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Overcoming Chemoresistance in Pancreatic Cancer by Targeting the Anti‐Apoptotic Mcl‐1 Protein
Author(s) -
Badejo Adeleke,
McCreary Jordan,
True Heather,
Weisenburger Alexis,
Davis Madison,
Malhotra Ashim
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1194.4
Subject(s) - raloxifene , apoptosis , pancreatic cancer , autophagy , cancer research , western blot , downregulation and upregulation , chemistry , cancer , pharmacology , medicine , tamoxifen , breast cancer , biochemistry , gene
Pancreatic cancer is highly aggressive with high mortality and poor 5‐year prognosis. Treatment is challenging due to substantial chemoresistance. The ability of cancers to limit the apoptotic response to drug treatment emphasizes the urgent need to develop drugs that can overcome chemoresistance. Overexpression of the anti‐apoptotic Mcl‐1 protein is associated with the development of chemoresistance in some cancers. Using the human pancreatic cancer cell line MIA PaCa‐2, we investigated the effect of the drug raloxifene on the regulation of Mcl‐1 in this model. The rationale for the use of raloxifene was the observation that it can cause apoptosis in cancer cell lines. We treated MIA PaCa‐2 cells with 0.05, 0.5, 5, and 20 μM raloxifene for 72 hours, followed by the preparation of whole cell lysates, which we used for Western Blot analysis. Our data show that: 1) raloxifene disrupted the protein‐protein interaction between the anti‐apoptotic Mcl‐1 and the pro‐apoptotic Bax, suggesting Bax homodimerization at the mitochondria, and possible activation of the apoptotic or autophagy response. To explain this, we investigated 1) the effect of raloxifene on the protein expression of the alternative splice variants of Mcl‐1, Mcl‐1 L or Mcl‐1 S ; and, 2) whether raloxifene‐induced protein mistrafficking of Mcl‐1. Our data showed no changes in Mcl‐1 L and Mcl‐1 S , although levels decreased in the Bax/Mcl‐1 complex, as indicated by co‐immunoprecipitation experiments. 4) Additionally, we investigated the effect of raloxifene on global transcription by screening its effect on NFκB, Nrf‐2, eif‐2α, phospho‐cdc2, and cyclin proteins. While raloxifene treatment did not induce any of these transcription factors, at high doses it induced the expression of SRSF‐5, but had no effect on the expression of SRSF‐1, which are proteins that regulate the differential splicing of the Mcl‐1 gene. It also induced cyclin proteins. To further investigate the specific role of raloxifene in the turnover of Mcl‐1 protein, we pre‐incubated MIA PaCa‐2 cells with 10 μM cycloheximide, a global translation inhibitor. Our data showed a reduction in Mcl‐1 in raloxifene treated samples compared to control, suggesting a role for estrogen receptor β in the de novo expression of Mcl‐1. 6) Additionally, raloxifene treatment induced the autophagy markers Beclin‐1 and LCIII‐B, indicating a correlation between the differential regulation of Mcl‐1 and autophagy. Taken together, these data suggest that the main mechanism for the action of raloxifene is competitive displacement of Mcl‐1 from the Bax/Mcl‐1 protein complex. Support or Funding Information This research was supported in part by Pacific University Research Incentive Grant and Collins Medical Trust grant to AM.