- Open Access
NF-κB activation enhances cell death by antimitotic drugs in human prostate cancer cells
Molecular Cancer volume 9, Article number: 182 (2010)
NF-κB is a transcription factor that promotes inhibition of apoptosis and resistance to chemotherapy. It is commonly believed that inhibition of NF-κB activity can increase sensitivity of cancer cells to chemotherapy. However, there is evidence that NF-κB activation can sensitize cells to apoptosis and that inhibition of NF-κB results in resistance to chemotherapy. In prostate cancer, it is not clear in the different cell types (androgen-dependent and castration-resistant) if activation or inhibition of NF-κB is required for stimulation of apoptosis by chemotherapy.
Our data indicate that the response of prostate cancer (PC) cells to the antimitotic drugs docetaxel (Doc) and 2-methoxyestradiol (2ME2) is dependent on the levels of NF-κB activity. In androgen-dependent LNCaP cells, Doc and 2ME2 treatment increased the low basal NF-κB activity, as determined by Western blot analysis of phospho-IκBα/p65, NF-κB promoter reporter assays, and p65 localization. Treatment of LNCaP cells with parthenolide, a pharmacologic inhibitor of NF-κB, or introduction of dominant-negative IκBα, or an shRNA specific for p65, a component of the NF-κB heterodimer, blocked apoptosis induced by Doc and 2ME2. In castration-resistant DU145 and PC3 cells, Doc and 2ME2 had little effect on the high basal NF-κB activity and addition of parthenolide did not enhance cell death. However, the combination of Doc or 2ME2 with betulinic acid (BA), a triterpenoid that activates NF-κB, stimulated apoptosis in LNCaP and non-apoptotic cell death in DU145 and PC3 cells. Increased sensitivity to cell death mediated by the Doc or 2ME2 + BA combination is likely due to increased NF-κB activity.
Our data suggest that the combination of antimitotic drugs with NF-κB inhibitors will have antagonistic effects in a common type of PC cell typical of LNCaP. However, combination strategies utilizing antimitotic drugs with BA, an activator of NF-κB, will universally enhance cell death in PC cells, notably in the aggressive, castration-resistant variety that does not respond to conventional therapies.
NF-κB, originally discovered as a transcription factor that regulates the immune system, is now known to be widely expressed in almost all cells and mediates multiple signaling pathways including cell proliferation and survival . A common form of NF-κB is a heterodimer consisting of p65 (RelA) and p50 proteins that exists as an inactive IκBα-bound form in the cytoplasm of unstimulated cells. Activation of the NF-κB pathway by a variety of inducers including cytokines, growth factors, UV light and DNA-damaging drugs often occurs by increasing the phosphorylation of IκBα by the IκB kinase (IKK) complex. This results in ubiquitination and rapid degradation of IκBα by the 26S proteasome, allowing for the increase of nuclear NF-κB DNA binding activity and transcriptional activation of its target genes, including Inhibitor of Apoptosis (IAP) family members XIAP, IAP-1, IAP-2 and anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL . Therefore, NF-κB activation is often associated with increased survival of cancer cells and resistance to chemotherapy. Accordingly, there are several candidate inhibitors of NF-κB activity that are in development as anti-cancer therapy [2–4].
However, the role of NF-κB in cancer progression and in anti-cancer therapeutics is complex, as there is also evidence to suggest that NF-κB activation can sensitize cells to apoptosis [5, 6]. For example, inhibition or loss of NF-κB activity blocks p53-mediated apoptosis, suggesting that inhibition of NF-κB in p53 positive tumors may weaken the therapeutic response . Furthermore, activation of NF-κB by UV light and doxorubicin converts it into an active repressor of the anti-apoptotic genes XIAP and Bcl-xL . Additional evidence supporting a pro-apoptotic role for NF-κB in cancer chemotherapy comes from the observation that the retinoid-related compounds 3-Cl-AHPC and CD437 require activation of NF-κB in order to induce apoptosis in DU145 and PC3 castration-resistant prostate cancer (CRPC) cells [9, 10]. Exposure of CRPC cells to 3-Cl-AHPC or CD437 enhances the expression of the pro-apoptotic Death Receptor (DR) 4 and 5 genes. An NF-κB binding site located in intron 1 of the DR5 promoter is important for positive regulation by NF-κB . Activation of NF-κB by betulinic acid (BA), a naturally occurring pentacyclic triterpenoid small molecule with anticancer properties, is also required for induction of apoptosis in tumor cells .
The clinical progression of prostate cancer (PC) involves the transition from androgen-dependent cancer, which can be successfully treated with androgen-ablation therapy, to a castration-resistant cancer with few treatment options . One of the critical factors in the progression to CRPC is the increased activity of NF-κB and its promotion of apoptotic inhibition [14–16]. It is not clear in the different types of PC cells (androgen-dependent and castration- resistant) whether activation or inhibition of NF-κB is required for stimulation of apoptosis by chemotherapy. Since PC consists of a heterogeneous mixture of cell types, it is important to better understand the mechanisms of the effect of chemotherapy on NF-κB activity in different PC cell lines in order to increase therapeutic response [17, 18].
In this report, we investigate the effects of the antimitotic drugs docetaxel (Doc) and 2-methoxyestradiol (2ME2) on NF-κB activity and induction of cell death in androgen-responsive and castration-resistant PC cell lines. Doc is now one of the most effective anti-cancer drugs and is FDA approved for the treatment of prostate, breast, gastric, head and neck, and non-small cell lung cancers [19, 20]. 2ME2, an endogenous metabolite of estradiol, is being investigated in clinical trials as an anti-cancer agent . Both Doc and 2ME2 have been reported to increase NF-κB activity in tumor cell lines including PC, but whether this stimulates or antagonizes apoptosis appears to be dependent on the specific tumor cell type [22, 23].
Studies have shown constitutive NF-κB DNA binding and transcriptional activity in DU145 and PC3 CRPC cells but not in androgen-dependent LNCaP cells [24–26]. Our data indicated that both Doc and 2ME2 increased NF-κB activity in LNCaP cells and that inhibition of NF-κB was able to block treatment-induced apoptosis. Doc and 2ME2 treatment had little effect on NF-κB activity in DU145 and PC3 cells and the addition of an NF-κB inhibitor did not stimulate cell death in these cells. In contrast, addition of BA increased NF-κB activity and stimulated Doc- and 2ME2-mediated apoptosis in LNCaP and caspase-independent cell death in DU145 and PC3 cells.
Materials and methods
2ME2 was obtained from EntreMed, Inc. and Doc from Aventis Pharmaceuticals. Parthenolide and 4'-6-Diamidino-2-phenylindole (DAPI) were purchased from Calbiochem; BA from Calbiochem, Biomol, or AG Scientific; Trypan blue (0.4%) from Invitrogen; and Coomassie blue from EMD Chemicals, Inc.
Human PC cell lines LNCaP, DU145, and PC3 were obtained from the American Type Culture Collection . LN-AI is a castration-resistant subline of LNCaP, which was spontaneously derived in our laboratory . These cells express androgen receptor (AR) and prostate-specific antigen (PSA), similar to LNCaP. DU145 and PC3 cells do not express AR or PSA. All cells were maintained in RPMI 1640 medium (Invitrogen) with 5% fetal bovine serum (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin (Invitrogen). Media for LN-AI/dnI clones 7, -20, and LN-AI/neo cells also contained 200 μg/ml G418 (Invitrogen).
PC cells were cultured in media containing 2ME2 (5 μM), Doc (1 nM), parthenolide (10 μM), BA (10 μM) or DMSO control for varying times (24-72 h). In all the experiments, adherent and non-adherent cells were pooled for further analysis.
Western Blot Analysis
Preparation of total protein lysates was done as previously described . Preparation of nuclear extracts was done using NE-PER nuclear extraction reagents as per manufacturer's instructions (Pierce Biotechnology). After separation of 25-50 μg protein by SDS-PAGE, proteins were transferred by electrophoresis to Immobilon-P membrane and incubated in 5% nonfat dry milk, TBS, and 0.1% Tween-20 for 1 h. Antibodies specific for phospho-(Ser32/36) IκBα (9246), IκBα (9242), phospho (Ser536)-p65 (3031), cleaved PARP (Asp 214), and XIAP (2042) from Cell Signaling; p65 (C-20), p53 (DO-1), and AIF/N-terminus (E-1) from Santa Cruz Biotechnology; and AIF/C-terminus (A7549) from Sigma-Aldrich were diluted 1/1,000-1/3,000 in 5% nonfat dry milk, TBS, and 0.1% Tween-20 and incubated overnight at 4°C. Membranes were washed in TBS and 0.1% Tween-20 and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1/3,000 dilution; Santa Cruz) for 1 h, washed in TBS and 0.1% Tween-20, and analyzed by exposure to X-ray film using enhanced chemiluminescence plus (ECL plus, GE Healthcare Bio-Sciences Corp). Staining of total protein with Coomassie blue was used as a protein loading control. X-ray films were scanned using an Epson Perfection 2450 Photo scanner.
NF-κB Reporter Assay
To measure NF-κB transcription activity, we used a plasmid containing the luciferase reporter gene regulated by four copies of NF-κB cis-acting elements linked to TATA box from the thymidine kinase promoter (NF-κB-TA/luc; Clontech). TA/Luc is the negative control plasmid without NF-κB elements. Plasmids were co-transfected with CMV/β-galactosidase (Clontech) into LNCaP and PC3 cells using FuGene 6 HD transfection reagent (Roche), as previously described . After 24 h, transfected cells were grown in the presence or absence of 5 μM 2ME2 for 24 and 72 h and the luciferase and β-gal activities determined. The NF-κB luciferase activity relative values as light units/β-gal were divided by TA/luc values and expressed as fold above TA/luc from 3 independent experiments done in duplicate.
Adherent and non-adherent cells were harvested, fixed in formalin for 5 min, applied to slides by smearing, air dried, rinsed with PBS, and blocked with M.O.M. (Vector Laboratories) or goat serum for 20 min. For double immunofluorescence, after first immunostaining for p65 (1:50 dilution; 30 min), we used biotinylated anti-rabbit IgG (1/200 dilution; Vector Laboratories) for 20 min, fluorescein Avidin DCS (1/300 dilution; Vector Laboratories) for 5 min., followed by avidin/biotin blocking for 15 min, immunostaining with nucleolin (H-250; Santa Cruz Biotechnology) for 30 min, biotinylated anti-rabbit IgG, Texas Red DCS, and mounting media with DAPI stain (Vector Laboratories). AIF was immunostained using AIF/N-terminus (1:25 dilution), biotinylated anti-mouse IgG (1/200 dilution; Vector Laboratories), Fluorescein Avidin DCS, and mounting media with propidium iodide (PI) (Vector Laboratories). Color images were acquired using a Nikon Eclipse 90i fluorescence microscope with FITC/Texas Red filters and merged using Adobe Photoshop 7.
DAPI Apoptosis and Trypan Blue Exclusion Assays
The DAPI staining apoptosis assay was done as previously described . Changes in apoptosis in cells treated with drugs were determined as percentage of apoptotic cells (densely stained and fragmented chromatin) from 3-6 independent experiments done in duplicate. Minimal apoptosis was detected in control treated cells (<0.5%). For the trypan exclusion assay, treated and control prostate cancer cells were harvested, resuspended in growth media, diluted 1:1 in 0.4% trypan blue, dead blue and live non-blue cells immediately counted using a hemacytometer, and the % dead blue cells determined from at least 3 independent experiments done in duplicate.
Annexin-FITC/Propidium Iodide (PI) Flow Cytometry
For the annexin apoptosis assay, we used the ApoAlert Annexin V-FITC Apoptosis kit (Clontech). LN-AI and DU145 cells were resuspended in binding buffer followed by the addition of annexin V-FITC and PI. After 20 min., cells were analyzed by flow cytometry using a Coulter XL flow cytometer and the percentage of annexin+ and PI+ cells determined using WinMDI version 2.8.
Stable Transfection of Dominant Negative IκBα
To inhibit endogenous NF-κB activity, we obtained the pCMV-IκBαM plasmid (Clontech) expressing dominant negative IκBα containing Ser to Ala mutations at positions 32 and 36, which cannot be phosphorylated and degraded . LN-AI cells (90% confluent) were co-transfected with pCMV-IκBαM and pCMVneo (for drug selection) using FuGene 6 HD following the manufacturer's instructions. The negative control was transfection with pCMVneo alone. Cells were initially grown in media with 400 μg/ml G418 (Invitrogen), colonies selected, and clones that express dominant negative-IκBα (dnI) compared to pCMVneo negative control cells clones were identified by Western blot (migrates faster than endogenous wild type IκBα).
Lentiviral Transduction of LNCaP and DU145 with shRNA Against p65 and AIF
The shRNA design, lentivirus production and infection were done as previously described . The following DNA oligonucleotides (Operon Technologies) targeting p65 and AIF were cloned into pLKO.1 lentivirus vector: shp65-1: GGCGGATTGAGGAGAAACGTAAACTCG AGTTTACGTTTC TCCTCAATCCGTTTTTG; shp65-2: CCGGCCTGAGGCTATAACTCG CCTACTCGAGTAGGCGA GTTATAGCCTCAGGTTTTTG; shAIF-1: CCGGCCTGGAAA TAGACTCAGATTT CTCGAGAAATCTGAGTCTATTTCCAGGTTTTTG; shAIF-2: CCGG CTGCATGCTTCTACGATATAACTCGAG TTATATCGTAGA AGCATGCAGTTTTTG. The control shRNA was targeted against GFP. LNCaP/shp65-2, LNCaP/shAIF-2, LNCaP/shGFP, DU145/sh p65-1, DU145/shAIF-1, and DU145/shGFP were further analyzed.
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)
RNA was isolated from prostate cancer cells using QIAshredder and RNeasy miniprep kit (Qiagen Inc.). All RNAs were treated with RNase-free DNase (Ambion) to remove possible DNA contamination. The following DNA oligonucleotides (Operon Technologies) were used for RT-qPCR: AIFsh sense 5'-TCATGCCCACTGTCCTGTAAGT-3' and antisense 5'-CCATGG TCCAGTTGCTGAGGT-3' (239 amplicon) ; IκBα sense 5'-CTCCGAGACTTTCGAGG AAATAC-3' and antisense 5'-GCCATTGTAGTTGGTAGCCTTCA-3' (135 amplicon) ; A20 sense 5'-AAGCTGTGAAGATACGGGAGA-3' and antisense 5'-CGATGAGGGCTTT GTGGATGAT-3' (159 amplicon) ; DR5 sense 5'-AAGACCCTTGTGCTCGTTGT-3' and antisense 5'-AGGTGGACACAATCCCTCTG-3' (144 amplicon) ; and the reference gene ribosomal protein, large, P0 (RPL0) sense 5'-GCAATGTTGCCAGTGTCTG-3' and antisense 5'-GCCTTGACCTTTTCAGCAA-3' (141 amplicon) . cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad) and qPCR with Brilliant II Sybr Green QPCR Kit at 30 sec at 95°C, 1 min at 55°C, and 30 sec at 72°C for 40 cycles using a MX3005 qPCR system (Stratagene). Crossing point values from logarithmic amplification profiles for target genes were divided by values from the RPL0 reference gene from RNA samples and presented as fold above control treated cells analyzed five times from three independent experiments. Each product was confirmed for the expected size by agarose gel electrophoresis.
Statistical differences between drug-treated and control PC cells were determined by two-tailed Student's t-test with P < 0.05 considered significant.
To evaluate the effect of the antimitoic drugs 2ME2 and Doc on NF-κB activity in human PC cells, we used androgen-dependent LNCaP and castration-resistant LN-AI, DU145, and PC3 cells. LNCaP and LN-AI cells contain wild-type p53 and exhibit higher sensitivity to 2ME2 and Doc apoptosis relative to DU145 and PC3, which are p53 mutated or null and thus are more resistant to apoptosis [27, 28, 35–37].
Antimitotic Drugs Activate NF-κB in LNCaP Cells
To determine the effect of 2ME2 and Doc on NF-κB, we analyzed phosphorylation of IκBα and of p65 at the serine 536 position, which can enhance the transcriptional activity of NF-κB and lower its affinity to IκBα . Western blot analysis of LNCaP cells treated with 5 μM 2ME2 for 24 h resulted in increased phospho-IκBα (decrease in total IκBα) and phospho-p65 (no change in total p65) relative to control treated cells (Fig. 1A). In DU145 and PC3 cells, which unlike LNCaP cells have constitutively active NF-κB, there were no differences in phospho-IκBα or phospho-p65 in 2ME2-treated cells relative to control cells (Fig. 1A). In addition, total IκBα protein decreased in 2ME2 or Doc-treated LNCaP and LN-AI cells but not in DU145 cells (Fig. 1B).
To determine the effect of 2ME2 on NF-κB transcriptional activity, we used a plasmid containing the luciferase reporter regulated by NF-κB cis-acting elements. The results showed that 2ME2 increased NF-κB activity ~2-fold in LNCaP cells at 24 and 72 h compared to control treated cells (Fig. 1C). In contrast, 2ME2 slightly decreased NF-κB activity 1.5-fold in PC3 cells at 24 h but not at 72 h. The results also showed a 7.3-fold higher basal NF-κB activity in PC3 compared to LNCaP cells.
Nucleolar Localization of p65 in LNCaP cells Treated with Anitmitotic Drugs
We used fluorescence immunocytochemistry to determine if p65 localizes to the nucleus after 2ME2 treatment of LNCaP cells. In control cells, p65 localized in the cytoplasm but after 24 h treatment with 5 μM 2ME2, some cells appeared to have p65 localization to the nucleolus, similar to a previous report . Double fluorescence immunocytochemistry confirmed the presence of several cells with localization of p65 to the nucleolus, as revealed by merged images of the nucleolar marker nucleolin (Texas Red) with p65 (FITC), and DAPI nuclear stain (blue) (Fig. 2). There was also co-localization of p65 and nucleolin in the cytoplasm (yellow), likely due to the disruption of the nucleolus and nuclear membrane by antimitotic drugs. The nucleolus is normally disassembled during mitosis and reassembled after cell division . These results suggest that in LNCaP cells treated with antimitotic drugs, p65 can localize to the nucleolus, which has previously been shown to be important in increasing apoptosis in colon cancer cells treated with aspirin .
NF-κB Inhibition Blocks Apoptosis Induced by Antimitotic Drugs
To determine whether 2ME2- or Doc-mediated activation of NF-κB in LNCaP cells is important for stimulating apoptosis, we used the NF-κB inhibitor parthenolide . Treatment of LNCaP cells with 10 μM parthenolide lowered apoptosis induced by 2ME2 or Doc, as determined by the DAPI apoptosis assay and decreased levels cleaved PARP protein (Fig. 3). Parthenolide lowered the 2ME2- or Doc-mediated increase in phospho-IκBα and phospho-p65, suggesting inhibition of NF-κB activity. These results indicate that 2ME2- or Doc-mediated increase in NF-κB activity is important for induction of apoptosis in LNCaP cells. Similar results were obtained in LN-AI cells (result not shown).
To further investigate molecular changes involved in why inhibition of NF-κB reduced 2ME2- or Doc-mediated apoptosis, we analyzed the expression of p53 and XIAP. p53, the most commonly mutated gene in human cancers, can mediate the apoptosis response to chemotherapy . Overexpression of IAP family members such as XIAP blocks apoptosis and increases drug resistance . Similar to our previous results, 2ME2 and Doc increased p53 and decreased XIAP proteins in LNCaP and LN-AI cells [28, 35]. However, parthenolide blocked the 2ME2 and Doc-induced changes in p53 and XIAP relative to control levels (Fig. 3). These results suggest that the 2ME2- or Doc-mediated increase in NF-κB activity correlates with increased p53 and decreased XIAP, conditions that favor the induction of apoptosis.
Dominant Negative IκBα and p65 Knockdown Inhibit 2ME2 and Doc Apoptosis
To further determine if there is a role for activation of NF-κB in 2ME2- or Doc-mediated apoptosis, we isolated stably transfected LN-AI clones expressing dominant negative IκBα (dnI) . LN-AI/dnI clones 7 and 20 and negative control LN-AI/neo cells were treated with 2ME2 or Doc for 48 h and the effect on apoptosis determined by DAPI and cleaved PARP protein levels. Results showed decreased apoptosis in LN-AI/dnI-7 and 20 compared to LN-AI/neo cells (Fig. 4). As expected, there was an increase in endogenous phospho-IκBα in all cells treated with 2ME2 or Doc for 24 h but phospho-dnI was not detected (faster migrating band in total IκBα blot; Fig. 4B). Similar results were obtained in LNCaP cells stably expressing dnI (result not shown) or shRNA targeting p65 (Fig. 4C). These results further support an important role for NF-κB activation-mediated increase in apoptosis by treatment with 2ME2 or Doc in LN-AI and LNCaP cells.
Betulinic Acid (BA), an NF-κB Activator, Stimulates Cell Death in All Prostate Cancer Cells Treated with Antimitotic Drugs
Our results showed that the NF-κB inhibitor parthenolide antagonized apoptosis mediated by antimitotic drugs in LNCaP and LN-AI cells. In DU145 or PC3 cells, parthenolide had little effect on 2ME2- or Doc-mediated cell death, as shown by the trypan blue exclusion assay (Fig. 5). Thus, we determined whether addition of an NF-κB activator could increase apoptosis by antimitotic drugs in all types of PC cells. BA is a natural pentacyclic triterpenoid proposed to activate NF-κB . Combination of 2ME2 or Doc with 10 μM BA increased apoptosis in LN-AI cells compared to the single drugs, as determined by DAPI, cleaved PARP protein levels, and annexin V-FITC/PI flow cytometry (Figs. 6, 7). BA further increased phospho-IκBα and phospho-p65 and decreased total IκBα compared to 2ME2 or Doc alone in LN-AI and DU145 cells, suggesting enhancement of NF-κB activity (Fig. 6). Similar results were obtained in LNCaP cells (results not shown). In addition, BA increased NF-κB DNA binding in LNCaP and DU145 cells as determined by EMSA (result not shown). In DU145 and PC3 cells, despite increased fragmented and condensed nuclei (DAPI), BA +2ME2 or Doc did not increase cleaved PARP (substrate for active caspase) (Fig. 6 and not shown), suggesting caspase-independent cell death.
Further supporting a role for activation of NF-κB in the 2ME2/Doc + BA combination is the observation that the LN-AI/dnI clones underwent less apoptosis compared to the LN-AI/neo negative control cells (Fig. 8A). Knockdown of p65 in LNCaP and DU145 also lowered cell death and cleaved PARP (Fig. 8B). In addition, pretreatment of DU145 cells with parthenolide (10 μM) for 24 h followed by 2ME2/Doc + BA/parthenolide lowered cell death compared to DU145 cells treated without parthenolide (Fig. 8C). Furthermore, PC cells treated with BA alone or in combination with 2ME2 or Doc decreased IκBα and XIAP protein levels (Fig. 6). These results suggest that enhancement of NF-κB activity by BA plays a role in increasing cell death in PC cells treated with antimitotic drugs.
Because cleaved PARP levels are not increased in DU145 or PC3 cells treated with the 2ME2/Doc + BA combination, we assessed whether there were any differences in total cell death by the trypan blue exclusion assay. Results indicated a greater extent of cell death in the 2ME2/Doc + BA combination compared to 2ME2, Doc, or BA treatment alone (Fig. 8D). This result further suggests that BA increases caspase-independent cell death in DU145 and PC3 cells.
Apoptosis-Inducing Factor (AIF) Increases Cell Death by 2ME2/Doc + BA
To investigate potential downstream effectors of increased cell death mediated by BA, we analyzed the expression of apoptosis-inducing factor (AIF), the first identified protein involved in caspase-independent cell death . Upon cytotoxic insult, AIF is cleaved, released from the mitochondria to the cytoplasm and translocates to the nucleus where it causes caspase-independent DNA fragmentation and cell death [32, 43]. Western blotting indicated that DU145 and LNCaP cells treated with 2ME2/Doc + BA increased nuclear AIF compared to single drug treatment and control (Fig. 9A). Increased nuclear AIF was also detected by immunofluorescent cell staining of DU145 cells treated with 2ME2/Doc + BA and compared to control cells (Fig. 9C).
In addition, we investigated the expression of AIFshort (AIFsh) protein, an alternative transcription start site coding for AIF corresponding only to the C-terminal pro-death portion that translocates to the nucleus to induce cell death . Western blot and RT-qPCR analysis showed that LNCaP cells treated with 2ME2/Doc + BA or BA alone increased AIFsh protein and mRNA (2-5-fold) compared to 2ME2 and Doc alone (Fig. 9B, D). IκBα, A20, and DR5 mRNAs, genes known to be regulated by NF-κB  also increased in LNCaP cells treated with the 2ME2/Doc + BA combination (Fig. 10). In DU145 and PC3 cells, there was also an increase in AIFsh protein after treatment with 2ME2/Doc + BA or BA alone (Fig. 9B).
Finally, we analyzed if shRNA knockdown of AIF has any effect on cell death induced by the 2ME2/Doc + BA combination in LNCaP and DU145 cells. In LNCaP cells, AIF shRNA reduced both total AIF and AIFsh protein and lowered cell death compared to control LNCaP/shGFP cells (Fig. 9E). In DU145 cells, AIF knockdown also lowered cell death and cleaved PARP by the 2ME2/Doc + BA combination (result not shown). We suggest that the NF-κB activator BA increases expression of AIFsh and stimulates caspase-independent cell death in apoptosis resistant PC cells such as DU145.
Treatment of cancer cells with chemotherapeutic drugs often results in substantial heterogeneity in the response to NF-κB activity. In some cases, NF-κB activation by chemotherapeutic drugs elicits a pro-survival cellular response and combination with inhibitors of NF-κB improves efficacy [1, 3, 4]. However, depending on the type of drug or cancer cell, activation of NF-κB can elicit a pro-death response . Our results indicated that improving the cell death response to 2ME2 and Doc in PC cells depends on stimulating rather than inhibiting NF-κB activity. In contrast to what was observed with NF-κB inhibitors, combination of 2ME2 or Doc with BA, an activator of NF-κB, increased cell death in androgen-responsive as well as castration-resistant PC cell lines. Therefore, our data suggests that a chemotherapy combination strategy utilizing antimitotic drugs with BA is likely to be a more universally effective chemotherapeutic strategy for PC.
Our data suggest that the combination of antimitotic drugs with NF-κB inhibitors will have antagonistic effects in a common type of PC cell typical of LNCaP and LN-AI. Support for this observation comes from a report demonstrating that bortezomib, a proteasome inhibitor that lowers NF-κB activity by blocking degradation of IκBα, inhibits Doc-induced apoptosis in LNCaP cells . More importantly, recent clinical trials indicate that patients with CRPC have no added benefit from bortezomib above Doc in one study and some antitumor activity in another study [45, 46]. It is not yet known, however, if more specific inhibitors of NF-κB in combination with antimitotic drugs will have a better therapeutic effect clinically, especially since constitutive NF-κB activity is very prominent in CRPC [14–16].
Our results are similar to a previous report showing that inhibition of NF-κB with dnI or the NF-κB inhibitor BAY 117082 blocks 2ME2-induced apoptosis in LNCaP cells . Others have shown that 2ME2 can inhibit NF-κB in PC3 and medulloblastoma/glioma cell lines and blocking the Doc increase in NF-κB can enhance apoptosis in a variety of cancer cell lines [47–51]. Overall, the heterogeneity implicated in the NF-κB response to anti-cancer drugs is dependent on the specific type of drug and cancer cell.
In LNCaP and LN-AI cells, the requirement of 2ME2 and Doc to activate NF-κB and increase apoptosis may depend upon the p53 tumor suppressor protein . There is evidence suggesting a link between activation of NF-κB and the ability of p53 to induce apoptosis [7, 52]. Our results in LNCaP cells indicated that 2ME2 increased nucleolar localization of p65 (Fig. 2). Nucleolar localization of p65 has been previously reported in colon cancer cells treated with aspirin and a model is proposed that the nucleolus sequesters p65 and inhibits its antiapoptotic functions . Interestingly, the ARF tumor suppressor protein is localized to the nucleolus and after activation by oncogenes can prevent Hdm2 from targeting p53 for degradation and therefore increases the stability of p53 . In addition, ARF can modulate p65 transcriptional activity to repress antiapoptotic genes in a p53 independent manner . One of the effects of antimitotic drugs is the disruption of the nucleolus, the release of ARF, and sequestration of Hdm2, which then leads to the stabilization of p53 and subsequent induction of apoptosis . Our results suggest that investigating the mechanistic basis of p65 nucleolar localization is likely to yield significant insights regarding how to optimize the cytotoxic antitumor action of antimitotic drugs.
It is known that the ability of BA to kill cancer cells occurs by multiple signaling pathways including through activation of NF-κB [12, 56]. One potential mechanism for NF-κB activation and increase in apoptosis by BA is the degradation of IκBα and XIAP (Fig. 6). Activation of selective proteasome-dependent degradation of Sp1, 3, and 4 transcription factors controlling the proangiogenic gene VEGF and the antiapoptosis gene survivin by BA has been recently reported .
The pro-death effects of BA are independent of p53, which is a desirable characteristic for any agent utilized for the treatment of advanced PC, which frequently lacks functional p53 . Our data show that despite a decrease in p53 protein, BA can still increase apoptosis or cell death in all PC cells (Fig. 6). Our results also suggest that BA combined with 2ME2 or Doc increases cell death in a caspase-independent manner (Figs. 6, 7B, 8D). We suggest that one of the factors that allow DU145 cells to overcome the defect in the apoptosis pathway (mutant p53/null Bax) is the increased nuclear translocation AIF/AIFsh to mediate non-apoptotic cell death.
Combination chemotherapy is required to further improve the survival of patients with CRPC. A prevailing strategy has been to inhibit the NF-κB response in order to block its pro-survival effect and improve drug efficacy. In this study, we demonstrated that in PC cells such as LNCaP and LN-AI, activation of NF-κB by the antimitotic agents 2ME2 or Doc is important for increasing apoptosis. In addition, when 2ME2 or Doc is combined with an NF-κB activator such as BA, there is effective induction of cell death in all the PC cells analyzed. We are currently investigating whether other NF-κB activators will also mediate increased cell death by antimitotic drugs. The combination of antimitotic agents with NF-κB activators may promote the pro-death responses in a greater variety of PC cells, a requirement for increased therapeutic efficacy.
castration-resistant prostate cancer
dominant negative IκBα
green fluorescent protein
real time quantitative polymerase chain reaction
electrophoretic mobility shift assay.
Hayden MS, Ghosh S: Shared principles in NF-kappaB signaling. Cell. 2008, 132: 344-62. 10.1016/j.cell.2008.01.020
Voorhees PM, Dees EC, O'Neil B, Orlowski RZ: The proteasome as a target for cancer therapy. Clin Cancer Res. 2003, 9: 6316-25.
Verma IM: Nuclear factor (NF)-kappa B proteins: therapeutic targets. Ann Rheum Dis. 2004, 63 (Suppl 2): ii57-ii61. 10.1136/ard.2004.028266
Kim HJ, Hawke N, Baldwin AS: NF-kappaB and IKK as therapeutic targets in cancer. Cell Death Differ. 2006, 13: 738-47. 10.1038/sj.cdd.4401877
Baud V, Karin M: Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov. 2009, 8: 33-40. 10.1038/nrd2781
Perkins ND, Gilmore TD: Good cop, bad cop, the different faces of NF-kappaB. Cell Death Differ. 2006, 13: 759-72. 10.1038/sj.cdd.4401838
Ryan KM, Ernst MK, Rice NR, Vousden KH: Role of NF-kappaB in p53-mediated programmed cell death. Nature. 2000, 404: 892-7. 10.1038/35009130
Campbell KJ, Rocha S, Perkins ND: Active repression of antiapoptotic gene expression by RelA(p65) NF-kappa B. Mol Cell. 2004, 13: 853-65. 10.1016/S1097-2765(04)00131-5
Farhana L, Dawson MI, Fontana JA: Apoptosis induction by a novel retinoid-related molecule requires nuclear factor-kappaB activation. Cancer Res. 2005, 65: 4909-17. 10.1158/0008-5472.CAN-04-4124
Jin F, Liu X, Zhou Z, Yue P, Lotan R, Khuri FR, Chung LW, Sun SY: Activation of nuclear factor-kappaB contributes to induction of death receptors and apoptosis by the synthetic retinoid CD437 in DU145 human prostate cancer cells. Cancer Res. 2005, 65: 6354-63. 10.1158/0008-5472.CAN-04-4061
Shetty S, Graham BA, Brown JG, Hu X, Vegh-Yarema N, Harding G, Paul JT, Gibson SB: Transcription factor NF-kappaB differentially regulates death receptor 5 expression involving histone deacetylase 1. Mol Cell Biol. 2005, 25: 5404-16. 10.1128/MCB.25.13.5404-5416.2005
Kasperczyk H, La Ferla-Bruhl K, Westhoff MA, Behrend L, Zwacka RM, Debatin KM, Fulda S: Betulinic acid as new activator of NF-kappaB: molecular mechanisms and implications for cancer therapy. Oncogene. 2005, 24: 6945-56. 10.1038/sj.onc.1208842
Hadaschik BA, Gleave ME: Therapeutic options for hormone-refractory prostate cancer in 2007. Urol Oncol. 2007, 25: 413-9.
Ross JS, Kallakury BV, Sheehan CE, Fisher HA, Kaufman RP, Kaur P, Gray K, Stringer B: Expression of nuclear factor-kappa B and I kappa B alpha proteins in prostatic adenocarcinomas: correlation of nuclear factor kappa-B immunoreactivity with disease recurrence. Clin Cancer Res. 2004, 10: 2466-72. 10.1158/1078-0432.CCR-0543-3
Sweeney C, Li L, Shanmugam R, Bhat-Nakshatri P, Jayaprakasan V, Baldridge LA, Gardner T, Smith M, Nakshatri H, Cheng L: Nuclear factor-kappaB is constitutively activated in prostate cancer in vitro and is overexpressed in prostatic intraepithelial neoplasia and adenocarcinoma of the prostate. Clin Cancer Res. 2004, 10: 5501-7. 10.1158/1078-0432.CCR-0571-03
Jin RJ, Lho Y, Connelly L, Wang Y, Yu X, Saint Jean L, Case TC, Ellwood-Yen K, Sawyers CL, Bhowmick NA, Blackwell TS, Yull FE, Matusik RJ: The nuclear factor-kappaB pathway controls the progression of prostate cancer to androgen-independent growth. Cancer Res. 2008, 68: 6762-9. 10.1158/0008-5472.CAN-08-0107
Roudier MP, True LD, Higano CS, Vesselle H, Ellis W, Lange P, Vessella RL: Phenotypic heterogeneity of end-stage prostate carcinoma metastatic to bone. Hum Pathol. 2003, 34: 646-53. 10.1016/S0046-8177(03)00190-4
Shah RB, Mehra R, Chinnaiyan AM, Shen R, Ghosh D, Zhou M, Macvicar GR, Varambally S, Harwood J, Bismar TA, Kim R, Rubin MA, Pienta KJ: Androgen-independent prostate cancer is a heterogeneous group of diseases: lessons from a rapid autopsy program. Cancer Res. 2004, 64: 9209-16. 10.1158/0008-5472.CAN-04-2442
Petrylak DP: The treatment of hormone-refractory prostate cancer: docetaxel and beyond. Rev Urol. 2006, 8 (Suppl 2): S48-S55.
Docetaxel information, National Cancer Institute. http://www.cancer.gov/cancertopics/druginfo/docetaxel
Sutherland TE, Anderson RL, Hughes RA, Altmann E, Schuliga M, Ziogas J, Stewart AG: 2-Methoxyestradiol--a unique blend of activities generating a new class of anti-tumour/anti-inflammatory agents. Drug Discov Today. 2007, 12: 577-84. 10.1016/j.drudis.2007.05.005
Zhang H, Morisaki T, Nakahara C, Matsunaga H, Sato N, Nagumo F, Tadano J, Katano M: PSK-mediated NF-kappaB inhibition augments docetaxel-induced apoptosis in human pancreatic cancer cells NOR-P1. Oncogene. 2003, 22: 2088-96. 10.1038/sj.onc.1206310
Shimada K, Nakamura M, Ishida E, Kishi M, Konishi N: Roles of p38- and c-jun NH2-terminal-kinase mediated pathways in 2-methoxyestradiol-induced p53 induction and apoptosis. Carcinogenesis. 2003, 24: 1067-75. 10.1093/carcin/bgg058
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, 8: 7389-94. 10.1038/sj.onc.1203160.
Gasparian AV, Yao YJ, Kowalczyk D, Lyakh LA, Karseladze A, Slaga TJ, Budunova IV: The role of IKK in constitutive activation of NF-kappaB transcription factor in prostate carcinoma cells. J Cell Sci. 2002, 115: 141-51.
Suh J, Payvandi F, Edelstein LC, Amenta PS, Zong WX, Gélinas C, Rabson AB: Mechanisms of constitutive NF-kappaB activation in human prostate cancer cells. Prostate. 2002, 52: 183-200. 10.1002/pros.10082
van Bokhoven A, Varella-Garcia M, Korch C, Johannes WU, Smith EE, Miller HL, Nordeen SK, Miller GJ, Lucia MS: Molecular characterization of human prostate carcinoma cell lines. Prostate. 2003, 57: 205-25. 10.1002/pros.10290
Gomez LA, de las Pozas A, Perez-Stable C: Sequential combination of flavopiridol and docetaxel reduces the levels of XIAP and AKT proteins and stimulates apoptosis in human LNCaP prostate cancer cells. Mol Cancer Ther. 2006, 5: 1216-26. 10.1158/1535-7163.MCT-05-0467
Reiner T, de las Pozas A, Parrondo R, Perez-Stable C: Progression of prostate cancer from a subset of p63 positive basal epithelial cells in FG/Tag transgenic mice. Mol Cancer Res. 2007, 5: 1171-79. 10.1158/1541-7786.MCR-07-0024
Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U: Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science. 1995, 267: 1485-8. 10.1126/science.7878466
Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, Sabatini DM, Chen IS, Hahn WC, Sharp PA, Weinberg RA, Novina CD: Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 2003, 9: 493-501. 10.1261/rna.2192803
Delettre C, Yuste VJ, Moubarak RS, Bras M, Lesbordes-Brion JC, Petres S, Bellalou J, Susin SA: AIFsh, a novel apoptosis-inducing factor (AIF) pro-apoptotic isoform with potential pathological relevance in human cancer. J Biol Chem. 2006, 281: 6413-27. 10.1074/jbc.M509884200
Wang X, Seed B: A PCR primer bank for quantitative gene expression analysis. Nucl Acids Res. 2003, 31: 1-8. e154, 10.1093/nar/gkg120
Dydensborg AB, Herring E, Auclair J, Tremblay E, Beaulieu JF: Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon. Am J Physiol Gastrointest Liver Physiol. 2006, 290: G1067-74. 10.1152/ajpgi.00234.2005
Perez-Stable CM: 2-Methoxyestradiol and paclitaxel have similar effects on the cell cycle and induction of apoptosis in prostate cancer cells. Cancer Lett. 2006, 231: 49-64. 10.1016/j.canlet.2005.01.018
Gomez LA, de las Pozas A, Reiner T, Burnstein K, Perez-Stable C: Increased expression of cyclin B1 sensitizes prostate cancer cells to apoptosis induced by chemotherapy. Mol Cancer Ther. 2007, 6: 1534-1543. 10.1158/1535-7163.MCT-06-0727
Reiner T, de las Pozas A, Gomez LA, Perez-Stable C: Low dose combination of 2-methoxyestradiol and docetaxel can block prostate cancer cells in mitosis and induce apoptosis. Cancer Lett. 2009, 276: 21-31. 10.1016/j.canlet.2008.10.026
Stark LA, Dunlop MG: Nucleolar sequestration of RelA (p65) regulates NF-kappaB-driven transcription and apoptosis. Mol Cell Biol. 2005, 25: 5985-6004. 10.1128/MCB.25.14.5985-6004.2005
Boisvert FM, van Koningsbruggen S, Navascués J, Lamond AI: The multifunctional nucleolus. Nat Rev Mol Cell Biol. 2007, 8: 574-85. 10.1038/nrm2184
Salminen A, Lehtonen M, Suuronen T, Kaarniranta K, Huuskonen J: Terpenoids: natural inhibitors of NF-kappaB signaling with anti-inflammatory and anticancer potential. Cell Mol Life Sci. 2008, 65: 2979-99. 10.1007/s00018-008-8103-5
Vassilev LT: p53 Activation by small molecules: application in oncology. J Med Chem. 2005, 48: 4491-9. 10.1021/jm058174k
Wright CW, Duckett CS: Reawakening the cellular death program in neoplasia through the therapeutic blockade of IAP function. J Clin Invest. 2005, 115: 2673-8. 10.1172/JCI26251
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G: Molecular characterization of mitochondrial apoptosis-inducing factor. Nature. 1999, 397: 441-6. 10.1038/17135
Canfield SE, Zhu K, Williams SA, McConkey DJ: Bortezomib inhibits docetaxel-induced apoptosis via a p21-dependent mechanism in human prostate cancer cells. Mol Cancer Ther. 2006, 5: 2043-50. 10.1158/1535-7163.MCT-05-0437
Hainsworth JD, Meluch AA, Spigel DR, Barton J, Simons L, Meng C, Gould B, Greco FA: Weekly docetaxel and bortezomib as first-line treatment for patients with hormone-refractory prostate cancer: a Minnie Pearl Cancer Research Network phase II trial. Clin Genitourin Cancer. 2007, 5: 278-83. 10.3816/CGC.2007.n.004
Dreicer R, Petrylak D, Agus D, Webb I, Roth B: Phase I/II study of bortezomib plus docetaxel in patients with advanced androgen-independent prostate cancer. Clin Cancer Res. 2007, 13: 1208-15. 10.1158/1078-0432.CCR-06-2046
Shimada K, Nakamura M, Ishida E, Kishi M, Matsuyoshi S, Konishi N: The molecular mechanism of sensitization to Fas-mediated apoptosis by 2-methoxyestradiol in PC3 prostate cancer cells. Mol Carcinog. 2004, 39: 1-9. 10.1002/mc.10158
Kumar AP, Garcia GE, Orsborn J, Levin VA, Slaga TJ: 2-Methoxyestradiol interferes with NF kappa B transcriptional activity in primitive neuroectodermal brain tumors: implications for management. Carcinogenesis. 2003, 24: 209-16. 10.1093/carcin/24.2.209
Nakahara C, Nakamura K, Yamanaka N, Baba E, Wada M, Matsunaga H, Noshiro H, Tanaka M, Morisaki T, Katano M: Cyclosporin-A enhances docetaxel-induced apoptosis through inhibition of nuclear factor-kappaB activation in human gastric carcinoma cells. Clin Cancer Res. 2003, 9: 5409-16.
Li Y, Ahmed F, Ali S, Philip PA, Kucuk O, Sarkar FH: Inactivation of nuclear factor kappaB by soy isoflavone genistein contributes to increased apoptosis induced by chemotherapeutic agents in human cancer cells. Cancer Res. 2005, 65: 6934-42. 10.1158/0008-5472.CAN-04-4604
Shanmugam R, Jayaprakasan V, Gokmen-Polar Y, Kelich S, Miller KD, Yip-Schneider M, Cheng L, Bhat-Nakshatri P, Sledge GW, Nakshatri H, Zheng QH, Miller MA, DeGrado T, Hutchins GD, Sweeney CJ: Restoring chemotherapy and hormone therapy sensitivity by parthenolide in a xenograft hormone refractory prostate cancer model. Prostate. 2006, 66: 1498-511. 10.1002/pros.20482
Fujioka S, Schmidt C, Sclabas GM, Li Z, Pelicano H, Peng B, Yao A, Niu J, Zhang W, Evans DB, Abbruzzese JL, Huang P, Chiao PJ: Stabilization of p53 is a novel mechanism for proapoptotic function of NF-kappaB. J Biol Chem. 2004, 279: 27549-59. 10.1074/jbc.M313435200
Weber JD, Taylor LJ, Roussel MF, Sherr CJ, Bar-Sagi D: Nucleolar Arf sequesters Mdm2 and activates p53. Nat Cell Biol. 1999, 1: 20-6. 10.1038/8991
Rocha S, Campbell KJ, Perkins ND: p53- and Mdm2-independent repression of NF-kappa B transactivation by the ARF tumor suppressor. Mol Cell. 2003, 12: 15-25. 10.1016/S1097-2765(03)00223-5
Rubbi CP, Milner J: Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses. EMBO J. 2003, 22: 6068-77. 10.1093/emboj/cdg579
Fulda S, Kroemer G: Targeting mitochondrial apoptosis by betulinic acid in human cancers. Drug Disc Today. 2009, 14: 885-890. 10.1016/j.drudis.2009.05.015.
Chintharlapalli S, Papineni S, Ramaiah SK, Safe S: Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res. 2007, 67: 2816-23. 10.1158/0008-5472.CAN-06-3735
Fulda S, Friesen C, Los M, Scaffidi C, Mier W, Benedict M, Nuñez G, Krammer PH, Peter ME, Debatin KM: Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res. 1997, 57: 4956-64.
This work was supported by Veterans Affairs Merit Review 086906 (CPS) and a University of Miami/Sylvester Cancer Center Papanicolaou Corps Developmental Cancer Research Grant (PR). We thank Dr. Ramiro Verdun for critical reading of the manuscript and helpful suggestions; Irving Vidaurre and Dr. George McNamara for imaging assistance; Kevin Curtis for assistance with RT-qPCR; Asmita Patel for assistance with plasmid preparation; Ron Hamelik for assistance with flow cytometry; and Drs. Bernard Roos and Guy Howard for support.
The authors declare that they have no competing interests.
RP and AP carried out all of the Western blots. TR carried out the immunofluorescence experiments. CPS carried out the cell culture, drug treatments, the transfection/luciferse assays, the DAPI/trypan blue/annexin-PI assays, isolation of LN-AI/dnI clones, and lentiviral transduction of shRNA. AP carried out and analyzed all of the RT-qPCR data. RP, AP, TR, PR, and CPS participated in the design of the experiments and critical review of the manuscript. CPS conceived of the study, coordinated and supervised the project, and wrote and edited the manuscript. All the authors read and approved the final manuscript.
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Parrondo, R., Pozas, A.d.l., Reiner, T. et al. NF-κB activation enhances cell death by antimitotic drugs in human prostate cancer cells. Mol Cancer 9, 182 (2010). https://doi.org/10.1186/1476-4598-9-182
- Prostate Cancer Cell
- LNCaP Cell
- DU145 Cell
- Betulinic Acid