J Gastrointest Surg (2012) 16:1333–1340 DOI 10.1007/s11605-012-1913-7
ORIGINAL ARTICLE
Dimethylamino Parthenolide Enhances the Inhibitory Effects of Gemcitabine in Human Pancreatic Cancer Cells Bryan K. Holcomb & Michele T. Yip-Schneider & Joshua A. Waters & Joal D. Beane & Peter A. Crooks & C. Max Schmidt
Received: 16 November 2010 / Accepted: 21 February 2011 / Published online: 23 May 2012 # 2012 The Society for Surgery of the Alimentary Tract
Abstract Introduction Gemcitabine is standard treatment for pancreatic cancer but has limited clinical benefit due to chemoresistance. Nuclear factor-kappaB (NF-κB) can promote chemoresistance and is therefore an attractive therapeutic target. We hypothesize that NF-κB suppression with the novel, orally bioavailable inhibitor dimethylamino parthenolide (DMAPT) will sensitize pancreatic cancer cells to gemcitabine. Methods BxPC-3, PANC-1, and MIA PaCa-2 human pancreatic cancer cell lines were treated with gemcitabine and/or DMAPT. Effects on the NF-κB pathway were determined by electrophoretic mobility shift assay, ELISA, or Western blot. Proliferation and apoptosis were measured by cell counts and ELISA, respectively. The effect of gemcitabine in vivo was determined using a MIA PaCa-2 heterotopic xenograft model. Results Gemcitabine induced NF-κB activity in BxPC-3, PANC-1, and MIA PaCa-2 cells and decreased the level of the NFκB inhibitor IκBα in BxPC-3 and PANC-1 cells. DMAPT prevented the gemcitabine-induced activation of NF-κB. The combination of DMAPT/gemcitabine inhibited pancreatic cancer cell growth more than either agent alone. Gemcitabine also induced intratumoral NF-κB activity in vivo. Conclusions DMAPT enhanced the anti-proliferative effects of gemcitabine in association with NF-κB suppression in pancreatic cancer cells in vitro. Furthermore, gemcitabine induced NF-κB activity in vivo, thus supporting the evaluation of NF-κB-targeted agents to complement gemcitabine-based therapies. Keywords Pancreatic cancer . NF-κB . DMAPT . Parthenolide and gemcitabine Bryan K. Holcomb and Michele T. Yip-Schneider contributed equally to this paper. B. K. Holcomb : M. T. Yip-Schneider (*) : J. A. Waters : J. D. Beane : C. M. Schmidt (*) Department of Surgery, Indiana University School of Medicine, 980 W. Walnut St., Building R3, Rm. 541C, Indianapolis, IN 46202, USA e-mail:
[email protected] e-mail:
[email protected] C. M. Schmidt Department of Biochemistry/Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA C. M. Schmidt Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA
C. M. Schmidt Indiana University Cancer Center, Indianapolis, IN, USA
C. M. Schmidt Richard L. Roudebush VA Medical Center, Indianapolis, IN, USA
P. A. Crooks University of Kentucky, University of Arkansas for Medical Sciences, Little Rock, AR, USA
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Introduction Pancreatic cancer is the fourth most common cause of cancer mortality in USA.1 Over 43,000 patients will be newly diagnosed and 36,800 of these will succumb to the disease in this year.1 The prognosis remains grim with a 5-year overall survival following initial diagnosis of only 6 %.2 Even when diagnosed early and amenable to operative resection, the 5-year overall survival is approximately 20 %.2 Currently, the only hope for cure is operative therapy, yet only 15 % of patients will be operative candidates at presentation.3–5 Despite advances in surgery, radiotherapy, and chemotherapy, more than 90 % of patients with pancreatic cancer die of chemoinsensitive disease. Even the most effective chemotherapeutic drug, gemcitabine (2′,2′-difluorodeoxycytidine, Gemzar), is able to induce only a 5 % response rate.6 The lack of effective therapy has motivated the search for new ways of combating this aggressive malignancy. One strategy is to target signaling pathways that lead to aberrant growth in cancer cells, for example the pathway regulated by the transcription factor nuclear factor kappaB (NF-κB). NF-κB is normally found in the cytoplasm in an inactive form, bound to the inhibitor proteins IκB-α or IκB-β. Activation of NF-κB by the classical pathway involves IκB kinase phosphorylation of IκB on serines 32 and 36 that targets IκB for degradation by the 26S proteosome.7 Once this occurs, then NF-κB is free to translocate into the nucleus, bind DNA and initiate transcription. NF-κB has attracted recent interest as a promising therapeutic target because it is constitutively active in human pancreatic cancer, and its activity is elevated in pancreatic cancer cell lines that are resistant to chemotherapy.8–10 In addition, NF-κB has been implicated in regulating the expression of genes that promote cell survival, angiogenesis, and invasion, such as Bcl-XL, VEGF, and MMP.11 We have previously shown that parthenolide, a sesquiterpene lactone isolated from the medicinal herb feverfew (Tanacetum parthenium), inhibits growth and NF-κB activity in human pancreatic cancer cells in vitro.12 Although effective in vitro, parthenolide is relatively insoluble. Thus, analogs were synthesized and screened resulting in the identification of the watersoluble, orally bioavailable analog dimethylamino parthenolide (DMAPT).13 In animal models of pancreatic cancer, DMAPT inhibits NF-κB activity and shows therapeutic promise in combination with sulindac or celecoxib in vivo.14,15 We and others have reported that the chemotherapeutic agent gemcitabine induces NF-κB activity in pancreatic cancer cells in vitro, suggesting that NF-κB may play a role in chemoresistance to gemcitabine.8,11,16–18 We hypothesize that pharmacologic suppression of NF-κB will enhance the anti-tumor effects of gemcitabine. In the present study, we investigate the effect of the novel inhibitor DMAPT in combination with gemcitabine on the NF-κB pathway, as well as cell growth and apoptosis in pancreatic cancer cells
J Gastrointest Surg (2012) 16:1333–1340
in vitro. We show that DMAPT not only suppresses gemcitabine-induced NF-κB activation but also sensitizes pancreatic cancer cells to the anti-proliferative effects of gemcitabine, indicating that the level of NF-κB activity is involved in determining the gemcitabine response. Furthermore, in a heterotopic xenograft model, we demonstrate that gemcitabine exposure activates NF-κB in pancreatic tumor cells, suggesting that NF-κB suppression may be a viable strategy to improve the anti-tumor effects of gemcitabine by countering both innate and acquired resistance mechanisms.
Methods Cells and Treatments BxPC-3, MIA PaCa-2, and PANC-1 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained as recommended. Gemcitabine (GEMZAR®; Eli Lilly, Indianapolis, IN, USA) was dissolved in sterile water and stored at 4 °C. DMAPT13 was synthesized by reaction of parthenolide (Sigma-Aldrich, St. Louis, MO) with dimethylamine (Sigma-Aldrich, St. Louis, MO, USA), isolated as the fumarate salt, dissolved in sterile water, and stored at −20 °C. Electrophoretic Mobility Shift Assays Cells were plated in 6-well plates and treated the following day for 24 or 48 h with vehicle (water), gemcitabine, DMAPT, or the combination. Whole cell lysates were prepared and incubated with radiolabeled probes specific for NF-κB or OCT-1 (Promega, Madison, WI, USA). DNAprotein complexes were separated by electrophoresis and then visualized by autoradiography. Western Blotting Cells were plated in 6-well plates and treated with vehicle or gemcitabine the following day. After the specified treatment time, the cells were lysed in radioimmunoprecipitation assay buffer (PBS, 1 % NP40, 0.5 % sodium deoxycholate, 0.1 % SDS, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, and 1 mmol/L Na3VO4), and the supernatants were obtained. Cell lysates (10 μg protein) were separated by SDS-PAGE on 4–20 % gradient gels (Invitrogen, Carlsbad, CA, USA) and transferred to Immobilon P membranes (Millipore, Billerica, MA, USA). The blots were probed with total IκB-α (Cell Signaling Technology, Beverly, MA) or actin (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies according to the manufacturer's protocol. Protein expression was detected with enhanced chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA).
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Cell Counts
Statistical Analysis
The cells were plated in 6-well plates. Twenty-four hours later, the cells were treated with vehicle, gemcitabine, DMAPT, or the combination. After 72 h of treatment, trypan blue-excluded cell counts were performed in duplicate using a hemocytometer. Cell growth was expressed relative to vehicle-treated cells (100 %).
Statistical analysis was performed using Microsoft Excel or GraphPad Prism. Continuous variables were evaluated using the Student's t test. P
