Intratumor microbiome features reveal antitumor potentials of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a rare malignancy with a high prevalence in China. This study aimed to characterize the ICC tissues' bacterial metagenomics signature and explore its antitumor potential for cancer. In this study, 16S rRNA sequencing was carried out on 99 tissues to characterize the features of intratumoral microbiota, followed by single-cell RNA sequencing (scRNA-seq) and multilevel validation. The presence of microbial DNA in tissues was determined using staining, fluorescence in situ hybridization (FISH), and transmission electron microscopy (TEM). A Gram-positive aerobic bacterium, identified as Staphylococcus capitis, was cultured from fresh tissues. Meanwhile, scRNA-seq showed that intratumoral bacteria could be present in multiple cell types. Using 16S rRNA sequencing, we identified a total of 2,320,287 high-quality reads corresponding to 4,594 OTU (operational taxonomic units) sequences. The most abundant bacterial orders include Burkholderiales, Pseudomonadales, Xanthomonadales, Bacillales and Clostridiales. Alpha and Beta diversity analysis revealed specific features in different tissues. In addition, the content of Paraburkholderia fungorum was significantly higher in the paracancerous tissues and negatively correlated with CA199 (Carbohydrate antigen199) levels. The results of in vitro and in vivo experiments suggest that P. fungorum possesses an antitumor activity against tumors. Metabolomics and transcriptomics showed that P. fungorum could inhibit tumor growth through alanine, aspartate and glutamate metabolism. We determined the characteristic profile of the intratumoral microbiota and the antitumor effect of P. fungorum in ICC.

2-Deoxy-D-glucose and combined 2-Deoxy-D-glucose/albendazole exhibit therapeutic efficacy against Echinococcus granulosus protoscoleces and experimental alveolar echinococcosis.

2-Deoxy-D-glucose (2-DG) is a glucose analog used as a promising anticancer agent. It exerts its effects by inhibiting the glycolytic energy metabolism to deplete cells of energy. The larval stage of Echinococcus relies on glycolysis for energy production. Therefore, in this study, we investigated the in vitro and in vivo efficacy of 2-DG against the larval stage of Echinococcus granulosus and E. multilocularis. 2-DG exhibited significant time- and dose-dependent effects against in vitro cultured E. granulosus protoscoleces and E. multilocularis metacestodes. A daily oral administration of 500 mg/kg 2-DG in E. multilocularis-infected mice effectively reduced the weight of metacestodes. Notably, the combination treatment, either 2-DG (500 mg/kg/day) + albendazole (ABZ) (200 mg/kg/day) or 2-DG (500 mg/kg/day) + half-dose of ABZ (100 mg/kg/day), exhibited a potent therapeutic effect against E. multilocularis, significantly promoting the reduction of metacestodes weight compared with the administration of 2-DG or ABZ alone. Furthermore, the combination significantly promoted apoptosis of the cells of metacestodes and inhibited glycolysis in metacestodes, compared with the administration of 2-DG or ABZ alone. In conclusion, 2-DG exerts an effective activity against the larval stage of Echinococcus. Thus, it may be a promising anti-Echinococcus drug, and its combination with ABZ may provide a new strategy for the treatment of echinococcosis in humans.

Effects of Echinococcus multilocularis metacestodes infection and drug treatment on the activities of biotransformation enzymes in mouse liver.

The changes of biotransformation enzymes will substantially affect the host's ability to metabolize drugs and other xenobiotic compounds. In order to further elucidate this process and promote the development in treatment of echinococcosis, we investigated the effects of Echinococcus multilocularis infection and drug treatment on biotransformation enzymes in mouse liver. In microsomal and cytosolic fractions, from the six activities assayed, significant decrease of glutathione S-transferases (GST) activity and significant increase of 7-pentoxyresorufin (PROD) and NADPH-cytochrome P450 reductase (CPR) activity were observed in the mice infected with E. multilocularis metacestodes. In addition, after six weeks treatment of albendazole in E. multilocularis infected mice, significant decreased GST activity and significant increase of 7- ethoxyresorufin (EROD), PROD, and particularly 3-fold higher 7-methoxyresorufin (MROD) activity were observed. The 3-bromopyruvate treated mice only exhibited significantly lower GST activity. Our results demonstrate that E. multilocularis metacestodes infection can affect the activities of main hepatic biotransformation enzymes and such alterations of activity may further affect the hepatic biotransformation of xenobiotics. Moreover, albendazole and 3-bromopyruvate, the promising potential drug against Echinococcus, affected different hepatic biotransformation enzymes and may affect their metabolism. The findings will help to develop rational treatments with less side effects and promote the development of more efficient treatments against E. multilocularis.

Quantitative acetylome analysis reveals histone modifications that may predict prognosis in hepatitis B-related hepatocellular carcinoma.

Lysine acetylation (Kac) as an important posttranslational modification of histones is essential for the regulation of gene expression in hepatocellular carcinoma (HCC). However, the atlas of whole acetylated proteins in HCC tissues and the difference in protein acetylation between normal human tissues and HCC tissues are unknown. In this report, we characterized the proteome and acetyl proteome (acetylome) profile of normal, paracancerous, and HCC liver tissues in human clinical samples by quantitative proteomics techniques. We identified 6781 acetylation sites of 2582 proteins and quantified 2492 acetylation sites of 1190 proteins in normal, paracancerous, and HCC liver tissues. Among them, 15 proteins were multiacetylated with more than 10 lysine residues. The histone acetyltransferases p300 and CBP were found to be hyperacetylated in hepatitis B virus pathway. Moreover, we found that 250 Kac sites of 214 proteins were upregulated and 662 Kac sites of 451 proteins were downregulated in HCC compared with normal liver tissues. Additionally, the acetylation levels of lysine 120 in histone H2B (H2BK120ac), lysine 18 in histone H3.3 (H3.3K18ac), and lysine 77 in histone H4 (H4K77ac) were increased in HCC. Interestingly, the higher levels of H2BK120ac, H3.3K18ac, and H4K77ac were significantly associated with worse prognosis, such as poorer survival and higher recurrence in an independent clinical cohort of HCC patients. Overall, this study lays a foundation for understanding the functions of acetylation in HCC and provides potential prognostic factors for the diagnosis and therapy of HCC.

Molecular characterization and serodiagnostic potential of Echinococcus granulosus hexokinase.

BACKGROUND: Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus (sensu stricto), is a life-threatening but neglected zoonosis. Glycolytic enzymes are crucial molecules for the survival and development of E. granulosus. The aim of this study was to investigate the molecular characterization, immunogenicity, tissue distribution and serodiagnostic potential of E. granulosus hexokinase (EgHK), the first key enzyme in the glycolytic pathway. METHODS: EgHK was cloned and expressed in Escherichia coli. Specific serum antibodies were evaluated in mice immunized with recombinant EgHK (rEgHK). The location of EgHK in the larval stage of E. granulosus was determined using fluorescence immunohistochemistry, and the potential of rEgHK as a diagnostic antigen was investigated in patients with CE using indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Recombinant EgHK could be identified in the sera of patients with CE and in mouse anti-rEgHK sera. High titers of specific immunoglobulin G were induced in mice after immunization with rEgHK. EgHK was mainly located in the tegument, suckers and hooklets of protoscoleces and in the germinal layer and laminated layer of the cyst wall. The sensitivity and specificity of the rEgHK-ELISA reached 91.3% (42/46) and 87.8% (43/49), respectively. CONCLUSIONS: We have characterized the sequence, structure and location of EgHK and investigated the immunoreactivity, immunogenicity and serodiagnostic potential of rEgHK. Our results suggest that EgHK may be a promising candidate for the development of vaccines against E. granulosus and an effective antigen for the diagnosis of human CE.

In vitro effects of lonidamine and 6-aminonicotinamide against Echinococcus granulosussensu stricto and Echinococcus multilocularis.

Echinococcosis is a zoonotic disease caused by cestode species of the genus Echinococcus, which demonstrates considerable medical and veterinary concerns. The development of novel drugs for echinococcosis treatment is urgently needed. In this study, we demonstrated that lonidamine (LND) and 6-aminonicotinamide (6-AN) exhibited considerable in vitro effects against both larval- and adult-stage of E. granulosussensu stricto (s. s.) and E. multilocularis. The combination of LND and 6-AN exhibited a significantly higher activity than the single drug treatment. These results highlight the therapeutic potential of LND, 6-AN and the combination of LND and 6-AN for the treatment of echinococcosis.

Development of a novel TLR8 agonist for cancer immunotherapy.

Toll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. Several TLRs are being pursued as cancer drug targets. We discovered a novel, highly potent and selective small molecule TLR8 agonist DN052. DN052 exhibited strong in vitro cellular activity with EC50 at 6.7 nM and was highly selective for TLR8 over other TLRs including TLR4, 7 and 9. DN052 displayed excellent in vitro ADMET and in vivo PK profiles. DN052 potently inhibited tumor growth as a single agent. Moreover, combination of DN052 with the immune checkpoint inhibitor, selected targeted therapeutics or chemotherapeutic drugs further enhanced efficacy of single agents. Mechanistically, treatment with DN052 resulted in strong induction of pro-inflammatory cytokines in ex vivo human PBMC assay and in vivo monkey study. GLP toxicity studies in rats and monkeys demonstrated favorable safety profile. This led to the advancement of DN052 into phase 1 clinical trials.

In vitro and in vivo effects of 3-bromopyruvate against Echinococcus metacestodes.

While searching for novel anti-echinococcosis drugs, we have been focusing on glycolysis which is relied on by Echinococcus for energy production and intermediates for other metabolic processes. The aim of this study was to investigate the potential therapeutic implication of glycolytic inhibitors on Echinococcus. Our results demonstrate that at an initial concentration of 40 µM, all inhibitors of glycolysis used in the current experiment [3-bromopyruvate (3-BrPA), ornidazole, clorsulon (CLS), sodium oxamate and 2,6-dihydroxynaphthalene (NA-P2)] show considerable in vitro effects against Echinococcus granulosus protoscoleces and Echinococcus multilocularis metacestodes. Among them, 3-BrPA exhibited the highest activity which was similar to that of nitazoxanide (NTZ) and more efficacious than albendazole (ABZ). The activity of 3-BrPA was dose dependent and resulted in severe ultrastructural destructions, as visualized by electron microscopy. An additional in vivo study in mice infected with E. multilocularis metacestodes indicates a reduction in parasite weight after the twice-weekly treatment of 25 mg/kg 3-BrPA for 6 weeks, compared to that of the untreated control. In particular, in contrast to ABZ, the administration of 25 mg/kg 3-BrPA did not cause toxicity to the liver and kidney in mice. Similarly, at the effective dose against Echinococcus larvae, 3-BrPA showed no significant toxicity to human hepatocytes. Taken together, the results suggest that interfering with the glycolysis of the parasite may be a novel chemotherapeutical option and 3-BrPA, which exhibited a remarkable activity against Echinococcus, may be a promising potential drug against cystic echinococcosis (CE) and alveolar echinococcosis (AE).

In vitro efficacy of ampelopsin against Echinococcus granulosus and Echinococcus multilocularis.

The metacestode stage of Echinococcus granulosus and Echinococcus multilocularis cause cystic echinococcosis and alveolar echinococcosis, respectively, which result in severe medical and veterinary problems. In this study, as an exploration of novel treatment agents against echinococcosis, we demonstrated that ampelopsin (AMP), which is extracted from Ampelopsis grossedentata and has been clinically used for treatments of various types of diseases including cancers for a long time, exhibited profound in vitro effect against E. granulosus protoscoleces and E. multilocularis metacestodes. Furthermore, in vitro cytotoxicity assay also demonstrated that AMP at the effective dose against E. granulosus protoscoleces and E. multilocularis metacestodes did not show significant toxicity to human hepatocytes. These results suggest that AMP has the potential as an alternative agent against echinococcosis.

Quercetin Inhibits the Migration and Invasion of HCCLM3 Cells by Suppressing the Expression of p-Akt1, Matrix Metalloproteinase (MMP) MMP-2, and MMP-9.

BACKGROUND Quercetin is a natural bioactive flavonoid that is present in a wide variety of vegetables and fruits and exhibits a promising anti-metastasis property in various human cancer cells. However, the effect of quercetin on human HCCLM3 cells is unclear. MATERIAL AND METHODS In the current study, a wound-healing assay was performed using quercetin-treated HCCLM3 cells to further explore whether quercetin affects the motility of human HCCLM3 cells. Transwell assay was used to explore the potential effect of quercetin in HCCLM3 cells on cell migration and cell invasion. Western blotting analysis was used to explore the expression of p-Akt1, MMP-2, and MMP-9 in quercetin-treated HCCLM3 cells. RESULTS The wound-healing time was delayed in quercetin-treated HCCLM3 cells, and the ability to migrate and invade was inhibited in quercetin-treated human HCCLM3 cells. Moreover, the protein levels of p-Akt1, MMP-2, and MMP-9 were down-regulated in quercetin-treated HCCLM3 cells, as detected by Western blotting. CONCLUSIONS Our data show that quercetin attenuated cell migration and invasion by suppressing the protein levels of p-Akt1, MMP-2, and MMP-9 in HCCLM3 cells.

[Effect of Echinococcus multilocularis Infection on the Activities of Drug-metabolizing Enzymes in Gerbil Liver].

Objective: To study the effect of Echinococcus multilocularis infection on the activities of drug-metabolizing enzymes in gerbil liver. Methods: Ten gerbils （Meriones unguiculatus） were randomly divided into 2 groups, the experimental group in which each animal was intraperitoneally injected with 300 µl E. multilocularis cyst homogenates （containing about 600 protoscoleces）, and the control group in which each animal received 300 µl saline（i.p.）. Five months after infection, the animals were sacrificed by cervical dislocation, and livers were collected. The liver microsomal and cytosolic fractions were obtained by differential centrifugation. Protein concentration was measured by the BCA method. CYP450 and Cyt b5 contents in the microsomal fraction were measured with differential spectroscopy. Activities of 7-ethoxyresorufin（EROD） and 7-methoxyresorufin（MROD） were measured by fluorescence spectrometry. The activities of NADPH-cytochrome C reductase（NCR）, glutathione-S-transferase （GST）, and flavine monooxygenases （FMO） were measured by UV-visible spectrophotometry. Results: The protein content of cytosolic fractions and liver microsomes in experimental group was （11.089±1.277） and （3.212±0.924） mg/ml， those in control group was （12.459±1.625） and （3.894±0.395） mg/ml， respectively. The contents of CYP450 and Cyt b5 in the experimental group [（0.508±0.142）, （0.515±0.077） nmol/mg protein, respectively] were both significantly lower than those in the control [（0.647±0.090）, （0.596±0.051） nmol/mg protein]（P<0.05）. The GST activity decreased significantly in the experimental group ［（1.766±0.339）×103 nmol/（mg·min）］ compared with the control ［（2.001±0.160）×103 nmol/（mg·min）］（P<0.05）. But the FMO and NCR activities increased significantly in the experimental group ［（1.142±0.327） nmol/（mg·min） and （0.602±0.162）×103 nmol/（mg·min）, respectivelyï¼½ compared with the control ［（0.882±0.150） nmol/（mg·min） and （0.442±0.082）×103 nmol/（mg·min）］（P<0.05）. However, there were no significant differences in EROD and MROD activities between the two groups (P>0.05). Conclusion: The activities of FMO and NCR markedly increase, while that of GST significantly decreases in the gerbil liver after E. multilocularis infection.

[Clinicopathologic features of extrapulmonary inflammatory myofibroblastic tumor].

OBJECTIVE: To investigate the clinicopathologic features, immunohistochemic phenotypes and genetic alterations of extrapulmonary inflammatory myofibroblastic tumor (IMT) and the correlation with prognosis. METHODS: Thirty cases of IMT with follow-up were analyzed morphologically and immunohistochemically. ALK FISH was also performed to determine the ALK gene status. RESULTS: Patients ranged in age from 12 to 73 years (mean 43.4 years). The male-to-female ratio was 1.0: 1.1. The tumors were located in various anatomical sites including gastrointestinal tract, liver, spleen, kidney, pelvic, retroperitoneum, mediastinum etc. Histologically, the majority of cases were composed of spindled fibroblastic and myofibroblastic cells accompanied by an inflammatory infiltrate of plasma cells, lymphocytes, and eosinophils. Most cases with aggressive behavior had features including prominent nucleoli and edematous myxoid background. Lymphohistiocytic reactions were usually absent. Some cases showed multinucleation, nuclear pleomorphism and mitoses. One case demonstrated epithelioid morphology with round-to-epithelioid cells. The immunohistochemical study showed vimentin, SMA, CK, desmin, and ALK were expressed in 100% (30/30), 70% (21/30), 13% (4/30), 27% (8/30), and 27% (8/30) of IMT, respectively. Diffuse cytoplasmic ALK staining was detected in seven cases. One case (containing round-to-epithelioid cells) demonstrated ALK nuclear membrane staining, coupled with positive reaction for CD30 and negative reaction for LCA. EMA, CD34, CD117 and S-100 protein, and MyoD1 were negative for all cases. Six ALK protein positive cases harbored ALK gene rearrangement, but not the remaining 22 cases. Follow-up data were available in 21 patients. After initial resection, 14 patients were alive with no evidence of disease, while 4 patients were alive with tumor recurrence and 3 patients died of the disease. CONCLUSIONS: Most IMT with aggressive behavior have features including prominent nucleoli, edematous myxoid background, and positive expression of ALK. Lymphohistiocytic reaction is usually absent. ALK may be a potential novel therapeutic target for IMT.