Pharmacokinetics, tissue distribution, and excretion study of GL-V9 and its glucuronide metabolite 5-O-glucuronide GL-V9 in Sprague-Dawley rats.

The objective of this study is to explore the pharmacokinetics, tissue distribution, and excretion patterns of GL-V9 and its glucuronide metabolite, 5-O-glucuronide GL-V9, following the administration of GL-V9 to Sprague-Dawley (SD) rats. In this research, we developed and validated rapid, sensitive, and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods for quantifying GL-V9 and 5-O-glucuronide GL-V9 in various biological samples, including SD rat plasma, tissue homogenate, bile, urine, and feces. Quantification of GL-V9 and 5-O-glucuronide GL-V9 in plasma, tissue homogenate, bile, urine, and feces was performed using the validated LC-MS/MS methods. The bioavailability of GL-V9 in SD rats ranged from 6.23% to 7.08%, and both GL-V9 and 5-O-glucuronide GL-V9 exhibited wide distribution and rapid elimination from tissues. The primary distribution tissues for GL-V9 and 5-O-glucuronide GL-V9 in rats were the duodenum, liver, and lung. GL-V9 was predominantly excreted in urine, while 5-O-glucuronide GL-V9 was primarily excreted in bile. GL-V9 exhibited easy absorption and rapid conversion to its glucuronide metabolite, 5-O-glucuronide GL-V9, following administration.

Peroxidase-encapsulated Zn/Co-zeolite imidazole framework nanosheets on ZnCoO nanowire array for detecting H2O2 derived from mitochondrial superoxide anion.

In this work, we have developed a nanocomposite consisting of horseradish peroxidase (HRP)-encapsulated 2D Zn-Co zeolite imidazole framework (ZIF) nanosheets strung on a ZnCoO nanowire array on a Ti support (denoted as 2D-Zn/Co-ZIF(HRP)|ZnCoO|Ti). This nanocomposite was then applied to constructing an electrochemical biosensor for detecting H2O2 derived from O2∙- released by mitochondria in living cells. This sensing platform shows excellent catalytic performance towards H2O2, attributable to the enzyme/metal-catalytic effect of HRP and Zn/Co-ZIF. The unique nano-string structure alleviates the aggregation of Zn/Co-ZIF nanosheets, readily exposes the catalytic active sites, protects the bioactivity of HRP, and reduces the charge/mass transfer pathway within Zn/Co-ZIF. The 2D-Zn/Co-ZIF(HRP)|ZnCoO|Ti biosensor offers two linear ranges of 0.2-10 µ M and 10-1100 µ M, a limit of detection of 0.082 µ M, a sensitivity of 3.3 mA mM-1 cm-2, good selectivity and stability over 40 days for H2O2 detection. After treating with specific mitochondrial complex inhibitors, the chronoamperometric results at the 2D-Zn/Co-ZIF(HRP)|ZnCoO|Ti confirmed complex I and III within the mitochondria electron transfer chain as the main electron leakage sites. This biosensor may contribute to the development of diagnostic health-care devices that shed light on the precaution and even treatment of oxidative stress diseases.

Photoelectrochemical detection of superoxide anions released from mitochondria in HepG2 cells based on the synergistic effect of MnO2@Co3O4 core-shell p-n heterojunction.

The detection and comparison of the amount of superoxide anion (O2.-) released by different complexes in mitochondrial electron transport chain (ETC) can locate the main electron leakage sites in mitochondria. In order to realize this, we designed an ultrasensitive photoelectrochemical (PEC) sensor by in situ hydrothermal growth of MnO2 nanosheets on Co3O4 nanowires array modified Ti substrate (NWA|Ti). Due to the formation of a core-shell p-n heterojunction with high specific surface area, tight surface contact and plentiful oxygen vacancies (OVs), MnO2@Co3O4 NWA|Ti possesses a strong visible light absorption, high charges transfer and separation ability. The proposed PEC sensor exhibited a wide linear range of 0.1-50000 nM and a low detection limit of 0.025 nM towards H2O2. Due to the rapid conversion of O2.- to H2O2 inside mitochondria, the PEC sensor can indirectly monitor the electron leakage in the ETC. Specifically, four selected mitochondrial inhibitors specifically inhibited the corresponding complex in mitochondria extracted from living HepG2 cells (hepatocellular carcinoma cells), and the H2O2 levels converted from O2.- was measured by the PEC sensor. It is evident that IQ (ubiquinone binding) site of complex I and Qo (ubiquinol oxidation) site of complex III are the key sites at which electron leakage occurred. This study could provide meaningful information for the diagnosis and treatment of certain disease caused by oxidative stress due to the electron leakage.

Dissecting the heterogeneities of the tumor microenvironment between metastatic and nonmetastatic primary colorectal cancer patients by single-cell RNA sequencing.

AIMS: One of the main factors hampering the long-term prognosis of colorectal cancer (CRC) patients is distant metastasis. However, the driving factors of CRC metastasis have not been clarified at the single-cell level, which limits the in-depth study of accurate prediction and prevention of CRC metastasis to improve the prognosis. MATERIALS AND METHODS: Heterogeneities in the tumor microenvironment (TME) between metastatic and nonmetastatic CRC were investigated by single-cell RNA (scRNA) sequencing data. In detail, 50,462 single cells from 20 primary CRC samples, including 40,910 cells from nonmetastatic CRC (M0 group) and 9552 cells from metastatic CRC (M1 group), were systematically analyzed in this study. KEY FINDINGS: Based on the single-cell atlas, we revealed that cancer cells and fibroblasts accounted for relatively high proportions in metastatic CRC compared with nonmetastatic CRC. Moreover, two specific cancer cell subtypes (FGGY+SLC6A6+ and IGFBP3+KLK7+ cancer cells) and three specific fibroblast subtypes (ADAMTS6+CAPG+, PIM1+SGK1+ and CA9+UPP1+ fibroblasts) in metastatic CRC were identified. The functional and differentiation characteristics of these specific cell subclusters were elucidated by enrichment and trajectory analyses. SIGNIFICANCE: These results provide fundamental knowledge for future in-depth research to screen effective methods and drugs to predict and prevent CRC metastasis to improve prognosis.

Identification of the molecular characteristics associated with microsatellite status of colorectal cancer patients for the clinical application of immunotherapy.

Background: Mismatch repair-proficient (pMMR) microsatellite stability (MSS) in colorectal cancer (CRC) indicates an unfavorable therapeutic response to immunotherapy with immune checkpoint inhibitors (ICIs). However, the molecular characteristics of CRC patients with pMMR MSS remain largely unknown. Methods: Heterogeneities between mismatch repair-deficient (dMMR) microsatellite instability (MSI) and pMMR MSS CRC patients were investigated at the single-cell level. Next, an MSS-related risk score was constructed by single-sample gene set enrichment analysis (ssGSEA). The differences in immune and functional characteristics between the high- and low-score groups were systematically analyzed. Results: Based on the single-cell RNA (scRNA) atlas, an MSS-specific cancer cell subpopulation was identified. By taking the intersection of the significant differentially expressed genes (DEGs) between different cancer cell subtypes of the single-cell training and validation cohorts, 29 MSS-specific cancer cell marker genes were screened out for the construction of the MSS-related risk score. This risk score signature could efficiently separate pMMR MSS CRC patients into two subtypes with significantly different immune characteristics. The interactions among the different cell types were stronger in the MSS group than in the MSI group, especially for the outgoing signals of the cancer cells. In addition, functional differences between the high- and low-score groups were preliminarily investigated. Conclusion: In this study, we constructed an effective risk model to classify pMMR MSS CRC patients into two completely different groups based on the specific genes identified by single-cell analysis to identify potential CRC patients sensitive to immunotherapy and screen effective synergistic targets.

The Prognostic Value of a lncRNA Risk Model Consists of 9 m6A Regulator-Related lncRNAs in Hepatocellular Carcinoma (HCC).

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. Although the RNA modification N6-methyladenine (m6A) has been reported to be involved in HCC carcinogenesis, early diagnostic markers and promising personalized therapeutic targets are still lacking. In this study, we identified that 19 m6A regulators and 34 co-expressed lncRNAs were significantly upregulated in HCC samples; based on these factors, we established a prognostic signal of HCC associated with 9 lncRNAs and 19 m6A regulators using LASSO Cox regression analysis. Kaplan-Meier survival estimate revealed correlations between the risk scores and patients' OS in the training and validation dataset. The ROC curve demonstrated that the risk score-based curve has satisfactory prediction efficiency for both training and validation datasets. Multivariate Cox's proportional hazard regression analysis indicated that the risk score was an independent risk factor within the training and validation dataset. In addition, the risk score could distinguish HCC patients from normal non-cancerous samples and HCC samples of different pathological grades. Eventually, 232 mRNAs were co-expressed with these 9 lncRNAs according to GSE101685 and GSE112790; these mRNAs were enriched in cell cycle and cell metabolic activities, drug metabolism, liver disease-related pathways, and some important cancer related pathways such as p53, MAPK, Wnt, RAS and so forth. The expression of the 9 lncRNAs was significantly higher in HCC samples than that in the neighboring non-cancerous samples. Altogether, by using the Consensus Clustering, PCA, ESTIMATE algorithm, LASSO regression model, Kaplan-Meier survival assessment, ROC curve analysis, and multivariate Cox's proportional hazard regression model analysis, we established a prognostic marker consisting of 9 m6A regulator-related lncRNAs that markers may have prognostic and diagnostic potential for HCC.

Identification of biomarkers for hepatocellular carcinoma based on single cell sequencing and machine learning algorithms.

Hepatocellular carcinoma (HCC) remains one of the most lethal cancers around the world. Precision oncology will be crucial for further improving the prognosis of HCC patients. Compared with traditional bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) enables the transcriptomes of a great deal of individual cells assayed in an unbiased manner, showing the potential to deeply reveal tumor heterogeneity. In this study, based on the scRNA-seq results of primary neoplastic cells and paired normal liver cells from eight HCC patients, a new strategy of machine learning algorithms was applied to screen core biomarkers that distinguished HCC tumor tissues from the adjacent normal liver. Expression profiles of HCC cells and normal liver cells were first analyzed by maximum relevance minimum redundancy (mRMR) to get a top 50 signature gene feature. For further analysis, the incremental feature selection (IFS) method and leave-one-out cross validation (LOOCV) were conducted to build an optimal classification model and to extract 21 potentially essential biomarkers for HCC cells. Our results provided new insights into HCC pathogenesis that might be valuable for HCC diagnosis and therapy.

Portulaca oleracea extract reduces gut microbiota imbalance and inhibits colorectal cancer progression via inactivation of the Wnt/ß-catenin signaling pathway.

BACKGROUND: Portulaca oleracea is a known medicinal plant with antioxidant, anti-inflammatory, and anticancer activities, and it may also function an important role in colorectal cancer (CRC). PURPOSE: We probed into study the critical function of Portulaca oleracea extract (POE) in CRC and the related downstream factors. METHODS: Azoxymethane (AOM) and dextransodiumsulfate (DSS) were used to induce mouse models of CRC, which were then administered different doses of POE to evaluate the therapeutic effects of POE on CRC. Diversity, abundance, and function of gut microbiota were analyzed. Moreover, the potential molecular targets of POE inhibiting CRC development were determined. Expression of c-Myc and cyclin D1 as well as CRC cell proliferation and apoptosis was detected. RESULTS: POE treatment inhibited AOM/DSS-induced CRC development in mice and ameliorated gut microbial imbalance. Bioinformatic analysis revealed marked differences in the gut microbiota between CRC samples and normal samples and that 20 differential microbiota may be involved in CRC development through the Wnt signaling pathway. Additionally, c-Myc and cyclin D1 were identified to be the key downstream target genes of the Wnt/ß-catenin signaling pathway. In vitro data revealed that POE played a suppressive role in the proliferation of CRC cells by reducing the expression of c-Myc and cyclin D1 and inactivating the Wnt/ß-catenin signaling pathway. CONCLUSION: This study underlines that POE reduces gut microbiota imbalance and inhibits CRC development and progression via inactivation of the Wnt/ß-catenin signaling pathway and downregulation of c-Myc and cyclin D1 expression, which is expected to be a potential biomarker for CRC.

The lncRNA CASC2 Modulates Hepatocellular Carcinoma Cell Sensitivity and Resistance to TRAIL Through Apoptotic and Non-Apoptotic Signaling.

The immune cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been widely concerned as a tumor therapy because of its ability of selective triggering cancer cell apoptosis; nevertheless, hepatocellular carcinoma (HCC) exhibits acquired resistance to TRAIL-induced apoptosis. In the present study, tumor-suppressive lncRNA cancer susceptibility candidate 2 (CASC2) was downregulated in HCC tissues and cell lines; HCC patients with lower CASC2 expression predicted a shorter overall survival rate. In vitro, CASC2 overexpression dramatically repressed HCC cell proliferation and inhibited cell apoptosis; in vivo, CASC2 overexpression inhibited subcutaneous xenotransplant tumor growth. CASC2 affected the caspase cascades and NF-κB signaling in TRAIL-sensitive [Huh-7 (S) and HCCLM3 (S)] or TRAIL-resistant cell lines [Huh-7 (R) and HCCLM3 (R)] in different ways. In Huh-7 (S) and HCCLM3 (S) cells, CASC2 affected cell apoptosis through the miR-24/caspase-8 and miR-221/caspase-3 axes and the caspase cascades. miR-18a directly targeted CASC2 and RIPK1. In Huh-7 (R) and HCCLM3 (R) cells, CASC2 affected cell proliferation through the miR-18a/RIPK1 axis and the NF-κB signaling. RELA bound to CASC2 promoter region and inhibited CASC2 transcription. In conclusion, CASC2 affects cell growth mainly via the miR-24/caspase-8 and miR-221/caspase-3 axes in TRAIL-sensitive HCC cells; while in TRAIL-resistant HCC cells, CASC2 affects cell growth mainly via miR-18a/RIPK1 axis and the NF-κB signaling. These outcomes foreboded that CASC2 could be a novel therapeutic target for further study of HCC-related diseases.

Safety and Feasibility of Robotic Natural Orifice Specimen Extraction Surgery in Colorectal Neoplasms During the Initial Learning Curve.

Aim: To analyze the learning curve (LC) for robotic natural orifice specimen extraction surgery (NOSES) for colorectal neoplasms and evaluate safety and feasibility during the initial LC. Method: Patients who consecutively underwent robotic NOSES performed by two surgeons between March 2016 and October 2019 were analyzed retrospectively. The operation time was evaluated using the cumulative sum method to analyze the LC. The clinicopathological data before and after the completion of LC were extracted and compared to evaluate safety and feasibility. Results: In total, 99 and 66 cases were scheduled for robotic NOSES by Prof. Yao and Prof. Li, respectively. The peak points of LC were observed at the 42nd and 15th cases of Yao and Li, respectively, then operation time began to decrease. Only the operation time for Yao before the completion of LC (213.3 ± 67.0 min) was longer than that after the completion of LC (143.8 ± 33.3 min). For Yao nor for Li, other indices, such as postoperative hospital stay, intraoperative blood loss, conversion to laparotomy, incidence of anastomotic leakage, reoperation rate, and 90-day mortality rate lacked significant statistical differences(P > 0.05). In terms of feasibility, the number of lymph nodes harvested, positive resection margin rate, and total cost before and after the completion of LC had no significant statistical difference (P > 0.05). Conclusion: The cases before the completion of LC for robotic NOSES in colorectal neoplasms varied from 15 cases to 42 cases. Robotic NOSES is safe and feasible during the initial LC.

CASC2/miR-24/miR-221 modulates the TRAIL resistance of hepatocellular carcinoma cell through caspase-8/caspase-3.

Hepatocellular carcinoma is one of the most common solid tumors in the digestive system. The prognosis of patients with hepatocellular carcinoma is still poor due to the acquisition of multi-drug resistance. TNF Related Apoptosis Inducing Ligand (TRAIL), an attractive anticancer agent, exerts its effect of selectively inducing apoptosis in tumor cells through death receptors and the formation of the downstream death-inducing signaling complex, which activates apical caspases 3/8 and leads to apoptosis. However, hepatocellular carcinoma cells are resistant to TRAIL. Non-coding RNAs, including long non-coding RNAs (lncRNAs) and miRNAs have been regarded as major regulators of normal development and diseases, including cancers. Moreover, lncRNAs and miRNAs have been reported to be associated with multi-drug resistance. In the present study, we investigated the mechanism by which TRAIL resistance of hepatocellular carcinoma is affected from the view of non-coding RNA regulation. We selected and validated candidate miRNAs, miR-24 and miR-221, that regulated caspase 3/8 expression through direct targeting, and thereby affecting TRAIL-induced tumor cell apoptosis TRAIL resistance of hepatocellular carcinoma. In addition, we revealed that CASC2, a well-established tumor suppressive long non-coding RNA, could serve as a "Sponge" of miR-24 and miR-221, thus modulating TRAIL-induced tumor cell apoptosis TRAIL resistance of hepatocellular carcinoma. Taken together, we demonstrated a CASC2/miR-24/miR-221 axis, which can affect the TRAIL resistance of hepatocellular carcinoma through regulating caspase 3/8; through acting as a "Sponge" of miR-24 and miR-221, CASC2 may contribute to improving hepatocellular carcinoma TRAIL resistance, and finally promoting the treatment efficiency of TRAIL-based therapies.

RIP-1/c-FLIPL Induce Hepatic Cancer Cell Apoptosis Through Regulating Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL).

BACKGROUND Almost all hepatic cancer cells have resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. c-FLIPL and RIP-1 are apoptotic negative regulatory factors. This study investigated the role of c-FLIPL and RIP-1 in hepatic cancer cell resistance to TRAIL-induced apoptosis. MATERIAL AND METHODS HepG2 cells were treated by TRAIL, RIP-1 siRNA, and/or BY11-7082. Cell viability was detected by MTT assay. Cell apoptosis was tested by flow cytometry. DISC component proteins, RIP-1, and p-p65 were measured by Western blot. Caspase-8 and caspase-3 were determined by spectrophotometry. RESULTS Single TRAIL treatment showed no significant impact on cell proliferation and apoptosis. HepG2 cells expressed high levels of RIP1 and c-FLIPL, while a high concentration of TRAIL upregulated RIP-1 and c-FLIPL expression but not DR4 and DR5. Single TRAIL treatment did not obviously activate caspase-8 and caspase-3. RIP-1 or c-FLIPL siRNA markedly induced cell apoptosis and enhanced caspase-8 and caspase-3 activities. Combined transfection obviously increased apoptotic cells. TRAIL markedly upregulated RIP-1 expression and enhanced p-p65 protein. Downregulating RIP-1 and/or BAY11-7082 significantly reduced NF-kB transcriptional activity, blocked cells in G0/G1 phase, weakened proliferation, elevated caspase-8 and caspase-3 activities, and promoted cell apoptosis. CONCLUSIONS TRAIL can enhance RIP1 and c-FLIPL expression in HepG2 cells. High expression of RIP1 and c-FLIPL is an important reason for TRAIL resistance. Downregulation of RIP1 and c-FLIPL can relieve caspase-8 suppression, activate caspase-3, and promote cell apoptosis. TRAIL mediates apoptosis resistance through upregulating RIP-1 expression, enhancing NF-kB transcriptional activity, and weakening caspase activity.

Protective effect of RIP and c-FLIP in preventing liver cancer cell apoptosis induced by TRAIL.

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the tumor necrosis factor superfamily that can induce tumor selective death by up-regulating death receptor 4 (DR4) and DR5 expression. The study aimed to explore the role of RIP and c-FLIP genes in TRAIL induced liver cancer cell HepG2 and Hep3B apoptosis and related mechanism. RIP and c-FLIP silenced HepG2 and Hep3B cell model were established through siRNA. Western blot was applied to test c-FLIP, RIP, DR4, DR5, FADD, Caspase-3/8/9, ERK1/2, and DFF45 protein expression. Caspase-8 kit was used to detect Caspase-8 expression. Flow cytometry was performed to measure cell apoptosis rate. Acid phosphatase method was applied to determine cell cycle. TRAIL had no significant effect on Caspase-3/8/9, DR4, DR5, ERK1/2, and DFF45 protein expression, but up-regulated c-FLIP and RIP protein expression and reduced FADD expression level. After treated by the chemotherapy drug mitomycin and adriamycin, c-FLIP and RIP expression decreased significantly, while FADD increased. After knockout c-FLIP and RIP gene, HepG2 and Hep3B cell apoptosis rate induced by TRAIL increased obviously. Meanwhile, cell subG1 percentage increased markedly and exhibited G1 phase growth retardation. In addition, after two kinds of gene knockout, Caspase-8 was activated and produce Caspase-3 P20 and P24, leading DFF45 appeared DNA fragment P17 and P25. c-FLIP and RIP can inhibit Caspase-8 activation and prompting HepG2 and Hep3B resistant to cell apoptosis induced by TRAIL.

Inhibitory effect of geraniol in combination with gemcitabine on proliferation of BXPC-3 human pancreatic cancer cells.

OBJECTIVE: To investigate the inhibitory effect of geraniol alone, or in combination with gemcitabine, on the proliferation of BXPC-3 pancreatic cancer cells. METHODS: BXPC-3 cells were treated under different conditions: with geraniol at 10, 20, 40, 80 and 160 µmol/l each for 24 h, 48 h or 72 h; with 20 µmol/l geraniol for 24 h or 0 h before 20 µmol/l gemcitabine for 24 h; with 20 µmol/l geraniol for 24 h, 48 h and 72 h following 20 µmol/l gemcitabine for 24 h; or with 20 µmol/l gemcitabine alone as a control. Cell proliferation was assessed and changes in cell morphology were assessed by light and fluorescence microscopy. Apoptosis was detected using flow cytometry. RESULTS: Geraniol inhibited BXPC-3 cell proliferation in a time- and dosa-dependent manner. Geraniol alone or combined with gemcitabine induced BXPC-3 cell apoptosis. BXPC-3 inhibition rates with combined treatment were 55.24%, 50.69%, 49.83%, 41.85% and 45.27% following treatment with 20 µmol/l geraniol for 24 h or 0 h before 20 µmol/l gemcitabine for 24 h, or 20 µmol/l geraniol for 24 h, 48 h and 72 h, following 20 µmol/l gemcitabine for 24 h, respectively. CONCLUSION: Geraniol inhibited the proliferation of BXPC-3 cells. Geraniol significantly increased the antiproliferative and apoptosis-inducing effects of gemcitabine on BXPC-3 cells. Maximum inhibition of BXPC-3 cells was achieved with geraniol treatment for 24 h before gemcitabine treatment.