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Abstract: Caffeine has been reported to prevent hepatocarcinogenesis. We investigated the molecular mechanisms by which caffeine inhibits the growth of hepatocellular carcinoma (HCC) cells. We found that caffeine inhibited the proliferation of HCC cells via cell cycle arrest independent of apoptosis. We revealed a novel signalling axis for caffeine involving activation of the mitogen-activated ERK-regulating kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway that resulted in the downstream up-regulation of epidermal growth factor receptor (EGFR), although the MEK/ERK/EGFR signalling pathway was not involved in the growth inhibitory effect of caffeine. Our data reveal that caffeine could be a promising candidate for the treatment of patients with HCC.
Hepatocellular carcinoma (HCC) is one of the most common solid tumours and the third-leading cause of death from cancer worldwide, and its incidence is rising . Although ablative therapies including ethanol injection and radiofrequency ablation as well as surgery have been applied to patients with early stage HCC, the prognosis of these patients is still unsatisfactory. It is particularly hampered by high recurrence rates because most patients with HCC also have persistent hepatitis B and C infections, which are associated with increased risk of HCC . No effective therapeutic agents are currently available, especially when HCC is diagnosed at the advanced stages. Given the poor outcome of patients with HCC, many researchers have been trying to clarify the exact molecular mechanisms of hepatocarcinogenesis. However, effective therapeutic compounds targeting key molecules involved in hepatocarcinogenesis have not been discovered yet.
Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in many popular beverages, including cocoa, tea and coffee. It may well be the most frequently ingested neuroactive drug in the world . There is a growing body of evidence that caffeine has beneficial effects on the liver. For example, consumption of coffee was associated with a lower incidence of chronic liver disease  and a reduced risk of HCC [5–9], and this effect was presumably mediated by caffeine, In addition, preventative effects of caffeine in rodent HCC models have been demonstrated [10–12]. However, the molecular mechanisms by which caffeine exerts beneficial effects on hepatocarcinogenesis are poorly defined. Therefore, we sought to examine the biological effects of caffeine on HCC cells and to clarify their molecular mechanisms. We found that caffeine inhibited the growth of HCC cells by mechanisms independent of apoptosis and by cell cycle arrest at the G0/G1 phase. We also found that caffeine activated the mitogen-activated ERK-regulating kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway, which was responsible for enhanced expression of epidermal growth factor receptor (EGFR).
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- Materials and Methods
Caffeine can be found in many popular beverages, including cocoa, tea and coffee. Although caffeine is most commonly used as a stimulant to prevent sleepiness and as a remedy for pain , there is a mounting body of in vitro evidence suggesting caffeine inhibits the growth of a variety of cancer cells through cell cycle arrest and the induction of apoptosis [3,16–18], and that caffeine enhances the toxicity of radiation and chemotherapeutic drugs . In clinical settings, high consumption of caffeine has been associated with beneficial effects on the liver, including a lower incidence of chronic liver disease  and a reduced risk of HCC [5–9].
In the present study, we investigated the molecular mechanisms and targets of the antiproliferative effect of caffeine on HCC cells. We demonstrated that treatment with caffeine at concentrations of 1 mM to 20 mM inhibited the growth of HCC cells. We used caffeine at concentrations mostly similar to those utilized in previous in vitro studies; however, to achieve a 2-mM blood level of caffeine, over 100 cups of coffee intake would be required . Therefore, our data in which high doses of caffeine were applied to cells may not be clinically relevant. However, we believe that our work could be a precedent for elucidation of the molecular mechanisms involved in the antiproliferative action of caffeine on HCC cells.
Caffeine has been reported to affect cell cycle function and to induce apoptosis in pancreatic cancer  and neuroblastoma  cells. However, our data revealed that the growth inhibitory effect of caffeine on HCC cells was associated with cell cycle arrest alone, not apoptosis. The molecular mechanisms by which caffeine inhibits cancer cell growth may be distinct depending on the cell types. The exact molecular targets of caffeine-mediated cell cycle regulation need to be further clarified, but may include cyclins and cyclin-dependent kinases.
We found that escalating doses of caffeine activated two MAPKs, MEK/ERK1/2 and p38MAPK, in HepG2 cells, a subset of HCC cells. Because these MAPKs have often been associated with growth modulation of cancer cells, including HCC cells, in positive or negative manners depending on the cellular context [13,22], we investigated the role of activated MAPKs in HepG2 cells. Contrary to our expectation, when MEK/ERK1/2 and p38MAPK were inhibited by U0126 and SB202190, respectively, growth inhibition of HepG2 cells by caffeine was not significantly modulated. Therefore, neither p38MAPK nor MEK/ERK1/2 molecules appear to play a role in growth inhibition by caffeine. The exact biological function of these MAPKs needs to be delineated in future studies.
We investigated the expression of EGFR when HCC cells were treated with caffeine, because EGFR has been often linked to the proliferation of a variety of cells, including HCC [23,24]. The expression level of EGFR was significantly enhanced after caffeine treatment of HepG2 cells, the same cells in which we observed ERK1/2 activation. Because our finding that caffeine-increased EGFR expression was novel, we further investigated the upstream molecules responsible for enhanced EGFR expression. Because ERK1/2 activation was observed 24 hr prior to the up-regulation of EGFR, which peaked at 48 hr, we hypothesized that ERK1/2 activation might be upstream of EGFR expression, although this scenario is contrary to the accepted mechanism . To our surprise, but in accord with our hypothesis, EGFR expression was totally abrogated by the pre-treatment of HepG2 cells with U0126, a MEK inhibitor. To our knowledge, there have been only few reports demonstrating that MAPKs regulate upstream molecules in their signalling pathways, including findings that ERK1/2 regulated Ras  and p38MAPK regulated EGFR .
Although we did not directly suppress EGFR expression by employing genetic approaches such as small interfering RNA, we were able to inhibit EGFR expression using the MEK inhibitor U0126. By taking advantage of this novel signalling axis, we were able to examine a potential role for EGFR in the growth inhibitory effect of caffeine. Our data revealed that the growth inhibitory effect of caffeine was not significantly modulated when EGFR expression was inhibited by U0126. Thus, we conclude that the novel MEK/ERK/EGFR signalling pathway is not involved in growth inhibition by caffeine. Because EGFR has been associated with cell differentiation, motility and angiogenesis in addition to cell growth regulation , we speculate that the enhanced expression of EGFR by caffeine might play a role in the modulation of one or more of these other functions. To clarify the precise role of the MEK/ERK/EGFR signalling pathway after activation by caffeine, further experiments are necessary.