Background
Lung cancer is the leading cause of cancer deaths in the world with over one million cases diagnosed every year. Multiple options for the treatment of lung cancer have been described, including surgery, chemotherapy and radiation; however, therapeutic efficacy is typically transient and mostly absent with advanced disease [
1,
2]. Therefore, the need for more rational approaches to lung cancer therapy is essential.
Chrysin (5, 7-dihydroxyflavone) is a naturally occurring flavonoid possessing a broad range of pharmacological activities and is widely found in fruits, vegetables, honey and propolis [
3,
4]. Recent studies have shown that chrysin in diets is a promising biological anti-cancer agent. Chrysin has been demonstrated to induce apoptosis of human myeloid leukemia cells via activation of caspases and inactivation of Akt [
5]. Our previous studies demonstrated that chrysin and its derivatives exhibited significant anticancer effect against gastric cancer SGC-7901 cells and colorectal cancer HT-29 cells [
6,
7]. However, administration of chrysin alone is insufficient. The combination of chrysin with other anti-cancer agents or modifications to its structure may improve the biological activity of chrysin.
Our previous studies showed that inhibition of proliferation and induction of apoptosis by 5,7-dihydrox-8-nitrochrysin and 8-bromo-7-methoxychrysin in human gastric carcinoma SGC-7901 cells and hepatocellular carcinoma cells, respectively, was stronger than that of the lead compound chrysin [
8,
9]. 5-allyl-7-gen-difluoromethoxychrysin (AFMC) is another important derivative of chrysin. We previously reported that AFMC inhibited the proliferation of the ovarian cancer CoC1 cell line and hepatocellular carcinoma HepG2 cell line [
10,
11]. However, whether AFMC possesses antitumor effects on human non-small cell lung cancer (NSCLC) cells and the molecular mechanisms of its action remain to be determined.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily, which includes potent inducers of apoptosis in a wide variety of cancer cells [
12‐
14]. In particular, TRAIL is considered as the most promising anticancer agent in the TNF superfamily because of its selective cytotoxicity against tumor cells versus normal primary cells. TRAIL induces apoptosis in various cancer cells through its interaction with death receptor 5 (DR5), which contains a cytoplasmic death domain capable of recruiting apoptosis signaling molecules and inducing apoptosis [
15‐
21]. However, many tumor cells are resistant to TRAIL-induced apoptosis [
22,
23]. Various cancer therapeutic agents have been demonstrated to augment TRAIL-induced apoptosis through induction of DR5 expression [
24‐
30], indicating that DR5 expression levels might be involved in resistance to TRAIL. Thus, induction of DR5 expression could enhance cytotoxicity and apoptosis mediated by TRAIL.
In this study, we investigated the apoptotic effects of AFMC in combination with TRAIL on NSCLC cells. We show for the first time that AFMC synergistically enhances TRAIL-mediated apoptosis in NSCLC cells through up-regulating DR5 expression. Thus, our findings raise the possibility that combined use of AFMC and TRAIL could be a candidate therapy for the treatment of human NSCLC.
Discussion
Flavonoids are a broad class of plant polyphenols that have important pharmacological effects. Recent studies have shown that chrysin and its derivatives possess strong anticancer activities. Methylated polyphenol derivatives have been reported to possess stronger bioactivities and higher hepatic metabolic stability and intestinal absorption than the lead compound [
40,
41], making these derivatives more favorable than the unmethylated lead compound for development as potential cancer chemopreventive agents. Our previous studies showed that AFMC, a derivative of chrysin, potently inhibited the proliferation of ovarian cancer CoC1 cells and human hepatocellular carcinoma HepG2 cells in a dose-dependent manner [
11,
42]. In this study, we found that AFMC sensitized A549 cells to TRAIL-induced apoptosis and enhanced the cytotoxic effects of TRAIL but had little effect on human embryo lung WI-38 cells (Figures
1 and
6). Combined treatment with subtoxic concentrations of AFMC and TRAIL induced caspase-3 and -8 activation and apoptosis of A549 cells (Figure
2). Activation of caspases plays an important role in apoptosis triggered by various proapoptotic signals [
42,
43]. Two major apoptotic pathways exist: one involving signals transduced through death receptors and one dependent on signals from the mitochondria [
9,
42]. Both pathways activate the caspase cascade, leading to cleavage of cellular substrates and the morphological and biochemical manifestations of apoptosis. The activation of caspase-8 and -9 has been documented to play central roles in mediating apoptosis through death receptors and the mitochondrial pathway, respectively [
9,
42]. However, caspase-8 can activate caspase-9-mediated apoptotic pathways via activation or cleavage of Bid protein [
42,
43]. Therefore, our results suggest that the induction of apoptosis in A549 cells by AFMC and TRAIL might involve death signals transduced through death receptors. As expected, we found that AFMC increased the expression of DR5 in a concentration- and time-dependent manner in A549 cells but not in WI-38 cells. In addition, we demonstrated that AFMC enhanced TRAIL-induced apoptosis in human lung cancer A549 cells through upregulation of DR5. Collectively, our findings could yield new insights into maximizing the efficiency of human lung carcinoma treatments while minimizing side effects to normal tissues.
Lung cancer represents the leading cause of cancer deaths in the world [
1,
2]. NSCLC accounts for approximately 75% of lung cancers [
44]. About 50% of NSCLC patients are initially diagnosed with advanced or metastatic disease, and the treatment of choice is palliative chemotherapy. Apoptosis induction is one of the major objectives of modern cancer therapy, for which an important goal is to specifically induce apoptosis in malignant tumor cells but not in normal cells. For this reason, TRAIL has recently received much attention because it preferentially kills tumor cells while leaving normal cells unaffected. However, a considerable number of tumors are resistant to TRAIL-induced apoptosis [
17,
21]. Various conventional chemotherapeutic agents have been demonstrated to synergistically augment TRAIL-induced apoptosis through induction of DR5 expression. In this regard, AFMC is an attractive agent for inducing DR5 expression and sensitizing TRAIL-resistant A549 cells to TRAIL.
Our study has important implications in cancer treatment. As mentioned above, AFMC enables to overcome the resistance to TRAIL in NSCLC. Furthermore, more than half of all malignant tumors possess an inactivating mutation in the
p53 gene and
p53 modulates the sensitivity to conventional anticancer agents [
45,
46]. Although H157 cells harbored inactivated
p53 [
32], combined treatment with AFMC and TRAIL is also useful for
p53-deficient tumor cells.
Activation of death receptor pathway is a new strategy for targeted therapy of cancer. Our previous study demonstrate AFMC induced apoptosis of human hepatocellular carcinoma cells via inhibition of NF-κB. Studies by Srivastava et al. shown that MS-275, a histone deacetylase (HDAC) inhibitors, could sensitize TRAIL-resistant breast cancer xenografts through the involvement of both cell-extrinsic and cell-intrinsic pathways of apoptosis[
47] and resveratrol regulates the expression of FOXO's target genes such as Bim, TRAIL, DR4, DR5, cyclin D1 and p27/KIP1 [
48,
49]. However, how AFMC induces DR5 upregulation and enhances TRAIL-induced apoptosis in NSCLC cells need to be further investigated.
Clinically, resistance to apoptosis is a major obstacle in chemotherapeutic treatments of cancers. The ability of AFMC to sensitize A549 cells to TRAIL-induced apoptosis at subtoxic concentrations makes AFMC a potentially effective preventative and/or therapeutic agent against NSCLC. However, the potential clinical implications of our studies will depend on whether or not AFMC can be safely administered to patients in high enough doses to be pharmacologically active. Oral flavonoids cannot be utilized in the clinic because of poor bioavailability because of rapid metabolism in the liver and intestinal wall. However, Walle
et al[
41]. reported that dimethoxyflavone (DMF) was clearly detected in plasma with a peak concentration of 2.5 ± 0.8 μM (mean ± SEM) at 1 h after gavage (5 mg/kg each) to rats. In that study, the area under the plasma concentration-time curve was 58.8 μg • ml
-1 • min. DMF was also clearly detected in the liver, lung and kidney tissues with concentrations in the liver exceeding those in the plasma by as much as seven-fold (16.5 ± 5 μM) 1 h after administration. The liver, which showed the highest accumulation of DMF, might be a site where DMF exerts its greatest activity. Therefore, additional
in vivo studies are needed to evaluate the applicability of AFMC as a chemopreventive and/or therapeutic agent for cancer.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ZHX, MFQ and FL performed the majority of experiments. JGC participated in its design and coordination and was also involved in revising the manuscript. JSZ conceived of the study and wrote the manuscript. All authors read and approved the final manuscript.