Regulating Sirtuin 3-mediated mitochondrial dynamics through vanillic acid improves muscle atrophy in cancer-induced cachexia

Abstract

Cancer cachexia is a cancer-associated disease characterized by gradual body weight loss due to pathologic muscle and fat loss, but effective treatments are still lacking. Here, we investigate the possible effect of vanillic acid (VA), known for its antioxidant, anti-inflammatory, and anti-obesity effects, on mitochondria-mediated improvement of cancer cachexia. We utilized cachexia-like models using CT26 colon cancer and dexamethasone. VA improved representative parameters of cancer cachexia including body weight loss and increased serum intereukin-6 levels. VA also attenuated muscle loss in the tibialis anterior and gastrocnemius muscles, inhibited proteolytic markers including muscle RING-finger protein-1 (MURF1) and muscle atrophy F-box (MAFbx) and improved mitochondrial function through alteration of sirtuins 3 (SIRT3) and mitofusin 1 (MFN1). Importantly, silencing the SIRT3 gene abolished the effect of VA, indicating that SIRT3 is important in the mechanism of action of VA. Overall, we suggest using VA as a novel therapeutic agent that can fundamentally treat and recover muscle atrophy in cancer cachexia patients.

Fig. 1. Effects of VA on cancer cachexia parameters in CT26-induced cachectic mice.

A Experimental scheme of the in vivo study is shown. The animals were subcutaneously inoculated with 5 × 10⁵ CT26 colorectal carcinoma cells in the dorsal region, except for the NC group. VA administration (100 mg/kg of body weight) via oral gavage started one week after cancer injection. The control groups (NC group and CT26 group) were administered distilled water. The vehicle or VA was fed five times per week for three weeks. The artwork was created on biorender.com. B, C Changes in body weight and final body weight were measured. D After sacrificing the mice, tumor-free body weight was calculated by subtracting the tumor weight from the final body weight. E Average food and water intake were measured. F Tissue weights of the heart, lung, BAT, liver, and spleen were measured. G Serum levels of CRP, IL-6, and TNF-α were measured. All data are expressed as mean ± S.E.M. (n = 6). Statistical differences were calculated by one-way ANOVA with post hoc Tukey’s test. ^#p < 0.05 vs. NC mice; ^*p < 0.05 vs. CT26 mice. VA vanillic acid, NC normal control, BAT brown adipose tissue.

Fig. 2. Effects of VA on muscle mass and myofiber diameter in CT26-induced cachectic mice.

A Latency to fall was measured by a rotarod test. B Representative images of GAS and TA in three groups are shown. Tissue weights of C GAS and D TA were measured. Paraffin-embedded E GAS and F TA sections from the NC, CT26, and VA groups were stained with H&E (magnification ×200 and ×400). Myofiber diameters and size distribution were evaluated. All data are expressed as means ± S.E.M. of three or more independent experiments. Statistical differences were calculated by one-way ANOVA with post hoc Tukey’s test. ^#p < 0.05 vs. NC mice; ^*p < 0.05 vs. CT26 mice. VA vanillic acid, NC normal control, GAS gastrocnemius, TA tibialis anterior.

Fig. 3. Effects of VA on MAFbx, MURF1, and AKT expression in TA of CT26-induced cachectic mice.

TA was immunostained with antibodies for A MAFbx and B MURF1. The slides were counter-stained with DAPI for visualization of the cell nucleus (magnification ×400, scale bar = 75 μm). C Average fluorescence intensity was quantified using ImageJ software and presented as a graph. D, E Protein levels of D proteolytic markers (MAFbx, MURF1, AKT) and E inflammation markers (STAT3, NF-κB) were measured in TA by western blot analysis. Results were expressed relative to GAPDH. All values are the means ± S.E.M. of three or more independent experiments. Statistical differences were evaluated using an unpaired t-test and a subsequent post hoc one-tailed Mann–Whitney U test. ^#p < 0.05 vs. NC mice; ^*p < 0.05 vs. CT26 mice. VA vanillic acid, NC normal control, TA tibialis anterior.

Fig. 4. Effects of VA on SIRTs and mitochondrial function in TA of CT26-induced cachectic mice.

A TA was immunostained with antibodies for SIRT3 and MFN1 and counter-stained with DAPI for visualization of the cell nucleus (magnification ×400). Fluorescence intensity was quantified using ImageJ software and presented as bar graphs. B Protein levels of SIRT1, SIRT3, PGC1α, MFN1, and DRP1 in TA were measured by western blot analysis. C Protein levels of OXPHOS complexes in TA were measured by western blot analysis. Results were expressed relative to GAPDH. All values are the means ± S.E.M. of three or more independent experiments. Statistical differences were evaluated using an unpaired t-test and a subsequent post hoc one-tailed Mann–Whitney U test. ^#p < 0.05 vs. NC mice; ^*p < 0.05 vs. CT26 mice. VA vanillic acid, NC normal control, TA tibialis anterior.

Fig. 5. Effects of VA on MAFbx and MURF1 expression in CM (CT26)-treated C2C12 cells.

A C2C12 myoblasts were incubated with VA at the indicated concentrations (0.1–100 μM) and cell viability after 24 h was assessed by a MTS assay. C2C12 myoblasts were differentiated in the absence or presence of CM (CT26). VA was treated at the indicated concentrations (10 μM and 100 μM). B mRNA expressions of Fbxo32, Trim63, and Myostatin were analyzed by Real-Time RT-PCR assays. Results were expressed relative to Gapdh. C Myofibers were immunostained with the antibody for MYH and counter-stained with DAPI for visualization of the cell nucleus (magnification ×100 and ×400). D Average diameter of C2C12 myofibers were measured using Fig. 5C. E Myofibers were stained with H&E (magnification ×100, scale bar = 275 μm). F, G Protein levels of F p4EBP1, MAFbx, MURF1, and G STAT3 were measured by western blot analysis. Results were expressed relative to GAPDH. All values are the means ± S.E.M. of three or more independent experiments. Statistical differences were evaluated using an unpaired t-test and a subsequent post hoc one-tailed Mann–Whitney U test. ^#p < 0.05 vs. CM (−); ^*p < 0.05 vs. CM (CT26). VA vanillic acid, CM (CT26) CT26-derived conditioned medium.

Fig. 6. Effects of VA on SIRT3 and mitochondrial dynamics-related factors in CM (CT26)-treated C2C12 cells.

A Myofibers were immunostained with antibodies for MitoTracker and SIRT3, and counter-stained with DAPI for visualization of the cell nucleus (magnification ×400). Fluorescence intensity was quantified using ImageJ software and presented as bar graphs. B Intracelluar ATP levels were measured C Oxygen consumption was analyzed. D The mRNA expressions of Sirt3, Ppargc1a, Nfe2l2, Mfn1, Opa1, and Dnm1l were analyzed by Real-Time RT-PCR assays. Results were expressed relative to Gapdh. E Protein levels of SIRT3, PGC1α, NRF1, MFN1, and DRP1 were measured by western blot analysis. F Protein levels of OXPHOS complexes were measured by western blot analysis. Results were expressed relative to GAPDH. All values are the means ± S.E.M. of three or more independent experiments. Statistical differences were evaluated using an unpaired t-test and a subsequent post hoc one-tailed Mann–Whitney U test. ^#p < 0.05 vs. CM (-); ^*p < 0.05 vs. CM (CT26). VA vanillic acid, CM (CT26) CT26-derived conditioned medium.

Fig. 7. Abolished effect of VA on the improvement of muscle loss in siSirt3 and CM (CT26)-treated C2C12 cells.

A After treatment with siSirt3 (10 pM) in C2C12 cells, the SIRT3 protein level was measured by western blot analysis. Results were expressed relative to GAPDH. B Myofibers were immunostained with the antibody for SIRT3 and counter-stained with DAPI for visualization of the cell nucleus (magnification ×400). C Myofibers were stained with H&E (magnification ×100, scale bar = 275 μm). Average diameter of C2C12 myofibers were measured. D The mRNA levels of Sirt3, Ppargc1a, Nfe2l2, Mfn1, and Opa1 were analyzed by Real-Time RT-PCR. Results were expressed relative to Gapdh. E Myofibers were immunostained with MitoTracker and counter-stained with DAPI for visualization of cell nucleus (magnification ×400). F Fluorescence intensity for MitoTracker was quantified using ImageJ software and presented as a bar graph. G Myofibers were immunostained with antibodies for MURF1 and counter-stained with DAPI for visualization of cell nucleus (magnification ×400). H Fluorescence intensity for MURF1 was quantified using ImageJ software and presented as a bar graph. All values are the means ± S.E.M. of three or more independent experiments. Statistical differences were evaluated using an unpaired t-test and a subsequent post hoc one-tailed Mann–Whitney U test. ^*p < 0.05 vs. CM (CT26); ^&p < 0.05 vs. CM (CT26) with VA. VA vanillic acid, CM (CT26) CT26-derived conditioned medium.

Fig. 8. Effects of VA on muscle atrophy in Dexa-induced cachectic mice and Dexa-treated C2C12 cells.

A Experimental scheme of the in vivo study is shown. The mice were injected Dexa (20 mg/kg of body weight) intraperitoneally for 5 days, except for the NC group. VA administration (100 mg/kg of body weight) via oral gavage started 2 days after Dexa injection. The control groups (NC group and Dexa group) were administered distilled water. The vehicle or VA was fed 3 days and sacrificed two days later. The artwork was created on biorender.com. B Body weight was measured. C Representative images of GAS, TA, Soleus, and EDL in three groups are shown. D Tissue weights of GAS, TA, soleus, and EDL were measured. E C2C12 myoblasts were differentiated in the absence or presence of Dexa 100 μM. VA was treated at the indicated concentrations (10 μM and 100 μM). mRNA expressions of Trim63, Fbxo32, Sirt3, Dnm1l, and Mfn1 was analyzed by Real-Time RT-PCR assays. Results were expressed relative to Gapdh. All values are the means ± S.E.M. of three independent experiments or more independent experiments. Statistical differences were evaluated using one-way ANOVA with post hoc Tukey’s test or an unpaired t-test and a subsequent post hoc one-tailed Mann–Whitney U test. ^#p < 0.05 vs. NC mice or non-treated; ^*p < 0.05 vs. Dexa mice or Dexa 100 μM. VA vanillic acid, NC normal control, Dexa dexamethasone, GAS gastrocnemius, TA tibialis anterior, EDL extensor digitorum longus.