Boxin Xue

Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death.

Castration‑resistant prostate cancer (CRPC) is difficult to treat in current clinical practice. Hypoxia is an important feature of the CRPC microenvironment and is closely associated with the progress of CRPC invasion. However, no research has been performed on the immune escape of CRPC from NK cells. The present study focused on this subject. Firstly, when the CRPC cell lines C4‑2 and CWR22Rv1 were induced by hypoxia, the expression of the UL16 binding protein (ULBP) ligand family of natural killer (NK) group 2D (NKG2D; ULBP‑1, ULBP‑2 and ULBP‑3) and MHC class I chain‑related proteins A and B (MICA/MICB) decreased. NKG2D is the main activating receptor of NK cells. Tumor cells were then co‑cultured with NK cells to conduct NK cell‑mediated cytotoxicity experiments, which revealed the decreased immune cytolytic activity of NK cells on hypoxia‑induced CRPC cells. In exploring the mechanism behind this observation, an increase in programmed death‑ligand 1 (PD‑L1) expression in CRPC cells induced by hypoxia was observed, while the addition of PD‑L1 antibody effectively reversed the expression of NKG2D ligand and enhanced the cytotoxic effect of NK cells on CRPC cells. In the process of exploring the upstream regulatory factors of PD‑L1, inhibition of the Janus kinase (JAK)1,2/signal transducer and activator of transcription 3 (Stat3) signaling pathway decreased the expression of PD‑L1 in CRPC cells. Finally, it was observed that combined inhibition of JAK1,2/PD‑L1 or Stat3/PD‑L1 was more effective than inhibition of a single pathway in enhancing the immune cytolytic activity of NK cells. Taking these results together, it is thought that combined inhibition of the JAK1,2/PD‑L1 and Stat3/PD‑L1 signaling pathways may enhance the immune cytolytic activity of NK cells toward hypoxia‑induced CRPC cells, which is expected to provide novel ideas and targets for the immunotherapy of CRPC.

BACKGROUND: High levels of the post-translational modification O-GlcNAcylation (O-GlcNAc) are found in multiple cancers, including bladder cancer. Autophagy, which can be induced by stress from post-translational modifications, plays a critical role in maintaining cellular homeostasis and regulating tumorigenesis. The impact of O-GlcNAcylation on autophagy in bladder cancer remains unclear. Here, we evaluate the change in autophagic activity in response to O-GlcNAcylation and explore the potential mechanisms. METHODS: O-GlcNAcylation levels in bladder cancer cells were altered through pharmacological or genetic manipulations: treating with 6-diazo-5-oxo-norleucine (DON) or thiamet-G (TG) or up- and downregulation of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA). Autophagy was determined using fluorescence microscopy and western blotting. Co-immunoprecipitation (Co-IP) assays were performed to evaluate whether the autophagy regulator AMP-activated protein kinase (AMPK) was O-GlcNAc modified. RESULTS: Cellular autophagic flux was strikingly enhanced as a result of O-GlcNAcylation suppression, whereas it decreased at high O-GlcNAcylation levels. Phosphorylation of AMPK increased after the suppression of O-GlcNAcylation. We found that O-GlcNAcylation of AMPK suppressed the activity of this regulator, thereby inhibiting ULK1 activity and autophagy. CONCLUSION: We characterized a new function of O-GlcNAcylation in the suppression of autophagy via regulation of AMPK. GRAPHICAL ABSTRACT: Blockage of O-linked GlcNAcylation induces AMPK dependent autophagy in bladder cancer cells.

Sphingosine kinases (SphK), including SphK1 and SphK2, are important enzymes promoting progression of prostate cancer. SKI-178 is a novel and highly potent SphK1/2 dual inhibitor. We here tested the potential anti-prostate cancer cell activity of SKI-178. Bioinformatics analyses and results from local tissues demonstrated that that both SphK1 and SphK2 are upregulated in human prostate cancer tissues. Ectopic overexpression of SphK1 and SphK2, by lentiviral constructs, promoted primary prostate cancer cell proliferation and migration. In primary human prostate cancer cells and immortalized cell lines, SKI-178 potently inhibited cell viability, proliferation, cell cycle progression and cell migration, causing robust cell death and apoptosis. SKI-178 impaired mitochondrial functions, causing mitochondrial depolarization, reactive oxygen species production and ATP depletion.SKI-178 potently inhibited SphK activity and induced ceramide production, without affecting SphK1/2 expression in prostate cancer cells. Further, SKI-178 inhibited Akt-mTOR activation and induced JNK activation in prostate cancer cells. Contrarily, a constitutively-active Akt1 construct or the pharmacological JNK inhibitors attenuated SKI-178-induced cytotoxicity in prostate cancer cells. In vivo, daily intraperitoneal injection of a single dose of SKI-178 potently inhibited PC-3 xenograft growth in nude mice. SphK inhibition, ceramide production, ATP depletion and lipid peroxidation as well as Akt-mTOR inactivation and JNK activation were detected in PC-3 xenograft tissues with SKI-178 administration. Together, targeting SphK1/2 by SKI-178 potently inhibited prostate cancer cell growth in vitro and in vivo.