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Cancer Res. Author manuscript; available in PMC Apr 1, 2011.
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PMCID: PMC2848907
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A NADPH oxidase dependent redox signaling pathway mediates the selective radiosensitization effect of parthenolide in prostate cancer cells

Abstract

Cancer cells are usually under higher oxidative stress than normal cells are. We hypothesize that introducing additional ROS insults or suppressing antioxidant capacity may selectively enhance cancer cell killing by oxidative-stress generating agents through stress overload or stress sensitization, while normal cells may be able to maintain redox homeostasis under exogenous ROS by adaptive response. Here, we demonstrate that parthenolide (PN), a sesquiterpene lactone, selectively exhibits a radiosensitization effect on prostate cancer PC3 cells but not on normal prostate epithelial PrEC cells. PN causes oxidative stress in PC3 cells but not in PrEC cells, as determined by the oxidation of the ROS-sensitive probe H2DCFDA and intracellular reduced thiol and disulfide levels. In PC3 but not PrEC cells, PN activates NADPH oxidase leading to a decrease in the level of reduced thioredoxin, activation of PI3K/Akt and consequent FOXO3a phosphorylation, which results in the downregulation of FOXO3a targets, antioxidant enzyme manganese superoxide dismutase (MnSOD) and catalase. Importantly, when combined with radiation, PN further increases ROS levels in PC3 cells, while it decreases radiation-induced oxidative stress in PrEC cells, possibly by increasing GSH level. Together, the results demonstrate that PN selectively activates NADPH oxidase and mediates intense oxidative stress in prostate cancer cells by both increasing ROS generation and decreasing antioxidant defense capacity. The results support the concept of exploiting the intrinsic differences in the redox status of cancer cells and normal cells as targets for selective cancer killing.

Keywords: parthenolide, radiation, prostate cancer, NADPH oxidase, oxidative stress

Introduction

Selectively killing cancer without harming normal tissue is a fundamental challenge in cancer therapy. Elevated oxidative stress and aberrant redox homeostasis are frequently observed in cancer cells compared to their normal cell counterparts. For example, prostate cancer cells often have increased reactive oxygen species (ROS) generation from mitochondria [1] or NADPH oxidase [2], and decreased antioxidant enzymes, such as MnSOD, CuZnSOD and catalase [3, 4]. A small shift toward an oxidizing condition in cells may lead to elevated proliferation and induction of adaptive response. However, a high oxidizing condition often results in cell injury and cell death. Persistent high ROS in cancer cells often leads to increased cell proliferation and adaptive responses that may contribute to tumorigenesis, metastasis and treatment resistance. Further exposure to exogenous ROS is hypothesized as pushing tumor cells, which already have high constitutive oxidative stress levels, to cell death, while normal cells may still maintain redox homeostasis through adaptive responses. Therefore, regulating intracellular redox state may represent an ideal strategy to selectively sensitize cancer cells to oxidative stress-inducing therapy, such as radiotherapy.

Parthenolide is a sesquiterpene lactone derived from the traditional herbal medicine feverfew. The biological activity of parthenolide is thought to be mediated through its α-methylene-γ-lactone moiety, which can react with nucleophiles, especially with cysteine thiol groups, in a Michael addition reaction. Thiols (-SH) are important in integrating intracellular redox changes with cellular signaling transduction pathways. Several regulatory proteins, such as kinases, phosphatases and transcription factors, have cysteines on their active sites. Oxidation and reduction of cysteine thiols effect protein functions or act as the molecular switch for their downstream signaling cascades [5]. The chemical properties of parthenolide make it a good candidate for modifying cellular redox signaling and give it great potential in cancer therapy. Oxidative stress has been shown to be a major mechanism for parthenolide-induced cell death [6]. Our previous study showed that parthenolide sensitizes human prostate cancer cells to radiation treatment through inhibiting the NF-κB pathway [7]. However, whether the radiosensitization effect of parthenolide is selective to prostate cancer cells but not normal prostate cells, and whether parthenolide differentially regulates intracellular redox state in cancer and normal cells, are unknown.

NADPH oxidase is an important source of ROS, which accounts, at least partially, for increased levels of ROS in prostate cancer [2, 8]. The first discovered NADPH oxidase is phagocyte NADPH oxidase. It is a multisubunit enzyme localized in cell membranes, consisting of membrane-bound components (gp91phox and p22phox) and cytosolic components (p47phox, p67phox, p40phox and Rac) that translocate to the membrane upon activation. Homologues of gp91phox (Nox2), including Nox1-5, Duox1 (dual oxidase) and Duox2, have been identified and named Nox (NADPH oxidase) proteins in non-phagocytic cells. Their activation requires p47phox paralog Noxo1 (Nox organizer 1) and p67phox paralog Noxa1 (Nox activator 1), or calcium binding [9]. Nox proteins catalyze the transfer of an electron to O2 to generate O2·, which is then dismutated to H2O2. It has been shown that prostate tumor is more likely (86%) to have Nox1 staining than benign prostate tissue is (62%) [8, 10] and that Nox-induced ROS is an important contributor to X-ray radiation induced cell death [11, 12]. Whether parthenolide activates NADPH oxidase in prostate cancer cells is unknown.

In a previous study, we found that parthenolide activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in prostate cancer cells [7]. While activated Akt is known to inhibit apoptosis and promote cell survival, recent studies suggest that the PI3K/Akt pathway may induce oxidative stress and trigger cell death under certain conditions [13]. The mechanisms by which Akt induces ROS may involve stimulation of mitochondrial oxidative metabolism and reduction of antioxidant defense via FOXO suppression.

The FOXO transcription factors are mammalian homologues of DAF-16, which regulates longevity in Caenorhabditis elegans. Among the four FOXO family members (FOXO1, FOXO3a, FOXO4 and FOXO6), FOXO1 and FOXO3a are the most highly expressed FOXO proteins in human prostate [14]. The downstream targets of FOXOs may be involved in cell cycle and cell death regulation, differentiation and development; cellular stress response, and energy metabolism control [15, 16]. Overexpression of FOXO1 and FOXO3a in prostate cancer cell line provokes apoptosis [17]. However, FOXOs also extend mammalian lifespan by protecting cells against oxidative stress-induced cell death [18]. As the cellular functions of FOXOs are diverse and in some cases antagonistic, it is postulated that the activity of FOXOs is differentially regulated in response to various types or intensities of external stimuli [19]. Phosphorylation of FOXO by Akt is inhibitory phosphorylation, which allows the chaperone protein 14-3-3 to bind to FOXOs in the nucleus and enhances FOXO nuclear export, leading to cytoplasmic sequestration. On the contrary, phosphorylation of FOXO by MST1 (mammalian Ste20-like kinase) and JNK (c-jun terminal kinase) is activatory phosphorylation, which disrupts 14-3-3 binding and triggers FOXO nuclear translocation. Radiation induces FOXO3a nuclear translocation and activates FOXO3a activity [20, 21]. Activation of FOXO3a by radiation may induce apoptosis by upregulating its targets, FasL and Bim [20]; but it may also protect cells against oxidative damage and genotoxic stress by upregulating antioxidant proteins, such as MnSOD, catalase, peroxiredoxin III and sestrin 3 [13, 18, 22, 23], and promoting DNA damage repair [21, 24, 25].

In this study, we explored how parthenolide differently modulates intracellular redox status in prostate cancer and normal prostate cells.

Materials and Methods

Cell culture and treatment

Human prostate cancer cell lines PC3 and DU145 were obtained from ATCC (Manassas, VA) and cultured as previously described [7]. Human normal prostate epithelial PrEC cells were purchased from Lonza (Walkersville, MD) and maintained in PrEGM medium (Lonza). Parthenolide, NADPH oxidase inhibitor diphenylene iodonium (DPI, Sigma) and PI3K inhibitor wortmannin (Cell signaling) were dissolved in DMSO.

MTT assay

Cells were treated with parthenolide for 24 hours, and exposed to radiation or were sham-irradiated. Twenty-four hours after radiation, parthenolide-containing medium was replaced with normal culturing medium for a total parthenolide treatment of 48 hours. After four cell doubling times [26] (approximately 4 days for PC3 cells and 8 days for PrEC cells), MTT assay was performed as previously described [7].

Cell growth curve

Cells were treated as described above for MTT assay. The mean number of cells/well was obtained every other day after radiation from the triplicate average. The results were plotted on a log-linear scale and fitted into exponential growth curve fit.

DCF assay

DCF assay was performed using carboxy-H2DCFDA (invitrogen, sensitive to oxidation) and oxidized carboxy-DCFDA (invitrogen, insensitive to oxidation) as optimized by Wan [27] et al. The fluorescence in cells preloaded with carboxy-H2DCFDA was normalized to that in cells preloaded with carboxy-DCFDA ( ratio of H2DCFDA/DCFDA) to control for the cell number, dye uptake, and ester cleavage differences between different treatment groups.

Detection of reduced thiols and disulfides

Protein thiols were labeled by 3-N-maleimido-propionyl biocytin (MPB) and detected by avidin-biotin technology on the blots as previously described by Bayer [28] et al. To detect protein disulfides, samples were first treated with N-ethylmaleimide (NEM, Sigma) to block the free thiol groups. Then 2-mercaptoethanol (ME, Sigma) was added to reduce the disulfide bond. The NEM-blocked, ME-reduced protein was then treated with MPB for disulfides labeling. Labeled proteins were subjected to SDS-PAGE, followed by detection with HRP-conjugated streptavidin.

NADPH oxidase activity assay

This assay was performed as described previously by Cui and Douglas [29]. Photoemission generated by the reaction of superoxide radical and lucigenin in terms of RLU was measured every minute for 15 minutes. Reaction velocity was calculated as the change of RLU per minute per μg protein.

GSH assay

Total GSH (GSHt) and GSSG were measured using the recycling assay of the Glutathione Assay Kit (Cayman Chemical). The amount of reduced GSH was calculated by subtracting the amount of GSSG from total GSH (GSHt - 2GSSG).

Western blot analysis

Western blot analysis was performed as previously described [7] using corresponding antibodies against Akt, Phospho-Akt (Ser 473), FOXO3a and Phospho-FOXO3a (Ser 253) (Cell Signaling), actin (Sigma), Nox1 (Santa Cruz), catalase (Santa Cruz) and MnSOD (Upstate). Representative blots and quantification from three independent experiments are shown.

Knocking down Nox1 using siRNA

Cells were transiently transfected with Nox1 siRNA (Santa Cruz) and control siRNA by using Oligofectamine™ (Invitrogen).

Electrophoretic mobility shift assay (EMSA)

Double-stranded oligonucleotides corresponding to the MnSOD promoter region containing consensus FOXO3a binding element (DBE, Daf-16 family protein binding element) [18] (5′-TTCTGACGTCTGTAAACAAGCCCAGCCCTT-3′) were labeled with [32P] ATP. The assay was performed as previously described [7].

Chromatin immunoprecipitation (ChIP assay)

Cells were collected and processed using ChIP-IT kit (Active Motif). Fixed protein/DNA complexes were sheared and precipitated using anti-FOXO3a antibody (Cell Signaling). The MnSOD promoter fragment containing DBE was amplified. The sequences of primer set were: upper-strand primer, 5′-CACCCCAACACGTAGCCCTAGTTACATTC-3′; and lower-strand primer, 5′- CTAGGCTTCCGGTAAGTGGAATGGGAAAAC-3′.

SOD mimetic treatment and colony survival assay

Cells were treated with parthenolide or DMSO for 24 hours prior to radiation exposure. Twenty-four hours after radiation, normal culture media replaced the media containing parthenolide. SOD mimetic (MnTE-2-PyP5+) was added concurrently with parthenolide and kept in the medium throughout. Colony survival assay was conducted as previously described [7].

FOXO3a transient transfection and determination of cell survival by trypan blue exclusion assay

Cells were transfected with HA-FOXO3a-WT (wild-type), HA-FOXO3a-TM (triple mutant) (from Addgene [15]) or pECE vector control plasmid, using Lipofectamine™ 2000 (Invitrogen). HA-FOXO3a-TM has three mutations at Akt phosphorylation sites (Thr32, Ser253, and Ser315), leading to constitutively active FOXO3a. After transfection, the medium was replaced with complete medium with or without parthenolide and incubated overnight. Cells were then treated with 6 Gy radiation or were sham-irradiated. Forty-eight hours after radiation, the cells were processed for trypan blue exclusion assay and collected for Western blot analysis.

Statistical analysis

Statistical analysis was performed using either Student's t test (for two-group comparison) or one-way ANOVA (for multiple-group comparison). Data were reported as mean ± SE.

Results

The radiosensitization effect of parthenolide is selective to prostate cancer PC3 cells but not normal prostate epithelial PrEC cells

Previously, we showed that parthenolide synergistically enhances the sensitivity of prostate cancer cells to radiation treatment using colony survival assay [7]. In the present study, we compared the effect of parthenolide in prostate cancer PC3 cells and normal prostate epithelial PrEC cells. Because PrEC cells did not form colonies in vitro, we performed MTT assay under the growth condition that provided a relative cell survival comparable with the clonogenic assays [7]. PrEC cells are more resistant to parthenolide-induced cytotoxicity than PC3 cells are (Fig. 1A). The sub-cytotoxic dose of parthenolide (1μmol/L) was chosen to study the combination effect of parthenolide and radiation on cell survival. As shown in Fig. 1B, 6 Gy radiation decreases cell viability in PC3 cells (by 53%) and in PrEC cells (by 41%). Parthenolide 1μmol/L reduces cell viability and enhances radiation-induced cytotoxicity in PC3 cells but not in PrEC cells. Comparisons of growth curves for these two cell lines also reveal a selective radiosensitization effect of parthenolide (Fig. 1C) in PC3 cells. Radiation 6 Gy decreases cell growth rate by approximately 50% in both cell lines (Fig. 1D). Parthenolide alone decreases cell number without obvious change in growth rate in both cell lines. A combination of parthenolide and radiation shows radiosensitization in PC3 cells by both decreased cell number and lower growth rate. However, the growth rate in the combination treatment group is the same as the untreated group for PrEC cells.

Figure. 1
The radiosensitization effect of parthenolide is selective to prostate cancer PC3 cells but not normal prostate epithelial PrEC cells

Parthenolide induces oxidative stress in PC3 cells but not in PrEC cells

We then explored potential determinants for the selectivity of parthenolide's effect. Since oxidative stress has been shown to be the major mechanism for both parthenolide [6] and radiation-induced cell death, we compared the effect of parthenolide on cellular ROS level in PC3 and PrEC cells by DCF assay. As shown in Fig. 2A, radiation significantly increases normalized carboxy-H2DCFDA fluorescence, a general indicator of cellular ROS level, in both PC3 and PrEC cells. However, parthenolide alone increases ROS level in prostate cancer PC3 cells but not normal prostate PrEC cells. When combined with radiation, parthenolide further elevates the cellular ROS level in PC3 cells, consistent with the radiosensitization effect. Interestingly, in PrEC cells, parthenolide decreases radiation-induced ROS level (significant at 5 μmol/L), suggesting an antioxidant response.

Figure. 2
Parthenolide induces oxidative stress in PC3 cells but not in PrEC cells

Parthenolide exerts its effect mainly by targeting thiol groups, which may lead to the depletion of intracellular glutathione and protein thiols, and induction of ROS [6, 30]. We therefore detected the presence of protein thiols and disulfides in PC3 and PrEC cells by MPB labeling. As shown in Fig. 2B, parthenolide 5 μmol/L significantly decreases reduced protein thiols and increases disulfides staining in PC3 cells. The decrease in the reduced protein thiols may result from the direct reaction of parthenolide with protein thiols or the oxidation of thiol groups due to parthenolide-induced oxidative stress. Interestingly, in PrEC cells, parthenolide does not significantly change the protein thiols and disulfides levels, which is consistent with the selective induction of oxidative stress in PC3 cells.

Parthenolide activates NADPH oxidase in PC3 cells but not in PrEC cells

Oxidative stress is the imbalance between prooxidants and antioxidants. Both increased production of ROS and decreased antioxidants can lead to oxidative stress. One major source of ROS generation in prostate cancer cells is NADPH oxidase [2, 8]. We measured NADPH oxidase activity to probe whether it is involved in parthenolide-induced oxidative stress in prostate cancer PC3 cells. Our results (Fig. 2C) show that parthenolide enhances NADPH oxidase activity dose-dependently in PC3 cells, which can be inhibited by DPI, a NADPH oxidase inhibitor. However, in PrEC cells, NADPH oxidase activity is not significantly affected by parthenolide.

Parthenolide decreases reduced thioredoxin (Trx) in PC3 cells as a downstream event of NADPH oxidase activation but increases glutathione (GSH) in PrEC cells

Since parthenolide targets thiols, we also detected two important thiol-containing small molecule antioxidants, GSH and Trx. As shown in Fig. 3A, total GSH level is slightly increased in PC3 cells and significantly increased in PrEC cells by parthenolide. The reduced GSH/GSSG ratio is not significantly changed by parthenolide in PC3 cells but is increased 2.4 fold in PrEC cells, which may lead to the protective effect against radiation-induced oxidative stress observed by DCF assay. Our result is contrary to a report that parthenolide can deplete intracellular GSH in cancer cells [6]. Since the reactivities of thiol groups are inversely related to their pKa, parthenolide may more readily react with protein thiols that have low pKa than with GSH which has a high pKa of 8.8. The induction of GSH may be due to the activation of the Nrf2/ARE (antioxidant/electrophile response element) pathway [31], because Keap1, the negative regulator of Nrf2, contains numerous low pKa cysteines [32].

Figure. 3
Effect of parthenolide on thiol-containing antioxidants GSH and Trx

Globally decreased protein thiols have been observed in PC3 cells but not in PrEC cells after parthenolide treatment (Fig. 2B). We examined reduced Trx, the active form of Trx, by using Trx antibody to pull down the thiol-labeled protein sample. Parthenolide significantly decreases reduced Trx in PC3 cells without altering the Trx protein amount (Fig. 3B). However, in the presence of DPI, the decrease caused by parthenolide in the reduced Trx is abolished, suggesting that this is a downstream event of NADPH oxidase activation. It is likely that Trx was oxidized by Nox-derived ROS.

Activation of NADPH oxidase by parthenolide is upstream of PI3K/Akt activation in PC3 cells

The PI3K/Akt pathway has been known to be activated by growth factors and oxidative stress [33], so we tested whether activation of the PI3K/Akt pathway is a downstream event of NADPH oxidase activation in PC3 cells. The PI3K inhibitor wortmannin prevents activation of Akt by parthenolide, as indicated by the decrease of p-Akt/Akt ratio. However, activation of NADPH oxidase by parthenolide is not affected (Fig. 4A). We then identified Nox1 as the major Nox isoform in PC3 cells by real-time PCR (data not shown). Knocking down Nox1 by siRNA inhibits both parthenolide-induced NADPH oxidase activation and Akt activation (Fig. 4B), suggesting Nox1-dependent NADPH oxidase activation by parthenolide is upstream of PI3K/Akt activation. This was further confirmed by using NADPH oxidase inhibitor DPI. In the presence of DPI, Akt activation by parthenolide and phosphorylation of FOXO3a, the downstream target of Akt kinase, are both prevented (Fig. 4C).

Figure. 4
Activation of NADPH oxidase by parthenolide is upstream of PI3K/Akt activation in PC3 cells

Activation of Akt by parthenolide induces FOXO3a phosphorylation and suppresses its downstream targets, antioxidant enzymes MnSOD and catalase, in prostate cancer cells but not in PrEC cells

FOXO3a is a main target of activated Akt. There are three conserved Akt phosphorylation sites on FOXO3a: Thr32, Ser253 and Ser315 [34]. Consistent with our previous study [7], parthenolide increases Akt phosphorylation in prostate cancer PC3 and DU145 cells (Fig. 5A). Consequently, the phosphorylation on Ser253 in FOXO3a is increased by parthenolide in a dose-dependent manner. Radiation only slightly increases Akt and FOXO3a phosphorylation. This may be explained by the fact that radiation only induces transient activation of Akt, which peaks at 1 hour after radiation and then drops at 6 hours after radiation [7]. In DU145 cells, the total FOXO3a level is increased after radiation treatment, consistent with Yang's observation in osteosarcoma cells [20]. Parthenolide decreases total FOXO3a level in DU145 cells, which may be due to Akt activation induced FOXO3a degradation by proteasome [35]. While FOXO3a phosphorylation increases in prostate cancer cells, in normal prostate epithelial PrEC cells, the combination of parthenolide with radiation does not enhance but slightly decreases FOXO3a phosphorylation.

Figure. 5
Activation of Akt by parthenolide induces FOXO3a inhibitory phosphorylation and suppresses its downstream targets, MnSOD and catalase, in prostate cancer cells but not in PrEC cells

Akt-mediated phosphorylation has been shown to induce FOXO3a cytoplasmic sequestration and thereby suppress its DNA binding activity. This is confirmed by EMSA (Fig. 5B). Radiation enhances FOXO3a DNA binding activity in PC3 cells. Parthenolide inhibits FOXO3a DNA binding dose-dependently in PC3 cells but not in PrEC cells. ChIP assay verified that FOXO3a indeed binds to the promoter of its target gene. As shown in Fig. 5C, radiation enhances FOXO3a binding to the MnSOD promoter region, which is suppressed by parthenolide, consistent with the EMSA result.

FOXO3a regulates a wide range of target genes. Since radiation kills cells, in part through generation of ROS, we detected FOXO3a targets, antioxidant enzymes catalase and MnSOD, in prostate cancer PC3 and DU145 cells (Fig. 5D). MnSOD protein level is increased after radiation in both cell lines, but is suppressed by parthenolide. Parthenolide also decreases catalase levels dose-dependently in both cell lines. Consistent with FOXO3a DNA binding activity, MnSOD and catalase levels in PrEC cells are not changed by parthenolide.

Suppression of antioxidant enzymes by parthenolide contributes to its radiosensitization effect

To confirm the role of antioxidant enzymes in the selective radiosensitization effect of parthenolide, we treated PC3 cells with a SOD mimetic, MnTE-2-PyP5+. SOD mimetic partially abolishes the radiosensitization effect of parthenolide in PC3 cells as determined by colony survival assay (Fig. 6A).

Figure. 6
Suppression of antioxidant enzymes by parthenolide is involved in its radiosensitization effect

We then overexpressed FOXO3a in PC3 cells to investigate whether overexpression of FOXO3a can rescue cells from parthenolide-induced radiosensitization effect by induction of antioxidant enzymes. Overexpression of FOXO3a-WT and FOXO3a-TM decreases survival of untreated cells, possibly due to the induction of apoptotic targets of FOXO3a [19]. We therefore normalized all untreated cell viability to 100% to eliminate basal survival differences among three different transfection groups. After normalization, we observed that FOXO3a overexpression does not significantly affect cell sensitivity to radiation treatment. However, overexpression of FOXO3a, especially constitutively active FOXO3a-TM, in PC3 cells significantly confers cellular resistance to parthenolide's effect (Fig. 6B). The expression of exogenous HA-FOXO3a was confirmed by Western blot. The basal levels of antioxidant enzymes, catalase and MnSOD, are higher when active FOXO3a-TM is overexpressed (Fig. 6C). These data demonstrate that FOXO3a plays an important role in maintaining cellular antioxidant enzymes, catalase and MnSOD, which are involved in the radiosensitization effect of parthenolide.

Discussion

The selective cytotoxicity of parthenolide to cancer cells has been reported in human acute myelogenous leukemia stem and progenitor cells [36]. Our current study confirms these findings in prostate cancer cells and extends to demonstrate that the radiosensitization effect of parthenolide is selective to prostate cancer cells but not normal prostate epithelial PrEC cells (Fig. 1). Our results also indicate that parthenolide “rejuvenates” irradiated normal prostate cells since cell growth rate after radiation is restored to the untreated control level when combined with parthenolide treatment. Parthenolide decreases radiation-induced ROS in PrEC cells (Fig. 2A), which correlates with increased GSH levels (Fig. 3A). Thus, the apparent antioxidant property of parthenolide in normal prostate cells may be due, in part, to the increased GSH level and may account for the “rejuvenation” of irradiated PrEC cells.

The selective targeting of cancer cells by parthenolide is of great interest. Our study shows for the first time that differential modulation of intracellular redox state by parthenolide in prostate cancer and normal prostate cells is involved in its selective radiosensitization effect. Cancer cells and normal cells have different redox statuses, which may be targeted for selective cancer killing. Prostate cancer PC3 cells have higher Nox1 and lower MnSOD, catalase (supplemental Figure 1) and GSH levels (Fig. 3A) compared with normal prostate PrEC cells, which may be responsible for the high oxidative stress levels in prostate cancer cells. In response to the constitutively high oxidative stress in prostate cancer cells, elevated proliferation and induction of adaptive response are observed; for example, higher Trx level in prostate cancer cells (supplemental Figure 2) and constitutively active NF-κB [37], which may be involved in treatment resistance [38-40]. Parthenolide further increases oxidative stress in prostate cancer cells by activation of NADPH oxidase and suppression of antioxidants Trx, MnSOD and catalase. The redox imbalance in prostate cancer cells caused by parthenolide enhances cellular sensitivity to oxidative stress-inducing radiotherapy (Fig. 6D). However, in normal prostate cells, due to high GSH (Fig. 3A) and glutathione-S-transferase (GSTs) [41] levels, parthenolide may more readily conjugate with GSH under the catalysis of GSTs and then be exported outside the cells, leading to cellular resistance to parthenolide.

How parthenolide activates Nox remains unknown. Since the amount of Nox1 is not increased (Fig. 4B) by parthenolide, parthenolide may activate Nox by facilitating the assembly of the multisubunit enzyme complex. The full activation of Nox1 requires p22phox, Noxo1, Noxa1 and Rac1 [9]. It has been shown that modulation of cysteine thiol can activate Ras [42, 43]. Since Rac1 is a member of the Ras superfamily of GTPases, it is possible that parthenolide may modify the cysteine thiol on Rac1 and increase its activity. In addition to direct oxidative damage, Nox-derived ROS plays an important role in redox signaling due to its highly regulated activation. Activation of Nox can activate PI3K/Akt by the oxidation and inactivation of phosphatases that dephosphorylate PI3K or AKT kinase by Nox-derived ROS. The PI3K/Akt/FOXO3a cascade leads to downregulation of the antioxidant enzymes MnSOD and catalase, which is also involved in the oxidative stress induced by parthenolide.

In summary, our study demonstrates that in prostate cancer cells, parthenolide induces dramatic oxidative stress via NADPH oxidase activation. On one hand, NADPH oxidase activation not only increases ROS generation, but it also decreases reduced Trx or acts as a second messenger to activate the PI3K/Akt pathway, leading to a decrease in antioxidant defense capacity via FOXO3a suppression. On the other hand, parthenolide increases GSH levels but does not activate NADPH oxidase in normal prostate epithelial cells. Thus, the selective induction of oxidative stress by parthenolide in prostate cancer cells accounts for its selective radiosensitization effect. Our results also imply that modulating intracellular redox state might be an ideal way for cancer therapy to achieve selective cancer killing.

Supplementary Material

Acknowledgments

This work was supported by NIH grant CA49797 and CA115801. We thank Dr. Ines Batinic-Haberle for kindly providing the SOD mimetic used in this study.

References

1. Petros JA, Baumann AK, Ruiz-Pesini E, et al. mtDNA mutations increase tumorigenicity in prostate cancer. Proc Natl Acad Sci U S A. 2005;102(3):719–24. [PMC free article] [PubMed]
2. Kumar B, Koul S, Khandrika L, Meacham RB, Koul HK. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res. 2008;68(6):1777–85. [PubMed]
3. Baker AM, Oberley LW, Cohen MB. Expression of antioxidant enzymes in human prostatic adenocarcinoma. Prostate. 1997;32(4):229–33. [PubMed]
4. Bostwick DG, Alexander EE, Singh R, et al. Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraepithelial neoplasia and cancer. Cancer. 2000;89(1):123–34. [PubMed]
5. Winterbourn CC, Hampton MB. Thiol chemistry and specificity in redox signaling. Free Radic Biol Med. 2008;45(5):549–61. [PubMed]
6. Wen J, You KR, Lee SY, Song CH, Kim DG. Oxidative stress-mediated apoptosis. The anticancer effect of the sesquiterpene lactone parthenolide. J Biol Chem. 2002;277(41):38954–64. [PubMed]
7. Sun Y, St Clair DK, Fang F, et al. The radiosensitization effect of parthenolide in prostate cancer cells is mediated by nuclear factor-kappaB inhibition and enhanced by the presence of PTEN. Mol Cancer Ther. 2007;6(9):2477–86. [PMC free article] [PubMed]
8. Lim SD, Sun C, Lambeth JD, et al. Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate. 2005;62(2):200–7. [PubMed]
9. Lambeth JD, Kawahara T, Diebold B. Regulation of Nox and Duox enzymatic activity and expression. Free Radic Biol Med. 2007;43(3):319–31. [PMC free article] [PubMed]
10. Arnold RS, He J, Remo A, et al. Nox1 expression determines cellular reactive oxygen and modulates c-fos-induced growth factor, interleukin-8, and Cav-1. Am J Pathol. 2007;171(6):2021–32. [PMC free article] [PubMed]
11. Liu Q, He X, Liu Y, et al. NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death. Biochem Biophys Res Commun. 2008;377(3):775–9. [PubMed]
12. Tateishi Y, Sasabe E, Ueta E, Yamamoto T. Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation. Biochem Biophys Res Commun. 2008;366(2):301–7. [PubMed]
13. Nogueira V, Park Y, Chen CC, et al. Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis. Cancer Cell. 2008;14(6):458–70. [PMC free article] [PubMed]
14. Kim D, Cheng GZ, Lindsley CW, Yang H, Cheng JQ. Targeting the phosphatidylinositol-3 kinase/Akt pathway for the treatment of cancer. Curr Opin Investig Drugs. 2005;6(12):1250–8. [PubMed]
15. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999;96(6):857–68. [PubMed]
16. Burgering BM. A brief introduction to FOXOlogy. Oncogene. 2008;27(16):2258–62. [PubMed]
17. Modur V, Nagarajan R, Evers BM, Milbrandt J. FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression. Implications for PTEN mutation in prostate cancer. J Biol Chem. 2002;277(49):47928–37. [PubMed]
18. Kops GJ, Dansen TB, Polderman PE, et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature. 2002;419(6904):316–21. [PubMed]
19. Calnan DR, Brunet A. The FoxO code. Oncogene. 2008;27(16):2276–88. [PubMed]
20. Yang JY, Xia W, Hu MC. Ionizing radiation activates expression of FOXO3a, Fas ligand, and Bim, and induces cell apoptosis. Int J Oncol. 2006;29(3):643–8. [PMC free article] [PubMed]
21. Tsai WB, Chung YM, Takahashi Y, Xu Z, Hu MC. Functional interaction between FOXO3a and ATM regulates DNA damage response. Nat Cell Biol. 2008;10(4):460–7. [PMC free article] [PubMed]
22. Nemoto S, Finkel T. Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway. Science. 2002;295(5564):2450–2. [PubMed]
23. Chiribau CB, Cheng L, Cucoranu IC, Yu YS, Clempus RE, Sorescu D. FOXO3A regulates peroxiredoxin III expression in human cardiac fibroblasts. J Biol Chem. 2008;283(13):8211–7. [PMC free article] [PubMed]
24. Tran H, Brunet A, Grenier JM, et al. DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science. 2002;296(5567):530–4. [PubMed]
25. Hollander MC, Sheikh MS, Bulavin DV, et al. Genomic instability in Gadd45a-deficient mice. Nat Genet. 1999;23(2):176–84. [PubMed]
26. Slavotinek A, McMillan TJ, Steel CM. Measurement of radiation survival using the MTT assay. Eur J Cancer. 1994;30A(9):1376–82. [PubMed]
27. Wan XS, Zhou Z, Kennedy AR. Adaptation of the dichlorofluorescein assay for detection of radiation-induced oxidative stress in cultured cells. Radiat Res. 2003;160(6):622–30. [PubMed]
28. Bayer EA, Safars M, Wilchek M. Selective labeling of sulfhydryls and disulfides on blot transfers using avidin-biotin technology: studies on purified proteins and erythrocyte membranes. Anal Biochem. 1987;161(2):262–71. [PubMed]
29. Cui XL, Douglas JG. Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells. Proc Natl Acad Sci U S A. 1997;94(8):3771–6. [PMC free article] [PubMed]
30. Zhang S, Ong CN, Shen HM. Critical roles of intracellular thiols and calcium in parthenolide-induced apoptosis in human colorectal cancer cells. Cancer Lett. 2004;208(2):143–53. [PubMed]
31. Herrera FMV, Rodriguez-Blanco J, García-Santos G, Antolín I, Rodriguez C. Intracellular redox state regulation by parthenolide. Biochem Biophys Res Commun. 2005;332(2):321–5. [PubMed]
32. Copple IM, Goldring CE, Kitteringham NR, Park BK. The Nrf2-Keap1 defence pathway: role in protection against drug-induced toxicity. Toxicology. 2008;246(1):24–33. [PubMed]
33. Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol. 2002;192(1):1–15. [PubMed]
34. Kashii Y, Uchida M, Kirito K, et al. A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction. Blood. 2000;96(3):941–9. [PubMed]
35. Plas DR, Thompson CB. Akt activation promotes degradation of tuberin and FOXO3a via the proteasome. J Biol Chem. 2003;278(14):12361–6. [PubMed]
36. Guzman ML, Rossi RM, Karnischky L, et al. The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells. Blood. 2005;105(11):4163–9. [PMC free article] [PubMed]
37. Palayoor ST, Youmell MY, Calderwood SK, Coleman CN, Price BD. Constitutive activation of IkappaB kinase alpha and NF-kappaB in prostate cancer cells is inhibited by ibuprofen. Oncogene. 1999;18(51):7389–94. [PubMed]
38. Powis G, Mustacich D, Coon A. The role of the redox protein thioredoxin in cell growth and cancer. Free Radic Biol Med. 2000;29(34):312–22. [PubMed]
39. Nordberg J, Arner ES. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med. 2001;31(11):1287–312. [PubMed]
40. Ahmed KM, Li JJ. NF-kappa B-mediated adaptive resistance to ionizing radiation. Free Radic Biol Med. 2008;44(1):1–13. [PMC free article] [PubMed]
41. Bostwick DG, Meiers I, Shanks JH. Glutathione S-transferase: differential expression of alpha, mu, and pi isoenzymes in benign prostate, prostatic intraepithelial neoplasia, and prostatic adenocarcinoma. Hum Pathol. 2007;38(9):1394–401. [PubMed]
42. Adachi T, Pimentel DR, Heibeck T, et al. S-glutathiolation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells. J Biol Chem. 2004;279(28):29857–62. [PubMed]
43. Heo J, Campbell SL. Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide. J Biol Chem. 2005;280(13):12438–45. [PubMed]
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