Sigma-2 receptor/TMEM97 agonist PB221 as an alternative drug for brain tumor

Abstract

Background: There are limited effective drugs that can reach the brain to target brain tumors, in particular glioblastoma, which is one of the most difficult cancers to be cured from. Because the overexpression of the sigma-2 receptor is frequently reported in glioma clinical samples and associated with poor prognosis and malignancy, we herein studied the anti-tumor effect of the sigma-2 receptor agonist PB221 (4-cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperidine) on an anaplastic astrocytoma tumor model based on previous encouraging results in pancreatic cancer and neuroblastoma SK-N-SH cells.

Methods: The expression of the sigma-2 receptor, transmembrane protein 97 (TMEM97), in ALTS1C1 and UN-KC6141 cell lines was measured by RT-PCR and quantitative RT-PCR. The binding of sigma-2 receptor fluorescent ligands PB385 (6-[5-[3-(4-cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-tetrahydronaphthalen-5-yloxy]-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexanamine) and NO1 (2-{6-[2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one-5-yloxy]hexyl}-5-(dimethylamino)isoindoline-1,3-dione) was examined by flow cytometry and the fluorescent plate reader. The antitumor activity of PB221 was initially examined in the murine brain tumor cell line ALTS1C1 and then in the murine pancreatic cell line UN-KC6141. The potential therapeutic efficacy of PB221 for murine brain tumors was examined by in vitro migration and invasion assays and in vivo ectopic and orthotopic ALTS1C1 tumor models.

Results: The IC₅₀ of PB221 for ALTS1C1 and UN-KC6141 cell lines was 10.61 ± 0.96 and 13.13 ± 1.15 μM, respectively. A low dose of PB221 (1 μM) significantly repressed the migration and invasion of ALTS1C1 cells, and a high dose of PB221 (20 μM) resulted in the apoptotic cell death of ALTS1C1 cells. These effects were reduced by the lipid antioxidant α-tocopherol, but not by the hydrophilic N-acetylcysteine, suggesting mitochondrial oxidative stress is involved. The in vivo study revealed that PB221 effectively retarded tumor growth to 36% of the control tumor volume in the ectopic intramuscular tumor model and increased the overall survival time by 20% (from 26 to 31 days) in the orthotopic intracerebral tumor model.

Conclusions: This study demonstrates that the sigma-2 receptor agonist PB221 has the potential to be an alternative chemotherapeutic drug for brain tumors with comparable side effects as the current standard-of-care drug, temozolomide.

Fig. 1

The responses of brain and pancreatic cell lines to the cytotoxicity of PB221 and binding affinity with selective sigma-2 receptor agonist ligand, NO1. (a) The survival curve of various cell lines in response to a range of PB221 and PB28 concentration for 3 days was measured by MTT assay. The IC₅₀ for murine astrocytoma ALTS1C1 cells and murine pancreatic UN-KC6141 cells was 10.61 and 13.13 μM, respectively. ALTS1C1* open circle curve is the responsive curve of ALTS1C1 to PB28 and the IC₅₀ is 11.78 μM. (b) The binding affinity of ALTS1C1 and UN-KC6141 cells with selective sigma-2 receptor ligand, NO1, was examined by fluorescent plate reader and the relative fluorescent unit (RFU) was measured with the excitation of 390 nm and emission at 525 nm

Fig. 2

The expression of TMEM97 by ALTS1C1 and UN-KC6141 cells. The expression of TMEM97 mRNA by normal brain cells, ALTS1C1 and UN-KC6141 cells was assessed by (a) RT-PCR and (b) quantitative PCR (Q-PCR). The difference (ΔCt) between the Ct of the gene transcript and the endogenous control β-actin determined the gene expression level

Fig. 3

Effects of PB221 on brain tumor cell migration and invasion. (a) Represented pictures of migration assay illustrate the retarded cell migration rate of ALTS1C1 cells following 1 μM PB221 treatment for 16 h. Scale bar = 100 μm. (b) A summary graph for the dose and time effects of PB221 on the migration distance of ALTS1C1 cells. *: P < 0.05 compared with control. (c) Represented pictures of invasion assay reveal the decrease of invasion cells following various doses of PB221 treatment for 16 h. (d) A summary graph for the dose effects of PB221 on ALTS1C1 cell invasion ability. ***: P < 0.001 compared with control

Fig. 4

The effect of anti-oxidant on PB221 effects on ALTS1C1 (a) A summary graph reveals the level of mitochondria superoxide in brain tumor cell lines, U87-MG and ALTS1C1 following PB221 stimulation was measured by the fluorescent intensity of MitoSoxTM Red using flow cytometry. (b) Represented pictures of migration and invasion assays illustrate the effect of anti-oxidant α-tocopherol and NAC on PB221 (1 μM)-inhibited migration and invasion ability of ALTS1C1 cells. Scale bar = 100 μm. (c) A summary graph shows the preventing effect of 10 μM of α-tocopherol, but not NAC, on PB221 (1 μM)-inhibited migration and invasion ability of ALTS1C1 cells. (d) A summary graph reveals the preventing effect of 10 μM of α-tocopherol, but not NAC, on PB221 (20 μM)-induced apoptotic cell death of ALTS1C1 cells assayed by flow cytometry. *: P < 0.05. **: P < 0.01. ***: P < 0.001

Fig. 5

The in vivo anti-tumor effects of PB221 against ALTS1C1 tumors. (a) The tumor growth curves of intramuscular ALTS1C1 tumors following the treatment of PB221 and TMZ. One dose of TMZ (2 mg/mouse/injection) and 4 doses of PB221 (2 mg/mouse/injection) were intraperitoneally injected into tumor-bearing mice at day 10 following the tumor inoculation as indicated by the arrows in the graph. The data represented the average tumor volume in each group from one representative experiment of three independent experiments. *: P < 0.05. **: P < 0.01. ***: P < 0.001. (b) The change of mouse body weight following the intramuscular tumor implantation and the treatment of 4 doses of PB221 (2 mg/mouse/injection) and one dose of TMZ (2 mg/mouse/injection). *: P < 0.05. (c) The animal survival curve for mouse-bearing intracranial ALTS1C1 tumor and the treatment of 5 doses of PB221 (1 mg/mouse/injection) and one dose of TMZ (2 mg/mouse/injection). **: P < 0.01; ****: P < 0.0001. (d) The change of mouse body weight following the intracranial tumor inoculation and the treatment of 5 doses of PB221 (1 mg/mouse/injection) and one dose of TMZ (2 mg/mouse/injection). *: P < 0.05. **: P < 0.01