Plumbagin ameliorates LPS-induced acute lung injury by regulating PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling pathways.

Acute lung injury (ALI) is a major pathophysiological problem characterized by severe inflammation, resulting in high morbidity and mortality. Plumbagin (PL), a major bioactive constituent extracted from the traditional Chinese herb Plumbago zeylanica, has been shown to possess anti-inflammatory and antioxidant pharmacological activities. However, its protective effect on ALI has not been extensively studied. The objective of this study was to investigate the protective effect of PL against ALI induced by LPS and to elucidate its possible mechanisms both in vivo and in vitro. PL treatment significantly inhibited pathological injury, MPO activity, and the wet/dry ratio in lung tissues, and decreased the levels of inflammatory cells and inflammatory cytokines TNF-α, IL-1ß, IL-6 in BALF induced by LPS. In addition, PL inhibited the activation of the PI3K/AKT/mTOR signalling pathway, increased the activity of antioxidant enzymes CAT, SOD, GSH and activated the Keap1/Nrf2/HO-1 signalling pathway during ALI induced by LPS. To further assess the association between the inhibitory effects of PL on ALI and the PI3K/AKT/mTOR and Keap1/Nrf2/HO-1 signalling, we pretreated RAW264.7 cells with 740Y-P and ML385. The results showed that the activation of PI3K/AKT/mTOR signalling reversed the protective effect of PL on inflammatory response induced by LPS. Moreover, the inhibitory effects of PL on the production of inflammatory cytokines induced by LPS also inhibited by downregulating Keap1/Nrf2/HO-1 signalling. In conclusion, the results indicate that the PL ameliorate LPS-induced ALI by regulating the PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling, which may provide a novel therapeutic perspective for PL in inhibiting ALI.

Effect of Modified Forms of Sodium Humate PowHumus on the Balance of Arginine in Peritoneal Macrophages of Intact Mice.

Substances of silver nanoparticles dialyzed through a 13 kDa membrane, synthesized in a medium of humic ligands modified with hydroquinone and 2-hydroxynaphthoquinone from PowHumus brown coal, specifically enhance the M2 properties of peritoneal macrophages due to inhibition of NO synthase and significant activation of arginase, thus enhancing anti-inflammatory properties of cells. In small, but effective concentrations, they do not have cytotoxic properties and do not contain pyrogenic impurities. The studied humates are able to influence the mechanisms of immune response formation and are an effective means for correcting inflammation and regeneration.

A comprehensive Study of Juglone's Effect on Polyphenol Oxidase in Cucumber: In Vitro Experiments and Computational Docking and Dynamics Insights.

This study explores the impact of juglone on cucumber (Cucumis sativus cv. Beith Alpha), scrutinizing its effects on seed germination, growth, and the polyphenol oxidase (PPO) enzyme's activity and gene expression. Employing concentrations ranging from 0.01 to 0.5 mM, we found juglone's effects to be concentration-dependent. At lower concentrations (0.01 and 0.1 mM), juglone promoted root and shoot growth along with germination, whereas higher concentrations (0.25 and 0.5 mM) exerted inhibitory effects, delineating a threshold for its allelopathic influence. Notably, PPO activity surged, especially at 0.5 mM in roots, hinting at oxidative stress involvement. Real-time PCR unveiled that juglone modulates PPO gene expression in cotyledons, peaking at 0.1 mM and diminishing at elevated levels. Correlation analyses elucidated a positive link between juglone-induced root growth and cotyledon PPO gene expression but a negative correlation with heightened root enzyme activity. Additionally, germination percentage inversely correlated with root PPO activity, while PPO activities positively associated with dopa and catechol substrates in both roots and cotyledons. Molecular docking studies revealed juglone's selective interactions with PPO's B chain, suggesting regulatory impacts. Protein interaction assessments highlighted juglone's influence on amino acid metabolism, and molecular dynamics indicated juglone's stronger, more stable binding to PPO, inferring potential alterations in enzyme function and stability. Conclusively, our findings elucidate juglone's dose-dependent physiological and biochemical shifts in cucumber plants, offering insights into its role in plant growth, stress response, and metabolic modulation.

Estimation for Raw Material Plants of a Henna Product Using LC-High Resolution MS and Multivariate Analysis.

Henna is a plant-based dye obtained from the powdered leaf of the pigmented plant Lawsonia inermis, and has often been used for grey hair dyeing, treatment, and body painting. As a henna product, the leaves of Indigofera tinctoria and Cassia auriculata can be blended to produce different colour variations. Although allergy from henna products attributed to p-phenylenediamine, which is added to enhance the dye, is reported occasionally, raw material plants of henna products could also contribute to the allergy. In this study, we reported that raw material plants of commercial henna products distributed in Japan can be estimated by LC-high resolution MS (LC-HRMS) and multivariate analysis. Principal Component Analysis (PCA) score plot clearly separated 17 samples into three groups [I; henna, II; blended henna primarily comprising Indigofera tinctoria, III; Cassia auriculata]. This grouping was consistent with the ingredient lists of products except that one sample listed as henna was classified as Group III, indicating that its ingredient label may differ from the actual formulation. The ingredients characteristic to Groups I, II, and III by PCA were lawsone (1), indirubin (2), and rutin (3), respectively, which were reported to be contained in each plant as ingredients. Therefore, henna products can be considered to have been manufactured from these plants. This study is the first to estimate raw material plants used in commercial plant-based dye by LC-HRMS and multivariate analysis.

In-vitro Studies of Extracts of Plumbago Zeylanica in Cancer Cell Lines.

Despite increased use of early detection methods and more aggressive treatment strategies, the worldwide incidence of colorectal cancer is still on the rise. Consequently, it remains urgent to identify novel agents with enhanced efficacy in prevention and/or therapeutic protocols. Our studies focused on the use of Plumbagin, a natural phytochemical that showed promising results against other tumor types, to determine its effectiveness in blocking the proliferation and survival of colon cancer cells in experimental protocols mimicking the environment in primary tumors (attached culture conditions) and in circulating tumor cells (unattached conditions). Under both experimental settings, exposure of HCT116 cells to Plumbagin concentrations in the low micromolar range resulted in cell cycle arrest at the G1 phase, apoptosis via the mitochondrial cell death pathway, and increased production of reactive oxygen species. The cell cycle effects were more noticeable in attached cells, whereas the induction of cell death was more evident in unattached cells. These effects were consistent with the nature and the magnitude of the alterations induced by Plumbagin on the expression levels of a set of proteins known to play key roles in the regulation of cell cycle dynamics, apoptosis mechanisms and cell proliferation. In light of its previously reported lack of toxicity on normal colon cells and the striking anti-survival effect on colon cancer cells observed in our study, Plumbagin should be considered a promising drug for the treatment of colon cancer.

Shikonin blocks CAF-induced TNBC metastasis by suppressing mitochondrial biogenesis through GSK-3ß/NEDD4-1 mediated phosphorylation-dependent degradation of PGC-1α.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore. METHODS: We investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan-Meier plotter database and clinical breast cancer tissue samples. RESULTS: We demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3ß-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo. CONCLUSIONS: Our findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.

Self-Delivery Nanobooster to Enhance Immunogenic Cell Death for Cancer Chemoimmunotherapy.

Inducing immunogenic cell death (ICD) is a promising strategy for cancer immunotherapy. Shikonin (SHK), a naphthoquinone compound from Lithospermum erythrorhizon, can stimulate antitumor immunity by inducing ICD. Nevertheless, the immunogenicity of tumor cells killed by SHK is weak. Endoplasmic reticulum (ER) stress is an important intracellular pathway of the ICD effect. Curcumin (CUR) can directly induce ER stress by disrupting Ca2+ homeostasis, which might enhance SHK-induced ICD effect. A self-delivery ICD effect nanobooster (CS-PEG NPs) was developed by the self-assembly of SHK (ICD inducer) and CUR (ICD enhancer) with the assistance of DSPE-PEG2K for cancer chemoimmunotherapy. CS-PEG NPs possessed effective CT26 tumor cell cellular uptake and tumor accumulation ability. Moreover, enhanced cytotoxicity against tumor cells and apoptosis promotion were achieved due to the synergistic effect of CUR and SHK. Notably, CS-PEG NPs induced obvious Ca2+ homeostasis disruption, ER stress, and ICD effect. Subsequently, the neoantigens produced by the robust ICD effect in vivo promoted dendritic cell maturation, which further recruited and activated cytotoxic T lymphocytes. Superior antitumor efficacy and systemic antitumor immunity were observed in the CT26-bearing BALB/c mouse model without side effects in major organs. This study offers a promising self-delivery nanobooster to induce strong ICD effect and antitumor immunity for cancer chemoimmunotherapy.

Inhibitory Effects and Mechanisms of the Novel Shikonin Derivative DMAKO-20 on Melanoma Metastasis and Invasion.

BACKGROUND: Cutaneous melanoma is a malignant tumor with an increasing incidence, prone to recurrence and metastasis. This study aims to explore the effects and mechanisms of the novel shikonin derivative 5,8-dimethyl alkannin oxime derivative (DMAKO-20) on the metastasis and invasion of melanoma cells. METHODS: The inhibitory effects of DMAKO-20 on the melanoma cell line A375 were investigated through Cell Counting Kit-8 (CCK-8), Transwell and angiogenesis experiments. Network pharmacology and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to explore potential sites and pathways involved in this process. Additionally, quantitative polymerase chain reaction (qPCR) and Western blot experiments were conducted before and after drug treatment to verify the expression trends of related pathways and proteins. RESULTS: DMAKO-20 demonstrated selective inhibition of proliferation, invasion and migration of melanoma cells at low concentrations. The WNT pathway appears to be implicated in this process, as DMAKO-20 effectively attenuates its activation, consequently reducing matrix metalloproteinase 9 (MMP9) and Cellular Communication Network Factor 1 (CCN1)/cysteine-rich angiogenic inducer 61 (CYR61) levels. Such modulation inhibits melanoma dissemination and invasion into other tissues. CONCLUSION: DMAKO-20 exhibits the capability to suppress metastasis and invasion of melanoma cells, suggesting its potential for clinical application as an adjuvant therapy against melanoma.

A dual responsive nitric oxide / ß-lapachone co-delivery platform for redox imbalance-enhanced tumor therapy.

Nitric oxide (NO) / ß-Lapachone (Lap) combined therapy by causing oxidative stress is an effective tumor therapy strategy. Herein, a dual-responsive lipid nanoparticles (LNPs) LSNO for NO / Lap co-delivery were constructed from the zinc-coordinated lipid (DSNO(Zn)) and the hydrophobic drug Lap in the presence of helper lipids (DOPE and DSPE-PEG2000). The zinc-coordinated structure in LSNO might elevate the Zn2+ content in tumor cells, contributing to antioxidant imbalance. The fluorescent assays proved the light-triggered NO release and fluorescent self-reporting abilities of LSNO. In addition, the LNPs had good drug release behavior under high concentration of GSH, indicating the NO / drug co-delivery capacity. In vitro antitumor assays showed that the NO / Lap combination treatment group could induce more significant tumor cell growth inhibition and cell apoptosis than individual NO or Lap treatment. The following mechanism studies revealed that NO / Lap combination treatment led to distinct oxidative stress by producing reactive oxygen species (ROS) and peroxynitrite anion (ONOO-). On the other hand, the intracellular redox balance could be further disrupted by Lap-induced NADPH consumption and Zn2+ / NO-induced reductase activities downregulation, thus promoting the degree of cell damage. Besides, it was also found that NO and Lap could directly damage nuclear DNA and induce mitochondrial dysfunction, thereby leading to caspase-3 activation and tumor cell death. These results proved that LSNO could serve as a promising multifunctional tumor therapy platform.

The peptidyl-prolyl isomerase Pin1 controls GM-CSF-induced priming of NADPH oxidase in human neutrophils and priming at inflammatory sites.

The production of superoxide anions and other reactive oxygen species (ROS) by neutrophils is necessary for host defense against microbes. However, excessive ROS production can induce cell damage that participates in the inflammatory response. Superoxide anions are produced by the phagocyte NADPH oxidase, a multicomponent enzyme system consisting of two transmembrane proteins (gp91phox/NOX2 and p22phox) and four soluble cytosolic proteins (p40phox, p47phox, p67phox and the small G proteins Rac1/2). Stimulation of neutrophils by various agonists, such as the bacterial peptide formyl-Met-Leu-Phe (fMLF), induces NADPH oxidase activation and superoxide production, a process that is enhanced by the pro-inflammatory cytokines such as GM-CSF. The pathways involved in this GM-CSF-induced up-regulation or priming are not fully understood. Here we show that GM-CSF induces the activation of the prolyl cis/trans isomerase Pin1 in human neutrophils. Juglone and PiB, two selective Pin1 inhibitors, were able to block GM-CSF-induced priming of ROS production by human neutrophils. Interestingly, GM-CSF induced Pin1 binding to phosphorylated p47phox at Ser345. Neutrophils isolated from synovial fluid of patients with rheumatoid arthritis are known to be primed. Here we show that Pin1 activity was also increased in these neutrophils and that Pin1 inhibitors effectively inhibited ROS hyperproduction by the same cells. These results suggest that the prolyl cis/trans isomerase Pin1 may control GM-CSF-induced priming of ROS production by neutrophils and priming of neutrophils in synovial fluid of rheumatoid arthritis patients. Pharmacological targeting of Pin1 may be a valuable approach to the treatment of inflammation.

In vivo transgenic studies confirm the critical acylation function of LeBAHD56 for shikonin in Lithospermum erythrorhizon.

KEY MESSAGE: LeBAHD56 is preferentially expressed in tissues where shikonin and its derivatives are biosynthesized, and it confers shikonin acylation in vivo. Two WRKY transcriptional factors might regulate LeBAHD56's expression. Shikonin and its derivatives, found in the roots of Lithospermum erythrorhizon, have extensive application in the field of medicine, cosmetics, and other industries. Prior research has demonstrated that LeBAHD1(LeSAT1) is responsible for the biochemical process of shikonin acylation both in vitro and in vivo. However, with the exception of its documented in vitro biochemical function, there is no in vivo genetic evidence supporting the acylation function of the highly homologous gene of LeSAT1, LeBAHD56(LeSAT2), apart from its reported role. Here, we validated the critical acylation function of LeBAHD56 for shikonin using overexpression (OE) and CRISPR/Cas9-based knockout (KO) strategies. The results showed that the OE lines had a significantly higher ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than the control. In contrast, the KO lines had a significantly lower ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than controls. As for its detailed expression patterns, we found that LeBAHD56 is preferentially expressed in roots and callus cells, which are the biosynthesis sites for shikonin and its derivatives. In addition, we anticipated that a wide range of putative transcription factors might control its transcription and verified the direct binding of two crucial WRKY members to the LeBAHD56 promoter's W-box. Our results not only confirmed the in vivo function of LeBAHD56 in shikonin acylation, but also shed light on its transcriptional regulation.

Targeting mTOR and survivin concurrently potentiates radiation therapy in renal cell carcinoma by suppressing DNA damage repair and amplifying mitotic catastrophe.

BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Deucravacitinib and shikonin combination therapy ameliorates imiquimod-induced psoriasis in mice.

INTRODUCTION: TYK2 inhibitors and traditional natural drugs as promising drugs for psoriasis therapy are receiving increasing attention. They both affect different molecules of JAK/STAT pathway, but it is currently unclear whether their combination will enhance the effect on psoriasis. In this study, we used imiquimod (IMQ)-induced psoriasis mouse model to investigate the therapeutic effects of the combined administration of deucravacitinib (TYK2 inhibitor) and shikonin. METHODS: Aldara cream containing 5% IMQ was used to topically treat the dorsal skin of each mouse for a total of six consecutive days to induce psoriasis. The psoriasis area and severity index (PASI) scores were recorded every day. On the 7th day, skin tissues were taken for histopathological examination and the content of cytokines in skin were evaluated. The frequency of immune cells in peripheral blood, spleen and skin were detected through flow cytometry. RESULTS: Compared to the vehicle control group, the psoriasis symptoms and immune disorder improved significantly in the combination therapy group and deucravacitinib treatment group on the 7th day, and the expressions of p-STAT3 and Ki67 in skin were reduced as well. Moreover, the combined treatment of deucravacitinib and shikonin for psoriasis was superior to the monotherapy group, especially in inhibiting abnormal capillaries proliferation, reducing immune cells infiltration and decreasing the concentration of IL-12p70 in skin. CONCLUSION: The combination of deucravacitinib and shikonin is a promising clinical application.

Naphthoquinone-Quinolone Hybrids with Antitumor Effects on Breast Cancer Cell Lines-From the Synthesis to 3D-Cell Culture Effects.

Breast cancer stands as one of the foremost cause of cancer-related deaths globally, characterized by its varied molecular subtypes. Each subtype requires a distinct therapeutic strategy. Although advancements in treatment have enhanced patient outcomes, significant hurdles remain, including treatment toxicity and restricted effectiveness. Here, we explore the anticancer potential of novel 1,4-naphthoquinone/4-quinolone hybrids on breast cancer cell lines. The synthesized compounds demonstrated selective cytotoxicity against Luminal and triple-negative breast cancer (TNBC) cells, which represent the two main molecular types of breast cancer that depend most on cytotoxic chemotherapy, with potency comparable to doxorubicin, a standard chemotherapeutic widely used in breast cancer treatment. Notably, these derivatives exhibited superior selectivity indices (SI) when compared to doxorubicin, indicating lower toxicity towards non-tumor MCF10A cells. Compounds 11a and 11b displayed an improvement in IC50 values when compared to their precursor, 1,4-naphthoquinone, for both MCF-7 and MDA-MB-231 and a comparable value to doxorubicin for MCF-7 cells. Also, their SI values were superior to those seen for the two reference compounds for both cell lines tested. Mechanistic studies revealed the ability of the compounds to induce apoptosis and inhibit clonogenic potential. Additionally, the irreversibility of their effects on cell viability underscores their promising therapeutic utility. In 3D-cell culture models, the compounds induced morphological changes indicative of reduced viability, supporting their efficacy in a more physiologically relevant model of study. The pharmacokinetics of the synthesized compounds were predicted using the SwissADME webserver, indicating that these compounds exhibit favorable drug-likeness properties and potential as antitumor agents. Overall, our findings underscore the promise of these hybrid compounds as potential candidates for breast cancer chemotherapy, emphasizing their selectivity and efficacy.

ß-Lapachone promotes the recruitment and polarization of tumor-associated neutrophils (TANs) toward an antitumor (N1) phenotype in NQO1-positive cancers.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in most solid cancers, emerging as a promising target for tumor-selective killing. ß-Lapachone (ß-Lap), an NQO1 bioactivatable drug, exhibits significant antitumor effects on NQO1-positive cancer cells by inducing immunogenic cell death (ICD) and enhancing tumor immunogenicity. However, the interaction between ß-Lap-mediated antitumor immune responses and neutrophils, novel antigen-presenting cells (APCs), remains unknown. This study demonstrates that ß-Lap selectively kills NQO1-positive murine tumor cells by significantly increasing intracellular ROS formation and inducing DNA double strand breaks (DSBs), resulting in DNA damage. Treatment with ß-Lap efficiently eradicates immunocompetent murine tumors and significantly increases the infiltration of tumor-associated neutrophils (TANs) into the tumor microenvironment (TME), which plays a crucial role in the drug's therapeutic efficacy. Further, the presence of ß-Lap-induced antigen medium leads bone marrow-derived neutrophils (BMNs) to directly kill murine tumor cells, aiding in dendritic cells (DCs) recruitment and significantly enhancing CD8+ T cell proliferation. ß-Lap treatment also drives the polarization of TANs toward an antitumor N1 phenotype, characterized by elevated IFN-ß expression and reduced TGF-ß cytokine expression, along with increased CD95 and CD54 surface markers. ß-Lap treatment also induces N1 TAN-mediated T cell cross-priming. The HMGB1/TLR4/MyD88 signaling cascade influences neutrophil infiltration into ß-Lap-treated tumors. Blocking this cascade or depleting neutrophil infiltration abolishes the antigen-specific T cell response induced by ß-Lap treatment. Overall, this study provides comprehensive insights into the role of tumor-infiltrating neutrophils in the ß-Lap-induced antitumor activity against NQO1-positive murine tumors.

Buparvaquone Induces Ultrastructural and Physiological Alterations Leading to Mitochondrial Dysfunction and Caspase-Independent Apoptotic Cell Death in Leishmania donovani.

Leishmaniasis is a neglected tropical disease (endemic in 99 countries) caused by parasitic protozoa of the genus Leishmania. As treatment options are limited, there is an unmet need for new drugs. The hydroxynaphthoquinone class of compounds demonstrates broad-spectrum activity against protozoan parasites. Buparvaquone (BPQ), a member of this class, is the only drug licensed for the treatment of theileriosis. BPQ has shown promising antileishmanial activity but its mode of action is largely unknown. The aim of this study was to evaluate the ultrastructural and physiological effects of BPQ for elucidating the mechanisms underlying the in vitro antiproliferative activity in Leishmania donovani. Transmission and scanning electron microscopy analyses of BPQ-treated parasites revealed ultrastructural effects characteristic of apoptosis-like cell death, which include alterations in the nucleus, mitochondrion, kinetoplast, flagella, and the flagellar pocket. Using flow cytometry, laser scanning confocal microscopy, and fluorometry, we found that BPQ induced caspase-independent apoptosis-like cell death by losing plasma membrane phospholipid asymmetry and cell cycle arrest at sub-G0/G1 phase. Depolarization of the mitochondrial membrane leads to the generation of oxidative stress and impaired ATP synthesis followed by disruption of intracellular calcium homeostasis. Collectively, these findings provide valuable mechanistic insights and demonstrate BPQ's potential for development as an antileishmanial agent.

Extracellularly secreted cysteine derived from cystine regulates oxidative and electrophilic stress in HepG2 cells.

While cysteine (CysSH) is known to be exported into the extracellular space, its biological significance is not well understood. The present study examined the movement of extracellular CysSH using stable isotope-labeled cystine (CysSSCys), which is transported into cells and reduced to CysSH. Exposure of HepG2 cells to 100 µM stable isotope-labeled CysSSCys resulted in 70 µM labeled CysSH in cell medium 1 h after CysSSCys exposure. When the cell medium was collected and incubated with either hydrogen peroxide (H2O2) or atmospheric electrophiles, such as 1,2-naphthoquinone, 1,4-naphthoquinone and 1,4-benzoquinone, CysSH in the cell medium was almost completely consumed. In contrast, extracellular levels of CysSH were unaltered during exposure of HepG2 cells to H2O2 for up to 2 h, suggesting redox cycling of CysSSCys/CysSH in the cell system. Experiments with and without changing cell medium containing CysSH from HepG2 cells revealed that oxidative and electrophilic modifications of cellular proteins, caused by exposure to H2O2 and 1,2-naphthoquinone, were significantly repressed by CysSH in the medium. We also examined participation of enzymes and/or antioxidants in intracellular reduction of CysSSCys to CysSH. These results provide new findings that extracellular CysSH derived from CysSSCys plays a role in the regulation of oxidative and electrophilic stress.

Targeting BIRC5 as a therapeutic approach to overcome ASXL1-associated decitabine resistance.

Hypomethylating agents (HMAs) are widely employed in the treatment of myeloid malignancies. However, unresponsive or resistant to HMAs occurs in approximately 50 % of patients. ASXL1, one of the most commonly mutated genes across the full spectrum of myeloid malignancies, has been reported to predict a lower overall response rate to HMAs, suggesting an essential need to develop effective therapeutic strategies for the patients with HMA failure. Here, we investigated the impact of ASXL1 on cellular responsiveness to decitabine treatment. ASXL1 deficiency increased resistance to decitabine treatment in AML cell lines and mouse bone marrow cells. Transcriptome sequencing revealed significant alterations in genes regulating cell cycle, apoptosis, and histone modification in ASXL1 deficient cells that resistant to decitabine. BIRC5 was identified as a potential target for overcoming decitabine resistance in ASXL1 deficient cells. Furthermore, our experimental evidence demonstrated that the small-molecule inhibitor of BIRC5 (YM-155) synergistically sensitized ASXL1 deficient cells to decitabine treatment. This study sheds light on the molecular mechanisms underlying the ASXL1-associated HMA resistance and proposes a promising therapeutic strategy for improving treatment outcomes in affected individuals.

Tumor-associated macrophage enhances PD-L1-mediated immune escape of bladder cancer through PKM2 dimer-STAT3 complex nuclear translocation.

Tumor-associated macrophages (TAMs) are important components of the tumor microenvironment (TME) and strongly associated with poor prognosis and drug resistance, including checkpoint blockade immunotherapy in solid tumor patients. However, the mechanism by which TAM affects immune metabolism reprogramming and immune checkpoint signalling pathway in the TME remains elusive. In this study we found that transforming growth factor-beta (TGF-ß) secreted by M2-TAMs increased the level of glycolysis in bladder cancer (BLCA) and played important role in PD-L1-mediated immune evasion through pyruvate kinase isoenzymes M2 (PKM2). Mechanistically, TGF-ß promoted high expression of PKM2 by promoting the nuclear translocation of PKM2 dimer in conjunction with phosphorylated signal transducer and activator of transcription (p-STAT3), which then exerted its kinase activity to promote PD-L1 expression in BLCA. Moreover, SB-431542 (TGF-ß blocker) and shikonin (PKM2 inhibitor) significantly reduced PD-L1 expression and inhibited BLCA growth and organoids by enhancing anti-tumor immune responses. In conclusion, M2-TAM-derived TGF-ß promotes PD-L1-mediated immune evasion in BLCA by increasing the PKM2 dimer-STAT3 complex nuclear translocation. Combined blockade of the TGF-ß receptor and inhibition of PKM2 effectively prevent BLCA progression and immunosuppression, providing a potential targeted therapeutic strategy for BLCA.