Analyses of the possible anti-tumor effect of yokukansan

  • Cheolsun HanEmail author
  • Miho Kawata
  • Yusuke Hamada
  • Takashige Kondo
  • Junna Wada
  • Katsunori Asano
  • Hitoshi Makabe
  • Katsuhide Igarashi
  • Naoko Kuzumaki
  • Michiko Narita
  • Hiroyuki Kobayashi
  • Minoru NaritaEmail author
Original Paper


The Kampo medicine yokukansan (YKS) has a wide variety of properties such as anxiolytic, anti-inflammatory and analgesic effects, and is also thought to regulate tumor suppression. In this study, we investigated the anti-tumor effect of YKS. We used Lewis lung carcinoma (LLC)-bearing mice that were fed food pellets containing YKS and then performed a fecal microbiota analysis, a microarray analysis for microRNAs (miRNAs) and an in vitro anti-tumor assay. The fecal microbiota analysis revealed that treatment with YKS partly reversed changes in the microbiota composition due to LLC implantation. Furthermore, a miRNA array analysis using blood serum showed that treatment with YKS restored the levels of miR-133a-3p/133b-3p, miR-1a-3p and miR-342-3p following LLC implantation to normal levels. A TargetScan analysis revealed that the epidermal growth factor receptor 1 signaling pathway is one of the major target pathways for these miRNAs. Furthermore, treatment with YKS restored the levels of miR-200b-3p and miR-200c-3p, a recognized mediator of cancer progression and controller of emotion, in the hypothalamus of mice bearing LLC. An in vitro assay revealed that a mixture of pachymic acid, saikosaponins a and d and isoliquiritigenin, which are all contained in YKS, exerted direct and additive anti-tumor effects. The present findings constitute novel evidence that YKS may exert an anti-tumor effect by reversing changes in the fecal microbiota and miRNAs circulating in the blood serum and hypothalamus, and the compounds found in YKS could have direct and additive anti-tumor effects.


Yokukansan Tumor MicroRNA Microbiota Hypothalamus 



Control food


Cytotoxic T lymphocyte-associated protein 4


Epidermal growth factor receptor






Lewis lung carcinoma




Nucleus accumbens


Non-small cell lung cancer






YKS-treated food



We thank H. Kazamatsuri, M. Nakahama, W. Eguchi, and J.L. Waddington for their support. This work was supported by grants from MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan)-Supported Program for the Strategic Research Foundation at Private Universities, 2014–2018, S1411019.

Author contributions

Minoru N designed the research. CH, YH, TK, NK, KA, HM and Michiko N performed experiments. YH, TK, KA, HM and KI analyzed the data. HK, NK, Michiko N and Minoru N supervised the research. CH, MK and Minoru N wrote the paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Sarkar DK, Murugan S, Zhang C, Boyadjieva N (2012) Regulation of cancer progression by β-endorphin neuron. Cancer Res 72:836–840. CrossRefGoogle Scholar
  2. 2.
    Powell ND, Tarr AJ, Sheridan JF (2013) Psychosocial stress and inflammation in cancer. Brain Behav Immun 30:41–47. CrossRefGoogle Scholar
  3. 3.
    Cao DD, Li L, Chan WY (2016) MicroRNAs: key regulators in the central nervous system and their implication in neurological diseases. Int J Mol Sci 17:842. CrossRefGoogle Scholar
  4. 4.
    Imai S, Saeki M, Yanase M, Horiuchi H, Abe M, Narita M, Kuzumaki N, Suzuki T, Narita M (2011) Change in MicroRNAs associated with neuronal adaptive responses in the nucleus accumbens under neuropathic pain. J Neurosci 31:15294–15299. CrossRefGoogle Scholar
  5. 5.
    Saisu H, Igarashi K, Narita M, Ikegami D, Kuzumaki N, Wajima K, Nakagawa T, Narita M (2015) Neuropathic pain-like stimuli change the expression of ribosomal proteins in the amygdala: genome-wide search for a “pain-associated anxiety-related factor”. Jpn J Pharm Palliat Care Sci 8:47–57Google Scholar
  6. 6.
    Zhang HF, Xu LY, Li EM (2014) A family of pleiotropically acting microRNAs in cancer progression, miR-200: potential cancer therapeutic targets. Curr Pharm Des 20:1896–1903CrossRefGoogle Scholar
  7. 7.
    Narita M, Shimura E, Nagasawa A, Aiuchi T, Suda Y, Hamada Y, Ikegami D, Iwasawa C, Arakawa K, Igarashi K, Kuzumaki N, Yoshioka Y, Ochiya T, Takeshima H, Ushijima T, Narita M (2017) Chronic treatment of non-small-cell lung cancer cells with gefitinib leads to an epigenetic loss of epithelial properties associated with reductions in microRNA-155 and -200c. PLoS One 12:e0172115. CrossRefGoogle Scholar
  8. 8.
    Schwabe RF, Jobin C (2013) The microbiome and cancer. Nat Rev Cancer 13:800–812. CrossRefGoogle Scholar
  9. 9.
    Nelson MH, Diven MA, Huff LW, Paulos CM (2015) Harnessing the microbiome to enhance cancer immunotherapy. J Immunol Res. Google Scholar
  10. 10.
    Zitvogel L, Ayyoub M, Routy B, Kroemer G (2016) Microbiome and anticancer immunosurveillance. Cell 165:276–287. CrossRefGoogle Scholar
  11. 11.
    Erdman SE, Poutahidis T (2017) Gut microbiota modulate host immune cells in cancer development and growth. Free Radic Biol Med 105:28–34. CrossRefGoogle Scholar
  12. 12.
    Ikarashi Y, Mizoguchi K (2016) Neuropharmacological efficacy of the traditional Japanese Kampo medicine yokukansan and its active ingredients. Pharmacol Ther 166:84–95. CrossRefGoogle Scholar
  13. 13.
    Yamaguchi T, Tsujimatsu A, Kumamoto H, Izumi T, Ohmura Y, Yoshida T, Yoshioka M (2012) Anxiolytic effects of yokukansan, a traditional Japanese medicine, via serotonin 5-HT1A receptors on anxiety-related behaviors in rats experienced aversive stress. J Ethnopharmacol 143:533–539. CrossRefGoogle Scholar
  14. 14.
    Fujiwara H, Han Y, Ebihara K, Awale S, Araki R, Yabe T, Matsumoto K (2017) Daily administration of yokukansan and keishito prevents social isolation-induced behavioral abnormalities and down-regulation of phosphorylation of neuroplasticity-related signaling molecules in mice. BMC Complement Altern Med 17:195. CrossRefGoogle Scholar
  15. 15.
    Tamano H, Kan F, Oku N, Takeda A (2010) Ameliorative effect of Yokukansan on social isolation-induced aggressive behavior of zinc-deficient young mice. Brain Res Bull 83:351–355. CrossRefGoogle Scholar
  16. 16.
    Katahira H, Sunagawa M, Watanabe D, Kanada Y, Katayama A, Yamauchi R, Takashima M, Ishikawa S, Hisamitsu T (2017) Antistress effects of Kampo medicine “Yokukansan” via regulation of orexin secretion. Neuropsychiatr Dis Treat 13:863–872. CrossRefGoogle Scholar
  17. 17.
    Furuya M, Miyaoka T, Tsumori T, Liaury K, Hashioka S, Wake R, Tsuchie K, Fukushima M, Ezoe S, Horiguchi J (2013) Yokukansan promotes hippocampal neurogenesis associated with the suppression of activated microglia in Gunn rat. J Neuroinflamm 10:145. CrossRefGoogle Scholar
  18. 18.
    Ebisawa S, Andoh T, Shimada Y, Kuraishi Y (2015) Yokukansan improves mechanical allodynia through the regulation of interleukin-6 expression in the spinal cord in mice with neuropathic pain. Evid Based Complement Alternat Med 2015:870687. CrossRefGoogle Scholar
  19. 19.
    Agalioti T, Giannou AD, Krontira AC, Kanellakis NI, Kati D, Vreka M, Pepe M, Spella M, Lilis I, Zazara DE, Nikolouli E, Spiropoulou N, Papadakis A, Papadia K, Voulgaridis A, Harokopos V, Stamou P, Meiners S, Eickelberg O, Snyder LA, Antimisiaris SG, Kardamakis D, Psallidas I, Marazioti A, Stathopoulos GT (2017) Mutant KRAS promotes malignant pleural effusion formation. Nat Commun 16:15205. CrossRefGoogle Scholar
  20. 20.
    Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, Fluckiger A, Messaoudene M, Rauber C, Roberti MP, Fidelle M, Flament C, Poirier-Colame V, Opolon P, Klein C, Iribarren K, Mondragon L, Jacquelot L, Qu B, Ferrere G, Clémenson C, Mezquita L, Masip JR, Naltet C, Brosseau S, Kaderbhai C, Richard C, Rizvi H, Rizvi H, Levenez F, Galleron N, Quinquis B, Pons N, Ryffel B, Minard-Colin V, Gonin P, Soria JC, Deutsch E, Loriot Y, Ghiringhelli F, Zalcman G, Goldwasser F, Escudier B, Hellmann MD, Eggermont A, Raoult D, Albiges L, Kroemer G, Zitvogel L (2017) Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science 359:91–97. CrossRefGoogle Scholar
  21. 21.
    Ueki T, Mizoguchi K, Yamaguchi T, Nishi A, Ikarashi Y, Hattori T, Kase Y (2015) Yokukansan increases 5-HT1A receptors in the prefrontal cortex and enhances 5-HT1A receptor agonist-induced behavioral responses in socially isolated mice. Evid Based Complement Alternat Med 25:25. Google Scholar
  22. 22.
    Mizoguchi K, Ikarashi Y (2017) Multiple psychopharmacological effects of the traditional Japanese Kampo medicine Yokukansan, and the brain regions it affects. Front Pharmacol 8:149. Google Scholar
  23. 23.
    Nishi A, Yamaguchi T, Sekiguchi K, Imamura S, Tabuchi M, Kanno H, Nakai Y, Hashimoto K, Ikarashi Y, Kase Y (2012) Geissoschizine methyl ether, an alkaloid in Uncaria hook, is a potent serotonin1A receptor agonist and candidate for amelioration of aggressiveness and sociality by yokukansan. Neuroscience 207:124–136. CrossRefGoogle Scholar
  24. 24.
    Fiorentino L, Ancoli-Israel S (2007) Sleep dysfunction in patients with cancer. Curr Treat Options Neurol 9:337–346CrossRefGoogle Scholar
  25. 25.
    Vétizou M, Pitt JM, Daillère R, Lepage P, Waldschmitt N, Flament C, Rusakiewicz S, Routy B, Roberti MP, Duong CP, Poirier-Colame V, Roux A, Becharef S, Formenti S, Golden E, Cording S, Eberl G, Schlitzer A, Ginhoux F, Mani S, Yamazaki T, Jacquelot N, Enot DP, Bérard M, Nigou J, Opolon P, Eggermont A, Woerther PL, Chachaty E, Chaput N, Robert C, Mateus C, Kroemer G, Raoult D, Boneca IG, Carbonnel F, Chamaillard M, Zitvogel L (2015) Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science 350:1079–1084. CrossRefGoogle Scholar
  26. 26.
    Wu M, Wu Y, Deng B, Li J, Cao H, Qu Y, Qian X, Zhong G (2016) Isoliquiritigenin decreases the incidence of colitis-associated colorectal cancer by modulating the intestinal microbiota. Oncotarget 7:85318–85331. Google Scholar
  27. 27.
    Ciardiello F, Tortora G (2003) Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur J Cancer 39:1348–1354CrossRefGoogle Scholar
  28. 28.
    Forcella M, Oldani M, Epistolio S, Freguia S, Monti E, Fusi P, Frattini M (2017) Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: effect of the plasma membrane-associated NEU3. PLoS One 12:e0187289. CrossRefGoogle Scholar
  29. 29.
    Morodomi Y, Okamoto T, Maehara Y (2017) Reply to “EGFR mutation in patients with lung adenosquamous cell carcinoma”. Ann Surg Oncol 24(3):676. CrossRefGoogle Scholar
  30. 30.
    Bethune G, Bethune D, Ridgway N, Xu Z (2010) Epidermal growth factor receptor (EGFR) in lung cancer: an overview and update. J Thorac Dis 2:48–51Google Scholar
  31. 31.
    Pancewicz-Wojtkiewicz J (2016) Epidermal growth factor receptor and notch signaling in non-small-cell lung cancer. Cancer Med 5:3572–3578. CrossRefGoogle Scholar
  32. 32.
    Zhang W, Liu HT (2002) MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 12:9–18CrossRefGoogle Scholar
  33. 33.
    Brantley EC, Benveniste EN (2008) Signal transducer and activator of transcription-3: a molecular hub for signaling pathways in gliomas. Mol Cancer Res 6:675–684. CrossRefGoogle Scholar
  34. 34.
    Fung TC, Olson CA, Hsiao EY (2017) Interactions between the microbiota, immune and nervous systems in health and disease. Nat Neurosci 20:145–155. CrossRefGoogle Scholar
  35. 35.
    Meister B, Herzer S, Silahtaroglu A (2013) MicroRNAs in the hypothalamus. Neuroendocrinology 98:243–253. CrossRefGoogle Scholar
  36. 36.
    Geaghan M, Cairns MJ (2015) MicroRNA and posttranscriptional dysregulation in psychiatry. Biol Psychiatry 78:231–239. CrossRefGoogle Scholar
  37. 37.
    Jin J, Cheng Y, Zhang Y, Wood W, Peng Q, Hutchison E, Mattson MP, Becker KG, Duan W (2012) Interrogation of brain miRNA and mRNA expression profiles reveals a molecular regulatory network that is perturbed by mutant huntingtin. J Neurochem 123:477–490. CrossRefGoogle Scholar
  38. 38.
    Wang Y, Wang Y, Yang GY (2013) MicroRNAs in cerebral ischemia. Stroke Res Treat 2013:276540. Google Scholar
  39. 39.
    Zhu J, Chen Z, Tian J, Meng Z, Ju M, Wu G, Tian Z (2017) miR-34b attenuates trauma-induced anxiety-like behavior by targeting CRHR1. Int J Mol Med 40:90–100. CrossRefGoogle Scholar
  40. 40.
    Li C, Liu Y, Liu D, Jiang H, Pan F (2016) Dynamic alterations of miR-34c expression in the hypothalamus of male rats after early adolescent traumatic stress. Neural Plast 2016:5249893. Google Scholar
  41. 41.
    Jadhav SP, Kamath SP, Choolani M, Lu J, Dheen ST (2014) microRNA-200b modulates microglia-mediated neuroinflammation via the cJun/MAPK pathway. J Neurochem 130:388–401. CrossRefGoogle Scholar
  42. 42.
    Beclin C, Follert P, Stappers E, Barral S, Nathalie C, de Chevigny A, Magnone V, Lebrigand K, Bissels U, Huylebroeck D, Bosio A, Barbry P, Seuntjens E, Cremer H (2016) miR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition. Sci Rep 6:35729. CrossRefGoogle Scholar
  43. 43.
    Gapter L, Wang Z, Glinski J, NG KY (2005) Induction of apoptosis in prostate cancer cells by pachymic acid from Poria cocos. Biochem Biophys Res Commun 332(4):1153–1161CrossRefGoogle Scholar
  44. 44.
    Ling H, Zhang Y, Ng KY, Chew EH (2011) Pachymic acid impairs breast cancer cell invasion by suppressing nuclear factor-kappaB-dependent matrix metalloproteinase-9 expression. Breast Cancer Res Treat 126(3):609–620. CrossRefGoogle Scholar
  45. 45.
    Chen S, Swanson K, Eliaz I, McClintick JN, Sandusky GE, Sliva D (2015) Pachymic acid inhibits growth and induces apoptosis of pancreatic cancer in vitro and in vivo by targeting ER stress. PLoS One 10(4):e0122270. CrossRefGoogle Scholar
  46. 46.
    Ma J, Liu J, Lu C, Cai D (2015) Pachymic acid induces apoptosis via activating ROS-dependent JNK and ER stress pathways in lung cancer cells. Cancer Cell Int 15:78. CrossRefGoogle Scholar
  47. 47.
    Lu C, Ma J, Cai D (2017) Pachymic acid inhibits the tumorigenicity of gastric cancer cells by the mitochondrial pathway. Anticancer Drugs 28(2):170–179. CrossRefGoogle Scholar
  48. 48.
    Kim BM, Hong SH (2011) Sequential caspase-2 and caspase-8 activation is essential for saikosaponin a-induced apoptosis of human colon carcinoma cell lines. Apoptosis 16(2):184–197. CrossRefGoogle Scholar
  49. 49.
    Chen JC, Chang NW, Chung JG, Chen KC (2003) Saikosaponin-A induces apoptotic mechanism in human breast MDA-MB-231 and MCF-7 cancer cells. Am J Chin Med 31(3):363–377CrossRefGoogle Scholar
  50. 50.
    Hsu YL, Kuo PL, Chiang LC, Lin CC (2004) Involvement of p53, nuclear factor kappaB and Fas/Fas ligand in induction of apoptosis and cell cycle arrest by saikosaponin d in human hepatoma cell lines. Cancer Lett 213(2):213–221CrossRefGoogle Scholar
  51. 51.
    Hsu YL, Kuo PL, Lin CC (2004) The proliferative inhibition and apoptotic mechanism of Saikosaponin D in human non-small cell lung cancer A549 cells. Life Sci 75(10):1231–1242CrossRefGoogle Scholar
  52. 52.
    Liu RY, Li JP (2014) Saikosaponin-d inhibits proliferation of human undifferentiated thyroid carcinoma cells through induction of apoptosis and cell cycle arrest. Eur Rev Med Pharmacol Sci 18(17):2435–2443Google Scholar
  53. 53.
    Li Y, Cai T, Zhang W, Zhu W, Lv S (2017) Effects of Saikosaponin D on apoptosis in human U87 glioblastoma cells. Mol Med Rep 16(2):1459–1464. CrossRefGoogle Scholar
  54. 54.
    Hsu YL, Kuo PL, Chiang LC, Lin CC (2004) Isoliquiritigenin inhibits the proliferation and induces the apoptosis of human non-small cell lung cancer a549 cells. Clin Exp Pharmacol Physiol 31(7):414–418CrossRefGoogle Scholar
  55. 55.
    Ii T, Satomi Y, Katoh D, Shimada J, Baba M, Okuym Y, Nishino H, Kitamura N (2004) Induction of cell cycle arrest and p21(CIP1/WAF1) expression in human lung cancer cells by isoliquiritigenin. Cancer Lett 207(1):27–35CrossRefGoogle Scholar
  56. 56.
    Jung SK, Lee MH, Lim DY, Kim JE, Singh P, Lee SY, Jeong CH, Lim TG, Chen H, Chi YI, Kundu JK, Lee NH, Lee CC, Cho YY, Bode AM, Lee KW, Dong Z (2014) Isoliquiritigenin induces apoptosis and inhibits xenograft tumor growth of human lung cancer cells by targeting both wild type and L858R/T790M mutant EGFR. J Biol Chem 289(52):35839–35848. CrossRefGoogle Scholar

Copyright information

© The Japanese Society of Pharmacognosy 2019

Authors and Affiliations

  • Cheolsun Han
    • 1
    • 2
    • 3
    Email author
  • Miho Kawata
    • 3
    • 4
  • Yusuke Hamada
    • 3
    • 5
  • Takashige Kondo
    • 3
  • Junna Wada
    • 3
  • Katsunori Asano
    • 3
  • Hitoshi Makabe
    • 3
  • Katsuhide Igarashi
    • 5
  • Naoko Kuzumaki
    • 3
    • 5
  • Michiko Narita
    • 3
  • Hiroyuki Kobayashi
    • 1
    • 2
  • Minoru Narita
    • 3
    • 5
    Email author
  1. 1.Department of Hospital AdministrationJuntendo University School of MedicineTokyoJapan
  2. 2.Center of Advanced Kampo Medicine and Clinical ResearchJuntendo University School of MedicineTokyoJapan
  3. 3.Department of PharmacologyHoshi University School of Pharmacy and Pharmaceutical ScienceTokyoJapan
  4. 4.Department of PhysiologyFujita Health UniversityToyoakeJapan
  5. 5.Life Science Tokyo Advanced Research Center (L-StaR)Hoshi University School of Pharmacy and Pharmaceutical ScienceTokyoJapan

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