Phospholipid analysis of two influenza A virus-infected cell lines differing in their viral replication kinetics.

Fluctuations in phospholipid composition in infected cells during influenza A virus replication were analyzed using two different susceptible host cell lines: H292 cells, exhibiting a rapid cytopathic effect, and A549 cells, exhibiting a retarded cytopathic effect. Microarray analysis demonstrated that A549 cells recognized influenza A virus invasion, expression of pathogen recognition genes was affected, and antiviral genes were activated. On the other hand, H292 cells did not display such an antiviral state, and in these cells, rapid virus amplification and a rapid cytopathic effect were observed. Levels of ceramide, diacylglycerol, and lysolipids were higher in virus-infected cells than in the corresponding mock-infected cells at the later stages of infection. The accumulation of these lipids in IAV-infected cells occurred together with viral replication. The relationship between the characteristic features of ceramide, diacylglycerol, and lysolipid in the plasma membrane, where enveloped viruses are released, and their role in viral envelope formation are discussed. Our results indicate that viral replication disturbs cellular lipid metabolism, with consequences for viral replication kinetics.

Palladium-catalyzed/copper-mediated carbon-carbon cross-coupling reaction for synthesis of 6-unsubstituted 2-aryldihydropyrimidines.

Dihydropyrimidines (DPs) show a wide range of biological activities for medicinal applications. Among the DP derivatives, 2-aryl-DPs have been reported to display remarkable pharmacological properties. In this work, we describe a method for the synthesis of hitherto unavailable 6-unsubstituted 2-aryl-DPs by Pd-catalyzed/Cu-mediated carbon-carbon cross-coupling reaction of 1-Boc 2-methylthio-DPs with organostannane reagents. The Boc group of the substrate significantly increases the substrate reactivity. Aryl tributylstannanes having various substituents such as MeO, Ph, CF3, CO2Me, and NO2 groups smoothly afforded the corresponding products in high yields. Various heteroaryl tributylstannanes having 2-, or 3-thienyl, 2-, or 3-pyridinyl groups were also applicable to the reaction. Regarding the substituents at the 4-position, the reactions of DPs bearing various aryl and alkyl substituents proceeded smoothly to give the desired products. The Boc group of the products was removed under a standard acidic condition to produce N-unsubstituted DP as a mixture of the tautomers in quantitative yields. The synthetic procedure was also applied to 4,4,6-trisubstituted 2-methylthio-DP to give novel 2,4,4,5,6-pentasubstituted DP. Therefore, the Pd-catalyzed/Cu-mediated reaction should help expand the DP-based molecular diversity, which would impact biological and pharmacological studies.

Heat shock-induced heme oxygenase-1 expression in a mouse hepatoma cell line is dependent on HSF1 and modified by NRF2 and BACH1.

The induction mechanism of heme oxygenase-1 (HO-1) by heat shock (HS) is still unknown. Here, we discovered that HS activates the HO-1 expression in a mouse hepatoma cell line (Hepa 1-6). Knockdown experiments showed that the HS-induced HO-1 expression was dependent on HS factor 1 (HSF1). A chromatin immunoprecipitation (ChIP) assay demonstrated that the HS-activated HSF1 bound to the HS elements (HSEs) in the upstream enhancer 1 region (E1). Unexpectedly, HS also facilitates the BTB and CNC homology 1 (BACH1) binding to the Maf recognition elements (MAREs) in E1. We examined the effects of a catalytically inactive CRISPR-associated 9 nucleases (dCas9) with short guide RNAs (sgRNAs), and demonstrated that the HSF1 binding to HSEs in E1 was indispensable for the HS-induced HO-1 expression. Heme treatment (HA) dissociates BACH1 from MAREs and facilitated the binding of nuclear factor-erythroid-2-related factor 2 (NRF2) to MAREs. Following treatment with both HS and HA, the HO-1 induction and the HSF1 binding to HSEs in E1 were most notably observed. These results indicate that the HS-induced HO-1 expression is dependent on the HSF1 binding to HSEs in E1, although modulated by the BACH1 and NRF2 binding to MAREs within the same E1.

A Comparative Study of Patient-Derived Tumor Models of Pancreatic Ductal Adenocarcinoma Involving Orthotopic Implantation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is associated with very poor prognoses. Therefore, new therapies and preclinical models are urgently needed. In the present study, we sought to develop more realistic experimental models for use in PDA research. METHODS: We developed patient-derived xenografts (PDXs), established PDX-derived cell lines (PDCLs), and generated cell line-derived xenografts (CDXs), which we integrated to create 13 matched "trios" - i.e., patient-derived tumor models of PDA. We then compared and contrasted histological and molecular alterations between these three model systems. RESULTS: Orthotopic implantation (OI) of the PDCLs resulted in tumorigenesis and metastases to the liver and peritoneum. Morphological comparisons of OI-CDXs and OI-PDXs with passaged tumors revealed that the histopathological features of the original tumor were maintained in both models. Molecular alterations in PDX tumors (including those to KRAS, TP53, SMAD4, and CDKN2A) were similar to those in the respective PDCLs and CDX tumors. When gene expression levels in the PDCLs, ectopic tumors, and OI tumors were compared, the distant metastasis-promoting gene CXCR4 was specifically upregulated in OI tumors, whose immunohistochemical profiles suggested epithelial-mesenchymal transition and adeno-squamous trans-differentiation. CONCLUSION: These patient-derived tumor models provide useful tools for monitoring responses to antineoplastic agents and for studying PDA biology.

Sites of flexor-pronator muscle injury and relationship between ulnar collateral ligament injury and flexor-pronator muscle injury in baseball players: a retrospective cohort study.

BACKGROUND: Ulnar collateral ligament (UCL) and flexor-pronator muscle (FPM) injuries are common in baseball players. However, the sites of FPM injuries and the relationship between UCL and FPM injuries in baseball players have not been fully clarified. The purpose of this study was to identify the sites of FPM injuries and to determine the relationships of location and severity of UCL injury with the presence of FPM injuries in baseball players. METHODS: UCL and FPM injuries were diagnosed using magnetic resonance imaging in 99 baseball players. The sites of FPM injuries were identified on coronal, sagittal, and axial images. UCL injury severity was classified into four grades: chronic changes, low-grade partial tear, high-grade partial tear, and complete tear. UCL injury location was classified as proximal UCL tear or distal UCL tear. All images were assessed by a musculoskeletal radiologist and an orthopedic surgeon. RESULTS: Combined UCL and FPM injuries were observed in 45 of 99 players, of which 40 of 45 (89%) involved injury of the flexor digitorum superficialis (FDS). All FDS injuries were in the deep layer of the muscle belly. There was no significant difference between the severity of UCL injury and presence of FPM injuries (P = .352). There was a significant association of distal UCL tears with FPM injuries (P < .001). CONCLUSION: FDS injury occurs most commonly in the muscle belly of the second and fifth digits. There may be no relationship between the severity of UCL injury and presence of FPM injury in baseball players. FPM injuries may be a contributing factor in the failure of nonoperative management of distal UCL tears in baseball players.

Synthesis of 4,4-Disubstituted 3,4-Dihydropyrimidin-2(1H)-ones and -thiones, the Corresponding Products of Biginelli Reaction Using Ketone, and Their Antiproliferative Effect on HL-60 Cells.

An efficient synthetic method for novel 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5 and -thiones 6 was developed. The cyclocondensation reaction of O-methylisourea hemisulfate salt 11 with 8 gives a tautomeric mixture of dihydropyrimidines 12 and 13 following acidic hydrolysis of the cyclized products to produce 5 in high yields. Thionation reaction of 5 at the 2-position smoothly proceeds to give 2-thioxo derivatives 6. These compounds 5 and 6, corresponding to the products of a Biginelli-type reaction using urea or thiourea, a ketone and a 1,3-dicarbonyl compound, have long been inaccessible and hitherto unavailable for medicinal chemistry. These methods are invaluable for the synthesis of 5 and 6, which have been inaccessible by conventional methods. Therefore, the synthetic methods established in this study will expand the molecular diversity of their related derivatives. These compounds were also assessed for their antiproliferative effect on a human promyelocytic leukemia cell line, HL-60. Treatment of 10 µM 6b and 6d showed high inhibitory activity similarly to 1 µM all-trans retinoic acid (ATRA), indicating that the 2-thioxo group and length of two alkyl substituents at the 4-position are strongly related to activity.

Heme oxygenase-1 induction by heat shock in rat hepatoma cell line is regulated by the coordinated function of HSF1, NRF2 and BACH1.

The mechanism of heme oxygenase-1 (HO-1) induction by heat shock (HS) loading remains unclear. Here, we investigated the contribution of transcription factors to HS-induced HO-1 expression, using a rat hepatoma cell line (H-4-II-E). Our results demonstrated that HS treatment resulted in a marked induction of HO-1. Immunohistochemical analysis showed a slight mismatch in the expression levels of HO-1 and HSP70 by HS among cells, suggesting a conflict between multiple induction mechanisms. We observed HS-induced nuclear localization of, not only phosphorylated HSF1 but also NRF2, which is a typical transcription factor activated by oxidative stress. HSF1 knockdown in H-4-II-E markedly reduced HO-1 induction by HS, while NRF2 knockdown resulted in a partial effect. The chromatin immunoprecipitation assay demonstrated that HS loading resulted in significant binding of HSF1 to the HSE in the promoter proximal region of HO-1 gene and another HSE located close to the Maf recognition element (MARE) in the -4 kb upstream enhancer region 1, where NRF2 also bound, together with basic leucine zipper transcription factor 1, a negative transcription factor of HO-1. These observations indicate that HO-1 induction by HS is mainly mediated by HSF1 binding to the proximal HSE. NRF2 binding to MARE by HS is predominantly suppressed by an increased binding of BACH1.

Sixteen Different Types of Lipid-Conjugated siRNAs Containing Saturated and Unsaturated Fatty Acids and Exhibiting Enhanced RNAi Potency.

SiRNAs are strong gene-silencing agents that function in a target sequence-specific manner. Although siRNAs might one day be used in therapy for intractable diseases such as cancers, a number of problems with siRNAs must first be overcome. In this study, we developed 16 different types of lipid-conjugated siRNAs (lipid-siRNAs) that could effectively inhibit the expression of target genes. We determined the hybridization properties, cellular uptake efficacies, and RNAi potencies of the resulting lipid-siRNAs. The lipid-siRNAs exhibited a mild interaction with Lipofectamine RNAiMAX (LFRNAi) as a transfection reagent, and a high membrane permeability was observed in all lipid-siRNAs-LFRNAi complexes; the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed an especially good cellular uptake efficacy. The in vitro RNAi effect of lipid-siRNAs targeted to a ß-catenin gene exhibited a strong RNAi potency compared with those of unmodified siRNAs. In particular, the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed excellent RNAi potencies with prolonged effectivities. Interestingly, the RNAi potencies of conjugate siRNAs containing 18 carbon chains with a trans-form (elaidic acid and trans-vaccenic acid) were inferior to those of the carbon chains with a cis-form (oleic acid and cis-vaccenic acid). These lipid-siRNAs can solve the many problems hindering the clinical application of siRNAs.

ADAR2 Regulates Malignant Behaviour of Mesothelioma Cells Independent of RNA-editing Activity.

BACKGROUND/AIM: Malignant pleural mesothelioma (MPM) is an intractable cancer, and causes of its malignant transformation are not well known. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme that converts adenosine into inosine in double-stranded RNAs potentially involved in malignant development. MATERIALS AND METHODS: To examine the role of ADAR1 and ADAR2 in MPM, small interfering RNAs (siRNAs) against ADAR1 or ADAR2 were used. RESULTS: Transfection of siRNA against ADAR2 suppressed proliferation, motility, and invasiveness of MPM cells expressing both ADAR1 and ADAR2; however, siRNA against ADAR1 did not affect these cellular activities. Overexpression of ADAR2, that was incapable of binding to RNA, suppressed growth, motility, and invasion of MPM cells. However, overexpression of ADAR2 that had no enzyme activity did not alter the malignant properties of MPM cells. CONCLUSION: Enhancement of the malignant characteristics of cultured MPM cells via ADAR2 was independent of RNA-editing activity.

Visualization of the Redox Status of Cytosolic Glutathione Using the Organelle- and Cytoskeleton-Targeted Redox Sensors.

Glutathione is a small thiol-containing peptide that plays a central role in maintaining cellular redox homeostasis. Glutathione serves as a physiologic redox buffer by providing thiol electrons for catabolizing harmful oxidants and reversing oxidative effects on biomolecules. Recent evidence suggests that the balance of reduced and oxidized glutathione (GSH/GSSG) defines the redox states of Cys residues in proteins and fine-tunes their stabilities and functions. To elucidate the redox balance of cellular glutathione at subcellular resolution, a number of redox-sensitive green fluorescent protein (roGFP) variants have been developed. In this study, we constructed and functionally validated organelle- and cytoskeleton-targeted roGFP and elucidated the redox status of the cytosolic glutathione at a subcellular resolution. These new redox sensors firmly established a highly reduced redox equilibrium of cytosolic glutathione, wherein significant deviation was observed among cells. By targeting the sensor to the cytosolic and lumen sides of the Golgi membrane, we identified a prominent redox gradient across the biological membrane at the Golgi body. The results demonstrated that organelle- and cytoskeleton-targeted sensors enable the assessment of glutathione oxidation near the cytosolic surfaces of different organelle membranes.

High Mannose Binding Lectin (PFL) from Pseudomonas fluorescens Down-Regulates Cancer-Associated Integrins and Immune Checkpoint Ligand B7-H4.

Pseudomonas fluorescens lectin (PFL), which belongs to the high mannose (HM)-binding OAAH (Oscillatoria agardhii agglutinin homologue) lectin family, induces cancer cell death. However, the detailed mechanisms underlying this process have not yet been elucidated. We found that PFL decreased various integrins as well as EGFR in cancer cells by promoting internalization and autophagic degradation of these molecules, subsequently inducing caspase-8 dependent cell apoptosis. As revealed by an ex vivo angiogenesis assay using the rat aortic model, PFL inhibited neovascularization in a dose-dependent manner, which was potentially mediated by down-regulation of endothelium integrins. Interestingly, PFL also down-regulated B7-H4 in cancer cells, which has been implicated as a negative regulator of T cell-mediated immunity. We found that B7-H4 co-localized with ß3 integrin in MKN28 gastric cancer cells. siRNA silencing of B7-H4 in MKN28 cells decreased expression of ß3 integrin, suggesting physical and functional association between these molecules. Direct interaction of PFL with integrin αvß3 or B7-H4 was examined by surface plasmon resonance analysis, which detected high affinity glycan-dependent binding to PFL. These investigations suggest that PFL interaction with cell surface integrins is a key process for the anti-cancer activities of PFL.

Development and characterization of a cancer cachexia model employing a rare human duodenal neuroendocrine carcinoma-originating cell line.

Cancer cachexia interferes with therapy and worsens patients' quality of life. Therefore, for a better understanding of cachexia, we aimed to establish a reliable cell line to develop a cachexia model. We recently established and characterized the TCC-NECT-2 cell line, derived from a Japanese patient with poorly differentiated neuroendocrine carcinoma of the duodenum (D-NEC). Subcutaneous xenograft of TCC-NECT-2 cells in mice resulted in tumor formation, angiogenesis, and 20% incidence of body weight (BW)-loss. Subsequently, we isolated a potent cachexia-inducing subline using stepwise selection and designated as AkuNEC. Orthotopic and s.c. implantation of AkuNEC cells into mice led to diminished BW, anorexia, skeletal muscle atrophy, adipose tissue loss, and decreased locomotor activity at 100% incidence. Additionally, orthotopic implantation of AkuNEC cells resulted in metastasis and angiogenesis. Serum IL-8 overproduction was observed, and levels were positively correlated with BW-loss and reduced adipose tissue and muscle volumes in tumor-bearing mice. However, shRNA knockdown of the IL-8 gene did not suppress tumor growth and cachexia in the AkuNEC model, indicating that IL-8 is not directly involved in cachexia induction. In conclusion, AkuNEC cells may serve as a useful model to study cachexia and D-NEC.

Development and Biological Analysis of a Novel Orthotopic Peritoneal Dissemination Mouse Model Generated Using a Pancreatic Ductal Adenocarcinoma Cell Line.

OBJECTIVES: Peritoneal dissemination (PD) is an important cause of morbidity and mortality among patients with pancreatic ductal adenocarcinoma (PDAC). We sought to develop and characterized a novel PD mouse model by using a previously established PDAC cell line TCC-Pan2. METHODS: TCC-Pan2 cell line was characterized for growth rate, tumor markers, histology, and somatic mutations. TCC-Pan2 cells were implanted orthotopically to produce PD. TCC-Pan2 cells from these metastatic foci were expanded in vitro and then implanted orthotopically in mice. This PD model was used for comparing the antitumor effect of paclitaxel and NK105. RESULTS: Orthotopically implanted TCC-Pan2 cells caused tumor formation and PD with high frequency in mice. A potent metastatic subline-Pan2M-was obtained. NK105 exerted a stronger antitumor effect than paclitaxel against Pan2M cells harboring a luciferase gene (Pan2MmLuc). Notably, the survival rate on day 80 in the Pan2MmLuc mouse model was 100% for the NK105 group and 0% for the paclitaxel group. CONCLUSION: TCC-Pan2 cell line and Pan2MmLuc PD model can serve as useful tools for monitoring the responses to antineoplastic agents and for studying PDAC biology.

Antitumor effect of palmitic acid-conjugated DsiRNA for colon cancer in a mouse subcutaneous tumor model.

In this study, we synthesized Dicer-substrate siRNA conjugated with palmitic acid at the 5'-end of the sense strand (C16-DsiRNA), and examined its RNAi effect on ß-catenin as a target gene in a colon cancer cell line, HT29Luc, both in vitro and in vivo. We examined the in vitro RNAi effect in HT29Luc cells and found that C16-DsiRNA strongly inhibited expression of the ß-catenin gene in comparison with non-modified DsiRNA. Also, high membrane permeability of C16-DsiRNA was exhibited, and it was confirmed that most of the C16-DsiRNA was localized in cytoplasm of HT29Luc cells. In regard to the in vivo RNAi effect, C16-DsiRNA complexed with Invivofectamine targeting the ß-catenin gene was locally administered to a subcutaneous tumor formed by implantation of HT29Luc cells into the subcutis of nude mice; we evaluated the effect by measuring the bioluminescence increase, which reflects tumor growth, using an in vivo imaging system. As a result, C16-DsiRNA strongly inhibited the growth of tumors formed in subcutis of nude mice compared with non-modified DsiRNA, and this in vivo RNAi effect lasted up to 15 days. Our results suggest that C16-DsiRNA should be vigorously pursued as a novel nucleic acid medicine for clinical treatment of cancer.

Establishment of a novel cell line from a rare human duodenal poorly differentiated neuroendocrine carcinoma.

Poorly differentiated neuroendocrine carcinoma of the duodenum (D-NEC) is a rare cancer with poor prognosis. However, a D-NEC cell line has not yet been established to study the disease. We established a cell line, TCC-NECT-2, from the ascites tumor of a 59-year-old male Japanese patient with D-NEC. TCC-NECT-2 was positive for neuroendocrine markers, chromogranin A (CGA), cluster of differentiation 56 (CD56/NCAM), synaptophysin (SYN/p38), and neuron specific enolase (NSE). Cells exhibited retinoblastoma (RB) protein loss. Orthotopic implantation of TCC-NECT-2 cells into nu/nu mice resulted in tumor formation (incidence = 83.3%) with neuroendocrine characteristics, metastasis, and weight loss. BRAFV600E and TP53 mutations and C-MYC gene amplification were also observed in TCC-NECT-2. BRAFV600E-expressing TCC-NECT-2 cells were sensitive to BRAF inhibitor vemurafenib, and especially dabrafenib, in vitro, and were strongly inhibited in a dose-dependent manner. Dabrafenib treatment (30 mg/kg) in a xenograft model for 14 days significantly suppressed tumor growth (percent tumor growth inhibition, TGI% = 48.04). An enhanced therapeutic effect (TGI% = 95.81) was observed on combined treatment of dabrafenib and irinotecan (40 mg/kg). Therefore, TCC-NECT-2, the first reported cell line derived from D-NEC, might serve as a useful model to study the basic biology of D-NEC and translational applications for treatment.

NRF2 and HSF1 coordinately regulate heme oxygenase-1 expression.

Heme oxygenase-1 (HO-1) is an inducible enzyme responding to various stresses and has cytoprotective activities. Although HO-1 has been referred to as heat shock protein (HSP) 32, the heat-mediated induction of HO-1 varies among different species and cell lines. We examined the effects of heat shock on HO-1 expression in mouse embryonic fibroblast (MEF) cells deficient in heat shock factor 1 (HSF1) or nuclear factor-erythroid-2-related factor 2 (NRF2). Heme-induced expression of HO-1 was 2-fold higher in Hsf1-/- cells than in the wild-type cells at both mRNA and protein levels. In Nrf2-/- cells, heme-induced expression of HO-1 was not detected. In contrast, HO-1 expression was markedly induced by heat shock at 40-42 °C in Nrf2-/- cells while the wild-type cells were not responsive. The heat-induced expression of HO-1 in Nrf2-/- cells were almost completely diminished by transfection of siRNA against Hsf1 gene. These results suggest that HSF1 and NRF2 suppress heme-induced and heat-induced HO-1 expression, respectively.

Biomechanical Analysis of Internal Bracing for Treatment of Medial Knee Injuries.

The internal brace technique uses a high-strength suture tie to augment injured tissues or a primary repair, allowing early rehabilitation. Anatomic repair with internal bracing is a novel and promising treatment for femoral-sided medial knee avulsion injuries of the medial collateral ligament and posterior oblique ligament. Unfortunately, biomechanical and clinical data are lacking. To evaluate this technique compared with other treatment options, 3 assays of 9 cadaveric matched pairs (54 knees) were tested to failure at 30° under valgus load in a biomechanical testing apparatus. The primary outcome measure was moment at failure (Nm), with secondary outcome measures of stiffness (Nm/°), valgus angulation at 10 Nm (°), and valgus angulation at failure (°). Repair with internal bracing was compared with the intact state, repair alone, and allograft reconstruction. The mean moment to failure (62.5±24.9 Nm) for internal bracing was significantly lower than that for intact specimens (107.2±39.7 Nm) (P=.009). Mean moment to failure and valgus angle at failure were significantly greater for internal bracing (95±31.9 Nm) than for repair (73.4±27.6 Nm) (P=.05). Internal bracing was similar to reconstruction for the primary outcome measure (53.5±26.3 Nm vs 66.9±28.8 Nm) (P=.227) and for all secondary outcome measures. These findings indicate that posteromedial knee repair with internal bracing for femoral-sided avulsions is superior to repair alone and is similar to allograft reconstruction for all parameters measured; however, this technique did not recreate biomechanical properties equivalent to the intact state. [Orthopedics. 2016; 39(3):e532-e537.].

High mannose-binding Pseudomonas fluorescens lectin (PFL) downregulates cell surface integrin/EGFR and induces autophagy in gastric cancer cells.

BACKGROUND: Pseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated. METHODS: Cell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated. RESULTS: The strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth. CONCLUSION: PFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs.

In Vivo RNAi Efficacy of Palmitic Acid-Conjugated Dicer-Substrate siRNA in a Subcutaneous Tumor Mouse Model.

Short interfering RNAs are used in RNA interference technology and are powerful tools for target gene silencing in a sequence-specific manner. In this study, we synthesized Dicer-substrate siRNAs consisting of 27-nt double-stranded RNAs conjugated with palmitic acid at the 5'-end of the sense strand and investigated their RNA interference efficacies in vitro and in vivo. The palmitic acid-conjugated 27-nt DsiRNAs (C16-Dsi27RNAs) were prepared by our simple synthesis strategy and achieved a good yield. C16-Dsi27RNAs showed enhanced in vitro RNA interference potency compared with not only non-modified Dsi27RNAs but also cholesterol-conjugated Dsi27RNAs against both an exogenous enhanced green fluorescent protein and the endogenous vascular endothelial growth factor gene in a human scirrhous-type gastric cancer cell line that stably expressed the enhanced green fluorescent protein gene (GCIY-eGFP). Additionally, C16-Dsi27RNAs had potent gene silencing activity against both enhanced green fluorescent protein and vascular endothelial growth factor as target genes in a subcutaneous tumor mouse model generated from GCIY-eGFP cells administered by intratumoral injection. These results suggest that the C16-Dsi27RNAs will be useful next-generation RNA interference molecules that can overcome the problems associated with RNA interference technology.

All-trans retinoic acid enhances cytotoxic effect of T cells with an anti-CD38 chimeric antigen receptor in acute myeloid leukemia.

We reported that T cells with anti-CD38-chimeric antigen receptors (CAR) eliminated B-cell lymphoma cells expressing CD38. To employ anti-CD38-CAR against acute myeloid leukemia (AML) blasts not expressing CD38, it is necessary to induce or increase the intensity of CD38 expression. A lactate dehydrogenase (LDH)-releasing assay and flow cytometry showed that anti-CD38-CAR T cells were cytotoxic against AML lines (THP-1 and CMK) expressing high CD38 levels (>99%), in time- and number of effector-dependent manners. In other AML lines (KG1, U937 and HL60) partially expressing CD38, CD38+ AML cells were killed by CD38-specific T cells, but CD38- AML cells remained survived. Intriguingly, 10 nM all-trans retinoic acid (ATRA) augmented CD38 expression in KG1, U937 and HL60 cells and primary leukemic cells from AML patients. Moreover, the withdrawal of ATRA from the medium decreased CD38 expression in AML cells. Killing effects of anti-CD38-CAR T cells against AML lines and AML cells were limited without ATRA, whereas CD38-specific T cells enhanced cytotoxicity on AML cells by ATRA in association with enhanced CD38 expression. These results indicate that anti-CD38-CAR T cells eliminate AML cells through CD38 expression induced by ATRA.