Background: Paracetamol exerts toxic effects on liver cells through its metabolism into N-acetyl-p-benzoquinone imine (NAPQI), which is detoxified by conjugation with cellular glutathione (GSH). Once GSH is depleted, NAPQI stimulates a range of oxidative reactions that result in cell necrosis. The aim of the present investigation is to find a new strategy that would selectively protect normal hepatic tissues and sensitize liver cancer cells to the toxic effects of paracetamol or its metabolite. This may lead to the development of a targeted therapy for liver cancer. Methods: The anti-proliferative effects of paracetamol and buthionine sulfoximine BSO (a glutathione depleting agent) alone and in combination on the liver cancer cells HepG2 and normal rat hepatocytes were investigated by sulphorhodamine-B assay. Effects on cell cycle regulation and induction of apoptosis were tested by flow cytometry. The level of prostaglandin expression was measured by ELISA. Results: The present study showed that both agents alone or in combination have anti-proliferative effects on both cell types. Surprisingly, BSO showed a cytoprotective effects on normal hepatocytes treated with high concentrations (1.75 and 2 mM) of paracetamol. This was confirmed by cell cycle analysis that recorded decreased fraction of sub-G1 cells indicating reduction of apoptosis in normal hepatocytes. Analysis of prostaglandin E2 revealed differential effects of paracetamol on normal and liver cancer cells. A significant increase in PGE2 level over the control was observed in normal hepatocytes whereas a significant decrease was seen in HepG2 cells after treatment with paracetamol. Conclusion: These results indicate that combination of paracetamol/BSO has differential effects on liver cancer cells and normal hepatocytes, which opens the avenue for a new effective and selective combination for management of liver cancer.
Being the current leading cause of death worldwide, cancer is considered as one of the major health problems. Primary cancers of the liver represent the fifth most common malignancy worldwide and the second most common cause of death from cancer [
In addition to its ability to treat pain and fever, Paracetamol was found to induce apoptosis and necrosis in various types of cultured liver carcinoma cells specially HepG2 cell line [
Human liver carcinoma cell line (HepG2) and primary normal rat hepatocytes were used in this study. HepG2 cell line was purchased from American Type Culture collection and was maintained in Dulbeco’s Modified Eagle’s Medium with phenol red (DMEM, Lonza, Belgium) supplemented with 10% fetal bovine serum (Cambrex, Belgium), penicillin/streptomycin (Lonza, Belgium). The Primary liver cells were isolated from male rat liver by two-step collagenase perfusion method according to Seglen [
Cytotoxicity of paracetamol alone (Bristol-Myers Squibb Pharmaceuticals Ltd, Anagni, Italy) or in combination with DL-Buthionine-(S, R)-Sulfoximine (Sigma, USA) was determined using sulpho Rhodamine-B (SRB) method as previously described [
Cell cycle and apoptosis were analyzed by flowcytometry as previously described [
The level of prostaglandin E2 (PGE2) in control and treated cells was measured colorimetrically using PGE2 assay kit purchased from R & D Systems (Minneapolis, USA). It is an accurate competitive immunoassay method that was done according to the manufacturer’s instructions. Briefly, 105 cells were seeded in 25 cm2-cell culture flasks and treated after 24 h with either paracetamol alone or in combination with BSO then flasks were incubated at 37˚C. After 48 h, cell culture supernatants were collected and centrifuged. This cell extract was used to measure PGE2 quantitatively by mixing in a 96-well plate with the reagents supplied with the kit. The PGE2 standard dilution series was made from the highest standard stock (supplied with the kit). The color development is stopped, and the absorbance is read at 450 nm using a spectrophotometric ELISA microplate reader (Meter tech. S960, USA).
Statistical analysis, data fitting and graphics were performed by the Prism 5.03 computer program (GraphPad Software, USA). Data are given as mean ± SEM of at least three independent experiments. Multiple comparisons were carried out using one way analysis of variance (ANOVA) followed by LSD (least significant difference) post-hoc test. Statistical significance was acceptable at a level of p < 0.05.
Survival of hepatoma (HepG2) cells was reduced following treatment with paracetamol alone in a concentration-dependant manner. Addition of BSO enhanced the anti-proliferative effect of paracetamol on HepG2 cells (
On the other hand, the addition of BSO potentiated the cytotoxic effect of paracetamol on normal hepatocytes at low (1.00, 1.25 and 1.5 mM) but not at high concentrations (1.75 and 2 mM) of paracetamol (
In order to understand the mechanisms underlying the anti-proliferative effects of
paracetamol and BSO and to understand the mechanism of their interaction, effects on cell cycle regulation and induction of apoptosis were measured.
Treatment of HepG2 cells with BSO alone for 48 h showed no remarkable changes in cell cycle distribution compared with control cells while treatment with paracetamol alone for the same period arrested cells at G1 phase. Upon combining BSO and paracetamol, a complete G1 block was noticed (
Longer exposure to drugs (72 and 96 hr) revealed a fraction of subG1 cells indicative of apoptosis. This SubG1 area was greatly increased upon treatment of HepG2 with paracetamol alone and in combination with BSO for 72 and 96 h (
Control primary normal rat hepatocytes showed a high fraction of subG1 cells which may be attributed to the use of collagenase and hyaluronidase in cells isolation. After 48 hr treatment with paracetamol, a strong amplification of the subG1 phase was noticed denoting the occurrence of apoptosis and/or necrosis. However, pretreatment of
Cell line | Drug | Duration | Viability % (from 0 - 1) |
---|---|---|---|
HepG2 | No drug control | 48 hr | 1.000 |
HepG2 | BSO (1 mM) | 48 hr | 0.414579 |
HepG2 | Paracetamol (2 mM) | 48 hr | 0.375722 |
HepG2 | BSO + Para (1 mM + 2 mM) | 48 hr | 0.335655 |
Hepatocytes | No drug control | 48 hr | 1.000 |
Hepatocytes | BSO (1 mM) | 48 hr | 0.55816 |
Hepatocytes | Paracetamol (2 mM) | 48 hr | 0.535 |
Hepatocytes | BSO + Para (1 mM + 2 mM) | 48 hr | 0.831072 |
hepatocytes with BSO surprisingly decreased that subG1 area obviously indicating a cytoprotective effect (
Cell line | Drug | Duration | Sub G1 phase % | G0/G1 phase % | S phase % | G2/M % |
---|---|---|---|---|---|---|
HepG2 | No drug control | 48 hr | 0% | 75.54% | 24.35% | 0.11% |
HepG2 | BSO | 48 hr | 0% | 74.19% | 25.48% | 0.34% |
HepG2 | Paracetamol | 48 hr | 0% | 92.31% | 7.69% | 0% |
HepG2 | BSO + Para | 48 hr | 0% | 99.80% | 0.13% | 0.07% |
HepG2 | No drug control | 72 hr | 6.15% | 70.77% | 23.08% | 0% |
HepG2 | BSO | 72 hr | 8.02% | 69.14% | 22.84% | 0% |
HepG2 | Paracetamol | 72 hr | 37.20% | 53.62% | 6.28% | 2.90% |
HepG2 | BSO + Para | 72 hr | 24.20% | 30.57% | 29.30% | 15.92% |
HepG2 | No drug control | 96 hr | 23.27% | 59.12% | 15.72% | 1.89% |
HepG2 | BSO | 96 hr | 6.31% | 56.76% | 36.94% | 0% |
HepG2 | Paracetamol | 96 hr | 79.42% | 18.65% | 1.93% | 0% |
HepG2 | BSO + Para | 96 hr | 54.88% | 8.13% | 36.99% | 0% |
Normal Hepatocytes | No drug control | 48 hr | 27.34% | 67.63% | 4.32% | 0.72% |
Normal Hepatocytes | BSO | 48 hr | 29.17% | 62.50% | 8.33% | 0% |
Normal Hepatocytes | Paracetamol | 48 hr | 40.06% | 53.78% | 6.16% | 0% |
Normal Hepatocytes | BSO + Para | 48 hr | 22.05% | 71.79% | 5.13% | 1.03% |
Quantitative analysis of PGE2 level in normal hepatocytes showed significant increase in PGE2 level over the control after all treatment schedules except for the combination of paracetamol and BSO. That combination, on the contrary, showed a significant decrease compared with control, BSO- or paracetmol-treated cells (
On the other hand, in HepG2 cells a totally reversed effect was noticed. A significant decrease in the level of PGE2 was observed after all treatment regimens except for both drugs combination which showed a significant increase compared with those of the control, BSO- or paracetmol-treated HepG2 cells (
Despite the great advance achieved in cancer therapy in the last decade, management of some types of malignant diseases such as liver cancer remains a great challenge. Side
Cell line | Drug | Duration | PGE2 concentration (pg/ml) | Statistical Significance |
---|---|---|---|---|
HepG2 | No drug control | 0 hr | 149.997 | d |
HepG2 | No drug control | 48 hr | 228.837 | d |
HepG2 | BSO | 48 hr | 173.979 | a, c, d |
HepG2 | Paracetamol | 48 hr | 129.096 | a, b, d |
HepG2 | BSO + Para | 48 hr | 201.213 | a, b, c, d |
Hepatocytes | No drug control | 0 hr | 186.042 | d |
Hepatocytes | No drug control | 48 hr | 219.579 | d |
Hepatocytes | BSO | 48 hr | 408.201 | a, c, d |
Hepatocytes | Paracetamol | 48 hr | 555.594 | a, b, d |
Hepatocytes | BSO + Para | 48 hr | 315.072 | a, b, c, d |
effects of cancer therapeutics limit the usefulness of many anticancer agents. Therefore, there is a great interest in developing agents with different effects on malignant and normal cells or using additional components that may preferentially sensitize cancer cells to the effect of anticancer agents. Paracetamol that is approved long time ago as analgesic antipyretic is a well established hepatotoxic at high doses. This effect is attributed to its metabolite NAPQI that accumulates after depletion of glutathione stores. The aim of the present study is to investigate the cellular effects of therapeutic- or low-dose paracetamol treatment on hepatoma cells versus normal rat hepatocytes in the presence and absence of the selective GSH-depleting agent BSO. The ultimate goal is to establish a strategy or a paracetamol-based combination therapy that may improve the outcome of liver cancer therapy with least effects on normal hepatocytes. Our results showed that both paracetamol and BSO either alone or in combination have anti-proliferative effects on both liver cancer (HepG2) and on normal rat hepatocytes. A previous study stated that HepG2 cell line constitutively expresses CYP2E1, CYP1A2 and CYP3A4, all are involved in paracetamol metabolism [
BSO either alone or in combination with low concentrations of paracetamol (1, 1.25, 1.5 mM) was also able to sensitize normal hepatocytes to paracetamol cytotoxic effects. Surprisingly, BSO showed a reversed action when combined with higher concentrations of paracetamol (1.75 and 2 mM). At 2 mM paracetamol, BSO caused a cytoprotective effect on normal hepatocytes and cells showed enhanced survival. Cell cycle analysis of normal hepatocytes revealed that either BSO or paracetamol alone caused an increased sub G1 phase compared with control cells confirming the incidence of apotosis/necrosis or both. On the contrary, their combined effect in normal hepatocytes showed a collapsed sub G1 area, similar to the one of untreated cells, denoting lesser cytotoxicity. This acquired cytoprotective property supported the results of SRB assay which confirmed the ability of BSO to reverse the cytotoxic action of higher doses of paracetamol in normal hepatocytes. This phenomenon might be explained by the report of Syng-ai et al. [
Incubation time is recognized as a reliable factor that may affect the cytotoxic effects of paracetamol, BSO, and their combination. In the present study, the best cytotoxic effects were achieved after exposure of cells to drug combinations for of 72 and 96 hours. This may be due to the direct activation by paracetamol/BSO of endonucleases leading to induction of cell death. This finding comes in line with Manov et al. [
Paracetamol is an inhibitor of cyclooxygenase enzymes that may affect the level of prostaglandin synthesis. Therefore, the role of prostglandins (PGs) in the cytotoxicity of BSO and paracetamol was investigated. PGs, especially PGE2, have been implicated as modulators of tumor metastasis [
As a fact, inhibition of COX enzyme by paracetamol and non steroidal anti inflammatory drugs (NSAIDs) is the main reason for the inhibition of PGs synthesis. Unlike aspirin, inhibition of cyclooxygenase (COX) activity by paracetamol is biphasic and requires glutathione and hydroquinone as cofactors [
In conclusion, paracetamol alone is more cytotoxic to the liver cancer HepG2 cells than the normal hepatocytes. Addition of BSO to paracetamol showed differential effects on cancer and normal liver cells. Enhancement of paracetamol cytotoxicity on liver cancer cells was observed at low and high paracetamol concentrations while its effect on paracetamol toxicity on normal hepatocytes was dependent on paracetamol concentration with increased cytotoxicity at low but increased protection at high paracetamol concentration. This finding if confirmed on other liver cancer and normal cells may open the avenue for a novel combination with more toxicity to liver cancer cells and protection of normal liver cells.
Conflict of Interest: All authors declare that have no conflict of interest.
Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.
Sayour, M.E., El Salam, R.M.A., Elyamany, M.F., El Sayed, A.M. and El-Awady, R.A. (2016) Combination of Paracetamol and the Glutathione De- pleting Agent Buthionine Sulfoximine Show Differential Effect on Liver Cancer Cells and Normal Hepatocytes. Pharmacology & Phar- macy, 7, 443-458. http://dx.doi.org/10.4236/pp.2016.711051