Intracellular ATP levels are a pivotal determinant of chemoresistance in colon cancer cells - PubMed (original) (raw)

. 2012 Jan 1;72(1):304-14.

doi: 10.1158/0008-5472.CAN-11-1674. Epub 2011 Nov 14.

Federico Tozzi, Jinyu Chen, Fan Fan, Ling Xia, Jinrong Wang, Guang Gao, Aijun Zhang, Xuefeng Xia, Heather Brasher, William Widger, Lee M Ellis, Zhang Weihua

Affiliations

Intracellular ATP levels are a pivotal determinant of chemoresistance in colon cancer cells

Yunfei Zhou et al. Cancer Res. 2012.

Abstract

Altered metabolism in cancer cells is suspected to contribute to chemoresistance, but the precise mechanisms are unclear. Here, we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP, and enhanced HIF-1α-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1α and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater "ATP debt" defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing that intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment.

©2011 AACR.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Characterization of cross-chemoresistant cancer cell lines

A, The drug resistant cells were continuously maintained in media with 2 μM oxaliplatin. Before plating for each experiment, media without drug was used for two passages. HT29-OxR and HCT116-OxR cells grew more slowly than their parental cells. See also Table S1. B, Survival inhibition curves from MTT assays showed a chemoresistant phenotype of HT29-OxR and HCT116-OxR cells to oxaliplatin (top panels) and 5-FU (bottom panels). C, HT29-OxR and HCT116-OxR showed decreased PARP cleavage (and thus decreased apoptosis) under 24h treatment with oxaliplatin (top panels) and 5-FU (bottom panels). Actin served as a loading control. All experiments were repeated at least three times. Representative data is shown.

Figure 2

Figure 2. Measurement of intracellular ATP and mitochondrial activity

A, HT29-OxR and HCT116-OxR cells displayed more than a 2-fold increase in intracellular ATP levels compared with their parental cells. B, HT29-OxR and HCT116-OxR cells consumed oxygen at a significantly higher rate than their parental cells as indicated by oxygen consumption rate (OXR, pmole/min). C, HT29-OxR and HCT116-OxR cells displayed morphologic changes as examined by TEM. Bar: 2 μm. At least 20 individual cells were analyzed for each cell type. Representative images are shown. D, Purified mitochondria from HT29-OxR and HCT116-OxR showed decreased substrate ATP production ability from both complex I (glutamate/malate as substrates) and complex II (succinate as a substrate) compared with that of their parental cells. All experiments were repeated at least three times. Representative data is shown.

Figure 3

Figure 3. Characterization of glycolytic activity and HIF-1α activity

A, HT29-OxR and HCT116-OxR cells consumed glucose at a higher rate than their parental cells. B, HT29-OxR and HCT116-OxR cells produced lactate at a higher rate than their parental cells. C, Key glycolytic enzymes GLUT1, HK2, LDHA, and HIF-1α were upregulated in HT29-OxR and HCT116-OxR cells compared with their parental (Par) cells, as shown by Western blot. D, HT29-OxR and HCT116-OxR produced more VEGFA into cell culture medium than their parental cells (top panel), which correlated with VEGFA mRNA level by qPCR (bottom panel). All experiments were repeated at least three times. Representative data is shown.

Figure 4

Figure 4. ATP regulates HIF-1α expression and induces a drug-resistant phenotype

A, Ratio between absolute cellular ADP and AMP amount decreased in HT29-OxR and HCT116-OxR cells, as shown by HPLC-MS measurement. B, PFK enzyme activity is elevated in HT29-OxR and HCT116-OxR cells compared with parental cells. The enzyme activity is reflected by the decrease of absorbance at 340nm (the Y-axis, unit) over the indicated time course (the X-axis, minute). (Baseline, blank control). C, ATP liposomal delivery to parental HT29 and HCT116 cells induced resistance to oxaliplatin under 72h treatment, as shown by MTT assay. D, ATP liposomal delivery to HT29-OxR and HCT116-OxR cells decreased HIF-1α expression and reverted glycolytic enzyme HK2 expression. All experiments were repeated at least three times. Representative data is shown.

Figure 5

Figure 5. ATP depletion sensitizes drug-resistant cells to chemoreagent

A, 3-Bromopyruvate (3-BrPA) depleted cellular ATP level in HT29-OxR and HCT116-OxR cells after 24h of treatment, as measured by ATP luminescent activity. B, Pre-treatment of 3-BrPA (30μM) for 24h sensitized HT29-OxR and HCT116-OxR cells to oxaliplatin, as shown in 72h MTT assays. C, Pre-treatment of 3-BrPA (30μM) for 24h sensitized HT29-OxR and HCT116-OxR cells to 5-FU, as shown in 72h MTT assays. All experiments were repeated at least three times. Representative data is shown.

References

    1. Wilson TR, Longley DB, Johnston PG. Chemoresistance in solid tumours. Ann Oncol. 2006;17(10):x315–24. - PubMed
    1. Cree IA. Chemosensitivity and chemoresistance testing in ovarian cancer. Curr Opin Obstet Gynecol. 2009;21:39–43. - PubMed
    1. Wan YW, Sabbagh E, Raese R, Qian Y, Luo D, Denvir J, et al. Hybrid models identified a 12-gene signature for lung cancer prognosis and chemoresponse prediction. PLoS One. 2010;5:e12222. - PMC - PubMed
    1. Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol. 2005;205(2):275–92. - PubMed
    1. Lemasters JJ, Qian T, He L, Kim JS, Elmore SP, Cascio WE, et al. Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy. Antioxid Redox Signal. 2002;4:769–81. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources