Prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay

AIM： To investigate the prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and rnicronucleus assay. METHODS： Clonogenic assay, flow cytometry, and CB micronuclei assay were used to survey the cell survival rate, radiation-induced apoptosis and rnicronucleus frequency of hepatocarcinorna cell lines SMMC-7721, HL-7702, and HepG2 after being irradiated by X-ray at the dosage ranging 0-8 Gy. RESULTS： After irradiation, there was a dose-effect relationship between rnicronucleus frequency and radiation dosage among the three cell lines （P〈0.05）. A positive relationship was observed between apoptosis and radiation dosage among the three cell lines. The HepG2 cells had a significant correlation （P〈0.05） but apoptosis incidence had a negative relationship with rnicronucleus frequency. There was a positive relationship between apoptosis and radiation dosage and the correlation between 5MMC-7721 and HL-7702 cell lines had a significant difference （P〈0.01）. After irradiation, a negative relationship between cell survival rate and radiation dosages was found among the three cell lines （P〈0.01）. There was a positive relationship between cell survival rate and rnicronucleus frequency （P〈0.01）. No correlation was observed between apoptosis and cell survival rate. CONCLUSION： The radiosensiUvity of hepatocarcinoma cells can be reflected by apoptosis and rnicronuclei. Detection of apoptosis and rnicronuclei could enhance the accuracy for predicting radiosensitivity.

Protein profile of human hepatocarcinoma cell line SMMC-7721:Identification and functional analysis

AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS： Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis （2DE）. The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION： Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

EXPRESSION OF ONCOGENES DURING INDUCED DIFFERENTIATION OF HUMAN HEPATOCARCINOMA CELL LINE

There was no detectable expression of c-fos,but a little c-myc,high c-fms and mederate high IGF-ⅡmRNA in the untreated human hepatocarcinoma cell SMMC- 7721.After treatment with 10 μmol/L retinoic acid or 0.5 mmol/L dibutyryl cyclic-3',5'adenosine monophosphate(db-cAMP),the c-fos was transiently expressed within 20- 60mins.If the treatment of RA or db-cAMP prolonged to 1-5 days, the transcriptions of c- myc were increased,reaching the highest level on the 2nd and 4th day.Simultaneously the transcriptions of c- fms and IGF- Ⅱwere gradually decreased.On the 5th day of the treatment,c-fms and IGF-ⅡmRNA were decreased to 32% and 14%respectively of the control (untreated cell) value by RA,and 35% and 22%respectively by db-cAMP.The biological significance of the above mentioned results was discussed.

EFFECT OF ACUPUNCTURE ON IL 2-IFN-NKC IMMUNOREGULATORY NETWORK IN MICE WITH TRANSPLANTED HEPATOCARCINOMA

: To study the effect of acupuncture on interleukin 2 (IL 2)-interferon(IFN)-natural killer cells (NKC) immunoregulatory network of mice with transplanted hepatocarcinoma(HAC) in order to provide new evidence for acupuncture treatment of hypofunction of immune system. Methods:The 28 HAC-vaccinated BALB/C mice are randomly divided into control group (n=14) and acupunctrue group (n=14). In the latter group bilateral'Dazhui' (BL 11) and 'Zusanili' (ST 36) which are located according to the same positions indicated in Comparative Anatomy of Macro-animals, are needled once every day, twelve sessions altogether. Twenty-four hours after the needling treatment the mice are killed and the spleen is taken out to be made into cell suspension for assaying concentrations of IL2 (MTT method) and NKC (colorimetric method) respectively. Obital Serum IFN is determined by using (CPE microplate staining), and the tumor mass is taken out and balanced with an analytical balance (1/10000) to calculate the tumor inhibition rate according to the formula. Results: The tumor weight of the mice of aupuncture group is obviously decreased (inhibition rate 43.06%) while the activity of the IL2 and NKC, and the IFN tiler are increased greatly, which have significant differences compared with those of control group (P>0.01). Conclusion:The results of the study show that acupuncture can strengthen the positive immunoregulatory function of the IL2 -IFN-NKC network in immune hypofunction mice bearing HAC.

Role of CD80 in stimulating T lymphocyte activation

AIM： To observe biological characteristics of hepatocarcinoma cells before and after CD80 transfection and to compare the effect of CD80-transfected hepatocarcinoma cells on T lymphocyte activation. METHODS： Retro virus vector carrying CD80 gene was transfected into HepG2 cells to establish CD80transfected hepatocarcinoma cells （HepG2/hCD80）. Flow cytometry （FCM） was performed to detect CD80 expression in the transfected cells. RT-PCR was used to evaluate CD80 expression at mRNA level. In the presence of anti-CD3 mAb, the proliferation of T lymphocyte was observed by M＇n＇. Meanwhile, the expression of activated molecule marker CD25 was analyzed through FCM. RESULTS： A stable cell line HepG2/hCD80 expressing the human CD80 was established. Growth curve showed that the molecule CD80 could obviously decrease the growth of tumor cells. HepG2/hCD80 was evidenced to have a potency to enhance T cell proliferation and upregulate CD25 expression. CONCLUSION： CD80 transfection can lower malignant phenotype of hepatocarcinoma cells. CD80 transfection has a down-regulatory effect to activated T cells in vitro.

Relationship of HepG2 cell sensitivity to continuous low dose-rate irradiation with ATM phosphorylation

Objective: To investigate the change of ATM phosphorylation in HepG2 cells and its effect on HepG2 cell survival under a continuous low dose-rate irradiation. Methods: HepG2 cells were exposed to equivalent doses of irradiation deliv- ered at either a continuous low dose-rate (7.76 cGy/h) or a high dose-rate (4500 cGy/h). The ATM phosphorylated proteins and surviving fraction of HepG2 cell after low dose-rate irradiation were compared with that after equivalent doses of high dose-rate irradiation. Results: The phosphorylation of ATM protein was maximal at 0.5 Gy irradiation delivered at either a high dose-rate or a continuous low dose-rate. As the radiation dose increased, the phosphorylation of ATM protein decreased under continuous low dose-rate irradiation. However, the phosphorylation of ATM protein was remained stable under high dose-rate irradiation. When the phosphorylation of ATM protein under continuous low dose-rate irradiation was equal to that under high dose-rate irradiation, there was no significant difference in the surviving fraction of HepG2 cells between two ir- radiation methods (P > 0.05). When the phosphorylation of ATM protein significantly decreased after continuous low dose-rate irradiation compared with that after high dose-rate irradiation, increased amounts of cell killing was found in low dose-rate irradiation (P < 0.01). Conclusion: Continuous low dose-rate irradiation increases HepG2 cells radiosensitivity compared with high dose-rate irradiation. The increased amounts of cell killing following continuous low dose-rate exposures are associated with reduced ATM phosphorylated protein.

Therapeutic Effects of All Trans-retinoitc Acid Combined with Transarterial Chemoembolization on Walker-256 Hepatoma in Rats

In order to investigate the inhibitory effects of all trans-retinoitc acid(ATRA)on differentiation and apoptosis of Walker-256 hepatocellular carcinoma cells and the therapeutic effects of ATRA combined with transarterial chemoembolization(TACE)on rat Walker-256 transplanted hepatocarcinoma,Walker-256 hepatocarcinoma cell lines were treated with ATRA at different concentrations.After culture for 48h,the inhibitory rate of cell proliferation was determined by MTT assay;the changes of Fas and Bcl-2mRNA expres...

Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L_(6))

In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells,a new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(Hca-P/L_(6))was established and the effect of curcumin on biological behavior of Hca-P/L_(6) was observed.Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential(Hca-P)was subcutaneously inoculated into the medioventral line of a mouse 615 and thefirst generation of metastatic tumor cells of inguinal lymph node(Hca-P/L_(1))was obtained.Then,Hca-P/L_(1) was screened by the route of mouse foot pad subcutaneously!lymph node!scale-up culture in vitro!mouse foot pad subcuta-neously forfive times consecutively.The sensitivity of two murine ascites hepatocarcinoma cell lines(Hca-P and Hca-P/L_(6))and two anchorage-dependent human hepato-carcinoma cell lines(SMC7721 and HepG_(2))to curcumin were studied by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay after these cells had been pretreated by curcumin at the concentration of 15–240μmol/L for 48 h.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15μmol/L in vitro,the effect of curcumin against cell proliferation of Hca-P and Hca-P/L6 was observed by inverted micro-scope,cell growth curve and cell population doubling time;the effects of curcumin on cell cycles of Hca-P/L6 and Hca-P were studied byflow cytometry(FCM).The results showed Hca-P/L_(6) spreading to the lymph nodes at multiple sites in mice was screened from Hca-P.The lymph node metastatic rate was 100%.Curcumin had significant growth inhibiting effect on both murine ascites and human hepatocarcinoma cell lines in a dose-dependent manner(P<0.05).At concentrations of 30–120μmol/L,curcu-min had more inhibition on murine ascites hepatocarci-noma cell lines than on human anchorage-dependent hepatocarcinoma cell lines.At concentrations of 60–240μmol/L,curcumin had more inhibition on Hca-P/L_(6) with the 50%inhibitory concentration(IC50)of 51.48μmol/L than on Hca-P with IC50 of 90.87μmol/L.After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days,the proliferations of Hca-P/L_(6) and Hca-P were inhibited(P<0.05)in a time-dependent manner(P<0.01)and the population doubling time of Hca-P/L6 and Hca-P was prolonged(P<0.01),and curcumin had more inhibition on Hca-P/L6 than on Hca-P(P<0.05).After pretreatment by 15μmol/L curcumin for 48 h,the morphous of Hca-P/L_(6) was influenced more seriously than that of Hca-P and the cell cycle was redistributed with Hca-P/L6 being blocked in the S phase and Hca-P in the S and G_(2)/M phases.Hca-P/L_(6) was validated to be more sensitive to curcumin than Hca-P.Hca-P/L_(6) is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.