Author Affiliations: School of Life Sciences (Wang SS, Xie X, Wong CST and Fung MC); Department of Anatomical and Cellular Pathology, Prince of Wales Hospital (Choi PY), the Chinese University of Hong Kong, Hong Kong, China

Corresponding Author: Ming Chiu Fung, MD, School of Life Sciences, the Chinese University of Hong Kong, Room EG07, Science Center, Hong Kong, China (Tel: 852-39436147; Fax: 852-26035745; Email: mingchiufung@cuhk.edu.hk)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60042-4

Published online March 27, 2014.

 

 

Contributors: WSS designed the model and wrote the manuscript; WSS and CPY performed the experiments; WCST revised manuscript. All authors have read and approved the final manuscript. FMC is the guarantor.

Funding: This study was supported by grants from the funding from the University Grants Committee of the Hong Kong Special Administrative Region, China (AoE/B-07/99), Lee Lysan Foundation, and Lo Kwee-Seong Biomedical Research Endowment Fund.

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

BACKGROUND: Cancer relapse, associated with increased drug resistance and rate of metastasis, often follows completion of chemotherapy but the cancer escape mechanisms are still incompletely understood. Percutaneous ethanol injection (PEI) has been used for treating hepatocellular carcinoma (HCC) for decades, while the recurrence after PEI treatment remains a major limitation. Recent evidence mounted that cancer cells could survive from chemical induced apoptosis, suggesting a potential route through which cancer relapse may occur. This study focuses on the consequence of HepG2 recovery from ethanol-induced apoptotic event.

 

METHODS: The model of HepG2 recovery from ethanol-induced apoptotic event was established by live cell imaging, BrdU assay and Western blotting. MTT assay, wound healing assay and invasion assay were used to investigate the behavior of HepG2 after recovery.

 

RESULTS: HepG2 cells could recover from ethanol-induced apoptosis. These cells changed their behaviors such as drug resistance, mobility and invasiveness. On average, the recovered HepG2 cell clones were found to be 46% more resistant to ethanol and 84% higher in mobility. The recovered clones became 58.2% more sensitive to 5-fluorouracil.

 

CONCLUSIONS: HepG2 cells can recover from ethanol-induced apoptotic event. These cells became more resistant to ethanol and more invasive. Although the recovered cell clones were more resistant to ethanol, they became more sensitive to 5-fluorouracil treatment.

 

(Hepatobiliary Pancreat Dis Int 2014;13:293-300)

 

KEY WORDS: cancer recurrence; apoptosis; recovery; liver cancer; percutaneous ethanol injection; 5-fluorouracil

Cancers often respond well to chemotherapy initially but end up with cancer recurrence which remains to be a major cause of cancer deaths. Common features of recurrent cancers include increased mobility, stronger drug resistance, and quicker rate of metastasis.^[1-3] There are multiple proposed theories explaining cancer recurrences, for example, diet, physical activity and cancer stem cell.^[4-6] In addition, residual cancer cells surviving chemotherapy is thought to be the major cause of cancer recurrence.^[1] Percutaneous ethanol injection (PEI) treatment in primary hepatocellular carcinoma (HCC) has been widely used for decades, and is proved to be initially effective against small-sized HCC tumors (smaller than 3 cm in diameter), but is associated with a 5-year recurrence rate ranging from 33% to 43% depending on the sizes of the tumor.^{[7, 8]} Until now, the exact mechanisms of cancer escape, particularly after PEI treatment in HCC remain debatable. In this study, we explored the mechanism of HCC relapse by investigating HepG2 cells treated by ethanol in vitro.

 

Recently, our laboratory discovered that both normal and cancer cells could recover from apoptosis even after they had passed the critical checkpoints, so called "point of no return" in apoptosis,^[9-12] including the activation of apoptotic protease caspase-3. Despite initiating the apoptosis determinants, the apoptotic cells regained normal cellular morphologies and functions after removal of apoptotic inducers. The dying cells failed to recover only when they underwent nuclear fragmentation, which seemed to be the terminal event.^[9] Such recovery from apoptotic cell death was also observed in the liver, heart, brain cells, and NIH3T3 fibroblast cell line, indicating that apoptotic recovery could be a general built-in tactic for cell-fate determination. In this study, we hypothesized that HepG2 can recover from ethanol-induced apoptosis, and such recovery might be one of the reasons causing cancer recurrence after PEI treatment. To test this hypothesis, we study the induction and recovery process in vitro. Furthermore, the cellular properties including drug resistance and mobility of HepG2 cells which underwent apoptosis were further explored.

 

 

Human liver cancer HepG2 cells (from American Type Culture Collection, Manassas, USA), were cultured in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated FBS (Hyclone, Thermo Scientific, USA), at 37 �� under the atmosphere of 5% CO₂. The cells were seeded on tissue culture plates until their density reached 70% confluence before being subjected to each experiment. Apoptotic stimuli ethanol (Scharlau, Barcelona, Spain), cisplatin (PHARMACHEMIE BV), doxorubicin (EBEWE Pharma, Austria), docetaxol (Hospira Australia Pty Ltd., Australia), and 5-fluorouracil (5-Fu) (EBEWE Pharma, Austria) were applied to the cells.

 

Human liver cancer HepG2 cells were cultured on a thermo-cell culture FCS2 chamber (Bioptech, Houston, USA) mounted onto the adapter in the stage of an inverted microscope Cell Observer (Carl Zeiss, New York, USA). The cell morphology was observed by a differential interference contrast (DIC) microscope. The drugs were introduced to the cell culture chamber through the perfusion tubes (Bioptech) to induce apoptosis. Then, the apoptotic cells were washed and cultured with fresh medium. The untreated cancer cells served as controls.

 

Three mg protein per lane was separated on a 10% or 12% SDS-PAGE gel and transferred onto a Hybond ECLs membrane (Amersham Biosciences, Chalfont St Giles, UK). After 1 hour blocking, the membrane was incubated overnight at 4 �� with 1:1000 diluted primary antibody [mouse anti-β-tubulin (Santa Cruz Biotechnology sc-55529, USA); rabbit anti-Caspase 3 and rabbit anti-PARP (Cell signaling 9642s, Danvers, MA, USA)]. The membrane was washed for three times and incubated for 1 hour in the corresponding horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (GE healthcare, NA934V, USA) at 1:5000 dilution. After washed for three times, signal was detected with the ECL Western blotting detection system (Amersham Biosciences, USA).

 

MTT (Sigma, St Louis, USA) assay and colorimetric BrdU cell proliferation enzyme-linked immunosorbent assay (Roche, Indianapolis, USA) were performed according to the protocols. The results of the assays were read by a SpectraMax 250 microplate reader (Molecular Devices Corp, Concord, ON, Canada).

 

Individual recovered HepG2 cell clones were prepared by limiting dilution cloning in 96-well plates. To avoid more than one cell in the same well, the density of cell was diluted to 1 cell/200 µL and 100 µL of the diluted cells with medium was added to each well in a 96-well plate (48 cells in each 96-well plate). Each well was replaced with fresh medium every 3-4 days at the beginning. After 5-10 days, wells with two or three cell clones were discarded. Single cell clones were transferred to bigger well or flask for continuous culture.

 

The 6-well culture plates were coated 2 g/L collagen in PBS. HepG2 cells were pretreated with 10 µg/mL mitomycin C (Sigma), which inhibited cell division. HepG2 cells, trypsinized with 2.5 g/L trypsin, were re-cultured (5×10⁵ cells/well) in DMEM high glucose medium containing 100 mL/L FBS for 20 hours to obtain confluent monolayer. The cells in 6-well culture plates were scratched with the tip of 200 µL pipet, and re-cultured in the incubator at 37 �� for 24 hours. At the time points of day 1, 2, 3, 4 and 5 after scratch, five visions (×200) of each well were photographed under a microscope and observed for scratch healing.

 

BioCoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA) coated with a thin layer of MATRIGEL basement membrane matrix were rehydrated and inserted into the 24-well plate which was pre-uploaded with tissue culture medium including 10% FBS. Then 0.5 mL of 1×10⁵ cells/mL HepG2 cell suspension was added into the chambers. After incubation for 24 hours, non-invading cells were removed by scrubbing the membrane twice. The invasive cells that were able to degrade the basement membrane matrix and migrate from the upper to the lower compartment were fixed and stained with 0.005% crystal violet. The invasion index was calculated as the ratio of the percentage of invasion treated cell to the percentage of invasion of a treated cell migrating through control insert membrane with no matrigel. To demonstrate the change in mobility, the invasion index between apoptosis-recovered cells and untreated cancer cells was compared.

 

All results were presented as means±SEM. Statistical analyses were performed by Student's t test using GraphPad Prism software. For the comparison among more than two groups, one-way analysis of variance (ANOVA) was used. The Tukey's procedure was applied for multiple comparisons when ANOVA indicated significant difference between or within groups. A P value less than 0.05 was considered statistically significant.

 

 

Toxicity of ethanol is dose- and time-dependent. According to previous reports and our observation, ethanol at high concentration (>40 g/L) predominantly led to necrosis in HCC cells while at low concentration (23.67-39.45 g/L) induced apoptosis.^{[7, 13, 14]} Although 95% ethanol was used in PEI injection, it will be diluted to diffuse to the outer part of the injection site. The dosage response of HepG2 cells to ethanol was evaluated (Fig. 1). Ethanol at 39.45 g/L led to a mortality of 95% in 24 hours; 36.30 g/L of ethanol was optimal for apoptotic induction, cellular shrinkage and nuclear condensation in 24 hours. However, they regained normal morphologies after putting the cells back to fresh medium (Fig. 2). The recovery of HepG2 cells from apoptosis was then further verified by regain of proliferative ability through BrdU assay (Fig. 3A). In addition to morphological changes, biochemical markers of apoptosis were also investigated. Cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 were decreased after recovery from apoptosis as detected by Western blotting analysis (Fig. 3B).^{[9, 10]} Our results demonstrated that HepG2 cells could recover from ethanol-induced apoptotic event morphologically and biochemically.

 

To explore the consequences of HepG2 cells recovered from ethanol-induced apoptosis, cell response to ethanol was first assayed. Individual cell clones recovered from apoptosis were isolated by limiting dilution. We obtained 30 HepG2 clones recovered from ethanol induction ("recovered clones" for short). Fifteen of them were randomly selected and their half maximal inhibitory concentration (IC50) of ethanol were determined by MTT assay (Fig. 4). Their IC50s were 19.7±4.0 g/L (14.2-28.0), while the IC50s of untreated control cell clones were 14.8±1.7 g/L (12.8-17.8). Except for R23, IC50s of all recovered clones were significantly higher than those of controls (P<0.001).

 

We also explored whether the recovered clones were resistant to other drugs including doxorubicin, docetaxel, cisplatin, and 5-Fu. The values of IC50 in response to chemotherapeutic drugs are summarized in Table. For doxorubicin, docetaxel and cisplatin, some cell clones showed an increased resistance but others displayed an increased sensitivity. Interestingly, all of the recovered clones became more sensitive to 5-Fu (Fig. 5). The IC50s of 5-Fu in different control cell clones were 1520.0±365.4 µmol/L (1001.0 to 2612.0); whereas in recovered cell clones, IC50s were 615.3±351.7 µmol/L (138.0 to 1497.0), (P<0.001). Except for R21 and R22 clones, IC50 of 5-Fu among recovered cell clones were significantly lower than that of the untreated control (P<0.001) (Fig. 5D). Results showed that the average IC50 of recovered clones decreased by 63% when compared to untreated control. These results revealed that the ethanol and 5-Fu combined chemotherapeutic treatment might improve the elimination of the residual HCC cells and hence hedge against cancer recurrence.

 

Mobility and invasiveness were also studied in recovered cell clones. Wound healing assay (also termed as in vitro scratch assay) was used to test the mobility of ten recovered cell clones. Our results showed that after five days, six of the recovered clones closed the wound while control clones did not (Fig. 6A). Nine of the recovered cell clones significantly increased the pace of cell movement compared to that of controls (P<0.05). Furthermore, the invasiveness of five clones (clone R4, R14, R15, R20 and R33) with relatively higher wound healing rates was verified by invasion assay. Cancer cells that pass through membrane covering with extracellular matrix component are generally regarded as more aggressive (higher invasiveness).^{[15, 16]} The average invasion index of the recovered cell clones was found to be 1.8-fold higher than that of the controls (Fig. 6B). These results demonstrated that HepG2 cells recovered from apoptosis had a higher mobility and some of them showed stronger invasiveness properties.

 

 

For decades, PEI had been widely used as an effective therapy for the patients with small size liver tumor. PEI has also been used in patients waiting for liver transplantation in order to delay tumor progression and to minimize the risk of list exclusion.^[17] Local tumor recurrence is the major limitation of ethanol treatment, and the five-year survival rate still needs to be improved.^{[18, 19]} In this study, we used ethanol as an apoptotic inducer in vitro to treat HepG2 cells as a model to mimic ethanol treatment of patients with HCC. Different concentration of ethanol may lead to diverse cellular response in HepG2 cells. The cells might experience necrosis or apoptosis. Moreover, ethanol can easily be metabolized in vivo.^[20] The decrease in ethanol concentration could cause apoptosis or even minor effect on the HCC cells instead of necrosis.^{[7, 21]} This may be one of the reasons for the failure of ethanol treatment. We found that apoptotic HepG2 cells can recover after the removal of ethanol as indicated by their morphology and biochemical markers (Figs. 2, 3). Our observations were consistent with previous studies in other cell types.^{[9, 10, 22]}

 

Cells were previously found to bear DNA damages after recovery from apoptosis.^{[9, 23]} As cancer cells recovered from apoptosis, they may bare genetic alterations, thus lead to modifications of their phenotypes, such as drug resistance, mobility and invasiveness. It was suggested that acquired mutations during apoptosis might lead to drug resistance.^[24] Tang et al^[10] demonstrated that recovered NIH3T3 formed foci and proliferated in soft agar, indicating the loss of contact inhibition and gain of anchorage independent growth. We therefore hypothesized that the HepG2 cells recovered from apoptosis may have altered phenotypes of drug resistance, mobility and invasiveness. To test this hypothesis, the individual cell clones recovered from apoptosis were isolated and analyzed. Except for R23, all of the recovered cell clones showed an increase in resistance to ethanol (Fig. 4). To determine whether the recovered cell clones were also resistant to other drugs, we tested doxorubicin, docetaxel, cisplatin and 5-Fu. Doxorubicin inhibits the activity of topoisomerase II while docetaxel binds to microtubules and interferes with cell division.^[25] Cisplatin is an alkylating agent that binds to and causes crosslinking of DNA.^[26] 5-Fu is a pyrimidine analog, which can inhibit thymidylate synthase.^[27] As shown in Table, three of the drugs tested displayed altered responses in different cell clones. Some of the clones were more resistant than others; some of them were even more sensitive. These might be attributed to different sets of mutation acquired after recovery from apoptotic event in different cell clones. Nevertheless, most of the recovered cell clones expressed improved sensitivity to 5-Fu. These results indicated that ethanol and 5-Fu combined chemotherapy may enhance the elimination of residual HCC cells and thus decrease the cancer recurrence.

 

There are three aspects affecting cancer response to 5-Fu: thymidylate synthase, dihydropyrimidine dehydrogenase, and p53.^[27] p53 is a well-known tumor suppress gene maintaining the DNA integrity of a cell. Bunz et al^[28] reported that p53-deficient cells were sensitized to the effects of DNA-damaging agents while p53 disruption rendered cells resistant to 5-Fu. A number of clinical studies demonstrated that p53 overexpression correlated with resistance to 5-Fu.^{[29, 30]} All of these evidences indicated that p53 is involved in cancer response to 5-Fu. The correlation between p53 and 5-Fu sensitivity after HepG2 cells are recovered from ethanol-induced apoptosis needs further investigation.

 

The similar combination therapy was studied by Jang et al^[31] who showed that the combination of epirubicin, cisplatin, 5-Fu and PEI had better therapeutic efficacy compared with transarterial chemoembolization, which can restrict blood supply and deliver drugs directly to the target tissue. Kawamura et al^[32] also demonstrated that combined transarterial chemoembolization and PEI is an effective option for large size HCC. Taken together, multiple drug combinations synergize ethanol efficacy in cancer therapy.

 

Other changes found in recovered cancer cells were the increases in mobility and invasiveness. Our wound healing assay demonstrated that 90% of the recovered cell clones showed a significant increase in mobile pace compared with controls (Fig. 6A).^[33] Also, 50% of recovered clones showed 1.8-fold higher in invasion compared with that of controls (Fig. 6B). These behaviors might be due to the increased proteolytic enzymes and the alterations in cell adhesiveness. Cancer cells with increased invasiveness can often invade nearby tissue. The metastatic ability of cancer cells recovered from apoptosis requires further investigation.

 

In conclusion, this study confirmed that HepG2 cells can recover from ethanol-induced apoptosis and this might be one of the reasons of treatment failure in clinical practice. The recovered cells increased ethanol resistance and mobility compared with controls. Another important finding is that the recovered clones become more sensitive to 5-Fu. These results indicated that the combination of ethanol and 5-Fu might be useful in preventing cancer recurrence.

 

 

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