HL156 compounds, novel metformin derivatives, inhibit angiogenesis through AMPK mediated downregulation FOXM1 in gastric cancer cells

Dept. of Medical Science/석사Metformin, which is widely used as an anti-diabetic drug, has recently gained significant attention as a potential anti-cancer agent. Although a couple of potential mechanisms explaining anti-tumor effects have been suggested, its detailed molecular mechanisms remain elusive. HL156 compounds are novel biguanide derivatives based on metformin formula with more potent anti-tumor effects than metformin. In cell based assays, HL156 activates AMP-activated kinase (AMPK) more potently than metformin in various cancer cell lines.Our preliminary analysis of Reverse phase protein array (RPPA) data from gastric cancer patients indicated that proteins related to regulating angiogenesis were increased in tumors of patients with poor prognosis. In addition, a pilot in vivo efficacy study showed that HL156 significantly suppressed metastasis in breast cancer xenograft models.Motivated by these, we hypothesized that HL156 would inhibit angiogenesis that is one of the critical steps of metastatic process. Further, the anti-angiogenic mechanism of HL156 would be mediated through AMPK activation which downregulated oncogenic transcription factor FOXM1. Culturing HUVEC cells in the conditioned media treated with HL156 compounds to gastric cancer cells downregulated in vitro endothelial cell network formation. In the same conditioned media, the expression of VEGFA, a major angiogenic factor, was prominently suppressed. Also, the expression of FOXM1, a pleiotropic oncogenic transcription factor, was inhibited by HL156, which is correlated with activation of AMPK. To further interrogate the molecular mechanisms by which FOXM1 was down-regulated, we assessed CDK4/6 expression in gastric cancer cells after HL 156 treatment. CDK4/6, which phosphorylates and activates FOXM1, was downregulated leading to the decrease of phospho-FOXM1 (Ser35) level. Electrophoretic mobility shift assay showed that HL156 treatment repressed the FOXM1 binding to the VEGFA promoter.Taken together, these results suggest that HL156 alleviates tumor angiogenesis by VEGFA inhibition through AMPK mediated CDK4/6 - FOXM1 downregulation in gastric cancer cells.ope

The different role of intratumoral and peritumoral lymphangiogenesis in gastric cancer progression and prognosis

BACKGROUND: Tumor-induced lymphangiogenesis plays a crucial role in metastasis and tumor progression. However, the significance of intratumoral lymphovascular density (I-LVD) and peritumoral lymphovascular density (P-LVD) has been controversial in gastric cancer. The purpose of this study was to investigate the differences of clinicopathologic characteristics with respect to I-LVD and P-LVD in gastric cancer. METHODS: Samples of I-LVD and P-LVD from 66 patients who had undergone radical gastrectomy for gastric cancer were assessed after staining with D2-40, an immunostaining marker for lymphatic endothelium. The mean number of lymphatic vessels in three hotspots was calculated in intratumoral and peritumoral areas. RESULTS: The peritumoral lymphatics were enlarged with dilated lumens compared to the intratumoral lymphatics. I-LVD was positively correlated with diffuse gastric cancer subtype, tumor stage, lymphovascular invasion, tumor node metastasis stage, and overall survival (P < 0.05). P-LVD was associated with lymphovascular invasion, node stage, and disease-free survival (P < 0.05). CONCLUSIONS: We conclude that P-LVD had an important role in lymph node metastasis, while I-LVD was more associated with depth of tumor invasion. However, both LVDs contributed to gastric cancer progression and prognosis.ope

Inhibition of Wntless/GPR177 suppresses gastric tumorigenesis

Wntless/GPR177 functions as WNT ligand carrier protein and activator of WNT/beta-catenin signaling, however, its molecular role in gastric cancer (GC) has remained elusive. We investigated the role of GPR177 in gastric tumorigenesis and provided the therapeutic potential of a clinical development of anti-GPR177 monoclonal antibodies. GPR177 mRNA expression was assessed in GC transcriptome data sets (GSE15459, n = 184; GSE66229, n = 300); protein expression was assessed in independent patient tumor tissues (Yonsei TMA, n = 909). GPR177 expression were associated with unfavorable prognosis [log-rank test, GSE15459 (P = 0.00736), GSE66229 (P = 0.0142), and Yonsei TMA (P = 0.0334)] and identified as an independent risk predictor of clinical outcomes: GSE15459 [hazard ratio (HR) 1.731 (95% confidence interval; CI; 1.103- 2.715), P = 0.017], GSE66229 [HR 1.54 (95% CI, 1.10-2.151), P = 0.011], and Yonsei TMA [HR 1.254 (95% CI, 1.049- 1.500), P = 0.013]. Either antibody treatment or GPR177 knockdown suppressed proliferation of GC cells and sensitized cells to apoptosis. And also inhibition of GPR177 suppresses in vitro and in vivo tumorogenesis in GC cells and inhibits WNT/beta-catenin signaling. Finally, targeting and inhibition of GPR177 with antibody suppressed tumorigenesis in PDX model. Together, these results suggest GPR177 as a novel candidate for prognostic marker as well as a promising target for treatment of GC patients. [BMB Reports 2018; 51(5): 255-260].ope

위암줄기세포에서 치료 표적으로서 미토콘드리아 활성산소 항상성 조절 기전

Increased oxidative phosphorylation (OXPHOS) and reactive oxygen species (ROS) levels are inherently linked. ROS are essential signaling molecules, with detrimental effects when produced in excess during chemotherapy, leading to cell death. Cancer stem-like cells (CSCs) are subpopulation of tumor cells resistant to chemotherapy, highly invasive and metastatic, driving malignant cancer behavior. In this study, we demonstrated that CSCs exhibit increased OXPHOS but paradoxically low ROS levels. Considering the detrimental effects of large amounts of ROS, CSCs have developed potential mechanisms for quenching excess ROS to maintain redox homeostasis. We aimed to investigate the distinct metabolic features and mechanisms of ROS regulation in gastric CSCs and explore potential therapeutic strategies targeting CSCs. Human gastric cancer cell lines, AGS and MKN1, were subjected to liquid chromatography/mass spectrometry-based metabolomic and microarray analyses. Mitochondrial properties such as mitochondrial mass, membrane potential, and ROS were assessed by flow cytometry analysis. CSCs with increased OXPHOS levels maintained low ROS levels by coupling FoxM1-dependent Prx3 expression and fatty acid oxidation-mediated NADPH regeneration. Thus, interventions targeting ROS homeostasis in CSCs may be a useful strategy for targeting this drug-resistant tumor cell subpopulation. 암 줄기세포는 일반 암 세포와는 달리 스스로 재생하고 다른 세포로 분화 할 수 있는 무제한 재생능력을 가져 전이의 주요 원인으로 작용하며 높은 약물 저항성을 나타낸다. 특히 일부 환자들은 이런 암 줄기세포가 활성화 되면서 항암제 치료가 듣지 않는 ‘강한 저항성’을 보이기도 한다. 기존 항암요법으로는 치료 할 수 없는 난치성 암이 이 경우에 해당된다. 최근 암 줄기세포는 미토콘드리아 기능이 일반 암 세포보다 향상 되어 Glycolysis 보다 OXPHOS에 의존해 ATP를 생성하는 것이 다양한 암종에서 공통적으로 보고된 바 있다. 그러나 암 줄기세포를 타겟 하는 정확한 메커니즘에 대해서는 잘 알려져 있지 않다. 본 연구에서는 미토콘드리아 활성 산소 항상성의 조절 기전을 규명 함으로써, 암 줄기 세포에서 임상적으로 가장 큰 문제점인 약물 저항성을 타겟 할 있는 메커니즘적 근거를 제시하고자 한다. 암 줄기세포에서 미토콘드리아 기능이 향상되어 있음에도 불구하고 활성 산소가 낮게 유지되는 것을 확인 할 수 있었다. 항산화 효소 중 미토콘도리아에 존재하는 Prx3의 발현이 암 줄기 세포에서 높은 것을 확인하였으며, 전사 인자인 FoxM1에 의해 Prx3 발현이 조절 됨을 확인하였다. 또한 암 줄기세포는 지방산 산화를 통한 에너지 대사 과정에서 미토콘드리아에 존재하는 IDH2와 MTHFD2에 의존적으로 NADPH를 재생성 함으로써 활성 산소 항상성을 유지함을 확인 할 수 있었다. 본 연구는 표준 항암치료에 저항성이 있는 암 줄기세포의 잠재적 생존 원리를 규명했으며, 미토콘드리아 활성산소 항상성 유지 메커니즘을 표적으로 난치성 악성 암을 치료할 수 있는 실험적 근거를 제시하였다.open박

DNAJC14 샤페로닌 조절에 의한 H723R 돌연변이 펜드린의 발현 및 기능 회복

In East Asia, the most prevalent mutation in SLC26A4/Pendrin, H723R (His723Arg), causes protein folding defect in pendrin, resulting in an autosomal recessive type of genetic hearing loss (DFNB4). There is no curative treatment for hearing loss caused by H723R mutation. The aim of the current study was to rescue H723R-pendrin expression and activity by the activation of DNAJC14 chaperonin through Flavivirus inoculation or overexpression of DNAJC14. We found that toxin-attenuated Japanese encephalitis virus (JEV), which is a Flavivirus, rescued surface expression and anion exchange activity of H723R-pendrin, in vitro. We established a human H723R-pendrin transgenic mouse model (hH723R Tg) with a mouse PDS knock-out background, that only expresses human H723R-pendrin in the inner ear (determined by Pax2-cre), which mimics human DFNB4. When hH723R Tg was crossed with DNAJC14 overexpressed mice, cochlear hydrops was reduced, expression of pendrin in the endolymphatic duct was increased, and outer hair cells in the cochlea were more preserved, compared to human H723R Tg. Furthermore, with DNAJC14 overexpression, stria vascularis and spiral ligaments were thicker and K+ channel KCNJ10 expression contributing to endocochlear potential generation was more abundant. It can be concluded that DNAJC14 rescues the pathology of misfolding in H723R-pendrin and may be a potential therapeutic target for genetic hearing loss. 유전성 난청은 500 ~ 1000 명의 어린이 중 1 명 꼴로 발생하는 매 우 만연한 질병이며, 선천성 난청의 절반 이상이 유전적 변이에 의해 유발된다. 그 중에서도 특히, SLC26A4/펜드린 p.H723R(His723Arg) 돌연 변이가 동아시아에서 유전성 난청을 일으키는 가장 흔한 원인 변이이 고, 이 돌연변이는 단백 접힘 이상을 초래하여 세포막으로 도달하지 못하는 것으로 알려져 있다. 그러나, 아직 유전성 난청 환자들을 위한, 임상적으로 치료 가능한 치료법이 없다. 그래서 본 연구로, H723R 펜 드린의 비전형적 단백 수송 경로를 통한 H723R 돌연변이 펜드린이 세포막으로 도달할 수 있도록, Flavivirus 또는, DNAJC14의 과발현을 통한 DNAJC14 조절현상을 이용하여, H723R 돌연변이 펜드린을 교정 하고자 하였다. Flavivirus의 JEV를 treat하여, DNAJC14를 세포주에서 과발현시켜, p.H723R 펜드린의 세포막 발현이 증가하는 것을, surface biotinylation assay와 immunocytochemistry를 통해 확인하였고, 펜드린의 기능인, anion exchange activity가 향상되는 것을 Cl-와 HCO3 - 의 exchange activity를 측정하여 확인하였다. 또한, hH723R 펜드린 마우스 모델에 DNAJC14를 과발현시킨 마우 스 모델을 제작하였고, 이를 통해 내이 내 H723R 돌연변이 펜드린에 의한 조직학적 퇴화가 개선되는 것을 확인하였다. 먼저, 난청을 가진 펜드린 knock-out 마우스의 주된 특징인 증가되어 있던 hydrops의 크 기가 현저히 줄어드는 것을 H&E를 통해 확인하였고, 청력회복까지는 미치지 못했지만 stria vascularis와 stria ligament의 두께가 증가한 것으 로 보아 구조적 퇴화를 막아주는 것을 확인하였고, endolymphatic duct 에 hH723R 펜드린의 발현이 증가되어있는 것과 외유모세포의 죽음을 막아주는 것 또한 확인하였다. 결론적으로, 본 연구를 통해 DNAJC14의 활성화 또는 과발현이 H723R 돌연변이 펜드린의 세포막 발현 및 음이온 교환 기능을 증가 시킨다는 것을 확인하였고, 나아가 SLC26A4/Pendrin 돌연변이에 의한 유전성 난청의 치료제 개발에 대한 해결의 실마리를 찾는데 도움이 될 것으로 생각된다.open석

Genetic Inheritance of Late-Onset, Down-Sloping Hearing Loss and Its Implications for Auditory Rehabilitation

OBJECTIVES: Late-onset, down-sloping sensorineural hearing loss has many genetic and nongenetic etiologies, but the proportion of this commonly encountered type of hearing loss attributable to genetic causes is not well known. In this study, the authors performed genetic analysis using next-generation sequencing techniques in patients showing late-onset, down-sloping sensorineural hearing loss with preserved low-frequency hearing, and investigated the clinical implications of the variants identified. DESIGN: From a cohort of patients with hearing loss at a tertiary referral hospital, 18 unrelated probands with down-sloping sensorineural hearing loss of late onset were included in this study. Down-sloping hearing loss was defined as a mean low-frequency threshold at 250 Hz and 500 Hz less than or equal to 40 dB HL and a mean high-frequency threshold at 1, 2, and 4 kHz greater than 40 dB HL. The authors performed whole-exome sequencing and segregation analysis to identify the genetic causes and evaluated the outcomes of auditory rehabilitation in the patients. RESULTS: There were nine simplex and nine multiplex families included, in which the causative variants were found in six of 18 probands, demonstrating a detection rate of 33.3%. Various types of variants, including five novel and three known variants, were detected in the MYH14, MYH9, USH2A, COL11A2, and TMPRSS3 genes. The outcome of cochlear and middle ear implants in patients identified with pathogenic variants was satisfactory. There was no statistically significant difference between pathogenic variant-positive and pathogenic variant-negative groups in terms of onset age, family history of hearing loss, pure-tone threshold, or speech discrimination scores. CONCLUSIONS: The proportion of patients with late-onset, down-sloping hearing loss identified with potentially causative variants was unexpectedly high. Identification of the causative variants will offer insights on hearing loss progression and prognosis regarding various modes of auditory rehabilitation, as well as possible concomitant syndromic features.restrictio

Optimal positive end-expiratory pressure to prevent anaesthesia-induced atelectasis in infants: A prospective, randomised, double-blind trial

Background: Paediatric patients have a particularly high incidence of anaesthesia-induced atelectasis. Applying positive end-expiratory pressure (PEEP) with an alveolar recruitment manoeuvre has been substantially studied and adopted in adults; however, few studies have been conducted in children. Objective: We compared the effects of three levels of PEEP (3, 6 and 9 cmH2O) on anaesthesia-induced atelectasis measured by ultrasound in infants between 6 and 12 months of age who were undergoing general anaesthesia. Design: A prospective, randomised, double-blind trial. Setting: Department of Anaesthesia, single centre, South Korea, from May 2019 to March 2020. Patients: Children who were 6 to 12 months of age, whose American Society of Anesthesiologists (ASA) physical status was 1 or 2, whose height and weight were within two standard deviations of those of their peers, and who were scheduled for elective urological or general surgery were included in the study. Main outcome measures: The primary outcome was the lung ultrasound score at the end of the procedure. The secondary outcomes included dynamic compliance, peak inspiratory pressure, driving pressure, cardiac index, mean arterial pressure and heart rate before and after applying PEEP. Results: The mean lung ultrasound score at the end of operation was 12.8 at PEEP 6 cmH2O and 12.1 at PEEP 9 cmH2O. Both were significantly lower than 18.4 at PEEP 3 cmH2O (P = 0.0002 and 0.00003, respectively). However, there was no significant difference between the scores of PEEP 6 cmH2O and PEEP 9 cmH2O. The Δ cardiac index (the cardiac index after PEEP - the cardiac index at 3 cmH2O of PEEP) was comparable among the three groups. Conclusion: To reduce anaesthesia-induced atelectasis measured by ultrasound in healthy infants undergoing low abdominal, genitourinary or superficial regional operations, 6 cmH2O of PEEP was more effective than 3 cmH2O. PEEP of 9 cmH2O was comparable with 6 cmH2O. Trial registration: ClinicalTrials.gov identifier NCT03969173.restrictio

Comparison of Monotherapy Versus Combination of Intravenous Ibuprofen and Propacetamol (Acetaminophen) for Reduction of Postoperative Opioid Administration in Children Undergoing Laparoscopic Hernia Repair: A Double-Blind Randomized Controlled Trial

Background: Extensive efforts have been made toward reducing postoperative opioid use in children. In this study, we assessed whether propacetamol, or a nonsteroidal anti-inflammatory drug (NSAID), or their combination could effectively reduce opioid use in children after laparoscopic inguinal hernia repair. Methods: This randomized, double-blind clinical trial included 159 children aged 6 months to 6 years. Children were allocated into 1 of the following 3 groups: group I was treated with 10 mg·kg-1 ibuprofen, group P was treated with 30 mg·kg-1 propacetamol, and group I + P was treated with both drugs in their respective concentrations. If the face-legs-activity-crying-consolability (FLACC) score was ≥4 during the postanesthesia care unit stay, 1.0 µg·kg-1 fentanyl was administered as a rescue analgesic. The number of patients who received rescue fentanyl in the postanesthesia care unit was defined as the primary outcome; this was analyzed using the χ2 test. The secondary outcomes included the FLACC and the parents' postoperative pain measure (PPPM) scores until the 24-hour postoperative period. Results: Among the 144 enrolled patients, 28.6% in group I, 66.7% in group P, and 12.8% in group I + P received rescue fentanyl in the postanesthesia care unit (P < .001). The highest FLACC score was lower in group I + P than in either group I or P (P = .007 and P < .001, respectively). Group I + P presented significantly lower PPPM scores than group P at 4 and 12 hours postoperative (P = .03 and .01, respectively). Conclusions: The use of ibuprofen plus propacetamol immediately following laparoscopic hernia repair surgery in children resulted in the reduced use of an opioid drug compared with the use of propacetamol alone. Trial registration: ClinicalTrials.gov NCT03352362.restrictio