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. 2013;8(1):e54342.
doi: 10.1371/journal.pone.0054342. Epub 2013 Jan 17.

Characterization of adult α- and β-globin elevated by hydrogen peroxide in cervical cancer cells that play a cytoprotective role against oxidative insults

Xiaolei Li  1 , Zhiqiang Wu, Yao Wang, Qian Mei, Xiaobing Fu, Weidong Han
Affiliations

Characterization of adult α- and β-globin elevated by hydrogen peroxide in cervical cancer cells that play a cytoprotective role against oxidative insults

Xiaolei Li et al. PLoS One. 2013.

Abstract

Objectives: Hemoglobin (Hgb) is the main oxygen and carbon dioxide carrier in cells of erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. However, Hgb is also expressed in nonerythroid cells. In the present study, the expression of Hgb in human uterine cervix carcinoma cells and its role in cervical cancer were investigated.

Methodology: The expression level of Hgb in cervical cancer tissues was assessed by quantitative reverse transcriptase-PCR (qRT-PCR). We applied multiple methods, such as RT-PCR, immunoblotting, and immunohistochemical analysis, to confirm Hgb expression in cervical cancer cells. The effects of ectopic expression of Hgb and Hgb mutants on oxidative stress and cell viability were investigated by cellular reactive oxygen species (ROS) analysis and lactate dehydrogenase (LDH) array, respectively. Both Annexin V staining assay by flow cytometry and caspase-3 activity assay were used, respectively, to evaluate cell apoptosis.

Results: qRT-PCR analysis showed that Hgb-α- (HBA1) and Hgb-β-globin (HBB) gene expression was significantly higher in cervical carcinoma than in normal cervical tissues, whereas the expression of hematopoietic transcription factors and erythrocyte specific marker genes was not increased. Immunostaining experiments confirmed the expression of Hgb in cancer cells of the uterine cervix. Hgb mRNA and protein were also detected in the human cervical carcinoma cell lines SiHa and CaSki, and Hgb expression was up-regulated by hydrogen peroxide-induced oxidative stress. Importantly, ectopic expression of wild type HBA1/HBB or HBA1, rather than mutants HBA1(H88R)/HBB(H93R) unable to bind hemo, suppressed oxidative stress and improved cell viability.

Conclusions: The present findings show for the first time that Hgb is expressed in cervical carcinoma cells and may act as an antioxidant, attenuating oxidative stress-induced damage in cervical cancer cells. These data provide a significant impact not only in globin biology but also in understanding of cervical cancer pathogenesis associated with oxidative stress.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Elevated expression of HBA1 and HBB in human cervical cancer tissues.
(A) To confirm the specificity of the HBA1 and HBB primers for qRT-PCR, expression of HBA1 and HBB was examined in human bone marrow and peripheral blood cells. Retrotranscriptase free (–) as a negative control, H2O as a system control. Mr, Marker. (B) cDNA from cervical cancer samples was prepared and analyzed for expression of HBA1 and HBB by RT-PCR. PCR products were separated on 2% agarose gels and visualized with ethidium bromide. GAPDH was used as a loading control. H2O as a system control, T, represents primary tumour samples. The expression levels of HBA1 and HBB were measured by qRT-PCR in 20 human cervical cancer specimens and 10 normal cervical tissue controls. Quantification of the indicated normalized HBA1 and HBB levels (log10) of the total unpaired normal cervix (n = 10) and cervical cancer (n = 20) samples are shown. The tukey boxes represent the upper and lower quartiles divided by the median and whiskers are the largest and smallest values, excluding outliers represented by circles. All differences were statistically significant at P<0.05 (C and D).
Figure 2
Figure 2. The Hgb protein is expressed in carcinoma tissues of the uterine cervix.
Sections from paraffin-embedded human cervical cancer tissues were subjected to either H&E staining or immnunohistochemical analysis with HBA1 and HBB antibodies. Cervical cancer samples showed well-differentiated squamous cell carcinoma (A and B). S, stroma. Cytoplasmic staining of HBA1 and HBB was detected in biopsy samples from uterine cervical tissues (D and F). A non-specific IgG monoclonal antibody diluted with PBS was used as a negative control (C and E). Double-immunostaining against p16INK4A, a specific marker of cervical cancer, demonstrated that Hgb was expressed in cervical cancer cells (I, K, M and O). White dashed boxes were digitally magnified (J, L, N and P). Immunofluorescence without primary antibody revealed negligible signals in cervical cancer cells (G and H).
Figure 3
Figure 3. Expression of HBA1 and HBB in cultured cervical cancer cells.
(A) RT-PCR amplification of HBA1 and HBB from SiHa, CaSki cells and human blood RNA. The HBA1 and HBB transcripts were clearly amplified from the human cervical cancer cell lines, SiHa and CaSki. RNA extracted from blood was used as a positive control and GAPDH was detected as a loading control. Amplicon identities were confirmed by sequencing. (B) HBA1 and HBB were analyzed by western blotting. Peripheral blood leukocytes (PBL) were used as a positive control and β-actin was detected as a loading control. (C) Cell lysates from SiHa and CaSki cells were immunoprecipitated and immunoblotted with the indicated antibodies. A clear band of 17 kDa was enriched from SiHa and CaSki lysates by immunoprecipitation using an anti-human Hgb antibody. Non-specific IgG was used as immunoprecipitation control. Cytoplasmic staining of HBA1 and HBB was detected in cervical cancer cells (D and E, F and G). Double-immunostaining with p16INK4A, a specific marker of cervical cancer for cytology and histological diagnosis, demonstrated that Hgb was expressed by cervical cancer cells (H–J). Inserts are magnified images of selected areas (small squares).
Figure 4
Figure 4. Up-regulation of Hgb expression by oxidative stress.
(A) HBA1 and HBB mRNA expression in SiHa cells was induced by H2O2 treatment. SiHa cells were treated with H2O2 (1 mM) for 24 h and Hgb mRNA levels were determined by qRT-PCR. Hgb levels in controls were normalized to 1. Data were expressed as mean ± SD (n = 3). ** P<0.01. (B) HBA1 and HBB protein expression was induced by H2O2 treatment. Whole cell lysates obtained from SiHa cells treated with or without H2O2 (1 mM, 48 h) were analyzed for HBA1 and HBB levels. β-actin was used as a loading control. (C) HBA1 and HBB mRNAs were up-regulated by H2O2 treatment in HEK293 cells. HEK293 cells were treated with H2O2 (1 mM, 36 h). Reverse transcription PCR products were separated on 2% agarose gels and visualized with ethidium bromide. GAPDH was used as a loading control. (D) Dose and time dependency of the response of SiHa cells to H2O2 treatment. SiHa cells were treated with a series of concentrations (0, 0.25, 0.5 and 1 mM) of H2O2 for 8, 16, 24 or 36 h. Relative HBA1 mRNA level was determined by qRT-PCR. Data were expressed as mean ± SD (n = 3). Values were normalized to the HBA1 level in the control (0 mM, 8 h), which was set to 1. One-way ANOVA test for multiple comparisons was used to analyze the differences between treatments. ** P<0.01; *** P<0.001, when compared to 0 mM treatment.
Figure 5
Figure 5. Effect of HBA1/HBB, HBA1 and HBA1H88R/HBBH93R overexpression on H2O2 levels and superoxide anion in SiHa cells undergoing oxidative stress.
(A) Whole cell lysates from SiHa cells transfected with a control plasmid or HBA1 and HBB expression plasmids were analyzed with an anti-Hb antibody to confirm the overexpression of globin proteins. Control and HBA1/HBB- overexpressing cells were stained with fluorogenic probes to detect intracellular H2O2 and superoxide anion and exposed to extracellular H2O2 (1 mM) for 10 min. Immunostaining confirmed that the intracellular generation of H2O2 (B) and superoxide anion (C) was suppressed in HBA1/HBB-overexpressing cells. Flow cytometry analysis confirmed these results (D and E) and showed that the intracellular levels of H2O2 and superoxide anion were higher in cells exposed to extracellular stimuli than in untreated cells (B and C). Quantitative analyses confirmed that the intracellular generation of H2O2 (F) and superoxide anion (G) was inhibited in HBA1/HBB-overexpressing cells (black column) more than in control cells (gray column; P<0.05). Quantitative analyses confirmed that the intracellular generation of ROS was reduced in HBA1-overexpressing cells (black column, H), but not in HBA1H88R/HBBH93R-overexpressing cells (black column, I) more than in control vector-overexpressing cells (gray column). *P<0.05; **P<0.01; N P>0.05.
Figure 6
Figure 6. Either HBA1/HBB or HBA1, but not HBA1H88R/HBBH93R overexpression protects cells against oxidative stress-induced damage.
(A) The viability of SiHa cells treated with 0, 0.5, and 1 mM H2O2 for 12, 24 and 36 h as extracellular oxidative stress was evaluated by the LDH release assay. SiHa cells transfected with the HBA1/HBB-expressing vectors (black column) showed increased resistance against H2O2-induced cell compared to cells transfected with the control vector (gray column). Similar experiments were preformed in both HBA1-overexpression cells and HBA1H88R/HBBH93R-overexpression cells (B). SiHa cells were transfected with the indicated plasmids. Forty-eight hours later, cells were treated with 1 mM H2O2 or left untreated for 22 h. The percentage of apoptotic cells was monitored by Annexin V staining followed by FACS analysis (C). SiHa cells were transfected with the indicated plasmids and forty hour later, were treated as in (A), Caspase-3 activity in different group was measured using specific caspase substrate AcDEVD-pNA as described under MATERIALs AND METHODs. Values of the absorption were expressed as control which was set as 1 (D and E). All experiments were performed in triplicate and were repeated at least three times, and the results are expressed as means ± S.E.M.*P<0.05; **P<0.01; N P>0.05.
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References

    1. Hardison RC (1996) A brief history of hemoglobins: Plant, animal, protist, and bacteria. Proc Natl Acad Sci U S A 93: 5675–5679. - PMC - PubMed
    1. Schechter AN (2008) Hemoglobin research and the origins of molecular medicine. Blood 112: 3927–3938. - PMC - PubMed
    1. Wittenberg BA, Wittenberg JB (1989) Transport of oxygen in muscle. Annu Rev Physiol 51: 857–878. - PubMed
    1. Schmidt M, Gerlach F, Avivi A, Laufs T, Wystub S, et al. (2004) Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia. Journal of Biological Chemistry 279: 8063–8069. - PubMed
    1. Burmester T, Weich B, Reinhardt S, Hankeln T (2000) A vertebrate globin expressed in the brain. Nature 407: 520–523. - PubMed

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This research was supported by the grants from the National Natural Science Foundation of China (website is http://www.nsfc.gov.cn/Portal0/default 124.htm) (No. 31201033, 31270820, 81230061 and 81001184) and was partially supported by the grants from National Basic Science and Development Programme of China (website is http://www.973.gov.cn/Default_3.aspx) (No. 2012CB518103 and 2012CB518105). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.