Novel patient-derived xenograft and cell line models for therapeutic screening in NF2-associated schwannoma.

Treatment of schwannomas in patients with neurofibromatosis type 2 (NF2) is extremely unsatisfactory, and innovative therapeutic approaches are urgently needed. However, the lack of clinically relevant NF2-associated schwannoma models has severely hampered drug discovery in this rare disease. Here we report the first establishment and characterization of patient-derived xenograft (PDX) and cell line models of NF2-associated schwannoma, which recapitulates the morphological and histopathological features of patient tumors, retain patient NF2 mutations, and maintain gene expression profiles resembling patient tumor profiles with the preservation of multiple key signaling pathways commonly dysregulated in human schwannomas. Using gene expression profiling, we identified elevated PI3K/AKT/mTOR networks in human NF2-associated vestibular schwannomas. Using high-throughput screening of 157 inhibitors targeting the PI3K/AKT/mTOR pathways in vitro, we identified a dozen inhibitors (such as BEZ235, LY2090314, and AZD8055) with significant growth-suppressive effects. Interestingly, we observed that three cell lines displayed differential therapeutic responses to PI3K/AKT/mTOR inhibitors. Furthermore, we demonstrated that two orally bioavailable inhibitors, AZD8055 and PQR309, suppressed NF2-associated schwannoma growth both in vitro and in vivo. In conclusion, our novel patient-derived models of NF2-associated schwannoma closely mimic the phenotypes and genotypes of patient tumors, making them reliable preclinical tools for testing novel personalized therapies. © 2022 The Pathological Society of Great Britain and Ireland.

Design, Synthesis, and Bioactivity of Cyclic Lipopeptide Antibiotics with Varied Polarity, Hydrophobicity, and Positive Charge Distribution.

Twenty-three polymyxin analogs with variations at nine amino acid positions were synthesized and assessed for antimicrobial activity and renal cytotoxicity. Compounds M2, 14, S2, and 16 (MIC = 0.125-4 µg/mL) had similar or stronger activities against susceptible and drug-resistant strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii compared to polymyxin B (MIC = 1-2 µg/mL). Most synthesized compounds (50% cytotoxic concentration, CC50 ≥ 200 µg/mL) exhibited lower cytotoxicity than polymyxin B (CC50 = 99 ± 6 µg/mL). Polymyxin S2 showed high plasma stability in vitro and strong efficacy in a mouse systemic infection model (ED50 = 0.9 mg/kg) against NDM-1-producing Klebsiella pneumoniae, suggesting that it is a potential candidate for drug development. The activity and cytotoxicity results indicated that the amino acids at positions 2, 3, 6, and 7 might be replaced. Effects on activity and cytotoxicity linked to changes in the number of positively charged amino acids varied among different cyclopeptide skeletons, but the underlying mechanisms are unknown.

Nrf2 mediates the resistance of human A549 and HepG2 cancer cells to boningmycin, a new antitumor antibiotic, in vitro through regulation of glutathione levels.

NF-E2-related factor 2 (Nrf2) is a transcription factor and a pivotal factor in the induction of the cell defense system. Recent reports show that Nrf2 plays critical roles in tumor heterogeneity and drug resistance. In the present study we investigated whether and how Nrf2 mediated the resistance of human cancer cells to boningmycin (BON), a new antitumor antibiotic of the bleomycin family. We showed that in the expression levels of Nrf2 in human non-small lung cancer A549 cells were much higher than those in human hepatoblastoma HepG2 cells, and their resistance to BON was opposite to Nrf2 expression (the IC50values of BON in A549 cells and HepG2 cells were 5.97 and 0.61 µmol/L, respectively). Similar results were observed with the anticancer agent cisdiamminedichloroplatinum (DDP), which was used as a positive control. In A549 cells, Nrf2 mRNA knockdown significantly increased their susceptibilities to BON and DDP. An enhanced resistance to BON and DDP was observed in HepG2 cells after overexpression of the wild-type Nrf2 protein. Treatment with a specific Nrf2 inhibitor, luteolin, significantly sensitized A549 cells to BON and DDP and increased BON- or DDP-induced apoptosis. The total levels of glutathione (GSH), the final product of the Nrf2 signaling pathway, were much higher in A549 cells than those in HepG2 cells. Supplementation of GSH in HepG2 cells significantly decreased their susceptibility to BON and DDP, wheras depleting GSH with the specific inhibitor L-buthionine sulfoximine in A549 cells significantly increased their susceptibility to BON and DDP. Our results demonstrate that Nrf2 mediates the resistance to BON through regulating glutathione levels in A549 cells and HepG2 cells.

Synthesis and Bioactivity Investigation of the Individual Components of Cyclic Lipopeptide Antibiotics.

In this paper, 26 natural polymyxin components and a new derivative S2 were synthesized, and their differences in efficacy and toxicity have been investigated. Almost all of the synthesized components showed strong activity against both susceptible and resistant strains of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. The toxicities were obviously different between the components. Only some of the components were tested for toxicity in vivo. Compounds E2, E2-Val, A2, M2, D2, and S2 showed obviously lower renal cytotoxicity and acute toxicity than polymyxins B and E. The in vivo nephrotoxicity of E2, M2, and S2 was similar to that of polymyxin E. Compound S2, with four positive charges, was especially interesting as it possessed both increased efficacy and decreased toxicity. The SAR and toxicity studies indicated that further structural modification could concentrate on polymyxin S. The results also indicated that S2 could be a new drug candidate.

[Tumor heterogeneity and drug resistance of targeted antitumor agents].

The popular application of targeted antitumor agents has greatly improved the efficacies of tumor therapy. However, some patients develop drug resistance after the administration with them for six to twelve months, leading to the failure of treatment. The cause of it is mainly due to tumor heterogeneity. The genesis of tumor heterogeneity is closely associated with tumor stem cells, genetic instability, cell competition and stochastic events. There are a lot of mechanisms involved in the drug resistance to the targeted agents. Tumor heterogeneity and drug resistance are great challenges for precision oncology and are taken account for the process of research and development of new antitumor agents.

[Mechanisms of yanshu injection for overcoming multidrug resistance in breast carcinoma MCF-7 cells: an experimental research].

OBJECTIVE: To investigate the mechanism of Yanshu Injection (YI) for overcoming multidrug resistance in breast carcinoma MCF-7 cells. METHODS: Human breast carcinoma MCF-7 cells and doxorubicin-resistant MCF-7/DOX cells were treated with YI. Its inhibition on the cell proliferation was detected by MTT assay. The fluorescence intensity of doxorubicin was detected by flow cytometry. The expression of apoptosis related protein and P-glycoprotein were examined by immunoblotting after treated by YI. RESULTS: The inhibitory action of YI on MCF-7/DOX cells was similar to that of MCF-7 cells, indicating no cross resistance (P > 0.05). 1/16 YI could obviously induce the apoptosis of MCF-7 cells and DOX cells. 1/256 YI +5 nmol/L doxorubicin and 1/128 YI +5 nmol/L doxorubicin could reduce the survival rate of MCF-7/ DOX resistant cells from 86.8% to 74.6% (P < 0.05) and 71.6% (P < 0.01) respectively, showing obvious synergistic effect. Besides, the accumulation of doxorubicin was increased after treated by YI in the MCF-7/ DOX cells. Results of immunoblotting indicated that reduction of P-glycoprotein expression was detected in MCF-7/DOX cells after exposure to YI. CONCLUSION: YI could overcome the multidrug resistance in breast carcinoma MCF-7 cells possibly through reducing the expression of P-glycoprotein.

Puerarin inhibits angiotensin II-induced cardiac hypertrophy via the redox-sensitive ERK1/2, p38 and NF-κB pathways.

AIM: To investigate the effects of puerarin (Pue), an isoflavone derived from Kudzu roots, on angiotensin II (Ang II)-induced hypertrophy of cardiomyocytes in vivo and in vitro. METHODS: C57BL/6J mice were infused with Ang II and treated with Pue (100 mg·kg(-1)·d(-1), po) for 15 d. After the treatment, systolic blood pressure (SBP) and left ventricular wall thickness were assessed. The ratios of heart weight to body weight (HW/BW) and left ventricular weight to body weight (LVW/BW) were determined, and heart morphometry was assessed. Expression of fetal-type genes (ANP, BNP and ß-MHC) in left ventricles was measured using semi-quantitative RT-PCR. Mouse primary cardiomyocytes were treated with Pue (50, 100, 200 µmol/L), then exposed to Ang II (1 µmol/L). ROS level was examined with flow cytometry, the binding activity of NF-κB was determined using EMSA. Western blot was used to measure the levels of ERK1/2, p38 and NF-κB pathway proteins. [(3)H]leucine incorporation was used to measure the rate of protein synthesis. RESULTS: Oral administration of Pue significantly suppressed Ang II-induced increases in the myocyte surface area, HW/BW, LVW/BW, SBP and left ventricular wall thickness. Furthermore, Pue significantly suppressed Ang II-induced increases in ANP, BNP and ß-MHC expression in the left ventricles in vivo. Treatment of cardiomyocytes with Pue (50-500 µmol/L) did not affect the viability of cardiomyocytes in vitro. Pretreatment of cardiomyocytes with Pue dose-dependently inhibited Ang II-induced increases in ROS production, NF-κB binding activity, protein synthesis and cell breadth. Furthermore, pretreatment with Pue significantly suppressed Ang II-induced activation of ERK1/2, p38 and the NF-κB pathway proteins and the expression of ANP and ß-MHC in cardiomyocytes. The positive drug valsartan exerted similar effects on Ang II-induced cardiac hypertrophy in vivo and in vitro. CONCLUSION: Pue attenuates Ang II-induced cardiac hypertrophy by inhibiting activation of the redox-sensitive ERK1/2, p38 and the NF-κB pathways.

[Study of a novel compound 2460A with activities produced by fungus].

With IL-6R as target, a new compound 2460A was identified from fungus using HTS screening model. The taxonomics of the produced strain was confirmed to be Trichoderma hazianum rifai after sequencing analysis of rDNA-ITS (internal transcribed spacer). Results showed that this compound has a binding activity on IL-6R competed with IL-6, thus it is a new ligand of IL-6R originating from microbe. With MTT assay, the anti-tumor activities of 2460A were demonstrated on CM126 and HT-29 cell lines separately, the IC50 are 2.17 x 10(-5) mol x L(-1) and 1.8 x 10(-5) mol x L(-1) respectively. The compound affected lightly the HT-29 cell cycle at S phase. Studies for the anti-tumor activity of 2460A in vivo are in progress in our lab.

[Characteristics of boningmycin induced cellular senescence of human tumor cells].

Cellular senescence is one of the important steps against tumor. This study was to observe the characteristics of boningmycin induced senescence of human tumor cells. MIT method and clone formation assay were used to detect the growth-inhibitory effect. Cellular senescence was detected with senescence-associated beta-galactosidase staining. Cell cycle distribution and accumulation of intracellular reactive oxygen species (ROS) were analyzed with flow cytometry. Protein expression was detected by Western blotting. The results showed that the growth-inhibitory effect of boningmycin was obviously stronger on human oral epithelial carcinoma KB cells than that on non-small cell lung cancer A549 cells. Comparison to the similar action of doxorubicin, that boningmycin induced the features of cellular senescence in both cell lines, its due to the arrest at G2/M phase and an increase of ROS level. The molecular senescence marker P21 increased significantly after boningmycin treatment at a dosage of 0.1 micromol x L(-1), whereas a higher concentration of it induced apoptosis. The results indicated that cellular senescence induced by boningmycin was one of its mechanisms in tumor suppression.

[Mechanism of Chinese herbal compound Zilongjin for antagonizing multi-drug resistance of tumor cells].

OBJECTIVE: To explore the mechanism of action of Zilongjin (ZLJ) in antagonizing multi-drug resistance (MDR) of tumor cells. METHODS: MDR tumor cells, including human breast cancer cell line MCF-7 and MCF-7/DOX, and human oral epithelial cancer cells KB and KBV200, were treated with ZLJ. The inhibition of ZLJ on cell proliferation was determined with MTT assay; cell cycle and fluorescence dye Rhodamine 123 intensity were detected by flow cytometry; and the expression of related proteins was examined by Western blot. RESULTS: IC50 values in MDR cells after ZLJ treatment were similar to those in sensitive cells; MDR cells showed no cross resistance to ZLJ. Flow cytometric analysis showed that the cell cycles of either sensitive or MDR cells were arrested at S phase after exposure to ZLJ. Using ZLJ singly showed a weak inhibition on MDR of MCF-7/ DOX and KBV200 cells, but when used in combining with doxorubicin or vincistine, it evidently increased their cytotoxicity. Expression of P-glycoprotein in MCF-7/DOX cells decreased after ZLJ treatment in a time-dependent manner. Western blot showed that ZLJ could cause the apoptosis marker protein PARP cleavage to initiate the apoptotic pathway. CONCLUSIONS: The proliferation of tumor cells with MDR could be inhibited by ZLJ and they show no cross resistance to ZLJ. The inhibitory effect is related to the activation of apoptotic pathway and the decrease of P-glycoprotein expression.

Role of prosurvival molecules in the action of lidamycin toward human tumor cells.

OBJECTIVE: Lidamycin, an enediyne antibiotic, leads to apoptosis and mitotic cell death of human tumor cells at high and low concentrations. The reason why tumor cells have distinct responses to lidamycin remains elusive. This study was to elucidate if cellular prosurvival molecules are involved in these responses. METHODS: Cleavage of chromatin and DNA was observed by chromatin condensation and agarose gel electrophoresis. Accumulation of rhodamine 123 in lidamycin-treated cells was assayed by flow cytometry. Cell multinucleation was detected by staining with Hoechst 33342. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively. RESULTS: SIRT1 deacetylase remained unchanged in 0.5 nmol/L lidamycin whereas cleavage occurred when apoptosis was induced by lidamycin. Increased FOXO3a, SOD-1 and SOD-2 expression and transient phosphorylation of ERK were detected after exposure of human hepatoma BEL-7402 cells to 0.5 nmol/L lidamycin. High expressions of SIRT1 and Akt were found in colon carcinoma HCT116 p53 knock-out cells exposed to lidamycin. Degradation of PARP and p53 by lidamycin as a substitute for SIRT1 and Akt was confirmed with caspase inhibitor Q-VD-OPh and proteasome inhibitor MG132. Resistance to lidamycin-induced DNA cleavage was observed in breast cancer doxorubicin-resistant MCF-7 cells. This was not induced by P-glycoprotein as no accumulation of rhodamine 123 was detected in the resistant cells following exposure to lidamycin. In contrast to sensitive MCF-7 cells, a lower multinucleation rate for the resistant cells was measured following exposure to equal concentrations of lidamycin. CONCLUSIONS: Cellular prosurvival molecules, such as SIRT1, Akt, SOD-1, SOD-2 and other unknown factors can influence the action of lidamycin on human tumor cells.

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.

[Mechanism of apoptosis induced by SIRT1 deacetylase inhibitors in human breast cancer MCF-7 drug-resistant cells].

The mechanism of apoptosis induced by SIRT1 deacetylase inhibitors in both human breast cancer MCF-7 and MCF-7 doxorubicin-resistant cells was studied. MTT assay was used to detect growth-inhibitory effect on the cells. Protein expression was detected by Western blotting. Chromatin condensation was detected by a fluorescent microscope after Hoechst 33342 staining. Cell cycle distribution was analyzed with flow cytometry. Apoptotic cells were detected with Annexin V staining. Nicotinamide (NAM) and Sirtinol, two SIRT1 deacetylase inhibitors, exhibited the similar growth-inhibitory effects on MCF-7/DOX cells and MCF-7 cells, but no potentiation of DOX activities. The arrest at G2/M phase was detected by flow cytometry in both MCF-7 and MCF-7/DOX cells after NAM treatment. Activation of caspase pathway in MCF-7 cells, such as the cleavages of PARP, caspase-6, -7, -9, were observed after exposure to NAM 50 mmol x L(-1), accompanied by the occurrence of chromatin condensation and Annexin V positive cells. However, the cleavages of PARP, caspase-6 and -7 in MCF-7/DOX cells delayed after exposure to NAM for 24 h and obviously increased at 48 h with appearance of chromatin condensation and Annexin V positive cells. SIRT1 deacetylase inhibitors show no cross resistance to MCF-7 drug-resistant cells, and the similar growth-inhibitory actions of them to MCF-7 sensitive and drug-resistant cells by which it is mediated by activation of apoptotic caspase pathway.

[Effects of lidamycin on apoptotic gene expressions and cytoskeleton in human hepatoma bel-7402 cells].

BACKGROUND & OBJECTIVE: Lidamycin, one of enediyne antitumor antibiotics, was isolated by our institute. It is the focus of intense due to its unique chemical structure and potent cytotoxicities to tumor cells in vivo and in vitro. In addition to cleavage of DNA, effect of lidamycin on apoptotic gene expressions and cytoskeleton may involve in its high activities. To further elucidate its mechanism, we have observed the effect of lidamycin on the above-mentioned factors in human hepatoma bel-7402 cells. METHODS: The gene expressions were determined by Northern blot and dot blotanalysis. The changes of microfilament and microtubule were detected by indirect immunofluorescent method and the apoptotic cells were detected with mitochondria-specific apoptotic dye Mitosensor. RESULTS: The obvious increment of c-myc and c-fos gene expressions and the inhibition of N-ras gene expression in bel-7402 cells were observed following lidamycin treatment (0.1-10 nmol/L) for 8 h. The arrangement of microfilament in the cells became regular and was similar to nonmalignant normal cells, but there was no effect on microtubule when the bel-7402 cells were treated with lidamycin 10 nmol/L for 8 h. No apoptotic cells were detected with Mitosensor in the lidamycin-treated cells for 8 h. CONCLUSION: Lidamycin at lower concentrations can markedly affect the expression of the apoptosis related genes and the distribution of the cytoskeleton in the hepatoma bel-7402 cells. The results are helpful to elucidate the molecular mechanism of potent cytotoxicities of lidamycin to tumor cells.

Effects of lidamycin on genomic DNA in human hepatoma BEL-7402 cells.

AIM: To further study the effect of enediyne antibiotic lidamycin (C1027) on genomic DNA in human hepatoma BEL-7402 cells. METHODS: The DNA patterns were detected by agarose gel electrophoresis. The gene damage was revealed by Southern hybridization. The DNA repair was determined by neutral agarose gel electrophoresis. The lidamycin-cleaved sites were determined with superhelix GEM plasmid. RESULTS: The DNA ladder patterns were observed after the BEL-7402 cells were treated with lidamycin 1 micromol/L for 15, 30, 60 min. The extrons and introns of active N-ras gene in the BEL-7402 cells were easily damaged by lidamycin at low concentrations, but no damage in the silent IL-2 gene was observed. It was difficult to repair DNA breaks after the BEL-7402 cells were treated with lidamycin. The cleaved sites of GEM plasmid treated with lidamycin were -OH HO-. CONCLUSION: Lidamycin can significantly damage genomic DNA in the hepatoma BEL-7402 cells. The results make it helpful to elucidate the molecular mechanism of potent cytotoxicities of lidamycin to tumor cells.