A major concern in the clinical application of tumor necrosis factor related apoptosis‐inducing ligand (TRAIL) in tumors is the development of resistance. Therefore, agents that can potentially restore TRAIL sensitivity are important therapeutic targets for cancer treatment. Herein, we evaluated lanatoside c and digoxin, both of which are widely used cardiac glycosides (CGs), for their ability to sensitize human hepatocellular carcinoma cells (Huh‐7 and HepG2) through TRAIL‐induced apoptosis. CGs functionalize TRAIL as shown by its effect on intracellular reactive oxygen species (ROS) generation, which damages mitochondrial integrity and thereby confers intrinsic apoptotic caspase cascade during combined treatment. Caspase activation is dependent on ROS as shown by the ability of CGs to generate ROS and the ROS‐N‐acetylcysteine (NAC) relationship, which inhibits apoptosis during cotreatment by preventing the formation of caspase‐8 and ‐3. Furthermore, CGs triggered p38MAPK phosphorylation and NAC pre‐exposure blocked p38MAPK phosphorylation, which demonstrated that p38MAPK was dependent upon ROS generation. Additionally, CGs were found to be potent inducers of AMPK‐mediated protective autophagy as pharmacological and genetic autophagy inhibition reached the higher threshold of TRAIL‐mediated apoptosis. Finally, CGs downregulated the expression of the antiapoptotic protein Bcl‐2 and increased the translocation of proapoptotic protein cytochrome c, thereby inducing apoptosis. Collectively, these results indicate that CGs potentiate the enhanced cytotoxic capacity to TRAIL through ROS generation, p38MAPK phosphorylation, cell survival protein downregulation, and protective autophagy inhibition.
Sphingosine 1‑phosphate (S1P) belongs to a significant group of signaling sphingolipids and exerts most of its activity as a ligand of G‑protein‑coupled receptors. In our previous study, S1P demonstrated a novel biological activity with the anti‑adipogenesis of 3T3‑L1 preadipocytes. In the present study, we identified a possible mechanism of S1P‑mediated anti‑adipogenic effects, particularly in target pathways of the S1P receptors, including S1P1 and S1P2. The mRNA levels of S1P1 and S1P2 receptors were increased by MDI media treatment, whereas S1P treatment highly induced S1P2 but not S1P1 receptor protein in adipocytes. Triglyceride accumulation assay using an agonist and antagonist of S1P receptors revealed that S1P2 receptor was only involved in S1P‑mediated anti‑adipogenic effects. Furthermore, pharmacological inhibition of S1P2 signals completely retrieved S1P‑mediated downregulation of the transcriptional levels of peroxisome proliferator‑activated receptor γ, CCAAT/enhancer binding protein α and adiponectin, which are markers of adipogenic differentiation. This study demonstrated that S1P2 receptor signals may regulate the S1P‑mediated anti‑adipogenic differentiation and also identifies the S1P2 receptor as a possible mechanism of anti‑adipogenic differentiation.
Abstract. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator that regulates multiple signals through S1P receptors responsible for biological responses. In particular, the S1P 2 receptor has distinct roles in the S1P-mediated differentiation of certain cell types. The present study was the first, to the best of our knowledge, to report the role of the S1P 2 receptor in the adipocyte differentiation of 3T3-L1 pre-adipocytes. In order to investigate the influence of S1P 2 receptors in the anti-adipogenic effects of S1P, S1P 2 receptor silencing and overexpression of were used. S1P 2 overexpression with adenoviral vectors inhibited adipogenesis and inhibited the expression of peroxisome proliferator-activated receptor γ (PPARγ), adiponectin and CCAAT/enhancer binding protein-α, which were upregulated following incubation in differentiation media. Furthermore, S1P completely lost its ability to impair adipogenic differentiation following silencing of S1P 2 . Silencing of the S1P 2 receptor additionally blocked the downregulation of PPARγ protein and phospho-c-Jun N-terminal kinase protein induced by S1P treatment. In conclusion, the present study demonstrated that the S1P 2 receptor is a key signaling molecule in the S1P-dependent inhibition of adipogenic differentiation and additionally suggested that selective targeting of S1P 2 receptors may have clinical applications for the treatment of obesity.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential target for cancer therapy, owing to its ability to selectively kill cancer cells without causing significant toxicity to normal cells. However, due to the lack of death receptor expression, cancer cells can become highly resistant to TRAIL. Hence, it is vital to develop agents that restore TRAIL efficacy. Sertraline is an antidepressant drug with anticancer properties. To the best of our knowledge, this is the first study to demonstrate that sertraline inhibits autophagic flux and increases the expression of death receptor 5 (DR5) on TRAIL-resistant lung cancer cells. Inhibition of autophagy using autophagy inhibitors 3-methyladenine and chloroquine upregulated the expression of DR5 and enhanced TRAIL-induced apoptosis, as confirmed by the increase of pro-apoptotic proteins caspase-8 and caspase-3. Silencing DR5 expression using DR5 small interfering RNA prevented sertraline-induced TRAIL-mediated apoptosis, indicating the role of DR5 in TRAIL-mediated apoptosis. Overall, sertraline enhanced TRAIL-mediated apoptosis via the downregulation of AMP-activated protein kinase phosphorylation, resulting in the inhibition of autophagic flux, upregulation of DR5 expression, and activation of the apoptotic caspase cascade. These data suggested that sertraline could be used to sensitize human lung cancer cells to TRAIL, while also serving as a therapeutic option in cancer patients with depression.
Tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) is toxic against transformed tumor cells. Cornification is the terminal differentiation of keratinocytes and a specific form of programmed cell death caused by TRAIL that occurs in keratinocytes. Apoptosis can also be triggered when TRAIL induces expression of keratinocyte differentiation markers. The present study reported that hypoxia inhibits TRAIL‑induced apoptosis due to autophagic flux. It is well known that hypoxia activates autophagy in keratinocytes and reduces p62 protein levels. The present study demonstrated that hypoxia inhibited TRAIL‑mediated apoptosis and induced autophagic flux in HaCaT cells. In addition, autophagic flux‑inactivating reagents, including 3‑methyladenine and chloroquine, increased the TRAIL sensitivity of HaCaT cells exposed to hypoxia. In conclusion, these results indicated that inactivating autophagy increased TRAIL sensitivity in hypoxic HaCaT cells. Autophagy inhibitors may be beneficial in therapies using TRAIL against skin cancers.
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