Inhibition of Akt phosphorylation attenuates resistance to TNF-α cytotoxic effects in MCF-7 cells, but not in their doxorubicin resistant derivatives

Document Type : Original Article


1 Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

3 Pharmaceutical Research Center, Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Acquisition of TNF-α resistance plays role in the onset and growth of malignant tumors. Previous studies have demonstrated that MCF-7 cell line and its doxorubicin resistant variant MCF-7/Adr are resistant against the cytotoxic effects of TNF-α. In this study, we investigated the role of Akt activation in resistance of MCF-7 and MCF-7/Adr against TNF-α cytotoxicity.
Materials and Methods: The role of Akt activation in TNF-α cytotoxicity was investigated by MTT cell viability assay following treatment of the cells with the chemical inhibitor of Akt activation with or without TNF-α treatment. Phosphorylation of Akt at Ser473 before and after 72 hr TNF-α treatment  was also determined by western blot.
Results: TNF-α treatment led to enhancement of Akt Ser473 phosphorylation. Treatment of MCF-7 cells with TNF-α along with Akt-inhibitor agent, tricribine, attenuated Akt Ser473 phosphorylation and sensitized these cells to the cytotoxic effects of TNF-α in a dose and time dependent manner while tricribine treatment did not cause any significant cytotoxicity in MCF-7/Adr cells alone or in combination with TNF-α.
Conclusion: These results demonstrate that Akt phosphorylation plays pivotal role in the resistance of MCF-7 cells against TNF-α-induced cytotoxicity while it might play no significant role in the resistance of MCF-7/Adr cells against TNF-α.


1. Beutler B. The role of tumor necrosis factor in health and disease. J Rheumatol Suppl 1999; 57:16-21.
2. Chu W-M. Tumor necrosis factor. Cancer Lett 2013; 328:222-225.
3. Van Herreweghe F, Festjens N, Declercq W, Vandenabeele P. Tumor necrosis factor-mediated cell death: to break or to burst, that’s the question. Cell Mol Life Sci 2010; 67:1567-1579.
4. Stein U, Walther W. Cytokine-mediated reversal of multidrug resistance. Multiple Drug Resistance in Cancer 2: Springer; 1998. p. 271-282.
5. Borsellino N, Crescimanno M, Flandina C, Flugy A, D'Alessandro N. Combined activity of interleukin-1 alpha or TNF-alpha and doxorubicin on multidrug resistant cell lines: evidence that TNF and DXR have synergistic antitumor and differentiation-inducing effects. Anticancer Res 1993; 14:2643-2648.
6. Cao W, Ma SL, Tang J, Shi J, Lu Y. A combined treatment TNF-α/doxorubicin alleviates the resistance of MCF-7/Adr cells to cytotoxic treatment. BBA Mol Cell Res 2006; 1763:182-187.
7. Bauer B, Hartz AM, Miller DS. Tumor necrosis factor alpha and endothelin-1 increase P-glycoprotein expression and transport activity at the blood-brain barrier. Mol Pharmacol 2007; 71:667-675.
8. Belliard AM, Lacour B, Farinotti R, Leroy C. Effect of tumor necrosis factor-alpha and interferon-gamma on intestinal P-glycoprotein expression, activity, and localization in Caco-2 cells. J Pharm Sci 2004; 93:1524-1536.
9. Bertilsson PM, Olsson P, Magnusson K-E. Cytokines influence mRNA expression of cytochrome P450 3A4 and MDRI in intestinal cells. J Pharm Sci 2001; 90:638-646.
10. Cusack JC. Rationale for the treatment of solid tumors with the proteasome inhibitor bortezomib. Cancer Treat Rev 2003; 29:21-31.
11. Burt R, Poirier M, Link C, Bohr V. Antineoplastic drug resistance and DNA repair. Ann Oncol 1991; 2:325-334.
12. Duffy M. The war on cancer: are we winning? Tumour Biol 2013; 34:1275-1284.
13. Gimenez-Bonafe P, Tortosa A, Perez-Tomas R. Overcoming drug resistance by enhancing apoptosis of tumor cells. Curr Cancer Drug Tar 2009; 9:320-340.
14. Gjertsen BT, Logothetis CJ, McDonnell TJ. Molecular regulation of cell death and therapeutic strategies for cell death induction in prostate carcinoma. Cancer Metast Rev 1998; 17:345-351.
15. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP–dependent transporters. Nat Rev Cancer 2002; 2:48-58.
16. Moscow JA, Townsend AJ, Goldsmith ME, Whang-Peng J, Vickers PJ, Poisson R, et al. Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer. Proc Natl Acad Sci USA 1988; 85:6518-6522.
17. Lu D, Huang J, Basu A. Protein kinase Cepsilon activates protein kinase B/Akt via DNA-PK to protect against tumor necrosis factor-alpha-induced cell death. J Biol Chem 2006; 281:22799-22807.
18. Osaki M, Oshimura Ma, Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 2004; 9:667-676.
19. Knuefermann C, Lu Y, Liu B, Jin W, Liang K, Wu L, et al. HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells. Oncogene 2003; 22:3205-3212.
20. W Grunt T, L Mariani G. Novel approaches for molecular targeted therapy of breast cancer: interfering with PI3K/AKT/mTOR signaling. Curr Cancer Drug Tar 2013; 13:188-204.
21. Nahta R, O’Regan RM. Therapeutic implications of estrogen receptor signaling in HER2-positive breast cancers. Breast Cancer Res Treat 2012; 135:39-48.
22. Mosaffa F, Kalalinia F, Parhiz BH, Behravan J. Tumor necrosis factor alpha induces stronger cytotoxicity in ABCG2-overexpressing resistant breast cancer cells compared with their drug-sensitive parental line. DNA Cell Biol 2011; 30:413-418.
23. Zyad A, Benard J, Tursz T, Clarke R, Chouaib S. Resistance to TNF-alpha and adriamycin in the human breast cancer MCF-7 cell line: relationship to MDR1, MnSOD, and TNF gene expression. Cancer Res 1994; 54:825-831.
24. Meuillet EJ. Novel Inhibitors of AKT: Assessment of a different approach targeting the pleckstrin homology domain. Curr Med Chem 2011; 18:2727-2742.
25. Cheng JQ, Lindsley CW, Cheng GZ, Yang H, Nicosia SV. The Akt/PKB pathway: molecular target for cancer drug discovery. Oncogene 2005; 24:7482-7492.
26. Vahdati Hassani F, Naseri V, Razavi BM, Mehri S, Abnous K, Hosseinzadeh H. Antidepressant effects of crocin and its effects on transcript and protein levels of CREB, BDNF, and VGF in rat hippocampus. DARU 2014; 22:16-16.
27. Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PR, Reese CB, et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 1997; 7:261-269.
28. Bartholomeusz C, Gonzalez-Angulo AM. Targeting the PI3K signaling pathway in cancer therapy. Expert Opin Ther Targets 2012; 16:121-130.
29. Ciraolo E, Morello F, Hirsch E. Present and future of PI3K pathway inhibition in cancer: perspectives and limitations. Curr Med Chem 2011; 18:2674-2685.
30. Blajecka K, Borgstrom A, Arcaro A. Phosphatidylinositol 3-kinase isoforms as novel drug targets. Curr drug targets 2011; 12:1056-1081.
31. Clark AS, West K, Streicher S, Dennis PA. Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Mol Cancer Ther 2002; 1:707-717.
32. Kim D, Dan HC, Park S, Yang L, Liu Q, Kaneko S, et al. AKT/PKB signaling mechanisms in cancer and chemoresistance. Front Biosci 2005; 10:975-957.
33. Suh BY, Jung JJ, Park N, Seong CH, Im HJ, Kwon Y, et al. Induction of steroid sulfatase expression by tumor necrosis factor-alpha through phosphatidylinositol 3-kinase/Akt signaling pathway in PC-3 human prostate cancer cells. Exp Mol Med 2011; 43:646-652.
34. Faurschou A, Gniadecki R. TNF-alpha stimulates Akt by a distinct aPKC-dependent pathway in premalignant keratinocytes. Exp Dermatol 2008; 17:992-997.
35. Radeff-Huang J, Seasholtz TM, Chang JW, Smith JM, Walsh CT, Brown JH. Tumor necrosis factor-α-stimulated cell proliferation is mediated through sphingosine kinase-dependent Akt activation and cyclin D expression. J Biol Chem 2007; 282:863-870.