A synthetic compound, 1,5-bis(2-methoxyphenyl)penta- 1,4-dien-3-one (B63), induces apoptosis and activates endoplasmic reticulum stress in non-small cell lung cancer cells

Authors

  • Jian Xiao,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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    • J. X. and Y. W. contributed equally to this work.

  • Yi Wang,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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    • J. X. and Y. W. contributed equally to this work.

  • Jing Peng,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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  • Lu Guo,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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  • Jie Hu,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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  • Menghua Cao,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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  • Xie Zhang,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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  • Hanqing Zhang,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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  • Zhouguang Wang,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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  • Xiaokun Li,

    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
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  • Shulin Yang,

    1. Institute of Bioengineering, Nanjing University of Science and Technology, Nanjing, People's Republic of China
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  • Huiling Yang,

    1. Department of Pathophysiology, Sun Yat-Sen University, Guangzhou, People's Republic of China
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  • Guang Liang

    Corresponding author
    1. Bioorganic & Medicinal Chemistry Research Center, Zhejiang Key Laboratory of Biotechnology Pharmaceutical Engineering, School of Pharmacy, Wenzhou Medical College, Wenzhou, People's Republic of China
    2. Institute of Bioengineering, Nanjing University of Science and Technology, Nanjing, People's Republic of China
    • Bioorganic & Medicinal Chemistry Research Center, School of Pharmacy, Wenzhou Medical College, University Town, Chashan, Wenzhou City, Zhejiang 325035, People's Republic of China
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    • Tel.: 86-577-86699892, Fax: 86-577-86689983


Abstract

Endoplasmic reticulum (ER) stress-induced cancer cell apoptosis has become a novel signaling target for the development of therapeutic drugs for cancer treatment. Curcumin, a dietary phytochemical, exhibits growth-suppressive activity against cancer cells via multitarget mechanisms. However, the low stability and poor pharmacokinetics significantly limit its clinical applications. Thus, we designed and synthesized a novel monocarbonyl analog of curcumin, 1,5-bis(2-methoxyphenyl) penta-1,4-dien-3-one (B63). This compound exhibited a higher chemical stability in cultural medium and a better intracellular profile than curcumin. Treatment with B63 potently induced apoptosis of human non-small cell lung cancer (NSCLC) cells in a dose-responsive manner, while exhibiting no cytotoxicity in normal lung fibroblast cells. Its antitumor effect was associated with the ER stress-mediated apoptotic pathway and, ultimately, the activation of the caspase cascades. However, curcumin at the same concentrations did not cause ER stress in H460 cells. Further, C/EBP homologous protein knockdown by siRNA attenuated B63-induced cell apoptosis, indicating that the apoptotic pathway is ER stress-dependent. In vivo, the volume and weight of the tumor were reduced significantly by pretreating the H460 tumor cells with B63 before implantation. Taken together, these insights on the novel compound B63, from both chemical and biological perspectives, may provide a novel anticancer candidate for the treatment of NSCLC.

Lung cancer is one of the most common cancers worldwide. Despite advances in chemotherapy, the prognosis and treatment of lung cancer are poor, especially for non-small cell lung cancer (NSCLC).1 The need for the development of new therapeutics and agents for the management of NSCLC is especially urgent. Natural products have been the main source of healthcare agents and new drug development.2 Curcumin is a phenolic compound isolated from the plant Curcuma Longa, which has long been used to treat various diseases in China and India.3 Curcumin exhibits anticancer effects in various types of cancer in vitro and in vivo, including NSCLC.4 Its anticancer activity is attributed partly to its ability to arrest the cell cycle, induce apoptotic activity and inhibit the proliferation and metastasis of tumor cells.5–7 Recently, it was reported that curcumin exerts its proapoptotic effects by inducing endoplasmic reticulum (ER) stress in human leukemia HL-60 cells, human lung carcinoma NCI-H460 cells, A549 cells and mouse melanoma cells.8–11 Although curcumin is remarkably nontoxic and has promising anticancer activities in vitro and in vivo, preclinical and clinical studies indicate that its poor bioavailability and pharmacokinetic profiles have limited its application in anticancer therapy significantly.12–14

The common way for developing new drugs is to formulate synthetic chemical compounds that are analogs to molecularly targeted chemotherapeutic drugs. Recently, extensive work has been done to modify curcumin chemically to identify potential analogs with better bioavailability and antitumor activities.7, 15–17 Among the synthetic curcumin analogs, EF-24 has been tested in clinical trials as a tumor-therapeutic candidate.18 Our previous study resulted in a series of monocarbonyl analogs of curcumin without the β-diketone moiety. These analogs exhibited enhanced stability and improved pharmacokinetic profiles, as well as potential antitumor activity in vitro.19 In our study, a new monocarbonyl curcumin analog, 1,5-bis(2-methoxyphenyl)penta-1,4-dien-3-one (B63), was designed and developed (Fig. 1), and we hypothesized that it may exhibit a larger cellular uptake than curcumin due to its structural stability and decreased polarity. Our data demonstrated that, due to the enhanced cellular uptake and the induction of ER stress-dependent apoptosis, B63 shows stronger antitumor potential than curcumin against NSCLC both in vitro and in vivo.

Abbreviations

ATCC: American Type Culture Collection; ATF: activating transcription factor; CHOP: C/EBP homologous protein; DMSO: dimethyl sulfoxide; ER: Endoplasmic reticulum; GRP: Glucose-regulated protein; NSCLC: non-small cell lung cancer; PCR: polymerase chain reaction; PI: propidium iodide; TG: thapsigargin; UPR: unfolding protein response; XBP: X-box binding proteins-1

Material and Methods

Synthesis of compound B63 (Fig. 1)

An amount of 7.5 mmol of acetone was added to a solution of 15 mmol of 2-methoxybenzaldehyde in methanol (10 ml). The solution was stirred at room temperature for 20 min, followed by the dropwise addition of NaOCH3/CH3OH (1.5 ml, 7.5 mmol). The mixture was stirred at room temperature and monitored with thin layer chromatography. When the reaction was complete, the residue was poured into saturated NH4Cl solution and filtered. The precipitate was washed with water and cold ethanol and dried in vacuum. The solid was purified by chromatography over silica gel using CH2Cl2/CH3OH as the eluent to yield the compound. Before use in biological experiments, compound B63 was recrystallized from CH2Cl2/CH3CH2OH, and HPLC was used to determine the purity of the compound (98.67%).

Treatment of NSCLC with B63 and curcumin

Human NSCLC cell lines (NCI-H460 and H358) and lung fibroblast CD199-Lu cells obtained from the American Type Culture Collection (ATCC) were grown in RPMI 1640 medium, which contained 10% FBS in a humidified environment at 37°C with 5% CO2. B63 and curcumin were dissolved in dimethyl sulfoxide (DMSO). The cells were treated with various concentrations of B63 and curcumin, i.e., 5, 10 and 20 μM, for 24 hr. Control cultures were treated with DMSO and processed similarly.

Stability and uptake of B63 and curcumin in H460 cells

Uptake of B63 was evaluated in 90% confluent cells seeded onto six-well plates. The cells were treated with 20 μM B63 and curcumin in medium. HPLC was used to determine the concentration accurately. After specific intervals of time, the cells were washed twice with 2 ml of cold PBS and then harvested with 200 μl of water. B63 and curcumin were extracted from the suspended cells by the addition of acetonitrile, and their concentrations were determined by HPLC method. The protein content was determined using a Bio-Rad protein assay reagent. The intracellular concentrations of the drugs were normalized as μg/mg protein. An Agilent LC (1,200 series) was used for the HPLC analysis. The mobile phase consisted of acetonitrile and water (50/50 to 60/40 v/v in 15 min). Chromatographic separation was obtained using a Beckman C18 reverse-phase column (5 μm, 4.6 mm × 25 cm) at room temperature at a flow rate of 1 ml/min. B63 and curcumin elution was monitored at a wavelength of 310 nm and 330 nm, respectively.

MTT assay

H460, H358 and CD199-Lu cells were seeded in a 96-well plate for 24 hr and then treated with different doses (5, 10 and 20 μM) of B63 and curcumin for 12 hr or 24 hr. This treatment was followed by the MTT treatment (5 mg/ml) of the cells in each well for 4 hr at 37°C, as described earlier. The MTT was aspirated, and 100 μl of DMSO were added to each well; absorbance at 570 nm was read by a plate reader. Each treatment was done in triplicate, the mean of the three values was determined, and the results were expressed as percent of control.

Apoptosis rate analysis

H460 cells were plated on 60-mm plates overnight and then treated with varying doses (5, 10 and 20 μM) of B63 and curcumin for 12 hr. The cells were washed with PBS three times and then digested by 0.25% tryptan-EDTA. After the solution was centrifuged, the cells were suspended in 0.5 ml of PBS. Then, the cells were stained with Annexin V and propidium iodide (PI) in the presence of 100 mg/mL of RNAse and 0.1% Triton X-100 for 30 min at37°C. Flow cytometric analysis was performed using a fluorescence-activated cell sorter.

Assay of ER calcium pools

H460 cells were seeded on 22 × 40-mm coverslips and treated with vehicle, curcumin and B63, for 24 hr. The cells were loaded with 4 μM Fura-2 AM and 0.3% Plurinic F-127 in HBSS at 37°C for 120 min and incubated in HBSS for an additional 30 min. Then, they were mounted on the stage of an Axioskop 2 plus upright fluorescence microscope equipped with a 40× objective. After washing three times with HBSS without Ca2+ and Mg2+, the cells were stimulated with 100 nM of thapsigargin (TG). Before and after the addition of TG, fluorescent images were collected at 15-sec intervals through a cooled camera that was attached to an image intensifier, an epifluorescent light source, a 515-nm dichroic beam splitter and a 535-nm emission filter. The 340/380 ratios of individual cells in these images were analyzed using TILLvisION version 3.1 imaging software.

RNA isolation and real-time quantitative PCR

Total mRNA was isolated from cells using an Ambion RNAqueous kit after treatment with compounds or control DMSO. The High-Capacity cDNA Archive Kit was used to obtain first-strand cDNAs of mRNAs. The mRNA levels of C/EBP homologous protein (CHOP), XBP-1, ATF4 and GPR78 were quantified by specific gene expression assay kits and primers on a multicolor, real-time polymerase chain reaction (PCR) detection system (Eppendorf, Hamburg, Germany) and normalized to internal control β-actin mRNA. PCR primer sequences: CHOP, sense, 5′-CTGAATCTGCACCAAGCATGA-3′, antisense, 5′-AAGGTGGGTAGTGTGGCCC-3′; XBP-1, sense, 5′-GCGCCTCACGCACCTG-3′, antisense, 5′-GCTGCTACTCTGTTTTTCAGTTTCC-3′; ATF-4, sense 5′-TGGCTGGCTGTGGATGG-3′, antisense, 5′-TCCCGGAGAAGGCATCCT-3′; GRP78, sense 5′-TCCTGCGTCGGCGTGT-3′, antisense, 5′-GTTGCCCTGATCGTTGGC-3′; Actin, sense 5′-CCTGGCACCCAGCACAAT-3′, antisense, 5′-GCCGATCCACACGGAGTACT-3′.

Western blot

GRP78, CHOP, XBP-1, Caspase-3 and Caspase-9 expression and phosphorylation were examined by Western blotting assay based on previous publications.20 Briefly, the cells were lysed, supernatants were collected and cytosol or nuclear proteins were extracted using known methods. After being resolved on tris-HCl polyacrylamide gels at 120 V, the proteins were transferred to a polyvinylidene difluoride blotting membrane, and the membranes were probed with specific polyclonal antibody. All antibodies were purchased from Santa Cruz (Santa Cruz, CA). Equal loading was verified by determining protein concentration using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The same amount of protein from protein lysates was used for electrophoretic analysis. β-actin was used as the standard.

Construction of lentiviral siRNA for CHOP

The sense sequence of the siRNA cassettes specifically targeting the nucleotides of CHOP was designed through siRNA Target Finder (Ambion, Austin, TX). A two-step PCR strategy was performed using two separate reverse primers to generate an siRNA expression cassette consisting of human U6 promoter and a hairpin siRNA cassette plus terminator and subcloned into pGL3.7 vector, which encodes the CMV-promoted enhanced green fluorescent protein (EGFP) marker as an internal control. The resulting lentiviral siRNA vector was confirmed by restriction enzyme digestion and DNA sequencing. The sequence of CHOP siRNA is 5′-GCAGGAAATCGAGCGCCTGAC-3′. The recombinant lentiviruses were produced by transient transfection of H460 cells using FuGene 6 Transfection reagent (Roche, Nutley, NJ). Briefly, H460 cells were cultured in a high-glucose DMEM medium, supplemented with 10% FBS, penicillin/streptomycin (100 U/ml) and 500 μg/ml of G418. The subconfluent cells in a 10-cm culture dish were cotransfected with lentiviral vector (10 μg), lentiviral packaging vectors pRSV-REV (2 μg) and pMDLg/pRRE (5 μg) and the vesicular stomatitis virus G glycoprotein (VSVG) expression vector pMD2G (3 μg). The viruses were collected from the culture supernatants on days two and three post-transfection, concentrated by ultracentrifugation for 1.5 hr at 25,000 rpm and suspended in PBS. Titers were determined by infecting H460 cells with serial dilutions of concentrated lentivirus and counting EGFP-expressing cells after 48 hr under fluorescent microscopy.

Tumor growth in nude mice

All animal experiments complied with Sun Yat-Sen University's Policy on the Care and Use of Laboratory Animals. Five-week-old female BALB/c nude mice (from the Animal Center at Sun Yat-Sen University, Guangzhou) were maintained in the animal facility at Sun Yat-Sen University. The animals were housed at a constant room temperature with a 12/12-hr light/dark cycle and fed a standard rodent diet and water. The mice were divided into four experimental groups with six mice in each group. H460 cells were treated with curcumin (20 μM), B63 (10 and 20 μM), or vehicle (DMSO) for 12 hr. After treatment, cells were harvested, normalized and 3 × 106 in 0.2 ml PBS was injected subcutaneously into the right flank of each mouse. Beginning on day eight after cell inoculation, tumor volumes were measured every 2 days by measuring the length (l) and width (w). The volume was calculated as lw2π as described in the literature.21, 22 At the end of 24 days, the mice were sacrificed, and the tumors were weighed.

Statistical analysis

All assays were performed at least three times, and the levels of all measured parameters were expressed as mean standard error (S.E.). Statistical comparisons between treatments with B63 and curcumin versus DMSO control were based on the t-test method.

Results

The intercellular concentration of the designed B63 in the H460 cells was greater than that of curcumin, and B63 was more stable as well.

Due to the deletion of the β-diketone moiety and the phenolic OH groups in the molecular structure, we hypothesized that B63 may possess a more stable structure in the cell culture medium and may get inside the tumor cells more easily than curcumin. To study the kinetics of the cellular uptake of B63 and curcumin in the H460 cells during the 24-hr treatment, we determined the levels of curcumin and B63 in the cells using the HPLC method described in the Method section. Meanwhile, the content of B63 and curcumin in the culture medium also was determined using the HPLC method to compare their stability in medium. Figure 2a indicates an excellent chromatographic specificity and the solid methodology for HPLC determination of B63 and curcumin. Figure 2b shows the concentration-time profiles of B63 and curcumin in culture medium. Curcumin underwent rapid degradation in the medium. More than 50% of curcumin degraded after a 4-hr incubation period, while more than 90% of the original B63 content was retained after a 12-hr incubation period in the medium. After being incubated for 24 hr, both curcumin and B63 were degraded completely. Figure 2c, presented as the ratio of pmol of the compound per mg of total protein in the cells, illustrates the cellular uptake profiles of curcumin and B63 in the H460 cells. B63 was taken up rapidly by the cells. It is interesting to note that the amount of B63 taken up by the H460 cells was much greater than that of curcumin after a 15-min treatment. After being incubated for 12 hr, the cellular content of curcumin vanished, while the concentration of B63 in the H460 cells remained high. These data suggested that a chemical modification in curcumin delayed its degradation and promoted cellular uptake in vitro. It is possible that a high and persistent intracellular concentration of B63 may contribute to its better antitumor effects against cancer cells.1

Figure 1.

Design and synthesis of B63.

Figure 2.

Time-concentration curves of B63 and curcumin in the culture medium and in H460 cells. (a) Using the developed HPLC methods, the chromatographic peaks of B63 and curcumin have been well identified. (b and c) H460 cells were treated with B63 and curcumin for indicated times. The cultural medium and cells were collected, respectively, and the concentrations of B63 and curcumin were detected by HPLC methods.

B63 exhibits more potent induction of NSCLC cell death than curcumin

We determined the effects of B63 and curcumin on cell viability in two NSCLC cell lines, i.e., H460 and H358. Treatment of B63 for 12 hr showed slight inhibition against NSCLC cell proliferation, while 24-hr treatment with B63 significantly induced cell death in a dose-dependent manner in both H460 and H358 (Fig. 3a). In contrast, curcumin at the same concentrations showed no significant inhibition against either cell line. Curcumin has been found to be safe in clinical trials, and dose-limiting toxicity was not observed in many studies. To test the cytotoxicity of B63 on normal cells, lung fibroblast cells (CD199-Lu cells) were treated with B63 and curcumin at 5, 10 and 20 μM for 24 hr. As shown in Figure 3b, neither B63 nor curucmin affected CD199-Lu cell survival, indicating that B63, like curcumin, has no significant toxicity for normal cells. Similar results also were observed in the in vivo safety assay (Supporting Information Figs. S1–S3). These data demonstrated that B63 exhibited more potent inhibition of cell viability than curcumin in both NSCLC cell lines.

Figure 3.

The effects of B63 on tumor cell growth and apoptosis. The effects of B63 and curcumin on cell growth in (a) H460 and H358 cells and (b) normal lung fibroblast CD199-Lu cells as determined by MTT assay. The data were obtained from three independent experiments performed in triplicate. (ce) The effects of B63 and curcumin on the induction of apoptosis in H460 cells as determined by flow cytometry. H460 cells were treated with B63 and curcumin at indicated concentrations for 12 hr, then stained with annexin V and PI, followed by detection using flow cytometry. Similar results were obtained in three independent experiments. Representative data are shown (c), and the percentage of cells with early apoptosis (d) and late apoptosis (e) also are shown. The mean of the three values was calculated, and the results were expressed as percentage of vehicle (DMSO) control. Data are presented as the mean ± S.E. Statistically significant, *p < 0.05, t-test, vs. vehicle control; #p < 0.05, t-test, vs. curcumin group in the same concentration. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

B63 induces NSCLC cell apoptosis

H460 cells appear to be more sensitive than H358 cells when exposed to B63. B63 showed the antiproliferative rates of 73.9% (at 20 μM) and 57.1% (at 10 μM) for H460 cells, while the rates were 56.7% (at 20 μM) and 33.6% (at 10 μM) for H358 cells (Fig. 3a). Next, we assessed the effects of B63 and curcumin on the induction of H460 cell apoptosis by flow cytometry. The results (Figs. 3c–3e) showed that B63 increased cell apoptosis that was dependent on dosage after 12 hr of treatment. B63 at 20 μM significantly induced a greater H460 cell apoptosis rate (78%) than curcumin (67%). This demonstrated that B63 was involved in the apoptotic induction that caused cell death.

B63 depletes ER calcium stores

To investigate the possible mechanism by which B63 induces H460 cell apoptosis, we assessed the effects of B63 and curcumin on ER calcium stores in H460 cells using a fluorescent calcium indicator. After treatment with 20 μM curcumin or B63 for 6 hr, cells were loaded with fura-2/AM and switched to a calcium-free medium. ER calcium release induced by 100 nM TG was recorded by fluorescence microscopy. Even at a concentration of 20 μM, curcumin had a slight effect on ER calcium stores (Fig. 4a). Treatment of H460 cells with B63 decreased the ER calcium content in a dose-dependent manner, and B63-treated cells remarkably reduced the stress response to TG, which suggested that ER calcium stores were depleted by the B63 treatment (Fig. 4b). Our data showed that treatment with B63 resulted in a rapid release of calcium from ER to cytosol as early as 1 hr after treatment in H460 cells, indicating that B63 may next activate unfolding protein response (UPR) to trigger the ER-related apoptotic pathway.

Figure 4.

The effects of B63 on endoplasmic reticulum calcium store and ER stress pathway activation. Depletion of endoplasmic reticulum calcium stores by B63 and curcumin. (a) Assessment of ER calcium stores in H460 cells treated with curcumin (20 μM) or B63 (20 μM) for 6 hr. Representative tracings of the Fura-2 fluorescence ratio of 340/380 nm in an individual cell for each treatment group before and after the addition of 10 μM thapsgargin are shown. (b) Assessment of ER calcium stores in H460 cells treated with indicated concentrations of B63 (5, 10 and 20 μM) for 6 hr. Similar results were obtained in three independent experiments. Dose-dependent activation of the ER stress pathway by B63 in H460 cells. (ch). Cells were treated with vehicle, B63, or curcumin at indicated concentrations for 12 hr. (c) The cytoplasmic protein was extracted, and GRP78 was examined by Western blot. TG was used as a positive control and lamin B was shown as a control for equal loading. (d) The nuclear protein was extracted and XBP-1 and CHOP were examined by Western blot. B19 was used as a positive control and actin was shown as a control for equal loading. (eh) The total RNA was extracted and the mRNA levels of GRP-78 (E), ATF-4 (F), XBP-1 (G) and CHOP (h) were examined. All RT-qPCR results were calculated and represented as the percent of vehicle control, and actin was used as a control for equal loading in the RT-qPCR experiments (*p < 0.05; **p < 0.01, vs. vehicle group). (i) H460 cells were incubated with vehicle, B63, or curcumin at indicated concentrations for 24 hr. The total protein was extracted and caspase-3 and caspase-9 were examined by Western blot. Actin was shown as a control for equal loading. Similar results were obtained in three independent experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

B63 activates ER stress-mediated apoptotic pathway

We further examined the possibility that B63 induces H460 cell apoptosis through an ER stress-mediated pathway. Glucose-regulated protein 78 (GRP78) is reported as the gatekeeper to the activation of the ER stress.23 First, we measured the protein expression of GRP78 in H460 cells after treatment with B63 or curcumin. TG was used as a GRP78-activating positive control. As shown in Figure 4c, GRP78 in cytosol was dose-dependently increased 12 hr after B63 treatment, while no significant increase could be observed in the curcumin-treated groups. Following the gatekeeper, we tested the expressions of the downstream transcriptional factors X-box binding proteins-1 (XBP-1) and CHOP in B63- or curcumin-treated H460 cells through Western blot. A compound B19, which was reported to be able to activate UPR signaling in our previous publication,20 was used as a positive control. The result also revealed that exposing H460 cells to B63 (20 μM) for 12 hr noticeably increased XBP-1 and CHOP levels in the nuclei in a dose-dependent manner, while curcumin at the same concentration had no effect on XBP-1 and CHOP protein profiles (Fig. 4d). Similar results were obtained using real-time qPCR. As shown in Figures 4e–4h, exposing H460 cells to B63 for 12 hr noticeably increased the mRNA expression of GRP-78, activating transcription factor 4 (ATF-4), XBP-1 and CHOP in a dose-dependent manner, while curcumin at the same concentrations had no effect on these mRNA levels. Especially, B63 treatment at 20 μM led to a 37-fold increase of CHOP mRNA expression (Fig. 4h). These results indicate that the B63-induced ER stress has been developed into the commitment phase toward apoptosis. Next, we examined the B63-induced ER stress-mediated apoptotic downstream events after commitment. The Western blot in Figure 4i indicates that the cleavage and activation of caspase-3 and caspase-9 were observed after treatment of H460 cells with 10 and 20 μM B63 for 24 hr. However, at these concentrations, curcumin was not able to activate caspase 3 and 9 in H460 cells. This result indicated that the caspase signals are associated with the ER stress-mediated apoptotic pathway induced by B63. Overall, our data demonstrated the capacity of B63 to activate proteins in initiation, commitment and execution phases of the ER stress-apoptosis cascade.

Reduction of CHOP expression inhibits B63-induced H460 cell death

To further confirm that ER stress plays a key role in the induction of H460 cell apoptosis by B63, lentiviral siRNA for CHOP gene was constructed. H460 cells were infected with serial dilutions of concentrated lentivirus, and the titers were determined after 48 hr by counting EGFP-expressing cells under fluorescence microscopy. Figure 5a shows that >65% of the H460 cells were transfected with lentiviral siRNA. The reduction of CHOP expression was confirmed by Western blot assay. After being transfected with lentiviral siRNA for CHOP, CHOP expression was significantly reduced in B63-treated cells compared to the vector-transfected control group (Fig. 5b). Furthermore, to confirm that the reduction of CHOP expression negatively affected B63-induced H460 cell death, we treated CHOP siRNA-transfected H460 cells with B63 and curcumin. The results in Figures 5c and 5d show that, when CHOP expression in H460 cells was silenced, cell apoptosis induced by B63 was reduced significantly compared to the control group (p < 0.05). Since CHOP is an extremely key protein in ER stress, these results indicated that B63-induced cell apoptosis is, at least partly, mediated by the ER stress pathway.

Figure 5.

CHOP knockdown inhibits B63-induced H460 cell apoptosis. H460 cells were transfected with CHOP siRNA virus using the method described in the experimental section. Forty-eight hours after transfection, the CHOP- and EGFP-expressing cells were counted using fluorescent microscopy (a). Cells were treated with B63 (10 μM) or curcumin (10 μM) for 6 hr and then CHOP expression was determined by Western blotting with actin as a control for equal loading (b). Cells were treated with B63 (10 μM) or curcumin (10 μM) for 24 hr, respectively. The figures were obtained by microscope with 20× amplification (c). The cell survival was determined using MTT assay, and the data were calculated as percent of the viability of nontransfected and DMSO-treated cells (d) (*p < 0.01, significantly different from DMSO-treated control virus group; ##p < 0.01, significantly different from B63-treated control virus group). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

B63 inhibits tumor growth in vivo

To verify the possible antitumor activity of B63, we performed a tumorigenicity assay in BALB/c nude mice. H460 cells were treated with B63 (10 μM or 20 μM), curcumin (20 μM) or vehicle (DMSO) for 12 hr and injected subcutaneously into the right flank of nude mice, and tumor formation was assayed. The tumorigenesis was dramatically lower in cells treated with 20 μM B63 than that in control cells that were treated with the vehicle. As shown in Figures 6a and 6b, a significant inhibition of tumor growth by B63 pretreatment at 20 μM, with treated versus control (T/C) values of 63.2% in tumor volume (p < 0.01) and 64.3% in tumor weight (p < 0.01), was observed on day 24 after treatment, respectively. Although there is no statistical significance (p = 0.087) between the group treated with 10 μM B63 and the vehicle control group, pretreatment with B63 still demonstrated a dose-dependent reduction in tumorigenesis. These data showed a potent inhibitory effect of B63 on H460 tumor growth in vivo, which was possibly associated with ER-stress-mediated cell injury.

Figure 6.

Pretreatment of B63 inhibits the tumorigenesis of H460 cells in nude mice. H460 cells were cultured in the presence and absence of B63 (10 and 20 μM) or curcumin (20 μM) for 12 hr. Cells were harvested and subcutaneously injected into the flank region of female nude mice. The mice were fed normally. The volumes of the tumors were monitored beginning on day 8 and concluding on day 24 after implantation. The graph of tumor volume over a 24-day period is shown (a). At the end of 24 days, the mice were sacrificed and the tumors were weighed. (b). Bars, S. D.; **p < 0.01, significantly different from the vehicle control group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Discussion

Curcumin is a natural polyphenol derived from the Curcuma longa, commonly called turmeric. Extensive research over the past 20 years has indicated that this polyphenol is a highly pleiotropic molecule that is capable of preventing and treating various cancers.4, 24 However, the anticancer potential of curcumin is affected severely by its limited systemic and target tissue bioavailability and rapid metabolism.12–14 A large volume of literature has reported the structural modifications of curcumin and the anticancer potential of its analogs.25 In vitro, curcumin is unstable at pH values above 6.5 because of the highly reactive β-diketone moiety.26 In our previous article, a series of monocarbonyl analogs of curcumin was designed by deleting the highly reactive β-diketone moiety in the structure of curcumin, which is considered to be responsible for the in vitro instability and the in vivo pharmacokinetic disadvantages.27 Our previous data demonstrated the significantly improved pharmacokinetic profiles of these monocarbonyl analogs of curcumin.27 Although the fact that curcumin decomposes rapidly in phosphate buffer and in serum-free medium has been demonstrated by Wang et al.,28 the kinetic profiles of curcumin in serum-containing medium and cells are still unknown. Our data in Figure 2 demonstrate a poor stability in the cultural medium and the weak intracellular kinetics of curcumin in H460 cells. In the research reported in this article, we designed the structure of B63 employing an acetone as a central linker to replace β-diketone. In addition, the loss of polarity benefits the penetration of drugs into the membranes of the cells, which contributes to greater cell uptake. The high polarity of the phenolic hydroxyl group contributes to curcumin's weak cellular penetration, and the considerable phenolic glucuronidation and sulfation of curcumin also are responsible for its rapid metabolism in cells.12 The B63 compound we developed possesses two nonpolar methoxyl groups instead of polar hydroxyl groups. The decrease of molecular polarity may help promote the penetration of B63 into cells and decrease the intercellular metabolism (Fig. 1). Rational design contributes to the enhanced chemical stability and improved cellular uptake profile of compound B63 compared to curcumin (Fig. 2). As a result, a higher and persistent intracellular concentration of B63 may support its better proapoptotic effects at a relatively low concentration.

ER plays a critical role in the regulation of protein synthesis, folding and trafficking. Many signals have been reported to disrupt the ER function and induce ER stress, which is associated with an accumulation of unfolded or misfolded proteins in the ER. The ER stress response is a balance between prosurvival and proapoptotic signaling pathways.29 When the prosurvivor responses fail, cells undergo apoptosis,29, 30 which seems to be the case in B63-treated H460 cells. The ER is also well known to regulate intracellular calcium (Ca2+) levels. Alterations in Ca2+ homeostasis or accumulation of misfolded proteins in ER can cause ER stress and ultimately lead to apoptosis.31 Perturbation of ER calcium homeostasis is expected to activate ER stress and induce UPR-mediated apoptosis.32 TG depletes the ER calcium stores and activates the UPR in many mammalian cells.33 Our data demonstrated that B63 treatment for 6 hr induced a gradual increase in the cytosolic Ca2+ concentration in a dose-dependent manner (Fig. 4). Although Wu et al. recently reported that curcumin at concentrations >30 μM induced apoptosis in H460 cells involving the ER stress signaling pathway,9 we were unable to find any evidence of such an effect using 20 μM curcumin on the Ca2+ profile and ER stress response. Thus, the ability of B63 to activate ER stress at lower concentrations is probably related to the improved cellular uptake of B63.

Then, the exact molecular mechanism by which B63 causes ER stress-mediated cell apoptosis was revealed by degrees. As a gatekeeper of ER stress, GRP78 activation triggers the cell apoptotic signaling pathway.34 We found that B63 could increase GRP78 protein expression in H460 cells 12 hr after treatment (Fig. 4c). Subsequently, ATF-6 is activated and moves to the nucleus and induces genes of XBP1 and ATF-4. The XBP-1 and ATF-4 activation have been reported to increase CHOP gene expression, triggering ER stress-specific cascade for implementation of apoptosis.29, 30 The upregulations of both XBP-1 and CHOP in the nuclei of H460 cells after B63 treatment for 12 hr (Fig. 4d) indicate that the B63-induced ER stress has been developed into the commitment phase toward apoptosis. Subsequently, we further validated the B63-induced ER stress activation by monitoring the mRNA expression of four commitment genes, i.e., GRP78, ATF-4, XBP-1 and CHOP, at 12 hr after B63 treatment (Figs. 4e–4h). All upstream signals ultimately lead to caspase activation to finish the execution of ER stress-induced apoptosis.35 Cleavage and activation of procaspase-3 and procaspase-12 have been observed in different studies on ER stress-induced human cancer cell apoptosis.35–37 Two groups reported that caspase-12 can directly trigger caspase-9 activation and apoptosis independently of the mitochondrial cytochrome c/Apaf-1 pathway.38, 39 Figure 4i demonstrates that B63 was able to induce the cleavage and activation of caspase-3 and caspase-9. Overall, these data showed that the main markers associated with the ER stress response are rapidly induced by B63 treatment, suggesting that B63-induced apoptosis is coupled to the ER stress signaling.

We do not yet know whether B63-induced cell apoptosis is ER stress-dependent or not. CHOP induction is probably most sensitive to ER stress response, and CHOP is considered as a marker of commitment of ER stress-induced apoptosis.40 Transfection of cells with CHOP or GRP78 siRNA reduced ER stress-mediated human colon cancer cell apoptosis.41 Knockdown of CHOP by specific siRNAs also attenuated the desipramine-induced42 and gamma-tocotrienol-induced ER stress-mediated apoptotic cascade.43 To show direct evidence for the role of ER stress in B63-induced apoptosis, CHOP siRNA transfection was used to compare B63-induced ER-stress-dependent and non-ER-stress-dependent apoptosis. Figure 5 solidly supports the conclusion that B63-induced apoptosis is, at least partially, ER stress-dependent.

Considering the mechanistic insights, our data related to cell viability assay also validated that H460 and H358 cells are sensitive to treatment by compound B63 at concentrations ranging from 2.5 to 20 μM, while curcumin did not exhibit good activity at the same concentrations (Fig. 3). Importantly, we observed no cytotoxic effects of B63 treatment in normal lung CCD199-Lu fibroblasts, indicating that B63, like the leading curcumin, may be safe for use with normal cells. Besides the cellular effects, we have shown that B63 is highly effective at inhibiting tumor growth in a tumor model using nude mice (Fig. 6). We also observed that B63 exhibited a high level of safety in mice (Supporting Information Figs. S1–S3).

In summary, a new, monocarbonyl analog of curcumin, B63, exhibited antitumor effects on human NSCLC both in vitro and in vivo via an ER stress-mediated mechanism. However, despite the data supporting our hypothesis, other apoptotic mechanisms may be also involved in B63-induced apoptosis. Caspase-3 and caspase-9 activations also play important roles in the mitochondria-mediated apoptotic pathway.44, 45 In addition, the leading curcumin has been reported to exert anticaner effects by multitargeting mechanisms.4–7 Therefore, since this work was focused only on ER stress-mediated apoptosis, further studies are necessary to establish this concept definitively and to evaluate the in vivo pharmacodynamics of B63 as a candidate. Based on rational drug design, B63 possesses a higher chemical stability and a better intracellular profile than the leading curcumin. It also exhibits no cytotoxicity against normal human lung fibroblast cells. Taken together, these mechanistic insights and our rational structure design of the novel compound B63 support that it should be explored further to develop safe and effective anticancer agents for the treatment of NSCLC.

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