Metformin-induced energy deficiency leads to the inhibition of lipogenesis in prostate cancer cells

Abstract

The deregulation of lipid metabolism is a hallmark of tumor cells, and elevated lipogenesis has been reported in prostate cancer. Metformin, a drug commonly prescribed for type II diabetes, displays antitumor properties. Here, we show that metformin inhibits lipogenesis in several prostate cancer cell lines. In LNCaP cells, this effect parallels the decrease of key lipogenic proteins: ACC (acetyl-CoA carboxylase), FASN (fatty acid synthase) and SREBP1c (sterol regulatory element binding protein-1c), whereas there is no modification in DU145 and PC3 cells. Despite the relatively high level of lipogenic proteins induced by the overexpression of a constitutively active form of SREBP1c or treatment with androgens, metformin is still able to inhibit lipogenesis. Metformin does not alter the concentration of malonyl-CoA (the fatty acid precursor), and it only slightly decreases the NADPH levels, which is a co-factor required for lipogenesis, in LNCaP. Finally, we show that the inhibitory effect of metformin on lipogenesis is primarily due to a cellular energy deficit. Metformin decreases ATP in a dose-dependent manner, and this diminution is significantly correlated with the inhibition of lipogenesis in LNCaP and DU145. Indeed, the effect of metformin is linked to changes in the ATP content rather than the regulation of protein expression. Our results describe a new mechanism of action for metformin on prostate cancer metabolism.

Keywords: ATP; cancer metabolism; lipogenesis; metformin; prostate cancer.

Figure 1. Metformin inhibits lipogenesis in prostate cancer cells

The cells were treated for 24 h with 5 mM metformin before measuring the incorporation of the [³H] acetate into the lipids, as described in the Materials and Methods section. The results are representative of four independent experiments, ***p < 0.005.

Figure 2. The effect of metformin on the lipogenic proteins

(A) A simplified schematic representation of lipogenesis. ACC: acetyl-CoA carboxylase, FASN: fatty acid synthase, AMPK: AMP activated protein kinase. (B) The cells were treated with 5 mM metformin (M) for 24 h, and the mRNA levels of ACC, FASN and SREBP1c were determined as described in the Materials and Methods section. **p < 0.01; ***p < 0.005. (C) An immunoblot of the indicated proteins in the cells treated with 5 mM metformin for 24 h (M). The blots are representative of three independent experiments.

Figure 3. Metformin represses SREBP1c-stimulated lipogenesis

(A) LNCaP cells were infected with a control adenoviral construct (AdCTL) or an adenoviral construct expressing the constitutive form of SREBP1c (AdSREBP1c CA). After 24 h, the cells were treated with 5 mM metformin (M) for 24 h, and the lipogenesis was observed using [³H] acetate as previously described. (B) An immunoblot of the key lipogenic proteins in the LNCaP cells treated as described in A. **p < 0.01; ***p < 0.005.

Figure 4. Metformin inhibits androgen-stimulated lipogenesis

(A) An immunoblot of the lipogenic proteins in the LNCaP cells treated with 1 or 10 nM R1881 for 48 h and 5 mM metformin for 24 h. (B) The LNCaP cells were treated for 48 h with 1 or 10 nM R1881 and then with 5 mM metformin for 24 h before the quantification of the lipogenesis. **p < 0.01; ***p < 0.05.

Figure 5. Metformin does not affect the malonyl-CoA and NADPH concentration

(A, B) LNCaP and DU145 were treated with 5 mM metformin, 5 mM AICAR (for malonyl-CoA), or 20 mM 2-DG (for NADPH) for 24 h. The concentrations of the malonyl-CoA and NADPH were determined as described in the Materials and Methods section. The graphs represent the results as the fold change versus CTL. **p < 0.01; ***p < 0.05.

Figure 6. Metformin induces a state of energy deficiency, which correlates with a decrease in lipogenesis

(A, B) LNCaP and DU145 were treated with increasing concentrations of metformin (0.25 to 5 mM). The ATP content (left panel), expressed as the % of the control, and lipogenesis were monitored. The correlation between the lipogenesis and the ATP content is shown in the left panel. The results are representative of three independent experiments.