SF1670 | P2 Receptor Signal

Exosomes derived from normal human bronchial epithelial cells down-regulate proliferation and migration of hydroquinone-transformed malignant recipient cells via up-regulating PTEN expression

Zhenwei Lian, Zuqing Hu, Hongyi Xian, Ran Jiang, Haoyu Huang, Yunxia Jiang, Zhongdaixi Zheng,
R. Stephen Lloyd, Jianhui Yuan, Yan Sha, Sanming Wang, Dalin Hu

PII: S0045-6535(19)32736-5
DOI: https://doi.org/10.1016/j.chemosphere.2019.125496
Reference: CHEM 125496

To appear in: Chemosphere

Received Date: 03 October 2019
Accepted Date: 26 November 2019
Please cite this article as: Zhenwei Lian, Zuqing Hu, Hongyi Xian, Ran Jiang, Haoyu Huang, Yunxia Jiang, Zhongdaixi Zheng, R. Stephen Lloyd, Jianhui Yuan, Yan Sha, Sanming Wang, Dalin Hu, Exosomes derived from normal human bronchial epithelial cells down-regulate proliferation and migration of hydroquinone-transformed malignant recipient cells via up-regulating PTEN expression, Chemosphere (2019), https://doi.org/10.1016/j.chemosphere.2019.125496

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Exosomes derived from normal human bronchial epithelial cells down-regulate proliferation and migration of hydroquinone-transformed malignant recipient cells via up-regulating PTEN expression
Zhenwei Lian1,#, Zuqing Hu1,2,#, Hongyi Xian1,#, Ran Jiang1, Haoyu Huang1, Yunxia Jiang1, Zhongdaixi Zheng1, R. Stephen Lloyd3, Jianhui Yuan4, Yan Sha5, Sanming Wang6, Dalin Hu1,⁎
ImageDepartment of Environmental Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, 510515, China
2 Department of Medicine, Jiamusi University, Jiamusi, 154007, China

3 Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, 3181 S. W. Sam Jackson Park Rd, Portland, Oregon, 97239, USA
4 Nanshan District Center for Disease Control and Prevention, Shenzhen, 518054, China
5 Institute of Occupational Disease, Shenzhen Prevention and Treatment Center for Occupational Disease, Shenzhen, 518020, China
6 Faculty of Health Sciences, University of Macau, Taipa, Macau SAR, China

* Correspondence to: Dalin Hu, Department of Environmental Health, School of Public Health, Southern Medical University, Guangdong Province, Guangzhou, 510515, China. Email: [email protected]; Tel: +86-20-62789126.
# These authors contributed equally to this paper.
Abstract

ImageThe gene encoding the tumor suppressor, phosphatase and tensin homolog (PTEN), located on chromosome 10, is frequently expressed at low levels in various tumors, resulting in the stimulation of cell proliferation and migration. However, the role of exosomal PTEN in cell-cell communication during the progress of benzene-induced carcinogenesis remains unclear. The goal of this study was to explore whether exosomes derived from normal human bronchial epithelial cells (16HBE) could transmit PTEN to hydroquinone-transformed malignant recipient cells (16HBE-t) and its possible effects on cell proliferation and migration. Consistent with PTEN expression being down-regulated in transformed cells, we found that its expression was significantly decreased in 16HBE-t relative to 16HBE cells and that purified exosomes secreted by 16HBE, up-regulated PTEN levels in recipient 16HBE-t cells. Thus, down-regulating their proliferation and migration. Further, when exosomes derived from 16HBE cells that had been treated with the PTEN inhibitor SF1670, were incubated with recipient 16HBE-t cells, they exhibited decreased PTEN levels, with a corresponding increase in their proliferation and migration. In conclusion, our study demonstrates that exosomes derived from 16HBE cells can down-regulate proliferation and migration of recipient 16HBE-t cells via transferring PTEN.
Key words: Benzene; toxicity; exosomes; PTEN; proliferation; migration.
1. Introduction

Phosphatase and tensin homolog on chromosome 10 (PTEN) is a typical tumor suppressor gene and its signal transduction pathway can be regulated by both phosphatase-dependent and -independent mechanisms (Freeman et al., 2003; Hopkins and Parsons, 2014). While its tumor-suppressor activity is mainly attributed to its lipid phosphatase activity (Song et al., 2012; Worby and Dixon, 2014). In the meanning time, it also exhibits protein phosphatase activity and regulates important biologicalprocesses, including inhibition of cell migration and arrest of cell cycle (Putz et al., 2012; Zhu et al., 2015; Brandmaier et al., 2017).Accumulated evidence suggests that somatic mutations, gene silencing, and epigenetic modification can result in the inactivation and dysfunction of PTEN, which in turn, increases cell proliferation and reduces cell death that are both critical in the progression of tumorigenesis (Lee et al., 2015; Milella et al., 2015; Yu et al., 2018). Inactivation and dysfunction of PTEN in somatic cell are frequently found in a variety of patients with cancer diseases, such as glioblastoma, colon cancer and endometrial cancer (Valeri et al., 2014; Benitez et al., 2017; Zhang et al., 2017). In addition, there is growing evidence that there exists a quantitative association between levels of PTEN gene expression and protein in the progress of various tumors.Benzene is a high volume industrial compound that is widely used worldwide (Snyder et al., 1993; Wang et al., 2014; Moro et al., 2015) and due to its high volatility, it is also a ubiquitous environmental pollutant (Fishbein, 1984; Abplanalp et al., 2017). While the International Agency for Research on Cancer (IARC) has classified benzene as a known human carcinogen, the fundamental mechanisms underlying its carcinogenesis has not been fully elucidated (Sheets et al., 2004; Snyder, 2012; Warden et al., 2018).

In this study, we evaluated the levels of PTEN in normal human bronchial epithelial cell line (16HBE) and hydroquinone (an active metabolite of benzene) – transformed malignant 16HBE cell line (16HBE-t). At the same time, we isolated exosomes of 16HBE, characterized and co-cultured the exosomes with 16HBE-t, and

assessed the effects of exosomal PTEN on proliferation and migration of recipient 16HBE-t cells. Data suggested that the level of PTEN was significantly decreased in 16HBE-t as compared to 16HBE cells, and exosomes derived from 16HBE down-regulated proliferation and migration of 16HBE-t cells via up-regulating their PTEN expression.
2. Materials and methods

2.1 ImageChemicals

HQ-transformed malignant 16HBE cell line (16HBE-t) was constructed using the methods as reported previously (Jiang et al., 2019). Cells were cultured in RMPI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10 U mL-1 penicillin and streptomycin at 37 ℃ with 5% CO2. When cells reached 80% confluence adherent, they were incubated with 0.25% EDTA-Trypsin and divided into two culture plates. Culture medium was replaced every other day.
2.3 ImageSoft agarose assay

16HBE-t cell line was confirmed by soft agarose assay. Briefly, 2 mL per well of 0.6% (v/v) normal melting agarose (NMA) was added onto 6-well plates at 4 ℃ to solidify as base layer. A total of 4 × 104 cells were re-suspended in a 6 mL mixture of fully supplemented 1640 medium and low melting agarose (LMA, 0.3%, v/v), then 1 mL was added per well onto the base layer and cultured at 37℃, 5% CO2. After 21 d of culture, the colony forming efficiency was counted.
2.4 Exosome purification

Transmission electron microscopic (TEM) was used to verify the morphological characteristic of exosomes. Exosomes derived from cell culture medium were enriched through ultracentrifugation and re-suspended in 20 μL PBS. A total of 2 μL of the exosome solution were applied to each carbon-coated copper grids and dried for 10 min at room temperature (RT). The prepared samples were negatively stained with 1% phosphotungstic acid (pH 7.0) for 5 min. The characteristic of exosomes were visualized using a transmission electron microscope (JEM-1200EX, Japan) operating at 80 kV.
2.6 16HBE-t cells co-cultured with exosomes

5 × 105 cells per well were added into 6-well plates, cultured at 37 ℃ with 5% CO2 for 24 h. After that, the cells were washed three times with PBS and co-cultured with 60 μL exosome suspension for about 48 h.
2.7 Confocal laser scanning microscopic analysis

Briefly, 50 μL exosomes suspension and 2 μL PKH67 were respectively diluted in 500 μL diluent C solution, mixed and incubated at 37 ℃ for 5 min. 1 ml of PBS containing 0.5% (w/v) bovine serum albumin (BSA) was added to terminate the labeling reaction. The labeled exosomes were re-enriched by ultra-centrifugation (120,000 × g for 60 min at 4 ℃) and re-suspended in 50 μL PBS, and then incubated with recipient cells for 3 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were fixed with 4% paraformaldehyde in PBS for 30 min, washed three times with PBS. Exosome up-taking was observed using a Confocal laser scanning microscope (Zeiss, LSM700B, Germany).
2.8 Quantitative RT-PCR analysis on PTEN

The levels of PTEN mRNA expressed in different cells were evaluated by using quantitative RT-PCR analysis. Total RNA was extracted by using Trizol (Invitrogen, USA) reagent following the manufacturer’s protocol. The concentration and purification of total RNA were assessed by Nanodrop 2000 machine (Thermo, USA). A total of 500 pg of total RNA was used to synthesize cDNA and qPCR RT Kit was used to perform reverse transcription. Sybr Green qPCR Master Mix was used to conduct PCR amplification. PTEN detection program was as follows: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s. Relative expression levels were calculated by using the comparative threshold cycle (2-ΔΔCT) method, in which GAPDH was used to normalize the level of PTEN. The related primer sequences were as follows:
PTEN Forward 5′-CGGCAGCATCAAATGTTTCAG-3′ PTEN Reverse 5′-AACTGGCAGGTAGAAGGCAACTC-3′ GAPDH Forward: 5′-ACCACGGTGCACGCCATCAC-3′ GAPDH Reverse: 5′-TCCACCACCCTGTTGCTGTA-3′
2.9 Western blot

Tris-buffered saline with Tween-20 (TBST) and 3% BSA for 4 h at RT, PVDF membranes were sequentially incubated with rabbit anti-PTEN (1:500 dilution) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenasen (GAPDH, 1:5,000 dilution) overnight at 4 ℃. Membranes were washed with TBST (3 × 10 min), incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution) for 2 h at 37 ℃. Enhanced chemiluminescence was performed for immunodetection analysis.
2.10 ImageCCK-8 analysis on cell proliferation

After 1 × 107 cells per well were seeded onto 6-well culture plates and cultured for 24 h, a straight scratch was made on the plates using a pipette tip. Plates were washed three times with PBS to remove cell-debris. The scratch was observed under a microscope at different times (0, 6, 12, 24 h) at the same area. Image J software (National institutes of health, USA) was used to detect the width of the scratch. The cell migration ability was measured by detecting the half-width of the scratch and by subtracting this value from its initial half-width.
2.12 Statistical analysis
Statistical analyses were performed by using SPSS 13.0 (SPSS Inc, USA). Data were showed as mean ± standard deviation. Analysis of variance (ANOVA) and student’s t-test were used to determine the differences between groups. P < 0.05 was defined as statistically significant.
3. Result

3.1 16HBE-t cell line construction

After being treated with 25 μM HQ for about 29 weeks (The 40th generation), 16HBE transformation was achieved. Cells showed a morphological characteristic of anchorage-independent growth (Fig. 1). Transformed 16HBE cells (16HBE-t) formed typical colonies in soft agarose. The colony formation efficiency was about 6.25% (Fig. 2). 16HBE transformation assay.A: Normal 16HBE; B – C: The 15th generation of 16HBE (The 15th G-16HBE) and the 30th generation of 16HBE (The 30th G-16HBE) treated with hydroquinone (HQ); D: The morphological characteristic of anchorage – independent growth of transformed cells (the 40th generation of 16HBE cells).

Confirmation of HQ-transformed malignant cells with soft agarose assay.

A: 16HBE-t cells formed typical colony in soft agarose medium; B: Colony formation rate of 16HBE cells in transformation assay (* p < 0.05).
3.2 Exosomal morphology under TEM

To confirm the ability of 16HBE-t to uptake the exosomes derived from normal 16HBE cells, exosomes were isolated and stained with PKH67 and co-cultured with 16HBE-t cells. After 3 hours’ incubation, a punctuated green fluorescence appeared in the cytoplasm of 16HBE-t cells (Fig. 3, B, C, D).TEM and confocal microscopic morphology of exosomesA: Exosomal morphology under TEM (Scale bar = 100 nm); B:16HBE-t cell nuclei stained with DAPI (Blue; Scale bar = 28 μm); C: 16HBE exosomes stained with PKH67 (Green; Scale bar= 28 μm); D: Exosomes uptake by 6HBE-t (Scale bar = 28 μm).

3.4 Exosomes up-regulate PTEN expression of 16HBE-t

qRT-PCR and western blot analyses were used to evaluate the level of PTEN expression, in which the comparative threshold cycle (2-ΔΔCT) method and Gray analysis were separately adopted to assess the level of PTEN mRNA and protein. The results showed that the PTEN mRNA and protein of 16HBE-t cells were significantly decreased as compared to 16HBE cells (p < 0.05). Interestingly, when 16HBE-t cells ingested exosomes derived from 16HBE cells (16HBE-t-Exos), the level of PTEN mRNA and protein were significantly higher than that of 16HBE-t (p < 0.05) (Fig. 4).Our data suggested that exosomes derived from 16HBE cells up-regulated the PTEN expression of 16HBE-t. The expression level of PTEN in different groups of cells.a: qRT-PCR analysis on PTEN mRNA (* P < 0.05); b: Western blot analysis on PTEN protein.

3.5 16HBE exosomes down-regulated the cell proliferation of 16HBE-t via PTEN pathway
CCk-8 analysis was used to assess the effects of 16HBE exosomes on proliferation of 16HBE-t cells. The results showed that proliferation of 16HBE-t cells was significantly higher relative to 16HBE cells. The proliferation of 16HBE-t-Exos cells was significantly decreased as compared to the group of 16HBE-t cells. Interestingly, when the PTEN signal in 16HBE-t-Exos cells was blocked by using inhibitor SF1670, their proliferation was significantly higher than that of 16HBE-t-Exos cells. These results suggested that 16HBE exosomes were involved in down-regulating proliferation of recipient 16HBE-t cells through PTEN pathway (Fig. 5).The proliferation of different groups of cells (* P <0.05).3.6 16HBE exosomes down-regulate cell migration of 16HBE-t via PTEN pathwayRelative rates of 16HBE-t and 16HBE cell migration were measured using a technique in which after cell plating, a scratch was made on the plate, debris removed and growth into the cell-free area measured. This assay revealed that the migration distance of 16HBE-t cells was significantly increased as compared with 16HBE. Further, the cell migration distance of 16HBE-t-Exos cells was significantly decreased as compared with the group of 16HBE-t cells. Interestingly, the cell migration distance of 16HBE-t-Exos-inhibitor was significantly longer than that of 16HBE-t-Exos. This result indicated that 16HBE exosomes down-regulated the cell migration of 16HBE-t via PTEN pathway (Fig. 6-7).The microscopic images of scratch in different groups of cells.A: 16HBE cell migration; B: 16HBE-t cell migration; C: 16HBE-t-Exos cell migration; D:16HBE-t-Exos-inhibitor cell migration. Cell migration distance of different groups of cells (* p < 0.05).
4. Discussion

Benzene is an important industrial chemical and widely used in the manufacture of perfume, medicine, gasoline additive, and paint (Snyder et al., 1993; Minciullo et al., 2014; Moro et al., 2015). And is an ubiquitous air contaminate that is asssociated with hematotoxic effects and can causes leukemia (Snyder, 2012; Nourozi et al., 2018).
However, the mechanisms underlying its carcinogenesis remain unclear.In the present study, we observed that the expression of PTEN in hydroquinone-transformed 16HBE cells (16HBE-t) was significantly decreased as compared to the normal control group (16HBE). Down-regulation of PTEN is an important biological event in the progress of tumorigenesis, which is frequently occur in various kinds of cancer (Hopkins et al., 2014; Lee et al., 2018), including breast carcinomas, acute myeloid leukemia, glioblastoma, colon, non-small cell lung cancer (NSCLC), endometrial cancers and melanoma (Noguera et al., 2013; Kechagioglou et al., 2014; Perez-Ramirez et al., 2015; Benitez et al., 2017). The frequency of PTEN loss or inactivating mutations ranges from 30% – 40% in sporadic breast carcinomas, 5% – 30% in sporadic colorectal cancers, 30% – 60% in melanomas , 24% – 44% in NSCLC (Marsit et al., 2005), 24% in early NSCLC (Soria et al., 2002) and up to 75% in acute myeloid leukemia (AML) (Cheong et al., 2003). However, at the present time, the possible molecular mechanisms for PTEN alteration is still unknown. It may include, but not be limited to the biological progress of transcriptional regulation, post-transcriptional modification, and protein–protein interactions (Milella et al., 2015).It’s well known that cells can communicate with one another following genetic or environmental stimuli, and thus transmit messages by way of transferring of signaling molecules including hormones, cytokines, growth factors and neurotransmitters and transcriptional regulators (Camussi et al., 2010; Catalano et al., 2013; Gonzalez and Medici, 2014; Camacho et al., 2017; Chiodoni et al., 2019). Interestingly, in early 1981, the discovery was made that membrane micro-vesicles exfoliated from normal and cancer cells, exosomes, might have certain physiological functions (Trams et al., 1981). Subsequently, researchers shown that exosomes widely participate in various kinds of biological processes, including immune response, antigen-presenting, cells growth, and cell invasion (Harris et al., 2015; Hock et al., 2017; Barros et al., 2018). Specifically, exosomes can carry and transmit crucial intercellular signal molecules thus, reprograming phenotypes of recipient cells, with the potential to modulate carcinogenic consequences (Quesenberry et al., 2015; Barile and Vassalli, 2017). Our study found that exosomes derived from normal 16HBE cells increased the expression level of PTEN in recipient 16HBE-t cells and down-regulated their proliferation and migration. Furthermore, when exosomal PTEN was blocked by using inhibitor SF1670, recipient 16HBE-t cells exhibited decreased PTEN level, and their proliferation and migration increased correspondingly. Thus, the intercellular communication of exosomal PTEN between normal 16HBE and 16HBE-t cells may be an important biological event in the progress of carcinogenesis of benzene.PTEN, as a typical tumor suppressor, can regulate signal transduction pathways by both phosphatase-dependent and -independent mechanisms (Leslie et al., 2007). Itstumor-suppressor activity is mainly attributed to lipid phosphatase activity, i.e., it is mainly involved in the stable maintenance of phosphatidylinositol-3-kinase (PI3K)/Akt cascade (Perez-Ramirez et al., 2015; Lee et al., 2018), thus indirectly inhibits the downstream targets, such as GSK3, FoxO and Bcl-2 that are involved in controlling cell metabolism, proliferation and survival.

5. Conclusion

ImageIn conclusion, this investigation suggests that exosomes derived from 16HBE cells can down-regulate proliferation and migration of recipient 16HBE-t cells via transferring PTEN, which may be an important event in the progress of toxicity of benzene.
Conflict of Interest Disclosure

The authors did not report any conflict of interests.

Acknowledgment

This work was supported by National Natural Science Foundation of China (21677066); Science and Technology Project of Guangdong Province (2014A020212206); Science and Technology Project of Guangzhou (201803030027); Science and Technology Project of Shenzhen city (JCYJ20160428173527959; JCYJ20160427111040009).
Author contribution

Dalin Hu: Conceptualization, Methodology, Supervision, Communication with editor, etc.; Zhenwei Lian, Zuqing Hu, Hongyi Xian, Ran Jiang, Haoyu Huang: Methodology, Investigation, Data Curation, Resources, Writing – Original Draft; R. Stephen Lloyd, Jianhui Yuan, Yan Sha, Sanming Wang : Writing – Review & Editing; Yunxia Jiang,

Zhongdaixi Zheng: Writing Editing.

References:

Abplanalp, W., DeJarnett, N., Riggs, D.W., Conklin, D.J., McCracken, J.P., Srivastava, S., Xie, Z., Rai, S., Bhatnagar, A., O’Toole, T.E., 2017. Benzene exposure is associated with

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Exosomes derived from normal human bronchial epithelial cells down-regulate proliferation and migration of hydroquinone-transformed malignant recipient cells via up-regulating PTEN expression

Zhenwei Lian, Zuqing Hu, Hongyi Xian, Ran Jiang, Haoyu Huang, Yunxia Jiang, Zhongdaixi Zheng,
R. Stephen Lloyd, Jianhui Yuan, Yan Sha, Sanming Wang, Dalin Hu

PII: S0045-6535(19)32736-5
DOI: https://doi.org/10.1016/j.chemosphere.2019.125496
Reference: CHEM 125496

To appear in: Chemosphere

ImageExosome, as nano-sized (30-100 nm) lipid bilayer-enclosed vesicle, plays a key role in inter-cellular communication stimulated by genes or environment (Raposo and Stoorvogel, 2013; Tan et al., 2013; Ruivo et al., 2017). Exosome contains specific bioactive molecules, such as mRNAs, proteins, lipids, and microRNAs, and can exist in various body fluids including blood, saliva, breast milk, and urine (Colombo et al., 2014; Whiteside, 2016; Sun et al., 2017). Exosomes released by different types of cells can be absorbed by adjacent cells or distant organs, and be involved in many important biological events, such as proliferation, immune modulation, apoptosis, tumor metastasis, cardiovascular and infectious diseases (Kahlert and Kalluri, 2013; Schorey and Harding, 2016; Zhang et al., 2018; Zamani et al., 2019). In recent years, the role of exosomes in the pathogenesis of tumors has attracted significant attention due to role in shuttling bioactive molecules between cells and specifically within the tumor microenvironment, they may have a profound biological impact on the occurrence, development and migration of tumors (Wang et al., 2016; Harada et al., 2017).
Phosphatase and tensin homolog on chromosome 10 (PTEN) is a typical tumor suppressor gene and its signal transduction pathway can be regulated by both phosphatase-dependent and -independent mechanisms (Freeman et al., 2003; Hopkins and Parsons, 2014). While its tumor-suppressor activity is mainly attributed to its lipid phosphatase activity (Song et al., 2012; Worby and Dixon, 2014). In the meanning time, it also exhibits protein phosphatase activity and regulates important biological
processes, including inhibition of cell migration and arrest of cell cycle (Putz et al., 2012; Zhu et al., 2015; Brandmaier et al., 2017).
ImageAccumulated evidence suggests that somatic mutations, gene silencing, and epigenetic modification can result in the inactivation and dysfunction of PTEN, which in turn, increases cell proliferation and reduces cell death that are both critical in the progression of tumorigenesis (Lee et al., 2015; Milella et al., 2015; Yu et al., 2018). Inactivation and dysfunction of PTEN in somatic cell are frequently found in a variety of patients with cancer diseases, such as glioblastoma, colon cancer and endometrial cancer (Valeri et al., 2014; Benitez et al., 2017; Zhang et al., 2017). In addition, there is growing evidence that there exists a quantitative association between levels of PTEN gene expression and protein in the progress of various tumors.
Benzene is a high volume industrial compound that is widely used worldwide (Snyder et al., 1993; Wang et al., 2014; Moro et al., 2015) and due to its high volatility, it is also a ubiquitous environmental pollutant (Fishbein, 1984; Abplanalp et al., 2017). While the International Agency for Research on Cancer (IARC) has classified benzene as a known human carcinogen, the fundamental mechanisms underlying its carcinogenesis has not been fully elucidated (Sheets et al., 2004; Snyder, 2012; Warden et al., 2018).
In this study, we evaluated the levels of PTEN in normal human bronchial epithelial cell line (16HBE) and hydroquinone (an active metabolite of benzene) – transformed malignant 16HBE cell line (16HBE-t). At the same time, we isolated exosomes of 16HBE, characterized and co-cultured the exosomes with 16HBE-t, and
assessed the effects of exosomal PTEN on proliferation and migration of recipient 16HBE-t cells. Data suggested that the level of PTEN was significantly decreased in 16HBE-t as compared to 16HBE cells, and exosomes derived from 16HBE down-regulated proliferation and migration of 16HBE-t cells via up-regulating their PTEN expression.
2. Materials and methods

2.1 ImageChemicals

Chemicals and reagents were obtained from the following suppliers, and unless otherwise stated, were used according to manufacturers’ protocols: RMPI 1640 medium (Gibco, USA), fetal bovine serum (FBS, Gibco, USA), phosphate buffer saline (PBS, Meilunbio, China), penicillin and streptomycin (Hyclone, USA), 0.25% EDTA-Trypsin (TE, Solarbio, China), hydroquinone (HQ, Macklin, China), Cell Counting Kit-8 (CCK-8; Dojindo, Japan), Trizol reagent (Invitrogen, USA), qPCR RT Kit (DBI, German), Sybr Green qPCR Master Mix (DBI, German), PKH67 green fluorescence labelling kit (BestBio, China), 4′,6-diamidino-2-phenylindole (DAPI, BestBio, China), phenylmethanesulfonyl fluoride (PMSF, Solarbio, China), bicinchoninic acid (BCA, Beyotime, China), bovine serum albumin (BSA, Biotopped, China), PTEN inhibitor (SF1670, TargetMol, China), low melting pointing agarose (LMPA, Macklin, China), normal melting point agarose (NMPA, Macklin, China), Ethanol (Aladdin, China), 4% paraformaldehyde (Solarbio, China).
2.2 Cell transformation and culture
HQ-transformed malignant 16HBE cell line (16HBE-t) was constructed using the methods as reported previously (Jiang et al., 2019). Cells were cultured in RMPI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10 U mL-1 penicillin and streptomycin at 37 ℃ with 5% CO2. When cells reached 80% confluence adherent, they were incubated with 0.25% EDTA-Trypsin and divided into two culture plates. Culture medium was replaced every other day.
2.3 ImageSoft agarose assay

A total of 100 mL of cell culture medium was collected and centrifuged at 300 × g for 20 min at 4 ℃ to pellet the cells. The supernatant was centrifuged at 120,000 × g for 60 min to pellet the exosomes, that were re-suspended in PBS and filtered through a 0.22 μm filter to remove the particles larger than 200 nm. Samples were centrifuged again at 120,000 × g for 60 min. The PBS was discarded and the exosomes were re-suspended in 150 μL PBS for further experiment.
2.5 Transmission electron microscopic (TEM) analysis
ImageTransmission electron microscopic (TEM) was used to verify the morphological characteristic of exosomes. Exosomes derived from cell culture medium were enriched through ultracentrifugation and re-suspended in 20 μL PBS. A total of 2 μL of the exosome solution were applied to each carbon-coated copper grids and dried for 10 min at room temperature (RT). The prepared samples were negatively stained with 1% phosphotungstic acid (pH 7.0) for 5 min. The characteristic of exosomes were visualized using a transmission electron microscope (JEM-1200EX, Japan) operating at 80 kV.
2.6 16HBE-t cells co-cultured with exosomes

ImageThe levels of PTEN mRNA expressed in different cells were evaluated by using quantitative RT-PCR analysis. Total RNA was extracted by using Trizol (Invitrogen, USA) reagent following the manufacturer’s protocol. The concentration and purification of total RNA were assessed by Nanodrop 2000 machine (Thermo, USA). A total of 500 pg of total RNA was used to synthesize cDNA and qPCR RT Kit was used to perform reverse transcription. Sybr Green qPCR Master Mix was used to conduct PCR amplification. PTEN detection program was as follows: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s. Relative expression levels were calculated by using the comparative threshold cycle (2-ΔΔCT) method, in which GAPDH was used to normalize the level of PTEN. The related primer sequences were as follows:
PTEN Forward 5′-CGGCAGCATCAAATGTTTCAG-3′ PTEN Reverse 5′-AACTGGCAGGTAGAAGGCAACTC-3′ GAPDH Forward: 5′-ACCACGGTGCACGCCATCAC-3′ GAPDH Reverse: 5′-TCCACCACCCTGTTGCTGTA-3′
2.9 Western blot

A total of 1 × 107 cells were collected and lysed in Radio immunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF). The concentration of total protein was measured with bicinchoninic acid (BCA) protein assay. 60 μg protein was subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (0.45 μm; Millipore). After being blocked in
Tris-buffered saline with Tween-20 (TBST) and 3% BSA for 4 h at RT, PVDF membranes were sequentially incubated with rabbit anti-PTEN (1:500 dilution) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenasen (GAPDH, 1:5,000 dilution) overnight at 4 ℃. Membranes were washed with TBST (3 × 10 min), incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution) for 2 h at 37 ℃. Enhanced chemiluminescence was performed for immunodetection analysis.
2.10 ImageCCK-8 analysis on cell proliferation

3.1 16HBE-t cell line construction

ImageImageAfter being treated with 25 μM HQ for about 29 weeks (The 40th generation), 16HBE transformation was achieved. Cells showed a morphological characteristic of anchorage-independent growth (Fig. 1). Transformed 16HBE cells (16HBE-t) formed typical colonies in soft agarose. The colony formation efficiency was about 6.25% (Fig. 2).
Fig. 1 16HBE transformation assay.

A: Normal 16HBE; B – C: The 15th generation of 16HBE (The 15th G-16HBE) and the 30th generation of 16HBE (The 30th G-16HBE) treated with hydroquinone (HQ); D: The morphological characteristic of anchorage – independent growth of transformed cells (the 40th generation of 16HBE cells).

ImageImageFig. 2 Confirmation of HQ-transformed malignant cells with soft agarose assay.

A: 16HBE-t cells formed typical colony in soft agarose medium; B: Colony formation rate of 16HBE cells in transformation assay (* p < 0.05).
3.2 Exosomal morphology under TEM

Exosomes secreted by 16HBE were isolated as previously described with and characterized by using TEM. The electron micrography showed that the exosomes appeared a typical double-layer membrane structure. Through comparison to a 100 nm scale bar, the size range of the exosomes were 60-120 nm (Fig. 3, A).
3.3 Exosomes uptake by 16HBE-t cells
To confirm the ability of 16HBE-t to uptake the exosomes derived from normal 16HBE cells, exosomes were isolated and stained with PKH67 and co-cultured with 16HBE-t cells. After 3 hours’ incubation, a punctuated green fluorescence appeared in the cytoplasm of 16HBE-t cells (Fig. 3, B, C, D).

ImageImageFig. 3 TEM and confocal microscopic morphology of exosomes

A: Exosomal morphology under TEM (Scale bar = 100 nm); B:16HBE-t cell nuclei stained with DAPI (Blue; Scale bar = 28 μm); C: 16HBE exosomes stained with PKH67 (Green; Scale bar
= 28 μm); D: Exosomes uptake by 6HBE-t (Scale bar = 28 μm).

3.4 Exosomes up-regulate PTEN expression of 16HBE-t

qRT-PCR and western blot analyses were used to evaluate the level of PTEN expression, in which the comparative threshold cycle (2-ΔΔCT) method and Gray analysis were separately adopted to assess the level of PTEN mRNA and protein. The results showed that the PTEN mRNA and protein of 16HBE-t cells were significantly decreased as compared to 16HBE cells (p < 0.05). Interestingly, when 16HBE-t cells ingested exosomes derived from 16HBE cells (16HBE-t-Exos), the level of PTEN mRNA and protein were significantly higher than that of 16HBE-t (p < 0.05) (Fig. 4).
Our data suggested that exosomes derived from 16HBE cells up-regulated the PTEN expression of 16HBE-t.

ImageImageFig. 4 The expression level of PTEN in different groups of cells.

a: qRT-PCR analysis on PTEN mRNA (* P < 0.05); b: Western blot analysis on PTEN protein.

ImageImageFig. 5 The proliferation of different groups of cells (* P <0.05).

3.6 16HBE exosomes down-regulate cell migration of 16HBE-t via PTEN pathway
Relative rates of 16HBE-t and 16HBE cell migration were measured using a technique in which after cell plating, a scratch was made on the plate, debris removed and growth into the cell-free area measured. This assay revealed that the migration distance of 16HBE-t cells was significantly increased as compared with 16HBE. Further, the cell migration distance of 16HBE-t-Exos cells was significantly decreased as compared with the group of 16HBE-t cells. Interestingly, the cell migration distance of 16HBE-t-Exos-inhibitor was significantly longer than that of 16HBE-t-Exos. This result indicated that 16HBE exosomes down-regulated the cell migration of 16HBE-t via PTEN pathway (Fig. 6-7).

ImageImageImage

Fig. 6 The microscopic images of scratch in different groups of cells.

A: 16HBE cell migration; B: 16HBE-t cell migration; C: 16HBE-t-Exos cell migration; D:

16HBE-t-Exos-inhibitor cell migration.

Fig.7 Cell migration distance of different groups of cells (* p < 0.05).
4. Discussion

ImageIn the present study, we observed that the expression of PTEN in hydroquinone-transformed 16HBE cells (16HBE-t) was significantly decreased as compared to the normal control group (16HBE). Down-regulation of PTEN is an important biological event in the progress of tumorigenesis, which is frequently occur in various kinds of cancer (Hopkins et al., 2014; Lee et al., 2018), including breast carcinomas, acute myeloid leukemia, glioblastoma, colon, non-small cell lung cancer (NSCLC), endometrial cancers and melanoma (Noguera et al., 2013; Kechagioglou et al., 2014; Perez-Ramirez et al., 2015; Benitez et al., 2017). The frequency of PTEN loss or inactivating mutations ranges from 30% – 40% in sporadic breast carcinomas, 5% – 30% in sporadic colorectal cancers, 30% – 60% in melanomas , 24% – 44% in NSCLC (Marsit et al., 2005), 24% in early NSCLC (Soria et al., 2002) and up to 75% in acute myeloid leukemia (AML) (Cheong et al., 2003). However, at the present time, the possible molecular mechanisms for PTEN alteration is still unknown. It may include, but not be limited to the biological progress of transcriptional regulation, post-transcriptional modification, and protein–protein interactions (Milella et al., 2015).
ImageIt’s well known that cells can communicate with one another following genetic or environmental stimuli, and thus transmit messages by way of transferring of signaling molecules including hormones, cytokines, growth factors and neurotransmitters and transcriptional regulators (Camussi et al., 2010; Catalano et al., 2013; Gonzalez and Medici, 2014; Camacho et al., 2017; Chiodoni et al., 2019). Interestingly, in early 1981, the discovery was made that membrane micro-vesicles exfoliated from normal and cancer cells, exosomes, might have certain physiological functions (Trams et al., 1981). Subsequently, researchers shown that exosomes widely participate in various kinds of biological processes, including immune response, antigen-presenting, cells growth, and cell invasion (Harris et al., 2015; Hock et al., 2017; Barros et al., 2018). Specifically, exosomes can carry and transmit crucial intercellular signal molecules thus, reprograming phenotypes of recipient cells, with the potential to modulate carcinogenic consequences (Quesenberry et al., 2015; Barile and Vassalli, 2017). Our study found that exosomes derived from normal 16HBE cells increased the expression level of PTEN in recipient 16HBE-t cells and down-regulated their proliferation and migration. Furthermore, when exosomal PTEN was blocked by using inhibitor SF1670, recipient 16HBE-t cells exhibited decreased PTEN level, and their proliferation and migration increased correspondingly. Thus, the intercellular communication of exosomal PTEN between normal 16HBE and 16HBE-t cells may be an important biological event in the progress of carcinogenesis of benzene.
PTEN, as a typical tumor suppressor, can regulate signal transduction pathways by both phosphatase-dependent and -independent mechanisms (Leslie et al., 2007). Its
tumor-suppressor activity is mainly attributed to lipid phosphatase activity, i.e., it is mainly involved in the stable maintenance of phosphatidylinositol-3-kinase (PI3K)/Akt cascade (Perez-Ramirez et al., 2015; Lee et al., 2018), thus indirectly inhibits the downstream targets, such as GSK3, FoxO and Bcl-2 that are involved in controlling cell metabolism, proliferation and survival.
5. Conclusion

The authors did not report any conflict of interests.

Acknowledgment

Zhongdaixi Zheng: Writing Editing.
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Image
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

⦁ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
ImageZhenwei Lian, Zuqing Hu, Hongyi Xian, Ran Jiang, Haoyu Huang, Yunxia Jiang, Zhongdaixi Zheng, R. Stephen Lloyd, Jianhui Yuan, Yan Sha, Sanming Wang, Dalin Hu
Highlights:

1) PTEN was significantly decreased in hydroquinone – transformed cells (16HBE-t)
2) Exosomes from normal cells (16HBE) could be ingested by 16HBE-t

3) Exosomes from 16HBE elevated the PTEN level of receipt 16HBE-t

ImagePTEN
4) Exosomes from 16HBE down-regulated proliferation and migration of 16HBE-t via transfer of
ImageJournal Pre-proof

Exosomes derived from normal human bronchial epithelial cells down-regulate proliferation and migration of hydroquinone-transformed malignant recipient cells via up-regulating PTEN expression

Zhenwei Lian, Zuqing Hu, Hongyi Xian, Ran Jiang, Haoyu Huang, Yunxia Jiang, Zhongdaixi Zheng,
R. Stephen Lloyd, Jianhui Yuan, Yan Sha, Sanming Wang, Dalin Hu

PII: S0045-6535(19)32736-5
DOI: https://doi.org/10.1016/j.chemosphere.2019.125496
Reference: CHEM 125496

To appear in: Chemosphere

2.1 ImageChemicals

3.1 16HBE-t cell line construction

ImageImageFig. 2 Confirmation of HQ-transformed malignant cells with soft agarose assay.

A: 16HBE-t cells formed typical colony in soft agarose medium; B: Colony formation rate of 16HBE cells in transformation assay (* p < 0.05).
3.2 Exosomal morphology under TEM

ImageImageFig. 3 TEM and confocal microscopic morphology of exosomes

3.4 Exosomes up-regulate PTEN expression of 16HBE-t

qRT-PCR and western blot analyses were used to evaluate the level of PTEN expression, in which the comparative threshold cycle (2-ΔΔCT) method and Gray analysis were separately adopted to assess the level of PTEN mRNA and protein. The results showed that the PTEN mRNA and protein of 16HBE-t cells were significantly decreased as compared to 16HBE cells (p < 0.05). Interestingly, when 16HBE-t cells ingested exosomes derived from 16HBE cells (16HBE-t-Exos), the level of PTEN mRNA and protein were significantly higher than that of 16HBE-t (p < 0.05) (Fig. 4).
Our data suggested that exosomes derived from 16HBE cells up-regulated the PTEN expression of 16HBE-t.

ImageImageFig. 4 The expression level of PTEN in different groups of cells.

a: qRT-PCR analysis on PTEN mRNA (* P < 0.05); b: Western blot analysis on PTEN protein.

ImageImageFig. 5 The proliferation of different groups of cells (* P <0.05).

ImageImageImage

Fig. 6 The microscopic images of scratch in different groups of cells.

A: 16HBE cell migration; B: 16HBE-t cell migration; C: 16HBE-t-Exos cell migration; D:

16HBE-t-Exos-inhibitor cell migration.

Fig.7 Cell migration distance of different groups of cells (* p < 0.05).
4. Discussion

The authors did not report any conflict of interests.

Acknowledgment

Image
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

1) PTEN was significantly decreased in hydroquinone – transformed cells (16HBE-t)
2) Exosomes from normal cells (16HBE) could be ingested by 16HBE-t

3) Exosomes from 16HBE elevated the PTEN level of receipt 16HBE-t

ImagePTEN
4) Exosomes from 16HBE down-regulated proliferation and migration of 16HBE-t via transfer of
ImageJournal Pre-proof

Exosomes derived from normal human bronchial epithelial cells down-regulate proliferation and migration of hydroquinone-transformed malignant recipient cells via up-regulating PTEN expression

Zhenwei Lian, Zuqing Hu, Hongyi Xian, Ran Jiang, Haoyu Huang, Yunxia Jiang, Zhongdaixi Zheng,
R. Stephen Lloyd, Jianhui Yuan, Yan Sha, Sanming Wang, Dalin Hu

PII: S0045-6535(19)32736-5
DOI: https://doi.org/10.1016/j.chemosphere.2019.125496
Reference: CHEM 125496

To appear in: Chemosphere

2.1 ImageChemicals

CCK-8 analysis was used to detect the effects of 16HBE exosomes on proliferation of transformed 16HBE cells. 2 × 103 cells per well of different groups of cells were seeded onto 96-well culture plates, separately incubated at 37 ℃ with 5% CO2 for 1 to 7 d, followed by addition of 10 μL CCK-8 was added to each well and incubated for 3 h at 37 ℃. Spectrophotometer (BioTek, USA) was used to detect the optical density (OD) values. All of the experimental samples were repeated five times.
2.11 Scratch analysis on cell migrationAfter 1 × 107 cells per well were seeded onto 6-well culture plates and cultured for 24 h, a straight scratch was made on the plates using a pipette tip. Plates were washed three times with PBS to remove cell-debris. The scratch was observed under a microscope at different times (0, 6, 12, 24 h) at the same area. Image J software (National institutes of health, USA) was used to detect the width of the scratch. The cell migration ability was measured by detecting the half-width of the scratch and by subtracting this value from its initial half-width.
2.12 Statistical analysis

Statistical analyses were performed by using SPSS 13.0 (SPSS Inc, USA). Data were showed as mean ± standard deviation. Analysis of variance (ANOVA) and student’s t-test were used to determine the differences between groups. P < 0.05 was defined as statistically significant.
3. Result

3.1 16HBE-t cell line construction

After being treated with 25 μM HQ for about 29 weeks (The 40th generation), 16HBE transformation was achieved. Cells showed a morphological characteristic of anchorage-independent growth (Fig. 1). Transformed 16HBE cells (16HBE-t) formed typical colonies in soft agarose. The colony formation efficiency was about 6.25% (Fig. 2).A: Normal 16HBE; B – C: The 15th generation of 16HBE (The 15th G-16HBE) and the 30th generation of 16HBE (The 30th G-16HBE) treated with hydroquinone (HQ); D: The morphological characteristic of anchorage – independent growth of transformed cells (the 40th generation of 16HBE cells).Confirmation of HQ-transformed malignant cells with soft agarose assay.A: 16HBE-t cells formed typical colony in soft agarose medium; B: Colony formation rate of 16HBE cells in transformation assay (* p < 0.05).

3.2 Exosomal morphology under TEM

Exosomes secreted by 16HBE were isolated as previously described with and characterized by using TEM. The electron micrography showed that the exosomes appeared a typical double-layer membrane structure. Through comparison to a 100 nm scale bar, the size range of the exosomes were 60-120 nm (Fig. 3, A).
3.3 Exosomes uptake by 16HBE-t cellTo confirm the ability of 16HBE-t to uptake the exosomes derived from normal 16HBE cells, exosomes were isolated and stained with PKH67 and co-cultured with 16HBE-t cells. After 3 hours’ incubation, a punctuated green fluorescence appeared in the TEM and confocal microscopic morphology of exosomesA: Exosomal morphology under TEM (Scale bar = 100 nm); B:16HBE-t cell nuclei stained with DAPI (Blue; Scale bar = 28 μm); C: 16HBE exosomes stained with PKH67 (Green; Scale bar= 28 μm); D: Exosomes uptake by 6HBE-t (Scale bar = 28 μm).

3.4 Exosomes up-regulate PTEN expression of 16HBE-t

qRT-PCR and western blot analyses were used to evaluate the level of PTEN expression, in which the comparative threshold cycle (2-ΔΔCT) method and Gray analysis were separately adopted to assess the level of PTEN mRNA and protein. The results showed that the PTEN mRNA and protein of 16HBE-t cells were significantly decreased as compared to 16HBE cells (p < 0.05). Interestingly, when 16HBE-t cells ingested exosomes derived from 16HBE cells (16HBE-t-Exos), the level of PTEN mRNA and protein were significantly higher than that of 16HBE-t (p < 0.05) (Fig. 4).Our data suggested that exosomes derived from 16HBE cells up-regulated the PTEN expression of 16HBE-t. The proliferation of different groups of cells (* P <0.05).

Benzene is an important industrial chemical and widely used in the manufacture of perfume, medicine, gasoline additive, and paint (Snyder et al., 1993; Minciullo et al., 2014; Moro et al., 2015). And is an ubiquitous air contaminate that is asssociated with hematotoxic effects and can causes leukemia (Snyder, 2012; Nourozi et al., 2018).
However, the mechanisms underlying its carcinogenesis remain unclearn the present study, we observed that the expression of PTEN in hydroquinone-transformed 16HBE cells (16HBE-t) was significantly decreased as compared to the normal control group (16HBE). Down-regulation of PTEN is an important biological event in the progress of tumorigenesis, which is frequently occur in various kinds of cancer (Hopkins et al., 2014; Lee et al., 2018), including breast carcinomas, acute myeloid leukemia, glioblastoma, colon, non-small cell lung cancer (NSCLC), endometrial cancers and melanoma (Noguera et al., 2013; Kechagioglou et al., 2014; Perez-Ramirez et al., 2015; Benitez et al., 2017). The frequency of PTEN loss or inactivating mutations ranges from 30% – 40% in sporadic breast carcinomas, 5% – 30% in sporadic colorectal cancers, 30% – 60% in melanomas , 24% – 44% in NSCLC (Marsit et al., 2005), 24% in early NSCLC (Soria et al., 2002) and up to 75% in acute myeloid leukemia (AML) (Cheong et al., 2003). However, at the present time, the possible molecular mechanisms for PTEN alteration is still unknown. It may include, but not be limited to the biological progress of transcriptional regulation, post-transcriptional modification, and protein–protein interactions (Milella et al., 2015).It’s well known that cells can communicate with one another following genetic or environmental stimuli, and thus transmit messages by way of transferring of signaling molecules including hormones, cytokines, growth factors and neurotransmitters and transcriptional regulators (Camussi et al., 2010; Catalano et al., 2013; Gonzalez and Medici, 2014; Camacho et al., 2017; Chiodoni et al., 2019). Interestingly, in early 1981, the discovery was made that membrane micro-vesicles exfoliated from normal and cancer cells, exosomes, might have certain physiological functions (Trams et al., 1981). Subsequently, researchers shown that exosomes widely participate in various kinds of biological processes, including immune response, antigen-presenting, cells growth, and cell invasion (Harris et al., 2015; Hock et al., 2017; Barros et al., 2018). Specifically, exosomes can carry and transmit crucial intercellular signal molecules thus, reprograming phenotypes of recipient cells, with the potential to modulate carcinogenic consequences (Quesenberry et al., 2015; Barile and Vassalli, 2017). Our study found that exosomes derived from normal 16HBE cells increased the expression level of PTEN in recipient 16HBE-t cells and down-regulated their proliferation and migration. Furthermore, when exosomal PTEN was blocked by using inhibitor SF1670, recipient 16HBE-t cells exhibited decreased PTEN level, and their proliferation and migration increased correspondingly. Thus, the intercellular communication of exosomal PTEN between normal 16HBE and 16HBE-t cells may be an important biological event in the progress of carcinogenesis of benzene.

PTEN, as a typical tumor suppressor, can regulate signal transduction pathways by both phosphatase-dependent and -independent mechanisms (Leslie et al., 2007). Itstumor-suppressor activity is mainly attributed to lipid phosphatase activity, i.e., it is mainly involved in the stable maintenance of phosphatidylinositol-3-kinase (PI3K)/Akt cascade (Perez-Ramirez et al., 2015; Lee et al., 2018), thus indirectly inhibits the downstream targets, such as GSK3, FoxO and Bcl-2 that are involved in controlling cell metabolism, proliferation and survival.

5. Conclusion

The authors did not report any conflict of interests.

Acknowledgment

Zhongdaixi Zheng: Writing Editing.
References:
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