MicroRNA-34a targets Forkhead box j2 to modulate differentiation of endothelial progenitor cells in response to shear stress.

Flow shear stress plays important roles in modulating differentiation of endothelial progenitor cells (EPCs). MicroRNAs are crucial for diverse cellular processes, but the expressions and functions of microRNAs in EPCs responding to mechanical stimuli remain unclear. We sought to determine the effects of microRNA-34a (miR-34a) and a novel target Forkhead box j2 (Foxj2) on shear stress-induced EPC differentiation. Human umbilical cord blood-derived EPCs were exposed to laminar shear stress of 15dyn/cm(2) with parallel plate flow chamber system. Real time RT-PCR showed that shear stress significantly increased miR-34a expression, which was accompanied by the endothelial differentiation of EPCs. Whereas Foxj2, a putative target of miR-34a predicted by multiple algorithms, was suppressed in this process. Dual luciferase reporter assays, as well as miR-34a mimics and inhibitor treatment were used to confirm the interplay between miR-34a and Foxj2. Our results revealed an inverse correlation of miR-34a and Foxj2 expressions implicated in the endothelial differentiation of EPCs. MiR-34a contributed to this process by up-regulating the expressions of endothelial cell markers, and down-regulating smooth muscular cell markers. In addition, Foxj2 overexpression attenuated endothelial differentiation of EPCs, while Foxj2 siRNA had the opposite effect. These data suggested a unique mechanism that shear stress induces the expression of miR-34a, which targets to Foxj2 and promotes endothelial differentiation of EPCs. The results provide new insights into miR-34a/Foxj2 on shear stress-induced EPC differentiation.

Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer.

OBJECTIVE: The effects of arsenic trioxide (As2O3) on hepatocellular carcinoma have been documented widely. Autophagy plays dual roles in the survival and death of cancer cells. Therefore, we investigated the exact role of autophagy in As2O3-induced apoptosis in liver cancer cells. METHODS: The viability of hepatoma cells was determined using the MTT assay with or without fetal bovine serum. The rate of apoptosis in liver cancer cells treated with As2O3 was evaluated using flow cytometry, Hoechst 33258 staining, and TUNEL assays. The rate of autophagy among liver cancer cells treated with As2O3 was detected using immunofluorescence, Western blot assay and transmission electron microscopy. RESULTS: Upon treatment with As2O3, the viability of HepG2 and SMMC-7721 cells was decreased in a time- and dose-dependent manner. The apoptosis rates of both liver cancer cell lines increased with the concentration of As2O3, as shown by flow cytometry. Apoptosis in liver cancer cells treated with As2O3 was also shown by the activation of the caspase cascade and the regulation of Bcl-2/Bax expression. Furthermore, As2O3 treatment induced autophagy in liver cancer cells; this finding was supported by Western blot, immunofluorescence of LC3-II and beclin 1, and transmission electron microscopy. In liver cancer cells, As2O3 inhibited the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway that plays a vital role in both apoptosis and autophagy. The PI3K activator SC-79 partially reversed As2O3-induced autophagy and apoptosis. Furthermore, inhibiting autophagy with 3-methyladenine partially reversed the negative effects of As2O3 on cell viability. Serum starvation increased autophagy and amplified the effect of As2O3 on cell death. CONCLUSION: As2O3 induces apoptosis and autophagy in liver cancer cells. Autophagy induced by As2O3 may have a proapoptotic effect that helps to reduce the viability of liver cancer cells. This study provides novel insights into the effects of As2O3 against liver cancer. Please cite this article as: Deng ZT, Liang SF, Huang GK, Wang YQ, Tu XY, Zhang YN, Li S, Liu T, Cheng BB. Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer. J Integr Med. 2024; 22(3): 295-302.

SIRT1 and Connexin40 Mediate the normal shear stress-induced inhibition of the proliferation of endothelial cells co-cultured with vascular smooth muscle cells.

BACKGROUND: Shear stress imposed by blood flow directly impacts endothelial cells (ECs), which are simultaneously influenced by neighboring vascular smooth muscle cells (VSMCs). However, the mechanisms by which shear stress and VSMCs modulate EC proliferation remain to be elucidated. METHODS: ECs, cultured alone or co-cultured with VSMCs, were subjected to a normal level of laminar shear stress (NSS) of 15 dyne/cm(2) or kept under static conditions by using a parallel-plate flow chamber system, respectively. RESULTS: BrdU incorporation assay and flow cytometry revealed that NSS inhibited EC proliferation with or without VSMCs. Western blot analysis demonstrated that NSS down-regulated the expression of Connexin40 (Cx40) in both ECs cultured alone and ECs co-cultured with VSMCs, accompanied by the increased expression of SIRT1. Moreover, salermide, an inhibitor of SIRT1, as well as SIRT1-specifc siRNA transfection inhibited the effect of NSS on EC proliferation and Cx40 expression. In contrast, resveratrol, a SIRT1 activator, induced an alteration in ECs similar to the application of NSS. CONCLUSION: NSS inhibits the proliferation of ECs via SIRT1 and Cx40 in the presence or absence of VSMCs. The data suggest that NSS plays a protective role in vascular homeostasis by maintaining EC proliferation at a normal level.

Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/ß-catenin pathway under hypoxia.

OBJECTIVE: Jiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment. METHODS: The chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/ß-catenin pathway. RESULTS: According to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/ß-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/ß-catenin pathway. CONCLUSION: JR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/ß-catenin pathway under hypoxic conditions. Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/ß-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474-486.

Association of SIRT1 expression with shear stress induced endothelial progenitor cell differentiation.

Shear stress imposed by blood flow is crucial for differentiation of endothelial progenitor cells (EPCs). Histone deacetylase SIRT1 has been shown to play a pivotal role in many physiological processes. However, association of SIRT1 expression with shear stress-induced EPC differentiation remains to be elucidated. The present study was designed to determine the effect of SIRT1 on EPC differentiation induced by shear stress, and to seek the underlying mechanisms. Human umbilical cord blood-derived EPCs were exposed to laminar shear stress of 15 dyn/cm(2) by parallel plate flow chamber system. Shear stress enhanced EPC differentiation toward endothelial cells (ECs) while inhibited to smooth muscle cells (SMCs). The expressions of phospho-Akt, SIRT1 and histone H3 acetylation (Ac-H3) in EPCs were detected after exposure to shear stress for 2, 6, 12, and 24 h, respectively. Shear stress significantly activated Akt phosphorylation, augmented SIRT1 expression and downregulated Ac-H3. SIRT1 siRNA in EPCs diminished the expression of EC markers, but increased the expression of SMC markers, and resulted in upregulation of Ac-H3. Whereas, resveratrol, an activator of SIRT1, had the opposite effects on both EPC differentiation and histone H3 acetylation. Wortmannin, an inhibitor of PI3-kinase, suppressed endothelial differentiation of EPCs, decreased SIRT1, and upregulated Ac-H3 expression. In addition, SIRT1 promoted tube formation of EPCs in matrix gels. These results provided a mechanobiological basis of shear stress-induced EPC differentiation into ECs and suggest that PI3k/Akt-SIRT1-Ac-H3 pathway is crucial in such a process.

[Effects of different assemblages with components of Shengmai Powder on glucocorticoid receptor in liver of thermal injured rat].

OBJECTIVE: To study the effects of different assemblages formed by components of Shengmai Powder (SMP) on glucocorticoid receptor (GR) in liver of thermal injured rat to find the optimal ratio of assembling for GR regulation. METHODS: With a orthogonal design adopted, the dosage of each component of SMP, including the total saponins of Ginseng (G), the water extract of lilyturf root (L), and the water extract of schisandra fruit (S), was ranked in three levels, namely, no participating, low dosage (G 7.1 mg, L 17.2 mg, S 9.6 mg), high dosage (G 14.2 mg, L 34.4 mg, S 19.2 mg). The components were assigned by L9(3(4)) orthogonal table and grouped, the best assembling ratio was determined through direct and variance analysis. RESULTS: After being acted by the different assemblages, the maximum binding volume of GR in rat's liver cell suspension was 161 +/- 26 fmol/mg protein in group 1, 271 +/- 40 fmol/mg protein in group 2, 166 66 fmol/mg protein in group 3, 222 +/- 45 fmol/mg protein in group 4, 192 +/- 26 fmol/mg protein in group 5, 194 +/- 23 fmol/mg protein in group 6, 166 +/- 15 fmol/mg protein in group 7, 165 +/- 47 fmol/mg protein in group 8 and 211 +/- 79 fmol/mg protein in group 9. The optimal GR level appeared during the dosage of G, L and S was 7.1 mg, 17.2 mg and 19.2 mg, respectively. CONCLUSION: The best assembling ratio of SMP for regulating GR in the liver of thermal injured rat was G:L:S = 3:3:4.

Comparable effects of Jiedu Granule, a compound Chinese herbal medicine, and sorafenib for advanced hepatocellular carcinoma: A prospective multicenter cohort study.

OBJECTIVE: Sorafenib has been extensively used for the treatment of advanced hepatocellular carcinoma (HCC), and Chinese herbal medicine has also been used to manage advanced HCC. The present work evaluates the effectiveness and safety of Jiedu (JD) Granule, a compound of traditional Chinese herbal medicine, side-by-side with sorafenib for the treatment of advance HCC. METHODS: Patients with advanced HCC receiving treatment with JD Granule or sorafenib were enrolled from December 2014 to March 2018. The primary endpoint was overall survival (OS). The secondary endpoints were progression-free survival (PFS) and safety. Propensity score matching (PSM) analysis was used to control for possible selection bias from the study group allocation process. RESULTS: Of the 325 patients included, 161 received JD Granule and 164 received sorafenib. No significant differences were found in OS or PFS among patients receiving JD Granule compared to sorafenib (P > 0.05). Median OS of the two study groups was 6.83 months (95% confidence interval [CI]: 5.83-9.47) in the group receiving JD Granule and 8 months (95% CI: 6.67-9.80) in the group receiving sorafenib, with half-, 1- and 2-year survival rates of 53.6%, 31.2% and 13.2% vs 60.1%, 35.5% and 14.2%, respectively. Even after PSM, the median survival time did not differ between the JD Granule group (9.03 months; 95% CI: 6.37-14.2) and the sorafenib group (7.93 months; 95% CI: 6.5-9.97), with comparable half-, 1- and 2-year survival rates. The most common adverse events (AEs) were diarrhea (13.7%) and fatigue (5.6%) in the JD Granule group, and hand-foot skin reaction (46.3%) and diarrhea (36.6%) in the sorafenib group. The JD Granule was more cost-effective than sorafenib treatment for advanced HCC. CONCLUSION: Compared to sorafenib, JD Granule was more cost-effective and caused fewer AEs for the treatment of Chinese patients with advanced HCC.

[Effects of a formula of components from Shengmai Powder on glucocorticoid receptor in rats after thermal injury].

OBJECTIVE: To investigate the effects of a formula of components from Shengmai Powder, a compound traditional Chinese herbal medicine, on glucocorticoid receptor (GR) in rats after thermal injury. METHODS: A total of 32 male SD rats were randomly assigned into normal control group, untreated group, ginsenosides group and components group, with 8 rats in each group. Rats in the normal control group were intragastrically administered with normal saline (NS) at room temperature once daily. Rats in the untreated group were treated with NS before thermal injury, and rats in the components group and ginsenosides group were once daily treated with a mixture of aqueous extracts of Ophiopogonis Japoni, Fructus schizandrae Chinensis and ginsenosides and ginsenosides respectively. Rats were administered for one week. After the last administration, rats in the untreated group and treated groups underwent thermal injury for one hour, and then were sacrificed immediately by decapitation. Blood serum was collected, and the serum corticosterone (CS) and adrenocorticotropic hormone (ACTH) levels were determined with enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) respectively. The liver, lung and kidney homogenates were used to determine the GR binding capacity by radioligand receptor binding assay. The results were analyzed by one point analysis. RESULTS: GR binding capacities in liver, lung and kidney cytosols in the untreated group were obviously lower than those in the normal control group (P<0.01). The GR binding capacities in the liver and lung cytosols in the components group and the ginsenosides group were significantly higher than those in the untreated group (P<0.01), but no significant difference was found in kidney cytosol. Compared with the ginsenosides group, GR binding capacity in liver cytosol in the components group was increased (P<0.01), but there were no noticeable differences when compared with the GR binding capacities in lung and kidney cytosols. Serum CS and ACTH levels of the normal rats were (66+/-16)microg/L and (59+/-18) ng/L respectively. There were significant differences in CS and ACTH levels between the normal control group and the other groups (P<0.01), in which the serum CS and ACTH levels were (113+/-33)microg/L and (125+/-20) ng/L, (123+/-26) microg/L and (110+/-30) ng/L and (118+/-17) microg/L and (115+/-35) ng/L respectively. But there was no significant difference between the untreated group and the other treated groups. CONCLUSION: The formula of components from Shengmai Powder can enhance the effect of ginsenosides in up-regulating GR in rats after thermal injury.

Mycoplasma contamination-mediated attenuation of plasmid DNA transfection efficiency is augmented via L-arginine deprivation in HEK-293 cells.

Mycoplasma infection is the most prevalent contamination in cell culture. Analysis of cell culture in laboratories from different countries shows that mycoplasma contamination ranges from 15% to 80% and, in some cases, even reaches 100% (Chernov et al., 2014). Whilst mycoplasma infection is not visible to the naked eye in cell culture, the consequences of mycoplasma contamination have been shown to induce a number of cellular changes, for example, increased resistance to chemotherapeutic drugs. Therefore, any results obtained from tissue culture studies, in the presence of mycoplasma contamination, potentially render the data invalid (Kim et al., 2015; Gedye et al., 2016). As such, mycoplasmas are not harmless bystanders and cannot be ignored in in vitro studies.

Anxiolytic and anti-depressant effects of hydroalcoholic extract from Erythrina variegata and its possible mechanism of action.

BACKGROUND: Erythrina variegata has been widely used as a traditional medicine. OBJECTIVE: The study was designed to evaluate the anxiolytic and anti-depressant effects of an extract from Erythrina variegata. METHODS: The extract was evaluated for anxiolytic and anti-depressant action using the elevated plus maze, light/dark box, open field, forced swimming and tail suspension tests in mice. The mechanism of action was further elucidated using high-performance liquid chromatography with fluorescence detection methods to assay the levels of five neurotransmitters in brain. RESULTS: The extract exhibited significant increase in the percentage of the open arms entries and the time spent in the open arms in the elevated plus maze test. The results of the light/dark box test revealed a significant increase in the amount of time spent in the light chamber. Extract- treated mice also produced significant increase in the number of crossings and rearings in the open field test. In the forced swimming and tail suspension tests, the extract was able to promote significant decrease in the immobility time. In addition, the extract significantly altered the levels of five neurotransmitters in the brain tissue. CONCLUSION: These findings suggest that Erythrina variegata presents potential anxiolytic and anti-depressant activity, and the mechanism may be related to the alteration of neurotransmitter levels.

Efficacy of complementary and integrative medicine on health-related quality of life in cancer patients: a systematic review and meta-analysis.

Complementary and integrative medicine (CIM) has been used for improving health-related quality of life (HRQOL) in patients with cancer. The objective of this review is to evaluate the effects of CIMs on the HRQOL of cancer patients. We identified randomized controlled trials (RCTs) involving patients with cancer at any stage by retrieving electronic databases from the inception to February 14, 2018 (Systematic Review Registration: PROSPERO CRD42018091609). The main outcomes were HRQOL scores and related domains such as physical well-being scores. The standardized mean difference was used for the analysis and heterogeneity was assessed with the I 2 statistic. A Bayesian framework was used to estimate the ranking order of efficacy in HRQOL change. Finally, 34 RCTs with 3,010 patients were included. As a whole, the results showed clearly superior efficacy of CIM in improving HRQOL. For different domains of HRQOL, different CIM interventions may play different roles. The ranking order of efficacy in change HRQOL was qigong plus mindfulness, Chinese herbal medicine, multimodal complementary medicine, qigong, nutritional supplement, mindfulness, acupuncture, yoga, and massage, and it was different among different domains. There was no evidence of publication bias. In conclusion, CIM may improve the HRQOL of cancer patients. More studies, especially focusing on male cancer patients, are needed to increase the confidence level of our findings.

[Regulatory effect of ginsenoside on glucocorticoid receptor in mice with ischemic liver damage].

OBJECTIVE: To study whether ginsenoside (GS) can regulate the glucocorticoid receptor (GR) in mice with ischemic liver damage, and to preliminarily observe its dose-effect relationship for providing an experimental bases in seeking a new way to relieve the damage from view of GR. METHODS: Adult male SD mouse was used to establish liver ischemia model, and different doses (100, 50, and 25 mg/kg) of GS was given via gastric infusion before modeling. The maximal GR binding capacity (Bmax) of liver and the level of GR mRNA expression in liver were dynamically determined at various time points (2 h, 6 h, 12 h and 24 h) after modeling. RESULTS: Compared with the normal control group, GR Bmax and GR mRNA expression in model rats were lower obviously (P < 0.01). As compared with the control group, GR Bmax and GR mRNA expression in model rats treated with 50 mg/kg GS significantly raised at 2 h, 6 h, 12 h (P < 0.01), while the changes in modeling rats treated with other two doses of GS were of no statistical significance. CONCLUSION: GS in dose of 50 mg/kg can elevate the GR Bmax of liver and the level of GR mRNA expression in liver of rats with ischemic damage.

Prognostic Role of Platelet-to-Lymphocyte Ratio in Hepatocellular Carcinoma with Different BCLC Stages: A Systematic Review and Meta-Analysis.

The role of platelet-to-lymphocyte ratio (PLR) in the prognosis of hepatocellular carcinoma (HCC) patients with different Barcelona Clinic Liver Cancer (BCLC) stages remains controversial. This systematic review and meta-analysis aimed to determine the efficacy of PLR on HCC prognosis. Five electronic databases were searched for clinical trials focusing on the role of PLR in the prognosis of HCC. A total of 297 potential studies were initially identified, and 9 studies comprising 2449 patients were finally enrolled to evaluate the association between the pretreatment PLR and clinical outcomes of overall survival (OS), disease-free survival (DFS), and event occurrence in patients with HCC in different BCLC stages. An elevated pretreatment PLR indicated unfavorable worse OS (HR = 1.73; 95% CI: (1.46, 2.04); P < 0.00001) and DFS (HR = 1.30; 95% CI: (1.06, 1.60); P = 0.01). Subgroup analysis indicated that high PLR indicated poor OS among BCLC-B/C patients without heterogeneity, while PLR in BCLC-A patients indicated high statistical heterogeneity with I2 value of 78%. As for the correlation between PLR and event occurrence, high PLR was related to poor clinical event occurrence only among BCLC-C patients, though obvious heterogeneity was observed in all different BCLC stages. In conclusion, PLR may be a significant biomarker in the prognosis of HCC in different BCLC stages.

Progress in research on the effects of traditional Chinese medicine on the tumor microenvironment.

Tumor microenvironment (TME) has received more and more attention as modern medical research has begun to understand its importance in tumorigenesis. The occurrence, development, metastasis and drug resistance of tumors are closely related to TME. TME is a complicated system, including nontumor cells, their secreted cytokines, extracellular matrix, among other components. The concepts of wholism and multitarget regulation in traditional Chinese medicine (TCM) make it well suited to the regulation of TME. In this paper, the authors reviewed the progress of TME research and the effect of TCM on TME, providing some views of Chinese medicine in antitumor research.

Design, Synthesis and Evaluation of the Antidepressant and Anticonvulsant Activities of Triazole-Containing Benzo[d]oxazoles.

BACKGROUND: Epilepsy and depression are two of the common diseases seriously threatening life and health of human. A shared neurobiological substrate led to the bidirectional relationship and high comorbid occurrence of the two disorders. Recently, an increasing number of patients with epilepsy (PWE) require some form of antidepressant medication. However, most of the available antidepressants are inadequate for PWE for some reasons. So, the search for novel and increasingly effective drugs with anticonvulsant and antidepressant activities is necessary. METHODS: A series of 2-substituted-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazoles (5a-p) were designed and synthesized. Their anticonvulsant activities were evaluated using maximal electroshock shock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models in mice. Their antidepressant activities were screened with the forced swimming test (FST). RESULTS: All the compounds showed anti-MES activities in different degree, among which 5g and 5j were the most promising one with ED50 value of 31.7 and 12.7 mg/kg, respectively. What's more, 5g and 5j also exhibited nice anti-scPTZ activities and low neurotoxicity. Interestingly, these compounds also showed good antidepressant activities in FST. And the efficacy of 5g were also confirmed by a tail suspension test and a open field test. The pretreatment of thiosemicarbazide (an inhibitor of γ- aminobutyric acid synthesis enzyme) significantly increased the ED50 of 5g in MES and reversed the reductions in the immobility time of 5g in FST. CONCLUSION: Triazole-containing benzo[d]oxazole is a good skeleton to develop compounds with both anticonvulsant and antidepressant activities. We have got the compound 5g, which display remarkable antidepressant and anticonvulsant activities, and the GABAergic system was involved in the action mechanism of 5g.

[Establishment of a multidrug-resistant cell line BEL-7402/5-FU of human hepatocellular carcinoma and its biological characteristics].

OBJECTIVE: To establish a multidrug-resistant cell line BEL-7402/5-FU of hepatocellular carcinoma (HCC). METHODS: BEL-7402/5-FU was induced by pulse therapy combined with continuous stepwise exposure to 5-fluorouracil in vitro. MTT assay was used to determine its multidrug resistance (MDR). Biological characteristics of the BEL-7402/5-FU cell line were observed including morphological changes, cell growth curve, population doubling time, plate cloning efficiency, adherence rate, cell cycle distribution, chromosome and tumorigenicity. Accumulation amount of adriamycin (ADM) in cytoplasm was measured by flow cytometry. The protein expression of thymidylate synthase (TS) was evaluated by immuno-cytochemical method. RESULTS: The acquired MDR cell line of BEL-7402/5-FU was established successfully. The BEL-7402/5-FU cells showed cross-resistance to ADM, vincristine (VCR), methotrexate (MTX) and oxaliplatin (OHP), whereas still sensitive to hydroxycamptothecin (HCPT). The BEL-7402/5-FU cells tended to grow in clusters in vitro. It was found that the population doubling time of BEL-7402/5-FU cells was longer than that of its parental cells. The plate cloning efficiency and the adherence rate of BEL-7402/5-FU cells at the 2nd and 3rd hour were both lower than those of the parental cells. The distributing proportion of BEL-7402/5-FU cells in G(0)/G(1) phase was less than that of the parental cells, whereas the distributing proportion of BEL-7402/5-FU cells in S phase was higher than that of the parental cells. The accumulation amount of ADM in cytoplasm of BEL-7402/5-FU cells was significantly lower while the expression level of TS protein of which was highly up-regulated as compared with those of the parental cells. CONCLUSION: Establishment of the human HCC cell line BEL-7402/5-FU might be beneficial to the studies of 5-Fluorouracil acquired MDR mechanisms and the selection of reversal modifiers.

[A cecum hookworm patient with main symptom of cough].

This paper reports a cecal hookworm patient whose main symptoms are dry cough, dyspnea and eosinophilia.

Reversal effect of bufalin on multidrug resistance in human hepatocellular carcinoma BEL-7402/5-FU cells.

Multidrug resistance (MDR) is a major obstacle to chemotherapy in patients with hepatocellular carcinoma (HCC). To overcome MDR and improve chemotherapeutic efficacy, novel reversal agents with higher efficacy and lower toxicity are urgently needed for HCC. The present study was designed to examine the potential reversal activity of bufalin, a toxic ligand isolated from the traditional Chinese medicine 'Chansu' and to elucidate the possible related mechanisms. A multidrug-resistant HCC cell line, BEL-7402/5-FU, was used as the cell model. The working concentration of bufalin as an effective reversal agent, and the cell viability in the reversal experiments were determined by MTT assay. The effects of bufalin at a non-cytotoxic dose on cell cycle distribution, apoptosis and drug efflux pump activity were measured by flow cytometry. Qualitative observation of apoptosis was also carried out by confocal microscopy. Furthermore, the effects of bufalin on the expression of potential genes involved in MDR of BEL-7402/5-FU cells, including thymidylate synthase (TS), P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), B-cell lymphoma-extra large (Bcl-xL) and Bcl-2-associated X protein (Bax), were determined using real-time PCR and western blot analysis. The results showed that bufalin at a concentration of 1 nM enhanced the chemosensitivity of BEL-7402/5-FU cells to 5-FU with a reversal fold of 3.8 which was similar to that of 1 µM verapamil. Bufalin significantly arrested the cell cycle at the G0/G1 phase, induced apoptosis through an increase in the Bax/Bcl-xL ratio, inhibited drug efflux pump activity via downregulation of MRP1, and reduced the expression of TS in BEL-7402/5-FU cells. The present study revealed that bufalin effectively reversed MDR in BEL-7402/5-FU cells through multiple pathways. The combination of bufalin with cytotoxic drugs may serve as a promising strategy for the chemotherapy of HCC.

Characterization and resistance mechanisms of a 5-fluorouracil- resistant hepatocellular carcinoma cell line.

PURPOSE: The chemoresistance of human hepatocellular carcinoma (HCC) to cytotoxic drugs, especially intrinsic or acquired multidrug resistance (MDR), still remains a major challenge in the management of HCC. In the present study, possible mechanisms involved in MDR of HCC were identified using a 5-fluorouracil (5-FU) -resistant human HCC cell line. METHODS: BEL-7402/5-FU cells were established through continuous culturing parental BEL-7402 cells, imitating the pattern of chemotherapy clinically. Growth curves and chemosensitivity to cytotoxic drugs were determined by MTT assay. Doubling times, colony formation and adherence rates were calculated after cell counting. Morphological alteration, karyotype morphology, and untrastructure were assessed under optical and electron microscopes. The distribution in the cell cycle and drug efflux pump activity were measured by flow cytometry. Furthermore, expression of potential genes involved in MDR of BEL-7402/5-FU cells were detected by immunocytochemistry. RESULTS: Compared to its parental cells, BEL-7402/5-FU cells had a prolonged doubling time, a lower mitotic index, colony efficiency and adhesive ability, and a decreased drug efflux pump activity. The resistant cells tended to grow in clusters and apparent changes of ultrastructures occurred. BEL-7402/5-FU cells presented with an increased proportion in S and G2/M phases with a concomitant decrease in G0/G1 phase. The MDR phenotype of BEL-7402/5-FU might be partly attributed to increased drug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase (TS), resistance to apoptosis by augmentation of the Bcl-xl/Bax ratio, and intracellular adhesion medicated by E-cadherin (E-cad). P-glycoprotein (P-gp) might play a limited role in the MDR of BEL-7402/5-FU. CONCLUSION: Increased activity or expression of MRP1, Bcl-xl, TS, and E-cad appear to be involved in the MDR mechanism of BEL-7402/5-FU.