Saikosaponin-D enhances radiosensitivity of hepatoma cells under hypoxic conditions by inhibiting hypoxia-inducible factor-1α.

BACKGROUND: Our previous study revealed that the combination of Saikosaponin-d ( SSd) and radiation is more effective in the treatment of liver cancer than the application of either of these monotherapeutic methods. However, the molecular mechanisms of the radiosensitizing effect of SSd on liver cancer remained ill defined. METHODS: Cells were treated with different interventions; afterward, cell viability, apoptosis, and cell survival of SMMC-7721 and HepG2 hepatoma cells were examined. Xenograft tumor models were established by subcutaneously injecting SMMC-7721 cells. The molecular mechanism was assessed by western blot. RESULTS: SSd dose-dependently increased radiosensitivity of hepatoma cells under hypoxic condition. The growth inhibitory effect of the combined treatment was correlated with cell apoptosis. Further mechanistic analysis indicated that SSd induced the upregulation of p53 and Bax as well as the downregulation of Bcl-2 by attenuating HIF-1α expression under hypoxic condition. These effects were enhanced when the HIF-1α inhibitor PX-478 was introduced. In vivo data also presented a more significant suppression of tumor xenograft growth from the combined therapy than from either of the monotherapeutic methods. CONCLUSIONS: Our study provides evidence for a radiosensitizing effect of SSd on hepatoma cells under hypoxic conditions by inhibiting HIF-1α expression. Thus, SSd can be used as a potential sensitizer in hepatoma radiotherapy. © 2014 S. Karger AG, Basel.

Saikosaponin-d increases the radiosensitivity of smmc-7721 hepatocellular carcinoma cells by adjusting the g0/g1 and g2/m checkpoints of the cell cycle.

BACKGROUND: Saikosaponin-d (SSd), a monomer terpenoid purified from the Chinese herbal drug Radix bupleuri, has multiple effects, including anticancer properties. However, the effect of SSd on tumors exposed to radiation is largely unknown. To investigate the radiosensitizing effect of SSd and its possible mechanism, we combined SSd with radiation therapy to treat SMMC-7721 hepatocellular carcinoma cells under oxia and hypoxia. METHODS: Cell growth, apoptosis, and cell cycle distribution were examined after treatment with SSd alone, radiation alone, and their combinations under oxia and hypoxia. The protein and mRNA levels of p53, Bcl2, and BAX were measured using western blot analysis and RT-PCR, respectively. RESULTS: Treatment with SSd alone and radiation alone inhibited cell growth and increased apoptosis rate at the concentration used. These effects were enhanced when SSd was combined with radiation. Moreover, SSd potentiated the effects of radiation to induce G0/G1 arrest in SMMC-7721 cells, and reduced the G2/M-phase population under hypoxia. However, under oxia, SSd only potentiated the effects of radiation to induce G0/G1 arrest, but not G2/M-phase arrest. These effects of SSd alone, radiation alone, and their combination, were accompanied by upregulated expression of p53 and BAX and downregulation of Bcl2 expression under oxia and hypoxia. CONCLUSION: SSd potentiates the effects of radiation on SMMC-7721 cells; thus, it is a promising radiosensitizer. The radiosensitizing effect of SSd may contribute to its effect on the G0/G1 and G2/M checkpoints of the cell cycle.

Increased expression of chondroitin sulphate proteoglycans in rat hepatocellular carcinoma tissues.

AIM: To investigate the expression of chondroitin sulphate proteoglycans (CSPGs) in rat liver tissues of hepatocellular carcinoma (HCC). METHODS: Thirty male Sprague Dawley rats were randomly divided into two groups: control group (n = 10) and HCC model group (n = 20). Rats in the HCC model groups were intragastrically administrated with 0.2% (w/v) N-diethylnitrosamine (DEN) every 5 d for 16 wk, whereas 0.9% (w/v) normal saline was administered to rats in the control group. After 16 wk from the initiation of experiment, all rats were killed and livers were collected and fixed in 4% (w/v) paraformaldehyde. All tissues were embedded in paraffin and sectioned. Histological staining (hematoxylin and eosin and Toluidine blue) was performed to demonstrate the onset of HCC and the content of sulphated glycosaminoglycan (sGAG). Immunohistochemical staining was performed to investigate the expression of chondroitin sulphate (CS)/dermatan sulphate (DS)-GAG, heparan sulphate (HS)-GAG, keratan sulphate (KS)-GAG in liver tissues. Furthermore, expression and distribution of CSPG family members, including aggrecan, versican, biglycan and decorin in liver tissues, were also immunohistochemically determined. RESULTS: After 16 wk administration of DEN, malignant nodules were observed on the surface of livers from the HCC model group, and their hepatic lobule structures appeared largely disrupted under microscope. Toluidine blue staining demonstrated that there was an significant increase in sGAG content in HCC tissues when compared with that in the normal liver tissues from the control group [0.37 ± 0.05 integrated optical density per stained area (IOD/area) and 0.21 ± 0.01 IOD/area, P < 0.05]. Immunohistochemical studies demonstrated that this increased sGAG in HCC tissues was induced by an elevated expression of CS/DS (0.28 ± 0.02 IOD/area and 0.18 ± 0.02 IOD/area, P < 0.05) and HS (0.30 ± 0.03 IOD/area and 0.17 ± 0.02 IOD/area, P < 0.01) but not KS GAGs in HCC tissues. Further studies thereby were performed to investigate the expression and distribution of several CSPG components in HCC tissues, including aggrecan, versican, biglycan and decorin. Interestingly, there was a distinct distribution pattern for these CSPG components between HCC tissues and the normal tissues. Positive staining of aggrecan, biglycan and decorin was localized in hepatic membrane and/or pericellular matrix in normal liver tissues; however, their expression was mainly observed in the cytoplasm, cell membranes in hepatoma cells and/or pericellular matrix within HCC tissues. Semi-quantitative analysis indicated that there was a higher level of expression of aggrecan (0.43 ± 0.01 and 0.35 ± 0.03, P < 0.05), biglycan (0.32 ± 0.01 and 0.25 ± 0.01, P < 0.001) and decorin (0.29 ± 0.01 and 0.26 ± 0.01, P < 0.05) in HCC tissues compared with that in the normal liver tissues. Very weak versican positive staining was observed in hepatocytes near central vein in normal liver tissues; however there was an intensive versican distribution in fibrosis septa between the hepatoma nodules. Semi-quantitative analysis indicated that the positive rate of versican in hepatoma tissues from the HCC model group was much higher than that in the control group (33.61% and 21.28%, P < 0.05). There was no positive staining in lumican and keratocan, two major KSPGs, in either normal or HCC liver tissues. CONCLUSION: CSPGs play important roles in the onset and progression of HCC, and may provide potential therapeutic targets and clinical biomarkers for this prevalent tumor in humans.

Inhibitory effect of emodin and Astragalus polysaccharide on the replication of HBV.

AIM: To evaluate the anti-viral effect of emodin plus Astragalus polysaccharide (APS) in hepatitis B virus (HBV) transgenic mice. METHODS: Sixty HBV transgenic mice (HBV TGM) whose weight varied between 18 and 24 g were randomly divided into 3 groups, with 20 mice in each group. Group A was the normal control, where the mice were treated with physiological saline; group B was the positive control where the mice were treated with lamivudine solution (100 mL/kg per day). Group C was the experimental group where the mice were treated with physiological saline containing emodin and APS (57.59 mg/kg per day and 287.95 mg/kg per day, respectively). The mice were treated daily for 3 wk. After 1 wk recovery time, the mice were sacrificed and serum as well as liver tissues were collected for ELISA and histological examination. RESULTS: After 21 d treatment, HBV DNA levels in group B and group C significantly declined when compared with group A (P < 0.05). However, a significant increase in HBV DNA content was observed in group B, whereas this phenomenon was not observed in group C. A reduction in the contents of HBsAg, HBeAg and HBcAg in the mice from group B and C was observed when compared with group A. CONCLUSION: Emodin and APS have a weak but persistent inhibitory effect on HBV replication in vivo, which may function as a supplementary modality in the treatment of hepatitis B infection.

[Angiogenesis inhibitory effect of saikosaponin-d on chicken embryo].

OBJECTIVE: To investigate the inhibitory effects of saikosaponin-d (SSd) on angiogenesis in chicken embryos and its mechanism of action. METHODS: Chorioallantoic membrane (CAM) model was established successfully in 86 chicken embryos. They were divided into 4 groups after fenestration: the three SSd treated groups (A, B and C) treated with high (20 microg/mL, n = 16), middle (10 microg/mL, n = 19) and low (5 microg/mL, n = 25) dose of SSd respectively, and the control group treated with 0.01 mol/L PBS (n = 26). The drug or reagent was administered by grafting 20 microL onto the surface of CAM. After incubation for 3 days, the vessel growth was recorded by digital photography; inflammatory cells were counted under light microscope with HE staining, and the positive rate of angiogenesis reaction was calculated by Leica image analyzer. RESULTS: On the 6th day of the embryonic age, vessels in the chicken embryo CAM showed a radial growing in spok-wheel pattern around the gelatin sponges with lateral axis running through it. Whereas after 3 days of SSd treatment, the angiogenesis reduced significantly with vague microvessels around the sponge, and vascular truncation and absence revealed. Microscopic examinations showed that the number of microvessels and infiltrated inflammatory cells in the sponge and peripheral CAM mesenchyme in the SSd groups were less than those in the control group, especially on vessels of medium and small size (P < 0.05, P < 0.01, respectively), but was insignificant on great vessels (P > 0.05). Correlation analysis revealed no correlation between the number of the great vessels in CAM and the infiltrated inflammatory degrees (r = 0.117, P > 0.05), but the increase of small vessels in CAM was positively correlated with that of inflammatory cells (r = 0.971, P < 0.01). CONCLUSIONS: SSd could inhibit the physiological angiogenesis of chicken embryoe, especially for the medium and small vessels, while there was no significant effect on great vessels (P > 0.05). Its mechanism of action may be related to its inhibition on leukocyte migration and activation.

Protective effects of emodin and astragalus polysaccharides on chronic hepatic injury in rats.

BACKGROUND: Chinese medicine plays an important role in hepatoprotective treatment. This study was conducted to investigate the protective effects of emodin and astragalus polysaccharides (APS) in a rat model of chronic hepatic injury. METHODS: Chronic hepatic injury was induced by hypodermic injection of an olive oil solution containing 40% carbon tetrachloride (CCl(4)) twice a week, in addition to a diet of 79.5% maizena, 20% fat, 0.5% cholesterol, and 10% alcohol in the drinking water ad libitum for 12 weeks. Meanwhile, the rats were exposed to different concentrations of emodin (40 mg x kg(-1) x d(-1)), APS (200 mg x kg(-1) x d(-1)), combination drug (emodin 40 mg x kg(-1) x d(-1) combined with APS 200 mg x kg(-1) x d(-1)) and colchicine (0.1 mg x kg(-1) x d(-1)) in parallel by oral gavage (once a day for 12 weeks). At the end of 12 weeks, blood serum and liver tissue were taken. Serum was collected to determine the levels of total bilirubin (TBIL), alanine transaminase (ALT), aspartate transaminose (AST), and albumin (ALB). Liver and spleen indexes were assayed, followed by the measurements of the liver associated enzyme superoxide dismutase (SOD) and malondialdehyde (MDA). Histopathological changes were studied using optical microscopy. RESULTS: Splenohepatomegalia was alleviated and serum levels of TBIL and ALT were reduced in the groups treated with emodin and APS when compared to the control group. In addition, the ALB level in the APS and combination groups was higher. Similarly, the SOD activity of liver homogenates was significantly higher in the groups treated with emodin and APS, while administration of the herbal derivatives prevented the elevation in MDA levels. Histological analysis showed that the APS and combination groups significantly ameliorated the hepatic injury. CONCLUSIONS: Co-administration of emodin and APS demonstrated a synergistic action in reducing ALT and restoring ALB in the serum from a rat model of chronic hepatic injury. Emodin and APS may ameliorate the CCl(4)-induced hepatic injury in rats by elevating antioxidant-enzyme activities and reducing lipid peroxidation.

Inhibitory effects of saikosaponin-d on CCl4-induced hepatic fibrogenesis in rats.

AIM: To investigate the suppressive effect of saikosaponin-d (SSd) on hepatic fibrosis in rats induced by CCl(4) injections in combination with alcohol and high fat, low protein feeding and its relationship with the expression of nuclear factor-kappaB (NF-kappaB), tumor necrosis factor-alpha (TNF-alpha) and interleukins-6 (IL-6). METHODS: Hepatic fibrosis models were induced by subcutaneous injection of CCl(4) at a dosage of 3 mL/kg in rats. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses (1.0, 1.5 and 2.0 mg/kg) once daily for 6 wk in combination with CCl(4), while the control group received olive oil instead of CCl(4). At the end of the experiment, rats were anesthetized and killed (except for 8 rats which died during the experiment; 2 from the model group, 3 in high-dose group, 1 in medium-dose group and 2 in low-dose group). Hematoxylin and eosin (HE) staining and Van Gieson staining were used to examine the changes in liver pathology. The levels of alanine aminotransferase (ALT), triglyeride (TG), albumin (ALB), globulin (GLB), hyaluronic acid (HA) and laminin (LN) in serum and the content of hydroxyproline (HYP) in liver were measured by biochemical examinations and radioimmuneoassay, respectively. In addition, the expression of TNF-alpha and IL-6 in liver homogenate was evaluated by enzyme-linked immunosorbent assay (ELISA) and the levels of NF-kappaBp65 and I-kappaBalpha in liver tissue were analyzed by Western blotting. RESULTS: Both histological examination and Van Gieson staining demonstrated that SSd could attenuate the area and extent of necrosis and reduce the scores of liver fibrosis. Similarly, the levels of ALT, TG, GLB, HA, and LN in serum, and the contents of HYP, TNF-alpha and IL-6 in liver were all significantly increased in model group in comparison with those in control group. Whereas, the treatment with SSd markedly reduced all the above parameters compared with the model group, especially in the medium group (ALT: 412 +/- 94.5 IU/L vs 113.76 +/- 14.91 IU/L, TG: 0.95 +/- 0.16 mmol/L vs 0.51 +/- 0.06 mmol/L, GLB: 35.62 +/- 3.28 g/L vs 24.82 +/- 2.73 g/L, HA: 42.15 +/- 8.25 ng/mL vs 19.83 +/- 3.12 ng/mL, LN: 27.56 +/- 4.21 ng/mL vs 13.78 +/- 2.57 ng/mL, HYP: 27.32 +/- 4.32 mug/mg vs 16.20 +/- 3.12 mug/mg, TNF-alpha: 4.38 +/- 0.76 ng/L vs 1.94 +/- 0.27 ng/L, IL-6: 28.24 +/- 6.37 pg/g vs 12.72 +/- 5.26 pg/g, respectively, P < 0.01). SSd also decreased ALB in serum (28.49 +/- 4.93 g/L vs 37.51 +/- 3.17 g/L, P < 0.05). Moreover, the expression of NF-kappaB p65 in the liver of treated groups was lower than that in model groups while the expression of I-kappaBalpha was higher in treated group than in model group (P < 0.01). The expression of NF-kappaBp65 and TNF-alpha had a positive correlation with the level of HA in serum of rats after treatment with CCl(4) (r = 0.862, P < 0.01; r = 0.928, P < 0.01, respectively). CONCLUSION: SSd attenuates CCl(4)-induced hepatic fibrosis in rats, which may be related to its effects of hepato-protective and anti-inflammation properties, the down-regulation of liver TNF-alpha, IL-6 and NF-kappaBp65 expression and the increased I-kappaBalpha activity in liver.

Inhibitory effect of Huangqi Zhechong decoction on liver fibrosis in rat.

AIM: To assess the inhibitory effect of Huangqi Zhechong decoction on hepatic fibrosis in rats induced by CCl(4) plus alcohol and high fat low protein diet. METHODS: Male SD rats were randomly divided into hepatic fibrosis model group, control group and 3 treatment groups consisting of 12 rats in each group. Except for the normal control group, all the rats were subcutaneously injected with CCl(4) at a dosage of 3 mL/kg. In 3 treated groups, either high-dose group (9 mL/kg), or medium-dose group (6 mL/kg), or low-dose group (3 mL/kg) was daily gavaged with Huangqi Zhechong decoction, and saline vehicle was given to model and normal control rats. Enzyme-linked immunosorbent assay (ELISA) and biochemical examinations were used to determine the changes of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN), type-III-procollagen-N-peptide (PIIIP), and type IV collagen content in serum, and hydroxyproline (Hyp) content in liver after sacrificing the rats. Pathologic changes, particularly fibrosis were examined by hematoxylin and eosin (HE) and Van Gieson staining. RESULTS: Compared with the model control group, serum ALT, AST, HA, LN, PIIIP and type IV collagen levels dropped markedly in Huangqi Zhechong decoction groups, especially in the medium-dose Huangqi Zhechong decoction group (1 954+/-576 U/L vs 759+/-380 U/L, 2 735+/-786 U/L vs 1 259+/-829 U/L, 42.74+/-7.04 ng/mL vs 20.68+/-5.85 ng/mL, 31.62+/-5.84 ng/mL vs 14.87+/-1.45 ng/mL, 3.26+/-0.69 ng/mL vs 1.47+/-0.46 ng/mL, 77.68+/-20.23 ng/mL vs 25.64+/-4.68 ng/mL, respectively) (P<0.05). The Hyp content in liver tissue was also markedly decreased (26.47+/-11.24 mg/mgprot vs 9.89+/-3.74 mg/mgprot) (P<0.01). Moreover, the stage of the rat liver fibrosis in Huangqi Zhechong decoction groups was lower than that in model group, and more dramatic drop was observed in medium-dose Huangqi Zhechong decoction group (P<0.01). CONCLUSION: Huangqi Zhechong decoction can inhibit hepatic fibrosis resulted from chronic liver injure, retard the development of cirrhosis, and notably ameliorate the liver function. It may be a safe and effective therapeutic drug for patients with fibrosis.