Original ArticlesFLLL12 induces apoptosis in lung cancer cells through a p53/p73-independent but death receptor 5-dependent pathway
Introduction
Lung cancer represents ~13% of all cancers with approximately 224,000 new cases and 159,000 deaths in the United States in 2014 [1]. Lung cancer remains the top leading cause of cancer related mortality because of low survival rate and high prevalence of advanced stage disease at diagnosis [2]. Extensive worldwide epidemiological data have established cigarette smoking (direct as well as second hand) as the key cause of lung cancers [3], [4]. Occupational exposures to environmental pollutants are other causes of lung cancers. Non-small cell lung cancer (NSCLC) is the most prevalent subtype and accounts for 85% of all lung cancer cases, and is relatively resistant to chemotherapy and radiation. The treatment of lung cancer still remains a challenge due to lack of effective screening tools for early detection, the presence of smoking-related comorbid illnesses, and the inherent molecular heterogeneity [5]. Despite advances in conventional surgical procedures, radiotherapy, and chemotherapy, the 5 year survival rate for lung cancer remains almost unchanged at ~15% [6]. Moreover, radiation and chemotherapy are frequently associated with serious side effects including pneumonitis and/or long-term fibrosis, renal, otologic, and bone marrow-suppressive sequelae [7]. Thus, there is an increasing emphasis on strategies to maximize tumor control, prolong overall survival, minimize chemotherapy side effects, and improve quality of life. As a result, there has been continued investigation into potential alternative and less toxic therapies for lung cancer, with the aim of achieving a more favorable clinical outcome while reducing treatment morbidity.
The safety of natural dietary agents present in fruits, vegetables and spices has been established through years of human consumption and these agents have drawn increasing attention when designing anti-cancer drugs [8]. Curcumin, the most abundant component of the spice turmeric and isolated from the rhizome of the Indian medicinal plant Curcuma longa, is one such compound extensively investigated for its potential anti-cancer effects [7], [9], [10]. Accumulated evidence suggests that curcumin affects a variety of biological pathways involved in apoptosis, tumor proliferation, chemo- and radiotherapy sensitization, tumor invasion, and metastases [7], [9], [11], [12]. However, low bioavailability, limited tissue distribution, rapid metabolism and subsequent excretion from the body limit the potential use of curcumin in cancers [13]. To circumvent these problems, numerous analogs of curcumin have been synthesized. For example, the potential activity of recently synthesized curcumin analogs (2E,6E)-2,6-bis(thiophen-3-methylene) cyclohexanone (AS), (3E,5E)-3,5-bis(thiophen-3-methylene)-tetrahydrothiopyran-4-one (FS), A501, and GO-Y031 has been studied in prostate cancers, NSCLC cells and gastric cancers [14], [15], [16].
Successful elimination of cancer cells from the body depends on the induction of cell death. Apoptosis is the major form of cell death and serves as one of the most powerful tools in the development of cancer chemotherapeutics. Defects in apoptosis are implicated in both tumorigenesis and drug resistance, and are the major causes of treatment failures [17]. Apoptosis can be initiated by two major pathways: either through mitochondria (intrinsic pathway) or through death receptors located in the cell surface (extrinsic pathway). In the case of extrinsic apoptosis, activation of death receptors via ligand binding or drugs initiates the formation of the death inducing signaling complex (DISC). DISC formation involves recruitment of the adaptor protein FADD to the receptor via the death domain (DD) and the inactive pro-caspases 8 and 10. This facilitates the activation and self-processing of caspases 8 and 10, leading to their release into the cytoplasm, where they activate effector caspases 3 and 7, leading to the induction of apoptosis [18]. Activated caspase-8 can also cleave the Bcl-2-family member Bid, which links the extrinsic pathway to the intrinsic pathway. Death receptor 5 (DR5) is a tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptor that activates apoptosis via the extrinsic apoptosis pathway [19]. Recent studies have shown that cancer cells resistant to TRAIL can be sensitized by co-treatment with classical chemotherapeutic drugs. This synergism is partially due to the ability of chemotherapeutic drugs to induce DR5 expression [20], [21], [22]. Hence, targeting the expression of DR5 and induction of apoptosis by anti-cancer drugs has drawn special attention for cancer therapy.
In the current study, we investigated the mechanism of FLLL12-induced apoptosis in lung cancer cell lines. FLLL12 is a potent synthetic curcumin analog [23]. The anti-cancer efficacy of FLLL12 has been tested against breast, prostate, colorectal and pancreatic cancers [23], [24], [25], and shown to be ~10-fold more potent than natural curcumin and to possess selective activity against cancer cells. FLLL12 induces apoptosis of these cancer cells by inhibition of two major survival pathways, AKT and STAT3. However, the detailed mechanism underlying FLLL12-induced apoptosis is not fully understood. Moreover, FLLL12 has never been tested against lung cancer cells. In the present study, for the first time, we investigated the anti-tumor effects of FLLL12 in lung cancers and showed that depending on the cell line, FLLL12 is 5–10-fold more potent than curcumin and induces apoptosis. Moreover, we further explored the fact that FLLL12, but not curcumin, induces DR5-mediated apoptosis in lung cancer cell lines.
Section snippets
Cell lines
Human lung cancer cell lines used in this study were obtained from Dr. Sun's laboratory (Emory University). All cell lines were authenticated through genotyping and maintained in RPMI medium supplemented with 5% heat-inactivated fetal bovine serum in a 37 °C and 5% CO2 humidified incubator. Generation and maintenance of the premalignant cell lines 1799 and 1198 were described elsewhere [26].
Reagents
Curcumin was purchased from Sigma Chemical (St. Louis, MO, USA) and FLLL12 was obtained from Dr. James R.
FLLL12 is 5–10-fold more potent than curcumin and induces apoptosis
Although the antitumor effects of FLLL12 have been investigated in prostate, breast, pancreatic and colon cancer cell lines and compared with those of curcumin [23], [24], [25], the agent has never been evaluated in lung cancer cell lines. In order to explore the mechanism of anti-tumor effect of FLLL12 in lung cancers, we first assessed the sensitivity of different premalignant and malignant lung cancer cell lines to FLLL12 versus curcumin by comparing IC50 values measured using SRB assays at
Discussion
Treatment outcomes in advanced or metastatic NSCLC remain frustrating, with low long-term survival rates. Overall, five-year survival for lung cancer patients is ~15%, and patients with advanced stage disease often die within 12 months [6]. Although recent advances in molecularly targeted treatments have made lung cancer therapy more personalized, the benefits are typically lost within months to a few years [42]. Long-term disease control requires activation of the apoptotic machinery that
Authors' contributions
Conception and design: Haque and Amin.
Development of methodology: Haque, Amin, and Rahman.
Acquisition of data: Haque and Amin.
Analysis and interpretation of data: Haque, Amin, Chen, and Shin.
Writing, review and/or revision of the manuscript: Haque, Amin, Fuchs, Saba, Khuri and Shin.
Study supervision: Amin.
Funding
This study was supported by R03CA171663, R03CA159369 and Robbins Scholar Award of Winship Cancer Institute of Emory University.
Conflict of interest
The authors declare no potential conflict of Interest.
Acknowledgments
We are thankful to Dr. Anthea Hammond and Shi-Yong Sun for carefully editing the manuscript and providing the luciferase construct, respectively.
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