Signal Transductions of BEAS-2B Cells in Response to Carcinogenic

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Signal Transductions of BEAS-2B Cells in Response to Carcinogenic PM2.5 Exposure Based on Microfluidic System Lulu Zheng, Sixiu Liu, Guoshun Zhuang, Jian Xu, Qi Liu, Xinlian Zhang, Congrui Deng, Zhigang Guo, Wang Zhao, Tingna Liu, Yiqi Wang, Yuxiao Zhang, Jing Lin, Qiongzhen Wang, and Guodong Sui Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 30, 2017

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Analytical Chemistry

Signal Transductions of BEAS-2B Cells in Response to Carcinogenic PM2.5 Exposure Based on Microfluidic System Authors: Lulu Zheng1, Sixiu Liu1*, Guoshun Zhuang1, Jian Xu1, Qi Liu1, Xinlian Zhang1, Congrui Deng1, Zhigang Guo1, Wang Zhao1, Tingna Liu1, Yiqi Wang1, Yuxiao Zhang1, Jing Lin1, Qiongzhen Wang1 & Guodong Sui1, 2* 1

Shanghai Key laboratory of Atmospheric Particle Pollution Prevention (LAP3), Department

of Environmental Science & Engineering, Fudan University, 220 Handan Road, Shanghai 200433, P.R. China. 2

Institute of Biomedical Science, Fudan University, Shanghai 200433, P.R.China.

Correspondence: Email: [email protected]; [email protected] ABSTRACT PM2.5 (particulate matter less than 2.5 micrometers in diameter) is considered as a harmful carcinogen. Determining the precise relationship between the chemical constituents of PM2.5 in the air and cancer progression could aid the treatment of environment related disease and establishing risk reduction strategies. Herein, we used transcriptomics (RNA-seq) and integrated microfluidic system to identify the global gene expression and differential target proteins expression induced by ambient fine particles collected from the heavy haze in China. The results clearly indicated that cancer related pathways exhibited the strongest dysregulation. The ambient fine particles could uptake into the cells by pinocytosis, mainly promoting the PI3K-Akt pathway, FGF/FGFR/MAPK/VEGF signaling, and JAK-STAT pathway, leading to evading apoptosis, sustained angiogenesis, and cell proliferation, which are the most important hallmarks of cancer. And fine particles also have been demonstrated to create intracellular reactive oxygen species (ROS) and mitochondrial ROS, change intracellular free Ca2+, and induce apoptosis, which are all key players in mediating cancer progression. It was observed by Transmission Electron Microscopy (TEM) that the particles

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from the haze could enter the mitochondria, resulting in the disturbance of the mitochondrial membrane and disruption of the mitochondria, and these particles can even enter inside the nucleus. It was also found in our study Organic (OC, PAHs) and metals (Zn, As, V) compounds of fine particles were more closely associated with the exacerbation of cancer and secondary aerosols generated by traffic had the largest impact on cancer related signal transductions. INTRODUCTION Air pollution pervades many urban areas of developing countries around the world as well as the megacities

1

in developed countries. As the biggest developing country and the second

largest economy, China has been particularly hard hit by the severe haze pollution over the past decade 2. It has been epidemiologically proved that exposure to outdoor air pollution could cause lung cancer. IARC (International Agency for Research on Cancer) announced that outdoor air pollution is a major environmental causes of cancer and is classified as a carcinogen 3. The present study indicated that fine particle toxicity is mainly thought to be mediated through the oxidative stress, mitochondrion disruption, and apoptosis 4. It is important to understand how fine particle affects cancer progression on the molecular level, but there is little direct evidence regarding the carcinogenicity of fine particle and its effects on cancer related signal transduction networks. Current research has indicated that several components of fine particle are mainly responsible for negative health effects 5. Little attention has been paid to the relationship between the components of fine particle and the molecular mechanisms of cancer, especially the relationships between the source of fine particle and the bio-signal transduction related to cancer. It has been difficult to directly determine those relationships, because fine particles are collected with a filter, which is normally inadequate to allow the accurate chemical analysis of its multiple components and performing biosignal transduction detection 6.


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However, the microfluidic technology, could help to solve this problem, due to its capacity to handle smaller samples, compared with traditional technologies

7,8

. Additionally,

whole-transcriptome sequencing can reveal the complex genomic landscape, transcriptome dynamics, and signaling networks. These two techniques are essential for quickly exploring the carcinogenic effects of fine particles. In the present study, RNA-seq and the integrated microfluidic chip were used to analyze the relationship between the components of fine particle and signal transductions underlying cancer induced by fine particles, as well as the sources of fine particles. Ambient fine particles samples were collected during the haze period in Shanghai, China. The human lung epithelial cell line BEAS-2B was cultured on microfluidic chips

9

for examining the

contribution rates of different components of fine particle for cytotoxicity. We designed and fabricated a microfluidic device consisting of liquid gradient generators, cell culture chambers, and an immune-detection chip in which the processes of fine particle dilution, cell culture, cell stimulation, and immunoassay could occur (Figure 1, S1). We clarified the cancer related signal pathways of BEAS-2B Cells in response to carcinogenic PM2.5 exposure. It was found that fine particle could enter cell through pinocytosis and trigger cancer related signal transductions network. Five components (OC, PAHs, Zn, As, V), mainly from traffic emission of anthropogenic emissions, had more closely relationship with cancer progression.

EXPERIMENTAL SECTION Chemical analysis of PM2.5. Ions analysis were measured using Ion Chromatography (IC, Dionex ICS 3000, USA); Elements analysis were performed by an inductively coupled plasma optical emission spectroscopy (ICP-OES, SPECTRO, Germany); Carbonaceous aerosol analysis were conducted using the Thermal/Optical Carbon Analyzer (DRI Model 2001A); PAHs and n-alkanes were measured by GC-MSD (Agilent GC 6890N coupled with



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5975C MSD). PM2.5 resources were apportioned by using US EPA PMF receptor model in this study. Details can be found in Supporting Information ‘Materials and Methods’. Gene expression changes induced by PM2.5. RNA-seq was performed using the Illumina sequencing platform and pathway analysis was performed using the KEGG database and the GO database. Details can be found in Supporting Information ‘Materials and Methods’. Microfluidic chip system. Protein detection was conducted by the microfluidic system (Figure 1, S1). The system was composed of twelve uniform structures (each unit contained parallel cell culture module, a concentration gradient generator (CGG)) and an immunodetection chip. The cell culture chambers are designed for cell culture with perfusion module. The immunodetection chip could be used to for protein microarray assays. Details description can be found in Supporting Information ‘Materials and Methods’. Cellular staining with fluorescent dyes and flow cytometer. ROS production, Ca2+ levels and cell apoptosis were measured by flow cytometry. Details can be found in Supporting Information ‘Materials and Methods’. TEM analysis. TEM was performed after BEAS-2B cells cultured exposed to 200 µg/mL PM2.5 for 24 h. Details can be found in Supporting Information ‘Materials and Methods’. Statistical analyses. The concentration of chemical composition (NO3-, SO42-, NH4+, Cl-, K+, C2O42-, Ca, Fe, Al, Mg, Zn, Pb, Mn, Cu, Ba, Ni, As, V, OC, EC, PAHs, and Alkanes) of 49 PM2.5 samples were considered to be the independent variables. The protein expression (nuclear factor kappa B (NF-κB), heme oxygenase-1(HO-1), quinone oxidoreductase (NQO1),

phosphorylated

c-Jun

NH2-terminal

kinases

(P-JNK),

nuclear

factor

erythroid-2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGFA), P53, mouse double minute 2 homolog (MDM2), serine/threonine-protein kinases (AKT1), FGF receptor (FGFR1), fibroblast growth factor 14 (FGF14), tumor necrosis factors alpha (TNF-α), Caspase 1(CASP1), interleukin 6 (IL-6), interleukin 1 beta (IL-1β)) levels measured by the


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protein microarray were considered to be the dependent variables. The PM2.5 chemical composition and the protein expression levels were introduced in the methods section (lmg: R2 contribution averaged over orderings among regressors). Weighted analysis of the contribution of the independent variables on the dependent variables was conducted. The analyses was performed in R version 2.10.1 using the MGCV package 10.

Figure 1. Optical micrograph and structure of the chip. (A) Illustration of the microfluidic system. (B) Optical micrograph of the microfluidic system. (C) Single unit structure composed of a concentration gradient generation（CGG）and cell culture chamber. RESULTS AND DISCUSSION Deregulated pathways and genes induced by PM2.5. Whole transcript analysis was performed using RNA-seq. The results indicated that fine particles have a significant and extensive effect on the gene expression of BEAS-2B cells. Dysregulated transcripts were considered to be significantly affected when P-value < 0.05. A total of 48240 transcripts were sequenced, and 759 transcripts were found to be significantly dysregulated when treated with a high-dose (80 µg/mL) of PM2.5. Further statistical analysis was focused on the pathways in which the significantly dysregulated transcripts are involved. The KEGG pathways (Table



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S1) and GO categories (Table S2) were analyzed to examine relationships among all of the dysregulated genes

11

. The strongest signal transductions that respond to PM2.5 were cancer

pathways (Table S1). The main genes contributing to the dysregulation of these pathways included AKT1, FGF14, FGFR1, VEGFA, CASP3, and MDM2 (Table S1). The subsequent dysregulated KEGG pathways were colorectal cancer, bladder cancer and the Jak-STAT signaling pathway (Table S1). These pathways have been reported to be related to cancer promoting processes

12

. The GO categories showed the activation of the Wnt receptor

signaling pathway, which triggers the activation cancer pathways (Table S2)

13

. The second

most important dysregulated KEGG pathway was the endocytosis pathway (Table S1), which consists of clathrin-dependent endocytosis and a clathrin-independent pathway 14,15. Caveolin and TGFβR involved in these pathways were deregulated in this study (Figure S4). Proteins expression levels detection after exposure to PM2.5. The fluorescent probe Rh-123 (50 µM) was used to test the gradient generated by the dilution network of microfluidic chip (Figure S1). The fluorescence intensity of Rh-123 that was proportional to the concentration was monitored by fluorescence microscopy to verify the gradient dilution generated by the CGG structure (Figure 1C). The gradient concentration experiments were compared with the theoretical calculations to validate the performance of the dilution network 16. The results showed the correlation coefficient at 0.97 (Figure S2), indicating that the microfluidic device did work. Schematic illustrations of the immunoassay for measuring target proteins levels are showed in Figure. 2A. For the microarray assay, the mean of three repeated spots was considered to be the final calculated value. A positive result of a spot in the protein array was defined as (1) signal-to-noise of each protein spot >1.5 and (2) (signal-to-noise of the protein spot with sample) / (signal-to-noise of the protein spot with blank) >1.5 (Figure 2E)17.


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Different concentrations of PM2.5 at dilutions of 100, 80, 60, 40, 20, 0 µg/ml were added the BEAS-2B cells in the microfluidic chip (Figure 2B). After, and the protein expression levels of HO-1, P-JNK, and MDM2 were measured by ELISA. When induced by incremental amounts of PM2.5, a PM2.5 dose of ≥ 20 µg/mL was found to activate HO-1. P-JNK activation required a PM2.5 dose of ≥ 40 µg/mL, and MDM2 expression showed a significant decline at a dose of ≥ 60 µg/mL. These results demonstrate that at dose ≥ 60 µg/mL, all of the proteins of interest were activated, which is consistent with the results of the immunodetection chip. A concentration of 60 µg/mL was verified reasonably well for all measurements using immunodetection chip (Figure 2B). BEAS-2B cells exposed to 49 PM2.5 samples for 24 hours were measured by immunochip assay. The target protein expression levels of HO-1 were tested by traditional enzyme-linked immune sorbent assay (ELISA) to verify the results of the immunochip assay. Linear regression analysis of R2 values were 0.8 (Figure 2C), indicating that there was a highly significant positive correlation between these two methods (r=0.9) (Figure 2D). Moreover, only 1.8µg PM2.5 (30µL, the concentration is 60µg/mL) sample were needed to measure the 15 proteins by Immunedetection chip, compared with 12mg (20mL, the concentration is 60µg/mL) PM2.5 sample consumption by traditional ELISA. Fifteen proteins were measured by the protein microarray based microfluidic system. The results show that ten proteins, including Akt1, VEGFA, P53, NQO1, P-JNK, HO-1, IL-6, IL-1β, NF-κB, and Nrf2, were up regulated. Five proteins, TNF-α, CASP1, FGFR, FGF14 and MDM2, were down regulated, as identified by KEGG pathway analysis and GO category analysis (Figure S4). These proteins are found in the Nrf2/ARE signaling pathway, the NF-κB cascade, the mitogen-activated protein kinase (MAPK) signaling pathway, and the PI3K-AKT signaling pathways.………………………………………………………………..



Figure 2. Immunoassay. (A) Diagram of the protein immunoassay for the detection of targeted proteins. (B) BEAS-2B cells were treated with different concentrations of PM2.5 at dilutions of 100, 80, 60, 40, 20, 0 µg/ml. The protein expression levels of HO-1, P-JNK, and


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MDM2 were measured by ELISA. (C-D) Target protein (HO-1) measured by ELISA and immunodetection chip, and results comparison. (C) The correlation coefficient of HO-1 between immunodection chip and ELISA, r=0.9. (D) R2 values of HO-1. R2=0.8. (E) Fluorescence image of target proteins by microarray on immunodetection chip.

Figure 3. Electron micrographs of the effects of PM2.5 on BEAS-2B cells after 24 h exposure (A) and (B) untreated cells. (C), (D), (E) and (F) BEAS-2B cells exposed to PM2.5. (P: the presence of PM2.5; M: mitochondria; N: nucleus). TEM analysis after PM2.5 exposure. The analysis of BEAS-2B cells with TEM demonstrated that the mitochondria were subcellular targets of fine particles. PM2.5 was deposited inside damaged mitochondria after exposure (Figure 3C). Disrupted mitochondrial cristae appeared as cellular vacuoles. It also showed that PM2.5 lodged inside the nucleus, and the cell membrane architecture was damaged (Figure 3E). These results offer direct evidence of the cytotoxicity induced by fine particles.



Figure 4. Change in intercellular ROS levels, mitochondrial ROS levels, intercellular calcium levels and apoptosis levels in response to PM2.5 treatment in BEAS-2B cells. (A) A histogram showing the fold increase of mean fluorescence intensity (MFI) in BEAS-2B cells stained with DCFH-DA and treated with 60 µg/mL PM2.5 for 24 h. (B) A histogram showing the fold increase of MFI in BEAS-2B cells stained with MitoSOX Red and treated with PM2.5 (60 µg/mL) for 24 h. (C) A histogram showing the fold increase of MFI in BEAS-2B cells stained with FLOU-3 and treatment with PM2.5 (60 µg/mL) for 24 h. (D) Analysis of PM2.5 apoptosis in BEAS-2B cells by flow cytometry using Annexin V/PI staining. Upper right: Annexin V+/PI+ (late apoptosis and necrosis); lower right: Annexin V+/PI- (early apoptosis); lower left: Annexin V-/PI- (Normal). ROS, cellular calcium [Ca2+] and apoptosis levels detection after PM2.5 exposure. DCFH is modified by ROS into a highly fluorescence derivative (DCF)18. The fold-increase of the mean fluorescence intensity (MFI) represented the ratio of PM2.5-treated cells to control cells (Figure 4A). A dose of 60 µg/mL PM2.5 exposure resulted in a significant increase in the production of ROS. MitoSOX Red is a fluorogenic dye that is highly selective for superoxide in the mitochondria of live cells18. PM2.5 exposure led to a significant increase in the


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MitoSOX Red MFI (Figure 4B). These results suggested that PM2.5 induces cell-mediated and mitochondria-mediated ROS production. Changes in intracellular free calcium concentration can be induced by oxidative stress

18

,

potentially resulting in cellular toxicity. Cellular staining with FLOU-3 reflects the intracellular [Ca2+] concentration. A dose of 60 µg/mL PM2.5 exposure induced a significant increase in FLOU-3 fluorescence (Figure 4C). These data demonstrate that PM2.5 could influence the oxidative stress cellular response via a Ca2+-regulated pathway, which was known to be linked to cytotoxicity. RNA sequencing demonstrated that the apoptosis pathway was activated. To confirm that PM2.5 treatment could induce apoptosis in BEAS-2B cells, the proportion of cells in apoptosis in the total cell population was measured by the Annexin V-FITC/PI assay

19

using flow

cytometry. After incubation with 100 µg/mL of PM2.5, the percentage of Annexin V-FITC+/PI- cells (early apoptosis) significant increased 65.5% compared to the control group. The percentage of Annexin V-FITC+/PI+ cells (late apoptosis and necrosis) increased 12.1% compared to the control group (Figure 4D). Analysis of the cancer related pathways in response to PM2.5 based on the results of the gene, protein, ROS, cellular calcium [Ca2+] and apoptosis levels detection combined with TEM observation. Based on the above results, we can infer that the cancer related pathways were activated by fine particles. PM2.5 was observed to interact with cell receptors, such as TGFβR (Figure 5), leading to cellular pinocytosis. It is evident that PM2.5 was able to interact with cells membrane and membrane receptors TGFβR, which was aberrant regulated by RNA-seq analysis (Figure S4). Clathrin-dependent endocytosis was initiated via activated TGFβR, fine particles were internalized through inward budding of plasma membrane regions named clathrin-coated pits. Then clathrin-coated vesicles are developed through endosomes and lysosomes (Figure 5). Clathrin-independent uptake of PM2.5 is modulated via



plasma membrane regions named caveolae. These membrane buds are rich in caveolin, which is deregulated by RNA-seq analysis in our study and necessary for biogenesis of caveolae (Figure S4). Fine particles from the caveola vesicle can be transferred into caveosomes and endoplasmatic reticulum (ER). It could be inferred that the fine particles from PM2.5 could cross the plasma membrane by pinocytosis 15. Subsequently PM2.5 promote the dysregulation of ROS levels and a series of signal transductions.

Figure 5. PM2.5 mediated activation of signal transductions. PM2.5 was observed to interact with cell receptors, leading to cellular phenotypes such as evading apoptosis, sustained angiogenesis, and cell proliferation. Genes marked red, purple and sky blue are members of cancer pathways. Genes marked with dark blue are involved in inflammation. Genes marked with grey are involved in anti-oxidant defense. PM2.5 was observed, through flow cytometric detection, to induce the generation intracellular ROS and mitochondrial ROS (Figure 4A, 4B). More evidence indicated that ROS are capable of functioning as second messengers, promoting cell proliferation, metabolism and cancer development

20,21

. It is clear that ROS could trigger the Nrf2 pathway, NF-κB signaling, the


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MAPK cascade and the PI3K/AKT pathway, which are key players in the mediation cell survival and cancer procession 15. Herein, the main effects of these cancer related signal transductions were listed as following: 1. Evading apoptosis. PM2.5 interact with FGFR, leading to evading apoptosis through the activation of the PI3K-Akt signaling pathway (Figure 5), which can inhibit apoptosis and is linked to many human malignancies 22. This apoptosis failure could result in the development of cancer and is one of the ten hallmarks of cancer 23. Additionally, MDM2, a negative p53 regulator involved in the PI3K-Akt signaling pathway, plays a vital role in cancer progression and prognosis. Our results showed that the oncogene AKT, which is a member of PI3K/Akt pathway, was up regulated and MDM2 was down regulated (Figure S4). Evading apoptosis were also mediated by the activation of mitochondrial pathways and the Wnt signaling pathway (Figure 5). B-cell lymphoma 2 (Bcl-2), a proto-oncogene and contributor to cancer formation and procession, was down regulated (Figure S4). Bcl-2 functions in two different cellular compartments, the ER and the mitochondria. In the ER, Bcl-2 alters Ca2+ signaling

24

. According to flow cytometer analysis, Ca2+ levels were

significantly increased (Figure 4C). 1,4,5-trisphosphate-sensitive calcium-release channel activity was dysregulated according to the GO category analysis of this study (Table S2); Bcl-2 modulates Ca2+ levels via this channel. Bcl-2 family proteins mediate Ca2+ release as well as its subsequent uptake by mitochondria. These two steps are crucial in the regulation of cytochrome c (Cyt c) release, and Cyt c leads to the proteolytic maturation of caspase-3 and caspase-9, ultimately resulting in apoptosis deregulation

25

. RNA-seq showed that

caspase-3 was down regulated (Figure S4). GO category analysis showed that the regulation of the release of Cyt c from mitochondria, which mediates apoptosis, was activated (Table S2). Apoptosis that was confirmed by flow cytometer (Figure 4D) is a crucial step in lung injury, and its deregulation can lead to the progression of more serious diseases, such as



cancer23. The Wnt signaling pathway is frequently found to be dysregulated in cancer. The Wnt5A gene was up regulated, and TCF/LEF was down regulated (Figure S4). Wnt5A, an aberrant expression of the Wnt ligand-protein, secreted downstream of components of this pathway, such as β-catenin, which accumulates and forms complexes with TCF/LEF to modulate the transcriptional induction of target genes 26,27. 2. Sustained angiogenesis. Members of the FGF family are proangiogenic signals, which are implicated in sustaining tumor angiogenesis when their expression is dysregulated. FGF signals are transduced via FGF receptors (FGFRs), and FGFRs are preferentially aberrant expressed in cancer 28. FGF14 and FGFR1 were down regulated in our study (Figure S4). The FGF/FGFR pathway activates downstream of the MAPK signal transduction (Figure 5). The MAPK pathway includes the stress-activated JNKs (Table S2), which were found to be upregulated in this study (Figure S4). The important role of JNK in cancer progression has been shown in recent studies 29. The MAPK pathway is dysregulated in a variety of human cancers and frequently implicated with sustained angiogenesis; moreover, this pathway is induced during the multistage development of cancer in humans and animal models 30. The MAPK pathway can alter the expression of VEGFA (Figure 5). VEGFA, a well-known angiogenesis inducer, was found to be up regulated in this study (Figure S4). The VEGFA gene is involved in new blood vessel formation and in the homeostatic survival of endothelial cells. It has been confirmed that VEGF gene expression can be upregulated by both hypoxia and oncogene signaling

23

. PM2.5 also influenced gene transcription through redox-sensitive

transcription factors, such as NF-κB, that was up regulated in this study (Figure S4) involved in a network in modulating angiogenesis. 3. Cell proliferation. Our RNA-seq results showed that the JAK-STAT pathway was activated after PM2.5 exposure (Table S1). This pathway plays an essential role in cell proliferation in different cancers. In addition, members of the STAT family have been


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detected in many human cancers

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, the effects of these proteins on cell proliferation and

survival have been well investigated. Under certain circumstances, STAT5, which was significantly dysregulated in our study (Figure S4), was able to protect against apoptosis and promote proliferation. The STAT proteins were phosphorylated by the Jaks and then activate transcription of downstream target genes (Figure 5). NF-κB family genes are considered to be critical regulators of genes that function in cell proliferation and inflammation. The NF-κB cascade is an important dysregulated GO pathway (Table S2). Inflammation is capable of enhancing cancer progression and tumorigenesis because it supplies molecules, such as ROS and growth factors that contribute proliferative signaling and so on, to the tumor microenvironment. Evidence has indicated that the NF-κB family regulates and activates key molecules that are connected with diseases of inflammation and cancer 31. In conclusion, we clarified the cancer related pathways were responded to PM2.5. PM2.5 enter cell through pinocytosis and trigger cancer related signal transductions network. It is known that PM2.5 consists of organic constituents and transition metals that generate ROS in the intracellular and the mitochondria (Figure 4A, 4B). ROS can play a pivotal role in apoptotic machinery by accelerating mitochondrial dysfunction and regulation Cyt c releases from mitochondria in this study (Table S2). ROS accumulation overwhelms the cell’s antioxidant defense, leading to a state of redox disequilibrium, which is termed oxidative stress. GO category analysis showed that pathways were simulated in response to chemical stimulus, organic substances and oxygen levels (Table S2). Oxidative stress has various effects on cell signaling pathways, such as the Nrf2/ARE pathway, MAPK signal transduction, and NF-κB cascade (Figure 5). PM2.5 can trigger a cell response through interaction with FGFR, which modulates the activation of the PI3K/AKT pathway and MAPK cascade 32. Evidence indicated that PM2.5 is



capable of stimulating Nrf2 mediated against ROS-induced oxidative stress via the PI3K signaling pathway. ROS production is linked to the activation of the PI3K/AKT pathway and MAPK cascade; these pathways phosphorylate Nrf2, and activated Nrf2 translocate to the nucleus and binds to the ARE to activate the downstream target genes HO-1 and NQO133. Protein analysis results showed that these proteins were up regulated after PM2.5 exposure (Figure S4). However, recent studies have indicated that high levels of Nrf2 facilitate cancer formation, and Nrf2 and Nrf2-mediated other phase II enzymes play a positive role in tumorigenesis 34-35. PI3K is capable of transmitting oncogenic signals to AKT to mediate tumorigenesis via downstream targets. AKT modulates tumor suppressor P53, which plays a vital role in carcinogenesis and was up regulated after exposure to fine particle in our study (Figure S4). AKT activates the NF-κB cascade, exhibiting a complicated network in modulating tumor growth and angiogenesis. NF-κB was able to regulate TNF and IL-6 to activate VEGF 36. NF-κB and IL-6 were up regulated and TNF was down regulated in our study (Figure S4). IL-6 is a pro-inflammatory cytokine that is multifunctional and plays a vital role in tumor proliferation. IL-6 is essential for the activation of the different pathways, such as JAK/STAT, PI3K, and MAPK, that induce the expression of downstream proteins, leading to inflammation and cancer progression

37

. Thus, the results clearly showed that PM2.5 could

stimulate signal transduction networks related to cancer development. Determine the contribution rates of PM2.5 components for each dysregulated protein. Forty-nine PM2.5 samples were collected between October 2011 and August 2012 in Shanghai, The daily PM2.5 concentrations ranged from 35 to 381 (mean 90) µg m-3, much higher than the newly defined gradeII level of the Chinese ambient air quality standards for PM2.5 (35µg m-3). Twenty-two components, i.e. OC, PAHs, elemental carbon (EC), NO3-,


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SO42-, NH4+, Cl-, K+, C2O42-, Ca, Fe, Al, Mg, Zn, Pb, Mn, Cu, Ba, Ni, As, V, and Alkanes, found in the PM2.5 samples were systematically analyzed to determine their contribution rates for each dysregulated protein (Figure S5). Additionally, the content of each component in PM2.5 and their relations were analyzed to determine the sources of these components (Figure S6). The apportioned five sources and the PMF factor profiles of the concentration and contributions of each component are shown in Figure S6. Secondary sources contributed 50%, 28%, 23%, 24%, and 43% of the measured OC, PAHs, NO3-, SO42-, C2O42-, respectively.

Figure 6. Link between the dysregulated proteins involved in cancer signaling pathways and the contributing components of PM2.5 with their sources in Shanghai over four seasons. The wider of the connecting lines represented the more contribution rates, the widest line means that the components have the most important contribution to activated the related proteins, the middle wide line means that the components have the second important contribution to the related proteins, the narrow line means that the components have the third important contribution to the related proteins. The sizes of the circle stands for the mass percent of components of PM2.5, the bigger is the diameter of circle, the larger is the mass percent of responding components.



Additionally, secondary sources contributed 50% of the measured alkane, 43% of the measured EC, and 33% of the measured As. High loadings of OC, NO3-, and SO42- were also observed. Biomass burning contributed 47% of the measured PAHs, 43% of the measured As, and 49% of all measured NH4+. Biomass burning also had a high loading of NH4+. Oil combustion contributed 80% of the measured V. Crustal sources accounted for 62% of the measured Pb and 57% of the measured Mn, This source had a high OC loading and moderate contributions from EC, Al, Fe, and NH4+. This type of profile is considered to be associated with crustal sources. Sea Salt contributed 56% of the measured Ca. The orderly rank of contribution rate for cytotoxicity is Zn, PAHs, OC, Mn, V, Ba, Pb, Cu, and EC (Figure S7, the contribution rate for each target protein of the components listed in figure 6 was more than 9%). The contribution rate of Zn and PAHs was approximately 7%. Nine components (Zn, PAHs, OC, Mn, V, Ba, Pb, Cu, EC) accounted for over 50% of the cytotoxicity effects. Figure 6 shows the relationship between the detected dysregulated proteins and the contributing components in PM2.5 with their sources in Shanghai over four seasons. The results showed that OC was crucial for the abnormal expression of 5 proteins [NQO1 (11%), HO-1 (16%), P53 (9%), AKT1 (11%), and IL-6 (14%)]. NQO1 and HO-1 are members of the Nrf2/ARE signaling pathway, and P53 and AKT are members of P13-AKT signaling pathway. IL-6 participates in the Jak-STAT signaling pathway, the NF-κB cascade and the other cancer pathways. All of these pathways are associated with cancer progression. It was worth to emphasize that PAHs were the most important components that induced abnormal expression of 4 proteins [HO-1 (10%), CASP1 (17%), NF-κB (9%), and P53 (22%)]. Dysregulation of Nrf2 and HO-1 demonstrated that PAHs play a vital role in the activation of the Nrf2/ARE signaling pathway. CASP1 and NF-κB are members of the NF-κB cascade. P53 is involved in cancer progression via P13-AKT signal transduction.


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Traffic emissions and biomass burning accounted for 28% and 42%, respectively, of the total PAHs in PM2.5 (Figure S6). Zn was also very important in activating 4 dysregulated proteins [FGF14 (23%), FGFR1 (12%), VEGFA (11%), and MDM2 (20%)] (Figure 6). The results showed that Zn and As played crucial roles in the activation of FGF/FGFR/MAPK/VEGF signaling, and PI3K-AKT signal transduction was also promoted by Zn. All of these pathways are relevant in cancer progression. V played a vital role in inducing abnormal expression of 4 proteins [P-JNK (10%), caspase1 (13%), TNF-α (11%), and FGFR1 (16%)] (Figure 6). These proteins are connected with the activation of the NF-κB cascade and FGF/FGFR/MAPK/VEGF signaling, which are involved in cancer progression. BEAS-2B cells responded strongly to the V content. Mn induced the abnormal expression of 4 proteins [(NQO1 (10%), P-JNK (14%), IL-1β (12%), and TNF-α (14%)) (Figure 6) involved in the NF-κB cascade and the MAPK cascade. Ba, Pb, and Mn dysregulated P-JNK and IL-1β, which are also members of the NF-κB cascade and the MAPK cascade (Figure 6). The connection between the PM2.5 components and the related diseases showed that organic compounds (OC, PAHs) and several metals (Zn, As, V) were vital to the promotion of cancer progression and induced the related signaling pathways that exacerbate cancer progression (Figure 6). Organic compounds (OC, 16 PAHs) and metals (Zn, As, V) were the major components of PM2.5, and most of these components were generated by anthropogenic pollution (Figure S6). OC and PAHs were mostly generated by traffic emissions, and traffic sources contributed 49.6% of the measured OC; PAHs were also largely from traffic emissions, which accounted for 28% of the total PAHs in PM2.5 (Figure S6). Previous studies reported that PAHs were mostly generated by traffic emissions in Shanghai 38. V is mainly generated by oil combustion, which contributed 80% of the total V in PM2.5 in Shanghai (Figure S6). The emissions from oil combustion in diesel motor vehicles and ships



contributed to a significant part of the PM2.5 in Shanghai 39, as the Shanghai port is the largest shipping port in the world. Therefore, V was primarily generated by traffic sources, especially ship traffic. As was mostly derived from coal combustion, and Zn was at least partially generated by anthropogenic emissions. Therefore, these results suggest that the increasing rate of cancer in Shanghai and in China are highly related to the anthropogenic emissions, especially traffic emissions. CONCLUSIONS In summary, the integrated microfluidic system including cell-culture, PM2.5 stimulation and immunodetection developed in this study could rapidly conduct high-throughput protein detection. This system required minimal amount of PM2.5 and could detect a large number of proteins. The required PM2.5 solution sample was＜1.8 µg, much less than the 12 mg sample required for ELISA. This is the first report of the using the microfluidic chip and RNA-seq analysis of BEAS-2B cells to examine cytotoxicity in response to PM2.5. According to the results of the gene, protein, ROS, cellular calcium [Ca2+] and apoptosis levels detection combined with TEM analysis. PM2.5 could enter the cells via pinocytosis and interact with cell membrane receptors to induce signaling pathways networks related to cancer growth. It is known that PM2.5 consists of organic constituents and transition metals. How the components of PM2.5 activate the signal transductions related cancer growth, and the sources of these deleterious constitutes is little known. Herein, we also studied the contribution rates of different components of PM2.5 for related diseases. Five components (OC, PAHs, Zn, As, V), mainly from traffic emission of anthropogenic emissions, had more closely relationship with cancer progression. The findings of this study may provide tremendous value to study in a broader field, such as environmental, medical and biological sciences.


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ASSICIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.anal-chem. Site description, Sampling, Components analysis, PMF modelling, Microfluidic fabrication and operation, Flow cytometry, TEM, Supplementary Figures and Tables. ACKNOWLEDGMENTS This study was funded by National Natural Science Foundation of China (NSFC, grant number: 21577020, 21577019, 21377026 and 21377027). REFERENCES (1) Parrish, D. D.; Zhu, T. Science 2009, 326, 674-675. (2) Huang, R. J.; Zhang, Y.; Bozzetti, C.; Ho, K. F.; Cao, J. J.; Han, Y.; Daellenbach, K. R.; Slowik, J. G.; Platt, S. M.; Canonaco, F.; Zotter, P.; Wolf, R.; Pieber, S. M.; Bruns, E. A.; Crippa, M.; Ciarelli, G.; Piazzalunga, A.; Schwikowski, M.; Abbaszade, G.; Schnelle-Kreis, J.; Zimmermann, R.; An, Z.; Szidat, S.; Baltensperger, U.; Haddad, I. E.; Prevot, A. S. H.

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for TOC only


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Signal Transductions of BEAS-2B Cells in Response to Carcinogenic

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