Suppression of transcription factor NF-kappaB activity by Bcl-2 protein in NIH3T3 cells: implication of a novel NF-kappaB p50-Bcl-2 complex for the anti-apoptotic function of Bcl-2

Bcl-2 can suppress apoptosis by controlling genes that encode proteins required for programmed cell death and by interference with peroxidative damage. Overexpression of Bcl-2 in NIH3T3 cells can prevent GSNO-induced (S-nitrosoglutathione-induced) apoptosis. The experimental results indicated that activation of NF-kappaB by GSNO is involved in inducing apoptosis. Surprisingly, we found that Bcl-2 delayed the release of IkB by formation of a Bcl-2-NF-kappaB complex (p50-p65-IkappaB) in the cytoplasm during cell apoptosis. Furthermore, a novel Bcl-2-p50 complex was found in the nucleus. These features were only observed in Bcl-2-transfected cells but not in the parental NIH3T3 cells. Overexpression of Bcl-2 suppressed the levels of c-myc, a target gene of NF-kappaB, and influenced the DNA-binding activity of NF-kappaB during GSNOinduced apoptosis. We suggest that the Bcl-2-p50 complex inhibits NF-kappaB DNA-binding activity by competing with the p65-p50 heterodimer for the DNA-binding site in the nucleus. Finally, it has been demonstrated that the anti-apoptotic potential of Bcl-2 may be attributed to its complexing with p50 in the nucleus that leads to blockage of nuclear gene expression.     

Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-kappaB preservation

We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-kappaB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-kappaB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-kappaB which revealed that protein levels of the RelA subunit of NF-kappaB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-kappaB protein levels and activity in the nucleus, indicating that preservation of NF-kappaB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis.     

Blockade of preS2 down-regulates the apoptosis of HepG2.2.15 cells induced by TRAIL

The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Our previous studies showed that both the whole HBV genome and its HBx protein enhanced TRAIL-induced hepatocyte apoptosis. We report here that preS2-containing viral proteins are also potent regulators of TRAIL-induced apoptosis. HBV-transfected hepatoma cell line, HepG2.2.15, pretreated with antisense oligonucleotide against preS2 gene, showed a lower sensitivity towards TRAIL-induced apoptosis. However, this effect might not be related with the expression level of TRAIL receptors. These results establish that besides HBx, preS2 viral proteins are also involved in enhancing TRAIL-induced hepatocyte apoptosis. The novel role of preS2 would be useful to further unravel the mechanisms of imbalanced apoptosis during HBV infection and provides a potential therapeutic target for HBV-related diseases.     

Hypoosmolarity influences the activity of transcription factor NF-kappaB in rat H4IIE hepatoma cells

The influence of anisoosmolarity on NF-kappaB binding activity was studied in H4IIE rat hepatoma cells. Hypoosmolarity induced a sustained NF-kappaB binding activity whereas the hyperosmotic NF-kappaB response was only minor. Hypoosmotic NF-kappaB activation was accompanied by degradation of the inhibitory IkappaB-alpha. Protein kinase C, PI(3)-kinase, reactive oxygen intermediates and the proteasome apparently participate in mediating the hypoosmotic effect on NF-kappaB. Hypoosmolarity plus PMA induced, amplified and prolonged IkappaB-alpha degradation and NF-kappaB binding activity. Transforming growth factor beta-induced apoptosis was diminished by hypoosmolarity. However, this anti-apoptotic effect was probably not related to NF-kappaB activation.     

Lignans from Saururus chinensis inhibiting the transcription factor NF-kappaB

The sesquineolignans, saucerneol D and saucerneol E were isolated from the roots of Saururus chinensis together with four known lignans, manassantin A, manassantin B, (-)-saucerneol methyl ether, and (+)-saucernetin. Structure elucidation was based on the analysis of spectroscopic data and anti-inflammatory activity was studied using HeLa cells transfected with NF-kappaB reporter construct. All compounds except for (+)-saucernetin inhibited NF-kappaB dependent reporter gene expression with IC50 values of 2.5-16.9 microM.     

Plumbagin-induced apoptosis of human breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2

Breast cancer remains the major cause of cancer-related deaths in women world-wide. The heterogeneity of breast cancer has further complicated the progress of target-based therapies. Triple negative breast cancers, lacking estrogen receptor, progesterone receptor and the Her-2/neu (ErbB2), represent a highly aggressive breast cancer subtype, that are difficult to treat. Pleiotropic agents, such as those found in nature, can target receptor-positive as well as receptor-negative cancer cells, suggesting that such agents could have significant impact in breast cancer prevention and/or therapy. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is one such agent which has anti-tumor activity against several cancers. However, its mechanism of action against breast cancer is not clearly understood. We hypothesized that plumbagin may act as an effective agent against breast cancer especially triple negative breast cancer. We tested our hypothesis using ER-positive MCF-7 and ER-negative MDA-MB-231 (triple negative) breast cancer cells, and we found that plumbagin significantly inhibits the growth of breast cancer cells with no effect on normal breast epithelial cells. We also found that plumbagin induces apoptosis with concomitant inactivation of Bcl-2 and the DNA binding activity of NF-kappaB. Bcl-2 over-expression resulted in attenuation of plumbagin-induced effects, suggesting that the inhibition of cell growth and induction of apoptosis by plumbagin is in part due to inactivation of NF-kappaB/Bcl-2 pathway. To our knowledge, this is the first report, showing mechanistic and cancer cell specific apoptosis-inducing effects of plumbagin in breast cancer cells, suggesting the potential role of plumbagin in the prevention and/or treatment of breast cancer.