Atezolizumab Induces Necroptosis and Contributes to Hepatotoxicity of Human Hepatocytes.

Atezolizumab is an immune checkpoint inhibitor (ICI) targeting PD-L1 for treatment of solid malignancies. Immune checkpoints control the immune tolerance, and the adverse events such as hepatotoxicity induced by ICIs are often considered as an immune-related adverse event (irAE). However, PD-L1 is also highly expressed in normal tissues, e.g., hepatocytes. It is still not clear whether, targeting PD-L1 on hepatocytes, the atezolizumab may cause damage to liver cells contributing to hepatotoxicity. Here, we reveal a novel mechanism by which the atezolizumab induces hepatotoxicity in human hepatocytes. We find that the atezolizumab treatment increases a release of LDH in the cell culture medium of human hepatocytes (human primary hepatocytes and THLE-2 cells), decreases cell viability, and inhibits the THLE-2 and THLE-3 cell growth. We demonstrate that both the atezolizumab and the conditioned medium (T-CM) derived from activated T cells can induce necroptosis of the THLE-2 cells, which is underscored by the fact that the atezolizumab and T-CM enhance the phosphorylation of RIP3 and MLKL proteins. Furthermore, we also show that necrostatin-1, a necrosome inhibitor, decreases the amount of phosphorylated RIP3 induced by the atezolizumab, resulting in a reduced LDH release in the culture media of the THLE-2 cells. This finding is further supported by the data that GSK872 (a RIP3 inhibitor) significantly reduced the atezolizumab-induced LDH release. Taken together, our data indicate that the atezolizumab induces PD-L1-mediated necrosome formation, contributing to hepatotoxicity in PD-L1+-human hepatocytes. This study provides the molecular basis of the atezolizumab-induced hepatotoxicity and opens a new avenue for developing a novel therapeutic approach to reducing hepatotoxicity induced by ICIs.

A Fully-Human Antibody Specifically Targeting a Membrane-Bound Fragment of CADM1 Potentiates the T Cell-Mediated Death of Human Small-Cell Lung Cancer Cells.

Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer and the leading cause of global cancer-related mortality. Despite the earlier identification of membrane-proximal cleavage of cell adhesion molecule 1 (CADM1) in cancers, the role of the membrane-bound fragment of CAMD1 (MF-CADM1) is yet to be clearly identified. In this study, we first isolated MF-CADM1-specific fully human single-chain variable fragments (scFvs) from the human synthetic scFv antibody library using the phage display technology. Following the selected scFv conversion to human immunoglobulin G1 (IgG1) scFv-Fc antibodies (K103.1-4), multiple characterization studies, including antibody cross-species reactivity, purity, production yield, and binding affinity, were verified. Finally, via intensive in vitro efficacy and toxicity evaluation studies, we identified K103.3 as a lead antibody that potently promotes the death of human SCLC cell lines, including NCI-H69, NCI-H146, and NCI-H187, by activated Jurkat T cells without severe endothelial toxicity. Taken together, these findings suggest that antibody-based targeting of MF-CADM1 may be an effective strategy to potentiate T cell-mediated SCLC death, and MF-CADM1 may be a novel potential therapeutic target in SCLC for antibody therapy.

Rationally designed redirection of natural killer cells anchoring a cytotoxic ligand for pancreatic cancer treatment.

The emergence of T-cell engineering with chimeric antigen receptors (CARs) has led to attractive therapeutics; however, autologous CAR-T cells are associated with poor clinical outcomes in solid tumors because of low safety and efficacy. Therefore, the aim of our study was to develop a CAR therapy with enhanced cytotoxicity against solid cancer using allogeneic NK cells. In this study, we engineered "off-the-shelf" NK cells to redirect them towards pancreatic ductal adenocarcinoma (PDAC) by improving their target-specific cytotoxic potential. By integrated bioinformatic and clinicopathological analyses, folate receptor alpha (FRα) and death receptor 4 (DR4) were significantly highly expressed in patient-derived tumor cells. The combined expression of FRα and DR4/5 was associated with inferior clinical outcomes, therefore indicating their use as potential targets for biomolecular treatment. Thus, FRα and DR4 expression pattern can be a strong prognostic factor as promising therapeutic targets for the treatment of PDAC. For effective PDAC treatment, allogeneic CAR-NK cells were reprogrammed to carry an apoptosis-inducing ligand and to redirect them towards FRα and initiate DR4/5-mediated cancer-selective cell death in FRα- and DR4/5-positive tumors. As a result, the redirected cytotoxic ligand-loaded NK cells led to a significantly enhanced tumor-selective apoptosis. Accordingly, use of allogeneic CAR-NK cells that respond to FRα and DR4/5 double-positive cancers might improve clinical outcomes based on personal genome profiles. Thus, therapeutic modalities based on allogeneic NK cells can potentially be used to treat large numbers of patients with optimally selective cytotoxicity.

A carrier-free multiplexed gene editing system applicable for suspension cells.

Genetically engineered cells via CRISPR/Cas9 system can serve as powerful sources for cancer immunotherapeutic applications. Furthermore, multiple genetic alterations are necessary to overcome tumor-induced immune-suppressive mechanisms. However, one of the major obstacles is the technical difficulty with efficient multiple gene manipulation of suspension cells due to the low transfection efficacy. Herein, we established a carrier-free multiplexed gene editing platform in a simplified method, which can enhance the function of cytotoxic CD8+ T cells by modulating suspension cancer cells. Our multiple Cas9 ribonucleoproteins (RNPs) enable simultaneous disruption of two programmed cell death 1 (PD-1) ligands, functioning as negative regulators in the immune system, by accessing engineered Cas9 proteins with abilities of complexation and cellular penetration. In addition, combination with electroporation enhanced multiple gene editing efficacy, compared with that by treatment of multiple Cas9 RNPs alone. This procedure resulted in high gene editing at multiple loci of suspension cells. The treatment of multiple Cas9 RNPs targeting both ligands strongly improved Th1-type cytokine production of cytotoxic CD8+ T cells, resulting in synergistic cytotoxic effects against cancer. Simultaneous suppression of PD-L1 and PD-L2 on cancer cells via our developed editing system allows effective anti-tumor immunity. Furthermore, the treatment of multiple Cas9 RNPs targeting PD-L1, PD-L2, and TIM-3 had approximately 70-90% deletion efficacy. Thus, our multiplexed gene editing strategy endows potential clinical utilities in cancer immunotherapy.

Anticancer effects of methanol extract of Myrmecodia platytyrea Becc. leaves against human hepatocellular carcinoma cells via inhibition of ERK and STAT3 signaling pathways.

Myrmecodia platytyrea Becc., a member of the Rubiaceae family, is found throughout Southeast Asia and has been traditionally used to treat cancer. However, there is limited pharmacological information on this plant. We investigated the anticancer effects of the methanol extract of Myrmecodia platytyrea Becc. leaves (MMPL) and determined the molecular mechanisms underlying the effects of MMPL on metastasis in human hepatocellular carcinoma (HCC) cells. MMPL dose-dependently inhibited cell migration and invasion in SK­Hep1 and Huh7 cells. In addition, MMPL strongly suppressed the enzymatic activity of matrix metalloproteinases (MMP­2 and MMP­9). Diminished telomerase activity by MMPL resulted in the suppression of both telomerase activity and telomerase-associated gene expression. The levels of urokinase-type plasminogen activator receptor (uPAR) expression as well as the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) were also attenuated by MMPL. The above results collectively suggest that MMPL has anticancer effects in HCC and that MMPL can serve as an effective therapeutic agent for treating human liver cancer.

Dual-specificity phosphatase 5 acts as an anti-inflammatory regulator by inhibiting the ERK and NF-κB signaling pathways.

Although dual-specificity phosphatase 5 (DUSP5), which inactivates extracellular signal-regulated kinase (ERK), suppresses tumors in several types of cancer, its functional roles remain largely unknown. Here, we show that DUSP5 is induced during lipopolysaccharide (LPS)-mediated inflammation and inhibits nuclear factor-κB (NF-κB) activity. DUSP5 mRNA and protein expression increased transiently in LPS-stimulated RAW 264.7 cells and then returned to basal levels. DUSP5 overexpression in RAW 264.7 cells suppressed the production of pro-inflammatory tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), whereas knockdown of DUSP5 increased their expression. Investigation of two major inflammatory signaling pathways, mitogen-activated protein kinase (MAPK) and NF-κB, using activator protein-1 (AP-1) and NF-κB reporter plasmids, respectively, showed that NF-κB transcription activity was downregulated by DUSP5 in a phosphatase activity-independent manner whereas AP-1 activity was inhibited by DUSP5 phosphatase activity towards ERK,. Further investigation showed that DUSP5 directly interacts with transforming growth factor beta-activated kinase 1 (TAK1) and inhibitor of κB (IκB) kinases (IKKs) but not with IκBα. DUSP5 binding to IKKs interfered with the association of TAK1 with IKKs, suggesting that DUSP5 might act as a competitive inhibitor of TAK1-IKKs association. Therefore, we propose that DUSP5 negatively regulates ERK and NF-κB in a phosphatase activity-dependent and -independent manner, respectively.

Scaffold Role of DUSP22 in ASK1-MKK7-JNK Signaling Pathway.

Mitogen-activated protein kinases (MAPKs) are involved in a variety of intracellular events such as gene expression, cell proliferation, and programmed cell death. MAPKs are activated by dual phosphorylation on threonine and tyrosine residues through sequential activation of protein kinases. Recent studies have shown that the protein kinases involved in MAPK signal transductions might be organized into signaling complexes by scaffold proteins. These scaffold proteins are essential regulators that function by assembling the relevant molecular components in mammalian cells. In this study, we report that dual-specificity phosphatase 22 (DUSP22), a member of the protein tyrosine phosphatase family, acts as a distinct scaffold protein in c-Jun N-terminal kinase (JNK) signaling. DUSP22 increased the phosphorylation in the activation loop of JNK regardless of its phosphatase activity but had no effect on phosphorylation levels of ERK and p38 in mammalian cells. Furthermore, DUSP22 selectively associated with apoptosis signal-regulating kinase 1 (ASK1), MAPK kinase 7 (MKK7), and JNK1/2. Both JNK phosphorylation and JNK-mediated apoptosis increased in a concentration-dependent manner regardless of DUSP22 phosphatase activity at low DUSP22 concentrations, but then decreased at higher DUSP22 concentrations, which is the prominent feature of a scaffold protein. Thus, our data suggest that DUSP22 regulates cell death by acting as a scaffold protein for the ASK1-MKK7-JNK signal transduction pathway independently of its phosphatase activity.

Methanol extracts of Xanthium sibiricum roots inhibit inflammatory responses via the inhibition of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) in murine macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Xanthium sibiricum has been used as a traditional Chinese medicine for the treatment of appendicitis, bronchitis, arthritis, and other inflammatory ailments. However, its pharmacological activity related to an anti-inflammatory effect remain unknown. This present study aims to investigate the anti-inflammatory effect of methanol extracts of X. sibiricum roots (MXS), and to further determine its underlying mechanism of action in order to assess the medicinal value of X. sibiricum roots. MATERIALS AND METHODS: To assess the anti-inflammatory activity of MXS in lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages, the production of nitric oxide (NO) was measured using the Griess reagent system. The levels of pro-inflammatory cytokines and mediators were quantified using an Enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Subsequently, immunoblotting analyses were employed to detect inflammatory mediators as well as to elucidate the underlying regulatory mechanisms suppressed by MXS. RESULTS: MXS inhibited LPS-stimulated NO production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages within the non-cytotoxic concentration range (50-400 µg/ml). In addition, mRNA and protein levels of pro-inflammatory cytokines such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were significantly suppressed by MXS at the concentration of 400 µg/ml. Furthermore, MXS (200 µg/ml) clearly reduced the phosphorylation levels of the inhibitor of kappa Bα (IκBα) and signal transducer and activator of transcription 3 (STAT3), without affecting changes in the phosphorylation levels of mitogen-activated protein kinases (MAPKs). When five major components (betulin, betulinic acid, ß-sitosterol, stigmasterol, and scopoletin) of MXS were separately investigated, stigmasterol and ß-sitosterol seemed to play major inhibitory roles in the LPS-induced production of inflammatory mediators such as NO, IL-6, and TNF-α. CONCLUSION: Our results demonstrate that MXS has an anti-inflammatory property in LPS-stimulated RAW 264.7 macrophages, and its anti-inflammatory activity is exerted by the regulation of nuclear factor-κB (NF-κB) and STAT3 signaling pathways.

Anti-inflammatory effects of Crataeva nurvala Buch. Ham. are mediated via inactivation of ERK but not NF-κB.

ETHNOPHARMACOLOGICAL RELEVANCE: Crataeva nurvala Buch. Ham. is an important medicinal plant in India, and its extracts and components were used to treat various inflammatory diseases, such as urinary tract infection, rheumatoid arthritis, and colitis. However, no systemic studies about anti-inflammatory effects of Crataeva nurvala Buch. Ham. and its underlying mechanisms of action have been reported. This study aimed to explore the anti-inflammatory effects of ethanol extracts of Crataeva nurvala Buch. Ham. (ECN). MATERIALS AND METHODS: The non-cytotoxic and maximal effective concentration of ECN was determined by measuring the formation of formazan from water-soluble tetrazolium salt in living cells. The inhibitory effect of ECN on nitric oxide (NO) synthesis was measured using Griess reagent, and Enzyme-linked immunosorbent assay (ELISA) was used to measure secreted tumor necrosis factor (TNF)-α and interleukin (IL)-6 protein levels. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting analysis were used to assess the mRNA and protein expression of each inflammatory mediator or relating signaling protein, respectively. RESULTS: A non-cytotoxic concentration of ECN (≤200 µg/ml) significantly reduced the production of NO and IL-6, but not TNF-α, in lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Decreased production of NO by ECN was correlated with reduced expression of iNOS at the mRNA and protein levels. However, cyclooxygenase (COX)-2 expressions at mRNA and protein level were not regulated by ECN. The mRNA expression of IL-6 and IL-1ß, but not TNF-α, was also inhibited by ECN treatment in LPS-stimulated RAW 264.7 macrophages. Reduced production of inflammatory mediators by ECN was followed by decreased activity of mitogen-activated protein kinases (MAPKs), especially extracellular signal-regulated kinase (ERK), but not nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). CONCLUSIONS: These results indicate that ECN inhibits LPS-induced inflammatory responses via negative regulation of ERK in murine macrophages, suggesting that ECN is a candidate for alleviating severe inflammation.

Methanol extracts of Euphorbia cooperi inhibit the production of inflammatory mediators by inhibiting the activation of c­Jun N­terminal kinase and p38 in murine macrophages.

Numerous Euphorbiaceae plants have been used for the treatment of diseases, including liver diseases, asthma and rheumatism. The present study evaluated the effect of methanol extracts from Euphorbia cooperi (MEC), a member of the Euphorbiaceae plant family, on the production of inflammatory cytokines interleukin (IL)­6 and tumor necrosis factor (TNF)­α, nitric oxide (NO) as well as the activation of mitogen­activated protein kinase and nuclear factor (NF)­κB signaling. Non­cytotoxic concentrations of MEC significantly reduced the production of NO and IL­6, but not TNF­α, in lipopolysaccharide (LPS)­stimulated RAW 264.7 macrophages. The decreased production of NO by MEC was due to alleviated expression of inducible NO synthase. Reporter assays with cells treated with MEC demonstrated reduced activator protein­1 (AP-1) activity, while NF­κB activity was not reduced. Furthermore, the phosphorylation levels of c­Jun N­terminal kinase (JNK) and p38 were suppressed by MEC while phosphorylation levels of inhibitor of κB were not reduced by MEC, suggesting that MEC­mediated inactivation of JNK and p38 is the underlying regulatory mechanism for inflammatory mediators in LPS­stimulated RAW 264.7 macrophages.