PK 11195 differentially affects cell survival in human wild-type and 18 kDa translocator protein-silenced ADF astrocytoma cells

Gliomas are the most common brain tumours with a poor prognosis due to their aggressiveness and propensity for recurrence. The 18 kDa translocator protein (TSPO) has been demonstrated to be greatly expressed in glioma cells and its over-expression has been correlated with glioma malignance grades. Due to both its high density in tumours and the pro-apoptotic activity of its ligands, TSPO has been suggested as a promising target in gliomas. With the aim to evidence if the TSPO expression level alters glioma cell susceptibility to undergo to cell death, we analysed the effects of the specific TSPO ligand, PK 11195, in human astrocytoma wild-type and TSPO-silenced cell lines. As first step, TSPO was characterised in human astrocytoma cell line (ADF). Our data demonstrated the presence of a single class of TSPO binding sites highly expressed in mitochondria. PK 11195 cell treatment activated an autophagic pathway followed by apoptosis mediated by the modulation of the mitochondrial permeability transition. In TSPO-silenced cells, produced by siRNA technique, a reduced cell proliferation rate and a decreased cell susceptibility to the PK 11195-induced anti-proliferative effect and mitochondrial potential dissipation were demonstrated respect to control cells. In conclusion, for the first time, PK 11195 was demonstrated to differentially affect glioma cell survival in relation to TSPO expression levels. These results encourage the development of specific-cell strategies for the treatment of gliomas, in which TSPO is highly expressed respect to normal cells.     

The 18-kDa Translocator Protein (TSPO) Disrupts Mammary Epithelial Morphogenesis and Promotes Breast Cancer Cell Migration

Mitochondria play important roles in cancer progression and have emerged as viable targets for cancer therapy. Increasing levels of the outer mitochondrial membrane protein, 18-kDa translocator protein (TSPO), are associated with advancing breast cancer stage. In particular, higher TSPO levels are found in estrogen receptor (ER)-negative breast tumors, compared with ER-positive tumors. In this study, we sought to define the roles of TSPO in the acquisition of breast cancer malignancy. Using a three-dimensional Matrigel culture system, we determined the impact of elevated TSPO levels on mammary epithelial morphogenesis. Our studies demonstrate that stable overexpression of TSPO in mammary epithelial MCF10A acini drives proliferation and provides partial resistance to luminal apoptosis, resulting in enlarged acinar structures with partially filled lumen that resemble early stage breast lesions leading to breast cancer. In breast cancer cell lines, TSPO silencing or TSPO overexpression significantly altered the migratory activity. In addition, we found that combination treatment with the TSPO ligands (PK 11195 or Ro5-4864) and lonidamine, a clinical phase II drug targeting mitochondria, decreased viability of ER-negative breast cancer cell lines. Taken together, these data demonstrate that increases in TSPO levels at different stages of breast cancer progression results in the acquisition of distinct properties associated with malignancy. Furthermore, targeting TSPO, particularly in combination with other mitochondria-targeting agents, may prove useful for the treatment of ER-negative breast cancer.     

Evidence for complex binding profiles and species differences at the translocator protein (TSPO) (18 kDa)

The translocator protein (TSPO) (18 kDa) is an emerging drug target for the treatment of numerous pathologies including cancer and neurodegenerative disease. However, our limited knowledge of TSPO binding site(s) has hindered the development of TSPO ligands with potential therapeutic effects. We have synthesized a series of pyrrolobenzoxazepines (1-10) to better characterize the interaction of ligands with the TSPO across species, and to determine their functional profiles. All ligands 1-10 displaced the binding of [3H]PK 11195 to the TSPO at nanomolar concentrations, with discrepancies in binding affinity between rat and human TSPO. Interestingly, non-linear regression analysis revealed that some ligands bound to the protein with a Hill slope not equal to 1.0, suggesting possible additional TSPO binding sites with allosteric effects. However, this trend was not conserved between rat and human. When tested for their effects on pregnenolone production in rat C6 glioma cells, nitric oxide release in murine microglia, and cell proliferation in human MCF-7 breast cancer cells, the pyrrolobenzoxazepines (40 μM) displayed functional effects which did not correlate to the binding trend observed in competition assays. We propose that consideration of species differences and binding site cooperativity, plus optimization of currently accepted functional assays, will aid in the development of drugs targeting TSPO that can be used as therapeutics for human disease.     

Translocator Protein-Mediated Stabilization of Mitochondrial Architecture during Inflammation Stress in Colonic Cells

Chronic inflammation of the gastrointestinal tract increasing the risk of cancer has been described to be linked to the high expression of the mitochondrial translocator protein (18 kDa; TSPO). Accordingly, TSPO drug ligands have been shown to regulate cytokine production and to improve tissue reconstruction. We used HT-29 human colon carcinoma cells to evaluate the role of TSPO and its drug ligands in tumor necrosis factor (TNF)-induced inflammation. TNF-induced interleukin (IL)-8 expression, coupled to reactive oxygen species (ROS) production, was followed by TSPO overexpression. TNF also destabilized mitochondrial ultrastructure, inducing cell death by apoptosis. Treatment with the TSPO drug ligand PK 11195 maintained the mitochondrial ultrastructure, reducing IL-8 and ROS production and cell death. TSPO silencing and overexpression studies demonstrated that the presence of TSPO is essential to control IL-8 and ROS production, so as to maintain mitochondrial ultrastructure and to prevent cell death. Taken together, our data indicate that inflammation results in the disruption of mitochondrial complexes containing TSPO, leading to cell death and epithelia disruption. Significance: This work implicates TSPO in the maintenance of mitochondrial membrane integrity and in the control of mitochondrial ROS production, ultimately favoring tissue regeneration.     

4'-Acetoamido-4-hydroxychalcone, a chalcone derivative, inhibits glioma growth and invasion through regulation of the tropomyosin 1 gene

Chalcones are precursors of flavonoids and have been shown to have anti-cancer activity. Here, we identify the synthetic chalcone derivative 4′-acetoamido-4-hydroxychalcone (AHC) as a potential therapeutic agent for the treatment of glioma. Treatment with AHC reduced glioma cell invasion, migration, and colony formation in a concentration-dependent manner. In addition, AHC inhibited vascular endothelial growth factor-induced migration, invasion, and tube formation in HUVECs. To determine the mechanism underlying the inhibitory effect of AHC on glioma cell invasion and migration, we investigated the effect of AHC on the gene expression change and found that AHC affects actin dynamics in U87MG glioma cells. In actin cytoskeleton regulating system, AHC increased tropomyosin expression and stress fiber formation, probably through activation of PKA. Suppression of tropomyosin expression by siRNA or treatment with the PKA inhibitor H89 reduced the inhibitory effects of AHC on glioma cell invasion and migration. In vivo experiments also showed that AHC inhibited tumor growth in a xenograft mouse tumor model. Together, these data suggest that the synthetic chalcone derivative AHC has potent anti-cancer activity through inhibition of glioma proliferation, invasion, and angiogenesis and is therefore a potential chemotherapeutic candidate for the treatment of glioma.     

Nordy, a synthetic lipoxygenase inhibitor, inhibits the expression of formylpeptide receptor and induces differentiation of malignant glioma cells

We recently found that formylpeptide receptor (FPR), a G-protein-coupled receptor that mediates chemotaxis of phagocytic leukocytes induced by bacterial peptide N-formyl-methionyl-leucyl-phenylalanine, is expressed by malignant human glioma cells and promotes tumor growth and angiogenesis. In this study, we examined the effect of Nordy, a novel chiral lipoxygenase inhibitor which was synthesized based on the structure of a natural nordihydroguaiaretic acid, on the expression of FPR by human glioblastoma cells. We found that FPR was expressed at the protein level by highly malignant human glioma cell lines U87 and BT325, and a rat glioma cell line C6. The expression level of FPR was correlated with the degree of the malignancy of tumor cells. The poorly differentiated glioma cell line U87 expressed the highest level of FPR. In U87 glioma cells, the expression of FPR was attenuated at the protein level by Nordy treatment for 48 (P<0.05). Nordy did not affect FPR mRNA expression in U87 cells. In addition, Nordy treatment seemed to promote glioma cell differentiation, as evidenced by their reduced expression of vimentin and increased expression of GFAP. Our results suggest that Nordy was capable of reducing the level of malignancy of glioma cells.     

Increased expression of platelet-derived growth factor associated protein-1 is associated with PDGF-B mediated glioma progression

The current treatment therapies available for malignant gliomas are inadequate. There is an urgent need to develop more effective therapies by characterizing the molecular pathogenesis of the disease. Over expression of platelet-derived growth factor (PDGF) ligands and receptors have been reported in malignant gliomas. Platelet-derived growth factor associated protein-1 (PDAP-1) is reported to modulate the mitogenic activity of PDGF ligands, but to date, there is no information concerning its role in PDGF-mediated glioma cell proliferation. This study aimed to characterize the role of PDAP-1 in PDGF-mediated glioma proliferation. The expression of PDAP-1 was observed to be significantly increased (p< 0.05) in grade IV glioma tissue and cell lines compared to grade III. siRNA-mediated knockdown of PDAP-1 reduced the expression of PDGF-B and its downstream genes (Akt1/Protein kinase B (PKB) and phosphoinositide-dependent kinase-1 (PDK1) by up to 50%. In PDAP-1 knockdown glioma cells, more than a twofold reduction was also observed in the level of phosphorylated Akt. Interestingly, knockdown of PDAP-1 in combination with PDGF-B antibody inhibited glioma cell proliferation through activation of Caspase 3/7 and 9. We also demonstrate that PDAP-1 co-localizes with PDGF-B in the cytoplasm of glioma cells, and an interaction between both of the proteins was established. Collectively, these findings suggest that the expression of PDAP-1 is associated with disease malignancy, and its inhibition reduced the proliferation of malignant glioma cells through down-regulation of PDGF-B/Akt/PDK1 signaling. Thus, this study establishes PDAP-1 as an effecter of PDGF signaling in glioma cells and suggests that it could also be a promising therapeutic target.     

Clinical and biological significance of Cdk10 in hepatocellular carcinoma

Cyclin-dependent kinase 10 (Cdk10) is a Cdc2-related kinase and plays an essential role in the progression from the G2 to M phase of the cell cycle. However, relative little is known about its expression pattern, clinical relevance, and biological function in hepatocellular carcinoma (HCC). In the present study, we investigated the mRNA and protein expression levels of Cdk10 in 127 pairs of HCC samples and adjacent nontumorous liver tissues and evaluated its clinical significance. Additionally, we assessed the effects of restoration of Cdk10 on cell proliferation and drug sensitivity in HCC cells. We showed that the Cdk10 mRNA and protein expression was markedly decreased in HCC samples compared to adjacent nontumorous liver tissues. Quantitative real-time polymerase chain reaction and immunohistochemical studies revealed that reduced Cdk10 expression was significantly associated with alpha-fetoprotein levels, tumor size, and tumor stage. Ectopic expression of Cdk10 reduced HCC cell proliferation, blocked the cell cycle at the G0-G1 phase, as well as inhibited cell migration and anchorage-independent growth. Additionally, Cdk10 overexpression enhanced the chemosensitivity of HCC cells to cisplatin and epidoxorubicin, two chemotherapeutic agents commonly used in HCC. These data collectively demonstrate that reduced Cdk10 expression is closely linked to HCC development and progression. Restoration of its expression may have therapeutic benefits in treating this malignancy.     

Silencing of the MEG3 gene promoted anti-cancer activity and drug sensitivity in glioma

Aberrant expression of MEG3 has been shown in various cancers. The purpose of this study is to evaluate the effect of MEG3 on glioma cells and the use of potential chemotherapeutics in glioma by modulating MEG3 expression. Cell viability, migration and chemosensitivity were assayed. Cell death was evaluated in MEG3 overexpressing and MEG3 suppressed cells. MEG3 expression was compared in patient-derived glioma cells concerning IDH1 mutation and WHO grades. Silencing of MEG3 inhibited cell proliferation and reduced cell migration while overexpression of MEG3 promoted proliferation in glioma cells. MEG3 inhibition improved the chemosensitivity of glioma cells to 5-fluorouracil (5FU) but not to navitoclax. On the other hand, there is no significant effect of MEG3 expression on temozolamide (TMZ) treatment which is a standard chemotherapeutic agent in glioma. Suppression of the MEG3 gene in patient-derived oligodendroglioma cells also showed the same effect whereas glioblastoma cell proliferation and chemosensitivity were not affected by MEG3 inhibition. Further, as a possible cell death mechanism of action apoptosis was investigated. Although MEG3 is a widely known tumour suppressor gene and its loss is associated with several cancer types, here we reported that MEG3 inhibition can be used for improving the efficiency of known chemotherapeutic drug sensitivity. We propose that the level of MEG3 should be evaluated in the treatment of different glioma subtypes that are resistant to effective drugs to increase the potential effective drug applications.     

Preclinical TSPO Ligand PET to Visualize Human Glioma Xenotransplants: A Preliminary Study

Current positron emission tomography (PET) imaging biomarkers for detection of infiltrating gliomas are limited. Translocator protein (TSPO) is a novel and promising biomarker for glioma PET imaging. To validate TSPO as a potential target for molecular imaging of glioma, TSPO expression was assayed in a tumor microarray containing 37 high-grade (III, IV) gliomas. TSPO staining was detected in all tumor specimens. Subsequently, PET imaging was performed with an aryloxyanilide-based TSPO ligand, [¹⁸F]PBR06, in primary orthotopic xenograft models of WHO grade III and IV gliomas. Selective uptake of [¹⁸F]PBR06 in engrafted tumor was measured. Furthermore, PET imaging with [¹⁸F]PBR06 demonstrated infiltrative glioma growth that was undetectable by traditional magnetic resonance imaging (MRI). Preliminary PET with [¹⁸F]PBR06 demonstrated a preferential tumor-to-normal background ratio in comparison to 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG). These results suggest that TSPO PET imaging with such high-affinity radiotracers may represent a novel strategy to characterize distinct molecular features of glioma growth, as well as better define the extent of glioma infiltration for therapeutic purposes.     

microRNA-34a is tumor suppressive in brain tumors and glioma stem cells

We recently found that microRNA-34a (miR-34a) is downregulated in human glioma tumors as compared to normal brain, and that miR-34a levels in mutant-p53 gliomas were lower than in wildtype-p53 tumors. We showed that miR-34a expression in glioma and medulloblastoma cells inhibits cell proliferation, G1/S cell cycle progression, cell survival, cell migration and cell invasion, but that miR-34a expression in human astrocytes does not affect cell survival and cell cycle. We uncovered the oncogenes c-Met, Notch-1 and Notch-2 as direct targets of miR-34a that are inhibited by miR-34a transfection. We found that c-Met levels in human glioma specimens inversely correlate with miR-34a levels. We showed that c-Met and Notch partially mediate the inhibitory effects of miR-34a on cell proliferation and cell death. We also found that mir-34a expression inhibits in vivo glioma xenograft growth. We concluded that miR-34a is a potential tumor suppressor in brain tumors that acts by targeting multiple oncogenes. In this extra view, we briefly review and discuss the implications of these findings and present new data on the effects of miR-34a in glioma stem cells. The new data show that miR-34a expression inhibits various malignancy endpoints in glioma stem cells. Importantly, they also show for the first time that miR-34a expression induces glioma stem cell differentiation. Altogether, the data suggest that miR-34a is a tumor suppressor and a potential potent therapeutic agent that acts by targeting multiple oncogenic pathways in brain tumors and by inducing the differentiation of cancer stem cells.     

TSPO ligand PK11195 and MAPK inhibitor UO126 modulate TSPO expression in melanoma cells

TSPO (translocator protein) is an intracellular protein involved in the regulation of cell proliferation, differentiation, and apoptosis. However, the mechanisms of TPSO regulation are poorly understood. We have studied the action of MAPK inhibitor UO126 and TSPO specific ligand PK11195 on the TSPO expression level in melanoma cells. It was found that, in nanomolar concentrations, PK11195 increased the TPSO level in skin melanoma cells. This result was confirmed with UO126 inhibitor by real-time PCR and immunocytochemistry. A combination of PK11195 and UO126 activated apoptotic death of melanoma cells. The data obtained may be used to regulate the functional activity of TPSO-mediated processes.     

Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity

Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.     

Overexpression of Annexin A2 promotes proliferation by forming a Glypican 1/c-Myc positive feedback loop: prognostic significance in human glioma

In order to set up a reliable prediction system for the tumor grade and prognosis in glioma patients, we clarify the complicated crosstalk of Annexin A2 (ANXA2) with Glypican 1 (GPC1) and demonstrate whether combined indexes of ANXA2 and GPC1 could improve the prognostic evaluation for glioma patients. We found that ANXA2-induced glioma cell proliferation in a c-Myc-dependent manner. ANXA2 increased the expression of GPC1 via c-Myc and the upregulated GPC1 further promoted the c-Myc level, forming a positive feedback loop, which eventually led to enhanced proliferation of glioma cells. Both mRNA and protein levels of ANXA2 were upregulated in glioma tissues and coincided with the overexpression of GPC1. Besides, we utilized tissue microarrays (TMAs) and immunohistochemistry to demonstrate that glioma patients with both high expression of ANXA2 and GPC1 tended to have higher rate of tumor recurrence and shorter overall survival (OS). In conclusion, the overexpression of ANXA2 promotes proliferation of glioma cells by forming a GPC1/c-Myc positive feedback loop, and ANXA2 together with its downstream target GPC1 could be a potential “combination biomarker” for predicting prognosis of glioma patients.Subject terms: CNS cancer, Prognostic markers     

Binding of NIR-conPK and NIR-6T to Astrocytomas and Microglial Cells: Evidence for a Protein Related to TSPO

PK 11195 and DAA1106 bind with high-affinity to the translocator protein (TSPO, formerly known as the peripheral benzodiazepine receptor). TSPO expression in glial cells increases in response to cytokines and pathological stimuli. Accordingly, [¹¹C]-PK 11195 and [¹¹C]-DAA1106 are recognized molecular imaging (MI) agents capable of monitoring changes in TSPO expression occurring in vivo and in response to various neuropathologies.Here we tested the pharmacological characteristics and TSPO-monitoring potential of two novel MI agents: NIR-conPK and NIR-6T. NIR-conPK is an analogue of PK 11195 conjugated to the near-infrared (NIR) emitting fluorophore: IRDye 800CW. NIR-6T is a DAA1106 analogue also conjugated to IRDye 800CW.We found that NIR-6T competed for [³H]-PK 11195 binding in astrocytoma cell homogenates with nanomolar affinity, but did not exhibit specific binding in intact astrocytoma cells in culture, indicating that NIR-6T is unlikely to constitute a useful MI agent for monitoring TSPO expression in intact cells. Conversely, we found that NIR-conPK did not compete for [³H]-PK 11195 binding in astrocytoma cell homogenate, but exhibited specific binding in intact astrocytoma cells in culture with nanomolar affinity, suggesting that NIR-conPK binds to a protein distinct, but related to, TSPO. Accordingly, treating intact astrocytoma cells and microglia in culture with cytokines led to significant changes in the amount of NIR-conPK specific binding without corresponding change in TSPO expression. Remarkably, the cytokine-induced changes in the protein targeted by NIR-conPK in intact microglia were selective, since IFN-γ (but not TNFα and TGFβ) increased the amount of NIR-conPK specific binding in these cells.Together these results suggest that NIR-conPK binds to a protein that is related to TSPO, and expressed by astrocytomas and microglia. Our results also suggest that the expression of this protein is increased by specific cytokines, and thus allows for the monitoring of a particular subtype of microglia activation.     

The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents

Although human cells exposed to DNA-methylating agents undergo mismatch repair (MMR)-dependent G(2) arrest, the basis for the linkage between MMR and the G(2) checkpoint is unclear. We noted that mitogen-activated protein kinase p38alpha was activated in MMR-proficient human glioma cells exposed to the chemotherapeutic methylating agent temozolomide (TMZ) but not in paired cells made MMR deficient by expression of a short inhibitory RNA (siRNA) targeted to the MMR protein Mlh1. Furthermore, activation of p38alpha in MMR-proficient cells was associated with nuclear inactivation of the cell cycle regulator Cdc25C phosphatase and its downstream target Cdc2 and with activation of the G(2) checkpoint, actions which were suppressed by the p38alpha/beta inhibitors SB203580 and SB202590 or by expression of a p38alpha siRNA. Finally, pharmacologic or genetic inhibition of p38alpha increased the sensitivity of MMR-proficient cells to the cytotoxic actions of TMZ by increasing the percentage of cells that underwent mitotic catastrophe as a consequence of G(2) checkpoint bypass. These results suggest that p38alpha links DNA MMR to the G(2) checkpoint and to resistance to chemotherapeutic DNA-methylating agents. The p38 pathway may therefore represent a new target for the development of agents to sensitize tumor cells to chemotherapeutic methylating agents.