Antrodia cinnamomea Extract Attenuates Cisplatin-Induced Muscle Atrophy, Apoptosis, and Cell Growth Suppression

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Introduction
Cancer patients receiving chemotherapy sufer from side efects of nausea, diarrhea, anorexia, and extensive loss of skeletal muscle mass. Among these symptoms, muscle wasting is a hallmark of cachexia induced by chemotherapeutic drugs [1,2]. Te antineoplastic drugs, cisplatin and other platinum-based drugs, are widely used in cancer treatment. Platinum-DNA adducts formed in cisplatin-treated cells induce cytotoxicity [3]. Despite killing cancer cells, cisplatin-induced muscle atrophy is reported to be caused by modulating IGF-1 signaling, proinfammatory cytokines, oxidative stresses, autophagy, abnormal proteolysis through ubiquitin-proteasome pathway, and increase of apoptosis as well as cell cycle arrest [4,5].
A cell cycle is a series of events that occur when a cell proliferates, which includes the process of DNA replication and division into two individual cells. It contains four stages, G1/S/G2/M phases, and these stages are regulated by various factors including cyclins, cyclin-dependent kinases (cdk), cdk modulators, tumor suppressor proteins, and transcriptional factors [9][10][11]. Te activation of the p53 tumor suppressor contributes to the induction of cell growth arrest gene p21 Cip1/Waf1 [12]. It was recognized that p53 and p21 are crucial factors in modulating cell cycle arrest and apoptosis [12]. An earlier study indicated that p53 participates in apoptosis during unloading-induced muscle atrophy [13]. Chronic activation of p53 induces muscle atrophy consistent with sarcopenia in aging muscle [14]. Limb immobilization induces muscle wasting and increases p53 expression, whereas the knockout p53 gene is partially resistant to immobilization-induced muscle atrophy [15]. In addition, it has been demonstrated that p21 was specifcally upregulated by myostatin, a negative regulator of muscle growth in myoblasts [16], and mRNA levels of both myostatin and p21 were increased in cisplatin-treated mice [5]. Tese results reveal that the p53/p21 signal cascade plays a crucial role in inducing muscle loss.
Antrodia cinnamomea (AC, synonyms: Antrodia camphorata; Taiwanofungus camphoratus) is a potential medicinal fungus that grows in the inner cavity of Cinnamomum kanehirai, an endemic plant of Taiwan [17,18]. AC is considered to have potential properties for anticancer activity [18][19][20][21][22][23][24][25], anti-infammation [26], antioxidative stresses [27][28][29], and attenuating alcoholic liver injury [18,30]. Tese protective characters come from the mycelium or fruiting bodies of AC through water or alcohol extraction [30,31] and unique triterpenoids isolated from AC [32]. In addition, earlier studies indicated that AC extract possesses potential anticachexia and antifatigue efects in chemotherapeutic drug-treated mice [33,34]. However, the molecular mechanism by which AC attenuates chemotherapeutic drug-induced muscle wasting remains unclear. In this study, we established a cisplatininduced sarcopenia mice model and found that ethanol extracts of AC (EEAC) have a protective efect on the attenuation of cisplatin-caused muscle wasting. We further demonstrated that EEAC ameliorates cisplatin/gemcitabine (C/G)-suppressed myotube formation and attenuates both C/G-reduced cell proliferation and apoptosis of C2C12 cells. Finally, we found that C/G increased p53 and p21 levels, but this efect was signifcantly attenuated by EEAC in C2C12 cells. Taken together, our fndings provide evidence that AC extract can be used as a potential dietary supplement used for ameliorating chemotherapy-induced muscle atrophy.

EEAC and Reagents
. EEAC used in this study was kindly provided by the Balay Biotechnology Corporation, Taipei, Taiwan. Te components of EEAC were analyzed and confrmed by the HPLC system consisting of a Spectra SYSTEM P1000 quipped with Spectra SYSTE AS3000 autosampler, Surveyor PDA plus detector, and a reversephase column (C18, 4.6 × 250 mm, Termo, BDS HYPERSIL). Te marker triterpenoids standards of ACF, including antcin A, B, C, H, K, dehydrosulphurenic acid, and dehydroeburicoic acid, were used as external standards to identify the specifc components in EEAC. Te quantitative LC-MS/MS analysis to determine the concentrations of 7 marker triterpenoids in EEAC was performed by ABM International Lab Inc. (New Taipei City, Taiwan). cis-Diammineplatinum (II) dichloride (cisplatin), gemcitabine hydrochloride, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).

Experimental Animals.
All experimental procedures performed in this study conform to the guidelines and regulations approved by the Institutional Animal Care and Use Committee of the National Health Research Institutes (NHRI-IACUC-108120-A) of Taiwan. 8-to 10-week-old C57BL/6 mice were purchased from the National Laboratory Animal Center and housed in a specifc pathogen-free facility of the National Health Research Institutes of Taiwan. Mice were randomized into four groups including control (CTR, n � 6), EEAC (n � 6), cisplatin alone (cisplatin, n � 6), and EEAC combined with cisplatin (EEAC + cisplatin, n � 6). Mice were fed with EEAC (30 mg/kg body weight) by oral gavage once daily for 8 days. At day 3, muscle atrophy was induced by intraperitoneal injection of cisplatin (40 mg/ kg body weight) once daily for 6 days [35]. Control mice were injected with DMSO diluted in an equal volume of phosphate-bufered saline solution. Te mice were sacrifced at day 8, and the mass of the quadriceps femoris was measured after harvesting.

Cell Culture and C2C12 Diferentiation. LLC1 mouse
Lewis lung carcinoma cell line and mouse myoblast C2C12 cells were obtained from the ATCC (Rockville, Maryland, USA). Both cells were maintained in Dulbecco's modifed Eagle's medium (DMEM) (Invitrogen-Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Invitrogen-Gibco) and penicillin-streptomycin (100 U/ml, Invitrogen-Gibco). For diferentiation of C2C12 myoblasts into myotubes, confuence C2C12 cells were incubated in diferentiation medium (DMEM containing 2% horse serum and penicillin-streptomycin) with EEAC and/or C/G, and the medium was changed once every day for six days. Diferentiated C2C12 cells were fxed and permeabilized with iced methanol (Sigma-Aldrich) and then incubated in 50 nM NH 4 Cl (Sigma-Aldrich) for 15 min. Fixed cells were incubated with MHC antibody (Termo Fisher Scientifc, Waltham, MA, USA) at 4°C overnight followed by incubation with HRP-conjugated secondary antibody (Termo Fisher Scientifc). Te expression of MHC was visualized with a DAB substrate kit (Termo Fisher Scientifc). Te nuclei were stained with hematoxylin (Sigma-Aldrich), and the values of fusion index were indicated by the ratio of MHC-positive nuclei number to total nuclei number of total cells.

Western Blot Analysis.
For determination of protein levels, cells were harvested and lysed by iced RIPA bufer (Millipore, Temecula, CA, USA) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Lysates were centrifuged at 15,000 rpm for 10 minutes. Protein concentrations of supernatant were determined using Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were loaded on gradient SDS-PAGE gels. Gels were transferred to 0.22 μM NC membranes (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) and then probed with specifc antibodies against cleaved caspase-3, cyclin D, p53, p21 (Cell Signaling, Danvers, MA, USA), and β-actin (Sigma-Aldrich). Te membranes were immersed in 0.1% PBST containing horseradish peroxidase-conjugated secondary antibodies (Cell Signaling), and the protein levels were determined using enhanced chemiluminescence reagents (Millipore).

Real-Time Quantitative PCR (RT-qPCR). Total RNA
was isolated by RNAzol ® RT (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's protocol, and cDNA was synthesized using the ABI RT Kit (ABI Applied Biosystems, Waltham, MA, USA). Te cDNA was applied to real-time quantitative PCR instruments (ABI ViiA 7, ABI Applied Biosystems) using SYBR Green (Kapa Biosystems, Woburn, MA, USA). Primer sequences for qPCR are used in this study of p53 (F: GAGGCCGGCTCT GAGTATACC; R: GGCAGGCACAAACACGAACC);p21 (F: CCAATCCTGGTGATGTCCGA; R: AGTCAAAGT TCCACCGTTCTCG); and GAPDH (F: CGCTCTCTGCTC CTCCTGTT; R: CCATGGTGTCTGAGCGATGT). All PCRs were performed in triplicate. Raw data were analyzed by using QuantStudio ™ Real-Time PCR Software. Te quantity of the specifc genes was normalized with GAPDH of the same sample. Te fold change of ΔΔCt was determined as the ratio compared to each control sample.
2.6. Statistical Analysis. Data were analyzed by Student's ttest between diferent groups, with p < 0.05 considered statistically signifcant.

EEAC Ameliorated the Cisplatin-Induced Muscle Loss in
Mice. Te quality and compositions of EEAC used in this study were confrmed and analyzed by HPLC.  (Table 1).
To evaluate the protective efcacy of EEAC on chemotherapeutic drugs-induced muscle wasting, we established a mice muscle atrophy model caused by cisplatin. C57BL/6 mice (8-10 weeks old) were fed with EEAC (30 mg/ kg) or ethanol control by oral gavage once daily for 8 days. At day 3, mice were intraperitoneally injected with cisplatin (40 mg/ml) once daily for 6 days [35] (illustrated scheme in Figure 1(a)). Injection of cisplatin dramatically reduced the body weight of mice, whereas oral gavage of EEAC has no signifcant efect on the fuctuation of body weight. On day 8, the mice were sacrifced, and the mass of quadriceps femoris was measured. Cisplatin injection signifcantly reduced the mass of mouse quadriceps when compared with the control and EEAC groups (Figure 1(b)). Intriguingly, EEAC treatment ameliorates cisplatin-induced muscle loss of the quadriceps (Figure 1(b)). Tese results reveal that EEAC may play a protective role in chemotherapy-induced muscle atrophy.

EEAC Impaired C/G-Suppressed Myotube Formation of C2C12 Cells.
To investigate the efect of EEAC and chemotherapeutic drugs-regulated myotube formation, mouse myoblast C2C12 cells were treated with 2% horse serum for 6 days to diferentiate into myotubes (representative image in Figure 2(a)). Myosin heavy chain (MHC)-positive multinucleated cells were examined by immunohistochemistry staining with its specifc antibody, and the nuclei were stained with hematoxylin of diferentiated elongated myotubes (Figure 2(b)). Te values of the fusion index demonstrating the capability of myotube formation were compared within diferently treated groups (control, EEAC, C/G, and EEAC + C/G). We found that chemotherapeutic drugs C/G inhibited myotube formation whereas EEAC possessed no signifcant efect on myoblast (Figures 2(a)-2(c)). Intriguingly, C/G-suppressed myotube formation was ameliorated by EEAC treatment (Figure 2(c)).

Diferential Efects of EEAC on C/G-Regulated Cell Proliferation and Apoptosis in LLC1 and C2C12 Cells.
To investigate the suppressive efects of C/G on myoblasts and cancer cells, C2C12 and LLC1 cells were treated with C/G and/or EEAC for 24 hours, and the viable cell number was calculated by 0.4% trypan blue staining (Figure 3(a)). EEAC partially reduced cell numbers of LLC1, but this reduction was not signifcant on C2C12 cells (Figure 3(b)). EEAC synergized with C/G to reduce the cell number of LLC1 cells, but EEAC attenuated the suppressive efect caused by C/G on C2C12 cells (Figure 3(b)). We then examined whether   EEAC modulates C/G-induced apoptosis and cell cycle progression in LLC1 and C2C12 cells by Western blot analysis. Te level of cleaved caspase-3 was abundantly increased by C/G treatment, but this efect was signifcantly attenuated by EEAC in both LLC1 and C2C12 cells (Figure 4(a)). Intriguingly, EEAC inhibited cyclin D expression in LLC1 cells regardless of C/G treatment (Figure 4(b), upper panel). Meanwhile, EEAC applied alone had no efect, but EEAC impaired C/G-reduced cyclin D expression in C2C12 cells (Figure 4(b), lower panel). Tese results indicated that EEAC possesses diferential efects on apoptosis and cell cycle repression in LLC1 and C2C12 cells.

EEAC Attenuated C/G-Induced p53 and p21
Expression of C2C12 Cells. It has been reported that p53 and p21 play crucial factors in regulating cell cycle arrest, thereby promoting muscle atrophy. To elucidate whether p53 and p21 expressions are involved in EEAC and C/G-regulated muscle atrophy, we determined p53 and p21 expression in EEAC and C/G-treated C2C12 cells by Western blot analysis and qPCR. We found that C/G abundantly induced p53 and p21 expression in C2C12 cells. EEAC alone had no efect, but it signifcantly attenuated C/G-induced p53 and p21 expression of C2C12 cells (Western blot analysis in Figure 5(a); qPCR analysis in Figure 5(b)). Tese results suggest that EEAC protects C/G-induced muscle atrophy which is potentially mediated by regulating p53/ p21 signaling.

Discussion
Many studies had demonstrated that AC extract exhibits anticancer activity by reducing cancer cell proliferation and tumor growth. A sulfated galactoglucan isolated from AC and ethanol extract of AC fruiting bodies suppressed cancer cell migration by inhibiting the signaling pathways of metalloproteinases, ERK, JNK, p38, PI3K/Akt, and TGF/FAK/Slug in human lung cancer cells [20,36,37]. Te ingredients isolated from AC, including 4-acetylantroquinonol B, antrocin, and antrodan, have been demonstrated to arrest cell proliferation and suppress cell migration and invasion of  cisplatin and 1 μM gemcitabine), and EEAC combined with C/ G (EEAC + C/G) for 24 hours. (b) EEAC synergized with C/G to reduce the number of LLC1 cells but attenuated the suppressive efect caused by C/G on C2C12 cells. * p < 0.05; * * p < 0.01; * * * p < 0.001. hepatocellular carcinoma, lung carcinoma, bladder cancer, and breast cancer cells [32,[38][39][40][41]. Moreover, the anticancer efects of AC extract include synergizing chemotherapeutic drugs to increase apoptosis and cell cycle arrest of cancer cells, thereby enhancing the sensitivity of chemotherapy [19,20,25,38,[42][43][44]. AC also enhances the chemotherapy sensitivity of 5-FU of colon cancer [22]. In addition, AC targets cancer-initiating cells by increasing cancer cell differentiation, thereby reducing cancer stemness and tumorigenesis by regulation of STAT3/Src signaling in neck squamous cell carcinoma cells and upregulation of miR-142-3p in colon cancer [22,45].
Results from many studies reveal the potential molecular mechanism of how chemotherapeutic drugs induce cell cycle repression and apoptosis. It has been reported that cisplatin and gemcitabine cause cell cycle arrest and apoptosis through activation of JNK, p38, c-histone H2AX, DNA damage response proteins Chk-1/Chk-2, p53, PARP, and caspase-3 in various cell types [46][47][48][49][50]. In this study, we focused on elucidating the efects of EEAC on chemotherapeutic drug-arrested cancer cells and induced muscle atrophy. Because muscle wasting is one of the common cachectic efects caused by chemotherapy, we investigated whether EEAC possesses antimuscle atrophy efects induced by C/G and its potential regulatory mechanism. We frst found that cisplatin-induced mass loss of quadriceps femoris in mice was restored by EEAC (Figure 1(b)). Tis fnding was supported by an earlier study which indicated that AC extract has an anticachectic efect in lung tumor-bearing mice with chemotherapy [45]. In addition, our fndings indicated that C/G treatment increased levels of cleaved caspase-3 whereas EEAC attenuated this apoptotic efect in both LLC1 and C2C12 cells (Figure 4(a)). Intriguingly, EEAC partially restored C/G-reduced cyclin D expression in C2C12 but not in LLC1 cells (Figure 4(b)). Tese results indicated a diferential regulatory mechanism of EEAC on cisplatin-induced apoptosis and cell cycle arrest between cancer and muscle myoblast cells.
In this study, we showed that EEAC attenuated chemotherapeutic drug-induced muscle atrophy in both in vitro and in vivo models. We found that C/G suppressed myotube diferentiation and elongated myotube formation of C2C12 cells, whereas EEAC signifcantly restored this efect ( Figure 2). Although EEAC protected C/G-induced apoptosis in both LLC1 and C2C12 cells, EEAC induced cell arrest of LLC1 cells but attenuated C/G-suppressed cyclin D expression in C2C12 cells (Figure 4(b)). In addition, we provide a potential mechanism of EEAC to protect C/Ginduced muscle atrophy by inhibiting p53/p21 signaling. However, the mechanism behind how EEAC modulating p53/p21 activation abrogates C/G-induced muscle atrophy needs further investigation.

Conclusion
Chemotherapy or sarcopenia-induced muscle loss is repaired by myoblast proliferation and diferentiation into myotubes. In this study, we provide evidence that AC extract attenuates cisplatin-induced muscle wasting, apoptosis, and cell cycle repression of C2C12 cells. AC extract is a potential natural supplement for attenuating chemotherapy-induced muscle atrophy.  Figure 5: EEAC attenuated C/G-induced p53 and p21 expression of C2C12 cells. C2C12 cells were treated with control vehicle, EEAC (10 μg/ml), C/G (1 μM cisplatin and 1 μM gemcitabine), and EEAC combined with C/G (EEAC + C/G) for 24 hours. (a) Expressions of p53 and p21 were determined by Western blotting analysis. (b) mRNA levels of p53 and p21 were examined by qPCR. * * * p < 0.001; * (or * * ) p < 0.01.

Data Availability
Te data (including fgures and table) used to support the fndings of this study are included within the article.

Conflicts of Interest
All authors declare that there are no conficts of interest.

Authors' Contributions
Shu-Man Liang and Chien-Liang Kuo contributed equally to this study.