Diterpenoid Vinigrol specifically activates ATF4/DDIT3-mediated PERK arm of unfolded protein response to drive non-apoptotic death of breast cancer cells.

Vinigrol is a natural diterpenoid with unprecedented chemical structure, driving great efforts into its total synthesis in the past decades. Despite anti-hypertension and anti-clot ever reported, comprehensive investigations on bioactions and molecular mechanisms of Vinigrol are entirely missing. Here we firstly carried out a complete functional prediction of Vinigrol using a transcriptome-based strategy coupled with multiple bioinformatic analyses and identified "anti-cancer" as the most prominent biofunction ahead of anti-hypertension and anti-depression/psychosis. Broad cytotoxicity was subsequently confirmed on multiple cancer types. Further mechanistic investigation on several breast cancer cells revealed that its anti-cancer effect was mainly through activating PERK/eIF2α arm of unfolded protein response (UPR) and subsequent non-apoptotic cell death independent of caspase activities. The other two branches of UPR, IRE1α and ATF6, were functionally irrelevant to Vinigrol-induced cell death. Using CRISPR/Cas9-based gene activation, repression, and knockout systems, we identified the essential contribution of ATF4 and DDIT3, not ATF6, to the death process. This study unraveled a broad anti-cancer function of Vinigrol and its underlying targets and regulatory mechanisms. It paved the way for further inspection on the structure-efficacy relationship of the whole compound family, making them a novel cluster of PERK-specific stress activators for experimental and clinical uses.

Caspase inhibitor zVAD-fmk protects against acute pancreatitis-associated lung injury via inhibiting inflammation and apoptosis.

BACKGROUND/OBJECTIVES: Pulmonary apoptosis is an important pathogenic mechanism of acute lung injury induced by many factors. This study aims to investigate whether the caspase inhibitor zVAD-fmk has a protective effect against lung injury in the severe acute pancreatitis model (SAP) in rats. METHODS: Seventy-two Sprague-Dawley rats were randomly divided into Sham, SAP, and SAP + zVAD-fmk groups. The SAP model was established by injection of 5% sodium taurocholate into the pancreatic duct. Animals were sacrificed at 3 h, 6 h, 12 h, and 24 h after operation and then HE staining analysis was performed to assess the lung injury. ELISA was used to detect the activity of myeloperoxidase (MPO) and the concentrations of tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). Western blotting was used to detect the expression of cleaved caspase-3 in the lung tissues. RESULTS: Rats in SAP group showed obvious lung injury through pathologic examination. Pretreatment with zVAD-fmk significantly inhibited a post-SAP increase in the activation of MPO, TNF-α, IL-1ß, and caspase-3, and decreased lung injury induced by SAP as determined by the pathologic score. CONCLUSION: Our results suggest that apoptosis plays an important role in acute pancreatitis-associated lung injury (APALI), and inhibition of caspase activity may represent a new therapeutic approach for the treatment of APALI.

Cellular autophagy, an unbidden effect of caspase inhibition by zVAD-fmk.

zVAD-fmk is a widely used pan-caspase inhibitor that blocks apoptosis but has undesirable side effects, including autophagy. In this issue, Needs et al. propose that zVAD-fmk induces autophagy by inhibiting the N-glycanase NGLY1 rather than caspases. NGLY1 is essential for the ERAD response and patients with inactivating mutations in NGLY1 present with neurodevelopmental defects and organ dysfunction. The ability of NGLY1 to inhibit basal levels of autophagy may contribute to this pathology. This study demonstrates possible crosstalk between protein turnover and autophagy while also underscoring the importance of specificity when using chemical tools to interrogate these pathways. Comment on https://doi.org/10.1111/febs.16345.

Betulinic Acid for cancer treatment and prevention.

Betulinic acid is a natural product with a range of biological effects, for example potent antitumor activity. This anticancer property is linked to its ability to induce apoptotic cell death in cancer cells by triggering the mitochondrial pathway of apoptosis. In contrast to the cytotoxicity of betulinic acid against a variety of cancer types, normal cells and tissue are relatively resistant to betulinic acid, pointing to a therapeutic window. Compounds that exert a direct action on mitochondria present promising experimental cancer therapeutics, since they may trigger cell death under circumstances in which standard chemotherapeutics fail. Thus, mitochondrion-targeted agents such as betulinic acid hold great promise as a novel therapeutic strategy in the treatment of human cancers.

Smac mimetic primes apoptosis-resistant acute myeloid leukaemia cells for cytarabine-induced cell death by triggering necroptosis.

The prognosis for patients with acute myeloid leukaemia (AML) is still poor, thus calling for novel treatment strategies. Here, we report that the small-molecule Smac mimetic BV6, which antagonizes Inhibitor of Apoptosis (IAP) proteins, acts in concert with cytarabine (AraC) to trigger cell death in AML cells in a highly synergistic manner (combination index 0.02-0.27). Similarly, BV6 cooperates with AraC to trigger cell death in primary AML samples, underscoring the clinical relevance of our findings. Molecular studies reveal that the TNFα-blocking antibody Enbrel significantly reduces BV6/AraC-induced cell death, demonstrating that an autocrine/paracrine TNFα loop mediates cell death. Furthermore, BV6 and AraC synergize to induce loss of mitochondrial membrane potential, caspase activation and DNA fragmentation, consistent with apoptotic cell death. Nevertheless, the caspase inhibitor zVAD.fmk fails to protect against BV6/AraC-induced cell death. Intriguingly, this cell death upon caspase inhibition is significantly reduced by pharmacological inhibition of two key components of necroptosis signaling, i.e. by RIP1 kinase inhibitor Necrostatin-1 or MLKL inhibitor NSA. Thus, BV6 sensitizes AML cells to AraC-induced cell death and overcomes apoptosis resistance by triggering necroptosis as alternative form of cell death. These findings have important implications for Smac mimetic-based strategies to bypass apoptosis resistance of AML.

Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene.

This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3-30 µM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10-30 µM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H2DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by 32P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.

A novel quinazolinone derivative induces cytochrome c interdependent apoptosis and autophagy in human leukemia MOLT-4 cells.

Crosstalk between apoptosis and autophagy is budding as one of the novel strategies in the cancer therapeutics. The present study tinted toward the interdependence of autophagy and apoptosis induce by a novel quinazolinone derivative 2,3-dihydro-2-(quinoline-5-yl) quinazolin-4(1H)-one structure [DQQ] in human leukemia MOLT-4 cells. DQQ induces cytochrome c arbitrated apoptosis and autophagy in MOLT-4 cells. Apoptosis induces by DQQ was confirmed through a battery of assay e.g. cellular and nuclear microscopy, annexin-V assay, cell cycle analysis, loss of mitochondrial membrane potential and immune-expression of cytochrome c, caspases and PARP. Furthermore, acridine orange staining, LC3 immunofluorescence and western blotting of key autophagy proteins revealed the autophagic potential of DQQ. A universal caspase inhibitor, Z-VAD-FMK and cytochrome c silencing, strongly inhibited the DQQ induce autophagy and apoptosis. Beclin1 silencing through siRNA partially reversed the cell death, which was not as significant as by cytochrome c silencing. Although, it partially reversed the PARP cleavage induced by DQQ, indicating the role of autophagy in the regulation of apoptosis. The present study first time portrays the negative feedback potential of cytochrome c regulated autophagy and the importance of quinazolinone derivative in discovery of novel anticancer therapeutics.