Sporoderm-broken spores of Ganoderma lucidum modulate hepatoblastoma malignancy by regulating RACK1-mediated autophagy and tumour immunity.

Hepatoblastoma (HB), a primary liver tumour, is notorious for its high metastatic potential and poor prognosis. Ganoderma lucidum, an edible mushroom species utilized in traditional Chinese medicine for addressing various tumour types, presents an intriguing avenue for HB treatment. However, the effectiveness of G. lucidum in managing HB and its underlying molecular mechanism necessitates further exploration. Standard in vitro assays were conducted to evaluate the impact of sporoderm-broken spores of G. lucidum (SBSGL) on the malignant characteristics of HB cells. The mechanism of SBSGL in treating HB and its tumour immunomodulatory effects were explored and validated by various experiments, including immunoprecipitation, Western blotting, mRFP-GFP-LC3 adenovirus transfection and co-localization analysis, as well as verified with in vivo experiments in this regard. The results showed that SBSGL effectively inhibited the malignant traits of HB cells and suppressed the O-GlcNAcylation of RACK1, thereby reducing its expression. In addition, SBSGL inhibited immune checkpoints and regulated cytokines. In conclusion, SBSGL had immunomodulatory effects and regulated the malignancy and autophagy of HB by regulating the O-GlcNAcylation of RACK1. These findings suggest that SBSGL holds promise as a potential anticancer drug for HB treatment.

Babao Dan is a robust anti-tumor agent via inhibiting wnt/ß-catenin activation and cancer cell stemness.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese Medicine (TCM) is being increasingly used worldwide due to its diverse efficacy and relatively low side effects. Babao Dan (BBD) is a well-known TCM formula that is currently used for the effective treatment of various cancers, however its underlying molecular mechanism remains unknown. AIM OF THE STUDY: Tumor growth and tumor recurrence are characterized by two distinct populations of cells, namely the well-differentiated cancer cells composing the majority of tumor bulk, and cancer stem cells (CSCs) involved in tumor relapse, which are both strongly associated with excessive activation of Wnt/ß-catenin signaling. Our study aims to elucidate the underlying molecular mechanisms associated with the anti-tumor proliferative effects of Babao Dan (BBD). MATERIALS AND METHODS: We used a hepatoblastoma cell line HepG2 with stem cell-like traits that harbors a constitutively active mutant of ß-catenin in order to study the anti-tumor ability of BBD via targeting Wnt/ß-catenin signaling. RESULTS: BBD robustly attenuated both the intrinsic and extrinsic activation of Wnt/ß-catenin pathway in HepG2 hepatoblastoma cells, as well as Wnt target genes. Moreover, BBD significantly inhibited both the proliferation of well-differentiated cancer cells, as well as the stem-like property of CSCs as evidenced by EpCAM, a Wnt target gene and a novel marker of cancer cell stemness. In addition, mice administered with BBD using HepG2 cell line derived xenograft model had marked reductions in tumor size and weight, as well as significantly decreased expressions of Wnt target genes and cancer cell stemness. CONCLUSION: Our findings elucidated the underlying molecular mechanisms associated with the robust anti-tumor effects of BBD via potent inhibition of Wnt/ß-catenin signaling, and implicate its use in the clinical treatment of cancers.

Integrated Genomic Analysis Identifies Driver Genes and Cisplatin-Resistant Progenitor Phenotype in Pediatric Liver Cancer.

Pediatric liver cancers (PLC) comprise diverse diseases affecting infants, children, and adolescents. Despite overall good prognosis, PLCs display heterogeneous response to chemotherapy. Integrated genomic analysis of 126 pediatric liver tumors showed a continuum of driver mechanisms associated with patient age, including new targetable oncogenes. In 10% of patients with hepatoblastoma, all before three years old, we identified a mosaic premalignant clonal expansion of cells altered at the 11p15.5 locus. Analysis of spatial and longitudinal heterogeneity revealed an important plasticity between "hepatocytic," "liver progenitor," and "mesenchymal" molecular subgroups of hepatoblastoma. We showed that during chemotherapy, "liver progenitor" cells accumulated massive loads of cisplatin-induced mutations with a specific mutational signature, leading to the development of heavily mutated relapses and metastases. Drug screening in PLC cell lines identified promising targets for cisplatin-resistant progenitor cells, validated in mouse xenograft experiments. These data provide new insights into cisplatin resistance mechanisms in PLC and suggest alternative therapies. SIGNIFICANCE: PLCs are deadly when they resist chemotherapy, with limited alternative treatment options. Using a multiomics approach, we identified PLC driver genes and the cellular phenotype at the origin of cisplatin resistance. We validated new treatments targeting these molecular features in cell lines and xenografts.This article is highlighted in the In This Issue feature, p. 2355.

Huaier Extract Induces Apoptosis in Hepatoblastoma Cells Via the MEK/ERK Signaling Pathway.

BACKGROUND/AIM: Huaier extract, whose main active constituent is the proteoglycan, has anti-tumor activity in several types of malignancies. In this study, we aimed to elucidate the effect of Huaier extract in hepatoblastoma cells. MATERIALS AND METHODS: The effect of Huaier extract on the proliferation of human hepatoblastoma cell lines HepG2 and HuH-6, was examined. RESULTS: Incubation with Huaier extract resulted in a marked, dose-dependent decrease in hepatoblastoma cell viability. Huaier extract induced S phase arrest in hepatoblastoma cells and upregulated the expression of the cell cycle related proteins cyclin D1 and cyclin D3. It also induced apoptosis in hepatoblastoma cells. Additionally, it significantly suppressed the activity of p-ERK and p-MEK. CONCLUSION: Huaier extract inhibits proliferation, and induces cell apoptosis and cell cycle arrest via the MEK-ERK pathway in hepatoblastoma cells. Huaier extract may act as a complementary agent for treating hepatoblastoma.

The Collagen Gel Droplet-embedded Culture Drug Sensitivity Test in Relapsed Hepatoblastoma.

There are few treatment options for patients with unresectable or refractory hepatoblastoma which has failed to respond to the standard treatment. The rarity of the disease and lack of experimental materials have hampered the development of new treatments. In this study, the collagen gel droplet-embedded culture drug sensitivity test was used to evaluate the effectiveness of the multikinase inhibitors sorafenib and sunitinib, and other drugs, in relapsed hepatoblastoma tumor tissues. Tumor samples from 6 patients with relapsed hepatoblastoma were tested for drug sensitivity by the collagen gel droplet-embedded culture drug sensitivity test; evaluable results were obtained from 5 of them. All samples were judged to be sensitive to sorafenib with a 50% growth inhibitory concentration (IC50) of 0.5 to 3.1 µg/mL. Sunitinib did not achieve IC50 in 2 of 3 samples within the tested concentration range based on clinically observed serum concentrations. In the drug combination assay using a hepatoblastoma cell line, sorafenib showed synergistic effects with SN-38, an active metabolite of irinotecan. Our results provide the basic science background warranting future clinical trials of a combination of sorafenib and irinotecan for relapsed or refractory hepatoblastoma.

Athyrium multidentatum (Doll.) Ching extract induce apoptosis via mitochondrial dysfunction and oxidative stress in HepG2 cells.

Athyrium multidentatum (Doll.) Ching (AMC), a unique and nutritious potherb widely distributed in china, has been extensively used in traditional Chinese medicine. Previous studies indicated that AMC extract exhibited antioxidant and antitumor properties. However, the chemical composition of AMC and molecular mechanism of AMC toxicity to HepG2 cells have not yet been elucidated. Hence, this study aimed to investigate the chemical compositions and the underlying mechanisms of the antiproliferative and apoptotic effects of AMC on HepG2. HPLC-MS analysis showed that AMC contain five compounds with chlorogenic acid accounting for 43 percent. Also, AMC strongly inhibited the cell growth and induced apoptosis and cell cycle arrest in HepG2 cells by significantly upregulating the protein expressions of Fas, Fas-L, Bax/Bcl-2, cyto-c, cleaved caspase-3, and PARP in a dose-dependent manner, which indicates AMC induces apoptosis in HepG2 cells through both intrinsic and extrinsic pathways. Moreover, AMC provoked the production of ROS, H2O2, and NO, modulating the PI3K/Akt, MAPK, NFκB and Nrf2 pathways and their downstream transcriptional cascades, ultimately evoked oxidative stress and apoptosis in HpeG2 cells. Further in vivo experiments demonstrated that AMC significantly suppressed the tumor growth, suggesting that AMC may be a novel promising agent for hepatocellular carcinoma treatment.

Complementary roles of ß-catenin and glutamine synthetase immunostaining in diagnosis of chemotherapy-treated and untreated hepatoblastoma.

BACKGROUND/PURPOSE: This study aimed to evaluate the expression of ß-catenin and its downstream target glutamine synthetase (GS) in hepatoblastoma (HB), and to evaluate the use of these two markers for diagnosing HB. METHODS: Eighteen untreated HBs and 22 HBs resected after neoadjuvant chemotherapy were analyzed using ß-catenin and GS immunostaining. RESULTS: We detected nuclear ß-catenin immunostaining in nearly all untreated HBs, including in fetal and embryonal epithelial components and in mesenchymal elements. We also observed diffuse GS expression in the epithelial component; however, it was frequently absent in embryonal and mesenchymal areas. In HBs resected after neoadjuvant chemotherapy, we recognized four histological patterns: fetal, hepatocellular-carcinoma-like, clear-cell, and normal-liver-like. All these patterns displayed diffuse GS expression. Fetal pattern showed diffuse nuclear ß-catenin immunostaining. Nuclear ß-catenin immunostaining was weak in the hepatocellular-carcinoma-like and clear-cell patterns. In normal-liver-like area, ß-catenin expression was only located in the cell membrane. CONCLUSION: The results suggest that nuclear ß-catenin expression and diffuse GS immunostaining are the hallmarks of HB. Although epithelial and mesenchymal components of HB display nuclear ß-catenin staining, this expression is attenuated following chemotherapy-induced cell maturation. GS immunostaining is especially useful for the assessment of section margins after neoadjuvant chemotherapy.

Optimization of a novel chelerythrine-loaded magnetic Fe3 O4 /chitosan alpha-ketoglutaric acid system and evaluation of its anti-tumour activities.

OBJECTIVES: A novel magnetic targeting anti-tumour drug delivery system (Fe3 O4 /KCTS-CHE) was designed using magnetic Fe3 O4 /chitosan alpha-ketoglutaric acid (Fe3 O4 /KCTS) as carrier and chelerythrine (CHE) as an anti-tumour drug model. Moreover, the anti-tumour activities and mechanisms of Fe3 O4 /KCTS-CHE were investigated. METHODS: The preparation conditions of Fe3 O4 /KCTS-CHE microspheres were optimized by response surface methodology (RSM). The CHE drug release kinetics was evaluated by fitting the experimental data to standard release equations. The inhibitive activities of Fe3 O4 /KCTS-CHE microspheres against the HepG2 cells were estimated using MTT assay in vitro, and the mechanisms were studied using Hoechst 33258 staining. KEY FINDINGS: The optimum preparation conditions were 11.68 : 1 for Fe3 O4 /KCTS:CHE ratio, 4 : 1 for oil/water ratio and 50.03 min for the ultrasonic time. The drug loading content and entrapment efficiency under the optimal conditions were 23.3% and 50.9%. The best fit was Higuchi model for the microspheres. The inhibitive rate on HepG2 cells of Fe3 O4 /KCTS-CHE nanoparticles varied from 30.19 ± 2.64% to 70.46 ± 6.42% at different concentrations from 50 to 400 mg/l in 72 h. CONCLUSION: Fe3 O4 /KCTS-CHE exhibited effective anti-tumour activities against the HepG2 cells and induced cell apoptosis in HepG2 cells. Fe3 O4 /KCTS-CHE possess a high drug loading efficiency and entrapment efficiency, which are a new matrix for controlling release of drugs and a promising candidate for targeted drug delivery.

Icariside II-induced mitochondrion and lysosome mediated apoptosis is counterbalanced by an autophagic salvage response in hepatoblastoma.

In this study, the anti-cancer effect of Icariside II (IS), a natural plant flavonoid, against hepatoblastoma cells and the underlying mechanisms were investigated. The in vitro and in vivo studies show that IS decreased the viability of human hepatoblastoma HepG2 cells in a concentration- and time-dependent manner and inhibited tumor growth in mice transplanted with H22 liver carcinomas. IS impaired mitochondria and lysosomes as evidenced by signs of induced mitochondrial and lysosomal membrane permeabilization, resulting in caspase activation and apoptosis. SQSTM1 up-regulation and autophagic flux measurements demonstrated that IS exposure also impaired autophagosome degradation which resulted in autophagosome accumulation, which plays a pro-survival role as the genetic knockdown of LC3B further sensitized the IS-treated cells. Electron microscopy images showed that autophagosome engulfs IS-impaired mitochondria and lysosomes, thus blocking cytotoxicity induced by further leakage of the hydrolases from lysosomes and pro-apoptosis members from mitochondria. In conclusion, these data suggest that IS plays multiple roles as a promising chemotherapeutic agent that induces cell apoptosis involving both mitochondrial and lysosomal damage.

Gamma-tocotrienol treatment increased peroxiredoxin-4 expression in HepG2 liver cancer cell line.

BACKGROUND: To determine the antiproliferative effect of gamma-tocotrienol (GTT) treatment on differential protein expression in HepG2 cells. METHODS: HepG2 cells were treated with 70 µM GTT for 48 hours and differentially expressed protein spots were determined by two-dimensional electrophoresis (2DE), identified by MALDI-TOF mass spectrometer (MS) and validated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: GTT treatment on HepG2 cells showed a total of five differentially expressed proteins when compared to their respective untreated cells where three proteins were down-regulated and two proteins were up-regulated. One of these upregulated proteins was identified as peroxiredoxin-4 (Prx4). Validation by qRT-PCR however showed decreased expression of Prx4 mRNA in HepG2 cells following GTT treatment. CONCLUSIONS: GTT might directly influence the expression dynamics of peroxiredoxin-4 to control proliferation in liver cancer.

Efficacy of preoperative transcatheter arterial chemoembolization combined with systemic chemotherapy for treatment of unresectable hepatoblastoma in children.

PURPOSE: The purpose of this study was to evaluate, retrospectively, the clinical efficacy of preoperative transcatheter arterial chemoembolization (TACE) combined with systemic chemotherapy for unresectable hepatoblastoma. MATERIALS AND METHODS: Five boys and three girls (mean age 15.2 months) were treated with preoperative TACE combined with systemic chemotherapy for unresectable hepatoblastomas. Mean tumor diameter and mean alfa-fetoprotein (AFP) level were 11.8 cm and 549,386 ng/mL, respectively. Pretreatment, the extent of disease (PRETEXT) was: II, 1; III, 6; IV, 1. For all patients, preoperative systemic chemotherapy was administered before TACE. At each TACE, carboplatin and adriamycin mixed with iodized oil were infused into the feeding arteries. Tumor response and prognosis after treatment were evaluated. RESULTS: TACE resulted in few Grade 1 adverse effects (AEs), without G3 or more AEs, according to CTACAE 3.0. Mean tumor shrinkage was 60.9%, and the mean AFP decrease from initial levels was 94.8%. In all cases TACE combined with systemic chemotherapy enabled subsequent safe and complete surgical resection. After a mean follow-up of 59 months, tumor-free survival was 75%. CONCLUSION: Preoperative TACE combined with systemic chemotherapy was effective in inducing surgical resectability of unresectable hepatoblastoma.

Dynamic monitoring of the cytotoxic effects of protoberberine alkaloids from Rhizoma Coptidis on HepG2 cells using the xCELLigence system.

AIM: To investigate the cytotoxic effects of the six protoberberine alkaloids (PAs) from Rhizoma Coptidis on HepG2 cells. METHOD: A systematic screening was conducted to investigate the dynamic response of HepG2 cells to the PAs using the impedance-based xCELLigence system. Cisplatin was selected as the positive control. The real time, concentration-response curves and the 50% inhibitory concentrations (IC50) were acquired to evaluate the anticancer activity of the PAs. RESULTS: All of the six PAs inhibited cell growth and induce death in HepG2 cells in a time- and concentration-dependent manner. The IC50 values of cisplatin, berberine, columbamine, coptisine, epiberberine, jatrorrhizine, and palmatine were 5.13, 42.33, 226.54, 36.90, 302.72, 383.54, and 456.96 µg·mL(-1), respectively. The results obtained using the xCELLigence system corresponded well with those of the conventional methods. CONCLUSION: The xCELLigence system is a reliable and efficient tool for real-time screening of the cytotoxic effect of compounds in cell-based in vitro assays. Coptisine and berberine, with methylenedioxy group at C2 and C3 on the phenyl ring showed stronger effect.than the other four PAs. However, compared with cisplatin, the six PAs didn't show obvious cytotoxic effect on HepG2 cells. These results provided some useful data for the evaluation of the anticancer compounds, and the clinical application of traditional Chinese medicine.

Involvement of substance p/neurokinin-1 receptor in the analgesic and anticancer activities of minimally toxic fraction from the traditional Chinese medicine Liu-Shen-Wan in vitro.

Liu-Shen-Wan (LSW), an ancient preparation used to treat localized infection with pain, was recently reported to possess anticancer activity. The mechanism responsible for LSW's analgesic and anticancer activity is unclear. In the present study, we obtained a LSW supernatant (LSWS) fraction from ultrasound-assisted ethanol extraction (yield 15.9%) which proved to be safer than LSW in terms of hepatotoxicity. The LSWS (1 and 10 µg/mL) exhibited a potent inhibitory effect on the bradykinin-evoked rapid release of substance P from dorsal root ganglion (DRG) cells. At concentrations of 0.1 µg/mL and higher, the LSWS resulted in a concentration-related growth inhibitory effect on HepG2, a representative cancer cell lines. The LSWS significantly down-regulated the neurokinin-1 (NK-1) receptor expression in both HepG2 and bradykinin-treated DRG cells. In addition to the NK-1 receptor-dependent growth inhibition in HepG2 cells (0.1-100 µg/mL), the LSWS induced mitochondria-mediated apoptosis at a higher concentration (1-100 µg/mL). In conclusion, we recently isolated a safer LSW fraction which maintained its analgesic and anticancer activity, and found that the substance P/NK-1 receptor system was partly responsible for these effects. Our findings will be useful for developing more effective and less toxic LSW preparations.

Study on in vitro anti-tumor activity of Bidens bipinnata L. extract.

We studied the in vitro anti-tumor activity of Bidens Bipinnata L. extract. MTT assay was used to investigate the inhibitory effect of different concentrations of the extracts on human hepatocellular carcinoma (HepG2) cell lines and human cervical carcinoma (Hela) cell lines, and the IC50 values were calculated. The Bidens Bipinnata L. extract had different degrees of inhibitory effects on these two cells, and when exposure time was 48 h, the inhibition rate reached its peak, with IC50 values of 14.80 µg/mL and 13.50 µg/mL respectively. The Bidens Bipinnata L. extract had a good inhibitory effect on human HepG2 cell lines and Hela cell lines, and thus has certain development prospects.

Imaging analysis of hepatoblastoma resectability across neoadjuvant chemotherapy.

PURPOSE: Hepatoblastomas often require neoadjuvant chemotherapy to facilitate partial hepatectomy, which necessitates freedom of tumor borders from the confluence of hepatic veins (COHV), portal vein bifurcation (PVB), and retrohepatic inferior vena cava (IVC). This study aimed to clarify the effect of incremental neoadjuvant cycles on the AHEP0731 protocol criteria of hepatoblastoma resectability. METHODS: Hepatoblastoma responses to neoadjuvant chemotherapy were analyzed among patients (n=23) treated at two children's hospitals between 1996 and 2010. Using digital imaging data, ellipsoid and point-based models were created to measure tumor volume regression and respective distances from tumor borders nearest to the COHV, PVB, and IVC. RESULTS: Hepatoblastoma volumes regressed with incremental neoadjuvant chemotherapy cycles (p<0.001). Although tumor borders regressed away from the COHV (p=0.008), on average only 1.1mm was gained. No change from tumor borders to the PVB was detected (p=0.102). Distances from tumor borders to the IVC remained stable at one hospital (p=0.612), but increased only 0.15 mm every 10 days of therapy at the other (p=0.002). Neoadjuvant chemotherapy induced slightly more tumors to meet the threshold vascular margin of 1cm (baseline to completion): COHV, 11 (47.8%) to 17 (73.9%; p=0.058); PVB, 11 (47.8%) to 15 (65.2%; p=0.157); and IVC, 4 (17.4%) to 10 (43.5%; p=0.034). No differences were detected in demographic or disease-specific characteristics between patients who did or did not achieve this 1-cm margin after conclusion of chemotherapy. CONCLUSION: Hepatoblastoma volumes regress significantly with increasing neoadjuvant chemotherapy cycles. However, tumors often remain anchored to the major hepatic vasculature, showing marginal improvement in resectability criteria.

Effect of sorafenib combined with cytostatic agents on hepatoblastoma cell lines and xenografts.

BACKGROUND: Sorafenib has recently been shown to reduce tumour growth in hepatoblastoma (HB) xenografts. The effect of a combined administration with cytostatic agents was now investigated. METHODS: Cell viability after treatment with sorafenib and different cytostatic agents was evaluated in two HB cell lines (HUH6 and HepT1) using MTT assay. ERK signalling was investigated by western blot, NOXA expression by rt-PCR, and formation of DNA adducts using immunocytology. NMRI mice bearing subcutaneous HUH6-derived tumours were treated with sorafenib alone or in combination with cisplatin. Tumour progression, viability, apoptosis, and vascularisation were monitored by tumour volume, AFP levels, TUNEL assay, and CD31 immunostaining, respectively. RESULTS: The combination of sorafenib and cisplatin led to a remarkable decrease in cell viability. The cisplatin-induced enhanced ERK1/2 activation, but not NOXA expression and the formation of DNA adducts was partly abrogated by sorafenib. In HB xenografts, both, sorafenib and alternated application of sorafenib and cisplatin significantly reduced tumour growth (P<0.05). Levels of AFP were lower in both treated groups (P=0.08). Relative apoptotic areas were increased (P=0.003). Mean vascular density was the lowest in the sorafenib/CDDP group (P=0.02). CONCLUSION: The combination of sorafenib with cisplatin might be a promising treatment option for high risk or recurrent HB.

MAPK signaling pathways regulate mitochondrial-mediated apoptosis induced by isoorientin in human hepatoblastoma cancer cells.

Isoorientin (ISO) (CAS RN: 4261-42-1) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum. ISO is able to induce apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cells, however, the effects of ISO on MAPK signaling pathways remain unknown. The present study investigated the effects of ISO on this pathway, and the roles of MAPK kinases on mitochondrial-mediated apoptosis in HepG2 cells. The results showed that ISO induced cell death in a dose- and time-dependent manner, and induction apoptosis is main cause for ISO-induced cytotoxicity in HepG2 cells. ISO significantly inhibited the levels of ERK1/2 kinase and increased the expression of JNK and p38 kinases. Furthermore, U0126 (an ERK1/2 inhibitor) significantly enhanced the ISO-induced the Bax/Bcl-2 ratio, the release of cytochrome c to the cytosol fraction, and the levels of cleaved caspase-3. While SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) markedly prevented the expression of these proteins induced by ISO. Furthermore, the ROS inhibitor (NAC) notably promoted the inhibited effect of ISO on the ERK1/2 kinase. NAC also suppressed the p-JNK and p-p38, but failed to reverse the effects of ISO. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells through inactivating ERK1/2 kinase and activating JNK and p38 kinases, and ROS stimulated by ISO is able to activate the MAPK singaling pathway as the upstream signaling molecules. Initiating event of the mitochondrial-mediated apoptosis induced by ISO is MAPK signals.

Induction of cytochrome P450 3A by Shexiang Baoxin Pill and its main components.

The expression of cytochrome P450 is regulated by both endogenous factors and xenobiotics including chemical drugs and natural medicines. Induction on cytochrome P450 can reduce the therapeutic efficacy from drugs inactivated by this enzyme system, but may increase the efficacy or lead to intoxication for prodrugs. Shexiang Baoxin Pill (SBP) is a traditional Chinese medicine widely used for the treatment of angina pectoris and myocardial infarction in China and other oriental countries. To assess the potential of SBP to alter the activity and expression of cytochrome P450 3A (CYP3A) extensively involved in drug metabolism, we investigated the enzyme-inducing effects of SBP in HepG2 cells and in rats. The results showed that treatment with SBP increased the enzyme activity, mRNA levels and protein expression of CYP3A4 in a concentration-dependent manner in HepG2 cells. Moreover, treatment with SBP enhanced the activities and mRNA expressions of CYP3A1 and CYP3A2 ex vivo in rats. Furthermore, we utilized HepG2 cell line to identify individual components in SBP as potential inducers of CYP3A4. It was found that bufalin, cinobufagin, and resibufogenin were novel CYP3A4 inducers. Among them, bufalin and cinobufagin significantly promoted the CYP3A4 enzyme activity, mRNA and protein levels, with the maximal induction challenging or exceeding that of the induction by rifampicin, indicating that they might play a critical role in CYP3A4 enzyme-inducing effects of SBP. In addition, the metabolic studies with specific inhibitors of CYP isoforms suggested that the three CYP3A4 inducers in SBP are also the substrates for the enzyme. Overall, our results show that SBP contains constituents that can potently induce CYP3A and suggest that this traditional Chinese medicine should be examined clinically for potential drug metabolic interactions.

Treatment effects of the multikinase inhibitor sorafenib on hepatoblastoma cell lines and xenografts in NMRI-Foxn1 nu mice.

BACKGROUND: Multidrug resistance is a major reason for poor treatment results in advanced hepatoblastoma (HB). Several alternative treatment options are currently under investigation to improve the prognosis of affected patients AIMS: This study aimed to analyse the impact of sorafenib on the viability of HB cells and xenotransplanted HB tumours. METHODS: Cell viability and apoptosis were evaluated in two HB cell lines (HUH6 and HepT1) after treatment with sorafenib using MTT and Caspase 3 activation assay. Extracellular signal-regulated kinase (ERK) phosphorylation was investigated using Western blot. In addition, sorafenib (30 mg/kg) was administered orally to NMRI mice bearing subcutaneous HUH6 derived tumours. Tumour progression and viability were monitored by tumour volume and α-fetoprotein (AFP) levels, and apoptosis was assessed using TUNEL assay. Tumour angiogenesis and mean vascular density (MVD) was determined using CD31 staining, ERK phosphorylation was detected using indirect immunofluorescence. RESULTS: Treatment with sorafenib led to decreased ERK phosphorylation, reduced cell viability and induction of apoptosis in HepT1 and HUH6 cells. In HB xenografts, sorafenib significantly reduced tumour growth compared with control (P < 0.05). AFP levels were lower in the sorafenib group (P = 0.07). Relative apoptotic areas detected using TUNEL assay were increased (P = 0.003). CD31 staining revealed inhibition of angiogenesis, and mean vascular density was lower in the sorafenib group (P = 0.02). ERK phosphorylation was reduced in tumours tissues after sorafenib treatment. CONCLUSION: Treatment with sorafenib led to a potent inhibition of cell viability, tumour progression and angiogenesis. Sorafenib might therefore also be a promising treatment option for high risk or recurrent HB.