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.

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.

[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.

The Ccr4-not protein complex regulates the phase of the Neurospora circadian clock by controlling white collar protein stability and activity.

In the Neurospora circadian negative feedback loop, white collar 1 (WC-1) and WC-2 form the WC complex that activates frequency (frq) transcription. Here we show that Not1 is a WC-interacting protein and is important for maintaining WC levels. The not1 transcript displays a circadian oscillation with a similar phase as frq. Down-regulation of not1 leads to low levels of WC-1 and WC-2 and a delayed circadian phase as a result of increased protein degradation and increased WC activity. Protein purification of Not1 shows that it is part of the Neurospora Ccr4-Not complex. ccr4 is a clock-controlled gene and is regulated directly by the WC complex. Down-regulation of ccr4 results in a phase delay and period lengthening of the clock. Together, our findings suggest that the Ccr4-Not complex participates in the Neurospora clock function by interacting with and regulating the WC complex.

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.

[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.

Hepatoprotection of berberine against hydrogen peroxide-induced apoptosis by upregulation of Sirtuin 1.

Berberine (BBR) has been suggested to be a hepatoprotective agent for oxidative-stress-related liver diseases because of its antioxidant activity. However, the antioxidant mechanisms of BBR are still not fully understood. In the present study, the protective effect of BBR was evaluated, and the underlying molecular mechanisms were investigated in hepatic cell line L02. Results from cell viability and apoptosis assay showed that in cells exposed to hydrogen peroxide (H2 O2 ), the pretreatment of 12 µM BBR could increase cell viability by 19.10 ± 7.40% and reduce apoptotic cells by 7.91 ± 0.78%. A significant change in the expression levels of sirtuin 1 (SIRT1) and apoptosis-related proteins was also observed in the BBR-pretreated hepatocytes under exposure to H2 O2 . Furthermore, BBR exhibited a time-dependent effect on upregulation of SIRT1 in L02 cells. This study demonstrated that the protective effect of BBR against H2 O2 -induced apoptosis was associated with regulation of SIRT1 in hepatic cell line L02, which provided a possible explanation for its antioxidant activity, and implied an application of BBR for the therapeutic relevance in oxidative-stress-related liver diseases.

Boningmycin induces AMPK-mediated endoplasmic reticulum-associated degradation of PD-L1 protein in human cancer cells.

Anti-PD-1/PD-L1 monoclonal antibodies have displayed remarkable clinical benefits and revolutionized the treatment of multiple tumor types, but the low response rates and immune-related adverse events limit their application, which promoting the development of small molecule agents to improve the efficacy of PD-1/PD-L1 blockade therapy. Boningmycin (BON), a new small molecule belonging to bleomycin (BLM) family, exhibits potent anticancer activity in vitro and in vivo, as well as negligible lung toxicity, thereby can be an alternative of BLM. However, understandings about the anticancer mechanism of BLM-related compounds are extremely rare, it remains unclear if they affect PD-L1 level in a manner similar to that of other antitumor drugs. In this study, we discover that BON significantly reduces PD-L1 protein level in NCI-H460 and HT-1080 cells. Meanwhile, BON decreases the protein level of PD-L1 in a tumor xenograft model of NCI-H460 cells. Nevertheless, the mRNA level is not influenced after BON exposure. Furthermore, BON-induced PD-L1 reduction is proteasome- dependent. By using specific inhibitors and RNA interference technology, we confirm that the decline of PD-L1 protein by BON is mediated by AMPK-activated endoplasmic reticulum-associated degradation pathway, which is like to the action of metformin. Last but not the least, BON has synergism on gefitinib in vitro and in vivo. In conclusion, it is the first report demonstrating that BON decreases PD-L1 protein level through AMPK-mediated endoplasmic reticulum-associated degradation pathway. These findings will benefit the clinical transformation of BON and aid in the elucidation of molecular mechanism of BLM-related compounds.

Potent antitumor actions of the new antibiotic boningmycin through induction of apoptosis and cellular senescence.

Boningmycin, a new antibiotic of the bleomycin family, is isolated from the fermentation broth of Streptomyces verticillus var. pingyangensis n.sp. This study aimed to evaluate its antitumor actions and mechanism. The results showed that boningmycin exhibited potent inhibitory effects on several human solid tumor cells and that it was stronger than bleomycin. The administration of boningmycin inhibited the growth of human hepatoma HepG2 xenografts in nude mice, with more efficacy than that of bleomycin. Boningmycin led to an increase of the reactive oxygen species involving iron and caused G2/M phase accumulation in the HepG2 and human breast cancer MCF-7 cells. Two types of cell death, apoptosis and senescence, were detected after exposure to boningmycin. The accumulation of sub-G1 phase cells, an index of apoptosis, and the activation of caspase apoptotic pathways were detected after treatment with higher concentrations of boningmycin. Low concentrations of boningmycin led to a senescent phenotype with an increase in senescence-associated ß-galactosidase activity and the time-dependent increase of p21, p27, and p53 expressions from 48 to120 h. Taken together, the results showed that boningmycin exhibits potent antitumor actions through the induction of apoptosis and cellular senescence.

[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.

Decrease of ABCB1 protein expression and increase of G1 phase arrest induced by oleanolic acid in human multidrug-resistant cancer cells.

Oleanolic acid (OA) is a natural compound that can be found in a number of edible and medicinal plants and confers diverse biological actions. However, the direct target of OA in human tumor cells remains poorly understood, preventing its application in clinical and health settings. A previous study revealed that overexpression of caveolin-1 in human leukemia HL-60 cells can increase its sensitivity to OA. The present study aimed to investigate the effects of OA on the doxorubicin-resistant human breast cancer MCF-7 cell line (MCF-7/DOX), harringtonine-resistant human leukemia HL-60 cells (HL-60/HAR) and their corresponding parental cell lines. Western blotting was performed to measure protein expression levels, whilst Cell Counting Kit-8 (CCK-8) assays, cell cycle analysis (by flow cytometry) and apoptosis assays (with Annexin V/PI staining) were used to assess drug sensitivity. CCK-8 assay results suggested that MCF-7/DOX cells, which overexpress the caveolin-1 protein, have similar OA susceptibility to their parent line. In addition, sensitivity of MCF-7/DOX cells to OA was not augmented by knocking down caveolin-1 using RNA interference. HL-60/HAR cells exhibited a four-fold increased sensitivity to OA compared with that in their parental HL-60 cells according to CCK-8 assay. Both of the resistant cell lines exhibited higher numbers of cells at G1 phase arrest compared with those in their parent lines, as measured via flow cytometry. Treatment of both MCF-7 cell lines with 100 µM OA for 48 h induced apoptosis, with increased effects observed in resistant cells. However, no PARP-1 or caspase-3 cleavage was observed, with some positive Annexin V staining found after HL-60/HAR cells were treated with OA, suggesting that cell death occurred via non-classical apoptosis or through other cell death pathways. It was found that OA was not a substrate of ATP-binding cassette subfamily B member 1 (ABCB1) in drug-resistant cells, as indicated by the accumulation of rhodamine 123 assessed using flow cytometry. However, protein expression of ABCB1 in both of the resistant cell lines was significantly decreased after treatment with OA in a concentration-dependent manner. Collectively, these results suggest that OA could reduce ABCB1 protein expression and induce G1 phase arrest in multidrug-resistant cancer cells. These findings highlight the potential of OA for cancer therapy.

[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.

Distinct characteristics of dasatinib-induced pyroptosis in gasdermin E-expressing human lung cancer A549 cells and neuroblastoma SH-SY5Y cells.

Dasatinib, a multikinase inhibitor, is used in the treatment of chronic myeloid leukemia and was developed to overcome imatinib resistance. Its mechanism of action involves the induction of apoptosis, autophagy and necroptosis. However, it remains unclear whether dasatinib can induce pyroptosis. In the present study, gasdermin E (GSDME)-expressing SH-SY5Y and A549 cells were chosen for investigation. Typical pyroptotic features, such as cleavage of GSDME protein, leakage of lactate dehydrogenase and large bubbled morphology, were observed in both cell lines after exposure to dasatinib. The generation of GSDME fragments was inhibited by specific caspase-3 inhibitor zDEVD in SH-SY5Y cells and pan-caspase inhibitor zVAD in A549 cells. Moreover, distinct characteristics of pyroptosis were observed in A549 cells, which occurred only with a high percentage of Annexin V/propidium iodide double-stained cells and low level of GSDME protein cleavage. The sensitivity of A549 cells to dasatinib is significantly reduced by increasing cell numbers. The elevation of GSDMD and GSDME protein levels was induced by low concentrations of dasatinib, which was not influenced by the reduction of p53 protein with RNA interference. In conclusion, to the best of our knowledge, this is the first study to report that dasatinib can induce pyroptosis in tumor cells and increase the protein levels of GSDMD and GSDME in a p53-independent manner.

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.

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.

Comparison of Clinical, Histopathological, and Genomic Features Between Malignant Peripheral Nerve Sheath Tumors and Cellular Schwannomas of the Eighth Cranial Nerve: A Case Series.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) and cellular schwannomas (CSs) of the eighth cranial nerve are exceedingly rare. The purpose of the present study was to evaluate clinical and genetic characterization of these rare tumors. METHODS: The clinical and radiological features were analyzed retrospectively. The histopathological characteristics were assessed by hematoxylin and eosin staining and immunohistochemistry. Genomic abnormalities were evaluated using array comparative genomic hybridization. RESULTS: Of the 1287 surgeries for vestibular schwannomas from 2014 to 2017, 2 were for MPNSTs and 5 were for CSs. The mean age at diagnosis was older for patients with MPNSTs (57.0 ± 4.2 years) than that of patients with CS (35.8 ± 9.4 years; P = 0.03). Two patients with MPNST died of tumor recurrence. None of the patients with CS died. The 2-year overall and progression-free survival of patients with MPNSTs were worse than those for patients with CSs (overall survival, 50.0% ± 35.4% vs. 100%, P = 0.027; progression-free survival, 0% vs. 100%; P = 0.012). The Ki-67 index for the MPNSTs (29.0% ± 3.5%) was greater than that for the CSs (10.3% ± 3.1%; P = 0.001). The common alterations in MPNSTs mainly included gains of chromosomes 7p, 8p, 9q, 12, and 17 and loss of heterozygosity of 1p, 6 and 9p. The common alterations in CSs included gain of 4p16.3, loss of heterozygosity of 2p15-14, and 22q11.1-13.3. CONCLUSIONS: To the best of our knowledge, the present study is the first high-resolution genomic analysis of MPNSTs and CSs of the eighth cranial nerve and has shown a significant difference that might be more accurate to distinguish between these 2 types of rare tumors.

Isolation and characterization of antibiotic NC0604, a new analogue of bleomycin.

NC0604, a new analogue of bleomycin, was isolated from fermentation broth of Streptomyces verticillus var. pingyangensis n.var. The structure of NC0604 was elucidated by spectroscopic analyses. NC0604 had the same kernel structure as bleomycin, but a different terminal amine moiety determined as amidepropyl spermidine. NC0604 exhibited antibacterial activity against a wide range of bacterial species and showed cytotoxicity in vitro against human HepG(2), KB, MCF-7, HCT116, BGC-823 and MCF-7/DOX cells with IC(50) values of 1.18, 1.21, 1.41, 1.83, 2.02, 1.45 muM, respectively. The antitumor activity of NC0604 against these cells was 3~9 times higher than that of bleomycin; and the pulmonary toxicity of NC0604 was much lower than that of bleomycin.

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.