PURPOSE: Pim kinases are highly conserved serine/threonine kinases, and different expression patterns of each isoform (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancers, including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effects of AZD1208 as a single agent and in combination with an Akt inhibitor in gastric cancer cells. MATERIALS AND METHODS: The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assays. The underlying mechanism was also examined by western blotting, immunofluorescence assay, and cell cycle analysis. RESULTS: AZD1208 treatment decreased gastric cancer cell proliferation rates and induced autophagy only in long-term culture systems. Light chain 3B (LC3B), a marker of autophagy, was increased in sensitive cells in a dose-dependent manner with AZD1208 treatment, which suggested that the growth inhibition effect of AZD1208 was achieved through autophagy, not apoptosis. Moreover, we found that cells damaged by Pim inhibition were repaired by activation of the DNA damage repair pathway, which promoted cell survival and led the cells to become resistant to AZD1208. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines. CONCLUSION: Treatment with AZD1208 alone induced considerable cell death through autophagy in gastric cancer cells. Moreover, the combination of AZD1208 with an Akt inhibitor showed synergistic antitumor effects through regulation of the DNA damage repair pathway.
Apoptosis ; Autophagy ; Blotting, Western ; Cell Cycle ; Cell Death ; Cell Line ; Cell Proliferation ; Cell Survival ; DNA Damage ; Fluorescent Antibody Technique ; Humans ; Phosphotransferases ; Protein Isoforms ; Stomach Neoplasms

PURPOSE: Overexpression of cyclooxygenase 2 (COX-2) is thought to promote survival of transformed cells. Transforming growth factor beta (TGF-beta) exerts anti-proliferative effects on a broad range of epithelial cells. In the current study, we investigated whether TGF-beta can regulate COX-2 expression in A549 human lung adenocarcinoma cells, which are TGF-beta-responsive and overexpress COX-2. MATERIALS AND METHODS: Western blotting, Northern blotting, and mRNA stability assays were performed to demonstrate that COX-2 protein and mRNA expression were suppressed by TGF-beta. We also evaluated the effects of tristetraprolin (TTP) on COX-2 mRNA using RNA interference. RESULTS: We demonstrated that COX-2 mRNA and protein expression were both significantly suppressed by TGF-beta. An actinomycin D chase experiment demonstrated that COX-2 mRNA was more rapidly degraded in the presence of TGF-beta, suggesting that TGF-beta-induced inhibition of COX-2 expression is achieved via decreased mRNA stability. We also found that TGF-beta rapidly and transiently induced the expression of TTP, a well-known mRNA destabilizing factor, before suppression of COX-2 mRNA expression was observed. Using RNA interference, we confirmed that increased TTP levels play a pivotal role in the destabilization of COX-2 mRNA by TGF-beta. Furthermore, we showed that Smad3 is essential to TTP-dependent down-regulation of COX-2 expression in response to TGF-beta. CONCLUSION: The results of this study show that TGF-beta down-regulated COX-2 expression via mRNA destabilization mediated by Smad3/TTP in A549 cells.
Adenocarcinoma ; Blotting, Northern ; Blotting, Western ; Cyclooxygenase 2 ; Dactinomycin ; Down-Regulation ; Epithelial Cells ; Humans ; Lung ; Lung Neoplasms* ; RNA Interference ; RNA Stability ; RNA* ; RNA, Messenger ; Transforming Growth Factor beta* ; Tristetraprolin

Purpose: Endocrine therapy is a standard treatment for hormone receptor-positive breast cancer, which accounts for 60%–75% of all breast cancer. Hormone receptor positivity is a prognostic and predictive biomarker in breast cancer. Approximately 50%–80% of breast cancer is also positive for androgen receptor (AR), but the prognostic and predictive value of AR expression in breast cancer is controversial. Here, we investigated AR expression and its prognostic value in patients with surgically resected breast cancer in Korea. Methods: We retrospectively reviewed the medical records of patients who had surgically resected breast cancer to collect AR expression data and other clinicopathological data. The optimal cut-off for AR positivity was determined using a receiver operating characteristic curve analysis. Results: We reviewed 957 patients with surgically resected breast cancer from June 2012 to April 2013. The median follow-up was 62 months, and relapse events occurred in 101 (10.6%) patients. Unlike the cut-off value of 1% or 10% in previous reports, 35% was determined to be best for predicting relapse-free survival (RFS) in this study. At the cut-off value of 35%, 654 (68.4%) patients were AR-positive. AR expression was more prevalent in luminal A (87.6%) and luminal B (73.1%) types than in human epidermal growth factor receptor 2-positive (56.2%) or triple-negative (20.6%) types. AR expression of ≥ 35% was significantly related to longer RFS in a multivariate analysis (hazard ratio, 0.430; 95% confidence interval, 0.260–0.709; p = 0.001). Conclusion We propose a cut-off value of 35% to best predict RFS in patients with surgically resected breast cancer. AR expression was positive in 68.4% of patients, and AR positivity was found to be an independent prognostic factor for longer RFS.

PURPOSE: KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo. MATERIALS AND METHODS: The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. RESULTS: KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. CONCLUSION: KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.
Aneuploidy ; Animals ; Breast Neoplasms ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line ; Fluorescent Antibody Technique ; Heterografts ; In Vitro Techniques ; Mice ; Microtubules ; Mitosis* ; src-Family Kinases* ; Triple Negative Breast Neoplasms ; Tubulin ; Wound Healing