Abnormally activated wingless/integrated signaling modulates tumor-associated macrophage polarization and potentially promotes hepatocarcinoma cell growth

In this article,we comment on the article by Huang et al.The urgent development of new therapeutic strategies targeting macrophage polarization is critical in the fight against liver cancer.Tumor-associated macrophages(TAMs),primarily of the M2 subtype,are instrumental in cellular communication within the tumor microenvironment and are influenced by various signaling pathways,including the wingless/integrated(Wnt)pathway.Activation of the Wnt signaling pathway is pivotal in promoting M2 TAMs polarization,which in turn can exacerbate hepatocarcinoma cell proliferation and migration.This manuscript emphasizes the burgeoning significance of the Wnt signaling pathway and M2 TAMs polarization in the pathogenesis and progression of liver cancer,highlighting the potential therapeutic benefits of inhibiting the Wnt pathway.Lastly,we point out areas in Huang et al’s study that require further research,providing guidance and new directions for similar studies.

Ascorbic Acid Promotes Arsenic-induced Cytotoxicity in Human Hepatocarcinoma Cells and Their Underlying Mechanisms

Objective: To study synergistic effect with Ascorbic acid(AA) on arsenic trioxide inducing human Hepatocarcinoma cell apoptosis, and provide theoretical basis for promoting human Hepatocarcinoma cell apoptosis induced by arsenic trioxide(AT). Methods: Human Hepatocarcinoma cell line BEL-7402 being cultured in vitro, the effect of AT and (or) AA on its growth inhibition and its two intracellular signal molecules was evaluated separately using MTT and Western blot. Results: AT at a few μmol/L concentration could suppress abnormal proliferation of human hepatocarcinoma cells, and initiate their apoptosis by activation of caspase-3, and activate extracellular-signal regulated kinases (ERKs), which were dependent on the dosage of AT conspicuously. The effect of AA on BEL-7402 was not significant; However, AA could effectively enhance AT-induced hepatocarcinoma cell apoptosis and lesion severity through activation of caspase-3 but not ERKs. Conclusion: Caspase-3 and ERKs proteins could involve in arsenic-induced hepatocarcinoma cell apoptosis and differentiation respectively as intracellular signaling molecules; The effect between AT and AA on hepatocarcinoma is synergistic, which further inhibits cell growth and induces apoptosis in human hepatocarcinoma cells through activation of caspase-3 but not ERKs.

Antineoplastic Effect of Calcium Channel Blocker-Verapamil and 5-Fluorouracil Intraperitoneal Chemotherapy on Hepatocarcinoma-Bearing Rats

Objective To study the antineoplastic effect of the calcium channel blocker verapamil and 5-fluorouracil intraperitoneal chemotherapy onhepatocarcinoma-bearing rats, and examine the action between calcium channel blockers and cytotoxic drugs.Methods We adopted the method of subcapsular implantation of carcinoma tissues of walker-256 in the left liver lobe as a model of livercarcinoma-bearing rats. All experimental animals were divided into four groups. On the sixth day post implantation, in group A (controlgroup) 6 ml of saline was injected intraperitoneally once a day for 3 days. In group B (single chemotherapy group) 6 ml of 5-Fu 75 mg/kg was injected intraperitoneally once a day for 3 days. In group C (combination of treatment group) both 5-Fu (75 mg/kg) and verapamil(25 mg/kg) were administered simultaneously as in A and B. In group D (simple verapamil group) only 6 ml of verapamil (25 mg/kg)was administered as above.Results Compared with groups A, B and D, The volume of cancer and the contents of liver cancer DNA and protein were significantlyreduced. The rates of inhibiting cancer (89.9% in group C and 35.4% in group B) were significantly increased in group C. Group C hadsignificantly long survival time compared to groups A, B and D ( P < 0.05) . By light microscopy, a number of focal necroses were foundin cancer tissue in group C.Conclusion Calcium channel blockers can enhance the antineoplastic effect of 5-Fu intraperitoneal chemotherapy to liver cancer ; Theuse of verapamil can not increase the toxicity of 5-Fu.

Polymorphism of p16INK4a gene and rare mutation of p15INK4b gene exon2 in primary hepatocarcinoma

INTRODUCTION Hepatocellular carcinoma(HCC)is the mostcommon cause of death from cancer in China.Themechanisms of hepatocarcinogenesis are not yetknown clearly,p16INK4a gene,the multiple tumorsuppressor gene 1(MTS1),encodes P16 protein,which acts as an inhibitor by binding directly toCDK4 and CDK6 and preventing its association

Traditional Chinese medicine for prevention and treatment of hepatocarcinoma: From bench to bedside

Traditional Chinese medicine(TCM) has played a positive role in the management of hepatocarcinoma. Hepatocarcinoma patients may present Qi-stagnation, damp-heat, blood stasis, Qi-deficiency, Yin-deficiency and other TCM syndromes(Zheng). Modern treatments such as surgery, transarterial chemoembolization(TACE) and high intensity focus ultrasound treatment would influence the manifestation of TCM syndromes. Herbs with traditional efficacy of tonifying Qi, blood and Yin, soothing liver-Qi stagnation, clearing heat and detoxifying and dissolving stasis, have been demonstrated to be potent to prevent hepatocarcinogenesis. TCM has been widely used in all aspects of integrative therapy in hepatocarcinoma, including surgical resection, liver transplantation, TACE, local ablative therapies and even as monotherapy for middle-advanced stage hepatocarcinoma. Clinical practices have confirmed that TCM is effective to alleviate clinical symptoms, improve quality of life and immune function, prevent recurrence and metastasis, delay tumor progression, and prolong survival time in hepatocarcinoma patients. The effective mechanism of TCM against hepatocarcinoma is related to inducing apoptosis, autophagy, anoikis and cell senescence, arresting cell cycle, regulating immune function, inhibiting metastasis and angiogenesis, reversing drug resistance and enhancing effects of chemotherapy. Along with the progress of research in this field, TCM will contribute more to the prevention and treatment of hepatocarcinoma.

Could metabolic syndrome lead to hepatocarcinoma via non-alcoholic fatty liver disease?

It was estimated that from 2002 to 2008 the risk of developing cancer increased a quarter-fold in men and two-fold in women due to excessive BMI. Obesity, metabolic syndrome and type 2 diabetes mellitus are strictly related and are key pathogenetic factors of non-alcoholic fatty liver disease (NAFLD), the most frequent liver disease worldwide. The most important consequence of the &#x0201c;metabolic epidemics&#x0201d; is the probable rise in the incidence of hepatocarcinoma (HCC), and NAFLD is the major causative factor. Adipose tissue is not merely a storage organ where lipids are preserved as an energy source. It is an active organ with important endocrine, paracrine, and autocrine actions in addition to immune functions. Adipocytes produce a wide range of hormones, cytokines, and growth factors that can act locally in the adipose tissue microenvironment and systemically. In this article, the main roles of insulin growth factor (IGF)-1 and IGF-2 are discussed. The role of IGF-2 is not only confined to HCC, but it may also act in early hepato-carcinogenesis, as pre-neoplastic lesions express IGF-2 mRNA. IGF-1 and IGF-2 interact with specific receptors (IGF-1R and IGF-2R). IGF-1R is over-expressed in in vitro and in animal models of HCC and it was demonstrated that IGF ligands exerted their effects on HCC cells through IGF-1R and that it was involved in the degeneration of pre-neoplastic lesions via an increase in their mitotic activity. Both IGF-2R and TGF &#x003b2;, a growth inhibitor, levels are reduced in human HCC compared with adjacent normal liver tissues. Another key mechanism involves peroxisome proliferator-activated receptor (PPAR)&#x003b3;. In in vitro studies, PPAR&#x003b3; inhibited various carcinomas including HCC, most probably by regulating apoptosis via the p21, p53 and p27 pathways. Finally, as a clinical consequence, to improve survival, efforts to achieve a &#x0201c;healthier diet&#x0201d; should be promoted by physicians and politicians.

Innate immunity and hepatocarcinoma:Can toll-likereceptors open the door to oncogenesis?

Hepatocarcinoma(HCC) is a highly prevalent cancer worldwide and its inflammatory background was established long ago.Recent studies have shown that innate immunity is closely related to the HCC carcinogenesis.An effective innate immunity response relies on the tolllike receptors(TLR) found in several different liver cells which,through different ligands and many signaling pathways can elicit,not only a pro-inflammatory but also an oncogenic or anti-oncogenic response.Our aim was to study the role of TLRs in the liver oncogenesis and as a consequence their value as potential therapeutic targets.We performed a systematic review of PubMed searching for original articles studying the relationship between HCC and TLRs until March 2015.TLR2 appears to be a fundamental stress-sensor as its absence reveals an augmented tendency to accumulate DNAdamages and to cell survival.However,pathways are still not fully understood as TLR2 up-regulation was also associated to enhanced tumorigenesis.TLR3 has a wellknown protective role influencing crucial processes like angiogenesis,cell growth or proliferation.TLR4 works as an interesting epithelial-mesenchymal transition's inducer and a promoter of cell survival probably inducing HCC carcinogenesis even though an anti-cancer role has already been observed.TLR9's influence on carcinogenesis is also controversial and despite a potential anticancer capacity,a pro-tumorigenic role is more likely.Genetic polymorphisms in some TLRs have been found and its influence on the risk of HCC has been reported.As therapeutic targets,TLRs are already in use and have a great potential.In conclusion,TLRs have been shown to be an interesting influence on the HCCs microenvironment,with TLR3 clearly determining an antitumour influence.TLR4 and TLR9 are considered to have a positive relationship with tumour development even though,in each of them anti-tumorigenic signals have been described.TLR2 presents a more ambiguous role,possibly depending on the stage of the inflammationHCC axis.

Telomere and telomerase in chronic liver disease and hepatocarcinoma

The pathogenesis of liver cirrhosis is not completely elucidated.Although in the majority of patients,the risk factors may be identified in B and C viral hepatitis,alcohol intake,drugs or fatty liver disease,there is a small percentage of patients with no apparent risk factors.In addition,the evolution of chronic liver disease is highly heterogeneous from one patient to another.Among patient with identical risk factors,some rapidly progress to cirrhosis and hepatocellular carcinoma(HCC)whereas others have a benign course.Therefore,a genetic predisposition may contribute to the development of cirrhosis and HCC.Evidence supporting the role of genetic factors as a risk for cirrhosis has been accumulating during the past years.In addition to the results from epidemiological studies,polymorphisms studies and data on twins,the concept of telomere shortening as a genetic risk factor for chronic liver disease and HCC has been proposed.Here we review the literature on telomerase mutations,telomere shortening and liver disease including hepatocellular carcinoma.

TIMP-3 gene transfection suppresses invasive and metastatic capacity of human hepatocarcinoma cell line HCC-7721

BACKGROUND: Tissue inhibitor of metalloproteinases (TIMPs) can restrain the tumor growth by their protein property and matrix metalloproteinases (MMPs) inhibition. There are currently four known TIMP family members-TIMP-1, 2, 3, 4. We determined whether increasing levels of TIMP-3 expression could suppress the malignant phenotype of human hepatocarcinoma cell line HCC-7721. METHODS: A recombinant expression vector, which contained full-length cDNA of human TIMP-3, was constructed and transfected into the human hepatocarcinoma cell line HCC-7721 by the lipofectamine technique. In vitro and in vivo tests such as Western blotting, immunohistochemistry as well as xenografting in nude mice were used to analyze expression levels of TIMP and MMP, and changes in malignant phenotype after the gene transfection. RESULTS: TIMP-3 expression in TIMP-3 gene-transfected HCC-7721 cells was upregulated as assessed by Western blotting. The ability of in vitro invasion through a Boyden chamber was significantly decreased compared to controls. Following subcutaneous injection into nude mice, the TIMP-3 transfected cells suppressed primary tumor growth, as characterized by reduced tumor weight, size and microvasculature as well as maintaining the extracellular matrix. CONCLUSION: The results suggest that upregulation of TIMP-3 expression in HCC-7721 cells inhibits invasion capacity in vitro as well as tumorigenic and metastatic potential in nude mice.

Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip

AIM: In order to obtain lymphogenous metastasisassociated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential.METHODS: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip() MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics.RESULTS: Out of the 14 500 known genes investigated,110 (0.8%) were up regulated at least 23 fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 23 fold. According to the Gene Ontology and TreeView analysis, the 110genes were further classified into two groups: differential biological process profile and molecular function profile.CONCLUSION: Using high-throughput gene chip method,a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription,chaperone activity, motor activity, protein kinase activity,receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation.Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments.ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.

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.

Development and characterization of multidrug resistant human hepatocarcinoma cell line in nude mice

AIM： To establish a multidrug resistant （MDR） cell subline from the human hepatocardnoma cell line （HepG2） in nude mice. METHODS： HepG2 cell cultures were incubated with increasing concentrations of adriamycin （ADM） to develop an ADM-resistant cell subline （HepG2/ADM） with crossresistance to other chemotherapeutic agents. Twenty male athymic BALB/c-nu/nu mice were randomized into HepG2/nude and HepG2/ADM/nude groups （10 in each group）. A cell suspension （either HepG2 or HepG2/ADM） was injected subcutaneously into mice in each group. Tumor growth was recorded, and animals were sacrificed 4-5 wk after cell implantation. Tumors were prepared for histology, and viable tumor was dispersed into a single-cell suspension. The IC50 values for a number of chemotherapeutic agents were determined by 2, 3-bis （2-methoxy-4-nitro-5-sulfophenyl）-2H-tetrazolium-5-carboxanilide inner salt （MTT） assay. Rhodamine-123 retention/efflux and the level of resistance-associated proteins were determined by flow cytometry. The mRNA expression of mdr1, mrp and Irp genes was detected using reverse transcriptase polymerase chain reaction （RT-PCR） in HepG2/nude and HepG2/ADM/nude groups. RESULTS： The appearances of HepG2/nude cells were slightly different from those of HepG2/ADM/nude cells. Similar tumor growth curves were determined in both groups. A cross-resistance to ADM, vincristine, cisplatin and 5-fluorouracil was seen in HepG2/ADM/nude group. The levels of P-glycoprotein and multidrug resistanceassociated proteins were significantly increased. The mRNA expression levels of mdrl, mrp and/rp were higher in HepG2/ADM/nude cells. CONCLUSION： ADM-resistant HepG2 subline in nude mice has a cross resistance to chemotherapeutic drugs. It may be used as an in vivo model to investigate the mechanisms of MDR, and explore the targeted approaches to overcoming MDR.

Cell cycle and radiosensitivity of progeny of irradiated primary cultured human hepatocarcinoma cells

AIM： To evaluate the change of growth characteristics and radiosensitivity of irradiated primary cultured human hepatocarcinoma cells. METHODS： All tumor tissue samples were obtained from 39 hepatocarcinoma patients with a mean age of 49.6 years （range 22-76 years）. We divided the samples into irradiated group and non-irradiated group and measured their plating efficiency （PE）, population doubling time （PDT）, radiosensitivity index SF2 and cell RESULTS： The PDT of primary culture of hepatocardnoma cells was 91.0±6.6 h, PE was 12.0±1.4%, SF2 was 0.41±0.05%. The PDT of their inadiated progeny was 124.8±5.8 h, PE was 5.0±0.7%, SF2 was 0.65±0.09%. The pdmary cultured human hepatocarcinoma cells showed significant S reduction and G^2 arrest in a dose-dependent manner. The progeny of irradiated primary cultured hepatocarcinoma cells grew more slowly and its radiosensitivity increased. CONCLUSION： The progeny of irradiated primary cultured human hepatocarcinoma cells grows more slowly and its radiosensitivity increases.

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.

Effects of dendritic cells from cord blood CD34^+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocartinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11d vs 7 d,P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

Purification and characterization ofα-L-fucosidase from human primary hepatocarcinoma tissue

AIM: To purify and characterizeα-L-fucosidase from human liver cancer tissue and to detect the localization ofα-L-fucosidase in tumor tissue. METHODS: Cation exchange chromatography on CM-52 and ultrafiltration were used to separateα-Lfucosidase (AFU) from crude extract of liver cancer tissue. 4-methylumbelliferyl-α-L-fucopyranoside was used as a fluorescent substrate to quantify the purified AFU activity in each step. A polyclonal antibody (pAb) against the purified AFU was obtained by anion exchange chromatography on DEAE-52 after ammonium sulfate fractionation and ultrafiltration. Immuohistochemical staining was used to observe the expression of AFU in malignant and adjacent liver tissues. RESULTS: Humanα-L-fucosidase was purified 74-fold to apparent homogeneity with 15% yield. SDSPAGE indicated the presence of one subunit of molecular weight of 55 Ku. The specific activity of AFU in pooled fraction by chromatography was 10085 IU/mg. Western blot analysis indicated that the pAb could recognize one protein band of molecular weight of 55 Ku. The expression of AFU was observed in cytoplasm membrane of liver cancer tissue but not in that of adjacent tissue. CONCLUSION: The purifiedα-L-fucosidase from primary hepatocarcinoma (PHC) is different in its properties fromα-L-fucosidase in human other organs. The polyclonal antibody prepared in this experiment can be applied to the diagnosis of PHC.

An Antisense Plasmid Targeting Survivin Expression Induces Apoptosis and Sensitizes Hepatocarcinoma Cells to Chemotherapy

To explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cells in vitro . Survivin mRNA structure region was amplified by RT PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINE TM 2000 (LF2000), with low concentration of 5 fluorouracil (5 Fu) added. Survivin protein was detected by Western blot, the growth activity was measured by MTT, and apoptosis was detected by Flow Cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase 3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5 Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase 3 activity were increased in antisense survivin transfected + 5 Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.

Antitumor and synergistic effect of Chinese medicine “Bushen huayu jiedu recipe” and chemotherapy on transplanted animal hepatocarcinoma

AIM： To investigate the antitumor and synergistic effect of Chinese medicine “Bushen huayu jiedu recipe” （recipe for invigorating the kidney, removing blood stasis and toxic substances） and chemotherapy on mice hepatocarcinoma. METHODS： Bushen huayu jiedu recipe （BSHYJDR） consisting of Chinese Cassia Bark, Psoralea, Zedoary, Rhubarb, etc. is equal to 1.5 g/mL liquid of originated herbs after being decoded, filtered, and concentrated. Kunming mice, weighing 18-22 g, were injected with 0.2 mL ascitic hepatocarcinoma H22 containing 1 × 10^7 cells/mL into armpit of the right forelimb of mice. After 24 h, the mice were weighed and randomly divided into tumor-bearing model control group, cisplatin （DDP） group, BSHYJDR high dosage group, low dosage BSHYJDR group, DDP combined with high and low dosage BSHYJDR group, 10 mice in each group. DDP group received injection intraperitoneally （ip） at the dosage of 1 mg/kg （equal to 1/10 LD50）, once a day for 4 d. High and low dosage BSHYJDR groups received intragastric BSHYJDR at the dosages of 26.6 and 13.3 g/kg （20 and 10 times each of clinical adult dosage） respectively, while tumor-bearing model group received the equal volume of distilled water once a day for 10 d. On the 11^th d, the mice were weighed and killed, then the tumor was dissected and weighed, the repression rate （RR） was calculated according to the mean weight of tumor （MWT）. RESULTS： Compared to the model group （MWT： 1.30±0.73）, DDP group （MWT： 0.41±0.09, RR： 68.46%） had a significant difference in the inhibition of hepatocarcinoma H22 （P〈0.01）. High dosage BSHYJDR group （MWT： 0.69±0.29, RR： 46.92%） also had a significant difference in inhibition （P〈0.05）, while no difference was found in low dosage BSHYJDR group （MVVT： 0.85±0.34, RR： 34.62%） （P〉0.05）. When DDP was combined with high dosage BSHYJDR （MWT： 0.29±0.17, RR： 77.69%） and low dosage BSHYJDR （MWT： 0.38±0.21, RR： 70.77%） respectively, we could see improvement of the inhibition effect of DDP on transplanted hepatocarcinoma H22. DDP combined with high dosage BSHYJDR had a significant difference （P〈0.001） compared to DDP, while DDP combined with low dosage BSHYJDR only had a little improvement that is not remarkable. CONCLUSION： Chinese medicine BSHYJDR in combination with chemotherapy can inhibit transplanted hepatocarcinorna in mice.

Therapeutic efficacy and bone marrow protection of the mdr1 gene and over-dose chemotherapy with doxorubicin for rabbits with VX2 hepatocarcinoma

BACKGROUND : Malignant tumors are common diseases threatening to the health and life of human being. Clinically, the multidrug resistance of tumor cells and bone marrow depression caused by chemotherapeutic agents are the main obstacles to the treatment of tumors, and both are related to the mdr1 gene. The over expression of the mdr1 gene in tumor cells contributes to the multidrug resistance of malignant tumor cells. With little expression of the mdr1 gene, bone marrow cells particularly susceptible to multidrug resistance-sensitive agents, which cause serious toxicity in bone marrow. This study was undertaken to assess therapeutic efficacy of transplantation of bone marrow mononuclear cells transferred with the mdr1 gene and over-dose chemotherapy with doxorubicin for VX2 hepatocarcinoma of rabbits. METHODS: The mdr1 gene was transferred into the bone marrow mononuclear cells of rabbits, which was co- cultured with retroviral vector-containing supernatant, and the cells were autotransplanted into a rabbit model with VX2 hepatocarcinoma. After chemotherapy with doxorubicin, the protective effects of the mdr1 gene and therapeutic efficacy of over-dose chemotherapy were observed. RESULTS: The mdr1 gene was transferred successfully into the bone marrow mononuclear cells, with a transduction efficiency of 35%. After autotransplantation, the mdr1 gene was expressed functionally in bone marrow with a positive rate of 8%, indicating that the gene played animportant role in bone marrow protection. The rabbits with VX2 hepatocarcinoma, which had received the mdr1 gene-transduced cells, survived after chemotherapy with a 3-fold dose of adriamycin, and their white blood cell counts were (4.26±1.03)×104/L. Since hepatocarcinoma cells were eradicated, the survival time (97.00±46.75 d) of the rabbits was extended (P<0.05) and the healing rate of the tumor was increased (P<0.05). CONCLUSIONS: The transferring of the mdr1 gene into bone marrow mononuclear cells could confer chemoprotection to bone marrow, and over-dose chemotherapy could be prescribed for the treatment of malignant tumors.

Inhibitory Effect of Melatonin on the Growth of H22 Hepatocarcinoma Cells by Inducing Apoptosis

Whether melatonin not only inhibits the growth of H22 hepatocarcinoma cells but also induces apoptosis in vitro was assessed. The anti proliferative effects of melatonin on tumor cells was observed by MTT assay and tumor cells growth curve assay. And the apoptosis of the cells was studied by acridine orange fluorescence assay and flow cytometry. The cell cycle of the tumor cells was also observed by flow cytometry. It was found that melatonin could significantly inhibit the growth of H22 hepatocarcinoma cells. Incubated with melatonin, chromatin condensation of the tumor cells was observed by fluorescence microscopy. Compared with control, the percentage of apoptotic cells was increased, and the proportion of G 0/S increased but that of G 2/M decreased. It was suggested that melatonin could directly inhibit the growth of H22 hepatocarcinoma cells by inducing apoptosis and extending the length of cell cycle of the tumor cells.