XFab-α4-1BB/CD40L fusion protein activates dendritic cells, improves expansion of antigen-specific T cells, and exhibits antitumour efficacy in multiple solid tumour models.

BACKGROUND: Additional immunotherapies are still warranted for non-responders to checkpoint inhibitors with refractory or relapsing cancers, especially for patients with "cold" tumours lacking significant immune infiltration at treatment onset. We developed XFab-α4-1BB/CD40L, a bispecific antibody targeting 4-1BB and CD40 for dendritic cell activation and priming of tumour-reactive T cells to inhibit tumours. METHODS: XFab-α4-1BB/CD40L was developed by engineering an anti-4-1BB Fab arm into a CD40L trimer based on XFab® platform. Characterisation of the bispecific antibody was performed by cell-based reporter assays, maturation of dendritic cell assays, and mixed lymphocyte reactions. The abilities of antigen-specific T-cell expansion and antitumour efficacy were assessed in syngeneic mouse tumour models. Toxicological and pharmacodynamic profiles were investigated in non-human primates. RESULTS: XFab-α4-1BB/CD40L demonstrated independent CD40 agonistic activity and conditional 4-1BB activity mediated by CD40 crosslinking, leading to dendritic cell maturation and T-cell proliferation in vitro. We confirmed the expansion of antigen-specific T cells in the vaccination model and potent tumour regression induced by the bispecific antibody alone or in combination with gemcitabine in vivo, concomitant with improved tumour-reactive T-cell infiltration. XFab-α4-1BB/CD40L showed no signs of liver toxicity at doses up to 51 mg/kg in a repeated-dose regimen in non-human primates. CONCLUSIONS: XFab-α4-1BB/CD40L is capable of enhancing antitumour immunity by modulating dendritic cell and T-cell functions via targeting 4-1BB agonism to areas of CD40 expression. The focused, potent, and safe immune response induced by the bispecific antibody supports further clinical investigations for the treatment of solid tumours.

Anti-c-MET Fab-Grb2-Gab1 Fusion Protein-Mediated Interference of c-MET Signaling Pathway Induces Methuosis in Tumor Cells.

Bio-macromolecules have potential applications in cancer treatment due to their high selectivity and efficiency in hitting therapeutic targets. However, poor cell membrane permeability has limited their broad-spectrum application in cancer treatment. The current study developed highly internalizable anti-c-MET antibody Fab fusion proteins with intracellular epitope peptide chimera to achieve the dual intervention from the extracellular to intracellular targets in tumor therapy. In vitro experiments demonstrated that the fusion proteins could interfere with the disease-associated intracellular signaling pathways and inhibit the uncontrolled proliferation of tumor cells. Importantly, investigation of the underlying mechanism revealed that these protein chimeras could induce vacuolation in treated cells, thus interfering with the normal extension and arrangement of microtubules as well as the mitosis, leading to the induction of methuosis-mediated cell death. Furthermore, in vivo tumor models indicated that certain doses of fusion proteins could inhibit the A549 xenograft tumors in NOD SCID mice. This study thus provides new ideas for the intracellular delivery of bio-macromolecules and the dual intervention against tumor cell signaling pathways.

Integral Equation Prediction of the Structure of Alternating Copolymer Nanocomposites near a Substrate.

The packing structure and phase behavior of polymer-nanoparticle mixtures under confinement play an important role in developing strategies for rational design of nanomaterials. However, understanding the microscopic dispersion and aggregation mechanism of polymer nanocomposites is a great challenge through experimental techniques. In this work, the microscopic structure of alternating copolymer nanocomposites (ACNs) near a substrate is investigated systematically through extension of the inhomogeneous polymer reference interaction site model (PRISM) theory. In order to characterize the flexibility and internal chain stiffness of copolymers, a semiflexible chain model is introduced to describe the intramolecular correlations between different monomers. Based on the bridge functionals derived from the fluids density functional theory, the modified hypernetted chain closure is integrated with the PRISM equation to form a full theoretical framework to capture the density distributions of ACNs. The influence of the particle volume fraction, nanoparticle diameter, and adsorption strengths between different interaction sites on the packing structure of ACNs under confinement is analyzed and discussed in detail. With the increase of the particle volume fraction, the size asymmetry between nanoparticles and copolymer monomers can greatly influence the density profiles of ACNs near a substrate. Increasing the nanoparticle diameter, the density distribution of nanoparticles experiences a process from absorbing onto the solid surface to segregating from the wall to larger distances. With increasing the adsorption strength between copolymers and nanoparticles, the density distribution of nanoparticles decreases, which is similar to the case of nanoparticles containing attractive interactions. All these characteristics of ACNs show that the current inhomogeneous PRISM theory can give a detailed description of the packing behavior of different segments. Predictive approaches could be desired and developed for design control of alternating copolymer nanocomposites under confinement.

Phase separation of comb polymer nanocomposite melts.

In this work, the spinodal phase demixing of branched comb polymer nanocomposite (PNC) melts is systematically investigated using the polymer reference interaction site model (PRISM) theory. To verify the reliability of the present method in characterizing the phase behavior of comb PNCs, the intermolecular correlation functions of the system for nonzero particle volume fractions are compared with our molecular dynamics simulation data. After verifying the model and discussing the structure of the comb PNCs in the dilute nanoparticle limit, the interference among the side chain number, side chain length, nanoparticle-monomer size ratio and attractive interactions between the comb polymer and nanoparticles in spinodal demixing curves is analyzed and discussed in detail. The results predict two kinds of distinct phase separation behaviors. One is called classic fluid phase boundary, which is mediated by the entropic depletion attraction and contact aggregation of nanoparticles at relatively low nanoparticle-monomer attraction strength. The second demixing transition occurs at relatively high attraction strength and involves the formation of an equilibrium physical network phase with local bridging of nanoparticles. The phase boundaries are found to be sensitive to the side chain number, side chain length, nanoparticle-monomer size ratio and attractive interactions. As the side chain length is fixed, the side chain number has a large effect on the phase behavior of comb PNCs; with increasing side chain number, the miscibility window first widens and then shrinks. When the side chain number is lower than a threshold value, the phase boundaries undergo a process from enlarging the miscibility window to narrowing as side chain length increases. Once the side chain number overtakes this threshold value, the phase boundary shifts towards less miscibility. With increasing nanoparticle-monomer size ratio, a crossover of particle size occurs, above which the phase separation is consistent with that of chain PNCs. The miscibility window for this condition gradually narrows while the other parameters of the PNCs system are held constant. These results indicate that the present PRISM theory can give molecular-level details of the underlying mechanisms of the comb PNCs. It is hoped that the results can be used to provide useful guidance for the future design control of novel, thermodynamically stable comb PNCs.

Integral equation theory for atactic polystyrene nanocomposite melts with a multi-site model.

In this work, a multi-site chain model was incorporated into the polymer reference interaction site model to investigate the structure and properties of atactic polystyrene (aPS) melt and the structural correlations of dilute spherical nanoparticles dissolved in aPS melt. The theoretically calculated X-ray scattering intensities, solubility parameters and intermolecular correlation functions of aPS and its nanocomposites are found to be in agreement with the corresponding molecular simulation and experimental data. The theory was further employed to investigate the distribution functions of different size effects of aPS-nanoparticle system with consideration of the potential of mean force and depletion force. The aggregation of large nanoparticles increases with the increase of the nanoparticle-site size ratio in the infinitely dilute limit. The results show that the present theory can be used to investigate the structure of aPS melt and its nanocomposite, and give a further understanding of the filler dispersion and aggregation. All the observations indicate molecular-level details of the underlying mechanisms, providing useful information for the future design control of new aPS-nanocomposite materials with tailored properties.

Structure and effective interactions of comb polymer nanocomposite melts.

In this work, the structure and effective interactions of branched comb polymer nanocomposite (PNC) melts are investigated by using the polymer reference interaction site model (PRISM) integral equation theory. It is observed that the nanoparticle contact (bridging) aggregation is formed when the nanoparticle-monomer attraction strength is relatively weak (large) in comb PNCs. The organization states of aggregation for the moderate nanoparticle-monomer attraction strength can be well suppressed by the comb polymer architecture, while the bridging structure for relatively large attraction is obviously promoted. With the increase of the particle volume fraction, the organization states of bridging-type structure become stronger and tighter; however, this effect is weaker than that of the nanoparticle-monomer attraction strength. When the particle volume fraction and moderate nanoparticle-monomer attraction strength are fixed, the effects of degree of polymerization, side chain number, side chain length, and nanoparticle-monomer size ratio on the organization states of PNC melts are not prominent and the nanoparticles can well disperse in comb polymer. All the observations indicate that the present PRISM theory can give a detailed description of the comb PNC melts and assist in future design control of new nanomaterials.

IMT030122, A novel engineered EpCAM/CD3/4-1BB tri-specific antibody, enhances T-cell recruitment and demonstrates anti-tumor activity in mouse models of colorectal cancer.

Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.

Preclinical characterization of a Fab-like CD3/CLDN18.2 XFab® bispecific antibody against solid tumors.

The claudin 18.2(CLDN18.2) antigen is highly expressed in gastric mucosa epithelial cells and frequently expressed in malignant tumors. Positive clinical outcomes have popularized claudin 18.2 as a novel cellular and antibody therapeutic. Here, we designed a bispecific antibody-ZWB67 using the XFab® platform, aimed at redirecting CD3+ effector T cells to CLDN18.2+ target cells or tissues. Physicochemical characterization, binding properties, T cell stimulatory activity, and T cell-dependent cellular cytotoxicity of ZWB67 were evaluated in dosage intervals using antigens of CD3 and target cells expressing CLDN18.2 or CD3. Then, the anti-tumor activity was assessed in humanized CD3EDG mice bearing MC-38-hCLDN18.2 tumors. Our data demonstrate that ZWB67 specifically binds to the human CD3e antigen (KD = 1.04E-08 M) and binds more strongly to CLDN18.2+ cells than to CD3+ cells (4.3- to 9.2-fold difference). ZWB67 showed good activity in the luciferase reporter system and exhibited dose-dependent activation, cytotoxicity of T cells, and cytokine release when co-cultured with CLDN18.2+ cells and CD3+ T cells. ZWB67 also exhibited high in vivo efficacy in the MC-38-hCLDN18.2 xenograft mouse model. In conclusion, the novel anti-CLDN18.2 × anti-CD3 bispecific antibody exhibited low affinity for anti-CD3, highly specific binding, potent cytotoxicity, and anti-tumor activity. These data provide a basis for future preclinical and clinical development of this therapeutic strategy.

Proteomic profiling revealed the functional networks associated with mitotic catastrophe of HepG2 hepatoma cells induced by 6-bromine-5-hydroxy-4-methoxybenzaldehyde.

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe, such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3σ and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe.

HIV-1 Tat regulates cyclin B1 by promoting both expression and degradation.

Cyclin B1, an important cell cycle regulator, was up-regulated in lymphocytes of human immunodeficiency virus (HIV)-infected patients. However, the mechanism of cyclin B1 up-regulation and the effects of the up-regulation on the host cells remain unclear. Here, we show that HIV-encoded Tat protein regulates cyclin B1 levels in two different ways: first, Tat stimulates the transcription of cyclin B1, which increases cyclin B1 levels and promotes the cells apoptosis; and second, Tat stimulates polyubiquitination-mediated degradation of cyclin B1 through binding to the N-terminal of cyclin B1 (aa 61-129) that is just downstream of the D box, which prevents excessive levels of cyclin B1 in the cells. These results suggest that Tat-regulating cyclin B1 affects the status of HIV: Tat stimulates cyclin B1 expression to slow down the host cell cycle progress and to promote the host cell apoptosis, which might facilitate HIV release; Tat stimulates cyclin B1 degradation to prevent overaccumulation of cyclin B1, which might facilitate HIV replication. Taken together, our results reveal for the first time how HIV-Tat regulates cyclin B1 and keeps its balance in the cells.

Structure of poly(ethylene glycol)-water mixture studied by polymer reference interaction site model theory.

In this work, the polymer reference interaction site model is applied to investigate the structure of poly(ethylene glycol) (PEG) aqueous solution with the strong hydrogen-bond interactions. In the theoretical model, the renormalized technique of electrostatic potentials is combined with our recently proposed multisite semiflexible chain model to describe the inter- and intramolecular correlations. To test the model for the description of hydrogen bonding, the intermolecular correlation functions of water, ethylene glycol (EG), and EG-water binary mixture are calculated. The results are in good agreement with the corresponding simulation or experimental data. The validated model is then employed to predict the intermolecular correlation functions of different sites of the PEG and its aqueous solution. Another priority of the model is that it can obtain the corresponding direct correlation functions directly.

[Combination with SN-38 on human colon cancer LoVo cells].

OBJECTIVE: To observe the anti-proliferation effect of bevacizumab and SN-38 (active metabolite of irinotecan), and investigate the possible mechanisms of these two agents. METHODS: Human colon cancer LoVo cells were cultured under hypoxic conditions. Inhibition of cell proliferation was evaluated by MTT assay. The drug modulation on HIF-1alpha, VEGF, ERK and AKT were assessed by the following assays. The mRNA expression of HIF-1alpha and VEGF were measured by RT-PCR. The protein expression of HIF-1alpha, ERK and AKT were evaluated by Western blot analysis, and VEGF by ELISA assay. RESULTS: Among different combination schedules, Bevacizumab given after SN-38 show most synergistic anti-proliferation effect. Under hypoxic conditions, the expression of HIF-1alpha and VEGF increased as time accumulated, Bevacizumab combined with SN-38 almost completely inhibited the expression of HIF-1alpha and VEGF. Moreover, the MAP kinase pathway was involved in the drug modulation of HIF-1alpha and VEGF. CONCLUSION: These findings suggest the anti-proliferation effect of bevacizumab and SN-38 was schedule-dependent, and the synergistic effect of Bevacizumab and SN-38 was related to drug modulation of the HIF-1alpha and MAP kinase pathway.

DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein.

BACKGROUND: C-Myc is a short-lived oncoprotein that is destroyed by ubiquitin-mediated proteolysis. Dysregulated accumulation of c-Myc commonly occurs in human cancers. Some of those cases with the dysregulated c-Myc protein accumulation are attributed to gene amplification or increased mRNA expression. However, the abnormal accumulation of c-Myc protein is also a common finding in human cancers with normal copy number and transcription level of c-Myc gene. It seems that the mechanistic dysregulation in the control of c-Myc protein stabilization is another important hallmark associated with c-Myc accumulation in cancer cells. Here we report a novel mechanistic pathway through which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulates the stability of c-Myc protein. RESULTS: Firstly, siRNA-mediated silencing of DNA-PKcs strikingly downregulated c-Myc protein levels in HeLa and HepG2 cells, and simultaneously decreased cell proliferation. The c-Myc protein level in DNA-PKcs deficient human glioma M059J cells was also found much lower than that in DNA-PKcs efficient M059K cells. ATM deficiency does not affect c-Myc expression level. Silencing of DNA-PKcs in HeLa cells resulted in a decreased stability of c-Myc protein, which was associated the increasing of c-Myc phosphorylation on Thr58/Ser62 and ubiquitination level. Phosphorylation of Akt on Ser473, a substrate of DNA-PKcs was found decreased in DNA-PKcs deficient cells. As the consequence, the phosphorylation of GSK3 beta on Ser9, a negatively regulated target of Akt, was also decreased, and which led to activation of GSK 3beta and in turn phosphorylation of c-Myc on Thr58. Moreover, inhibition of GSK3 activity by LiCl or specific siRNA molecules rescued the downregulation of c-Myc mediated by silencing DNA-PKcs. Consistent with this depressed DNA-PKcs cell model, overexpressing DNA-PKcs in normal human liver L02 cells, by sub-chronically exposing to very low dose of carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), increased c-Myc protein level, the phosphorylation of Akt and GSK3 beta, as well as cell proliferation. siRNA-mediated silencing of DNA-PKcs in this cell model reversed above alterations to the original levels of L02 cells. CONCLUSION: A suitable DNA-PKcs level in cells is necessary for maintaining genomic stability, while abnormal overexpression of DNA-PKcs may contribute to cell proliferation and even oncogenic transformation by stabilizing the c-Myc oncoprotein via at least the Akt/GSK3 pathway. Our results suggest DNA-PKcs a novel biological role beyond its DNA repair function.

Improved radial distribution functions for Coulomb charged fluid based on first-order mean spherical approximation.

In this work, we solve the Ornstein-Zernike equation in a simple, analytical, and consistent manner to obtain the like and unlike radial distribution functions (RDFs) for charged fluids. To improve mean spherical approximation (MSA) solutions, the direct correlation functions both for the density and charge contributions are modified with the Yukawa potential, respectively. On the basis of the contact values of RDFs and excess internal energy of the system, we construct correlated equations to cope with the potential parameters. Thus obtained equations are solved with the first-order MSA method. The resulting like and unlike RDFs are in good agreement with molecular simulation data within a wide range of densities and temperatures.

Radioprotection of 4-hydroxy-3,5-dimethoxybenzaldehyde (VND3207) in culture cells is associated with minimizing DNA damage and activating Akt.

Vanillin is a naturally occurring compound and food-flavoring agent with antioxidant and antimutagenic activities. In present study, we explored the radioprotective effect of a novel vanillin derivative VND3207 (4-hydroxy-3,5-dimethoxybenzaldehyde). VND3207 has a much higher potential in scavenging hydroxyl radical and superoxide radical than vanillin as indicated in the ESR spin-trapping measurement, and it can effectively protect plasmid DNA against 10-50 Gy gamma-ray induced breaks in vitro at the concentrations as low as 10-20 microM. Using human lymphoblastoid AHH-1 cells and human fibroblastoid HFS cells, we demonstrated that VND3207 at 5-40 microM concentrations significantly attenuated the inhibition of proliferation and occurrence of apoptosis produced by 1-8 Gy gamma-irradiation. In the cultured cells, VND3207 significantly decreased the initial production and residual level of DNA double-strand breaks (DSBs) induced by 2 or 8 Gy irradiation. Treatment of VND3207 enhanced the level of DNA-PKcs protein, a critical component of DNA DSB repair pathway in the cells with or without gamma-irradiation. Consistently, the phosphorylation of Akt protein, a mediator of survival signal, as well as its substrate GSK3beta was concurrently increased by VND3207. Our results suggest that VND3207 has radioprotection effect through its capabilities as a powerful antioxidant, in minimizing DNA damage, and activating survival signal Akt pathway, and it may be of value in the development of radioprotective compounds.

Dose-dependent expression changes of early response genes to ionizing radiation in human lymphoblastoid cells.

The sensitivity of cancer cells as well as normal cells in response to ionizing radiation (IR) is believed to be associated with the early inducible expression of specific genes. Using cDNA microarray technology, here we explored and compared the global transcriptional changes in human lymphoblastoid AHH-1 cells irradiated with 0.05-, 0.2-, 0.5-, 2.0- and 10-Gy doses of gamma-rays 4 h after exposure. A dose as low as 0.05 Gy was efficient in inducing a transcriptional response including the up-regulation of 25 genes, some of which are involved in signal transduction pathways, e.g. BMPR2, GPR124, MAPK8IP2 and AGGF1, and the down-regulation of 18 genes. Expression of some genes was altered only at a specific dose. Most importantly, we discovered a number of radiation-response genes, e.g. DNA repair gene XPC, tumor protein p53 inducible protein 3 gene (TP53I3), immediate early response 5 gene, whose transcriptional levels were increased or depressed by IR in a dose-dependent trend within the dose range 0.05-10 Gy. The dose-dependent induced expression of TP53I3 and XPC was confirmed by Northern blot analyses. Using quantitative real-time PCR, we further confirmed that XPC gene induction was dose dependent as well as time dependent, reaching a peak 4 h post-2 Gy and 10 h post-0.05 Gy. The maximum induced expression level of the XPC gene was higher after 2 Gy (3.2-fold) than 0.05 Gy (1.93-fold). The identification of these radiation-inducible genes, especially those exhibiting a dose-dependent response, not only expands our knowledge of the mechanisms underlying the diverse biological effects induced by IR, but provides candidates for developing novel biomarkers of radiation injury.

HIV-1 Tat depresses DNA-PK(CS) expression and DNA repair, and sensitizes cells to ionizing radiation.

PURPOSE: There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. METHODS AND MATERIALS: Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and gamma-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. RESULTS: The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and gammaH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy gamma-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G(2)/M arrest as compared with parental cells. In addition, the G(2)/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. CONCLUSION: HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS cancer patients to radiotherapy.

Vanillin derivative 6-bromine-5-hydroxy-4-methoxybenzaldehyde-elicited apoptosis and G2/M arrest of Jurkat cells proceeds concurrently with DNA-PKcs cleavage and Akt inactivation.

Vanillin, a naturally occurring food component, has been reported to have anti-mutagenic and anti-metastatic potentials, and to inhibit DNA-PKcs activity. However, vanillin itself exhibits very weak antiproliferative activity. We explored the effects of bromovanin (6-bromine-5-hydroxy-4-methoxybenzaldehyde), a novel vanillin derivative, on survival and cell-cycle progression of human Jurkat leukemia cells. Treatment with >10 microM bromovanin significantly elicited apoptosis and G2/M arrest in Jurkat cells in a dose- and time-dependent manner. Bromovanin-induced DNA double-strand breaks (DSB) were demonstrated by means of comet assay as well as detection of phosphorylated H2AX, a sensitive indicator of DNA DSBs. Immuno-hybridization analysis revealed that the cleavage of procaspase-3 and DNA-PKcs occurred concurrently with bromovanin-induced apoptosis. Furthermore, phosphorylated Akt protein (Ser473), which is catalyzed by DNA-PKcs, as well as phosphorylated GSK3beta (a substrate of activated Akt), markedly decreased in bromovanin-treated Jurkat cells, suggesting that bromovanin leads to inactivation of Akt pathway via cleaving DNA-PKcs. These multiple effects, associated with the regimen of cancer therapeutic strategies, make bromovanin very appealing for future development as a novel anticancer drug.

Baicalin exerts protective effects against lipopolysaccharide-induced acute lung injury by regulating the crosstalk between the CX3CL1-CX3CR1 axis and NF-κB pathway in CX3CL1-knockout mice.

Acute lung injury (ALI) as a serious diseases with high mortality and is considered a threat to human health and life. A number of studies have focused on the treatment and prevention of lung injury. However, the molecular mechanisms responsible for the development of lung injury are not yet fully understood, and this has impeded the development of effective drugs and treatment strategies. Hence, in the present study, mice with lipopolysaccharide (LPS)­induced ALI were used as a model to investigate the crosstalk between the CX3CL1-CX3CR1 axis and the nuclear factor (NF)-κB signaling pathway in the process of lung injury. CX3CL1-knockout (CX3CL1-KO or CX3CL1-/-) mice were used to examine the role of the CX3CL1-CX3CR1 axis in LPS-induced lung injury. We used baicalin, a natural product, to investigate its anti-inflammatory effects and its protective effects against lung injury. Western blot analysis, reverse transcription-quantitavie PCR (RT-qPCR), immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and the analysis of biochemical indicators were used to determine the key signaling pathway involved in the development of lung injury. The results indicated that, on the one hand, baicalin exerted potent anti-inflammatory effects by inhibiting the activation of the CX3CL1-CX3CR1 axis and NF-κB, thus preventing the the crosstalk between the CX3CL1­CX3CR1 axis and NF-κB pathway. In addition, the phosphorylation of AKT, which was significantly induced by LPS-induced ALI through the CX3CL1-CX3CR1 axis, was inhibited by treatment with baicalin. On the other hand, we further investigated the role of the CX3CL1-CX3CR1 axis in lung injury. We determined the diffrences in the expression levels of CX3CR1 between wild-type (WT) and CX3CL1-/- mice in order to establish its association with lung injury. Our results indicated that CX3CL1 may be the central and major indicator in the process of lung injury, which mediates the CX3CR1 receptor to activate AKT and further promote NF-κB activation. These findings demonstrate that the crosstalk between the CX3CL1-CX3CR1 axis and NF-κB signaling pathway plays a direct role in LPS-induced lung injury. The inhibition of the activation of the CX3CL1-CX3CR1 axis may thus suppress the development of ALI. In addition, baicalin inhibited the crosstalk between the CX3CL1-CX3CR1 axis and NF-κB pathway in mice with LPS-induced ALI. Thus, treatment with baicalin may be a potential therapeutic strategy for ALI.

Methods for PTEN in Stem Cells and Cancer Stem Cells.

PTEN (phosphatase and tensin homologue) is the first tumor suppressor identified to have phosphatase activity and its gene is the second most frequently deleted or mutated tumor-suppressor gene associated with human cancers. Germline PTEN mutations are the cause of three inherited autosomal dominant disorders. Phosphatidylinositol 3,4,5,-triphosphate (PIP3), the product of the PI3 kinase, is one of the key intracellular targets of PTEN's phosphatase activity, although PTEN's phosphatase-independent activities have also been identified. PTEN is critical for stem cell maintenance, which contributes to its controlled tumorigenesis. PTEN loss leads the development of cancer stem cells (CSCs) that share properties with somatic stem cells, including the capacity for self-renewal and multi-lineage differentiation. Methods to isolate and functionally test stem cells and CSCs are important for understanding PTEN functions and the development of therapeutic approaches to target CSCs without having adverse effects on normal stem cells. Here, we describe protocols for the isolation and functional analysis of PTEN deficient embryonic stem cells, hematopoietic stem cells and leukemia-initiating cells (LICs), neural stem cells, and prostate stem cells and CSCs.