Human ß-casein-derived peptide BCCY-1 improved the intestinal barrier integrity by regulating the TLR4/eNOS/3-Nitrotyrosine axis.

Necrotizing enterocolitis (NEC) is a lethal gastrointestinal disease affecting premature infants. Although earlier studies have highlighted protective effects of milk-derived peptides against NEC, the role of the human ß-casein-derived peptide BCCY-1 in intestinal barrier protection has never been investigated. Here, we showed that BCCY-1 alleviated the phenotype of NEC, reduced intestinal expression of Toll-like receptor 4 (TLR4) and interleukin-6, and improved the intestinal barrier integrity. NEC-associated multi-organ injury and impaired bone marrow hematopoiesis were also attenuated by BCCY-1. Metabolic screening revealed significant changes in intestinal metabolites in the NEC and NEC + BCCY-1 groups. Further analysis disclosed inhibition of 3-Nitrotyrosine formation due to the preservation of endothelial nitric oxide synthase (eNOS) activity, which was associated with the interactions between BCCY-1 and lipopolysaccharides, leading to disruption of TLR4 signaling. Our findings suggested that BCCY-1 improved intestinal barrier integrity through modulating the TLR4/eNOS/3-Nitrotyrosine axis, highlighting its potential role in the maintenance of intestinal health.

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OBJECTIVES: To study the effect of gut microbiota on hematopoiesis in a neonatal rat model of necrotizing enterocolitis (NEC). METHODS: Neonatal Sprague-Dawley rats were randomly divided into a control group and a model group (NEC group), with 6 rats in each group. Formula milk combined with hypoxia and cold stimulation was used to establish a neonatal rat model of NEC. Hematoxylin and eosin staining was used to observe the pathological changes of intestinal tissue and hematopoiesis-related organs. Routine blood tests were conducted for each group. Immunohistochemistry was used to observe the changes in specific cells in hematopoiesis-related organs. Flow cytometry was used to measure the changes in specific cells in bone marrow. 16S rDNA sequencing was used to observe the composition and abundance of gut microbiota. RESULTS: Compared with the control group, the NEC group had intestinal congestion and necrosis, damage, atrophy, and shedding of intestinal villi, and a significant increase in NEC histological score. Compared with the control group, the NEC group had significantly lower numbers of peripheral blood leukocytes and lymphocytes (P<0.05), nucleated cells in the spleen, thymus, and bone marrow, and small cell aggregates with basophilic nuclei in the liver (P<0.05). The NEC group had significant reductions in CD71+ erythroid progenitor cells in the liver, CD45+ lymphocytes in the spleen and bone marrow, CD3+ T lymphocytes in thymus, and the proportion of CD45+CD3-CD43+SSChi neutrophils in bone marrow (P<0.05). There was a significant difference in the composition of gut microbiota between the NEC and control groups, and the NEC group had a significant reduction in the abundance of Ligilactobacillus and a significant increase in the abundance of Escherichia-Shigella (P<0.05), which replaced Ligilactobacillus and became the dominant flora. CONCLUSIONS: Multi-lineage hematopoietic disorder may be observed in a neonatal rat model of NEC, which may be associated with gut microbiota dysbiosis and abnormal multiplication of the pathogenic bacteria Escherichia-Shigella.

CPT1A-mediated fatty acid oxidation confers cancer cell resistance to immune-mediated cytolytic killing.

Although tumor-intrinsic fatty acid ß-oxidation (FAO) is implicated in multiple aspects of tumorigenesis and progression, the impact of this metabolic pathway on cancer cell susceptibility to immunotherapy remains unknown. Here, we report that cytotoxicity of killer T cells induces activation of FAO and upregulation of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme of FAO in cancer cells. The repression of CPT1A activity or expression renders cancer cells more susceptible to destruction by cytotoxic T lymphocytes. Our mechanistic studies reveal that FAO deficiency abrogates the prosurvival signaling in cancer cells under immune cytolytic stress. Furthermore, we identify T cell-derived IFN-γ as a major factor responsible for induction of CPT1A and FAO in an AMPK-dependent manner, indicating a dynamic interplay between immune effector cells and tumor targets. While cancer growth in the absence of CPT1A remains largely unaffected, established tumors upon FAO inhibition become significantly more responsive to cellular immunotherapies including chimeric antigen receptor-engineered human T cells. Together, these findings uncover a mode of cancer resistance and immune editing that can facilitate immune escape and limit the benefits of immunotherapies.

Myeloid-derived suppressor cells: Are they involved in gestational diabetes mellitus?

Gestational diabetes mellitus (GDM) is currently the most common metabolic complication during pregnancy, with an increasing prevalence worldwide. Maternal immune dysregulation might be partly responsible for the pathophysiology of GDM. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells, emerging as a new immune regulator with potent immunosuppressive capacity. Although the fate and function of these cells were primarily described in pathological conditions such as cancer and infection, accumulating evidences have spotlighted their beneficial roles in homeostasis and physiological conditions. Recently, several studies have explored the roles of MDSCs in the diabetic microenvironment. However, the fate and function of these cells in GDM are still unknown. The current review summarized the existing knowledges about MDSCs and their potential roles in diabetes during pregnancy in an attempt to highlight our current understanding of GDM-related immune dysregulation and identify areas where further study is required.

SRA inhibition improves antitumor potency of antigen-targeted chaperone vaccine.

We have previously demonstrated that scavenger receptor A (SRA) acts as an immunosuppressive regulator of dendritic cell (DC) function in activating antitumor T cells. Here we investigate the potential of inhibiting SRA activity to enhance DC-targeted chaperone vaccines including one that was recently evaluated in melanoma patients. We show that short hairpin RNA-mediated SRA silencing significantly enhances the immunogenicity of DCs that have captured chaperone vaccines designed to target melanoma (i.e., hsp110-gp100) and breast cancer (i.e., hsp110-HER/Neu-ICD). SRA downregulation results in heightened activation of antigen-specific T cells and increased CD8+ T cell-dependent tumor inhibition. Additionally, small interfering RNA (siRNA) complexed with the biodegradable, biocompatible chitosan as a carrier can efficiently reduce SRA expression on CD11c+ DCs in vitro and in vivo. Our proof-of-concept study shows that direct administration of the chitosan-siRNA complex to mice promotes chaperone vaccine-elicited cytotoxic T lymphocyte (CTL) response, culminating in improved eradication of experimental melanoma metastases. Targeting SRA with this chitosan-siRNA regimen combined with the chaperone vaccine also leads to reprogramming of the tumor environment, indicated by elevation of the cytokine genes (i.e., ifng, il12) known to skew Th1-like cellular immunity and increased tumor infiltration by IFN-γ+CD8+ CTLs as well as IL-12+CD11c+ DCs. Given the promising antitumor activity and safety profile of chaperone vaccine in cancer patients, further optimization of the chitosan-siRNA formulation to potentially broaden the immunotherapeutic benefits of chaperone vaccine is warranted.

Irreversible tyrosine kinase inhibitors induce the endocytosis and downregulation of ErbB2.

Erb-b2 receptor tyrosine kinase 2 (ErbB2) is an oncogene that frequently overexpressed in a subset of cancers. Anti-ErbB2 therapies have been developed to treat these types of cancers. However, less is known about how anti-ErbB2 drugs affect the trafficking and degradation of ErbB2. We demonstrate that the reversible and irreversible tyrosine kinase inhibitors (TKIs) differentially modulate the subcellular trafficking and downregulation of ErbB2. Only the irreversible TKIs can induce the loss of ErbB2 expression, which is not dependent on proteasome or lysosome. The irreversible TKIs promote ErbB2 endocytosis from plasma membrane and enhance the ErbB2 accumulation at endosomes. The endocytosis of ErbB2 is mediated by a dynamin-dependent but clathrin-independent mechanism. Blocking of ErbB2 endocytosis can impair the TKI-induced ErbB2 downregulation.

Pharmacologic Inhibition of SHP2 Blocks Both PI3K and MEK Signaling in Low-epiregulin HNSCC via GAB1.

Preclinical and clinical studies have evidenced that effective targeted therapy treatment against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, re-activation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found HNSCC was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCC, resulting in downregulation of mTORC signaling and anti-tumor effects across several HNSCC mouse models, including an HPV+ patient-derived xenograft (PDX). Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2-GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. We demonstrate HNSCCs were highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin.

High-risk neuroblastoma with NF1 loss of function is targetable using SHP2 inhibition.

Reoccurring/high-risk neuroblastoma (NB) tumors have the enrichment of non-RAS/RAF mutations along the mitogen-activated protein kinase (MAPK) signaling pathway, suggesting that activation of MEK/ERK is critical for their survival. However, based on preclinical data, MEK inhibitors are unlikely to be active in NB and have demonstrated dose-limiting toxicities that limit their use. Here, we explore an alternative way to target the MAPK pathway in high-risk NB. We find that NB models are among the most sensitive among over 900 tumor-derived cell lines to the allosteric SHP2 inhibitor SHP099. Sensitivity to SHP099 in NB is greater in models with loss or low expression of the RAS GTPase activation protein (GAP) neurofibromin 1 (NF1). Furthermore, NF1 is lower in advanced and relapsed NB and NF1 loss is enriched in high-risk NB tumors regardless of MYCN status. SHP2 inhibition consistently blocks tumor growth in high-risk NB mouse models, revealing a new drug target in relapsed NB.

Genomic screening reveals ubiquitin-like modifier activating enzyme 1 as a potent and druggable target in c-MYC-high triple negative breast cancer models.

Triple negative breast cancer (TNBC) accounts for over 30% of all breast cancer (BC)-related deaths, despite accounting for only 10% to 15% of total BC cases. Targeted therapy development has largely stalled in TNBC, underlined by a lack of traditionally druggable addictions like receptor tyrosine kinases (RTKs). Here, through full genome CRISPR/Cas9 screening of TNBC models, we have uncovered the sensitivity of TNBCs to the depletion of the ubiquitin-like modifier activating enzyme 1 (UBA1). Targeting UBA1 with the first-in-class UBA1 inhibitor TAK-243 induced unresolvable endoplasmic reticulum (ER)-stress and activating transcription factor 4 (ATF4)-mediated upregulation of proapoptotic NOXA, leading to cell death. c-MYC expression correlates with TAK-243 sensitivity and cooperates with TAK-243 to induce a stress response and cell death. Importantly, there was an order of magnitude greater sensitivity of TNBC lines to TAK-243 compared to normal tissue-derived cells. In five patient derived xenograft models (PDXs) of TNBC, TAK-243 therapy led to tumor inhibition or frank tumor regression. Moreover, in an intracardiac metastatic model of TNBC, TAK-243 markedly reduced metastatic burden, indicating UBA1 is a potential new target in TNBC expressing high levels of c-MYC.

Venetoclax-based Rational Combinations are Effective in Models of MYCN-amplified Neuroblastoma.

Venetoclax is a small molecule inhibitor of the prosurvival protein BCL-2 that has gained market approval in BCL-2-dependent hematologic cancers including chronic lymphocytic leukemia and acute myeloid leukemia. Neuroblastoma is a heterogenous pediatric cancer with a 5-year survival rate of less than 50% for high-risk patients, which includes nearly all cases with amplified MYCN We previously demonstrated that venetoclax is active in MYCN-amplified neuroblastoma but has limited single-agent activity in most models, presumably the result of other pro-survival BCL-2 family protein expression or insufficient prodeath protein mobilization. As the relative tolerability of venetoclax makes it amenable to combining with other therapies, we evaluated the sensitivity of MYCN-amplified neuroblastoma models to rational combinations of venetoclax with agents that have both mechanistic complementarity and active clinical programs. First, the MDM2 inhibitor NVP-CGM097 increases the prodeath BH3-only protein NOXA to sensitize p53-wild-type, MYCN-amplified neuroblastomas to venetoclax. Second, the MCL-1 inhibitor S63845 sensitizes MYCN-amplified neuroblastoma through neutralization of MCL-1, inducing synergistic cell killing when combined with venetoclax. Finally, the standard-of-care drug cocktail cyclophosphamide and topotecan reduces the apoptotic threshold of neuroblastoma, thus setting the stage for robust combination efficacy with venetoclax. In all cases, these rational combinations translated to in vivo tumor regressions in MYCN-amplified patient-derived xenograft models. Venetoclax is currently being evaluated in pediatric patients in the clinic, including neuroblastoma (NCT03236857). Although establishment of safety is still ongoing, the data disclosed herein indicate rational and clinically actionable combination strategies that could potentiate the activity of venetoclax in patients with amplified MYCN with neuroblastoma.

MYCN-Amplified Neuroblastoma Is Addicted to Iron and Vulnerable to Inhibition of the System Xc-/Glutathione Axis.

MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer-related deaths. Therapy improvements for this subtype of cancer are a high priority. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1. Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. This dependence creates a marked vulnerability to targeting the system Xc-/glutathione (GSH) pathway with ferroptosis inducers. This reliance can be exploited through therapy with FDA-approved rheumatoid arthritis drugs sulfasalazine (SAS) and auranofin: in MYCN-amplified, patient-derived xenograft models, both therapies blocked growth and induced ferroptosis. SAS and auranofin activity was largely mitigated by the ferroptosis inhibitor ferrostatin-1, antioxidants like N-acetyl-L-cysteine, or by the iron scavenger deferoxamine (DFO). DFO reduced auranofin-induced ROS, further linking increased iron capture in MYCN-amplified neuroblastoma to a therapeutic vulnerability to ROS-inducing drugs. These data uncover an oncogene vulnerability to ferroptosis caused by increased iron accumulation and subsequent reliance on the system Xc-/GSH pathway. SIGNIFICANCE: This study shows how MYCN increases intracellular iron levels and subsequent GSH pathway activity and demonstrates the antitumor activity of FDA-approved SAS and auranofin in patient-derived xenograft models of MYCN-amplified neuroblastoma.

A human ß-casein-derived peptide BCCY-1 modulates the innate immune response.

In this study, we report a novel peptide corresponding to the sequence of human ß-casein (named BCCY-1), which was identified in our previous peptidome analysis of human milk and has great immunomodulatory activity. The results revealed that peptide BCCY-1, but not the scrambled version, enhanced monocyte migration without obvious toxicities. This selective effect was mediated via increased production of chemokines by peptide stimulated monocytes. Moreover, BCCY-1 exerted its modulatory effects by activating nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling. The abundances of peptide BCCY-1 and the peptides partially encompassing its fragment were found to be lower in preterm milk than in term milk. Our study may lead to new insights into the immunoregulatory effects of casein-derived peptides and facilitate the discovery of novel peptide-based food and pharmaceutical products.

Rat BAT xenotransplantation recovers the fertility and metabolic health of PCOS mice.

Polycystic ovarian syndrome (PCOS) is a major severe ovary disorder affecting 5-10% of reproductive women around the world. PCOS can be considered a metabolic disease because it is often accompanied by obesity and diabetes. Brown adipose tissue (BAT) contains abundant mitochondria and adipokines and has been proven to be effective for treating various metabolic diseases. Recently, allotransplanted BAT successfully recovered the ovarian function of PCOS rat. However, BAT allotransplantation could not be applied to human PCOS; the most potent BAT is from infants, so voluntary donors are almost inaccessible. We recently reported that single BAT xenotransplantation significantly prolonged the fertility of aging mice and did not cause obvious immunorejection. However, PCOS individuals have distinct physiologies from aging mice; thus, it remains essential to study whether xenotransplanted rat BAT can be used for treating PCOS mice. In this study, rat-to-mouse BAT xenotransplantation, fortunately, did not cause severe rejection reaction, and significantly recovered ovarian functions, indicated by the recovery of fertility, oocyte quality, and the levels of multiple essential genes and kinases. Besides, the blood biochemical index, glucose resistance, and insulin resistance were improved. Moreover, transcriptome analysis showed that the recovered PCOS F0 mother following BAT xenotransplantation could also benefit the F1 generation. Finally, BAT xenotransplantation corrected characteristic gene expression abnormalities found in the ovaries of human PCOS patients. These findings suggest that BAT xenotransplantation could be a novel therapeutic strategy for treating PCOS patients.

Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes.

OBJECTIVES: Little is known about the roles of integral membrane proteins beyond channels, carriers or receptors in meiotic oocytes. The transmembrane protein Fam70A was previously identified as a likely "female fertility factor" in Fox3a-knockout mouse ovaries where almost all follicles underwent synchronous activation and the mice became infertile very early. However, whether Fam70A functions in oocyte meiosis remains unknown. Therefore, the present study aimed to address this question. MATERIALS AND METHODS: Co-immunoprecipitation, immunogold labelling-electron microscopy, co-localization and yeast two-hybrid assays were used to verify the interaction. Antibody or small interfering RNA transfection was used to deplete the proteins. Immunofluorescence, immunohistochemistry and live tracker staining were used to examine the localization or characterize phenotypes. Western blot was used to examine the protein level. RESULTS: Fam70A was enriched in oocyte membranes important for normal meiosis. Fam70A depletion remarkably disrupted spindle assembly, chromosome congression and first polar body extrusion, which subsequently increased aneuploidy and abnormal fertilization. Moreover, Fam70A directly bound Wnt5a, the most abundant Wnt member within oocytes. Depletion of either Fam70A or Wnt5a remarkably increased adenomatous polyposis coli (APC), which stabilizes active ß-catenin and microtubules. Consequently, depletion of either Fam70A or Wnt5a remarkably increased p-ß-catenin (inactive form) and acetylated tubulin, while APC knockdown remarkably decreased these two. Furthermore, Fam70A depletion remarkably reduced Akt phosphorylation. CONCLUSIONS: Fam70A regulates meiosis and quality of mouse oocytes through both canonical and non-canonical Wnt5a signalling pathways.

Immunomodulatory significance of natural peptides in mammalians: Promising agents for medical application.

Modulation of immune responses by immunoregulatory agents, such as the natural or synthetic immunomodulatory peptides, has been suggested as a potential strategy to modulate immune system against infection and other immune-related diseases. These compositionally simple peptides have attracted much attention for many drug developers, due to their high activity, low toxicity and clear target specificity. Host defence peptides and milk-derived peptides are two kinds of natural immunomodulatory peptides which have been widely studied in mammalians. They could participate at the interface of innate and adaptive immunity by regulating immune effector cells. This review summarizes the recent advances in host defence peptides and milk-derived peptides as well as their general characteristics, immunomodulatory functions and possible applications.

Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression.

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 µM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 µM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

Sorting Nexin 5 Controls Head and Neck Squamous Cell Carcinoma Progression by Modulating FBW7.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide. Long-term survival rates in patients with HNSCC have not increased significantly in the past 30 years. Therefore, looking for novel molecular targets that control HNSCC progression is urgently required to improve the treatment of HNSCC. Here, we identified Sorting Nexin 5 (SNX5) as a new regulator that plays an oncogenic function in HNSCC progression. Analyzing HNSCC patients' data from the Cancer Genome Atlas (TCGA) indicates that the expression levels of SNX5 in HNSCC are significantly elevated compared to normal tissues. Furthermore, higher SNX5 expression correlates with a worse prognosis for HNSCC patients. These results suggest that SNX5 has an oncogenic role. Consistently, loss of SNX5 in HNSCC cells dramatically reduces colony formation and significantly decreases tumor growth in xenograft mouse models. SNX5 interacts with the tumor suppressor F-box/WD repeat-containing protein 7 (FBW7), an E3 ubiquitin ligase that mediates ubiquitination and degradation of oncoproteins such as c-Myc, NOTCH1, and Cyclin E1. By interacting with FBW7, SNX5 inhibits FBW7-mediated oncoproteins ubiquitination. In this way, SNX5 decreases the FBW7-mediated oncoproteins degradation to promote HNSCC progression.

EGFR tyrosine kinase inhibitors differentially affect autophagy in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide. The majority of HNSCCs overexpress Epidermal Growth Factor Receptor (EGFR), an essential receptor tyrosine kinase (RTK) that promotes HNSCC growth and metastasis. Therefore, EGFR has been used as an important therapeutic target to treat HNSCC. Inhibition of EGFR stimulates autophagy in cancer cells. However, the role of autophagy in EGFR inhibitor-induced cancer suppression is still in a debate. Here, we reveal that the first- and the second-generation EGFR tyrosine kinase inhibitors (TKIs) differentially affect HNSCC autophagy. The second-generation EGFR TKIs have much stronger effects on autophagy than the first-generation TKIs. The second-generation EGFR TKIs not only promote autophagy initiation signaling but also block autophagic flux by disturbing the lysosomes function, indicating a novel mechanism by which EGFR TKIs modulate cancer cell autophagy. Blocking the initiation of autophagy does not affect the second-generation EGFR TKI-induced HNSCC growth suppression. This suggests that the anti-growth effect of the second-generation EGFR TKIs on HNSCC is not dependent on autophagy.

Design, synthesis and biological evaluation of hybrids of ß-carboline and salicylic acid as potential anticancer and apoptosis inducing agents.

A novel series of hybrids (7a-l, 8a-l) from ß-carboline and salicylic acid (SA) were designed and synthesized, and their in vitro biological activities were evaluated. Most of the hybrids displayed potent antiproliferative activity against five cancer cell lines in vitro, showing potencies superior to 5-FU and harmine. In particular, compound 8h selectively inhibited proliferation of liver cancer SMMC-7721 cells but not normal liver LO2 cells, and displayed greater inhibitory selectivity than intermediate 5h and SA. 8h also induced cancer cell apoptosis in an Annexin V-FITC/propidium iodide flow cytometry assay, and triggered the mitochondrial/caspase apoptosis by decreasing mitochondrial membrane potential which was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation levels of the caspase cascade in a concentration-dependent manner. Our findings suggest that the ß-carboline/SA hybrids may hold greater promise as therapeutic agents for the intervention of human cancers.

Type I γ Phosphatidylinositol Phosphate 5-Kinase i5 Controls the Ubiquitination and Degradation of the Tumor Suppressor Mitogen-inducible Gene 6.

Mitogen-inducible gene 6 (Mig6) is a tumor suppressor, and the disruption of Mig6 expression is associated with cancer development. Mig6 directly interacts with epidermal growth factor receptor (EGFR) to suppress the activation and downstream signaling of EGFR. Therefore, loss of Mig6 enhances EGFR-mediated signaling and promotes EGFR-dependent carcinogenesis. The molecular mechanism modulating Mig6 expression in cancer remains unclear. Here we demonstrate that type I γ phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme producing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), stabilizes Mig6 expression. Knockdown of PIPKIγi5 leads to the loss of Mig6 expression, which dramatically enhances and prolongs EGFR-mediated cell signaling. Loss of PIPKIγi5 significantly promotes Mig6 protein degradation via proteasomes, but it does not affect the Mig6 mRNA level. PIPKIγi5 directly interacts with the E3 ubiquitin ligase neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1). The C-terminal domain of PIPKIγi5 and the WW1 and WW2 domains of NEDD4-1 are required for their interaction. The C2 domain of NEDD4-1 is required for its interaction with PtdIns(4,5)P2 By binding with NEDD4-1 and producing PtdIns(4,5)P2, PIPKIγi5 perturbs NEDD4-1-mediated Mig6 ubiquitination and the subsequent proteasomal degradation. Thus, loss of NEDD4-1 can rescue Mig6 expression in PIPKIγi5 knockdown cells. In this way, PIPKIγi5, NEDD4-1, and Mig6 form a novel molecular nexus that controls EGFR activation and downstream signaling.