ANTICANCER RESEARCH 36: 3383-3394 (2016) Enhanced TLR4 Expression on Colon Cancer Cells After Chemotherapy Promotes Cell Survival and Epithelial–Mesenchymal Transition Through Phosphorylation of GSK3β YOON HEE CHUNG1 and DAEJIN KIM2 1Department of Anatomy, Chung-Ang University College of Medicine, Seoul, Republic of Korea; 2Department of Anatomy, Inje University College of Medicine, Busan, Republic of Korea Abstract. Background: Phosphorylation of glycogen dependent apoptosis, and blocked the expression of cancer synthase kinase 3β (GSK3β) by phosphatidyl-inositide 3- stem cell markers and invasive characteristics in LPS- kinase (PI3K)/protein kinase B (AKT) or inhibition of GSK3β stimulated drug-treated cells. In addition, the ERK-specific with small-molecule inhibitor attenuates cell survival and inhibitor, PD98059, triggered the apoptosis of TLR4- proliferation and increases apoptosis in most cancer cell activated drug-exposed colon cancer cells, whereas there was lines. In this study, we investigated the role of phosphorylated no effect on the expression of epithelial–mesenchymal GSK3β activated by enhanced toll-like receptor 4 (TLR4) transition markers or GSK3β phosphorylation. Conclusion: expression in drug-treated colon cancer cells as a model of These results suggest that TLR4-induced GSK3β and ERK post-chemotherapy cancer cells. Materials and Methods: The phosphorylation independently controls cancer cell survival effect of TLR4 stimulation on metastasis and apoptosis in and regulation of GSK3β and ERK after chemotherapy, drug-exposed colon cancer cells was determined by real-time making TLR4 a critical target for reducing drug resistance polymerase chain reaction (PCR) and immunoblotting. and metastasis in patients with colon cancer. Results: Despite the induction of apoptosis after treatment with oxaliplatin and 5-fluorouracil, lipopolysaccharide (LPS) Toll-like receptor 4 (TLR4) is expressed in various types of stimulation via increased TLR4 in drug-treated cancer cells human cancer, including prostate, pancreatic, liver, colon, effectively inhibited apoptosis through up-regulation of and breast cancer (1-5). TLR4, the receptor for expression of anti-apoptosis-related B-cell lymphoma 2 lipopolysaccharide (LPS), primarily induces inflammatory (BCL2) family proteins [X-linked inhibitor of apoptosis cytokines in immune cells, but it is also involved in protein (XIAP), BCL2, and survivin] and drug-resistance carcinogenesis and cancer cell survival (1). Furthermore, proteins [multidrug-resistance protein 1 (MDR1), multidrug LPS can modify cytokine levels of the tumor resistance-associated protein (MRP)1/2/3]. LPS-mediated microenvironment to promote tumor growth, invasion and signaling in drug-treated cancer cells elevated the expression metastasis in vitro and in vivo (6). Adjuvant chemotherapy of phosphorylated GSK3β, extracellular signal–regulated with 5-fluorouracil (5-FU) and leucovorin is commonly kinase (ERK), and the p65 subunit of nuclear factor kappa- adopted to reduce the risk of recurrence in patients with light-chain-enhancer of activated B-cells (NF-ĸB). advanced colon cancer after surgery (7). 5-FU induces TLR4 Pharmacological inhibition of GSK3β (using SB216763) expression in colon cancer cells and synergistically enhances reduced phosphorylation of GSK3β, re-activated caspase- apoptotic cell death. In addition, the drug significantly induces the expression of TLR4 regardless of stimulation with LPS (8). TLR4 and interleukin (IL-6) up-regulation is a common feature of colorectal cancer and high expression of Correspondence to: Daejin Kim, MD, Ph.D., Department of TLR4 and myeloid differentiation (MyD) 88-mediated Anatomy, Inje University College of Medicine, 75, Bokji-ro, Jin- signaling are associated with liver metastasis and poor gu, Busan 47392, Republic of Korea. Tel: +82 518906637, Fax: +82 51896-6634, e-mail: [email protected] prognosis (6). Based on these results, the exact role of increased TLR4 expression after anticancer drug treatment Key Words: TLR4, epithelial–mesenchymal transition, EMT, GSK- of cancer cells is still unclear and the role of LPS-induced 3β, colorectal cancer. TLR4 signaling after chemotherapy is also controversial. 0250-7005/2016 $2.00+.40 3383 ANTICANCER RESEARCH 36: 3383-3394 (2016) GSK3β is a ubiquitously expressed serine/threonine kinase were maintained in RPMI-1640 medium, supplemented with 10% that regulates glycogen synthesis (9). The activity of GSK3β is fetal bovine serum (FBS) (HyClone, Logan, UT, USA), regulated by the phosphorylation of N-terminal serine-9 (p- streptomycin, and glutamine at 37˚C in 5% CO2. LPS, oxaliplatin and 5-FU were purchased from Sigma-Aldrich (St. Louis, MO, GSK3βSer9) (14). The phosphorylation of GSK3β is necessary USA). LY294002 (PI3K/AKT inhibitor), PD98059 (ERK inhibitor), to induce inflammatory responses in LPS-stimulated and SB216763 (GSK3β inhibitor) were purchased from Calbiochem macrophages through increased nuclear translocation and (San Diego, CA, USA). accumulation of β-catenin (10). LPS-mediated GSK3β phosphorylation, or inactivation, in macrophages enhances Analysis of apoptosis by flow cytometry. Cells (1.5×105 cells/ml) production of the pro-inflammatory cytokine interferon-β (11). were cultured in 6-well plates and pre-treated with either oxaliplatin The dephosphorylated and active form of GSK3β in resting (5 μM) or 5-FU (10 μM) singly for 8 h and then treated with LPS (500 ng/ml) for an additional 18 h. For comparison, cells were cells regulates cell activation, differentiation, and survival treated either with oxaliplatin (5 μM) or 5-FU (10 μM) alone for 8 (12). GSK3β, as a tumor suppressor, also inhibits a diverse h, subsequently washed and then incubated with complete medium group of pro-oncogenic substrates, such as cyclin D1, c-JUN, for an additional 18 h. The percentages of cells undergoing c-MYC and cAMP response element-binding protein (CREB) apoptosis were determined by flow cytometry using fluorescein via induction of phosphorylation (13). In addition, inactivation isothiocyanate (FITC)-labeled annexin-V (BD Biosciences, San by phosphorylation of GSK3β has been reported in many Diego, CA, USA) and 7-aminoactinomycin D (7-AAD) (BD cancer cells (15, 16). Biosciences). Cells were harvested, rinsed with phosphate-buffered saline (PBS), and resuspended in 100 μl of 1× annexin-V binding Many studies have demonstrated that pharmacological buffer. Next, 3 μl of annexin-V-fluorescein isothiocyanate (FITC) inhibitors of GSK3β, such as thiadiazolidinone or SB216763, and 3 μl of 7-AAD were added, and cells were incubated at room can prevent proliferation and induce apoptosis of tumor cells, temperature for 15 min in the dark with gentle vortex. The stained including multiple myeloma and ovarian cancer cell lines (17, cells were analyzed using a FACSCalibur flow cytometer (BD 18). The phosphorylation of GSK3β by protein kinase B Biosciences) equipped with CellQuestpro software (BD (PKB), also known as AKT, was also found to cause a dose- Biosciences). and time-dependent decrease in cell proliferation after TLR4 stimulation, while inhibition of GSK3β with lithium chloride Quantitative real-time polymerase chain reaction (PCR). Total cellular RNA was extracted using an RNeasy Mini Kit (Qiagen, reversed LPS-mediated growth inhibition in normal intestinal Hilden, Germany) according to the manufacturer’s protocol. cDNA enterocytes (19). The levels of active GSK3β expression in was made from 2 μg total RNA using oligo(dT) (Bioneer, Daejeon, colon cancer cells and patients with colorectal cancer are Korea) and reverse transcriptase (Bioneer). Quantitation of mRNA higher than in their normal counterparts (20), whereas the levels was measured using an ABI7300 real-time PCR system inactive form, phosphorylated GSK3βSer9, is frequently (Applied Biosystems, Foster City, CA, USA) and SYBR Green detected at high levels in normal tissues (20). Inhibition of Master Mix kit (Takara, Tokyo, Japan) with specific primer sets for: GSK3β leads to attenuated proliferation and increased X-linked inhibitor of apoptosis protein (XIAP) (upstream primer, 5’- GTG CCA CGC AGT CTA CAA ATT CTG G; downstream primer, apoptosis by blocking the nuclear factor-ĸB (NF-ĸB) 5’-CGT GCT TCA TAA TCT GCC ATG GAT GG), B-cell lymphoma activation in pancreatic and colon cancer cells (21, 22). 2 (BCL2) (upstream primer, 5’-GGA TTG TGG CCT TCT TTG AG; GSK3β is also significantly more activated in drug-resistant downstream primer, 5’-CAG CCA GGA GAA ATC AAA CAT); and versus responsive patients after treatment with 5-FU (23). survivin (upstream primer, 5’-GAT TTG AAT CGC GGG ACC CGT Based on these results, the precise role of GSK3β in different TG; downstream primer, 5’-TCA AGA CAA AAC AGG AGC ACA types of cancer and under diverse conditions remains unclear. GT). A specific primer set for β-actin (upstream primer, 5’-ATC CAC In the present study, we investigated whether an increase GAA ACT ACC TTC AA; downstream primer, 5’-STC CAC ACG GAG TAC TTG C) was used as a control. The relative mRNA in TLR4 expression in drug-treated cancer cells regulates quantification was calculated using the arithmetic formula 2–ΔΔCq, colon cancer cell survival after exposure to LPS. We also where ΔCq was the difference between the threshold cycle of a given examined subsequent GSK3β-mediated activation of target cDNA and an endogenous reference cDNA. downstream factors in drug-treated colorectal cancer cells, including NF-ĸB and mitogen-associated protein kinase Detection of PI3K, GSK3β, and