5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and Prostatic-Neoplasms

We investigated the importance of HMGN5, a nuclear protein that binds to nucleosomes, unfolds chromatin, and affects transcription, in the LNCaP prostate cancer cell line. We also examined the molecular mechanisms that promote apoptosis of LNCaP cells after infection with small interfering RNA (siRNA) targeting HMGN5 (siRNA-HMGN5). The androgen-dependent LNCaP human prostate cancer cells were infected with siRNA-HMGN5. Apoptosis was detected using the Annexin V-PE/7-AAD double staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. Mitochondrial membrane potential was measured by JC-1 staining. HMGN5 and GAPDH mRNA expression were determined using real-time PCR. Bcl-2 and other apoptosis-related protein levels were determined by Western blot analysis. Caspase activity was measured by cleavage of the caspase substrate. Infection with siRNA targeting HMGN5 efficiently and specifically reduced the HMGN5 expression in LNCaP cells. The downregulation of HMGN5 induced remarkable apoptosis of LNCaP cells and resulted in the reduction of mitochondrial membrane potential. The induction of cell apoptosis was accompanied by the upregulation of Bax, the Bax/Bcl-2 ratio and the activation of caspase3. The HMGN5-targeted siRNA was effective in downregulating the expression of HMGN5 in androgen-dependent prostate cancer cells and inducing cell apoptosis via the regulation of a caspase-related mitochondrial pathway and Bcl-2 family proteins. This study suggests that HMGN5 may be a potential molecular target with therapeutic relevance for the treatment of prostate cancer.

Topics: Apoptosis; Benzimidazoles; Carbocyanines; Cell Line, Tumor; Cell Survival; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Therapy; HMGN Proteins; Humans; Male; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Trans-Activators

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1-Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.

Topics: Adenoviridae; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Benzimidazoles; Carbocyanines; Cell Line; Cell Line, Tumor; Cell Membrane Permeability; Cytochromes c; Fluorescent Dyes; HCT116 Cells; Humans; Kidney; Male; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Models, Biological; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Transgenes