Pseudolaric Acid B Inhibits FLT4-induced Proliferation and Migration in Non-small Cell Lung Cancer.

OBJECTIVES: Non-Small Cell Lung Cancer (NSCLC) has attracted much attention on account of the high incidence and mortality of cancers. Vascular Endothelial Growth Factor Receptor 3 (VEGFR3/FLT4), which is a highly expressed receptor in NSCLC, greatly regulates cancer proliferation and migration. Pseudolaric Acid B (PAB) is a diterpenoid acid with antitumor activity isolated from Pseudolarix kaempferi. This study aimed to explore the inhibitory effect of PAB targeting FLT4 in NSCLC. METHODS: Cell membrane chromatography was used to evaluate the affinity of PAB binding on FLT4. NCIH1299 cells were used in this study, and an MTT assay was performed to determine the anti-proliferation effect of PAB. Cell cycle analysis was conducted to study the cycle arrest of PAB. Wound healing and Transwell assays assessed the rate of cell migration. Western blot analysis evaluated the expression of related proteins. RESULTS: PAB showed strong affinity to FLT4 with a KD value of 3.01 × 10- 6 M. Targeting FLT4 by PAB inactivated downstream P38MAPK and PI3K/AKT pathways, which inhibited the proliferation of NCI-H1299 cells. Meanwhile, PAB promoted G2/M phase arrest by influencing CyclinB1 and CDK1 complex formation to inhibit NCI-H1299 cell growth, but the effect was attenuated by knocking down the FLT4. Besides, PAB regulated MMP9 secretion through the Wnt/ß-catenin signaling pathway to inhibit NCI-H1299 cell migration. However, the ability of PAB to inhibit migration was significantly weakened by FLT4 knockdown in NCI-H1299 cells. CONCLUSION: PAB can inhibit the proliferation and migration of NSCLC cells through targeting FLT4 and is expected to be a promising FLT4 inhibitor for NSCLC treatment.

In vitro pharmacokinetic behavior in lung of harringtonine, an antagonist of SARS-CoV-2 associated proteins: New insights of inhalation therapy for COVID-19.

BACKGROUND: Recent studies have shown that harringtonine (HT) could specifically bind with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and host cell transmembrane serine protease 2 (TMPRSS2) to block membrane fusion, which is an effective antagonist for SARS-CoV-2. PURPOSE: Our study focused on in-depth exploration of in vitro pharmacokinetic characteristics of HT in lung. METHODS: HPLC-fluorescence detection method was used to detect changes of HT content. Incubation systems of lung microsomes for phase I metabolism and UGT incubation systems for phase II metabolism were performed to elucidate metabolites and metabolic mechanisms of HT, and then the metabolic enzyme phenotypes for HT were clarified by chemical inhibition method and recombinant enzyme method. Through metabolomics, we comprehensively evaluated the physiological dynamic changes in SD rat and human lung microsomes, and revealed the relationship between metabolomics and pharmacological activity of HT. RESULTS: HPLC-fluorescence detection method showed strong specificity, high accuracy, and good stability for rapid quantification of HT. We confirmed that HT mainly underwent phase I metabolism, and the metabolites of HT in different species were all identified as 4'-demethyl HT, with metabolic pathway being hydrolysis reaction. CYP1A2 and CYP2E1 participated in HT metabolism, but as HT metabolism was not NADPH dependent, the esterase HCES1 in lung also played a role. The main KEGG pathways in SD rat and human lung microsomes were cortisol synthesis and secretion, steroid hormone biosynthesis and linoleic acid metabolism, respectively. The downregulated key biomarkers of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME suggested that HT could prevent immunosuppression and interfere with infection and replication of SARS-CoV-2. CONCLUSION: HT was mainly metabolized into 4'-demethyl HT through phase I reactions, which was mediated by CYP1A2, CYP2E1, and HCES1. The downregulation of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME were key ways of HT against SARS-CoV-2. Our study was of great significance for development and clinical application of HT in the treatment of COVID-19.

Dauricine Inhibits Non-small Cell Lung Cancer Development by Regulating PTEN/AKT/mTOR and Ras/MEK1/2/ERK1/2 Pathways in a FLT4-dependent Manner.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is still a solid tumor with high malignancy and poor prognosis. Vascular endothelial growth factor receptor 3 (FLT4, VEGFR3) is overexpressed in NSCLC cells, making it a potential target for NSCLC treatment. In this study, we aimed to explore the anti-cancer effects of dauricine on NSCLC cells and its mechanism targeting FLT4. METHODS: We found that dauricine inhibited the growth of NCI-H1299 cells by blocking the cycle in the G2/M phase through flow cytometry analysis. In addition， dauricine also inhibited the migration of NCI-H1299 cells by wound healing assay and transwell migration assay. More importantly, our empirical analysis found the anti-cancer effect of dauricine on NCI-H1299 cells and the protein level of FLT4 had a distinctly positive correlation, and this effect was weakened after FLT4 knockdown. RESULTS: It is suggested that dauricine suppressed the growth and migration of NCI-H1299 cells by targeting FLT4. Furthermore, dauricine inhibited FLT4 downstream pathways, such as PTEN/AKT/mTOR and Ras/MEK1/2/ERK1/2, thereby regulating cell migration-related molecule MMP3 and cell cycle-related molecules (CDK1, pCDK1-T161, and cyclin B1). CONCLUSION: Dauricine may be a promising FLT4 inhibitor for the treatment of NSCLC.

HPLC-fluorescence detection for stability of harringtonine, and identification of degradation products by UPLC-Q-TOF-MS.

Harringtonine (HT) is an anticancer alkaloid early extracted and isolated from cephalotaxus fortunei Hook. f., also has various pharmacological activities such as antiviral, antibacterial, antimalarial, anti-inflammatory, antioxidant, herbicidal and insecticidal. However, the factors affecting the stability of HT, the main degradation sites and mechanisms involved in its disposal process in vivo have not yet been elucidated. This study utilized HPLC-fluorescence detection method to establish a simple quantitative detection method for HT with good accuracy, precision, and high sensitivity. Temperature and pH were the main factors affecting the stability of HT, which underwent significant degradation in high temperature and alkaline environments because of the occurrence of hydrolysis reactions. In isolated biological homogenates of SD rats, except gastrointestinal tract, HT was degraded in other sites, especially respiratory, mainly in airway and lungs, and systemic metabolism, mainly in livers, spleens, and kidneys. Through UPLC-Q-TOF-MS, three forced degradation products were identified as 4'-demethyl HT, cephalotaxine, and dehydrated HT, respectively. However, the degradation product in isolated biological homogenates of SD rats was only 4'-demethyl HT due to the relatively mild environment. Our findings contributed to a necessary study basis for HT in terms of structural optimization, dosage form selection, storage and transportation.

[Corrigendum] ND­09 inhibits chronic myeloid leukemia K562 cell growth by regulating BCR­ABL signaling.

Subsequently to the publication of this paper, the authors have realized that an error was made during the compilation of Fig. 2A as it appeared on p. 4. Essentially, the partial Q2­3 images of the '1.56 µm' group were inadvertently copied across to the Q2­3 images of the '3.12 µm' group, leading to the cell number of the Q2­3 quadrant being the same for both the 1.56 µm and the 3.12 µm groups, and also leading the total cell number of the 3.12 µm group being calculated as 106.97%, which was clearly incorrect (the total percentage should have added up to 100%). The corrected version of Fig. 2, showing the correct data for the Q2­3 images in the '3.12 µm' group, is shown on the next page. Note that this error did not significantly affect the results or the conclusions reported in this paper, and all the authors agree with the publication of this Corrigendum. The authors are grateful to the Editor of Oncology Reports for allowing them this opportunity to publish a corrigendum, and apologize to the readership for any inconvenience caused. [Oncology Reports 46: 136, 2021; DOI: 10.3892/or.2021.8087].

Serum Proteomics Combined with Metabolomics Analysis Explore the Molecular Biological Characteristics of Eruptive Syringoma.

Background: Eruptive syringoma (ES) is a clinical variant of the appendageal tumor syringoma. Around 75% of ES arise in the head or neck, which makes them unsightly. ES is common in patients with amyloidosis, diabetes, and Down's syndrome, suggesting that it may be associated with potential systemic effects. ES is a rare tumor with the unclear pathogenesis and no effective treatment. Methods: A PubMed search of ES was conducted. Plasma samples of patients with ES were acquired from the Department of Dermatology at Xi'an Jiaotong University's Second Affiliated Hospital. After removing highly abundant proteins, plasma samples were subjected to proteomics and metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: LC-MS/MS revealed 71 differentially expressed proteins and 18 differentially abundant metabolites. The functional analysis highlighted the importance of complement binding, coagulation, secretory granules and vesicle lumen. Further, the study revealed 15 hub genes associated with FGG, GC, APOE, FGA, FGB, C4A, C3, CRP, C4B, FLNA, TAGLN2, ANXA5, MYL6, MYL12B, and TLN1 organized into three clusters. The seed genes in each cluster were GC, FLNA, and MYL6. In addition, glycol metabolism was associated with variable abundance of serum metabolites, which explains the relatively high rate of ES among diabetics. Conclusion: This study suggests that immunological inflammation and tumor glycol metabolism may play significant role in the pathophysiology of ES.

Proteomic and Phosphoproteomic Analyses Reveal the Oncogenic Role of PTK7-NDRG1 Axis in Non-small-cell Lung Cancer Cell Resistance to AZD9291.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the most important chemotherapeutics for non-small-cell lung cancer (NSCLC) therapy. The resistance to EGFR-TKIs is one of the biggest obstacles to NSCLC outcome. In this study, taking advantage of phospho- and proximal proteomic techniques, we analyzed the network rearrangement in cell lines responding to AZD9291 treatment and found that cell-cell adhesion was dramatically enhanced in AZD9291-resistant cells. Further analysis revealed that protein tyrosine kinase 7 (PTK7) expression was significantly elevated. Knockdown or overexpression assays showed that PTK7 played a critical role in improving cell adhesion, which enhanced drug resistance. Because PTK7 is a membrane-localized pseudokinase, the proximal labeling probe BirA* was fused to reveal PTK7-interacting proteins. We found that PTK7 interacted with and stabilized NDRG1, which is located predominantly adjacent to adherens junctions. Downregulation of PTK7 or NDRG1 eliminated the resistance of H1975-resistant (H1975-R) and PC9-resistant (PC9-R) cells to AZD9291, suggesting that the PTK7-NDRG1 axis might be a potential target to eliminate the EGFR-TKI resistance during NSCLC therapy.

Thebaine induces anaphylactic reactions via the MRGPRX2 receptor pathway on mast cells.

Morphine derivatives are clinically important anesthetic and sedative drugs, which often show anaphylactic side effects. Mas-related G-protein coupled receptor member X2 (MRGPRX2) triggers mast cell degranulation, which is important process in anaphylactic reactions. MRGPRX2-HEK293 and LAD2 cell membrane chromatographic (CMC) models were used to screen morphine derivatives binding to MRGPRX2. Furthermore, most morphine derivatives significantly enhanced Ca2+ mobilization. More importantly, thebaine was found to effectively promote histamine release. Thebaine induced the increased release of ß-hexosaminidase and high secretion level of cytokines, confirming that thebaine could further trigger anaphylactic reactions and promote subsequent inflammatory reactions. Moreover, the ability of thebaine inducing degranulation and the release of allergenic mediators in mast cells was significantly decreased after MRGPRX2 knockdown, which proved that MRGPRX2 is the key media for thebaine-induced anaphylactic reactions. Significant hind paw swelling and hypothermia in mice after injecting thebaine suggested that thebaine could trigger anaphylactic reactions in vivo.

Echinacoside inhibits the proliferation, migration, invasion and angiogenesis of ovarian cancer cells through PI3K/AKT pathway.

Echinacoside is a group of natural compounds extracted from medicinal plants Cistanche and Echinacea, which has neuroprotective, antiaging, immunomodulatory and anticancer effects, but its specific role and mechanism in tumor remains partially unclear. To our knowledge, it was the first time to reported the effect of Echinacoside in ovarian cancer. Colony formation, TUNEL staining, Transwell and tube formation assays were conducted to analyze the proliferation, apoptosis, invasion and tube formation abilities of serous ovarian carcinoma cells (SKOV3 and OVCAR-3), respectively. The expressions of apoptosis-, invasion- and PI3K/AKT pathway-related proteins were measured by western blotting. In addition, PI3K agonist (740Y-P) was used to assess the regulatory effect of Echinacoside on PI3K/AKT signaling in ovarian cancer. Finally, the anti-tumor effect of Echinacoside on SKOV3-xenografted mice was evaluated by xenograft tumor mouse model. Our results demonstrated Echinacoside concentration-dependently reduced the proliferation, migration and angiogenesis of ovarian cancer cells, whereas promoted apoptosis. Moreover, western blotting revealed that Echinacoside suppressed the growth of ovarian cancer cells by downregulating the phosphorylation levels of PI3K, AKT and mTOR, which could be partially reversed by 740Y-P. Further, in vivo results showed that Echinacoside could effectively alleviate the tumor growth of xenograft mice, accompanied by the decrease of PI3K/AKT signaling. In general, our results demonstrate that Echinacoside could reduce the ovarian cancer progression through inhibition of PI3K/AKT pathway, suggesting that Echinacoside may be a new treatment option for ovarian cancer.

Novel Compound, ND-17, Regulates the JAK/STAT, PI3K/AKT, and MAPK Pathways and Restrains Human T-lymphoid Leukemia Development.

BACKGROUND: T cell acute lymphoblastic leukemia (T-ALL) is an invasive hematological malignant disorder of T cell progenitors. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway plays an important role in the development of T-ALL and in the inhibition of the key molecule, JAK2, and could suppress T-ALL cell proliferation. OBJECTIVE: The objective of this study was to investigate the in vitro anti-tumor effects of a novel nilotinib derivative, ND-17, on cancer cell lines via its interactions with JAK2. METHODS: The effects of ND-17 on cell proliferation and on cell cycle and apoptosis were evaluated using the tetrazolium assay and flow cytometry, respectively. In addition, the ND-17/JAK2 binding interactions were evaluated using surface plasmon resonance and western blot analyses. RESULTS: ND-17 exerted the greatest inhibitory effects on T-ALL cells amongst all hematological cancer cell lines tested. Flow cytometric analysis indicated that ND-17 blocked the cell cycle at the S phase in T-ALL cells. Nilotinib did not significantly inhibit T-ALL cell growth or regulate the cell cycle. Preliminary investigations revealed that the regulation of cyclin-dependent kinases/cyclins was attributed to ND-17-induced cell cycle arrest. Furthermore, ND-17 could bind to JAK2 with strong affinity, and more importantly, ND-17 bound to the ATP pocket of JAK2 in a manner similar to the potent inhibitor. Thus, ND-17 treatment exhibited a prominent effect in inhibiting the phosphorylation of JAK2 in T-ALL cells. An increase in the phosphorylation of JAK2 was observed in interleukin-6- stimulated Jurkat cells, which was reversed by ND-17 treatment. Meanwhile, the combination of TG- 101348 and ND-17 led to further improvement in inhibiting the phosphorylation of JAK2. Moreover, the transfection and knockdown of JAK2 altered the inhibitory effect of ND-17 on Jurkat cell viability. In addition, ND-17 treatment suppressed the JAK/STAT, phosphatidylinositol-3-kinase/protein kinase B/mechanistic target of rapamycin, and mitogen-activated protein kinase/extracellular signal-regulated protein kinases 1 and 2 signaling pathways. CONCLUSION: These findings suggest that ND-17 could be a promising JAK2 inhibitor for the treatment of T-ALL.

ND-16: A Novel Compound for Inhibiting the Growth of Cutaneous T Cell Lymphoma by Targeting JAK2.

OBJECTIVE: Cutaneous T cell lymphoma (CTCL) is a kind of extranodal non-Hodgkin Tcell lymphoma without healable treatment in the clinic. JAK2 amplification in CTCL patients makes it a potential target for CTCL treatment. In the present study, we aimed to evaluate the anticancer effect of ND-16, a novel nilotinib derivate, on CTCL cells and the underlying mechanism targeting JAK2. METHODS AND RESULTS: We found that ND-16 was capable of regulating JAK2 and had a selective inhibitory effect on CTCL H9 cells. The surface plasmon resonance and molecular docking study indicated ND-16 bound to JAK2 with a high binding affinity. Further investigation revealed that ND-16 inhibited the downstream cascades of JAK2, including STATs, PI3K/AKT/mTOR, and MAPK pathways, followed by regulation of Bcl-2 family members and cell cycle proteins CDK/- Cyclins. Flow cytometry analysis confirmed these results that ND-16-treated H9 cells showed cell apoptosis and cell cycle arrest at S-phase. CONCLUSION: ND-16 may be of value in a potential therapy for the management of CTCL.

ß2-adrenergic receptor affinity chromatography with an interaction force analysis model: A method for analysis of active compounds targeting ß2-adrenergic receptor.

Asthma is one of the most prevalent diseases worldwide, and ß2-adrenergic receptor (ß2AR) agonists have been reported to be highly effective bronchodilators against this disease. In this study, we successfully constructed a novel CHO-ß2AR affinity chromatography (CHO-ß2AR/AC), which was evaluated by infrared spectroscopic and scanning electron microscope (SEM) analysis. In addition, CHO-ß2AR/AC model exhibited good selectivity and reliability with the relative standard deviation smaller than 5.6% after 30 days. Furthermore, an interaction force analysis model was developed based on CHO-ß2AR/AC. The results showed that the interaction force analysis model (Φ•E•pKa) exhibited a strong correlation with equilibrium dissociation constant (KD) (r2=0.9284, p=0.002) and a good correlation with logarithm of half-maximum effective concentration (pEC50) values (r2=0.7135, p=0.034). In addition, a pool of clinically approved drugs was screened by this CHO-ß2AR/AC model. Codeine wasfound to bind to and activate ß2AR with KD value of 4.10 × 10-7 M, leading to increased cyclic adenosine monophosphate (cAMP) production with EC50 of 6.49 × 10-7 M and reduction of intracellular Ca2+ concentration, which in turn relaxes bronchial contraction with EC50 of 2.62 × 10-6 M. Furthermore, the KD value and pEC50 of codeine were within the 95% prediction range of the interaction force analysis model. The results indicate that the CHO-ß2AR/AC with interaction force analysis model constructed in this study can be used to effectively and rapidly screen active compounds targeting ß2AR.

ND­09 inhibits chronic myeloid leukemia K562 cell growth by regulating BCR­ABL signaling.

Chronic myeloid leukemia (CML) accounts for approximately 15% of new adult leukemia cases. The fusion gene BCR­ABL is an important biological basis and target for CML. In the present study, a novel compound, ND­09, was developed and its inhibitory effect and mechanism of action on CML growth were evaluated using RT­PCR and western blot analysis. The results showed that ND­09 demonstrated a high level of inhibitory action toward CML cells overexpressing BCR­ABL and induced K562 cell apoptosis through the mitochondrial pathway. Notably, combined ND­09 and BCR­ABL siRNA treatment could better inhibit cell proliferation and induce apoptosis in K562 cells. Furthermore, this growth effect of BCR­ABL siRNA could be fully rescued by transfection with BCR­ABL. ND­09 exhibited a good fit within BCR­ABL and occupied its ATP­binding pocket, thus altering BCR­ABL kinase activity. Therefore, ND­09 downregulated the phosphorylation of BCR­ABL and ABL, ultimately inhibiting the downstream signaling pathways in K562 cells. These findings suggest that ND­09 induces growth arrest in CML cells by targeting BCR­ABL.