Autophagy in Hepatocytes during Distant Tumor Growth.

Structural changes in the liver of CBA mice were studied during the development of experimental hepatocarcinoma-29 inoculated into the hip. A decrease in the volume density of hepatocyte cytoplasm, mitochondria, endoplasmic reticulum, and lipid inclusions and an increase in the volume density of lysosomal structures during tumor growth were observed. All stages of intracellular autophagy were recorded by the method of electron microscopy. These stages included the appearance of autophagosomes, autophagolysosomes, and secondary lysosomes in the hepatocyte cytoplasm. Fragments of cytoplasm, glycogen rosettes, mitochondria, and fragments of endoplasmic reticulum with ribosomes were found in autophagosomes. The obtained data indicate the development of non-selective autophagy in the liver during distant tumor growth in aimed at the maintenance of intracellular homeostasis in hepatocytes and energy and trophic homeostasis of organism.

The combination effects of Shen-Ling-Bai-Zhu on promoting apoptosis of transplanted H22 hepatocellular carcinoma in mice receiving chemotherapy.

ETHNOPHARMACOLOGICAL RELEVANCE: Shen-Ling-Bai-Zhu Powder (SLBZP) is a classic traditional Chinese medical formula that has been used for several decades in the treatment of patients with gastrointestinal malignancies. Whether SLBZP is best employed as single agent or adjunctive therapy has yet to be determined as does the mechanism whereby SLBZP exerts its anti-tumor effects. AIM OF THE STUDY: To investigate the effects of SLBZP alone and in combination with Cytoxan (CTX) on tumor growth, malignant cell apoptosis and Akt/Nuclear Factor kappa B (NF-КB) signaling in a murine model of hepatocellular carcinoma (HCC) receiving chemotherapy. MATERIALS AND METHODS: Sixty-four adult mice developed HCC following subcutaneous inoculation with H22 hepatocellular carcinoma cells. Seven days later, all received chemotherapy with CTX (200mg/kg) once. Mice were then randomized into eight study groups (N=8/group). Three groups were treated with different concentrations of SLBZP alone (6.00, 3.00, 1.5g/kg), three with SLBZP (6.00, 3.00, 1.5g/kg) plus CTX (20mg/kg), one with CTX (20mg/kg) alone (positive control), and one with physiologic saline (untreated, negative control). All groups were treated for 14 days. Tumor size, histology and serum or tissue levels and/or mRNA expression of PDGF-BB, VEGF, Ang-1, Ang-2, NF-КB, B-cell lymphoma-2 (Bcl-2); B-cell lymphoma-extra large (Bcl-xL); X-linked inhibitor of apoptosis (XIAP), Survivin, Caspase-3, Caspase-9, Caspase-7, Akt and phosphorylated Akt expression were documented at the end of treatment. RESULTS: Compared to untreated negative controls, tumor sizes were decreased in the CTX alone, SLBZP (M)+CTX and SLBZP (H)+CTX groups (-52%,-53% and -58% respectively). Tumor cell density was decreased in all treated groups but most apparent in the SLBZP (H)+CTX group. Electron microscopic evidence of apoptosis was also most apparent in this group. Serum and/or tissue levels and expression of PDGF-BB, VEGF, Ang-1, Ang-2, their downstream signaling proteins and anti-apoptotic markers were lowest and pro-apoptotic markers highest in SLBZP (H)+CTX treated mice. CONCLUSIONS: In this chemotherapy-induced animal model of HCC, SLBZP was most efficacious as adjunctive therapy and appears to act by inhibiting tumor growth promoters and anti-apoptotic proteins while enhancing pro-apoptotic proteins.

Hepatocellular carcinoma mouse models: Hepatitis B virus-associated hepatocarcinogenesis and haploinsufficient tumor suppressor genes.

The multifactorial and multistage pathogenesis of hepatocellular carcinoma (HCC) has fascinated a wide spectrum of scientists for decades. While a number of major risk factors have been identified, their mechanistic roles in hepatocarcinogenesis still need to be elucidated. Many tumor suppressor genes (TSGs) have been identified as being involved in HCC. These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors: the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele. Hepatitis B virus (HBV) is one of the most important risk factors associated with HCC. Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor, one advantage of mouse models for HBV/HCC research is the numerous and powerful genetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs. Here, we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner. Discoveries obtained using mouse models will have a great impact on HCC translational medicine.

Modulation of the unfolded protein response impedes tumor cell adaptation to proteotoxic stress: a PERK for hepatocellular carcinoma therapy.

BACKGROUND: Functional disturbances of the endoplasmic reticulum (ER) lead to activation of the unfolded protein response (UPR), which is involved in the consecutive steps of carcinogenesis. In human hepatocellular carcinoma (HCC), the UPR is shown to be activated; however, little is known about the UPR kinetics and effects of UPR modulation in HCC. METHODS: We sequentially monitored the UPR over time in an orthotopic mouse model for HCC and explored the effects of UPR modulation on cell viability and proliferation in vitro and in the mouse model. RESULTS: The expression of ER-resident chaperones peaked during tumor initiation and increased further during tumor progression, predominantly within the nodules. A peak in Ire1 signaling was observed during tumor initiation. The Perk pathway was activated during tumor progression, and the proapoptotic target Chop was upregulated from week 5 and continued to rise, especially in the tumors. The Atf6 pathway was modestly activated only after tumor initiation. Consistent with the UPR activation, electron microscopy demonstrated ER expansion and reorganization in HCC cells in vivo. Strikingly, under ER stress or hypoxia, the Perk inhibitor and not the Ire1 inhibitor reduced cell viability and proliferation via escalating proteotoxic stress in vitro. Notably, the Perk inhibitor significantly decreased tumor burden in the mouse model. CONCLUSION: We provide the first evaluation of the UPR dynamics in a long-term cancer model and identified a small molecule inhibitor of Perk as a promising strategy for HCC therapy.

Changes in cell ultrastructure and inhibition of JAK1/STAT3 signaling pathway in CBRH-7919 cells with astaxanthin.

Astaxanthin (AST), a xanthophylls carotenoid, possesses significant anticancer effects. However, to date, the molecular mechanism of anticancer remains unclear. In the present research, we studied the anticancer mechanism of AST, including the changes in cell ultrastructure, such as the mitochondrion, rough endoplasmic reticulum (RER), Golgi complex, and cytoskeleton, the inhibition of Janus kinase 1(JAK1)/transduction and the activators of the transcription-3 (STAT3) signaling pathway using rat hepatocellular carcinoma CBRH-7919 cells. Cell apoptosis was evaluated and the expressions of JAK1, STAT3, non-metastasis23-1 (nm23-1), and apoptotic gene like B-cell lymphoma/leukemia-2 (bcl-2), B-cell lymphoma-extra large (bcl-xl), proto-oncogene proteins c myc (c-myc) and bcl-2- associated X (bax) were also examined. The results showed that AST could induce cancer cell apoptosis. Under transmission electron microscope, the ultrastructure of treated cells were not clearly distinguishable, the membranes of the mitochondrion, RER, Golgi complex were broken or loosened, and the endoplasmic reticulum (ER) was degranulated. Cytoskeleton depolymerization of the microtubule system led to the collapse of extended vimentin intermediate filament bundles into short agglomerations with disordered distributions. AST inhibited the expression of STAT3, its upstream activator JAK1, and the STAT3 target antiapoptotic genes bcl-2, bcl-xl, and c-myc. Conversely, AST enhanced the expressions of nm23-1 and bax. Overall, our findings demonstrate that AST could induce the apoptosis of CBRH-7919 cells, which are involved in cell ultrastructure and the JAK1/STAT3 signaling pathway.

Chitosan-alginate scaffold culture system for hepatocellular carcinoma increases malignancy and drug resistance.

PURPOSE: Hepatocellular carcinoma (HCC) is a prevalent solid malignancy. Critically needed discovery of new therapeutics has been hindered by lack of an in vitro cell culture system that can effectively represent the in vivo tumor microenvironment. To address this need, a 3D in vitro HCC model was developed using a biocompatible, chitosan-alginate (CA) scaffold cultured with human HCC cell lines. METHODS: The correlation between the cell function, such as secretion of growth factors and production of ECM in vitro, and the tumor growth and blood vessel recruitment in vivo was investigated. RESULTS: HCC cells grown on 3D CA scaffolds demonstrated morphological characteristics and increased expression of markers of highly malignant cells. Implantation of CA scaffolds cultured with human HCC cells in mice showed accelerated tumor growth. Histology revealed marked differences in morphology and organization of newly formed blood vessels between tumors produced by different pre-cultured conditions. Resistance to doxorubicin was significantly pronounced in CA scaffold-cultured HCC cells compared to 2D or Matrigel cultured HCC cells. CONCLUSIONS: This 3D model of HCC, with its ability to more closely mimic the in vivo tumor behavior, may serve as an invaluable model for study and application of novel anticancer therapeutics against HCC.

Evaluation of centrosome abnormalities and p53 inactivation in chemical induced hepatocellular carcinogenesis.

UNLABELLED: Abnormal centrosome frequently found in human cancer is a major cause of mitotic defects and chromosome instability in cancer cells. Centrosome duplication is controlled in a cell cycle-specific manner, whereas cancer cells with dysregulation of centrosome duplication can survive and reenter the cell cycle through defective cell cycle checkpoint systems. Although numerous studies showed that centrosome amplification can be readily induced by loss or mutational inactivation of p53, however, the role of centrosomally localized p53 in the regulation of centrosome duplication had been enigma. To investigate the role of centrosome and p53 in the in vivo carcinogenesis, we performed immunofluorescence and Western blot analysis, respectively, to detect the alteration of centrosome and p53 status as well as immunohistochemical assay to detect cell proliferation in diethyl nitrosoamine (DENA) induced rat hepatocellular carcinoma (HCC). The frequencies of the centrosome abnormalities in HCC lesions were significantly higher than that of in their preneoplasitc counterparts as well as cell proliferation expression profile. Intriguingly, there was no correlation between centrosome abnormalities and cell proliferation. As for p53, the level of p53 increased in inflammation lesion, but decreased in hepatocirrhosis lesion, even undetectable in HCC lesion. These findings may imply that in inflammatory lesions aberration centrosome occurred irrespective of p53 background. However, the significantly increased percentage of cells with abnormal centrosome in hepatocirrhosis, particularly in HCC lesion concomitant with p53 inactivation and increased cell proliferation rate might synergistically contribute to carcinogenesis. Taken together, centrosome abnormalities were an early event prior to p53 inactivation in the time course of carcinogenesis, suggesting that p53 inactivation may not be the cause of centrosome aberration and centrosome may be a susceptible organelle responding to cellular insults. KEYWORDS: centrosome, p53, hepatocellular carcinoma, cell proliferation.

Changes of splenic macrophage during the process of liver cancer induced by diethylnitrosamine in rats.

BACKGROUND: It is generally accepted that spleen plays a complex role in the tumor immunity, which would change in the different periods of cancer. In this study, we investigated the changes in the function of splenic macrophage (Mphi) in different stages of liver cancer induced by diethylnitrosamine (DEN) in rats. The aim was to support the characteristics of "two-way" and "phase" of spleen in tumor immunity. METHODS: The model of pulmonary metastasis of liver cancer was established in forty male SD rats by DEN. In the 8th, 13th and 16th week, 10 rats were randomly chosen and sacrificed, and divided into cirrhosis, liver cancer and pulmonary metastasis groups depending on the pathological result, respectively. The other 10 rats were taken as control group. The Mphi was isolated by anchoring cultivation. The changes in ultrastructure, phagocytosis, cytokine secretion, antigen processing and presenting, and viability of splenic Mphi were detected by transmission electron microscopy, Vybrant(TM) Phagocytosis Assay, DQ(TM) Ovalbumin, and rat TNF-alpha ELISpot kits. RESULTS: Under the electron microscope, the Mphi in the control group had some pseudopodium-like prominences, and mitochondria, ribosome, rough endoplasmic reticulum, lysosome can be found in the cytoplasm, and phagocytized RBC. In the liver cirrhosis and liver cancer group, Mphi had more prominences, meanwhile much more mitochondria, ribosome, rough endoplasmic reticulum, lysosome can be found in the cytoplasm, especially in the liver cancer group. In the pulmonary metastasis group, the Mphi was swelling, with few organelle. As compared to the control group, the function of splenic Mphi increased in cirrhosis and cancer groups, but decreased in metastasis group (phagocytosis rate: (84.7 +/- 1.9)%, (89.5 +/- 3.1)%, and (36.0 +/- 2.6)% vs (75.6 +/- 1.7)%, P < 0.05, P < 0.01; viability: (1.53 +/- 0.15)%, (1. +/- 0.14)%, and (1.12 +/- 0.29)% vs (1.48 +/- 0.17)%, P < 0.05, P < 0.01; TNF-alpha secretion: (741.0 +/- 52.9)%, (1126.2 +/- 174.5)%, and (313.8 +/- 50.8)% vs (626.6 +/- 24.6)%, P < 0.05, P < 0.01; positive cell rate of antigen processing and presenting: (24.03 +/- 1.87)%, (27.95 +/- 2.63)%, and (10.46 +/- 2.16)% vs (16.45 +/- 1.86)%, P < 0.01). CONCLUSIONS: In the stage of cirrhosis and early cancer, the immune functions of splenic Mphi were reinforced. It may promote the non-specificity tumor immunity. On opposite, in the stage of pulmonary metastasis, the immune functions of splenic Mphi were impaired. It may lead to the decrease of tumor immunity.

[Anti-proliferative and apoptosis-inducing activity of Scutellaria barbate containing serum on mouse's hepatoma H22 cells].

OBJECTIVE: To investigate the effects of Scutellaria barbate extract (ESB) on suppressing proliferation and inducing apoptosis of mouse hepatoma H22 cells. METHODS: H22 cells cultured in vitro were divided into 5 groups: blank control group, ESB in high, medium, low dose groups and 5-Fu group. H22 cells were cultured in media with serum containing different concentrations of ESB and blank serum. The proliferation of H22 cells was determined by microculture tetrazolium (MTT) assay. Fluorescence microscopy was utilized to observe the apoptosis of H22 cells by staining with Hoechst 33258. The cell cycle and apoptosis were analyzed by flow cytometry (FCM). RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observerd in a dose and time dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48 hours. Among the various phases of cell cycle, the percentage of cells in S phase decreased significantly, while the percentage of cells in G1 phase increased. Drug-containing serum showed positive effect on cell apoptosis. The apoptosis rate of blank control group, ESB in low, medium, high dose groups and 5-Fu group were 0.51%, 1.07%, 3.15%, 7.83%, 11.26%, respectively. CONCLUSION: ESB containing serum can inhibit proliferation and induce apoptosis of H22 cells in vitro.

[Apoptosis induced by the C21 sterols in Baishouwu and its mechanism of action in hepatoma].

This study is to investigate the effect of the C21 sterols on inducing apoptosis of hepatocellular cancer cells and its potential mechanism. The transplanted model of hepatoma substantiality (Heps) was established in mice, and the mice were divided into four groups: negative controls group and C21 sterols groups (10, 20, 40 mg x kg(-1)) , treated with drugs separately once a day for 9 days. Then the mice were sacrificed, the tumor growth inhibition rate (IR) was calculated and tumor tissue samples were taken and examined under electron microscope. The tumor cells were harvested and cell viability or apoptosis was analyzed by acridine orange and ethidium bromide (AO/EB) stain. B-cell lymphoma/leukemia-2 gene (bcl-2) in tumor cells was inspected by immunohistochemistry. After treatment with C21 sterols (10, 20, 40 mg x kg(-1)), inhibitory effect on the transplanted Heps was observed. The IR was 34.79%, 47.08% and 50.23%, respectively. Apoptosis induced by the C21 sterols was observed, low growth density and some apoptotic cells were observed in tumor under the electron microscope. The expression of bcl-2 gene on tumor cells decreased in the C21 sterols groups, but the percentage of positive area is higher in 40 mg x kg(-1) group than that in 20 mg x kg(-1) group, which differed from apoptosis results. Inhibiting the excessive expression of bcl-2 gene to promote apoptosis may be one of anti-tumor mechanisms for the C21 sterols in Baishouwu.

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 morphological changes 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.

[Antitumor effects of nobiletin on Heps and its mechanism].

AIM: To study the inhibitory effect and mechanism of nobiletin on Heps tumor bearing mice. METHODS: Models of Heps tumor bearing mice were established. The inhibitory rates of tumor growth were calculated, the apoptosis morphology of tumor tissue was observed. The T lymphocyte transformation capacity was tested by MTT assay, the TNFalpha and IL-2 production were measured by LDH kits. RESULTS: Nobiletin could significantly inhibit Heps tumor growth. The inhibitory rates were 42.14% - 65.09% (P < 0.01). The morphology of tumor tissues in nobiletin group had typical characters of necrosis and apoptosis through transmission electron microscope. Nobiletin could stimulate T lymphocyte transformation and the production of TNFalpha and IL-2. CONCLUSION: Nobiletin has obvious antitumor effect on Heps, the main mechanism is to enhance the cellular immune function and induce apoptosis of tumor tissue.

Tyroserleutide tripeptide affects calcium homeostasis of human hepatocarcinoma BEL-7402 cells.

This study aimed to observe the effects of tyroserleutide (tyrosyl-seryl-leucine, YSL) on the growth of human hepatocarcinoma BEL-7402 that was transplanted into nude mice, and explore its anti-tumor mechanism preliminarily. YSL, at doses of 80 microg x kg(-1) x d(-1), 160 microg x kg(-1) x d(-1) and 320 microg x kg(-1) x d(-1) significantly inhibited the growth of the human hepatocarcinoma BEL-7402 tumor in nude mice, producing inhibition of 21.66%, 41.34%, and 34.78%, respectively. Ultra structure of BEL-7402 tumor in nude mice showed that YSL could induce tumor cells apoptosis and necrosis, cell organelle mitochondria and endoplasmic reticulum damage, and calcium overload. By confocal laser scanning microscopy and flow cytometry, we found that 10 microg/mL YSL rapidly induced an increase of the concentration of cytoplasmic free calcium in BEL-7402 cells in vitro, and maintained high concentrations of cytoplasmic free calcium for 1 h. Then the calcium concentration began to decrease after 2 h, and was lower than that of the control group at 4 h and 24 h (p < 0.05). YSL also decreased the mitochondrial transmembrane potential of BEL-7402 cells in vitro, but had no effect on the calcium homeostasis or mitochondrial transmembrane potential of Chang liver hepatocytes. So affecting calcium homeostasis, then inducing apoptosis and necrosis may be a mechanism by which YSL inhibits the tumor growth in animal model.

Redifferentiation of human hepatoma cell induced by 6-(p-chlorophenyl)-3-[1-(p-chlorophenyl)-5-methyl-1 H-1,2,3-triazol-4-yl]-s-triazolo[3,4-b]-1,3,4-thiadiazole (TDZ).

6-(p-Chlorophenyl)-3-[1-(p-chlorophenyl)-5-methyl-1 H-1,2,3-triazol-4-yl]-s-triazolo[3,4-b]-1,3,4-thiadiazole (TDZ) is a derivative of various substituted s-triazolo[3,4-b]-1,3,4-thiadiazoles, which are associated with diverse pharmacological activities. However, the antitumor activity of TDZ is not well understood. To evaluate its role on tumor cell lines, we have examined the effect of TDZ on two tumor lines: human hepatoma cell (SMMC-7721) in vitro and Sarcoma180 tumor (S180) in vivo. The cytotoxicity of TDZ on human hepatoma cells was assessed using the MTT assay. The inhibition on tumor growth was evaluated by means of trypan blue exclusion test in vitro, and using a Sarcoma180 tumor (S180) animal model in vivo. A scanning electronic microscope was used to discover the morphological changes on cell surface, cell electrophoresis was employed to determine the changes of cell surface negative charges, and alpha-fetoprotein was applied as a biomarker of hepatoma. The effect of TDZ on DNA synthesis was determined by a [3H]-thymidine incorporation assay, and cell cycle distribution by flow cytometry. The IC50 value of TDZ on SMMC-7721 cells was 52.9 microg/ml (48 h). However, TDZ could inhibit the growth of SMMC-7721 cells at concentrations far lower than the IC50 value. Treated with the same low concentrations of TDZ, microvilli on the surface of SMMC-7721 cells decreased obviously, electrophoresis rate of cells reduced from 2.14 microm ms(-1) x V(-1) x cm(-1) of control to 1.54 and 1.56 microm x s(-1) x V-1 x cm(-1), the content of AFP dropped from 205.14 +/- 6.41 ng x mg(-1) Pr to 115.68 +/- 3.47 and 78.57 +/- 2.35 ng mg(-1) Pr, and the DNA replication was inhibited by 26.8% and 45.2%. These results indicated that TDZ may inhibit proliferation of cancer cells by reversing SMMC-7721 cells malignant phenotypic characteristics and inducing redifferentiation. Flow cytometry showed that TDZ-treated cells resulted in a higher proportion of cells in S phase compared with untreated cells, and only when the concentration reached 64 microg/ml, the apoptosis could happen at the rate of 4.2%. Detection of the inhibition of Sarcoma 180 tumor growth in vivo showed that TDZ reduced the tumor weight and 69.08% of the growth was inhibited. TDZ could inhibit the proliferation of tumors in vitro and in vivo; the possible antitumor mechanism might be inducing redifferentiation at a lower dosage on vitro.

Cadmium chloride-induced DNA and lysosomal damage in a hepatoma cell line.

Cadmium is a toxic metal and no uniform mechanism of toxicity has so far been proposed. The aim of this study was to investigate the biochemical effects of cadmium chloride in a rat hepatoma cell line (HTC cells) and the cellular events mediating DNA damage. HTC cells were exposed to various concentrations of cadmium chloride for 5 and 8 h and lysosomal damage was assessed with the neutral red assay (NR) and fluorescence microscopy. Mitochondrial integrity was assessed from ATP levels and DNA damage determined with the single cell gel electrophoresis/comet assay. The formation of reactive oxygen species (ROS) was also determined under the same experimental conditions with the dichlorofluorescein assay. Cytotoxicity was assessed with the LDH leakage assay and the levels of glutathione were measured and correlated with the other effects. The results indicate that lysosomal damage occurs at a lower concentration of cadmium chloride (20 microM) than DNA damage (500 microM) in HTC cells. The latter effect was accompanied by an increase of reactive oxygen species without any significant LDH leakage whereas lysosomal damage was significant as determined by the neutral red assay and confirmed with fluorescence microscopy. The effect of CdCl2 on mitochondria and glutathione levels were observed at concentrations or incubation times higher than the ones required to induce lysosomal damage. The data suggest that DNA damage may be due to the formation of reactive oxygen species. It is possible that cadmium induced lysosomal damage is an earlier event than DNA damage and can mediate other cellular events that lead to cell death.

Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.

The majority of hepatic intracellular triacylglycerol (TG) is mobilized by lipolysis followed by reesterification to reassemble TG before incorporation into a very-low-density lipoprotein (VLDL) particle. Triacylglycerol hydrolase (TGH) is a lipase that hydrolyzes TG within hepatocytes. Immunogold electron microscopy in transfected cells revealed a disparate distribution of this enzyme within the endoplasmic reticulum (ER), with particularly intense localization in regions surrounding mitochondria. TGH is localized to the lumen of the ER by the C-terminal tetrapeptide sequence HIEL functioning as an ER retention signal. Deletion of HIEL resulted in secretion of catalytically active TGH. Mutation of HIEL to KDEL, which is the consensus ER retrieval sequence in animal cells, also resulted in ER retention and conservation of lipolytic activity. However, KDEL-TGH was not as efficient at mobilizing lipids for VLDL secretion and exhibited an altered distribution within the ER. TGH is a glycoprotein, but glycosylation is not required for catalytic activity. TGH does not hydrolyze apolipoprotein B-associated lipids. This suggests a mechanism for vectored movement of TGs onto developing VLDL in the ER as TGH may mobilize TG for VLDL assembly, but will not access this lipid once it is associated with VLDL.

[Establishment and biological characteristics of orthotopic transplantation tumor model of hepatocellular carcinoma in mice].

OBJECTIVE: To study the feasibility of the establishment of the orthotopic transplantation tumor model of hepatocellular carcinoma in mice and its tumor biological characteristics. METHODS: H22 cells of hepatocellular carcinoma were inoculated to form ectopic transplanted model in mice by subcutaneous injection. Then the subcutaneous tumors were implanted into the liver of mice, and the orthotopic transplantation tumor model of hepatocellular carcinoma was established. RESULTS: The successful rate of the orthotopic transplantation tumor model was 95.6% and the spontaneous metastatic rate was 81.8%, the rate of mass ascites was 40.9% and the natural extinctive rate was 0%. The natural survival time in the orthotopic transplantation tumor model was 28 days and the proliferation of tumor in transplanted model was accelerated after 2 weeks or so. CONCLUSION: The orthotopic transplantation tumor model in mice is an ideal model for studying the metastatic mechanism and screening anti-tumor drugs for liver cancer, just because of its high successful rate and high spontaneous metastatic rate with no natural extinction.

Telomere dynamics determine episodes of anticancer drug resistance in rat hepatoma cells.

Clinical and experimental observations indicate that resistance to anticancer drugs may be spontaneously reversible over time, but the mechanisms of this reversal are unknown. The resistance of cultured hepatoma cells to methotrexate (MTX) and cisplatin was followed for 9 months. Cells were exposed to three treatments: MTX 200 nM for 24 h or 15 nM continuously and cisplatin 50 microM for 2 h. We investigated the relation between the temporal pattern of cell resistance and the previously reported fluctuations in cell proliferation rate, telomere length and telomerase activity. Spontaneous major peaks in resistance to each drug fell in time windows of 2-3 months (60-70 population doublings) and were at different times for each drug. The frequency of the fluctuations in drug resistance was the same as that of variations in cell growth rate, but amplitudes were unrelated. By contrast, resistance was directly related to telomere length dynamics in the same cells. MTX resistance occurred when telomeres shortened and cisplatin resistance when they were elongated. Furthermore, peaks of resistance to the continuous treatment with MTX were observed at 350-bp intervals of mean telomere length (9.06, 9.41, and 9.76 kbp) during the two 2-month phases of telomere shortening. Statistical analysis demonstrates the sinusoidal relationship between intermittent MTX resistance and telomere length. Possibly, erosion of telomeres encroaches on periodically spaced nucleosomal proteins, defining the onset of resistance phases. This evidence that resistance of tumoral cells to anticancer drugs may be intermittent and that onset of resistance is dictated by telomere length has major implications for clinical practice.

Inositol-specific phospholipase C in low and fast proliferating hepatoma cell lines.

Inositol lipid cycle, among the pletora of signalling events, is directly involved in cell growth. It is located both in the cytoplasm and in the nucleus. Disturbances may cause uncontrolled proliferation of the cell and ultimately cancer. The phosphatidyl inositol phospolipase C (PLC) is a key enzyme in the hydrolysis of polyphosphoinositides (PIs) and could be differently involved in the normal and pathological cell growth. We report immunochemical and immunocytochemical demonstrations that the PLC isoforms are present in both cytoplasmic and nuclear compartments of low and fast proliferating hepatoma cells. The PLC activity is increased in fast proliferating cells, in which PLC delta1 and to a greater extent PLC delta4 are more expressed at cytosolic level, suggesting an involvement of PI specific PLCs in the progression of cell cycle and in the control of cell proliferation and possibly of neoplastic cell growth.