Characterizing the molecular mechanisms of acquired temozolomide resistance in the U251 glioblastoma cell line by protein microarray.

Acquired chemoresistance refers to tumor cells gradually losing their sensitivity to anticancer drugs during the course of treatment, resulting in tumor progression or recurrence. This phenomenon, which has deleterious outcomes for the patient, has long been observed in patients with glioblastoma receiving temozolomide (TMZ)-based radiochemotherapy. Currently, the mechanisms for acquired TMZ chemoresistance are not fully understood. In the present study, a TMZ-resistant cell line U251R with a 4-fold 50% inhibition concentration compared with its TMZ-sensitive parent cell line was isolated by incremental long-time TMZ treatment in the human glioblastoma cell line U251. Fluorescence-activated cell sorting analysis indicated G2/M arrest and a lower proportion of cells in the S phase, accompanied by a decreased apoptosis rate in the U251R cell line compared with the parental U251 cell line. In addition, a sphere-formation assay indicated an increased self-renewal capacity in U251R cells. Furthermore, a high-throughput protein microarray unveiled more than 200 differentially expressed proteins as potential molecular targets accounting for acquired TMZ resistance. Subsequent bioinformatics analysis illustrated the molecular and signaling networks and revealed the central role of SRC. Immunoblotting and reverse-transcription quantitative polymerase chain reaction analysis further confirmed the expressional upregulation of SRC family kinases. Moreover, SRC knockdown led to partial reversal of TMZ resistance in the U251R cell line and sensitization in the U373 cell line. These data helped to develop a comprehensive understanding of survival strategies, particularly with respect to pro-stemness regulation, which could be potential targets for overcoming TMZ resistance.

Toll-like receptor-4/p38 MAPK signaling in the dorsal horn contributes to P2X4 receptor activation and BDNF over-secretion in cancer induced bone pain.

Our previous research suggested that the P2X4 receptor (P2X4R) expression in microglia was involved in the activation of toll-like receptor-4 (TLR4) in the dorsal horn in the rat model of cancer induced bone pain (CIBP). In this study, we focused on whether TLR4- mitogen-activated protein kinases, p38 (p38 MAPK) contributes to P2X4R activation and brain-derived neurotrophic factor (BDNF) over-secretion in CIBP. In in vitro experiment, the results showed that BDNF expression evoked by ATP stimulation was dependent on TLR4-p38. In in vivo experiment, the results demonstrated that an intrathecal injection of TLR4 siRNA alleviated nociception induced by lipopolysaccharide (LPS) plus ATP or CIBP with decreased expression of P2X4R, TLR4, BDNF, interleukin-6 (IL-6) and phosphorylated-p38 MAPK (p-p38 MAPK). Moreover, injection with p38MAPK inhibitor SB203580 resulted in an identical pattern compared with treatment with TLR4 siRNA. Our results demonstrate that the activation of TLR4-p38MAPK-P2X4R signaling in microglial possibility plays an important role in the process of nociceptive transmission in CIBP, suggesting new mechanism and potential therapeutic targets for CIBP.

AKT2-knockdown suppressed viability with enhanced apoptosis, and attenuated chemoresistance to temozolomide of human glioblastoma cells in vitro and in vivo.

The AKT2 kinase (protein kinase Bß) is overexpressed in high-grade gliomas. Upregulation of the AKT2 gene has been previously observed in glioblastoma patients suffering from chemotherapy failure and tumor progress. In this study, we aimed to evaluate the effect of AKT2 on viability and chemoresistance in the human glioblastoma cell line U251. The U251 cell line was stably transfected with short hairpin RNA (shRNA) targeting AKT2. U251 cells underexpressing AKT2 were then examined for viability with temozolomide (TMZ) treatment, and tested for cell apoptosis both in vitro and in tumor-implanted mice. Next, expressions of several chemoresistance-related molecules were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analysis. The results showed that the 50% inhibitory concentration (IC50) of AKT2 shRNA-transfected cells was significantly lower compared with Lenti-GFP-transfected and nontransfected controls and that the tumor growth of the AKT2-shRNA and TMZ combined-treated mice was obviously suppressed in either mass or volume. Concomitantly, the apoptosis of TMZ-treated tumor cells was significantly enhanced after knockdown of AKT2, as measured by flow cytometry and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. Furthermore, AKT2-inhibition in TMZ-treated glioblastoma U251 cells upregulated apoptotic effector caspase-3, whereas it downregulated antiapoptotic protein Bcl-2, DNA repairing protein MGMT, and drug efflux pump protein MRP1. Our study identified AKT2 as an important gene in presenting chemoresistance in glioblastoma, and a potential target to potentiate the clinical effect of chemotherapy in glioma treatment.

MCP-1 stimulates spinal microglia via PI3K/Akt pathway in bone cancer pain.

Accumulating evidence suggests that chemokine monocyte chemoattractant protein-1 (MCP-1) is significantly involved in the activation of spinal microglia associated with pathological pain, at the same time that the phosphatidylinositol 3-kinase/Protein Kinase B (PI3K/Akt) pathway localized in spinal microglia is involved in both neuropathic and inflammatory pain. However, whether there is a connection between MCP-1 and the PI3K/Akt pathway and in their underlying mechanisms in bone cancer pain (BCP) has not yet been elucidated. In the current study, we investigated the expression changes of p-Akt in microglia and OX-42 (microglia marker) after being stimulated with MCP-1 in vitro, as well as in a BCP model that was established by an intramedullary injection of mammary gland carcinoma cells(Walker 256 cells) into the tibia of rats. We observed a significant increase in expression levels of p-Akt and OX-42 in microglia as well as in spinal dorsal horns of BCP rats. Furthermore, the intrathecal administration of an anti-MCP-1 neutralizing antibody or PI3K inhibitor LY294002 reduced the expression of p-Akt or OX-42, and LY294002 attenuated the mechanical allodynia of BCP rats. These results suggest that MCP-1 may stimulate spinal microglia via the PI3K/Akt pathway in BCP.

P2X4 receptor in the dorsal horn partially contributes to brain-derived neurotrophic factor oversecretion and toll-like receptor-4 receptor activation associated with bone cancer pain.

Previous studies have suggested that the microglial P2X7 purinoceptor is involved in the release of tumor necrosis factor-α (TNFα) following activation of toll-like receptor-4 (TLR4), which is associated with nociceptive behavior. In addition, this progress is evoked by the activation of the P2X4 purinoceptor (P2X4R). Although P2X4R is also localized within spinal microglia in the dorsal horn, little is known about its role in cancer-induced bone pain (CIBP), which is in some ways unique. With the present rat model of CIBP, we demonstrate a critical role of the microglial P2X4R in the enhanced nociceptive transmission, which is associated with TLR4 activation and secretion of brain-derived neurotrophic factor (BDNF) and TNFα in the dorsal horn. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, P2X4R small interfering RNA (siRNA) was administered intrathecally, and real-time PCR, Western blots, immunofluorescence histochemistry, and ELISA were used to detect the expression of P2X4R, TLR4, OX-42, phosphorylated-p38 MAPK (p-p38), BDNF, and TNFα. Compared with controls, intrathecal injection of P2X4R siRNA could prevent nociceptive behavior induced by ATP plus lipopolysaccharide and CIBP and reduce the expression of P2X4R, TLR4, p-p38, BDNF, and TNFα. In addition, the increase of BDNF protein in rat microglial cells depended on P2X4 receptor signaling, which is partially associated with TLR4 activation. The ability of microglial P2X4R to activate TLR4 in spinal cord leading to behavioral hypersensitivity and oversecretion of BDNF could provide an opportunity for the prevention and treatment of CIBP.

Expression of R132H mutational IDH1 in human U87 glioblastoma cells affects the SREBP1a pathway and induces cellular proliferation.

Sterol regulatory element-binding protein-1a (SREBP1a) is a member of the SREBP family of transcription factors, which mainly controls homeostasis of lipids. SREBP1a can also activate the transcription of isocitrate dehydrogenase 1 (IDH1) by binding to its promoter region. IDH1 mutations, especially R132H mutation of IDH1, are a common feature of a major subset of human gliomas. There are few data available on the relationship between mutational IDH1 expression and SREBP1a pathway. In this study, we investigated cellular effects and SREBP1a pathway alterations caused by R132H mutational IDH1 expression in U87 cells. Two glioma cell lines, stably expressing mutational (U87/R132H) or wild type (U87/wt) IDH1, were established. A cell line, stably transfected with pcDNA3.1(+) (U87/vector), was generated as a control. Click-iT EdU assay, sulforhodamine B assay, and wound healing assay respectively showed that the expression of R132H induced cellular proliferation, cell growth, and cell migration. Western blot revealed that SREBP1 was increased in U87/R132H compared with that in U87/wt. Elevated SREBP1a and several its target genes, but not SREBP1c, were detected by real-time polymerase chain reaction in U87/R132H. All these findings indicated that R132H mutational IDH1 is involved in the regulation of proliferation, growth, and migration of glioma cells. These effects may partially be mediated by SREBP1a pathway.

Activation of spinal TDAG8 and its downstream PKA signaling pathway contribute to bone cancer pain in rats.

Bone cancer pain is difficult to treat and has a strong impact on the quality of life of patients. Few therapies have emerged because the molecular mechanisms underlying bone cancer pain are poorly understood. Recently, T-cell death-associated gene 8 (TDAG8) has been shown to participate in complete Freund's adjuvant-induced chronic inflammatory pain. In this study, we aimed to examine whether TDAG8 and its downstream protein kinase A (PKA) pathway are involved in bone cancer pain. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Spinal TDAG8 expression was increased after inoculation with tumor cells. Intrathecal TDAG8 siRNA attenuated bone cancer pain behaviors during the initiation and maintenance phases; there were also concomitant decreases in TDAG8 mRNA and protein levels in spinal cord. Moreover, we found spinal PKA and phosphorylated cAMP response element-binding (pCREB) protein levels were up-regulated in the rat model of bone cancer pain. Knockdown of TDAG8 resulted in reduced bone cancer pain-induced spinal PKA and pCREB protein expression in two procedures. Furthermore, intrathecal H-89 (a PKA inhibitor) significantly attenuated bone cancer pain behaviors in rats. Our results suggest a causal relationship between TDAG8 expression and the initiation and maintenance of bone cancer pain. Activation of spinal TDAG8 contributes to bone cancer pain through the PKA signaling pathway in rats. These findings may lead to novel strategies for the treatment of bone cancer pain.

Preparation, characterization, and assessment of the antiglioma effects of liposomal celastrol.

The role of celastrol in the treatment of cancer has been an area of growing interest. To circumvent the issues of low solubility, poor bioavailability, and systemic toxicity of celastrol, we prepared liposomal celastrol using the thin-film dispersion method. We characterized particle size, encapsulation efficiency, and pharmacological parameters of liposomal celastrol. The drug concentration in plasma and tissues was measured using LC-MS/MS. In addition, the sulforhodamine B assay was used to determine the 50% inhibiting concentration. We assessed the effects of the compound in SHG-44 glioma subcutaneous xenografts in BALB/c nude mice. To compare the toxic effects of liposomal and free celastrol, the weight as well as hematologic, heart, liver, and kidney parameters were measured weekly and the morphology of organ tissues was observed pathologically. We found that liposomal celastrol had high encapsulation efficiency (71.67%) and liposomal celastrol had a higher C(max) and area under the curve, longer t(1/2), and better biodistribution than free celastrol. A cytotoxicity assay indicated that free celastrol had lower 50% inhibiting concentration values than the liposomal celastrol; however, treatment of subcutaneous xenografts with 1 mg/kg of liposomal celastrol induced greater antitumor activity than free celastrol at an equimolar concentration. In addition, a 4 mg/kg dose of liposomal celastrol had fewer severe side effects than free celastrol at the same dose. In this study, we found that the use of liposomes as a carrier of celastrol increased the bioavailability and reduced the side effects of the compound. Our findings suggest that liposomal celastrol should be further investigated in the clinical setting.

Brain-derived neurotrophic factor modulates N-methyl-D-aspartate receptor activation in a rat model of cancer-induced bone pain.

Brain-derived neurotrophic factor (BDNF) released within the spinal cord induces phosphorylation of N-methyl-D-aspartate (NMDA) receptors on the spinal cord neurons. This process is necessary for maintaining pain hypersensitivity after nerve injury. However, little is known about the role of BDNF and NMDA receptors in cancer-induced bone pain (CIBP), whose features are unique. This study demonstrates a critical role of the BDNF-modulated NMDA subunit 1 (NR1) in the induction and maintenance of behavioral hypersensitivity in a rat model of CIBP, both in the spinal cord and in the dorsal root ganglia (DRG). We selectively suppressed BDNF expression by RNA interference (RNAi) using intrathecal administration of BDNF small interfering RNA (siRNA). Then, we assessed mechanical threshold and spontaneous pain in CIBP rats. Real-time PCR, Western blotting, and fluorescent immunohistochemical staining were used to detect BDNF or NR1 both in vivo and in vitro. BDNF and phospho-NR1 were expressed under CIBP experimental conditions, with expression levels peaking at day 6 (BDNF) or 9 (NR1). Intrathecal BDNF siRNA prevented CIBP at an early stage of tumor growth (days 4-6). However, at later stages (days 10-12), intrathecal BDNF siRNA only attenuated, but did not completely block, the established CIBP. BDNF-induced NMDA receptor activation in the spinal cord or DRG leads to central sensitization and behavioral hypersensitivity. Thus, BDNF might provide a targeting opportunity for alleviating CIBP.

Minocycline-induced reduction of brain-derived neurotrophic factor expression in relation to cancer-induced bone pain in rats.

Previous studies have suggested that the release of brain-derived neurotrophic factor (BDNF) from microglia in spinal cord is necessary for maintaining pain hypersensitivity after nerve injury. However, little is known about its role in cancer-induced bone pain (CIBP), which is in some ways unique. This study demonstrates a critical role of minocycline (a potent inhibitor of microglial activation)-modulated BDNF in the induction and maintenance of behavioral hypersensitivity in a rat model of CIBP. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, minocycline was administered intrathecally from day 4 to day 6 (early stage) or from day 10 to day 12 (later stage), after carcinoma cell inoculation. Real-time PCR, Western blots, and double immunofluorescence were used to detect the expression of OX-42 (marker of activated microglia), phosphorylated p38-MAPK (p-p38), and BDNF. We found that intrathecal minocycline could prevent CIBP at an early stage of tumor growth (from day 4 to day 6). However, at the late stage (from day 10 to day 12), intrathecal minocycline had no effect. Moreover, the expression of OX-42 and BDNF under CIBP, peaking on day 6, were all reduced after minocycline injection from day 4 to day 6. The ability of minocycline-induced reduction of BDNF in the induction of behavioral hypersensitivity could provide an opportunity for alleviating CIBP.

Cancer-induced bone pain sequentially activates the ERK/MAPK pathway in different cell types in the rat spinal cord.

BACKGROUND: Previous studies have demonstrates that, after nerve injury, extracellular signal-regulated protein kinase (ERK) activation in the spinal cord-initially in neurons, then microglia, and finally astrocytes. In addition, phosphorylation of ERK (p-ERK) contributes to nociceptive responses following inflammation and/or nerve injury. However, the role of spinal cells and the ERK/MAPK pathway in cancer-induced bone pain (CIBP) remains poorly understood. The present study analyzed activation of spinal cells and the ERK/MAPK pathway in a rat model of bone cancer pain. RESULTS: A Sprague Dawley rat model of bone cancer pain was established and the model was evaluated by a series of tests. Moreover, fluorocitrate (reversible glial metabolic inhibitor) and U0126 (a MEK inhibitor) was administered intrathecally. Western blots and double immunofluorescence were used to detect the expression and location of phosphorylation of ERK (p-ERK). Our studies on pain behavior show that the time between day 6 and day 18 is a reasonable period ("time window" as the remaining stages) to investigate bone cancer pain mechanisms and to research analgesic drugs. Double-labeling immunofluorescence revealed that p-ERK was sequentially expressed in neurons, microglia, and astrocytes in the L4-5 superficial spinal cord following inoculation of Walker 256 cells. Phosphorylation of ERK (p-ERK) and the transcription factor cAMP response element-binding protein (p-CREB) increased in the spinal cord of CIBP rats, which was attenuated by intrathecal injection of fluorocitrate or U0126. CONCLUSIONS: The ERK inhibitors could have a useful role in CIBP management, because the same target is expressed in various cells at different times.

[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.

Isocitrate dehydrogenase mutations may be a protective mechanism in glioma patients.

Gliomas are the most common human brain tumours and can be classified into four grades based on clinical and pathological criteria. A recent cancer genome-sequencing project revealed that more than 70% of low-grade gliomas bear mutations in one of two NAD(+)-dependent isocitrate dehydrogenase enzymes, namely, IDH1 and IDH2. Based on the findings that glioma-derived mutations in IDH1 can inhibit the catalytic activity of the enzyme, induce HIF-1α, and can produce 2-hydroxyglutarate, two research groups speculated that the IDH mutations may contribute to the promotion of tumorigenesis in gliomas. However, they cannot fully explain the phenomenon that patients harbouring such mutations usually have better outcomes than those with the wild-type IDH genes. This fact leads us to hypothesize that the IDH mutations are not the origin of gliomas but a subsequent protective mechanism that interferes with the metabolism of the tumour cells, making these cells fragile and susceptible to cell death. This process finally helps patients who harbour such IDH mutations to survive. Therefore, contrary to the proposals of other researchers, we speculate that any interventions that correct the impaired function of the mutant IDHs, such as the use of cell-permeable α-ketoglutarate derivatives, may not cure gliomas and may even worsen the disease.

CD40 is a regulator for vascular endothelial growth factor in the tumor microenvironment of glioma.

CD40 is expressed in many tumor cells, however, its role in tumor biology is yet to be demonstrated. In the present study, we investigated the role of CD40 in gliomas. In vivo, we evaluated CD40 expression in 95 glioma tissues and 10 non-tumorous brain tissues and investigated the relationship between histopathological parameters, vascular density, and vascular endothelial growth factor (VEGF) expressions. In vitro, we aimed to understand the biological relevance of CD40 and VEGF in glioma cell lines. The results clearly demonstrated that CD40 expression, including membranous and cytoplasmic staining, was significantly higher in poorly differentiated and well differentiated gliomas than in the non-tumorous brain tissues (P=0.045 and P=0.043, respectively). In gliomas, the expression of CD40 was significantly correlated with tumor size, VEGF expressions and microvessel density (MVD) (P=0.022, P=0.023 and P=0.0316, respectively). In the in vitro study, stimulation of human glioma cells by CD40 ligation induced the expression and secretion of VEGF and was blocked by anti-CD40 monoclonal antibody. These observations provide evidence that CD40 ligation supports the expression and secretion of VEGF and may be involved in neovascularization of gliomas, they also suggest that CD40 and VEGF may be useful biomarkers for evaluating the risk of developing gliomas, and may also be used as a target for therapy.

Prourokinase mutant that induces highly effective clot lysis without interfering with hemostasis.

Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300-->His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by approximately 600 microg/kg M5 versus approximately 1200 microg/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.