Piperlongumine exerts cytotoxic effects against cancer cells with mutant p53 proteins at least in part by restoring the biological functions of the tumor suppressor

  • Authors:
    • Debasish Basak
    • Surendra R. Punganuru
    • Kalkunte S. Srivenugopal
  • View Affiliations

  • Published online on: February 3, 2016     https://doi.org/10.3892/ijo.2016.3372
  • Pages: 1426-1436
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Piperlongumine (PL), a small molecule alkaloid present in black pepper (Piper longum), has been reported to kill tumor cells irrespective of their p53 gene status, however, the mechanisms involved are unknown. Since p53 is a redox-sensitive protein, we hypothesized that the redox imbalance induced by PL may affect the structure and/or function of the mutant p53 protein and promote cell death. We used two human colon cancer cell lines, the HT29 and SW620 which harbor the R273H DNA contact abrogatory mutation in p53. PL treatment induced significant ROS production and protein glutathionylation with a concomitant increase in Nrf-2 expression in both cell lines. Surprisingly, immunoprecipitation with wt-p53 specific antibodies (PAb1620) or direct western blotting showed a progressive generation of wild-type-like p53 protein along with a loss of its mutant counterpart in PL-treated HT29 and SW620 cells. Moreover, the EMSA and DNA-affinity blotting revealed a time-dependent restoration of DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes, MDM2 and Bax. PL, while cytotoxic by itself, also increased the cell killing by many anticancer drugs. In nude mice bearing the HT29 tumors, PL alone (7.5 mg/kg daily) produced a 40% decrease in tumor volume, which was accompanied by diminished intratumoral mutant p53 protein levels. The antitumor efficacy of BCNU or doxorubicin in HT29 xenografts was highly potentiated by PL, followed by expression of apoptotic proteins. These clinically-relevant findings suggest that PL-induced oxidative milieu facilitates a weak functional restoration of mutant p53 through protein glutathionylation and contributes to the increased drug sensitivity.

References

1 

Trachootham D, Alexandre J and Huang P: Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach? Nat Rev Drug Discov. 8:579–591. 2009. View Article : Google Scholar : PubMed/NCBI

2 

Sosa V, Moliné T, Somoza R, Paciucci R, Kondoh H and LLeonart ME: Oxidative stress and cancer: An overview. Ageing Res Rev. 12:376–390. 2013. View Article : Google Scholar

3 

Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, Tolliday NJ, Golub TR, Carr SA, Shamji AF, et al: Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature. 475:231–234. 2011. View Article : Google Scholar : PubMed/NCBI

4 

Adams DJ, Dai M, Pellegrino G, Wagner BK, Stern AM, Shamji AF and Schreiber SL: Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs. Proc Natl Acad Sci USA. 109:15115–15120. 2012. View Article : Google Scholar : PubMed/NCBI

5 

Han JG, Gupta SC, Prasad S and Aggarwal BB: Piperlongumine chemosensitizes tumor cells through interaction with cysteine 179 of IκBα kinase, leading to suppression of NF-κB-regulated gene products. Mol Cancer Ther. 13:2422–2435. 2014. View Article : Google Scholar : PubMed/NCBI

6 

Polyak K, Xia Y, Zweier JL, Kinzler KW and Vogelstein B: A model for p53-induced apoptosis. Nature. 389:300–305. 1997. View Article : Google Scholar : PubMed/NCBI

7 

Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P and Olivier M: Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: Lessons from recent developments in the IARC TP53 database. Hum Mutat. 28:622–629. 2007. View Article : Google Scholar : PubMed/NCBI

8 

Olivier M, Hollstein M and Hainaut P: Hollstein M and Hainaut P: TP53 mutations in human cancers: Origins, consequences, and clinical Use. Cold Spring Harb Perspect Biol. 2:1–17. 2010. View Article : Google Scholar

9 

Basu A and Haldar S: The relationship between BcI2, Bax and p53: Consequences for cell cycle progression and cell death. Mol Hum Reprod. 4:1099–1109. 1998. View Article : Google Scholar

10 

Bykov VJN and Wiman KG: Mutant p53 reactivation by small molecules makes its way to the clinic. FEBS Lett. 588:2622–2627. 2014. View Article : Google Scholar : PubMed/NCBI

11 

Wiman KG: Strategies for therapeutic targeting of the p53 pathway in cancer. Cell Death Differ. 13:921–926. 2006. View Article : Google Scholar : PubMed/NCBI

12 

Saha MN, Qiu L and Chang H: Targeting p53 by small molecules in hematological malignancies. J Hematol Oncol. 6:232013. View Article : Google Scholar : PubMed/NCBI

13 

Bykov VJN, Issaeva N, Shilov A, Hultcrantz M, Pugacheva E, Chumakov P, Bergman J, Wiman KG and Selivanova G: Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat Med. 8:282–288. 2002. View Article : Google Scholar : PubMed/NCBI

14 

Bykov VJN, Issaeva N, Zache N, Shilov A, Hultcrantz M, Bergman J, Selivanova G and Wiman KG: Reactivation of mutant p53 and induction of apoptosis in human tumor cells by maleimide analogs. J Biol Chem. 280:30384–30391. 2005. View Article : Google Scholar : PubMed/NCBI

15 

Tang X, Zhu Y, Han L, Kim AL, Kopelovich L, Bickers DR and Athar M: CP-31398 restores mutant p53 tumor suppressor function and inhibits UVB-induced skin carcinogenesis in mice. J Clin Invest. 117:3753–3764. 2007. View Article : Google Scholar : PubMed/NCBI

16 

Zache N, Lambert JMR, Rökaeus N, Shen J, Hainaut P, Bergman J, Wiman KG and Bykov VJN: Mutant p53 targeting by the low molecular weight compound STIMA-1. Mol Oncol. 2:70–80. 2008. View Article : Google Scholar

17 

Demma M, Maxwell E, Ramos R, Liang L, Li C, Hesk D, Rossman R, Mallams A, Doll R, Liu M, et al: SCH529074, a small molecule activator of mutant p53, which binds p53 DNA binding domain (DBD), restores growth-suppressive function to mutant p53 and interrupts HDM2-mediated ubiquitination of wild-type p53. J Biol Chem. 285:10198–10212. 2010. View Article : Google Scholar : PubMed/NCBI

18 

Yu X, Vazquez A, Levine AJ and Carpizo DR: Allele-specific p53 mutant reactivation. Cancer Cell. 21:614–625. 2012. View Article : Google Scholar : PubMed/NCBI

19 

Bykov VJN, Lambert JMR, Hainaut P and Wiman KG: Mutant p53 rescue and modulation of p53 redox state. Cell Cycle. 8:2509–2517. 2009. View Article : Google Scholar : PubMed/NCBI

20 

Velu CS, Niture SK, Doneanu CE, Pattabiraman N and Srivenugopal KS: Human p53 is inhibited by glutathionylation of cysteines present in the proximal DNA-binding domain during oxidative stress. Biochemistry. 46:7765–7780. 2007. View Article : Google Scholar : PubMed/NCBI

21 

Yusuf MA, Chuang T, Bhat GJ and Srivenugopal KS: Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines. Free Radic Biol Med. 49:908–917. 2010. View Article : Google Scholar : PubMed/NCBI

22 

Liu Y, Asch H and Kulesz-Martin MF: Functional quantification of DNA-binding proteins p53 and estrogen receptor in cells and tumor tissues by DNA affinity immunoblotting. Cancer Res. 61:5402–5406. 2001.PubMed/NCBI

23 

Gallogly MM and Mieyal JJ: Mechanisms of reversible protein glutathionylation in redox signaling and oxidative stress. Curr Opin Pharmacol. 7:381–391. 2007. View Article : Google Scholar : PubMed/NCBI

24 

Ghezzi P, Bonetto V and Fratelli M: Thiol-disulfide balance: From the concept of oxidative stress to that of redox regulation. Antioxid Redox Signal. 7:964–972. 2005. View Article : Google Scholar : PubMed/NCBI

25 

Klatt P and Lamas S: Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur J Biochem. 267:4928–4944. 2000. View Article : Google Scholar : PubMed/NCBI

26 

Al-Sawaf O, Clarner T, Fragoulis A, Kan YW, Pufe T, Streetz K and Wruck CJ: Nrf2 in health and disease: Current and future clinical implications. Clin Sci (Lond). 129:989–999. 2015. View Article : Google Scholar

27 

Brandt R and Keston AS: Synthesis of diacetyldichlorofluorescin: A stable reagent for fluorometric analysis. Anal Biochem. 11:6–9. 1965. View Article : Google Scholar : PubMed/NCBI

28 

Wang Z and Sun Y: Targeting p53 for novel anticancer therapy. Transl Oncol. 3:1–12. 2010. View Article : Google Scholar : PubMed/NCBI

29 

Rainwater R, Parks D, Anderson ME, Tegtmeyer P and Mann K: Role of cysteine residues in regulation of p53 function. Mol Cell Biol. 15:3892–3903. 1995. View Article : Google Scholar : PubMed/NCBI

30 

Li Y and Prives C: Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function? Oncogene. 26:2220–2225. 2007. View Article : Google Scholar : PubMed/NCBI

31 

Bonsing BA, Corver WE, Gorsira MCB, van Vliet M, Oud PS, Cornelisse CJ and Fleuren GJ: Specificity of seven monoclonal antibodies against p53 evaluated with Western blotting, immunohistochemistry, confocal laser scanning microscopy, and flow cytometry. Cytometry. 28:11–24. 1997. View Article : Google Scholar : PubMed/NCBI

32 

Wang PL, Sait F and Winter G: The ‘wildtype’ conformation of p53: Epitope mapping using hybrid proteins. Oncogene. 20:2318–2324. 2001. View Article : Google Scholar : PubMed/NCBI

33 

Gannon JV, Greaves R, Iggo R and Lane DP: Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. EMBO J. 9:1595–1602. 1990.PubMed/NCBI

34 

Niture SK, Velu CS, Bailey NI and Srivenugopal KS: S-thiolation mimicry: Quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography. Arch Biochem Biophys. 444:174–184. 2005. View Article : Google Scholar : PubMed/NCBI

35 

Punganuru SR, Madala HR, Venugopal SN, Samala R, Mikelis C and Srivenugopal KS: Design and synthesis of a C7-aryl piperlongumine derivative with potent antimicrotubule and mutant p53-reactivating properties. Eur J Med Chem. 107:233–244. 2016. View Article : Google Scholar

36 

Piskounova E, Agathocleous M, Murphy MM, Hu Z, Huddlestun SE, Zhao Z, Leitch AM, Johnson TM, DeBerardinis RJ and Morrison SJ: Oxidative stress inhibits distant metastasis by human melanoma cells. Nature. 527:186–191. 2015. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

April 2016
Volume 48 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Basak, D., Punganuru, S.R., & Srivenugopal, K.S. (2016). Piperlongumine exerts cytotoxic effects against cancer cells with mutant p53 proteins at least in part by restoring the biological functions of the tumor suppressor. International Journal of Oncology, 48, 1426-1436. https://doi.org/10.3892/ijo.2016.3372
MLA
Basak, D., Punganuru, S. R., Srivenugopal, K. S."Piperlongumine exerts cytotoxic effects against cancer cells with mutant p53 proteins at least in part by restoring the biological functions of the tumor suppressor". International Journal of Oncology 48.4 (2016): 1426-1436.
Chicago
Basak, D., Punganuru, S. R., Srivenugopal, K. S."Piperlongumine exerts cytotoxic effects against cancer cells with mutant p53 proteins at least in part by restoring the biological functions of the tumor suppressor". International Journal of Oncology 48, no. 4 (2016): 1426-1436. https://doi.org/10.3892/ijo.2016.3372