Dapagliflozin promotes browning of white adipose tissue through the FGFR1-LKB1-AMPK signaling pathway.

BACKGROUND: Obesity is associated with a wide variety of metabolic disorders that impose significant burdens on patients and society. The "browning" phenomenon in white adipose tissue (WAT) has emerged as a promising therapeutic strategy to combat metabolic disturbances. However, though the anti-diabetic drug dapagliflozin (DAPA) is thought to promote "browning," the specific mechanism of this was previously unclear. METHODS: In this study, C57BL/6 J male mice were used to establish an obesity model by high-fat diet feeding, and 3T3-L1 cells were used to induce mature adipocytes and to explore the role and mechanism of DAPA in "browning" through a combination of in vitro and in vivo experiments. RESULTS: The results show that DAPA promotes WAT "browning" and improves metabolic disorders. Furthermore, we discovered that DAPA activated "browning" through the fibroblast growth factor receptors 1-liver kinase B1-adenosine monophosphate-activated protein kinase signaling pathway. CONCLUSION: These findings provide a rational basis for the use of DAPA in treating obesity by promoting the browning of white adipose tissue.

Efficacy and safety of thoracic radiotherapy for extensive stage small cell lung cancer after immunotherapy in real world.

The immunotherapy combined chemotherapy has been the standard treatment strategy for extensive-stage small lung cancer (ES-SCLC). The CREST trial reported consolidative thoracic radiotherapy (cTRT) improved overall survival (OS) for ES-SCLC with intrathoracic residual after chemotherapy. In this study, patients with ES-SCLC who received immunotherapy were assigned to receive either TRT or no TRT. TRT significantly improved progression-free survival (PFS), local recurrence-free survival (LRFS) and OS with well tolerated toxicity. Further sub-cohort analysis, TRT significantly improved LRFS in patients with oligo-metastasis and without liver metastasis.

Association between diet quality scores and risk of overweight and obesity in children and adolescents.

BACKGROUND: This study examined the associations of diet quality assessed by Healthy Eating Index 2015 (HEI-2015), Alternative Healthy Eating Index 2010 (AHEI-2010), Mediterranean Diet (MedDiet) and overweight/obesity in children and adolescents. METHODS: This cross-sectional study used data of participants aged 2-19 years from National Health and Nutrition Examination Survey (NHANES) 2005-2018. The weighted logistic regression model was adopted to explore the association between diet quality scores and overweight, obesity in children and adolescents. Subgroup analysis was also performed based on sex. RESULTS: A total of 9,724 participants were included in children group (2-11 years old), and 5,934 were adolescent group (12-19 years old). All participants were divided into based on the BMI-for-age: underweight and normal, overweight and obesity groups. After adjusting for age, race, poverty-income ratio, maternal smoking during pregnancy and total energy, HEI-2015 and MedDiet scores were related to the risk of overweight in children, and only MedDiet scores remained associated with a decreased risk of obesity in children. MedDiet scores were associated with a decreased risk of overweight, obesity in adolescents, respectively, after adjusting age, sex, race, poverty-income ratio, cotinine, total energy and physical activity. The similar results in male participants were also found. CONCLUSION: Higher MedDiet scores were associated with lower the risk of overweight and obesity, respectively, particularly for male children and adolescents. The higher HEI-2015 scores were also related to the risk of overweight in children.

Iron deficiency downregulates ENPEP to promote angiogenesis in liver tumors.

The abnormal iron metabolism in liver cancer leads to iron deficiency in tumor tissues. We previously found that iron deficiency promoted liver cancer metastasis, but the mechanisms were not fully understood. In the present study, we identified that the angiogenesis-associated glutamyl aminopeptidase (ENPEP) was consistently decreased in iron-deficient liver tissues, iron-deficient liver tumors, and iron-deprived liver cancer cells. Interestingly, the lower expression of ENPEP was correlated with the poor prognosis of liver cancer patients, while the biomarkers of angiogenesis, CD31 and CD34, were increased in tumor tissues. In vivo imaging of liver-orthotopically implanted and tail vein-injected liver cancer cells showed that iron deficiency increased the pulmonary metastasis of liver cancer. The angiogenesis in iron-deficient tumors was enhanced, and the expression of ENPEP was decreased. Silencing ENPEP expression increased the migration of liver cancer cells and the proliferation of cocultured HUVECs. By sequence analysis, we found that the transcription factor SP1 possessed abundant binding sites in the ENPEP promoter region. Its combination with ENPEP promoters was verified by chromatin immunoprecipitation. The inhibition of SP1 by mithramycin A effectively restored the expression of ENPEP, which was decreased by iron deficiency. In conclusion, these results revealed that iron deficiency in liver tumors decreased the expression of ENPEP by SP1 and increased the angiogenesis and metastasis of liver tumors, which further explained the mechanism by which iron deficiency promoted liver cancer metastasis.

Integrative genomic analysis of drug resistance in MET exon 14 skipping lung cancer using patient-derived xenograft models.

Background: Non-small cell lung cancer (NSCLC) driven by MET exon 14 skipping (METex14) occurs in 3-4% of NSCLC cases and defines a subset of patients with distinct characteristics. While MET targeted therapy has led to strong clinical results in METex14 patients, acquired drug resistance seemed to be unavoidable during treatment. Limited information is available regarding acquired resistance during MET targeted therapy, nor has there been any report on such patient-derived xenografts (PDXs) model facilitating the research. Methods: We describe a patient case harboring METex14 who exhibited drug resistance after treatment with crizotinib. Subcutaneous xenografts were generated from pretreatment and post-resistance patient specimens. PDX mice were then treated with MET inhibitors (crizotinib and tepotinib) and EGFR-MET bispecific antibodies (EMB-01 and amivantamab) to evaluate their drug response in vivo. DNA and RNA sequencing analysis was performed on patient tumor specimens and matching xenografts. Results: PDXs preserved most of the histological and molecular profiles of the parental tumors. Drug resistance to MET targeted therapy was confirmed in PDX models through in vivo drug analysis. Newly acquired MET D1228H mutations and EGFR amplificated were detected in patient-resistant tumor specimens. Although the mutations were not detected in the PDX, EGFR overexpression was observed in RNA sequencing analysis indicating possible off-target resistance through the EGFR bypass signaling pathway. As expected, EGFR-MET bispecific antibodies overcome drug resistant in the PDX model. Conclusions: We detected a novel MET splice site deletion mutation that could lead to METex14. We also established and characterized a pair of METex14 NSCLC PDXs, including the first crizotinib resistant METex14 PDX. And dual inhibition of MET and EGFR might be a therapeutic strategy for EGFR-driven drug resistance METex14 lung cancer.

Loss of SLC46A1 decreases tumor iron content in hepatocellular carcinoma.

It is interesting that high iron is an independent inducer or cofactor of hepatocellular carcinoma (HCC) while the amount of iron is decreased in the liver tumor tissues. Due to the previous findings that iron deficiency promoted HCC metastasis, it is of significance to identify the underlying mechanism of iron deficiency in HCC. The tumor iron content and expressions of iron-metabolic molecules were observed in the primary liver cancers of rats and mice. The molecules that changed independently of iron were identified by comparing the expression profiles in the human HCC tissues and iron-deprived HCC cells. The downstream effects of these molecules on regulating intracellular iron content were investigated in vitro and further validated in vivo. Both in primary liver cancers of rats and mice, we confirmed the decreased iron content in tumor tissues and the altered expressions of iron-metabolic molecules, including transferrin receptor 1 (TfR1), six-transmembrane epithelial antigen of prostate 3 (STEAP3), divalent metal transporter 1 (DMT1), SLC46A1, ferroportin, hepcidin, and ferritin. Among these, STEAP3, DMT1, and SLC46A1 were altered free of iron deficiency. However, only silence or overexpression of SLC46A1 controlled the intracellular iron content of HCC cells. The interventions of STEAP3 or DMT1 could not change the intracellular iron content. Lentivirus-mediated regain of SLC46A1 expression restored the iron content in orthotopically implanted tumors, with correspondingly changes in the iron-metabolic molecules as iron increasing. Conclusion: Taken together, these results suggest that the loss of SLC46A1 expression leads to iron deficiency in liver tumor tissues, which would be an effective target to manage iron homeostasis in HCC.

Novel chlorpromazine derivatives as anti-endometrial carcinoma agents with reduced extrapyramidal side effects.

As the traditional conservative remedy for endometrial carcinoma (EC), progesterone has great limitations due to its poor performance, and a new strategy is urgently needed. Our previous work revealed that the antipsychotic drug chlorpromazine (CPZ) has stronger antitumor activity on EC than progesterone does, which may provide a promising conservative alternative for EC patients. Unfortunately, the severe extrapyramidal symptoms (EPSs) at concentrations (>5 mg/kg) that are required for anticarcinoma activity limited its repurposing. Therefore, a series of novel CPZ derivatives were designed and synthesized to avoid EPS and retain its antitumor activity. Among them, 11·2HCl and 18 displayed greater inhibitory activity by modulating SOS1. Notably, even at a dose of 100 mg/kg, 11·2HCl/18 had little effect on the extrapyramidal system. In conclusion, 11·2HCl and 18 greatly repressed the malignant features of endometrial carcinoma and decreased extrapyramidal side effects compared with the original drug CPZ.

The Logistics of Medication and Patient Flow in Video-Based Virtual Clinics During a Sudden COVID-19 Outbreak in Taiwan: Observational Study.

BACKGROUND: The COVID-19 pandemic was well controlled in Taiwan until an outbreak in May 2021. Telemedicine was rapidly implemented to avoid further patient exposure and to unload the already burdened medical system. OBJECTIVE: To understand the effect of COVID-19 on the implementation of video-based virtual clinic visits during this outbreak, we analyzed the logistics of prescribing medications and patient flow for such virtual visits at a tertiary medical center. METHODS: We retrospectively collected information on video-based virtual clinic visits and face-to-face outpatient visits from May 1 to August 31, 2021, from the administrative database at National Taiwan University Hospital. The number of daily new confirmed COVID-19 cases in Taiwan was obtained from an open resource. RESULTS: There were 782 virtual clinic visits during these 3 months, mostly for the departments of internal medicine, neurology, and surgery. The 3 most common categories of medications prescribed were cardiovascular, diabetic, and gastrointestinal, of which cardiovascular medications comprised around one-third of all medications prescribed during virtual clinic visits. The number of virtual clinic visits was significantly correlated with the number of daily new confirmed COVID-19 cases, with approximately a 20-day delay (correlation coefficient 0.735; P<.001). The patient waiting time for video-based virtual clinic visits was significantly shorter compared with face-to-face clinic visits during the same period (median 3, IQR 2-6 min vs median 20, IQR 9-42 min; rank sum P<.001). Although the time saved was appreciated by the patients, online payment with direct delivery of medications without the need to visit a hospital was still their major concern. CONCLUSIONS: Our data showed that video-based virtual clinics can be implemented rapidly after a COVID-19 outbreak. The virtual clinics were efficient, as demonstrated by the significantly reduced waiting time. However, there are still some barriers to the large-scale implementation of video-based virtual clinics. Better preparation is required to improve performance in possible future large outbreaks.

Gap junctions mediate glucose transfer to promote colon cancer growth in three-dimensional spheroid culture.

In vivo tumor growth is characterized by a necrotic core generated by oxygen and nutrients gradients, which is replicated by in vitro three-dimensional (3D) tumor spheroids but not traditional two-dimensional cell monolayers. Gap junctions provide direct communication between adjacent cells and play a critical role in cancer development, but their effects are still debatable. In this study, we found that connexin 43 (Cx43) reduced the area of necrotic core in colon cancer 3D spheroids, thus providing a growth advantage. This impact is dependent on gap junction channel functions, as the channel blocker carbenoxolone or connexin channel death mutant reverses this effect. Additionally, enhanced glucose uptake was detected in Cx43-overexpressing spheroids, along with upregulated mTOR, downregulated AMPK signaling, increased ATP content, and enhanced oxygen consumption rate. Furthermore, the xenograft mouse model confirmed the growth advantage of Cx43 in vivo. RNAseq data and clinical information from The Cancer Genome Atlas (TCGA) database indicated a more heterogeneous expression pattern of Cx43 in colon cancer compared to normal colon tissue, and higher Cx43 level is associated with worse clinical outcomes. Our data suggest a novel function of connexin in tumor growth, that gap junctions may provide nutrients transmitting routes in lieu of vasculature to meet the increasing metabolic requirement of solid tumors.

Postoperative delirium in geriatric patients with hip fractures.

Postoperative delirium (POD) is a frequent complication in geriatric patients with hip fractures, which is linked to poorer functional recovery, longer hospital stays, and higher short-and long-term mortality. Patients with increased age, preoperative cognitive impairment, comorbidities, perioperative polypharmacy, and delayed surgery are more prone to develop POD after hip fracture surgery. In this narrative review, we outlined the latest findings on postoperative delirium in geriatric patients with hip fractures, focusing on its pathophysiology, diagnosis, prevention, and treatment. Perioperative risk prediction, avoidance of certain medications, and orthogeriatric comprehensive care are all examples of effective interventions. Choices of anesthesia technique may not be associated with a significant difference in the incidence of postoperative delirium in geriatric patients with hip fractures. There are few pharmaceutical measures available for POD treatment. Dexmedetomidine and multimodal analgesia may be effective for managing postoperative delirium, and adverse complications should be considered when using antipsychotics. In conclusion, perioperative risk intervention based on orthogeriatric comprehensive care is the most effective strategy for preventing postoperative delirium in geriatric patients with hip fractures.

PD-L1 Protein Expression and Gene Amplification Correlate with the Clinicopathological Characteristics and Prognosis of Lung Squamous Cell Carcinoma.

PURPOSE: To investigate PD-L1 protein expression and gene amplification in lung squamous cell carcinoma (LUSC) and analyse their correlation with the clinicopathological characteristics and prognosis of LUSC patients. PATIENTS AND METHODS: Tissue samples from 164 LUSC patients were collected. PD-L1 protein was detected by immunochemistry (IHC), and PD-L1 gene amplification was investigated by fluorescence in situ hybridization in LUSC patients. RESULTS: The positive expression rate of PD-L1 in LUSC was 47.6% (78/164), and the amplification rate of PD-L1 was 6.7% (11/164); both rates were higher than those of paratumor tissue. Both PD-L1 positive expression and gene amplification were correlated with clinical stage and lymph node metastasis (P<0.05). PD-L1 protein expression, PD-L1 gene amplification, late stage, lymph node metastasis and distant metastasis were significantly correlated with the prognosis of patients. Among these factors, late stage, lymph node metastasis, PD-L1 protein expression and PD-L1 gene amplification were independent prognostic factors for LUSC. CONCLUSION: Positive PD-L1 protein expression and gene amplification are involved in the malignant progression and metastasis of LUSC. Both PD-L1 protein expression and gene amplification are associated with poor prognosis.

Chlorpromazine Sensitizes Progestin-Resistant Endometrial Cancer Cells to MPA by Upregulating PRB.

Medroxyprogesterone acetate (MPA) is the main conservative treatment for endometrial cancer (EC) patients desirable to preserve fertility and those who cannot suffer from surgery. Considering the high incidence of progestin resistance and recurrence of MPA treatment, we reproposed antipsychotics chlorpromazine (CPZ) as a new strategy for both progestin-sensitive and -resistant endometrial cancer. Cytobiology experiments indicated that CPZ could significantly suppress proliferation, migration/invasion and induce apoptosis in Ishikawa (ISK) and KLE EC cell lines. And xenograft mouse models were constructed to validate the antitumor effect and toxicity of CPZ in-vivo. CPZ inhibited the growth at a low dose of 3mg/kg and the mice exhibited no signs of toxicity. Next, concomitant treatment and sequential treatment with CPZ and MPA were proceeded to analysis the synergistic effect in EC cells. Concomitant treatment only performed a limited synergistic effect on apoptosis in ISK and KLE cells. Nevertheless, sequential treatment showed favorable synergistic effects in progestin-resistant KLE cells. Finally, a stable MPA-resistant cell line shRNA was established to explore the mechanism of CPZ reversing progestin resistance. Immunoblot data showed that CPZ inhibited the activation of PI3K/AKT signal in ISK and KLE cells and upregulated PRB expression in progestin-resistant cells, by which CPZ overcame progestin resistance to MPA. Thus, CPZ might act as a candidate drug for conservative treatment and sequential treatment with CPZ and MPA could be a suitable therapeutic option for progestin resistant patients.

Low KRT15 expression is associated with poor prognosis in patients with breast invasive carcinoma.

Although keratin 15 (KRT15) has been indicated to be overexpressed in several types of tumor, its role in breast invasive carcinoma (BRCA) has so far remained elusive. The aim of the present study was to explore KRT15 expression in BRCA based on data obtained from The Cancer Genome Atlas and The Genotype-Tissue Expression. KRT15 expression was compared using a Wilcoxon rank-sum test. Functional enrichment analysis was performed to reveal the biological roles and pathways of KRT15. The association between KRT15 expression and immune-cell infiltration was evaluated via single-sample gene set enrichment analysis (ssGSEA). To investigate the relationship between clinicopathological features and KRT15 expression, the prognostic value of KRT15 and other clinical factors was evaluated using Cox regression analysis and Kaplan-Meier (KM) plots. Subgroup prognostic analysis was also performed using forest plots and KM curves. Finally, a tissue microarray was used to assess KRT15 expression in BRCA tissues. KRT15 expression was significantly lower in BRCA tissues compared with that in normal tissues. Functional enrichment analysis suggested that KRT15-related genes were primarily enriched in the transmembrane transporter complex, cornification and ligand-receptor interactions. Increased KRT15 was associated with several tumor-suppressive pathways. ssGSEA revealed that high KRT15 expression was significantly associated with natural killer-cell, B-cell and mast-cell infiltration. Significant associations were observed between low KRT15 expression and advanced stage clinicopathological factors, as well as unfavorable overall survival (OS) and disease-specific survival. Multivariate Cox regression analysis suggested that KRT15 was an independent prognostic factor for OS (P=0.039; hazard ratio, 0.590; 95% CI, 0.358-0.974). Subgroup prognostic analysis demonstrated that low KRT15 was a reliable predictor of poor OS. Immunohistochemistry of a tissue microarray indicated that positive KRT15 expression rates were significantly higher in normal tissues compared with those in the BRCA tissues. In conclusion, low KRT15 expression was significantly associated with poor prognosis in patients with BRCA. Thus, KRT15 may serve an important role in BRCA progression and may be used as a promising prognostic marker for diagnostic and prognostic analyses in patients with BRCA.

Altered gene expression in glycolysis-cholesterol synthesis axis correlates with outcome of triple-negative breast cancer.

Identification of molecular subtypes of clinically resectable triple-negative breast cancer (TNBC) is of great importance to achieve better clinical outcomes. Inter- and intratumor metabolic heterogeneity improves cancer survival, and the interaction of various metabolic pathways may affect treatment outcome of TNBC. We speculated that TNBC can be categorized into prognostic metabolic subtype according to the expression changes of glycolysis and cholesterol synthesis. The genome, transcriptome, and clinical data were downloaded from the Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium and subsequently analyzed by integrated bioinformatics methods. Four subtypes, namely, glycolytic, cholesterogenic, quiescent, and mixed, were classified according to the normalized median expressions of the genes involved in glycolysis and cholesterol synthesis. In the four subtypes, the cholesterogenic type was correlated with the shortest median survival (log rank P = 0.044), while patients with high-expressed glycolytic genes tended to have a longer survival. Tumors with PIK3CA amplification and dynein axonemal heavy chain 2 deletion exhibited higher expressions of cholesterogenic genes than other mutant oncogenes. The expressions of mitochondrial pyruvate carrier MPC1 and MPC2 were the lowest in quiescent tumor, and MPC2 expression was higher in cholesterogenic tumor compared with glycolytic or quiescent tumor (t-test P < 0.001). Glycolytic and cholesterogenic gene expressions were related to the expressions of prognostic genes in some other types of cancers. Classification of glycolytic and cholesterogenic pathways according to metabolic characteristics provides a new understanding to previously identified subtypes of TNBC and could improve personalized treatments based on tumor metabolic profiles.

Mild iron overload induces TRIP12-mediated degradation of YY1 to trigger hepatic inflammation.

Increasing populations are found to bear mild hepatic iron overload (HIO) due to unhealthy lifestyles, metabolic diseases, etc., whether this mild but chronic HIO induces hepatic inflammation is unknown. In the present study, mice receiving a 12-months 0.3% dextran-iron diet show mild HIO with no detectable oxidative damages in the liver but have infiltrated macrophages and increased IL-6, TNFα, AST and ALT since 6-months. The HNF4α/miR-122/CCL2 pathway, identified by our previous studies to induce macrophages infiltration, is initiated by chronic mild HIO. After excluding the role of DNA methylation, a modified transcription factor microarray is applied to find that transcription factor YY1 is responsible for HIO-decreased HNF4α expression. Then the E3 ubiquitin ligase TRIP12 is identified by an immunoprecipitation coupled LC-MS/MS and proved to bind and ubiquitinate YY1, leading to its degradation. The overexpression or silence of YY1 in the liver regulates the HNF4α/miR-122/CCL2 pathway. More importantly, YY1 overexpression alleviates chronic mild HIO induced hepatic inflammatory responses. In conclusion, these results elucidate an oxidative-stress-independent, TRIP12/YY1/HNF4α/miR-122/CCL2 pathway of chronic mild HIO inducing hepatic inflammation, implying that effective measures in addition to antioxidants are needed for individuals at the risk of chronic mild HIO.

[Effect of CPEB4 on Migration and Cycle of Chronic Myeloid Leukemia Cell].

OBJECTIVE: To investigate the effects of CPEB4 on the migration and cycle of K562 cells and the changes of protein molecules that may be involved in the regulatory mechanism. METHODS: Western blot was used to detect the expression of CPEB4 in normal leukocytes and K562 cells. The overexpression plasmid pcDNA3.1(+)-His-CPEB4, silencing plasmid pPLK+Puro-CPEB4 shRNA were transfected into K562 cells by electroporation so as to change CPEB4. The transfection efficiency was detected by Western blot. Finally, the migration and cycle of different cells were detected by Transwell chamber and flow cytometry.Western blot was used to detect the expression changes of MMP2, MMP9, CDK4, CyclinD1 and P21 proteins. RESULTS: Compared with normal white blood cells, the expression of CPEB4 protein in K562 cells was significantly enhanced (P＜0.01); Compared with the control group, CPEB4-silenced K562 cells showed that the cell migration ability was significantly enhanced (P＜0.01); G0/G1 phase cell ratio reduced, G2/M phase cell ratio increased, and cell cycle progression accelerated（P＜0.01）, The expression levels of MMP2 (P＜0.05), MMP9 (P＜0.05), CDK4 (P＜0.01), CyclinD1 (P＜0.01) proteins increased significantly. The expression level of P21 protein significantly decreased (P＜0.01). The migration ability of K562 cells after CPEB4 overexpression was decreased (P＜0.01), the cell ratio of G0/G1 phase in the cell cycle increased, the cell proportion of S phase decreased and the cell cycle progression was arrested at G0/G1 phase (P＜0.01). The expression of P21 protein increased, MMP2 , MMP9, CDK4, CyclinD1 protein expression decreased significantly（P＜0.05-0.01）. CONCLUSION: CPEB4 can inhibit the migration of K562 cells and arrest cell cycle progression at G0/G1 phase. Its mechanism may be related with regulating the exprossion of MMP2, MMP9, CDK4, CyclinD1 and P21 proteins.

Inward Tension of Talin and Integrin-related Osmotic Pressure are involved Synergetically in the Invasion and Metastasis of Non-small Cell Lung Cancer.

The integrin receptor protein talin plays vital roles in intracellular chemical and mechanical activities, and it is implicated in the high invasion and poor prognosis of non-small cell lung cancer (NSCLC). To better understand the mechanism underlying the function of talin in NSCLC invasion and metastasis, a few newly designed tension probe based on Förster resonance energy transfer was used for real-time observation of tension changes in A549 cells. High NSCLC cell aggressiveness was found to be accompanied with inward talin and outward glial fibrillary acidic protein (GFAP) tensions, which are closely associated with microfilament (MF) force and intracellular osmotic potential. The increased osmotic pressure resulted from the production of intracellular protein nanoparticles and the related ion influx. Furthermore, integrin activation was found to adjust the talin and GFAP tensions. Disruption of the interaction between talin and MFs blocked the mechanical source of talin, reducing both talin tension and osmotic pressure and thus inhibiting NSCLC cell invasion and migration. Consequently, our study demonstrates that talin is involved in NSCLC invasion and migration via its inward tension and that the integrin pathway is correlated closely with protein-nanoparticle-induced outward osmotic pressure.

SLC46A1 contributes to hepatic iron metabolism by importing heme in hepatocytes.

BACKGROUND: Iron is finely regulated due to its vital roles in organisms and the peroxidase reactivity if excess. Solute Carrier Family 46 Member 1 (SLC46A1), also named PCFT or HCP1, is the main importer of heme­iron in the intestine, but has a high abundance in the liver. Since the liver has a central role in iron homeostasis, whether SLC46A1 regulates hepatic iron metabolism is of interest to be identified. METHODS: The recombinant adeno-associated virus vectors were used to hepatic-specifically inhibit SLC46A1 expression to observe its effects on hepatic iron metabolism. Then the abilities of SLC46A1 in importing heme and folate, and consequent alterations of iron content in hepatocytes were determined. Furthermore, effects of iron on SLC46A1 expression were investigated both in vitro and in vivo. RESULTS: The hepatocyte-specific inhibition of SLC46A1 decreases iron content in the liver and increases iron content in serum. Expressions of iron-related molecules, transferrin receptor 1, hepcidin and ferroportin, are correspondingly altered. Interestingly, free heme concentration in serum is increased, indicating a decreased import of heme by the liver. In hepatocytes, SLC46A1 is capable of importing hemin, increasing intracellular iron content. The import of hemin by SLC46A1 is unaffected by its other substrate, folate. Instead, hemin treatment decreases SLC46A1 expression, reducing the import of folate. In addition, SLC46A1 itself shows to be iron-responsive both in vivo and in vitro, making it available for regulating iron metabolism. CONCLUSION: The results elucidate that SLC46A1 regulates iron metabolism in the liver through a folate-independent manner of importing heme. The iron-responsive characters of SLC46A1 give us a new clue to link heme or iron overload with folate deficiency diseases.

Repurposing of antipsychotics perphenazine for the treatment of endometrial cancer.

Endometrial cancer (EC) is one of the most common and fatal gynecological cancers worldwide, but there is no effective treatment for the EC patients of progesterone resistance. Repurposing of existing drugs is a good strategy to discover new candidate drugs. In this text, perphenazine (PPZ), approved for psychosis therapy, was identified as a potential agent for the treatment of both progesterone sensitive and resistant endometrial cancer for the first time. Specifically, perphenazine exhibited good cell proliferation inhibition in Ishikawa (ISK) and KLE cell lines according to the CCK-8 assay and colony formation assay. It also reduced the cell migration of ISK and KLE cell lines in the light of the transwell migration assay. Annexin-V/PI double staining assay suggested that perphenazine could effectively induce ISK and KLE cell apoptosis. Moreover, results of western blot assay indicated perphenazine obviously inhibited the phosphorylation of Akt. Delightedly, PPZ also could significantly attenuate xenograft tumor growth at both 3 mg/kg and 15 mg/kg in mice without influencing the body weights.

S-Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid.

Heat shock protein 70 (Hsp70) proteins are a family of ancient and conserved chaperones. Cysteine modifications have been widely detected among different Hsp70 family members in vivo, but their effects on Hsp70 structure and function are unclear. Here, we treated HeLa cells with diamide, which typically induces disulfide bond formation except in the presence of excess GSH, when glutathionylated cysteines predominate. We show that in these cells, HspA1A (hHsp70) undergoes reversible cysteine modifications, including glutathionylation, potentially at all five cysteine residues. In vitro experiments revealed that modification of cysteines in the nucleotide-binding domain of hHsp70 is prevented by nucleotide binding but that Cys-574 and Cys-603, located in the C-terminal α-helical lid of the substrate-binding domain, can undergo glutathionylation in both the presence and absence of nucleotide. We found that glutathionylation of these cysteine residues results in unfolding of the α-helical lid structure. The unfolded region mimics substrate by binding to and blocking the substrate-binding site, thereby promoting intrinsic ATPase activity and competing with binding of external substrates, including heat shock transcription factor 1 (Hsf1). Thus, post-translational modification can alter the structure and regulate the function of hHsp70.