Completely Resectable (cT1-2) Esophageal Squamous Cell Carcinoma with Minimal Lymph Node Involvement (cN1): Is Neoadjuvant Chemoradiation Therapy the Only Viable Treatment Option?

BACKGROUND: Neoadjuvant chemoradiation therapy (nCRT) is recommended when lymph node metastasis is evident or strongly suspected on preoperative imaging studies, even for a completely resectable (cT1-2) tumor with minimal lymph node involvement (cN1). We evaluated the validity of upfront surgical approach in this patient group. METHODS: We retrospectively reviewed data from 247 patients with cT1-2 esophageal squamous cell carcinoma (ESCC) who underwent upfront radical esophagectomy followed by the pathology-based adjuvant treatment. Oncologic outcomes of cN1 patients were compared with those of cN0 patients. RESULTS: There were 203 cN0 and 44 cN1 patients. The lymph node yield was 62.0 (interquartile range [IQR], 51.0-76.0) in cN0 and 65.5 (IQR, 57.5-85.0) in cN1 patients (p = 0.033). The size of metastatic node was 0.6 cm (IQR, 0.4-0.9 cm) in cN0 and 0.8 cm (IQR, 0.5-1.3 cm) in cN1 patients (p = 0.001). Nodal upstaging was identified in 29.1% of cN0 and 40.9% of cN1 patients, whereas 18.2% of the cN1 had no actual lymph node metastasis (pN0). The 5-year disease-free survival rate was not significantly different between the groups (cN0, 74.4%; cN1, 71.8%; p = 0.529). Survival rates were closely correlated with pN stage, and a multivariate analysis revealed that pN2-3 stage was a risk factor for poor disease-free survival. CONCLUSIONS: Upfront radical surgery provided accurate nodal staging information, potentially sparing some cN1 patients from unnecessary nCRT while demonstrating comparable survival rates. It might be a valid option for the treatment of cT1-2N1 ESCC.

Enhanced osteogenic differentiation potential of Arnica montana and Bellis perennis in C3H10T1/2 multipotent mesenchymal stem cells.

BACKGROUND: Arnica montana and Bellis perennis are two medicinal plants that are thought to accelerate bone repair in homoeopathic literature. Mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to differentiate and regenerate bone or osteogenesis. Hence, we aimed to determine the role of Arnica montana and Bellis perennis on the osteogenic differentiation of the C3H10T1/2 stem cell line. METHODS AND RESULTS: The cell proliferation of Arnica montana and Bellis perennis was evaluated by MTT assay. Osteogenic differentiation of C3H10T1/2 was induced by the addition of ß-glycerophosphate, ascorbic acid and dexamethasone in the differentiation medium over 3 weeks. Cells were treated with Arnica montana and Bellis perennis individually as well as in combination. The osteogenic differentiation potential of Arnica montana and Bellis perennis to differentiate C3H10T1/2 into osteoblasts was measured by alkaline phosphatase activity, alizarin red staining and the expression of Osteocalcin using immunostaining and qRT-PCR. Arnica montana and Bellis perennis could enhance C3H10T1/2 cell proliferation at 1600 µg. Further, the compound showed the ability to augment osteogenesis as confirmed by increased expression of alkaline phosphatase and enhanced calcium accumulation as seen by the Alizarin Red staining and quantification. Enhanced osteogenesis was further supported by the increased expression of osteocalcin in the treated cells with individual and combined doses of Arnica montana and Bellis perennis. Therefore, the findings provide additional support for the positive impact of Arnica montana and Bellis perennis on bone formation. CONCLUSIONS: Our findings suggest that homoeopathic compounds Arnica montana and Bellis perennis can augment osteogenesis individually as well as in combination.

Assessment of the disruption effects of tetrabromobisphenol A and its analogues on lipid metabolism using multiple in vitro models.

Tetrabromobisphenol A (TBBPA), a widely-used brominated flame retardant, has been revealed to exert endocrine disrupting effects and induce adipogenesis. Given the high structural similarities of TBBPA analogues and their increasing exposure risks, their effects on lipid metabolism are necessary to be explored. Herein, 9 representative TBBPA analogues were screened for their interference on 3T3-L1 preadipocyte adipogenesis, differentiation of C3H10T1/2 mesenchymal stem cells (MSCs) to brown adipocytes, and lipid accumulation of HepG2 cells. TBBPA bis(2-hydroxyethyl ether) (TBBPA-BHEE), TBBPA mono(2-hydroxyethyl ether) (TBBPA-MHEE), TBBPA bis(glycidyl ether) (TBBPA-BGE), and TBBPA mono(glycidyl ether) (TBBPA-MGE) were found to induce adipogenesis in 3T3-L1 preadipocytes to different extends, as evidenced by the upregulated intracellular lipid generation and expressions of adipogenesis-related biomarkers. TBBPA-BHEE exhibited a stronger obesogenic effect than did TBBPA. In contrast, the test chemicals had a weak impact on the differentiation process of C3H10T1/2 MSCs to brown adipocytes. As for hepatic lipid formation test, only TBBPA mono(allyl ether) (TBBPA-MAE) was found to significantly promote triglyceride (TG) accumulation in HepG2 cells, and the effective exposure concentration of the chemical under oleic acid (OA) co-exposure was lower than that without OA co-exposure. Collectively, TBBPA analogues may perturb lipid metabolism in multiple tissues, which varies with the test tissues. The findings highlight the potential health risks of this kind of emerging chemicals in inducing obesity, non-alcoholic fatty liver disease (NAFLD) and other lipid metabolism disorders, especially under the conditions in conjunction with high-fat diets.

Identification of Novel GANT61 Analogs with Activity in Hedgehog Functional Assays and GLI1-Dependent Cancer Cells.

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.

Optimal measurement of gastric emptying of solids in gastroparesis or functional dyspepsia: evidence to establish standard test.

OBJECTIVE: Symptoms in gastroparesis (Gp) and functional dyspepsia (FD) overlap; using egg protein substitute to measure gastric emptying of solids (GES), ~40% of patients are reclassified from Gp to FD, and vice versa. Our aim was to assess inter-individual and intra-individual coefficients of variation (COV) in GES in symptomatic patients with Gp or FD with documented slow or normal GES, respectively. DESIGN: Scintigraphic GES (T1/2 and GE% at 2 and 4 hours) using a 320 kcal real egg meal (30% fat) was tested in the following: single measurements in 20 patients with diabetes mellitus (10 each type 1 and type 2); repeat GES to estimate COVintra measured: 3 days apart in 9 Gp, 4 weeks apart in 21 Gp and 18 with FD with normal GE assigned to placebo and in 70 patients at 94.3 weeks (median) apart. RESULTS: COVinter for GE% at 4 hours and GE T1/2 were respectively 14.2% and 23.5% in FD and 27.5% and 33% in Gp; COVintra for GE% at 4 hours and GE T1/2 up to 4 weeks apart were 23.4% and 37.9% in FD and 20.1% and 33% in Gp. GE% at 2 hours showed less consistent results. However, >85% retained original diagnosis as normal or delayed. From clinical GES to baseline research for Gp group, repeat GES (after treatment) showed the COVintra for GE% at 4 hours was 37.3% at median 94.3 weeks, with 26/70 changed diagnoses. CONCLUSION: The 320 kcal (30% fat) GES scintigraphic test provides consistent diagnosis in >85% and should be the standard test for suspected gastric emptying disorders.

Transmembrane anterior posterior transformation 1 regulates BMP signaling and modulates the protein stability of SMAD1/5.

The bone morphogenetic protein (BMP) signaling pathway plays pivotal roles in various biological processes during embryogenesis and adult homeostasis. Transmembrane anterior posterior transformation 1 (TAPT1) is an evolutionarily conserved protein involved in murine axial skeletal patterning. Genetic defects in TAPT1 result in complex lethal osteochondrodysplasia. However, the specific cellular activity of TAPT1 is not clear. Herein, we report that TAPT1 inhibits BMP signaling and destabilizes the SMAD1/5 protein by facilitating its interaction with SMURF1 E3 ubiquitin ligase, which leads to SMAD1/5 proteasomal degradation. In addition, we found that the activation of BMP signaling facilitates the redistribution of TAPT1 and promotes its association with SMAD1. TAPT1-deficient murine C2C12 myoblasts or C3H/10T1/2 mesenchymal stem cells exhibit elevated SMAD1/5/9 protein levels, which amplifies BMP activation, in turn leading to a boost in the transdifferentiation or differentiation processing of these distinct TAPT1-deficient cell lines changing into mature osteoblasts. Furthermore, the enhancing effect of TAPT1 deficiency on osteogenic differentiation of C3H/10T1/2 cells was observed in an in vivo ectopic bone formation model. Importantly, a subset of TAPT1 mutations identified in humans with lethal skeletal dysplasia exhibited gain-of-function activity on SMAD1 protein levels. Thus, this finding elucidates the role of TAPT1 in the regulation of SMAD1/5 protein stability for controlling BMP signaling.

AIP4 regulates adipocyte differentiation by targeting C/EBPα for ubiquitin-mediated proteasomal degradation.

Adipogenesis, that is, the formation of terminally differentiated adipocytes is intricately regulated by transcription factors where CCAAT/enhancer binding protein alpha (C/EBPα) plays a key role. In the current study, we demonstrate that E3 ubiquitin ligase AIP4 negatively regulates C/EBPα protein stability leading to reduced adipogenesis. While AIP4 overexpression in 3T3-L1 cells preadipocytes inhibited lipid accumulation when treated with differentiation inducing media (MDI), AIP4 depletion was sufficient to partially promote lipid accumulation even in the absence of MDI. Mechanistically, overexpression of AIP4 inhibited protein levels of both ectopically expressed as well as endogenous C/EBPα while catalytically inactive AIP4 failed. On the contrary, AIP4 depletion profoundly enhanced endogenous C/EBPα protein levels. The observation that AIP4 levels decrease with concomitant increase in C/EBPα levels during adipocyte differentiation further indicated that AIP4 negatively regulates C/EBPα levels. We further show that AIP4 physically interacts with C/EBPα and ubiquitinates it leading to its proteasomal degradation. AIP4 promoted K48-linked ubiquitination of C/EBPα while catalytically inactive AIP4-C830A failed. Taken together, our data demonstrate that AIP4 inhibits adipogenesis by targeting C/EBPα for ubiquitin-mediated proteasome degradation.

Reconsidering N component of cancer staging for T1-2N0-2M0 small-cell lung cancer: a retrospective study based on multicenter cohort.

BACKGROUND: The current nodal (pN) classification still has limitations in stratifying the prognosis of small cell lung cancer (SCLC) patients with pathological classifications T1-2N0-2M0. Thus. This study aimed to develop and validate a modified nodal classification based on a multicenter cohort. MATERIALS AND METHODS: We collected 1156 SCLC patients with pathological classifications T1-2N0-2M0 from the Surveillance, Epidemiology, and End Results database and a multicenter database in China. The X-tile software was conducted to determine the optimal cutoff points of the number of examined lymph nodes (ELNs) and lymph node ratio (LNR). The Kaplan-Meier method, the Log-rank test, and the Cox regression method were used in this study. We classified patients into three pathological N modification categories, new pN#1 (pN0-#ELNs > 3), new pN#2 (pN0-#ELNs ≤ 3 or pN1-2-#LNR ≤ 0.14), and new pN#3 (N1-2-#LNR > 0.14). The Akaike information criterion (AIC), Bayesian Information Criterion, and Concordance index (C-index) were used to compare the prognostic, predictive ability between the current pN classification and the new pN component. RESULTS: The new pN classification had a satisfactory effect on survival curves (Log-rank P < 0.001). After adjusting for other confounders, the new pN classification could be an independent prognostic indicator. Besides, the new pN component had a much more accurate predictive ability in the prognostic assessment for SCLC patients of pathological classifications T1-2N0-2M0 compared with the current pN classification in the SEER database (AIC: 4705.544 vs. 4731.775; C-index: 0.654 vs. 0.617, P < 0.001). Those results were validated in the MCDB from China. CONCLUSIONS: The multicenter cohort developed and validated a modified nodal classification for SCLC patients with pathological category T1-2N0-2M0 after surgery. Besides, we propose that an adequate lymph node dissection is essential; surgeons should perform and consider the situation of ELNs and LNR when they evaluate postoperative prognoses of SCLC patients.

Is the prognosis of T1-2N1 colon cancer the same as that of T1-2N0 colon cancer after curative surgery?

Objectives: The prognoses of T1-2N0 and T1-2N1 colon cancer after curative surgery remain unclear. This study compared the prognoses of patients with T1-2N0 and T1-2N1 colon cancer after curative surgery.Materials and methods: We retrospectively evaluated 307 consecutive patients with T1-2N0/1 colon cancer who underwent radical surgery at our hospital between January 2010 and December 2016. There were 266 patients with T1-2N0 colon cancer and 41 patients with T1-2N1 colon cancer. After excluding patients with <12 retrieved lymph nodes, 179 patients with T1-2N0 and 32 with T1-2N1 colon cancer were included in the cohort.Results: Overall survival and disease-free survival did not differ between the T1-2N0 and T1-2N1 groups (p = 0.498 and p = 0.681, respectively). Overall survival and disease-free survival were not significantly different between the T1-2N1 + no chemotherapy and T1-2N1 + chemotherapy groups (p = 0.740 and p = 0.765, respectively). Additionally, overall survival and disease-free survival did not differ between the T1-2N0, T1-2N1 + no chemotherapy, and T1-2N1 + chemotherapy groups (p = 0.757 and p = 0.877, respectively), even after excluding patients with <12 retrieved lymph nodes.Conclusions: T1-2N1 has a prognosis as good as that of T1-2N0 colon cancer after curative surgery. Moreover, further research is needed to investigate the efficacy of adjuvant FOLFOX chemotherapy in T1-2N1.

3-tert-Butyl-4-hydroxyanisole Perturbs Differentiation of C3H10T1/2 Mesenchymal Stem Cells into Brown Adipocytes through Regulating Smad Signaling.

3-tert-Butyl-4-hydroxyanisole (3-BHA), one of the most commonly used antioxidants in foodstuffs, has been identified as an environmental endocrine disruptor (EED) with obesogenic activity. Given the increasing concern on EED-caused dysfunction in lipid metabolism, whether 3-BHA could influence the development of brown adipocytes is worthy of being explored. In this study, the effect of 3-BHA on the differentiation of C3H10T1/2 mesenchymal stem cells (MSCs) into brown adipocytes was investigated. Exposure to 3-BHA promoted lipogenesis of the differentiated cells, as evidenced by the increased intracellular lipid accumulation and elevated expressions of adipogenic biomarkers, including peroxisome proliferator-activated receptor γ (PPARγ), Perilipin, Adiponectin, and fatty acid binding protein 4 (FABP4). Surprisingly, the thermogenic capacity of the differentiated cells was compromised as a result of 3-BHA exposure, because neither intracellular mitochondrial contents nor expressions of thermogenic biomarkers, including uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), cell-death-inducing DNA fragmentation factor α subunit-like effector A (CIDEA), and PR domain containing 16 (PRDM16), were increased by this chemical. The underlying molecular mechanism exploration revealed that, in contrast to p38 MAPK, 3-BHA stimulation induced phosphorylation of Smad1/5/8 in an exposure time-dependent manner, suggesting that this chemical-triggered Smad signaling was responsible for the shift of C3H10T1/2 MSC differentiation from a brown to white-like phenotype. The finding herein, for the first time, revealed the perturbation of 3-BHA in the development of brown adipocytes, uncovering new knowledge about the obesogenic potential of this emerging chemical of concern.