Effect of melittin on programmed necrosis of human hepatocellular carcinoma cell line SMMC￣7721 / 解剖学报

Objective To investigate the lethal effect of melittin on human hepatocellular carcinoma cell line SMMC-7721 and its possible mechanism. Methods MTT assay was used to investigate the killing effect of different concentrations melittin on human hepatocellular carcinoma cells SMMC-7721. Meanwhile, the inhibitory effect of specific programmed necrosis inhibitors necrostain-1(Nec-1) on melittin killing SMMC-7721 cells was detected. Programmed cell necrosis observed by Hoechst 33342 and PI double staining. The necrotosis rate was detected by flow cytometry. Ultrastructural changes of cell was detected by transmission electron microscopy. The expression of receptor interacting protein 1(RIP1) was detected by Western blotting. Results Compared with the control group, the proliferation activity of SMMC-7721 cells was significantly decreased after treatment with different concentrations of melittin for 24 hours (P < 0. 05) . Cells stained in dark blue and red. Cell membrane integrity was destroyed, organelle swelling, organelle membrane was destroy, that demonstrates cell was necrosis. Westen blotting result showed an increased proportion of RIP1 expression in SMMC-7721 cells. Compared with the melittin group, cell proliferation activity was significantly increased, cell necrotosis rate was decreased, and intracellular RIP1 expression was down-regulated in the Nec-1 pretreatment group. Conclusion Melittin induces cell death in SMMC-7721 cells through the RIP1-mediated programmed necrosis pathway.

The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line

Purpose: This trial studies the feasibility and potential utility of stereotactic body radiation therapy in patients with unresectable liver metastasis. Aims: (1) The aim of this study is to assess the local response of the liver lesions poststereotactic body radiation therapy regarding number and size of lesions and (2) to evaluate the toxicity to organ (s) at risk. Materials and Methods: A total of 15 patients were enrolled in this study from November 2014 to October 2015. The inclusion criteria for this study were patients having 1–3 liver metastasis from any solid tumor except germ cell tumor or lymphoma with no evidence of progressive disease (PD) outside the liver. A planning four dimensional-computed tomography (CT) scan was taken. Planning target volume was generated by giving margin of 5 mm. Dose prescribed was 36 Gy in 3#. Response was defined by CT abdomen done at 3 and 6 months poststereotactic body radiation therapy as per RECIST guideline (v1.1). Results: At 3 months poststereotactic body radiation therapy, five patients had partial response, five patients had stable disease, and five patients had PD as per RECIST criteria. Out of 20 assessable lesions, 16 were controlled at 3 months poststereotactic body radiation therapy. The actuarial local control rate was 86% at 3 months and 77% at 6 months poststereotactic body radiation therapy. The median progression free survival was 7 months. Two patients experienced Grade 2 gastric toxicity and one patient experienced Grade 2 small bowel toxicity. No cases of radiation-induced liver disease were observed. Conclusions: This trial examines the feasibility of stereotactic body radiotherapy to liver metastasis in the Indian scenario. It shows excellent tolerability and is a safe therapeutic option for inoperable patients, showing good local control

miR-375 affects the proliferation and invasion of hepatoma cells via targetingYAP / 中国肿瘤生物治疗杂志

@# Objective: To investigate the mechanisms of miR-375 affecting the proliferation and invasion of hepatoma cells via targeting YAP (Yes-associated protein). Methods: The cancerous tissues and corresponding para-cancerous tissues of 70 patients with hepatocellular carcinoma (HCC) who underwent surgical resection at the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine from January 2015 to December 2016, as well as the hepatoma cell lines SMMC-7721, Hb611, HepG2 and BEL-7405 were collected for this study. qPCR method was used to detect the expression level of miR-375 in collected HCC tissues and different hepatoma cell lines; Dual luciferase reporter gene assay was used to verify the interaction between miR-375 and YAP; The relationship between miR-375 and clinicopathological features of HCC patients was also analyzed; MTT assay was used to detect the effect of miR375 on the proliferation of hepatoma cells; Transwell invasion assay was used to detect the invasive ability of hepatoma cells after inhibiting the expression of miR-375; Western blotting was used to detect the expression of YAP in HepG2 cells. The nude mouse model of subcutaneously transplanted xenograft was established, and the tumor volume and mass of transplanted hepatoma cells were detected after inhibiting the expression of miR-375. The expression of YAP in xenograft of nude mice was detected by immunohistochemistry and Western blotting. Results: The expression of miR-375 and YAP in HCC tissues was significantly higher than that in para-cancerous tissues (all P<0.05). The expression of miR-375 in HepG2 cells was the highest (P<0.05). miR-375 could specifically bind to the 3' UTR of YAP and regulate the expression activity of YAP. After inhibiting the expression of miR-375, the proliferation and invasion abilities of HepG2 cells were reduced (all P<0.05); The tumor volume and mass of transplanted xenografts were significantly reduced (both P<0.05); The expression of YAP protein in the transplanted xenografts was down-regulated (P<0.05). Conclusion: miR-375 plays an important role in the occurrence and development of liver cancer, and can influence the malignant biological behaviors of hepatoma cells by targeting and regulating the expression ofYAP.

Study on Synthesis and Anti-tumor Activity of 6 Kinds of Tectorigenin Mannich Base Derivatives / 中国药房

OBJECTIVE： To conduct structural modification of tectorigenin to search for new compounds with anti-tumor activity. METHODS： Tectorigenin was used as a lead compound， and then added into amine reagents as ethanolamine， methylamine， ethylamine， dimethylamine， diethylamine， n-propylamine and formaldehyde solution. Tectorigenin Mannich base derivatives were synthesized by mannich reaction with as the lead compound. The structures of the derivatives were identified according to IR， UV， MS and NMR data. Solubility of tectorigenin and its derivatives were investigated by solubility test method. MTT assay was used to investigate the inhibitory effects of tectorigenin and its derivatives on the proliferation of human colon cancer cell line HCT116， human lung cancer cell line A549 and human hepatoma cell line HepG2， and half inhibitory concentration （IC50） was calculated. The inhibition rate of tectorigenin and its derivatives （100 mg/kg） on H22 hepatoma-bearing mice in vivo was studied. RESULTS： Totally of 6 kinds of tectorigenin mannich base derivatives were synthesized， such as 8-（N-hydroxyethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-methyl）-methyleneamino-5，7，4′-trihydroxy-6- methoxyisoflavone， 8-（N， N-diethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N， N-dimethyl）-methyleneamino- 5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-ethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-propyl）- methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone （compounds 1-6 in turn）. Compared with tectorigenin， the water solubility of six derivatives was significantly improved， and the solubility was 5-20 times higher than that of tectorigenin. IC50 of compounds 1， 3 and 5 to HCT116 cells were （34.82±3.27）， （16.21±4.13）， （33.12±3.25） μmol/L， which were stronger than that of tectorigenin [（45.23±5.74） μmol/L]； IC50 of compounds 1， 3 and 5 to A549 cells were （37.05±5.74）， （26.88±4.52）， （30.13±6.23） μmol/L， which were stronger than that of tectorigenin [（53.24±6.34） μmol/L]； IC50 of compounds 1， 3 and 5 to HepG2 cells were （23.74±1.45）， （18.96±2.34）， （30.95±2.87） μmol/L， which were stronger than that of tectorigenin [（48.98±2.58） μmol/L]. Compounds 1， 3 and 5 showed higher inhibition rates （55.51％， 57.20％ and 49.15％） than tectorigenin （33.05％） on H22 hepatoma-bearing mice， respectively. The other three compounds had no obvious advantage over tectorigenin in anti-tumor activity. CONCLUSIONS： In this study， compounds 1， 3 and 5 of six tectorigenin mannich base derivatives synthesized in this study have stronger antitumor activity than tectorigenin.

Inhibitory Effect of siRNA Expression Vector Inhibiting IGF2 Gene on the Proliferation of Hepatoma Cell Line Huh-7 / 中国药房

OBJECTIVE:To investigate the inhibitory effect of siRNA expression vector inhibiting human insulin-like growth factor 2(IGF2)gene on the proliferation of hepatoma cell line Huh-7. METHODS:siRNA expression vector pGL3-hAFP-hTERT-siRNA3(“siRNA3”)which inhibited IGF2 gene by dual promoter regulation of recombinant human alpha-foetoprotein(hAFP)and human telomerase reverse transcriptase(hTERT)was transfected into the Huh-7 cell and normal hepatocyte L-02,and then a nega-tive control group(vector pGL3-hAFP-hTERT)and a blank control group were set up. IGF2 mRNA expression was detected by re-al-time fluorescent quantitative polymerase chain reaction 48 h after transfection into the cells in all groups;the activity of the cells by the microplate reader 0,24,48 and 72 h thereafter;and the cell cycle and apoptosis by the flow cytometer 48 h thereafter,and the changes in the protein levels of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in the cell were detected by Western blot. RESULTS:Compared with the negative control group and blank control group,IGF2 mRNA expression in the Huh-7 cell transfected with siRNA3 was obviously weaker;at 48 and 72 h after transfection,the activity of Huh-7 cell signigicantly reduced, Huh-7 cells at G1 phase obviously increased and those at S phase markedly decreased;the occurrence of early,late and total apopto-sis in Huh-7 cells apparently increased,and the protein expression of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in cells significantly weakened,with statistically significance(P0.05). CONCLUSIONS:siRNA which inhibited IGF2 gene by dual promoter regulation of recombinant hAFP and hTERT can specially inhibit IGF2 gene expression and the prolifer-ation of Huh-7 cells,which may be involved with down-regulated protein expression of cell proliferation-associated gene PCNA, cell cycle control-associated genes Cyclin E2,Cyclin D2 and Cdc2 and apoptosis regulation-associated gene Bcl-2 as a result of down-regulated IGF2 mRNA expression and protein expres-sion.

Inhibition of proliferation of 5-FU combined with thermotherapy in human hepatoma cell line / 中国综合临床

Objective To investigate the inhibition rate of cell proliferation, cell apoptosis rate and their effects on the cell cycle proceeding of the SSMC7721 cell line when 5-FU combined with thermotherapy is induced into the cells, and then provide theoretical bases to the combined therapy of hepatocellular carcinoma. Methods The inhibition rate of cell proliferation was detected by the MTT under different conditions, the cell cycle proceeding and the cell apoptosis rate was detected by flow cytometry and the subcellular structure was detected by the electronmicroscope. Results The cell inhibition rate of the thermotherapy group, 5-FU group and the combinedgroup were 18.4% ,28. 3% and 52. 7% ,respectively. The inhibition rates in the latter two groups were significantly different to the thermotherapy group. The results of flow cytometry showed that the cell numbers increased in G1 stage decreased in S stage,and increased in G2/M stage;the cell apoptosis rate increased. There was significant difference between different groups(P ＜ 0.01 or P ＜0.05). The results of the electronmicroscop showed that the nuclear chromatins agglutinated in the borderline and the mitochondriums became swelled. Conclusions The 5-FU combined with thermotherapy could significantly improve the inhibition rate of cell proliferation, inhibit the cell cycle proceeding from G1 stage to S stage, and induce cells apoptosis and change the subcellular structures in the SSMC7721 cell line.

Effects of extracellular ATP on apoptosis of human esophageal carcinoma Eca-109 and hepatoma SMMC-7721 cells / 基础医学与临床

Objective To study the effects of adenosine triphosphate (ATP) on proliferation of human squamous esophageal carcinoma Eca-109 and hepatoma SMMC-7721 cells in vitro and the underlying mechanism. MethodsMTT assay was used to determine the proliferation of tumor cells. The AO/EB double stained cells were observed under fluorescence microscope. The effects of ATP on the cell cycle, apoptotic rate and apoptosis-related protein were detected by flow cytometry. Results ATP showed inhibitory effects on Eca-109 and SMMC-7721 cells at the concentration of 0.01～0.3 mmol/L. Exposure to 0.3 mmol/L ATP for 72 h, some of SMMC-7721 cells displayed morphological changes of apoptosis, but Eca-109 cells did not show the characteristics of apoptosis markedly. There was no significant change in the apoptotic rate and apoptosis-related protein of the two tumor cell lines treated with ATP 0.03, 0.1 and 0.3 mmol/L for 72 h respectively. The proportion of Eca-109 cells in G0/G1-phase of cellcycle was significantly increased, meanwhile the proportion of Eca-109 cells in S-phase and proliferation index value was significantly decreased by treatment with 0.3 mmol/L ATP. Conclusion ATP inhibited Eca-109 cell proliferation by changing the distribution of cell cycle phase, and its mechanism might not related to apoptosis, but for SMMC-7721 cell, the inhibition of cell proliferation induced by ATP was not related to the change in cell cycle.

Study of Mechanism of Apoptotic Signal Transduction in Human Hepatic Carcinoma Cell Lines Induced by TGF-?1 / 中国普外基础与临床杂志

Objective To investigate the apoptosis induced by TGF-?1 in human hepatic carcinoma cell lines and its relationship with p53 gene and Smad. Methods Three human hepatic carcinoma cell lines which involving in various status of the p53 gene were used in this study. TGF-?1-induced apoptosis in hepatic carcinoma cell lines was measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. To study the mechanism of TGF-?1-induced apoptosis, these cell lines were transfected with a TGF-?1-inducible luciferase reporter plasmid containing Smad 4 binding elements (SBE) and luciferase gene using Lipofectamine 2000, then treated with TGF-?1, relative luciferase activity was assayed. Results Of three cell lines studied with TUNEL assay, TGF-?1 induced apoptosis was observed in HepG2 cells (wild type p53). Huh-7 (mutant p53) and Hep3B (deleted p53) cell lines showed less apoptosis. Luciferase activity assay indicated that the response to TGF-?1 induction in HepG2 cells was increased dramatically but was not significant in Huh-7 and Hep3B cell lines. Conclusion HepG2 cells seem to be highly susceptible to TGF-?1-induced apoptosis compared with Hep3B and Huh-7 cell lines. Smad 4 is a central mediator of the TGF-?1 signal transduction pathway.

Inhibition of growth and induction of apoptosis in human hepatoma cell line by tanshinone ⅡA / 第三军医大学学报

Objective To investigate the effect of tanshinone ⅡA on growth and apoptosis in human hepatoma cell line BEL 7402 in vitro . Methods The human hepatoma cell line BEL 7402 was treated with tanshinone ⅡA at various concentrations for 72 h. Cytotoxicity was evaluated by MTT assay, apoptosis related alterations in morphology ascertained by cytochemical staining(Hoechst 33258) and transmission electron microscopy(TEM). Apoptotic rate was quantified by flow cytometry (FCM). Results Tanshinone ⅡA inhibited the growth of hepatoma cells in a dose dependent manner, with IC 50 values of 6.28 ?g/ml. After treatment with 1～10 ?g/ml tanshinone ⅡA for 72 h, BEL 7402 cell apoptosis with nuclear chromatin concentration and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. FCM analysis showed hypodiploid peaks on histogram and the apoptotic rates at 5 ?g/ml concentration for 12, 24, 36, 48 and 72 h were (2.32?0.16)%, (3.01?0.35)%, (3.87? 0.43 )%, (6.73?0.58)% and (20.85?1.74)% respectively, which were all significantly higher than that of control group (1.07?0.13)%. Conclusion Tanshinone ⅡA can induce the apoptosis of human hepatoma cell line BEL 7402 in vitro , which may be related to the mechanism of growth inhibition of the human hepatoma cell line.

5-FU Induces Apoptosis of Fas (+), HepG2 Cells Via Activation of Fas-mediated Caspase and Mitochondria Dysfunction / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: In order to investigate the role of Fas on the chemosensitivity of cancer cells in regards to chemotherapeutic agents, the Fas/FasL signaling pathway of apoptosis was explored in human hepatoma cells. MATERIALS AND METHODS: Fas expression of hepatoma cells including Chang, Huh7, HepG2, and Hep3B cells, was determined by RT-PCR and flow cytometry analysis. Cell viability was measured by MTT assay and apoptosis was assessed by DNA fragmentation assay. The catalytic activity of the caspase-family proteases including caspase-3, 6, 8, and 9 proteases, was tested using fluorogenic biosubstrates. The expression of apoptotic mediators including cytochrome c, PARP, and Bcl2 family proteins were measured from cytosolic and mitochondrial compartments. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. RESULTS: Fas mRNA was constitutively expressed in Chang and HepG2 as defined as Fas (+) cells, but not in Huh7 and Hep3B cells, defined as Fas (-) cells. Fas (+) cells were markedly sensitive to 5-FU whereas Fas (-) cells were resistant and able to survive. 5-FU increased Fas expression of Fas (+) HepG2 cells and simultaneously resulted in apoptotic death, characterized by the ladder-pattern fragmentation of genomic DNA. Moreover, it increased the catalytic activity of caspase-8 protease, which eventually cleaved the Bid into truncated Bid which translocated into mitochondria only in Fas (+) cells. It also increased the caspase-9 protease activity with Bax expression, cytosolic release of cytochrome c, and mytochondrial dysfunction only in Fas (+) HepG2 cells. Furthermore, 5-FU increased the enzymatic activity of caspase-3 protease with PARP digestion in HepG2 cells. CONCLUSION: 5-FU exerted cytotoxicity against hepatoma cells via activation of Fas-mediated apoptotic signaling including caspase cascades and mytochondrial dysfunction. Our data suggests that Fas may be an important modulator of the chemosensitivity of cancer cells vis- -vis anticancer chemotherapeutic agents.

Effects of Interleukin-2 Transduction into the Human Hepatoma Cell Lines Using Retroviral Vector / 대한암학회지

PURPOSE: We compared the differences between parent hepatoma cell lines and interleukin-2 (IL-2) transduced hepatoma cell lines using N2A/IL-2 and LNC/IL-2 retrovirus with regards to in vitro sensitivity to peripheral blood monocytes and in vivo tumorigenic activity. MATERIALS AND METHODS: Retroviral vector and producer cell line were constructed and IL-2 gene was transduced into the human hepatoma cell lines (SK-Hep1, Hep-G2, Hep-3B). IL-2 secretion after IL-2 transduction was measured by ELISA. MTT assay for in vitro sensitivity to peripheral blood monocytes was performed and the tumorigenic activity was observed in BALB/c mice and nude mice. RESULTS: IL-2 secretion was 186 pg/10 degrees C cells/24 hrs in SK-Hep1 cell line and was 147 pg/10 (6) cells/24 hrs in Hep-3B cell line with N2A/IL-2 retroviral vector and was 55,000 pg/10 (6) cells/24 hrs with LNC/IL-2 retroviral vector. In vitro sensitivity to peripheral blood monocytes was increased by 163.8~254% in IL-2 transduced hepatoma cell lines (Hep -3B/N2A/IL-2, Hep-G2/N2A/IL-2) compared to those of the parent cell lines. The tumorigenicity was observed in 1 of 3 BALB/c mice and all 3 nude mice. Simultaneous injection of 1 X 10 (7) cells of the parent cell line (Hep-3B) into the right flank and IL-2 transduced cell line (Hep-3B/LNC/IL-2) into the left flank of the three BALB/c mice and of 5 X 10 (5) cells for the three nude mice resulted in a complete regression of the IL-2 modified tumor cell line (Hep-3B/LNC/IL-2) in 3 weeks and the parent cell line (Hep-3B) in 5 weeks. But, after the injection of 1.5 X 10 (7) cells for other five nude mice, the tumor of the IL-2 transduced hepatoma cell line (Hep-3B/LNC/IL-2) was gradually disappeared, and the tumor of the parent hepatoma cell line (Hep-3B) was initially decreased and then gradually regrew 20 days later. CONCLUSION: IL-2 transduced hepatoma cell lines secreting IL-2 became more sensitive to peripheral blood monocytes and resulted in the increased antigenicity to the tumors formed by IL-2 transduced hepatoma cell line and parent cell line, and finally resulted in the regression of the tumors in experimental animals.

SYNERGISTIC EFFECT OF HYPERTHERMIA ON PHOTOCARCINORIN PHOTODYNAMlC THERAPY / 第二军医大学学报

In this paper, we describe a synergistic effect of hyperthermia on photocarcino-rin photodynamic therapy. The results suggest that the synergistic action of hyperthermia and photodynamic reaction was markedy better than that of hyperthermia alone plus photocarcinorin alone. with hyperthermia treatment (42℃, 30 min) alone, the fraction of cells inactivated was 7%, while with photodynamic effect alone(1J/cm2) that was 30% (?5%).when the same dosage of irradiation was applied after a rise in temperature, the fraction of cells inactivated increased to 80% (?5%) and when temperature was raised to the same amount after irradiation the fraction of cells inactivated increased to 92%. It can be seen that hyperthermia enhances, the response of tumor cells to photocarcinorin photodynamic effect in a synergistic way.