Evaluation of the anti-tumor effects of lactate dehydrogenase inhibitor galloflavin in endometrial cancer cells
verfasst von:
Xiaoyun Han, Xiugui Sheng, Hannah M Jones, Amanda L Jackson, Joshua Kilgore, Jessica E Stine, Monica N Schointuch, Chunxiao Zhou, Victoria L Bae-Jump
High rates of aerobic glycolysis represent a key mechanism by which endometrial cancer cells consume glucose as its primary energy source. The up-regulated glycolytic pathway is a common therapeutic target whose inhibition has implications for anti-tumor activity in cancer cells. The present study was aimed at evaluating the potential of a novel lactate dehydrogenase (LDH) inhibitor, Galloflavin, as a therapeutic agent for endometrial cancer. Our results revealed that Galloflavin effectively inhibited cell growth in endometrial cancer cell lines and primary cultures of human endometrial cancer through its involvement in multiple signaling pathways that regulate metabolism, cell cycle, apoptosis, cell stress and metastasis.
The online version of this article (doi:10.1186/s13045-014-0097-x) contains supplementary material, which is available to authorized users.
Xiaoyun Han and Xiugui Sheng contributed equally to this work.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Manuscript editing: CZ VBJ. Conceived and designed the experiments: CZ VBJ. Performed the experiments: XH XS HMJ ALJ JK MNS Analyzed the data: CZ VBJ. Contributed reagents/materials/analysis tools: XH XS CZ VBJ. Wrote the paper: CZ VBJ. All authors read and approved the final manuscript.
To the Editor
Galloflavin (GF), which is synthesized from gallic acid, is a new lactate dehydrogenase inhibitor that inhibits both the A and B isoforms of LDH [1]. By serving as a competitive inhibitor with NADH for LDH, GF has been shown to disrupt aerobic glycolysis and decrease cell viability effectively across many cancer cell types, including breast, colon and liver cancers as well as Burkitt lymphoma [2-4]. To investigate the effect of GF on endometrial cancer cell growth, we utilized the Ishikawa and ECC-1 cells exhibiting high rates of glycolysis to identify the potential of GF. GF significantly reduced LDHA activity, inhibited cell proliferation and reduced colony formation in a dose dependent manner (Figure 1, Additional file 1). The IC50 values for the ECC-1 and Ishikawa cells were 25 uM and 43 uM after 72 hours of treatment, respectively. We next confirmed that GF was responsible for the activation of the mitochondrial apoptosis pathway, accompanied by an increase in cleaved caspase3 and a decrease in MCL-1 and BCL-2 protein expression (Additional file 2: Figure S1). Cell cycle analysis showed minimal G1 phase arrest in the ECC-1 cells and G2 arrest in Ishikawa cells after 24 hours of treatment (Additional file 3: Figure S2), thus, suggesting that GF induces cell cycle changes by altering different checkpoints in different endometrial cancer cells. After treatment with GF for 24 hours, both cell lines had a reduced ability to adhere to laminin-1 as well as decreased migratory capacity as evaluated by a transwell assay. In addition, E–cadherin increased while Slug proteins decreased after treatment with GF (Additional file 4: Figure S3). GF was also shown to increase reactive oxygen species (ROS) and mitochondrial DNA damage after 24 hours of treatment (Additional file 5: Figure S4), indicating that an increase in ROS production and mitochondrial DNA damage might also be involved in the anti-tumorigenic effects of GF in endometrial cancer cells.
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Cancer cells maintain a significant level of mitochondrial oxidative phosphorylation (OXPHOS) capacity to rapidly switch from glycolysis to OXPHOS during carcinogenesis and cell energy stress [5,6]. The point of balance between glycolysis and mitochondrial OXPHOS fluctuates depending on changes in the cancer cells microenvironment. Inhibition of LDHA activity by GF resulted in a decreased rate of glucose uptake and ATP production, with subsequent increased pyruvate dehydrogenase (PDH) protein expression and production of pyruvate (Additional file 6: Figure S5). These findings confirm a direct effect of GF on the glucose metabolism by impairing cytosolic glycolysis in the endometrial cancer cells. Since glycolysis and OXPHOS are tightly coupled processes [7], we noted that GF increased glutaminase protein expression, and enhanced Krebs cycle activity, by increasing the production of malate, another Krebs cycle intermediate, after 16 hours of treatment (Additional file 7: Figure S6). GF was effective in inhibition of cell proliferation in 6 of the 8 primary cultures of endometrial cancer with IC50 values ranging from 20-53 uM. A linear regression model showed the level of LDHA protein but not c-Myc was related to the sensitivity to GF in primary cultures and endometrial cancer cell lines (Figure 2). Moreover, we found that GF decreased c-Myc expression in a dose-dependent manner after 24 hours of treatment. Given that c-Myc transcriptionally induces expression and activity of LDHA [8], the inhibition of c-Myc by JQ1 (a c-Myc inhibitor) synergistically increased the inhibitory effects of GF at different concentrations in GF-sensitive and GF-resistant cells (Additional file 7: Figure S6). These results suggest a causal link between GF treatment and down-regulation of c-Myc expression in endometrial cancer cells (Additional file 8: Figure S7). In conclusion, our study indicated that targeted inhibition of LDH by GF has promising anti-tumor activity in endometrial cancer cell lines and primary cultures of endometrial cancer cells. We believe our present study demonstrates that inhibition of LDH by GF decreases cell proliferation, invasion, and glycolytic metabolism while promoting cell stress and apoptosis in endometrial cancer cells. These findings provide a molecular basis for the use of LDH inhibitors in the treatment of endometrial cancer.
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Acknowledgements
This work was generously supported by NIH/NCI 1K23CA143154-01A1 and the Steelman fund.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
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The authors declare that they have no competing interests.
Authors’ contributions
Manuscript editing: CZ VBJ. Conceived and designed the experiments: CZ VBJ. Performed the experiments: XH XS HMJ ALJ JK MNS Analyzed the data: CZ VBJ. Contributed reagents/materials/analysis tools: XH XS CZ VBJ. Wrote the paper: CZ VBJ. All authors read and approved the final manuscript.
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