Antitumor effect of polyphyllin D on liver metastases of neuroblastoma.

PURPOSE: We previously reported that polyphyllin D, a main component of the traditional Chinese medicinal herb Paris polyphylla, exhibited anticancer effects in vitro against human neuroblastoma cells. The aims of this investigation was to examine the presence or absence of in vivo anti-metastasis effects of polyphyllin D were to establish a liver metastasis model of neuroblastoma and to evaluate the anti-metastasis effects of polyphyllin D. METHODS: Subcutaneous and intraperitoneal tumors, and metastasis models were established in immune-deficient BALB/c nude and BALB/c Rag-2/Jak3 double-deficient (BRJ) mice using the human neuroblastoma cell lines IMR-32, LA-N-2, or NB-69. For evaluating polyphyllin D activity, we used a mouse model of liver metastasis with the IMR-32 cells line injected through the tail vein. We analyzed the livers number and area of liver tumors in of the phosphate buffer solution- and polyphyllin D-treated groups. RESULTS: Liver metastasis and intraperitoneal dissemination models were successfully established in immune-deficient BRJ mice using the three human neuroblastoma cell lines. In the liver metastasis, the model of IMR-32 cells, we found that polyphyllin D suppressed both the number and total area of metastatic foci the average number of metastatic foci, average focus areas, and number of cleaved caspase-3-positive cells were significantly lower in the polyphyllin D group (p = 0.016, 0.020, 0.043, respectively). CONCLUSIONS: We developed a mouse models of neuroblastoma metastasis and demonstrated for the first time that polyphyllin D has an antitumor effect on neuroblastoma liver metastases.

Arctigenin induces apoptosis in human hepatoblastoma cells.

PURPOSE: Arctigenin has been shown to have anti-tumor effects in various types of cancers. This study was conducted to verify these effects in the human-derived hepatoblastoma cell line, HUH-6 clone 5 (hereinafter, HUH-6). METHODS: Arctigenin was added to cultured HUH-6 cells, and cellular activity was evaluated by MTS assay. To determine the relationship between reduced cellular activity and apoptosis, we measured the activities of caspase 3/7, 8, and 9 and conducted flow cytometry with Annexin V/PI staining. RESULTS: The MTS assay revealed that cellular activity decreased after arctigenin treatment in a concentration-dependent manner (IC50 = 4 µM). To investigate apoptosis induction, activity assays of caspase 3/7, 8, and 9 were performed. While caspase 3/7 and 8 exhibited high activity, caspase 9 showed no activity. Thus, apoptosis induction may have involved the action of tumor necrosis factor receptor 1 (TNFR1). Flow cytometry conducted with Annexin V/PI staining revealed the occurrence of early apoptosis. CONCLUSION: We found that arctigenin has anti-tumor effects in HUH-6 cells in a concentration-dependent manner. Arctigenin may have exerted its anti-tumor effect by inducing apoptosis via TNFR1, which recruits Complex IIa to activate caspase 8 and 3/7. These results may be useful for developing therapeutic agents for hepatoblastoma.

Clinical course of human herpesvirus 6 infection in pediatric living donor liver transplantation.

Differentiation between active and latent viral infection is critical for analysis of HHV-6-associated disease. HHV-6 infection has been associated with several clinical manifestations; however, the precise role of HHV-6 in pediatric LDLT remains unclear. This retrospective cohort study included 33 pediatric patients who received LDLT. All of the recipients were monitored for HHV-6 infection using viral isolation and real-time PCR. HHV-6 infection was observed in 14 of 33 (42.4%) recipients, and HHV-6B infection occurred within 2 weeks after LDLT in 10 of 14 (71.4%) recipients. HHV-6 was isolated from 10 of 33 (30.3%) recipients. Multivariate analysis showed that independent predictors of HHV-6B infection were age (OR 0.975; 95% CI 0.943-0.999; P = .041), PELD (OR 1.091; P = .038), and biliary atresia (OR 16.48; P = .035). The occurrence of unexplained fever was significantly higher in recipients with HHV-6B infection (11/14) compared with uninfected recipients (6/19) (P = .013). Additionally, ALT levels at 8 and 9 weeks after transplantation were significantly higher in the recipients with HHV-6B infection. Younger age, high MELD/PELD score, and biliary atresia as an underlying disease were identified as risk factors for viral infection.

Polyphyllin D, a steroidal saponin in Paris polyphylla, induces apoptosis and necroptosis cell death of neuroblastoma cells.

PURPOSE: Neuroblastoma is a refractory pediatric malignant solid tumor. The previous studies demonstrated that Polyphyllin D, the main constituent of Paris polyphylla, a traditional Chinese medicine, exerts an anti-tumor effect on many tumors. However, its effects against neuroblastomas are unclear. METHODS: We examined the anti-tumor effect of polyphyllin D in human neuroblastoma using IMR-32 and LA-N-2 cells, which exhibit MYCN gene amplification, and NB-69 cells, which do not exhibit MYCN gene amplification. RESULTS: All cell lines showed reduced cell viability in response to polyphyllin D treatment. No caspase-3/-7, -8, and -9 activity was observed in IMR-32 and LA-N-2 cells treated with polyphyllin D. In contrast, activation of caspase-3/-7, and -8 activity was observed in NB-69 cells. When polyphyllin D and specific inhibitors of RIPK3 involved in necroptosis were added to IMR-32 and LA-N-2 cell lines, polyphyllin D-induced cell death was inhibited. CONCLUSION: Together, this indicates that the underlying mechanism of polyphyllin D-induced cell death in NB-69 cells is apoptosis, whereas the cell death of IMR-32 and LA-N-2 cells occurs by necroptosis. We continue research on this topic and look forward the discovery of a new therapeutic agent for neuroblastoma.

Successful living donor liver transplantation for classical maple syrup urine disease.

MSUD is an autosomal recessive condition characterized by a deficiency in the enzyme, BCKDH, which catalyzes the breakdown of BCAAs. If left untreated, MSUD can result in mental retardation, central nervous system disorders, and even death. Most patients with MSUD are treated with a restricted protein diet and milk from which BCAAs have been removed. LT has been shown effective in patients with MSUD. This report describes the case of a 15-month-old boy who received a liver graft from his mother. Transplantation was successful, and the patient was then able to ingest a normal diet. Despite episodes of acute rejection, chylous ascites, and high fever (40 °C), he has shown no evidence of MSUD recurrence. These findings indicate that patients with MSUD can be successfully treated by LDLT, even when the donor is a heterozygous carrier of a mutated BCKDH gene.

Nuclear receptor gene alteration in human induced pluripotent stem cells with hepatic differentiation propensity.

AIM: Human induced pluripotent stem (hiPS) cells are an alternative cell source of regenerative medicine for liver disease. Because variations in hepatic differentiation efficacy among hiPS cells exist, it is important to select a hiPS cell line with hepatic differentiation propensity. In addition, nuclear receptors (NR) regulate essential biological processes including differentiation and development. In this study, we identified the hiPS cell line with hepatic differentiation propensity and examined expression levels of 48 NR during this process. METHODS: We screened 28 hiPS cell lines, which are established from various tissues of healthy persons with various reprogramming methods, using a three-step differentiation method, and examined expression levels of 48 NR by quantitative real-time polymerase chain reaction during the differentiation process in the selected cells. RESULTS: hiPS-RIKEN-2B and hiPS-RIKEN-2F cells have hepatic differentiation propensity. Differentiation propensity towards endoderm was affected by donor origin but not by reprogramming methods or cell type of origins. Expression levels of NR were closely associated with those of hepatic differentiation markers. Furthermore, expression patterns of NR were categorized as five patterns. In particular, seven NR such as chicken ovalbumin upstream promoter transcription factor 1, retinoic acid receptor α, peroxisome proliferator-activated receptor-γ, progesterone receptor, photoreceptor cell-specific nuclear receptor, tailless homolog orphan receptor and glucocorticoid receptor were identified as the genes of which expression gradually goes up with differentiation. CONCLUSION: These findings will be useful for not only elucidating mechanisms of hepatic differentiation of hiPS cells but also cell-based therapy for liver diseases.

Impact of Portal Flow on the Prognosis of Children With Congenital Portosystemic Shunt: A Multicentric Observation Study in Japan.

BACKGROUND: Although congenital portosystemic shunts (CPSSs) are increasingly being recognized, the optimal treatment strategies and natural prognosis remain unclear, as individual CPSSs show different phenotypes. METHODS: The medical records of 122 patients who were diagnosed with CPSSs at 15 participating hospitals in Japan between 2000 and 2019 were collected for a retrospective analysis based on the state of portal vein (PV) visualization on imaging. RESULTS: Among the 122 patients, 75 (61.5%) showed PV on imaging. The median age at the diagnosis was 5 months. The main complications related to CPSS were hyperammonemia (85.2%), liver masses (25.4%), hepatopulmonary shunts (13.9%), and pulmonary hypertension (11.5%). The prevalence of complications was significantly higher in patients without PV visualization than in those with PV visualization (P < 0.001). Overall, 91 patients (74.6%) received treatment, including shunt closure by surgery or interventional radiology (n = 82) and liver transplantation (LT) or liver resection (n = 9). Over the past 20 years, there has been a decrease in the number of patients undergoing LT. Although most patients showed improvement or reduced progression of symptoms, liver masses and pulmonary hypertension were less likely to improve after shunt closure. Complications related to shunt closure were more likely to occur in patients without PV visualization (P = 0.001). In 25 patients (20.5%) without treatment, those without PV visualization were significantly more likely to develop complications related to CPSS than those with PV visualization (P = 0.011). CONCLUSION: Patients without PV visualization develop CPSS-related complications and, early treatment using prophylactic approaches should be considered, even if they are asymptomatic. LEVEL OF EVIDENCE: Level III.

1,3-Migrative Ring Expansion of Spiroindolenines to Azepino[3,4-b]indoles.

We serendipitously found an unprecedented 5-to-7-membered ring expansion of 2-alkylspiroindolenines to azepinoindoles mediated by n-tetrabutylammonium fluoride. The starting materials can be easily prepared by the hypoiodite-catalyzed oxidative dearomative spirocyclization of indole derivatives. Mildly basic conditions and electron-deficient protecting groups for the amines were found to be crucial to promoting chemoselective reactions. Moreover, the ring expansion of aniline-derived spiroindolenines proceeds smoothly under much milder conditions using only a catalytic amount of cesium carbonate.

Appendiceal atresia causing recurrent right lower quadrant pain without inflammation.

An 11-year-old girl presented with recurrent right lower quadrant (RLQ) pain. There was no evidence of inflammation and appendiceal swelling except at the initial onset. The repeated presence of a small amount of ascites at the time of abdominal pain triggered the performance of exploratory laparoscopy. Intraoperative examination revealed a non-inflamed, unswollen appendix with a cord-like atretic segment at the middle part and an appendectomy was performed. At 46 months follow-up, she remained asymptomatic. In patients with recurrent RLQ pain of unknown cause, it is necessary to consider diagnostic laparoscopy while keeping appendiceal atresia in mind as a differential diagnosis.

Evaluation of Ki-67 as prognostic factor for pediatric neuroblastoma and the possibility of molecular-targeted drugs with VEGF and PDGFR.

BACKGROUND: Neuroblastoma (NB) is a pediatric malignant solid tumor characterized as refractory cancer with poor prognosis. The Mitosis-Karyorrhexis Index (MKI) is a prognostic factor but is prone to observer bias. The usefulness of MKI with Ki-67, as a marker of malignancy, was investigated. The efficacy of molecular-targeted therapeutic agents with fewer side effects in tumors has been studied. Molecular-targeted therapy targets include vascular endothelial growth factor (VEGF), involved in tumor angiogenesis; c-Kit, receptor of Kit/stem cells involved in tumor growth, vasculature, and lymphangiogenesis; platelet-derived growth factor receptor (PDGFR); and B-Raf proto-oncogene, serine/threonine kinase (BRAF), involved in the RAS protein-mediated mitogen-activated protein kinase pathway. Therefore, expression profiles of these factors and growth inhibitory effects of molecular-targeted drugs against NB were investigated. METHODS: Ten frozen NB tissue samples collected from January 1993 to December 2017 were evaluated immunohistochemically for Ki-67 and VEGF. c-Kit, PDGFR, and BRAF expression levels were evaluated using enzyme-linked immunosorbent assays; relationships between these factors and clinicopathological parameters of NB were analyzed. RESULTS: Eight patients with NB showed no amplification of MYCN (MYCN proto-oncogene, bHLH transcription factor). There were two cases of ganglioneuroblastoma (GNB). More NB cells were positive for Ki-67 than for GNB cells. VEGF expression was observed in all NB specimens and was stronger in stage IIB and higher. No BRAF or c-Kit activity was observed; PDGFR activity was greater in NB than in GNB (P=0.02). CONCLUSIONS: Thus, Ki-67 may help evaluate NB malignancy. As the first therapy for NB prevents amplification of MYCN, agents targeting PDGFR as well as VGFG can inhibit NB cell proliferation.

Disrupted plasma membrane localization of equilibrative nucleoside transporter 2 in the chemoresistance of human pancreatic cells to gemcitabine (dFdCyd).

Although the nucleoside pyrimidine analogue gemcitabine is the most effective single agent in the palliation of advanced pancreatic cancer, cellular resistance to gemcitabine treatment is a major problem in the clinical scene. To clarify the molecular mechanisms responsible for chemoresistance to gemcitabine, mRNA expression of the key enzymes including cytidine deaminase (CDA), deoxycytidine kinase (dCK), 5'-nucleotidase (NT5), equilibrative nucleoside transporter 1 and 2 (ENT1 and ENT2), dCMP deaminase (dCMPK), ribonucleotide reductase M1 and M2 (RRM1 and RRM2), thymidylate synthase (TS) and CTP synthase (CTPS) was examined. The interacellular uptake of gemcitabine was greatly impaired in the chemoresistant cell lines due to dysfunction of ENT1 and ENT2. Protein expression of ENT1 and ENT2 and their protein coding sequences were not altered. Immunohistochemical and western blot analyses revealed that localization of ENT2 on the plasma membrane was disrupted. These data suggest that the disrupted localization of ENT2 is one of causes of the impaired uptake of gemcitabine, resulting in a gain of chemoresistance to gemcitabine.

c-Jun N-terminal kinase activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of retinoic acid receptor α protein in hepatic cells.

We previously reported that impaired retinoid signaling causes hepatocellular carcinoma (HCC) through oxidative stress. However, the interaction between oxidative stress and retinoid signaling has not been fully understood. To address this issue, the effects of hydrogen peroxide on the transcriptional activity of RAR/RXR heterodimers, RARα and RXRα proteins and intracellular signaling pathways were examined. The transcriptional activity of RAR/RXR examined by the DR5-tk-Luc reporter assay was significantly suppressed. The RARα protein level began to decrease at 6 h after treatment and declined thereafter. However, RARα mRNA were not changed. Activation of extracellular regulated kinases (ERK), p38, c-Jun N-terminal kinase (JNK) and Akt was observed after treatment of hydrogen peroxide. SP600125, an inhibitor of JNK, reversed the RARα protein level reduced by hydrogen peroxide. Anisomycin, an activator of JNK, reduced RARα protein. Transfection of wild-type JNK-constitutive actively expressing plasmid, but not kinase-negative JNK-expressing plasmid caused reduction of RARα protein. Proteasomal degradation of RARα was observed after anisomycin treatment; however, the mutant RARα, of which phosphorylation sites are replaced with alanines, was not degradated. In hepatitis C virus (HCV)-related human liver tissues, phospho-JNK and RARα reciprocally expressed with the progression of liver disease. Finally, the staining of 8-OHdG and thioredoxin was increased with the disease progression. These data indicate that JNK activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of RARα, suggesting that a vicious cycle between aberrant retinoid signaling and oxidative stress accelerates hepatocarcinogenesis.

Reactive oxygen species and NADPH oxidase 4 induced by transforming growth factor ß1 are the therapeutic targets of polyenylphosphatidylcholine in the suppression of human hepatic stellate cell activation.

OBJECTIVE AND DESIGN: To clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs). MATERIAL: Using human LX-2 HSC cells, we examined the effects of PPC on expression of α-smooth muscle actin (α-SMA) and collagen 1, generation of ROS, Nox4 expression, p38 activation and cell proliferation, induced by transforming growth factor ß1 (TGFß1). RESULTS: PPC suppressed ROS which are induced by TGFß1, phosphorylation of p38MAPK, and expression levels of α-SMA and collagen 1 in a dose-dependent manner. Higher concentrations of PPC also suppressed Nox4 levels. CONCLUSION: These results suggest that ROS and Nox4 induced by TGFß1 are the therapeutic targets of PPC in the suppression of human hepatic stellate cell activation.

Novel ARG1 variants identified in a patient with arginase 1 deficiency.

We report a case of a 13-year-old boy with arginase 1 deficiency carrying a new variant in ARG1. Sanger sequencing identified the compound heterozygous variants: NM_000045.4: c.365G>A (p.Trp122*)/c.820G>A (p.Asp274Asn). Although not previously reported, the p.Asp274Asn variant is predicted to have strong pathogenicity because it is located in a highly conserved domain in the protein core and arginase activity in the patient was below measurement sensitivity.

Urgent intraoperative endovascular stent placement to resolve acute hepatic or portal venous obstruction during liver surgery: a case series.

BACKGROUND: Acute obstruction of the hepatic vein (HV) or the portal vein (PV), particularly when it occurs during liver surgery, is potentially fatal unless repaired swiftly. As surgical interventions for this problem are technically demanding and potentially unsuccessful, other treatment options are needed. CASE PRESENTATION: We report two cases of acute, surgically uncorrectable HV or PV obstruction during liver resection or living donor liver transplantation (LDLT), which was successfully treated with urgent intraoperative placement of endovascular stents using interventional radiology (IVR). In Case 1, a patient with colonic liver metastases underwent a non-anatomic partial hepatectomy of the segments 4 and 8 with middle hepatic vein (MHV) resection. Additionally, the patient underwent an extended right posterior sectionectomy with right hepatic vein (RHV) resection for tumors involving RHV. Reconstruction of the MHV was needed to avoid HV congestion of the anterior section of the liver. The MHV was firstly reconstructed by an end-to-end anastomosis between the MHV and RHV resected stumps. However, the reconstruction failed to retain the HV outflow and the anterior section became congested. Serial trials of surgical revisions including re-anastomosis, vein graft interposition and vein graft patch-plasty on the anastomotic wall failed to recover the HV outflow. In Case 2, a pediatric patient with biliary atresia underwent an LDLT and developed an intractable PV obstruction during surgery. Re-anastomosis with vein graft interposition failed to restore the PV flow and elongated warm ischemic time became critical. In both cases, the misalignment in HV or PV reconstruction was likely to have caused flow obstruction, and various types of surgical interventions failed to recover the venous flow. In both cases, an urgent IVR-directed placement of self-expandable metallic stents (SEMS) restored the HV or PV perfusion quickly and effectively, and saved the patients from developing critical conditions. Furthermore, in Cases 1 and 2, the SEMS placed were patent for a sufficient period of time (32 and 44 months, respectively). CONCLUSIONS: The IVR-directed, urgent, intraoperative endovascular stenting is a safe and efficient treatment tool that serves to resolve the potentially fatal acute HV or PV obstruction that occurs in the middle of liver surgery.

A new murine ileostomy model: recycling stool prevents intestinal atrophy in the distal side of ileostomy.

OBJECTIVES: Proximal stoma creation in neonates results in growth failure and distal intestinal atrophy. "Recycling stool" consists of stool injection from the proximal limb to the distal limb of a stoma. Because this method may prevent distal bowel atrophy and increase body weight, we investigated the effects of recycling stool upon distal intestinal mucosa by generating an ileostomy model in rats. METHODS: An ileostomy was created 5 cm proximal to the cecum in male Wistar/ST rats. Discharged stool or saline was injected into the distal limb, twice per day for 7 days. The intestinal adaptation was assessed by measuring the villus height and counting goblet cell number. Proliferation and apoptosis were analyzed by Ki67 and TUNEL immunostaining. RESULTS: The ratios of the height of the distal villi (D) to the that of proximal villi (P) were 0.97 (median [range] of D and P length: 421 [240-729] µm and 436 [294-638] µm, P<0.05) in the stool-injected group and 0.81 in the saline-injected group (442 [315-641] µm and 548 [236-776] µm, P<0.05). Compared with the saline-injected group, the stool-injected group showed elevated numbers of goblet cells (3.6 [2.0-7.6] vs. 4.9 [2.4-7.5] cells/100-µm villus length) and Ki67-positive cells (26.8% [13.8%-35.4%] vs. 40.1% [31.2%-45.7%]), along with a reduced number of apoptotic cells (5.0 [2.0-14.0] vs. 4.0 [1.0-9.0] cells/100-µm villus length). CONCLUSIONS: Recycling stool prevented distal intestinal atrophy; this experimental design may facilitate further studies concerning alternative methods to prevent intestinal atrophy and growth failure.

Case Report: Management of Cholestasis Associated With Congenital Syphilis.

Cholestasis is a rare but life-threatening complication of congenital syphilis. However, standard management methods for this disease have not been established. Here, we report a case of congenital syphilis presenting with progressively worsening cholestasis, and we review the clinical features and management practices. In these cases, differentiation from other diseases presenting with cholestasis during the neonatal period, such as biliary atresia, is critical. In this regard, operative cholangiogram and histopathological analysis of the liver are required. Moreover, comprehensive genetic analysis can be useful. Although there is no specific treatment for cholestasis associated with congenital syphilis, appropriate nutritional management and supplementation with fat-soluble vitamins, especially vitamin K, should be provided. The severity of liver fibrosis may affect the prognosis of cholestasis associated with congenital syphilis. Therefore, attention should be paid to liver fibrosis in these patients.

Boost radiosurgery for treatment of brain metastases after surgical resections.

BACKGROUND: We evaluated results of resection surgery followed by boost radiosurgery for the treatment of brain metastases. METHODS: We treated 21 patients (13 male, 8 female) with surgical resection (subtotal or total) followed by boost radiosurgery. The mean patient age was 61 years (range, 41-80 years); supratentorial lesions were treated in 12 patients, and posterior fossa lesions were treated in 9 patients. The most common primary cancers were lung (24%) and colon (24%). Fifty-three percent of patients had brain metastases only, whereas 47% had extracranial metastases. The radiosurgery dose plan was designed to radiate the operative cavity; the mean treatment volume (50% isodose) was 10.7 mL (range, 3.4-23.3 mL), and the mean marginal dose was 17 Gy (range, 13-20 Gy). RESULTS: Local control was achieved in 16 (76%) patients. However, new intracranial lesions developed in 10 patients, and meningeal carcinomatosis occurred in 5 patients. Local tumor recurrence occurred more often for patients treated with lower radiotherapy doses (<18 vs > or =18 Gy, P = .03), and meningeal carcinomatosis occurred more often in patients with posterior fossa lesions (P = 0.05). Gamma knife radiosurgery was performed in 13 patients, and whole-brain radiation was performed in 2 patients. No patients experienced symptomatic radiation injury, and the median survival time was 20 months. CONCLUSIONS: Although boost radiosurgery is less invasive and reduces morbidity, the radiosurgical dose must be higher than 18 Gy for the treatment to be most effective. Treatment of lesions of the posterior fossa must be considered carefully because of the higher frequency of meningeal carcinomatosis. Also, we recommend that the surgeons who operate on the metastatic tumors must try to decrease the resected cavity volume and to prevent cerebrospinal fluid dissemination at the operation for posterior fossa lesions.