Cyclizine induces cytotoxicity and apoptosis in macrophages through the extrinsic and intrinsic apoptotic pathways.

Cyclizine, an over-the-counter and prescription antihistamine, finds widespread application in the prevention and treatment of motion sickness, encompassing symptoms such as nausea, vomiting, dizziness, along with its effectiveness in managing vertigo. However, the overuse or misuse of cyclizine may lead to hallucinations, confusion, tachycardia, and hypertension. The molecular mechanisms underlying cyclizine-induced cytotoxicity and apoptosis remain unclear. During the 24 h incubation duration, RAW264.7 macrophages were exposed to different concentrations of cyclizine. Cytotoxicity was assessed through the lactate dehydrogenase assay. Flow cytometry employing annexin V-fluorescein isothiocyanate and propidium iodide was utilized to evaluate apoptosis and necrosis. Caspase activity and mitochondrial dysfunction were evaluated through a fluorogenic substrate assay and JC-1 dye, respectively. Flow cytometry employing fluorogenic antibodies was utilized to evaluate the release of cytochrome c and expression of death receptor, including tumor necrosis factor-α receptor and Fas receptor. Western blotting was utilized to evaluate the expression of the Bcl2 and Bad apoptotic regulatory proteins. The findings unveiled from the present study demonstrated that cyclizine exerted a concentration-dependent effect on RAW264.7 macrophages, leading to the induction of cytotoxicity, apoptosis, and necrosis. This compound further activated the intrinsic apoptotic pathway by inducing mitochondrial dysfunction, Bcl2/Bad exchange expression, cytochrome c liberation, and activation of caspases contained caspase 3, 8, and 9. Moreover, the activation of the extrinsic apoptotic pathway was observed as cyclizine induced the upregulation of death receptors and increased caspase activities. Based on our investigations, it can be inferred that cyclizine prompts cytotoxicity and apoptosis in RAW264.7 macrophages in a concentration-dependent manner by triggering both the intrinsic and extrinsic apoptotic pathways.

Effects of 4-nonylphenol on spermatogenesis and induction of testicular apoptosis through oxidative stress-related pathways.

This study tested the hypothesis that prepubertal exposure to 4-nonylphenol (NP) affects reproductive function in male rats. Twenty-four rats at five-weeks-old were randomly divided into four groups and treated with NP at varying concentrations (0, 5, 20, and 60mg/kg/2d) for thirty days by intra-peritoneal injection. 60mg/kg NP induced spermatogenic degeneration and pronounced deficits in epididymal sperm count, motility and function, whereas potentially stimulatory effects were observed at 5 NPmg/kg. Moreover, 60mg/kg NP resulted in a significant reduction in fructose, FSH and LH; induced apoptosis related to oxidative stress; inhibited mRNA and protein levels of Bcl-2 and PCNA; as well as the additional up-regulation of p53, Bax, Apaf-1, cytochrome c, cleaved-caspase-3, Fas and FasL expression. Our data suggest potentially hormetic effects of NP on spermatogenic function. High-dose NP impairs testicular development and function by reducing cell proliferation and inducing apoptosis involving oxidative stress-related p53-Bcl-2/Bax and -Fas/FasL pathways.

Andrographolide sensitizes the cytotoxicity of human colorectal carcinoma cells toward cisplatin via enhancing apoptosis pathways in vitro and in vivo.

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, has been shown to suppress the growth and invasion of human colorectal carcinoma (CRC) Lovo cells, and trigger apoptosis in vitro. The potential of Andro as a chemotherapeutic agent in CRC was evaluated by investigating its cytotoxic effects as a single agent or in coadministration with cisplatin (CDDP). Andro potentiated the cytotoxic effect of CDDP in Lovo cells through apoptosis. The molecular mechanism for these favorable cellular response was further investigated by analyzing the apoptotic profiles, protein levels, and mRNA expression patterns of several key genes after treatments of Andro or/and CDDP. Molecular results indicated that the effect of Andro alone might be mediated via both intrinsic and extrinsic apoptotic pathways in Lovo cells. The addition of Andro to CDDP induced synergistic apoptosis, which could be corroborated to the changes in protein and mRNA levels of Bax and Bcl-2, and the increased Fas/FasL association in these cells, resulting in increased release of cytochrome c, and activation of caspases. Pretreatment of Nok-1 monoclonal antibody, a Fas signaling inhibitor, or Bax inhibitor peptide V5 repressed the Andro-induced cleavage of procaspase and the sensitization to CDDP-induced apoptosis. Finally, the combination therapy of Andro with CDDP was evidenced by its synergistic inhibition on the growth of Lovo cells in xenograft tumor studies. The results indicate that Andro, in combination with chemotherapeutics, is likely to represent a potential therapeutic strategy for CRC.

Cadmium (Cd(2+)) exposure differentially elicits both cell proliferation and cell death related responses in SK-RC-45.

Cadmium (Cd(2+)) is a major nephrotoxic environmental pollutant, affecting mostly proximal convoluted tubule (PCT) cells of the mammalian kidney, while conditionally Cd(2+) could also elicit protective responses with great variety and variability in different systems. The present study was designed to evaluate the molecular mechanism of Cd(2+) toxicity on human PCT derived Renal Cell Carcinoma (RCC), SK-RC-45 and compare its responses with normal human PCT derived cell line, NKE. Exposure of SK-RC-45 cells with different concentrations of CdCl2 (e.g. 0, 10 and 20µM) in serum free medium for 24h generate considerable amount of ROS, accompanied with decreased cell viability and alternations in the cellular and nuclear morphologies, heat shock responses and GCLC mediated protective responses. Also phosphatidylserine externalization, augmentation in the level of caspase-3, PARP, BAD, Apaf1 and cleaved caspase-9 along with decreased expression of Bcl2 and release of cytochrome c confirmed that, Cd(2+) dose dependently induces solely intrinsic pathway of apoptosis in SK-RC-45, independent of JNK. Furthermore, the non-toxic concentration (10µM) of Cd(2+) induced nuclear translocation of Nrf2 and increased expression in the level of HO-1 enzyme suggesting that at the milder concentration, Cd(2+) induces protective signaling pathways. On the other hand, exposure of NKE to different concentrations of CdCl2 (e.g. 0, 10, 20, 30 and 50µM) under the same conditions elevate stronger heat shock and SOD2 mediated protective responses. In contrary to the RCC PCT, the normal PCT derived cell follows JNK dependent and extrinsic pathways of apoptosis. Cumulatively, these results suggest that Cd(2+) exposure dose dependently elicit both cell proliferative and cell death related responses in SK-RC-45 cells and is differentially regulated with respect to normal kidney epithelia derived NKE cells.