Potentiating antitumor effects of a combination therapy with lovastatin and butyrate in the Lewis lung carcinoma model in mice
Increasingly more evidence indicates that there is no single drug with satisfactory efficacy in cancer therapy and therefore combination therapy using two or more chemotherapeutic agents is one of the most common strategies used in current oncology. The toxicity or resistance of tumour cells, however, often limits application of classical chemotherapeutics. This draws attention to other agents with potential antitumor activity but decreased toxicity or with the ability to modulate cancer cells' response to classical chemotherapeutics.
Lovastatin has in recent years gained more attention as an anticancer agent. By inhibiting the activity of HMG-CoA reductase it negatively influences posttranslational modification of a number of proteins important in signal transduction, mainly by inhibition of their farnesylation or geranylgeranylation. The encouraging preclinical results3-5 were followed by clinical trials evaluating the efficacy of lovastatin in tumour therapy.6-8 Butyric acid and its derivatives were also shown to elicit anticancer effects in experimental tumour models.15, 16, 24 Oncological clinical trials with butyric acid derivatives have also been started.17, 18
In our in vitro studies lovastatin used in combination with butyrate showed potentiated antitumor effects against 3LL cells (Fig. 1). Based on the isobolanalysis, this drug interaction could be defined as synergy (Fig. 1b). Accumulation in the G2/M phase of the cell cycle (Fig. 2) and massive apoptosis (Figs. 3, 4) were observed in 3LL cells treated with lovastatin and particularly with lovastatin/butyrate combination. Although G1 arrest seems to be the most characteristic feature for lovastatin-treated cells1, 2 the delay of the progression through G2 has also been reported.1 The observed induction of apoptosis in 3LL cells after treatment with the combination of drugs was accompanied by an increase in both p53 levels and its downstream effector p21Waf1 (Fig. 5). The modulating effects of butyrate and lovastatin on p21Waf1 expression were previously reported.16, 25 Rather surprising effects of the combined treatment on the expression of cyclin D1 were observed. The block in G2/M phase was accompanied by the complete disappearance of cyclin D1 (which is required for S phase entry) in cells treated with both combinations. The dispensable role of cyclin D1 in transition from G1 to S phase, however, was already reported.26
Because butyrate metabolites in the cells may serve as precursors of HMG-CoA, we can speculate that synergistic effects are due to an increase in the cellular concentration of butyrate after HMG-CoA reductase inhibition by lovastatin. The accessibility of higher concentrations of intracellular butyrate or its metabolites may then increase the hyperacetylating effect on histones, which was reported to correlate with their apoptosis inducing activity.27 The use of butyrate and HMG-CoA reductase inhibitor mevastatin in vitro with potentiating antitumor effects was previously reported on colorectal carcinoma cells.19 The combination of butyrate derivative phenylacetate with lovastatin was described to exert cytostatic effects against glioma cells.28
Because butyrate has extremely short half-life, tributyrin (glycerol ester containing three moieties of butyric acid) was used in vivo as a butyrate prodrug. Our study in the murine model of syngeneic tumour demonstrates that both drugs used in combination are able to exert antitumor effects in vivo (Fig. 6). We consider direct induction of 3LL cells apoptosis as the main mechanism responsible for the tumour growth retardation in mice treated with both agents. Other mechanisms, however, could not be excluded. For example, the results of our previous studies suggested that systemic administration of butyrate24 or lovastatin9 demonstrate antiangiogenic activity.
As both lovastatin29 and butyrate30 have been suggested to modulate P-glycoprotein mediated multidrug resistance and both have been already shown to potentiate antitumor effects of 5-fluorouracil and cisplatin,5, 10, 31, 32 antitumor potential of lovastatin and butyrate therapy should be examined extensively in combination with classical chemotherapeutics.