as well as survival in starvation. These results suggest that manipulation of lipid droplet formation or TG storage in lung cancer cells could potentially decrease the progression of lung cancer. Further exploration of lipid biology in lung cancer could help design novel treatment strategies.

The potential and limitation of targeted chromosomal breakpoint sequencing for the ROS1 fusion gene identification in lung cancer.

ROS1 fusion genes are rare but important driver genes in lung cancer. Owing to their rarity, many clinicopathological features and treatment responses for each ROS1 fusion variant are still largely unknown and require further investigation. RNA is the preferable template for the ROS1 fusion gene screening, but deterioration of RNA in FFPE often makes the detection challenging. To resolve the difficulty, a targeted chromosomal breakpoint sequencing method was developed for searching the ROS1 fusion gene, and was compared with fluorescence in situ hybridization, immunohistochemistry, RT-qPCR using 260 lung cancer samples of Southern Taiwan. The results showed that ROS1-altered cases were present at low frequencies, did not share distinct clinicopathological features, and often carried other driver mutations. The performance of the targeted sequencing assay was superior to the RT-qPCR in ROS1 fusion gene identification when the cDNAs were from FFPE samples, but long-read DNA sequencing and fresh-frozen samples would be better to revolve all fusion genes. Precise determination of all ROS1 fusion variants and concomitant driver mutations using both genomic DNA and RNA would be required to help improve the treatment of patients with ROS1 alterations.

18F-Fluorodeoxyglucose PET/CT for Early Prediction of Outcomes in Patients with Advanced Lung Adenocarcinomas and EGFR Mutations Treated with First-Line EGFR-TKIs.

This study aims to investigate the role of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) in early prediction of response and survival following epithelial growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy in patients with advanced lung adenocarcinomas and EGFR mutations. Thirty patients with stage IIIB/IV lung adenocarcinomas and EGFR mutations receiving first-line EGFR-TKIs were prospectively evaluated between November 2012 and May 2015. EGFR mutations were quantified by delta cycle threshold (dCt). 18F-FDG PET/CT was performed before and 2 weeks after treatment initiation. PET response was assessed based on PET Response Criteria in Solid Tumors (PERCIST). Baseline and percentage changes in the summed standardized uptake value, metabolic tumor volume (bsumMTV and ΔsumMTV, respectively), and total lesion glycolysis of ≤5 target lesions/patient were calculated. The association between parameters (clinical and PET) and non-progression disease after 3 months of treatment in CT based on the Response Evaluation Criteria in Solid Tumors Version 1.1 (nPD3mo), progression-free survival (PFS), and overall survival (OS) were tested. The median follow-up time was 19.6 months. The median PFS and OS were 12.0 and 25.3 months, respectively. The PERCIST criteria was an independent predictor of nPD3mo (p = 0.009), dCt (p = 0.014) and bsumMTV (p = 0.014) were independent predictors of PFS, and dCt (p = 0.014) and ΔsumMTV (p = 0.005) were independent predictors of OS. 18F-FDG PET/CT achieved early prediction of outcomes in patients with advanced lung adenocarcinomas and EGFR mutations receiving EGFR-TKIs.

The effect of cullin 4A on lung cancer cell chemosensitivity to paclitaxel through p33ING1b regulation.

Cullin 4A (Cul4A) reportedly has oncogenic roles in several cancer types by regulating tumor suppressors through the ubiquitination and proteolysis of the tumor suppressor. In addition, Cul4A is associated with chemosensitivity to chemotherapy drugs. This study investigated the association between Cul4A and lung cancer cell chemosensitivity to paclitaxel, particularly with respect to the role of the p33 inhibitor of the growth 1 (p33ING1b) tumor suppressor. The results showed that the Cul4A knockdown upregulated the p33ING1b expression in lung cancer cells and increased the lung cancer cell and mice tumor xenograft chemosensitivity to paclitaxel. The Cul4A knockdown also inhibited the growth and increased the apoptosis in the tumor xenografts treated with paclitaxel. Notably, the p33ING1b overexpression increased the lung cancer cell chemosensitivity to paclitaxel, but the p33ING1b knockdown reduced the chemosensitivity. A further analysis demonstrated that Cul4A regulates the expression of p33ING1b through protein-protein interactions, ubiquitination, and protein degradation. In conclusion, the present findings suggest that Cul4A mediates the chemosensitivity of lung cancer cells to paclitaxel by regulating p33ING1b. These findings may offer novel insights into future therapeutic strategies for lung cancer that target Cul4A.

The Association of Annexin A1 and Chemosensitivity to Osimertinib in Lung Cancer Cells.

Annexin A1 (ANXA1) has been reported to promote tumor growth and resistance to chemotherapy drugs in lung cancer cells. In this study, we focused on the association of ANXA1 and chemosensitivity with a third generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), Osimertinib, in lung cancer cells with EGFR mutations. The overexpression of ANXA1 was observed in the lung cancer cells studied. The downregulation of ANXA1 with small interference RNA (siRNA) decreased the growth of lung cancer cells. In lung cancer cells with EGFR mutations, the knockdown of ANXA1 increased the chemosensitivity to Osimertinib, and decreased the tumorigenesis, invasion and migration of lung cancer cells. Further study showed that the knockdown of ANXA1 inhibited the phosphorylation of EGFR and down-stream Akt pathways and promoted apoptosis in lung cancer cells treated with Osimertinib. A mice xenograft lung cancer model was established in our study and showed that ANXA1 siRNA enhanced the effects of Osimertinib in vivo. Our study results showed that ANXA1 plays critical roles in chemosensitivity to EGFR-TKI in lung cancer cells with the EGFR mutation. Our efforts may be used in the development of lung cancer treatment strategies in the future.

Virtual Screening and In Vitro Evaluation of PD-1 Dimer Stabilizers for Uncoupling PD-1/PD-L1 Interaction from Natural Products.

Genetic mutations accumulated overtime could generate many growth and survival advantages for cancer cells, but these mutations also mark cancer cells as targets to be eliminated by the immune system. To evade immune surveillance, cancer cells adopted different intrinsic molecules to suppress immune response. PD-L1 is frequently overexpressed in many cancer cells, and its engagement with PD-1 on T cells diminishes the extent of cytotoxicity from the immune system. To resume immunity for fighting cancer, several therapeutic antibodies disrupting the PD-1/PD-L1 interaction have been introduced in clinical practice. However, their immunogenicity, low tissue penetrance, and high production costs rendered these antibodies beneficial to only a limited number of patients. PD-L1 dimer formation shields the interaction interface for PD-1 binding; hence, screening for small molecule compounds stabilizing the PD-L1 dimer may make immune therapy more effective and widely affordable. In the current study, 111 candidates were selected from over 180,000 natural compound structures through virtual screening, contact fingerprint analysis, and pharmacological property prediction. Twenty-two representative candidates were further evaluated in vitro. Two compounds were found capable of inhibiting the PD-1/PD-L1 interaction and promoting PD-L1 dimer formation. Further structure optimization and clinical development of these lead inhibitors will eventually lead to more effective and affordable immunotherapeutic drugs for cancer patients.

A highly sensitive and specific real-time quantitative PCR for BRAF V600E/K mutation screening.

Mutations that lead to constitutive activation of key regulators in cellular processes are one of the most important drivers behind vigorous growth of cancer cells, and are thus prime targets in cancer treatment. BRAF V600E mutation transduces strong growth and survival signals for cancer cells, and is widely present in various types of cancers including lung cancer. A combination of BRAF inhibitor (dabrafenib) and MEK inhibitor (trametinib) has recently been approved and significantly improved the survival of patients with advanced NSCLC harboring BRAF V600E/K mutation. To improve the detection of BRAF V600E/K mutation and investigate the incidence and clinicopathological features of the mutation in lung cancer patients of southern Taiwan, a highly sensitive and specific real-time quantitative PCR (RT-qPCR) method, able to detect single-digit copies of mutant DNA, was established and compared with BRAF V600E-specific immunohistochemistry. Results showed that the BRAF V600E mutation was present at low frequency (0.65%, 2/306) in the studied patient group, and the detection sensitivity and specificity of the new RT-qPCR and V600E-specific immunohistochemistry both reached 100% and 97.6%, respectively. Screening the BRAF V600E/K mutation with the RT-qPCR and V600E-specific immunohistochemistry simultaneously could help improve detection accuracy.

The potential chemical structure of anti-SARS-CoV-2 RNA-dependent RNA polymerase.

An outbreak of coronavirus disease 2019 (COVID-19) occurred in Wuhan and it has rapidly spread to almost all parts of the world. For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an important polymerase that catalyzes the replication of RNA from RNA template and is an attractive therapeutic target. In this study, we screened these chemical structures from traditional Chinese medicinal compounds proven to show antiviral activity in severe acute respiratory syndrome coronavirus (SARS-CoV) and the similar chemical structures through a molecular docking study to target RdRp of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that theaflavin has a lower idock score in the catalytic pocket of RdRp in SARS-CoV-2 (-9.11 kcal/mol), SARS-CoV (-8.03 kcal/mol), and MERS-CoV (-8.26 kcal/mol) from idock. To confirm the result, we discovered that theaflavin has lower binding energy of -8.8 kcal/mol when it docks in the catalytic pocket of SARS-CoV-2 RdRp by using the Blind Docking server. Regarding contact modes, hydrophobic interactions contribute significantly in binding and additional hydrogen bonds were found between theaflavin and RdRp. Moreover, one π-cation interaction was formed between theaflavin and Arg553 from the Blind Docking server. Our results suggest that theaflavin could be a potential SARS-CoV-2 RdRp inhibitor for further study.

MET exon 14 skipping mutations and gene amplification in a Taiwanese lung cancer population.

Somatic mutations of MET gene are emerging as important driver mutations for lung cancers. To identify the common clinicopathological features of MET exon 14 skipping mutations and amplification and clarify whether the two MET gene alterations cause protein overexpression were investigated using 196 lung cancer samples of Taiwan through real time-qPCR/sequencing, fluorescence in situ hybridization, and immunohistochemistry. The two MET gene alterations are both present in low frequency, ~1%, in the studied lung cancer population of Taiwan. MET exon 14 skipping mutations were identified from two early-stage patients, who were both relatively advanced in age, and did not carry other driver mutations. One was an adenocarcinoma and the other was a rare carcinosarcoma. Three gene amplifications cases were identified. Neither of the two MET gene alterations would lead to protein overexpression; hence, direct detection in nucleic acid level would be a preferred and straightforward solution for the identification of skipping mutations. The presence of MET exon 14 mutations in minor histological types of lung cancers urge to extend screening scope of this mutation in lung cancer and treatment response evaluation in clinical trials. These would be important next steps for the success of MET target therapy in clinical practice.

Comparative Analysis of Two Methods for the Detection of EGFR Mutations in Plasma Circulating Tumor DNA from Lung Adenocarcinoma Patients.

Mutations in the epidermal growth factor receptor (EGFR) are associated with various solid tumors. This study aimed to compare two methods for the detection of EGFR mutations in circulating tumor DNA (ctDNA) from lung adenocarcinoma (LUAD) patients and to evaluate the clinical significance of EGFR mutations in ctDNA. In this prospective cohort study, the EGFR mutation status of 77 patients with stage IIIB or IV LUAD was first determined using lung cancer tissue. The amplification refractory mutation system (ARMS) and single allele base extension reaction combined with mass spectroscopy (SABER/MassARRAY) methods were also used to detect EGFR mutations in plasma ctDNA from these patients and then compared using the EGFR mutation status in lung cancer tissue as a standard. Furthermore, the relationship between the presence of EGFR mutations in ctDNA after receiving first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy and survival was evaluated. The overall sensitivity and specificity for the detection of EGFR mutations in plasma ctDNA by ARMS and SABER/MassARRAY were 49.1% vs. 56% and 90% vs. 95%, respectively. The agreement level between these methods was very high, with a kappa-value of 0.88 (95% CI 0.77-0.99). Moreover, 43 of the patients who carried EGFR mutations also received first-line EGFR-TKI therapy. Notably, patients with EGFR mutations in plasma ctDNA had significantly shorter progression-free survival (9.0 months, 95% CI 7.0-11.8, vs. 15.0 months, 95% CI 11.7-28.2; p = 0.02) and overall survival (30.6 months, 95% CI 12.4-37.2, vs. 55.6 months, 95% CI 25.8-61.8; p = 0.03) compared to those without detectable EGFR mutations. The detection of EGFR mutations in plasma ctDNA is a promising, minimally invasive, and reliable alternative to tumor biopsy, and the presence of EGFR mutations in plasma ctDNA after first-line EGFR-TKI therapy is associated with poor prognosis.

Cul4A Modulates Invasion and Metastasis of Lung Cancer Through Regulation of ANXA10.

: Cullin 4A (Cul4A) is overexpressed in a number of cancers and has been established as an oncogene. This study aimed to elucidate the role of Cul4A in lung cancer invasion and metastasis. We observed that Cul4A was overexpressed in non-small cell lung cancer (NSCLC) tissues and the overexpression of Cul4A was associated with poor prognosis after surgical resection and it also decreased the expression of the tumor suppressor protein annexin A10 (ANXA10). The knockdown of Cul4A was associated with the upregulation of ANXA10, and the forced expression of Cul4A was associated with the downregulation of ANXA10 in lung cancer cells. Further studies showed that the knockdown of Cul4A inhibited the invasion and metastasis of lung cancer cells, which was reversed by the further knockdown of ANXA10. In addition, the knockdown of Cul4A inhibited lung tumor metastasis in mouse tail vein injection xenograft models. Notably, Cul4A regulated the degradation of ANXA10 through its interaction with ANXA10 and ubiquitination in lung cancer cells. Our findings suggest that Cul4A is a prognostic marker in NSCLC patients, and Cul4A plays important roles in lung cancer invasion and metastasis through the regulation of the ANXA10 tumor suppressor.

Survival-associated factors of first-line EGFR-tyrosine kinase inhibitor responders and non-responders in lung adenocarcinoma patients with common EGFR mutations.

The aim of the present retrospective cohort study was to elucidate the clinical presentation of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) responders and non-responders in lung adenocarcinoma patients with common EGFR mutations. The cohort included 131 lung adenocarcinoma patients with common exon 19 or exon 21 EGFR mutations, who were receiving first-line EGFR-TKI therapy. The patient characteristics, treatment regimen and outcomes were recorded and analyzed. Of the 131 patients, 104 (79.3%) responded to treatment, while 27 (20.7%) did not. A significantly longer median progression-free survival (PFS) [14.3, 95% confidence interval (CI): 12.2-18.4 vs. 5.7, 95% CI: 2.7-9.9 months; P<0.001] and overall survival (OS) (42.2, 95% CI: 28.1-58.1 vs. 11.5, 95% CI: 8.3-19.7 months; P<0.001) were observed in responders compared with non-responders. In responders, bone [hazard ratio (HR)=1.87, 95% CI: 1.11-3.20, P=0.021] and pleural (HR=2.40, 95% CI: 1.37-4.22, P=0.002) metastasis were independent factors of PFS. Exon 19 mutations (HR=0.38, 95% CI: 0.19-0.76, P=0.006), Eastern Cooperative Oncology Group performance status score ≥2 (HR=3.53, 95% CI: 1.42-8.75, P=0.007) and bone metastasis (HR=2.01, 95% CI: 1.05-3.85, P=0.034), were independent factors of OS. In non-responders, smoking (HR=3.97, 95% CI: 1.13-13.91, P=0.031) was an independent factor of PFS. Different survival-associated factors were observed between EGFR-TKI responders and non-responders. The development of new treatment strategies should be advocated in EGFR-TKI non-responders.