The anticancer effects of Aronia berry extract are mediated by Chk1 and p53 in colorectal cancer.

BACKGROUND: Aronia berry extracts (ABE) have recently been reported to possess significant anti-cancer effects in various malignancies, including colorectal cancer (CRC), due to their high polyphenolic content. However, the molecular mechanism(s) underlying the anti-cancer effects of ABE in CRC remain unclear, which is important to consider when considering their use as complementary medicine approaches in cancer. METHODS: We performed genome-wide transcriptomic profiling and pathway enrichment analysis to identify specific growth signaling pathways associated with ABE treatment in CRC cells. In addition, a series of systematic and comprehensive cell culture studies were performed to investigate the anti-cancer effects of ABE in SW480 and HCT116 CRC cell lines. Subsequently, these findings were validated in patient-derived 3D organoids (PDOs) models. RESULTS: Transcriptomic profiling analysis identified p53 signaling as one of the key enriched pathways mediating the anti-cancer activity of ABE. Analysis of public datasets revealed that Chk1, a key regulator of p53, was one of the critical targets of ABE in CRC. Chk1 and p53 activation was shown to be downregulated with ABE treatment, leading to the induction of cell cycle arrest (p = 0.003-0.014) and enhanced DNA damage (p = 0.015-0.026). Furthermore, these findings were validated in PDOs, where the ABE treatment resulted in significantly fewer and smaller PDOs in a concentration-dependent manner (p = 0.045 - <0.001). CONCLUSIONS: We firstly provide evidence for the role of the p53 signaling pathway as a mediator of the anti-cancer activity of ABE, which provides a rationale for its use as a safe and effective integrative medicine approach in CRC.

Blood flow assessment of gastric tube with indocyanine green fluorescence angiography and postoperative endoscopy during esophagectomy: indocyanine green enhancement time indicated congestion.

BACKGROUND: During esophagectomy, evaluation of blood supply to the gastric tube is critically important to estimate and avoid anastomotic complications. This retrospective study investigated the relationship between indocyanine green (ICG) fluorescence angiography during esophagectomy and postoperative endoscopy findings, especially mucosal color change. METHODS: This study retrospectively collected data from 86 patients who underwent subtotal esophagectomy and reconstruction using a gastric tube for esophageal cancer at the Tokyo Medical and Dental University between 2017 and 2020. The flow speed of ICG fluorescence in the gastric tube was evaluated during the operation. Additionally, the main root of ICG enhancement and pattern of ICG distribution in the gastric tube were evaluated. On postoperative day 1 (POD1), the change in the mucosal color to white, thought to reflect ischemia, or black, thought to reflect congestion of the proximal gastric tube, was evaluated. The correlations between these factors, clinical parameters, and surgical outcomes were evaluated. Univariate and multivariate analyses used logistic regression to identify the risk factors affecting mucosal color change. RESULTS: Multivariate analyses revealed that the only independent significant predictor of mucosal congestion on POD1 was the ICG enhancement time of the right gastric tube tip (odds ratio, 14.49; 95% confidential interval, 2.41-87.24; P = 0.004). CONCLUSIONS: This study indicated that the ICG enhancement time is related to venous malperfusion and congestion rather than arterial malperfusion and ischemia.

Intraoperative Recurrent Laryngeal Nerve Monitoring for Esophagectomy: A National Cohort Study.

BACKGROUND: Lymphadenectomy around the recurrent laryngeal nerve (RLN) is an essential part of curative surgery for esophageal cancer. Although several single-center studies have shown that intraoperative nerve monitoring (IONM) can avoid RLN palsy, this has not been confirmed in a large-scale multicenter study. This study used a national database to evaluate whether IONM can reduce postoperative RLN palsy during minimally invasive esophagectomy (MIE) for esophageal cancer. METHODS: We retrieved data of patients with esophageal cancer who underwent 3-field thoracoscopic or robotic MIE with cervical anastomosis with IONM(+) (502 patients) and without IONM(-) (4353 patients) from April 2020 to March 2022 from the Diagnosis Procedure Combination database in Japan. We used propensity score-matching analysis to compare the frequency of postoperative RLN palsy and respiratory complications between the IONM(+) group and IONM(-) group. RESULTS: The postoperative RLN palsy rate was significantly lower in the IONM(+) than IONM(-) group (odds ratio, 0.24; 95% CI, 0.13-0.46). The respiratory complication rate was also significantly lower in the IONM(+) than in the IONM(-) group (odds ratio, 0.66; 95% CI, 0.45-0.97). The anesthesia time was significantly longer in the IONM(+) group (regression coefficient, 60.1 minutes; 95% CI, 44.2-76.9 minutes). The length of postoperative hospitalization tended to be shorter in the IONM(+) than in the IONM(-) group (regression coefficient, -1.39 days; 95% CI, -3.91 to 1.14). CONCLUSIONS: This national cohort study showed that IONM during 3-field MIE for esophageal cancer was associated with a reduction of postoperative RLN palsy and respiratory complications.

Difference in TMPRSS2 usage by Delta and Omicron variants of SARS-CoV-2: Implication for a sudden increase among children.

It has been postulated from a combination of evidence that a sudden increase in COVID-19 cases among pediatric patients after onset of the Omicron wave was attributed to a reduced requirement for TMPRSS2-mediated entry in pediatric airways with lower expression levels of TMPRSS2. Epidemic strains were isolated from the indigenous population in an area, and the levels of TMPRSS2 required for Delta and Omicron variants were assessed. As a result, Delta variants proliferated fully in cultures of TMPRSS2-positive Vero cells but not in TMPRSS2-negative Vero cell culture (350-fold, Delta vs 9.6-fold, Omicron). There was no obvious age-dependent selection of Omicron strains affected by the TMPRSS2 (9.6-fold, Adults vs. 12-fold, Children). A phylogenetic tree was generated and Blast searches (up to 100 references) for the spread of strains in the study area showed that each strain had almost identical homology (>99.5%) with foreign isolates, although indigenous strains had obvious differences from each other. This suggested that the differences had been present abroad for a long period. Therefore, the lower requirement for TMPRSS2 by Omicron strains might be applicable to epidemic strains globally. In conclusion, the property of TMPRSS2-independent cleavage makes Omicron proliferate with ease and allows epidemics among children with fewer TMPRSS2 on epithelial surfaces of the respiratory organs.

Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH.

When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.

Thyroid hormone may predict treatment failure in Kawasaki disease.

BACKGROUND: In systemic inflammatory conditions, inflammatory cytokines can cause low thyroid hormone levels. There are no reports discussing the relation between thyroid hormone levels and response to treatment for Kawasaki disease. METHODS: We investigated 67 patients who underwent treatment in the acute phase of Kawasaki disease. We divided patients into two groups based on their response to initial intravenous immunoglobulin (IVIG) treatment: the responder group (n = 40), and the non-responder group (n = 27). The serum levels of the thyroid hormones free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) were compared before and after treatment in all patients, and between responder and non-responder groups. RESULTS: The FT3, FT4, and TSH levels were low before the initial treatment and increased significantly after treatment (p < 0.05). The FT3, FT4, and TSH levels before treatment were significantly lower in the non-responder group than in the responder group (p < 0.05). Logistic regression analysis suggested that the addition of pre-treatment FT4 values to Gunma score was useful in predicting treatment failure. CONCLUSIONS: Thyroid hormone and TSH levels were lower in the non-responder group than in the responder group in the initial IVIG treatment for Kawasaki disease. This study suggests that Kawasaki disease in the acute phase is associated with low thyroid hormone levels and TSH. It is possible that these hormone levels predict response to the initial IVIG.

Hypoxia-inducible factor-prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduces renal fibrosis in Dahl salt-sensitive rats.

OBJECTIVE: Although hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors have been developed for the treatment of renal anemia, their effects on cardiac and renal dysfunction remain unknown. We previously reported on Dahl salt-sensitive rats, in a rat model of salt-sensitive hypertension, that exhibited anemia and impaired expression of duodenal iron transporters after the development of hypertensive cardiac and renal dysfunction. Therefore, we investigated the effects of Roxadustat (FG-4592), an HIF-PH inhibitor, on anemia, iron regulation, and cardiac and renal dysfunction in Dahl salt-sensitive rats. METHODS: Six-week-old male Dahl salt-sensitive rats were fed a normal or high-salt diet for 8 weeks. A further subset of Dahl salt-sensitive rats, that were fed a high-salt diet, was administered Roxadustat for 8 weeks. RESULTS: Dahl salt-sensitive rats fed a high-salt diet developed hypertension, cardiac and renal dysfunction, and anemia after 8 weeks of feeding. Roxadustat increased hemoglobin and serum erythropoietin levels in Dahl salt-sensitive rats fed a high-salt diet. With regard to the iron-regulating system, Roxadustat lowered hepatic hepcidin gene expression and increased the gene expression of duodenal iron transporters, such as cytochrome b and divalent metal transporter 1 , in Dahl salt-sensitive rats fed a high-salt diet. Roxadustat did not affect the development of hypertension and cardiac hypertrophy in Dahl salt-sensitive rats with a high-salt diet; however, Roxadustat treatment attenuated renal fibrosis in these rats. CONCLUSIONS: Roxadustat ameliorated anemia with affecting the gene expression of the iron-regulating system, and did not affect cardiac hypertrophy but attenuated renal fibrosis in Dahl salt-sensitive rats fed a high-salt diet.

Infused juice concentrate of Japanese plum Prunus mume attenuates inflammatory vascular remodeling in a mouse model of hypertension induced by angiotensin II.

Fruit from the Prunus mume tree is a traditional food in Japan. Recently, bainiku-ekisu, an infused juice concentrate of Japanese Prunus mume, is attracting attention as a health promoting supplement. Angiotensin II (Ang II) plays a central role in development of hypertension. It has been reported that bainiku-ekisu treatment attenuates the growth-promoting signaling induced by Ang II in vascular smooth muscle cells. However, whether bainiku-ekisu has any effect on an animal model of hypertension remains unknown. Therefore, this study was designed to explore the potential anti-hypertensive benefit of bainiku-ekisu utilizing a mouse model of hypertension with Ang II infusion. Male C57BL/6 mice were infused with Ang II for 2 weeks and given 0.1% bainiku-ekisu containing water or normal water for 2 weeks with blood pressure evaluation. After 2 weeks, mice were euthanized, and the aortas were collected for evaluation of remodeling. Aortic medial hypertrophy was observed in control mice after Ang II infusion, which was attenuated in bainiku-ekisu group with Ang II infusion. Bainiku-ekisu further attenuated aortic induction of collagen producing cells and immune cell infiltration. Development of hypertension induced by Ang II was also prevented by bainiku-ekisu. Echocardiograph indicated protection of Ang II-induced cardiac hypertrophy by bainiku-ekisu. In vascular fibroblasts, bainiku-ekisu attenuated vascular cell adhesion molecule-1 induction, an endoplasmic reticulum stress marker, inositol requiring enzyme-1α phosphorylation, and enhancement in glucose consumption in response to Ang II. In conclusion, Bainiku-ekisu prevented Ang II-induced hypertension and inflammatory vascular remodeling. Potential cardiovascular health benefit to taking bainiku-ekisu should be further studied.

Ginseng mediates its anticancer activity by inhibiting the expression of DNMTs and reactivating methylation-silenced genes in colorectal cancer.

Developing safe and effective therapeutic modalities remains a critical challenge for improving the prognosis of patients with colorectal cancer (CRC). In this regard, targeting epigenetic regulation in cancers has recently emerged as a promising therapeutic approach. Since several natural compounds have recently been shown to be important epigenetic modulators, we hypothesized that Ginseng might exert its anticancer activity by regulating DNA methylation alterations in CRC. In this study, a series of cell culture studies were conducted, followed by their interrogation in patient-derived 3D organoid models to evaluate Ginseng's anticancer activity in CRC. Genome-wide methylation alterations were interrogated by undertaking MethylationEpic BeadChip microarrays. First, 50% inhibitory concentrations (IC50) were determined by cell viability assays, and subsequent Ginseng treatment demonstrated a significant anticancer effect on clonogenicity and cellular migration in CRC cells. Treatment with Ginseng potentiated cellular apoptosis through regulation of apoptosis-related genes in CRC cells. Furthermore, Ginseng treatment downregulated the expression of DNA methyltransferases (DNMTs) and decreased the global DNA methylation levels in CRC cells. The genome-wide methylation profiling identified Ginseng-induced hypomethylation of transcriptionally silenced tumor suppressor genes. Finally, cell culture-based findings were successfully validated in patient-derived 3D organoids. In conclusion, we demonstrate that Ginseng exerts its antitumorigenic potential by regulating cellular apoptosis via the downregulation of DNMTs and reversing the methylation status of transcriptionally silenced genes in CRC.

Andrographis Reverses Gemcitabine Resistance through Regulation of ERBB3 and Calcium Signaling Pathway in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, primarily due to intrinsic or acquired resistance to chemotherapy, such as Gemcitabine (Gem). Naturally occurring botanicals, including Andrographis (Andro), can help enhance the anti-tumorigenic therapeutic efficacy of conventional chemotherapy through time-tested safety and cost-effectiveness. Accordingly, we hypothesized that Andro might reverse Gem resistance in PDAC. The critical regulatory pathways associated with Gem resistance in PDAC were identified by analyzing publicly available transcriptomic profiling and PDAC tissue specimens. A series of systematic in vitro experiments were performed using Gem-resistant (Gem-R) PDAC cells and patient-derived 3D-organoids to evaluate the Andro-mediated reversal of Gem resistance in PDAC. Transcriptomic profiling identified the calcium signaling pathway as a critical regulator of Gem-resistance (Fold enrichment: 2.8, p = 0.002). Within this pathway, high ERBB3 expression was significantly associated with poor prognosis in PDAC patients. The combination of Andro and Gem exhibited superior anti-cancer potential in Gem-R PDAC cells through potentiating cellular apoptosis. The combined treatment down-regulated ERBB3 and decreased intracellular calcium concentration in Gem-R PDAC cells. Finally, these findings were successfully interrogated in patient-derived 3D-organoids. In conclusion, we demonstrate novel evidence for Andro-mediated reversal of chemoresistance to Gem in PDAC cells through the regulation of ERBB3 and calcium signaling.

An exosome-based liquid biopsy signature for pre-operative identification of lymph node metastasis in patients with pathological high-risk T1 colorectal cancer.

BACKGROUND: According to current guidelines, more than 70% of patients with invasive submucosal colorectal cancer (T1 CRC) undergo a radical operation with lymph node dissection, even though only ~ 10% have lymph node metastasis (LNM). Hence, there is imperative to develop biomarkers that can help robustly identify LNM-positive patients to prevent such overtreatments. Given the emerging interest in exosomal cargo as a source for biomarker development in cancer, we examined the potential of exosomal miRNAs as LNM prediction biomarkers in T1 CRC. METHODS: We analyzed 200 patients with high-risk T1 CRC from two independent cohorts, including a training (n = 58) and a validation cohort (n = 142). Cell-free and exosomal RNAs from pre-operative serum were extracted, followed by quantitative reverse-transcription polymerase chain reactions for a panel of miRNAs. RESULTS: A panel of four miRNAs (miR-181b, miR-193b, miR-195, and miR-411) exhibited robust ability for detecting LNM in the exosomal vs. cell-free component. We subsequently established a cell-free and exosomal combination signature, successfully validated in two independent clinical cohorts (AUC, 0.84; 95% CI 0.70-0.98). Finally, we developed a risk-stratification model by including key pathological features, which reduced the false positive rates for LNM by 76% without missing any true LNM-positive patients. CONCLUSIONS: Our novel exosomal miRNA-based liquid biopsy signature robustly identifies T1 CRC patients at risk of LNM in a preoperative setting. This could be clinically transformative in reducing the significant overtreatment burden of this malignancy.

Angiotensin II Type 1A Receptor Expressed in Smooth Muscle Cells is Required for Hypertensive Vascular Remodeling in Mice Infused With Angiotensin II.

BACKGROUND: Ang II (angiotensin II) type 1 (AT1) receptors play a critical role in cardiovascular diseases such as hypertension. Rodents have 2 types of AT1 receptor (AT1A and AT1B) of which knock-in Tagln-mediated smooth muscle AT1A silencing attenuated Ang II-induced hypertension. Although vascular remodeling, a significant contributor to organ damage, occurs concurrently with hypertension in Ang II-infused mice, the contribution of smooth muscle AT1A in this process remains unexplored. Accordingly, it is hypothesized that smooth muscle AT1A receptors exclusively contribute to both medial thickening and adventitial fibrosis regardless of the presence of hypertension. METHODS: About 1 µg/kg per minute Ang II was infused for 2 weeks in 2 distinct AT1A receptor silenced mice, knock-in Tagln-mediated constitutive smooth muscle AT1A receptor silenced mice, and Myh11-mediated inducible smooth muscle AT1A together with global AT1B silenced mice for evaluation of hypertensive cardiovascular remodeling. RESULTS: Medial thickness, adventitial collagen deposition, and immune cell infiltration in aorta were increased in control mice but not in both smooth muscle AT1A receptor silenced mice. Coronary arterial perivascular fibrosis in response to Ang II infusion was also attenuated in both AT1A receptor silenced mice. Ang II-induced cardiac hypertrophy was attenuated in constitutive smooth muscle AT1A receptor silenced mice. However, Ang II-induced cardiac hypertrophy and hypertension were not altered in inducible smooth muscle AT1A receptor silenced mice. CONCLUSIONS: Smooth muscle AT1A receptors mediate Ang II-induced vascular remodeling including medial hypertrophy and inflammatory perivascular fibrosis regardless of the presence of hypertension. Our data suggest an independent etiology of blood pressure elevation and hypertensive vascular remodeling in response to Ang II.

A liquid biopsy signature for predicting early recurrence in patients with gastric cancer.

BACKGROUND: Gastric cancer (GC) patients who experience recurrence within the first year following surgery (early recurrence [ER]) exhibit worse prognosis. Herein, we established a microRNA-based liquid biopsy assay to predict ER in GC patients. METHODS: A comprehensive biomarker discovery was performed by analysing miRNA expression profiling in 271 primary GC tumours. Thereafter, the expression of these biomarkers was validated in 290 GC cases, which included 218 tissues and 72 pre-treatment sera, from two independent institutions. RESULTS: A panel of 8 miRNAs was identified during the initial biomarker discovery, and this panel could robustly predict ER in a tissue-based clinical cohort (area under the curve [AUC]: 0.81). Furthermore, a model combining the miRNA panel, microsatellite instability (MSI) status and tumour size exhibited superior predictive performance (AUC: 0.86), and was defined as a Prediction of Early Recurrence in GC (PERGC) signature, which was successfully validated in another independent cohort (AUC: 0.82). Finally, the PERGC signature was translated into a liquid biopsy assay (AUC: 0.81), and a multivariate regression analysis revealed this signature to be an independent predictor for ER (odds ratio: 11.20). CONCLUSION: We successfully established a miRNA-based liquid biopsy signature that robustly predicts the risk of ER in GC patients.

Deep learning-based diagnostic model for predicting complications after gastrectomy.

BACKGROUND: Gastric cancer is one of the leading causes of cancer deaths, and gastrectomy with lymph node dissection is the mainstay of treatment. Despite clinician efforts and advances in surgical methods, the incidence of complications after gastrectomy remains 10%-20% including fatalities. To the best of our knowledge, this is the first report on utilization of a deep learning method to build a new artificial intelligence model that could help surgeons diagnose these complications. METHODS: A neural network was constructed with a total of 4000 variables. Clinical, surgical, and pathological data of patients who underwent radical gastrectomy at our institute were collected to maintain a deep learning model. We optimized the parameters of the neural network to diagnose whether these patients would develop complications after gastrectomy or not. RESULTS: Seventy percent of the data was used to optimize the neural network parameters, and the rest was used to validate the model. A model that maximized the receiver operating characteristics (ROC) area under the curve (AUC) for validation of the data was extracted. The ROC-AUC, sensitivity, and specificity of the model to diagnose all complications were 0.8 vs 0.7, 81% vs 50%, and 69% vs 75%, for the teaching and validation data, respectively. CONCLUSIONS: A predictive model for postoperative complications after radical gastrectomy was successfully constructed using the deep learning method. This model can help surgeons accurately predict the incidence of complications on postoperative day 3.

Endoplasmic Reticulum Chemical Chaperone 3-Hydroxy-2-Naphthoic Acid Reduces Angiotensin II-Induced Vascular Remodeling and Hypertension In Vivo and Protein Synthesis In Vitro.

Background Investigations into alternative treatments for hypertension are necessary because current treatments cannot fully reduce the risk for the development of cardiovascular diseases. Chronic activation of unfolded protein response attributable to the endoplasmic reticulum stress has been proposed as a potential therapeutic target for hypertension and associated vascular remodeling. Triggered by the accumulation of misfolded proteins, chronic unfolded protein response leads to downstream signaling of cellular inflammation and dysfunction. Here, we have tested our hypothesis that a novel chemical chaperone, 3-hydroxy-2-naphthoic acid (3HNA) can attenuate angiotensin II (AngII)-induced vascular remodeling and hypertension. Methods and Results Mice were infused with AngII for 2 weeks to induce vascular remodeling and hypertension with or without 3HNA treatment. We found that injections of 3HNA prevented hypertension and increase in heart weight body weight ratio induced by AngII infusion. Histological assessment revealed that 3HNA treatment prevented vascular medial thickening as well as perivascular fibrosis in response to AngII infusion. In cultured vascular smooth muscle cells, 3HNA attenuated enhancement in protein synthesis induced by AngII. In vascular adventitial fibroblasts, 3HNA prevented induction of unfolded protein response markers. Conclusions We present evidence that a chemical chaperone 3HNA prevents vascular remodeling and hypertension in mice with AngII infusion, and 3HNA further prevents increase in protein synthesis in AngII-stimulated vascular smooth muscle cells. Using 3HNA may represent a novel therapy for hypertension with multiple benefits by preserving protein homeostasis under cardiovascular stress.