Tumor Biology

, Volume 35, Issue 5, pp 4967–4976 | Cite as

Efficacy of acetylsalicylic acid (aspirin) in skin B16-F0 melanoma tumor-bearing C57BL/6 mice

  • Nikhil M. Vad
  • Shashi K. Kudugunti
  • Hezhen Wang
  • G. Jayarama Bhat
  • Majid Y. Moridani
Research Article


Several epidemiological studies show that aspirin can act as a chemopreventive agent and decrease the incidences of various cancers including melanoma. In this work, we investigated the in vitro and in vivo efficacy of acetylsalicylic acid (ASA) as an antimelanoma agent in B16-F0 cells and skin B16-F0 melanoma tumor mouse model. Our findings indicate that the IC50 (48 h) for ASA in B16-F0 melanoma cells was 100 μM and that ASA caused a dose- and time-dependent GSH depletion and increase in reactive oxygen species (ROS) formation in B16-F0 melanoma cells. Male C57BL/6 mice were inoculated s.c. with 1 × 106 B16-F0 melanoma cells. ASA (80, 100, and 150 mg/kg) was initiated on day 1 or day 7, or day 9 after cell inoculation and continued daily for 13, 7, and 5 days, respectively. Animals were weighed daily and sacrificed on day 13. The tumors were excised and weighed. The animals receiving 13 days of ASA therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 1 ± 12 %, 19 ± 22 %, and 50 ± 29 %, respectively. Animals receiving 7 days of therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 12 ± 14 %, 27 ± 14 %, and 40 ± 14 %, respectively. No significant tumor growth inhibition was observed with 5 days of therapy. ASA at 100 and 150 mg/kg caused significant tumor growth inhibition in C57BL/6 mice when administered for 13 and 7 days, respectively. The results obtained in this study are consistent with the recent epidemiologically based report that aspirin is associated with lower melanoma risk in humans.


Aspirin Melanoma Tyrosinase ASA B16-F0 C57BL/6 mice 



Aspirin, acetylsalicylic acid


Dulbecco’s Modified Eagle Medium


Fetal bovine serum


Phosphate-buffered saline


Thiobarbituric acid-reactive substances


Alanine aminotransferase



This work was supported partly by a grant from NIH (1R15CA122044-01A1) and the TTUHSC School of Pharmacy to M.Y.M and 5RO3CA133061-02 to G.J.B. Support from the Translational Cancer Research Seed Grant to G.J.B, funded as 2010 Research Initiative Center by the State of South Dakota, is also gratefully acknowledged.

Conflicts of interest



  1. 1.
    Saleem M, Maddodi N, Abu Zaid M, Khan N, Bin Hafeez B, Asim M, et al. Lupeol inhibits growth of highly aggressive human metastatic melanoma cells in vitro and in vivo by inducing apoptosis. Clin Cancer Res. 2008;14:2119–212. 14.CrossRefPubMedGoogle Scholar
  2. 2.
    Tawbi HA, Kirkwood JM. Management of metastatic melanoma. Semin Oncol. 2007;34:532–45.CrossRefPubMedGoogle Scholar
  3. 3.
    Naish S, Cooksey C, Riley P. Initial mushroom tyrosinase-catalysed oxidation product of 4-hydroxyanisole is 4-methoxy-ortho-benzoquinone. Pigment Cell Res. 1988;1:379–81.CrossRefPubMedGoogle Scholar
  4. 4.
    Naish S, Holden JL, Cooksey CJ, Riley PA. Major primary cytotoxic product of 4-hydroxyanisole oxidation by mushroom tyrosinase is 4-methoxy ortho benzoquinone. Pigment Cell Res. 1988;1:382–5.CrossRefPubMedGoogle Scholar
  5. 5.
    Moridani MY, Cheon SS, Khan S, O’Brien PJ. Metabolic activation of 4-hydroxyanisole by isolated rat hepatocytes. Drug Metab Dispos. 2002;30:1063–9.CrossRefPubMedGoogle Scholar
  6. 6.
    Riley PA, Cooksey CJ, Johnson CI, Land EJ, Latter AM, Ramsden CA. Melanogenesis-targeted anti-melanoma pro-drug development: effect of side-chain variations on the cytotoxicity of tyrosinase-generated ortho-quinones in a model screening system. Eur J Cancer. 1997;33:135–43.CrossRefPubMedGoogle Scholar
  7. 7.
    Vad NM, Yount G, Moore D, Weidanz J, Moridani MY. Biochemical mechanism of acetaminophen (APAP) induced toxicity in melanoma cell lines. J Pharm Sci. 2009;98:1409–25.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Vad NM, Yount G, Moridani MY. Biochemical mechanism of acetylsalicylic acid (aspirin) selective toxicity toward melanoma cell lines. Melanoma Res. 2008;18:386–99.CrossRefPubMedGoogle Scholar
  9. 9.
    Riley PA. Hydroxyanisole depigmentation: in-vitro studies. J Pathol. 1969;97:193–206.CrossRefPubMedGoogle Scholar
  10. 10.
    Harris RE, Beebe-Donk J, Doss H, Burr Doss D. Aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs in cancer prevention: a critical review of non-selective COX-2 blockade (review). Oncology Rep. 2005;13:559–83.Google Scholar
  11. 11.
    Kaiser J. Will an aspirin a day keep cancer away? Science. 2012;337:1471–3.CrossRefPubMedGoogle Scholar
  12. 12.
    Sahasrabuddhe VV, Gunja MZ, Graubard BI, Trabert B, Schwartz LM, Park Y, et al. Nonsteroidal anti-inflammatory drug use, chronic liver disease, and hepatocellular carcinoma. J Natl Cancer Inst. 2012;104:1808–14.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Veitonmaki T, Tammela TL, Auvinen A, Murtola TJ. Use of aspirin, but not other non-steroidal anti-inflammatory drugs is associated with decreased prostate cancer risk at the population level. Eur J Cancer. 2013;49:938–45.CrossRefPubMedGoogle Scholar
  14. 14.
    Gamba CA, Swetter SM, Stefanick ML, Kubo J, Desai M, Spaunhurst KM, et al. Aspirin is associated with lower melanoma risk among postmenopausal Caucasian women: the women’s health initiative. Cancer. 2013;119:1562–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Thun MJ, Jacobs EJ, Patrono C. The role of aspirin in cancer prevention. Nat Rev Clin Oncol. 2012;9:259–67.CrossRefPubMedGoogle Scholar
  16. 16.
    Sun Y, Chen J, Rigas B. Chemopreventive agents induce oxidative stress in cancer cells leading to COX-2 overexpression and COX-2-independent cell death. Carcinogenesis. 2009;30:93–9100.CrossRefPubMedGoogle Scholar
  17. 17.
    Lai MY, Huang JA, Liang ZH, Jiang HX, Tang GD. Mechanisms underlying aspirin-mediated growth inhibition and apoptosis induction of cyclooxygenase-2 negative colon cancer cell line SW480. World J Gastroenterol. 2008;14:4227–33.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Moridani MY, Moore M, Bartsch RA, Yang Y, Heibati-Sadati S. Structural toxicity relationship of 4-alkoxyphenols’ cytotoxicity towards murine B16-F0 melanoma cell line. J Pharm Pharm Sci. 2005;8:348–60.PubMedGoogle Scholar
  19. 19.
    Moridani MY. Biochemical basis of 4-hydroxyanisole induced cell toxicity towards B16-F0 melanoma cells. Cancer Lett. 2006;243:235–45.CrossRefPubMedGoogle Scholar
  20. 20.
    Wu X, Zeng H, Zhang X, Zhao Y, Sha H, Ge X, et al. Phosphatase of regenerating liver-3 promotes motility and metastasis of mouse melanoma cells. Am J Pathol. 2004;164:2039–54.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Moridani MY, Cheon SS, Khan S, O’Brien PJ. Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes. Chem Biol Interact. 2003;142:317–33.CrossRefPubMedGoogle Scholar
  22. 22.
    Moldeus P, Hogberg J, Orrenius S. Isolation and use of liver cells. Methods Enzymol. 1978;52:60–71.CrossRefPubMedGoogle Scholar
  23. 23.
    Shaik IH, Mehvar R. Rapid determination of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples. Anal Bioanal Chem. 2006;385:105–13.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Vad NM, Shaik IH, Mehvar R, Moridani MY. Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells. J Pharm Sci. 2008;97:1934–45.CrossRefPubMedGoogle Scholar
  25. 25.
    Siraki AG, Chan TS, O’Brien PJ. Application of quantitative structure-toxicity relationships for the comparison of the cytotoxicity of 14 p-benzoquinone congeners in primary cultured rat hepatocytes versus PC12 cells. Toxicol Sci. 2004;81:148–59.CrossRefPubMedGoogle Scholar
  26. 26.
    Qiao J, Wang H, Kottke T, White C, Twigger K, Diaz RM, et al. Cyclophosphamide facilitates antitumor efficacy against subcutaneous tumors following intravenous delivery of reovirus. Clin Cancer Res. 2008;14:259–69.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem. 1968;25:192–205.CrossRefPubMedGoogle Scholar
  28. 28.
    Wills ED. Mechanisms of lipid peroxide formation in animal tissues. Biochem J. 1966;99:667–76.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Kaur G, Tirkey N, Bharrhan S, Chanana V, Rishi P, Chopra K. Inhibition of oxidative stress and cytokine activity by curcumin in amelioration of endotoxin-induced experimental hepatoxicity in rodents. Clin Exp Immunol. 2006;145:313–21.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Carson III WE WM: Animal models of melanoma. In: Tumor models in cancer research. Humana Press Inc., 2002.Google Scholar
  31. 31.
    Kawai S, Yoshinari M, Matsumoto J, Kirinoki M, Aikawa M, Minami M, et al. Plasmodium coatneyi-infected erythrocytes bind to C32 amelanotic melanoma cells under static and flow conditions. J Vet Med Sci. 2003;65:375–80.CrossRefPubMedGoogle Scholar
  32. 32.
    Kelloff GJ, Boone CW, Crowell JA, Steele VE, Lubet R, Sigman CC. Chemopreventive drug development: perspectives and progress. Cancer Epidemiol Biomarkers Prev. 1994;3:85–98.PubMedGoogle Scholar
  33. 33.
    Roberts I: Lj, & jd. Morrow. Analgesic-antipyretic and antiinflammatory agents and drugs employed in the treatment of gout. Goodman & Gilman’s the pharmacological basis of therapeutics, 10th edition, International Edition, Editado por Hardman JG, Limbird LE y Gilman AG, McGraw-Hill:New York; 2001. 703–705.Google Scholar
  34. 34.
    Davison C. Salicylate metabolism in man. Ann N Y Acad Sci. 1971;179:249–68.CrossRefPubMedGoogle Scholar
  35. 35.
    Craig JO, Ferguson IC, Syme J. Infants, toddlers, and aspirin. Br Med J. 1966;1:757–61.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Dovizio M, Bruno A, Tacconelli S, Patrignani P. Mode of action of aspirin as a chemopreventive agent. Recent Results Cancer Res Fortschritte der Krebsforschung Progres dans les recherches sur le cancer. 2013;191:39–65.PubMedGoogle Scholar
  37. 37.
    Tiboni GM, Iammarrone E, Piccirillo G, Liberati M, Bellati U. Aspirin pretreatment potentiates hyperthermia-induced teratogenesis in the mouse. Am J Obstet Gynecol. 1998;178:270–9.CrossRefPubMedGoogle Scholar
  38. 38.
    Reddy BS, Rao CV, Rivenson A, Kelloff G. Inhibitory effect of aspirin on azoxymethane-induced colon carcinogenesis in F344 rats. Carcinogenesis. 1993;14:1493–7.CrossRefPubMedGoogle Scholar
  39. 39.
    Duperron C, Castonguay A. Chemopreventive efficacies of aspirin and sulindac against lung tumorigenesis in A/J mice. Carcinogenesis. 1997;18:1001–6.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  1. 1.Department of Pharmaceutical and Department of Biomedical Sciences, School of PharmacyTexas Tech University Health Sciences CenterAmarilloUSA
  2. 2.Department of Pharmaceutical SciencesSouth Dakota State University College of PharmacyBrookingsUSA
  3. 3.Department of PathologyMedical College of WisconsinMilwaukeeUSA

Personalised recommendations