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. 2008 Dec;47(12):964-73.
doi: 10.1002/mc.20447.

Betulinic acid suppresses constitutive and TNFalpha-induced NF-kappaB activation and induces apoptosis in human prostate carcinoma PC-3 cells

Thangaiyan Rabi  1 , Sanjeev Shukla, Sanjay Gupta
Affiliations

Betulinic acid suppresses constitutive and TNFalpha-induced NF-kappaB activation and induces apoptosis in human prostate carcinoma PC-3 cells

Thangaiyan Rabi et al. Mol Carcinog. 2008 Dec.

Abstract

Development of chemoresistance in androgen-refractory prostate cancer cells is partly due to constitutive activation of Rel/NF-kappaB transcription factors that regulate several cell survival and anti-apoptotic genes. In this study we examined whether betulinic acid (BetA), a pentacyclic triterpene from the bark of white birch, is effective in inhibiting NF-kappaB expression in androgen-refractory human prostate cancer cells exhibiting high constitutive NF-kappaB expression. Treatment of PC-3 cells with BetA inhibited DNA binding and reduced nuclear levels of the NF-kappaB/p65. BetA-mediated NF-kappaB inhibition involved decreased IKK activity and phosphorylation of IkappaBalpha at serine 32/36 followed by its degradation. Reporter assays revealed that NF-kappaB inhibition by BetA is transcriptionally active. These effects were found to correlate with a shift in Bax/Bcl-2 ratio and cleavage of poly(ADP)ribose polymerase more towards apoptosis. BetA also inhibited TNFalpha-induced activation of NF-kappaB via the IkappaBalpha pathway, thereby sensitizing the cells to TNFalpha-induced apoptosis. Our studies demonstrate that BetA effectively inhibits constitutive NF-kappaB activation and supports the rationale for targeting NF-kappaB through combination protocols with BetA in androgen-refractory prostate cancer.

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Figures

Figure 1
Figure 1. Constitutive NF-κB/p65 activation in human prostate cancer LNCaP, DU145 and PC-3 cells
A, immunoblotting for NF-κB/p65, IKKα, IκBa expression and its phosphorylation in whole cell lysates. B, DNA binding by EMSA. C, rate of phosphorylation of IκBα treated with 20 μM MG132 for 6 and 12 h. D, NF-κB DNA binding activity in all three cell lines treated with 20 μM MG132 for 12 h. The blot was stripped and reprobed with anti-tubulin antibody to ensure equal protein loading. EMSA was performed to identify the nuclear translocation of NF-κB/p65 and its binding to DNA. Data shown below the blots represents densitometry of the bands.
Figure 2
Figure 2. Effect of Betulinic acid on cell survival and apoptosis in human prostate carcinoma PC-3 cells
A, structure of Betulinic acid. B, microphotograph of cells treated with 20 μM BetA. C, percentage of cells survived after treatment with BetA for 24, 48 and 72 h with different concentrations as demonstrated by MTT assay. Representative data Mean ± SE, n=8 which was repeated twice with similar results. D, apoptosis in the lysates from PC-3 cells treated with different concentration of BetA for 24 h, values are represented as enrichment factor described in materials and methods section. Data, Mean ± SE, n=3 significantly different from control, *p<0.05; and **p<0.001.
Figure 3
Figure 3. Effect of Betulinic acid on NF-κB signaling pathway in human prostate carcinoma PC-3cells
A, immunoblotting for NF-κB/p65, IκBα, p-IκBα and IKKα in the cytosol and nuclear levels of NF-κB/p65 after treatment of cells with BetA in dose and time dependent manner. The blot was stripped and reprobed with anti-tubulin and anti-Oct-1 antibody to ensure equal protein loading in cytosolic and nuclear fractions. B, NF-κB/p65 DNA binding activity in PC-3 cells treated with 20 μM BetA for 24 h. EMSA was performed to identify nuclear translocation of NF-κB/p65 its binding to DNA. Data shown below the blots represents densitometry of the bands.
Figure 4
Figure 4. Effect of Betulinic acid on Bcl-2 family member proteins and apoptosis in human prostate cancer PC-3 cells
A, immunoblotting for Bax, Bcl-2, and PARP cleavage using lysates from PC-3 cells treated with various concentration of BetA for indicated time periods. The blot was stripped and reprobed with anti-tubulin antibody to ensure equal protein loading. Data shown below the blots represents densitometry of the bands. B, Bax/Bcl-2 ratio after densitometric analysis for proteins.
Figure 5
Figure 5. Effect of Betulinic acid on TNFα-induced NF-κB activation and apoptosis in human prostate cancer PC-3 cells
A, NF-κB/p65 DNA binding activity in PC-3 cells pretreated with 20 μM BetA for 16 h followed by TNFα (10 ng/ml) treatment for 30 min. B, immunoblotting for IκBα and p-IκBα in PC-3 cells after similar treatments. The blot was stripped and reprobed with anti-tubulin antibody to ensure equal protein loading. Data shown below the blots represents densitometry of the bands. C, NF-κB-dependent reporter gene assay after transient transfection of cells with the NF-κB-regulated reporter construct followed by incubation with 20 μM BetA for 16 h, and then stimulation with 10 ng/ml TNFα for 30 min. D, estimation of apoptosis by FACS analysis of annexin V-PI stained cells following similar treatment. Data, Mean ± SE, n=3 significantly different from control, **p<0.001.
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