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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.
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Male , Animals , Mice , Cisplatin/pharmacology , Carcinoma, Hepatocellular/genetics , MAP Kinase Signaling System , Beclin-1 , Apoptosis , Liver Neoplasms/genetics , Necrosis , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Line, Tumor , RNA, Messenger/metabolism , AutophagyABSTRACT
Objective:To investigate the expression of zinc finger protein 22 (ZNF22) gene in hepatocellular carcinoma (HCC) and its effect on tumor proliferation, apoptosis, invasion and metastasis of HCC.Methods:The expression of ZNF22 in 32 HCC specimens, and 371 HCC samples from the cancer genome atlas database were analyzed. ZNF22 knockdown and negative control SNU-449 and JHH-7 HCC cell lines were constructed. The effects of ZNF22 on HCC cells were observed by cell proliferation assay, plate clone formation assay, apoptosis assay, scratch healing assay, Transwell invasion assay, subcutaneous tumor formation, tail vein injection transfer, and small animal live imaging assay in nude mice.Results:The expression of ZNF22 gene is higher in HCC tissues than in paracellular carcinoma tissues, and the difference was statistically significant ( P<0.001). The growth rate of SNU-449 and JHH-7 cells in ZNF22 knockdown group was lower than that in control group, and the difference was statistically significant ( P<0.001). Compared with negative control group, the clone number formed by SNU-449 cells in ZNF22 knockdown group decreased (26±8 vs. 59±5, P<0.01), the level of apoptosis increased (6.60%±0.22% vs. 2.38%±0.30%, P<0.001), the migration rate decreased (14.47%±6.42% vs. 68.84%±8.01%, P<0.001), and the number of invasive cells decreased (48.00±2.23 vs. 179.00±4.81, P<0.001). There was no obvious tumor growth after subcutaneous injection of JHH-7 cells into nude mice in ZNF22 knockdown group, and the systemic fluorescence expression was lower than that of the negative control group, and the difference was statistically significant ( P<0.05). No metastases were observed on autopsy in knockdown group nude mice. Conclusion:ZNF22 is highly expressed in HCC while knockdowing ZNF22 gene inhibited the growth, proliferation, invasion, metastasis of HCC cells, and induced apoptosis of HCC cells.
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The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development, whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction. We previously demonstrated that in mice, the makorin-2 (Mkrn 2) gene is expressed exclusively in the testis and its deletion leads to male infertility. To understand the potential molecular mechanism, in this study, we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn 2. To identify specific gene(s) involved, we performed digital gene expression profiling (DGE) and pathway analysis via gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22 (PERP) expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels. GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility. Further, Gene Expression Omnibus (GEO) dataset analysis showed that p53, upstream of PERP, was upregulated in oligoasthenoteratozoospermia (OAT). These observations suggest that Mkrn 2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn 2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility.
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Aim: To investigate the effect of astragaloside IV on apoptosis of HT22 after oxygen and glucose deprivation/reoxygenation by regulating autophagy. Methods: HT22 cells were randomly divided into control, model, DMSO, AS-IV, AS-IV + N, AS-IV + 3-MA, 3-MA and Rapa group. Except for control group, cells in other groups were reoxygenated after 6 h of oxygen and glucose deprivation. Invertded microscope was employed to observe cell morphology. CCK-8 method was used to test cell survival rate. LDH method was applied to detect cell damage, and Bax, Bcl-2 immunofluorescence was used to detect apoptosis. Results: Compared with control, the synapses of cell bodies decreased, the cells shrank, the number of intercellular connections decreased, cell viability was significantly reduced, and LDH leakage and Bax/Bcl-2 significantly increased in model group (P <0. 01). Compared with model group, cell viability in AS-IV group and Rapa group markedly increased, and LDH leakage and Bax/ Bcl-2 significantly decreased (P < 0. 01); cell viability was significantly down-regulated, and Bax/Bcl-2 was markedly up-regulated in 3-MA group (P < 0. 01). Astragaloside IV +3-MA group had no significant difference compared with model group. Conclusion: Astragaloside IV protects OGD/R HT22 by inhibiting apoptosis via activating autophagy.
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Objective To investigate the expression of USP22 in cervical cancer cells,and the effects of USP22 on cell proliferation and chemosensitivity to cisplatin in cervical cancer cells. Methods Quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to detect the expression of USP22 mRNA in cervical cancer cells.Cell count kit-8,flow cytometry,and tumorigenesis were performed to detect the effects of USP22 on the proliferation,apoptosis,cycle cycle,and chemosensitivity of cervical cancer cells to cisplatin in vitro and in vivo. Results USP22 was upregulated in cervical cancer cells compared to human immortalized epidermal cells. Cell proliferation was inhibited,cell apoptosis was promoted,cell cycle was arrested in G1 stage and chemosensi-tivity to cisplatin was enhanced in cervical cancer cells transfected with USP22-siRNAs.Furthermore,tumorigene-sis assays proved tumor growth was inhibited and chemosensitivity to cisplatin was enhanced when USP22 was silenced in HeLa cells. Conclusion USP22 expression is upregulated in cervical cancer cells,and USP22 could promote cell proliferation and inhibit chemosensitivity to cisplatin in cervical cancer cells.
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Aim To study the inhibitory effect of volatile components in Oroxyli Semen on liver cancer and its possible mechanisms.Methods H22 bearing mouse model was used,the mice were divided into six groups:blank,model,positive (cytoxan,100 mg · kg-1),low-,mid-,and high-dose (17.5,35,and 70 mg · kg-1) volatile components groups,and then the mice were ig given once daily for consecutive 12 d.Then the tumor growth inhibitory rate,spleen and thymus indexes were calculated;the serum levels of IL-2,IL-6 were determined.HE staining was used to study of the apoptosis of the solid tumor.After treatment of SMMC-7721 cells with 0 ～ 1 g · L-1 of volatile components for 24,48 and 72 h,MTT assay was used to examine the proliferation.TUNEL method was applied to detect cell apoptosis,and RT-PCR method to detect Bax,Bcl-2,caspase-3 mRNA experssion.Results The inhibitory rate of volatile components high-dose on H22 bearing mice was 42.08％.The thymus index and the contents of serum IL-2 and IL-6 of H22 bearing mice were significantly higher than those in model group.Volatile components could significantly inhibit proliferation and induce apoptosis of SMMC-7721 cells,downregulate the expression of Bcl-2 mRNA,and up-regulate the expression of Bax,caspase-3 mRNA.Conclusions The volatile components in Oroxyli Semen have obvious anti-tumor activity in vitro and in vivo,and its mechanism may be related to enhancing immune system and promoting tumor cell apoptosis.
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OBJECTIVE: To investigate the effects of Rhaponticum uniflorum extract (RUE) on angiogenesis and apoptosis of H22 transplanted tumor tissue in mice. METHODS: Mice bearing H22 hepatoma cells were randomly assigned into 5 groups: model, high, medium and low dose RUE, and 5-fluorouracil (5-Fu) group. The intervention was lasted for 10 d. The pathological changes were detected with hematoxylin & eosin (HE) staining, the apoptosis of hepatoma cells were determined by DNA laddering method, the microvessel densities (MVD) were detected using immunohistochemical assay, and the protein expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2) and hypoxia-inducible factor-1α (HIF-1α) were detected by Western blot method. RESULTS: RUE treatment reduced the cell proliferation, aggravated the necrosis of transplanted tumor tissue, reduced DNA fragmentation of H22 hepatoma cells, decreased MVD of tumor tissue, and down-regulated the protein expression of VEGF, VEGFR2 and HIF-1α of the transplanted tumor tissue, as compared with the model group. CONCLUSION: RUE could exhibit anti-angiogenic and pro-apoptotic effects against H22 hepatoma cells in mice, and its anti-angiogenic mechanism is probably related to down-regulation of VEGF, VEGFR2 and HIF-1α proteins.
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Background and purpose:Quinonoids can change the cell cycle distribution of tumor cells, and effect the radiosensitizing. This study aimed to investigate the radiosensitization effects, cell cycle and apoptosis of cryptotanshinone on H22 hepatoma-bearing mice. Methods:The mouse hepatoma H22 model was established, then divided into blank control group, irradiation alone group, high dose of cryptotanshinone group, cryptotanshinone (low, medium and high)+IR groups. After irradiated, observed the growth of tumor’s conditions, record epigenetic tumor irradiation time, calculated the delay time of tumor growth and enhancement factor (EF). After 22 days, mice were killed, stripped tumor, and calculated the inhibition rate. The cell cycle distribution and apoptosis were measured by lfow cytometry. Results:Cryptotanshinone (low, medium and high) groups inhibited the tumor growth better than the blank group, and had the signiifcant radiosensitizing effect. The enhancement factor was 1.22, 1.43,2.19, respectively. Cells were treated with cryptotanshinone which had significant effects on cell cycle, and induced apoptosis, which indicated signiifcant G2/M phase arrest and a decrease in S phase. Conclusion:Cryptotanshinone inhibited the tumor growth and had the radiosensitizing effects on H22 hepatoma-bearing mice. One of the mechanism may be that it might make significant G2/M phase arrest and S phase decreased, and induced apoptosis. So cells were more sensitive to radiation.
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AIMS@#To investigate the antitumor effects of extracts from Oxytropis falcata on human hepatocellular carcinoma SMMC-7721 cells in vitro and in transplanted murine H22 tumors in vivo.@*METHODS@#Cell proliferation, cell cycle distribution and apoptosis in SMMC-7721 cells were determined and tumor growth inhibition in H22 tumors was investigated. Cell cycle distribution was analyzed by flow cytometry with propidium iodide (PI) and Annexin V-FITC/ PI double staining.@*RESULTS@#MTT assay revealed that essential oil and flavonoids of O. falcata (named EOOF and FOF) inhibited proliferation of SMMC-7721 cells in a dose-dependent manner. The IC50 value of EOOF and FOF were 0.115 and 0.097 mg·mL(-1), respectively. Cell cycle was arrested at G(1) phase, and induction of apoptosis occurred in SMMC-7721 cells when subjected to FOF. Growth inhibition in H22 solid tumors transplanted mice was significantly pronounced after being treated with FOF, and the inhibition ratio were 56.1% and 70.8% at the concentration of 30 and 60 mg·kg(-1).@*CONCLUSION@#The results suggest that FOF promotes apoptosis in SMMC-7721 cells and inhibits H22 tumor growth, resulting in a potential antitumor effect on hepatic cancer.
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Animals , Humans , Male , Mice , Antineoplastic Agents, Phytogenic , Apoptosis , Carcinoma, Hepatocellular , Drug Therapy , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Drugs, Chinese Herbal , Growth Inhibitors , Liver Neoplasms , Drug Therapy , Mice, Inbred ICR , Oxytropis , ChemistryABSTRACT
Objective To investigate the effects of overexpression of heat shock protein 22(HSP22) in hematopoietic malignant tumor cell lines.Methods A lentiviral system was used to mediate transduction of HSP22 complementary DNA-containing expression vector or empty vector into K562 and Namalwa cells.The transduction effeciency was tested by fluorescence microscope scan and flow cytometry.Semi-quantitative RT-PCR and Western blot were used to identify the expression levels of HSP22 mRNA and protein.Growth curve analysis,cell cycle analysis,colony-forming assay,tumor growth in nude mice and apoptosis analysis were used to evaluate the role of HSP22 in K562 and Namalwa cells.Results Lentivector expression systemmediated delivery of HSP22 into K562 and Namalwa cells can inhibit colony forming of K562 and Namalwa cells,the average numbers of colonies per well were 108,72,125 and 80 for K562-V,K562-H,Namalwa-V and Namalwa-H respectively (P =0.000 16 and 0.000 37 for K562 and Namalwa respectively).HSP22 transduction can also inhibit proliferation of Namalwa cells in vitro (P =0.015,0.042 and 0.048 for day 5,6 and 7 respectively) and K562 cells in vivo (P =0.022 for day 21).No significant difference in cell cycle and apoptosis was found in K562 and Namalwa cells compared with controls (all P ＞ 0.05).Conclusion Overexpression of HSP22 could inhibit the growth of hematopoietic malignant tumor cell lines K562 and Namalwa.
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Objective: To investigate the antitumor effect of volatile oil from Sinapis Albae Semen (VOSAS) on H22-bearing mice and to determine the mechanism. Methods: To establish the H22 implanted hepatocellular carcinoma animal model which was used to analyze the effect of VOSAS on the growth of transplanted tumor. Mice were divided into five groups 24 h after modeling: model, cytoxan (CTX, 25 mg/kg) positive control, low-, mid-, and high-dose (20, 40, and 80 mg/kg) VOSAS groups. The mice were ip administered once daily for 10 d. Morphological changes in H22 solid tumor cells were observed by both Hematoxylin-eosin (HE) and acridine orange (AO) staining. The expression of Bax and Bcl-2 in the tumor tissue was determined using immunohistochemistry. Results: VOSAS could inhibit the tumor growth and extend the life span of H22-bearing mice (P < 0.01); and it could also raise the expression of Bax while suppress the expression of Bcl-2; the antitumor effect of VOSAS on H22-bearing mice demonstrated a good dose-effect relationship, but the high-dose group of the volatile oil has obvious toxicity and side effects on the mice. Conclusion: VOSAS could inhibit the growth of H22 tumor cells and the mechanism may be related to up-regulating the expression of Bax and down-regulating the expression of Bcl-2, and the induction of apoptosis.
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Interleukin-22 (IL-22) is a new kind of eytokine discovered in 2000. The major sources of IL-22 are activated T1 -cells and NK-cells. Tissue cells at outer body barriers, i.e. of the skin, kidney, the di-gestive and respiratory systems all highly express IL-22R or respond to IL-22. IL-22 functions by promoting the anti-microbial defense, inducing phase reactants, protecting against damage and enhancing natural immu-nity. Furthermore, IL-22 mediates the proliferation, differentiation and apoptesis in cancer cells, that gives us a new idea about tumor therapy.
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Objective: To investigate the apoptosis-inducing effect of matrine on murine hepatocarcinoma cell line H22 in vivo and in vitro and explore the underlying mechanisms. Methods: The H22 cell apoptosis induced by matrine at the early stage was detected with Annexin V-FITC/PI double staining assay, The expressions of Bcl-2 and Bax proteins in H22 cells as well as in the BALB/c H22 xenograft tumor tissues were detected using immunohistochemical method. Transmission electron microscopy (TEM) was used to observe the ultramicro-structure alterations of H22 xenograft tumor cells in BALB/c mice. The effect of matrine on the kinetics of tumor growth after subcutaneous injection of H22 cells in BALB/c mice was observed and the tumor inhibition rate was calculated. Results: Annexin V staining detected early apoptosis of H22 cells after treatment with matrine 1.0 mg/mL and 1.5 mg/mL for 48 h. The apoptotic rates were 11.71% and 17.86%, respectively, both of which higher than that of control groups (P <0.05). The tumor inhibition rate was above 60% after matrine treatment. Immunohistochemistry analysis revealed that matrine increased Bax protein expression and reduced Bcl-2 protein expression in both H22 cells in vitro and xenograft tumor tissues in vivo. TEM demonstrated the existence of apoptotic cells and apoptotic bodies in H22 xenograft tumor tissues after matrine treatment. Conclusion: Matrine significantly suppresses tumor growth and induced apoptosis both in vitro and in vivo. The apoptosis-inducing effects is related with up-regulation of Bax protein and down-regulation of Bcl-2 protein.
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[Objective]To study the inhibitory effect of the extract of Rhizoma Amorphophallus,we set up the Kunming mices which were vaccinated with H22 tumor as experimental model,and explore the possibility mechanism of them.[Method]We adopted the mices vaccinated with H22 tumor as experimental model to observe the influence of different extracts on the antitumor ratio,and the cooperating antitumor effect together with 5-Fu.Using flow cytometry to detect the effect of petroleum ether extract on apoptotic cells.Using immunohistochemical method to observe the influence of this extract to the expression of Survivin and Bax(Bcl-2 associated protein x).[Result]The extract of petroleum ether of Rhizoma Amorphophalli had inhibitory effect of the growth on mice vaccinated with H22 tumor,and the possible mechanism was the downregulation of Survivin expression and upregulation of Bax expression to induce apoptosis of tumor cell.
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This study was performed to investigate mistletoe extract-induced apoptosis in oral squamous cell carcinoma. In vivo study, HN22 cells were xenografted in nude mice. After tumor was experimentally induced, mistletoe extract was directly injected on the tumor mass. The specimens were evaluated using light and transmission electron microscopes. In vitro study, HN22 cells were cultured and exposed to mistletoe extract. The cells were evaluated using transmissin electron microscope. To evaluate apoptotic cells, flow cytometric analysis was done. The results were obtained as follows: 1. Light microscopic view of tumor mass showed necrosis at 2-4 weeks. 2. Transmission electron micrographs of tumor mass showed apoptosis and necrosis. 3. In TEM view of cell lines, necrosis and apoptosis were shown with mistletoe extract at 300microgram/ml, apoptosis was shown with mistletoe extract at 100microgram/ml. 4. In flow cytometric analysis, early and late apoptosis was shown when using caspase-3Ab and annexin-V, but no significant change was noted when using mebstain and Apo2.7 Ab. In this study, mistletoe extract induced necrosis and apoptosis in the tumor mass was induced by HN22 cells, early and late apoptosis in vitro study. Mistletoe extract was likely to induce cell death in oral squamous cell carcinoma through apoptosis.
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Animals , Mice , Apoptosis , Carcinoma, Squamous Cell , Cell Death , Cell Line , Heterografts , Mice, Nude , Mistletoe , NecrosisABSTRACT
Objective:The purpose of this study is to evaluate the effects of a single-chain antibody against death receptor 5 (ZF1) on tumor growth and survival in murine H22 hepatocellular carcinoma tumor model.Methods:Killing effect of ZF1 on H22 cells was analyzed by MTT assay in vitro. The apoptosis rate of H22 cells induced by ZF1 was detected using Flow Cytometry assay. The transplanted model of H22 tumor was developed in mice. The mice were randomly divided into four groups, PBS group, ZF1 group, EPI group and combined treatment group of ZF1/EPI. Tumor growth and body weight changes were observed. After treatment over 13 days, the tumor tissue for HE staining and TUNNEL assay was performed to detect apoptosis.Results:The results showed that ZF1 could inhibit growth of H22 cells in a dose dependent manner. The growth inhibition rate was up to 84.5%. The results showed that ZF1 alone or in combination with ZF1/EPI, the tumor growth was significantly inhibited. HE staining and TUNNEL analysis showed that ZF1 could effectively induce apoptosis of tumor cells without toxic effects, especially in ZF1/EPI combined treatment group.Conclusion:It is showed that ZF1 induces a good inhibition on the proliferation of H22 cell, especially in combined treatment group of ZF1/EPI.
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Objective:To study the anti-tumor effects of matrine on murine hepatocarcinoma cell line H 22 in vitro and in vivo .Methods:Cytotoxicity effect of matrine on cultured H 22 cells was determined by MTT assay in vitro .Apoptosis of cultured H 22 cells induced by matrine were determined with Annexiin V-FITC/PI affinity assay.Tumor-bearing BALB/C mice were used to observe the inhibitory effects of matrine on H 22 cells in vivo .The ultra micro-structured changes of H 22 cells were observed by transmission electron microscope in tumor-bearing BALB/C mice after treated with matrine.The expressions of Bcl-2 & Bax protein were detected by immunohistochemical technique and the staining densities of Bcl-2 & Bax protein were quantitated through computerized image processing.The data were analyzed with one-way ANOVA by means of SPSS 10.0.Results:Matrine could obviously inhibit the growth and induce the apoptosis of cultured H 22 cells.Matrine also had significant anti-tumor activity,on mice bearing H 22 hepatoma.The inhibitory rates were above 60.7% and 62.5% in higher and lower doses groups,respectively ( P
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Objective:To explore the availability for tumor-derived gp96 to induce specific CTL activity of splenocytes in vitro.Methods:gp96 purified by the techniques for protein extraction and identified by SDS-PAGE gel electrophoresis and Western blot method; CD8+T cell induced by gp96 and CTL activity detected by flow cytometry, immunofluorescence technic and CCK-8 assay.Results:gp96 was identified by SDS-PAGE and Western blot; Analysis with FCM showed that the number of CD8+T cells(nearly 70%) was obviously increased after pulsed with gp96-peptide complexes as compared with that of control groups(35%,26%, P