Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway.

BACKGROUND: The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway. AIM: To confirm the effect of XHP on HCC and the possible mechanisms involved. METHODS: The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT-qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed. RESULTS: The following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-ß-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-ß-boswellic acid, 5ß-androstane-3,17-dione, and 3-acetyl-11-keto-ß-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights. CONCLUSION: XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

Shuyu pills inhibit immune escape and enhance chemosensitization in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by dysregulation of the immune microenvironment and the development of chemoresistance. Specifically, expression of the programmed cell death protein 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis, an immune checkpoint, may lead to tumour immune escape, resulting in disease progression. The latest research shows that tumour immune escape may be caused by the upregulation of PD-L1 mediated by hypoxia-inducible factor-1 alpha (HIF-1α), and simultaneous inhibition of HIF-1α and PD-L1 has the potential to enhance the host's antitumour immunity. Moreover, inhibition of the PD-1/PD-L1 axis may mitigate tumour chemoresistance. Shuyu pills (SYPs) contain immunity-enhancing and antitumour components, making them a potential HCC treatment. AIM: To investigate the efficacy of SYPs for HCC treatment via simultaneous HIF-1α and PD-L1 inhibition and the mechanism involved. METHODS: A subcutaneous xenograft tumour model was first established in BALB/c nude mice by the subcutaneous injection of 1 × 107 SMMC-7721 cells. Male mice (male, 5 weeks old; n = 24) were then randomly divided into the following four groups (n = 6): Control (0.9% normal saline), SYP (200 mg/kg), SYP + cisplatin (DDP) (200 mg/kg + 5 mg/kg DDP weekly via intraperitoneal injection), and DDP (5 mg/kg cisplatin weekly via intraperitoneal injection). The dose of saline or SYPs for the indicated mouse groups was 0.2 mL/d via intragastric administration. The tumour volumes and body weights of the mice were measured every 2 d. The mice were euthanized by cervical dislocation after 14 d of continuous treatment, and the xenograft tissues were excised and weighed. Western blot assays were used to measure the protein expression of HIF-1α, PD-1, PD-L1, CD4+ T cells, and CD8+ T cells in HCC tumours from mice. Quantitative reverse transcription polymerase chain reaction was used for real-time quantitative detection of PD-1, PD-L1, and HIF-1α mRNA expression. An immunofluorescence assay was conducted to examine the expression of CD4+ T cells and CD8+ T cells. RESULTS: Compared to mice in the control group, those in the SYP and SYP + DDP groups exhibited reduced tumour volumes and tumour weights. Moreover, the protein and mRNA expression levels of the oncogene HIF-1α and that of the negative immunomodulatory factors PD-1 and PD-L1 were decreased in both the SYP and SYP + DDP groups, with the decrease effects being more prominent in the SYP + DDP group than in the SYP group (HIF-1α protein: Control vs SYP, P = 0.0129; control vs SYP + DDP, P = 0.0004; control vs DDP, P = 0.0152, SYP + DDP vs DDP, P = 0.0448; HIF-1α mRNA: control vs SYP, P = 0.0009; control vs SYP + DDP, P < 0.0001; control vs DDP, P = 0.0003, SYP vs SYP + DDP, P = 0.0192. PD-1 protein: Control vs SYP, P = 0.0099; control vs SYP + DDP, P < 0.0001, SPY vs SYP + DDP, P = 0.0009; SYP + DDP vs DDP, P < 0.0001; PD-1 mRNA: control vs SYP, P = 0.0002; control vs SYP + DDP, P < 0.0001; control vs DDP, P = 0.0003, SPY vs SYP + DDP, P = 0.0003; SYP + DDP vs DDP, P = 0.0002. PD-L1 protein: control vs SYP, P < 0.0001; control vs SYP + DDP, P < 0.0001; control vs DDP, P < 0.0001, SPY vs SYP + DDP, P = 0.0040; SYP + DDP vs DDP, P = 0.0010; PD-L1 mRNA: Control vs SYP, P < 0.0001; control vs SYP + DDP, P < 0.0001; control vs DDP, P < 0.0001, SPY vs SYP + DDP, P < 0.0001; SYP + DDP vs DDP, P = 0.0014). Additionally, the quantitative and protein expression levels of CD4+ T cells and CD8+ T cells were simultaneously upregulated in the SYP + DDP group, whereas only the expression of CD4+ T cells was upregulated in the SYP group. (CD4+ T cell quantitative: Control vs SYP + DDP, P < 0.0001, SYP vs SYP + DDP, P = 0.0005; SYP + DDP vs DDP, P = 0.0002. CD4+ T cell protein: Control vs SYP, P = 0.0033; Control vs SYP + DDP, P < 0.0001; Control vs DDP, P = 0.0021, SYP vs SYP + DDP, P = 0.0004; SYP + DDP vs DDP, P = 0.0006. Quantitative CD8+ T cells: Control vs SYP + DDP, P = 0.0013; SYP vs SYP + DDP, P = 0.0347; SYP + DDP vs DDP, P = 0.0043. CD8+ T cell protein: Control vs SYP + DDP, P < 0.0001; SYP vs SYP + DDP, P < 0.0001; SYP + DDP vs DDP, P < 0.0001). Finally, expression of HIF-1α was positively correlated with that of PD-1/PD-L1 and negatively correlated with the expression of CD4+ T cells and CD8+ T cells. CONCLUSION: SYPs inhibit immune escape and enhance chemosensitization in HCC via simultaneous inhibition of HIF-1α and PD-L1, thus inhibiting the growth of subcutaneous xenograft HCC tumours.

T-cell lymphoma with rhabdomyolysis: case report and literature review.

Rhabdomyolysis refers to the destruction or disintegration of striated muscles. This syndrome is characterized by muscle breakdown and necrosis, resulting in the leakage of intracellular muscle constituents into the circulation and extracellular fluid. We report a rare case of rhabdomyolysis complicating multi-organ failure caused by T-cell lymphoma in a 32-year-old woman. The final diagnosis was rhabdomyolysis caused by peripheral T-cell lymphoma based on bone marrow aspirate and biopsy.

Potential Maintenance Mechanism in Atrial Fibrillation Patients Subject to Different Treatment Procedures: A Preliminary Study Based on a Human Atrial Model.

Two clinical ablation protocols, 2C3L and stepwise, have been routinely used in our group to treat atrial fibrillation (AF), but with a less than 60% long-term arrhythmia-free outcome achieved in patients. The goal of this study was to examine the underlying mechanism of low success in clinical outcome. MRI images from one patient were used to reconstruct a human atrial anatomical model, and fibrotic tissue was manually added to represent the arrhythmia substrate. AF was induced with standard protocols used in clinical practice. 2C3L and stepwise were then used to test the efficacy of arrhythmia termination in our model. The results showed that re-entries induced in our model could not be terminated by using either 2C3L or the stepwise protocol. Although some of the induced re-entries were terminated, others emerged in new areas. Ablation using only the 2C3L or stepwise method was not sufficient to terminate all re-entries in our model, which may partially explain the poor long-term arrhythmiafree outcomes in clinical practice. Our findings also suggest that computational heart modelling is an important tool to assist in the establishment of optimal ablation strategies.

T-cell lymphoma with rhabdomyolysis: case report and literature review

Rhabdomyolysis refers to the destruction or disintegration of striated muscles. This syndrome is characterized by muscle breakdown and necrosis, resulting in the leakage of intracellular muscle constituents into the circulation and extracellular fluid. We report a rare case of rhabdomyolysis complicating multi-organ failure caused by T-cell lymphoma in a 32-year-old woman. The final diagnosis was rhabdomyolysis caused by peripheral T-cell lymphoma based on bone marrow aspirate and biopsy.

The anti-tumor effects of cordycepin-loaded liposomes on the growth of hepatoma 22 tumors in mice and human hepatoma BEL-7402 cells in culture.

Liposomes have successfully been used for decades to encapsulate and protect drugs that are prone to deactivation in the body. The present study aimed to demonstrate the use of liposomes to encapsulate cordycepin, an adenosine analog that quickly loses its activity in vivo. The cordycepin-loaded liposomes were prepared by the ammonium sulfate gradient approach, and its in vitro and in vivo antitumour activities were evaluated using BEL-7402 cells and hepatocellular carcinoma H22 transplanted tumors, respectively. An MTT assay was used to observe the cytotoxicity of cells treated with cordycepin and cordycepin-loaded liposomes in vitro. High-content screening (HSC) was carried out using Hoechst 33342 to detect apoptotic cells and the ratio of cells in different cell cycle stages. The data demonstrated that both the cordycepin and the cordycepin-loaded liposomes resulted in clear cytotoxicity with IC50 values of 18.97 and 29.39 µg/mL, respectively. The latter showed significantly strong inhibitory effects on H22 tumor growth in mice, while the former did not show any inhibitory effects on tumor growth. In addition, the HSC assay showed that the cordycepin-loaded liposomes resulted in a higher rate of apoptosis than the cordycepin alone in BEL-7402 cells. Further data analysis revealed that the cells treated with cordycepin-loaded liposomes were predominately arrested at the G2/M phase (p < 0.05), while those treated with cordycepin alone were arrested in the G0/G1 phase (p < 0.05). In conclusion, these results suggest that liposomes can enhance and maintain the in vivo anti-tumor activity of cordycepin.

[Anticancer activity of Crocodylus siamensis blood in vitro and in vivo].

OBJECTIVE: To study the anticancer effects of the blood of Crocodylus siamensis in vitro and in vivo. METHODS: The inhibitory effects of serum and plasma of Crocodylus siamensis on proliferation of HepG2, BGC823, HeLa and SKOV3 cell were measured by MTT assay. The mouse S180 tumor model was used to evaluate the anti-tumor effect in vivo. RESULTS: High dosage serum and plasma of Crocodylus siamensis could inhibit the proliferation of HepG2, BGC823, HeLa and SKOV3 cell. The tumor inhibitory rate of high dosage blood of Crocodylus siamensis on S180 tumor was up to 57.55%. CONCLUSION: The blood of breeding Crocodylus siamensis has anticancer activity.

[Expression of DNA-dependent protein kinase catalytic subunit in adult acute leukemia and its significance].

This study was aimed to explore the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in adult acute leukemia and its correlation with clinical characteristics, karyotype and prognosis. Indirect immunofluorescent cytometry was used to detect the expression of DNA-PKcs in bone marrow mononuclear cells of 105 patients with acute leukemia before chemotherapy and 41 of them after 2 cycles of chemotherapy. Cytogenetic data were obtained from 26 of them by R band karyotypic analysis. The results showed that the expression of DNA-PKcs was correlated with higher WBC count level in peripheral blood (p < 0.05), but was not obviously associated with median age, gender, percentage of bone marrow blasts, clinical classification, median hemoglobin level and median platelet count (p > 0.05). The middle and strong positive expression of DNA-Pkcs in non-remission group was significantly higher than that in remission group (p < 0.05). The positive rate of DNA-PKcs in abnormal chromosome group was significantly higher than that in chromosome normal group (p < 0.05). It is concluded that the DNA-PKcs expression level is closely related with the increased WBC count, and the expression of DNA-PKcs is correlated also with karyotype and clinical prognosis in adult acute leukemia.

(E)-1-(2,4-Dihy-droxy-phen-yl)-3-(4-hydroxy-phen-yl)prop-2-en-1-one monohydrate.

In the title compound, C(15)H(12)O(4)·H(2)O, the two benzene rings are not coplanar, making a dihedral angle of 7.24 (16)°. An intra-molecular hy-droxy-carbonyl O-H⋯O hydrogen bond occurs. In the crystal, four inter-molecular O-H⋯O hydrogen bonds involving the hy-droxy residues, the carbonyl group and the water mol-ecule lead to the formation of a three-dimensional network. The supra-molecular structure is further stabilized by weak C-H⋯O inter-actions.

[Clinical significance of CRKL protein phosphorylation level in the treatment of chronic myeloid leukemia with imatinib].

OBJECTIVE: To investigate the adaptor protein CRKL phosphorylation level (p-CRKL) and its significance in chronic myeloid leukemia (CML) treated with imatinib. METHODS: ABL kinase domain was amplified by nested RT-PCR, domain point mutations analysis by direct sequencing, BCR-ABL mRNA level by real time-PCR, and p-CRKL level by flow cytometry in 52 bone marrow samples from 35 CML patients, and the relationship of p-CRKL level with ABL kinase domain mutation and with BCR-ABL mRNA level was analyzed. RESULTS: In the 15 imatinib-resistant patients, ABL domain point mutations were detected in 6 with 4 types of nucleotide substitutions: T315I (n = 3), Y253H (n = 1), E255K and F317L. The incidence of mutations in disease chronic phase (CP), accelerated phase (AP) and blast phase (BP) was 25.00%, 40.00% and 30.00%, respectively. The BCR-ABL mRNA level in newly diagnosed CML was higher than that in imatinib-responded patients (P = 0.01); and so did in imatinib-resistant patients than in imatinib-effective patients (P = 0.03). The level of BCR-ABL mRNA was not significantly different between newly diagnosed CML and imatinib-resistant patients. p-CRKL%, MFI showed a high degree of phosphorylation in newly diagnosed CML and imatinib-resistant patients (P = 5.130; P = 3.178). The level of p-CRKL % and MFI in newly diagnosed group was higher than that in imatinib responded group (P = 0.000; P = 0.01) and also higher in imatinib-effective group than in imatinib-resistant group (P = 0.000; P = 0.02). There was a positive correlation between the level of BCR-ABL expression and p-CRKL% (and the MFI of p-CRKL) (P < 0.05). CONCLUSION: It seems that p-CRKL detection might be helpful in predicting imatinib treatment outcomes.

[Effects of metallothionein on rifampicin (RFP)-induced hepatotoxicity in mice].

OBJECTIVE: To investigate the effect of metallothionein (MT) on rifampicin (RFP)-induced hepatotoxicity and the possible mechanisms. METHODS: Male MT- I / II null (MT-/-) and wild type (MT+/+) mice were randomly divided into 4 groups, respectively, and were orally administered RFP (50, 100 or 200 mg/kg) or equal volumes of solvent daily for 15 consecutive days. Levels of plasma alanine aminotransferase (ALT), alkaline phosphatase (ALP) and direct bilirubin (DB) were determined. Liver indexes were calculated and liver histopathologic changes were examined by hematoxylin and eosin (HE) staining. The content of glutathione (GSH) as well as the activities of glutathione peroxydase (GPx) and glutathione reductases (GR) were measured in the liver homogenates. RESULTS: RFP treatment induced significant increases in plasma ALT, AST and DB, as well as liver index. Significant histopathologic changes which were charactered as fatty degeneration in liver were noteced. Moreover, augmentations of GSH content and GR activity and attenuation of GPx activity were observed. More severe hepatic injuries in MT-/- mice were observed as compared to MT+/+ mice, which were evidenced by higher liver/body weight ratio and GR activity, lower GSH content and GPx activity, and more serious fatty degeneration. CONCLUSION: RFP-induced hepatotoxicity was associated with cholestasis and oxidative damage. MT -/- mice were more susceptible than MT +/+ mice to RFP-induced hepatotoxicity and the enhanced hepatotoxicity involves increased oxidative stress.

[Detection of ABL kinase domain point mutations in chronic myeloid leukemia patients receiving imatinib treatment].

OBJECTIVE: To analyze the frequency and clinical significance of ABL tyrosine kinase point mutations in chronic myeloid leukemia (CML) patients receiving imatinib treatment. METHODS: Nested reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on 40 bone marrow samples from 23 patients to amplify the ABL kinase domain, followed by direct sequencing and sequence homologous analysis. RESULTS: In the 23 patients analyzed, the ABL domain point mutations was detected in 7 patients who presented with 5 types of nucleotide changes, namely T315I(n=3), Y253H, E255K, F317L and G321W. The incidence of mutations in chronic phase (CP), accelerated phase (AP) and blast phase (BP) was 25.00%, 40.00% and 30.00%, respectively. For 6 of the 7 patients with mutations who were resistant to imatinib before sequencing, the daily drug dose had been increased to 600-800 mg daily for poor response to 400 mg/day imatinib. During the follow-up for 3-6 months, only the patient with F317L achieved major cytogenetic response (MCR), and the patient with Y253H and 1 of the 3 with T315I progressed to BP. The newly diagnosed patient with G321W IN cp achieved a complete hematologic remission and had a significant decrease of the proportion of BCR-ABL-positive cells. CONCLUSIONS: ABL kinase point mutation is an important mechanism of imatinib resistance. The type of mutations is associated with the level of resistance to imatinib, and detection of ABL kinase point mutations by direct sequencing may help estimate the prognosis and plan for therapeutic strategy adjustment.

[Analyzing genetic quality of BALB/c mouse strains in China by microsatellite marking].

Eleven BALB/c mouse strains of Beijing, Shanghai, Shenyang, Haerbin, Guangzhou, Chongqing and Changchun were monitored in order to assure the genetic quality of inbred BALB/c mouse strains in China and to estimate the credibility of microsatllite markers. Fourteen microsatellites loci on different chromosomes were investigated by PCR analysis. It showed that all these microsatellites DNA loci displayed single allelic gene band in mouse strains of Beijing, Shanghai and Haerbin. But the mice came from Shenyang, Guangzhou, Chongqing and Changchun had polymorphisms or heterozygosis, among which the Shenyang and Changchun strains showed polymorphisms and heterozygosis at two separate loci. Four loci showed polymorphisms or heterozygosis in one of the Guangzhou mouse strains. The Chongqing strains showed polymorphisms and heterozygosis at seven loci, including the D10Mit180 locus as compared with the Shanghai strains.