IMB0901 inhibits muscle atrophy induced by cancer cachexia through MSTN signaling pathway

Abstract

Background: Cancer cachexia as a metabolic syndrome can lead to at least 25% of cancer deaths. The inhibition of muscle atrophy is a main strategy to treat cancer cachexia. In this process, myostatin (MSTN) can exert a dual effect on protein metabolism, including inhibition of protein biosynthesis and enhancement of protein degradation. In this study, we will test the effect on muscle atrophy induced by cancer cachexia of IMB0901, a MSTN inhibitor.

Methods: Two high-throughput screening models against MSTN were developed. By screening, IMB0901, 2-((1-(3,4-dichlorophenyl)-1H-pyrazolo [3,4-d] pyrimidin-4-yl) amino) butan-1-ol, was picked out from the compound library. The in vitro cell model and the C26 animal model of muscle atrophy induced by cancer cachexia were used to determine the pharmacological activity of IMB0901. Whether IMB0901 could inhibit the aggravating effect of doxorubicin on muscle wasting was examined in vitro and in vivo.

Results: IMB0901 inhibited the MSTN promoter activity, the MSTN signaling pathway, and the MSTN positive feedback regulation. In atrophied C2C12 myotubes, IMB0901 had a potent efficiency of decreasing MSTN expression and modulating MSTN signaling pathway which was activated by C26-conditioned medium (CM). In C2C12 myotubes, the expressions of three common myotube markers, myosin heavy chain (MyHC), myogenic differentiation 1 (MyoD), and myogenin (MyoG), were downregulated by CM, which could be efficiently reversed by IMB0901 via reduction of ubiquitin-mediated proteolysis and enhancement of AKT/mTOR-mediated protein synthesis. In the C26 animal model, IMB0901 mitigated the weight loss of body, quadricep and liver, and protected the quadriceps cell morphology. Furthermore, IMB0901 decreased the expression of two E3 ligases Atrogin-1 and MuRF-1 in the quadriceps in vivo. At the cellular level, IMB0901 had no influence on anti-tumor effect of three chemotherapeutic agents (cisplatin, doxorubicin, and gemcitabine) and lowered doxorubicin-induced upregulation of MSTN in C2C12 myotubes. IMB0901 did not affect the inhibitory effect of doxorubicin on C26 tumor and delayed the weight loss of muscle and adipose tissue caused by C26 tumor and doxorubicin.

Conclusions: IMB0901 inhibits muscle atrophy induced by cancer cachexia by suppressing ubiquitin-mediated proteolysis and promoting protein synthesis. These findings collectively suggest that IMB0901 is a promising leading compound for the management of muscle atrophy induced by cancer cachexia.

Keywords: C26; C2C12; Cancer cachexia; Muscle atrophy; Myostatin.

Fig. 1

The effect of IMB0901 on MSTN promoter activity and MSTN signaling pathway. a The structural formula of IMB0901. b The cytotoxicity of IMB0901. MTT assay was used to detect the cell survival rate of HEK293T-MSTNP cells after 24-h treatment with IMB0901 at various concentrations. The values presented means ± SD from three experiments, each performed in triplicate. c The inhibition of IMB0901 on the MSTN promoter activity. Bright-Glo™ Luciferase Assay was applied to assess the luciferase activity of HEK293T-MSTNP cells after 24-h direct treatment with IMB0901 at various concentrations. The values were means ± SD from three experiments, each performed in triplicate. ^**P < 0.01 compared to the control group (0 μM). d The inhibition of IMB0901 on the MSTN positive feedback regulation. Bright-Glo™ Luciferase Assay was applied to assess the luciferase activity of MSTN-activated HEK293T-MSTNP cells after 24-h treatment with IMB0901 at various concentrations. The data presented means ± SD from three experiments, each performed in triplicate. *P < 0.05 compared to the control group, ^##P < 0.01 compared to the MSTN group. e The inhibition of IMB0901 on the MSTN signaling pathway. Bright-Glo™ Luciferase Assay was used to detect the luciferase activity of MSTN-activated HEK293-SBE cells after 24-h treatment with IMB0901 at various concentrations. The values were means ± SD from three different experiments, each performed in triplicate. ^**P < 0.01 compared to the control group, ^##P < 0.01 compared to the MSTN group

Fig. 2

The effect of IMB0901 in the in vitro model. a The effect of IMB0901 on the survival rate of C2C12 cells. MTT assay was used to detect the survival rate of C2C12 cells after 24-h and 48-h treatment with IMB0901 at various concentrations. The values presented means ± SD from three experiments, each performed in triplicate. b The inhibition of IMB0901 on the MSTN mRNA expression. Real-time PCR was used to detect the effect of IMB0901 on the upregulation of MSTN during C2C12 myotube atrophy induced by C26 cell supernatant at mRNA level. There were four groups in this experiment: control (no CM or IMB0901), CM (1/6 CM), IMB0901 (1/6 CM + 2.5 μM IMB0901), and IMB0901 (1/6 CM + 5 μM IMB0901), for the following data. The data presented means ± SD from three experiments, each performed in triplicate. **P < 0.01 compared to the control group, ^#P < 0.05 compared to the CM group. c The inhibition of IMB0901 on the MSTN protein expression. Western blotting was used to detect the effect of IMB0901 on the upregulation of MSTN during C2C12 myotube atrophy induced by C26 cell supernatant at protein level. The data presented means ± SD from three experiments. **P < 0.01 compared to the control group, ^##P < 0.01 compared to the CM group. d The inhibition of IMB0901 on the MSTN signaling pathway in the in vitro cell model. Western blotting was applied to assess the effect of IMB0901 on the phosphorylation level of SMAD and FOXO3a during the process of C2C12 myotube atrophy induced by C26 cell supernatant. The values presented means ± SD from three experiments. **P < 0.01 compared to the control group, ^#P < 0.05 and ^##P < 0.01 compared to the CM group

Fig. 3

The therapeutic effect of IMB0901 in the in vitro model. a The effect of IMB0901 on the expression of three myotube protein markers MyHC, MyoD, and MyoG in the in vitro cell model. Western blotting was used to detect the effect of IMB0901 on the expression of three myotube protein markers MyHC, MyoD, and MyoG during the process of C2C12 myotube atrophy induced by C26 cell supernatant. b The effect of IMB0901 on the cytomorphology of C2C12 myotube atrophy induced by C26 cell supernatant. Left: The cell morphology during the process of C2C12 myotube atrophy induced by C26 cell supernatant was recorded with the inverted microscopy (upper), and the MyHC expression in C2C12 myotubes was detected by immunofluorescence and the nuclei was stained by DAPI (down). Right: The number of nuclei per field was examined by Image J software (upper), and the myotube area percentage was calculated by the immunofluorescence area divided by the horizon area (down). The values presented means ± SD from three experiments. *P < 0.05 and **P < 0.01 compared to the control group, ^#P < 0.05 and ^##P < 0.01 compared to the CM group

Fig. 4

The mechanism of IMB0901 in the in vitro model. a The inhibitory effect of IMB0901 on two E3 ligases, Atrogin-1 and MuRF-1. Western blotting was used to detect the effect of IMB0901 on the expressions of two E3 ligases, Atrogin-1 and MuRF-1, during the process of C2C12 myotube atrophy induced by C26 cell supernatant. b The inhibitory effect of IMB0901 on the ubiquitin expression. Western blotting was used to detect the effect of IMB0901 on the ubiquitin expressions during the process of C2C12 myotube atrophy induced by C26 cell supernatant. c, d, e The positive effect of IMB0901 on phosphorylation level of AKT, P70S6K and mTOR in atrophied C2C12 myotubes. Western blotting was used to detect the phosphorylation of AKT, P70S6K and mTOR during the process of C2C12 myotube atrophy induced by C26 cell supernatant. The values were means ± SD from three independent experiments. *P < 0.05 and **P < 0.01 compared to the control group, ^#P < 0.05 and ^##P < 0.01 compared to the CM group

Fig. 5

The therapeutic effect of IMB0901 in the C26 animal model. a The effect of IMB0901 on the survival rate of C2C12 cells. MTT assay was used to detect the survival rate of C2C12 cells after 24-h and 48-h treatment with IMB0901 at various concentrations. The values presented means ± SD from three experiments, each performed in triplicate. b The effect of IMB0901 on C26 tumor weights at endpoint. c The effect of IMB0901 on the tumor growth in in the group of C26 animal model and IMB0901 treatment. d The body weights of BALB/c mice at day 0~20. e The carcass weights of BALB/c mice. f, g The mass of quadriceps and gastrocnemius muscles in the C26 animal model. h The effect of IMB0901 on the expressions of two E3 ligases, Atrogin-1 and MuRF-1. Western blotting was used to detect the effect of IMB0901 on the expressions of two E3 ligases, Atrogin-1 and MuRF-1, in the quadriceps tissue. i Representative images of an H&E-stained cross-section of quadriceps from BALB/c mice and the average cross-sectional area (CSA) of the quadriceps fibers in the C26 animal model. There were three groups in this experiment: control, C26, C26 + IMB0901. The values presented means ± SD from 10 animals. **P < 0.01 compared to the control group, ^#P < 0.05 and ^##P < 0.01 compared to the C26 group

Fig. 6

The effect of IMB0901 on the antitumor effect of doxorubicin in vitro and in vivo. a The effect of IMB0901 combined with doxorubicin on the cell survival rate of C26 cells. MTT assay was used to detect the cell survival rate of C26 cells after 24 h treatment with IMB0901 at various concentrations combined with doxorubicin. The values presented the mean ± SD from three experiments, each performed in triplicate. b The effect of IMB0901 on the MSTN expression change induced by doxorubicin in C2C12 myotubes. Western blotting was used to detect the expression of MSTN after 24-h treatment with IMB0901 and doxorubicin in C2C12 myotubes. The values were means ± SD from three experiments. *P < 0.05 compared to the control group, ^#P < 0.05 compared to the DOX group. c The body weights of BALB/c mice at day 0~20. Doxorubicin was administered twice with 0.2 ml each at a dose of 10 mg/kg via the intraperitoneal injection. d The carcass weights of BALB/c mice. e–h The mass of quadriceps, gastrocnemius muscles, WAT, and BAT in the C26 and doxorubicin model. There were four groups in this experiment: control, C26, C26 + DOX, and C26 + DOX + IMB0901. The data were means ± SD from 10 animals. *P < 0.05 and **P < 0.01 compared to the control group, ^#P < 0.05 and ^##P < 0.01 compared to the C26 + DOX group

Fig. 7

The mechanism schematic presentation of IMB0901 in inhibiting muscle atrophy induced by cancer cachexia