The anticancer properties of SLs has attracted a great deal of interest. Various SLs have been shown to execute their antitumor capability via suppression of inflammatory responses, prevention of metastasis and induction of apoptosis in cell culture and animal models [3,4]. Xanthatin investigated here is an outstanding SL compound in terms of antitumor activity [5,6]. Moreover, xanthatin showed strong gastric protective activity [14] and exhibited little or no toxicity to animals, with an LD₅₀ value of 800 mg/kg [15], so xanthatin may be a highly effective inhibitor of tumor cell proliferation. However, the molecular basis for this selectivity toward cancer cells remains to be defined. Until recently, there has been an investigation addressing the mechanisms underlying xanthatin’s antitumor effects in MDA-MB-231 breast cancer cells that were p53 mutant [16]. We herein selected A549 lung cancer cells that were p53 wild type to investigate xanthatin effects on cell cycle progression and apoptosis, and the involved molecular pathways. These findings could further explain the selectivity of xanthatin implicated in cancer chemotherapy.

Our study demonstrated that xanthatin selectively induced G2/M arrest in A549 cells, leading to decreased cell viability and growth in a dose- and time-dependent manner. It is known that cell growth is controlled by cell cycle progression, a highly regulated process [17,18]. The standard cell cycle is divided into four non-overlapping phases, namely G1, S, G2 and M phases in sequence. Each phase has checkpoints that cause cell cycle arrest and activation of repair mechanisms [19,20]. Unlike normal cells that rely on the G1 checkpoint to protect against DNA damage, cancer cells are more dependent on the G2 checkpoint for DNA damage repair [21]. These insights give rise to the concept that cell cycle G2 checkpoint could be a specific target for cancer therapy. Both Chk1 and Chk2 are essential components in the G2 checkpoint via phosphorylating Cdc25c in response to DNA damage [22]. Actually, much evidence has suggested that Chk2 and/or Chk1 inhibitors may be good candidates for therapeutic G2 checkpoint abrogation implicated in cancer chemotherapy [23,24]. Our present study demonstrated that xanthatin was such an agent that selectively induced G2/M arrest in A549 cells, which was attributed to downregulation of Chk1 and Chk2 and decreased phosphorylation of CDC2. Our findings were in agreement with the established molecular recognitions and strongly suggested that the antitumor properties of xanthatin were consistent with the criterion for developing selective agents for cancer treatment.

To further examine the underlying mechanisms, we investigated xanthatin’s effects on apoptosis. Drug-induced cell cycle arrest can cause inefficient repair, leading to survival of genomically unstable cancer cells, or apoptosis if the damage is unrepairable. To switch the balance toward cell death with simultaneous abrogation of the DNA repair functions can enhance chemotherapy sensitization [25]. Some signaling molecules share function between cell proliferation and cell death machinery. We here found that xanthatin dose-dependently activated p53 concomitant with significant apoptosis in A549 cells. The mechanistic data indicated that p53 activation by xanthatin upregulated the ratio of Bax/Bcl-2, and subsequently promoted procaspase-9 cleavage and activated caspase-3. Many studies have pointed to a central role for p53 in balancing proliferation and apoptosis [10]. As a primary component of the G2 checkpoint, p53 not only conducts DNA damage signals and suppresses G2/M transition, but also is required for apoptosis induction. Through its transcriptional activities, p53 regulates the balance between the proapoptotic genes like Bax and the antiapoptotic genes like Bcl-2 [26]. These events lead to caspase activation and apoptosis. Our present data suggested the involvement of p53-involved mitochondria apoptosis pathway in xanthatin’s effects. Moreover, we also ruled out the involvement of the extrinsic apoptosis pathway, because xanthatin had no effects on procaspase-8 expression. This probably accounted for xanthatin’s selectivity in the mode of action.

NF-κB is shown to stimulate cell survival and proliferation, and its increased activity is positively associated with many cancer types including lung cancer [27]. Thus to further gain insights into the mechanisms of xanthatin-induced apoptosis, we investigated the expressions of key proteins in NF-κB pathway. We demonstrated that xanthatin inhibited the phosphorylation of NF-κB p65 subunit. Xanthatin also inhibited TNFα induced NF-κB (p65) translocation. These effects could disrupt NF-κB translocation into the nucleus to transactivate the genes involved in cell proliferation. Disruption of NF-κB pathway by xanthatin contributed to its inhibitory effects on cell cycle progression and apoptosis induction in A549 cells. These molecular insights could make xanthatin a more valuable candidate for cancer chemotherapy given that invasion and metastasis of tumor cell are closely related to inflammation, while NF-κB is indeed a key player in inflammation-induced tumor metastasis. There is evidence that the basal NF-κB activity is often low or required for cell differentiation rather than oncogenesis [8]. This could be the reason why the agents derived from sesquiterpene lactones, shown to be potential NF-κB inhibitors, selectively target tumor cells [28]. Targeting NF-κB pathway via which inflammation contributes to tumor progression and metastasis could lead to innovative approach for treating cancer [27]. Our investigation demonstrated that xanthatin disrupted NF-κB signaling in A549 cells. To determine its precise antitumor role and associations with inflammatory system requires further investigations especially the in vivo evidence.

The whole plant of Xanthium sibiricum Patrin ex Widder. (X. strumarium L.) (Asteraceae) was collected in Xuzhou city of Jiangsu Province, China, in May 2009 and authenticated by Jianwei Chen (College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China). Xanthatin was isolated and purified from air-dried aerial parts of X. sibiricum by our group as previously described [29] and the purity exceeded 95% as determined by a HPLC method. Xanthatin was dissolved in dimethyl sulfoxide (DMSO) for all experiments in this study.

RPMI-1640 medium and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). Primary antibodies against p-Rb, p-CDC2, caspase-3, caspase-8, caspase-9, NF-κB, p-NF-κB, IκBα, p-IκBα were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against CDC2, Chk1, Chk2, Rb, Bax, Bcl-2, Cytochrome C, GAPDH and Histon H3, goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were obtained from Bioworld (St. Louis Park, MN, USA). Primary antibody against p53 was obtained from Signalway Antibody (Jiangsu, China).

The NSCLC cell line A549 (Chinese Academy of Science, Shanghai, China) was cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO₂ at 37 °C. Cells were passaged every 4–5 days.

A549 cells were cultured in RPMI-1640 medium until mid-log phase for experimental use. DMSO used as control in all experiments (0.5%) or xanthatin at indicated concentrations (final concentration of 5, 10, 20, 40 μM) were added to the culture medium. After incubation for 12, 24 and 48 h, images of the cell morphology were taken with an inverted microscope (Olympus IX-70, Tokyo, Japan).

The effects of xanthatin on proliferation of A549 cells were assessed using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Briefly, A549 cells at mid-log phase were seeded in 96-well plates (5 × 10³ per well) in 200 μL of medium. After 24 h incubation, cells were exposed to 0.5% DMSO or xanthatin at indicated concentrations for 12, 24 and 48 h. Then, MTS (final concentration of 333 μg/mL)/PMS (25 μM) (Promega, Madison, WI, USA) was added and the cells were incubated for 2 h at 37 °C. The spectrophotometric absorbance was measured by SPECTRAmax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) at 490 nm. Triplicate experiments were performed. The concentration of xanthatin resulting in 50% inhibition of control growth (IC₅₀) was calculated using PASW Statistics 18 for windows.

Cell cycle analysis was performed as described previously [30]. Cells were seeded in growth medium in 6-well plates (3 × 10⁵ per well) and were grown overnight at 37 °C in a humidified incubator with 5% CO₂. Cells were then treated with xanthatin at indicated concentrations for 6, 12, 24 and 48 h. They were harvested (including attached and detached cells) and fixed with 75% alcohol at 4 °C. Distribution of cells with different DNA content was analyzed using the Cellular DNA Flow Cytometric Analysis Kit (KeyGEN, Nanjing, China) according to the manufacture’s instructions. The percentage of cell cycle distribution was determined using a FACScan laser flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were analyzed using the software CELLQuest.

A549 cells were treated with xanthatin at indicated concentrations for 12 or 24 h. Then they were harvested (including attached and detached cells), washed and resuspended with PBS. Apoptotic cells were determined with an FITC Annexin V Apoptosis Detection Kit (KeyGEN, Nanjing, China) according to the manufacturer’s protocol. Apoptosis was analyzed by fluorescence microscope and FACScan laser flow cytometer. The data were analyzed using software (CELLQuest).

After xanthatin treatment (20 μM) for 0, 12 or 24 h, attached cells were washed twice with PBS and fixed with 4% formaldehyde at 4 °C for 30 min. The fixing solution was removed and cells were washed twice with PBS before staining with Hoechst 33258 (Beyotime, Nanjing, China). After staining for 10 min, cells were washed again and observed under a fluorescence microscope (Olympus IX-70, Tokyo, Japan). At least 20 fields were randomly selected and images were taken.

Whole cell proteins were extracted using radioimmunoprecipitation (RIPA) assay buffer supplemented with 1 mM PMSF and 1:100 dilution of Protease Inhibitor Cocktail (Bestbio, Shanghai, China). Protein concentration was determined using the BCA™ protein assay kit (Pierce, Rockford, IL, USA). Protein samples (50 μg) were separated on SDS-polyacrylamide gels (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 for 2 h, the membranes were incubated with primary antibodies at 1:200 to 1,000 dilutions with 5% BSA in TBST overnight at 4 °C. The blots were washed and incubated with HRP-conjugated secondary antibodies (1:10,000, Bioworld, St. Louis Park, MN, USA) for 1 h at room temperature. Membranes were visualized using enhanced chemiluminescence (Immobilon ECL, Millipore).

The effect of xanthatin on TNFα-induced nuclear translocation of p65 was examined using an immunocytochemical method. Cells were seeded in growth medium in 24-well plates (5 × 10⁴ per well) and grown overnight at 37 °C in a humidified incubator with 5% CO₂. Cells were pretreated with 40 μM xanthatin for 24 h before being stimulated with 20 ng/mL TNFα (Signalway, Jiangsu, China) for 10 min. Treated cells were washed with cold PBS followed by fixation with cold acetone for 10 min at 4 °C. Cells were permeabilized with 0.5% triton X-100 (Sigma, St. Louis, MO, USA) for 10 min at room temperature, washed with PBS and blocked with 1% bovine serum albumin (BSA) for 1 h. Rabbit anti-p65 antibody was added and incubated overnight at 4 °C. After washing with PBS for three times, goat anti-rabbit IgG-Tritc (ZHONGSHAN, Beijing, China) was added and incubated for 1 h at room temperature. Fluorescence cells were observed and photographed under a laser scanning confocal microscope (LEICA, Mannheim, Germany). The nuclear proteins after treating with xanthatin or/and TNF-α were extracted using Nuclear and Cytoplasmic Extraction Reagents (KeyGEN, Nanjing, China) following the instruction. The extracts were analyzed by Western blot assay using antibodies against p-NF-κB (p65). Histon H3 was used as reference.

All data are expressed as mean ± SD. Statistical comparisons between groups performed using 1-way ANOVA followed by Student’s t-test and a p value of less than 0.05 was considered statistically significant.