Abstract. It is evident based on literature that flavonoids from fruit can safely modulate cancer cell biology and induce apoptosis. Therefore, we investigated the anticancer activity of morin, a flavonoid which is plentiful in twigs of mulberry focusing on apoptosis, and its mechanisms. Morin upregulated the Fas receptor, and activates caspase-8, -9 and -3 in HCT-116 cells. Morin also activates Bid, and induced the loss of mitochondrial membrane potential (MMP, ∆Ψ m ) with Bax protein activation and cytochrome c release. In addition, morin induced ROS generation which was not blocked by N-acetylcysteine. Morin also suppressed Bcl-2 and cIAP-1, anti-apoptotic proteins, which may contribute to augmentation of morin-triggered apoptosis. As an upstream signaling pathway, suppressed Akt activity by morin was associated to apoptosis. This study suggests that morin induces caspase-dependent apoptosis through extrinsic pathway by upregulating Fas receptor as well as through the intrinsic pathway by modulating Bcl-2 and IAP family members, and ROS generation, and that Akt is the critical upstream signaling that regulates the apoptotic effect of morin in human colon cancer HCT-116 cells.

We examined the anti-cancer effect of genistein, a soy-derived isoflavone, in human bladder transitional cell carcinoma T24 cells. According to our data, genistein induced G2/M phase arrest of the cell cycle and apoptosis. Genistein down-regulated the levels of cyclin A and cyclin B1, but up-regulated the levels of p21WAF1/CIP1, cyclin-dependent kinase (Cdk) inhibitor, that was complexed with Cdc2 and Cdk2. Furthermore, genistein induced the activation of caspases (caspase-3, -8 and -9), and cleavage of poly (ADP-ribose) polymerase cleavage. However, genistein-induced apoptosis was significantly inhibited by a pan-caspase inhibitor, indicating that the induction of apoptosis by genestein was caspase-dependent. In addition, genistein increased the cytosolic release of cytochrome c by increasing the Bax/Bcl-2 ratio and destroying mitochondria integrity. Moreover, genistein inactivated the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, while LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of genistein. Genistein further increased the accumulation of reactive oxygen species (ROS), which was significantly suppressed by N-acetyl cysteine (NAC), a ROS scavenger, and in particular, NAC prevented genistein-mediated inactivation of PI3K/Akt signaling, G2/M arrest and apoptosis. Therefore, the present results indicated that genistein promoted apoptosis induction in human bladder cancer T24 cells, which was associated with G2/M phase cell cycle arrest via regulation of ROS-dependent PI3K/Akt signaling pathway.

Licochalcone A (LCA) is a chalcone that is predominantly found in the root of Glycyrrhiza species, which is widely used as an herbal medicine. Although previous studies have reported that LCA has a wide range of pharmacological effects, evidence for the underlying molecular mechanism of its anti-cancer efficacy is still lacking. In this study, we investigated the anti-proliferative effect of LCA on human bladder cancer cells, and found that LCA induced cell cycle arrest at G2/M phase and apoptotic cell death. Our data showed that LCA inhibited the expression of cyclin A, cyclin B1, and Wee1, but increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, and increased p21 was bound to Cdc2 and Cdk2. LCA activated caspase-8 and -9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. Additionally, LCA increased the Bax/Bcl-2 ratio, and reduced the integrity of mitochondria, which contributed to the discharge of cytochrome c from the mitochondria to the cytoplasm. Moreover, LCA enhanced the intracellular levels of reactive oxygen species (ROS); however, the interruption of ROS generation using ROS scavenger led to escape from LCA-mediated G2/M arrest and apoptosis. Collectively, the present data indicate that LCA can inhibit the proliferation of human bladder cancer cells by inducing ROS-dependent G2/M phase arrest and apoptosis.

Mammalian thioredoxin reductase (TrxR) plays a vital role in restoring cellular redox balance disrupted by reactive oxygen species (ROS) generation and oxidative damage. Here, we evaluated whether auranofin, a selective inhibitor of TrxR, could serve as a potential anti-cancer agent through its selective targeting of TrxR activity in Hep3B hepatocellular carcinoma cells. Auranofin treatment reduced the TrxR activity of these cells and induced apoptosis, which were accompanied by up-regulation of death receptors (DRs) and activation of caspases, as well as promotion of proteolytic degradation of poly(ADP-ribose)-polymerase. Treatment with a pan-caspase inhibitor reversed the auranofin-induced apoptosis and growth suppression, indicating that auranofin may induce apoptosis through a caspase-dependent mechanism involving both the intrinsic and extrinsic apoptotic pathways. Auranofin also significantly altered mitochondrial function, promoting mitochondrial membrane permeabilization and cytochrome c release by regulating Bcl-2 family proteins; these events were accompanied by an accumulation of ROS. Inhibition of ROS generation with the ROS quencher significantly attenuated the inactivation of TrxR in auranofin-treated cells and almost completely suppressed the auranofin-induced up-regulation of DRs and activation of caspases, thereby preventing auranofin-induced apoptosis and loss of cell viability. Taken together, these findings indicate that auranofin inhibition of TrxR activity in Hep3B cells activates ROS- and caspase-dependent apoptotic signaling pathways and triggers cancer cell death.

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Retinal pigment epithelial (RPE) cells occupy the outer layer of the retina and perform various biological functions. Oxidative damage to RPE cells is a major risk factor for retinal degeneration that ultimately leads to vision loss. In this study, we investigated the role of spermidine in a hydrogen peroxide (H2O2)-induced oxidative stress model using human RPE cells. Our findings showed that 300 μM H2O2 increased cytotoxicity, apoptosis, and cell cycle arrest in the G2/M phase, whereas these effects were markedly suppressed by 10 μM spermidine. Furthermore, spermidine significantly reduced H2O2-induced mitochondrial dysfunction including mitochondrial membrane potential and mitochondrial activity. Although spermidine displays antioxidant properties, the generation of intracellular reactive oxygen species (ROS) upon H2O2 insult was not regulated by spermidine. Spermidine did suppress the increase in cytosolic Ca2+ levels resulting from endoplasmic reticulum stress in H2O2-stimulated human RPE cells. Treatment with a cytosolic Ca2+ chelator markedly reversed H2O2-induced cellular dysfunction. Overall, spermidine protected against H2O2-induced cellular damage by blocking the increase of intracellular Ca2+ independently of ROS. These results suggest that spermidine protects RPE cells from oxidative stress, which could be a useful treatment for retinal diseases.

No abstract

The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.