A STUDY OF THE BIOLOGICAL CHARACTERISTICS OF HUMAN HEPATOMA XENOGRAFTS IN NUDE MICE AFTER IRRADIATION

Cell growth kinetics and changes in AFP in nude mice with human hepatoma xenografts were evaluated using the flow cytometry method. After receiving 10 Gy of radiation, the mice showed a marked delay in tumor growth; approximately 1 Gy of radiation caused a tumor growth delay of one day. Irradiation altered various phases of the cell cycle. An acute and temporary block of G2 cells was characteristic; FCM measurements demonstrated that about 58% of cells were blocked in the G2 phase and this blocking effect lasted 90 hours after an irradiation of 10 Gy. This indicated that human hepatoma xenografts in nude mice were quite sensitive to irradiation. It was also noted that the AFP decreased for 96 hours after irradiation. Changes in G2 cells after irradiation may be closely related to changes in AFP.

Effects of targeting magnetic drug nanoparticles on human cholangiocarcinoma xenografts in nude mice

BACKGROUND: Targeting is a new therapeutic tool for malignant tumor as a result of combining nanotechnology with chemotherapeutics. The aim of our study was to investigate the effects of magnetic nanoparticles enveloping a chemotherapeutic drug on human cholangiocarcinoma xenografts in nude mice. METHODS: The human cholangiocarcinoma xenograft model was established in nude mice with the QBC939 cell line. The nude mice were randomly assigned to 7 groups. 0.9% saline or magnetic nanoparticles, including high (group 2), medium (group 4) and low (group 5) dosages, were given to nude mice through the tail vein 20 days after the QBC939 cell line was implanted. Calculations were made 35 days after treatment in order to compare the volumes, inhibition ratios and growth curves of the tumors in each group. Mice in each group were sacrificed randomly to collect tumor tissues and other organs for electron microscopy and pathological examination. RESULTS: The high and medium dosage groups were significantly different from the control group (P<0.05). The tumor inhibition ratios for the high, medium and low dosage groups were 39.6%, 14.6% and 7.9%, respectively. The tumor growth curve of groups 5, 4, and 2 changed slowly in turn. The high and medium groups showed cell apoptosis under an electron microscope. CONCLUSION: Magnetic nanoparticles can inhibit the growth of human cholangiocarcinoma xenografts in nude mice.

Antitumor effects of artesunate on human breast carcinoma MCF-7 cells and IGF-IR expression in nude mice xenografts

Purpose： The objective of this study was to investigate the anti-tumor effects and analyze the mechanism of artesunate （ART） action on breast cancer in vivo using tumor transplanted nude mice. Methods： The human breast tumor cell line MCF-7 was transplanted into nude mice, and the animals were treated with various doses of ART alone or in combination with cyclophosphamide （CTX） or normal saline （NS）. The tumor inhibitory effects were observed and compared, and the ultrastructural morphology of the transplanted tumor cells was observed by electron microscopy. The apoptosis rates and cell cycle status were detected by flow cytometry （FCM）. The expression of apoptosis-related proteins p53, Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry and IGF-IR was detected by western blot. The expression correlation for these proteins was also analyzed. Results： The tumor inhibition rates in the low dose ART group, high dose ART group, CTX group and combined drug therapy group were （24.39±10.20）%, （40.24±7.02）%, （57.01±5.84）% and （68.29±5.1）%, respectively. The cell cycle was arrested in phase G0/Gt after treatment with ART. The expression of Bcl-2 was significantly reduced, and the expression levels of Bax and Caspase-3 were significantly increased in the ART group compared to the negative control saline group. There was no significant difference detected in p53 expression. The Bcl-2 level was negatively related to Bax and Caspase-3. The western blotting results showed IGF-IR downregulation. Conclusions： ART inhibits the growth of MCF-7 breast tumor cell xenografts in nude mice. The anti-tumor mechanism of ART for human breast carcinoma in nude mice might be correlated with the alteration of apoptosis related protein expression, which may further induce apoptosis and inhibit cell proliferation.

Effect of antidepressants on body weight, ethology and tumor growth of human pancreatic carcinoma xenografts in nude mice

AIM: To investigate the effects of mirtazapine and fluoxetine, representatives of the noradrenergic and specific serotonergic antidepressant (NaSSA) and se- lective serotonin reuptake inhibitor (SSRI) antidepres- sant respectively, on body weight, ingestive behavior, locomotor activity and tumor growth of human pancre- atic carcinoma xenografts in nude mice. METHODS: A subcutaneous xenograft model of hu- man pancreatic cancer cell line SW1990 was estab- lished in nude mice. The tumor-bearing mice were ran- domly divided into mirtazapine group [10 mg/(kg·d)], fluoxetine group [10 mg/(kg·d)] and control group (an equivalent normal saline solution) (7 mice in each group). Doses of all drugs were administered orally, once a day for 42 d. Tumor volume and body weight were measured biweekly. Food intake was recorded once a week. Locomotor activity was detected weekly using an open field test (OFT). RESULTS: Compared to the fluoxetine, mirtazapine significantly increased food intake from d 14 to 42 and attenuated the rate of weight loss from d 28 to 42 (t = 4.38, P < 0.05). Compared to the control group, food intake was significantly suppressed from d 21 to 42 and weight loss was promoted from d 35 to 42 in the fluoxetine group (t = 2.52, P < 0.05). There was a significant difference in body weight of the mice after removal of tumors among the three groups. The body weight of mice was the heaviest (13.66 ± 1.55 g) in the mirtazapine group and the lightest (11.39 ± 1.45 g) in the fluoxetine group (F(2,12) = 11.43, P < 0.01). The behavioral test on d 7 showed that the horizontal and vertical activities were significantly increased in the mirtazapine group compared with the fluoxetine and control groups (F(2,18) = 10.89, P < 0.01). These effects disappeared in the mirtazapine and fluoxetine groups during 2-6 wk. The grooming activity was higher in the mirtazapine group than in the fluoxetine group (10.1 ± 2.1 vs 7.1 ± 1.9 ) (t = 2.40, P < 0.05) in the second week. There was no significant difference in tumor vol- ume and tumor weight of the three groups. CONCLUSION: Mirtazapine and fluoxetine have no effect on the growth of pancreatic tumor. However, mirtazapine can significantly increase food intake and improve nutrition compared with fluoxetine in a pan- creatic cancer mouse model.

Effects of recombinant human growth hormone on growth of human gastric carcinoma xenograft model in nude mice

AIM： To study effects of recombinant human growth hormone （rhGH） on growth of a human gastric carcinoma cell in vivo. METHODS： Experimental mice were divided into control group, rhGH group, oxaliplatin （L-OHP） group and rhGH＋L-OHP group. Cultured human gastric carcinoma cells BGC823 were inoculated into right axilla of nude mice and carcinoma xenograft model was established successfully. Inhibitory rate of xenograft tumor growth was estimated by measuring tumor volume; expression of proliferating cell nuclear antigen （PCNA）, Bax and Bcl-2 proteins of xenograft tumor was detected using immunohistochemical S-P method. RESULTS： Tumor growth inhibitory rate, the positive expression rate of PCNA, Bax and Bcl-2 were 49.3%, 58.2%, 65.2% and 59.2% in rhGH＋L-OHP group respectively; 46.6%, 62.5%, 59.7% and 64.7% in L-OHP group; 5.0%, 82.7%, 23.2% and 82.2% in rhGH group and 0, 77.8%, 23.5% and 80.3% in control group. There was significant difference between rhGH＋L-OHP group （or L-OHP group ） and control group or rhGH group （P 〈 0.05）, whereas there were no significant differences （P 〉 0.05） between L-OHP group and rhGH＋L-OHP group and between rhGH group and control group. CONCLUSION： rhGH does not accelerate the proliferation of human gastric cancer cell in vivo.

Effects and possible anti-tumor immunity of electrochemotherapy with bleomycin on human colon cancer xenografts in nude mice

AIM: To evaluate the anti-tumor effects and possible involvement of anti-tumor immunity of electrochemotherapy (ECT) employing electroporation and bleomycin in human colon cancer xenografts in nude mice, and to establish the experimental basis for clinical application of ECT. METHODS: Forty nude mice, inoculated subcutaneously human colon cancer cell line LoVo for 3 wk, were allocated randomly into four groups: B+E+ (ECT), B+E- (administration of bleomycin alone), B-E+ (administration of electric pulses alone), and B-E- (no treatment). Tumor volumes were measured daily. The animals were killed on the 7* d, the weights of xenografts were measured, and histologies of tumors were evaluated. Cytotoxicity of spleen natural killer (NK) and lymphokine-activated killer (LAK) cells was then assessed by lactic dehydrogenase release assay. RESULTS: The mean tumor volume of group B+E+ was statistically different from the other three groups after the treatment (F= 36.80, P<0.01). There was one case of complete response, seven cases of partial response (PR) in group B+E+, one case of PR in group B+E- and group B-E+ respectively, and no response was observed in group B-E-. The difference of response between group B+E+ and the other three groups was statistically significant (X2 = 25.67,P<0.01). Histologically, extensive necrosis of tumor cells with considerable vascular damage and inflammatory cells infiltration were observed in group B+E+. There was no statistical difference between the cytotoxicity of NK and LAK cells in the four treatment groups. CONCLUSION: ECT significantly enhances the chemosensitivity and effects of chemotherapy in human colon cancer xenografts in nude mice, and could be a kind of novel treatment modality for human colon cancer. The generation of T-cell-dependent, tumor-specific immunity might be involved in the process of ECT.

RADIOIMMUNOLOCALIZATION OF XENOGRAFTED HUMAN GASTRIC ADENOCARCINOMA WITH ^(131)I-LABELED MONOCLONAL ANTIBODY RWS_(4) IN NUDE MICE

The monoclonal antibody (MAb) RWS4 specific to membrane-associated antigen of human gastric adenocarcinoma was purified by protein A-Sepharose 4B affinity chromatography and labeled with 131I by chloramine-T method. 131-RWS,, was injected (65 μCi/10μg/0.2 ml, intraperitoneally) into the stomach cancer-bearing nude mice (solid tumor about 1 cm in diameter), and its biodistribution was studied by SPECT and gamma-counter over a peroid of 7 days. A clear image of transplanted tumor was observed on the 4th day, and the image became more clear on the 6th day. After SPECT scanning, the animals were killed on the 3rd to 7th day separately and radioactivity was detected in various organs. The ratios of T/NT were calculated. The results were shown as follows: tumor/blood, was 3.41±0.29 on the 6th day and the tumor/other organs (liver, spleen, stomach, lung, heart, kidney and brain etc.) were>3. The specificity of the 131I-RWS4 was 7.74±0.65.

Inhibitory effects of transfected Bcl-XL antisense oligodeoxynucleotide to proliferation and growth in esophageal carcinoma cell line and human esophageal carcinoma xenograft of nude mice

Objective： The aim of our study was to investigate the biological effects of Bcl-XL antisense oligodeoxynucleotide （ASODN） transfected into cultured esophageal carcinoma cells and human esophageal carcinoma xenograft in nude mice. Methods： Cationic liposome-mediated ASODN was used to transfect esophageal carcinoma cells. RT-PCR, Western blot, MTT assay, flow cytometry, and in situ apoptosis cells detection （TUNEL detection） were used to systematically study the biological effects of transfected cells both in vitro and in vivo. Results： In this study, the results showed that the proliferation of esophageal carcinoma cells in ASODN group decreased significantly when compared with the control group （P 〈 0.05）, at 57.3% Bcl-XL mRNA inhibitory rate, and a significant decreasing of Bcl-XL protein expression, at the apoptosis rates of （31.1 ＋ 5.8）% and 35.0% by flow cytometry and TUNEL assay respectively （P 〈 0.01, when compared with control groups）. It also showed that the growth of human esophageal carcinoma in nude mice of ASODN group was significantly inhibited （P 〈 0.05）, together with a significant decreased expression level of Bcl-XL mRNA and protein, and an induced tumor cell apoptosis in nude mice. Conclusion： Our result indicates BcI-XL ASODN can effectively inhibit the proliferation of esophageal carcinoma cells in vitro and tumor growth in vivo. The suppression of Bcl-XL expression by ASODN may offer both a therapeutic approach and an important theoretic foundation for gene therapy against esophageal carcinoma.

Identification of label-retaining cells in human gastric cancer xenograft in nude mice

Objective:This study has investigated the existence of label-retaining cell and its distribution in gastric cancer,in the hope that this information will assist investigations on gastric cancer stem cells.Methods:The gastric carcinoma cell line BGC-823 was labeled with BrdU in vitro and then engrafted into the right axilla of nude mice,which developed tumors.Label-retaining cells were quantified by immunohistochemical methods.Results:BrdU positive cells constituted about 96%of the cells in xenograft tumors after 10 days.Subsequently,BrdU positive cells gradually decreased,at the 80th day,labelretaining cells steadily occupied about 0.5%.This set of population cell localized in the margin of cancer nests,which had no difference in cellular morpha.Conclusion:The study demonstrates the presence of label-retaining cells in human gastric cancer xenografts in nude mice and the label-retaining cells may be related with cancer stem cells,which are most likely the cause for spread,metastasis and recurrence.

Effects of Ethyl Acetate Extract of Phyllanthus reticulatus Leaves on Autophagy-related Proteins Beclin-1,ATG5 and LC3 in Nude Mice Xenograft Model of HCC

[Objectives]To explore the effects of ethyl acetate extract of Phyllanthus reticulatus leaves on autophagy-related proteins Beclin-1,ATG5 and LC3 by immunohistochemistry,and to preliminarily explore their effects on autophagy.[Methods]BEL-7404 Hepatocellular Carcinoma(HCC)nude mice model was established,and blank group(same volume of pure water),positive control group(20 mg/kg fluorouracil),high dose drug group(600 mg/kg),and medium dose drug group(300 mg/kg),and low dose drug group(150 mg/kg)were set up.After 2 weeks of intragastric administration,the nude mice were sacrificed,and the tumor tissues were taken out,processed by immunohistochemistry,and then made into paraffin sections.Photos were taken under an optical microscope(10×40),and evaluation and analysis were performed with the aid of the Image-Pro Plus 6.0 image analysis software.Differences were calculated using SPSS 20.0 software.The effects of drugs on autophagy-related proteins LC3,Beclin-1 and ATG5 were observed.[Results]Compared with the blank group,the medium and high dose groups of ethyl acetate extract of P.reticulatus leaves had the effect of promoting the increase of autophagy-related proteins LC3,Beclin-1 and ATG5(P<0.05).However,there was no significant difference between the low dose group of ethyl acetate extract of P.reticulatus leaves and the blank group(P>0.05).[Conclusions]The ethyl acetate extract of P.reticulatus leaves has a promoting effect on autophagy-related proteins LC3,Beclin-1,and ATG5.

ABCG2-overexpressing S1-M1-80 cell xenografts in nude mice keep original biochemistry and cell biological properties

S1-M1-80 cells,derived from human colon carcinoma S1 cells,are mitoxantrone-selected ABCG2-overexpressing cells and are widely used in in vitro studies of multidrug resistance(MDR).In this study,S1-M1-80 cell xenografts were established to investigate whether the MDR phenotype and cell biological properties were maintained in vivo.Our results showed that the proliferation,cell cycle,and ABCG2 expression level in S1-M1-80 cells were similar to those in cells isolated from S1-M1-80 cell xenografts(named xS1-M1-80 cells).Consistently,xS1-M1-80 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan,but remained sensitive to the non-ABCG2 substrate cisplatin.Furthermore,the specific ABCG2 inhibitor Ko143 potently sensitized xS1-M1-80 cells to mitoxantrone and topotecan.These results suggest that S1-M1-80 cell xenografts in nude mice retain their original cytological characteristics at 9 weeks.Thus,this model could serve as a good system for further investigation of ABCG2-mediated MDR.

Tumor radioimmunoimaging of chimeric antibody in nude mice with hepatoma xenograft

IM To study the radioimmunoimaging (RAII) using the human/mouse chimeric Ab to evaluate its targeting activity in animal models.METHODS To chimeric Ab was labeled with 131I. RAII was performed at different intervals after injection of radiolabeled Abs in nude mice with human hepatoma xenograft, and tissue distribution of radioactivity was measured. Comparison was made in the chimeric Ab between the single segment Ab and previous murine mAb against HBxAg.RESULTS The experimental objects developed tumorpositive image after 2 days of radiolabeled Abs injection, and the peak accumulation of radioactivity fell on the 7th day. The tumor/liver ratioactivity of the chimeric Ab, single segment Ab, antiHBx mAb, and the control group was 281±021, 244±016, 460±019, and 096±014, respectively.CONCLUSION The genetic engineering Abs have a considerable targeting activity which can be used as a novel humanized vector in the targeting treatment of liver cancer..

Tumour Localization of A Mitomycin C-Monoclonal Antibody MGb_2 Conjugate in Nude Mice with Human Gastric Carcinoma Xenografts

MGb2 is a monoclonal antibody againstgastric cancer.It can be well localized in thetumour tissue and has been successfully used inthe radioimmunoimaging in the patients withgastric cancer.The conjugate of MGb2 withchemotherapeutic drug mitomycin C（MMC）al-so shows highly selective cytotoxic effect uponhuman gastric cancer cells in vitro.

^(131)I-labelled anti-hepatoma monoclonal antibodies in targeting therapy of hepatoma xenografts in nude mice

Anti-hepatoma monoclonal antibodies HAb8,HAb18 IgG and HAb18 F（ab’）2were labelled with[131I]according to chloramine T method.The labelling rate andradioactivity were 51% and 9.58×104Bq/mg,respectively.After administration of the[131I]-labelled conjugates through mouse tail vein,hepatoma xenografts were clearly im-aged,Localization index was 2.15,3.09,and 6.99 for HAb8,HAb18 IgG and HAb18F（ab’）2,respectively.A total of 16 hepatoma-bearing nude mice（SMMU-LTNM）were di-vided into 4 groups.When xenografts reached 0.3cm in diameter,the treatment was be-gun by injection of the[131I]-conjugates through mouse tail vein.After 2 weeks of treat-ment,obvious inhibitory or killing effects on the xenografts were found.The tu-mor-bearing mice were killed during the period from 14 to 21d after treatment.Xenografts as well as other organs were sampled and viewed under light microscope ac-cording to routine procedure.The vital organs,such as heart,liver,spleen,lung,kidneyand brain showed no pathological lesions.In hepatoma xenografts,some tumor cellmembranes were found,with obscure cytoplasmic structure and increased eosinophilia,representing necrotic alterations.

Targeting Effect Study of ￣(3) H-Mitoxantrone Nanosphereson Hepatocellular Carcinoma(HCC) Model in Nude Mice

The distribution of ￣(3)H-mitoxantrone polybutyl cyanoacrylate nanospheres(￣(3)H-DHAQ-PBCA-NS)in the viscera,muscle and tumors of human hepatocellular carcinoma (HCC)model in nude mice was studied with liquid scintillation counting techniique. The results showed that the ￣(3)H-DHAQ-PBCA-NS had remarkable liver targeting effect. The content of ￣(3)H-DHAQ-PBCA-NSin liver and heterotopic liver tumor was found to be 71.31±10. 49% of total amount of drug in animal body. It was also found that the content of ￣(3)H-DHAQ-PBCA-NS in liver was higher than that in liver tissue, and the content of ￣(3)H-DHAQ-PBCA-NS in annpit tumor was higher than that in armpit muscle tissue,but had no significant difference;It provides an ideal preparation for the DHAQ admini-stration.

Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphologicalchanges were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR. CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.

Effects of endostatin on expression of vascular endothelial growth factor and its receptors and neovascularization in colonic carcinoma implanted in nude mice

AIM:To investigate the antiangiogenic effects of endostatin on colonic carcinoma cell line implanted in nude mice and its mechanism. METHODS:Nude mice underwent subcutaneous injection with LS-174t colonic carcinoma cell line to generate carcinoma and were randomly separated into two groups.Mice received injection of vehicle or endostatin every day for two weeks. After the tumor was harvested,the tumor volumes were determined,and the expressions of CD34,VEGF and FIk-1 were examined by immunohistochemical method. RESULTS:Tumor volume was significantly inhibited in the endostatin group(84.17%)and tumor weight was significantly inhibited in the endostatin group(0.197±0.049) compared to the control group(1.198±0.105)(F=22.56, P=0.001),microvessel density(MVD)was significantly decreased in the treated group(31.857±3.515)compared to the control group(100.143±4.290)(F=151.62,P<0.001). Furthermore,the expression of FIk-1 was significantly inhibited in the treated group(34.29%) ompared to the control group(8.57%)(X^2=13.745,P=0.001).However no significant decrease was observed in the expression of vascular endothelial growth factor(VEGF)between these two groups(X^2=0.119,P=0.730). CONCLUSION:Endostatin can inhibit tumor growth and angiogenesis by blocking Vegf/FIk-1 pathway.This experiment provides the theory basis for developing a new anti-carcinoma drug through studying the properties of anti-angiogenesis inhibitors.

Change in expression of apoptosis genes after hyperthermia,chemotherapy and radiotherapy in human colon cancer transplanted into nude mice

AIM:To investigate the change in expression of p53 ,Bcl-2 ,and Bax genes in human colon cancer cells transplanted into nude mice after hyperthermia,chemotherapy,radiotherapy,thermochemotherapy,thermoradiotherapy and thermochemoradiotherapy. METHODS:Human colon cancer cell line (HT29) was transplanted into the hind limbs of nude mice. Under laboratory simulated conditions of hyperthermia (43℃,60 min),the actual radiation doses and doses of mitomycin C (MMC) were calculated in reference to the clinical radiotherapy for human rectal cancer and chemotherapy prescription for colon cancer. The mice were divided into 6 groups according to the treatment approaches:hyperthermia,chemotherapy,radiotherapy,thermochemotherapy,thermoradiotherapy,and thermochemoradiotherapy. The mice were sacrificed at different time points and the tumor tissue was taken for further procedures. The morphologic changes in membrane,cytoplasm and nuclei of tumor cells of p53,Bcl-2,and Bax after treatment,were observed by immunohistochemistry staining. RESULTS:All of the six treatment modalities down-regulated the expression of p53,Bcl-2 and up-regulated the expression of Bax at different levels. The combined therapy of hyperthermia,with chemotherapy,and/or irradiation showed a greater effect on down-regulating the expression of p53 (0.208 ± 0.009 vs 0.155 ± 0.0115,P < 0.01) and Bcl-2 (0.086 ± 0.010 vs 0.026 ± 0.0170,P < 0.01) and up-regulating Bax expression (0.091 ± 0.0013 vs 0.207 ± 0.027,P < 0.01) compared with any single therapy.CONCLUSION:Hyperthermia enhances the effect of radio-and chemotherapy on tumors by changing the expression of apoptosis genes,such as p53,Bcl-2 and Bax.

Anticancer activity of genistein on implanted tumor of human SG7901 cells in nude mice

AIM： To investigate genistein-induced apoptosis of implanted tumors of SG7901 cells in nude mice, and the relationship between this apoptosis and expression of Bcl-2 and Bax. METHODS： Establishing a transplanted tumor model by injecting human SG7901 cells into subcutaneous tissue of nude mice. Genistein （0.5, 1 and 1.5 mg/kg） was directly injected adjacent to the tumor, six times at 2-d intervals. Then, changes in tumor volume were measured continuously and tumor inhibition rate of each group was calculated. We observed the morphological alterations by transmission electron microscopy （TEN）, measured the apoptotic rate by the TUNEL staining method, and detected the expression of apoptosisregulated gene Bcl-2 and bax by immunohistochemical staining and RT-PCR. RESULTS： Genistein 0.5, 1 and 1.5 mg/kg significantly inhibited carcinoma growth when it was injected near the tumor by 10.8%, 29.9% and 39.6%, respectively. Genistein induced implanted tumor cells to undergo apoptosis, with apoptotic characteristics seen by TEM. The apoptosis index was increased progressively with increasing genistein dose （28.9% ± 1.2%, 33.8% ±1.6% and 37.7% ±1.2%）. The positive rate of Bcl-2 protein was decreased progressively （11.9%± 0.9%, 5.9%± 0.7% and 4.2% ±0.6%）, and the positive rate of bax protein was increased progressively （0.9% ±1.7%, 24.9% ±0.8% and 29.6% ± 1.7%） by immunohistochemical staining, with increasing dose of genistein. The density of Bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR. CONCLUSION： Genistein was able to induce apoptosisof transplanted tumor cells. This apoptosis may be mediated by down-regulation of the apoptosis-regulated gene Bcl-2 and up-regulation of apoptosis-regulated gene bax.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Wheat Germ Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells in nude mice was established to evaluate anticarcinogenic activities, and the apoptosis-regulated mechanism effect of aqueous extract of fermented wheat germ with Lactobacillus plantarum dy-1 （LFWGE）. Methods The HT-29 cells were transplanted via subcutaneous injection of 1×10^7cells into the right flank of each nude mouse. Then, nude mice were treated for 30 d with LFWGE （high-dose 2 g/kg/d; low-dose 1 g/kg/d） and for 7 d with 5-fluorouracil （5-FU, 25 mg/kg/d） by gavage and intraperitoneal injection, respectively. An inhibition of tumor growth was observed. Results Tumor volume and weights decreased significantly in both groups of nude mice treated with LFWGE. In addition, the cell apoptosis rate of the LFWGE group （2 g/kg/d, 60.2%＋4.4%; 1 g/kg/d, 58.6%＋6.9%） was significantly higher than that of the control group （11.5%＋1.6%） and 5-FU group （32.1%＋3.5%） as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot method further confirmed these enhancing apoptosis and growth inhibition effects. The involvement of LFWGE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, Caspase-3, and CyclinD1. Conclusion The results showed that LFWGE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be as a natural nutrient supplements or chemopreventive agent in the treatment of human colon cancer.