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NF-kappaB activation enhances cell death by antimitotic drugs in human prostate cancer cells.

Parrondo R, de las Pozas A, Reiner T, Rai P, Perez-Stable C - Mol. Cancer (2010)

Bottom Line: In castration-resistant DU145 and PC3 cells, Doc and 2ME2 had little effect on the high basal NF-kappaB activity and addition of parthenolide did not enhance cell death.Our data suggest that the combination of antimitotic drugs with NF-kappaB 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-kappaB, will universally enhance cell death in PC cells, notably in the aggressive, castration-resistant variety that does not respond to conventional therapies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Geriatric Research, Education, and Clinical Center and Research Service, Bruce W, Carter Veterans Affairs Medical Center, Miami, FL 33125, USA.

ABSTRACT

Background: NF-kappaB is a transcription factor that promotes inhibition of apoptosis and resistance to chemotherapy. It is commonly believed that inhibition of NF-kappaB activity can increase sensitivity of cancer cells to chemotherapy. However, there is evidence that NF-kappaB activation can sensitize cells to apoptosis and that inhibition of NF-kappaB 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-kappaB is required for stimulation of apoptosis by chemotherapy.

Results: 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-kappaB activity. In androgen-dependent LNCaP cells, Doc and 2ME2 treatment increased the low basal NF-kappaB activity, as determined by Western blot analysis of phospho-IkappaBalpha/p65, NF-kappaB promoter reporter assays, and p65 localization. Treatment of LNCaP cells with parthenolide, a pharmacologic inhibitor of NF-kappaB, or introduction of dominant-negative IkappaBalpha, or an shRNA specific for p65, a component of the NF-kappaB 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-kappaB 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-kappaB, 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-kappaB activity.

Conclusions: Our data suggest that the combination of antimitotic drugs with NF-kappaB 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-kappaB, will universally enhance cell death in PC cells, notably in the aggressive, castration-resistant variety that does not respond to conventional therapies.

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NF-κB activator BA stimulates 2ME2 and Doc-mediated apoptosis in LN-AI and DU145 cells. DAPI apoptosis assay (% condensed fragmented nuclei) showing that BA (B; 10 μM) increases 2ME2 (M; 5 μM) and Doc (D; 1 nM) apoptosis (MB and DB) in LN-AI (24 h) and DU145 (72 h) compared to M, B, and control cells. n = 6-8, three to four independent experiments; *, P < 0.004. Western blot analysis shows greater cleaved PARP in MB and DB compared to M, D, or B alone in LN-AI but not in DU145 cells. MB and DB increases P-IκBα/IκBα and P-p65/p65 in LN-AI and DU145 cells at 24 h compared to M, D, B, and control cells, suggesting activation of NF-κB. MB and DB decreases IκBα, p53 and XIAP compared to M and D alone; B alone also decrease IκBα, p53, and XIAP compared to control cells. Coomassie blue stain of total protein is loading control.
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Figure 6: NF-κB activator BA stimulates 2ME2 and Doc-mediated apoptosis in LN-AI and DU145 cells. DAPI apoptosis assay (% condensed fragmented nuclei) showing that BA (B; 10 μM) increases 2ME2 (M; 5 μM) and Doc (D; 1 nM) apoptosis (MB and DB) in LN-AI (24 h) and DU145 (72 h) compared to M, B, and control cells. n = 6-8, three to four independent experiments; *, P < 0.004. Western blot analysis shows greater cleaved PARP in MB and DB compared to M, D, or B alone in LN-AI but not in DU145 cells. MB and DB increases P-IκBα/IκBα and P-p65/p65 in LN-AI and DU145 cells at 24 h compared to M, D, B, and control cells, suggesting activation of NF-κB. MB and DB decreases IκBα, p53 and XIAP compared to M and D alone; B alone also decrease IκBα, p53, and XIAP compared to control cells. Coomassie blue stain of total protein is loading control.

Mentions: 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 [12]. 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.


NF-kappaB activation enhances cell death by antimitotic drugs in human prostate cancer cells.

Parrondo R, de las Pozas A, Reiner T, Rai P, Perez-Stable C - Mol. Cancer (2010)

NF-κB activator BA stimulates 2ME2 and Doc-mediated apoptosis in LN-AI and DU145 cells. DAPI apoptosis assay (% condensed fragmented nuclei) showing that BA (B; 10 μM) increases 2ME2 (M; 5 μM) and Doc (D; 1 nM) apoptosis (MB and DB) in LN-AI (24 h) and DU145 (72 h) compared to M, B, and control cells. n = 6-8, three to four independent experiments; *, P < 0.004. Western blot analysis shows greater cleaved PARP in MB and DB compared to M, D, or B alone in LN-AI but not in DU145 cells. MB and DB increases P-IκBα/IκBα and P-p65/p65 in LN-AI and DU145 cells at 24 h compared to M, D, B, and control cells, suggesting activation of NF-κB. MB and DB decreases IκBα, p53 and XIAP compared to M and D alone; B alone also decrease IκBα, p53, and XIAP compared to control cells. Coomassie blue stain of total protein is loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: NF-κB activator BA stimulates 2ME2 and Doc-mediated apoptosis in LN-AI and DU145 cells. DAPI apoptosis assay (% condensed fragmented nuclei) showing that BA (B; 10 μM) increases 2ME2 (M; 5 μM) and Doc (D; 1 nM) apoptosis (MB and DB) in LN-AI (24 h) and DU145 (72 h) compared to M, B, and control cells. n = 6-8, three to four independent experiments; *, P < 0.004. Western blot analysis shows greater cleaved PARP in MB and DB compared to M, D, or B alone in LN-AI but not in DU145 cells. MB and DB increases P-IκBα/IκBα and P-p65/p65 in LN-AI and DU145 cells at 24 h compared to M, D, B, and control cells, suggesting activation of NF-κB. MB and DB decreases IκBα, p53 and XIAP compared to M and D alone; B alone also decrease IκBα, p53, and XIAP compared to control cells. Coomassie blue stain of total protein is loading control.
Mentions: 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 [12]. 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.

Bottom Line: In castration-resistant DU145 and PC3 cells, Doc and 2ME2 had little effect on the high basal NF-kappaB activity and addition of parthenolide did not enhance cell death.Our data suggest that the combination of antimitotic drugs with NF-kappaB 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-kappaB, will universally enhance cell death in PC cells, notably in the aggressive, castration-resistant variety that does not respond to conventional therapies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Geriatric Research, Education, and Clinical Center and Research Service, Bruce W, Carter Veterans Affairs Medical Center, Miami, FL 33125, USA.

ABSTRACT

Background: NF-kappaB is a transcription factor that promotes inhibition of apoptosis and resistance to chemotherapy. It is commonly believed that inhibition of NF-kappaB activity can increase sensitivity of cancer cells to chemotherapy. However, there is evidence that NF-kappaB activation can sensitize cells to apoptosis and that inhibition of NF-kappaB 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-kappaB is required for stimulation of apoptosis by chemotherapy.

Results: 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-kappaB activity. In androgen-dependent LNCaP cells, Doc and 2ME2 treatment increased the low basal NF-kappaB activity, as determined by Western blot analysis of phospho-IkappaBalpha/p65, NF-kappaB promoter reporter assays, and p65 localization. Treatment of LNCaP cells with parthenolide, a pharmacologic inhibitor of NF-kappaB, or introduction of dominant-negative IkappaBalpha, or an shRNA specific for p65, a component of the NF-kappaB 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-kappaB 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-kappaB, 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-kappaB activity.

Conclusions: Our data suggest that the combination of antimitotic drugs with NF-kappaB 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-kappaB, will universally enhance cell death in PC cells, notably in the aggressive, castration-resistant variety that does not respond to conventional therapies.

Show MeSH
Related in: MedlinePlus