A Combination of Amaranth Protein Hydrolysate and Korean Mint Extract Ameliorates Cisplatin-Induced Nephrotoxicity and Cachexia in CT26 Tumor-Bearing BALB/c Mice

TL;DR

A combination of amaranth protein hydrolysate and Korean mint extract reduces kidney damage and muscle loss in mice undergoing chemotherapy for cancer.

Contribution

A novel combination therapy using amaranth protein hydrolysate and Korean mint extract is shown to alleviate cisplatin-induced nephrotoxicity and cachexia in tumor-bearing mice.

Findings

AKE reduced body weight loss and systemic inflammation in cisplatin-treated tumor-bearing mice.

AKE preserved skeletal muscle and adipose tissue mass and improved renal function markers.

AKE modulated protein turnover pathways and suppressed renal apoptosis in mice.

Abstract

Background/Objectives: Cancer cachexia involves progressive skeletal muscle and adipose tissue loss, which is further aggravated by cisplatin chemotherapy via increased systemic inflammation, tissue catabolism, and renal toxicity. The present study aimed to evaluate whether a combination of amaranth protein hydrolysate and Agastache rugosa extract (AKE) could attenuate cisplatin-associated cachexia and nephrotoxicity in CT26 tumor-bearing mice. Methods: Cancer cachexia was induced by subcutaneous CT26 cell inoculation in 6-week-old male BALB/c mice, followed by a 7-day tumor establishment period. Cisplatin was then administered intraperitoneally, and AKE (125 or 250 mg/kg/day) was given daily by oral gavage for 14 days. Results: AKE administration significantly alleviated cisplatin-induced body weight loss and systemic inflammation, accompanied by preservation of skeletal muscle and adipose tissue mass, as well as increased myofiber cross-sectional area and adipocyte size. AKE markedly reduced serum inflammatory cytokines, blood urea nitrogen, and creatinine levels, indicating protection against cisplatin-induced renal injury. Mechanistically, AKE suppressed renal apoptosis through inhibition of mitogen-activated protein kinase signaling. In skeletal muscle, AKE attenuated muscle atrophy by modulating protein turnover pathways, including downregulation of muscle-specific ubiquitin ligases and restoration of Akt/mTOR and FoxO3a signaling. Furthermore, AKE mitigated adipose tissue wasting by suppressing AMP-activated protein kinase-dependent browning and restoring adipogenic signaling involved in lipid storage and differentiation. Conclusions: These findings demonstrate that AKE confers comprehensive protection against cisplatin-induced cachexia and nephrotoxicity by coordinately preserving muscle and adipose tissue and attenuating renal injury, suggesting its potential as a functional nutritional strategy to alleviate chemotherapy-associated tissue wasting.

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1. Introduction

Cancer continues to be a major global health challenge, causing around 10 million deaths each year [1]. While treatment options now include targeted therapies and immunotherapies, traditional cytotoxic chemotherapy remains essential for many solid tumors. Cisplatin, a widely used platinum-based chemotherapeutic agent, is a first-line drug for lung, bladder, ovarian, head and neck, and colorectal cancers [2]. Its antitumor action is primarily mediated through DNA cross-linking, transcription and replication inhibition, and apoptosis induction in rapidly proliferating cancer cells [3]. Despite its remarkable antitumor activity, cisplatin treatment is often accompanied by severe toxicities, which remain a major clinical challenge.

Dose-limiting nephrotoxicity is a critical treatment-related adverse event that severely limits the clinical use of cisplatin. The incidence of acute kidney injury (AKI) has been reported in approximately 20–30% of patients treated with cisplatin [4]. Furthermore, cisplatin tends to accumulate in proximal tubular epithelial cells, causing mitochondrial dysfunction, oxidative stress, inflammation, and apoptotic cell death, which ultimately compromise renal filtration function [5]. Beyond its well-characterized nephrotoxicity, cisplatin also exacerbates cancer-associated cachexia, which is defined by progressive muscle wasting accompanied by adipose tissue reduction [6]. It accelerates muscle protein breakdown while suppressing protein synthesis, thereby activating catabolic pathways [7,8]. Additionally, cisplatin promotes adipose tissue loss by increasing thermogenesis, inducing adipose browning, and inhibiting adipogenesis, collectively reducing lipid storage capacity [9,10]. These combined catabolic effects contribute to functional decline, reduced treatment tolerance, and poor prognosis in cancer-bearing hosts [11]. Therefore, there is growing interest in developing effective therapeutic strategies using medicinal plants or dietary sources that can simultaneously reduce cisplatin-induced organ toxicity and systemic metabolic dysfunction.

Amaranthus spp., a pseudocereal cultivated across Central and South America, is characterized by a high protein content, particularly albumins and globulins. Notably, it exhibits a well-balanced amino acid profile enriched in lysine, an essential amino acid often limited in conventional cereals [12]. Enzymatic hydrolysis of amaranth seed proteins further enhances their bioavailability and functional properties, including anti-inflammatory, antioxidant, and metabolic regulatory effects [13]. Given that cancer-associated cachexia is characterized by impaired protein anabolism and reduced availability of essential amino acids, amaranth protein hydrolysates may help compensate for anabolic substrate insufficiency during chemotherapy. Meanwhile, Korean mint (Agastache rugosa), a traditional medicinal herb in East Asia, has shown efficacy in preventing muscle atrophy in immobilization-induced models [14]. The major flavonoid component, tilianin, has demonstrated nephroprotective effects by reducing oxidative stress, inflammation, and apoptosis in a renal ischemia–reperfusion injury model, as indicated by decreases in blood urea nitrogen (BUN), creatinine, and tumor necrosis factor-alpha (TNF-α) [15]. These properties suggest that A. rugosa may attenuate inflammation-driven catabolic signaling, a key mechanism underlying cisplatin-induced muscle wasting. Accordingly, a combination of amaranth protein hydrolysate and A. rugosa extract was hypothesized to exert complementary protective effects by simultaneously suppressing catabolic signaling and supporting muscle protein maintenance, thereby ameliorating cisplatin-induced nephrotoxicity and cancer-related muscle wasting. Few studies to date have examined dietary-based combination strategies that target both cisplatin-induced renal injury and systemic cachexia in tumor-bearing models. Therefore, this study aimed to determine whether a combination of amaranth protein hydrolysate and Korean mint extract (AKE) could ameliorate cisplatin-induced nephrotoxicity and cachexia in CT26 tumor-bearing BALB/c mice. Specifically, we examined the effects of AKE on systemic inflammation, muscle wasting, adipose tissue loss, and renal dysfunction to evaluate its potential as a multi-target supportive intervention during chemotherapy.

2. Materials and Methods

2.1. Preparation of AKE

Amaranth protein hydrolysate powder was supplied by Daesang Wellife Co., Ltd. (Seoul, Republic of Korea). Enzymatic hydrolysis of amaranth powder was performed using a two-step proteolytic process. Briefly, Alcalase (2.4 Anson units/g; Novozymes, Copenhagen, Denmark) was applied at an enzyme-to-substrate (E:S) ratio of 0.3% (w/w) and incubated for 2 h at 50 °C. This was followed by further hydrolysis with Flavourzyme (500 leucine aminopeptidase units/g; Novozymes) at an E:S ratio of 0.1% (w/w) for an additional 2 h under the same temperature conditions. Enzyme inactivation was achieved by incubating the reaction mixture at 85 °C for 10 min. The obtained hydrolysate was subsequently lyophilized and pulverized to yield a fine powder.

The Korean mint (A. rugosa) extract powder was provided by Bolak (Hwasung, Republic of Korea). Dried aerial parts of A. rugosa were pulverized and extracted with distilled water at 95 °C for 4 h. The extract was filtered, concentrated using a vacuum rotary evaporator at 65 °C, and subsequently spray-dried with dextrin at an extract-to-dextrin ratio of 8:2 (w/w). The combined formulation, designated as AKE, was prepared by blending the amaranth protein hydrolysate powder and A. rugosa extract powder at a weight ratio of 5:1 (w/w). HPLC analysis identified tilianin as the principal marker compound in the A. rugosa extract, with a concentration of 0.76% (w/w). The 5:1 (w/w) blending ratio was selected based on preliminary in vitro observations demonstrating biological activity of the combined formulation.

2.2. Cell Culture

CT26 murine colon carcinoma cells were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in Dulbecco’s Modified Eagle Medium (Welgene, Seoul, Republic of Korea) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin–streptomycin (100 U/mL penicillin, 100 mg/L streptomycin). Cultures were incubated at 37 °C in 5% CO_2_ with controlled humidity.

2.3. Animal Study

Male BALB/c mice (6 weeks old) were supplied by Samtako Bio Korea (Osan, Republic of Korea). The animals were certified as specific pathogen-free (SPF) by the vendor and were confirmed to be healthy upon arrival. At baseline (week 0), the body weight was 22.19 ± 0.95 g (range, 20.5–24.0 g). Mice were housed under standard laboratory conditions (23 ± 1 °C, 12 h light/dark cycle) with ad libitum access to food and water. After a 1-week acclimation period, baseline body weights were measured, and the 30 mice were stratified by body weight and randomly allocated into five experimental groups (n = 6 per group) based on a computer-generated randomization sequence to ensure comparable mean body weights across groups: (I) normal control (NC), (II) cancer cachexia (CC), (III) CC treated with cisplatin (CIS), (IV) CC treated with cisplatin plus 125 mg/kg AKE (AKEL), and (V) CC treated with cisplatin plus 250 mg/kg AKE (AKEH). Each group consisted of six mice housed three per cage (two cages per group). The individual mouse was defined as the experimental unit for all in vivo measurements and statistical analyses, except for cage-level parameters (e.g., food intake), for which the cage was considered the experimental unit. Sample size determination was performed with G*Power (v3.1.9.4) for a one-way ANOVA, with parameters set at f = 0.70, α = 0.05, and 80% power. Change in body weight was defined as the primary outcome measure, as it reflects the overall severity of cancer-induced cachexia and is widely used as a primary efficacy endpoint in preclinical cachexia models. The effect size was estimated based on previously reported between-group differences in body weight in comparable tumor-bearing mouse models. All other measured parameters were considered secondary endpoints. To induce cancer cachexia, mice in all groups except the NC group were subcutaneously inoculated with 1 × 10^6^ CT26 colon carcinoma cells into the right flank on day 1. Following a 7-day tumor implantation period, cisplatin was administered intraperitoneally at a dose of 1 mg/kg every 3 days from day 8 to day 21. During the same period, AKE was administered once daily by oral gavage at doses of 125 mg/kg (AKEL) or 250 mg/kg (AKEH). To minimize potential environmental confounding effects, cage positions were rotated weekly, and all experimental procedures and outcome assessments were conducted according to a predefined standardized protocol. Body weight and tumor volume were monitored every other day throughout the experimental period. Tumor dimensions were recorded using a digital caliper, and the volume was calculated based on the modified ellipsoid formula: 1/2 × (length × width^2^). On day 22, all mice were euthanized, and tissues were rapidly excised and collected. At the experimental endpoint, mice were deeply anesthetized with 1.25% tribromoethanol (Avertin, 200–250 mg/kg) administered via intraperitoneal injection (i.p.). Adequate anesthetic depth was confirmed by the absence of the pedal withdrawal reflex prior to blood collection by cardiac puncture. Following blood collection, animals were euthanized by cervical dislocation while under deep anesthesia, in accordance with institutional and national guidelines for the care and use of laboratory animals. Tumors, kidneys, skeletal muscles (quadriceps [QD], gastrocnemius [GA], soleus [SOL], tibialis anterior [TA], and extensor digitorum longus [EDL]), as well as adipose tissues (epididymal white adipose tissue [eWAT], subcutaneous white adipose tissue [sWAT], and brown adipose tissue [BAT]), were carefully dissected and weighed. Animal experiments were conducted with authorization from the Yonsei University IACUC (Seoul, Republic of Korea; Approval No. IACUC-202411-1959-02). This study adhered to the principles of replacement, reduction, and refinement (3Rs). An in vivo model was required to evaluate the systemic effects of AKE on cancer-induced cachexia. Reduction was achieved through a priori sample size calculation and optimized experimental design to minimize animal use while maintaining statistical validity. Refinement measures included the use of anesthesia during invasive procedures and routine monitoring for signs of distress. Investigator blinding was not feasible because routine oral gavage, body weight recording, and behavioral observations required hands-on involvement throughout the study. However, image analysis, histological scoring, and data analysis were independently reviewed and verified by an investigator blinded to group allocation. No predefined humane endpoints were established because the study procedures were not expected to cause notable pain or distress. Animals were closely observed during routine oral administration to monitor overall health status. All animal experiments adhered to institutional animal care guidelines, and appropriate measures were implemented to minimize distress and discomfort.

2.4. Histological Analysis

Kidney, GA muscle, and eWAT tissues were fixed in 10% formalin, embedded in paraffin, and sectioned. Sections were then stained with hematoxylin and eosin (H&E) and visualized using a CK40 inverted microscope (Olympus, Tokyo, Japan) equipped with a T500 camera (eXcope, Daejeon, Republic of Korea). The grading system of kidney injury was scored as follows: 0 (normal kidney); 1 (0–5% injury, minimal damage); 2 (5–25% injury, mild damage); 3 (25–75% injury, moderate damage); and 4 (75–100% injury, severe damage). ImageJ software (version 1.47, National Institutes of Health, Bethesda, MD, USA) was used to quantify the cross-sectional area (CSA) of skeletal muscle fibers and the adipocyte area in adipose tissue sections. Histological analyses were conducted with n = 4 per group due to tissue section quality limitations (e.g., damaged or insufficient sections), unrelated to statistical exclusion.

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

Following cardiac puncture, whole blood was left to clot at ambient temperature for 1 h and then centrifuged (1500× g, 20 min, 4 °C). The resulting serum was collected and preserved at −80 °C. Concentrations of TNF-α, interleukin (IL)-6, and IL-1β, were quantified using ELISA kits (ABclonal, Woburn, MA, USA) according to the manufacturer’s instructions. Serum creatinine and BUN, indicators of renal toxicity, were measured using ELISA kits from ABclonal and Elabscience (Wuhan, China), respectively. Optical density was read at 450 nm using a VERSAMAX microplate reader (Molecular Devices, Sunnyvale, CA, USA), and concentrations were calculated by interpolation from standard curves. Serum-based analyses were performed with n = 4 per group due to limited serum volume obtained from certain tumor-bearing mice. No samples were excluded based on statistical criteria.

2.6. Western Blot Analysis

Tissue extracts from GA muscle and eWAT were prepared by homogenization in NP-40 lysis buffer supplemented with 0.2% protease inhibitors. After centrifugation (14,000 rpm, 10 min, 4 °C), the clarified supernatants were obtained, and total protein levels were quantified by the Bradford method. For Western blotting, six individual samples per group were initially collected (n = 6 per group). Due to limited protein yield, lysates from two mice within the same group were pooled to generate one biological replicate, resulting in three pooled biological replicates per group (n = 3). This approach was applied consistently across all groups. Equal amounts of protein were loaded per lane in a consistent order (NC, CC, CIS, AKEL, and AKEH) across all blots. After denaturation in 5× SDS-PAGE loading buffer (Biosolution, Suwon, Republic of Korea), equal protein amounts were resolved by 10% SDS-PAGE and electrotransferred onto 0.45 μm nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). Membranes were blocked with 5% skim milk (LPS Solution, Daejeon, Republic of Korea) for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies against phosphorylated and total forms of extracellular signal-regulated kinase (p-ERK and ERK), c-Jun N-terminal kinase (p-JNK and JNK), p38 mitogen-activated protein kinase (p-p38 and p38), forkhead box O3a (p-FoxO3a and FoxO3a), phosphoinositide 3-kinase (p-PI3K and PI3K), protein kinase B (p-Akt and Akt), mammalian target of rapamycin (p-mTOR and mTOR), and AMP-activated protein kinase (p-AMPK and AMPK), as well as peroxisome proliferator-activated receptor gamma (PPARγ) (Cell Signaling Technology, Beverly, MA, USA). Additional primary antibodies against cleaved caspase-3, Bcl-2–associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), muscle RING-finger protein-1 (MuRF1), muscle atrophy F-box (atrogin-1), uncoupling protein 1 (UCP1), CCAAT/enhancer-binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), and sterol regulatory element-binding protein 1 (SREBP1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). When target proteins had similar molecular weights, membranes were stripped and re-probed according to standard protocols. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) for 2 h at 4 °C. Bands were visualized using enhanced chemiluminescence reagents (Chembio, Hanam, Republic of Korea) and imaged using a G:BOX EF system (Syngene, Cambridge, UK). ImageJ software (National Institutes of Health) was used to identify band intensities. Loading controls were detected on the same membrane used for target protein detection, and band intensities were normalized to the corresponding loading controls.

2.7. Statistical Analysis

Statistical comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test, while unpaired. All statistical analyses were carried out using GraphPad Prism software (version 10.0; GraphPad Software, La Jolla, CA, USA). Results are expressed as mean values ± standard error of the mean (SEM), and statistical significance was defined as p < 0.05.

3. Results

3.1. AKE Alleviates Cisplatin-Induced Body Weight Loss and Systemic Inflammation in CT26 Tumor-Bearing Mice

To investigate the therapeutic effects of AKE on cisplatin-induced cachexia, changes in body weight and tumor-free body weight were evaluated in CT26 tumor-bearing mice. Compared with the NC group, CT26 tumor implantation significantly reduced both overall and tumor-free body weights in the CC group. One-way ANOVA revealed a significant overall difference among groups (F(4,30) = 12.65, p < 0.0001, η^2^ = 0.63). Moreover, cisplatin treatment further aggravated body weight loss in tumor-bearing mice, as evidenced by a significantly greater reduction in body weight in the CIS group relative to the CC group (mean difference = −2.67 g, 95% CI: −4.40 to −0.94 g), reflecting an exacerbation of the cachectic phenotype. In contrast, oral administration of AKE significantly alleviated cisplatin-induced weight loss in both the AKEL and AKEH groups, leading to a partial restoration of body weight toward levels observed in the CC group (Figure 1A). Tumor volume and tumor weight were significantly decreased in all cisplatin-treated groups (CIS, AKEL, and AKEH) compared with the CC group, thereby confirming the antitumor efficacy of cisplatin. Nevertheless, AKE supplementation did not compromise the antitumor efficacy of cisplatin, as tumor weight did not differ significantly between the AKE-treated groups and the CIS group (Figure 1B).

To further assess systemic inflammation and explore the mechanisms underlying the anti-cachectic effects of AKE, serum levels of pro-inflammatory cytokines were analyzed. The concentrations of TNF-α, IL-6, and IL-1β were significantly elevated in the CC group compared with the NC group, indicating activation of cachexia-associated inflammatory signaling. Cisplatin treatment further amplified this inflammatory response, as reflected by significantly higher cytokine levels in the CIS group. In contrast, AKE administration substantially attenuated cisplatin-induced increases in TNF-α, IL-6, and IL-1β, with cytokine levels in both the AKEL and AKEH groups approaching those observed in the CC group (Figure 1C). Collectively, these findings indicate that AKE alleviates cisplatin-exacerbated cachexia, at least in part, by preserving body mass and modulating systemic inflammatory signaling, without compromising antitumor efficacy.

3.2. AKE Mitigates Cisplatin-Induced Nephrotoxicity in CT26 Tumor-Bearing Mice

Nephrotoxicity is a major adverse effect associated with cisplatin treatment. To elucidate the renoprotective effects of AKE against cisplatin-induced nephrotoxicity, serum creatinine and BUN levels were evaluated as functional indicators of renal toxicity. Cisplatin treatment resulted in a marked increase in both serum creatinine and BUN levels in CT26 tumor-bearing mice compared with the CC group. CT26 tumor implantation alone (CC group) did not significantly alter serum creatinine levels compared with the NC group, whereas BUN levels were significantly elevated, indicating that tumor burden was associated with selective alterations in renal function markers. In contrast, AKE supplementation significantly attenuated cisplatin-induced elevations in serum creatinine and BUN levels in both the AKEL and AKEH groups. Although serum creatinine and BUN levels in the AKE-treated groups remained higher than those in the CC group, they were substantially lower than those observed in the CIS group (Figure 2A). Despite these functional changes, kidney weight did not differ significantly among the experimental groups, and no gross alterations in kidney mass were observed (Figure 2B).

Consistent with these functional findings, histological evaluation of kidney sections revealed pronounced renal cortical tubular injury in the CIS group, whereas no apparent structural abnormalities were observed in the NC and CC groups. Semi-quantitative assessment of renal tubular injury showed a significant increase in kidney injury scores following cisplatin treatment. Importantly, AKE administration significantly reduced kidney injury scores in both the AKEL and AKEH groups, although the scores remained slightly elevated compared with those in the NC and CC groups (Figure 2C).

3.3. AKE Mitigates Cisplatin-Induced Renal Injury by Modulating Mitogen-Activated Protein Kinase (MAPK) Signaling and Apoptosis in CT26 Tumor-Bearing Mice

To investigate the molecular basis of AKE-mediated protection against cisplatin-induced nephrotoxicity, we examined whether AKE modulates renal MAPK signaling, a key pathway implicated in stress-induced kidney injury. The phosphorylation levels of ERK, JNK, and p38 were comparable between the NC and CC groups, indicating that CT26 tumor implantation alone did not activate MAPK signaling in the renal tissue. In contrast, cisplatin treatment markedly enhanced the phosphorylation of ERK, JNK, and p38, reflecting robust activation of renal stress signaling pathways. However, AKE supplementation significantly suppressed the cisplatin-induced phosphorylation of all three MAPKs. Although phosphorylation levels in the AKE-treated groups remained elevated relative to the CC group, they were substantially lower than those observed in the CIS group, indicating partial attenuation of MAPK activation by AKE.

Given that sustained MAPK activation is closely associated with the induction of apoptosis, we next assessed the expression of apoptosis-related proteins in the kidney tissue. Consistent with the activation of MAPK signaling, cisplatin-treated mice exhibited pronounced upregulation of the pro-apoptotic markers Bax and cleaved caspase-3, accompanied by a marked reduction in the anti-apoptotic protein Bcl-2. In contrast, the expression levels of these apoptosis-related proteins did not differ between the NC and CC groups, confirming that tumor burden alone was insufficient to induce renal apoptosis. AKE administration significantly reduced Bax and cleaved caspase-3 expression while partially restoring Bcl-2 levels compared with the CIS group, indicating suppression of cisplatin-induced apoptotic signaling (Figure 3B). Taken together, these results demonstrate that AKE mitigates cisplatin-induced renal injury by attenuating MAPK stress signaling and modulating apoptosis-related pathways in kidney tissue.

3.4. AKE Reduces Cisplatin-Induced Muscle Atrophy Through Modulation of Muscle Protein Turnover Pathways in CT26 Tumor-Bearing Mice

Given the exacerbation of skeletal muscle wasting by cisplatin in cancer cachexia, we examined whether AKE protects against cisplatin-induced muscle atrophy in CT26 tumor-bearing mice. Cisplatin treatment significantly reduced skeletal muscle mass in the CIS group compared with the NC and CC groups, as reflected by marked decreases in the weights of multiple skeletal muscles, including QD, GA, SOL, TA, and EDL. In contrast, AKE supplementation significantly attenuated cisplatin-induced muscle loss, with muscle weights in both the AKEL and AKEH groups significantly higher than those in the CIS group (Figure 4A). Consistently, histological analysis of GA muscle revealed a pronounced reduction in muscle fiber CSA in the CIS group, whereas AKE preserved muscle fiber morphology and CSA in a dose-dependent manner, approaching CC levels (Figure 4B).

Considering the established role of FoxO3 in muscle protein degradation during cachexia and chemotherapy, we next assessed FoxO3 signaling and downstream ubiquitin–proteasome markers. Cisplatin markedly reduced FoxO3 phosphorylation and increased the levels of the muscle-specific E3 ubiquitin ligases MuRF1 and atrogin-1 in the CIS group relative to the CC group, indicating activation of FoxO3-dependent proteolytic signaling. AKE significantly restored FoxO3 phosphorylation and suppressed the cisplatin-induced upregulation of MuRF1 and atrogin-1 (Figure 5A,B). Alongside the attenuation of catabolic signaling, anabolic pathways involved in muscle protein synthesis were also examined. Cisplatin treatment suppressed the PI3K/Akt/mTOR anabolic signaling axis, as evidenced by reduced phosphorylation of PI3K, Akt, and mTOR, whereas AKE supplementation partially reversed these effects by increasing the phosphorylation of PI3K, Akt, and mTOR compared with the CIS group (Figure 5C). Together, these results show that AKE attenuates cisplatin-induced skeletal muscle atrophy by suppressing FoxO3-mediated proteolysis while preserving PI3K/Akt/mTOR-dependent anabolic signaling, thereby maintaining skeletal muscle mass and structure.

3.5. AKE Attenuates Cisplatin-Induced Adipose Tissue Wasting by Suppressing Browning and Restoring Adipogenic Signaling in CT26 Tumor-Bearing Mice

Considering the involvement of adipose tissue wasting in cancer cachexia, we examined whether AKE attenuates cisplatin-associated adipose tissue depletion in CT26 tumor-bearing mice. Adipose tissue weights did not differ between the NC and CC groups, whereas cisplatin treatment markedly reduced the weights of eWAT, sWAT, and BAT in the CIS group. In contrast, AKE supplementation significantly attenuated cisplatin-induced adipose tissue loss, resulting in higher adipose tissue weights compared with the CIS group (Figure 6A). Consistent with these changes in tissue mass, histological analysis of eWAT revealed a pronounced reduction in adipocyte size following cisplatin treatment, whereas AKE administration preserved adipocyte morphology, with adipocyte size and structure approaching those observed in the CC group (Figure 6B).

To further elucidate the molecular mechanisms underlying these morphological and mass-related changes, browning- and adipogenesis-related signaling pathways in adipose tissue were examined. Cisplatin treatment significantly increased AMPK activation, along with upregulation of the thermogenic markers PGC-1α and UCP1, indicating enhanced adipose tissue browning. In parallel, cisplatin markedly suppressed the expression of the adipogenic transcription factors PPARγ, SREBP1, and C/EBPα, reflecting impaired adipogenic capacity. In contrast, AKE supplementation attenuated cisplatin-induced AMPK-associated browning signaling and restored the expression of adipogenic markers toward levels observed in the CC group (Figure 7A–C). Collectively, AKE attenuated cisplatin-induced adipose tissue wasting by suppressing AMPK-associated browning and restoring adipogenic signaling, contributing to improved adipose tissue homeostasis during chemotherapy-associated cachexia.

4. Discussion

Cisplatin-induced renal injury is a critical contributor to the metabolic and inflammatory dysfunction that exacerbates cancer cachexia. Cisplatin-driven nephrotoxicity has been reported to involve oxidative stress, activation of MAPK signaling, and tubular apoptosis [4,16]. In our previous work (manuscript under review), we demonstrated that AKE effectively attenuated tumor-induced cachexia in the absence of chemotherapy by modulating inflammatory pathways and metabolic signaling in skeletal muscle and adipose tissue. Building on these findings, the present study was designed to extend the therapeutic relevance of AKE to a more severe cachectic condition compounded by cisplatin treatment. Using a CT26 tumor-bearing mouse model treated with cisplatin, we evaluated whether AKE could mitigate chemotherapy-induced renal injury as well as the combined effects of cancer- and chemotherapy-associated skeletal muscle and adipose tissue wasting. Our results demonstrate that AKE confers multi-organ protective effects without compromising anticancer efficacy, highlighting its potential as a multi-target supportive intervention for the management of chemotherapy-associated cachexia.

We employed a combination of amaranth protein hydrolysate and Korean mint extract to address the multifactorial pathophysiology of cisplatin-induced cachexia. This condition is characterized by metabolic insufficiency, systemic inflammation, and multi-organ dysfunction, making it difficult to manage with a single therapeutic approach [17,18,19]. On this basis, we designed a combination strategy that simultaneously targets nutritional deficiency and inflammation-driven tissue damage. Adequate intake of high-quality protein and essential amino acids is a cornerstone of supportive care in cancer-associated cachexia, with clinical nutrition guidelines recommending increased protein intake to preserve lean body mass and counteract catabolic wasting [20,21,22]. Accordingly, amaranth seed protein was selected as a nutritionally valuable plant-derived protein source with a balanced essential amino acid profile and high digestibility. Enzymatic hydrolysis further enhances its bioavailability by providing readily absorbable amino acids and bioactive peptides, thereby compensating for anabolic substrate insufficiency during chemotherapy [23,24,25,26]. In parallel, Korean mint extract was incorporated to counteract inflammation- and cytotoxicity-driven tissue injury associated with cisplatin treatment. This extract, rich in tilianin, has demonstrated cytoprotective and anti-apoptotic effects in renal injury models, supporting its relevance as a complementary component to mitigate chemotherapy-induced organ damage [27]. Thus, the combination of amaranth protein hydrolysate and Korean mint extract was designed to simultaneously support anabolic substrates and suppress inflammation-driven catabolic stress, highlighting AKE as a rational multi-target intervention for chemotherapy-associated cachexia.

Here, we demonstrate that even a relatively low dose of cisplatin, selected to minimize mortality in severely cachectic tumor-bearing mice, was sufficient to induce marked renal dysfunction, as evidenced by significant elevations in BUN and creatinine levels. These findings are in line with previous reports identifying cisplatin as a potent inducer of renal injury markers [28]. Histological assessment further revealed pronounced renal damage accompanied by activation of the ERK, JNK, and p38 MAPK pathways, as well as increased expression of apoptotic markers, consistent with the established mechanisms of cisplatin nephrotoxicity [4]. Notably, AKE administration partially restored kidney architecture, attenuated MAPK phosphorylation, and suppressed the pro-apoptotic proteins Bax and cleaved caspase-3 while restoring the anti-apoptotic protein Bcl-2, indicating meaningful reduction in cisplatin-induced tubular apoptosis rather than complete renal recovery. In this context, kidney weight showed no statistically significant change in the current study, despite reports of increased kidney weight following cisplatin-induced nephrotoxicity in other models [29]. This discrepancy may be attributable to the relatively low cisplatin dose used here, which was carefully selected to account for the severe systemic wasting characteristic of tumor-bearing cachectic mice. Considering that tilianin, a major flavonoid constituent of A. rugosa, has documented anti-inflammatory and anti-apoptotic activity in renal injury models [15], similar molecular mechanisms may contribute to the renoprotective effects of AKE observed in the present study. Collectively, these findings suggest that AKE has potential as a modulator of cisplatin-induced nephrotoxicity; however, because renal damage was not fully reversed, further evaluation in AKI and chronic kidney injury (CKI) models is warranted.

Cisplatin treatment in CT26 tumor-bearing mice substantially accelerated the loss of skeletal muscle and adipose tissue. This finding is consistent with clinical and preclinical evidence showing that chemotherapeutic agents exacerbate tumor-induced systemic inflammation, metabolic dysregulation, and tissue wasting [30,31]. In line with these observations, the anti-cachectic effects of AKE appear to be mediated primarily through suppression of systemic inflammation, as evidenced by reduced circulating levels of TNF-α, IL-6, and IL-1β. These pro-inflammatory cytokines are well-established drivers of muscle proteolysis and adipose tissue catabolism; therefore, their attenuation provides a plausible mechanistic basis for protection against chemotherapy-associated cachexia [32,33].

In skeletal muscle, cisplatin enhanced FoxO3a activation and increased the expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, reflecting activation of the ubiquitin–proteasome system and disruption of muscle protein homeostasis [34,35]. Concomitantly, cisplatin suppressed PI3K/Akt/mTOR signaling, consistent with previous studies demonstrating that pro-inflammatory cytokines such as TNF-α inhibit anabolic pathways and reduce protein synthesis capacity [36,37]. Our findings show that AKE treatment counteracted these catabolic alterations by restoring FoxO3a phosphorylation, downregulating MuRF1 and atrogin-1 expression, and reactivating PI3K/Akt/mTOR signaling, thereby favoring the preservation of muscle protein balance. These molecular alterations translated into phenotypic improvements, including increased muscle fiber CSA and muscle mass in AKE-treated mice, supporting the protective role of AKE against cisplatin-induced muscle wasting. These observations are consistent with previous studies demonstrating that diverse natural bioactive compounds, such as daidzein and linalool, confer protection against muscle wasting by targeting conserved regulators of muscle protein turnover [38,39].

Likewise, AKE attenuated adipose tissue wasting, another hallmark of cachexia. In cachectic conditions, cisplatin is known to enhance AMPK activation and induce browning markers such as PGC1-α and UCP1, reflecting adaptive responses to inflammatory and metabolic stress [40,41]. Concomitantly, the downregulation of adipogenic transcription factors including PPARγ, C/EBPα, and SREBP1 is consistent with cytokine-driven suppression of adipogenesis [42,43]. In this study, cisplatin treatment in CC mice similarly promoted AMPK activation and browning-associated gene expression while suppressing adipogenic regulators, indicating a shift toward increased energy expenditure and impaired lipid storage. By contrast, AKE administration counteracted these alterations by attenuating AMPK activation, reducing browning-related gene expression, and restoring adipogenic transcription factors, thereby preserving adipocyte integrity, maintaining lipid stores, and limiting excessive energy dissipation.

Collectively, the present study reveals that AKE exerts multi-organ protective effects against cachexia aggravated by both tumor burden and cisplatin chemotherapy. AKE ameliorates key drivers of renal dysfunction as well as muscle and fat deterioration by suppressing systemic inflammation, modulating MAPK- and FoxO3a-dependent stress signaling, restoring anabolic pathways, and preventing adipose tissue browning. This coordinated regulation of inflammatory and metabolic pathways positions AKE as a promising candidate for the management of chemotherapy-associated cachexia, despite its partial renoprotective effects. Nevertheless, further studies incorporating a range of cisplatin doses and CKI models will be essential to fully define and validate its therapeutic potential.

5. Conclusions

In summary, AKE, a combination of amaranth protein hydrolysate and Korean mint extract, exerted coordinated multi-organ protective effects in cisplatin-treated CT26 tumor-bearing mice, alleviating both nephrotoxicity and cancer-associated cachexia. AKE significantly attenuated cisplatin-induced renal injury, skeletal muscle atrophy, and adipose tissue wasting. These protective effects were mediated by distinct tissue-specific mechanisms. In the kidney, AKE attenuated ERK-, JNK-, and p38 MAPK-driven apoptotic signaling, resulting in improved renal function. In skeletal muscle, AKE suppressed FoxO3a-dependent catabolic pathways while restoring PI3K/Akt/mTOR signaling. In adipose tissue, AKE inhibited AMPK-mediated browning and restored the expression of adipogenic transcription factors. Furthermore, AKE reduced circulating pro-inflammatory cytokine levels, thereby alleviating inflammatory and metabolic drivers of tissue wasting. Together, these preclinical findings suggest that AKE represents a promising supportive nutritional intervention to mitigate cisplatin-induced nephrotoxicity and cachexia during chemotherapy.