2-Methoxyestradiol Reverses the Pro-Carcinogenic Effect of L-Lactate in Osteosarcoma 143B Cells

Abstract

Background/aim: According to the reverse Warburg effect, tumor cells may metabolize lactate as an energy source and shuttle L-lactate to neighboring cancer cells, adjacent stroma, and vascular endothelial cells, thus inducing metabolic reprogramming. An increased tumor L-lactate level strictly correlates with increased metastasis, tumor recurrence and a poor outcome. A potent anticancer agent that may act on L-lactate activated cells is 2-metoxyestradiol. Thus, the aim of the study was to evaluate whether a potent anticancer agent, 2-methoxyestradiol, is able to reverse L-lactate-induced metabolic reprogramming in osteosarcoma 143B cells.

Materials and methods: We used flow cytometry in order to determine cell death, autophagy, expression of KI-67, mitochondrial membrane depolarization. We performed cell proliferation assay in order to determine cell viability and cell migration assay to determine invasive potential of osteosarcoma cells. While, CalcuSyn software was used in order to evaluate the interaction between 2-methoxyestradiol and L-lactate.

Results: We demonstrated that 2-methoxyestradiol abolished L-lactate-induced migration and proliferation of osteosarcoma cells. Moreover, we observed that this effect was associated with regulation of Ki-67 and induction of autophagy.

Conclusion: 2-Methoxyestradiol is a potent anticancer agent also under metabolic reprogramming conditions.

Keywords: 2-Methoxyestradiol; L-lactate; osteosarcoma; tumor microenvironment.

Figure 1. Experimental design. Osteosarcoma 143B cells were first treated with L-lactate to induce the reverse Warburg effect. Afterword, the cells were continuously treated with L-lactate and/or 2-ME. Cells treated separately with 2-ME and control cells were also used.

Figure 2. Effects of L-lactate in low and high glucose medium on osteosarcoma cell viability (A). Effect of 2-ME, L-lactate and combination on proliferation of osteosarcoma 143B cells (B-D). The inhibition of osteosarcoma proliferation was determined using the MTS assay. The values are the means±SE of three independent experiments (N=6 replicate cultures). The absence of an error bar denotes a line thickness greater than the error. **p<0.001, ***p<0.0001, ****p<0.00001 versus control cells (C).

Figure 3. Antagonistic effects between L-lactate and 2-ME. The data obtained from the MTS assay were analyzed using CalcuSyn software version 2.0 (Biosoft) and presented in the form of a Median-Effect Plot (A) and a Dose-Effect curve (B).

Figure 4. Antagonistic effects between L-lactate and 2-ME. The data obtained from the MTS assay were analyzed using CalcuSyn software version 2.0 (Biosoft) and presented in the form of an algebraic estimate (fractional effect) (A), and a Fa-CI plot (B).

Figure 5. Impact of 2-ME and L-lactate separately or in combination on the migratory potential of osteosarcoma cells (A), Ki-67-positive cells (B), and the induction of autophagy (C). The values are the means±SE of three independent experiments (N=6 replicate cultures). The absence of an error bar denotes a line thickness greater than the error. **p<0.001, ***p<0.0001, ****p<0.00001 versus control cells (C).

Figure 6. Impact of 2-ME and L-lactate separately or in combination on the mitochondrial membrane depolarization (A), the induction of cell death (B, C). The values are the means±SE of three independent experiments (N=6 replicate cultures). The absence of an error bar denotes a line thickness greater than the error. **p<0.001, ***p<0.0001, ****p<0.00001 versus control cells (C).