RNA interference targeting sensitive-to-apoptosis gene potentiates doxorubicin- and staurosporine-induced apoptosis of PC3 cells.

Several anticancer agents exert their cancer cell killing effects by generating reactive oxygen species (ROS). Thus, a combination of ROS-producing agents and the inhibition of ROS elimination promotes the death of cancer cells. The sensitive to apoptosis gene (SAG) protein, a redox-inducible protein and potential ROS scavenger, protects mammalian cells from redox agent-induced apoptosis. In the present study, we found that silencing of SAG expression in human prostate cancer PC3 cells by transfection with SAG small-interfering RNA (siRNA) markedly enhanced susceptibility to doxorubicin- and to staurosporine-induced apoptotic cell death. Furthermore, pre-treatment with the thiol antioxidant N-acetylcysteine suppressed increases in ROS and apoptosis. This study suggests that knockdown of SAG augments the apoptosis of PC3 cells exposed to doxorubicin or staurosporine presumably by increasing intracellular ROS levels.

Differential pathways for calcium influx activated by concanavalin A and CD3 stimulation in Jurkat T cells.

Sustained increase in [Ca(2+)](c) (Δ[Ca(2+)](c)) is a critical early signal from T-cell receptor (TCR/CD3). In general, Ca(2+)-release activated Ca(2+) channels (CRAC) are responsible for the Ca(2+) influx and Δ[Ca(2+)](c) after TCR/CD3 stimulation. However, T cells also express Ca(2+)-permeable nonselective cation channels such as TRPM2 and TRPC. Gd(3+) is a relatively selective blocker for CRAC at micromolar concentrations. Here, Jurkat T cells were used to investigate the Gd(3+)-resistant Ca(2+) influx (Δ[Ca(2+)](c,Gd)) induced by concanavalin A (ConA, 1 µg/ml), a widely used mitogenic agent for T cells, or by anti-CD3 Ab (αCD3). αCD3-induced Δ[Ca(2+)](c) was partly (~60%) inhibited by 1 µM Gd(3+) while thapsigargin-induced Δ[Ca(2+)] was almost completely abolished. ConA-induced Δ[Ca(2+)] was mostly inhibited by 1 µM Gd(3+) during the early phase (<30 s of ConA application) and became resistant during the late phase (>2 min). Induction of Δ[Ca(2+)](c,Gd) by αCD3 and ConA was inhibited by 2-aminoethoxydiphenyl borate (2-APB) and by N-(p-amylcinnamoyl) anthranilic acid, indicating that TRPM2 and TRPC are involved in this process. Treatment with Pyr-3, a TRPC3-specific inhibitor, potently suppressed Δ[Ca(2+)](c,Gd) by αCD3 (IC(50), 0.16 µM). Patch clamp experiments demonstrated that the TRPM2 channels were activated by ConA, and the TRPC-like channels were activated by αCD3. Our present study suggests that TRPM2 and TRPC3 are activated by ConA and TCR/CD3, respectively, in Jurkat T cells and are responsible for the induction of Δ[Ca(2+)](c,Gd).

Knockdown of sensitive to apoptosis gene by small interfering RNA enhances the sensitivity of PC3 cells toward actinomycin D and etoposide.

Actinomycin D and etoposide induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Sensitive to apoptosis gene (SAG) protein, a redox inducible zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species scavenger. The present report show that knockdown of SAG expression in PC3 cells greatly enhances apoptosis induced by actinomycin D and etoposide. Transfection of human prostate cancer PC3 cells with SAG small interfering RNA (siRNA) markedly decreased the expression of SAG, enhancing the susceptibility of actinomycin D- and etoposide-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that SAG may play an important role in regulating the apoptosis induced by actinomycin D and etoposide and the sensitizing effect of SAG siRNA on the apoptotic cell death of PC3 cells offers the possibility of developing a modifier of cancer chemotherapy.