TRADITIONAL CHINESE MEDICINE-BASED AGENT FOR CACHEXIA PREVENTION AND TREATMENT

Abstract

This invention discloses a traditional Chinese medicine, Mu Dan Pi (Moutan radicis cort), that has the potential to be used for the prevention or treatment of cachexia and muscle loss in cancer patients. The composition is a novel therapeutic agent for cachexia.

Description

FIELD OF THE INVENTION

The present invention discloses a traditional Chinese medicine (TCM)-based agent, Mu Dan Pi (MDP) (Moutan radicis cort), which can be used to prevent or treat tumor-induced muscle atrophy and cachexia in cancer patients. This agent is developed through an integrated, multi-tiered strategy involving both in vitro and in vivo muscle atrophy platforms from an in-house TCM library.

BACKGROUND OF THE INVENTION

A major challenge in the treatment of certain types of cancers, including those of pancreas, stomach, lung and colon, is the accompanying cancer-induce body weight loss, which is characterized by anorexia and loss of adipose tissues and skeletal muscle masses, in terms of cachexia. Given no broad consensus on definitions of cancer cachexia, the present of at least three features among the following five characteristics was considered as cachexia: decreased muscle strength, fatigue, anorexia or limited food intake, low fat-free mass index and abnormal biochemistry (e.g., increasing C-reactive protein, anemia, or low serum albumin), in addition to edema-free weight loss 5% (or a body mass index (BMI)<20.0 kg/m² in 12 months. As a result, the body weight loss due to cancer cachexia cannot be reversed by nutritional support, and has severe impacts on the morbidity, mortality, and quality of life of cancer patients. Although substantial advances have been made in understanding the multifactorial pathophysiology of cachexia, prevention and/or treatment of this debilitating disease remains an unmet medical need.

Currently, no approved targeted therapy is available for cachexia treatment. The semi-synthetic progestational steroid, megestrol, is used to ameliorate cachexia-associated symptoms.

In addition, a number of Kampo medicines (i.e., multi-component herbal extracts) have been commonly prescribed in Japan to alleviate fatigue and chronic weakness in cachexia patients, which act upon the immune system to improve inflammatory and nutritional status. More recently, although the hunger hormone ghrelin and ghrelin mimetics have received much attention in light of their potential to enhance appetite and quality of life, clinical evidence is lacking to support their use for the treatment of cachexia.

The advantage of TCMs over small-molecule targeted agents for the prevention and/or treatment of cachexia is multifold. First, the therapeutic utility of the polypharmacology (or network pharmacology) of TCMs is manifested by their long-standing history in the treatment of various chronic and complex diseases. Second, many TCMs could be consumed as dietary supplements on a daily basis for disease control and prevention. Third, TCMs are generally perceived in oriental societies as having fewer side effects, which might lead to better compliance in cancer patients with muscle atrophy.

SUMMARY OF THE INVENTION

In view of the above technical circumstances, the present invention provides a TCM composition for the treatment and/or prevention of cachexia with a definite clinical benefit, preparations thereof, and a method for preparing the same.

This invention discloses a TCM, Mu Dan Pi (MDP) that has the potential to be used for the prevention or treatment of cachexia in cancer patients. The MDP extract is prepared by soaking the TCM in 50% to 100% methanol or 50% to 100% ethanol.

The present invention also provides a method of treating or preventing cancer-induced cachexia, comprising the administration of effective amounts of the composition to a subject with tumor-associated cachexia, wherein the composition comprises a Moutan radicis cort or a Moutan radicis cort extract. The extract of MDP is prepared by soaking the TCM in 50% to 100% methanol or 50% to 100% ethanol.

The present invention further provides a composition for preventing or treating skeletal muscle atrophy in cancer patients, which is attributable to its ability to reverse tumor-induced reprogramming of muscle homeostasis-associated gene expression in skeletal muscles, thereby rescuing skeletal muscles from wasting. The extract of MDP is prepared by soaking the TCM in 50% to 100% methanol or 50% to 100% ethanol.

The present invention also provides a method treating or preventing cancer-induced losses of skeletal muscle mass, comprising the administration of effective amounts of the composition to a subject with tumor-associated skeletal muscle atrophy, which is attributable to its ability to reverse tumor-induced reprogramming of muscle homeostasis-associated gene expression in skeletal muscles, thereby rescuing skeletal muscles from wasting, wherein the composition comprises the Moutan radicis cort or extracts of Moutan radicis cort. The extract Mu Dan Pi (MDP) is prepared by soaking the TCM in 50% to 100% methanol or 50% to 100% ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate the effects of DMSO and different TCMs on C26CM-induced C2C12 myotube atrophy. FIG. 1A illustrates the experimental procedure for C26CM-induced atrophy of C2C12 myotubes. FIG. 1B illustrates the effects of DMSO and H3-14 on C26CM-induced atrophy of C2C12 myotubes. FIG. 1C illustrates myotube diameter of C26CM-induced C2C12 treated with different TCMs.

FIG. 2 illustrates the lack of protective effect of 24 TCM extracts on C26CM-induced atrophy of C2C12 myotubes.

FIGS. 3A-3D illustrate the time-dependent effects of MDP versus DR on age-associated mobility and/or total body contraction in C. elegans. FIG. 3A illustrates the mobility of C. elegans treated with DR. FIG. 3B illustrates the mobility of C. elegans treated with MDP. FIG. 3C illustrates the mobility of C. elegans treated with 100 μg/ml MDP. FIG. 3D illustrates the relative total body contraction in C. elegans treated with 100 μg/ml MDP.

FIGS. 4A-4E illustrate the effects of MDP with three different doses (MDP-L, MDP-M, and MDP-H) versus vehicle via oral gavage on the body weight, tumor volume, protecting hindlimb muscles of C-26 tumor-bearing mice, and the alert and active phenotype of C-26 tumor-bearing mice. FIG. 4A illustrates the change of body weight of C-26 tumor-bearing mice treated with three different doses of MDP. FIG. 4B illustrates the change of body weight w/o tumor of C-26 tumor-bearing mice treated with three different doses of MDP.

FIG. 4C illustrates the tumor volume of C-26 tumor-bearing mice treated with three different doses of MDP. FIG. 4D illustrates mean of mass normalized to control on three different sections of skeletal muscles (Quad, GC, and TA) of hindlegs of C-26 tumor-bearing mice treated with three different doses of MDP. FIG. 4E illustrates the alert and active phenotype of C-26 tumor-bearing mice treated with three different doses of MDP.

FIGS. 5A-5C illustrate the effects of DR with 100 mg/kg versus vehicle via oral gavage on body weight (w/o tumor) and tumor volume. FIG. 5A illustrates the change in body weight w/o tumor of C-26 tumor-bearing mice treated with 100 mg/kg DR. FIG. 5B illustrates the photograph of C-26 tumor-bearing mice treated with three different treatments (w/o tumor, vehicle, and DR). FIG. 5C illustrates tumor volume of C-26 tumor-bearing mice treated with 100 mg/kg DR.

FIGS. 6A-6F illustrate a duplicate experiment showing the effects of MDP with 1000 mg/kg (MDP-H) versus vehicle via oral gavage on the body weight, tumor volume, its ability to diminish cachexia-associated decreases in skeletal muscle weights, rescuing the fiber size distribution from shifting to smaller cross-sectional areas, and restore the forelimb grip strength at day 17 of C-26 tumor-bearing mice. FIG. 6A illustrates the change in body weight of C-26 tumor-bearing mice treated with 1000 mg/kg MDP (MDP-H). FIG. 6B illustrates the change in body weight w/o tumor of C-26 tumor-bearing mice treated with 1000 mg/kg MDP (MDP-H). FIG. 6C illustrates tumor volume of C-26 tumor-bearing mice treated with 1000 mg/kg MDP (MDP-H). FIG. 6D illustrates mean of mass normalized to control on three different sections of skeletal muscles (Quad, GC, and TA) of hindlegs of C-26 tumor-bearing mice treated with 1000 mg/kg MDP (MDP-H). FIG. 6E illustrates muscle fiber size of C-26 tumor-bearing mice treated with 1000 mg/kg MDP (MDP-H). FIG. 6F illustrates the forelimb grip strength of C-26 tumor-bearing mice treated with 1000 mg/kg MDP (MDP-H).

FIGS. 7A-7C illustrate the effects of MDP on serum IL-6 in Veh-versus MDP-H-treated C26-tumor-bearing mice and its cell viability to C26 cells. FIG. 7A illustrates the mean serum IL-6 levels in the serum of C26 tumor-bearing mice treated with 1000 mg/kg MDP (MDP-H). FIG. 7B illustrates the secretion of serum IL-6 level by C26 cells treated with three treatments (DMSO, 25 μg/ml MDP, and 50 μg/ml MDP). FIG. 7C illustrates cell viability of C26 cells treated with three treatments (DMSO, 25 μg/ml MDP, and 50 μg/ml MDP).

FIGS. 8A-8B illustrate the bioinformatic analysis of shotgun sequencing. FIG. 8A illustrates two dimensional projection of RNA-seq data from the three study groups (T/Veh, TF/Veh, T/MDP). FIG. 8B illustrates a Venn diagram of total differentially expressed genes from the three study groups (T/Veh, TF/Veh, T/MDP).

FIGS. 9A-9B illustrate the effects of MDP on skeletal muscle-related genes and protein expressions. FIG. 8A illustrates the relative expression level of different skeletal muscle-related genes and proteins from MDP-H-treated mice. FIG. 9B illustrates western blot analysis of the protein expression levels of MuRF1 and Atrogin-1 in skeletal muscles from vehicle- and MDP-H-treated mice (T/Veh, TF/Veh, T/MDP).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” are used interchangeably.

In one embodiment, the DR and MDP showed their abilities to suppress the inflammatory cytokine-induced atrophy effect of C2C12 myotubes.

In one embodiment, MDP ameliorates age-associated decreases in the mobility of C. elegans.

In one embodiment, the MDP prevents tumor-induced muscle wasting in C26 tumor-bearing mice.

In one embodiment, the MDP is effective in protecting hindlimb muscles, including quadriceps and tibialis anterior, against cancer-induced wasting.

In one embodiment, the MDP shows in vivo efficacy in protecting mice from C-26 tumor-induced body weight loss.

In another embodiment, the MDP diminishes cachexia-associated decreases in skeletal muscle weights.

In one embodiment, the MDP is able to rescue the fiber size distribution from shifting to smaller cross-sectional areas in cachectic muscles (P<0.05).

In one embodiment, the MDP reduces serum IL-6 levels.

In one embodiment, the MDP exerts the anti-cachectic effect by reversing tumor-induced reprogramming of muscle homeostasis-associated gene expression in skeletal muscle.

The present invention provides a method of treating or preventing cancer-induced cachexia, comprising the administration of effective amounts of a composition to a subject with cancer-induced cachexia, wherein the composition comprises a Moutan radicis cort or a Moutan radicis cort extract.

The present invention also provides a method of treating or preventing cancer-induced skeletal muscle weight losses, comprising the administration of effective amounts of a composition to a subject with cancer-induced muscle atrophy, wherein the composition comprises a Moutan radicis cort or a Moutan radicis cort extract.

In one embodiment, the Moutan radicis cort extract is prepared by soaking the Moutan radicis cort in 50% to 100% methanol or 50% to 100% ethanol.

In a preferred embodiment, the Moutan radicis cort extract is prepared by soaking the Moutan radicis cort in 70% methanol or 70% ethanol.

Example

The examples below are non-limited and are merely representative of various aspects and features of the present invention.

Example 1: C26CM-Induced Myotube Atrophy Model

The experimental design is depicted in FIG. 1A C2C12 myoblasts are exposed to 2% horse serum-containing differentiation medium (DM) for 4 days to facilitate their differentiation into myotubes, followed by treatment with C26CM for 4 days. At the end of this 8-day treatment, diameters of C26CM-treated C2C12 myotubes versus those of control cells (receiving DM only) are analyzed under light microscopy, of which the difference was quantified as a readout of C26CM-induced atrophy. In order to recapitulate the use of TCMs to delay the onset of muscle wasting, individual TCMs versus DMSO are added to culture media from the beginning throughout the course of experiment with daily replacement (H3-14 as another control), each of which was repeated four times. As shown, C26CM caused significant narrowing in C2C12 myotubes (FIG. 1B & FIG. 1C).

As shown in FIG. 1, the effects of different TCMs on C26CM-induced C2C12 myotube atrophy was shown. Schematic representation of the experimental design was shown in FIG. 1A. FIG. 1B was the image. FIG. 1C were the two quantitative analyses of image of FIG. 1B about the protective effect of Dioscoreae rhizome (DR), MDP, and three other representative TCMs on C26CM-induced atrophy of C2C12 myotubes of the representative experiments. Bar, means±S.D. (for data from two independent experiments Expt #1 and Expt #2).

Extracts of different TCMs, each at 25 and/or 50 μg/ml, were evaluated for their anti-atrophy activities, including Dioscoreae rhizome (DR), MDP, Sambuci chinensis radix et caulis (SCRC), Helminthostachydis radix et rhizome (HRR), Condonopsis radix (CR), Polygonati odorati rhizome, Glycyrrhizae radix et rhizome, Lilii bulbus, Citri sarcodactylis fructus, Euryales semen, Hordei fructus germinates, Siraitiae fructus, Pruni semen, Lycii fructus, Poria, Platycodonis radix, Bombycis chrysallidem, Alpiniae oxyphyllae Fructus, Nelumbinis semen, Polygonati rhizome, Sesami nigrum semen, Ziziphi spinosae semen, Coicis semen, Rubi fructus, Ginseng radix et rhizome, Amynthas et metaphire, Arctii radix, Portulacae herba, and Trionycis carapax. Among these TCM extracts, we found two widely used TCMs, DR and MDP, shared the ability of H3-14 to fully protect C2C12 myotubes from C26CM-induced atrophy (all Ps=0.0002 vis-à-vis C26CM control in the myotube atrophy platform, FIG. 1C), but others were either cytotoxic, or provided no protection, of which the representative data were shown in FIG. 1C.

Data of other TCMs were shown in FIG. 2. Bar, means±S.D. (N=4). #1 was Polygonati odorati rhizoma; #2 was Glycyrrhizae radix et rhizome; #3 was Lilii bulbus; #4 was Citri sarcodactylis fructus; #5 was Euryales semen; #6 was Hordei fructus germinates; #7 was Siraitiae fructus; #8 was Pruni semen; #9 was Lycii fructus; #10 was Poria; #11 was Platycodonis radix; #12 was Bombycis chrysallidem; #13 was Alpiniae oxyphyllae fructus; #14 was Nelumbinis semen; #15 was Polygonati rhizoma; #16 was Sesami nigrum semen; #17 was Ziziphi spinosae semen; #18 was Coicis semen; #19 was Rubi fructus; #20 was Ginseng radix et rhizoma; #21 was Amynthas et metaphire; #22 was Arctii radix; #23 was Portulacae herba; #24 was Trionycis carapax.

Example 2: C. elegans Mobility Testing of DR and MDP

In this phenotypic assay of nematode C, elegans, MDP and DR were dissolved in 1% DMSO-containing water at 10 mg/ml as stock solutions, and 100 μl of individual solutions versus vehicle control were evenly applied onto nematode growth medium (NGM) agar plates containing OP50 lawns (total volume of agar, 10 ml). After the solution was completely absorbed into agar, these OP50 plates were radiated under UV for 40 min, followed by seeding with about 50 synchronized eggs of CF512 worms. These plates were incubated at 25° C., and worm mobility was determined starting day 1 after adulthood every other day till day 7. Data, means±SEM. (n=170-420). *P<0.05; **P<0.01; ***P<0.0001 (t-test). The worms at different ages (day 1, 5, 9, 13 adults) were first incubated in drug-free solution and then in levamisole-containing solution for 10 minutes. The digital imaging system were used to quantify the length of the worm body using ImageJ. Data, means±SEM. (n>50). *P<0.05; **P<0.01; ***P<0.0001 (t-test).

As shown in FIG. 3A, there is no difference of time-dependent effects of Dioscorea radix (DR) as compared to control (vehicle) on age-associated mobility and/or total body contraction in C. elegans.

As shown in FIG. 3B, MDP could ameliorate age-associated decreases in C. elegans mobility relative to control.

As shown in FIG. 3C, the time-dependent effects of MDP showed parallel protective effects of mobility in a separate experiment with longer life-expectancy (body bending/second on day 1, 3, 5, 7 and 9).

As shown in FIG. 3D, MDP significantly delayed the age-associated loss of total body contraction (%) in muscle functions (relative total body contraction on day 1, 5, 9 and 13).

To sum up, MDP exhibited a unique ability to ameliorate age-associated decreases in worm mobility relative to control, but not for DR.

Example 3: MDP Efficacy in the C26 Model of Cancer Cachexia

The C-26 model is to confirm the in vivo anti-muscle wasting efficacy of MDP versus DR for their abilities to protect CD2F1 mice from C-26 tumor-induced body weight loss, which was reported to be associated with excessive IL-6 secretion by the tumor.

In the first set of experiments, the in vivo efficacy of MDP at three different doses (low dose: MDP-L, 100 mg/kg; medium dose: MDP-M, 500 mg/kg; high dose: MDP-H, 1,000 mg/kg) was evaluated via oral gavage once daily to CD2F1 mice starting at 7 days before C-26 tumor cells were implanted till mice were sacrificed at day 17. The body weight, tumor size, and food and water consumption of individual mice were measured every other day. At sacrifice, hindleg skeletal muscles were dissected and stored at −80° C. for further analysis after the weights were measured. The first set of experiment was shown in FIG. 4.

FIG. 4 illustrates the effects of MDP at three doses (MDP-L, MDP-H, and MDP-H), vehicle and control (H3-14) via oral gavage on the body weight (FIG. 4A & FIG. 4B) and tumor volume (FIG. 4C) of C-26 tumor-bearing mice. NC, tumor-free mice. Tumor weights were estimated based on the assumption that 1,000 mm³ equals to 1 grain of body weight. S.D. bars are not shown to avoid over congestion of these graphs. For statistical analysis, generalized linear mixed-effects models with random intercept for individual subject and fixed effects for treatment and days of the treatment were used to test for differences among groups, using the Tukey-Kramer correction for multiple comparisons. (a) Significant difference from the control group at p<0.05. (b) Significant difference from the vehicle group at p<0.05. In FIG. 4D, the effects of MDP at three doses, vehicle and H3-14 via oral gavage on three different sections of skeletal muscles of hindlegs of C-26 tumor-bearing mice was shown. NC, tumor-free mice. (a) and (b) denote significant difference from NC group and vehicle group, respectively (Kruskal-Wallis test, with Dunn's multiple-comparison test using Bonferroni adjustment, p<0.05). FIG. 4E shown the photographs of one representative mouse from these five groups at the study endpoint depicting the therapeutic effect of MDP-H and MDP-M in tumor-bearing mice, as shown by alertness, normal posture, smooth haircoat, and better body conditions, despite large tumor burden.

As shown in FIG. 4A, MDP-H was effective in ameliorating body weight losses in C-26 tumor-bearing mice.

As shown in FIG. 4B, MDP-H was effective in ameliorating body weight losses (without tumor) in C-26 tumor-bearing mice.

As shown in FIG. 4C, MDP-H showed a very modest tumor-suppressive effect on tumor growth.

As shown in FIG. 4D, MDP-M was effective in protecting hindlimb muscles C-26 tumor-bearing mice.

As shown in FIG. 4E, C-26 tumor-bearing mice treated with MDP-M and MDP-H exhibited an alert and active phenotype.

In the second set of experiments, the in vivo efficacy of DR at 100 mg/kg versus vehicle via oral gavage was evaluated, which was shown in FIG. 5.

FIG. 5 illustrated the effects of DR at 100 mg/kg versus vehicle via oral gavage on body weight (w/o tumor) (FIG. 5A) and tumor volume (FIG. 5C) of C-26 tumor-bearing mice (*P<0.05; n=3-6). Control, tumor-free mice. (FIG. 5B) Photographs of representative mice from each group at the study endpoint depicting lack of therapeutic effect of DR. (a) and (b) denote significant difference from the control group and the vehicle group, respectively (Generalized linear mixed-effects models with Tukey-Kramer correction for multiple comparisons).

As shown in FIG. 5A, DR at 100 mg/kg exacerbated body weight loss.

As shown in FIG. 5B, DR at 100 mg/kg caused deterioration of physical appearances relative to vehicle control.

As shown in FIG. 5C, DR at 100 mg/kg had no effect on tumor growth.

FIG. 6 illustrated a duplicate experiment showing the effects of MDP at 1000 mg/kg (MDP-H) versus vehicle via oral gavage on the body weight (FIG. 6A & FIG. 6B), tumor volume (FIG. 6C) of C-26 tumor-bearing mice. NC, tumor-free mice. Tumor weights were estimated based on the assumption that 1,000 mm³ equals to 1 grain of body weight. S.D. bars are not shown to avoid over congestion of these graphs. For statistical analysis, generalized linear mixed-effects models with random intercept for individual subject and fixed effects for treatment and days of the treatment were used to test for differences among groups, using the Tukey-Kramer correction for multiple comparisons. Significant difference from the control group at p<0.05. (b) Significant difference from the vehicle group at p<0.05. FIG. 6D shown the effects of MDP-H versus vehicle on three different sections of skeletal muscles of hindlegs of −26 tumor-bearing mice. Control, tumor-free mice. (a) and (b) denote significant difference from control group and vehicle group, respectively (Kruskal-Wallis test, with Dunn's multiple-comparison test using Bonferroni adjustment, p<0.05). FIG. 6E shown the effects of MDP-H on muscle fiber size in C-26 tumor-bearing mice. The cross-sectional areas of muscle fibers in gastrocnemius muscles represented as a frequency histogram. Five sections from the gastrocnemius obtained from each of five mice per treatment group were analyzed as described in the Methods section. Using multiple comparisons for the log-rank test, comparison between muscles from tumorbearing/vehicle and tumor-bearing/MDP mice showed statistical significance (P<0.001). Data are presented as means. FIG. 6F shown the effects of MDP on grip strength.

As shown in FIG. 6A, MDP-H showed protecting mice from C-26 tumor-induced body weight loss on tumor burden at day 17.

As shown in FIG. 6B, MDP-H showed protecting mice from C-26 tumor-induced body weight loss (without tumor) on tumor burden at day 17.

As shown in FIG. 6C, MDP-H showed no significant suppressive effect on tumor burden at day 17.

As shown in FIG. 6D, MDP-H showed its ability to diminish cachexia-associated decreases in skeletal muscle weights.

As shown in FIG. 6E, MDP-H was able to rescue the fiber size distribution from shifting to smaller cross-sectional areas in cachectic muscles through immunostaining with anti-dystrophin of GC myofibers followed by quantification of myofiber diameter (P<0.0001).

As shown in FIG. 6F, MDP-H was able to restore the forelimb grip strength at day 17 (P<0.001).

FIG. 7 shown the effect of MDP on serum IL-6 in Veh-versus MDP-H-treated C26-tumor-bearing mice. Wilcoxon rank sum test was used for statistical analysis (n=5). FIG. 7B shown the effects of MDP at 25 and 50 μg/mL on IL-6 production in C26 cell culture medium and FIG. 7C shown the viability of C26 cells. Bar, means±S.D. (for data obtained from three independent experiments for (B) and (C), where ****, P<0.0001.

As shown in FIG. 7A, the mean serum IL-6 (a major driver in the C-26 tumor model of cachexia) levels in MDP-H-treated C-26 tumor-bearing mice was lower but no statistically significant different from that of vehicle control (P=0.06).

As shown in FIG. 7B, the diminished serum IL-6 level was associated with the unique ability of MDP at 25 and 50 μg/mL to suppress IL-6 secretion into culture medium by C26 cells (P<0.001).

As shown in FIG. 7C, MDP at 25 and 50 μg/mL was non-cytotoxic to C26 cells.

Example 4: Whole Transcriptome Shotgun Sequencing (RNA-Seq) Analysis

Whole transcriptome shotgun sequencing (RNA-seq) analysis was conducted by a commercial vendor (Welgene Biotech; Taiwan). Subsequently, these RNA-seq data were subjected to principal component analysis (PCA) to interrogate transcriptome variations among these groups. This clustering of expression profiles suggests that MDP was able to shift the gene expression profile of cachectic skeletal muscles (T/Veh) to a state similar to that of non-cachectic muscles (TF/Veh). Furthermore, pair comparisons of RNA-seq data was analyzed the differences in gene expression profiles among individual groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) Knowledgebase described the related biological signaling pathways.

As shown in FIG. 8A, two-dimensional projection of RNA-seq data from the three study groups. The principal component analysis axes (Dim1, x-axis; Dim2, y-axis) emphasize the overall variation in RNA-seq data. As shown in FIG. 8B, each black dot represents a transformed gene expression value. Venn diagram of differentially expressed genes in each of the two pairwise comparisons of T/Veh versus TF/Veh and T/Veh versus T/MDP.

As shown in FIG. 8A, the principal component analysis (PCA) plot shows that the two-dimensional projection of the variation in the T/MDP group was much closer to that of the TF/Veh group than to that of the T/Veh group.

As shown in FIG. 8B, Venn diagram analysis reveals a total of 1849 differentially expressed genes shared by the two pairwise comparisons (center portion) that showed changed expression in the same direction.

The above bioinformatic analyses demonstrated the ability of MDP to reprogram the expression of genes associated with muscle homeostasis in cachectic skeletal muscles, which are reflected by the top twenty most up-versus down-regulated genes of the following Table 1 and Table 2.

TABLE 1
Top 20 most upregulated genes in skeletal muscles of MDP-
versus vehicle-treated C-26 tumor-bearing mice.
Log2
ratio
(MDP/	NCBI	Gene	Gene
Veh)	gene ID	name	description	Gene functions
17.3	100504362	Ccl21a	chemokine	Ccl21 acts as a chemoattractant of
			(C-C motif)	many types of immune cells via
			ligand 21A	the cell surface receptor CCR7.
			(serine)	CCR7 plays a role in regulating
				energy metabolism by
				suppressing brown adipose
				tissues, which is involved in the
				development of cancer cachexia
15.7	20293	Ccl12	chemokine	Ccl12/MCP-5 specifically
			(C-C motif)	attracts eosinophils, monocytes
			ligand 12;	and lymphocytes by signaling
			aka,	through the receptor CCR2,
			monocyte	which plays a critical role in
			chemotactic	muscle regeneration following
			protein 5	injury.
			(a.k.a.,
			MCP-5)
15.7	16846	Lep	leptin	Deficiency in leptin has been
				associated with reduced skeletal
				muscle mass in genetically
				engineered mice, and treatment
				with exogenous leptin could
				reverse the muscle loss by
				inhibiting myofibrillar protein
				degradation as well as enhancing
				muscle cell proliferation
6.63	16545	Kera	Keratocan	One of the small leucine-rich
				repeat proteoglycans (SLRPs)
				identified as FoxO-regulated
				transcripts downregulated in
				cachectic muscle; located in the
				ECM where they regulate the
				structure and integrity of the
				ECM.
6.61	545798	Tmem233	Transmembrane	Highly and specifically expressed
			protein 233	in skeletal muscles, of which the
				functions remain unclear.
6.05	12643	Chad	Chondroadheri	One of the small leucine-rich
				repeat proteoglycans (SLRPs)
				identified as FoxO-regulated
				transcripts downregulated in
				cachectic muscle; located in the
				ECM where they regulate the
				structure and integrity of the
				ECM. Chad is one of the most
				downregulated genes in the
				skeletal muscles of C26 tumor-
				bearing mice
5.50	16716	Ky	Kyphoscoliosis	This muscle-specific protein
			peptidase	plays a vital role in muscle
			(Ky)	growth; the absence of Ky protein
				leads to muscular dystrophy.
5.46	68709	Clip	Cartilage	The two isoforms of Cilp (Cilp-1
			intermediate	and Cilp-2), are components of
			layer	extracellular matrix of cartilage,
			protein	which play a role in cartilage
			(Clip)	scaffolding. Downregulation of
				Cilp is involved in joint
				pathologies such as osteoarthritis.
				Cilp is one of the most
				downregulated genes in the
				skeletal muscles of C26 tumor-
				bearing mice.
5.16	403183	Mettl21e	Methyltransferase	This skeletal muscle-specific
			like 21E	lysine methyltransferase acts an
				important modulator of
				autophagy-associated protein
				degradation in skeletal muscles,
				and ablation of Mettl21c in mice
				results in muscle weakness and
				disturbance of the protein
				degradation machinery.
5.15	238564	Mylk4	Myosin	One of the members of the
			light chain	Myosin light chain kinase
			kinase	(MLCK) family which act as
			family,	regulatory proteins in smooth
			member 4	muscle contraction. MYLK4
				expression was reported to
				increase in skeletal muscles after
				high-intensity intermittent
				exercise training.
5.12	66733	Kcng4	Potassium	Kcng4 is one of the most
			voltage-	downregulated genes in the
			gated	skeletal muscles of C26 tumor-
			channel,	bearing mice. Voltagegated
			subfamily	potassium (Kv) channels regulate
			G, member	diverse physiological functions,
			4	including neurotransmitter
				release, heart rate, insulin
				secretion, neuronal excitability,
				epithelial electrolyte transport,
				smooth muscle contraction, and
				cell volume.
4.90	18802	Plcd4	Phospholipase C,	Plcd4 is one of the most
			delta 4	downregulated genes in the
				skeletal muscles of C26 tumor-
				bearing mice. Most abundant in
				skeletal muscles, and responsible
				for the production of important
				second messengers, including
				inositol trisphosphate and
				diacylglycerol.
4.80		Vwa3b	von	Intracellular proteins with VWA
			Willebrand	domains are thought to function
			factor A	in transcription, DNA repair,
			domain	ribosomal and membrane
			containing	transport, and the proteasome.
			3B	Mutations in this gene are
				associated with Spinocerebellar
				ataxia.
4.51	103968	Plin1	Perilipin 1	Perilipin 1 promotes
				triacylglycerol storage under
				basal conditions by reducing the
				access of cytosolic lipases to
				triacylglycerol substrates stored
				in lipid droplets. Expression of
				perilipins in human skeletal
				muscle in vitro and in vivo in
				relation to diet, exercise and
				energy balance.
4.49	12377	Casq1	Calsequestrin 1	A Ca2+-binding protein that acts
				as a Ca2+ buffer within the
				sarcoplasmic reticulum (SR),
				helping storing Ca2+ in the
				cisterna of the SR after muscle
				contractions.
4.48	16420	Itgb6	Integrin	Itgb6 mRNA was found highly
			beta 6	enriched in skeletal muscles.
				Evidence suggests that Itgb6
				protein is upregulated post-
				transcriptionally in response to
				muscle injury, which might be
				involved in the remodeling of
				extracellular matrix via TGF
				signaling.
4.43	64103	Tnmd	Tenomodulin	A marker of tendons and
				ligaments that integrate
				musculoskeletal components, and
				its loss-of-function in mice leads
				to a phenotype with distinct signs
				of premature aging at the tissue
				and stem/progenitor cell levels.
4.37	17306	Sypl2	Synaptophy-	SYPL2 is thought to participate in
			sin-like 2	the excitation-contraction
				coupling process of skeletal
				muscle as SYPL2-null mice
				showed reduced muscle
				contractile force and altered triad
				junction structure and increased
				susceptibility to fatigue of the
				skeletal muscle.
4.32	74843	Mss51	MSS51	Mss51 was predominantly
			mitochondrial	expressed in skeletal muscles and
			translationa1	in those muscles dominated by
			activator	fast-Twitch fibers. In vitro, its
				expression was upregulated upon
				differentiation of C2C12
				myoblasts into myotubes.
4.22	433294	Mettl21c	Methyltransferase	Ablation of Mettl21c in mice
			like 21C	resulted in muscle weakness and
				disturbance of the protein
				degradation machinery.

TABLE 2
Top 20 most downregulated genes in skeletal muscles of
MDP-versus vehicle-treated C-26 tumor-bearing mice.
Log2
ratio
(MDP/	NCBI	Gene	Gene
Veh)	gene ID	name	description	Gene functions
−7.43	237320	Aldh8a1	Aldehyde	ALDH8A1 is involved in
			dehydrogenase	retinaldehyde metabolism,
			8	specifically the 9-cis retinal, and
			family,	in oxidation of aliphatic
			member A1	aldehydes and glutaraldehyde.
				Although ALDHs are reported to
				regulate skeletal muscle
				homeostasis in healthy
				individuals and patients with
				Duchenne muscular dystrophy,
				the exact role of ALDH8A1 in
				cachectic muscles remains
				unclear.
−5.85	16819	Lcn2	Lipocalin 2	One of the most upregulated
				genes in the skeletal muscles of
				C26 tumorbearing mice.
				Lipocalin was recently reported
				to be a pathologic mediator of
				cachexia through the
				melanocortin 4 receptor in the
				mediobasal hypothalamus. Its
				expression is closely associated
				with reduced food consumption
				and lean mass loss, and lipocalin
				2 knockout mice are protected
				from cachexia.
−5.07	17750	Mt2	Metallothionein	One of the most upregulated
				genes in the skeletal muscles of
				C26 tumorbearing mice.
				Concomitant abrogation of
				metallothioneins 1 and 2 resulted
				in activation of the Akt pathway
				and increases in myotube size,
				and ultimately in muscle strength
				mass and strength.
−5.04	13419	Dnase1	DNase I	The functional role of DNase I in
				cachectic muscles remains
				uncharacterized
−4.83	17748	Mt1	Metallothionein	Concomitant abrogation of
			1	metallothioneins 1 and 2 resulted
				in activation of the Akt pathway
				and increases in myotube size,
				and ultimately in muscle strength
				mass and strength.
−4.51	213053	Slc39a14	Solute	ZIP14 regulates zinc homeostasis
			carrier	in skeletal muscle, and represents
			family 39	a critical mediator of cancer-
			(zinc	induced cachexia by facilitating
			transporter),	zinc accumulation, leading to
			member	muscle atrophy and blocked
			14 (a.k.a.,	muscle regeneration.
			ZIP14)
−4.49	236690	Nyx	Nyctalopin	Nyctalopin is one of the SLRP
				members. Although it was
				reported to be essential for
				synaptic transmission in the cone
				dominated zebrafish retina, its
				role in cachectic muscles remains
				uncharacterized.
−4.37	74127	Krt80	Keratin 80	KRT80 is a filament protein that
				make up one of the major
				structural fibers of epithelial
				cells. However, its role in
				cachectic muscles remains
				uncharacterized.
−4.29	20717	Serpina3m	Serine (or	One of the most upregulated
			cysteine)	genes in the skeletal muscles of
			peptidase	C26 tumor bearing mice.
			inhibitor,	Serpina3m is likely involved in
			clade A,	neuromuscular junction
			member	maintenance and/or stability, as
			3M	its expression is induced and
				localized at the motor endplate
				following denervation, and this
				effect is augmented in a rodent
				model of enhanced reinnervation.
−4.22	16529	Kcnk5	Potassium	Inhibition of TASK2 during
			channel,	differentiation revealed a
			subfamily	morphological impairment of
			K, member	myoblast fusion accompanied by
			5 (a.k.a.,	a downregulation of maturation
			TASK2)	markers in C2C12 cells.
−4.11	55985	Cxcl13	Chemokine	A significant upregulation of
			(C-X-C	CXCL13 was found in the CNS
			motif)	of the amyotrophic lateral
			ligand 13	sclerosis mice, indicating a direct
				correlation between the activation
				of the chemokine and a faster
				disease progression.
−4.00	216725	Adamts2	A	ADAM-TS2 is also known as
			disintegrinlike	procollagen I N-proteinase (PC I-
			and	NP). ADAMTS2 is responsible
			metallopeptidase	for processing several types of
			(reprolysin	procollagen proteins.
			type) with	Procollagens are the precursors of
			thrombospondin	collagens, the proteins that add
			type 1	strength and support to many
			motif, 2	body tissues.
			(ADAM-TS2)
−3.93	11717	Ampd3	Adenosine	AMPD3 facilitates adenine
			monophosphate	nucleotide degradation in skeletal
			deaminase	muscles, which was found to
			3 (AMPD3)	accelerate protein degradation in
				C2C12 myotubesand to
				contribute to the pathophysiology
				of skeletal muscle atrophy.
−3.87	14085	Fah	Fumarylacetoacetate	This gene encodes the last
			hydrolase	enzyme in the tyrosine
				catabolism pathway, of which the
				role in cachectic muscles remain
				uncharacterized.
−3.81	13447	Doc2b	Double C2,	One of the most upregulated
			beta	genes in the skeletal muscles of
				C26 tumor bearing mice. Doc2b
				is a key positive regulator of
				Muncl8c-syntaxin 4-mediated
				insulin secretion as well as of
				insulin responsiveness in skeletal
				muscle, and thus a key effector
				for glucose homeostasis in vivo.
−3.69	93732	Acox2	Acyl-	ACOX2 encodes branched-chain
			Coenzyme	acylCoA oxidase, a peroxisomal
			A oxidase	enzyme believed to be involved
			2, branched	in the metabolism of branched-
			chain	chain fatty acids and bile acid
				intermediates
−3.66	432516	Myo1a	Myosin IA	Myosins are a superfamily of
				motor proteins best known for
				their roles in muscle contraction
				and in a wide range of other
				motility processes in eukaryotes.
−3.65	233011	Itpkc	Inositol	ITPKC catalyzes the
			1,4,5-	phosphorylation of inositol 1,4,5-
			trisphosphate	trisphosphate to 1,3,4,5-
			3-kinase	tetrakisphosphate, and has been
			C (ITPKC)	proposed as a prognostic and
				predictive biomarker of
				neoadjuvant chemotherapy for
				triple negative breast cancer.
−3.52	68738	Acss1	Acyl-CoA	ACSS1 is a mitochondrial matrix
			synthetase	enzyme that is strongly expressed
			short-chain	in heart, skeletal muscle and
			family	brown adipose tissue.
			member 1
			(ACSS1)
−3.41	16009	Igfbp3	Insulin-like	IGFBP-3 potently leads to
			growth	impaired myogenesis and
			factor	enhanced muscle protein
			binding	degradation, the major features of
			protein 3	muscle wasting, via IGF
			(IGFBP-3)	signaling inhibition.

As shown in FIG. 9, the effects of MDP on skeletal muscle-related genes and protein expressions. As shown in FIG. 9A, 5 upregulated (left) and 5 downregulated (right) genes in skeletal muscles from vehicle-treated versus MDP-H-treated mice (n=3 for each group) were shown by qPCR analysis. The fold increase and % expression on upregulated and downregulated genes were shown as relative expression levels of selected skeletal muscle-related genes of MDP-H-treated mice to the vehicle counterparts, respectively. Bar, means+S.D (n=3). As shown in FIG. 9B, western blot analysis of the protein expression levels of MuRF1 and Atrogin-1 in skeletal muscles from vehicle- and MDP-H-treated C26 tumor-bearing mice (T/Veh and T/MDP-H, respectively) vis-à-vis vehicle-treated tumor-free mice (TF/Veh) were shown, respectively. The forward primer and reverse primer of the quantitative RT-PCR was shown in the following Table 3.

TABLE 3
Primer sequences used for quantitative
RT-PCR analysis
Gene	Forward/
name	Reverse	Sequence (5’-3’)
18s	Forward	AGAAACGGCTACCACATCCA
rRNA		(SEQ ID NO: 1)
	Reverse	CCCTCCAATGGATCCTCGTT
		(SEQ ID NO: 2)
Kera	Forward	CAGCCACAGGACTCAACGG
		(SEQ ID NO: 3)
	Reverse	AGTAGGGAAACTGGGAGGACA
		(SEQ ID NO: 4)
LEP	Forward	AAGGGGCTTGGGTTTTTCCA
		(SEQ ID NO: 5)
	Reverse	CAGACAGAGCTGAGCACGAA
		(SEQ ID NO: 6)
Ky	Forward	ACAGTCAATGGGAAAGCCACA
		(SEQ ID NO: 7)
	Reverse	CTCCAGCTTCATCCCGTTCT
		(SEQ ID NO: 8)
Chad	Forward	CAACTCGTTTCGGACCATGC
		(SEQ ID NO: 9)
	Reverse	GATGTCGTTGTGGGACAGGT
		(SEQ ID NO: 10)
Mettl21e	Forward	GCCATCGGCCCTTGTTCTAT
		(SEQ ID NO: 11)
	Reverse	TAGCAATCACACGAGCACCA
		(SEQ ID NO: 12)
Trim63	Forward	AGGGACTAGCATAGGGCTCC
		(SEQ ID NO: 13)
	Reverse	TGACAATCGCCAGTCACACA
		(SEQ ID NO: 14)
Fbxo32	Forward	CGGGGTTTGTTTTCAGCAGG
		(SEQ ID NO: 15)
	Reverse	ACACAGACATTGCCTCCCAG
		(SEQ ID NO: 16)
Lcn2	Forward	TGAGTGTCATGTGTCTGGGC
		(SEQ ID NO: 17)
	Reverse	GAACTGATCGCTCCGGAAGT
		(SEQ ID NO: 18)
Mt1	Forward	CTGTCCTCTAAGCGTCACCA
		(SEQ ID NO: 19)
	Reverse	AGCAGCTCTTCTTGCAGGAG
		(SEQ ID NO: 20)
Ampd3	Forward	ACAACTGACCTGTCCTCCCT
		(SEQ ID NO: 21)
	Reverse	CAAAGCTCAGCCCGTTAGGA
		(SEQ ID NO: 22)

As shown in FIG. 9A, the changes in the expression levels of upregulated and downregulated genes from qPCR analysis paralleled that of RNA-seq analysis.

As shown in FIG. 9B, MDP-H was effective in suppressing the protein expression of Atrogin-1 and MuRF1 in C26 tumor-bearing mice to the basal levels noted in the control group upon Western blotting analysis.

While the invention has been described and exemplified in sufficient details for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of this invention.

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The processes and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

SEQUENCE LISTING
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<INSDQualifier id = ″q23″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>reverse primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q22″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>gatgtcgttgtgggacaggt</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″11″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q25″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>forward primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q24″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>gccatcggcccttgttctat</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″12″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q27″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>reverse primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q26″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>tagcaatcacacgagcacca</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″13″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q29″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>forward primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q28″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>agggactagcatagggctcc</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″14″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q31″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>reverse primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q30″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>tgacaatcgccagtcacaca</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″15″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q33″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>forward primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q32″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>cggggtttgttttcagcagg</INSDSeq_sequence>
</lNSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″16″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q35″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>reverse primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q34″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>acacagacattgcctcccag</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″17″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q37″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>forward primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q36″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>tgagtgtcatgtgtctgggc</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″18″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q39″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>reverse primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q38″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>gaactgatcgctccggaagt</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″19″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q41″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>forward primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q40″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>ctgtcctctaagcgtcacca</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″20″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q43″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>reverse primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q42″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>agcagctcttcttgcaggag</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″21″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>1..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q45″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>forward primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q44″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>acaactgacctgtcctccct</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
<SequenceData sequenceIDNumber = ″22″>
<INSDSeq>
<INSDSeq_length>20</INSDSeq_length>
<INSDSeq_moltype>DNA</INSDSeq_moltype>
<INSDSeq_division>PAT</INSDSeq_division>
<INSDSeq_feature-table>
<INSDFeature>
<INSDFeature_key>source</INSDFeature_key>
<INSDFeature_location>l..20</INSDFeature_location>
<INSDFeature_quals>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>other DNA</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q47″>
<INSDQualifier_name>note</INSDQualifier_name>
<INSDQualifier_value>reverse primer</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id = ″q46″>
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>synthetic construct</INSDQualifier_value>
</INSDQualifier>
</INSDFeature_quals>
</INSDFeature>
</INSDSeq_feature-table>
<INSDSeq_sequence>caaagctcagcccgttagga</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
</ST26SequenceListing>

Claims

1. A method of treating or preventing cancer-induced cachexia, comprising the administration of effective amounts of a composition to a subject with cancer-induced cachexia, wherein the composition comprises a Moutan radicis cort or a Moutan radicis cort extract.

2. The method of claim 1, wherein the Moutan radicis cort extract is prepared by soaking the Moutan radicis cort in 50% to 100% methanol or 50% to 100% ethanol.

3. The method of claim 2, wherein the Moutan radicis cort extract is prepared by soaking the Moutan radicis cort in 70% methanol or 70% ethanol.

4. A method of treating or preventing cancer-induced skeletal muscle weight losses, comprising the administration of effective amounts of a composition to a subject with cancer-induced muscle atrophy or muscle weight loss, wherein the composition comprises a Moutan radicis cort or a Moutan radicis cort extract.

5. The method of claim 4, wherein the Moutan radicis cort extract is prepared by soaking the Moutan radicis cort in 50% to 100% methanol or 50% to 100% ethanol.

6. The method of claim 5, wherein the Moutan radicis cort extract is prepared by soaking the Moutan radicis cort in 70% methanol or 70% ethanol.

7. The method of claim 4, wherein the composition upregulates the muscle homeostasis-associated gene, comprising Cc121a, Cc112, Lep, Kera, Tmem233, Chad, Ky, Clip, Mett121e, Mylk4, Kcng4, Plcd4, Vwa3b, Plin1, Casq1, Itgb6, Tnmd, Syp12, Mss51, Mett121c. 27 or combination thereof to reduce cancer-induced muscle atrophy.

8. The method of claim 4, wherein the composition downregulates the muscle homeostasis-associated gene, comprising Aldh8a1, Lcn2, Mt2, Dnase1, Mt1, Slc39a14, Nyx, Krt80, Serpina3m, Kcnk5, Cxcl13, Adamts2, Ampd3, Fah, Doc2b, Acox2, Myo1a, Itpkc, Acss1, Igfbp3. or combination thereof to reduce cancer-induced muscle atrophy.