Autologous Tumor-Infiltrating Lymphocyte Mono-Therapy Can Rapidly Shrank Tumor in Asian Patient with Stage III/IV Cervical Cancer: Two Cases Report.

Introduction: Tumor-infiltrating lymphocytes (TILs) therapy is one of the most promising adoptive T cell therapies, which has shown great clinical efficacy against several solid malignancies. Nevertheless, clinical response to TILs mono-therapy in Asian patients with recurrent cervical cancer has not been well reported. Case Presentation: Here, we report two patients who were diagnosed with metastatic cervical cancer and tumor progression following multiple conventional treatments. In particular, one of the patients has a history of severe myelosuppression after chemotherapy. The patients received lymphodepletion therapy, which consisted of cyclophosphamide (30mg/kg) for 2 days, followed by Fludarabine (25mg/m2) for 5 days, approximately 24 hr before receiving intravenous autologous TILs infusion. These two patients then received high doses of IL-2 for 10 days with the purpose of maintaining T cell survival and proliferation. Patient 1 experienced clinical partial response (PR) at 6 weeks post TILs infusion and a 33% tumor shrinkage at 12 weeks follow-up, and patient 2 was evaluated as stable disease (SD) at 6 weeks post treatment. Mild and manageable adverse events were observed and soon subsided after the TILs treatment. A time-course study examining the peripheral blood cell count and cytokine secretion demonstrated the persistence of infused TILs and long-term immune response. Conclusion: These results suggest that TILs mono-therapy can be a promising treatment strategy for Asian patients with late-stage metastatic cervical cancer even with severe myelosuppression. TILs infusion can induce persistence and a long-term systematic immune response that reversed peripheral CD4+T and CD8+T percentages implying that TILs infusion increased cytotic T cell responses, which is consistent with clinical responses in these patients. Trial registration number: NCT05366478.

Neoantigen vaccination induces clinical and immunologic responses in non-small cell lung cancer patients harboring EGFR mutations.

BACKGROUND: Neoantigen (NeoAg) peptides displayed at the tumor cell surface by human leukocyte antigen molecules show exquisite tumor specificity and can elicit T cell mediated tumor rejection. However, few NeoAgs are predicted to be shared between patients, and none to date have demonstrated therapeutic value in the context of vaccination. METHODS: We report here a phase I trial of personalized NeoAg peptide vaccination (PPV) of 24 stage III/IV non-small cell lung cancer (NSCLC) patients who had previously progressed following multiple conventional therapies, including surgery, radiation, chemotherapy, and tyrosine kinase inhibitors (TKIs). Primary endpoints of the trial evaluated feasibility, tolerability, and safety of the personalized vaccination approach, and secondary trial endpoints assessed tumor-specific immune reactivity and clinical responses. Of the 16 patients with epidermal growth factor receptor (EGFR) mutations, nine continued TKI therapy concurrent with PPV and seven patients received PPV alone. RESULTS: Out of 29 patients enrolled in the trial, 24 were immunized with personalized NeoAg peptides. Aside from transient rash, fatigue and/or fever observed in three patients, no other treatment-related adverse events were observed. Median progression-free survival and overall survival of the 24 vaccinated patients were 6.0 and 8.9 months, respectively. Within 3-4 months following initiation of PPV, seven RECIST-based objective clinical responses including one complete response were observed. Notably, all seven clinical responders had EGFR-mutated tumors, including four patients that had continued TKI therapy concurrently with PPV. Immune monitoring showed that five of the seven responding patients demonstrated vaccine-induced T cell responses against EGFR NeoAg peptides. Furthermore, two highly shared EGFR mutations (L858R and T790M) were shown to be immunogenic in four of the responding patients, all of whom demonstrated increases in peripheral blood neoantigen-specific CD8+ T cell frequencies during the course of PPV. CONCLUSIONS: These results show that personalized NeoAg vaccination is feasible and safe for advanced-stage NSCLC patients. The clinical and immune responses observed following PPV suggest that EGFR mutations constitute shared, immunogenic neoantigens with promising immunotherapeutic potential for large subsets of NSCLC patients. Furthermore, PPV with concurrent EGFR inhibitor therapy was well tolerated and may have contributed to the induction of PPV-induced T cell responses.

A Retrospective Study on Using a Novel Single Needle Cone Puncture Approach for the Iodine-125 Seed Brachytherapy in Treating Patients With Thoracic Malignancy.

BACKGROUND: Patients with progressive thoracic malignancy characterized by large irregular tumors with necrosis and life-threatening symptoms lack effective treatments. We set out to develop a single needle cone puncture method for the Iodine-125 seed (SNCP-125I) brachytherapy, and aim to report the initial results. METHODS: 294 patients with advanced thoracic malignancy were treated with local SNCP-125I brachytherapy between March 2009 and July 2020, followed by thorough evaluation of clinical outcome, overall survival (OS), progression-free survival (PFS) and procedure-related complications after treatment. RESULTS: The overall response rate (ORR) among the treated patients was 81.0% (238/294). Life-threatening symptoms due to tumor oppression, hemoptysis and large irregular tumor with necrosis were successfully alleviated after the SNCP-125I treatment with a remission rate at 91% to 94%. The median OS and PFS were 13.6 months and 5.8 months, respectively. Procedure-related side effects including pneumothorax (32/294), blood-stained sputum (8/294), subcutaneous emphysema (10/294), puncture site bleeding (16/294) and chest pain (6/294) were observed. Patients who were able to follow with chemotherapy or immunotherapy experienced extended OS and PFS, as compared with patients who opted to receive hospice care (16.5 months Vs. 11.2 months). Further pathological and immunological analysis showed that SNCP-125I induced tumor lymphocytes infiltration and long-term tumor necrosis. CONCLUSION: SNCP-125I brachytherapy effectively eliminates life-threatening symptoms due to local tumor oppression, hemoptysis and large irregular and necrotic tumors in patients with unresectable chest malignancy and significantly induces local tumor regression. SNCP-125I brachytherapy combines with chemotherapy significantly prolong OS and PFS compare with SNCP-125I brachytherapy alone.

Association of tumor mutation burden and epidermal growth factor receptor inhibitor history with survival in patients with metastatic stage III/IV non-small-cell lung cancer: A retrospective study.

OBJECTIVES: Lung cancer is the leading cause of cancer-related deaths worldwide. However, factors associated with the survival of patients with advanced non-small-cell lung cancer (NSCLC) who received only hospice care are largely unclear. In this study, we aimed to determine the prognostic factors correlated with survival in patients with advanced NSCLC who had undergone hospice care only. METHODS: A total of 102 patients with recurrent stage III/IV NSCLC after traditional treatment failure were investigated. Survival was measured from the date of enrollment to December 2019 or the time of death. Tumor tissues were collected, and DNA sequencing was performed to identify somatic mutations. Data on clinical factors of patients were collected and analyzed by univariate and multivariate analyses. Overall survival analysis was conducted using the Kaplan-Meier method. RESULTS: The 6-month, 1-year, and 2-year overall survival rates of the 102 patients with metastatic NSCLC were 17.65%, 3.92%, and 0.98%, respectively. The median overall survival of the 102 patients was 3.15 months. Tumor location in the peripheral lung, epidermal growth factor receptor (EGFR) inhibitor history, low tumor mutation load, adenocarcinoma, and poor performance status score were associated with prolonged survival compared with tumor location in the central lung, no EGFR inhibitor history, high tumor mutation load, squamous cell carcinoma, and good performance status score (p=0.045, p=0.003, p=0.045, p=0.021, and p=0.0003, respectively). CONCLUSIONS: EGFR inhibitor treatment history and tumor mutation load are risk factors for the overall survival of patients with stage III/IV NSCLC who have undergone only hospice care. These results provide a critical clinical basis for further study of nontraditional anti-tumor responses induced by EGFR inhibitors.

[Research progress of tannins in traditional Chinese medicines in recent ten years].

In this paper, the newly isolated tannins were sorted after a review of the literature concerning tannins in recent 10 years, and their research progress was summarized in terms of extraction, isolation, pharmacological activity and metabolism. Hydrolysable tannins and condensed tannins are the main structural types. Modern research shows that tannins have many pharmacological effects, such as bacteriostasis, antioxidation, antitumor, antivirus and blood glucose reduction, and have broad development prospects. They are usually extracted by water, ethanol and acetone and isolated and purified by macroporous resin and gel column chromatography. The packings commonly adopted for the column chromatography mainly included Sephadex LH-20, Diaion HP-20, MCI-gel CHP-20 and Toyopearl HW-40. Modern analytical techniques such as nuclear magnetic resonance spectroscopy(NMR), fast atom bombardment mass spectrometry(FAB-MS) and circular dichroism(CD) are generally used for the structural identification of tannins. Howe-ver, their isolation, purification and structural identification are still challenging. It is necessary to use a variety of high-throughput screening methods to explore their pharmacological activities and to explore the material basis responsible for their functions through experiments in vivo.

Association of tumor mutation burden and epidermal growth factor receptor inhibitor history with survival in patients with metastatic stage III/IV non-small-cell lung cancer: A retrospective study

OBJECTIVES: Lung cancer is the leading cause of cancer-related deaths worldwide. However, factors associated with the survival of patients with advanced non-small-cell lung cancer (NSCLC) who received only hospice care are largely unclear. In this study, we aimed to determine the prognostic factors correlated with survival in patients with advanced NSCLC who had undergone hospice care only. METHODS: A total of 102 patients with recurrent stage III/IV NSCLC after traditional treatment failure were investigated. Survival was measured from the date of enrollment to December 2019 or the time of death. Tumor tissues were collected, and DNA sequencing was performed to identify somatic mutations. Data on clinical factors of patients were collected and analyzed by univariate and multivariate analyses. Overall survival analysis was conducted using the Kaplan-Meier method. RESULTS: The 6-month, 1-year, and 2-year overall survival rates of the 102 patients with metastatic NSCLC were 17.65%, 3.92%, and 0.98%, respectively. The median overall survival of the 102 patients was 3.15 months. Tumor location in the peripheral lung, epidermal growth factor receptor (EGFR) inhibitor history, low tumor mutation load, adenocarcinoma, and poor performance status score were associated with prolonged survival compared with tumor location in the central lung, no EGFR inhibitor history, high tumor mutation load, squamous cell carcinoma, and good performance status score (p=0.045, p=0.003, p=0.045, p=0.021, and p=0.0003, respectively). CONCLUSIONS: EGFR inhibitor treatment history and tumor mutation load are risk factors for the overall survival of patients with stage III/IV NSCLC who have undergone only hospice care. These results provide a critical clinical basis for further study of nontraditional anti-tumor responses induced by EGFR inhibitors.

Effects of Astaxanthin on Inflammation and Insulin Resistance in a Mouse Model of Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a condition in which a hormone made by the placenta prevents the body from using insulin effectively. It is important to find an effective treatment. A mouse model of GDM was used to testify the effects of astaxanthin on glucose tolerance and insulin sensitivity. Production of inflammatory cytokines, reactive oxygen species (ROS), and glucose transporter type 4 (GLUT4) translocation and insulin-related signaling were measured in the presence of astaxanthin both in vivo and in vitro. It was found that astaxanthin improved insulin sensitivity, glucose tolerance, and litter size of offspring and reduced birth weight of offspring and inflammation in GDM mouse. Moreover, astaxanthin increased GLUT4 translocating to membrane without altering its secretion/expression and glucose uptake and consumption in C2C12 skeletal muscle cells. Furthermore, ROS generation and insulin-related signaling inhibited by tumor necrosis factor α was restored by astaxanthin. It is concluded that astaxanthin has the potential to attenuate GDM symptoms by regulating inflammation and insulin resistance in skeletal muscle of pregnant mice. Our findings suggest that astaxanthin could be a promising and effective molecule to treat GDM.

[Quality evaluation of Poria based on specific chromatogram and quantitative analysis of multicomponents].

HPLC specific chromatograms of Poria were established, and the concentrations of 10 triterpenoids(16α-hydroxydehydrotrametenolic acid, poricoic acid B, dehydrotumulosic acid, poricoic acid A, polyporenic acid C, poricoic acid AM, 3-O-acetyl-16α-hydroxydehydrotrametenolic acid, dehydropachymic acid, pachymic acid, and dehydrotrametenolic acid) were simultaneously determined. Chromatographic analysis was conducted on a Welch Ultimate XB C_(18) column(4.6 mm × 250 mm,5 µm). Acetonitrile solution(contain 3% tetrahydrofuran)(A) and 0.1% formic acid aqueous solution(B) were used as the mobile phase with gradient elution at a flow rate of 1.0 mL·min~(-1). The column temperature was 30 ℃ and the injection volume was 20 µL. The experimental data were analyzed by the SPSS 22.0 and GraphPad Prism 7.0. The established triterpenoids fingerprints were specific, and the 10 components were well separated and showed good linearity(r≥0.999 6) within the concentration ranges tested. The mean recoveries were between 98.53%-103.8%(RSD 1.7%-2.7%). The method was specific and repeatable, and could be used for identification and quality evaluation of Poria. The results showed that the contents of 10 triterpenoids were positively correlated with each other. The contents of 10 triterpenoids of samples collected from producing areas were higher than that collected from markets. The total contents of 10 triterpenoids of samples collected from Hubei and Yunnan province were slightly higher than that from Anhui province, but the contents of samples from Anhui province were varied in smaller ranges.

[Establishment of quality standard and discussion on standard decoction containing volatile oil pieces-Cinnamomi Ramulus pieces].

To prepare Cinnamomi Ramulus pieces standard decoction and establish its quality standard, provide quality reference for formula granules and other clinic non-traditional forms of medicines, and lay a foundation for standard decoction research for the pieces containing essential oil. 14 batches of Cinnamomi Ramulus pieces with different quality were collected from market and their extraction process was further improved based on the preparation principle of standard decoction to prepare the standard decoction of Cinnamomi Ramulus pieces. Then its transfer rate of Cinnamaldehyde, dry extract rate and pH value were calculated to evaluate its process stability; and a method for chromatographic fingerprint and content determination was also established. Results revealed that the dry extract rate for standard decoction of Cinnamomi Ramulus pieces was from 6.06%-8.95%, with an average value of 7.18%; the transfer rate of cinnamaldehyde was at the range of 29.6%-54.3%, with an average of 43.2%; and the pH value was at the range of 4.33-4.82. The fingerprint similarities between 14 batches of standard decoction of Cinnmomi Rammulus pieces and reference fingerprint were all>0.9. The established method for standard decoction was stable and its quality standard was perfect, suitable for evaluating the quality of standard decoction of Cinnanomi Ramulus pieces.

Aflatoxin B1-Induced Developmental and DNA Damage in Caenorhabditis elegans.

Aflatoxin B1 (AFB1) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB1 has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB1 is also a potent genotoxic mutagen that causes DNA damage in vitro and in vivo. However, the link between DNA damage and abnormal development and reproduction is unclear. To address this issue, we examined the DNA damage, germline apoptosis, growth, and reproductive toxicity following exposure to AFB1, using Caenorhabditis elegans as a study model. Results found that AFB1 induced DNA damage and germline apoptosis, and significantly inhibited growth and reproduction of the nematodes in a concentration-dependent manner. Exposure to AFB1 inhibited growth or reproduction more potently in the DNA repair-deficient xpa-1 nematodes than the wild-type N2 strain. According to the relative expression level of pathway-related genes measured by real-time PCR, the DNA damage response (DDR) pathway was found to be associated with AFB1-induced germline apoptosis, which further played an essential role in the dysfunction of growth and reproduction in C. elegans.

[Anti-tumor mechanism of Artemisia annua based on bioinformatics].

This paper was aimed to explore the relationship between Artemisia annua and tumor, and investigate its anti-tumor mechanism based on the bioinformatics molecular network analysis, text mining technique and other related methods. Text mining tool Polysearch database was used to get the information, and after formatting conversion, the information was imported into the bioinformatics analysis software Cytoscape3.2.1 for visualization and subsequent bioinformatics molecular network analysis .It was shown that the A. annua was associated with the tumor by 8 key proteins： TNF,VEGF,PI3K, ALDH1, Bcl-2, MicroRNA, p38 and CASP3 by text mining technique. The main biological processes involved in its anti-tumor effect included cell cycle, post-translational protein modification, cell cycle regulation, protein ubiquitination and organelle tissues regulation. The key network analysis showed that the action may be achieved by regulation of triglyceride metabolic process, positive regulation of triglyceride metabolic process, positive regulation of triglyceride catabolic process, regulation of budding cell apical bud growth, negative regulation of mitotic cell cycle, negative regulation of meiotic cell cycle, and positive regulation of transcription factors. The results showed that the anti-tumor effect of A. annua may be associated with regulating lipid metabolism of tumor cells, decomposing large amounts of lipids, releasing energy, reducing the rate of tumor cell division and accelerating tumor cell apoptosis.

[Exploration research on hepatotoxic constituents from Polygonum multiflorum root].

By observing the cytotoxic effects of anthraquinones on HepG2 cell and using the precision-cut liver slices technique to authenticate the cytotoxic constituents, the paper aims to explore the material basis of Polygonum multiflorum root to cause liver toxicity. Firstly, MTT method was used to detect the effect of 11 anthraquinone derivatives on HepG2 cell. Then, the clear cytotoxic ingredients were co-cultured with rat liver slices for 6h respectively, and the liver tissue homogenate was prepared. BCA method was used to determine the content of protein in the homogenate and continuous monitoring method was used to monitor the leakage of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamine amino transpeptidase (GGT) and lactate dehydrogenase (LDH). The toxic effect of these ingredients on liver tissue was tested by calculating the leakage rate of the monitored enzymes. As a result, rhein, emodin, physcion-8-O-ß-D-glucopyranoside and physcion-8-O-(6'-O-acetyl)-ß-D-glucopyranoside showed cytotoxic effects on HepG2 cell and their IC50 values were 71.07, 125.62, 242.27, 402.32 µmol•L⁻¹ respectively, but the other 7 compounds are less toxic and their IC50 values can not be calculated. The precision-cut liver slices tests showed that rhein group of 400 µmol•L⁻¹ concentration significantly increased the leakage rate of ALT, AST and LDH (P<0.01), and the rhein group of 100 µmol•L⁻¹ concentration only increased the leakage rate of LDH (P<0.05). With the increase of rhein concentration, the protein content in liver slices decreased significantly (P<0.05) with a certain range of does. Emodin group of 400 µmol•L⁻¹ concentration significantly increased the leakage rate of ALT, GGT and LDH (P<0.01). Physcion-8-O-ß-D-glucopyranoside group of 800 µmol•L⁻¹ concentration also significantly increased the leakage rate of ALT, AST and LDH (P<0.01 or P<0.05), but the group of 200 µmol•L⁻¹ concentration only significantly increased the LDH leakage (P<0.05). Along with the increase of the concentration of physcion-8-O-ß-D-glucopyranoside, the leakage rate of ALT, AST and LDH showed a trend of increase, but the protein content in liver slices was in decline. Furthermore, MTT reduction ability of liver slices significantly decreased (P<0.01) in the physcion-8-O-ß-D-glucopyranoside group of 800 µmol•L⁻¹ concentration. The results suggested that rhein, emodin and physcion-8-O-ß-D-glucopyranoside at high concentrations (≥400 µmol•L⁻¹) can produce some damage to the liver tissue. However, the exposure levels of these constituents are very low, so to reach the toxic concentration (400 µmol•L⁻¹ or 800 µmol•L⁻¹) an adult of 65 kg body weight will need at least a single oral 4 898 g, 339 g and 5 581 g of P.multiflorum root respectively, which is far from the statutory dose of crude P. multiflorum root (3-6 g) or its processed product (6-12 g). Therefore, the conclusion that anthraquinones are the prime constituents of the hepatotoxicity of P. multiflorum root are still not be proved.

[Effect of gefitinib on the migration of triple-negative breast cancer cell line MDA-MB-231 cells].

OBJECTIVE: To investigate the effect of gefitinib on the migration of triple-negative breast cancer cell line MDA-MB-231 cells. METHODS: Gefitinib was used in concentrations of 0 micromol/L, 0.1 micromol/L, 1 micromol/L, 10 micromol/L and 20 micromol/L, respectively. Phosphorylation levels of EGFR and Akt were analyzed by Western blot. The capability of migration was measured by scratch test and Boyden chamber assay. Microfilaments (cell skeleton ) remolding and polarization were evaluated by immunofluorescence microscopy. RESULTS: Comparing with the control group (0 micromol/L gefitinib), gefitinib effectively inhibited the phosphorylation of EGFR and its downstream key proteins, and the effect displayed an obvious dose-effect relationship. At 24 hours after wound scratch, the cell migration distance of each group with 0, 0.1, 1, 10, 20 micromol/L gefitinib was (36.3 +/- 4.0) microm, (30.3 +/- 3.8) microm, (26.8 +/- 3.3) microm, (17.0 +/- 2.6) microm, and (11.0 +/- 2.5) microm, respectively. At 3.5 hours after Boyden chamber assay, the cell count of each group with 0, 0.1, 1, 10, 20 micromol/L gefitinib was 69.2 +/- 7.0, 51.8 +/- 7.5, 43.8 +/- 8.7, 30.6 +/- 4.8, and 28.4 +/- 3.4, respectively. Compared with the control group (0 micromol/L gefitinib), gefitinib could significantly prolong the wound-healing time and decrease the migrating cell count (P < 0.05), and significantly inhibit the lamellipodium formation, cell skeleton remolding and changes of the cytoskeleton polarization. CONCLUSIONS: Gefitinib can reduce the migration capacity of triple-negative breast cancer cells through inhibiting phosphorylation of EGFR/PI3K/Akt pathway, suppressing the cell skeleton (microfilaments) remolding and changes of its polarization.

[Molecular mechanism of Aurora kinase inhibitor PHA739358 in inhibited proliferation and induced apoptosis of breast cancer cells].

OBJECTIVE: To explore the molecular mechanisms of Aurora kinase inhibitor PHA739358 in inhibited proliferation and in vitro induced apoptosis of breast cancer cells. METHODS: The in vitro cultured T47D cells in logarithmic growth phase were used. The effects of PHA739358 on cell proliferation were examined by MTT (methyl thiazolyl tetrazolium) assay. The variety of nuclear and spindle morphologies was examined by immunofluorescence. And G2/M arrest was determined by flow cytometry. The morphological changes of apoptotic cells were observed by fluorescent microscope. The levels of Aurora kinase relative protein expression phosphonate-AuroraA (p-AuroraA), AuroraA, phosphonate-histone H3 (p-histone H3), histone H3, cell cycle-specific protein expression CyclinB1, cell cycle regulative protein expression Cdc2, Cdc25c, phosphonate-Cdc2 (p-Cdc2), phosphonate-Cdc25c (p-Cdc25c) and apoptosis relative protein expression PARP, Bcl-2 and Bax were detected by Western blot. The apoptotic rate was examined by flow cytometer. RESULTS: PHA739358 obviously inhibited the proliferation of T47D cells after a 24 h or 48 h treatment in a dose-dependent and time-dependent manner. Their IC50 values were (3.44 ± 0.54) and (0.21 ± 0.67) µmol/L respectively. Flow cytometry showed that G2/M arrested in a dose-dependent manner. PHA739358 dose-dependently and time-dependently promoted the dissection of PARP (poly (ADP-ribose) polymerase); down-regulated the expressions of Bcl-2, p-AuroraA, p-histone H3, CyclinB1 and up-regulated the levels of Bax, p-Cdc25c, p-Cdc2, P21 and P53 protein. PHA739358 had no significant effects on the expressions of AuroraA and histone H3. Flow cytometry and fluorescence microscope showed that PHA739358 significantly induced apoptosis. Flow cytometry showed the rate of apoptosis significantly increased from 0.31% ± 0.03% to 40.6% ± 0.81%. CONCLUSION: The proliferation-inhibiting and apoptosis-inducing effects of PHA739358 may provide new therapeutic approaches of breast cancer.

A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death.

The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 h before through 24 h after the irradiation.

[Bcl-2 inhibitor ABT-737 enhances the cisplatin-induced apoptosis in breast cancer T47D cells].

OBJECTIVE: To study the effect of ABT-737 combined with cisplatin on apoptosis of breast cancer cell line T47D cells. METHODS: T47D cells cultured in vitro was used for this experiment. Cell proliferation was measured by MTT assay. The expression of apoptosis-related protein was determined by Western blot. Morphological changes of apoptotic cells were observed by fluorescence microscopy. The apoptosis rate was examined by flow cytometry. RESULTS: The MTT assay showed that ABT-737 significantly decreased the IC(50) of cisplatin in T47D cells [(26.00 ± 1.41) µmol/L of single cisplatin vs. (13.00 ± 1.11) µmol/L of combination (ABT-737 + cisplatin)]. As a single agent, ABT-737 did not inhibit the proliferation of T47D cells, but enhanced the inhibitory effect of cisplatin in a dose-dependent manner. The detection of the cleavage of PARP showed that ABT-737 lowered the doses of cisplatin to induce apoptosis and shortened the induction time of apoptosis in T47D cells. Compared with the single use of cisplatin, the combination of ABT-737 and cisplatin accelerated the cleavage of PARP and caspase3, but did not alter the expression levels of Bcl-2, Bcl-X(L), and Bax. Both flow cytometry and fluorescence microscopy showed that ABT-737 combined with cisplatin significantly increased the apoptosis induction in T47D cells (2.3% ± 0.1 % in the control, 30.0% ± 0.8% in the cisplatin alone, and 49.0% ± 0.5% in the cisplatin + ABT-737 groups, P < 0.05). CONCLUSION: The Bcl-2 inhibitor ABT-737 can significantly enhance cisplatin-induced apoptosis in human breast cancer T47D cells in vitro.

Myeloperoxidase-dependent oxidation of etoposide in human myeloid progenitor CD34+ cells.

Etoposide is a widely used anticancer drug successfully used for the treatment of many types of cancer in children and adults. Its use, however, is associated with an increased risk of development of secondary acute myelogenous leukemia involving the mixed-lineage leukemia (MLL) gene (11q23) translocations. Previous studies demonstrated that the phenoxyl radical of etoposide can be produced by action of myeloperoxidase (MPO), an enzyme found in developing myeloid progenitor cells, the likely origin for myeloid leukemias. We hypothesized, therefore, that one-electron oxidation of etoposide by MPO to its phenoxyl radical is important for converting this anticancer drug to genotoxic and carcinogenic species in human CD34(+) myeloid progenitor cells. In the present study, using electron paramagnetic resonance spectroscopy, we provide conclusive evidence for MPO-dependent formation of etoposide phenoxyl radicals in growth factor-mobilized CD34(+) cells isolated from human umbilical cord blood and demonstrate that MPO-induced oxidation of etoposide is amplified in the presence of phenol. Formation of etoposide radicals resulted in the oxidation of endogenous thiols, thus providing evidence for etoposide-mediated MPO-catalyzed redox cycling that may play a role in enhanced etoposide genotoxicity. In separate studies, etoposide-induced DNA damage and MLL gene rearrangements were demonstrated to be dependent in part on MPO activity in CD34(+) cells. Together, our results are consistent with the idea that MPO-dependent oxidation of etoposide in human hematopoietic CD34(+) cells makes these cells especially prone to the induction of etoposide-related acute myeloid leukemia.

Peroxidase activity of hemoglobin-haptoglobin complexes: covalent aggregation and oxidative stress in plasma and macrophages.

As a hemoprotein, hemoglobin (Hb) can, in the presence of H(2)O(2), act as a peroxidase. In red blood cells, this activity is regulated by the reducing environment. For stroma-free Hb this regulation is lost, and the potential for Hb to become a peroxidase is high and further increased by inflammatory cells generating superoxide. The latter can be converted into H(2)O(2) and feed Hb peroxidase activity. Haptoglobins (Hp) bind with extracellular Hb and reportedly weaken Hb peroxidase activity. Here we demonstrate that: (i) Hb peroxidase activity is retained upon binding with Hp; (ii) in the presence of H(2)O(2), Hb-Hp peroxidase complexes undergo covalent cross-linking; (iii) peroxidase activity of Hb-Hp complexes and aggregates consumes reductants such as ascorbate and nitric oxide; (iv) cross-linked Hb-Hp aggregates are taken up by macrophages at rates exceeding those for noncovalently cross-linked Hb-Hp complexes; (v) the engulfed Hb-Hp aggregates activate superoxide production and induce intracellular oxidative stress (deplete endogenous glutathione and stimulate lipid peroxidation); (vi) Hb-Hp aggregates cause cytotoxicity to macrophages; and (vii) Hb-Hp aggregates are present in septic plasma. Overall, our data suggest that under conditions of severe inflammation and oxidative stress, peroxidase activity of Hb-Hp covalent aggregates may cause macrophage dysfunction and microvascular vasoconstriction, which are commonly seen in severe sepsis and hemolytic diseases.

Phosphatidylserine targets single-walled carbon nanotubes to professional phagocytes in vitro and in vivo.

Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid "eat-me" signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells.

Cardiolipin deficiency leads to decreased cardiolipin peroxidation and increased resistance of cells to apoptosis.

Cardiolipin (CL), a unique mitochondrial phospholipid synthesized by CL synthase (CLS), plays important, yet not fully understood, roles in mitochondria-dependent apoptosis. We manipulated CL levels in HeLa cells by knocking down CLS using RNA interference and selected a clone of CL-deficient cells with approximately 45% of its normal content. ESI-MS analysis showed that the CL molecular species were the same in CL-deficient and CL-sufficient cells. CL deficiency did not change mitochondrial functions (membrane potential, reactive oxygen species generation, cellular ATP levels) but conferred resistance to apoptosis induced by actinomycin D (ActD), rotenone, or gamma-irradiation. During ActD-induced apoptosis, decreased CL peroxidation along with suppressed cytochrome (cyt) c release was observed in CL-deficient cells, whereas Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely bound cyt c (releasable under high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is catalyzed by CL/cyt c complexes and CL oxidation products are essential for cyt c release from mitochondria, our results suggest that CL deficiency prevents adequate assembly of productive CL/cyt c complexes and CL peroxidation, resulting in increased resistance to apoptosis.