The Radiosensitization Effect of Parthenolide in Prostate Cancer Cells Is Mediated by Nuclear Factor-KB Inhibition and Enhanced by the Presence of PTEN

2477 The radiosensitization effect of parthenolide in prostate cancer cells is mediated by nuclear factor-KB inhibition and enhanced by the presence of PTEN Yulan Sun,1 Daret K.St.Clair, 1 Fang Fang,2 sensitization effect of parthenolide is due, in part, to the Graham W.Warren, 2 Vivek M.Rangnekar, 1,2 inhibition of the NF-KB pathway. The presence of PTEN Peter A.Crooks, 3 and William H.St.Clair 2 enhances the radiosensitization effect of parthenolide, in part, by suppressing the absolute amount of activated 1Graduate Center for Toxicology, 2Department of Radiation p-Akt. [Mol Cancer Ther 2007;6(9):2477–86] Medicine, and 3College of Pharmacy, University of Kentucky, Lexington, Kentucky Introduction Prostate cancer is the most common cancer type and was Abstract the third leading cause of cancer death among U.S. men in Parthenolide hasbeen shownto have anti-inflammatory 2006 (1). Radiation therapy is frequently used to treat early and antitumor properties. However, whether and how stage and inoperable locally advanced prostate cancer. The parthenolide enhancestumor sensitivityto radiation outcome of radiation therapy can be greatly improved if therapy are unknown. In thisstudy,we show that higher doses of radiation are applied, especially for patients K K inhibition of the nuclear factor- B (NF- B) pathway isa with unfavorable tumors (i.e., stage T3 lesions, pretreat- common mechanism for the radiosensitization effect of ment prostate-specific antigen levels >10 ng/mL, or biopsy parthenolide in prostate cancer cells LNCaP, DU145, and Gleason scores z7). For these patients, the 5-year cancer- PC3. Parthenolide inhibitsradiation-induced NF- KB DNA- free survival rate increased from 41% to 75% when binding activity and the expression of its downstream radiation dose was increased to >7,200 cGy (2). However, target sod2, the gene coding for an important antiapop- the side effects and late complications resulting from high- totic and antioxidant enzyme (manganese superoxide dose radiation will increase to an unacceptable level, which dismutase) in the three prostate cancer cells. Different limits the use of high doses of radiation. Another problem susceptibilities to parthenolide’s effect are observed in in radiation therapy is that tumor cells develop radio- two radioresistant cancer cells, DU145 and PC3, with resistance. Therefore, it is important to find agents that DU145 cells showing higher sensitivity. This differential sensitize malignant tumor cells to radiation therapy, thus susceptibility to parthenolide is due, in part, to the fact minimizing radiation toxicity to surrounding organs by that in addition to NF-KB inhibition, parthenolide activates lowering effective therapeutic doses. the phosphatidylinositol-3-kinase/Akt prosurvival pathway Many mechanisms are involved in the development of in both cell lines. However, the activated Akt in DU145 radioresistance in tumor cells. One of the possible cellsiskept at a relatively low level compared with that mechanisms is activation of the nuclear factor-nB (NF-nB) in PC3 cellsdue to the presenceof functional PTEN. signaling pathway. It has been shown that approximately Transfection of wild-type PTEN into PTEN-null cells, two-thirds of the biological damage by radiation is due to PC3, confers the enhanced radiosensitization effect of free radical–mediated indirect action (3). NF-nB is a redox- parthenolide in PTEN-expressing cells. When PTEN ex- sensitive transcription factor family regulating cell survival pression is knocked down in DU145 cells, the cells and death. In response to radiation-induced reactive become more resistant to parthenolide’s effect. Taken oxygen species, the NF-nB pathway is activated (4). There together, these results suggest that parthenolide inhibits are five members in the NF-nB family: RelA (p65), RelB, the NF-KB pathway and activates the phosphatidylinositol- c-Rel, NF-nB1 (p105/p50), and NF-nB2 (p100/p52). NF-nB 3-kinase/Akt pathway in prostate cancer cells. The radio- is normally sequestered in the cytoplasm by its inhibitor InB family members in an inactive complex. Two NF-nB activation pathways exist. The classical pathway activates the InB kinase complex, which consists of IKKa, IKKh, and g n a Received 3/16/07; revised 6/18/07; accepted 7/10/07. IKK , leading to I B phosphorylation, ubiquitination, and Grant support: NIH grant CA49797. further degradation by the 26S proteasome. As a result, the The costs of publication of this article were defrayed in part by the p50:RelA dimer is released and translocated into the payment of page charges. This article must therefore be hereby marked nucleus. The alternative pathway activates the IKKa advertisement in accordance with 18 U.S.C. Section 1734 solely to homodimer, leading to the partial degradation of p100 indicate this fact. and activation of the p52:RelB dimer. Russell and Tofilon Requests for reprints: William H. St. Clair, Department of Radiation n Medicine, University of Kentucky, 800 Rose Street, Lexington, KY 40536. reported that radiation activates the classical NF- B Phone: 859-323-6486; Fax: 859-257-4931. E-mail: [email protected] pathway by selective degradation of plasma membrane– Copyright C 2007 American Association for Cancer Research. associated InBa (5). Our previous study shows that RelB is doi:10.1158/1535-7163.MCT-07-0186 also activated by radiation (6). Activation of NF-nB induces Mol Cancer Ther 2007;6(9). September 2007 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. 2478 Radiosensitization Effect of Parthenolide the transcription of its target genes, which are involved in three prostate cancer cell lines, including the radiosensitive antiapoptosis and tumor metastasis (7). Among numerous LNCaP cells and two radioresistant cell lines, DU145 NF-nB downstream targets, manganese superoxide dismu- (PTEN wild-type) and PC3 (PTEN null), to investigate tase (MnSOD) has been identified as a constitutively and whether PTEN cooperates with NF-nB in the radiosensiti- immediately accessible NF-nB target (8). MnSOD is a zation effect of parthenolide. nuclear-encoded primary antioxidant enzyme localized in the mitochondria. The known function of MnSOD is to remove superoxide radicals in mitochondria (9). Over- Materials and Methods expression of MnSOD is protective against radiation- Cell Culture and Treatment induced cell death (6, 10, 11). Human prostate cancer cell lines LNCaP, DU145, and We and others have shown that NF-nB is constitutively PC3 were obtained from American Type Culture Collec- activated in aggressive prostate cancer (6, 12), which may tion. LNCaP and PC3 cells were cultured in RPMI 1640 be responsible for the intrinsic radioresistance of some (Invitrogen) supplemented with 10% fetal bovine serum, prostate cancer cells. Thus, inhibition of the NF-nB pathway 1% penicillin and streptomycin mixture, 1 mmol/L of represents a target to enhance the sensitivity of prostate sodium pyruvate, 10 mmol/L of HEPES, 1% NEAA cancer to radiation therapy. mixture (Cambrex), 1% MEM vitamin mixture (Cellgro), Parthenolide is a major active component of the herbal and 2 mmol/L of L-glutamine. DU145 cells were cultured medicine feverfew (Tanacetum parthenium), which is con- in MEM (Sigma) supplemented with 10% fetal bovine ventionally used in Europe to treat inflammatory diseases serum, 1% penicillin and streptomycin mixture, 1 mmol/L such as fever, migraine, and arthritis (13). Parthenolide has of sodium pyruvate, and 0.1 mmol/L of nonessential amino j been shown to inhibit growth or induce apoptosis in a acids. Cells were grown in a 5% CO2 atmosphere at 37 C. number of tumor cell lines (14–17). More remarkably, it has The parthenolide stock solution (5 mmol/L) was prepared been shown that parthenolide is cytotoxic to hepatoma cells in DMSO and diluted in culture medium to the indicated and leukemia cells whereas sparing normal liver cells and final concentration for cell treatment. DMSO (0.1%) diluted hematopoietic cells (14, 16), suggesting that the cytotoxic in medium was used as vehicle control. A 130 kv X-ray effect of parthenolide may be selective for tumor cells. machine (Faxitron X-ray Corporation) was used to radiate Many mechanisms are postulated to be involved in the cells, with a dose rate of 89.7 cGy/min. antitumor effect of parthenolide, including inhibition of Clonogenic Survival Assay nucleic acid synthesis (18), depletion of thiols, induction of Cells were trypsinized and plated in triplicate into six- oxidative stress (14, 15), induction of mitochondria dys- well plates at different densities based on cell types and function (14), disruption of intracellular calcium equilibri- doses of radiation. LNCaP, DU145, and PC3 cells were um (15), induction of cell cycle G2-M phase arrest (14, 19), plated at a density of 2,000 to 10,000 cells/well, 100 to Jun sustained activation of c- -NH2-kinase (20, 21), and 600 cells/well, and 100 to 500 cells/well, respectively. Cells inhibition of NF-nB (21). It has also been shown that were treated with the indicated concentrations of parthe- parthenolide sensitizes cancer cells to various apoptosis- nolide or vehicle control for 24 h prior to exposure to the inducing agents mainly through inhibition of NF-nB (22). indicated doses of radiation. Twenty-four hours after However, whether parthenolide sensitizes cancer cells to radiation treatment, the medium containing parthenolide radiation-induced cell death and whether inhibition of was removed and cells were maintained in normal

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