[Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway].

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.

Possible mechanisms associated with immune escape and apoptosis on anti-hepatocellular carcinoma effect of Mu Ji Fang granules.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common digestive system cancers with high mortality rates worldwide. The main ingredients in Mu Ji Fang Granules (MJF) are alkaloids, flavonoids, and polysaccharides. MJF has been used in the clinical treatment of hepatitis, cirrhosis and HCC for more than 30 years. Few previous studies have focused on the mechanism of MJF on tumor immu-nology in the treatment of HCC. AIM: To explore the mechanism of action of MJF on tumor immunology in the treatment of HCC. METHODS: The absorbable ingredients of MJF were identified using Molecule Network related to High Performance Liquid Chromatography-Electron Spray Ionization-Time of Flight- Mass Spectrometry, and hub potential anti-HCC targets were screened using network pharmacology and pathway enrichment analysis. Forty male mice were randomly divided into the Blank, Model, and MJF groups (1.8, 5.4, and 10.8 g/kg/d) following 7 d of oral administration. Average body weight gain, spleen and thymus indices were calculated, tumor tissues were stained with hematoxylin and eosin, and Interferon gamma (IFN-γ), Tumor necrosis factor α (TNF-α), Interleukin-2, aspartate aminotransferase, alanine aminotransferase, alpha-fetoprotein (AFP), Fas, and FasL were measured by Enzyme-linked Immunosorbent Assay. Relevant mRNA expression of Bax and Bcl2 was evaluated by Real Time Quantitative PCR (RT-qPCR) and protein expression of Transforming growth factor ß1 (TGF-ß1) and Mothers against decapentaplegic homolog (SMAD) 4 was assessed by Western blotting. The HepG2 cell line was treated with 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL of MJF, and another 3 groups were treated with TGF-ß1 inhibitor (LY364947) and different doses of MJF. Relevant mRNA expression of TNF-α, IFN-γ, Bax and Bcl2 was evaluated by RT-qPCR and protein expression of TGF-ß1, SMAD2, p-SMAD2, SMAD4, and SMAD7 was assessed by Western blotting. RESULTS: It was shown that MJF improved body weight gain and tumor inhibition rate in H22 tumor-bearing mice, protected immune organs and liver function, reduced the HCC indicator AFP, affected immunity and apoptosis, and up-regulated the TGF-ß1/SMAD signaling pathway, by increasing the relative expression of TGF-ß1, SMAD2, p-SMAD2 and SMAD4 and decreasing SMAD7, reducing immune factors TNF-α and IFN-γ, decreasing apoptosis cytokines Fas, FasL and Bcl2/Bax, and inhibiting the effect of LY364947 in HepG2 cells. CONCLUSION: MJF inhibits HCC by activating the TGF-ß1/SMAD signaling pathway, and affecting immune and apoptotic cytokines, which may be due to MJF adjusting immune escape and apoptosis.

Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway / 中国中药杂志

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.

Effects of increasing efficacy and decreasing toxicity of ginkgo flavone aglycone on doxorubicin in the treatment of liver cancer / 中国药房

OBJECTIVE To study the effects of increasing efficacy and decreasing toxicity of ginkgo flavone aglycone （GA） on doxorubicin （DOX）in the treatment of liver cancer. METHODS A tumor bearing model was established by inoculating liver cancer cell H 22 into the right axillary skin of ICR mice. The successfully modeled mice were randomly divided into model control group，DOX group （2.5 mg/kg，once every other day ，via tail vein ），GA group （30 mg/kg，once a day ，gavage）and GA+DOX group（the usage was the same as single drug groups ），with 6 mice in each group. The administration cycle was 15 days. The general growth of mice in each group were observed ，body weight and tumor weight were measured ，and the inhibition rate of tumor was calculated. Jin’s formula was used to evaluate the effect of combined medication （Q）. The serum level of alpha-fetal protein（AFP），the pathological changes of tumor tissue ，cell apoptosis and the expression of platelet-endothelial cell adhesion molecule-1（CD31）were detected in each group. The cardiac index，serum levels of B-type natriuretic peptide （BNP）and N-terminal pro-brain natriuretic peptide （NT-pro BNP ），pathological changes of heart and myocardial fibrosis degree were also detected. RESULTS The percentage of body weight change （except for GA group ） and tumor weights of DOX group，GA group and GA + DOX group were all decreased significantly，compared with model control group （P＜0.05 or P＜0.01），while tumor weight of GA+DOX gro up was significantly lower than DOX group （P＜0.01）. Inhibitory rates of tumor in 3 administration groups were 54.29％，42.50％ and 89.29％ respectively，and Q of two-drug combination was 1.21. The tumor tissues of mice in each administration group were necrotic to varying degrees ；the serum level of AFP and the expression of CD31 in tumor tissue were decreased significantly ，compared with model control group （P＜0.05 or P＜0.01）；the percentage of necrosis area of tumor tissue and the positive rate of apoptosis （except for single drug groups ）were significantly increased （P＜0.05 or P＜0.01），while positive rate of apoptosis in GA+DOX group was significantly higher than DOX group （P＜0.05）. Cardiac index of mice in DOX group was significantly lower than model control group （P＜0.01）；serum levels of BNP and NT-pro BNP in DOX group and GA+ DOX group were significantly higher than model control group （P＜0.05 or P＜0.01）；pathological changes of heart and the degree of myocardial fibrosis in GA+DOX group were lower than DOX group. CONCLUSIONS GA combined with DOX show synergistic antitumor effect. GA can strengthen the apoptosis promoting effect of DOX ，and can help to reduce the cardiotoxicity of DOX.

The immunomodulatory effects of ginsenoside derivative Rh2-O on splenic lymphocytes in H22 tumor-bearing mice is partially mediated by TLR4.

PURPOSE: Previously, we reported the octyl ester derivative of ginsenoside Rh2 (Rh2-O) had better antitumor and immunomodulatory effects than Rh2 in H22 tumor-bearing mice. Therefore, this study further explored the effects of Rh2-O on splenic lymphocytes in H22 tumor-bearing mice and the underlying mechanism. METHODS: Wild type and Tlr4-/- mice were selected to establish the H22 tumor-bearing mice model. After the treatment of Rh2-O (10 mg/kg by gavage) for 15 days, the sizes of tumor were measured. Subsequently, the splenic lymphocytes were isolated and the activities (eg. cell proliferation, cytotoxicity and cytokine secretion) were evaluated. Then, the proteins and mRNA expression levels of TRAF6 and NF-ĸB p65 in splenic lymphocytes were examined. RESULTS: The results showed that Rh2-O administration enhanced the proliferative capacity and cytotoxicity of splenic lymphocytes, and the effects were Tlr4-associated. Compared to WT mice, the up-regulation of cytokines secretion (eg. IFN-γ, IL-2 and IL-4) in isolated splenic lymphocytes after Rh2-O administration was lower in Tlr4-/- mice. Moreover, the results showed Rh2-O increased the expression of TRAF6 and the level of endonuclear NF-ĸB p65, which was inhibited in Tlr4-/- mice (P < 0.05). CONCLUSION: Rh2-O could exert immunomodulatory effects on splenic lymphocytes with the partial participation of TLR4 in H22 tumor-bearing mice.

Yangyin Fuzheng Jiedu Prescription exerts anti-tumor immunity in hepatocellular carcinoma by alleviating exhausted T cells.

BACKGROUND: Yangyin Fuzheng Jiedu Prescription (YFJP), a formulated Chinese herbal medicine, has been used for several decades in the treatment of hepatocellular carcinoma (HCC). Previous studies have demonstrated its anti-tumor efficacy, but the mechanism of action remains uncharacterized. This study aims to evaluate the therapeutic effect of YFJP on H22 tumor-bearing mice. PURPOSE: This study aimed to evaluate the therapeutic effect of YFJP on H22 tumor-bearing mice. METHODS: A total of 50 male H22 tumor-bearing mice were randomly divided into 6 groups and continuous administered either different doses of YFJP or cyclophosphamide (CTX) or normal saline. for 2 weeks. The tumor appearance was observed by taking photos, and the tumor volume, weight, spleen and thymus index were calculated. Morphology of tumor infiltrating lymphocytes and the CD8+ T lymphocytes were detected through HE staining immunohistochemistry respectively. The frequency of CD3+, CD8+ T cell subsets and co-inhibitory receptors PD-1, TIGIT, Tim-3 on CD8+ T cell in spleen, peripheral blood and tumor tissue was performed by flow cytometry. Meanwhile, the killing and apoptotic functions of CD8+ T cells in tumor tissues were also detected by the same method. The levels of cytokines in peripheral blood were detected by Milliplex map mouse highs sensitivity T Cell kit. The expression of T cell transcription factor T-bet and Eomes in tumor tissues were observed by Western blot. RESULTS: We found that YFJP could effectively inhibit the solid tumor growth and spleen indexes, but showed little effect on the body weight in the established mouse model of HCC. Furthermore, we investigated the effect of YFJP on the phenotypic and functional changes of T cells. The results showed that YFJP could maintain the high ratio of CD3+ and CD8+ T cells in the peripheral blood, spleen, and tumor tissues while decreasing the expression of programmed cell death-1 (PD-1), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin domain and mucin domain-3 (Tim-3) in CD8+ T cells, respectively. Surprisingly, PD-1/Tim-3 double-positive T cells in the peripheral blood and tumor tissues were significantly decreased. Additionally, YFJP restored the cytotoxicity of tumor-infiltrating T cells and delayed their apoptosis in H22 tumor-bearing mice. In addition, treatment with YFJP significantly decreased the expression of inflammatory and immunosuppressive cytokines (including IL-1ß, IL-6, and IL-10) in the serum and tumor tissues whereas enhancing that of effector cytokines TNF-α, and IFN-γ. Moreover, T cell transcription factors T-bet increased and Eomes degraded in the tumor tissues upon YFJP treatment. CONCLUSION: In conclusion, these results demonstrated that YFJP could simultaneously exert anti-tumor immune response in H22 tumor-bearing mice by alleviating T cell exhaustion and immunosuppression.

[Anti-tumor and immune-modulating effect of Jiawei Sijunzi decoction in mice bearing hepatoma H22 tumor].

OBJECTIVE: To investigate the antitumor activity of Jiawei Sijunzi decoction and study its liver and kidney toxicity and its effect on the immune system in a tumor-bearing mouse model. METHODS: Hepatoma H22 tumor-bearing mouse models were randomized into model group, cyclophosphamide (CTX) group, and low-, moderate-, and high-dose Jiawei Sijunzi decoction groups (JW-L, JW-M, and JW-H groups, respectively). The antitumor activity of Jiawei Sijunzi decoction was assessed by calculating the tumor inhibition rate and pathological observation of the tumor tissues. Immunohistochemistry was used to detect the expressions of Bax, Bcl-2, Bax/Bcl-2 and caspase-3 in the tumors. The liver and kidney toxicity of Jiawei Sijunzi decoction was analyzed by evaluating the biochemical indicators of liver and kidney functions. The immune function of the tumor-bearing mice were assessed by calculating the immune organ index, testing peripheral blood routines, and detection of serum IL-2 and TNF-α levels using enzyme-linked immunosorbent assay. RESULTS: Compared with that in the model group, the tumor mass in CTX, JW-M and JW-H groups were all significantly reduced (P < 0.05) with cell rupture and necrosis in the tumors. Immunohistochemistry revealed obviously up-regulated expressions of Bax and caspase-3 and down- regulated expression of Bcl-2 protein with an increased Bax/Bcl-2 ratio in CTX, JW-M and JW-H groups. Treatment with Jiawei Sijunzi decoction significantly reduced Cr, BUN, AST and ALT levels, improved the immune organ index, increased peripheral blood leukocytes, erythrocytes and hemoglobin levels, and up-regulated the levels of TNF-α and IL-2 in the tumor-bearing mice. These changes were especially significant in JW-H group when compared with the parameters in the model group (P < 0.01). CONCLUSIONS: Jiawei Sijunzi decoction has a strong anti-tumor activity and can improve the liver and kidney functions of tumor-bearing mice. Its anti-tumor effect may be attributed to the up-regulation of Bax, caspase-3, TNF-α and IL-2 levels and the down-regulation of Bcl-2 expression as well as the enhancement of the non-specific immune function.

Anti-tumor and immune-modulating effect of decoction in mice bearing hepatoma H22 tumor / 南方医科大学学报

OBJECTIVE@#To investigate the antitumor activity of decoction and study its liver and kidney toxicity and its effect on the immune system in a tumor-bearing mouse model.@*METHODS@#Hepatoma H22 tumor-bearing mouse models were randomized into model group, cyclophosphamide (CTX) group, and low-, moderate-, and high-dose decoction groups (JW-L, JW-M, and JW-H groups, respectively). The antitumor activity of decoction was assessed by calculating the tumor inhibition rate and pathological observation of the tumor tissues. Immunohistochemistry was used to detect the expressions of Bax, Bcl-2, Bax/Bcl-2 and caspase-3 in the tumors. The liver and kidney toxicity of decoction was analyzed by evaluating the biochemical indicators of liver and kidney functions. The immune function of the tumor-bearing mice were assessed by calculating the immune organ index, testing peripheral blood routines, and detection of serum IL-2 and TNF-α levels using enzyme-linked immunosorbent assay.@*RESULTS@#Compared with that in the model group, the tumor mass in CTX, JW-M and JW-H groups were all significantly reduced ( < 0.05) with cell rupture and necrosis in the tumors. Immunohistochemistry revealed obviously up-regulated expressions of Bax and caspase-3 and down- regulated expression of Bcl-2 protein with an increased Bax/Bcl-2 ratio in CTX, JW-M and JW-H groups. Treatment with decoction significantly reduced Cr, BUN, AST and ALT levels, improved the immune organ index, increased peripheral blood leukocytes, erythrocytes and hemoglobin levels, and up-regulated the levels of TNF-α and IL-2 in the tumor-bearing mice. These changes were especially significant in JW-H group when compared with the parameters in the model group ( < 0.01).@*CONCLUSIONS@# decoction has a strong anti-tumor activity and can improve the liver and kidney functions of tumor-bearing mice. Its anti-tumor effect may be attributed to the up-regulation of Bax, caspase-3, TNF-α and IL-2 levels and the down-regulation of Bcl-2 expression as well as the enhancement of the non-specific immune function.

Effect on tumor growth inhibition activity of Gekkoswinhonis Guenther with different extent of broiling on H22 tumor-bearing mice / 药学学报

This study was designed to investigate the effect on tumor growth inhibition activity of lizards (Gekkoswinhonis Guenther) with different extent of broiling. Samples were prepared by a traditional drying method combined with broiling on clay tiles. Four groups of samples were all dried before broiling. Group A was without broiling; group B was mildly broiled; group C was moderately broiled; and group D was heavily broiled. Crispiness was detected by the sizes of the generated fragments of different groups and crispiness increased with broiling. Sensory evaluation of vision and olfaction was performed, and scores were generated by evaluators. Moderately broiled group had the highest total score in sensory evaluation. Water content and content of water-soluble extracts were detected according to Chinese Pharmacopoeia. With the increasing broiling extent, content of water-soluble extracts increased while water content decreased. Soluble protein concentration was detected by bicinchoninic acid (BCA) kit and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with the same crude drug content. Soluble protein concentration decreased with the increasing broiling extent. With equal loading of proteins at the same concentration, soluble protein diversity was detected by SDS-PAGE. Band difference was marked by red boxes. Soluble protein molecule weights showed significant difference with the increasing broiling extent. H22 tumor-bearing mice model was established and used to detect tumor growth inhibition rate and immune organ index. Life quality of mice was evaluated. Mice treated with Gekkoswinhonis Guenther had better appetites and higher average weights compared with positive control group treated with fluorouracil (5-FU). Animal experiments were approved by the Ethics Committee of Beijing University of Chinese Medicine. Group A had the highest tumor growth inhibition rate (34.11%), followed by Group B (29.14%) and Group D (28.43%), Group C (21.98%) had the lowest tumor growth inhibition rate, but sensory evaluation was on the contrary. These results indicated that moderately broiling improved sensory evaluation but reduced the tumor growth inhibition activity of Gekkoswinhonis Guenther. The best tumor growth inhibition activity appeared when water content was 7.71%.

Study on Anti-tumor Effects of Periplaneta americana Polypeptide PAP- 2 on H 22 Tumor-bearing Mice / 中国药房

OBJECTIVE： To investigate anti-tumor effects of Periplaneta americana polypeptide PAP-2 on H22 tumor-bearing mice. METHODS： The mice tumor-bearing model was established by subcutaneous injection of ascites of H22 hepatocellular carcinoma mice via axilla. 70 mice were randomly divided into model group （normal saline）， 5-FU group （positive drug control， 20 mg/kg）， P. americana extract skimmed cream group （200 mg/kg， calculated by extract）， CⅡ-3 group （polypeptide isolated from skimmed cream as main active ingredient， 200 mg/kg， calculated by extract） and polypeptide PAP-2 high-dose， medium-dose and low-dose groups （isolated from CⅡ-3， 200， 100， 50 mg/kg， calculated by monomer）， with 10 mice in each group. The mice in the 5-FU group were given intraperitoneal injection once every other day， while the mice in the other groups were given intragastric administration once a day， the administration cycle was 10 d. After medication， the changes of tumor were observed and the organs （spleen， thymus and liver） index were measured. Histopathological changes of tumor tissue were observed after HE staining. The contents of VEGF， IL-1β and IL-4 in serum were determined by ELISA. RESULTS： Skimmed cream， CⅡ-3 and different doses of PAP-2 could inhibit the growth of tumor in tumor-bearing mice to different extent and increase organ index， and PAP-2 showed a dose-effect relationship. The tumor inhibition rate （38.95％） of PAP-2 high dose group was significantly higher than those of skimmed cream group and CⅡ-3 group （P＜0.05）， which was close to that （40.87％） of 5-FU group （P＞0.05）. Spleen index， thymus index and liver index of mice in PAP-2 high dose group were significantly those of model group and CⅡ-3 group （P＜0.05）； and the liver index of mice in PAP-2 high dose group was significantly higher than that of skimmed cream group （P＜0.05）. In addition， PAP-2 could decrease the serum contents of VEGF and IL-4， and increased serum content of IL-1β， with high dose group showed significant difference compared with model group （P＜0.05）； the serum content of IL-1β of mice in PAP-2 high dose group was significantly higher the that of skimmed cream group and CⅡ-3 group （P＜0.05）， serum contnet of IL-4 in PAP-2 high dose group was significantly lower the  that of skimmed cream group and CⅡ-3 group （P＜0.05）， but the serum content in which was significantly lower than that of skimmed cream group and CⅡ-3 group（P＜0.05）. CONCLU- SIONS： P. americana polypeptide PAP-2 it has a certern anti-tumor effects on H22 tumor-bearing mice， and its can increase the index of organs of H22 tumor-bearing mice， decrease the contents of VEGF and IL-4 in serum， increase the content of IL-1β in serum.

Anti-tumor properties of anthocyanins from Lonicera caerulea 'Beilei' fruit on human hepatocellular carcinoma: In vitro and in vivo study.

In this study, the anthocyanin from Lonicera caerulea 'Beilei' fruit (ABL) was extracted and purified. The purified component (ABL-2) was then evaluated for its anti-tumor properties on human hepatoma cells (SMMC-7721) in vitro and the murine hepatoma cells (H22) in vivo. In vitro, ABL-2 not only significantly inhibited the growth of SMMC-7721 cells, but also remarkably blocked the cells' cycle in G2/M phase, inducing DNA damage and eventually leading to apoptosis. In vivo, ABL also killed tumor cells, inhibited tumor growth, and improved the survival status of H22 tumor-bearing mice. These effects were associated with an increase in the activities of antioxidase and a decrease in the level of lipid peroxidation, as evidenced by changes in SOD, GSH-Px, GSH, and MDA levels. In addition, ABL-2 also regulated the levels of immune cytokines including IL-2, IFN-γ, and TNF-α. These results revealed that ABL-2 exerts an effective anti-tumor effect by dynamically adjusting the REDOX balance and improving the immunoregulatory activity of H22 tumor-bearing mice. High performance liquid chromatography (HPLC) analysis revealed that cyanidin-3,5-diglucoside (8.16 mg/g), cyanidin-3-glucoside (387.60 mg/g), cyanidin-3-rutinoside (23.62 mg/g), and peonidin-3-glucoside (22.20 mg/g) were the main components in ABL-2, which may contribute to its anti-tumor activity.

A novel mechanism of tumor-induced thymic atrophy in mice bearing H22 hepatocellular carcinoma.

BACKGROUND: Thymic atrophy was discovered in tumor-bearing mice in recent years. METHODS: Flow cytometry was carried out including Annexin V-FITC/PI double staining, PI staining, Terminal dUTP nick-end labeling, CD3-FITC/CD19-PE and CD8-FITC/CD4-PE double staining. Enzyme-linked immunosorbent assay and polymerase chain reaction were also investigated. RESULTS: According to our experiments, we demonstrated that no signs of apoptosis in thymocytes were found in H22-bearing mice, while the proportions of CD4+ T cells and CD8+ T cells in thymuses were remarkably increased, the opposite tendency was found in peripheral bloods, and only CD3+CD8+ T cells were discovered in H22 solid tumors. We further discovered that the level of thymosin alpha 1 (Tα1) and the expression of Wnt4 in thymus of H22-bearing mice were significantly improved than control, which indicated the active proliferation and differentiation of thymocytes. Our study revealed that CD8+ T cells could not effectively eliminate H22 cells independently when CD4+ T cells were suppressed by tumors, while the body would only enhance the differentiation and maturation of T cells in thymuses and release them to solid tumor to reinforce antitumor immunocompetence, leading to a vicious cycle which finally led to thymic atrophy. CONCLUSION: Our data propose a novel mechanism of tumor-induced thymic atrophy regulated by abnormal immunoreaction and may provide new ideas for the immunotherapy of tumors.

Antitumor Effects of Ethanol Extracts from Hyptis Rhomboidea in H22 Tumor-bearing Mice.

BACKGROUND: Research the antitumor effects of ethanol extracts from Hyptis rhomboidea in H22 tumor-bearing mice. At the fist-stage of the experiments, the research team took MTT method to measure the antitumor activity in vitro, then selected the most inhibitory tumor cell strain as the test object of antitumor activity in vivo, established three models of a solid tumor H22 liver cancer, ascites tumor, and immunodeficiency in male mice. From inflammatory factor, liver toxicity, in vivo antioxidant index to observe antitumor activity of ethanol extracts from H. rhomboidea. MATERIALS AND METHODS: Hundred and twenty ICR male mice were used to establish three models of a solid tumor H22 liver cancer, ascites tumor, and immunodeficiency in male mice and models group of a solid tumor H22 liver cancer randomly divided into six groupshe normal control group, the model control group, the positive group (cyclophosphamide), the sample treated group (high - 1.300 g/kg, medium - 0.750 g/kg, low - 0.373 g/kg). The animals were sacrificed 15d after oral administration and tumors were taken out for the tumor weights and antitumor rates. Blood in eyeball was collected for the determination of aspartate transaminase, alanine transaminase, malondialdehyde (MDA), superoxide dismutase (SOD), interleukin (IL)-2, and tumor necrosis factor (TNF)-α in serum. Sections of tumor issue were prepared, and morphological changes in tumor tissue cells were observed using hematoxylin and eosin staining technique. RESULTS: The results showed that ethanol extracts from H. rhomboidea have a certain inhibitory effect on the digestive tumor cells. In solid tumor model, the inhibitory rate is up to 68.84% of the high dose of treated group from H. rhomboidea, and H. rhomboidea could improve the immune organ index, decrease the concentration of TNF-α and IL-2 in serum. In ascites tumor model, H. rhomboidea could slow down weight gain in mice and prolong the survival time; in immunodeficiency model, H. rhomboidea could improve the serum TNF-α and, IL-2 levels, increase SOD activity, and reduce MDA content, so as to achieve antitumor effect. CONCLUSIONS: Ethanol extracts from H. rhomboidea have obvious antitumor activity in vivo and can improve a tumor-burdened mice inflammation factors, improve the survival quality of H22 tumor mice, and enhance immunity and antitumor activity. SUMMARY: Ethanol extracts from Hyptis rhomboidea have obvious antitumor activity without obviously liver damageThe experiment results prompt that the antitumor activity probably caused by decreasing the inflammatory factors, improve the survival quality, enhance the abnormal cytokines and scavenging free radicals, and raising autoimmune function in H22 tumor bearing mice. Abbreviations used: H. rhomboidea: Hyptis rhomboidea; AST: Aspartate transaminase; ALT: Alanine transaminase; MDA: Malondialdehyde; SOD: Superoxide dismutase; IL-2: Interleukin 2; TNF-α: Tumor Necrosis Factor-α; CTX: Cyclophosphamide.

Antitumor Effects and Mechanism of n-butanol Fraction from Aril of Torreya grandis in H22 Mice.

BACKGROUND: To determine the antitumor effects and its mechanism of n-butanol fraction from aril of Torreya grandis (BFAT) on H22 mice models of liver cancer. MATERIALS AND METHODS: Sixty ICR male mice were used to establish H22 mice models of liver cancer and then randomly divided into six groups, the normal control group, the model control group, the positive group (cyclophosphamide [CTX]), the BFAT-treated group (high, 4 g/kg, medium, 2 g/kg, and low, 1 g/kg). The animals were sacrificed 15 days after oral administration, and tumors were taken out for the tumor weights and antitumor rates, while thymus and spleen were taken for thymus index and spleen index. Blood in eyeball was collected for the determination of aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (Alb), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase enzyme (GSH-Px), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), transforming growth factor-ß1 (TGF-ß1), and IL-10 in serum. Sections of tumor tissue were prepared, and morphological changes in tumor tissue cells were observed using hematoxylin and eosin staining technique. RESULTS: Compared with the model control group, the tumor inhibition rate of the high-dose administered group is 60.15%, which is quite closed to the effect of CTX. Moreover, the tumor weight is decreased, the indexes of spleen, thymus were increased significantly. Furthermore, the administration of BFAT significantly enhanced the activities of TNF-α, IL-2, SOD, and GSH-Px and reduced the levels of AST, ALT, MDA, Alb, TGF-ß1, and IL-10 (P < 0.01). CONCLUSIONS: The results demonstrated that n-butanol fraction from aril of T. grandis showed out antitumor activity without obviously liver damage through potentiating immunologic function and antioxidant activity of tumor-bearing mice and which may become one potential as anticancer drug alternatives or supplements. SUMMARY: High and medium groups could significant elevate the thymus and spleen indexes and the interleukin-2 and tumor necrosis factor-α level in serum of H22 micen-butanol fraction from aril of Torreya grandis (BFAT) could ameliorate the levels of aspartate aminotransferase, alanine aminotransferase and albumin to almost normal, and increase the concentrations of superoxide dismutase and glutathione peroxidase enzyme, decrease the malondialdehyde level in serum of mice significantlyBFAT may indirectly play the role of antitumor activity through improving immunologic functionBFAT had potent antitumor properties without obviously liver damage. Abbreviations used: DDP: Cisplatin; CTX: Cyclophosphamide; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; Alb: Albumin; MDA: Malondialdehyde; SOD: Superoxide dismutase; GSH-Px: Glutathione peroxide enzyme; TNF-α: Tumor necrosis factor-α; IL-2: Interleukin-2; TGF-ß1: Transforming growth factor-ß1; IL-10: Interleukin-10; HE: Hematoxylin and eosin; PBS: Phosphate-buffered saline; PFAT: Petroleum ether fraction from aril of Torreya grandis; EFAT: Ethyl acetate fraction from aril of Torreya grandis; BFAT: N-butanol fraction from aril of Torreya grandis.

[Effects of Shiquanyuzhentang on immunologic function of H22 tumor-bearing mouse].

OBJECTIVE: To investigate the effects of Shiquanyuzhentang (Traditional Chinese Medicine) on the immunologic function of tu-mor-bearing mice and its mechanism of antitumor. METHODS: Thirty SPF grade male Kunming mice transplanted with H22 hepatocellular carci-noma were divided into three groups randomly:model group,positive control group and Shiquanyuzhentang group(n=10). Another 10 mice were selected as normal control group. Normal control group and model group received normal saline and distilled water supplementation by 10 mL/kg everyday. Positive control group and Shiquanyuzhentang group received Shengyi(80 mg/ml)and Shiquanyuzhentang decoction at the doses of 8 g/kg and 18 g/kg respectively everyday. After 14 days of continuous administration, the mice were killed and the thymus, spleen index, tumor inhibition rate,peripheral blood leukocytes,lymphocyte content,cell percentage of T cell subsets CD3, CD4, CD8,interleukin 2 (IL-2)in serum,tumor necrosis factor-α(TNF-α),interferon ß(IFN-ß) content,lymphocyte proliferation ability and NK cell were measured. RESULTS: Compared with normal control group, the weight of mice and thymus and spleen index of the Shiquanyuzhentang group were increased significantly(P<0.05);leukocyte and lymphocyte CD3、CD4、CD8 and TNF-α were increased greatly(P<0. 05),IL-2 and IFN-ß were de-creased significantly(P<0.05). Compared with model group,thymus and spleen index of Shiquanyuzhentang group were increased significant-ly(P<0.05);leukocyte and lymphocyte CD3、CD4、IL-2、IFN-ß and TNF-α of Shiquanyuzhentang group were increased significantly(P<0. 05),CD8 content was decreased significantly(P<0.05);NK cell and lymphocyte proliferation were increased significantly(P<0.05). CONCLUSIONS: Shiquanyuzhentang can promote the growth of immune organs in mice bearing H22, enhance immune function and is beneficial to the recovery of tumor body.

A Research on the Anti-tumor Effects of Acid-Soluble Polysaccharides from Gloeostereum incamatum in H22 Tumor Bearing Mice / 世界科学技术-中医药现代化

This study aimed at exploring the inhibitory effect behind its mechanism on acid-soluble polysaccharides from G.incamatum in transplanted H22 tumor mice.Different indices,including tumor inhibitory rate,organ index of liver,thymus and spleen,IL-2,IFN-γ and TNF-α were detected for the evaluation of anti-tumor effects and the mechanism.Furthermore,HE staining and TUNEL assay were adopted to investigate the pathological changes of tumor tissue and cell apoptosis,respectively.As a result,the three dose groups of acidsoluble polysaccharides of G.incamatum successfully inhibited the proliferation of tumor cells,while organ indexes of spleen and thymus were improved and serum IL-2,IFN-γ and TNF-α increased.H&E staining and TUNEL assay showed the polysaccharides induced cell apoptosis,playing a significant role in the inhibition of tumor growth.In conclusion,acid-soluble polysaccharides of G.incamatum possessed significant anti-tumor effects,behind which the mechanism could be related to the regulation of immune regulation,cell apoptosis,and the protection of liver function.

Anti-tumor Effect of Ginsenoside Rg1 Pyrolysis Products (HPPRg1) on H22 Tumor-bearing Mice / 中国药学杂志

OBJECTIVE: To investigate the antitumor effect and molecular mechanism of ginsenoside Rg1 pyrolysis products (HPPRg1) on H22 tumor bearing mice. METHODS: To establish tumor model of transplanting H22 tumor-bearing mice and observe the anti-tumor effects of HPPRg1, H22 tumor-bearing mice were randomly divided into groups of control, model, cyclophosphamide (CTX, 30 mg·kg-1), low dosage of HPPRg1 (HPPRg1-L, 10 mg·kg-1), middle dosage of HPPRg1 (HPPRg1-H, 20 mg·kg-1) and high dosage of HPPRg1 (HPPRg1-H, 40 mg·kg-1) groups, respectively. Through evaluating inhibition rates of tumors, organ indices, and levels of TNF-α, IFN-γ and IL-2 to observe the anti-tumor effect of HPPRg1. In addition, H&E and Hoechst 33258 straining were used to observe the apoptosis of H22 tumor cell. RESULTS: Compared with the model group, the three dose groups of HPPRg1 can inhibit tumor proliferation. Mainly through the inhibition of tumor cell proliferation and pro-apoptosis to exert anti-tumor effect. CONCLUSION: HPPRg1 has a significantly inhibitory effect on H22 tumor-bearing mice, the mechanism may related to promote apoptosis of tumor cells and improve immunity.

Antitumor effect of alcohol extract of pileostegia tomentella on H22 tumor-bearing mice / 中国药学杂志

OBJECTIVE: To study the antitumor effect of Pileostegia tomentella 95% alcohol extract (PTAE) on H22 tumor-bearing mice and its possible mechanisms. METHODS: Sixty mice were chosen and mouse models bearing H22 solid tumor were established in fifty mice, and the others were as normal control. H22 tumor-bearing mice were randomly divided into five groups:model control group, fluorouracil group(20 mg·kg-1), PTAE high, middle and low-dose group(180, 90, 45 g·kg-1 of crude drug, respectively). The mice in treatment groups were intragastric administration respectively, meanwhile, the mice in normal control and model groups were treated with the same volume of distilled water, once a day for ten days. The blood was collected from eyeball in all mice, and the serum were separated and detected by ELISA for IL-2 and TNF-α. Then the mice were put to death. Their tumors, thymuses and spleens were separated and weighted, and the tumor inhibitory rates, thymus and spleen indexes were calculated. The pathological change of tumor tissue was observed. RESULTS: Compared with model control group, the tumor weights of PTAE high and middle-dose groups were significantly decreased (P<0.05, P<0.01), the tumor inhibitory rates were 37.44% and 38.46% respectively. The spleen index of PTAE middle-dose group was increased significantly (P<0.01). The level of IL-2 in serum of tumor-bearing mice in the PTAE high-dose group was increased significantly(P<0.05), and the level of TNF-α in serum (P<0.01) could be increased significantly in the PATE high, middle and low-dose groups. CONCLUSION: Pileostegia tomentella 95% alcohol extract has antitumor activity, its mechanism may be developed by immuno-regulation.

Anti-Tumor Effect of Steamed Codonopsis lanceolata in H22 Tumor-Bearing Mice and Its Possible Mechanism.

Although previous studies confirmed that steaming and the fermentation process could significantly improve the cognitive-enhancement and neuroprotective effects of Codonopsis lanceolata, the anti-tumor efficacy of steamed C. lanceolata (SCL) and what mechanisms are involved remain largely unknown. The present study was designed to evaluate the anti-tumor effect in vivo of SCL in H22 tumor-bearing mice. The results clearly indicated that SCL could not only inhibit the tumor growth, but also prolong the survival time of H22 tumor-bearing mice. Besides, the serum levels of cytokines, such as interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-2 (IL-2), were enhanced by SCL administration. The observations of Hoechst 33258 staining demonstrated that SCL was able to induce tumor cell apoptosis. Finally, immunohistochemical analysis revealed that SCL treatment significantly increased Bax expression and decreased Bcl-2 and vascular endothelial growth factor (VEGF) expression of H22 tumor tissues in a dose-dependent manner. Moreover, LC/MS analysis of SCL indicated that it mainly contained lobetyolin and six saponins. Taken all together, the findings in the present study clearly demonstrated that SCL inhibited the H22 tumor growth in vivo at least partly via improving the immune functions, inducing apoptosis and inhibiting angiogenesis.

Extraction and isolation of alkaloids of Sophora alopecuroides and their anti-tumor effects in H22 tumor-bearing mice.

BACKGROUND: Alkaloids of Sophora alopecuroides have good biological activity, and are widely used in clinical settings, which not only have pharmacological activities of anti-cancer, cancer suppression, as well as the inhibition, and killing of various microorganisms; but also possess extensive pharmacological effects on immune system, nervous system and cardiovascular system. The objective of this paper was to extract and isolate total alkaloids of Sophora alopecuroides (TASA), and to study their anti-tumor effects in H22 tumor-bearing mice. MATERIALS AND METHODS: TASA were extracted and isolated using thin-layer chromatography, and column chromatography; and the isolated compounds were analyzed using nuclear magnetic resonance. The inhibitory effects of TASA on tumor in H22-bearing mice were determined by MTT assay. RESULTS: Three compounds were isolated from Sophora alopecuroides L., which were matrine, oxymatrine and sophoridine, respectively. Meanwhile, mouse H22 sarcoma model was established and different doses of TASA apparently inhibited solid H22-tumor in mice; it inhibited the thymus, and spleen to some extent; the degree of inhibition was more obvious for the spleen. CONCLUSION: TASA has an anti-tumor effect in H22 tumor-bearing mice.