TRAIL and Celastrol Combinational Treatment Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells via Targeting Wnt/ß-catenin Signaling Pathway.

OBJECTIVE: To investigate the mechanistic basis for the anti-proliferation and anti-invasion effect of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) and celastrol combination treatment (TCCT) in glioblastoma cells. METHODS: Cell counting kit-8 was used to detect the effects of different concentrations of celastrol (0-16 µmol/L) and TRAIL (0-500 ng/mL) on the cell viability of glioblastoma cells. U87 cells were randomly divided into 4 groups, namely control, TRAIL (TRAIL 100 ng/mL), Cel (celastrol 0.5 µmol/L) and TCCT (TRAIL 100 ng/mL+ celastrol 0.5 µmol/L). Cell proliferation, migration, and invasion were detected by colony formation, wound healing, and Transwell assays, respectively. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to assess the levels of epithelial-mesenchymal transition (EMT) markers (zona occludens, N-cadherin, vimentin, zinc finger E-box-binding homeobox, Slug, and ß-catenin). Wnt pathway was activated by lithium chloride (LiCl, 20 mol/L) and the mechanism for action of TCCT was explored. RESULTS: Celastrol and TRAIL synergistically inhibited the proliferation, migration, invasion, and EMT of U87 cells (P<0.01). TCCT up-regulated the expression of GSK-3ß and down-regulated the expression of ß-catenin and its associated proteins (P<0.05 or P<0.01), including c-Myc, Cyclin-D1, and matrix metalloproteinase (MMP)-2. In addition, LiCl, an activator of the Wnt signaling pathway, restored the inhibitory effects of TCCT on the expression of ß-catenin and its downstream genes, as well as the migration and invasion of glioblastoma cells (P<0.05 or P<0.01). CONCLUSIONS: Celastrol and TRAIL can synergistically suppress glioblastoma cell migration, invasion, and EMT, potentially through inhibition of Wnt/ß-catenin pathway. This underlies a novel mechanism of action for TCCT as an effective therapy for glioblastoma.

LWJ-M30, a conjugate of DM1 and B6, for the targeted therapy of colorectal cancer with improved therapeutic effects.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide as well as a significant cause of mortality. The conventional treatment could cause serious side effects and induce drug resistance, recurrence and metastasis of cancers. Hence, specific targeting of cancer cells without affecting the normal tissues is currently an urgent necessity in cancer therapy. The emerging of peptide-drug conjugates (PDC) is regarded as a promising approach to address malignant tumors. LWJ-M30, a conjugate of DM1 and B6 peptide, targeted transferrin receptors (TfRs) on the surface of the CRC cells, showing a powerful anti-cancer effect. LWJ-M30 significantly inhibited the HCT116 cells proliferation and migration in vitro. LWJ-M30 also dramatically decreased the level of polymeric tubulin, while the disruption of microtubules caused the cell cycle to be arrested in the G2/M phase. LWJ-M30 induced the HCT116 cells apoptosis both in vivo and in vitro. The results in vivo demonstrated that LWJ-M30 could inhibit the HCT116 growth without affecting the mouse body weight. Taking these results together, our data indicated that LWJ-M30 could improve the therapeutic effects of DM1 while reducing the systemic toxicity in normal tissues.

XHL11, a novel selective EGFR inhibitor, overcomes EGFRT790M-mediated resistance in non-small cell lung cancer.

The first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib significantly improved the therapeutic effect in non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, the EGFRT790M mutation occurs and results in acquired resistance. Consequently, mutant selective third-generation EGFR TKIs represented by AZD9291 (Osimertinib) have been developed to offer more effective therapeutic treatment, but the clinical application is limited by the acquired resistance and the high costs. A series of 5-chloropyrimidine-2,4-diamine derivatives were synthesized and screened for in vitro antitumor activity on H1975 and A431 cells. XHL11 showed the strongest antineoplastic activity. Compared to AZD9291, XHL11 suppressed cellular proliferation and colony formation and induced apoptosis in H1975 cells with EGFRL858R/T790M mutation. In addition, XHL11 caused expression changes in EGFR and apoptosis-related pathways. Moreover, oral administration of XHL11 suppressed tumor progression in vivo in a H1975 subcutaneous xenograft model. These data demonstrated that XHL11 might be developed as a promising EGFR TKI for the therapeutic use of NSCLC patients.

Poor preoperative sleep quality is a risk factor for severe postoperative pain after breast cancer surgery: A prospective cohort study.

The aim of this study was to assess the effect of preoperative sleep quality on acute postoperative pain in breast cancer patients.The Pittsburgh Sleep Quality Index questionnaire (PSQI) was used to assess the overall sleep status of women scheduled for unilateral modified radical mastectomy in the past month. Based on the responses, patients were allocated to good sleep group or poor sleep group. Postoperatively, acute pain was assessed using the numerical rating score in the first 24 hours; in addition, the requirement of analgesics and the incidence of postoperative complications were recorded.A total of 108 breast surgery patients were enrolled. Based on the PSQI results, 55 (51%) patients were allocated to poor sleep group and 53 (49%) to good sleep group. Pain scores were similar in the 2 groups at the end of surgery (P = .589); however, poor sleep group reported higher postoperative pain scores than the good sleep group at 2 (P = .002), 6 (P < .001), 12 (P < .001), and 24 (P = .002) hours after surgery. The incidence of severe pain in the poor sleep group was higher than that in the good sleep group (27% vs 8%, P = .018), and the ratio of participants who required rescued analgesics was greater in the poor sleep group (52% vs 22%, P = .002). In addition, patients with poor sleep quality had more postoperative complications and longer hospital stay.In this study, breast cancer patients with poor preoperative sleep quality reported more severe postoperative pain, required more analgesics, experienced more complications, and had longer hospital stay.

The histone demethylase Jmjd3 regulates zebrafish myeloid development by promoting spi1 expression.

The histone demethylase Jmjd3 plays a critical role in cell lineage specification and differentiation at various stages of development. However, its function during normal myeloid development remains poorly understood. Here, we carried out a systematic in vivo screen of epigenetic factors for their function in hematopoiesis and identified Jmjd3 as a new epigenetic factor that regulates myelopoiesis in zebrafish. We demonstrated that jmjd3 was essential for zebrafish primitive and definitive myelopoiesis, knockdown of jmjd3 suppressed the myeloid commitment and enhanced the erythroid commitment. Only overexpression of spi1 but not the other myeloid regulators rescued the myeloid development in jmjd3 morphants. Furthermore, preliminary mechanistic studies demonstrated that Jmjd3 could directly bind to the spi1 regulatory region to alleviate the repressive H3K27me3 modification and activate spi1 expression. Thus, our studies highlight that Jmjd3 is indispensable for early zebrafish myeloid development by promoting spi1 expression.

The effect of intraperitoneal chemotherapy on early pain hyperalgesia in patients following elective laparoscopic transabdominal resection of rectal cancer.

BACKGROUND: Chemotherapy has been associated with hyperalgesia. This prospective study was designed to investigate the effect of intraperitoneal chemotherapy with lobaplatin on post-operative pain intensity and sufentanil requirements after laparoscopic transabdominal resection of rectal cancer. METHODS: Eighty subjects (40 subjects treated with intraperitoneal chemotherapy and 40 subjects without chemotherapy treatment) scheduled for laparoscopic transabdominal resection of rectal cancer were included in this study. All subjects received standardized anesthetic and patient-controlled analgesia using sufentanil for 72 h post-surgery, as the only analgesics. Pain intensity scores, cumulative sufentanil requirements and side effects were recorded until 72 h post-surgery. RESULTS: Following intraperitoneal chemotherapy, patients had a significantly higher total post-operative sufentanil requirement (193 µg vs. 142 µg; P = 0.008), significantly higher verbal rating scale post-surgery pain intensity scores at rest and with coughing (P < 0.05), and a significantly worse functional activity score (P < 0.05) over 72 h, compared with those without intraperitoneal chemotherapy. There were no post-operative differences in the incidence of side-effects (post-operative nausea [P = 0.189], vomiting [P = 0.311], pruritus [P = 0.263], respiratory depression [P = 1.000], and dizziness [P = 0.712]) between the two groups. CONCLUSION: Intraperitoneal chemotherapy is associated with significantly increased post-operative sufentanil requirements and pain intensity, suggesting chemotherapy-associated hyperalgesia.

Monocyte chemoattractant protein-1 contributes to morphine tolerance in rats with cancer-induced bone pain.

Cancer-induced bone pain can severely compromise the life quality of patients, while tolerance limits the use of opioids in the treatment of cancer pain. Monocyte chemoattractant protein-1 (MCP-1) is known to contribute to neuropathic pain. However, the role of spinal MCP-1 in the development of morphine tolerance in patients with cancer-induced bone pain remains unclear. The aim of the present study was to investigate the role of spinal MCP-1 in morphine tolerance in bone cancer pain rats (MTBP rats). Bone cancer pain was induced by intramedullary injection of Walker 256 cells into the tibia of the rats, while morphine tolerance was induced by continuous intrathecal injection of morphine over a period of 9 days. In addition, anti-MCP-1 antibodies were intrathecally injected to rats in various groups in order to investigate the association of MCP-1 with mechanical and heat hyperalgesia using the paw withdrawal threshold (PWT) and thermal withdrawal latency (TWL) tests, respectively. Furthermore, MCP-1 and CCR2 expression levels were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, and CCR2 expression levels were measured using RT-qPCR. The results indicated that MCP-1 and CCR2 expression levels were significantly increased in the spinal cord of MTBP rats. Intrathecal administration of anti-MCP-1 neutralizing antibodies was observed to attenuate the mechanical and thermal allodynia in MTBP rats. Therefore, the upregulation of spinal MCP-1 and CCR2 expression levels may contribute to the development of mechanical allodynia in MTBP rats. In conclusion, MCP-1/CCR2 signaling may serve a crucial role in morphine tolerance development in rats suffering from cancer-induced bone pain.

Pten regulates homeostasis and inflammation-induced migration of myelocytes in zebrafish.

BACKGROUND: Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) is frequently observed in hematopoietic malignancies. Although PTEN has been implicated in maintaining the quiescence of hematopoietic stem cells (HSCs), its role in hematopoiesis during ontogeny remains largely unexplored. METHODS: The expression of hematopoietic marker genes was analyzed via whole mount in situ hybridization assay in ptena and ptenb double mutant zebrafish. The embryonic myelopoiesis was characterized by living imaging and whole mount in situ immunofluorescence with confocal microscopy, as well as cell-specific chemical staining for neutrophils and macrophages. Analyses of the involved signaling pathway were carried out by inhibitor treatment and mRNA injection. RESULTS: Pten-deficient zebrafish embryos exhibited a strikingly increased number of myeloid cells, which were further characterized as being immune deficient. In accordance with this finding, the inhibition of phosphoinositide 3-kinase (PI3K) or the mechanistic target of rapamycin (mTOR) corrected the expansive myelopoiesis in the pten-deficient embryos. Further mechanistic studies revealed that the expression of cebpa, a critical transcription factor in myeloid precursor cells, was downregulated in the pten-deficient myeloid cells, whereas the injection of cebpa mRNA markedly ameliorated the dysmyelopoiesis induced by the loss of pten. CONCLUSIONS: Our data provide in vivo evidence that definitive myelopoiesis in zebrafish is critically regulated by pten via the elevation of cebpa expression.

Activated N-Ras signaling regulates arterial-venous specification in zebrafish.

BACKGROUND: The aberrant activation of Ras signaling is associated with human diseases including hematological malignancies and vascular disorders. So far the pathological roles of activated Ras signaling in hematopoiesis and vasculogenesis are largely unknown. METHODS: A conditional Cre/loxP transgenic strategy was used to mediate the specific expression of a constitutively active form of human N-Ras in zebrafish endothelial and hematopoietic cells driven by the zebrafish lmo2 promoter. The expression of hematopoietic and endothelial marker genes was analyzed both via whole mount in situ hybridization (WISH) assay and real-time quantitative PCR (qPCR). The embryonic vascular morphogenesis was characterized both by living imaging and immunofluorescence on the sections with a confocal microscopy, and the number of endothelial cells in the embryos was quantified by flow cytometry. The functional analyses of the blood circulation were carried out by fluorescence microangiography assay and morpholino injection. RESULTS: In the activated N-Ras transgenic embryos, the primitive hematopoiesis appeared normal, however, the definitive hematopoiesis of these embryos was completely absent. Further analysis of endothelial cell markers confirmed that transcription of arterial marker ephrinB2 was significantly decreased and expression of venous marker flt4 excessively increased, indicating the activated N-Ras signaling promotes the venous development at the expense of arteriogenesis during zebrafish embryogenesis. The activated N-Ras-expressing embryos showed atrophic axial arteries and expansive axial veins, leading to no definitive hematopoietic stem cell formation, the blood circulation failure and subsequently embryonic lethality. CONCLUSIONS: Our studies revealed for the first time that activated N-Ras signaling during the endothelial differentiation in vertebrates can disrupt the balance of arterial-venous specification, thus providing new insights into the pathogenesis of the congenital human vascular disease and tumorigenic angiogenesis.

Cannabinoid receptor 2 suppresses leukocyte inflammatory migration by modulating the JNK/c-Jun/Alox5 pathway.

BACKGROUND: The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. RESULTS: Cnr2 activation down-regulates 5-lipoxygenase (Alox5) expression by suppressing the JNK/c-Jun activation. CONCLUSION: The Cnr2-JNK-Alox5 axis modulates leukocyte inflammatory migration. SIGNIFICANCE: Linking two important regulators in leukocyte inflammatory migration and providing a potential therapeutic strategy for treating human inflammation-associated diseases. Inflammatory migration of immune cells is involved in many human diseases. Identification of molecular pathways and modulators controlling inflammatory migration could lead to therapeutic strategies for treating human inflammation-associated diseases. The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. Through a chemical genetic screen using a zebrafish model for leukocyte migration, we found that both an agonist of the Cnr2 and inhibitor of the 5-lipoxygenase (Alox5, encoded by alox5) inhibit leukocyte migration in response to acute injury. These agents have a similar effect on migration of human myeloid cells. Consistent with these results, we found that inactivation of Cnr2 by zinc finger nuclease-mediated mutagenesis enhances leukocyte migration, while inactivation of Alox5 blocks leukocyte migration. Further investigation indicates that there is a signaling link between Cnr2 and Alox5 and that alox5 is a target of c-Jun. Cnr2 activation down-regulates alox5 expression by suppressing the JNK/c-Jun activation. These studies demonstrate that Cnr2, JNK, and Alox5 constitute a pathway regulating leukocyte migration. The cooperative effect between the Cnr2 agonist and Alox5 inhibitor also provides a potential therapeutic strategy for treating human inflammation-associated diseases.

[The role of brain-derived neurotrophic factor in pain facilitation and spinal mechanism in rat model of bone cancer pain].

OBJECTIVE: To investigate the role of brain-derived neurotrophic factor (BDNF) in pain facilitation and spinal mechanisms in the rat model of bone cancer pain. METHODS: The bone cancer pain model was developed by inoculated Walker 256 mammary gland carcinoma cells into the tibia medullary cavity. Sixty SD female rats were divided into 5 groups (n = 12 each) randomly; group I: control group (sham operation); group II: model group; group III: control group + anti-BDNF intrathecal (i.t.); group IV: model group + control IgG i.t.; group V: model group + anti-BDNF i.t.. Anti-BDNF or control IgG was injected i.t. during 7 to 9th day. Von-Frey threshold was measured one day before operation and every 2 days after operation. On the 9th day after threshold tested, rats were sacrificed after i.t. injection of either anti-BDNF or control IgG, the lumbar 4-6 spinal cord was removed. The expression of the spinal BDNF and the phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) were detected by immunohistochemistry assay and Western-Blot. Co-expression pattern of BDNF and p-ERK1/2 were determined by double-labeling immunofluorescence. RESULTS: We demonstrated the coexistence of BDNF and p-ERK1/2 in the spinal cord of rats. From the 7 to 9th day after operation, von-Frey threshold in groups II and IV was significantly lower than that in group I and group V (P < 0.01), group V was remarkly higher than that in group IV (P < 0.01). The spinal BDNF and p-ERK1/2 expression in group II or IV were significantly increased compared with that in group I or V (P < 0.01), intrathecal anti-BDNF was significantly suppressed BDNF and p-ERK1/2 expression (P < 0.01). CONCLUSION: BDNF and p-ERK1/2 was coexistence in the spinal cord of rats, and it maybe involved in the bone cancer pain.

An evolutionarily conserved PTEN-C/EBPalpha-CTNNA1 axis controls myeloid development and transformation.

Loss of function of tumor suppressor genes, such as PTEN, CEBPAlpha, and CTNNA1 (encoding the alpha-catenin protein), has been found to play an essential role in leukemogenesis. However, whether these genes genetically interact remains largely unknown. Here, we show that PTEN-mammalian target of rapamycin signaling acts upstream to dictate the ratio of wild-type p42 C/EBPalpha to its dominant-negative p30 isoform, which critically determines whether p30 C/EBPalpha (lower p42/p30 ratio) or p42 C/EBPalpha (higher p42/p30 ratio) binds to the proximal promoter of the retained CTNNA1 allele. Binding of p30 C/EBPalpha recruits the polycomb repressive complex 2 to suppress CTNNA1 transcription through repressive H3K27me3 modification, whereas binding of p42 C/EBPalpha relieves this repression and promotes CTNNA1 expression through activating H3K4me3 modification. Loss of Pten function in mice and zebrafish induces myelodysplasia with abnormal invasiveness of myeloid progenitors accompanied by significant reductions in both wild-type C/EBPalpha and alpha-catenin protein. Importantly, frame-shift mutations in either PTEN or CEBPA were detected exclusively in the primary LICs with low CTNNA1 expression. This study uncovers a novel molecular pathway, PTEN-C/EBPalpha-CTNNA1, which is evolutionarily conserved and might be therapeutically targeted to eradicate LICs with low CTNNA1 expression.

Functional characterization of Lmo2-Cre transgenic zebrafish.

Cre/loxP system is a powerful tool to manipulate the genome. Transgenic animals expressing Cre recombinase in specific tissues or cells have been widely used for conditional gene targeting, lineage tracing, and other genetic analyses. In zebrafish, the transgenic line with stable expression of Cre in specific tissues and cell subtypes has not been generated and its functional activity remains to be defined. Here we report the establishment of a stable transgenic fish Tg(zlmo2:Cre), which specifically expresses Cre in the primitive hematopoietic progenitors and vascular endothelial cells, under the control of lmo2 promoter. Our result shows that the Cre expression pattern recapitulates the endogenous lmo2 expression pattern during embryogenesis. Crossing of the Tg(zlmo2:Cre) line with another established transgenic reporter line Tg(zlmo2:loxP-DsRed-loxP-EGFP), induces a robust recombination activity in hematopoietic progenitors and vascular endothelial cells. Thus, the Tg(zlmo2:Cre) transgenic line provides an invaluable tool to dissect genetic pathways in hematopoietic development and diseases.