Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy.

Oxidative damage to lens epithelial cells plays an important role in the development of age-related cataract, and the health of the lens has important implications for overall ocular health. As a result, there is a need for effective therapeutic agents that prevent oxidative damage to the lens. Thiol antioxidants such as tiopronin or N-(2-mercaptopropionyl)glycine (MPG), N-acetylcysteine amide (NACA), N-acetylcysteine (NAC), and exogenous glutathione (GSH) may be promising candidates for this purpose, but their ability to protect lens epithelial cells is not well understood. The effectiveness of these compounds was compared by exposing human lens epithelial cells (HLE B-3) to the chemical oxidant tert-butyl hydroperoxide (tBHP) and treating the cells with each of the antioxidant compounds. MTT cell viability, apoptosis, reactive oxygen species (ROS), and levels of intracellular GSH, the most important antioxidant in the lens, were measured after treatment. All four compounds provided some degree of protection against tBHP-induced oxidative stress and cytotoxicity. Cells treated with NACA exhibited the highest viability after exposure to tBHP, as well as decreased ROS and increased intracellular GSH. Exogenous GSH also preserved viability and increased intracellular GSH levels. MPG scavenged significant amounts of ROS, and NAC increased intracellular GSH levels. Our results suggest that both scavenging ROS and increasing GSH may be necessary for effective protection of lens epithelial cells. Further, the compounds tested may be useful for the development of therapeutic strategies that aim to prevent oxidative damage to the lens.

BACKGROUND: Haematopoietic stem cell (HSC) infusion has demonstrated short-term improvement in liver functions in patients with chronic liver disease. The combination of HSC with mesenchymal stem cells (MSCs), which has an immunomodulatory effect, may augment the effects and enhance the duration of improvements on liver functions. The aim of the present study was to assess the safety of infusing the combination of autologous HSCs and MSCs in decompensated liver cirrhosis.
METHODS: In phase I of the study, in vitro assessment was performed to observe the effect of coculturing MSCs with HSCs on their viability and cytokine profiles. Phase II of the study was to assess the safety of combination of stem cell infusions. Bone marrow (50 ml) was aspirated for MSC isolation and expansion using standard protocol. Patients received subcutaneous doses (n = 5) of granulocyte colony-stimulating factor (G-CSF) for stem cell mobilization followed by leukapheresis for harvesting HSCs using CliniMacs. HSCs and MSCs were infused through the hepatic artery under fluoroscopic guidance and were monitored for any adverse effects.
RESULTS: In vitro studies revealed 94% viable HSCs in coculture similar to monoculture. HSCs released only interleukin (IL)-8, whereas MSCs secreted IL-8 and IL-6 in monocultures, and both IL-8 and IL-6 were secreted in coculture. G-CSF administration- and bone marrow aspiration-related complications were not observed. Infusion of the cells through the hepatic artery was safe, and no postprocedural complications were noted.
CONCLUSIONS: The combination of autologous HSC and MSC infusion is a safe procedure in patients with decompensated liver cirrhosis, and the outcomes needed to be assessed in larger studies.
BACKGROUND: NCT04243681.

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Salvia miltiorrhiza Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out GSDME switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC via GSDME-dependent pyroptosis.

Obtaining T cells by reprogramming is one of the major goals in regenerative medicine. Here, we describe a protocol for generating functional T cells from Hoxb5-expressing pro/pre-B cells in vivo. This protocol includes the construction of Hoxb5 recombinant plasmids, retroviral packaging, isolation and viral transduction of pro/pre-B cells, cell transplantation, and phenotypic analysis of induced T cells. The procedure is reproducible and straightforward, providing an approach for generating induced T cells for translational research.

BACKGROUND: Licochalconce (LC) H is an artificial compound in the course of synthesizing LCC in 2013. So far, few studies on the effects of LCH have been found in the literature. Despite progress in treatment modalities for oral cancer, the cure from cancer has still limitations.
OBJECTIVE: The effects of LCH were investigated on human oral squamous cell carcinoma (OSCC) cells to elucidate its mechanisms.
METHODS: We explored the mechanism of action of LCH by which it could have effects on JAK2/STAT3 signaling pathway.
METHODS: To confirm LCH anti-cancer effect, analyzed were MTT assay, DAPI staining, soft agar, kinase assay, molecular docking simulation, flow cytometry and Western blotting analysis.
RESULTS: According to docking and molecular dynamics simulations, the predicted pose of the complex LCH and JAK2 seems reasonable and LCH is strongly bound to active JAK2 with opened activation loop. The LCH inhibitor is surrounded by specific ATP-binding pocket in which it is stabilized by forming hydrogen bonds and hydrophobic interactions. It is shown that LCH plays as a competitive inhibitor in an active state of JAK2. LCH caused a dose-dependent decrease in phosphorylation of JAK2 and STAT3. More interestingly, LCH suppressed JAK2 kinase activity in vitro by its direct binding to the JAK2. LCH significantly inhibited the JAK2/STAT3 signaling pathway, causing the down-regulation of target genes such as Bcl-2, survivin, cyclin D1, p21 and p27. In addition, LCH inhibited cell proliferation and colony formation of OSCC cells in a dose- and time-dependent manner, as well as induction of cell apoptosis through extrinsic and intrinsic pathway. The induction of apoptosis in OSCC cells by LCH was evident in the increased production of ROS, loss of mitochondrial membrane potential, release of cyto c, variation of apoptotic proteins and activation of caspase cascade.
CONCLUSIONS: LCH not only induces apoptosis in OSCC cells through the JAK/STAT3 signaling pathway but also inhibits cell growth. It is proposed that LCH has a promising use for the chemotherapeutic agent of oral cancer.

Cancer stem-like cells (CSCs) are a rare subpopulation of cancer cells capable of propagating the disease and causing cancer recurrence. In this study, we found that the cellular localization of PKB/Akt kinase affects the maintenance of CSCs. When Akt tagged with nuclear localization signal (Akt-NLS) was overexpressed in SKBR3 and MDA-MB468 cells, these cells showed a 10-15% increase in the number of cells with CSCs enhanced ALDH activity and demonstrated a CD44(+High)/CD24(-Low) phenotype. This effect was completely reversed in the presence of Akt-specific inhibitor, triciribine. Furthermore, cells overexpressing Akt or Akt-NLS were less likely to be in G0/G1 phase of the cell cycle by inactivating p21(Waf1/Cip1) and exhibited increased clonogenicity and proliferation as assayed by colony-forming assay (mammosphere formation). Thus, our data emphasize the importance the intracellular localization of Akt has on stemness in human breast cancer cells. It also indicates a new robust way for improving the enrichment and culture of CSCs for experimental purposes. Hence, it allows for the development of simpler protocols to study stemness, clonogenic potency, and screening of new chemotherapeutic agents that preferentially target cancer stem cells.
CONCLUSIONS: The presented data, (i) shows new, stemness-promoting role of nuclear Akt/PKB kinase, (ii) it underlines the effects of nuclear Akt on cell cycle regulation, and finally (iii) it suggests new ways to study cancer stem-like cells.

A wide variety of signaling transduction pathways contribute to tumorigenesis. Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family and its upregulation is closely correlated with tumor progression and prognosis of multiple cancer types, including colorectal cancer. However, the molecular mechanisms by which FOXQ1 promotes tumorigenesis, especially cancer cell invasion and metastasis in colorectal cancer, have not been fully elucidated. In the present study, we demonstrate that FOXQ1 is overexpressed in colorectal tumor tissues and its expression level is closely correlated with the stage and lymph node metastasis of colorectal cancer. In in vitro cultured SW480 colorectal cancer cells, knockdown of FOXQ1 expression by small interfering RNA greatly diminished the aggressive tumor behaviors of SW480 cells, including angiogenesis, invasion, epithelial-mesenchymal transition, and resistance to chemotherapy drug-induced apoptosis. Further mechanistic investigation showed that FOXQ1 silencing prevents the nuclear translocation of β-catenin, thus reducing the activity of Wnt signaling. Moreover, TGF-β1 induced the expression of FOXQ1 as well as the migration and invasion of SW480 cells, which was partially prevented following knockdown of FOXQ1. Our results demonstrate that FOXQ1 plays a critical role during the tumorigenesis of colorectal cancer and is a mediator of the crosstalk between Wnt and TGF-β signaling pathways. Our findings provide further insight into the cancer biology of colorectal cancer and suggest that FOXQ1 is a potential therapeutic target for the development of therapies for colorectal cancer.

Spermatozoa and lentiviruses are two of nature\'s most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies. Lentiviral vectors encoding Green Fluorescent Protein (GFP) were shown to be efficiently processed and expressed in sperm. When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts. This simple technique of directly transducing spermatozoa has potential to be a powerful tool for the study of early and pre-implantation development and could be used as a technique in transgenic development and vertical viral transmission studies.