Antiproliferative and differentiating effects of benzodiazepine receptor ligands on B16 melanoma cells

Kang

Journal Cellular Physiology

et al. 2003

The peripheral benzodiazepine receptor (PBR) has been known to have many functions such as a role in cell proliferation, cell differentiation, steroidogenesis, calcium flow, cellular respiration, cellular immunity, malignancy, and apoptosis. However, the presence of PBR has not been examined in mesenchymal stem cells. In this study, we demonstrated the expression of PBR in human bone marrow stromal cells (hBMSCs) and human adipose stromal cells (hATSCs) by RT-PCR and immunocytochemistry. To determine the roles of PBR in cellular functions of human mesenchymal stem cells (hMSCs), effects of diazepam, PK11195, and Ro5-4864 were examined. Adipose differentiation of hMSCs was decreased by high concentration of PBR ligands (50 microM), whereas it was increased by low concentrations of PBR ligands (<10 microM). PBR ligands showed a biphasic effect on glycerol-3-phosphate dehydrogenase (GPDH) activity. High concentration of PBR ligands (from 25 to 75 microM) inhibited proliferation of hMSCs. However, clonazepam, which does not have an affinity to PBR, did not affect adipose differentiation and proliferation of hMSCs. The PBR ligands did not induce cell death in hMSCs. PK11195 (50 microM) and Ro5-5864 (50 microM) induced cell cycle arrest in the G(2)/M phase. These results indicate that PBR ligands play roles in adipose differentiation and proliferation of hMSCs.

Section: Discussioncontrasting

confidence: 39%

Section: Discussionmentioning

confidence: 43%

Effects of peripheral benzodiazepine receptor ligands on proliferation and differentiation of human mesenchymal stem cells

Kang

Journal Cellular Physiology

et al. 2003

“…Besides its well-established function in the regulation of steroidogenesis (Papadopoulos et al, 1997), the abundance of PBR in cancers of the colon (Katz et al, 1990b;Maaser et al, 2002a), brain (Cornu et al, 1992), breast (Hardwick et al, 1999), ovary (Katz et al, 1990a), and liver (Venturini et al, 1998) suggests an additional role in tumorigenesis. The proliferation of breast cancer (Beinlich et al, 1999;Carmel et al, 1999), melanoma (Landau et al, 1998), Leydig's cell tumour (Garnier et al, 1993), astrocytoma (Neary et al, 1995), colorectal (Maaser et al, 2001), and oesophageal carcinoma was shown to be inhibited by PBR-specific ligands. In haematopoetic and epithelial cells, the antiproliferative effects of PBR-specific ligands were mediated by the induction of apoptosis and cell cycle arrest (Tanimoto et al, 1999;Maaser et al, 2001;Sutter et al, 2002).…”

mentioning

confidence: 99%

“…Likewise, a PBR-ligand-induced G1/S arrest was found in colorectal cancer cells (Maaser et al, 2001). Additionally, PBR ligands induce a cell cycle arrest at both major restriction points, the G1/Sand the G2/M junction (Carmel et al, 1999;Sänger et al, 2000) in breast cancer cells, whereas in lung and melanoma cells an accumulation is observed in the G2/M phase (Landau et al, 1998). However, the exact mechanism of PBR-ligand-mediated cell cycle arrest is not yet understood.…”

mentioning

confidence: 99%

Ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in oesophageal cancer cells: involvement of the p38MAPK signalling pathway

et al. 2003

Specific ligands of the peripheral benzodiazepine receptor (PBR) are known to induce apoptosis and cell cycle arrest in oesophageal cancer cells. However, the underlying mechanisms are still unknown. Here, we investigated the transcriptional alterations and activation of protein kinases in response to PBR-specific ligands. Using cDNA arrays, we examined the transcriptional effects of the PBR-specific ligand FGIN-1-27 in two oesophageal cancer cell lines, KYSE-140 (squamous cell carcinoma) and OE-33 (adenocarcinoma). In oesophageal cancer cells, FGIN-1-27 induced extensive changes in the expression of genes involved in the regulation of apoptosis and cell cycle. Both in oesophageal cancer cell lines (KYSE-140, OE-33) we observed a strong upregulation of the growth arrest and DNA-damage-inducible genes, gadd45 and gadd153, in response to PBR ligands. gadd genes are known to be induced by p38MAPK activation. Using Western blotting we detected a time-and dose-dependent phosphorylation of p38MAPK, which was found to be functionally involved in gadd induction, apoptosis, and cell cycle arrest. In conclusion, our data indicate that PBR-specific ligands cause apoptosis and cell cycle arrest by activation of the p38MAPK pathway and induction of gadd45 and gadd153.

“…This ligand effects a diverse range of physiological actions including inhibition of cell proliferation and respiratory control (Hirsch et al, 1989;Camins et al, 1995b), effects on mitochondrial protein and cholesterol translocation Wright and Reichenbecher, 1999), induction of cell differentiation (Landau et al, 1998), heat shock protein expression (Camins et al, 1995a), and facilitation of a diverse range of apoptotic stimuli (Pastorino et al, 1996;Hirsch et al, 1998;Verma et al, 1998). Consequently, a clearly defined physiological role for the PBR has remained elusive.…”

mentioning

confidence: 99%

Bcl-2 resistant mitochondrial toxicity mediated by the isoquinoline carboxamide PK11195 involves de novo generation of reactive oxygen species

et al. 2001

SummaryResistance to apoptosis is a major obstacle preventing effective therapy for malignancy. Mitochondria localized anti-death proteins of the Bcl-2 family play a central role in inhibiting apoptosis and therefore present valid targets for novel therapy. The peripheral benzodiazepine receptor (PBR) shares a close physical association with the permeability transition pore complex (PTPC), a pivotal regulator of cell death located at mitochondrial contact sites. In this study we investigated the cytotoxicity of the PBR ligand, PK11195, in the micromolar concentration range. PK11195 induced antioxidant inhibitable collapse of the inner mitochondrial membrane potential (∆Ψ m ) and mitochondrial swelling in HL60 human leukaemia cells, but not in SUDHL4 lymphoma cells (which exhibited a higher level of reduced glutathione and relative tolerance to chemotherapy or pro-oxidant induced ∆Ψ m dissipation). PK11195 induced the production of hydrogen peroxide that was not inhibited by Bcl-2 transfection, nor depletion of mitochondrial DNA. ROS production was however blocked by protonophore, implicating a requirement for ∆Ψ m . Our findings suggest that PK11195-induced cytotoxicity relies upon Bcl-2 resistant generation of oxidative stress; a process only observed at concentrations several orders of magnitude higher that required to saturate its receptor. 1397-1404 © 2001 Cancer Research Campaign doi: 10.1054/ bjoc.2001.1788, available online at http://www.idealibrary.com on http://www.bjcancer.com methyl-N-methyl-N-(1-methylpropyl)-isoquinoline carboxamide (PK11195), carbonylcyanide m-chlorophenylhdrazone (CCCP), propidium iodide and all cell culture reagents were purchased from Sigma-Aldrich Ltd, UK. FITC-conjugated mouse antihuman Bcl-2 monoclonal antibody, and the Dako intrastain fixation/permeabilization kit were purchased from Dako, UK. Cell culture and treatmentsHL60, KG1A, K652 cells stably transfected with the Bcl-2 gene (K562 B4) or vector only (K562 N2) (kindly provided by Dr DE Banker and Dr F Applebaum, The Fred Hutchinson Cancer Research Center, USA), and SUDHL4 lymphoma cell lines, were maintained in exponential suspension cultures in RPMI 1640 medium supplemented with 10% fetal calf serum, 5 mM glutamine, 100 µg ml -1 streptomycin and 100 U ml -1 penicillin. Cells were grown in a humidified atmosphere of 5% CO 2 /95% air at 37˚C. PK11195 was dissolved in ethanol at a stock concentration of 8.7 mg ml -1 , and added to cells at a 75 µM final concentration for 4 hours; vehicle alone was also used in medium. To test the effect of an antioxidant on PK11195 activity, cells were treated with 10 mM N-acetylcysteine for 45 minutes prior to treatment with PK11195.Cells were incubated with VP16, or ara-C at 10 µM final concentration for 24 hours, or with the thiol crosslinking prooxidant, diamide, at 100 µM concentration for 24 hours. The protonophore CCCP was used to dissipate ∆Ψ m by treating cells at a concentration of 10 µM for 15 minutes prior to treatment with PK11195. Depletion of mitochondrial DNAKG1A cells ...