LW-213 induces immunogenic tumor cell death via ER stress mediated by lysosomal TRPML1.

Dying tumor cells release biological signals that exhibit antigenicity, activate cytotoxic T lymphocytes, and induce immunogenic cell death (ICD), playing a key role in immune surveillance. We demonstrate that the flavonoid LW-213 activates endoplasmic reticulum stress (ERS) in different tumor cells and that the lysosomal calcium channel TRPML1 mediates the ERS process in human cellular lymphoma Hut-102 cells. Apoptotic tumor cells induced by ERS often possess immunogenicity. Tumor cells treated with LW-213 exhibit damage-associated molecular patterns (DAMPs), including calreticulin translocation to the plasma membrane and extracellular release of ATP and HMGB1. When co-cultured with antigen-presenting cells (APCs), LW-213-treated tumor cells activated APCs. Two groups of C57BL/6J mice were inoculated with Lewis cells: a "vaccine group", which demonstrated that LW-213-treated tumor cells promote the maturation of dendritic cells and increase CD8+ T cells infiltration in the tumor microenvironment and a "pharmacodynamic group", treated with a combination of LW-213 and PD1/PD-L1 inhibitor (BMS-1), which reduced tumor growth and significantly prolonged the survival time of mice in the "pharmacodynamic group". Therefore, LW-213 can be developed as a novel ICD inducer, providing a new concept for antitumor immunotherapy.

Biosensor-based active ingredient recognition system for screening TNF-α inhibitors from lotus leaves.

Erhuangquzhi granules (EQG) have been clinically proven to be effective in nonalcoholic steatohepatitis (NASH) treatment. However, the active components and molecular mechanisms remain unknown. This study aimed to screen active components targeting tumor necrosis factor α (TNF-α) in EQG for the treatment of NASH by a surface plasmon resonance (SPR) biosensor-based active ingredient recognition system (SPR-AIRS). The amine-coupling method was used to immobilize recombinant TNF-α protein on an SPR chip, the specificity of the TNF-α-immobilized chip was validated, and nine medicinal herbs in EQG were prescreened. Nuciferine (NF), lirinidine (ID), and O-nornuciferine (NNF) from lotus leaves were found and identified as TNF-α ligands by UPLC‒MS/MS, and the affinity constants of NF, ID, and NNF to TNF-α were determined by SPR experiments (Kd = 61.19, 31.02, and 20.71 µM, respectively). NF, ID, and NNF inhibited TNF-α-induced apoptosis in L929 cells, the levels of secreted IL-6 and IL-1ß were reduced, and the phosphorylation of IKKß and IκB was inhibited in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In conclusion, a class of new active small-molecule TNF-α inhibitors was discovered, which also provides a valuable reference for the material basis and mechanism of EQG action in NASH treatment.

LW-213 induces cell apoptosis in human cutaneous T-cell lymphomas by activating PERK-eIF2α-ATF4-CHOP axis.

Cutaneous T-cell lymphoma (CTCL) is characterized by a heterogeneous group of extranodal non-Hodgkin lymphomas, in which monoclonal T lymphocytes infiltrate the skin. LW-213, a derivative of wogonin, was found to induce cell apoptosis in chronic myeloid leukemia (CML). In this study, we investigated the effects of LW-213 on CTCL cells and the underlying mechanisms. We showed that LW-213 (1-25 µM) dose-dependently inhibited human CTCL cell lines (Hut-102, Hut-78, MyLa, and HH) with IC50 values of around 10 µM, meanwhile it potently inhibited primary leukemia cells derived from peripheral blood of T-cell lymphoma patients. We revealed that LW-213-induced apoptosis was accompanied by ROS formation and the release of calcium from endoplasmic reticulum (ER) through IP3R-1channel. LW-213 selectively activated CHOP and induced apoptosis in Hut-102 cells via activating PERK-eIF2α-ATF4 pathway. Interestingly, the degree of apoptosis and expression of ER stress-related proteins were alleviated in the presence of either N-acetyl cysteine (NAC), an ROS scavenger, or 2-aminoethyl diphenylborinate (2-APB), an IP3R-1 inhibitor, implicating ROS/calcium-dependent ER stress in LW-213-induced apoptosis. In NOD/SCID mice bearing Hut-102 cell line xenografts, administration of LW-213 (10 mg/kg, ip, every other day for 4 weeks) markedly inhibited the growth of Hut-102 derived xenografts and prolonged survival. In conclusion, our study provides a new insight into the mechanism of LW-213-induced apoptosis, suggesting the potential of LW-213 as a promising agent against CTCL.

LW-213, a newly synthesized flavonoid, induces G2/M phase arrest and apoptosis in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell neoplasm characterized by an uncontrolled proliferation of moderately and well differentiated cells of the granulocytic lineage. LW-213, a newly synthesized flavonoid compound, was found to exert antitumor effects against breast cancer through inducing G2/M phase arrest. We investigated whether LW-213 exerted anti-CML effects and the underlying mechanisms. We showed that LW-213 inhibited the growth of human CML cell lines K562 and imatinid-resistant K562 (K562r) in dose- and time-dependent manners with IC50 values at the low µmol/L levels. LW-213 (5, 10, 15 µM) caused G2/M phase arrest of K562 and K562r cells via reducing the activity of G2/M phase transition-related proteins Cyclin B1/CDC2 complex. LW-213 treatment induced apoptosis of K562 and K562r cells via inhibiting the expression of CDK9 through lysosome degradation, thus leading to the suppression of RNAPII phosphorylation, down-regulation of a short-lived anti-apoptic protein MCL-1. The lysosome inhibitor, NH4Cl, could reverse the anti-CML effects of LW-213 including CDK9 degradation and apoptosis. LW-213 treatment also degraded the downstream proteins of BCR-ABL1, such as oncoproteins AKT, STAT3/5 in CML cells, which was blocked by NH4Cl. In primary CML cells and CD34+ stem cells, LW-213 maintained its pro-apoptotic activity. In a K562 cells-bearing mice model, administration of LW-213 (2.5, 5.0 mg/kg, ip, every other day for 4 weeks) dose-dependently prolonged the survival duration, and significantly suppressed huCD45+ cell infiltration and expression of MCL-1 in spleens. Taken together, our results demonstrate that LW-213 may be an efficient agent for CML treatment.

Effects of heat and ultraviolet radiation on the oxidative stability of pine nut oil supplemented with carnosic acid.

The effects of carnosic acid (CA) of different concentrations (0.05, 0.1, and 0.2 mg/g) and two common antioxidants (butylated hydroxytoluene and α-tocopherol) on oxidative stability in pine nut oil at different accelerated conditions (heating and ultraviolet radiation) were compared. The investigation focused on the increase in peroxide and conjugated diene values, as well as free fatty acid and thiobarbituric acid-reactive substances. The changes in trans fatty acid and aldehyde compound contents were investigated by Fourier transform infrared spectroscopy, while the changes in pinolenic acid content were monitored by gas chromatography-mass spectrometry. The results show that CA was more effective in restraining pine nut oil oxidation under heating, UV-A and UV-B radiation, in which a dose-response relationship was observed. The antioxidant activity of CA was stronger than that of α-tocopherol and butylated hydroxytoluene. Pine nut oil supplemented with 0.2 mg/g CA exhibited favorable antioxidant effects and is preferable for effectively avoiding oxidation.

[Anatomical study and three dimensional image characteristic analysis of basicranial artery and its clinical significance].

OBJECTIVE: To observe and measure the inside diameter of basicranial arteries, the angulation of main arteries, the three dimensional image characteristic of internal carotid arteries and the anatomical variation of Willis circle. METHODS: The arteries of 30 formalin-fixed adult heads were injected with latex after which the caliber and characteristic of cerebral arteries were observed and measured. The three dimensional image characteristic of internal carotid arteries and its branches were measured using 3D-DSA. RESULTS: (1) Main artery caliber: origin of internal carotid artery (Left 5.12 +/- 1.48 mm; Right 5.11 +/- 1.42 mm); origin of middle cerebral artery (Left 2.93 +/- 1.44 mm; Right 2.92 +/- 1.46 mm); origin of anterior cerebral artery (Left 2.63 +/- 1.33 mm; Right 2.61 +/- 1.32 mm); origin of vertebral artery (Left 4.37 +/- 1.21 mm; Right 3.22 +/- 1.64 mm); origin of basilar artery (4.45 +/- 1.28 mm); origin of posterior cerebral artery (Left 2.62 +/- 1.36 mm; Right 2.61 +/- 1.22 mm). (2) The angulation of main arteries: C1, 2 of ICA and C4, 5 of ICA (Left 32 +/- 22 degrees; Right 36 +/- 28 degrees ); ICA and ACA (Left 43 +/- 26 degrees; Right 46 +/- 28 degrees). (3) The results show that anatomical and three dimensional image characteristic of internal carotid arteries have no difference (P > 0.05). (4) The anatomical variation of Willis circle: Type O (56.7%); Type A (16.7%); Type P (20.0%); Type AP (6.7%). CONCLUSIONS: It is helpful to measure the inside diameter of basicranial arteries for the selection of various catheter in interventional neuroradiology, to observe the angulation of main arteries and the three dimensional image characteristic of internal carotid arteries for the moulding of various catheter in endovascular therapy and to master the anatomical variation of Willis circle for decreasing complications of endovascular treatment and judging prognosis of cerebrovascular diseases.