Paeonia lactiflora root extract suppresses cancer cachexia by down-regulating muscular NF-κB signalling and muscle-specific E3 ubiquitin ligases in cancer-bearing mice

https://doi.org/10.1016/j.jep.2019.112222Get rights and content

Abstract

Ethnopharmacological relevance

The dried root of Paeonia lactiflora Pall. (Radix Paeoniae) has been traditionally used to treat various inflammatory diseases in many Asian countries.

Aim of the study

Cancer cachexia is a catabolic syndrome driven by inflammation and characterised by a loss of skeletal muscle. This study aimed to assess the effects of an ethanolic extract of Radix Paeoniae (RP) on cancer cachexia and elucidate its mechanism of action.

Material and methods

The anti-cachexic effect and mechanism of RP were examined in mouse models of cancer cachexia established in C57BL/6 mice by subcutaneously injecting Lewis lung carcinoma or MC38 colon carcinoma cells. Skeletal muscle tissues were analysed by RNAseq, real-time quantitative reverse transcription PCR, western blotting, and immunofluorescence microscopy. Megestrol acetate, which is recommended for the treatment of cachexia in cancer patients, was used as the comparator treatment in this study.

Results

In lung and colon cancer-bearing mice, RP significantly restored food intake and muscle mass, along with muscle function measured by grip strength and treadmill running time. In the skeletal muscle tissue of the cancer-bearing mice, RP suppressed NF-κB signalling and reduced inflammatory cytokines, including TNF-α, IL-6, and IL-1β; it also down-regulated the muscle-specific E3 ubiquitin ligases MuRF1 and MAFbx.

Conclusion

RP restored skeletal muscle function and mass in cancer-bearing mice by down-regulating the muscular NF-κB signalling pathway and muscle-specific E3 ubiquitin ligases. Our study indicates that RP is a potential candidate for development as a therapeutic agent against cancer cachexia.

Introduction

Cancer cachexia occurs in up to 80% of cancer patients and is the primary cause of death in 30% of all cancer cases. It is a multifactorial syndrome characterised by an ongoing loss of skeletal muscle often accompanied by adipose tissue loss that cannot be reversed by nutritional support, leading to functional muscle impairment. Its pathophysiology is driven by a combination of reduced food intake and abnormal protein metabolism (Fearon et al., 2011). Skeletal muscle protein depletion by enhanced degradation, i.e., muscle wasting, is the most common characteristic of cancer cachexia that contributes to muscle function impairment in cancer patients (Fearon et al., 2013). Thus, animal models of cancer cachexia are typically focused on muscle wasting (Penna et al., 2016).

Ubiquitin-dependent proteolysis has been identified as the main mechanism for muscle wasting in cancer cachexia (Jagoe and Goldberg, 2001). Recent studies indicated the involvement of two muscle-specific E3 ubiquitin ligases, muscle ring-finger-1 (MuRF-1) and muscle atrophy F-Box (MAFbx)/atrogin-1, which are highly expressed during muscle wasting. Specifically, MuRF-1 and MAFbx tended to be elevated in the skeletal muscle of cancer patients, and their expression levels were up-regulated in a cancer cachexia mouse model (Yuan et al., 2015).

Cachexia is assumed to be mediated by inflammation, i.e., inflammatory cytokines, produced by tumour cells or host tissues (Fearon et al., 2013). Inflammatory cytokines, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6, have been implicated in inducing cancer-related muscle wasting (Narsale and Carson, 2014; Suzuki et al., 2013). To date, however, many clinical trials attempting to reverse cachexia by targeting the immune system (anti-TNFα and anti–IL-6 therapies), stimulating the appetite (agonists of ghrelin receptor and cannabinoid receptor), and regenerating the muscle tissue (androgen receptor agonist and myostatin) have failed (Anderson et al., 2017). Thus, there is an urgent medical need for effective and safe therapeutics for cancer cachexia.

The dried root of Paeonia lactiflora Pall. (Radix Paeoniae) has been traditionally used for more than 1200 years in China, Korea, Japan, Taiwan, Thailand, and other south Asian countries for the treatment of inflammatory diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, and hepatitis) and associated symptoms (e.g. fever, dysmenorrhea, and muscle cramping and spasms) (He and Dai, 2011). Current studies found that P. lactiflora has many biological properties, including anti-pyretic, cerebral infraction-inhibitory, antioxidant, anti-inflammatory, anticonvulsant, anxiolytic, and lipid peroxidation-inhibitory activities (Lee et al., 2008).

Many studies reported that traditional herbal medicines containing P. lactiflora enhance the efficacy of anti-cancer therapies and improve the quality of life (QOL) of cancer patients. A meta-analysis of oxaliplatin-based chemotherapy of colorectal cancer combined with traditional herbal medicine showed that the use of herbal therapeutics containing Paeonia was associated with significantly increased tumour response rates (Chen et al., 2016). Jia-wei-xiao-yao-san, containing 10 herbal ingredients, including P. lactiflora, is among the top 10 herbal formulations prescribed for post-surgery colon cancer patients in Taiwan (Chao et al., 2014). Other traditional herbal formulations (Kampo in Japanese) containing P. lactiflora, such as TJ-48 or PHY906, have reportedly improved the QOL of cancer patients by attenuating chemotherapy-induced side effects (Cheng et al., 2012; Qi et al., 2010). PHY906, composed of four herbs including P. lactiflora, reportedly provided a safe and feasible therapy for advanced pancreatic carcinoma in combination with capecitabine (Saif et al., 2014). More recently, some studies indicated that traditional herbal medicines containing P. lactiflora inhibited cancer cachexia. For instance, Sipjeondaebo-tang (Juzen-taiho-to in Japanese), composed of 10 herbal ingredients including P. lactiflora, inhibited cancer anorexia/cachexia in CT-26 tumour-bearing mice (Choi et al., 2014) and prevented anorexia and weight loss in a cisplatin-induced mouse anorexia model (Woo et al., 2016). Ninjin'yeoito, consisting of 12 herbal ingredients including P. lactiflora, suppressed the loss of skeletal muscle function in B16BF6 melanoma-bearing mice (Ohsawa et al., 2018). To date, however, the effect of P. lactiflora as single-component treatment on cancer cachexia has not been assessed, and its mechanism of action is still unknown.

In this study, the suppressive effects of a P. lactiflora root ethanol extract on cancer cachexia and its mechanism of action were investigated in mouse models of cancer cachexia induced by either lung or colon cancer.

Section snippets

Reagents

Anti-IκB (cat# 9242), anti-phospho-IκB (Ser 32/36) (cat# 9246), and anti–NF–κB (cat# 8242) primary antibodies along with anti-mouse (cat# 7076S) and anti-rabbit (cat# 7074S) secondary antibodies, were purchased from Cell Signalling Technology (Danvers, MA, USA). Anti-MuRF1 (cat # 172,479) and anti-MAFbx (cat # 157,596) antibodies were obtained from Abcam (Cambridge, UK). Anti-MyHC antibody was from R&D Systems (MAB4470) (Minneapolis, MN, USA). Anti-β-actin (cat# 47,778) and anti-TATA box

RP restored skeletal muscle function and mass in LLC lung cancer-bearing mice

In this study, the effects of RP on cancer cachexia were analysed in mouse tumour models. Megestrol acetate (MA), which is the currently recommended treatment for cancer cachexia patients, was used as the comparator treatment in our study. LLC lung cancer-bearing mice were either mock-treated (control) or received daily oral doses of 50, 100, or 200 mg/kg of either RP (test article) or MA (comparator) for 2 weeks. After completion of treatment, the function and mass of skeletal muscle were

Discussion

Cancer cachexia, associated with up to 80% of patients with advanced cancer, is a significant cause of morbidity and mortality. Different tumours have varying propensities to induce cachexia. Cachexia is most commonly seen in subjects with lung and gastrointestinal cancers, in contrast to haematological and breast cancers, wherein it is rare (Gordon et al., 2005). Although there are several animal models of cancer cachexia, each model has its own merits and does not present all aspects of

Conflicts of interest

The authors declare no conflict of interests.

Author contribution statement

T.B., J.J., H.L., J.S., and S.C. conducted the animal experiments. M.P. conducted the RNAseq analyses. C.S., S.Y., and Y.Y. conceived the research and analysed the results. All authors reviewed the manuscript.

Acknowledgements

This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) of Korea (2017M3A9E4065192). Jaewoong Jang was supported by Chung-Ang University Research Grant in 2018. The authors thank the staffs of BIOCON for assistance with the histology experiments.

References (42)

  • S.C. Bodine et al.

    Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1

    Am. J. Physiol. Endocrinol. Metab.

    (2014)
  • M. Chen et al.

    Meta-analysis of oxaliplatin-based chemotherapy combined with traditional medicines for colorectal cancer: contributions of specific plants to tumor response

    Integr. Cancer Ther.

    (2016)
  • K.C. Cheng et al.

    The use of herbal medicine in cancer-related anorexia/cachexia treatment around the world

    Curr. Pharmaceut. Des.

    (2012)
  • Y.K. Choi et al.

    Effect of Sipjeondaebo-tang on cancer-induced anorexia and cachexia in CT-26 tumor-bearing mice

    Mediat. Inflamm.

    (2014)
  • S. Cohen et al.

    During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

    J. Cell Biol.

    (2009)
  • K. Fearon et al.

    Understanding the mechanisms and treatment options in cancer cachexia

    Nat. Rev. Clin. Oncol.

    (2013)
  • R.A. Frost et al.

    Skeletal muscle cytokines: regulation by pathogen-associated molecules and catabolic hormones

    Curr. Opin. Clin. Nutr. Metab. Care

    (2005)
  • V. Girish et al.

    Affordable image analysis using NIH Image/ImageJ

    Indian J. Cancer

    (2004)
  • J.N. Gordon et al.

    Cancer cachexia

    QJM

    (2005)
  • D.Y. He et al.

    Anti-inflammatory and immunomodulatory effects of paeonia lactiflora pall., a traditional Chinese herbal medicine

    Front. Pharmacol.

    (2011)
  • R.T. Jagoe et al.

    What do we really know about the ubiquitin-proteasome pathway in muscle atrophy?

    Curr. Opin. Clin. Nutr. Metab. Care

    (2001)
  • Cited by (28)

    • Paeonia lactiflora extract improves the muscle function of mdx mice, an animal model of Duchenne muscular dystrophy, via downregulating the high mobility group box 1 protein

      2022, Journal of Ethnopharmacology
      Citation Excerpt :

      Total RNA was isolated from the gastrocnemius and diaphragm tissues by using an RNeasy Kit (Qiagen; cat# 74106), and then 1 μg of the total RNA was reverse-transcribed using a cDNA Reverse Transcription kit (Applied Biosystems Inc.; cat# 43-688-13). The qPCR reactions were performed as described previously (Bae et al., 2020). Assay-on-Demand Gene Expression Products (Applied Biosystems) were used for the qPCR reactions of interleukin (IL)-6 (cat# Mm00446190_m1), IL-1β (cat# Mm00434228_m1), IL-1α (cat# Mm00439620_m1), interferon (IFN)-γ (cat# Mm01168134_m1), tumor necrosis factor (TNF)-α (cat# Mm00443258_m1), C–C motif chemokine ligand (CCL)2 (cat# Mm00441242_m1), CCL3 (cat# Mm00441259_m1), CCL4 (cat# 00443111_m1), CCL5 (cat# Mm01302428_m1), and 18 S ribosomal RNA (cat# Hs99999901_s1).

    • Identification of a novel anticancer mechanism of Paeoniae Radix extracts based on systematic transcriptome analysis

      2022, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      PR exerts anticancer effects by manipulating molecular mechanisms involved in well-known anticancer targets. PR reduces inflammatory cytokines in lung and colon carcinoma cells and suppresses the molecular NF-κB signaling pathway [13]. Moreover, it exerts anti-proliferative properties by decreasing certain cell cycle populations, predominantly in the G1 phase in bladder cancer cells [14].

    • A standardized herbal combination of Astragalus membranaceus and Paeonia japonica, protects against muscle atrophy in a C26 colon cancer cachexia mouse model

      2021, Journal of Ethnopharmacology
      Citation Excerpt :

      This dose was equivalent to 96–125 mg/kg in mice; thus, we selected 100 mg/kg for this study. For AM, PJ and APX, the same dose (100 mg/kg) was selected based on a previous study (Bae et al., 2020a) and the commonly recommended human dose (500 mg/day per adult). Food intake and body weight were measured daily in the morning (10:00) after injection of C26 adenocarcinoma cells.

    • Paeonia lactiflora extract suppresses cisplatin-induced muscle wasting via downregulation of muscle-specific ubiquitin E3 ligases, NF-κB signaling, and cytokine levels

      2021, Journal of Ethnopharmacology
      Citation Excerpt :

      The traditional herbal formulation PHY906, comprising four herbs including P. lactiflora, has been reported to reduce chemotherapy-induced toxicities and enhance the therapeutic indices of many chemotherapeutic agents in both preclinical and clinical studies (Liu and Cheng, 2012). In our previous study, we reported that P. lactiflora extract suppress muscle wasting in cancer-bearing mice (Bae et al., 2020). In the present study, the suppressive effects of a P. lactiflora ethanol extract on chemotherapy-induced muscle wasting and its mechanism of action were investigated in cisplatin-treated mice.

    View all citing articles on Scopus
    1

    These two authors contributed equally in this work.

    View full text