Auramine O, an incense smoke ingredient, promotes lung cancer malignancy

Abstract Burning incense to worship deities is a popular religious ritual in large parts of Asia, and is a popular custom affecting more than 1.5 billion adherents. Due to incomplete combustion, burning incense has been well recognized to generate airborne hazards to human health. However, the correlation between burning incense and lung cancer in epidemiological studies remains controversy. Therefore, we speculated that some unknown materials in incense smoke are involved in the initiation or progression of lung cancer. Based on this hypothesis, we identified a major compound auramine O (AuO) from the water‐soluble fraction of incense burned condensate using mass spectrometry. AuO is commonly used in incense manufacture as a colorant. Due to thermostable, AuO released from burned incenses becomes an unexpected air pollutant. AuO is classified as a Group 2B chemical by the International Agency of Research on Cancer (IARC), however, the damage of AuO to the respiratory system remains elusive. Our study revealed that AuO has no apparent effect on malignant transformation; but, it dramatically promotes lung cancer malignancy. AuO accumulates in the nucleus and induces the autophagy activity in lung tumor cells. AuO significantly enhances migration and invasive abilities and the in vitro and in vivo stemness features of lung tumor cells through activating the expression of aldehyde dehydrogenase family 1 member A1 (ALDH1A1), and ALDH1A1 knockdown attenuates AuO‐induced autophagy activity and blocks AuO‐induced lung tumor malignancy. In conclusion, we found that AuO, an ingredient of incense smoke, significantly increases the metastatic abilities and stemness characters of lung tumor cells through the activation of ALDH1A1, which is known to be associated with poor outcome and progression of lung cancer. For public health, reducing or avoiding the use of AuO in incense is recommended.

Water-soluble fraction in incense burned condensate (IBC) has long been ignored because organic matters are decomposed by combustion into small chemical compounds, most of which are organic-soluble or volatile such as PMs, PAHs, and VOCs. Whether water-soluble materials exist in incense smoke or they are correlated with cancer progression/malignancy is fully unknown. In this study, we utilized mass spectrometry (MS) to identify the water-soluble materials of IBC and found auramine O (AuO) is the major compound in them.
AuO (bis [4-(dimethylamino)phenyl] methaniminium chloride), a yellow dye, is commonly used in incense manufacture as a colorant. AuO has a high melting point of 2678C and thermostability, which results in the existence of AuO in incense smoke as an unexpected air pollutant.
Oral administration of high-dose AuO has been shown to induce liver tumors in both mice and rats 14 and cause genotoxicity in primary rat and human hepatocytes, 15

and the International Agency of
Research on Cancer (IARC) classified AuO into Group 2B as a possible carcinogen to humans. 16 To date, the exposure and damage of AuO to the respiratory system has never been recognized. Therefore, we investigated the carcinogenic potential of AuO for the initiation and progression of lung tumor. In this study, we found that AuO undergoes nuclear translocation and induces autophagy activity in lung tumor cells. AuO has no apparent effect on inducing lung tumor initiation.
However, AuO significantly increases cell migration and invasive abilities through activating the stemness of tumor cells, which finally leads to tumor malignancy.

| Incense and incense smoke collection
Incense sticks including Chen-Shiang (CS; agarwood) and Tan-Shiang (TS; sandalwood) were purchased from vendors surrounding Dajia Jenn Lann Temple (Taichung, Taiwan) and local incense manufacture (Taichung, Taiwan). Incense sticks (three CS and three TS) collected from vendors were used for the estimation of AuO content, whereas those (two CS and two TS) collected from local incense manufacture were used as reference because their AuO content was no detection.
The sampling device and sampling method have been described in an earlier publication. 17 Personal environmental monitor (SKC 761-203) with a size cut of 2.5 lm mounted with 37 mm Teflon filter (SKC 225-1709) was used for smoke particle sampling. After 5 min collection, the filters were cut into small pieces and extracted by 20 mL ddH 2 O. The extracted solution was lyophilized and resolved with assigned volume of ddH 2 O.

| Mass spectrometry
Materials extracted from IBC were assayed by Apex Qe FT-ICR MS equipped with a 9.4 T actively shielded magnet (Bruker Daltonics). The analysis was set in positive ion mode using an Apollo II electrospray source. The MS scan was set m/z 100 to 1000 with a mass resolution of 66,000 at m/z 400. The spectra were processed using Data

| Molecular modeling
The molecular modeling was performed to predict the binding affinity of AuO to DNA using Autodock software, and the similar structures of AuO were analyzed using Autodock 4.2 software with Lamarckian Genetic Algorithm. 20 The crystal structure of DNA was obtained from the protein data bank (PDB ID: 1Z3F) (http://www.rcsb.org/pdb/ home/home.do). 21 The substrates including ligands, water, and small molecules were removed from co-crystallized DNA. Polar hydrogens and Kallman united atom charges were added to the DNA for docking calculation using Autodock Tool 1.5.6 interfaces (ADT). 22,23 The optimization of AuO and its similar compounds were performed by using MMFF94 force field by ChemBio3D software (version 11.0; Cambridge Soft Corp.). Hydrogens and Gasteiger charges were added to the AuO for docking. 22 The Grid box calculated by AutoGrid program was centered at the binding site of co-crystal ligand of DNA with dimensions 68 3 64 3 44 Å grid points at spacing of 0.375Å. All docking parameters were set to default except for the following parameter: maximum number of energy evaluation increase to 25,000,000 per run.
The docking results were analyzed by using cluster analysis and were shown by ADT. The models of docking results were shown by Discovery Studio Visualizer 4.5 (Accelrys).

| Isothermal titration calorimetry
The energetic process of AuO-dsDNA association was measured by the isothermal titration calorimetry (ITC, TA Instrument). The titration of dsDNA (five GC pairs of DNA) with AuO was performed by adding AuO from a rotating syringe in 20 injections with an interval of 310 s.
All solutions were appropriately degassed to avoid the interference of bubble formation during the process of titration. The measurement in phosphate buffer was used as the background control to correct for the heat of dilution and deconvoluted on NanoAnalyze software (version 2.4.1) according to a single site binding model.

| Protein extraction and tryptic peptide preparation
Total proteins were extracted using RIPA lysis and extraction buffer (Thermo Fisher) and protein quantification was determined using the Bio-Rad Protein Assay by the measurement of absorbance at 595 nm.
Total 20 lg of protein was separated using 10% SDS-PAGE and divided into 10 gel fractions, which were then cut into small gel pieces

| Western blot analysis
After SDS-PAGE separation, proteins were transferred onto a PVDF membrane using electroblot at 400V at 48C for 3 h in 25 mmol/L Tris-HCl, 197 mmol/L glycine, and 13.3% (v/v) methanol. Membranes were blocked with 5% (w/v) skim milk in TBST for 1 h, and then incubated with primary antibodies by a gentle shaking at room temperature for overnight. After gently agitating in three TBST washes and one TBS wash for 15 min each, horseradish peroxidase (HRP)-conjugated secondary antibody was added to further incubate at RT for 1 h. Immunoreactive signals were revealed using an enhanced chemiluminescence substrate kit (NEN Life Science), which includes luminescent substance luminol that is oxidized to luminesce by HRP using H 2 O 2 as an oxidizing agent and enhancers that increases the light intensity, and the signals were recorded by developing photographic film under optimum exposure.

| Wound healing and matrigel invasion assay
The wound healing assay was used to study the migration ability of tumor cell in vitro. Tumor cells (70 mL; concentration: 3 3 10 5 cells/ mL) were added to Culture-Insert well (ibidi) and cultured for 24 h.
A "wound gap" was created by removal of Culture-Insert, and the "healing" of this gap by cell migration was recorded per 4 h until 24 h.
The migration area of tumor cells was measured using Image J software. The matrigel invasion assay was used to determine the inva- DMEM medium supplemented with 10% FBS was added as a chemoattractant to the lower half. After incubation at 378C for 24 h, tumor cells that passed through the insert were fixed with 3% formalin (Sigma-Aldrich) and stained with 0.2% crystal violet (Sigma-Aldrich).

| Spheroid formation assay
Spheroid formation assay was performed in 6 cm culture dish coated with 1% agarose. Tumor cells suspended in serum free medium were seeded at a density 5000 cells/dish and incubated in a humidified atmosphere of 5% CO 2 and 95% air at 378C for 7 days. The numbers of spheroid were counted manually at 7th day under microscope.

| Mouse model of subcutaneous inoculation
The animal procedure (2016-047) was approved by the Institutional Animal Care and Use Committee (IACUC) at CMUH. Lung cancer cell A549 was pretreated with 1 lM AuO for 7 days. Both AuO-treated and control A549 cells (1 3 10 6 ) were mixed with matrigel and injected into 5-week-old male BALB/c nude mice (purchased from BioLASCO, Taiwan). Left flank was injected with control, whereas right flank was injected with AuO-treated A549 cell. Tumor size was measured by caliper weekly for 6 weeks once tumors became visible. Tumor volume was calculated with the formula: (length 3 width 2 )/2. After animal sacrifice, tissue samples were fixed in formalin and embedded with paraffin, and then analyzed using immunohistochemical (IHC) staining. The IHC procedure followed our previously described method. 24 Briefly, rabbit anti-human ALDH1A1 antibody (#12035; Cell Signaling) was used to perform IHC staining by horseradish peroxidase-conjugated avidin-biotin complex from the Vectastain Elite ABC Kit (Vector Laboratories) and AEC chromogen (Vector Laboratories).

| Statistical analysis
Data are expressed as means 6 SD. The significance of difference was examined by Student's t-test (two-tailed). P < .05 was considered to be significant. To quantify the content of AuO in IBC and incense, we used AuO standard compound to construct a calibration curve using a Velos Pro MS (Thermo Fisher) coupled with a nano-HPLC system. The linear relationship for AuO quantity and peak-area intensity is calculated as  Table 1).

Confocal images showed that an increase of LC3 puncta is observed in
AuO-treated A549 cells in comparison to the control cells ( Figure 4B). Chloroquine (CQ) treatment is used as positive control because CQ inhibits both fusion of autophagosome with lysosome and lysosomal protein degradation, which leads to LC3B accumulation. 27 In addition, significantly higher levels of LC3B II/I ratio (a definitive autophagy biomarker) were observed in A549 cells within 5-day AuO treatment (Figure 4D). However, AuO does not have an obvious effect on enhancing autophagy activity in bronchial epithelial cell BEAS-2B ( Figure 4B,C).
These results are consistent with the confocal observations. Collectively, our data demonstrate that AuO exposure increases autophagy activity of lung cancer cell.

| AuO promotes lung cancer malignancy
Next, we determined whether AuO promotes lung cancer malignancy.
Two human lung cancer cell lines A549 and CL1-0 were used to examine their metastatic abilities after AuO treatment. Various doses of AuO were tested and 1 mM AuO was used for the following treatment because this dose shows a limited inhibition to cancer viability ( Figure 6A). After 7 culture days, both cancer cells increase their migration and invasive abilities ( Figure 6B,C). Moreover, AuO enhances the spheroid formation of lung cancer cells in number and size ( Figure 6D), suggesting that AuO could increase the stemness of lung tumor cells. These data indicate that AuO significantly promotes lung cancer malignancy.

| AuO increases the metastatic abilities of lung tumor cells through activating ALDH1A1 expression
To investigate the molecular effect of AuO on lung cancer malignancy, a comparative proteomic analysis was performed to determine the protein  Table 1. Of these, we noticed that aldehyde dehydrogenase family 1 member A1 (ALDH1A1), which has been characterized as a potential cancer stem cell (CSC) marker of lung cancer and its expression is associated with poor outcome and progression of lung cancer. 28,29 Western blot validation showed that AuO induces ALDH1A1 expression in a dose-dependent manner (Figure 7A). Although other CSC-related markers such as CD133 and CD44 were not included in the list, they also showed an increase of protein levels under AuO treatment in western blot assay ( Figure 7A). To evaluate the efficiency of AuO in ALDH1A1 induction, the shortest time and minimum dose of AuO treatment were determined in A549 cells. As presented in Figure 7B,C, AuO is able to induce ALDH1A1 at the minimum dose of 5 nM within 1 day, which is an achievable dose for people under daily exposure based on our previous estimation in temple workers.
To address whether AuO increases lung cancer metastatic abilities through ALDH1A1 activation, small hairpin RNAs (shRNAs) were used to knock down ALDH1A1 expression in A549 cells. Western blot confirmed that two shRNAs inhibit AuO-induced ALDH1A1 expression ( Figure 7D) and significantly abolish AuO-induced migration and invasive abilities in A549 cells ( Figure 7E,F). Moreover, because autophagy has been shown to be involved in modulating tumor cell motility and invasion, 30 we checked the effect of ALDH1A1 knockdown on AuOinduced autophagy activity. Compared with the data in Figure 4D, ALDH1A1 knockdown attenuates AuO-induced autophagy activity ( Figure 7G). These results suggest that ALDH1A1 plays an important role in mediating AuO-induced lung cancer metastasis.
FIG URE 6 AuO promotes lung cancer malignancy. A, A549 and CL1-0 cells were treated with various doses of AuO for 1 day and the cell viability was determined by MTT assay. After pretreated with 1 lM AuO for 7 days, AuO-treated and the control A549 and CL1-0 cells were used to perform the following analyses. B, Wound healing assay. C, Transwell assay. D, Spheroid formation assay. Experiments were performed in triplicate (mean 6 SD). *P < .05; **P < .01 [Color figure can be viewed at wileyonlinelibrary.com]

| D I SCUSSION
AuO is a multifunctional dye used in the coloring of paper, textile, leather, incense as well as acid-fast bacteria such as Mycobacterium tuberculosis. 31 Due to low price, high intensive color, and excellent stability, AuO has been found to extensively add in foodstuff and pharmaceuticals as an illegal additive. The abuse of AuO is regarded as an important public health issue because of its carcinogenic potential. 15 Therefore, AuO is forbidden in food matrices by many international food regulation acts, including European Union, 32 China, 33 Japan, 34 and the United States. 35  to predict the potential G-quadruplex sequence, we indeed find G-quadruplex sequences located at the promoter of ALDH1A1. However, the precise mechanism remains to be investigated.
Autophagy is an important mechanism used to clear pathogenic organism and deal with environmental stress such as starvation for maintaining cellular homeostasis. During the transformation of normal cells, autophagy protects genomic stability and inhibits the formation of chronic inflammatory microenvironment, thus preventing tumor generation. 47 Autophagy has a complex and context-dependent role in tumor cells, that is, autophagy suppresses primary tumor growth but is required for tumor maintenance and progression to advanced disease. 48 Additionally, recent investigations have suggested that autophagy promotes multiple steps in the metastatic cascade. 30 In this study, we find that AuO induces autophagy activity ( CSC defined as a subset of tumor cells with self-renewability involves in cancer initiation and progression. CSC is also associated with the resistance to radiation and chemotherapy as well as the metastasis of carcinoma, 49,50 thus increased CSC population has been viewed as a poor prognosis indicator of cancer patients. 51 ALDH1A1 overexpression has been known to associate with lung cancer malignancy. 29 Our study reveals that ALDH1A1 plays an important role in mediating AuO-induced stemness features and metastatic abilities of lung tumor cells, whereas ALDH1A1 knockdown significantly blocks these effects. In clinic, ALDH1A1 overexpression might be relevant to AuO exposure, especially in Asian countries with the custom of burning incense. Their correlation could be addressed clearly through epidemiological study in the future. In recent years, accumulating efforts have been put to develop drugs for targeting CSC. 52 Since ALDH1A1 is crucial for AuO-induced lung malignancy, ALDH1A1 targeting therapy could be benefit to the patients suffering from AuO exposure. 53,54 In conclusion, we identify AuO released from incense smoke as an unexpected air pollutant due to its thermostability. The damage of AuO to the respiratory system has never been awakened. In this study, we find that AuO promotes lung cancer malignancy through increasing autophagy activity and the stemness of lung cancer cells. AuO is currently used as an additive colorant in incense manufacture, to some degree meaning the control of its usage could be accessible. For public health, reducing or avoiding the use of AuO in incense is recommended.