Hepatoblastoma

Abstract: This study demonstrates that two anticancer drugs, taxol and doxorubicin (Dox), can kill human hepatoblastoma HepG2 cells in a dose-dependent manner via the induction of apoptosis. Characteristic events, including externalisation of phosphatidylserine, cytoplasmic shrinkage, chromatin condensation and DNA degradation, were observed in a large majority of the drug-treated cells. DNA fragmentation showed that a ladder of DNA fragments of approximately 200 bp multiples was observed in taxol-treated, but not in Dox-treated, cells. In addition, the expression patterns of Bcl-2 family members during taxol or Dox treatment were investigated. Results from Western blot analysis indicated that HepG2 cells did not express either the death repressor Bcl-2, or the death promoters Bcl-X^sub s^ and Bax. However, during the apoptotic process one death repressor, Bcl-X^sub L^, and two death promoters, Bak and Bad, were expressed. The expression levels of Bcl-X^sub L^ and Bak remained unchanged, whereas the level of Bad was down-regulated. As the ratio between death repressors and death promoters in the Bcl-2 family will determine the sensitivity of cells to apoptotic stimuli, the findings suggest that the changed expression patterns of Bcl-2 family proteins caused by anticancer drugs in liver cancer cells may be involved in chemoresistance.

Key words: Apoptosis. Bcl-2 family proteins, expression. Hepatoblastoma.

Introduction

Apoptosis (programmed cell death) is a genetically determined and precisely regulated cell-death process that occurs during normal cell development, in response to a number of external or internal signals. It can be induced by various physiological, biochemical and/or noxious stimuli.1 In comparison to cell necrosis, apoptosis is an active and energy-dependent process of gene-directed cellular self-destruction.2 An identifying marker of apoptosis, observed in many studies, is the breakdown of internucleosomal DNA into multiples of 200 bp fragments. Another characteristic feature is change in cell morphology, including cytoplasmic shrinkage, chromatin condensation, and apoptotic-body formation.3

Recent studies of apoptosis indicate that the balance between cell proliferation and cell death is disturbed in cancer cells, partially through inactivation or overexpression of genes involved in the regulation of cell death.4 These apoptotic regulatory genes are members of the Bcl-2 family, and are divided into two categories: death repressors and death promoters. The ratio of death repressors to death promoters determines a cell's destiny upon apoptotic stimuli.5 Furthermore, the over-activation of death repressor genes or the downregulation of death promoting genes may render tumour cells relatively more resistant to chemotherapy.6 Using a gene transfer approach to elevate Bcl-2 protein levels, it has been found that high levels of Bcl-2 protein render the tumour cells more resistant to chemotherapeutic agents in many types of tumours, including leukaemia,7 lung cancer,8 and neuroblastoma.9 Conversely, use of antisense oligonucleotides or antisense expression plasmids to reduce Bcl-2 protein levels can render leukaemia and lymphoma cells more sensitive to the cytotoxic effects of anticancer drugs such as doxorubicin (Dox).1 These observations provide strong evidence that Bcl-2 family proteins are important regulators of chemosensitivity and chemoresistance in cancer cells.

Liver cancer is particularly refractory to chemotherapy.10 This stubborn resistance may be caused by several factors, including heterogeneous tumour presentation, the inducible over-expression of the multidrug resistance gene, and/or inherent resistance by an unexplained mechanism.11 Bcl-2 family proteins may contribute to this inherent resistance, since they can affect the sensitivity of tumour cells to anticancer drugs via the regulation of apoptosis. For example, chemoresistance acquired from enhanced Bcl-2 expression during anticancer drug treatment has been observed in myeloma cells.12 Therefore, changes in the expression patterns of Bcl-2 family proteins caused by anticancer drugs may be involved in developing chemoresistance in liver cancer during chemotherapy. However, experimental evidence of the resistance of liver cancer to chemotherapy is scarce. Exploration of the expression levels of Bcl-2 family proteins during drug treatment may contribute not only to our understanding of their roles in the sensitivity of cells to anticancer drugs, but also to the development of new strategies for enhancing chemotherapy efficiency in liver cancer.

Chemotherapy is one of the major treatments for liver cancer. Taxol (or Paclitaxel) and Dox are used clinically to treat a variety of common solid tumours, including liver cancer, breast cancer13 and leukaemia.14,15,16 However, conventional cancer treatment is practically ineffective against human liver cancer. This is partially due to chemoresistance in the cancer cells. It is therefore important to understand the mechanisms of such resistance, in order to develop new strategies to enhance the efficiency of chemotherapy. To understand these mechanisms, the current study investigates the expression patterns of the Bcl-2 family proteins (Bcl-2, Bcl-X^sub L^, Bcl-X^sub S^, Bax, Bak and Bad) during taxol or Dox treatment in the human hepatoblastoma HepG2 cell line. The discrepancy observed in the expression levels of Bcl-2 family proteins may contribute to the development of chemoresistance in liver cancer.

Materials and methods

Cell culture and chemotherapeutic agents

The human hepatoblastoma cell-line HepG2 (ATCC, HB8065) was cultured in RPMI-1640 (Gibco) supplemented with 10% fetal calf serum (Gibco), 5% antibiotic and antimycotic solution (30 000 U/L penicillin and 30 mg/L streptomycin, pH 7.2 [Gibco]), and 2 mmol/L glutamine (Gibco). Cells were incubated at 37 deg C in 5% carbon dioxide at 95% humidity. The culture medium was changed twice weekly, and the cells were examined routinely for mycoplasma contamination. Cells growing logarithmically were used for all experiments.

Taxol (Paclitaxol, Sigma) was prepared freshly prior to each experiment. It was dissolved in Cremophor (David Bull Laboratories) to make a stock solution of 7.03 mol/L which was diluted with culture medium to obtain the concentrations required for the experiments. Doxorubicin (Dox, Ebewe) was diluted with culture medium to obtain the desired concentrations.

XTT dye reduction assay

Chemotherapy-induced cytotoxicity was determined by the XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide, sodium salt) (Sigma) dye reduction assay. The methodology described is a modification of the original XTT calorimetric assay.17 In principle, the number of viable cells is directly proportional to the production of formazan, measured spectrophometrically.

Annexin V binding assay

Phosphatidylserine redistribution in the early stages of apoptosis was detected using the Annexin-V-Fluos kit (Boehringer Mannheim). Cells seeded onto coverslips were treated with anticancer drugs, and then stained with the Annexin-V binding solution (15 min), following the manufacturer's instructions. Propidium iodide was used simultaneously to stain cells which were at a late stage of apoptosis. The cells were examined immediately with a Zeiss fluorescence microscope (488 nm excitation filter, and 515 nm long-pass filter).

DAPI staining

DAPI (4',6-diamidine-2'-phenylindole dihydrochloride) (Boehringer Mannheim) is a fluorescent dye which binds selectively to DNA to form strongly fluorescent DNA-DAPI complexes. DAPI was used according to the method suggested by the manufacturer. Briefly, cells seeded onto coverslips were treated with anticancer drugs, and then fixed (methanol/acetic acids, 3:1) for 10 min. After washing with PBS, the cells were stained with DAPI working solution ( 1 (mu)g/mL) for 10 min. Nuclear morphological changes were observed with a Zeiss fluorescence microscope (360 nm excitation filter, LP 430 nm barrier filter).

TdT-mediated dUTP nick-end labelling (TUNEL)

TUNEL staining was used to detect DNA degradation in situ in the relatively late stages of apoptosis. Cells seeded onto coverslips were treated with anticancer drugs, and subsequently fixed in 2% paraformaldehyde (Sigma) for 15 min. Apoptotic cells were labelled by the TUNEL reaction, using an in situ cell-death detection kit (Boehringer Mannheim).

DlVA fragmentation assay

Drug-treated and untreated cells were harvested, lysed with buffer (10 mmol/L Tris/HCI [pH 7.8], 5 mmol/L EDTA, 0.5% sodium dodecyl sulphate [SDS], 50 pg/mL proteinase K), and incubated at 45 deg C for 3 h. DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) twice and chloroform once, and then treated with 100 (mu)g/mL RNase A (Boehringer Mannheim) at 37C for 1 h. The DNA was further extracted with chloroform twice to ensure the removal of phenol. For DNA precipitation, the final concentration of NaCl was adjusted to 0.15 mol/L. The DNA was precipitated with 70% ethanol, dissolved in TE (Tris-EDTA) buffer, and analysed by 1.5% agarose gel electrophoresis. The resultant gel was stained with ethidium bromide solution (Sigma).

Measurement of cell viability

Cells were seeded (1.0 x 10^sup 5^ cells/well) in a six-well tissue-culture plate, and incubated at 37 deg C for 24 h. The culture medium was then replaced with the drugcontaining medium. The cell viability at each drug exposure time point was tested by Trypan blue dye exclusion.ls Each experiment was repeated twice, independently, and the data were plotted as mean SD.

Western blot analysis

Cells were harvested at each time point and lysed in a loading buffer (50 mmol/L Tris/HCI [pH 6.8], 100 mmol/L dithiothretol, 1% SDS and 10% glycerol). The quantity of protein in different samples was confirmed by Coomassie blue staining prior to Western blot analysis. Protein samples were separated on 10% SDS-polyacrylamide gel (SDS-PAGE) for Bcl-2 and Bcl-X^sub L^, and on 12% gel for Bcl-X^sub s^, Bax, Bak and Bad proteins. The protein bands were transferred to a nitrocellulose membrane (Amersham) in a transfer buffer (39 mmol/L glycine, 48 mmol/L Tris base, 0.037% SDS and 20% methanol). The membrane was treated with a blocking solution (PBS containing 5% low-fat dried milk and 0.1% Tween-20) at 37 deg C for one hour, and then at room temperature for one hour. After incubation with primary mouse monoclonal anti-human Bcl-2 (clone 124, 1 (mu)g/mL) (Cambridge Research Biochemicals) and secondary polyclonal goat antimouse IgG conjugated with horseradish peroxidase (1 in 1000 dilution) (Sigma), the presence of protein was detected by an enhanced chemiluminescence (ECL) detection kit (Amersham).

Other antibodies were used to repeat the same experiment, and included mouse monoclonal antihuman Bax (Calbiochem, AM13), and mouse monoclonal anti-human Bak (Calbiochem, AM03), rabbit polyclonal anti-human Bcl-X^sub S^ (Calbiochem, PC89), mouse monoclonal anti-human Bad (Transduction Laboratories, BB36429), mouse monoclonal antihuman Bcl-X (Transduction Laboratories, B61220), and goat anti-mouse IgG or goat polyclonal antirabbit IgG conjugated with horseradish peroxidase (Sigma).

Results

Cytotoxic effects of taxol and Dox on HepG2 cells

To test the effects of anticancer drugs on the expression of Bcl-2 family proteins during the apoptotic process, we first selected the appropriate drug concentrations and treatment duration for HepG2 cells. The cytotoxic effects of taxol and Dox on HepG2 cells appeared to be dose-dependent (Fig. 1), and the IC^sub 50^ (drug concentration that inhibited cell growth by 50%) values were 11.36 +/- 1.67 nmol/L for taxol and 620.10 +/- 78.12 nmol/L for Dox. Therefore, 50 nmol/L taxol and 5 (mu)mol/L Dox were selected for further experiments on HepG2 cells. Cells were also exposed to Cremophor to ensure that the solution would not affect the cells.

Induction of apoptosis in HepG2 cells by taxol utX Dax

No obvious morphological change was observed in the early stage (within one hour of induction) of apoptosis in human HepG2 cells. However, the plasma membrane of the cells became asymmetrical, due to translocation of phosphatidylserine from inside the plasma membrane to the outer layer, as detected by the Annexin-V binding assay (Fig. 2).

DAPI staining was used for both taxol-treated and Dox-treated HepG2 cells to verify morphological changes in the nucleus. When HepG2 cells were treated with 50 nmol/L taxol for 12 h, nuclear shrinkage was accompanied by chromatin condensation; whereas, in 5 (mu)mol/L Dox-treated cells, the major morphological change was nuclear shrinkage (Fig. 3). Untreated HepG2 cells showed no change in the nucleus, and the cells appeared normal in size (Fig. 3). TUNEL staining showed DNA degradation in nuclei of HepG2 cells after they were treated with taxol or Dox for 24 h (Fig. 4).

DNA fragmentation is another typical feature of apoptotic cells. Exposure of HepG2 cells to taxol for 24 h resulted in a characteristic ladder of DNA fragments of approximately 200 bp multiples (Fig. 5). This confirmed that taxol can induce apoptosis in HepG2 cells. In Dox-treated HepG2 cells, however, DNA ladder fragmentation was not demonstrated, even though various concentrations of the drug and different exposure times were tested.

The degree of morphological change in nuclei and the types of DNA fragmentation observed in HepG2 cells were different in taxol-induced and Doxinduced apoptosis. The differences indicated that the apoptotic pathway in HepG2 cells may vary with different drug treatments. However, the results showed that both taxol and Dox can induce apoptosis in HepG2 cells.

Viability of HepG2 cells during taxol and Dox treatment

In order to analyse the expression patterns of other Bcl-2 family proteins in HepG2 cells during the apoptotic process induced by taxol and Dox, cell viability was checked during the course (ranging from 0 to 72 h) of drug treatment. As shown in Fig. 6, the cell viability, as determined by Trypan blue exclusion, decreased dramatically in a time-dependent manner during 50 nmol/L taxol and 5 (mu)mol/L Dox treatment. Cell viability dropped to approximately 40% after 48 h, and therefore times up to 48 h were selected for testing the expression patterns of Bcl-2 family proteins.

HepG2 is a Bcl-2-, Bcl-Xs- and Bax-negative cell line

To investigate the Bcl-2 expression in HepG2 cells treated with taxol and Dox, cells were exposed to each drug for 48 h. Cell lysates from each time point were analysed by Western blot, which showed that the HepG2 cells did not express Bcl-2 protein (Fig. 7A) after treatment with taxol or Dox. Moreover, neither Bax nor Bcl-X^sub S^ was expressed in HepG2 cells (data not shown). The negative results for Bcl-2 and Bax expression contradicted those of a recent study19 which reported that Western blot analysis showed expression of Bcl-2 and Bax protein in HepG2 cells. In order to verify the results, a new HepG2 cell line (ATCC, HB8065) was used to repeat the Western blot experiment. Consistent results were obtained in several independent experimental trials, confirming that the HepG2 cells did not express Bcl-2, Bax or Bcl-X^sub S^ proteins.

Expression levels of otherf Bcl-2 family proteins in HepG2 cells

HepG2 cells were exposed to taxol or Dox for up to 48 h and the expression of other Bcl-2 family proteins (Bcl-X^sub L^, Bak, and Bad) was analysed by Western blot. Bcl-X^sub L^ and Bak remained unchanged (Fig. 7B and C), whereas Bad gradually decreased in HepG2 cells during taxol and Dox treatment (Fig. 7D). This indicated that Bad (death promoter) was down-regulated during the drug-induced apoptotic process.

Discussion

The human hepatoblastoma cell-line HepG2 was used to investigate the relationship between the expression patterns of Bcl-2 family proteins and apoptosis induced by taxol and Dox. The results indicated that both taxol and Dox cause cell death via apoptosis of HepG2 cells. During the apoptotic process, both taxol and Dox down-regulated the level of Bad protein. However, the levels of Bcl-X^sub L^ and Bak remained unchanged, and Bcl-2, Bcl-X^sub S^ and Bax were not expressed in the HepG2 cell line.

Taxol- and Dox-induced apoptosis in HepG2 cells

DNA fragmentation is a biochemical event which occurs during apoptosis. It is caused by the cutting action of activated endogenous endonuclease on DNA at internucleosomal sites or higher orders of chromatin structure, leading to the breakdown of genomic DNA into small (200 bp repeats) and/or large (50-300 kbp) fragments. Both types of DNA fragmentation can be present in a single apoptotic cell;20 however, under certain conditions only the large fragment is present.21 Our results showed that taxol induces small-type DNA fragmentation (200 bp repeats) in HepG2 cells (Fig. 5). Although this small-type DNA fragmentation was not observed in HepG2 cells treated with Dox, the Annexin-V binding assay and TUNEL and DAPI staining demonstrated different marked features of apoptosis in the Dox-treated cells. This suggested that Dox can induce apoptosis in HepG2 cells.25 Nuclear condensation was observed in Dox-treated HepG2 nuclei (Fig. 3), and chromatin condensation in taxoltreated HepG2 nuclei (Fig. 3).

These observed differences in characteristic apoptotic indicators, resulting from taxol or Dox treatment of HepG2 cells, suggest that the apoptotic pathway may vary with different drug treatments.

Role of Bcl-2 family proteins in HepG2 apoptosis

Bcl-2 protein protects the cells against apoptosis induced by many different stimuli, including taxol and Dox,23 and most cells express this protein. However, from the results presented here, HepG2 cells do not (Fig. 7A). Two other Bcl-2-negative cell-lines have also been discovered: the human prostate cancer cellline DU145(24) and the neuroblastoma cell-line Shep-1.(9) Interestingly, the DU145 cells did not express Bcl-2 protein but were reported to be insensitive to the apoptotic effect of taxol. The results remain unexplained; perhaps the insensitivity to taxol shown by DU145 cells was due to a specific resistance.24 Our results, however, showed that although HepG2 cells did not express Bcl-2 they did respond to the apoptotic effect of taxol. These findings support the theory that taxolinduced apoptosis in different cell types may be regulated by different mechanisms.

In Bcl-2-positive cell-lines, Bcl-2 family proteins regulate apoptosis through the formation of homodimers or heterodimers, as shown in a speculative model.25 In this model, it is proposed that Bax/Bax or Bak/Bak homodimers trigger cell death. Binding of Bcl-2 to Bax and binding of Bcl-X^sub L^ to Bak oppose the death promoter actions of Bax or Bak, resulting in enhanced cell survival. Bad displaces Bax or Bak from Bcl-2/Bax or Bcl-X^sub L^/Bak homodimers, leading to the promotion of cell death. Bcl-X^sub S^ antagonises Bcl-2 by binding to the Bcl-2 proteins. However, the presence of Bcl-2-negative cell lines suggests that the interaction patterns among Bcl-2 family proteins may not be consistent, and that the mechanism by which apoptosis is regulated by this family of proteins may be different in different kinds of cells. In HepG2 cells, our results suggested that other Bcl-2 family proteins (Bax, Bcl-X^sub S^) may be involved in the regulation of apoptosis in this type of cell.

Experiments on the human ovarian carcinoma cell line A2780, performed by Jones et al.26 showed that levels of Bcl-2, Bcl-X^sub L^, or the 24 kDa Bax did not change over a 72 h period after exposure to taxol or cisplatin, but up-regulation of Bak and the 21 kDa Bax did occur. In addition, taxol, but not cisplatin, increased Bak and 21 kDa Bax levels in cisplatin-resistant A2780/cp70 cells. All observations indicated that the levels of most Bcl-2 family proteins are not affected simultaneously during drug-induced apoptosis.

The present study showed that drugs can selectively influence the expression levels of Bcl-2 family proteins (i.e. Bcl-X^sub L^ and Bak remained unchanged during the apoptotic process, but Bad was down-regulated [Fig. 7]). This and other experimental evidence gives a clear indication that taxol can affect the expression patterns of Bcl-2 family proteins, but its influential effect differs with the type of cancer cell.

An increasing amount of experimental evidence obtained from in vitro and in vivo studies indicates that chemoresistance of cancer cells acquired during drug treatment contributes to refractory cancer. Overexpression of the multi-drug resistant gene (MDR 1) encoding P-glycoprotein (Pgp), caused by anticancer drugs, has been found in some cancer cells, including human hepatoma27 and human breast cancer cells.28 Pgp is a cell-surface ATPase which confers resistance to many of the most active anticancer drugs, including taxol and Dox.29 However, other mechanisms may be involved in this acquired chemoresistance, as it also occurred in the absence of Pgp expression.30

Recent studies have shown that Bcl-2 family proteins are associated with the development of drug resistance. Dox-treated myeloma cells with up-regulated Bcl-2 expression showed resistance to a second Dox exposure,31 implying that exposure to these agents may enhance Bcl-2 expression in myeloma cells and contribute to acquired chemoresistance. Another in vivo study in neuroblastoma patients suggested that the tumour cells show enhanced Bcl-2 expression after drug treatment.32

In addition to Bcl-2, other proteins in the Bcl-2 family are involved in desensitising tumour cells to anticancer agents. In the drug-resistant human lymphoma cell-line U-937, Bcl-XL is over-expressed, suggesting that it may participate in acquired resistance to chemotherapeutic agents.33

An over-expression of both Bcl-2 and Bcl-X^sub L^ is necessary to render cells resistant,34 and this is supported by the finding of a higher Bcl-2/Bax ratio in drugtreated B-cell chronic lymphocytic leukaemia.35 In addition, gene transfer-mediated elevation of the death promoter Bcl-X^sub S^ increased the sensitivity of MCF-7 breast cancer cells to chemotherapeutic drugs, including taxol.36 In the lymphoma cell-line U-937, cells which acquired resistance to cytotoxic drugs induced the death repressor Bcl-X^sub L^ protein.33

These observations indicate that Bcl-2 family proteins are involved in chemoresistance in several types of cancer cells. In the present study, the only change in expression of the Bcl-2 family proteins examined was the down-regulation of the pro-apoptotic protein Bad. This suggests that the alteration in expression pattern of Bcl-2 family proteins may make the cells more resistant to chemotherapeutic agents.

In summary, both taxol and Dox decreased the level of one Bcl-2 family death promoter (Bad) but did not influence Bcl-X^sub L^ and Bak levels in HepG2 cells. As the ratio between the death repressors and death promoters in the Bcl-2 family determines the relative sensitivity of the cells to apoptotic stimuli, the variations in expression profile of the proteins during anticancer drug treatment may be involved in the development of chemoresistance in liver cancer. The mechanisms by which anticancer drugs induce the up- and downregulation of Bcl-2 family proteins in liver cancer cells remain unknown, and further study is required to confirm that acquired chemoresistance is due to alterations in Bcl-2 family protein levels.

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DAN LUO, SAMUEL C.S. CHENG and YONG XIE

Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China

(Accepted 8 Febuary 1999)

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