Fine particulate matter PM2.5 generated by building demolition increases the malignancy of breast cancer MDA-MB-231 cells

PM 2.5 is associated with increased risk of mortality for a variety of cancers and all subjects, including breast cancer in females, and lung cancer in males. This study investigates the effects of water-extracted PM 2.5 on a triple-negative breast cancer (TNBC) cell line, MDA-MB-231, by sampling suspended particulates around a building demolition site. PM 2.5 particles were obtained using a high-flow TISCH sampler. Being water-soluble, they were extracted from sampled filters using an ultrasonic oscillator and then freeze-dried. The heavy metal components of soluble PM 2.5 particle was analyzed by ICP-MS. Cell viability was evaluated by MTT assay for cells that were exposed to PM 2.5 . Wound healing and transwell cell migration and invasion assays were used to measure cell motility and the invasiveness of cancer cells that had been exposed to PM 2.5 into a chemo-attractant substance. Possible mechanisms of cancer malignancy were analyzed by Western blot analysis. The results revealed that nearby 2.5 concentrations increased significantly during the deconstruction and the Cd, Cu, Pb, Zn and Cr contents of soluble PM 2.5 also significantly increased. Following exposure to , the survival rate of cancer was significantly higher than that the control group. Soluble PM 2.5 -treated cells also had a higher migration capacity, as determined by wound healing and transwell migration The signaling FAK/PI3K/AKT was more activated in PM 2.5 -treated cells than the control group. The data show that increased levels of Aurora B and Bcl-2 were associated with cell proliferation. Elevated levels of cathepsins D, β-catenin, N-cadherin, vimentin and MMP-9 were associated with breast cancer cell metastasis Conclusion Soluble PM 2.5 that is generated in building demolition may have a role in the promotion/progression of surviving in TNBC cells, increasing the malignancy of breast cancer. The prevention of environmental PM 2.5 from deconstruction is strongly recommended. a To identify the underlying molecular mechanism by which PM 2.5 acts on TNBC tumor cell malignancies, concentrations of PM 2.5 were measured during building demolition following the collection of smaller particles than PM 2.5 as exposure sources. In this investigation, invasive MDA-MB-231 cells were treated with water-soluble extracted PM 2.5 . PM 2.5 -induced cancer characteristics were studied by cell viability and migration assays. The results demonstrated the carcinogenic potential of PM 2.5 particles in building demolition environments to exacerbate the progression of tumor cells. These findings can improve our understanding of the need for optimal air quality management during building demolition to prevent cancer cell malignancy.


Introduction
The demolition of a building can produce large amounts of particulate matter (PM), usually seriously degrading ambient air quality in implosion areas (Beck et al. 2003). An extremely high concentration of PM that is generated during the demolition of buildings may be inhaled by field workers and people who live nearby (Farhad Azarmi 2016). The assessment of possible pathogenicity under such environmental exposure to specific substances with aerodynamic diameters of less than 2.5 µm (PM 2.5 ) remains an important issue. Studies of the impact of PM 2.5 at real-world demolition sites, especially on breast cancer, are still very limited.
The collapse of the New York World Trade Center (WTC) Twin Towers on September 11, 2001, led to an estimated release of 10 million tons of material, exposing more than 300,000 rescue workers and New York City (NYC) residents and local workers to WTC particulate matter (Aldrich et al., 2010;Claudio, 2001;Landrigan, 2001). After the collapse of the WTC building, many neighboring places were stuck in the initial 4 dust/smoke cloud (4 to 8 h) and Lower Manhattan was briefly exposed to PM 2.5 levels in air in the mg/m 3 (thousands of µg/m 3 ) range (Lippmann et al., 2015). The toxicological and physical properties of WTC-PM have been described elsewhere (Lioy et al., 2002;McGee et al., 2003). WTC-PM comprised mostly powered concrete, plastics and other hydrocarbons.
WTC-PM was found to be highly alkaline, with pH 9-11 (Lioy et al., 2006;McGee et al., 2003). Exposure to fine (PM 2.5 ) and coarse (PM 53 , > 53 microns) PM has been associated with the development of lung injuries and high sensitive immune responses (Rom et al., 2002;Weiden et al. 2015).
Evidence that causally links air pollution to breast cancer risk remains controversial. A recent study addressed increased breast cancer risk that is associated with environmental air pollutants, including nitrogen dioxide (NO 2 (Tagliabue et al., 2016). Mammographic breast density is a well-established strong risk indicator for breast cancer and women are at higher risk of developing breast cancer because they are exposed to a higher mean of PM 2.5 concentration (Yaghjyan et al., 2017). However, some works have found no significant correlation between breast cancer and PM 2.5 (Andersen et al., 2017;Hart et al., 2016;Reding et al., 2015). Interestingly, women who are estrogen receptor-positive (ER+) may develop breast cancer upon prolonged exposure to a xenoestrogenic compound, leading to the tumorigenesis of mammary epithelial cells (Huo et al., 2013). Positive correlations exist between exposure to environmental estrogenexpelling agents and hormone receptor-positive breast cancer risk, and between levels of cadmium compounds to which a person is exposed and risk of hormone receptor-negative 5 tumors (Liu et al., 2015). Approximately 15% of all invasive breast cancers are triplenegative breast cancers (TNBC) that lack estrogen receptor (ER), progesterone receptor (PR), and HER2 (human epidermal growth factor receptor 2) expression, and exhibit a distinct pattern of recurrence with unfavorable outcomes (Dent et al., 2007). To identify the underlying molecular mechanism by which PM 2.5 acts on TNBC tumor cell malignancies, concentrations of PM 2.5 were measured during building demolition following the collection of smaller particles than PM 2.5 as exposure sources. In this investigation, invasive MDA-MB-231 cells were treated with water-soluble extracted PM 2.5 . PM 2.5 -induced cancer characteristics were studied by cell viability and migration assays. The results demonstrated the carcinogenic potential of PM 2.5 particles in building demolition environments to exacerbate the progression of tumor cells. These findings can improve our understanding of the need for optimal air quality management during building demolition to prevent cancer cell malignancy.

Materials And Methods
Collection of the PM 2 . 5 that is generated by building demolition Airborne particles were obtained using a TISCH high-flow sampler (TE-6070) and a highvolume cascade impactor (TE-231, Tisch Environmental, Cleves, Ohio. USA). Suspended particulates enter the cascade impactor through the first set of parallel slots in the first stage. Particulates with high inertial force that are too large to pass to the next stage are impacted on the quartz fiber filter (Pall, USA) and the smaller particles remain in the air stream and travel to the next stage. The slots become successively smaller and most of the particulates eventually become impacted on one of the collection stages in the filter.
Beyond the last stage, the smallest particles will be collected on the backup filter, which will be weighed to determine PM content. The filter was dried at 50 o C for 24 h and then 6 incubated in a humidifier for 24 h. PM 10 − 2.5 (< 10 − 2.5 µm) and PM 2.5 (< 2.5 µm) were Preparation of water-soluble PM 2 . 5 extracts The sampled PM 2.5 filter was weighed; cut into small pieces, and then transferred into a 50 ml tube that contained enough double-distilled water for a 30 min sonication. The PM 2.5 suspension was centrifuged at 13,000 x g for 10 min at 4 o C and filtered using a 0.22 µm syringe filter. To obtain concentrated PM, the filtered suspension was dried in a vacuum dryer (VIRTIS) at 50 o C until completely dry. A total of 101.556 mg of PM 2.5 was estimated for initial sonication and 27.368 mg of PM 2.5 was recovered and dissolved in 100 mL double-distilled water to perform an in vitro assay. The control for the assay was prepared from a blank quartz fiber filter that was went through all the steps of extraction except for exposure to PM. Wound healing assay MDA-MB-231 cells were treated with 600 µg/mL of PM 2.5 or a control for 24 h and collected as described above. Cells were counted and adjusted to a concentration of 3 × 10 5 cells/mL. The culture-insert (ibidi 80206, Martinsried, Germany) was loaded into a 60 mm dish; then 100 µL of the prepared cells was added to both chambers to yield a total of 3 × 10 4 cells. The dish was maintained at 37 °C, 5% CO 2 for 24 h and then the culture-insert was carefully removed. The chambers were then rinsed using PBS solution. The first 8 photograph was taken as 0 h and 1% FBS in fresh medium (4 mL) was added to induce would healing. After 16 h of incubation, the medium was removed and rinsed in PBS, and then the second photograph was taken. Microscopic images of a representative field of the cell-free space were obtained at 0 and 16 h, and the numbers of cells were calculated in ImageJ software (Java 1.8.0_112, imagej.nih.gov).
Transwell migration assay MDA-MB-231 cells migration was characterized using a transwell migration assay with 24well hanging-inserts that were fitted with an 8 µm-pore-size membrane (Millicell Cell Culture Inserts Category No. MCEP24H48). A total of 5 × 10 4 serum free cells (200 µL) were seeded in triplicate in culture medium onto the apical surface of each hanging-insert and placed into wells that contained 10% FBS in culture medium (500 µL). The plate was incubated for 16 h and the lower surface of the insert was fixed with 100% methanol and stained with 0.4% crystal violet for 15 min. Non-migrating cells were removed from the upper surface using a cotton stick and the migrated cells were counted.

Western blot analysis
Proteins (20 ∝g) that had been separated by SDS-PAGE were transferred onto an Immobilon-P membrane that was then subjected to western blotting using a suitable primary antibody against human FAK (Gene Tex), p-FAK(Y925), p38, p-p38, ERK1/2, similarly been negatively correlated with PM 2.5 level. During the demolition process, the concentration of PM 2.5 at the collection point significantly exceeded that in the surrounding area ( Fig. 2A). Fourteen months later, PM 2.5 concentrations in the air at the collection point were normally lower than in the local surrounding area (Fig. 2B).
To assess the effect of PM 2.5 on the progression of breast cancer cells, MDA-MB-231 breast cancer cells were treated with water-extracted PM 2.5 for 48 h and analyzed using an MTT survival assay (Fig. 3A). The higher PM 2.5 concentrations resulted in a tumor cell viability of double that of the control group. The proliferation capacity of breast cancer cells was examined using a colony formation assay. After 24 h of exposure to 600 µg/mL PM 2.5 , the number of colonies of the PM 2.5 -treated breast cancer cells significantly exceeded that in the control group after seven days of incubation (Fig. 3B). These data provide strong evidence that water-extracted PM 2. Growing evidence shows that prolonged exposure to heavy metals is associated with a poor prognosis of cancer. The effects of MAPK and PI3K/AKT signaling pathways on heavy metal-induced carcinogenesis in tumors in the lung have been reported (Ohgami et al., 2015). Accordingly, metal species in water-soluble PM 2.5 that promote the development of cancer cells by activating the FAK/PI3K/AKT pathway, and thereby, enhance cell proliferation, migration, and metastasis. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that the amounts of the five heavy metals, cadmium (Cd), copper (Cu), lead (Pb), zinc (Zn) and chromium (Cr) were higher in the water-soluble PM 2.5 extracts that were generated by building demolition than in the post-control sample from the surrounding area ( Table 2). The data on the heavy metals were normalized using the environmental factors that are listed in Table 3. Interestingly, our hypothesis was supported by observations that increased concentrations of Cd and Cu in the water-soluble

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All data generated or analyzed during this study are included in this published article.