Mitochondrial dysfunction in cancer cells due to aberrant mitochondrial replication

Abstract

Warburg effect is a hallmark of cancer manifested by continuous prevalence of glycolysis and dysregulation of oxidative metabolism. Glycolysis provides survival advantage to cancer cells. To investigate molecular mechanisms underlying the Warburg effect, we first compared oxygen consumption among hFOB osteoblasts, benign osteosarcoma cells, Saos2, and aggressive osteosarcoma cells, 143B. We demonstrate that, as both proliferation and invasiveness increase in osteosarcoma, cells utilize significantly less oxygen. We proceeded to evaluate mitochondrial morphology and function. Electron microscopy showed that in 143B cells, mitochondria are enlarged and increase in number. Quantitative PCR revealed an increase in mtDNA in 143B cells when compared with hFOB and Saos2 cells. Gene expression studies showed that mitochondrial single-strand DNA-binding protein (mtSSB), a key catalyst of mitochondrial replication, was significantly up-regulated in 143B cells. In addition, increased levels of the mitochondrial respiratory complexes were accompanied by significant reduction of their activities. These changes indicate hyperactive mitochondrial replication in 143B cells. Forced overexpression of mtSSB in Saos2 cells caused an increase in mtDNA and a decrease in oxygen consumption. In contrast, knockdown of mtSSB in 143B cells was accompanied by a decrease in mtDNA, increase in oxygen consumption, and retardation of cell growth in vitro and in vivo. In summary, we have found that mitochondrial dysfunction in cancer cells correlates with abnormally increased mitochondrial replication, which according to our gain- and loss-of-function experiments, may be due to overexpression of mtSSB. Our study provides insight into mechanisms of mitochondrial dysfunction in cancer and may offer potential therapeutic targets.

FIGURE 1.

Proliferation rate and invasive potential of osteosarcoma cells. A, hFOB, Saos2, and 143B cells were seeded in 6-well plates at the density of 200,000 cells per well. After 24 or 48 h of incubation, cells were trypsinized, harvested, and counted using an automatic cell counter. B, hFOB, Saos2, and 143B cells were seeded onto either control or Matrigel-coated membranes of the invasion chambers at the density of 50,000 cells per chamber. After 48 h of incubation, cells that have penetrated through the membranes were stained with toluidine blue to assess the invasive potential. C, quantitative analysis of the invasive potential was performed by calculating the invasion index, as described under “Experimental Procedures.” Values represent the means ± S.E.; n = 3–4 independent wells. *, p < 0.05, as compared with hFOB cells.

FIGURE 2.

Oxygen consumption by osteosarcoma cells is significantly decreased. A, a graphic representation of the oxygen consumption rate is shown. 600,000 cells were suspended in 300 μl of medium at 37 °C in a sealed chamber, and oxygen consumption (Vresp.) was measured for 15 min with a Clark-type electrode. B, respiratory capacity of the cells as expressed in nmol of oxygen/min/1 million cells is shown. Values represent the means ± S.E.; n = 3 independent measurements. *, p < 0.05, as compared with hFOB cells.

FIGURE 3.

Changes in mitochondrial morphology of osteosarcoma cells in vitro and in vivo. Thin sections were prepared for electron microscopic analysis from pellets of hFOB, Saos2, and 143B cells (A) or tissue obtained from xenografted tumors (B, middle panel) and archival tissue blocks (B, right panel) as described under “Experimental Procedures.” In B, N and T stand for normal or tumor tissue, respectively. C, quantitative analysis of changes in mitochondrial morphology expressed as the ratio of the area occupied by mitochondria relative to the area of the cytosol is shown. Values represent the means ± S.E.; n = 30 cells per sample analyzed. *, p < 0.05, as compared with hFOB cells.

FIGURE 4.

Levels of mitochondrial DNA, mtSSB, and respiratory complexes in osteosarcoma cells. A, total cellular DNA was isolated and subjected to real-time PCR analysis to determine the amount of mtDNA in the studied cells. The data are expressed as a ratio between the amounts of ND1, an mtDNA-encoded gene, and β-actin, a nuclear-encoded gene. B, total cellular RNA was isolated and reverse-transcribed. Real-time PCR analysis was performed to determine expression levels of several genes implicated in mitochondrial biogenesis. Experimental data were normalized to the expression of GAPDH. C, mitochondria were isolated from hFOB and 143B cells, and mitochondrial proteins were separated using non-denaturing gel electrophoresis. The plot depicts densitometric changes in protein level of 143B cells compared with hFOB cells. D, isolated mitochondria was used for assay of enzyme activities of respiratory complexes I-IV in hFOB and 143B cells, as described under “Experimental Procedures.” The data are expressed as percentage of activity compared with hFOB cells. Values represent the means ± S.E.; n = 3 independent measurements. *, p < 0.05, as compared with hFOB cells.

FIGURE 5.

Effect of gain of mtSSB function on mitochondrial mass and function. A, shown is a representative microphotograph of an immunoblot demonstrating stable mtSSB overexpression in Saos2 cells. Cells were transfected with the pcDNA-mtSSB construct, and the levels of mtSSB protein were confirmed for at least five passages. B and C, shown is the effect of mtSSB overexpression on the amount of mtDNA (B) and oxygen consumption (C) in Saos2 cells. gDNA, genomic DNA. Mitochondrial DNA content and the rate of oxygen consumption were determined as described under “Experimental Procedures.” Values represent the means ± S.E.; n = 3 independent measurements. *, p < 0.05, as compared with non-transfected Saos2 cells.

FIGURE 6.

Effect of loss of mtSSB function on mitochondrial mass, oxygen consumption, and cell growth. A, shown are representative microphotographs of an immunoblot demonstrating stable mtSSB knockdown (KD) in 143B cells that was achieved by using a viral vector containing shRNA against human mtSSB. The knockdown was confirmed for at least 10 passages. B and C, shown is the effect of mtSSB knockdown on the amount of mtDNA (B) and oxygen consumption (Vresp.) (C) in 143B cells. Mitochondrial DNA content and the rate of oxygen consumption were determined as described under “Experimental Procedures.” D and E, shown is the effect of mtSSB loss-of-function on the growth of cancer cells in vitro and in vivo. D, wild-type 143B or 143B-KD-mtSSB cells were seeded in 6-well plates at the density of 100,000 cells per well. After 24 or 48 h of incubation, the cells were trypsinized, harvested, and counted using an automatic cell counter. E, wild-type 143B or 143B-KD-mtSSB cells were injected subcutaneously into mice. Tumor size was measured at 7, 14, and 21 days. Values represent the means ± S.E.; n = 4 independent measurements. *, p < 0.05, as compared with non-transfected 143B cells.