U. davidiana var. japonica (UJ) powder was ground to ultrafine particle size using an herbal medicine pulverizer (Delsa™Nano; Beckman Coulter Inc., Brea, CA, USA). Catechin-7-O-xyloside (C7Ox) was obtained through purification of an ethanol extract of UJ by using a Sep-Pak cartridge (Waters, Milford, MA, USA). The water-soluble tetrazolium salt (WST)-8 cell proliferation assay kit was obtained from Dojindo Laboratories (Kumamoto, Japan). Lactate dehydrogenase (LDH) cytotoxicity assay and caspase-6 colorimetric assay kits were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA) and Abcam (Cambridge, MA, USA), respectively. The Annexin V/PI apoptosis detection kit was from BD Biosciences (Bedford, MA, USA). Primary antibodies for cleaved-caspase-6, cleaved-poly(ADP-ribose) polymerase (PARP), CHOP and tubulin, and the secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).

The extract was dissolved in ethanol at a concentration of 10 mg/ml and diluted with 50% ethanol to a final concentration of 2 mg/ml, and 2 μl was analyzed using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). LC-MS/MS was performed using an LTQ Orbitrap XL ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated electrospray ionization source. Separation by ultra HPLC (UHPLC) was performed on a Thermo Accela LC system by using an Acquity BEH C18 column (1.7 μm, 2.1 × 150 mm; Waters). Mobile phase A contained water and mobile phase B contained acetonitrile; both contained 0.1% formic acid. Gradient elution at a flow rate of 0.4 ml/min was carried out as follows: 0–1 min with 1–5% B (linear gradient) and 1–10 min with 5–20% B (linear gradient). Full-scan mass spectra were obtained in the negative ion modes at a range m/z 100–1000. To identify the structures of the compounds, the data obtained from tandem mass spectrometry (MS/MS) analysis were compared with those from an MS/MS spectral library search (16).

The non-small cell lung cancer (NSCLC) cell line H1299 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL/Life Technologies) in a 5% CO₂ incubator at 37°C.

Cell cytotoxicity was assessed by measuring the optical density at 450 nm with a microplate reader (SpectraMax 190^®; Molecular Devices Corp., Sunnyvale, CA, USA) 2 h after the addition of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H-tetrazolium (WST-8) reagent solution according to the manufacturer’s guidelines. Plasma membrane integrity was assessed based on lactate dehydrogenase (LDH) leakage into the culture medium from cells. LDH leakage was determined by measuring the optical density at 490 nm.

After C7Ox treatment, cells were harvested and stained with propidium iodide (PI) and Annexin V (BD Biosciences) for 15 min at room temperature in binding buffer and then analyzed using flow cytometry (BD Biosciences). PI and Annexin V emissions were detected in the FL-2 and FL-1 channels, respectively. For each sample, data from 10,000 cells were recorded in list mode on logarithmic scales. Data analysis was conducted using CellQuest software (BD Biosciences).

Mitochondrial membrane potential was assessed using the cationic dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide) according to the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA). Images were collected using a Zeiss LSM 510 fluorescence photomicroscope (Carl Zeiss, Oberkochen, Germany). Visualization of JC-1 monomers (green fluorescence) and JC-1 aggregates (red fluorescence) was carried out using filter sets for fluorescein and rhodamine dyes, respectively, and analyzed using ImageJ software.

A caspase-6 activity assay was conducted using substrates of the color reporter molecule Val-Glu-Ile-Asp-p-nitroaniline (VEID-pNA), which is specific for caspase-6. Briefly, H1299 cells were collected for sampling and lysed on ice by using lysis buffer containing protease inhibitors. Lysates were centrifuged at 13,000 rpm for 10 min at 4°C, and the supernatant collected was used for the assay. Caspase-6 activity was measured using a microplate reader at 405 nm.

Total cell lysate and total tissue protein from lung cancer xenografts of mice were prepared according to a previous study (17). Equal amounts of protein were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were probed with antibodies against cleaved caspase-6, poly(ADP-ribose) polymerase (PARP), CHOP, tubulin and secondary antibodies were detected using Pierce ECL-Plus chemiluminescence kit (Thermo Fisher Scientific).

CHOP antisense oligonucleotides (GTCCTGTC TTCAGATGAATT) were synthesized by Genolution Pharmaceuticals, Inc. (Seoul, Korea). Irrelevant scrambled siRNA was used as a control. Cells were transfected with the siRNAs using Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

For the tumor model, H1299 cells (1×10⁶) were subcutaneously injected into the right flank of female BALB/c nude mice (6 weeks of age) by using a 28-gauge needle. One week later, after the appearance of implanted tumors, the mice were randomly divided into 2 groups: C7Ox group and vehicle group (n=5 mice per group). C7Ox was dissolved in ethanol (EtOH) and diluted with phosphate-buffered saline (PBS) (EtOH:PBS=2:8). C7Ox or vehicle (ie, 20% EtOH in PBS) was intraperitoneally administered once daily for 1 month. Tumor size was calculated by measuring the length and width of the tumor with a caliper. Tumor volume was calculated as follows: Tumor volume (mm³) = length × width²/2. Next, tumor tissues were collected and frozen at −80°C until use.

Total RNA from the tumor tissues was extracted using TRIzol reagent (Invitrogen). cDNA was synthesized using the ImProm-II™ reverse transcription system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. RT-PCR was performed using a Solgent PCR detection kit following the manufacturer’s instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primers used included the following: CHOP forward, GACCT GCAAGAGGTCCTG and reverse, CTACTTCCCTGGTCA GGC; GAPDH forward, 5′-GGGCTCATCTGAAGGGTGGT GCTA-3′ and reverse, 5′-GTGGACGCTGGGATGATGTTC TGG-3′. The following PCR cycle was used: 1 min of denaturation at 92°C, 30 sec of annealing at 58°C and 1 min of extension at 72°C. PCR was conducted for 28 cycles. PCR products were run on a 0.8% agarose gel and visualized under UV illumination.

The data are expressed as the means ± standard error (SE) of at least three independent experiments. Student’s t-test was used to assess differences between the two groups. The level of significance was set at P<0.01.

The 50-mg ethanol extract was loaded on Sep-Pak cartridges and eluted stepwise using 0, 10, 20, 30, 40 and 50% water-ethanol solvent (15 ml each). The 30% fraction was collected and concentrated for the next experiment. The purity of the isolated C7Ox was found to be 87% using ultra high-pressure liquid chromatography (UHPLC) at 280 nm (Fig. 1B). The ultrafine UJ ethanol extract was characterized for its major constituent compounds by simultaneous estimation using negative ion-mode tandem mass analysis (MS² and MS³). The major peak was identified as C7Ox ([M-H]⁻, m/z (mass-to-charge ratio) of 421.3) at RT at 5.37 min (Fig. 1). The sugar moiety was defined using MS² fragment ion analysis. Indeed, in the MS² spectrum, neutral losses of m/z 132 indicated the loss of a pentose. The aglycone structures of MS³ spectra were identified as catechin by MS/MS spectrum matching (16).

To investigate the cytotoxic effect of C7Ox on cancer cells, we used the WST assay to monitor the survival rate of C7Ox-exposed H1299 cells. Treatment of H1299 cells with C7Ox for 48 h caused dose-dependent decreases in cell survival (P<0.01; Fig. 2A). Next, we investigated whether C7Ox induces LDH release by measuring the activity of LDH released from the cytosol of damaged cells into the medium. As shown in Fig. 2B, LDH release into the culture medium was significantly elevated after C7Ox treatment in H1299 cells when compared with that in the vehicle-treated cells (P<0.01). These results indicated that C7Ox-treated cells underwent postapoptotic necrosis.

To determine whether this agent induces typical cancer cell apoptosis as well as necrosis, we treated H1299 cells with 400 μM C7Ox for 30 h. After treatment, the cells were harvested and apoptotic cells were examined by FACS analysis using Annexin V/PI staining. As shown in Fig. 2C, C7Ox treatment increased the early apoptotic population (stained by Annexin V only; quadrant 4) from 0.12 to 4.93% as well as the necrotic population (stained by PI only; quadrant 2) from 0.04 to 3.25% or the late apoptotic population (stained by Annexiv V + PI; quadrant 1) from 0.25 to 26.99%. These results indicated that the proportion of apoptotic cells as well as necrotic cells was significantly increased after C7Ox treatment.

Loss of mitochondrial membrane potential has been linked to the initiation and activation of the apoptotic process (18). To evaluate whether C7Ox triggers mitochondrial injury, the JC-1 probe was used to evaluate the changes in mitochondrial membrane potential during C7Ox treatment. In healthy cells, the dye accumulates in the mitochondria as aggregates with red fluorescence, whereas in apoptotic or dead cells, the dye remains in the cytosol as monomers with green fluorescence. As shown in Fig. 2D, C7Ox-treated cells exhibited a reduction in red emission intensity and a concomitant increase in green emission intensity when compared to vehicle-treated cells. These results indicated that C7Ox treatment induced H1299 cell apoptosis by disrupting the mitochondrial membrane potential.

Caspase activation and degradation of caspase substrates are key markers of apoptosis (7). We confirmed the onset of apoptosis by examining the protease activity using a colorimetric substrate (VEID-pNA) specific for caspase-6. As shown in Fig. 3A, C7Ox activated caspase-6 in a dose-dependent manner (P<0.01). We also examined whether the expression levels of apoptosis-related proteins were affected by C7Ox treatment at various time-points. Western blotting showed that C7Ox significantly increased the cleavages of caspase-6 and PARP proteins in H1299 cells (Fig. 3B).

Apoptotic cell death can be triggered by ER stress, and several pathways have been directly implicated in ER stress-induced apoptosis (10). One of the major components of the ER stress-mediated apoptotic pathway is CHOP expression (19). To investigate whether C7Ox treatment causes ER stress, CHOP expression was measured. Our results showed that C7Ox induced an increase in CHOP expression in a dose- and time-dependent manner (Fig. 4A and B).

Next, to evaluate the role of CHOP in C7Ox-induced cytotoxicity and apoptosis, we examined the effect of the knockdown of CHOP expression on cell viability, LDH release and caspase-6 activity. As shown in Fig. 5A, C7Ox treatment increased CHOP expression, and CHOP siRNA, but not scrambled siRNA, significantly downregulated CHOP expression providing evidence for the specificity of siRNA inhibition. C7Ox treatment significantly reduced cell viability, increased LDH release and caspase-6 activity, as expected (P<0.01; Fig. 5B–D). However, downregulation of CHOP expression by CHOP siRNA significantly attenuated C7Ox treatment-induced cell death, LDH release and caspase-6 activation (P<0.01; Fig. 5B–D). C7Ox treatment-induced apoptosis, measured by Annexin V/PI staining, was also attenuated by CHOP siRNA (data not shown). These results suggest that ER stress is responsible for C7Ox-induced apoptotic cell death and caspase-6 activation.

To further evaluate the anticancer activity of C7Ox in vivo, we treated BALB/c nude mice bearing subcutaneous H1299 cell-derived tumors with either C7Ox (20 mg or 50 mg/kg) or vehicle. Tumor growth was inhibited by C7Ox as compared to the vehicle (Fig. 6A) and there was no apparent change in body weight in the C7Ox-injected animals (Fig. 6B). We also measured the mRNA level of CHOP within the tumor tissue. CHOP mRNA levels were significantly upregulated in the tissues obtained from the C7Ox-treated group when compared with levels obtained from the vehicle-treated group (Fig. 6C). The cleaved caspase-6 protein expression was also upregulated in the tissues obtained from the C7Ox-treated group (data not shown). Our results suggest that C7Ox may be a potential antitumor agent for NSCLC and that ER stress signals are involved in the anticancer activity of C7Ox.