Development of nomograms to predict therapeutic response and prognosis of non-small cell lung cancer patients treated with anti-PD-1 antibody.

BACKGROUND: Anti-programmed death-1 (PD-1) antibody changed the treatment of non-small cell lung cancer (NSCLC), however, reliable predictive markers were lacking. We aimed to explore factors associated with response and survival, and develop predictive models. METHODS: This multicenter retrospective study included a training cohort (n = 92) and validation cohort (n = 111) with NSCLC patients received anti-PD-1 antibody monotherapy in eight Chinese hospitals, and a control cohort (n = 124) with NSCLC patients received chemotherapy. Logistic and Cox models were used to identify factors associated with response and survival respectively. Nomograms were developed based on significant factors, and evaluated by Concordance-index (C-index), area under the curve (AUC) and calibration curve. RESULT: In training cohort, smoking history (P = 0.027) and higher absolute lymphocyte count (P = 0.038) were associated with response. Female (P < 0.001), age ≥ 65 years (P = 0.004) and higher lactate dehydrogenase (LDH, P < 0.001) were associated with shorter progression-free survival (PFS). Higher LDH (P < 0.001) and derived neutrophil-to-lymphocyte ratio (P = 0.035) were associated with poorer overall survival (OS). While these factors were nonsignificant in chemotherapy cohort. Three nomograms to predict response at 6-week after treatment, PFS and OS at 6-, 12- and 18-months were developed, and validated in validation cohort. The C-indices of each nomogram in both cohorts were as follow (training vs validation): 0.706 vs 0.701; 0.728 vs 0.701; 0.741 vs 0.709; respectively. AUC showed a good discriminative ability. Calibration curves demonstrated a consistence between actual results and predictions. CONCLUSION: We developed predictive nomograms based on easily available factors to help clinicians early assess response and prognosis for NSCLC patients received anti-PD-1 antibody.

Disrupting phosphatase SHP2 in macrophages protects mice from high-fat diet-induced hepatic steatosis and insulin resistance by elevating IL-18 levels.

Chronic low-grade inflammation plays an important role in the pathogenesis of type 2 diabetes. Src homology 2 domain-containing tyrosine phosphatase-2 (SHP2) has been reported to play diverse roles in different tissues during the development of metabolic disorders. We previously reported that SHP2 inhibition in macrophages results in increased cytokine production. Here, we investigated the association between SHP2 inhibition in macrophages and the development of metabolic diseases. Unexpectedly, we found that mice with a conditional SHP2 knockout in macrophages (cSHP2-KO) have ameliorated metabolic disorders. cSHP2-KO mice fed a high-fat diet (HFD) gained less body weight and exhibited decreased hepatic steatosis, as well as improved glucose intolerance and insulin sensitivity, compared with HFD-fed WT littermates. Further experiments revealed that SHP2 deficiency leads to hyperactivation of caspase-1 and subsequent elevation of interleukin 18 (IL-18) levels, both in vivo and in vitro Of note, IL-18 neutralization and caspase-1 knockout reversed the amelioration of hepatic steatosis and insulin resistance observed in the cSHP2-KO mice. Administration of two specific SHP2 inhibitors, SHP099 and Phps1, improved HFD-induced hepatic steatosis and insulin resistance. Our findings provide detailed insights into the role of macrophagic SHP2 in metabolic disorders. We conclude that pharmacological inhibition of SHP2 may represent a therapeutic strategy for the management of type 2 diabetes.

Inhibition of AIM2 inflammasome-mediated pyroptosis by Andrographolide contributes to amelioration of radiation-induced lung inflammation and fibrosis.

Radiation-induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy, whereas no effective interventions are available. Andrographolide, an active component extracted from Andrographis paniculate, is prescribed as a treatment for upper respiratory tract infection. Here we report the potential radioprotective effect and mechanism of Andrographolide on RILI. C57BL/6 mice were exposed to 18 Gy of whole thorax irradiation, followed by intraperitoneal injection of Andrographolide every other day for 4 weeks. Andrographolide significantly ameliorated radiation-induced lung tissue damage, inflammatory cell infiltration, and pro-inflammatory cytokine release in the early phase and progressive fibrosis in the late phase. Moreover, Andrographolide markedly hampered radiation-induced activation of the AIM2 inflammasome and pyroptosis in vivo. Furthermore, bone marrow-derived macrophages (BMDMs) were exposed to 8 Gy of X-ray radiation in vitro and Andrographolide significantly inhibited AIM2 inflammasome mediated-pyroptosis in BMDMs. Mechanistically, Andrographolide effectively prevented AIM2 from translocating into the nucleus to sense DNA damage induced by radiation or chemotherapeutic agents in BMDMs. Taken together, Andrographolide ameliorates RILI by suppressing AIM2 inflammasome mediated-pyroptosis in macrophage, identifying Andrographolide as a novel potential protective agent for RILI.

Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis.

Aberrant activation of NLRP3 inflammasome has an important function in the pathogenesis of various inflammatory diseases. Although many components and mediators of inflammasome activation have been identified, how NLRP3 inflammasome is regulated to prevent excessive inflammation is unclear. Here we show NLRP3 inflammasome stimulators trigger Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) translocation to the mitochondria, to interact with and dephosphorylate adenine nucleotide translocase 1 (ANT1), a central molecule controlling mitochondrial permeability transition. This mechanism prevents collapse of mitochondrial membrane potential and the subsequent release of mitochondrial DNA and reactive oxygen species, thus preventing hyperactivation of NLRP3 inflammasome. Ablation or inhibition of SHP2 in macrophages causes intensified NLRP3 activation, overproduction of proinflammatory cytokines IL-1ß and IL-18, and increased sensitivity to peritonitis. Collectively, our data highlight that, by inhibiting ANT1 and mitochondrial dysfunction, SHP2 orchestrates an intrinsic regulatory loop to limit excessive NLRP3 inflammasome activation.

T lymphocyte SHP2-deficiency triggers anti-tumor immunity to inhibit colitis-associated cancer in mice.

Nonresolving inflammation is involved in the initiation and progression process of tumorigenesis. Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) is known to inhibit acute inflammation but its role in chronic inflammation-associated cancer remains unclear. The role of SHP2 in T cells in dextran sulfate sodium (DSS)-induced colitis and azoxymethane-DSS-induced colitis-associated carcinogenesis was examined using SHP2CD4-/- conditional knockout mice. SHP2 deficiency in T cells aggravated colitis with increased level of pro-inflammatory cytokines including IFN-γ and IL-17A. In contrast, the SHP2CD4-/- mice developed much fewer and smaller tumors than wild type mice with higher level of IFN-γ and enhanced cytotoxicity of CD8+ T cells in the tumor and peritumoral areas. At the molecular level, STAT1 was hyper-phosphorylated in T cells lacking SHP2, which may account for the increased Th1 differentiation and IFN-γ secretion. IFN-γ neutralization or IFN-γ receptor knockout but not IL-17A neutralization, abrogated the anti-tumor effect of SHP2 knockout with lowered levels of perforin 1, FasL and granzyme B. Finally, the expression of granzyme B was negatively correlated with the malignancy of colon cancer in human patients. In conclusion, these findings suggest a new strategy to treat colitis-associated cancer via targeting SHP2.

Hepatitis B virus X (HBx) play an anti-apoptosis role in hepatic progenitor cells by activating Wnt/ß-catenin pathway.

Increasing evidence has shown that normal stem cells may act as cancer-initiating cells and contribute to the development and progression of cancer. HBx has a close relationship with hepatocellular carcinoma, however, the role of HBx in hepatic progenitor cells (HPCs) is poorly understood. In this study, we sought to determine the role of HBx in regulating HPCs apoptosis and the underlying molecular mechanism(s) using HPCs derived from mouse fetal liver. The apoptotic ratio of HPCs infected with adenovirus-expressing HBx (Ad-HBx) was examined using flow cytometry. Results showed that the Ad-HBx treatment led to substantially decreased apoptotic ratio of HPCs, as confirmed by the Hoechst 33342 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Possible alterations of relative proteins were examined using Western blot and real-time PCR assays. The HBx expression in HPCs increased the expression levels of Bcl2 and Mcl1 while decreasing the expression levels of Bax and cleaved caspase-9 and -3. In addition, the mRNA and protein expression levels of ß-catenin were both increased. The ß-catenin protein were mainly accumulated in cytoplasm and tended to transfer into cell nucleus after Ad-HBx treatment. The over-expression of ß-catenin decreased the apoptotic ratio of HPCs and inhibited the expression of cleaved caspase-9 and -3 while blocking ß-catenin expression resulted in the opposite results. Taken together, our results strongly suggested that the HBx protein may inhibits apoptosis of hepatic progenitor cells, at least in part by activating the WNT/ß-catenin pathway. This provided a new insight into the molecular mechanism of HBx-mediated live carcinogenesis.

[Establishment of hepatic stem cell line stably expressing HBx protein and the effect of HBx on cell proliferation].

OBJECTIVE: To establish HP14.5 cell line stably expressing hepatitis B virus X (HBx) and detect the effect of HBx on cell proliferation, cell cycle and the wnt/ß-catenin signal pathway of hepatic progenitor cells. METHODS: The plasmid of pSEB-Flag-HBx and the packaging plasmid pAmpho were co-transfected into HEK 293T cells to construct the recombinant retrovirus carrying HBx gene. The recombinant retrovirus wan then transfected into mouse hepatic progenitor cells HP14.5. The blasticidin-resistant clones of HBx cells (HP14.5/HBx) were selected out and cultured as the experimental group, paralleled with the vector control HP14.5/Rv (HP14.5/pSEEB-Flag) and HP14.5 cell as negative control. The cell proliferation was tested by MTS assay, the cell cycle was measured by flow cytometry, the expression levels of cyclin D1 and c-myc mRNA were tested by real time PCR, and the expression leveIs of HBx, cyclin D1, c-myc, GSK3ß, p-GSK3ß (ser-9), ß-catenin proteins were examined by Western blotting. RESULTS: The expression of HBx at both mRNA and protein levels was positive in HP14.5/HBx cell line as confirmed by RT-PCR and Western blotting. Compared with the control groups, the proliferation of HP14.5/HBx cells increased significantly (P<0.05), and the expression level of cyclin D1 and c-myc mRNA rose significantly(P<0.05). G1-phase cell proportion was reduced while the proportion in S and G2 phases went up significantly (P<0.05). The expression levels of cyclin D1, c-myc, ß-catenin and p-GSK3ß (ser-9) proteins increased significantly (P<0.05)except GSK3ß (P>0.05). CONCLUSION: The hepatic stem cell line stably expressing HBx has been constructed successfully. HBx can promote the proliferation and malignant transformation of hepatic stem cells via the activation of Wnt/ß-catenin signal pathway.

[HBx induction of epithelial mesenchymal transition in mouse hepatic progenitor cells].

OBJECTIVE: To investigate the epithelial-mesenchymal transition (EMT) function of HBx in hepatic progenitor cells (HPCs). METHODS: The plasmids of pSEB-Flag-HBX and the packaging plasmids pAmpho were co-transfected in HEK 293T cells, and the recombinant retroviruses carrying HBx gene generated from the cell line were used to infect mouse HPCs HP14.5. We screened HP14.5 cells which stably expressed HBx by blasticidin to observe the morphological changes and investigate the epithelial and mesenchymal markers by real-time PCR and Western blotting. The metastatic capacity of HPCs infected with HBx was evaluated by wound-healing assay. RESULTS: HP14.5 cells with stable expression of HBx protein suffered morphological changes from polygonal to a spindle-like shape. The expressions of neural cadherin (N-cadherin), Snail and vimentin at both protein and mRNA levels significantly increased (P<0.05), but epithelial cadherin (E-cadherin) and cytokeratin-18 (CK18) decreased (P<0.05). In addition, the migratory potential was enhanced in HP14.5 cells infected with HBx. CONCLUSION: HBx can induce EMT and enhance the migratory potential of HPCs.

Parallel induction of cell proliferation and inhibition of cell differentiation in hepatic progenitor cells by hepatitis B virus X gene.

Increasing evidence has shown that normal stem cells may contribute to the development and progression of cancer by acting as cancer-initiating cells. The hepatitis B virus X (HBX) protein has been implicated in the hepatitis B virus (HBV)-associated liver carcinogenesis. However, the role of HBX in hepatic progenitor cells (HPCs) is poorly understood. In this study, we aimed to determine the role of HBX in regulating HPC proliferation and differentiation. Using MTT analysis, we showed that HPCs infected with adenovirus expressing HBX (Ad-HBX) grew more rapidly compared to HPCs infected with adenovirus expressing green fluorescent protein (Ad-GFP). To reveal the mechanism for the increased cell number after HBX treatment, we searched for possible alterations in the cell cycle and apoptosis by flow cytometry. We found that HBX treatment resulted in an increase in the S phase cell cycle fraction and a decrease in apoptosis. In addition, we examined the differentiation of HPCs infected with Ad-HBX and found that the HBX expression in HP14.5 cells led to an increased expression of early progenitor markers and a decreased expression of late hepatocyte markers. Furthermore, HBX inhibited glycogen synthesis in HP14.5 cells, indicating that HBX is capable of inhibiting terminal hepatic differentiation. Therefore, our results strongly suggest that HBX plays an important role in regulating HPC proliferation and differentiation. This is the potential mechanism of HBX-mediated liver carcinogenesis.

Tissue plasminogen activator and plasminogen activator inhibitor 1 contribute to sonic hedgehog-induced in vitro cerebral angiogenesis.

The molecular mechanisms underlying cerebral angiogenesis have not been fully investigated. Using primary mouse brain endothelial cells (MBECs) and a capillary-like tube formation assay, we investigated whether the sonic hedgehog (Shh) signaling pathway is coupled with the plasminogen/plasmin system in mediating cerebral angiogenesis. We found that incubation of MBECs with recombinant human Shh (rhShh) substantially increased the tube formation in naïve MBECs. This was associated with increases in tissue plasminogen activator (tPA) activation and reduction of plasminogen activator inhibitor 1 (PAI-1). Blockage of the Shh pathway with cyclopamine abolished the induction of tube formation and the effect of rhShh on tPA and PAI-1. Addition of PAI-1 reduced rhShh-augmented tube formation. Genetic ablation of tPA in MBECs impaired tube formation and downregulated of vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang1). Addition of rhShh to tPA-/- MBECs only partially restored the tube formation and upregulated Ang1, but not VEGF, although rhShh increased VEGF and Ang1 expression on wild-type MBECs. Complete restoration of tube formation in tPA-/- MBECs was observed only when both exogenous Shh and tPA were added. The present study provides evidence that tPA and PAI-1 contribute to Shh-induced in vitro cerebral angiogenesis.

Synthesis and biological evaluation of furoxan-based nitric oxide-releasing derivatives of glycyrrhetinic acid as anti-hepatocellular carcinoma agents.

A series of novel furoxan-based nitric oxide (NO)-releasing derivatives of glycyrrhetinic acid (GA) were designed, synthesized, and evaluated for their in vitro cytotoxicity against human hepatocellular carcinoma (HCC) and non-tumor liver cells. Five furoxan/GA hybrids, 7b-d, 7f, and 7g, displayed potent cytotoxicity against HCC cells (IC(50): 0.25-1.10 µM against BEL-7402 cells and 1.32-6.78 µM against HepG2 cells), but had a little effect on the growth of LO2 cells, indicating that these compounds had selective cytotoxicity against HCC cells. Furthermore, these compounds produced high concentrations of NO in HCC cells, but low in LO2 cells and treatment with hemoglobin partially reduced the cytotoxicity of the hybrid in HCC cells. Apparently, the high concentrations of NO produced by NO donor moieties and the bioactivity of GA synergistically contribute to the cytotoxicity, but the NO is a major player against HCC cells in vitro. Potentially, our findings may aid in the design of new chemotherapeutic reagents for the intervention of human HCC at clinic.

[Magnetic resonance imaging of ovarian carcinosarcoma: correlation to the clinicopathological findings].

OBJECTIVE: To investigate magnetic resonance imaging (MRI) characteristics of ovarian carcinosarcoma and the diagnostic value of MRI. METHODS: The MRI features of ovarian carcinosarcoma and clinical data of 5 patients with ovarian carcinosarcoma were reviewed. All the lesions were confirmed by surgery and pathological examination. RESULTS: MRI of ovarian carcinosarcoma in the 5 cases all showed large tumor mass and heterogeneous high-intensity on T2-weighted images and low-intensity on T1-weighted images, with laminar or stripe-like enhancement. Hemorrhage and necrosis were also displayed in some lesions. In two cases, the tumors invaded the greater omentum, sigmoid colon and the body of the uterus, with regional lymph node involvement. Pelvic effusion was observed in all the cases with pelvic hematocele in 1 case. CONCLUSION: MRI is useful in the detection and staging of ovarian carcinosarcoma.

Neurotrophic and growth factor gene expression profiling of mouse bone marrow stromal cells induced by ischemic brain extracts.

Treatment of rodents after stroke with bone marrow stromal cells (BMSCs) improves functional outcome. However, the mechanisms underlying this benefit have not been ascertained. This study focused on the contribution of neurotrophic and growth factors produced by BMSCs to therapeutic benefit. Rats were subjected to middle cerebral artery occlusion and the ischemic brain extract supernatant was collected to prepare the conditioned medium. The counterpart normal brain extract from non-ischemic rats was employed as the experimental control. Using microarray assay, we measured the changes of the neurotrophin associated gene expression profile in BMSCs cultured in different media. Furthermore, real-time RT-PCR and fluorescent immunocytochemistry were utilized to validate the gene changes. The morphology of BMSCs, cultured in the ischemic brain-conditioned medium for 12 h, was dramatically altered from a polygonal and flat appearance to a fibroblast-like long and thin cell appearance, compared to those in the normal brain-conditioned medium and the serum replacement medium. Forty-four neurotrophin-associated genes in BMSCs were identified by microarray assay under all three culture media. Twelve out of the 44 genes (7 neurotrophic and growth factor genes, 5 receptor genes) increased in BMSCs cultured in the ischemic brain-conditioned medium compared to the normal brain-conditioned medium. Real time RT-PCR and immunocytochemistry validated that the ischemic brain-conditioned medium significantly increased 6/7 neurotrophic and growth factor genes, compared with the normal brain-conditioned medium. These six genes consisted of fibroblast growth factor 2, insulin-like growth factor 1, vascular endothelial growth factor A, nerve growth factor beta, brain-derived neurotrophic factor and epidermal growth factor. Our results indicate that transplanted BMSCs may work as 'small molecular factories' by secreting neurotrophins, growth factors and other supportive substances after stroke, which may produce therapeutic benefits in the ischemic brain.

Axonal sprouting into the denervated spinal cord and synaptic and postsynaptic protein expression in the spinal cord after transplantation of bone marrow stromal cell in stroke rats.

We investigated whether compensatory reinnervation in the corticospinal tract (CST) and the corticorubral tract (CRT) is enhanced by the administration of bone marrow stromal cells (BMSCs) after experimental stroke. Adult male Wistar rats were subjected to permanent right middle cerebral artery occlusion (MCAo). Phosphate-buffered saline (PBS, control, n=7) or 3x10(6) BMSCs in PBS (n=8) were injected into a tail vein at 1 day postischemia. The CST of the left sensorimotor cortices was labeled with DiI 2 days prior to MCAo. Functional recovery was measured. Rats were sacrificed at 28 days after MCAo. The brain and spinal cord were removed and processed for vibratome sections for laser-scanning confocal analysis and paraffin sections for immunohistochemistry. Normal rats (n=4) exhibited a predominantly unilateral pattern of innervation of CST and CRT axons. After stroke, bilateral innervation occurred through axonal sprouting of the uninjured CRT and CST. Administration of BMSCs significantly increased the axonal restructuring on the de-afferented red nucleus and the denervated spinal motoneurons (p<0.05). BMSC treatment also significantly increased synaptic proteins in the denervated motoneurons. These results were highly correlated with improved functional outcome after stroke (r>0.81, p<0.01). We conclude that the transplantation of BMSCs enhances axonal sprouting and rewiring into the denervated spinal cord which may facilitate functional recovery after focal cerebral ischemia.

Allogeneic bone marrow stromal cells promote glial-axonal remodeling without immunologic sensitization after stroke in rats.

We evaluated the effects of allogeneic bone marrow stromal cell treatment of stroke on functional outcome, glial-axonal architecture, and immune reaction. Female Wistar rats were subjected to 2 h of middle cerebral artery occlusion. Rats were injected intravenously with PBS, male allogeneic ACI--or syngeneic Wistar--bone marrow stromal cells at 24 h after ischemia and sacrificed at 28 days. Significant functional recovery was found in both cell-treated groups compared to stroke rats that did not receive BMSCs, but no difference was detected between allogeneic and syngeneic cell-treated rats. No evidence of T cell priming or humoral antibody production to marrow stromal cells was found in recipient rats after treatment with allogeneic cells. Similar numbers of Y-chromosome+ cells were detected in the female rat brains in both groups. Significantly increased thickness of individual axons and myelin, and areas of the corpus callosum and the numbers of white matter bundles in the striatum were detected in the ischemic boundary zone of cell-treated rats compared to stroked rats. The areas of the contralateral corpus callosum significantly increased after cell treatment compared to normal rats. Processes of astrocytes remodeled from hypertrophic star-like to tadpole-like shape and oriented parallel to the ischemic regions after cell treatment. Axonal projections emanating from individual parenchymal neurons exhibited an overall orientation parallel to elongated radial processes of reactive astrocytes of the cell-treated rats. Allogeneic and syngeneic bone marrow stromal cell treatment after stroke in rats improved neurological recovery and enhanced reactive oligodendrocyte and astrocyte related axonal remodeling with no indication of immunologic sensitization in adult rat brain.

Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action.

In the present paper, we overview the discovery of new biological activities induced by ginsenoside Rg1 and Rb1 and discuss possible mechanisms of action. Both compounds could increase neural plasticity in efficacy and structure; especially Rg1, as one small molecular drug, can increase proliferation and differentiation of neural progenitor cells in dentate gyrus of hippocampus of normal adult mice and global ischemia model in gerbils. This finding has great value for treatment of Alzheimer's disease and other neurodegenerative disorders which is characterized by neurons loss. Increase of expression of brain derived neurotrophic factor, Bcl-2 and antioxidant enzyme, enhanced new synapse formation, inhibition of apoptosis and calcium overload are also important neuron protective factors. Rg1 and Rb1 have common effects, but there are some differences in pharmacology and mechanism. These differences may attribute to their different chemical structure. Rg1 is panaxtriol with two sugars, while Rb1 is panaxtriol with four sugars.

Structural analysis and optimization of NK(1) receptor antagonists through modulation of atropisomer interconversion properties.

We have previously described a series of antagonists that showed high potency and selectivity for the NK(1) receptor. However, these compounds also had the undesirable property of existing as a mixture of interconverting rotational isomers. Here we show that alteration of the 2-naphthyl substituent can modulate the rate of isomer exchange. Comparisons of the NK(1) receptor affinity for the various conformational forms has facilitated the development of a detailed NK(1) pharmacophore model.