Study of the cytotoxicity of asiaticoside on rats and tumour cells

Fatma J Al-Saeedi 1

Affiliations

doi: 10.1186/1471-2407-14-220

Abstract

Background: Cancer chemoprevention is considered one of the most promising areas in current cancer research, and asiaticoside, which is derived from the plant Centella asiatica, has a relative lack of systemic toxicity. The purpose of this study was to investigate whether asiaticoside is effective against 7,12-dimethylbenz(a)anthracene (DMBA)-induced carcinogenicity in vitro (MCF-7 and other cells) and in vivo (DMBA-induced rat cancer).

Methods: An MTT assay was performed involving the treatment of MCF-7 cells for 48 h with H2O2 alone and H2O2 + different asiaticoside concentrations. Flow cytometry was performed, and the level of caspase 3, tumour necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1) were quantified. Adult female Sprague-Dawley (SD) rats were divided into five groups designated I (control), II (DMBA-induced cancer), III (pre- and post-treatment with asiaticoside (200 μg/animal) in DMBA-induced cancer), IV (post-treatment with asiaticoside in DMBA-induced cancer), and V (treated with asiaticoside alone, drug control). Twelve weeks post-DMBA, rats developed mammary tumours. Rats either were sacrificed or imaged with MIBI. Histological examination of tumour tissues was performed. Tumour MIBI uptake ratios were determined. The data are expressed as the means ± standard deviation. Appropriate t-test and ANOVA statistical methods were used to compare data.

Results: The IC50 of asiaticoside for MCF-7 cells was determined to be 40 μM. Asiaticoside has potential for hydrogen peroxide cytotoxicity, and the caspase-3 activity increased with increasing asiaticoside dose in MCF-7 cells treated for 48 h. The expression of the cytokines TNF-α and IL-1β was significantly decreased and correlated with MIBI uptake ratios in vitro and in vivo after asiaticoside administration.

Conclusion: This study demonstrates that asiaticoside is effective in vitro and in vivo in inducing apoptosis and enhancing anti-tumour activity.

Figures

Figure 1 The experimental protocol.

Figure 1 The experimental protocol.

Figure 2 Raw data for the percentage of cell inhibition for a 48 h asiaticoside incubation period, showing no effects of asiaticoside on cell lines with the exception of the MCF-7 cell line upon MTT assay.

Figure 2 Raw data for the percentage of cell inhibition for a 48 h asiaticoside incubation period, showing no effects of asiaticoside on cell lines with the exception of the MCF-7 cell line upon MTT assay.

Figure 3 The percentage of asiaticoside cell inhibition over control for a 48 h incubation period and the potential for hydrogen peroxide cytotoxicity using an MTT assay for MCF-7 cells.

Figure 3 The percentage of asiaticoside cell inhibition over control for a 48 h incubation period and the potential for hydrogen peroxide cytotoxicity using an MTT assay for MCF-7 cells.

Figure 4 Percentage of fragmented DNA in response to the effects of asiaticoside.

Figure 4 Percentage of fragmented DNA in response to the effects of asiaticoside.

Figure 5 Percentage of fragmented DNA in response to the effects of asiaticoside using a caspase-3 fluorescence assay. To the right of the graph is shown a western blot for the same lysate prepared for ELISA and caspase 3 assays using MCF-7 and HaCaT cells.

Figure 5 Percentage of fragmented DNA in response to the effects of asiaticoside using a caspase-3 fluorescence assay. To the right of the graph is shown a western blot for the same lysate prepared for ELISA and caspase 3 assays using MCF-7 and HaCaT cells.

Figure 6 Histograms of DNA synthesis with 0, 20, 40 and 80 μm asiaticoside treatment (S phase; % gated S phase cells in cell cycle) as measured by flow cytometry.

Figure 6 Histograms of DNA synthesis with 0, 20, 40 and 80 μm asiaticoside treatment (S phase; % gated S phase cells in cell cycle) as measured by flow cytometry.

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References

  1.  
    1. World Health Organization, WHO. Cancer. Chapter 1 Burden. World Health Organization. http://www.who.int/nmh/publications/ncdreportchapter1.pdf.
  2.  
    1. Babu TD, Kuttan G, Padikkala J. Cytotoxic and antitumor properties of certain texa of umbelliferae with specific reference to Centella asiatica (L.) urban. J Ethnopharmacol. 1995;48:53–57. doi: 10.1016/0378-8741(95)01284-K. - DOI - PubMed
  3.  
    1. Cheng CL, Guo JS, Luk J, Koo MW. The healing effects of Centella extract and asiaticoside on acetic acid induced gastric ulcers in rats. Life Sci. 2004;74:2237–2249. doi: 10.1016/j.lfs.2003.09.055. - DOI - PubMed
  4.  
    1. Komarcević A. The modern approach to wound treatment. Med Pregl. 2000;53:363–368. - PubMed
  5.  
    1. Suguna L, Sivakumar P, Chandrakasan G. Effects of centella asiatica extract on dermal wound healing in rats. Indian J Exp Biol. 1996;34:1208–1211. - PubMed
  6.  
    1. Apparao MVR, Srinivasan K, Rao K. The effect of mandookparni (Centella asiatica) on the general mental ability (Medhya) of mentally retarded children. J Res Indian Med. 1973;8:9–16.
  7.  
    1. Oyedeji OA, Afolayan AJ. Chemical composition and antibacterial activity of the essential oil of Centella asiatica growing in South Africa. Pharm Biol. 2005;43:249–252. doi: 10.1080/13880200590928843. - DOI
  8.  
    1. Mook-Jung I, Shin JE, Yun SH, Huh K, Koh JY. Protective effects of asiaticoside derivatives against beta-amyloid neurotoxicity. J Neurosci Res. 1999;58:417–425. doi: 10.1002/(SICI)1097-4547(19991101)58:3<417::AID-JNR7>3.0.CO;2-G. - DOI - PubMed
  9.  
    1. Shukla A, Rasik AM, Dhawan BN. Asiaticoside-induced elevation of antioxidant levels in healing wounds. Phytother Res. 1999;13:50–54. doi: 10.1002/(SICI)1099-1573(199902)13:1<50::AID-PTR368>3.0.CO;2-V. - DOI - PubMed
  10.  
    1. Salami S, Karami TF. Biochemical studies of apoptosis induced by tamoxifen in estrogen receptor positive and negative breast cancer cell lines. Clin Biochem. 2003;36:247–253. doi: 10.1016/S0009-9120(03)00007-9. - DOI - PubMed
  11.  
    1. Ma D, Zhang Y, Yang T, Xue Y, Wang P. Isoflavone intake inhibits the development of 7,12-dimethylbenz(a)anthracene(DMBA)-induced mammary tumors in normal and ovariectomized rats. J Clin Biochem Nutr. 2014;54:31–38. doi: 10.3164/jcbn.13-33. doi:10.3164/jcbn.13-33. - DOI - PMC - PubMed
  12.  
    1. Miyata M, Furukawa M, Takahashi K, Gonzalez FJ, Yamazoe Y. Mechanism of 7, 12-Dimethylbenz[a]anthracene-induced immunotoxicity: Role of metabolic activation at the target organ. Jpn J Pharmacol. 2001;86:302–309. doi: 10.1254/jjp.86.302. - DOI - PubMed
  13.  
    1. Kinuya S, Bai J, Shiba K, Yokoyama K, Mori H. 99mTc-sestamibi to monitor treatment with antisense oligodeoxynucleotide complementary to MRP mRNA in human breast cancer cells. Ann Nucl Med. 2006;20:29–34. doi: 10.1007/BF02985587. - DOI - PubMed
  14.  
    1. Aloj L, Zannetti A, Caracó C, Del Vecchio S, Salvatore M. Bcl-2 overexpression prevents 99mTc-MIBI uptake in breast cancer cell lines. Eur J Nucl Med Mol Imaging. 2004;31:521–527. doi: 10.1007/s00259-003-1381-0. - DOI - PubMed
  15.  
    1. Rodrigues M, Chehne F, Kalinowska W, Berghammer P, Zielinski C. Uptake of 99mTc-MIBI and 99mTc-tetrofosmin into malignant versus nonmalignant breast cell lines. J Nucl Med. 2000;41:1495–1499. - PubMed
  16.  
    1. de Jong M, Bernard BF, Breeman WA, Ensing G, Benjamins H. Comparison of uptake of 99mTc-MIBI, 99mTc-tetrofosmin and 99mTc-Q12 into human breast cancer cell lines. Eur J Nucl Med. 1996;23:1361–1366. doi: 10.1007/BF01367592. - DOI - PubMed
  17.  
    1. Al-Saeedi FJ, Bitar M, Pariyani S. Effect of asiaticoside on 99mTc-tetrofosmin and 99mTc-sestamibi uptake in MCF-7 cells. J Nucl Med Technol. 2011;39:279–283. doi: 10.2967/jnmt.111.091868. - DOI - PubMed
  18.  
    1. Piwnica-Worms D, Kronauge JF, Chiu ML. Uptake and retention of hexakis (2-methoxy isobutyl isonitrile) technetium (I) in cultured chick myocardial cells: mitochondrial and plasma membrane potential dependence. Circulation. 1990;82:1826. doi: 10.1161/01.CIR.82.5.1826. 1838. - DOI - PubMed
  19.  
    1. Piwnica-Worms D, Chiu ML, Budding J. Functional imaging of multi drug-resistant P-glycoprotein with an organo-technetium complex. Cancer Res. 1993;53:977–984. - PubMed
  20.  
    1. Carlsson J, Nilsson K, Westermark B, Pontén J, Sundström C, Larsson E, Bergh J, Påhlman S, Busch C, Collins VP. Formation and growth of multicellular spheroids of human origin. Int J Cancer. 1983;31:523–533. doi: 10.1002/ijc.2910310502. - DOI - PubMed
  21.  
    1. Huang YH, Zhang SH, Zhen RX, Xu XD, Zhen YS. Asiaticoside inducing apoptosis of tumor cells and enhancing anti-tumor activity of vincristine. Ai Zheng. 2004;23:1599–1604. - PubMed
  22.  
    1. Gurfinkel DM, Chow S, Hurren R, Gronda M, Henderson C, Berube C, Hedley DW, Schimmer AD. Disruption of the endoplasmic reticulum and increases in cytoplasmic calcium are early events in cell death induced by the natural triterpenoid Asiatic acid. Apoptosis. 2006;11:1463–1471. doi: 10.1007/s10495-006-9086-z. - DOI - PubMed
  23.  
    1. Gnanapragasam A, Yogeeta S, Subhashini R, Ebenezar KK, Sathish V, Devaki T. Adriamycin induced myocardial failure in rats: protective role of Centella asiatica. Mol Cell Biochem. 2007;294:55–63. doi: 10.1007/s11010-006-9245-0. - DOI - PubMed
  24.  
    1. Wijeweera P, Arnason JT, Koszycki D, Merali Z. Evaluation of anxiolytic properties of Gotukola–(Centella asiatica) extracts and asiaticoside in rat behavioral models. Phytomedicine. 2006;13:668–676. doi: 10.1016/j.phymed.2006.01.011. - DOI - PubMed
  25.  
    1. Harris RE, Alshafie GA, Abou-Issa H, Seibert K. Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor. Cancer Res. 2000;60:2101–2103. - PubMed
  26.  
    1. Ouhtit A, Ismail MF, Othman A, Fernando A, Abdraboh ME, El-Kott AF, Azab YA, Abdeen SH, Gaur RL, Gupta I, Shanmuganathan S, Al-Farsi YM, Al-Riyami H, Raj MH. Chemoprevention of rat mammary carcinogenesis by spirulina. Am J Pathol. 2014;184:296–303. doi: 10.1016/j.ajpath.2013.10.025. doi: 10.1016/j.ajpath.2013.10.025. - DOI - PubMed
  27.  
    1. Hamdy SM, Latif AK, Drees EA, Soliman SM. Prevention of rat breast cancer by genistin and selenium. Toxicol Ind Health. 2012;28:746–757. doi: 10.1177/0748233711422732. doi:10.1177/0748233711422732. - DOI - PubMed
  28.  
    1. Kubatka P, Stollárová N, Škarda J, Žihlavníková K, Kajo K, Kapinová A, Adamicová K, Péč M, Dobrota D, Bojková B, Kassayová M, Orendáš P. Preventive effects of fluvastatin in rat mammary carcinogenesis. Eur J Cancer Prev. 2013;22:352–357. doi: 10.1097/CEJ.0b013e32835b385d. doi:10.1097/CEJ.0b013e32835b385d. - DOI - PubMed
  29.  
    1. Sharmila G, Athirai T, Kiruthiga B, Senthilkumar K, Elumalai P, Arunkumar R, Arunakaran J. Chemopreventive effect of quercetin in MNU and testosterone induced prostate cancer of Sprague–Dawley rats. Nutr Cancer. 2014;66:38–46. doi: 10.1080/01635581.2014.847967. doi:10.1080/01635581.2014.847967. - DOI - PubMed
  30.  
    1. Huggins C, Briziarelli G, Sutton H. Rapid induction of mammary carcinoma in the rat and the influence of hormones on the tumors. J Exp Med. 1959;109:25–41. doi: 10.1084/jem.109.1.25. - DOI - PMC - PubMed
  31.  
    1. Huggins C, Grand LC, Brillantes FP. Mammary cancer induced by a single feeding of polynuclear hydrocarbons, and its suppression. Nature. 1961;189:204–207. doi: 10.1038/189204a0. - DOI - PubMed
  32.  
    1. Thompson HJ, McGinley JN, Rothhammer K, Singh M. Rapid induction of mammary intraductal proliferations, ductal carcinoma in situ and carcinomas by the injection of sexually immature female rats with 1-methyl-1-nitrosourea. Carcinogenesis. 1995;16:2407–2411. doi: 10.1093/carcin/16.10.2407. - DOI - PubMed
  33.  
    1. Banerjee S, Bueso-Ramos C, Aggarwal BB. Suppression of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in rats by resveratrol: role of nuclear factor-kappaB, cyclooxygenase 2, and matrix metalloprotease 9. Cancer Res. 2002;62:4945–4954. - PubMed
  34.  
    1. Terada S, Uchide K, Suzuki N, Akasofu K, Nishida E. Induction of ductal carcinomas by intraductal administration of 7,12-dimethylbenz(a)anthracene in Wistar rats. Breast Cancer Res Treat. 1995;34:35–43. doi: 10.1007/BF00666489. - DOI - PubMed
  35.  
    1. Perumal BS, Sakharkar KR, Chow VT, Pandjassarame K, Sakharkar MK. Intron position conservation across eukaryotic lineages in tubulin genes. Front Biosci. 2005;10:2412–2419. doi: 10.2741/1706. - DOI - PubMed
  36.  
    1. Padmavathi R, Senthilnathan P, Chodon D, Sakthisekaran D. Therapeutic effect of paclitaxel and propolis on lipid peroxidation and antioxidant system in 7,12 dimethyl benz (a) anthracene-induced breast cancer in female Sprague Dawley rats. Life Sci. 2006;8:2820–2825. - PubMed
  37.  
    1. Shobi V, Goel HC. Protection against radiation-induced conditioned taste aversion by centella asiatica. Physiol Behav. 2001;73:19–23. doi: 10.1016/S0031-9384(01)00434-6. - DOI - PubMed
  38.  
    1. Sharma J, Sharma R. Radioprotection of Swiss albino mouse by centella asiatica extract. Phytother Res. 2002;16:785–786. doi: 10.1002/ptr.1069. - DOI - PubMed
  39.  
    1. Horne T, Pappo I, Cohen-Pour M, Baumer M, Orda R. 99Tc(m)-tetrofosmin scintimammography for detecting breast cancer: a comparative study with 99Tc(m)-MIBI. Nucl Med Commun. 2001;22:807–811. doi: 10.1097/00006231-200107000-00012. - DOI - PubMed
  40.  
    1. Söderlund V, Jonsson C, Bauer HC, Brosjö O, Jacobsson H. Comparison of technetium-99 m-MIBI and technetium-99 m-tetrofosmin uptake by musculoskeletal sarcomas. J Nucl Med. 1997;38:682–686. - PubMed
  41.  
    1. Gao J, Huang F, Zhang J, Zhu G, Yang M. Cytotoxic cycloartane triterpene saponins from Actaea asiatica. J Nat Prod. 2006;69:1500–1502. doi: 10.1021/np060113h. - DOI - PubMed
  42.  
    1. Beanlands RSB, Dawood F, Wen WH, McLaughlin PR, Butany J. Are the kinetics of technetium-99 m methoxyisobutyl isonitrile affected by cell metabolism and viability? Circulation. 1990;82:1802. doi: 10.1161/01.CIR.82.5.1802. 1814. - DOI - PubMed
  43.  
    1. Khalkhali I, Mena I, Jouanne E, Diggles L, Venegas R. Prone scintimammography in patients with suspicion of carcinoma of the breast. J Am Coll Surg. 1994;178:491–497. - PubMed
  44.  
    1. Khalkhali I, Cutrone J, Mena I, Diggles L, Venegas R. Technetium-99 m sestamibi scintimammography of breast lesions: clinical and pathological follow-up. J Nucl Med. 1995;36:1784–1789. - PubMed
  45.  
    1. Abdel Dayem MH, Scott AM, Macapinlac HA, El-Gazzar AH, Larson SM. In: Nuclear Medicine Annual. Freeman LM, editor. New York: Raven; 1994. Role of 201Tl chloride and 99mTc sestamibi in tumor imaging; pp. 181–234.
  46.  
    1. Palmedo H, Schomburg A, Grünwald F, Mallmann P, Krebs D. Technetium-99 m-MIBI scintimammography for suspicious breast lesions. J Nucl Med. 1996;37:626–630. - PubMed
  47.  
    1. Pappo I, Horne T, Weissberg D, Wasserman I, Orda R. The usefulness of MIBI scanning to detect underlying carcinoma in women with acute mastitis. Breast J. 2000;6:126–129. doi: 10.1046/j.1524-4741.2000.98107.x. - DOI - PubMed
  48.  
    1. Grivennikov SI, Karin M. Inflammatory cytokines in cancer: tumour necrosis factor and interleukin 6 take the stage. Ann Rheum Dis. 2011;70:i104–i108. doi: 10.1136/ard.2010.140145. doi:10.1136/ard.2010.140145. - DOI - PubMed
  49.  
    1. Zheng J, Zhang L, Wu M, Li X, Zhang L, Wan J. Protective effects of asiaticoside on sepsis-induced acute kidney injury in mice. Zhongguo Zhong Yao Za Zhi. 2010;35:1482–1485. - PubMed
  50.  
    1. Zhang Z, Qin DL, Wan JY, Zhou QX, Xiao SH. Effects of asiaticoside on the balance of inflammatory factors of mouse’s acute lung injury induced by LPS. Zhong Yao Cai. 2008;31:547–549. - PubMed
  51.  
    1. Zhang LN, Zheng JJ, Zhang L, Gong X, Huang H. Protective effects of asiaticoside on septic lung injury in mice. Exp Toxicol Pathol. 2011;63:519–525. doi: 10.1016/j.etp.2010.04.002. - DOI - PubMed
  52.  
    1. Pan S, Li T, Li Y. Effects of asiaticoside on cell proliferation and Smad signal pathway of hypertrophic scar fibroblasts. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2004;18:291–294. - PubMed
  53.  
    1. Nowwarote N, Osathanon T, Jitjaturunt P, Manopattanasoontorn S, Pavasant P. Asiaticoside induces type I collagen synthesis and osteogenic differentiation in human periodontal ligament cells. Phytother Res. 2013;27:457–462. doi: 10.1002/ptr.4742. doi:10.1002/ptr.4742. - DOI - PubMed
  54.  
    1. Pizzorno JE, Murray MT. Textbook of Natural Medicine. London: Churchill Livingstone Press; 1999.
  55.  
    1. Boiteau P, Nigeon-Dureuil M, Ratsimamanga AR. Action of asiaticoside on the reticuloendothelial tissue. Acad Sci Compt Rend. 1951;232:760–762.

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