Amelioration of Cancer Cachexia by Dalbergia odorifera Extract Through AKT Signaling Pathway Regulation

Abstract

Background/Objectives: Cancer cachexia is a multifactorial syndrome characterized by the progressive loss of skeletal muscle mass and adipose tissue. Dalbergia odorifer is widely used in traditional medicine in Korea and China to treat various diseases. However, its exact role and underlying mechanism in regulating cancer cachexia have not been elucidated yet. This research was conducted to investigate the effect of D. odorifer extract (DOE) in preventing the development of cancer-induced cachexia symptoms and figure out the relevant mechanisms. Methods: A cancer cachexia model was established in Balb/c mice using the CT26 colon carcinoma cell line. To evaluate the anti-cachexia effect of Dalbergia odorifer extract (DOE), CT26-bearing mice were orally administered with DOE at concentrations of 50 and 100 mg/kg BW for 14 days. C2C12 myotubes and 3T3L1 adipocytes were treated with 80% CT26 conditioned medium, DOE, and wortmannin, a particular AKT inhibitor to determine the influence of DOE in the AKT signaling pathway. Mice body weight, food intake, myofiber cross-sectional area, adipocyte size, myotube diameter, lipid accumulation, and relevant gene expression were analyzed. Results: The oral administration of DOE at doses of 50 and 100 mg/kg body weight to CT26 tumor-bearing mice resulted in a significant reduction in body weight loss, an increase in food intake, and a decrease in serum glycerol levels. Furthermore, DOE treatment led to an increase in muscle mass, larger muscle fiber diameter, and elevated expression levels of MyH2 and Igf1, while simultaneously reducing the expression of Atrogin1 and MuRF1. DOE also attenuated adipose tissue wasting, as evidenced by increased epididymal fat mass, enlarged adipocyte size, and upregulated Pparγ expression, alongside a reduction in Ucp1 and IL6 levels. In cachectic C2C12 myotubes and 3T3-L1 adipocytes induced by the CT26 conditioned medium, DOE significantly inhibited muscle wasting and lipolysis by activating the AKT signaling pathway. The treatment of wortmannin, a specific AKT inhibitor, effectively neutralized DOE's impact on the AKT pathway, myotube diameter, and lipid accumulation. Conclusions: DOE ameliorates cancer cachexia through the expression of genes involved in protein synthesis and lipogenesis, while suppressing those related to protein degradation, suggesting its potential as a plant-derived therapeutic agent in combating cancer cachexia.

Keywords: AKT; Dalbergia odorifer extract; adipose wasting; cancer cachexia; muscle atrophy.

Figure 1

Dalbergia odorifera plant extract improved the symptoms of cancer cachexia in CT26-bearing mice. A schematic representation of the experimental design is illustrated. In general, CT26 tumor cells were intravenously injected into 6-week-old Balb/c mice on day 7, and DOE (50, 100 mg/kg) was orally administrated for 14 days starting after 1 day of tumor inoculation. Healthy mice and the vehicle group received an equal volume of solvent. Body weight and food intake were recorded every day (A). Representative pictures of mice from the four groups at the end of the experiment (B) and the body weight curve show changes in body weight in the four treatment groups over the experiment (C). Mouse body weight (D), food consumption (E), and different organs were compared among the four groups. Values are expressed as the mean ± SEM. A one-way ANOVA was performed with * p < 0.05, ns: not significant.

Figure 2

DOE ameliorates skeletal muscle wasting in CT26 tumor-bearing mice. Representative images and weight of the gastrocnemius muscle collected from mice in the four groups (A). The hematoxylin and eosin (H&E) staining of gastrocnemius sections from the four groups with scale bars = 100 px (B). The minimal Feret’s diameter of 100 muscle fibers from each group was measured using ImageJ (C). The mRNA expressions of Atrogin1, MuRF, IL6, Myh2, and Igf1 in the skeletal muscles were assessed by qRT-PCR (n = 6). mRNA was normalized against GAPDH. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant versus the vehicle control (D). Western blot analysis of MuRF1, Atrogin-1, MYH2, FOXO3a, p-FOX3a, AKT, and p-AKT expression levels in the gastrocnemius muscles of different groups. GAPDH was used as the internal control (E).

Figure 3

DOE attenuates adipose tissue loss in CT26 tumor-bearing mice. Representative images and weight of epididymal fats collected from mice in the four groups (A). Glycerol was measured to evaluate the degree of lipolysis in the serum samples (B). The hematoxylin and eosin (H&E) staining of epididymal sections from the four groups with scale bars = 100 px (C). The cross-sectional area (CSA) of 100 adipocytes from each group was measured using ImageJ (D). mRNA expression levels of Ucp1, IL6, and Ppary were evaluated by RT-qPCR. mRNA was normalized against GAPDH (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant versus the vehicle control (E). Protein expressions of UCP1, PPARy, AKT, and p-AKT in epididymal fats were evaluated using Western blot, and GAPDH was used as the internal control (F).

Figure 4

DOE prevents the CM-induced atrophy of C2C12 myotubes. The toxicity of DOE to C2C12 myoblasts was checked by CCK-8 (A). Cell morphology of myotubes from different groups was visualized by LADD (20X magnification) (B) or ICC when nuclei were detected by DAPI (blue) and MYH2 by Alexa Flour 555 at 40X magnification (C). Myotube diameter was measured using ImageJ (D). qRT-PCR was conducted to evaluate the mRNA expression levels of Atrogin1, MuRF, IL6, Myh2, and Igf1 after 24 h of treatment with CM and DOE (E), and the protein expression levels of MuRF1, Atrogin1, MYH2, FOXO3a, p-FOXO3a, AKT, and p-AKT were evaluated using Western blot (F). A one-way ANOVA was carried out to determine significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant).

Figure 5

DOE prevents CM-induced lipolysis in 3T3L1 adipocytes. The cytotoxicity assay of the DOE to 3T3L1 preadipocytes was assessed using CCK-8 (A). Cell morphology was visualized by Oil Red O staining (B), and lipid accumulation was compared among the four groups (C). Glycerol was measured to evaluate the degree of lipolysis (D), and the mRNA expression levels of Ucp1, IL6, and Ppary were evaluated for all the groups (E). Protein levels of UCP1, PPARγ, AKT and p-AKT via Western blot (F). Data are presented as the mean ± SEM. Significance was determined by a one-way ANOVA (* p < 0.05, ** p < 0.01, **** p < 0.0001, ns: not significant).

Figure 6

PI3K inhibitor (wortmannin) inhibited the therapeutic effects of DOE in preventing muscle wasting. Wortmannin was added to inhibit the AKT signaling pathway. In general, 1 µM wortmannin was pretreated to cells 1 h before the addition of CM and DOE. After 24 h, cells were stained and harvested for further analysis. Representative images of myofibers were immunostained with the antibody for MYH2 (orange) and counter-stained with DAPI (blue) for visualization of the nucleus (20× magnification) (scale bar = 20 μm) (A), and C2C12 myotubes by LADD staining (20× magnification) (scale bar = 100 px) (B). The expression levels of Igf1, Irs1, Myh2, and Atrogin-1 mRNA in myotubes treated with 15 ug/mL DOE with or without wortmannin for 24 h were measured, and mRNA was normalized against GAPDH (C). The protein levels of AKT, p-AKT, Atrogin1, and MYH2 were measured via Western blot analysis (D). Data are presented as the mean ± SEM. The statistical significance was determined by a one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001, ns: not significant).

Figure 7

PI3K inhibitor (wortmannin) inhibited the therapeutic effects of DOE in promoting lipogenesis. Wortmannin was added to inhibit the AKT signaling pathway. In general, 1 µM wortmannin was pretreated to cells 1 h before the addition of CM and DOE. After 24 h, cells were stained and harvested for further analysis. Oil Red O staining (A) and lipid accumulation measurement (B) of 3T3L1 adipocytes after CM, DOE, and wortmannin treatments in different groups were conducted. The mRNA levels of Igf1, Irs1, Ppary, and Scd1 were evaluated using qPCR, and the results are expressed relative to GAPDH (C). Western blot analysis of the protein expression levels of AKT, p-AKT, and PPARγ in the 3T3-L1 mature adipocytes of different groups (D). Data are presented as the mean ± SEM. The statistical significance was determined by a one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001 or **** p < 0.0001, ns: not significant).

Figure 8

DOE is involved in the Akt signaling pathway to regulate protein synthesis and lipogenesis. Schematic illustration of the mechanism. A diagram of the proposed mechanism shows that DOE attenuates cachexia symptoms by targeting AKT via PI3K. DOE promotes the phosphorylation of PI3K and then AKT, leading to the downregulation of Atrogin1 and MuRF1 expression levels, and increasing mTOR activation, thus increasing protein synthesis and lipogenesis.