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Low concentrations of FA exhibits the Hormesis effect by affecting cell division and the Warburg effect
Ecotoxicology and Environmental Safety 183 (2019) 109576
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Low concentrations of FA exhibits the Hormesis effect by affecting cell division and the Warburg effect Jieran Ana,b, Fuhong Lia, Yujie Qina, Hongmao Zhanga, Shumao Dinga, a b
T
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Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, China Key Laboratory of Functional Dairy, Co-constructed by the Ministry of Education and Beijing Municipality, China Agricultural University, Beijing, China
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A B S T R A C T
Keywords: Formaldehyde (FA) The Hormesis effect Cell proliferation Cell division The Warburg effect
Formaldehyde (FA), a ubiquitous indoor environmental pollutant, has been classified as a carcinogen. There are many studies showed that low levels of FA could promote cell proliferation, however, little is known about the signal pathways. To determine the potential molecular mechanisms, human chronic myeloid leukemia cells (K562 cells) and human bronchial epithelial cells (16HBE cells) were exposed to different concentrations of FA. The data showed that FA at 0–125 μM or 0–60 μM promoted the proliferation of K562 cells or 16HBE cells respectively, indicating that FA did have the Hormesis effect. FA at 75 μM (K562 cells) and 40 μM (16HBE cells) significantly promoted cell proliferation, increased intracellular reactive oxygen species (ROS) levels, and decreased glutathione (GSH) content. At the same time, FA treatment induced a marked increase in the key molecules of cell division like CyclinD-cdk4 and E2F1. In addition, pyruvate kinase isozyme M2 (PKM2), glucose, glucose transporter 1 (GLUT1), lactic acid and lactate dehydrogenase A (LDHA) content in the Warburg effect were increased. Administering Vitamin E (VE), significantly disrupted cell division and disturbed the Warburg effect, effectively indicating the decrease of cell activity. Conclusively, these findings suggested that low concentrations of FA could promote cell proliferation by accelerating cell division process or enhancing the Warburg effect to embody the Hormesis effect.
1. Introduction Formaldehyde (FA), a common indoor air pollutant, is widely used in construction, furniture, textiles and numerous other applications (Zhang et al., 2016), having been defined as “human carcinogen” (Anonymous and Who, 2006). FA can enter the living body from thorough respiratory tract, causing reproductive toxicity (Duong et al., 2011), neurotoxicity (Qiang et al., 2014), immunotoxicity (Wen et al., 2016), respiratory toxicity (Wu et al., 2013) and other toxicities. It was researched that the levels of indoor FA could exist for a long time after remodeling (Zhang et al., 2007). Although the World Health Organization (WHO) allows no more than 0.1 mg/m3 FA in the residential environment, many of surveillances have revealed the exceeding indoor FA level in many countries, making the environmental FA condition as a globally public health concern.
At present, the researches about cytotoxicity induced by FA mostly concentrated on cell apoptosis or cell death. Although there did some reports show low concentrations of FA could promote cell proliferation, no reports brought to light on the molecular mechanisms. Tyihák et al. found that human colon cancer HT-29 cells showed cell death and apoptosis induced by 10 mM and 1 mM FA, while FA at 0.5 and 0.1 mM increased cell activity (Tyihák et al., 2001). In the related studies of our laboratory, we found that human embryonic kidney cell HEK293 cells proliferation rate was decreased once the levels of FA was more than 60 μM (Xu et al., 2007); and 10 μM of FA was found to be the optimal concentration for promoting Hela cells viability (Ke et al., 2014). Such a phenomenon that it’s the response of organisms to low-level exposures to a stressor opposes the response to high-level exposures can be called the Hormesis effect. Although there are more and more studies involved in its molecular mechanisms (Cheng-Run et al., 2010; Chun et al., 2013;
Abbreviations: FA, formaldehyde; K562 cells, human chronic myeloid leukemia cells; 16HBE cells, human bronchial epithelial cells; ROS, reactive oxygen species; PKM2, pyruvate kinase isozyme M2; GLUT1, glucose transporter 1; LDHA, lactate dehydrogenase A; VE, Vitamin E; WHO, the World Health Organization; TCA, tricarboxylic acid cycle; MTT, 3-(4, 5- dimethylthiazol-2-yl) - 2, 5 diphenyltetrazolium bromide); DMSO, dimethyl sulfoxide; DTNB, 5,5′-Dithiobis-(2-nitrobenzoic acid); OD, optical density; DCFH-DA, dichloro-dihydro-flurescein; PBS, phosphate buffer saline; GSH, glutathione; PMSF, Phenylmethanesulfonyl fluoride; RT-PCR, Real-time PCR; Ct, comparative threshold; one way ANOVA, one way analysis of variance; SD, standard deviation ⁎ Corresponding author. College of Life Sciences, Central China Normal University, Wuhan, China. E-mail address: [email protected] (S. Ding). https://doi.org/10.1016/j.ecoenv.2019.109576 Received 27 May 2019; Received in revised form 12 August 2019; Accepted 14 August 2019 Available online 26 August 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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2.3. Cell proliferation assays
Liang et al., 2016; Rialdi et al., 2016), the Hormesis effect was and is widely known for chemical compounds or radiation, which means that whether it will appear upon a variety of environmental factors including FA and the related molecular mechanisms remain underexplored. More than 20 cyclins which are closely related to cell proliferation, cell differentiation and carcinogenesis have been found in mammalian cells, playing important regulatory roles in cell division phase transition (Karunagaran et al., 2007). For a complete mitosis process, the correct combination between cyclins and cyclin-dependent kinases is critical, for example, what's important for the late stage of G1 is that the precise combination between CyclinD and CDK4 (Demichele et al., 2015). In addition, under normal circumstances, nearly all cells can produce enough energy for the growth or metabolism through tricarboxylic acid cycle (TCA). But even there is oxygen, the glycolysis in tumor cells will proceed to supply energy for rapid growth, which is the Warburg effect. There was study has pointed out that lactic acid, instead of glucose, is the cancer cell of energy providers (Hui et al., 2017). And recent studies also researched that several signaling pathways implicated in cell proliferation may regulate metabolic pathways that incorporate nutrients into biomass. So the better understanding of the mechanistic links between growth control and cellular metabolism may ultimately lead to better treatments for human cancer. In this study, human chronic myeloid leukemia cells (K562 cells) and human bronchial epithelial cells (16HBE cells) were exposure to different levels of FA, and then the cell viability and changes in cytokines associated with cell proliferation were measured. Furthermore, combined with the antioxidants Vitamin E (VE), the present study conducted a more in-depth discussion on the specific molecular mechanism between low concentrations of FA and cell proliferation — the Hormesis effect of FA, providing a scientific basis for the biosafety evaluation of FA and the treatment of FA-caused cancer or other illnesses.
Cell proliferation analysis was allowed us to establish the FA concentration which could significantly promote cell proliferation to use for the further experiments. The MTT (3-(4, 5- dimethylthiazol-2-yl) - 2, 5 diphenyltetrazolium bromide) assay is usually used to determine cell activity. Cells were seeded in a 96-well microtiter plate at an initial density of 1 × 105 cells per well and grown for 24 h in 90 μL medium before different levels of FA exposure. After 2 h, 10 μL of MTT (5 mg/ mL) (Sigma-Aldrich, St Louis, Missouri, USA) was added to each well at 37 °C for 4 h. A solution of 100 μL dimethyl sulfoxide (DMSO) was added in order to dissolve the crystal of formazan produced in wells (Perna et al., 2018). Last, the solution was read at 490 nm using an eon microplate spectrophotomete. The value of absorbance is proportional with the number of living cells. Each MTT assay was performed in triplicate. The viability of cells also was determined by CCK-8 kit (DOJINDO, Kumamoto Island, Japan) according to its instruction. CCK-8 reagent (10 μL) was added to each well of the plates when FA exposure was finished, and then cells were incubated at 37 °C for 4 h. Absorbance at a wavelength of 450 nm was measured with a microplate reader. The value of absorbance is proportional with the number of living cells. It should be performed three times independently. 2.4. ROS levels and GSH content measurements Intracellular ROS levels were determined by dichloro-dihydro-flurescein diacetate (DCFH, Sigma-Aldrich, St Louis, Missouri, USA) fluorescent assay descripted by Wu (Wu et al., 2013), with minor modifications. Briefly, 3.5 × 105 cells in good logarithmic growth phase were treated by FA, and then DCFH was added to each cell suspension at a final concentration of 10 μM. Cells were then washed twice with phosphate buffer saline (PBS) and resuspended in 600 μL PBS. A 100 μL aliquot of the cell suspension was transferred to a 96 well microplate and the fluorescence intensity was measured at 488 nm (excitation) and 525 nm (emission). The analyses were done within 3 h. Repeat 3 times for each group. Glutathione (GSH) is a major scavenger of ROS in tissues. The cell lysis buffer with Phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, Shanghai, China) was added to the treated cells at 4 °C for 1 h, then they were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was taken for testing. Basing on that thiols such as GSH can react with 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) in the dark and form yellow compounds, the content of GSH was conducted according to a previously described procedure with minor modifications (Yang et al., 2012). The absorbance was tested at 405 nm. The protein concentration was determined using the Lowry assay (Lowry et al., 1951). Repeat three times for each group. At the same time, cell viability was tested by above MTT method.
2. Materials and methods 2.1. Cell culture K562 cells and 16HBE cells (China Type Culture Collection (CCTCC), Wuhan, China) were cultivated in RPMI 1640 medium (Sigma-Aldrich, St Louis, Missouri, USA) with 10% fetal calf serum (FBS), 1% penicillin/streptomycin (Gibco, Life Technologies, Grand Island New York, USA) at 37 °C, 5% CO2 in a humidified incubator. When 80% confluent, K562 cells were discard two-thirds of the volume while 16HBE cells were enzymatically detached with trypsin-edta (Sigma-Aldrich, St Louis, Missouri, USA), then both of them were seeded in a new cell culture flasks. The medium was changed at least every 2 days. All of cells were harvested in the exponential growth phase for follow-up experimental operation.
2.5. RNA extraction and real- time PCR 2.2. Cell treatment
mRNA expression levels of target genes were analyzed using Realtime PCR (RT-PCR). Total cellular RNA was extracted from the control and reeated cells using the TRIzol® reagent (Invitrogen, U.S.) according to its manufacturer and quantified with a NanoDrop spectrophotometer. cDNAs were synthesized from 1 μg RNA using the High Capacity cDNA Reverse Transcriptase (Vazyme Biotech Co., Lid, China). RT-PCR was performed with a Chromo 4 detector using SYBR® Premix Ex Taq™ (Takara Bio, Otsu, Japan) and referred to the method described in Luisa et al. (2015). All primers used, are listed in the supplementary material and were synthesized by Genescript (Piscataway, New Jersey, USA; Supplementary Material). The amount of target cDNA was calculated by comparative threshold (Ct) method and expressed by means of the 2−ΔΔCt method (LIVAK, 2001) using the housekeeping gene β- actin or actin. The analyses were always carried out in triplicate
To explore the effects on cell proliferation induced by FA, 4% FA (Sigma-Aldrich, St Louis, Missouri, USA) was stored in accordance with the manufacturer's recommendations. It is diluted to different concentrations in serum-free RPMI 1640 medium to treat cells in logarithmic growth phase (0, 25, 50, 75, 100, 125, 150, 200 μM for K562 cells and 0, 20, 40, 60, 80, 100, 120 μM for 16HBE cells) for 2 h. Working solutions were prepared immediately before use. In the next experiment to explore the molecular mechanisms of the Hormesis of FA, Vitamin E (VE) (Sigma-Aldrich, St Louis, Missouri, USA) at 75 μM (for K562 cells) and 80 μM (for 16HBE cells) were prepared immediately with serum-free RPMI 1640 medium before use and added into cells half an hour before FA exposure. 2
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cells in the FA group showed a significant increase compared with the control group, while GSH content decreased significantly (Fig. 2B and F). Importantly, the treatment with VE declined the ROS fluorescence intensity but increased GSH levels in comparison with the FA group. ROS is a natural by-product of the normal metabolism of oxygen and plays an important role in cell signaling and homeostasis. To demonstrate whether low levels of FA-caused ROS was beneficial to cell proliferation, we analyzed the cells activity by MTT method and the data were shown in Fig. 2C, D, G and H. Not surprisingly, although cells vitality in the FA group was increased compared with the control group, it exhibited the opposite trend compared with the FA + VE group.
samples for each experimental point. 2.6. Detection of biomarkers Levels of CyclinD-cdk4, E2F1, pyruvate kinase isozyme M2 (PKM2), glucose, glucose transporter 1 (GLUT1), lactic acid, lactate dehydrogenase A (LDHA) were measured using commercial ELISA kits (BioTechne, Minnesota, USA), according to the specific manufacturer's instructions. In brief, standards with different concentrations were added to the standard wells. Firstly, 10 μL samples with 40 μL sample dilution were added to the wells that had been coated with the antibody. Next, the mixture incubated for 1 h, at 37 °C. Then the washing buffer was added to each well, left for 30s before being drained, and then patted dry. This step was repeated 5 times. 50 μL HRP-conjugate reagents was added to each well except the blank wells, and the progress of incubating or washing were consistent with above steps. 50 μL chromogen solution A and 50 μL chromogen solution B were added to each well, and kept in the dark for 10 min at 37 °C. Finally, 50 μL stop solution was added into each well. The absorbance at 450 nm of the plate was read. The specific assay protocol for determining glucose and lactic acid was based on the manufacturer's instructions of the kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). For glucose, the mixture of samples and the working solution were incubated for 15 min at 37 °C before the absorbance at 505 nm of the plate was analyzed. As for lactic acid, samples were mixed with the working solution and stored at 37 °Cfor 10 min. Then, stop solution was added to wells. The absorbance was tested at 530 nm.
3.3. Low concentrations of FA exposure accelerated cell division Cell division is closely related to cell proliferation. Therefore, we studied the variation of some molecules involved in cell division. FAtreated cells showed an up-regulation on the levels of CyclinD-cdk4 (Fig. 3A and D). Moreover, mRNA levels of Cyclin D1 and CDK4 had an FA-induced increase in two kinds of cells. Whereas the content and mRNA levels in the FA + VE group showed a downward trend when it was compared with the FA group. As a key biomolecule affected by CDK4, E2F1 variation was also measured. The results in Fig. 4 showed that FA resulted in the increasing E2F1 content and mRNA levels. Not surprisingly, regarding the effect induced by VE, the significantly decrease of ELISA and RT-PCR data could be looked. 3.4. Low concentrations of FA exposure enhanced the Warburg effect
2.7. Statistical analysis
After 2 h of exposure with FA, ELISA and RT-PCR were performed to evaluate some biomarkers involved in the Warburg effect such as PKM2, glucose, GLUT1, lactic acid and LDHA. It was obvious that in Fig. 5, when compared the no FA group with the FA group, intracellular PKM2 content increased (Fig. 5A and C) and FA also interfered on the mRNA levels (Fig. 5B and D). After the addition of VE, the data of ELISA and RT-PCR in the FA group tended to show a significant decrease in comparison to the FA + VE group. As a phenomenon which is associated with energy supply, the Warburg effect is so complex that there are various signal pathways in it. We first chose the most common “energy supplier” - glucose for test. For K562 cells, FA induced an increase in glucose content (Fig. 6A) and GLUT1 (Fig. 6B) compared with the no FA group, while VE caused a converse tendency by comparing the FA group with the FA + VE group. The data in 16HBE cells showed FA promoted GLUT1 mRNA level in association with the control group (Fig. 6F), leading the increase of glucose (Fig. 6D), but their levels treatment with VE dropped. For cancer cells with uncontrolled proliferation, lactic acid can also provide energy (Hui et al., 2017). Consequently, we measured certain biomarkers involved in lactic acid. As shown in Fig. 7A and D, intracellular lactic acid levels did experience an up-regulation tendency. Being the enzyme closely related to lactic acid, LDHA content and mRNA levels also showed significantly increase induced by FA exposure in comparison with the control group. Simultaneously, in Fig. 7, the decreasing tendency of above molecules was also obvious by comparing the FA group with FA + VE exposure.
The results were analyzed using the one way analysis of variance (one way ANOVA) with t-test by Graph Pad Prism 7.0 software. The data of ELISA assay were presented as mean while others were showed as mean ± standard deviation (SD) for the indicated number of independent determinations. The differences between groups were considered statistically significant when the P values was at least p < 0.05. All experiments were repeated at least three times and performed in triplicate. 3. Results 3.1. Low concentrations of FA promoted cells proliferation To investigate whether exposure to different concentrations of FA could have various effects on cell proliferation, we measured the proliferative activity of K562 cells by CCK-8 assay (Fig. 1A and B) and MTT assay (Fig. 1C and D). We looked that at a relatively low level (less than 125 μM FA), compared with the control group, the OD value and the proliferation rate of K562 cells were higher, and FA at 75 μM induced a significant increased cells viability. But with the FA concentrations growing, cells viability decreased and had an obvious drop in the 200 μM FA group. Could FA provide the same influence on normal cells? 16HBE cells were treated in the same manner to carry on the further exploration (Fig. 1E–H). After 2 h of exposure, FA at a concentration of higher than 60 μM decreased cells proliferative activity and there was an obvious dropping tendency in the 120 μM FA group. 40 μM FA could promoted cells proliferation significantly.
4. Discussion In exploring whether there was the Homersis effect in FA or not, we set the broader concentrations of FA, and it also contributed to the screening of the optimal concentration which promoted cell proliferation significantly. By analyzing cell viability, the data shown in Fig. 1 were well confirmed that low concentrations of FA could promote cell proliferation while high levels of FA inhibited cell activity. This was consistent with the characteristics of the Hormesis effect, indicating the existence of Hormesis effect in FA. However, as mentioned above,
3.2. Low concentrations of FA exposure increased ROS levels and decreased GSH content According to the results in Fig. 1, 75 μM FA and 40 μM FA were chosen as the efficient doses to explore the underlying mechanisms of the Hormesis of FA. In Fig. 2A and E, ROS levels in K562 cells or 16HBE 3
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Fig. 1. Cells vitality analysis after exposed to different concentrations of FA. The experimental data were analyzed by one way ANOVA with t-test and expressed as means ± standard deviation (Mean ± SD), n = 6. (A) OD value of K562 cells exposed to different concentrations of FA tested by CCK-8 assay; (B) Cell proliferation rate of K562 cells exposed to different concentrations of FA tested by CCK-8 assay; (C) OD value of K562 cells exposed to different concentrations of FA tested by MTT assay; (D) Cell proliferation rate of K562 cells exposed to different concentrations of FA tested by MTT assay; (E) OD value of 16HBE cells exposed to different concentrations of FA tested by CCK-8 assay; (F) Cell proliferation rate of 16HBE cells exposed to different concentrations of FA tested by CCK-8 assay; (G) OD value of 16HBE cells exposed to different concentrations of FA tested by MTT assay; (H) Cell proliferation rate of 16HBE cells exposed e to different concentrations of FA tested by MTT assay. *: p < 0.05, **: p < 0.01, ***: p < 0.001, compared with the control group.
differences from the experimental environment or the researchers; and the reason why different cells enjoyed different the most optimal level of FA might be the differences in tolerance to FA. Consequently, 75 μM FA (for K562 cells) as well as 40 μM FA (for 16 HBE cells) were selected for the next study. A previous study suggested that cellular responses to ROS could cause cytotoxicity or enhanced cell survival depending on the concentrations (Topaloglu et al., 2016). We found a significant difference in ROS levels between the control group and the FA group (Fig. 2A and E). VE is usually used as the effective inhibitor of ROS. In order to fully explore the relations between ROS variation and cell proliferation, we further measured the content of GSH, an important scavenger for free radicals in the body by adding VE. The data in Fig. 2B and F showed that low concentrations of FA increased ROS fluorescence intensity while decreased GSH levels. In addition, when compared with the FA
although there are many studies about the Hormesis effect, there is no report about it in FA. In general, once cell proliferation is out of control, the risk of suffering from cancer will increase greatly. A large number of epidemiological studies have shown that FA can induce some cancer including leukemia (Kwon et al., 2018), but there is no the definitive reports to reveal the molecular mechanism of FA-caused leukemia. As a common cell model for studying the occurrence, development and treatment of leukemia, K562 cells are selected as the object in our experiment. In addition, for humans, most tumors stem from epithelial cells, and the 16HBE cells can represent the initial phase of a multistage model of in vitro carcinogenesis. This is why we chose the two kinds of cells to explore the molecular mechanisms of low concentrations of FA-promoted cell proliferation in our experimentation. It was worth noting that we found that the same cells had different the most optimal level of FA to promote cell proliferation, probably due to
Fig. 2. The effect of low concentrations of FA exposure on ROS fluorescence intensity and GSH content. The experimental data were analyzed by one way ANOVA with t-test and expressed as Mean ± SD, n = 6. (A) ROS fluorescence intensity of K562 cells; (B) GSH content of K562 cells; (C) OD value of K562 cells tested by MTT assay; (D) Cell proliferation rate of K562 cells tested by MTT assay; (E) ROS fluorescence intensity of 16HBE cells; (F) GSH content of 16HBE cells; (G) OD value of 16HBE cells tested by MTT assay; (H) Cell proliferation rate of 16HBE cells tested by MTT assay. *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group.
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Fig. 3. The effect of low concentrations of FA exposure on intracellular CyclinDcdk4, CyclinD1 and CDK4. The experimental data were analyzed by one way ANOVA with t-test. (A) CyclinD-cdk4 content in K562 cells (The data were presented by Mean, n = 6); (B) CyclinD1 expression level in K562 cells (The data were presented by Mean ± SD, n = 3); (C) CDK4 expression level in K562 cells (Mean ± SD, n = 3); (D) CyclinD-cdk4 content in 16HBE cells (Mean, n = 6); (E) CyclinD1 expression level in 16HBE cells (Mean ± SD, n = 3); (F) CDK4 expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group.
some biochemical pathways that induce cell survival were further stimulated, helping FA fully to embody the Hormesis effect. Cell division is an essential part of cell cycle and a disorder of the normal cell cycle can lead tumors. Studies have shown that intracellular ROS content varies regularly with the cell division process (Liu and Liu, 2013). Therefore, based on the fact that low concentrations of FA treatment caused elevated levels of intracellular ROS in our text, we immediately determined the key factors involved in cell division. By comparing CyclinD-cdk4 content in the control group with its in the FA group, we found that FA increased the content of the factor and the mRNA levels, but they all decreased due to the addition of VE. This result was consistent with Blázquez-Castro's data that the photoactive compounds-caused ROS was associated with the Cyclin-D1 expression and effectively promoted cell proliferation (Blázquez-Castro et al., 2012) and the review from Zhou which showed that the production of
group, the changes of ROS and GSH in the FA + VE group also completely opposite. At present, most studies have revealed the adverse effects of ROS, but there are also reports showed that it was good for cell proliferation. The bovine granulosa cell proliferation rate increased by 1.2 times under hypoxic conditions (Shiratsuki et al., 2016); low doses of 2,2′,4,4′-tetrabromodiphenyl ether promoted human hepatoma HepG- 2 cell proliferation, while ROS levels were high than the control group (Wang et al., 2012). Consistent with them, in Fig. 2B, C, G and H, although ROS levels in the FA-treated group increased, the decrease of cell proliferation rate of two kinds of cells was also significant. Referring to previous study where it indicated that by the shock on NAS biomass would enhance intracellular and extracellular substances of microbial communities such as sludge communities (Sepehri and Sarrafzadeh, 2018), it reminded us that our results could show that due to the relatively low levels of ROS induced by low concentrations of FA,
Fig. 4. The effect of low concentrations of FA exposure on intracellular E2F1. The experimental data were analyzed by one way ANOVA with t-test. (A) E2F1 content in K562 cells (The data were presented by Mean, n = 6); (B) E2F1 expression level in K562 cells (The data were presented by Mean ± SD, n = 3); (C) E2F1 content in 16HBE cells (Mean, n = 6); (D) E2F1 expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group.
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Fig. 5. The effect of low concentrations of FA exposure on intracellular PKM2. The experimental data were analyzed by one way ANOVA with t-test. (A) PKM2 content in K562 cells (The data were presented by Mean, n = 6); (B) PKM2 expression level (The data were presented by Mean ± SD, n = 3); (C) PKM2 content in 16HBE cells (Mean, n = 6); (D) PKM2 expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group.
division, showing Hormesis effect. We know that cell metabolism is adapted to facilitate the uptake and incorporation of nutrients into the biomass needed to produce a new cell, so the more energy is necessary for the metabolism (Sepehri and Sarrafzadeh, 2018). In our experiment, we first measured the content of glucose and lactic acid which could supply energy. The transport of glucose requires the glucose transporter (GLUT), and GLUT1 is often overexpressed in cancer cells (Brown and Wahl, 2015). As shown in Fig. 6, we found that glucose content and GLUT1 levels increased after FA treatment, and lactic acid content also increased. LDHA catalyzes
intracellular ROS in cancer cells affected the normal cell metabolism and the function of cell cycle checkpoints, making tumor cells more proliferative (Zhou et al., 2014). In addition, in the signaling pathway involved in the CyclinD-cdk, the E2F family is non-negligible. It mainly concludes E2F1, E2F2 as well as E2F3, and the mutation of E2F1 can make the cell cycle blocked (Nevins, 2001). Therefore, E2F1 was selected as the object in this experiment. According to the data in Fig. 4, we found that FA exposure had the same effect on the changes of E2F1 and CyclinD-cdk4, indicating that low concentration of FA could promote cell proliferation by affecting the expression of key factors of cell
Fig. 6. The effect of low concentration of FA exposure on intracellular glucose and GLUT1. The experimental data were analyzed by one way ANOVA with ttest. (A) Glucose content in K562 cells (The data were presented by Mean, n = 6); (B) GLUT1 content in K562cells (Mean, n = 6); (C) GLUT1 expression level in K562 cells (The data were presented by Mean ± SD, n = 3); (D) Glucose content in 16HBE cells (Mean, n = 6); (E) GLUT1 content in 16HBE cells (Mean, n = 6); (F) GLUT1 expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group. 7
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Fig. 7. The effect of low concentrations of FA exposure on intracellular lactic acid and LDHA. The experimental data were analyzed by one way ANOVA with ttest. (A) Lactic acid content in K562 cells (The data were presented by Mean, n = 6); (B) LDHA content in K562 cells (Mean, n = 6); (C) LDHA expression level in K562 cells (The data were presented by Mean ± SD, n = 3); (D) Lactic acid content in16HBE cells (Mean, n = 6); (E) LDHA content in 16HBE cells (Mean, n = 6); (F) LDHA expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; ##p < 0.01, compared with the FA + VE group.
the conversion of pyruvate to lactic acid when oxygen is deficient (Hartmann et al., 2012). The results shown in Fig. 7 did indicate that the FA-caused the increase of LDHA content and mRNA levels were significant. It was also worth noting that the changes in lactic acid were more obvious than glucose. This might be because lactic acid was also formed form pyruvate produced by glucose oxidation. The Warburg effect, also known as aerobic glycolysis, has become one of the characteristics of tumor cells and requires various enzymes to regulate the whole process. A research has showed that PKM2 was a key rate-limiting enzyme which is often overexpressed in tumor cells (Sontakke et al., 2016). Just as the data shown in Fig. 5, we found that FA exposure increased PKM2 content. Combining the FA-induced changes in GLUT1 and LDHA, we showed that once PKM2 content was upregulated, it would enhanced transcription of downstream target genes. Referring to studies which have proposed that the Warburg effect confers direct signaling functions including the generation and modulation of ROS to tumor cells (Liberti and Locasale, 2016), we analyzed the data after the addition of VE. The decrease of all of above biomarkers demonstrated that when the low concentration of FA promoted cell proliferation, the ROS level caused it was just beneficial to the signal transduction process of cell proliferation. Overview of these results, it could be seen that low concentrations of FA promoted cell proliferation by enhancing the Warburg effect.
relationship between FA-promoted cell proliferation and FA-caused cancer or other illnesses. However, the present experiment was conducted independently and separately from the above two aspects. Considering that the molecular mechanisms involved in cell proliferation is complicated, it is necessary to explore whether there is crosstalk between the above signal paths in the deeper researches.
5. Conclusions
Appendix A. Supplementary data
From the above all analysis, we can summarize our results in some points: we demonstrated that different concentrations of FA had various effects on cell vitality and only FA at low levels could promote cell proliferation. Low concentrations of FA probably through accelerating cell division process and enhancing the Warburg effect promoted cell proliferation and embodied the Hormesis effect. The appropriate ROS content induced by FA at low concentrations might be related to the process. As we all know that once cell proliferation is out of control, cancer will be on onset, therefore the present study laid the foundation for the objective evaluation of FA and the comprehensive disclosure of the
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109576.
Conflicts of interest The authors declare no competing financial interest. Author contributions Jieran An conducted the whole experiment and finished the manuscript. Fuhong Li and Yujie Qin helped with preparing experimental data and with the literature review for the paper. Hongmao Zhang and Shumao Ding gave guidance to the experiment. Shumao Ding participated in every step of preparing this manuscript. Acknowledgements The work was supported by the National Natural Science Foundation of China (31772471).
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Low concentrations of FA exhibits the Hormesis effect by affecting cell division and the Warburg effect Jieran Ana,b, Fuhong Lia, Yujie Qina, Hongmao Zhanga, Shumao Dinga, a b
T
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Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan, China Key Laboratory of Functional Dairy, Co-constructed by the Ministry of Education and Beijing Municipality, China Agricultural University, Beijing, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Formaldehyde (FA) The Hormesis effect Cell proliferation Cell division The Warburg effect
Formaldehyde (FA), a ubiquitous indoor environmental pollutant, has been classified as a carcinogen. There are many studies showed that low levels of FA could promote cell proliferation, however, little is known about the signal pathways. To determine the potential molecular mechanisms, human chronic myeloid leukemia cells (K562 cells) and human bronchial epithelial cells (16HBE cells) were exposed to different concentrations of FA. The data showed that FA at 0–125 μM or 0–60 μM promoted the proliferation of K562 cells or 16HBE cells respectively, indicating that FA did have the Hormesis effect. FA at 75 μM (K562 cells) and 40 μM (16HBE cells) significantly promoted cell proliferation, increased intracellular reactive oxygen species (ROS) levels, and decreased glutathione (GSH) content. At the same time, FA treatment induced a marked increase in the key molecules of cell division like CyclinD-cdk4 and E2F1. In addition, pyruvate kinase isozyme M2 (PKM2), glucose, glucose transporter 1 (GLUT1), lactic acid and lactate dehydrogenase A (LDHA) content in the Warburg effect were increased. Administering Vitamin E (VE), significantly disrupted cell division and disturbed the Warburg effect, effectively indicating the decrease of cell activity. Conclusively, these findings suggested that low concentrations of FA could promote cell proliferation by accelerating cell division process or enhancing the Warburg effect to embody the Hormesis effect.
1. Introduction Formaldehyde (FA), a common indoor air pollutant, is widely used in construction, furniture, textiles and numerous other applications (Zhang et al., 2016), having been defined as “human carcinogen” (Anonymous and Who, 2006). FA can enter the living body from thorough respiratory tract, causing reproductive toxicity (Duong et al., 2011), neurotoxicity (Qiang et al., 2014), immunotoxicity (Wen et al., 2016), respiratory toxicity (Wu et al., 2013) and other toxicities. It was researched that the levels of indoor FA could exist for a long time after remodeling (Zhang et al., 2007). Although the World Health Organization (WHO) allows no more than 0.1 mg/m3 FA in the residential environment, many of surveillances have revealed the exceeding indoor FA level in many countries, making the environmental FA condition as a globally public health concern.
At present, the researches about cytotoxicity induced by FA mostly concentrated on cell apoptosis or cell death. Although there did some reports show low concentrations of FA could promote cell proliferation, no reports brought to light on the molecular mechanisms. Tyihák et al. found that human colon cancer HT-29 cells showed cell death and apoptosis induced by 10 mM and 1 mM FA, while FA at 0.5 and 0.1 mM increased cell activity (Tyihák et al., 2001). In the related studies of our laboratory, we found that human embryonic kidney cell HEK293 cells proliferation rate was decreased once the levels of FA was more than 60 μM (Xu et al., 2007); and 10 μM of FA was found to be the optimal concentration for promoting Hela cells viability (Ke et al., 2014). Such a phenomenon that it’s the response of organisms to low-level exposures to a stressor opposes the response to high-level exposures can be called the Hormesis effect. Although there are more and more studies involved in its molecular mechanisms (Cheng-Run et al., 2010; Chun et al., 2013;
Abbreviations: FA, formaldehyde; K562 cells, human chronic myeloid leukemia cells; 16HBE cells, human bronchial epithelial cells; ROS, reactive oxygen species; PKM2, pyruvate kinase isozyme M2; GLUT1, glucose transporter 1; LDHA, lactate dehydrogenase A; VE, Vitamin E; WHO, the World Health Organization; TCA, tricarboxylic acid cycle; MTT, 3-(4, 5- dimethylthiazol-2-yl) - 2, 5 diphenyltetrazolium bromide); DMSO, dimethyl sulfoxide; DTNB, 5,5′-Dithiobis-(2-nitrobenzoic acid); OD, optical density; DCFH-DA, dichloro-dihydro-flurescein; PBS, phosphate buffer saline; GSH, glutathione; PMSF, Phenylmethanesulfonyl fluoride; RT-PCR, Real-time PCR; Ct, comparative threshold; one way ANOVA, one way analysis of variance; SD, standard deviation ⁎ Corresponding author. College of Life Sciences, Central China Normal University, Wuhan, China. E-mail address: [email protected] (S. Ding). https://doi.org/10.1016/j.ecoenv.2019.109576 Received 27 May 2019; Received in revised form 12 August 2019; Accepted 14 August 2019 Available online 26 August 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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2.3. Cell proliferation assays
Liang et al., 2016; Rialdi et al., 2016), the Hormesis effect was and is widely known for chemical compounds or radiation, which means that whether it will appear upon a variety of environmental factors including FA and the related molecular mechanisms remain underexplored. More than 20 cyclins which are closely related to cell proliferation, cell differentiation and carcinogenesis have been found in mammalian cells, playing important regulatory roles in cell division phase transition (Karunagaran et al., 2007). For a complete mitosis process, the correct combination between cyclins and cyclin-dependent kinases is critical, for example, what's important for the late stage of G1 is that the precise combination between CyclinD and CDK4 (Demichele et al., 2015). In addition, under normal circumstances, nearly all cells can produce enough energy for the growth or metabolism through tricarboxylic acid cycle (TCA). But even there is oxygen, the glycolysis in tumor cells will proceed to supply energy for rapid growth, which is the Warburg effect. There was study has pointed out that lactic acid, instead of glucose, is the cancer cell of energy providers (Hui et al., 2017). And recent studies also researched that several signaling pathways implicated in cell proliferation may regulate metabolic pathways that incorporate nutrients into biomass. So the better understanding of the mechanistic links between growth control and cellular metabolism may ultimately lead to better treatments for human cancer. In this study, human chronic myeloid leukemia cells (K562 cells) and human bronchial epithelial cells (16HBE cells) were exposure to different levels of FA, and then the cell viability and changes in cytokines associated with cell proliferation were measured. Furthermore, combined with the antioxidants Vitamin E (VE), the present study conducted a more in-depth discussion on the specific molecular mechanism between low concentrations of FA and cell proliferation — the Hormesis effect of FA, providing a scientific basis for the biosafety evaluation of FA and the treatment of FA-caused cancer or other illnesses.
Cell proliferation analysis was allowed us to establish the FA concentration which could significantly promote cell proliferation to use for the further experiments. The MTT (3-(4, 5- dimethylthiazol-2-yl) - 2, 5 diphenyltetrazolium bromide) assay is usually used to determine cell activity. Cells were seeded in a 96-well microtiter plate at an initial density of 1 × 105 cells per well and grown for 24 h in 90 μL medium before different levels of FA exposure. After 2 h, 10 μL of MTT (5 mg/ mL) (Sigma-Aldrich, St Louis, Missouri, USA) was added to each well at 37 °C for 4 h. A solution of 100 μL dimethyl sulfoxide (DMSO) was added in order to dissolve the crystal of formazan produced in wells (Perna et al., 2018). Last, the solution was read at 490 nm using an eon microplate spectrophotomete. The value of absorbance is proportional with the number of living cells. Each MTT assay was performed in triplicate. The viability of cells also was determined by CCK-8 kit (DOJINDO, Kumamoto Island, Japan) according to its instruction. CCK-8 reagent (10 μL) was added to each well of the plates when FA exposure was finished, and then cells were incubated at 37 °C for 4 h. Absorbance at a wavelength of 450 nm was measured with a microplate reader. The value of absorbance is proportional with the number of living cells. It should be performed three times independently. 2.4. ROS levels and GSH content measurements Intracellular ROS levels were determined by dichloro-dihydro-flurescein diacetate (DCFH, Sigma-Aldrich, St Louis, Missouri, USA) fluorescent assay descripted by Wu (Wu et al., 2013), with minor modifications. Briefly, 3.5 × 105 cells in good logarithmic growth phase were treated by FA, and then DCFH was added to each cell suspension at a final concentration of 10 μM. Cells were then washed twice with phosphate buffer saline (PBS) and resuspended in 600 μL PBS. A 100 μL aliquot of the cell suspension was transferred to a 96 well microplate and the fluorescence intensity was measured at 488 nm (excitation) and 525 nm (emission). The analyses were done within 3 h. Repeat 3 times for each group. Glutathione (GSH) is a major scavenger of ROS in tissues. The cell lysis buffer with Phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, Shanghai, China) was added to the treated cells at 4 °C for 1 h, then they were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was taken for testing. Basing on that thiols such as GSH can react with 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) in the dark and form yellow compounds, the content of GSH was conducted according to a previously described procedure with minor modifications (Yang et al., 2012). The absorbance was tested at 405 nm. The protein concentration was determined using the Lowry assay (Lowry et al., 1951). Repeat three times for each group. At the same time, cell viability was tested by above MTT method.
2. Materials and methods 2.1. Cell culture K562 cells and 16HBE cells (China Type Culture Collection (CCTCC), Wuhan, China) were cultivated in RPMI 1640 medium (Sigma-Aldrich, St Louis, Missouri, USA) with 10% fetal calf serum (FBS), 1% penicillin/streptomycin (Gibco, Life Technologies, Grand Island New York, USA) at 37 °C, 5% CO2 in a humidified incubator. When 80% confluent, K562 cells were discard two-thirds of the volume while 16HBE cells were enzymatically detached with trypsin-edta (Sigma-Aldrich, St Louis, Missouri, USA), then both of them were seeded in a new cell culture flasks. The medium was changed at least every 2 days. All of cells were harvested in the exponential growth phase for follow-up experimental operation.
2.5. RNA extraction and real- time PCR 2.2. Cell treatment
mRNA expression levels of target genes were analyzed using Realtime PCR (RT-PCR). Total cellular RNA was extracted from the control and reeated cells using the TRIzol® reagent (Invitrogen, U.S.) according to its manufacturer and quantified with a NanoDrop spectrophotometer. cDNAs were synthesized from 1 μg RNA using the High Capacity cDNA Reverse Transcriptase (Vazyme Biotech Co., Lid, China). RT-PCR was performed with a Chromo 4 detector using SYBR® Premix Ex Taq™ (Takara Bio, Otsu, Japan) and referred to the method described in Luisa et al. (2015). All primers used, are listed in the supplementary material and were synthesized by Genescript (Piscataway, New Jersey, USA; Supplementary Material). The amount of target cDNA was calculated by comparative threshold (Ct) method and expressed by means of the 2−ΔΔCt method (LIVAK, 2001) using the housekeeping gene β- actin or actin. The analyses were always carried out in triplicate
To explore the effects on cell proliferation induced by FA, 4% FA (Sigma-Aldrich, St Louis, Missouri, USA) was stored in accordance with the manufacturer's recommendations. It is diluted to different concentrations in serum-free RPMI 1640 medium to treat cells in logarithmic growth phase (0, 25, 50, 75, 100, 125, 150, 200 μM for K562 cells and 0, 20, 40, 60, 80, 100, 120 μM for 16HBE cells) for 2 h. Working solutions were prepared immediately before use. In the next experiment to explore the molecular mechanisms of the Hormesis of FA, Vitamin E (VE) (Sigma-Aldrich, St Louis, Missouri, USA) at 75 μM (for K562 cells) and 80 μM (for 16HBE cells) were prepared immediately with serum-free RPMI 1640 medium before use and added into cells half an hour before FA exposure. 2
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cells in the FA group showed a significant increase compared with the control group, while GSH content decreased significantly (Fig. 2B and F). Importantly, the treatment with VE declined the ROS fluorescence intensity but increased GSH levels in comparison with the FA group. ROS is a natural by-product of the normal metabolism of oxygen and plays an important role in cell signaling and homeostasis. To demonstrate whether low levels of FA-caused ROS was beneficial to cell proliferation, we analyzed the cells activity by MTT method and the data were shown in Fig. 2C, D, G and H. Not surprisingly, although cells vitality in the FA group was increased compared with the control group, it exhibited the opposite trend compared with the FA + VE group.
samples for each experimental point. 2.6. Detection of biomarkers Levels of CyclinD-cdk4, E2F1, pyruvate kinase isozyme M2 (PKM2), glucose, glucose transporter 1 (GLUT1), lactic acid, lactate dehydrogenase A (LDHA) were measured using commercial ELISA kits (BioTechne, Minnesota, USA), according to the specific manufacturer's instructions. In brief, standards with different concentrations were added to the standard wells. Firstly, 10 μL samples with 40 μL sample dilution were added to the wells that had been coated with the antibody. Next, the mixture incubated for 1 h, at 37 °C. Then the washing buffer was added to each well, left for 30s before being drained, and then patted dry. This step was repeated 5 times. 50 μL HRP-conjugate reagents was added to each well except the blank wells, and the progress of incubating or washing were consistent with above steps. 50 μL chromogen solution A and 50 μL chromogen solution B were added to each well, and kept in the dark for 10 min at 37 °C. Finally, 50 μL stop solution was added into each well. The absorbance at 450 nm of the plate was read. The specific assay protocol for determining glucose and lactic acid was based on the manufacturer's instructions of the kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). For glucose, the mixture of samples and the working solution were incubated for 15 min at 37 °C before the absorbance at 505 nm of the plate was analyzed. As for lactic acid, samples were mixed with the working solution and stored at 37 °Cfor 10 min. Then, stop solution was added to wells. The absorbance was tested at 530 nm.
3.3. Low concentrations of FA exposure accelerated cell division Cell division is closely related to cell proliferation. Therefore, we studied the variation of some molecules involved in cell division. FAtreated cells showed an up-regulation on the levels of CyclinD-cdk4 (Fig. 3A and D). Moreover, mRNA levels of Cyclin D1 and CDK4 had an FA-induced increase in two kinds of cells. Whereas the content and mRNA levels in the FA + VE group showed a downward trend when it was compared with the FA group. As a key biomolecule affected by CDK4, E2F1 variation was also measured. The results in Fig. 4 showed that FA resulted in the increasing E2F1 content and mRNA levels. Not surprisingly, regarding the effect induced by VE, the significantly decrease of ELISA and RT-PCR data could be looked. 3.4. Low concentrations of FA exposure enhanced the Warburg effect
2.7. Statistical analysis
After 2 h of exposure with FA, ELISA and RT-PCR were performed to evaluate some biomarkers involved in the Warburg effect such as PKM2, glucose, GLUT1, lactic acid and LDHA. It was obvious that in Fig. 5, when compared the no FA group with the FA group, intracellular PKM2 content increased (Fig. 5A and C) and FA also interfered on the mRNA levels (Fig. 5B and D). After the addition of VE, the data of ELISA and RT-PCR in the FA group tended to show a significant decrease in comparison to the FA + VE group. As a phenomenon which is associated with energy supply, the Warburg effect is so complex that there are various signal pathways in it. We first chose the most common “energy supplier” - glucose for test. For K562 cells, FA induced an increase in glucose content (Fig. 6A) and GLUT1 (Fig. 6B) compared with the no FA group, while VE caused a converse tendency by comparing the FA group with the FA + VE group. The data in 16HBE cells showed FA promoted GLUT1 mRNA level in association with the control group (Fig. 6F), leading the increase of glucose (Fig. 6D), but their levels treatment with VE dropped. For cancer cells with uncontrolled proliferation, lactic acid can also provide energy (Hui et al., 2017). Consequently, we measured certain biomarkers involved in lactic acid. As shown in Fig. 7A and D, intracellular lactic acid levels did experience an up-regulation tendency. Being the enzyme closely related to lactic acid, LDHA content and mRNA levels also showed significantly increase induced by FA exposure in comparison with the control group. Simultaneously, in Fig. 7, the decreasing tendency of above molecules was also obvious by comparing the FA group with FA + VE exposure.
The results were analyzed using the one way analysis of variance (one way ANOVA) with t-test by Graph Pad Prism 7.0 software. The data of ELISA assay were presented as mean while others were showed as mean ± standard deviation (SD) for the indicated number of independent determinations. The differences between groups were considered statistically significant when the P values was at least p < 0.05. All experiments were repeated at least three times and performed in triplicate. 3. Results 3.1. Low concentrations of FA promoted cells proliferation To investigate whether exposure to different concentrations of FA could have various effects on cell proliferation, we measured the proliferative activity of K562 cells by CCK-8 assay (Fig. 1A and B) and MTT assay (Fig. 1C and D). We looked that at a relatively low level (less than 125 μM FA), compared with the control group, the OD value and the proliferation rate of K562 cells were higher, and FA at 75 μM induced a significant increased cells viability. But with the FA concentrations growing, cells viability decreased and had an obvious drop in the 200 μM FA group. Could FA provide the same influence on normal cells? 16HBE cells were treated in the same manner to carry on the further exploration (Fig. 1E–H). After 2 h of exposure, FA at a concentration of higher than 60 μM decreased cells proliferative activity and there was an obvious dropping tendency in the 120 μM FA group. 40 μM FA could promoted cells proliferation significantly.
4. Discussion In exploring whether there was the Homersis effect in FA or not, we set the broader concentrations of FA, and it also contributed to the screening of the optimal concentration which promoted cell proliferation significantly. By analyzing cell viability, the data shown in Fig. 1 were well confirmed that low concentrations of FA could promote cell proliferation while high levels of FA inhibited cell activity. This was consistent with the characteristics of the Hormesis effect, indicating the existence of Hormesis effect in FA. However, as mentioned above,
3.2. Low concentrations of FA exposure increased ROS levels and decreased GSH content According to the results in Fig. 1, 75 μM FA and 40 μM FA were chosen as the efficient doses to explore the underlying mechanisms of the Hormesis of FA. In Fig. 2A and E, ROS levels in K562 cells or 16HBE 3
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Fig. 1. Cells vitality analysis after exposed to different concentrations of FA. The experimental data were analyzed by one way ANOVA with t-test and expressed as means ± standard deviation (Mean ± SD), n = 6. (A) OD value of K562 cells exposed to different concentrations of FA tested by CCK-8 assay; (B) Cell proliferation rate of K562 cells exposed to different concentrations of FA tested by CCK-8 assay; (C) OD value of K562 cells exposed to different concentrations of FA tested by MTT assay; (D) Cell proliferation rate of K562 cells exposed to different concentrations of FA tested by MTT assay; (E) OD value of 16HBE cells exposed to different concentrations of FA tested by CCK-8 assay; (F) Cell proliferation rate of 16HBE cells exposed to different concentrations of FA tested by CCK-8 assay; (G) OD value of 16HBE cells exposed to different concentrations of FA tested by MTT assay; (H) Cell proliferation rate of 16HBE cells exposed e to different concentrations of FA tested by MTT assay. *: p < 0.05, **: p < 0.01, ***: p < 0.001, compared with the control group.
differences from the experimental environment or the researchers; and the reason why different cells enjoyed different the most optimal level of FA might be the differences in tolerance to FA. Consequently, 75 μM FA (for K562 cells) as well as 40 μM FA (for 16 HBE cells) were selected for the next study. A previous study suggested that cellular responses to ROS could cause cytotoxicity or enhanced cell survival depending on the concentrations (Topaloglu et al., 2016). We found a significant difference in ROS levels between the control group and the FA group (Fig. 2A and E). VE is usually used as the effective inhibitor of ROS. In order to fully explore the relations between ROS variation and cell proliferation, we further measured the content of GSH, an important scavenger for free radicals in the body by adding VE. The data in Fig. 2B and F showed that low concentrations of FA increased ROS fluorescence intensity while decreased GSH levels. In addition, when compared with the FA
although there are many studies about the Hormesis effect, there is no report about it in FA. In general, once cell proliferation is out of control, the risk of suffering from cancer will increase greatly. A large number of epidemiological studies have shown that FA can induce some cancer including leukemia (Kwon et al., 2018), but there is no the definitive reports to reveal the molecular mechanism of FA-caused leukemia. As a common cell model for studying the occurrence, development and treatment of leukemia, K562 cells are selected as the object in our experiment. In addition, for humans, most tumors stem from epithelial cells, and the 16HBE cells can represent the initial phase of a multistage model of in vitro carcinogenesis. This is why we chose the two kinds of cells to explore the molecular mechanisms of low concentrations of FA-promoted cell proliferation in our experimentation. It was worth noting that we found that the same cells had different the most optimal level of FA to promote cell proliferation, probably due to
Fig. 2. The effect of low concentrations of FA exposure on ROS fluorescence intensity and GSH content. The experimental data were analyzed by one way ANOVA with t-test and expressed as Mean ± SD, n = 6. (A) ROS fluorescence intensity of K562 cells; (B) GSH content of K562 cells; (C) OD value of K562 cells tested by MTT assay; (D) Cell proliferation rate of K562 cells tested by MTT assay; (E) ROS fluorescence intensity of 16HBE cells; (F) GSH content of 16HBE cells; (G) OD value of 16HBE cells tested by MTT assay; (H) Cell proliferation rate of 16HBE cells tested by MTT assay. *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group.
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Fig. 3. The effect of low concentrations of FA exposure on intracellular CyclinDcdk4, CyclinD1 and CDK4. The experimental data were analyzed by one way ANOVA with t-test. (A) CyclinD-cdk4 content in K562 cells (The data were presented by Mean, n = 6); (B) CyclinD1 expression level in K562 cells (The data were presented by Mean ± SD, n = 3); (C) CDK4 expression level in K562 cells (Mean ± SD, n = 3); (D) CyclinD-cdk4 content in 16HBE cells (Mean, n = 6); (E) CyclinD1 expression level in 16HBE cells (Mean ± SD, n = 3); (F) CDK4 expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group.
some biochemical pathways that induce cell survival were further stimulated, helping FA fully to embody the Hormesis effect. Cell division is an essential part of cell cycle and a disorder of the normal cell cycle can lead tumors. Studies have shown that intracellular ROS content varies regularly with the cell division process (Liu and Liu, 2013). Therefore, based on the fact that low concentrations of FA treatment caused elevated levels of intracellular ROS in our text, we immediately determined the key factors involved in cell division. By comparing CyclinD-cdk4 content in the control group with its in the FA group, we found that FA increased the content of the factor and the mRNA levels, but they all decreased due to the addition of VE. This result was consistent with Blázquez-Castro's data that the photoactive compounds-caused ROS was associated with the Cyclin-D1 expression and effectively promoted cell proliferation (Blázquez-Castro et al., 2012) and the review from Zhou which showed that the production of
group, the changes of ROS and GSH in the FA + VE group also completely opposite. At present, most studies have revealed the adverse effects of ROS, but there are also reports showed that it was good for cell proliferation. The bovine granulosa cell proliferation rate increased by 1.2 times under hypoxic conditions (Shiratsuki et al., 2016); low doses of 2,2′,4,4′-tetrabromodiphenyl ether promoted human hepatoma HepG- 2 cell proliferation, while ROS levels were high than the control group (Wang et al., 2012). Consistent with them, in Fig. 2B, C, G and H, although ROS levels in the FA-treated group increased, the decrease of cell proliferation rate of two kinds of cells was also significant. Referring to previous study where it indicated that by the shock on NAS biomass would enhance intracellular and extracellular substances of microbial communities such as sludge communities (Sepehri and Sarrafzadeh, 2018), it reminded us that our results could show that due to the relatively low levels of ROS induced by low concentrations of FA,
Fig. 4. The effect of low concentrations of FA exposure on intracellular E2F1. The experimental data were analyzed by one way ANOVA with t-test. (A) E2F1 content in K562 cells (The data were presented by Mean, n = 6); (B) E2F1 expression level in K562 cells (The data were presented by Mean ± SD, n = 3); (C) E2F1 content in 16HBE cells (Mean, n = 6); (D) E2F1 expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group.
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Fig. 5. The effect of low concentrations of FA exposure on intracellular PKM2. The experimental data were analyzed by one way ANOVA with t-test. (A) PKM2 content in K562 cells (The data were presented by Mean, n = 6); (B) PKM2 expression level (The data were presented by Mean ± SD, n = 3); (C) PKM2 content in 16HBE cells (Mean, n = 6); (D) PKM2 expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group.
division, showing Hormesis effect. We know that cell metabolism is adapted to facilitate the uptake and incorporation of nutrients into the biomass needed to produce a new cell, so the more energy is necessary for the metabolism (Sepehri and Sarrafzadeh, 2018). In our experiment, we first measured the content of glucose and lactic acid which could supply energy. The transport of glucose requires the glucose transporter (GLUT), and GLUT1 is often overexpressed in cancer cells (Brown and Wahl, 2015). As shown in Fig. 6, we found that glucose content and GLUT1 levels increased after FA treatment, and lactic acid content also increased. LDHA catalyzes
intracellular ROS in cancer cells affected the normal cell metabolism and the function of cell cycle checkpoints, making tumor cells more proliferative (Zhou et al., 2014). In addition, in the signaling pathway involved in the CyclinD-cdk, the E2F family is non-negligible. It mainly concludes E2F1, E2F2 as well as E2F3, and the mutation of E2F1 can make the cell cycle blocked (Nevins, 2001). Therefore, E2F1 was selected as the object in this experiment. According to the data in Fig. 4, we found that FA exposure had the same effect on the changes of E2F1 and CyclinD-cdk4, indicating that low concentration of FA could promote cell proliferation by affecting the expression of key factors of cell
Fig. 6. The effect of low concentration of FA exposure on intracellular glucose and GLUT1. The experimental data were analyzed by one way ANOVA with ttest. (A) Glucose content in K562 cells (The data were presented by Mean, n = 6); (B) GLUT1 content in K562cells (Mean, n = 6); (C) GLUT1 expression level in K562 cells (The data were presented by Mean ± SD, n = 3); (D) Glucose content in 16HBE cells (Mean, n = 6); (E) GLUT1 content in 16HBE cells (Mean, n = 6); (F) GLUT1 expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; #: p < 0.05, ##p < 0.01, compared with the FA + VE group. 7
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Fig. 7. The effect of low concentrations of FA exposure on intracellular lactic acid and LDHA. The experimental data were analyzed by one way ANOVA with ttest. (A) Lactic acid content in K562 cells (The data were presented by Mean, n = 6); (B) LDHA content in K562 cells (Mean, n = 6); (C) LDHA expression level in K562 cells (The data were presented by Mean ± SD, n = 3); (D) Lactic acid content in16HBE cells (Mean, n = 6); (E) LDHA content in 16HBE cells (Mean, n = 6); (F) LDHA expression level in 16HBE cells (Mean ± SD, n = 3). *: p < 0.05, **: p < 0.01, compared with the control group; ##p < 0.01, compared with the FA + VE group.
the conversion of pyruvate to lactic acid when oxygen is deficient (Hartmann et al., 2012). The results shown in Fig. 7 did indicate that the FA-caused the increase of LDHA content and mRNA levels were significant. It was also worth noting that the changes in lactic acid were more obvious than glucose. This might be because lactic acid was also formed form pyruvate produced by glucose oxidation. The Warburg effect, also known as aerobic glycolysis, has become one of the characteristics of tumor cells and requires various enzymes to regulate the whole process. A research has showed that PKM2 was a key rate-limiting enzyme which is often overexpressed in tumor cells (Sontakke et al., 2016). Just as the data shown in Fig. 5, we found that FA exposure increased PKM2 content. Combining the FA-induced changes in GLUT1 and LDHA, we showed that once PKM2 content was upregulated, it would enhanced transcription of downstream target genes. Referring to studies which have proposed that the Warburg effect confers direct signaling functions including the generation and modulation of ROS to tumor cells (Liberti and Locasale, 2016), we analyzed the data after the addition of VE. The decrease of all of above biomarkers demonstrated that when the low concentration of FA promoted cell proliferation, the ROS level caused it was just beneficial to the signal transduction process of cell proliferation. Overview of these results, it could be seen that low concentrations of FA promoted cell proliferation by enhancing the Warburg effect.
relationship between FA-promoted cell proliferation and FA-caused cancer or other illnesses. However, the present experiment was conducted independently and separately from the above two aspects. Considering that the molecular mechanisms involved in cell proliferation is complicated, it is necessary to explore whether there is crosstalk between the above signal paths in the deeper researches.
5. Conclusions
Appendix A. Supplementary data
From the above all analysis, we can summarize our results in some points: we demonstrated that different concentrations of FA had various effects on cell vitality and only FA at low levels could promote cell proliferation. Low concentrations of FA probably through accelerating cell division process and enhancing the Warburg effect promoted cell proliferation and embodied the Hormesis effect. The appropriate ROS content induced by FA at low concentrations might be related to the process. As we all know that once cell proliferation is out of control, cancer will be on onset, therefore the present study laid the foundation for the objective evaluation of FA and the comprehensive disclosure of the
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109576.
Conflicts of interest The authors declare no competing financial interest. Author contributions Jieran An conducted the whole experiment and finished the manuscript. Fuhong Li and Yujie Qin helped with preparing experimental data and with the literature review for the paper. Hongmao Zhang and Shumao Ding gave guidance to the experiment. Shumao Ding participated in every step of preparing this manuscript. Acknowledgements The work was supported by the National Natural Science Foundation of China (31772471).
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