Molecular and Cellular Biochemistry

, Volume 406, Issue 1–2, pp 53–62 | Cite as

Low inducible expression of p21Cip1 confers resistance to paclitaxel in BRAF mutant melanoma cells with acquired resistance to BRAF inhibitor

  • Gun-Hee Jang
  • Na-Yeon Kim
  • Michael Lee


The therapeutic efficacy of oncogenic BRAF inhibitor is limited by the onset of acquired resistance. In this study, we investigated the potential therapeutic effects of the mitotic inhibitor paclitaxel on three melanoma cell lines with differing sensitivity to the BRAF inhibitor. Of the two BRAF inhibitor-resistant cell lines, A375P/Mdr cells harboring the BRAF V600E mutant were resistant and the wild-type BRAF SK-MEL-2 cells were sensitive to paclitaxel. In particular, paclitaxel caused the growth inhibition of SK-MEL-2 cells to a much greater extent than it caused growth inhibition of A375P cells. Paclitaxel exhibited no significant effect on the phosphorylation of MEK-ERK in any cell lines tested, regardless of both the BRAF mutation and the drug resistance, implying that paclitaxel activity is independent of MEK-ERK inhibition. In A375P cells, paclitaxel treatment resulted in a marked emergence of apoptotic cells after mitotic arrest, concomitant with a remarkable induction of p21Cip1. However, paclitaxel only moderately increased the levels of p21Cip1 in A375P/Mdr cells, which exhibited a strong resistance to paclitaxel. The p21Cip1 overexpression partially conferred paclitaxel sensitivity to A375P/Mdr cells. Interestingly, we found an extremely low background expression level of p21Cip1 in SK-MEL-2 cells lacking normal p53 function, which caused much greater G2/M arrest than that seen in A375P cells. Taken together, these results suggest that paclitaxel may be an effective anticancer agent through regulating the expression of p21Cip1 for the treatment of BRAF mutant melanoma cells resistant to BRAF inhibitors.


Paclitaxel BRAF inhibitor-resistance Melanoma Mitotic arrest p21Cip1 Apoptosis 



This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2A10058897).

Supplementary material

11010_2015_2423_MOESM1_ESM.ppt (188 kb)
Supplementary material 1 (PPT 187 kb)
11010_2015_2423_MOESM2_ESM.docx (15 kb)
Supplementary material 2 (DOCX 15 kb)


  1. 1.
    Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W et al (2002) Mutations of the BRAF gene in human cancer. Nature 417:949–954PubMedCrossRefGoogle Scholar
  2. 2.
    Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, O’Dwyer PJ, Lee RJ, Grippo JF, Nolop K et al (2010) Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 363:809–819PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    McGettigan S (2014) Dabrafenib: a new therapy for use in BRAF-mutated metastatic melanoma. J Adv Pract Oncol 5:211–215PubMedCentralPubMedGoogle Scholar
  4. 4.
    Kim YK, Ahn SK, Lee M (2012) Differential sensitivity of melanoma cell lines with differing B-Raf mutational status to the new oncogenic B-Raf kinase inhibitor UI-152. Cancer Lett 320:215–224PubMedCrossRefGoogle Scholar
  5. 5.
    Rizos H, Menzies AM, Pupo GM, Carlino MS, Fung C, Hyman J, Haydu LE, Mijatov B, Becker TM, Boyd SC et al (2014) BRAF inhibitor resistance mechanisms in metastatic melanoma: spectrum and clinical impact. Clin Cancer Res 20:1965–1977PubMedCrossRefGoogle Scholar
  6. 6.
    Aplin AE, Kaplan FM, Shao Y (2011) Mechanisms of resistance to RAF inhibitors in melanoma. J Invest Dermatol 131:1817–1820PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, Johnson LA, Emery CM, Stransky N, Cogdill AP, Barretina J et al (2010) COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468:968–972PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, Chen Z, Lee MK, Attar N, Sazegar H et al (2010) Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468:973–977PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Ahn J-H, Lee M (2014) The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors. Mol Cell Biochem 392:239–247PubMedCrossRefGoogle Scholar
  10. 10.
    Chandarlapaty S (2012) Negative feedback and adaptive resistance to the targeted therapy of cancer. Cancer Discov 2:311–319PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Pratilas CA, Taylor BS, Ye Q, Viale A, Sander C, Solit DB, Rosen N (2009) (V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway. Proc Natl Acad Sci USA 106:4519–4524PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, Alvarado R, Ludlam MJ, Stokoe D, Gloor SL, Vigers G et al (2010) RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464:431–435PubMedCrossRefGoogle Scholar
  13. 13.
    Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, Hussain J, Reis-Filho JS, Springer CJ, Pritchard C et al (2010) Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140:209–221PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Poulikakos PI, Persaud Y, Janakiraman M, Kong X, Ng C, Moriceau G, Shi H, Atefi M, Titz B, Gabay MT et al (2011) RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480:387–390PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Zhou J, Giannakakou P (2005) Targeting microtubules for cancer chemotherapy. Curr Med Chem Anticancer Agents 5:65–71PubMedCrossRefGoogle Scholar
  16. 16.
    Blagosklonny MV, Schulte T, Nguyen P, Trepel J, Neckers LM (1996) Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res 56:1851–1854PubMedGoogle Scholar
  17. 17.
    Blagosklonny MV, Giannakakou P, El-Deiry WS, Kingston DG, Higgs PI, Neckers L, Fojo T (1997) Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res. 57:130–135PubMedGoogle Scholar
  18. 18.
    Ahn J-H, Eum K-H, Lee M (2009) The enhancement of Raf-1 kinase activity by knockdown of Spry2 is associated with high sensitivity to paclitaxel in v-Ha-ras-transformed NIH 3T3 fibroblasts. Mol Cell Biochem 332:189–197PubMedCrossRefGoogle Scholar
  19. 19.
    McGrogan BT, Gilmartin B, Carney DN, McCann A (2008) Taxanes, microtubules and chemoresistant breast cancer. Biochim Biophys Acta 1785:96–132PubMedGoogle Scholar
  20. 20.
    Horwitz SB, Cohen D, Rao S, Ringel I, Shen HJ, Yang CP (1993) Taxol: mechanisms of action and resistance. J Natl Cancer Inst Monogr 15:55–61PubMedGoogle Scholar
  21. 21.
    Ahn J-H, Lee M (2013) Autophagy-dependent survival of mutant B-Raf melanoma cells selected for resistance to apoptosis induced by inhibitors against oncogenic B-Raf. Biomol Ther 21:114–120CrossRefGoogle Scholar
  22. 22.
    Toyoshima H, Huter T (1994) p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78:67–74PubMedCrossRefGoogle Scholar
  23. 23.
    Marcus AI, Peters U, Thomas SL, Garrett S, Zelnak A, Kapoor TM, Giannakakou P (2005) Mitotic kinesin inhibitors induce mitotic arrest and cell death in taxol-resistant and -sensitive cancer cells. J Biol Chem 280:11569–11577PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Blagosklonny MV (2007) Mitotic arrest and cell fate: why and how mitotic inhibition of transcription drives mutually exclusive events. Cell Cycle 6:70–74PubMedCrossRefGoogle Scholar
  25. 25.
    Boya P, González-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Métivier D, Meley D, Souquere S, Yoshimori T et al (2005) Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 25:1025–1040PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, Tsujimoto Y (2004) Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol 6:1221–1228PubMedCrossRefGoogle Scholar
  27. 27.
    Holderfield M, Deuker MM, McCormick F, McMahon M (2014) Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond. Nat Rev Cancer 14:455–467PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Gottesman MM (2002) Mechanisms of cancer drug resistanace. Annu Rev Med 53:615–627PubMedCrossRefGoogle Scholar
  29. 29.
    Lito P, Rosen N, Solit DB (2013) Tumor adaptation and resistance to RAF inhibitors. Nat Med 19:1401–1409PubMedCrossRefGoogle Scholar
  30. 30.
    Yvon AM, Wadsworth P, Jordan MA (1999) Taxol suppresses dynamics of individual microtubules in living human tumor cells. Mol Biol Cell 10:947–959PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Li W, Fan J, Banerjee D, Bertino JR (1999) Overexpression of p21(waf1) decreases G2-M arrest and apoptosis induced by paclitaxel in human sarcoma cells lacking both p53 and functional Rb protein. Mol Pharmacol 55:1088–1093PubMedGoogle Scholar
  32. 32.
    Gorospe M, Cirielli C, Wang X, Seth P, Capogrossi MC, Holbrook NJ (1997) p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells. Oncogene 14:929–935PubMedCrossRefGoogle Scholar
  33. 33.
    Bartek J, Lukas J (2001) Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr Opin Cell Biol 13:738–747PubMedCrossRefGoogle Scholar
  34. 34.
    O’Connor PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M, Scudiero DA, Monks A, Sausville EA, Weinstein JN et al (1997) Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res 57:4285–4300PubMedGoogle Scholar
  35. 35.
    Wahl AF, Donaldson KL, Fairchild C, Lee FY, Foster SA, Demers GW, Galloway DA (1996) Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis. Nat Med 2:72–79PubMedCrossRefGoogle Scholar
  36. 36.
    Eum KH, Lee M (2011) Crosstalk between autophagy and apoptosis in the regulation of paclitaxel-induced cell death in v-Ha-ras-transformed fibroblasts. Mol Cell Biochem 348:61–68PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Division of Life Sciences, College of Life Sciences and BioengineeringIncheon National UniversityIncheonRepublic of Korea

Personalised recommendations