Analog of somatostatin vapreotide exhibits biological effects in vitro via interaction with neurokinin-1 receptor

Abstract

Objectives: Vapreotide, a synthetic analog of somatostatin, has analgesic activity most likely mediated through the blockade of neurokinin-1 receptor (NK1R), the substance P (SP)-preferring receptor. The ability of vapreotide to interfere with other biological effects of SP has yet to be investigated.

Methods: We studied the ability of vapreotide to antagonize NK1R in three different cell types: immortalized U373MG human astrocytoma cells, human monocyte-derived macrophages (MDM) and a human embryonic kidney cell line, HEK293. Both U373MG and MDM express endogenous NK1R while HEK293 cells, which normally do not express NK1R, are stably transformed to express human NK1R (HEK293-NK1R).

Results: Vapreotide attenuates SP-triggered intracellular calcium increases and nuclear factor-κB activation in a dose-dependent manner. Vapreotide also inhibits SP-induced interleukin-8 and monocyte chemotactic protein-1 production in HEK293-NK1R and U373MG cell lines. Vapreotide inhibits HIV-1 infection of human MDM in vitro, an effect that is reversible by SP pretreatment.

Conclusions: Our findings indicate that vapreotide has NK1R antagonist activity and may have a potential application as a therapeutic intervention in HIV-1 infection.

Figure 1. Aprepitant and vapreotide suppress SP-induced [Ca₂₊]_i increase in U373MG cells

A. The effect of different concentrations of aprepitant (10, 30 and 100 nM) on SP-induced intracellular calcium increase was measured in U373MG cells stimulated with concentrations of SP ranging from 10⁻¹¹ to 10⁻⁵ M. Each data point represents the average calcium concentrations measured in eight individual cells ± SD. B. U373MG cells were pretreated with different concentrations of vapreotide as indicated, followed by stimulation with 100 nM or 10 nM of SP. Representative tracings show the average intracellular calcium concentration for eight cells over 240 seconds of measurement.

Figure 2. Vapreotide attenuates SP-induced NF-κB activation and IL-8, EGR-1 and c-Fos mRNA up-regulation in HEK293-NK1R cells

A. HEK293-NK1R cells that coexpress NF-κB-luc were pre-incubated with or without aprepitant (1 μM), CP-96,345 (1 μM), or vapreotide (10 μM) for 30 min followed by stimulation with SP (0.1 μM) for 6 h. Mock treated cells were used as controls. The results are presented as fold-change in Relative Light Units (RLU) compared to the mock treated control which is defined as 1.0. B and C. HEK293-NK1R cells were pre-incubated with or without vapreotide (10 μM), aprepitant (1 μM), or mock treated as control for 30 min followed by 0.1 μM SP stimulation for 3 h. IL-8 (B), EGR-1 (C) and C-FOS (C) mRNA levels were determined by real-time PCR and are presented as fold-change of mock treated control, which is defined as 1.0. These results are representative of three independent experiments ± S.D. NF-kB activation and IL-8, EGR-1 and c-Fos expression were significantly increased in SP treated cultures (* p<0.01) versus mock-treated control, aprepitant-treated, CP-96,345-treated or vapreotide-treated cultures by Student’s t-test.

Figure 3. Vapreotide inhibits SP-induced IL-8 and MCP-1 expression in U373MG cells

A. and B. U373MG cells were treated with SP alone; vapreotide and SP; SSTR2 inhibitor CYN, vapreotide and SP; or aprepitant and SP. U373MG cells were pretreated with vapreotide (10 μM) or aprepitant (1 μM) for 30 min followed by incubation in the presence or absence of SP (0.1 μM) for 3 hours. When cells were treated with both CYN and vapreotide, the cells were first incubated with CYN (10 μM) for 30 min followed by vapreotide (10 μM) for additional 30 min, and then stimulated with SP (0.1 μM) for 3 hours. IL-8 mRNA (A) levels were determined by real-time PCR and are presented as fold-change of mock treated control, which is defined as 1.0. Secreted IL-8 (B) in culture medium was measured by EIA and the levels are presented as pg/ml. C. and D. U373MG cells were pre-incubated with or without vapreotide (10 μM), aprepitant (1 μM), or mock treated as controls for 30 min followed by 0.1 μM SP stimulation for 3 h for when measuring MCP-1 mRNA (C) or 18 h when measuring MCP-1 secretion in the culture supernatants (D). MCP-1 mRNA levels were determined by real-time PCR and are presented as fold of mock-treated control, which is defined as 1.0. Secreted MCP-1 in culture medium was measured by EIA and data are presented as pg/ml. These results are representative of three independent experiments ± S.D. SP treated cultures had significantly higher levels of IL-8 and MCP-1 (*p<0.01) compared to mock-treated control, aprepitant-treated, and CYN and vapreotide-treated cultures by Student’s t-test.

Figure 4. SP reverses vapreotide inhibition of HIV-1 replication in human MDM

7–10 day cultured MDM were pretreated with SP and/or vapreotide at the concentrations indicated for 2 hours followed by infection with HIV-1 for 7 days. HIV gag mRNA levels were quantified by real-time RT-PCR as copy numbers and normalized based on GAPDH levels. Representative data from one of three independent experiments ± S.D. are shown. GAG mRNA levels were significantly higher in SP treated cultures (* p<0.01) compared to mock treated control, vapreotide-treated or both vapreotide and SP-treated cultures by Student’s t-test.