5-fluorouracil (5-FU) and cisplatin (CDDP) (Sigma Aldrich, UK) were dissolved in double distilled water to yield working stocks at concentration of 20 mM and 2 mM respectively. The working stocks were stored at room temperature.

The parental human hepatocellular carcinoma (HEPG2) cell line was obtained from the European Collection of Animal Cell Cultures (ECACC). Cells were cryopreserved and then rapidly thawed and grown in RPMI 1640 medium (Invitrogen, UK), fully supplemented with 10% heatinactivated foetal bovine serum (FBS) (Invitrogen, UK), 1% glutamine (Invitrogen, UK) and 1% penicillin streptomycin (Invitrogen, UK). The physiological conditions of the cells were maintained at 5% CO₂ and 37˚C in a humidified atmosphere. Logarithmically growing cells were at the second passage when they were cryopreserved with a freezing medium containing 10% dimethylsulphoxide (DMSO) (Sigma Aldrich, UK) and 90% supplemented RPMI 1640 medium (Invitrogen, UK). The freezing vials containing the cells were cryo-preserved in a cryovial containing isopropanol and maintained at –80˚C for 24 hours; and the vials containing the parental cells were finally maintained as working stock at –196˚C in liquid nitrogen.

Cell viabilities in response to chemotherapeutic induction, and the degrees of cytotoxicity of CDDP and 5-FU on the cells were measured using cell counting kit (CCK-8) (Sigma Aldrich, UK). The CCK-8 is made up of Dojindo’s highly water-soluble tetrazolium salt, WST-8[2- (2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]; which produces a water-soluble, yellowish formazan dye upon reduction in an electron carrier [12]. The number of viable cells is directly proportional to the amount of the formazan dye generated in cells. Assays were done following manufacturer’s instruction. Briefly, cells were seeded at density of 10⁴ cells per well in 96-well plates and preincubated overnight for adherence at physiological conditions of 5% CO₂ and 37˚C, in a humidified atmosphere. The cells were then treated at concentrations of 0 µM to 20 µM for CDDP; and 0 µM to 100 µM for 5-FU. After 12 to 24 hours incubation, 10 µl of CCK-8 was added to each well and incubated at 37˚C for 2 hours; followed by the measurement of absorbance using an automated micro-plate reader ELx 800 (BioTek, UK) at 450 nm. Each experiment was done in triplicates and the results were the mean of at least three independent measurements and expressed as percentage of the absorbance of untreated control cells ± S.E.M.

The inhibitory concentration otherwise known as IC₅₀, is the optimal concentration at which the survival rate of HEPG2 cells reached the minimum following exposure to an appropriate anticancer drug [13]. Determination of this concentration (IC₅₀) is primordial to the selection of cells for any drug resistance. Briefly, cells at the logarithmic growth phase (80% confluence) were harvested and seeded at a density of 10⁴ cells per well in 96-well plates and incubated overnight for proper adherence. Subsequently, the drug-free medium was replaced with prewarmed fully supplemented fresh medium, containing 0 µM to 100 µM of 5-FU or 0 µM to 20 µM of CDDP. The cells were incubated for 24 hours, while maintaining the physiological conditions at 5% CO₂ and 37˚C temperature, in a humidified atmosphere. After 24 hours incubation, cell viability assessment was conducted colorimetrically using cell counting kit-8 (CCK-8) (Sigma Aldrich, UK). The optical densities (OD) of cells treated at different drug concentrations were measured at 450 nm using a micro-plate reader ELx 800 (BioTek, UK); and cell viability was calculated as a measure of the optical density (OD) of treated cells relative to the optical density of the untreated controls, excluding the OD of blank controls. Dose-dependent response curve was plotted and the respective inhibitory concentrations (IC₅₀) of CDDP and 5-FU were established.

The inhibitory concentrations (IC₅₀) were confirmed by the flow cytometric analysis of cell death using Annexin V-FITC kit (BD Biosciences, San Diego). Briefly, HEPG2 cells were grown to 80% confluence, and detached using 0.25% (w/v) trypsin in 5 mM EDTA (Invitrogen). The cells were seeded at the rate of 2 × 10⁵ per ml of RPMI 1640 medium (Invitrogen), supplemented with 10% foetal bovine serum (Invitrogen), 1% Penicillin streptomycin (Invitrogen), and 1% L-glutamine (Invitrogen). After 24 hour incubation in appropriate concentrations of CDDP (0 - 16 µM) or 5-FU (0 - 90 µM), under physiological conditions of 5% CO₂, 37˚C, in a humidified atmosphere; the cells were detached by trypsinization, washed and re-suspended in binding buffer. The cells were analysed for apoptosis following mixing with Annexin VFITC and propidium iodide (PI), and incubation in the dark for 5 minutes. Analysis was done using flow cytometer FACSCalibur (BD Biosciences, Europe).

In selection of the cells for resistance, clinical method of treatment was adopted. The parental HEPG2 cell line was treated in pulse, at the IC₅₀ of either CDDP or 5-FU for 4 to 6 hours. Induction was repeated 6 times, whilst allowing the cells to attain at least 70% confluence between induction intervals. After 6 complete cycles of induction, selected cells were maintained in drug-free RPMI 1640 medium containing appropriate supplements, and passaged at the attainment of 70% - 80% confluence. No further experiment was performed on the cells until after 4 weeks maintenance in drug-free medium, whilst testing the stability of the developed resistance.

The stabilities of the selected clones (HEPG2CR) and (HEPG2FR) were tested after 2 weeks, one month and 3 months of drug withdrawal. Briefly, the selected clones were harvested at exponential growth phase using 0.25% trypsin-EDTA. Cell counting was performed using heamatocytometer, and the cells were seeded in 96-well plate at the rate of 10⁴ cells per well, in triplicates. Plates were maintained at 37˚C in a humidified atmosphere of 5% CO₂. After overnight incubation for attachment, cells were incubated for 24 hours in RPMI 1640 medium containing appropriate concentration of CDDP (0 - 20 µM) or 5-FU (0 - 100 µM). Following the 24 hours incubation, the cells were further incubated for 2 hours in the presence of 10 µl of CCK-8 per well. The optical densities were measured and the new IC₅₀ was obtained from a dose-dependent cytotoxicity curve for each of the drugs as previously described. The difference between the IC₅₀ of the resistant clone and that of the parental cell line defines the degree of resistance.

To understand the effect of CDDP or 5-FU on HEPG2 cell viability and establish the degree of cytotoxicity due to these drugs, HEPG2 cell line was incubated for 24 hours with 0 µM to 20 µM of CDDP or 0 µM to 100 µM of 5-FU. Cell viabilities were measured by CCK-8 assay and HEPG2 cell line was found to be more susceptible to CDDP than 5-FU. Furthermore, with 5-FU, cytotoxicity was found to be directly proportional to dose and time. However, for CDDP, an approximately reciprocal degree of cytotoxicity was obtainable at both 12 hours treatment and 24 hours treatment with similar concentration (Figure 1).

To determine the concentration of CDDP or 5-FU beyond which HEPG2 cell survival was minimum, the cells were exposed to different concentrations of CDDP (0 µM to 20 µM) or 5-FU (0 µM to 100 µM) for 24 hours. The respective inhibitory concentration was calculated from a dose-dependent response curve, following analysis of cell viability by CCK-8 assay. For CDDP, cell survival was minimal beyond 4 µM (Figure 1), but in the case of 5-FU, cell survival was minimal beyond 50 µM (Figure 2). Hence, the IC₅₀ of CDDP was found to be 4 µM while that of 5-FU was 50 µM. The inhibitory concentrations were confirmed by the flow cytometric analyses of cell deaths due to CDDP or 5-FU (data not shown).

Following the establishment of the concentration of CDDP or 5-FU at which HEPG2 cell viability is reduced by 50%, the resistant sublines HEPG2CR and HEPG2FR were established by incubating the parental HEPG2 cell line at the IC₅₀ of CDDP (4 µM) or 5-FU (50 µM, whilst imitating the clinical method of treatment. The cells were found to have developed resistance to either CDDP or 5-FU following six intervals of induction. A test for resistance was conducted after 2 weeks of growth in drugfree medium, and a comparison of the IC₅₀ of the parental cells and those of the resistant cells revealed a remarkable increase. For CDDP, the IC₅₀ of HEPG2CR was double that of the parental cell line, hence a 100% increase (Figure 3). However, for 5-FU, the IC₅₀ of

HEPG2FR was found to be 65 µM as against the 50 µM of the parental cell line (Figure 4).