SUBSCRIBE
Search JIM Advanced Search

Journal of Chinese Integrative Medicine ›› 2013, Vol. 11 ›› Issue (6): 405-415.doi: 10.3736/jintegrmed2013057

• Research Article • Previous Articles     Next Articles

Diarylheptanoid-myricanone isolated from ethanolic extract of Myrica cerifera shows anticancer effects on HeLa and PC3 cell lines: Signalling pathway and drug-DNA interaction

Paul Avijita, Das Sreemantia, Das Jayeetaa, Samadder Asmitaa, Bishayee Kausika, Sadhukhan Ratanb, Anisur Rahman Khuda-Bukhsha()   

  1. a. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani,Kalyani-741235, India
    b. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani-741235, India
  • Received:2013-06-11 Accepted:2013-08-01 Online:2013-11-10 Published:2013-11-15

RichHTML

PDF (PC)

Like

Objective

To test if myricanone (C21H24O5), a cyclic diarylheptanoid, has anticancer effects on two different cancer cell lines HeLa and PC3. The present study was conducted with a note on the drug-DNA interaction and apoptotic signalling pathway.


Methods

Several studies like cytotoxicity, nuclear damage, annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)-labelled apoptotic assay and cell cycle arrest, immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) were used following standard protocols. Circular dichroism (CD) spectroscopy was also done to evaluate whether myricanone effectively interacted with DNA to bring about conformational changes that could strongly inhibit the cancer cell proliferation.


Results

Myricanone showed a greater cytotoxic effect on PC3 cells than on HeLa cells. Myricanone promoted G0/G1 arrest in HeLa cells and S phase arrest in PC3 cells. Nuclear condensation and annexin V-FITC/PI studies revealed that myricanone promoted apoptotic cell death. CD spectroscopic data indicated that myricanone had an interaction with calf thymus DNA that changed DNA structural conformation. RT-PCR and immunoblot studies revealed that myricanone activated the apoptotic signalling cascades through down-regulation of transcription factors like nuclear factor-κB (NF-κB) (p65), and signal transducers and activators of transcription 3 (STAT3); cell cycle regulators like cyclin D1, and survivin and other signal proteins like Bcl-2 and up-regulation of Bax, caspase-9 and caspase-3.


Conclusion

Myricanone induced apoptosis in both types of cancer cells by triggering caspase activation, and suppression of cell proliferation by down-regulation of NF-κB and STAT3 signalling cascades, which makes it a suitable candidate for possible use in the formulation of therapeutic agent for combating cancer.

Key words: Myrica, DiarylHeptanoids, Plant extracts, Apoptosis, NF-κB;, STAT3 transcription factor, Signal transduction

"

Figure 1

Mass spectroscopic data and chemical structure of myricanone"

Figure 2

Analysis of cytotoxic and apoptotic potentials of myricanoneCells were cultured in presence of various concentrations of myricanone (10-50 μg/mL) for 48 h. Cell proliferation was measured by MTT assay. IC50 value of myricanone on HeLa and PC3 cells were found to be 29.6 μg/mL and 18.4 μg/mL, respectively. Data are expressed as mean ± standard error of mean, n=3.HaCaT: immortal human keratinocyte line; PBMC: peripheral blood mononuclear cell; HeLa: human cervical cancer cell line; PC3: human prostate cancer cell line; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC50: half inhibitory concentration."

Figure 3

Morphological changes of control and D1-, D2-, and D3-treated HeLa and PC3 cells for 48 h of myricanone treatment (Phase contrast microscopy, ×200) Myricanone showed greater apoptotic effect on both HeLa and PC3 cells in a dose-dependent manner. In higher doses, more apoptotic round-shaped, cellular blebbing, and shrinked cells were observed in both cells.D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 4

Analysis of drug-DNA interaction and DNA damage induced by myricanone CD spectra of calf thymus DNA were incubated with myricanone.CT-DNA: calf thymus DNA; CD: circular dichroism."

Figure 5

Nuclear condensation assessment of control and drug-treated cells by DAPI staining (Fluorescence microscopy, ×200) After drug treatment the numbers of apoptotic nuclei were increased and more nuclear condensation was observed in both cells with respect to control cells. Arrows showed the apoptotic nuclei.DAPI: 4',6-diamidino-2-phenylindole. D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 6

DNA fragmentation assay of HeLa and PC3 cells treated with control and D1, D2, and D3. D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 7

Flow cytometric analysis of myricanone-mediated apoptosis in HeLa and PC3 cells by annexin V-FITC/PI double staining Differentiation of cell population was done on flow cytometer and was assessed as: viable cancer cells (annexin -ve; PI -ve); early apoptotic cancer cells (annexin +ve; PI -ve); late apoptotic cancer cells (annexin +ve; PI +ve); necrotic cells (PI +ve). Percentages of cell population were written in their respective quadrants. Samples were analyzed in Cyflogic (v.1.2.1) software and determination was based on mean fluorescence intensity of 10 000 events. Bar charts show the mean values of unaffected cells, early apoptotic cells, late apoptotic cells and necrotic cells.FITC: fluorescein isothiocyanate; PI: propidium iodide; D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 8

Flow cytometric analysis of the cell cycle in myricanone-treated HeLa and PC3 cells with respect to untreated cells In HeLa cells, arrest occurred at G0/G1 stage whereas in PC3 cells arrest occurred at S phase.D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 9

Effects of myricanone on mRNA and protein level expressions in HeLa and PC3 cells with respect to untreated cells A: RT-PCR analysis of Bax, Bcl-2 and caspase-3 of control and treated cells. mRNA expression analysis showed up-regulation of Bax, caspase-3 and down-regulation of Bcl-2 activity. B: Expression of Bax, Bcl-2, caspase-9 and caspase-3 of control and treated cells was analyzed by Western blot analysis. Significant up-regulation of Bax, caspase-3, and caspase-9 and down-regulation of Bcl-2 was found in myricanone-treated cells in a dose-dependent manner. The cleavage of caspase-3, which is the active form of caspase-3 was also observed in both cell lines. GAPDH acts as a housekeeping gene. The intensity of the control was normalized to 1, and the intensity of each band from treated cells is compared with the control. RT-PCR: reverse transcriptase-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 10

mRNA and protein level expressions in myricanone-treated HeLa and PC3 cells with respect to untreated cells A: RT-PCR analysis of IL-6, STAT3 and NF-κB. Expression analysis revealed significant down-regulation of IL-6, STAT3 and NF-κB expression when treated with myricanone with respect to control cells. B: Expression of STAT3, phosphorylated STAT3 (Tyrosine 705) and NF-κB (p65) of control and treated cells was analyzed by Western blotting. GAPDH acts as a housekeeping gene. STAT3 and NF-κB expression was found to be significantly down-regulated in myricanone-treated cells compared to untreated control cells. The intensity of the control was normalized to 1, and the intensity of each band from treated cells is compared with the control.NF-κB: nuclear factor-κB; STAT3: signal transducer and activator of transcription 3; IL-6: interleukin-6; RT-PCR: reverse transcriptase-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

Figure 11

RT-PCR analysis of cyclin D1 and survivin of control and treated cellsMyricanone-treated cells showed significant down-regulation of cyclin D1 and survivin expression when compared with untreated control cells. GAPDH acts as a housekeeping gene. The intensity of the control was normalized to 1, and the intensity of each band from treated cells is compared with the control.RT-PCR: reverse transcriptase-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; D1: 10 μg/mL; D2: 20 μg/mL; D3: 30 μg/mL."

[1] Lin WW, Karin MA . Cytokine-mediated link between innate immunity, inflammation, and cancer[J]. J Clin Invest, 2007,117(5):1175-1183
doi: 10.1172/JCI31537
[2] Barnes PJ, Karin M . Nuclear factor-κB: a pivotal transcription factor in chronic inflammatory diseases[J]. N Engl J Med, 1997,336(15):1066-1071
doi: 10.1056/NEJM199704103361506
[3] Ghosh S, Karin M . Missing pieces in the NF-κB puzzle[J]. Cell, 2002,109(Suppl):S81-S96
doi: 10.1016/S0092-8674(02)00703-1
[4] Masuda M, Suzui M, Yasumatu R, Nakashima T, Kuratomi Y, Azuma K, Tomita K, Komiyama S, Weinstein IB . Constitutive activation of signal transducers and activators of transcription 3 correlates with cyclin D1 overexpression and may provide a novel prognostic marker in head and neck squamous cell carcinoma[J]. Cancer Res, 2002,62(12):3351-3355
[5] Baud V, Karin M . Is NF-κB a good target for cancer therapy? Hopes and pitfalls[J]. Nat Rev Drug Discov, 2009,8(1):33-40
doi: 10.1038/nrd2781
[6] Lentz DL, Clark AM, Hufford CD, Meurer-Grimes B, Passreiter CM, Cordero J, Ibrahimi O, Okunade AL . Antimicrobial properties of Honduran medicinal plants[J]. J Ethnopharmacol, 1998,63(3):253-263
doi: 10.1016/S0378-8741(98)00100-7
[7] Paul BD, Rao GS, Kapadia GJ . Isolation of myricadiol, myricitrin, taraxerol, and taraxerone from Myrica cerifera L. root bark[J]. J Pharm Sci, 1974,63(6):958-959
doi: 10.1002/jps.2600630638
[8] Singh S, Aggarwal BB . Activation of transcription factor NF-κB is suppressed by curcumin(diferuloylmethane) [corrected][J]. J Biol Chem, 1995,270(42):24995-245000
doi: 10.1074/jbc.270.42.24995
[9] Akihisa T, Taguchi Y, Yasukawa K, Tokuda H, Akazawa H, Suzuki T, Kimura Y . Acerogenin M, a cyclic diarylheptanoid, and other phenolic compounds from Acer nikoense and their anti-inflammatory and anti-tumor-promoting effects[J]. Chem Pharm Bull (Tokyo), 2006,54(5):735-739
doi: 10.1248/cpb.54.735
[10] Lee CS, Jang ER, Kim YJ, Myung SC, Kim W, Lee MW . Diarylheptanoid hirsutenone enhances apoptotic effect of TRAIL on epithelial ovarian carcinoma cell lines via activation of death receptor and mitochondrial pathway[J]. Invest New Drugs, 2012,30(2):548-557
doi: 10.1007/s10637-010-9601-5
[11] Matsuda H, Yamazaki M, Matsuo K, Asanuma Y, Kubo M . Anti-androgenic activity of Myricae Cortex — isolation of active constituents from bark of Myrica rubra[J]. Biol Pharm Bull, 2001,24(3):259-263
doi: 10.1248/bpb.24.259
[12] Matsuda H, Morikawa T, Tao J, Ueda K, Yoshikawa M . Bioactive constituents of Chinese natural medicines. VII. Inhibitors of degranulation in RBL-2H3 cells and absolute stereostructures of three new diarylheptanoid glycosides from the bark of Myrica rubra[J]. Biol Pharm Bull, 2001,24(3):259-263
doi: 10.1248/bpb.24.259
[13] Schindler R, Dinarello CA . A method for removing interleukin-1- and tumor necrosis factor-inducing substances from bacterial cultures by ultrafiltration with polysulfone[J]. J Immunol Methods, 1989,116(2):159-165
doi: 10.1016/0022-1759(89)90199-3
[14] Samadder A, Chakraborty D, De A, Bhattacharyya SS, Bhadra K, Khuda-Bukhsh AR . Possible signaling cascades involved in attenuation of alloxan-induced oxidative stress and hyperglycemia in mice by ethanolic extract of Syzygium jambolanum: drug-DNA interaction with calf thymus DNA as target[J]. Eur J Pharm Sci, 2011,44(3):207-217
doi: 10.1016/j.ejps.2011.07.012
[15] Biswas R, Mandal SK, Dutta S, Bhattacharyya SS, Boujedaini N, Khuda-Bukhsh AR . Thujone-rich fraction of Thuja occidentalis demonstrates major anti-cancer potentials: evidences from in vitro studies on A375 cells[J]. Evid Based Complement Alternat Med, 2011,2011:568148
[16] Bhattacharyya SS, Paul S, Dutta S, Boujedaini N, Khuda-Bukhsh AR . Anti-oncogenic potentials of a plant coumarin(7-hydroxy-6-methoxy coumarin) against 7,12-dimethylbenz[a] anthracene-induced skin papilloma in mice: the possible role of several key signal proteins[J]. J Chin Integr Med, 2010,8(7):645-654
doi: 10.3736/jcim201007
[17] Hengartner MO . The biochemistry of apoptosis[J]. Nature, 2000,407(6805):770-776
doi: 10.1038/35037710
[18] Campbell KJ, Rocha S, Perkins ND . Active repression of antiapoptotic gene expression by RelA(p65) NF-κB[J]. Mol Cell, 2004,13(6):853-865
doi: 10.1016/S1097-2765(04)00131-5
[19] Barton BE, Karras JG, Murphy TF, Barton A, Huang HF . Signal transducer and activator of transcription 3(STAT3) activation in prostate cancer: Direct STAT3 inhibition induces apoptosis in prostate cancer lines[J]. Mol Cancer Ther, 2004,3(1):11-20
doi: 10.1186/1476-4598-3-11
[20] Chen CL, Hsieh FC, Lieblein JC, Brown J, Chan C, Wallace JA, Cheng G, Hall BM, Lin J . Stat3 activation in human endometrial and cervical cancers[J]. Br J Cancer, 2007,96(4):591-599
doi: 10.1038/sj.bjc.6603597
[21] Tamatani M, Che YH, Matsuzaki H, Ogawa S, Okado H, Miyake S, Mizuno T, Tohyama M . Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NF-κB activation in primary hippocampal neurons[J]. J Biol Chem, 1999,274(13):8531-8538
doi: 10.1074/jbc.274.13.8531
[22] Kawamura K, Sato N, Fukuda J, Kodama H, Kumagai J, Tanikawa H, Shimizu Y, Tanaka T . Survivin acts as an antiapoptotic factor during the development of mouse preimplantation embryos[J]. Dev Biol, 2003,256(2):331-341
doi: 10.1016/S0012-1606(02)00135-5
[23] Hodge DR, Hurt EM, Farrar WL . The role of IL-6 and STAT3 in inflammation and cancer[J]. Eur J Cancer, 2005,41(16):2502-2512
doi: 10.1016/j.ejca.2005.08.016
[24] Nielsen M, Kaestel CG, Eriksen KW, Woetmann A, Stokkedal T, Kaltoft K, Geisler C , Rpke C, Odum N.Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells[J]. Leukemia, 1999,13(5):735-738
doi: 10.1038/sj.leu.2401415
[25] Danial NN, Pernis A, Rothman PB . Jak-STAT signaling induced by the v-abl oncogene[J]. Science, 1995,269(5232):1875-1877
doi: 10.1126/science.7569929
[26] Trécul A, Morceau F, Dicato M, Diederich M . Dietary compounds as potent inhibitors of the signal transducers and activators of transcription(STAT) 3 regulatory network[J]. Genes Nutr, 2012,7(2):111-125
doi: 10.1007/s12263-012-0281-y
[27] Grivennikov SI, Karin M . Dangerous liaisons: STAT3 and NF-κB collaboration and crosstalk in cancer[J]. Cytokine Growth Factor Rev, 2010,21(1):11-19
doi: 10.1016/j.cytogfr.2009.11.005
[1] Maged Mohamed Maher Abou-Hashem, Dina Mohamed Abo-elmatty, Noha Mostafa Mesbah, Ahmed Mohamed Abd EL-Mawgoud. Induction of sub-G0 arrest and apoptosis by seed extract of Moringa peregrina (Forssk.) Fiori in cervical and prostate cancer cell lines. Journal of Integrative Medicine, 2019, 17(6): 410-422.
[2] Supakanya Kumkarnjana, Rutt Suttisri, Ubonthip Nimmannit, Apirada Sucontphunt, Mattaka Khongkow, Thongchai Koobkokkruad, Nontima Vardhanabhuti. Flavonoids kaempferide and 4,2′-dihydroxy-4′,5′,6′-trimethoxychalcone inhibit mitotic clonal expansion and induce apoptosis during the early phase of adipogenesis in 3T3-L1 cells. Journal of Integrative Medicine, 2019, 17(4): 288-295.
[3] Shriniwas S. Basaiyye, Sanjay Kashyap, Kannan Krishnamurthi, Saravanadevi Sivanesan. Induction of apoptosis in leukemic cells by the alkaloid extract of garden cress (Lepidium sativum L.). Journal of Integrative Medicine, 2019, 17(3): 221-228.
[4] Stefania Lamponi, Anna Maria Aloisi, Claudia Bonechi, Marco Consumi, Alessandro Donati, Gemma Leone, Claudio Rossi, Gabriella Tamasi, Luana Ghiandai, Ersilia Ferrini, Paolo Fiorenzani, Ilaria Ceccarelli, Agnese Magnani. Evaluation of in vitro cell and blood compatibility and in vivo analgesic activity of plant-derived dietary supplements. Journal of Integrative Medicine, 2019, 17(3): 213-220.
[5] Carlos R. Oliveira, Daniel G. Spindola, Daniel M. Garcia, Adolfo Erustes, Alexandre Bechara, Caroline Palmeira-dos-Santos, Soraya S. Smaili, Gustavo J.S. Pereira, André Hinsberger, Ezequiel P. Viriato, Maria Cristina Marcucci, Alexandra C.H.F. Sawaya, Samantha L. Tomaz, Elaine G. Rodrigues, Claudia Bincoletto. Medicinal properties of Angelica archangelica root extract: Cytotoxicity in breast cancer cells and its protective effects against in vivo tumor development. Journal of Integrative Medicine, 2019, 17(2): 132-140.
[6] Rohitash Jamwal. Bioavailable curcumin formulations: A review of pharmacokinetic studies in healthy volunteers. Journal of Integrative Medicine, 2018, 16(6): 367-374.
[7] Bruno José Martins Da Silva, Sandro Wilson Gomes Pereira, Ana Paula Drummond Rodrigues, José Luiz Martins Do Nascimento, Edilene Oliveira Silva. In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum. Journal of Integrative Medicine, 2018, 16(6): 404-410.
[8] Supakanya Kumkarnjana, Rutt Suttisri, Ubonthip Nimmannit, Thongchai Koobkokkruad, Chutichot Pattamadilok, Nontima Vardhanabhuti . Anti-adipogenic effect of flavonoids from Chromolaena odorata leaves in 3T3-L1 adipocytes. Journal of Integrative Medicine, 2018, 16(6): 427-434.
[9] Shohini Chakraborty, Nashra Afaq, Neelam Singh, Sukanta Majumdar. Antimicrobial activity of Cannabis sativa, Thuja orientalis and Psidium guajava leaf extracts against methicillin-resistant Staphylococcus aureus. Journal of Integrative Medicine, 2018, 16(5): 350-357.
[10] Bruno José Martins Da Silva, Amanda Anastácia Pinto Hage, Edilene Oliveira Silva, Ana Paula Drummond Rodrigues. Medicinal plants from the Brazilian Amazonian region and their antileishmanial activity: A review. Journal of Integrative Medicine, 2018, 16(4): 211-222.
[11] Eric Wei Chiang Chan, Siu Kuin Wong, Hung Tuck Chan . Casticin from Vitex species: A short review on its anticancer and anti-inflammatory properties. Journal of Integrative Medicine, 2018, 16(3): 147-152.
[12] Nisha Panth, Keshav Raj Paudel, Rajendra Karki. Phytochemical profile and biological activity of Juglans regia. Journal of Integrative Medicine, 2016, 14(5): 359-373.
[13] Musa Toyin Yakubu, Abdulquadri Funmbi Olutoye. Aphrodisiac activity of aqueous extract of Anthonotha macrophylla P. Beauv. leaves in female Wistar rats. Journal of Integrative Medicine, 2016, 14(5): 400-408.
[14] JiaXia, Li-qunHe, XiaoSu. Interventional mechanisms of herbs or herbal extracts on renal interstitial fibrosis. Journal of Integrative Medicine, 2016, 14(3): 165-173.
[15] Jesmin Mondal, Asmita Samadder, Anisur Rahman Khuda-Bukhsh. Psorinum 6× triggers apoptosis signals in human lung cancer cells. Journal of Integrative Medicine, 2016, 14(2): 143-153.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] Dong Yang, Yong-ping Du, Qing Shen, Wei Chen, Yan Yu, Guang-lei Chen. Expression of alpha-smooth muscle actin in renal tubulointerstitium in patients with kidney collateral stasis. Journal of Chinese Integrative Medicine, 2008, 6(1): 41-44
[2] Daniel Weber, Janelle M Wheat, Geoffrey M Currie. Inflammation and cancer: Tumor initiation, progression and metastasis,and Chinese botanical medicines. Journal of Chinese Integrative Medicine, 2010, 8(11): 1006-1013
[3] Hong Liu , Guo-liang Zhang, Li Shen , Zhen Zeng, Bao-luo Zhou, Cheng-hai Liu, Guang Nie . Application and evaluation of a pseudotyped virus assay for screening herbs for anti-H5Nl avian influenza virus. Journal of Chinese Integrative Medicine, 2010, 8(11): 1036-1040
[4] Rui Jin, Bing Zhang. A complexity analysis of Chinese herbal property theory: the multiple formations of herbal property (Part 1). Journal of Chinese Integrative Medicine, 2012, 10(11): 1198-1205
[5] Peng-fei Gao, Kenji Watanabe. Introduction of the World Health Organization project of the International Classification of Traditional Medicine. Journal of Chinese Integrative Medicine, 2011, 9(11): 1161-1164
[6] Yang Chen, Jian-hua Huang, You Ning, Zi-yin Shen. Icariin and its pharmaceutical efficacy: Research progress of molecular mechanism. Journal of Chinese Integrative Medicine, 2011, 9(11): 1179-1184
[7] Zhang-ling Zhou, Yue-bo Jiang, Gao-bo Li, Yu-hong Gao. Analysis of facial far-infrared thermogram of patients with acute facial neuritis. Journal of Chinese Integrative Medicine, 2011, 9(11): 1221-1225
[8] Li-fang Wang, Jin-feng Wang, Wei-fang Jin, Hong-fu Wang, Shao-fen Zhang, Jian-jun Gao. Effects of daidzein on steroid receptor coactivator-1 expression in MC3T3-E1 cells and the mechanism. Journal of Chinese Integrative Medicine, 2011, 9(11): 1248-1253
[9] Yi Chen, Wen-jian Wang. Relationships between insulin resistant and hypertension and traditional Chinese medicine intervention. Journal of Chinese Integrative Medicine, 2010, 8(12): 1114-1118
[10] Kai Qu, Tian-fang Wang, Yan Zhao, Xiao-lin Xue, Xiu-yan Wu, Hua-liang Deng, Lu Yang, Li-liYu , Hong Yue, Qing-guo Wang. Common syndrome factors of traditional Chinese medicine in chronic renal failure based on the questionnaire investigation among experts. Journal of Chinese Integrative Medicine, 2010, 8(12): 1147-1152
Journal of Integrative Medicine, ISSN: 2095-4964 CN 31-2083/R
Copyright © Journal of Integrative Medicine
Powered by Beijing Magtech Co. Ltd
沪公网安备31010602003416号      沪ICP备18017633号-3