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Garcinol from Garcinia indica Downregulates Cancer Stem-like Cell Biomarker ALDH1A1 in Nonsmall Cell Lung Cancer A549 Cells through DDIT3 Activation Jinhan Wang,†,‡ Liwen Wang,‡ Chi-Tang Ho,§ Kunsheng Zhang,‡ Qiang Liu,*,† and Hui Zhao*,‡ †

Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China ‡ Tianjin Key Laboratory of Food and Biotechnology, School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China § Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States S Supporting Information *

ABSTRACT: Nonsmall cell lung cancer (NSCLC) is the predominant type of lung cancer. Patients with NSCLC show high mortality rates because of failure to clean up cancer stem cells (CSCs). The anticancer activity of phytochemical garcinol has been identified in various cancer cell models. However, the effect of garcinol on NSCLC cell lines is still lacking. Of the NSCLC cell lines we tested, A549 cells were the most sensitive to garcinol. Interestingly, Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) was preferentially expressed in A549 cells and downregulated by the addition of garcinol. We also found that garcinol enriched DNA damage-inducible transcript 3 (DDIT3) and then altered DDIT3-CCAAT-enhancer-binding proteins beta (C/EBPβ) interaction resulting in a decreased binding of C/EBPβ to the endogenous ALDH1A1 promoter. Furthermore, garcinol’s inhibition of ALDH1A1 was identified in a xenograft mice model. Garcinol repressed ALDH1A1 transcription in A549 cells through alterations in the interaction between DDIT3 and C/EBPβ. Garcinol could be a potential dietary phytochemical candidate for NSCLCs patients whose tumors harbored high ALDH1A1 expression. KEYWORDS: garcinol, ALDH1A1, A549 cells, phytochemical, nonsmall cell lung cancer

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INTRODUCTION Lung cancer is one of the primary causes of cancer-related deaths in China, as well as worldwide. Among these lung cancer cases, approximately 85% is nonsmall cell lung cancer (NSCLC).1,2 Mortality rates in NSCLC patients remain high, despite the progress of novel targeted agents and chemotherapeutic regimens. These high mortality rates are attributed to residual lung cancer cells, referred to as cancer stem cells (CSCs), which possess similar properties as stem cells. For example, these cells can exist in quiescent stages, have the ability to self-renew, and are typically resistant to chemotherapeutics and radiotherapy, thus resulting in treatment failure.3 In recent years, increasing number of studies have aimed at discovering dietary phytochemicals for the prevention and targeting of cancer stem-like cells. The interest in this area has been raised due to the limited efficacy and various complications associated with conventional chemotherapy and radiotherapy.4 Indeed, natural phytochemicals, such as resveratrol, lycopene, and sulforaphane, possess not only a broad safety profile but also target multiple key signaling pathways for the treatment of heterogeneous populations of cancer cells, including CSCs.5 Furthermore, epidemiological and clinical studies have provided evidence that phytochemicals may reduce the risk of cancer and benefit patients.6−9 For example, the Mediterranean diet, which is rich in natural polyphenols, has been demonstrated to lower breast cancer incidence for people of Mediterranean descent.6 © 2017 American Chemical Society

In addition to the examples of natural phytochemicals described above, garcinol (C38H50O6, Figure 1A) is a polyisoprenylated benzophenone extracted from the fruit of Garcinia indica,10 which has been demonstrated to inhibit the growth of various types of cancer, including pancreatic, colon, breast, leukemia, and prostate cancer. It is rationalized that garcinol functions as an anticarcinogenic molecule through the mechanisms such as antioxidation, anti-inflammation, antiangiogenesis, and pro-apoptosis.11 As mentioned above, cancer stem cell-like cells are known to confer multidrug resistance. Interestingly, recent studies have indicated that garcinol is able to sensitize cancer cells to chemotherapeutic treatment. For instance, garcinol has been shown to sensitize human head and neck carcinoma cells to cisplatin treatment12 and human pancreatic adenocarcinoma cells to gemcitabine treatment.13 Thus, garcinol could act as a promising compound for targeting cancer stem cell-like cells. In the present study, we investigated the sensitivity to garcinol in a panel of NSCLC cell lines. Interestingly, A549 cells were found to be the most significantly sensitive to garcinol compared to any other NSCLC cell line used in this study. Furthermore, we noticed that A549 cells harbored higher expression levels of ALDH1A1, a putative stem cell marker for Received: Revised: Accepted: Published: 3675

January 24, 2017 April 18, 2017 April 18, 2017 April 18, 2017 DOI: 10.1021/acs.jafc.7b00346 J. Agric. Food Chem. 2017, 65, 3675−3683

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Journal of Agricultural and Food Chemistry

quantitatively analyzed depending on sequentially obtained images of migrating cells. RNA Isolation and Quantitative Real-Time PCR (qPCR). Quantitative PCR was carried out as reported previously19 to test the levels of ALDH1A1 and DDIT3 mRNA. Gene expression levels were analyzed following normalization to the gene GAPDH using the ΔΔCT method. Gene expression levels were expressed as relative mRNA levels compared to the internal control. The specific primer pairs used were: ALDH1A1, 5′-TGGCATGATTCAGTGAGTGG-3′ (forward) and 5′-ATAATAGTCGCCCCCTCTCG-3′ (reverse); DDIT3, 5′-TGCTTCTCTGGCTTGGCTGAC-3′ (forward) and 5′CTGGTTCTCCCTTGGTCTTCC-3′ (reverse); GAPDH, 5′CTGGTAAAGTGGATATTGTTGCCAT-3′ (forward) and 5′TGGAATCATATTGGAACATGTAAACC-3′ (reverse). Immunoprecipitation (IP) and Western Blot (WB) Analysis. According to previously described, cells were lysed in Nonidet P-40 lysis buffer.20 IP of soluble protein was carried out using anti-C/EBPβ (Ab15050, Ab32358) or anti-DDIT3 (2895P) antibodies. Protein samples were analyzed by immunoblot using the following specific primary antibodies: DDIT3 (15204-1-AP) and ALDH1A1 (60171-1lg) were purchased from Proteintech (Chicago, IL, USA); C/EBPβ (Ab15050, Ab35328) antibody was purchased from Abcam; DDIT3 (2895P) was purchased from Cell Signaling Technology (Boston, MA, USA). The antigen−antibody complexes were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Detection of ALDH Activity. For the evaluation of the express levels of ALDH1A1 in A549 cells exposed to garcinol, ALDEFLUOR (Stemcell Technologies, Vancouver BC, CA) staining was measured using the FITC channel of fluorescent activated cell sorting according to the manufacturing instruction. To mark the base-fluorescence, pretreatment with the ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used as a negative control. Chromatin Immunoprecipitation (ChIP) Assays. ChIP assays were performed according to the manufacturer’s protocol (P2078, Beyotime Co. Jiangsu, China) with minor modifications. Soluble chromatin was prepared as described20 and precipitated using anti-C/ EBPβ (Ab15050) or a control IgG antibody. The SYBR green-based real-time ChIP-qPCR was carried out with the BioRad CFX-96 RealTime PCR Detector System instrument (Bio-Rad Laboratories, Hercules, CA). The primers used for qPCR of the ALDH1A1 promoter20 and “off target” control. A relative fold enrichment was analyzed as described.21 The amplified PCR products were run on a 1% agarose gel and stained by ethidium bromide. Immunofluorescence. Briefly, cells were fixed and permeabilized in paraformaldehyde/methanol. Cells were then blocked with PBS containing 1% BSA for 1 h at room temperature to prevent nonspecific binding, followed by staining with primary antibodies specific for DDIT3 (2895P, CST) and C/EBPβ (Ab32358, Abcam). Cells were then incubated with secondary antibodies: antimouse IgG-FITC (SA00003-1, Proteintech, Chicago, IL, USA) and antirabbit IgG-Cy3 (SA00009-1, Proteintech, Chicago, IL, USA). Nuclei were stained with DAPI. Images were captured using the EVOSfl fluorescent microscope (Thermo Fisher Scientific, Waltham, MA, USA) with a DAPI filter, GFP filter, and RFP filter. RNA Interference. DDIT3 targeting siRNA (DDIT3 target sequences: GGCTCAAGCAGGAAATCGA) and negative control siRNA were purchased from Gene-Pharma (Shanghai, China) and transfected with lipofectamine RNAiMAX reagent (Invitrogen, Grand Island, NY, USA) according to kit protocol. After 48 h transfection, cells were exposed to the indicated concentration of garcinol for 24 h and harvested for RNA extraction and the ALDH activity assay. Xenograft Tumor Models. A549 cells (2 × 107 in 200 μL of PBS) were injected subcutaneously into the right flanks of 5-week-old NMRI (nu/nu) female mice. The tumor size was measured using a caliper, and the tumor volume was estimated via the following formula: tumor volume (mm3) = L × W2/2, where L refers to the length and W refers to the width. When the tumor size reached an average of 50 mm3, the mice were randomized into two groups and were received an intraperitoneal injection of either corn oil (control) or garcinol (15

Figure 1. Effect of garcinol on the viability of NSCLC cell lines. (A) Chemical structure and molecular weight (mol wt) of garcinol. (B) The viability of NSCLC cell lines was examined by an MTT assay. (C) Colonogenic survival was determined by the colonogenic assay. In the colony formation, cells were exposed to garcinol (4 μM) for 24 h, counted, plated in 60 mm dishes, and cultured for approximately 10 days. (D) Colony frequency after exposure to 2 μM garcinol. A colony formation assay shows that garcinol treatment decreased the cologenicty of A549 cells. Statistical comparisons are shown as *p < 0.05 and **p < 0.01, respectively.

identifying cancer stem cell-like cells in NSCLC cell lines.14 In addition, we observed that garcinol resulted in a downregulation of ALDH1A1. Mechanistically, we showed that garcinol repressed ALDH1A1 transcription through alterations in the interaction between DDIT3 and C/EBPβ.

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MATERIALS AND METHODS

Cell Culture and Garcinol Treatment. The human lung cancer cell lines A549, H460, H1299, H1650, H358, and HCC827 originally purchased from ATCC were stored in our Institute. Cells grew in RPMI-1640 medium to which 10% fetal calf serum (Gibco, Grand Island, NY, USA) had been added. Garcinol was isolated from Garcinia indica fruit rind according to the method described previously.15 Garcinol was dissolved in DMSO (Solarbio Science and Technology, Beijing, China) to a 100 mM stock concentration. For in vivo studies, garcinol was dissolved in 100% corn oil to 0.6 mg/L. The final DMSO concentration was accounted for less than 0.1% (v/v). 0.05% DMSO treated cells were taken as a vehicle control. Viability Assay. Viability was evaluated using an MTT assay as described previously.16 NSCLC cells were treated with different concentrations of garcinol, and cell viability was measured using a microplate reader (Synergy HT; Bio-Tek Inc., Winooski, VT, USA), with a 570 nm wavelength. Colony Formation Assay. Tumor cells (3 × 105 per well) were first received a 24 h seeding in six-well tissue culture plates. Next, cells were pretreated with garcinol for 24 h. Following garcinol treatment, cells were moved into 60 mm dishes at a density of 200 cells per well inch. Colony formation was then evaluated as described previously.17 Spheroid Formation Assay. The sphere formation assay was carried out as described previously.18 Briefly, A549 cells were seeded in tumorsphere medium which is prepared from RPMI1640 medium added with 0.4% BSA, 20 ng/mL EGF, 10 ng/mL bFGF, and N2 supplements (Invitrogen, Grand Island, NY, USA). The numbers and sizes of tumorsphere were measured on day 10. Wound Healing Assay. Upon A549 cells (5× 105) plated in 6well plates reached to about 80% confluence, a 200 μL pipet tip was applied to scrape a line within the confluent cells. When cells were exposed to garcinol for 24 h, the distance of wound closure was 3676

DOI: 10.1021/acs.jafc.7b00346 J. Agric. Food Chem. 2017, 65, 3675−3683

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Figure 2. Garcinol downregulates the expression of ALDH1A1 in A549 cells. (A) Specifically elevated mRNA levels of ALDH1A1 in A549 NSCLC cell line. (B) ALDH1A1 mRNA levels, (C) protein levels, and (D) percentage of ALDH+ cells from A549 cells treated with or without garcinol. (E) The number, (F) the frequency, and (G) the morphological observation of the A549 cells tumorsphere treated with or without garcinol. (H) Representative images displayed migration of A549 cells was significantly decreased with garcinol treatment. (I) Representative flow cytometry plots from the ALDEFLUOR assays showed that garcinol reduced the percentage of ALDH+ cells (black, gated) in A549 cells. *p < 0.05; **p < 0.01. ADC is the abbreviation for adenocarcinoma cell lines, and LCC is the abbreviation for large cell carcinoma cell lines. mg/kg), daily for 40 days. The mice experiments have been approved by the ethics committee of Tianjin University of Commerce and Certification Materials will be provided upon request. Immunohistochemistry. Immunohistochemistry staining was carried out according to kit instructions (PV-6000, ZSGB-NIO, China). Tissue sections were cut from blocks of formalin-fixed paraffin embedded xenografts. Four-micron thick tissue sections were stained with antibodies against Ki67, DDIT3, and ALDH1A1, used at 1:100 dilutions. Statistics. Data are displayed as the mean ± SD from 3−5 independent experiments. Differences between groups were performed using Student’s t test. A p < 0.05 was considered statistically significant. Statistical comparisons are shown as *p < 0.05 and **p < 0.01 respectively. Unless stated otherwise, the experiments were performed at least three times.

the colony formation assay consistently showed significantly decreased colony numbers in A549 cells (38.8% ± 10.3%) compared to H460 cells (89.7 ± 11.7%) (Figure 1C,D). Garcinol Downregulates the Expression of ALDH1A1 in A549. The results above indicate that the lung cancer A549 cell line is much more sensitive to garcinol treatment compared to other lung cancer cell lines. It has been shown that aldehyde dehydrogenase ALDH1A1 is preferentially expressed in A549 cells and plays a critical role in maintaining the stemness of A549 cells.22−24 In addition, ALDH1A1 is one of the stem celllike markers of NSCLC cell lines.25 Therefore, we hypothesize that ALDH1A1 plays a role in mediating the toxicological mechanism of garcinol in A549 cells. To study this hypothesis, we developed an in vitro screen using qPCR to analyze the expression levels of classic stem cell markers in lung cancer (Supplementary Figure 1 and Supplementary Table 1).26 As expected,27,28 we observed higher levels of ALDH1A1 mRNA (Figure 2A, Supplementary Figure 1 and Supplementary Table 1) in A549 cells compared with any other cell line utilized in this study. In the clinic, patients with NSCLC that harbored ALDH1A1 had a poor prognosis.29 ADLH1A1 is known to promote typical cancer stem cell-like phenotypes, such as increased cell

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RESULTS Effect of Garcinol on the Cellular Phenotype in Lung Cancer Cells. A panel of NSCLC cell lines (A549, H460, H1299, H1650, H358, and HCC827) was used to investigate the cytotoxic effect of garcinol in vitro using the MTT assay (Figure 1B). Of these NSCLC cell lines, A549 cells were found to be the most sensitive to garcinol treatment (Figure 1B). We found that, when A549 cells and H460 cells were treated with 2 μM garcinol for 24 h and cultured for approximately 10 days, 3677

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Journal of Agricultural and Food Chemistry migration, sphere-forming ability, and colony formation.14,30 We investigated the effect of garcinol on the expression level of ALDH1A1 in A549 cells. We found that A549 cells treated with garcinol (4 μM) for 24 h exhibited a significant decrease in the transcriptional expression of ADLH1A1 (Figure 2B). This result was further confirmed by Western blot analysis (Figure 2C, Supplementary Figure 2A). We next studied the effect of garcinol on the stemness phenotypes driven by ALDH1A1 in A549 cells. To test the tumorsphere forming capability, we cultured A549 cells in a three-dimensional microenvironment in either the absence or presence of garcinol (2 μM) for 24 h. Approximately 10 days following this treatment period, we counted the number of multicellular tumorspheres formed. As shown in Figure 2E−G, garcinol-treated A549 cells exhibited reduced sphere numbers and size compared with the control. In addition, we carried out wound healing assays to evaluate changes in cell migration capability. As shown in Figure 2H (Supplementary Figure 3), significantly fewer cells were able to migrate in the wound healing assay when exposed to garcinol (2 μM, 24 h). The ALDEFLUOR assay, which has been used to quantify the percentage of ALDH+ cells, has also been used to study cancer stem cell-like cells in a variety of tumors harboring ALDH1A1.31 As expected, garcinol treatment resulted in a strong reduction in the ALDH+ cell subpopulation from 12 to 4% (Figure 2D,I). These results indicate that garcinol may result in the downregulation of ALDH1A1 expression, thereby reducing the percentage of ALDH+ cells in A549 cells. Garcinol Treatment Reduces the Binding of C/EBPβ to the Endogenous ALDH1A1 Promoter. We have shown that garcinol treatment resulted in the downregulation of ALDH1A1 expression, a gene whose promoter contains a CCAAT box at position −75 to −71 upstream of the transcription start site (Figure 3A).32 Accordingly, it is known that CCAAT enhancer binding proteins β (C/EBPβ) are critical forALDH1A1 mRNA expression. In order to investigate the mechanism of ALDH1A1 mRNA modulation by garcinol treatment, we carried out ChIP studies of the ALDH1A1 promoter. Quantitative PCR analysis of ChIP with anti-C/ EBPβ antibodies from A549 cells revealed an effective occupancy of the C/EBPβ-binding site in untreated control cells (as compared to an isotype-matched mouse IgG). Garcinol treatment, on the other hand, was found to sharply reduce the occupancy of the C/EBPβ binding site by C/EBPβ (Figure 3B,C). These results strongly correlate with the observed effect of garcinol treatment on ALDH1A1 mRNA and protein expression levels (Figure 2B,C). Garcinol Treatment Increases Binding of DDIT3 to C/ EBPβ. Having identified a clear relationship between C/EBPβ occupation and ALDH1A1 mRNA turnover with garcinol treatment, we aimed to understand how garcinol treatment altered the C/EBPβ binding activity in A549 cells. In theory, C/EBPβ transcriptional modulation of the ALDH1A1 gene could be sequestered through protein−protein interactions to the stress responsor DDIT3.33−35 Coincidentally, garcinol treatment has been demonstrated to induce DDIT3 expression in human hepatocellular carcinoma Hep3B cells, resulting in cellular apoptosis.36 Thus, we speculated whether the relationship exists between the interaction between DDIT3 and C/ EBPβ and ALDH1A1 turnover. To investigate this question, we first aimed to understand whether garcinol treatment results in an increased binding of DDIT3 in A549 cells. As expected, garcinol treatment was found to up-regulate DDIT3 mRNA levels (Figure 4A), and this increase was found to match

Figure 3. Garcinol treatment reduces the binding of C/EBPβ to the endogenous ALDH1A1 promoter. (A) Schematic of ALDH1A1 promoter showing the location of the CCAAT box at −75 to −71 bp from the transcription start site. (B) Quantitative ChIP analysis indicated that garcinol treatment reduced the occupancy of the ALDH1A1 promoter by C/EBPβ and (C) PCR electrophoresis picture from a representative experiment. *p < 0.05; **p < 0.01.

increased protein levels observed (Figure 4B, Supplementary Figure 2B). In addition, the observed increased DDIT3 levels were found to match the decreased levels of ALDH1A1 in garcinol treated cells (Figure 2B,C). Next, we aimed to understand whether DDIT3 induced by garcinol acted as a dominant-negative member that prevents the activities of other C/EBPβ proteins by forming heterodimers.35 To address this question, we looked at colocalization of DDIT3 and C/EBPβ by immunostaining. Under standard culture conditions, A549 cells exhibited a cytoplasmic expression of DDIT3 (Figure 4C). However, following a 24 h garcinol treatment, DDIT3 localization was found to translocate from the cytosol to the nucleus, where it colocalized with C/EBPβ (Figure 4C). To further demonstrate the physical association between DDIT3 and C/EBPβ, we treated A549 cells with garcinol for 24 h. Following the garcinol treatment, we harvested the cell lysates and carried out coimmunoprecipitation experiments. The immunoprecipitated material showed increased amounts of C/EBPβ bound to DDIT3 upon garcinol treatment (Figure 4D, Supplementary Figure 2C). In addition, analysis of anti-C/ EBPβ precipitates showed that garcinol treatment resulted in increased binding of DDIT3 to C/EBPβ (Figure 4E, Supplementary Figure 2D), which strictly correlated with the observed decreased amount of C/EBPβ bound to the ALDH1A promoter (Figure 3B,C) and, ultimately, with the reduced levels of ALDH1A3 mRNA (Figure 2B,C). 3678

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Figure 4. Garcinol treatment increases binding of DDIT3 to C/EBPβ in A549 cells. Garcinol downregulated DDIT3 mRNA (A) and protein (B) synthesis in A549 cells. (C) Garcinol treatment (4 μM, 24 h) promoted transportation of DDIT3 to the nucleus in A549 cells (green, DDIT3; red, C/EBPβ; blue, DAPI). (D and E) Immunoprecipitation identified the physical interaction between C/EBPβ and DDIT3 under the stimulation of garcinol (4 μM, 24 h) in A549 cells. *p < 0.05; **p < 0.01.

To confirm whether the garcinol mediated reduction of ALDH1A1 expression was dependent on DDIT3, we used RNAi to knock down DDIT3 in A549 cells. As expected, the garcinol-mediated decrease in ALDH1A1 levels was prevented by DDIT3 siRNA but not by control siRNA (Figure 5A,B). A knockdown of DDIT3 was also found to reverse the ALDH+ cell subpopulation (Figure 5C,D) reduction induced by garcinol exposure. This suggests that ALDH1A1 mRNA levels and the ALDH+ cell subpopulation were reduced via modulation of DDIT3 in garcinol treated A549 cells. In essence, these findings strongly support the hypothesis that increased DDIT3 physically and functionally interacts with C/EBPβ to alter ALDH1A1 expression levels in A549 cells upon garcinol exposure. Garcinol Suppresses ALDH1A1 Expression in Tumor Tissues in Xenograft Mice Model. To verify whether the inhibition of ALDH1A1 expression and the resulting antitumor potential of garcinol treatment could be translated to an in vivo model, we used a xenograft model in which A549 cells were subcutaneously injected into the right flanks of athymicmice (Supplementary Figure 4A). Mice were randomized into two groups and were given a daily intraperitoneal injection of either corn oil (control) or garcinol (15 mg/kg) for a total of 40 days. The tumor volume of mice was recorded once every 10 days using calipers. As shown in Figure 6A, garcinol treatment resulted in a marked inhibition of A549 xenograft tumor growth (n = 5). Notably, there was no significant difference in the body weight of mice from either group compared to the vehicle control upon garcinol exposure (Supplementary Figure 4B).

This suggests that garcinol treatment does not result in severe toxicity to mice. By day 40 of garcinol treatment, control group tumors were observed to be twice as large as those of the garcinol treatment group (Figure 6A−C). The difference in tumor size was most likely due to a reduction in the proliferation of tumor cells in the garcinol group, as we observed decreased expression of the nuclear antigen Ki-67 by immunohistochemical analysis (Figure 6D). We further investigated the effect of garcinol treatment on ALDH1A1 and DDIT3 levels in tumor tissue and found that ALDH1A1 expression levels were downregulated, while DDIT3 expression levels were substantially increased in the garcinol treatment group compared to the control group (Figure 6D). These in vivo results are consistent with our in vitro observations described above.

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DISCUSSION According to recent data from population-based cancer statistics, lung cancer is one of the most serious public problems because it is the leading cause of cancer-related deaths worldwide,1 with more than one million mortalities each year. These statistics are especially true in China, where lung cancer has been the most common diagnosed cancer, as well as the leading cause of cancer-related deaths.37 Approximately 85% of all new lung cancer cases are diagnosed as NSCLC. With locally improved advances, NSCLC can be treated with conventional cancer therapies, including ionizing radiation and traditional antiproliferative chemotherapy. However, recurrence rates remain high at 30−50%, while overall 5-year survival rates remain low at approximately 7−20%. Several targeted therapies 3679

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Figure 5. Garcinol reduces ALDH1A1 mRNA levels by modulating DDIT3 levels in A549 cells. (A) Real-time PCR determined mRNA expression in cells transfected with DDIT3 targeting siRNA and negative control siRNA. (B) Knockdown of DDIT3 blunted the regulation of ALDH1A1 by garcinol (4 μM, 24 h) in A549 cells. (C) Knockdown of DDIT3 gained the increased percentage of ALDH+ cells from A549 cells with garcinol treatment and (D) a representative picture from flow cytometry analysis showed this effect. *p < 0.05; **p < 0.01.

have been developed for tumors harboring EGFR and ALK mutations. While these tumors exhibit very high fractional response rates, cancer cells ultimately become resistant to these targeted therapeutic approaches. In addition, these treatment strategies are connected with several adverse effects, including hair loss, pancytopenia, nausea and vomiting, and drug resistance. Thus, an improved treatment exhibiting increased efficacy and lower toxicity will be highly desirable. Prior studies have shown that tumors which harbor the ALDH1A1 gene indicate a poor clinical prognosis in the case of breast, pancreatic, and prostate cancers, as well as in lung cancer.38 This is especially true in the case where ALDH1A1 expression in lung cancer tissue is higher than that in the lymph node metastasis groups and the poorly differentiated tumor groups.39 In addition, it was found that ALDH1A1+ lung cancer cells displayed increased resistance to the radio-/ chemotherapeutic and targeted therapies, including cisplatin, gemcitabine, doxorubicin, vinorelbine, docetaxel, and EGFR inhibitor gefitinib.25,40 ALDH+ cancer cells were found to develop drug resistance, potentially through the ALDH-specific activity involved in metabolizing these drugs. ALDHs could possess the ability to oxidize the aldehyde group of chemotherapy drugs to carboxylic acid. This would result in the detoxification of the cytotoxic chemotherapy drugs, as they become metabolized into nontoxic metabolites. The knockdown of ALDH1A1 is connected with alterations in the proliferation and motility of cells, demonstrating that the target for ALDH enzymes in cell homeostasis may provide potential prevention against NSCLC. Natural products constitute a relatively neoteric strategy with strong attraction due to its potential for fewer side effects. Since

the 1970s, the drug discovery strategy has been based on screening a large quantity of candidates from natural and synthetic compounds, resulting in the modern and rational drug design in discovery process. In this study, we focus on the natural compound, garcinol, which is isolated from native Guttiferae plants growing in India and Southeast Asia.36 Numerous scientific studies on garcinol have demonstrated that this compound possesses a broad anticancer activity in addition to anti-inflammatory, antioxidant, and antibacterial activity. Our present knowledge surrounding anticancer mechanisms includes the induction of apoptosis, inhibition of the cell cycle, and a reduction of tumor angiogenesis and metastasis. In regard to lung cancer studies, the majority of research has primarily been carried out in vitro. For example, Yu et al. demonstrated that garcinol treatment triggered cell apoptosis of the lung cancer cell line, H1299, in a p53independent manner.41 Oike et al. showed that garcinol treatment radiosensitized lung cancer A549 cells through the inhibition of nonhomologous end joining.42 Interestingly, when we scanned a panel of NSCLC cell lines (A549, H460, H1299, H1650, H358, and HCC827) to investigate the cytotoxic effect of garcinol, A549 cells were observed to be the most sensitive to garcinol treatment. A549 cells exhibit characteristically high ALDH1A1 expression levels, and garcinol treatment was shown to significantly downregulate ALDH1A1 expression (Figure 7). This resulted in the inhibition of the malignant potential of A549 cells. In vivo, garcinol treatment was shown to result in a marked inhibition of A549 xenograft tumor growth. Furthermore, we explored the cellular mechanisms surrounding ALDH1A1 inhibition upon garcinol treatment. Our results suggest that, in A459 cells, garcinol treatment promotes the 3680

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Figure 6. Garcinol inhibits ALDH1A1 levels in vivo. A549 cells were subcutaneously injected into the right flanks of athymic mice, and mice were randomized into two groupscontrol group (corn oil) and garcinol group (15 mg/kg·day). Garcinol was administered orally for 40 days. (A) Volumes of subcutaneous A549 xenografts in mice treated by continual infusion with garcinol at a dose of 15 mg/kg/day or corn oil for the time shown. (B) Tumor weight at the end of treatment with garcinol. (C) On day 40, tumors were excised and photographed. (D) Immunohistochemical staining for Ki67, DDIT3, and ALDH1A1 in the mouse xenograft tumors of each group. All data are presented as mean ± SEM *p < 0.05; **p < 0.01.

preclinical and clinical experiments are needed to further pursue this avenue.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00346. Supplementary Table 1, Figure 1, Figure 2, Figure 3, Figure 4 (PDF)

Figure 7. Schematic representation of the proposed mechanism of garcinol-mediated reduction of ALDH1A1 activity in A549 cells.

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expression of the stress response protein DDIT3. Interestingly, some natural compounds also induced DDIT3 gene upregulation. For example, resveratrol induces DDIT3 expression following by the activation of the ER stress signature proteins, IRE1α and ATF6α, in time-dependent manner and results in ER-stress mediated apoptosis.43 Unlike resveratrol, 6-shogaol from ginger induces DDIT3 ER stress-mediated apoptosis depending on eIF2α.44 In theory, the transcriptional regulation of DDIT3 is complicated. Several cis-acting elements, including AARE1, AARE2, ERSE1, and ERSE2, are involved in the regulation of DDIT3. In addition, ATF4, pATF6(N), NF-kB, STAT3, and XBP-1 are identified to activate transcription of DDIT3.45 Therefore, it is worth investigating how garcinol regulates DDIT3 in this system in a future work. Anyhow, our research identifies that garcinol promotes DDIT3 which contributes to the regulation of ALDH1A1. Overall, the current results demonstrate that garcinol may represent a potential phytochemical drug for use against NSCLC tumors harboring the ALDH1A1 gene. Further

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Liwen Wang: 0000-0001-7135-3418 Hui Zhao: 0000-0002-7517-5054 Funding

This work was supported by the National Natural Science Foundation of China (nos. 31571832, 31671873, and 31670859); Natural Science Foundation of Tianjin (no. 15KPXM01SF056); Fundamental Research Funds for CAMS & PUMC (nos. 2016ZX310068 and 2016RC310017); Research Funds for the Innovation Team of IRM-CAMS (no. 1650); and Tianjin Innovative Research Team Grant (TD-125049). Notes

The authors declare no competing financial interest. 3681

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