The reversal effect of Ginsenoside Rh2 on drug resistance in human colorectal carcinoma cells and its mechanism.

Recent studies hint that Ginsenoside is involved in cancer prevention and treatment. In this study, we investigated the effect of Ginsenoside Rh2 on drug resistance in human colorectal carcinoma (CRC) cells and its mechanism. The resistance reversion effect of Ginsenoside Rh2 in CRC cells was analyzed using CCK-8 assay. After treating with Ginsenoside Rh2, the cell cycle distribution and cellular apoptosis were analyzed by flow cytometry, cell migration was determined by transwell migration assay, the expression of drug-resistance genes and proteins were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Ginsenoside Rh2 could enhance the cytotoxicity of 5-FU in drug-resistant CRC cells (LoVo/5-FU and HCT-8/5-FU). Treatment with Ginsenoside Rh2 could result in an increase of cell numbers in G0/G1 phase accompanied with a decrease in S-phase, and induced cellular apoptosis in drug-resistant CRC cells. In addition, the migration process and EMT process of drug-resistant CRC cells were suppressed by treatment of Ginsenoside Rh2. Compared to control group, expression of drug-resistance genes, such as MRP1, MDR1, LRP and GST, were negatively correlated to Ginsenoside Rh2. All these results indicated that Ginsenoside Rh2 could effectively reverse drug resistance in human colorectal carcinoma cell and its mechanism involved the prevention of cellular proliferation and migration, the promotion of cellular apoptosis and the alteration of drug-resistance genes, which suggested that Ginsenoside Rh2 may act as a promising candidate for drug resistance in human colorectal carcinoma chemotherapy.

Interferon-γ suppresses the proliferation and migration of human placenta-derived mesenchmal stromal cells and enhances their ability to induce the generation of CD4+CXCR5+Foxp3+Treg subset.

We investigate the effects of interferon (IFN)-γ on human placenta-derived mesenchymal stromal cells (hPMSCs), in particular, their adhesion, proliferation and migration and modulatory effects on the CD4+CXCR5+Foxp3+Treg subset. And we compared hPMSCs ability to induce the generation of different Treg subsets in response to treatment with IFN-γ. We found that IFN-γ suppressed the proliferation and migration for hPMSCs. The ability of hPMSCs to induce the generation of CD4+CXCR5+Foxp3+Treg subset was enhanced by IFN-γ. And maximal effectiveness of IFN-γ treated hPMSCs upon inducing the generation of Treg subsets was for CD4+CXCR5+Foxp3+Treg subset as compared with that of CD4+CD25+Foxp3+, CD8+CD25+Foxp3+, CD4+IL-10+ and CD8+IL-10+Treg subsets. These results have important implications for the development and application of hPMSCs in clinical use.

[Role of Autophagy in Acute Lymphoblastic Leukemia -Review].

Autophagy is an evolutionarily highly conservative lysosomal degradative process and closely associates with pathogenesis, process, treatment, drug resistance and relapse of acute lymphoblastic leukemia (ALL). Whether the autophagy displays resistance to chemotherapy or a tumor suppressor in ALL, it mainly depends on the context of autophagy in the cells. Understanding the different role of autophagy in different conditions for ALL, determing the autophagy signaling pathways and targeting combination with autophagy revulsants or inhibitors were significant for the therapy of ALL, particularly for the treatment of refractory/relapsed ALL patients. This review summarizes the role of autophagy in pathogenesis, developmant, drug resistance and treatment of ALL, providing some theoretical guidance of new drugs targeting autophagy for ALL therapy.

[Effect of Magnolol on Proliferation and Apoptosis of HL-60 Cells and Its Molecular Mechanism].

OBJECTIVE: To investigate the effect of magnolol on proliferation and apoptosis of HL-60 cells and its mechanism. METHODS: MTT assay was used to measure the proliferation of HL-60 cells after treatment with different concentration of magnolol (5, 10, 20, 40, 80 and 160 µg/ml). The morphological changes of HL-60 cells were examined by light microscopy, and DAPI staining was performed to observe the nuclear morphology of HL-60 cells. The early cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI double-staining. RT-PCR was carried out to examine the mRNA expression of BAX and BCL-2. Western blot was performed to detect the protein expression of caspase family. RESULTS: The magnolol inhibited HL-60 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose- and time- dependent manner (P < 0.05). HL-60 cells became small, even apoptotic bodies appeared after treatment with magnolol. In addition, nuclear condensation or fragmentation could be observed, which is the typical morphological features of apoptosis. When HL-60 cells were treated with 40 µg/ml of magnolol for 24 h, the ratio of early apoptotic cells reached to (11.7 ± 2.4) %, which was significant different from control (1.4 ± 1.1) % (P < 0.05). RT-PCR results showed that treatment of HL-60 cells with magnolol up-regulated the expression of BAX, whereas down-regulated the expression of BCL-2. Western blot results showed that the cleavages of caspase-3, -8 and -9 were significantly enhanced by magnolol. CONCLUSION: The magnolol can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells, which may occur through up-regulation of BAX, down-regulation of BCL-2 and the activation of caspases.

The treatment of solvent recovery raffinate by aerobic granular sludge in a pilot-scale sequencing batch reactor.

Mature aerobic granular sludge (AGS) was inoculated for the start-up of a pilot-scale sequencing batch reactor for the treatment of high concentration solvent recovery raffinate (SRR). The proportion of simulated wastewater (SW) (w/w) in the influent gradually decreased to zero during the operation, while volume of SRR gradually increased from zero to 10.84 L. AGS was successfully domesticated after 48 days, which maintained its structure during the operation. The domesticated AGS was orange, irregular, smooth and compact. Sludge volume index (SVI), SV30/SV5, mixed liquor volatile suspended solids/mixed liquor suspended solids (MLVSS/MLSS), extracellular polymeric substances, proteins/polysaccharides, average particle size, granulation rate, specific oxygen utilization rates (SOUR)H and (SOUR)N of AGS were about 38 mL/g, 0.97, 0.52, 39.73 mg/g MLVSS, 1.17, 1.51 mm, 96.66%, 47.40 mg O2/h g volatile suspended solids (VSS) and 8.96 mg O2/h g VSS, respectively. Good removal effect was achieved by the reactor. Finally, the removal rates of chemical oxygen demand (COD), total inorganic nitrogen (TIN), NH4+-N and total phosphorus (TP) were more than 98%, 96%, 97% and 97%, respectively. The result indicated gradually increasing the proportion of real wastewater in influent was a useful domestication method, and the feasibility of AGS for treatment of high C/N ratio industrial wastewater.

MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation.

Enterovirus A71 (EV-A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV-A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV-A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV-A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV-A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV-A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID50 ) values calculated in viral proliferations (ranged from 10(7.6) to 10(7.8) ); the TCID50 being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV-A71Jinan1002 amplifies more efficiently in MA104, Hep-2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV-A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV-A71.

The stability of aerobic granular sludge treating municipal sludge deep dewatering filtrate in a bench scale sequencing batch reactor.

Inoculated with mature aerobic granular sludge in a sequencing batch reactor, gradually increasing the proportion of municipal sludge deep dewatering filtrate in influent, aerobic granular sludge was domesticated after 84 days and maintained its structure during the operation. The domesticated AGS was yellowish-brown, dense and irregular spherical shape, average size was 1.49 mm, water content and specific density were 98.13% and 1.0114, the SVI and settling velocity were 40 ml/g and 46.5m/h. After 38 days, NO3(-)-N accumulated obviously in the reactor as lack of carbon sources. When adding 1-3g solid CH3COONa at 4.5 and 5.5h of each cycle from the 57th day, the removal rate of TN rose to above 90% after 20 days, where effective COD removal and denitrification were realized in a single bioreactor. Finally, the removal rates of COD, TP, TN and NH4(+)-N were higher than 95%, 88%, 96% and 99%.

Rapid cultivation of aerobic granular sludge in a pilot scale sequencing batch reactor.

Aerobic granular sludge which had good performance to pollutants removal was successfully cultivated within 18 days in a pilot scale sequencing batch reactor, about 25% mature aerobic granular sludge was inoculated when the setting time of activated sludge was reduced to 10 min. Anaerobic biological selector was implemented to inhibit filamentous bacteria overgrowth, where the maximum COD could reach to 1703.74 mg/L. The cultivated aerobic granular sludge was irregular and pale yellow, average particle size, SVI, SV30/SV5, PN/PS, EPS and water content were 1.58 mm, 67.64 mL/g, 0.91, 2.17, 268.90 mg EPS/g MLVSS and 98.16% on the 18th day. Mechanism of rapid granulation mainly included crystal nucleus hypothesis and selection pressure hypothesis. The inoculated aerobic granules could maintain stable under short setting time environment, making it directly as the crystal nucleus and the carriers for new particles without obvious disintegration, which eventually shortened the granulation time greatly.

[Effect of modified zuoguiwan on Th17/Treg subpopulation of estrogen deficiency induced bone loss mice].

OBJECTIVE: To observe the effect of Modified Zuoguiwan (MZ) on the balance between helper T cell subsets 17 (Th17) and regulatory T cell subsets (Treg) in estrogen deficiency induced bone loss mice and to explore its mechanism. METHODS: Totally 50 BALB/c mice were divided into the sham-operation group, the ovariectomy model group, the low dose MZ group, the middle dose MZ group, and the high dose MZ group by random digit table, 10 in each group. Mice in the low, middle, and high dose MZ groups were respectively administered with MZ at the daily dose of 7.25, 14.50, and 29.00 g/kg by gastrogavage, 0.5 mL each time for 12 successive weeks. Meanwhile, mice in the sham-operation group and the ovariectomy model group were administered with equal volume by gastrogavage, 0.50 mL each time. The serum estradiol (E2) level was assessed by enzyme linked immunosorbent assay (ELISA). Bone mineral density (BMD) of thigh bone was measured with dual energy X ray absorptiometry. In addition, the population of Th17/Treg subsets in spleen mononuclear cells was analyzed by extracellular and intracellular staining method using flow cytometry. Moreover, the mRNA expression of IL-17A and TGF-ß in the spleen mononuclear cells was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared with the sham-operation group, both E2 and BMD significantly decreased, the percentage of Th17 subset and Th17/Treg ratio both increased, the percentage of Treg subset obviously decreased, the expression of IL-17A mRNA significantly increased, and the expression of TGF-ß mRNA significantly decreased in the ovariectomy model group (all P < 0.05). Compared with the model group, BMD obviously increased, the percentage of Th17 subset and Th17/Treg ratio both decreased, the percentage of Treg subset obviously increased, the expression of IL-17A mRNA significantly decreased, and the expression of TGF-ß mRNA significantly increased in the middle dose MZ group and the high dose MZ group (all P < 0. 05). Correlation analyses showed that BMD was positively related to both the serum E2 level and the percentage of Treg subset (P < 0.05), but negatively related to the percentage of Th17 subset (P < 0.05). In addition, the serum E2 level was positively related to the percentage of Treg subset, but obviously negatively related to that of Th17 subset (P < 0.05). CONCLUSIONS: There was correlation between Th17/Treg imbalance and E2 deficient bone loss. MZ could decrease the proportion of Th17 subset, but elevate the proportion of Treg subset in E2 deficient bone loss mice. It could achieve therapeutic effect through adjusting the balance of Th17/Treg in E2 deficient bone loss mice.

[Molecular mechanism of HL-60 cell apoptosis induced by baicalin].

This study was aimed to investigate the effect of baicalin on proliferation and apoptosis of HL-60 cells and its mechanism. Cell proliferation was assayed by using Cell Counting Kit-8. The morphological changes of HL-60 cells were examined by light microscopy and nucleolus morphological changes were observed by fluorescent microscopy after Hoechst 33342 staining. The early cell apoptosis was detected by using flow cytometry with Annexin V-FITC/PI double staining. The expression of caspase-3, caspase-9, Bcl-2 and Bax mRNA was detected by RT-PCR and Western blot assay was carried out to examine Bax, Bcl-2, caspase-8 and cleaved caspase-3 expression. The results showed that Baicalin inhibited the proliferation of HL-60 cells in a time- and concentration-dependent manner. HL-60 cells exhibited typical morphological features (for example, cell shrinkage, membrane blebbing and formation of apoptotic bodies). Cell apoptosis in early stage could be detected, the expression of caspase-3, caspase-9 and Bax mRNA was obviously up-regulated, while the Bcl-2 expression down-regulated, and accordingly Bcl-2/Bax ratio decreased. Such results were consistent with the expression of these proteins. In addition, the expression of cleaved caspase-8 protein was induced significantly after treated with baicalin. It is concluded that baicalin can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells, which may occur through decreasing Bcl-2/Bax ratio by intrinsic pathway and through extrinsic pathway. It suggests that baicalin may be a promising drug for the therapy of acute myeloid leukemia.

[Effect of EBV immediate-early protein Zta on the cell cycle of Daudi cells and its mechanisms].

OBJECTIVE: To investigate the effect of EBV immediate-early protein Zta on cell cycle of Daudi cells and the involved mechanisms. METHODS: The expression vector encoding Zta was constructed and electroporated into Daudi cells. Flow cytometric analysis was used to detect the cell cycle, Western blot to the protein levels of p21, Rb and E2F-1. RESULTS: The vector was constructed successfully, the expression of Zta protein inhibited the proliferation of Daudi cells and promoted cell cycle from G(0)/G(1) phase \[(30.0 ± 3.4)%\] to S phase \[(47.7 ± 1.1)%\]. Meanwhile, Rb expression was significantly downregulated, E2F-1 and p21 expression upregulated by Zta. CONCLUSION: Zta could promote G(0)/G(1) phase to S phase transition in Daudi cells, which might be associated with the reduced expression of Rb and increased expression of E2F-1 and p21 protein.

Anti-proliferation Effect of Polypeptide Extracted from Scorpion Venom on Human Prostate Cancer Cells in vitro.

BACKGROUND: Prostate cancer is a major cause of cancer-related death in men. Therefore there has been considerable interest to explore neoadjuvant therapy. Polypeptide extracted from scorpion venom (PESV), originally obtained from the East-Asian scorpion Buthus martensi Karsch (BmK), is being studied for both prevention and treatment of various human malignancies including prostate cancer. METHODS: The present study was to investigate the effect of PESV on cell proliferation, cell cycle, and apoptosis in human androgen-independent prostate cancer cells DU-145 in vitro. RESULTS: PESV treatment on these cells resulted in a significantly dose-dependent growth inhibition with a G1 phase arrest at 40µg/mL after 48h treatment. PESV treatment strongly induced expression of p27 (Kip1), but resulted in a decrease in cyclin E, one of cyclins involved in G1 progression. In other studies, PESV treatment also induced high apoptosis index (AI), confirmed by TdTmediated dUTP-biotin nick-end labeling (TUNEL) assay. Further, the apoptosis induction by PESV (40µg/mL) in DU145 cells was associated with an increase of pro-apoptotic protein Bax. CONCLUSIONS: These results suggest that PESV modulates the expression of cell cycle-related and apoptosis-related proteins and induces growth inhibition and apoptosis of DU145 cells, providing a strong rationale for future studies to evaluate prevention or/and intervention strategies for PESV in pre-clinical prostate cancer models. KEYWORDS: Prostate cancer, PESV, cell proliferation, cell cycle, apoptosis.

Relationship between cytokines and leukocytosis in patients with APL induced by all-trans retinoic acid or arsenic trioxide.

Leukocytosis or hyperleukocytosis occurs during ATRA or arsenic trioxide differentiation therapy, which is related to the RA syndrome. The number of WBC often increased by ten or more times as high as that of pretreatment, around 7 to 20 days after treatment with ATRA or arsenic trioxide. Usually, when number of WBC tended to peak, there was concomitance with down-regulation of promyelocytes, up-regulation of myelocytes and more mature neutrophils. The same trend of classification of BM was observed in most of the patients with APL to whom leukocytosis happened. Although the mechanism of leukocytosis has not been demonstrated clearly, so far the proliferation hypothesis by cytokines and rheological hypothesis by adhesion molecules were taken into consideration. Otherwise, hypothesis about more divisions of differentiated myelocytes induced by ATRA or arsenic trioxide remains unclear. Usually, this kind of leukocytosis or hyperleukocytosis itself requires no additional cytotoxic treatment.

Immune tolerance in pancreatic islet xenotransplantation.

AIM: To observe the effect of tail vein injection with donor hepatocytes and/or splenocytes on the islet xenotransplantation rejection. METHODS: New-born male pigs and BALB/C mice were selected as donors and recipients respectively. Islet xenotransplantation was performed in recipients just after the third time of tail vein injection with donor hepatocytes and/or splenocytes. Macrophage phagocytosis, NK(natural killing cell) killing activity, T lymphocyte transforming function of spleen cells, antibody forming function of B lymphocytes, and T lymphocyte subsets were taken to monitor transplantation rejection. The effects of this kind of transplantation were indicated as variation of blood glucose and survival days of recipients. RESULTS: The results showed that streptozotocin (STZ) could induce diabetes mellitus models of mice. The pre-injection of donor hepatocytes, splenocytes or their mixture by tail vein injection was effective in preventing donor islet transplantation from rejection, which was demonstrated by the above-mentioned immunological marks. Each group of transplantation could decrease blood glucose in recipients and increase survival days. Pre-injection of mixture of donor hepatocytes and splenocytes was more effective in preventing rejection as compared with that of donor hepatocyte or splenocyte pre-injection respectively. CONCLUSION: Pre-injection of donor hepatocytes, splenocytes or their mixture before donor islet transplantation is a good way in preventing rejection.