NF-κB targeting by way of IKK inhibition sensitizes lung cancer cells to adenovirus delivery of TRAIL

BMC Cancer NF- B targeting by way of IKK inhibition sensitizes lung cancer cells to adenovirus delivery of TRAIL Cigdem Aydin 2 Ahter D Sanlioglu 2 Atil Bisgin 0 Burcak Yoldas 1 Levent Dertsiz 5 Bahri Karacay 4 Thomas S Griffith 3 Salih Sanlioglu 0 0 Department of Medical Genetics, Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics , Antalya, 07058 , Turkiye 1 Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics , Antalya, 07058 , Turkiye 2 Department of Medical Biology and Genetics, Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics , Antalya, 07058 , Turkiye 3 Department of Urology and Gene Therapy Center, University of Iowa , Iowa City, IA, 52242 , USA 4 Department of Pediatrics and Gene Therapy Center, University of Iowa , Iowa City, IA, 52242 , USA 5 Department of Thorasic Surgery, Akdeniz University Faculty of Medicine , Antalya, 07058 , Turkiye Background: Lung cancer causes the highest rate of cancer-related deaths both in men and women. As many current treatment modalities are inadequate in increasing patient survival, new therapeutic strategies are required. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells but not in normal cells, prompting its current evaluation in a number of clinical trials. The successful therapeutic employment of TRAIL is restricted by the fact that many tumor cells are resistant to TRAIL. The goal of the present study was to test a novel combinatorial gene therapy modality involving adenoviral delivery of TRAIL (Ad5hTRAIL) and IKK inhibition (AdIKKbKA) to overcome TRAIL resistance in lung cancer cells. Methods: Fluorescent microscopy and flow cytometry were used to detect optimum doses of adenovirus vectors to transduce lung cancer cells. Cell viability was assessed via a live/dead cell viability assay. Luciferase assays were employed to monitor cellular NF- B activity. Apoptosis was confirmed using Annexin V binding. Results: Neither Ad5hTRAIL nor AdIKKbKA infection alone induced apoptosis in A549 lung cancer cells, but the combined use of Ad5hTRAIL and AdIKKbKA significantly increased the amount of A549 apoptosis. Luciferase assays demonstrated that both endogenous and TRAIL-induced NF- B activity was down-regulated by AdIKKbKA expression. Conclusions: Combination treatment with Ad5hTRAIL and AdIKKbKA induced significant apoptosis of TRAILresistant A549 cells, suggesting that dual gene therapy strategy involving exogenous TRAIL gene expression with concurrent IKK inhibition may be a promising novel gene therapy modality to treat lung cancer. - Backround Lung cancer is the leading cause of cancer mortality in the world (31% for men and 26% for women of all cancer deaths) [1]. Despite the use of conventional multimodal treatment methods (chemotherapy, radiation, and surgery), the overall survival rate from lung cancer has improved little, with <15% of patients surviving >5 years [2]. Consequently, new therapeutic strategies, such as gene therapy, are being tested in preclinical and clinical settings. Knowing that apoptosis is a key mechanism in the regulation of tissue homeostasis, several members of the tumor necrosis factor (TNF) superfamily have been implicated in the process. TNF-related apoptosisinducing ligand (TRAIL), also known as Apo2L, was originally identified through its homology to TNF, FasL, and other members of the TNF superfamily [3,4]. Like most other members of the TNF superfamily of ligands, TRAIL is primarily expressed as a type II membrane protein of 33-35 kD [5]. To date, four human membrane-bound receptors for TRAIL have been identified: DR4/TRAIL-R1, DR5/TRAIL-R2/KILLER, TRID/DcR1/ TRAIL-R3, and DcR2/TRAIL-R4. Two of the membrane receptors, DR4 and DR5, contain the essential cytoplasmic death domain through which TRAIL can transmit an apoptotic signal. DcR1 and DcR2 can also bind TRAIL, but they appear to act as antagonistic receptors because they lack a functional death domain [6-9]. There are several reasons why TRAIL is of interest for people working on cancer gene therapy. TRAIL is unique in that it selectively induces apoptosis in tumor and transformed cells, but does not harm normal cells [10,11]. In addition, apoptosis induction in response to most DNA-damaging drugs usually requires functional tumor supressor p53 gene [12]. Because of the inactivation of p53 in more than 50% of human cancers during tumorigenesis, the tumors eventually display resistance to both radiotherapy and chemotherapy. TRAIL, however, can induce p53-independent apoptosis of cancer cells [13]. Despite this fact, a significant proportion of tumor cells display TRAIL resistance by a mechanism that is not yet fully understood [14,15]. Resistance to TRAIL-induced apoptosis, both in normal and cancer cells, was initially considered to be due to DcR1 and/or DcR2 expression, which compete with DR4 and DR5 for binding to TRAIL [6,16]. Apart from TRAIL receptor composition, [17,18] there are a number of other possible reasons why some cancer cells exhibit TRAIL resistance. For example, the presence of intracellular apoptosis inhibitory proteins (Bcl-xL, c-FLIP, cIAP etc.) or the loss of Bax and Bak function may lead to a TRAIL-resistant phenotype [14,19]. Interestingly, the engagement of DR4, DR5, and DcR2 can activate the NF- B pathway [20,21], and high levels of endogenous NF- B activity interfere with TRAIL-induced apoptosis. Thus, targeting the NF- B signaling pathway may help sensitize cancer cells to TRAIL. In this study, a complementary gene therapy modality using adenovirus-mediated delivery of an IKKbA mutant (AdIKKbKA) was deployed to test the extent to which NF- B inhibition sensitized lung cancer cells to TRAIL (Ad5hTRAIL). Methods Adenovirus Preparation Recombinant adenoviral vectors, AdEGFP [22], Ad5hTRAIL [23], AdIKKbKA [24], AdNF BLuc [25], and AdCMVLacZ [26,27], were amplified in 293 cells and purified by cesium chloride gradient. After vector purification, adenoviral vectors were kept at -80C in 10 mM Tris containing 20% glycerol. The titers of purified adenoviral stocks were measured to be 1013 DNA particles/ ml. AdIKKbKA encodes the dominant negative mutant form (K44A) of IKKb and forms inactive IKK complex so that IKKb does not phosphorylate IkB. IkBaSR produces dominant negative mutant form (S32A/S36A) of IkBa. Thus, the IKK complex cannot phosphorylate mutant IkBa from S32 and S36 residues. By doing so NF- B is always sequestered in cytoplasm. Both mutant proteins interfere with NF- B signaling at different levels of the signaling cascade. Cell Culture The human non-small cell lung carcinoma cell line A549 was obtained from American Type Culture Collection. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2.2 g/l sodium bicarbonate, 1 mM L-glutamine, and 1% penicillin-streptomycinamphoterisine mixture (PSA) using Thermo SteriCult incubators. The study was carried out in a (...truncated)