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. 2019 Sep 1;20(9):2763-2774.
doi: 10.31557/APJCP.2019.20.9.2763.

Signal Transduction of Improving Effects of Ibudilast on Methamphetamine Induced Cell Death

Reza Tahvilian  1 , Komail Amini  2 , Hossein Zhaleh  3
Affiliations

Signal Transduction of Improving Effects of Ibudilast on Methamphetamine Induced Cell Death

Reza Tahvilian et al. Asian Pac J Cancer Prev. .

Abstract

Objective: Interaction of methamphetamine and sigma (σ) receptors lead to up-regulation and activation of these receptors. The σ receptors induced apoptosis in some parts of the brain by increasing calcium, dopamine, ROS, mitochondrial pores and caspase activity. Ibudilast is a phosphodiesterase inhibitor and anti-inflammatory drug, which can decrease the inflammatory cytokines. Also, it has a neuroprotective effect. It seems that ibudilast can reduce the methamphetamine-induced cell death due to inhibition of σ receptors. Materials and Methods: There were seven treatments including; control: culture medium, Treatment 1: 1mM methamphetamine, Treatment 2: 1mM methamphetamine and 1nM ibudilast, Treatment 3: 1mM methamphetamine and 10nM ibudilast, Treatment 4: 1mM methamphetamine and 100nM ibudilast, Treatment 5: 1mM methamphetamine and 1uM ibudilast, Treatment 6: 1mM methamphetamine and 10uM ibudilast, and Treatment 7: 1mM methamphetamine and 100uM ibudilast. Finally, for inhibition of PKA, CREB, IP3 receptor, NMDA receptor, Sigma receptor antagonist, sigma receptor agonist, cells were preincubated with adding H89 dihydrochloride, 666-15, Heparin, Ketamine, BMY 14802, and Pentazocine. MTT and LDH tests were performed for cell viability and cytotoxicity measurement, respectively. In continuing, the caspase activity colorimetric assay kit used for caspase 3 activity diagnosis. Rhodamine-123 performed to detection of mitochondrial membrane potential. TUNEL test used to DNA fragmentation and apoptosis, Fura-2 used to Measurement of (Ca2+) ic and (Ca2+) m, and fluorescence microscope used to Measurement of antioxidant enzyme activities. Results: Ibudilast increased the cell viability and the rhodamine-123 absorbance in methamphetamin-treated PC12 cells. It reduced cell cytotoxicity, caspase 3 activity, ic and m Ca2+ concentration, (OH) generation and DNA fragmentation in all concentrations of 1 nM t0 100 μM (p<0.05) by the optimal concentration of 100 μM, between our tested treatments. Conclusion: Ibudilast as a phosphodiesterase inhibitor can reduce the methamphetamine-induced cell death due to inhibition of σ receptors through cAMP production.

Keywords: Ibudilast; Methamphetamine; PC12; cell death.

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Conflict of interest statement

There is no conflict of interest.

Figures

Figure 1
Figure 1
The Effects of Different Treatments on the Cell Viability of the Cells. CRL, culture medium; T1, 1mM methamphetamine; T2, 1mM methamphetamine and 1nM ibudilast; T3, 1mM methamphetamine and 10nM ibudilast; T4, 1mM methamphetamine and 100nM ibudilast; T5, 1mM methamphetamine and 1uM ibudilast; T6, 1mM methamphetamine and 10uM ibudilast; T7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 2
Figure 2
The Effects of Different Treatments on the Cell Cytotoxicity of the Cells. CRL, culture medium; T1, 1mM methamphetamine; T2, 1mM methamphetamine and 1nM ibudilast; T3, 1mM methamphetamine and 10nM ibudilast; T4, 1mM methamphetamine and 100nM ibudilast; T5, 1mM methamphetamine and 1uM ibudilast; T6, 1mM methamphetamine and 10uM ibudilast; T7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 3
Figure 3
The Effects of Different Treatments on the Cell Death Index. CRL, culture medium; T1,1mM methamphetamine; T2, 1mM methamphetamine and 1nM ibudilast; T3, 1mM methamphetamine and 10nM ibudilast; T4, 1mM methamphetamine and 100nM ibudilast; T5, 1mM methamphetamine and 1uM ibudilast; T6, 1mM methamphetamine and 10uM ibudilast; T7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 4
Figure 4
The Effects of Different Treatments on the Caspase-3 Activity of the Cells. CRL, culture medium; T1, 1mM methamphetamine; T2, 1mM methamphetamine and 1nM ibudilast; T3, 1mM methamphetamine and 10nM ibudilast; T4, 1mM methamphetamine and 100nM ibudilast; T5, 1mM methamphetamine and 1uM ibudilast; T6, 1mM methamphetamine and 10uM ibudilast; T7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 5
Figure 5
The Effects of Different Treatments on the Mitochondrial Membrane Potential (Rhodamine-123 Absorbance) of the Cells. CRL, culture medium; T1, 1mM methamphetamine; T2, 1mM methamphetamine and 1nM ibudilast; T3, 1mM methamphetamine and 10nM ibudilast; T4, 1mM methamphetamine and 100nM ibudilast; T5, 1mM methamphetamine and 1uM ibudilast; T6, 1mM methamphetamine and 10uM ibudilast; T7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 6
Figure 6
Determination of (Ca2+) m (A) and (Ca2+) ic (B) in Different Treated Cells. CRL, culture medium; T1, 1mM methamphetamine; T2, 1mM methamphetamine and 1nM ibudilast; T3, 1mM methamphetamine and 10nM ibudilast; T4, 1mM methamphetamine and 100nM ibudilast; T5, 1mM methamphetamine and 1uM ibudilast; T6, 1mM methamphetamine and 10uM ibudilast; T7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 7
Figure 7
the antioxidants and reduce agents of endogenous reactive oxygen species (ROS) production in treated cells. CRL, culture medium; T1, 1mM methamphetamine; T2, 1mM methamphetamine and 1nM ibudilast; T3, 1mM methamphetamine and 10nM ibudilast; T4, 1mM methamphetamine and 100nM ibudilast; T5, 1mM methamphetamine and 1uM ibudilast; T6, 1mM methamphetamine and 10uM ibudilast; T7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 8
Figure 8
The Effects of Different Inhibitors on the Cell Viability of the Cells. Control, culture medium; Treatment 1, 1mM methamphetamine; Treatment 7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 9
Figure 9
The Efects of Different Inhibitors on the Cell Cytotoxicity of the Cells. Control, culture medium; Treatment 1, 1mM methamphetamine; Treatment 7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05).
Figure 10
Figure 10
Determination of (Ca2+) c (A) and (Ca2+) m (B) in Different Inhibitor Treated Cells. Control, culture medium; Treatment 1, 1mM methamphetamine; Treatment 7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 11
Figure 11
The Antioxidants and Reduce Agents of Endogenous Reactive Oxygen Species (ROS) Production in Inhibitor Treated Cells. Control, culture medium; Treatment 1, 1mM methamphetamine; Treatment 7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
Figure 12
Figure 12
The Effects of Different Inhibitors on the Caspase-3 Activity of the Cells. Control, culture medium; Treatment 1, 1mM methamphetamine; Treatment 7, 1mM methamphetamine and 100uM ibudilast. All data represented by mean ± S.E.M (p<0.05)
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