Structure-activity relationship and biological evaluation of 12 N-substituted aloperine derivatives as PD-L1 down-regulatory agents through proteasome pathway.

Twenty-nine 12 N-substituted aloperine derivatives were synthesized and screened for suppression on PD-L1 expression in H460 cells, as a continuation of our work. Systematic structural modifications led to the identification of compound 6b as the most active PD-L1 modulator. Compound 6b could significantly down-regulate both constitutive and inductive PD-L1 expression in NSCLC cells, and successively enhance the cytotoxicity of co-cultured T cells against tumor cells at the concentration of 20 µM. Also, it exhibited a moderate in vivo anticancer efficacy against Lewis tumor xenograft with a stable PK and safety profile. The mechanism study indicated that 6b mediated the degradation of PD-L1 through a proteasome pathway, rather than a lysosome route. These results provided the powerful information for cancer immunotherapy of aloperine derivatives with unique endocyclic skeleton by targeting PD-L1 to activate immune cells to kill cancer cells.

Serum magnesium level as a predictor of acute kidney injury in patients with acute pancreatitis.

BACKGROUND: Decreased serum magnesium (Mg2+) is commonly seen in critically ill patients. Hypomagnesemia is significantly more frequent in patients with severe acute pancreatitis. Acute kidney injury (AKI) in patients with acute pancreatitis (AP) is associated with an extremely high mortality. The association underlying serum Mg2+ and AKI in AP has not been elucidated. AIM: To explore the association between serum Mg2+ on admission and AKI in patients with AP. METHODS: A retrospective observational study was conducted in a cohort of patients (n = 233) with AP without any renal injury before admission to our center from August 2015 to February 2019. Demographic characteristics on admission, severity score, laboratory values and in-hospital mortality were compared between patients with and without AKI. RESULTS: A total of 233 patients were included for analysis, including 85 with AKI. Compared to patients without AKI, serum Mg2+ level was significantly lower in patients with AKI at admission [OR = 6.070, 95%CI: 3.374-10.921, P < 0.001]. Multivariate logistic analysis showed that lower serum Mg2+ was an independent risk factor for AKI [OR = 8.47, 95%CI: 3.02-23.72, P < 0.001]. CONCLUSION: Our analysis indicates that serum Mg2+ level at admission is independently associated with the development of AKI in patients with AP and may be a potential prognostic factor.

Prognostic value of TP53 co-mutation status combined with EGFR mutation in patients with lung adenocarcinoma.

PURPOSE: TP53/EGFR co-mutation has been reported to affect the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma (LUAD). However, its impact on survival is unclear. In this analysis, we explored the prognostic effect of TP53/EGFR co-mutation in LUAD. METHODS: Clinical data and transcriptome sequencing of LUAD patients with matched genomic data were downloaded from the Cancer Genome Atlas (TCGA) database for overall survival (OS) analysis. Differential expression genes (DEGs) were recognized by R software and bioconductor package. Clusterprofiler was used for functional analysis. STRING was used for estimating PPI information and plug-in CytoHubba to screen hub modules in Cytoscape. The association between tumor mutation burden (TMB) and survival was also analyzed. RESULTS: OS was shorter for patients carrying TP53 mutation (MUT) than that of wild type (WT) (37.7 m vs 52.8 m; p = 0.040, HR = 1.38, 95% CI 1.01-1.89). Dual TP53/EGFR-MUT was associated with inferior OS compared with the dual WT/WT cohort (38.4 m vs 51.9 m; p = 0.023, HR 1.83, 95% CI 0.95-3.52). 316 DEGs between dual TP53/EGFR-MUT and dual WT/WT samples were obtained and functional analysis made known that DEGs were strikingly enriched in regulating the metabolism of important amino acids, cell division, cell cycle regulation, cell adhesion, and extracellular matrix composition. KEGG analysis discovered that DEGs were mainly enriched in signaling pathways such as PI3K-Akt, cytokine-cytokine receptor interaction, focal adhesions, and extracellular matrix receptor interaction. PPI network suggested that GPC3, CCL28, GPR37, and NPY genes were up-regulated in dual mutation samples. OS in the high TMB cohort was significantly better than that in the low TMB in patients with TP53 MUT(43.2 m vs 32.4 m; P = 0.007, HR = 0.52, 95% CI: 0.34-0.81), as well as in the combination of TP53 MUT and EGFR WT group (44.4 m vs 31.2 m; P = 0.021, HR = 0.55, 95% CI 0.34 - 0.89). CONCLUSIONS: TP53 MUT is a poor prognostic factor in LUAD patients, and the prognosis of TP53/EGFR co-mutation is worse. GPC3, CCL28, GPR37, and NPY may be novel prognostic markers and potential therapeutic targets for patients with dual TP53/EGFR mutation LUAD.

Co-occurring genetic alterations predict distant metastasis and poor efficacy of first-line EGFR-TKIs in EGFR-mutant NSCLC.

PURPOSE: To determine the frequency of co-occurring genes in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation and the predictive effect of co-mutations on the efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). METHODS: 54 patients with advanced NSCLC were tested for 422 clinically relevant genes by next-generation sequencing (NGS) before treatment. Among them, patients with EGFR mutation received first-line treatment of EGFR-TKIs. Progression-free survival (PFS) and objective response rate (ORR) were evaluated using Kaplan-Meier methods and compared between two groups using log-rank test. RESULTS: Among 24 EGFR mutant and 30 EGFR wild-type patients, co-mutation rate was lower in patients with EGFR mutation (62.5% [15/24] vs 93.3% [28/30], p = 0.005). There was lower frequency for co-alterations in BRAF (0% [0/24] vs 20% [7/30], p = 0.033), NF1 (4.2% [1/24] vs 30% [9/30], p = 0.038) and RAS-RAF-MAPK pathway genes (16.6% [4/24] vs 56.7% [17/30], p = 0.003) in EGFR mutation group. 24 patients with EGFR mutation received first-line treatment of gefitinib or erlotinib, with an ORR of 83.3% and a median PFS of 12.3 months (95% CI 10.00-14.60). Co-mutation was associated with shorter median PFS (10.2 months [95% CI 5.20-15.20] vs 15.3 months [95% CI 12.09-15.81]; HR 0.29 [95% CI 0.10-0.82]; p = 0.014) in EGFR mutation cohort. Among patients with EGFR mutation and distant metastasis, median PFS was decreased in those with co-mutations (6.3 months [95% CI 3.25-9.35] vs 22.0 months[95% CI 12.10-31.90]; HR 0.12 [95% CI 0.00-5.87]; p = 0.007) and frequency of PIK3CA (0% [0/12] vs 41.7% [5/12], p = 0.037) and PI3K/AKT/mTOR pathway genes (0% [0/12] vs 50% [6/12], p = 0.014) was lower. CONCLUSION: The presence of co-mutations was lower in the EGFR mutation patients and reduces the efficacy of EGFR-TKI, especially in patients with distant metastases. Lower frequency of co-mutation in PIK3CA and PI3K/AKT/mTOR pathway genes may be responsible for promoting metastasis and limiting the efficacy of EGFR-TKIs.

Discovery and evolution of aloperine derivatives as novel anti-filovirus agents through targeting entry stage.

Preventing filoviruses in the entry stage is an attractive antiviral strategy. Taking aloperine, a Chinese natural herb with an endocyclic skeleton, as the lead, 23 new aloperine derivatives were synthesized and evaluated for their anti-filovirus activities including ebola virus (EBOV) and marburg virus (MARV) using pseudotyped virus model. Structure-activity relationship (SAR) analysis indicated that the introduction of a 12N-dichlorobenzyl group was beneficial for the potency. Compound 2e exhibited the most potent anti-EBOV and anti-MARV effects both in vitro and in vivo. It also displayed a good pharmacokinetic and safety profile in vivo, indicating an ideal druglike feature. The primary mechanism study showed that 2e could block a late stage of viral entry, mainly through inhibiting cysteine cathepsin B activity of host components. We consider compound 2e to be a promising broad-spectrum anti-filovirus agent with the advantages of a unique chemical scaffold and a specific biological mechanism.

Synthesis and biological evaluation of new berberine derivatives as cancer immunotherapy agents through targeting IDO1.

To discover small-molecule cancer immunotherapy candidates through targeting Indoleamine 2,3-dioxygenase 1 (IDO1), twenty-five new berberine (BBR) derivatives defined with substituents on position 3 or 9 were synthesized and examined for repression of IFN-γ-induced IDO1 promoter activities. Structure-activity relationship (SAR) indicated that large volume groups at the 9-position might be beneficial for potency. Among them, compounds 2f, 2i, 2n, 2o and 8b exhibited increased activities, with inhibition rate of 71-90% compared with BBR. Their effects on IDO1 expression were further confirmed by protein level as well. Furthermore, compounds 2i and 2n exhibited anticancer activity by enhancing the specific lysis of NK cells to A549 through IDO1, but not cytotoxicity. Preliminary mechanism revealed that both of them inhibited IFN-γ-induced IDO1 expression through activating AMPK and subsequent inhibition of STAT1 phosphorylation. Therefore, compounds 2i and 2n have been selected as IDO1 modulators for small-molecule cancer immunotherapy for next investigation.

Synthesis and Identification of Novel Berberine Derivatives as Potent Inhibitors against TNF-α-Induced NF-κB Activation.

Twenty-three new berberine (BBR) analogues defined on substituents of ring D were synthesized and evaluated for their activity for suppression of tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-κB activation. Structure-activity relationship (SAR) analysis indicated that suitable tertiary/quaternary carbon substitutions at the 9-position or rigid fragment at position 10 might be beneficial for enhancing their anti-inflammatory potency. Among them, compounds 2d, 2e, 2i and 2j exhibited satisfactory inhibitory potency against NF-κB activation, with an inhibitory rate of around 90% (5 µM), much better than BBR. A preliminary mechanism study revealed that all of them could inhibit TNF-α-induced NF-κB activation via impairing IκB kinase (IKK) phosphorylation as well as cytokines interleukin (IL)-6 and IL-8 induced by TNF-α. Therefore, the results provided powerful information on further structural modifications and development of BBR derivatives into a new class of anti-inflammatory candidates for the treatment of inflammatory diseases.

Schedule-dependent cytotoxicity of sunitinib and TRAIL in human non-small cell lung cancer cells with or without EGFR and KRAS mutations.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients who do initially respond to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) may eventually develop resistance, which may at least partly be due to the acquisition of a secondary EGFR mutation (T790M). Additionally, it has been found that KRAS mutations may serve as poor prognostic biomarkers. Here, we aimed at establishing a suitable treatment regimen for the multi-target TKI sunitinib and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in NSCLC-derived cells with or without EGFR and KRAS mutations. METHODS: Four NSCLC-derived cell lines with or without EGFR and KRAS mutations were exposed to different sunitinib and TRAIL treatment regimens. Alterations in cell viability, cell cycle distribution, apoptosis, phosphorylation of AKT and expression of the death receptors DR4 and DR5 were evaluated using CCK8, flow cytometry and Western blotting assays, respectively. RESULTS: A synergistic cytotoxic effect was observed in all four cell lines treated with sunitinib (1 nM) followed by TRAIL (100 ng/ml), as well as after simultaneous treatment with both agents. We found that sunitinib enhances TRAIL-induced G0/G1-phase cell cycle arrest and blocks TRAIL-triggered activation of AKT as the underlying mechanism. In contrast, we observed antagonistic effects when sunitinib was administered after TRAIL to the cell lines tested. A decreased DR4 and DR5 expression was found to be correlated with this antagonism. CONCLUSION: From our data we conclude that administration of sunitinib followed by TRAIL, as well as a simultaneous administration of both agents, serve as favorable treatment regimens for NSCLC-derived cells, irrespective of their EGFR and/or KRAS mutation status.

Mutations of EGFR or KRAS and expression of chemotherapy-related genes based on small biopsy samples in stage IIIB and IV inoperable non-small cell lung cancer.

PURPOSES: Epidermal growth factor receptor (EGFR) and KRAS mutations may predict the outcome of targeted drug therapy and also may be associated with the efficacy of chemotherapy in patients with non-small cell lung cancer (NSCLC). This report investigated the relation of EGFR or KRAS mutation and expression of chemotherapy-related genes, including excision repair cross-complementing 1 (ERCC1), thymidylate synthetase (TYMS), ribonucleotide reductase subunit M1 (RRM1) and class III ß-tubulin (TUBB3), as a potential explanation for these observations. METHODS: A total of 143 patients with stage IIIB and IV NSCLC from bronchoscopy or percutaneous lung biopsy obtained tumor samples were analyzed concurrently for EGFR or KRAS mutations, and mRNA expression of ERCC1, TYMS, RRM1 and TUBB3. EGFR or KRAS mutations were detected with xTAG liquidchip technology (xTAG-LCT), and mRNA expression levels of four genes were detected by branched DNA-liquidchip technology (bDNA-LCT). RESULTS: Of 143 patients, 63 tumors were positive for EGFR-activating mutations, and 16 tumors were positive for KRAS mutations. EGFR-activating mutations are more frequent in females, adenocarcinoma and non-smokers patients, and KRAS mutations are more frequent in smoking patients. ERCC1 mRNA levels were significantly associated with histological type and tumor differentiation, whereas TYMS levels were significantly associated with age. NSCLC specimens that harboring EGFR-activating mutations are more likely to express low ERCC1 and high TUBB3 mRNA levels, whereas tumors from patients with NSCLC harboring KRAS mutation are more likely to express high ERCC1 mRNA levels. CONCLUSIONS: Mutations and expression of chemotherapy-related genes may provide a basis for the selection of suitable molecular markers for individual treatment in a population with stage IIIB and IV NSCLC.

Novel N-substituted sophoridinol derivatives as anticancer agents.

Using sophoridine (1) as the lead compound, a series of new N-substituted sophoridinic acid derivatives were designed, synthesized and evaluated for their cytotoxicity. SAR analysis indicated that introduction of a chlorobenzyl on the 12-nitrogen atom of sophoridinol might significantly enhance the antiproliferative activity. Of the newly synthesized compounds, sophoridinol analogue 9k exhibited a potent effect against six human tumor cell lines (liver, colon, breast, lung, glioma and nasopharyngeal). The mode of action of 9k was to inhibit the DNA topoisomerase I activity, followed by the G0/G1 phase arrest. It also showed a moderate oral bioavailability and good safety in vivo. Therefore, compound 9k has been selected as a novel-scaffold lead for further structural optimizations or as a chemical probe for exploring anticancer pathways of this kinds of compounds.

[S632A3 promotes LPS-induced IFN-beta production through inhibiting the activation of GSK-3beta].

LPS stimulation of macrophages production of IFN-beta plays a key role in innate immunity defending the microbial invasion. In this study, the effect of S632A3 promoting LPS-induced IFN-beta production and the underlying mechanism were investigated, mRNA level was measured by real-time PCR, cytokine production was determined by ELISA, GSK-3beta activity was investigated by kinase assay, protein phosphorylation and expression were evaluated by Western blotting. The results revealed that S632A3 significantly augmented IFN-beta production by LPS-stimulated macrophages. S632A3 inhibition of the activation of GSK-3beta, reduced the threonine 239 phosphorylation of transcription factor c-Jun but increased the total level of c-Jun in LPS-stimulated macrophages. Moreover, small interfering RNA-mediated knockdown of c-Jun level abrogated the ability of S632A3 to augment IFN-beta. The study thus demonstrates S632A3 being a new anti-inflammation lead compound and provides a molecular mechanism by which S632A3 promoted LPS-induced IFN-beta production in macrophages through inhibiting the activation of GSK-3beta.

Salidroside stimulates osteoblast differentiation through BMP signaling pathway.

Salidroside (SAL) is one of main active components of Rhodiola rosea L. and possesses diverse pharmacological effects. However, the direct role of SAL in bone metabolism remains elusive. In this study, effects of SAL on osteoblast differentiation of murine pluripotent mesenchymal cell line C3H10T1/2 and osteoblastic cell line MC3T3-E1 were examined. We first identified SAL as a potential BMP2 activator in a cell-based screening assay. SAL (0.5-10 µM) could slightly promote the proliferation and greatly increase the alkaline phosphatase (ALP) activity in both cells. Furthermore, SAL increased the mRNA expressions of osteoblast marker genes in either C3H10T1/2 or MC3T3-E1 cells after treatment for different time. Moreover, the mineralization of C3H10T1/2 cells assayed by Alizarin red S staining was dose-dependently increased by SAL. Mechanistically, SAL increased the mRNA level of genes involved in the regulation of BMP signaling pathway, including BMP2, BMP6 and BMP7 and enhanced the phosphorylation of Smad1/5/8 and ERK1/2. The osteogenic effect of SAL was abolished by BMP antagonist noggin or by BMP receptor kinase inhibitor dorsomorphin. Further in vivo study demonstrated that SAL reversed bone loss in ovariectomized rats. Collectively, our findings indicate that SAL regulates bone metabolism through BMP signaling pathway.

Advances and challenges in screening traditional Chinese anti-aging materia medica.

To provide a better service for senior health care, we summarized screening studies of traditional Chinese anti-aging materia medica (TCAM). We collected and analyzed literature of TCAM screening studies using the lifespan test and animal models of aging from 1984 to 2012. We found 26 screening methods for TCAM, and 153 single herbs or active ingredients of TCAM that have been screened out during the past 28 years. The cell lifespan test, the fruit fly lifespan test, and D-galactose aging model were the most widely used and intensively studied screening methods. However, the method for establishing the D-galactose aging model needs to be standardized, and the D-galactose aging model cannot completely be a substitute for the normal aging mouse model. Great success has been achieved in screening studies in TCAM. To further improve screening studies in TCAM, we suggest that the D-galactose aging model be incorporated into the lifespan test in the New Drugs of Traditional Chinese Medicine Research Guide.

Salidroside protects human fibroblast cells from premature senescence induced by H(2)O(2) partly through modulating oxidative status.

Although salidroside and salidroside-like compounds are considered as most critical constitutes needed and responsible for multiple therapeutic benefits of Rhodiola rosea L., including anti-aging, direct demonstration regarding the role of salidroside in anti-aging process is still deficient. In this study, we selected the H(2)O(2)-induced premature senescence model in human fetal lung diploid fibroblasts to investigate the protection of salidroside against aging in vitro and associated molecular mechanisms. We found that salidroside considerably reversed senescence-like phenotypes in the oxidant challenged model, including alterations of morphology, cell cycle, SA-ß-gal staining, DNA damage, as well as related molecules expression such as p53, p21 and p16. The protection occurred in a dose-dependent manner, with 5µM offering best efficacy. The proposed antioxidant property of the compound was confirmed in this cellular system, and thus at least partially accounted for the protection of the compound against premature senescence. Similar protection of salidroside against replicative senescence was observed as well. Interestingly, the regulation of senescence-related molecules by salidroside involved ROS-irrelevant mechanisms in both models. This finding presents salidroside as an attractive agent with potential to retard aging and attenuate age-related diseases in humans.

Design, synthesis, and cholesterol-lowering efficacy for prodrugs of berberrubine.

In order to enhance oral bioavailability of berberine (BBR) for its cholesterol-lowering efficacy in vivo, a series of ester or ether prodrugs of berberrubine (M1), which is an active metabolite of BBR after first-pass metabolism, were designed, semi-synthesized, and evaluated. Among these M1 prodrugs, compound 5g possessing palmitate at the 9-position showed a moderate LogP value and esterase hydrolysis rate for releasing M1 in blood. Its cholesterol-lowering efficacy in vivo was evaluated in hyperlipidemic SD rats. Compound 5g (100mg/kg/d) reduced blood CHO and LDL-c by 35.8% and 45.5%, respectively, similar to that by BBR. It also exhibited a good safety in rats with no side-effect on liver and kidney function. Therefore, the design of M1 prodrug appears to be an effective strategy to improve pharmacokinetic feature of BBR for its lipid-lowering efficacy in vivo.

Protective role of salidroside against aging in a mouse model induced by D-galactose.

OBJECTIVE: To investigate the protective effects of putative AGEs (advanced glycation endproducts) inhibitor salidroside against aging in an accelerated mouse aging model induced by D-galactose. METHODS: A group of 5-month-old C57BL/6J mice were treated daily with D-galactose, D-galactose combined with salidroside, salidroside alone, and control buffer for 8 weeks. At the end of the treatment, serum AGEs levels, neurological activities, expression of glial fibrillary acidic protein (GFAP) and neurotrophin-3 (NT-3) in the cerebral cortex, as well as lymphocyte proliferation and IL-2 production were determined. RESULTS: D-galactose induced mouse aging model was developed as described before. As expected, salidroside blocked D-galactose induced increase of serum AGEs levels. It also reversed D-galactose induced aging effects in neural and immune system, as evidenced by improving motor activity, increasing memory latency time, and enhancing lymphocyte mitogenesis and interleukin-2 (IL-2) production. Furthermore, elevated expression of GFAP and NT-3 in the aged model mice was also reduced upon salidroside treatment. CONCLUSION: Salidroside inhibits AGEs formation in vivo, which at least partially contributes to its anti-aging effect in D-galactose induced aging model.

Reversal of apoptotic resistance by Lycium barbarum glycopeptide 3 in aged T cells.

OBJECTIVE: To study whether Lycium barbarum glycopeptide 3 (LBGP3) affects T cell apoptosis in aged mice. METHODS: LBGP3 was purified with DEAE cellulose and Sephadex columns. Apoptotic "sub-G1 peak" was detected by flow cytometry and DNA ladder was resolved by agarose gel electrophoresis. Levels of IFN-gamma and IL-10 were measured with specific kits and mRNA expression was detected by RT-PCR. Apoptosis-related proteins of FLIP, FasL, and Bcl-2 were determined by Western blotting. RESULTS: LBGP3 was purified from Fructus Lycii water extracts and identified as a 41 kD glycopeptide. Treatment with 200 microg/mL LBGP3 increased the apoptotic rate of T cells from aged mice and showed a similar DNA ladder pattern to that in young T cells. The reversal of apoptotic resistance was involved in down-regulating the expression of Bcl-2 and FLIP, and up-regulating the expression of FasL. CONCLUSION: Lycium barbarum glycopeptide 3 reverses apoptotic resistance of aged T cells by modulating the expression of apoptosis-related molecules.

Structural and functional changes of immune system in aging mouse induced by D-galactose.

OBJECTIVE: To investigate the role of D-galactose, especially in the structural and functional changes of the immune system in aging. METHODS: Serum levels of advanced glycation end-products (AGE) were determined by ELISA method. Ultra-structures of thymus and spleen were detected by transmission electron microscopy. MTT method was used to determine the lymphocyte proliferation. IL-2 activity was determined by bioassay. Northern blot was used to detect the IL-2 mRNA levels. RESULTS: Serum AGE levels of D-galactose- (P < 0.01) and AGE-treated (P < 0.05) mice (n = 8) were increased significantly. The ultra-structures of thymus and spleen in D-galactose- and AGE-treated mice showed regressive changes similar to those in the aged control group. The lymphocyte mitogenesis and IL-2 activity of spleen were also decreased significantly (P < 0.01, n = 8). The change of IL-2 activity shown by Northern blot resulted from the change of mRNA expression. The AGE plus aminoguanidine group, however, showed no significant change in these parameters in comparison with the young control group (P < 0.01 or P < 0.05, n = 8). CONCLUSION: D-galactose and AGE lead to a mimic regression change of aging in the immune system in vivo.