Nuciferine Attenuates Cancer Cachexia-Induced Muscle Wasting in Mice via HSP90AA1

Abstract

Background: Around 80% of patients with advanced cancer have cancer cachexia (CC), a serious complication for which there are currently no FDA-approved treatments. Nuciferine (NF) is the main active ingredient of lotus leaf, which has anti-inflammatory, anti-tumour and other effects. The purpose of this work was to explore the target and mechanism of NF in preventing cancer cachexia-induced muscle atrophy.

Methods: The action of NF against CC-induced muscle atrophy was determined by constructing an animal model with a series of behavioural tests, H&E staining and related markers. Network pharmacology and molecular docking were used to preliminarily determine the mechanism and targets of NF against CC-induced muscle atrophy. The mechanisms of NF in treating CC-induced muscle atrophy were verified by western blotting. Molecular dynamics simulation (MD), drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) were used to validate the key target of NF.

Results: After 13 days of NF treatment, the reduction of limb grip strength and hanging time in LLC model mice increased by 29.7% and 192.2% (p ≤ 0.01; p ≤ 0.001). Gastrocnemius and quadriceps muscles weight/initial body weight (0.98 ± 0.11 and 1.20 ± 0.17) and cross-sectional area of muscle fibres (600-1600 μm²) of NF-treated mice were significantly higher than those of the model group (0.84 ± 0.10, 0.94 ± 0.09, 400-800 μm², respectively) (p ≤ 0.01; p ≤ 0.01; p ≤ 0.001). NF treatment also decreased the MyHC (myosin heavy chain) degradation and the protein levels of muscle-specific E3 ubiquitin ligases Atrogin1 and MuRF1 in the model group (p ≤ 0.001; p ≤ 0.01; p ≤ 0.05). Network pharmacology revealed that NF majorly targeted AKT1, TNF and HSP90AA1 to regulate PI3K-Akt and inflammatory pathways. Molecular docking predicted that NF bound best to HSP90AA1. Mechanism analysis demonstrated that NF regulated NF-κB and AKT-mTOR pathways for alleviating muscle wasting in tumour bearing mice. The results of MD, DARTS and SPR further confirmed that HSP90AA1 was the direct target of NF.

Conclusions: Overall, we first discovered that NF retards CC-induced muscle atrophy by regulating AKT-mTOR and NF-κB signalling pathways through directly binding HSP90AA1, suggesting that NF may be an effective treatment for cancer cachexia.

Keywords: AKT–mTOR; HSP90AA1; NF‐κB; cancer cachexia, muscle wasting; nuciferine.

FIGURE 1

NF improved the muscle strength of LLC tumour‐bearing mice. (a) Study design. Tumours were inoculated on Day −8, tumours grew on Day 0, NF treatment was given on Days 1–13 and mice were sacrificed on Day 14. (b–d) Grip strength test (GST). Grip strength on Days 0, 7 and 14. (e, f) Wire grip test (WGT). Frailty score and hanging time on WGT. The data are shown as the mean ± SD. n = 7 mice/group. **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 2

NF ameliorated the loss of tumour free body weight, muscle mass and skeletal muscle atrophy in mice bearing lung tumour: (a) The tumour free body weight. (b, c) Gastrocnemius muscle weight, quadriceps muscle weight to initial body weight (IBW). (d) Epididymal fat mass to initial body weight (IBW). (e) H&E‐stained sections of mouse muscle (scale bar = 50 μm), skeletal muscle myofiber cross‐sectional area and relative frequency. (f) Representative Western blots showing the expression of MYHC, Atrogin1, MuRF1 in the gastrocnemius muscle and relative protein levels in the experiments of MYHC, Atrogin1 and MuRF1. The data are shown as the mean ± SD. n = 7, 3 or 5 mice/group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 3

Network pharmacology analysis of NF on cancer cachexia. (a) The 2D structure of NF. (b) Common targets for the action targets of NF and CC. (c) PPI network diagram in which the size of the circle and the shade of the colour are arranged according to the degree. (d) The core target and the colour of the shade of the degree are arranged from largest to smallest. (e) KEGG enrichment analysis. (f) NF against cancer‐associated muscular dystrophy gene ontology enrichment analysis of potential targets. The top biological processes (BP), cellular components (CC) and molecular functions (MF) are shown as green, orange and purple bands, respectively.

FIGURE 4

Visualization was performed based on molecular docking results. NF docked with (a) HSP90AA1, (b) EGFR, (c) AKT1, (d) PPARG, (e) MMP9 and (f) ICAM1.

FIGURE 5

NF inhibited inflammation response of muscle via HSP90AA1/NF‐κB pathway in LLC tumour‐bearing mice. (a) Representative Western blots showing the expression of HSP90AA1, p‐IKKβ, p‐NF‐κB, IL‐6 and TNF‐α in the gastrocnemius muscle. (b–f) Relative protein levels in the experiments shown in A. The data were shown as the mean ± SD, n = 5. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 6

NF alleviated cancer cachexia‐induced muscle atrophy via AKT–mTOR signalling pathway. (a) Representative Western blots showing the expression of p‐AKT, AKT, p‐mTOR and mTOR in the gastrocnemius muscle. (b, c) Relative protein levels in the experiments shown in A. The data were shown as the mean ± SD, n = 5. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 7

The NF‐HSP90AA1 interaction was verified by molecular dynamics simulation, DARTS and SPR experiments. (a–c) The results of RMSD, RMSF and Rg for complex and HSP90AA1. (d) The SASA, number of hydrogen bonds of the complex and using the last 10‐ns molecular dynamics (MD) simulation locus; the residue energy decomposition of the MM‐PBSA binding energy of the complex was calculated. (e) DARTS assay in C2C12 myotubes. (f) SPR analysis.

FIGURE 8

Schematic diagram of the proposed mechanisms underlying the anticachexic effect of NF in mice bearing tumour. NF ameliorates muscle atrophy in mice bearing tumour by regulating NF‐κB and AKT/mTOR signalling pathway via directly binding HSP90AA1.