Introduction and Objective: Multidrug resistance phenotype (MDR) is characterized by overexpression of P-glycoprotein (Pgp/ABCB1) and related to chemotherapy cancer treatment failure. However, MDR is considered a multifactorial phenotype associated with molecular pathways deregulation, such as changes in apoptosis-associated proteins or transcriptional regulators expression. Y-box protein 1 (YB-1) and NFκB may regulate Pgp expression, acting as regulators of MDR1/ABCB1 gene. Studies have shown that Pgp and apoptosis-associated proteins expression may also contribute to multifactorial cancer resistance. Tumor necrosis factor-alpha (TNF-α) is an important cytokine that presents ambiguous function on cancer development, since it may act as death signaling or tumor growth factor. Also, TNF-α is a main activator of NFκB pathway. Therefore, the aim of this study was to investigate the role of Pgp expression, the proapoptotic protein TNF-α, and YB-1 and NFκB transcriptional factors in the contribution of multifactorial resistance phenotype in cancer cells.

Materials and Methods: In this study we used two cervical cancer cell lines: KB-3-1, parental cell line, and KB-C1, Pgp-positive cell line selected from KB-3-1 through increased doses of colchicine. KB-3-1 and KB-C1 cell lines were treated with doxorubicin, cisplatin, and colchicine for 24, 48, or 72 h and cell viability was analyzed by MTT assay. KB-3-1 and KB-C1 cell lines were treated with recombinant TNF-α (rTNF-α) for 30 min or 24 h and apoptosis index was measured by Annexin-V/PI staining using flow cytometry. Pgp expression, function, and subcellular localization were analyzed by Western blot, flow cytometry, and immunofluorescence, respectively. TNF-α, YB-1, and NFκB expression and subcellular localization were also investigated.

Results and Conclusion: Our data showed that KB-3-1 cells were sensitive to drug treatment while KB-C1 cells were resistant to doxorubicin and colchicine drugs, and it is probably related to a functional overexpression of Pgp. However, both cell lines showed sensitivity to high doses of cisplatin, a non-Pgp substrate. Then, we observed that KB-3-1 cells showed higher expression of YB-1 and NFκB/p65 subunit than KB-C1, but lower expression of NFκB/p105 subunit. We observed a perinuclear, nuclear, and cytoplasmatic subcellular distribution of NFκB in both cell lines. Also, YB-1 was detected in cytoplasm and nuclear foci in both cell lines, but apparently wide larger in KB-C1 than KB-3-1. KB-C1 cells exhibited cytoplasmatic and nuclear TNF-α distribution, while KB-3-1 cells showed mostly nuclear localization. Further, we observed low apoptosis rate following rTNF-α treatment in both cell lines. Besides that, Pgp expression was increased and showed strong membrane staining in KB-C1 cells treated with rTNF-α. Also, KB-3-1 and KB-C1 showed higher expression of TNF-α and cytoplasmatic localization after treatment with rTNF-α. In summary, our results suggest that Pgp expression in KB-C1 cell line might be regulated by YB-1 pathway. In addition, rTNF-α treatment changed Pgp expression in resistant cells, suggesting a possible role of TNF-α in supporting resistance phenotype. More data are required to understand the role of TNF-α protein in MDR phenotype.

Citation Format: Tandressa Berguetti, Paloma Souza, Raquel Maia. Role of TNF-α and transcriptional factors YB-1 and NFκB in P-glycoprotein expressing cancer cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B46.