Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May 21;8(5):e63402.
doi: 10.1371/journal.pone.0063402. Print 2013.

Enhanced heme function and mitochondrial respiration promote the progression of lung cancer cells

Jagmohan Hooda  1 , Daniela Cadinu, Md Maksudul Alam, Ajit Shah, Thai M Cao, Laura A Sullivan, Rolf Brekken, Li Zhang
Affiliations

Enhanced heme function and mitochondrial respiration promote the progression of lung cancer cells

Jagmohan Hooda et al. PLoS One. .

Abstract

Lung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer biology and therapeutics.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The levels of heme biosynthesis and heme biosynthetic enzymes are enhanced in lung cancer cells.
The normal HBEC30KT lung epithelial (HBEC) and NSCLC HCC4017 (HCC) cells were cultured, RNA and proteins were extracted. Transcript levels were detected by using quantitative real-time RT-PCR, and protein levels were detected by using Western blotting. (A) The levels of heme biosynthesis in normal and NSCLC cells. (B) The transcript level of heme biosynthetic enzyme ALAS1 in normal and NSCLC cells. (C) The transcript level of heme biosynthetic enzyme ALAS2 in normal and NSCLC cells. (D) The protein level of heme biosynthetic enzyme ALAS1 in normal and NSCLC cells. The protein level of β-actin in the samples was used for normalization. For statistical analysis, the levels in cancer cells were compared to the levels in normal cells, by using Welch 2-sample t-test. *, p value <0.05; **, p value <0.005.
Figure 2
Figure 2. The levels of heme transporters involved in heme uptake are enhanced in lung cancer cells.
The normal HBEC30KT lung epithelial (HBEC) and NSCLC HCC4017 (HCC) cells were cultured and treated as indicated. Protein extracts were prepared and the levels of HCP1 and HRG1 were detected by Western blotting. The protein level of β-actin in the samples was used for normalization. (A) The protein level of heme transporter HCP1 in normal and NSCLC cells. (B) The protein level of heme transporter HRG1 in normal and NSCLC cells. For statistical analysis, the levels in cancer cells were compared to the levels in normal cells, by using Welch 2-sample t-test. **, p value <0.005.
Figure 3
Figure 3. The levels of oxygen utilizing hemoproteins are enhanced in lung cancer cells.
The normal HBEC30KT lung epithelial (HBEC) and NSCLC HCC4017 (HCC) cells were cultured and treated as indicated. Protein extracts were prepared and the levels of hemoproteins were detected by Western blotting. The protein level of β-actin was used for normalization. (A) The protein level of cytoglobin in normal and NSCLC cells. (B) The protein level of cytochrome c in normal and NSCLC cells. (C) The protein level of CYP1B1 in normal and NSCLC cells. (D) The protein level of Cox-2 in normal and NSCLC cells. For statistical analysis, the levels in cancer cells were compared to the levels in normal cells, by using Welch 2-sample t-test. *, p value <0.05; **, p value <0.005.
Figure 4
Figure 4. The effect of lowering heme availability on the mitochondrial ALAS1 (A) and cytochrome c (B) proteins.
NSCLC HCC4017 (HCC) cells were cultured in regular medium (none) or in heme-depleted medium containing 0.5 mM succinyl acetone (SA). Cells were stained first with anti-ALAS1 or anti-cytochrome c antibodies, and then with FITC-conjugated goat anti-rabbit secondary antibody, MitoTracker, as well as DAPI. FITC, MitoTracker and DAPI fluorescent images were captured and are shown here. The scale bar indicates 10 µm.
Figure 5
Figure 5. The levels of heme biosynthetic enzymes, heme transporters, and oxygen-utilizing hemoproteins are elevated in various human tumor xenografts.
Lysates from the indicated human tumor xenografts were prepared, and the levels of the indicated proteins were detected by Western blotting. The protein level of β-actin was used for normalization. (A) The protein level of ALAS1 in HCC4017 cells and in various human tumor xenografts. (B) The protein level of HCP1 in HCC4017 cells and in various human tumor xenografts. (C) The protein level of HRG1 in HCC4017 cells and in various human tumor xenografts. (D) The protein level of cytoglobin in HCC4017 cells and in various human tumor xenografts. (E) The protein level of CYP1B1 in HCC4017 cells and in various human tumor xenografts. For statistical analysis, the levels in HCC4017 cells and tumors were compared to the levels in normal cells, by using Welch 2-sample t-test. *, p value <0.05; **, p value <0.005.
Figure 6
Figure 6. Reducing heme availability suppresses the intensified rate of oxygen consumption in lung cancer cells.
The normal HBEC30KT lung epithelial (HBEC) and NSCLC HCC4017 (HCC) cells were cultured and treated in normal medium (None), in medium with heme depleted (HD), in medium with heme depleted and succinyl acetone (HD+SA), and in medium with CCCP. Cells were collected and oxygen consumption rates were detected and plotted here. The values plotted were averages from at least three experiments. For statistical analysis, the levels in cancer cells were compared to the levels in normal cells, by using Welch 2-sample t-test. *, p value <0.05; **, p value <0.005.
Figure 7
Figure 7. Reducing heme availability suppresses lung cancer cell proliferation, colony formation, and migration.
Cells treated with succinyl acetone were also maintained in heme-depleted medium. (A) Reducing heme availability or mitochondrial function preferentially reduces NSCLC cancer cell proliferation. %live cells was calculated by dividing the number of treated cells with the number of untreated cells (seeded with the same number of cells). It shows the relative proliferative rates of treated cells (SA or SnPP) vs. untreated cells (None). (B) Reducing heme availability or mitochondrial function preferentially reduces NSCLC cell colony formation. (C) Reducing heme availability preferentially reduces NSCLC cell migration. For statistical analysis, the levels in cancer cells were compared to the levels in normal cells, by using Welch 2-sample t-test. **, p value <0.005.
Figure 8
Figure 8. Cartoon illustrating key bioenergetics changes in NSCLC cells.
Cancer cells exhibit enhanced expression levels of the rate-limiting heme biosynthetic enzyme, 5-aminolevulinic acid synthase (ALAS) and the heme uptake proteins HCP1 and HRG1. As such, heme availability in cancer cells is substantially increased, leading to increased production and levels of various oxygen-utilizing hemoproteins, such as cytochrome c, cytoglobin, Cox-2 and cytochrome P450. The increase in these hemoproteins ultimately leads to intensified oxygen consumption and the generation of cellular energy by respiration. This in turn causes increased cellular energy production, cell proliferation, migration and colony formation.
See this image and copyright information in PMC

References

    1. Minami S (1923) Respiration and glycolysis. Biochem. Z. 142: 94–113.
    1. Warburg O (1930) The Metabolism of Tumours. London: Constable & Co.
    1. Vander Heiden MG, Locasale JW, Swanson KD, Sharfi H, Heffron GJ, et al. (2010) Evidence for an alternative glycolytic pathway in rapidly proliferating cells. Science 329: 1492–9. - PMC - PubMed
    1. Hitosugi T, Kang S, Vander Heiden MG, Chung TW, Elf S, et al. (2009) Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Sci Signal 2: ra73. - PMC - PubMed
    1. Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang JK, et al. (2011) Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science 334: 1278–83. - PMC - PubMed

Publication types

MeSH terms