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Camalexin-induced apoptosis in prostate cancer cells involves alterations of expression and activity of lysosomal protease cathepsin D.

Smith B, Randle D, Mezencev R, Thomas L, Hinton C, Odero-Marah V - Molecules (2014)

Bottom Line: We therefore hypothesized that camalexin reduces cell viability in PCa cells via alterations in expression and activity of CD.Immunocytochemical analysis revealed CD relocalization from the lysosome to the cytosol subsequent to camalexin treatments, which was associated with increased protein expression of mature CD; p53, a transcriptional activator of CD; BAX, a downstream effector of CD, and cleaved PARP, a hallmark for apoptosis.Therefore, camalexin reduces cell viability via CD and may present as a novel therapeutic agent for treatment of metastatic prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research and Therapeutic development, Department of Biological Sciences, Clark Atlanta University, Atlanta, GA 30314, USA.

ABSTRACT
Camalexin, the phytoalexin produced in the model plant Arabidopsis thaliana, possesses antiproliferative and cancer chemopreventive effects. We have demonstrated that the cytostatic/cytotoxic effects of camalexin on several prostate cancer (PCa) cells are due to oxidative stress. Lysosomes are vulnerable organelles to Reactive Oxygen Species (ROS)-induced injuries, with the potential to initiate and or facilitate apoptosis subsequent to release of proteases such as cathepsin D (CD) into the cytosol. We therefore hypothesized that camalexin reduces cell viability in PCa cells via alterations in expression and activity of CD. Cell viability was evaluated by MTS cell proliferation assay in LNCaP and ARCaP Epithelial (E) cells, and their respective aggressive sublines C4-2 and ARCaP Mesenchymal (M) cells, whereby the more aggressive PCa cells (C4-2 and ARCaPM) displayed greater sensitivity to camalexin treatments than the lesser aggressive cells (LNCaP and ARCaPE). Immunocytochemical analysis revealed CD relocalization from the lysosome to the cytosol subsequent to camalexin treatments, which was associated with increased protein expression of mature CD; p53, a transcriptional activator of CD; BAX, a downstream effector of CD, and cleaved PARP, a hallmark for apoptosis. Therefore, camalexin reduces cell viability via CD and may present as a novel therapeutic agent for treatment of metastatic prostate cancer cells.

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Pepstatin A abrogates the effects of camalexin-mediated decrease in cell viability and PARP cleavage. C4-2 cells were treated with 25 and 50 μM camalexin only, or 25 and 50 μM camalexin plus 100 μM Pep A for 3 days and cell viability assayed using the MTS proliferation assay (A). Western blot analysis was done to examine p53, CD and cleaved PARP protein expression in untreated, camalexin-treated and camalexin plus Pep A- treated C4-2 cells, along with the densitometry of protein expression (B). Treatment for 3 days indicated that Pep A could significantly inhibit camalexin-mediated decrease in cell viability and increase in Bax and cleaved PARP protein expression, but does not significantly alter p53 and CD protein expression. Statistical analysis was done using ANOVA and Tukey’s Multiple Comparison as a Post Hoc Test. Values were expressed as mean ± S.E.M normalized to untreated controls, and 25 μM camalexin vs. 25 μM camalexin + 100 μM Pep A (* p < 0.05, ** p < 0.01). (N = 3). β-Actin was used as loading control and data are representative of at least three independent experiments.
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Figure 6: Pepstatin A abrogates the effects of camalexin-mediated decrease in cell viability and PARP cleavage. C4-2 cells were treated with 25 and 50 μM camalexin only, or 25 and 50 μM camalexin plus 100 μM Pep A for 3 days and cell viability assayed using the MTS proliferation assay (A). Western blot analysis was done to examine p53, CD and cleaved PARP protein expression in untreated, camalexin-treated and camalexin plus Pep A- treated C4-2 cells, along with the densitometry of protein expression (B). Treatment for 3 days indicated that Pep A could significantly inhibit camalexin-mediated decrease in cell viability and increase in Bax and cleaved PARP protein expression, but does not significantly alter p53 and CD protein expression. Statistical analysis was done using ANOVA and Tukey’s Multiple Comparison as a Post Hoc Test. Values were expressed as mean ± S.E.M normalized to untreated controls, and 25 μM camalexin vs. 25 μM camalexin + 100 μM Pep A (* p < 0.05, ** p < 0.01). (N = 3). β-Actin was used as loading control and data are representative of at least three independent experiments.

Mentions: We utilized the potent peptide inhibitor of CD activity, pepstatin A (Pep A), to confirm the role of this protease in camalexin-induced apoptosis in prostate cancer cells. We observed after 3 days that camalexin-mediated decrease in cell viability was blocked significantly by co-treatment with 100 μM Pep A plus 25 or 50 μM camalexin in C4-2 cells (Figure 6A). Additionally, treatment of C4-2 cells with Pep A significantly antagonized camalexin-mediated increase in Bax and PARP cleavage protein expression without significantly affecting p53 levels (Figure 6B). Hence, Pep A functions downstream of CD and our studies imply that p53 must be acting upstream of CD since it is not significantly affected by Pep A.


Camalexin-induced apoptosis in prostate cancer cells involves alterations of expression and activity of lysosomal protease cathepsin D.

Smith B, Randle D, Mezencev R, Thomas L, Hinton C, Odero-Marah V - Molecules (2014)

Pepstatin A abrogates the effects of camalexin-mediated decrease in cell viability and PARP cleavage. C4-2 cells were treated with 25 and 50 μM camalexin only, or 25 and 50 μM camalexin plus 100 μM Pep A for 3 days and cell viability assayed using the MTS proliferation assay (A). Western blot analysis was done to examine p53, CD and cleaved PARP protein expression in untreated, camalexin-treated and camalexin plus Pep A- treated C4-2 cells, along with the densitometry of protein expression (B). Treatment for 3 days indicated that Pep A could significantly inhibit camalexin-mediated decrease in cell viability and increase in Bax and cleaved PARP protein expression, but does not significantly alter p53 and CD protein expression. Statistical analysis was done using ANOVA and Tukey’s Multiple Comparison as a Post Hoc Test. Values were expressed as mean ± S.E.M normalized to untreated controls, and 25 μM camalexin vs. 25 μM camalexin + 100 μM Pep A (* p < 0.05, ** p < 0.01). (N = 3). β-Actin was used as loading control and data are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376535&req=5

Figure 6: Pepstatin A abrogates the effects of camalexin-mediated decrease in cell viability and PARP cleavage. C4-2 cells were treated with 25 and 50 μM camalexin only, or 25 and 50 μM camalexin plus 100 μM Pep A for 3 days and cell viability assayed using the MTS proliferation assay (A). Western blot analysis was done to examine p53, CD and cleaved PARP protein expression in untreated, camalexin-treated and camalexin plus Pep A- treated C4-2 cells, along with the densitometry of protein expression (B). Treatment for 3 days indicated that Pep A could significantly inhibit camalexin-mediated decrease in cell viability and increase in Bax and cleaved PARP protein expression, but does not significantly alter p53 and CD protein expression. Statistical analysis was done using ANOVA and Tukey’s Multiple Comparison as a Post Hoc Test. Values were expressed as mean ± S.E.M normalized to untreated controls, and 25 μM camalexin vs. 25 μM camalexin + 100 μM Pep A (* p < 0.05, ** p < 0.01). (N = 3). β-Actin was used as loading control and data are representative of at least three independent experiments.
Mentions: We utilized the potent peptide inhibitor of CD activity, pepstatin A (Pep A), to confirm the role of this protease in camalexin-induced apoptosis in prostate cancer cells. We observed after 3 days that camalexin-mediated decrease in cell viability was blocked significantly by co-treatment with 100 μM Pep A plus 25 or 50 μM camalexin in C4-2 cells (Figure 6A). Additionally, treatment of C4-2 cells with Pep A significantly antagonized camalexin-mediated increase in Bax and PARP cleavage protein expression without significantly affecting p53 levels (Figure 6B). Hence, Pep A functions downstream of CD and our studies imply that p53 must be acting upstream of CD since it is not significantly affected by Pep A.

Bottom Line: We therefore hypothesized that camalexin reduces cell viability in PCa cells via alterations in expression and activity of CD.Immunocytochemical analysis revealed CD relocalization from the lysosome to the cytosol subsequent to camalexin treatments, which was associated with increased protein expression of mature CD; p53, a transcriptional activator of CD; BAX, a downstream effector of CD, and cleaved PARP, a hallmark for apoptosis.Therefore, camalexin reduces cell viability via CD and may present as a novel therapeutic agent for treatment of metastatic prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research and Therapeutic development, Department of Biological Sciences, Clark Atlanta University, Atlanta, GA 30314, USA.

ABSTRACT
Camalexin, the phytoalexin produced in the model plant Arabidopsis thaliana, possesses antiproliferative and cancer chemopreventive effects. We have demonstrated that the cytostatic/cytotoxic effects of camalexin on several prostate cancer (PCa) cells are due to oxidative stress. Lysosomes are vulnerable organelles to Reactive Oxygen Species (ROS)-induced injuries, with the potential to initiate and or facilitate apoptosis subsequent to release of proteases such as cathepsin D (CD) into the cytosol. We therefore hypothesized that camalexin reduces cell viability in PCa cells via alterations in expression and activity of CD. Cell viability was evaluated by MTS cell proliferation assay in LNCaP and ARCaP Epithelial (E) cells, and their respective aggressive sublines C4-2 and ARCaP Mesenchymal (M) cells, whereby the more aggressive PCa cells (C4-2 and ARCaPM) displayed greater sensitivity to camalexin treatments than the lesser aggressive cells (LNCaP and ARCaPE). Immunocytochemical analysis revealed CD relocalization from the lysosome to the cytosol subsequent to camalexin treatments, which was associated with increased protein expression of mature CD; p53, a transcriptional activator of CD; BAX, a downstream effector of CD, and cleaved PARP, a hallmark for apoptosis. Therefore, camalexin reduces cell viability via CD and may present as a novel therapeutic agent for treatment of metastatic prostate cancer cells.

Show MeSH
Related in: MedlinePlus