Downregulation of NKG2DLs by TGF-ß in human lung cancer cells.

BACKGROUND: Transforming growth factor beta (TGF-ß) is a typical immuno-inhibitory cytokine and highly secreted by lung cancer cells. It was supposed that its immunosuppressive effects to NK cell might be related with the altered expression of activating and inhibitory molecules in lung cancer cells. In this study, we examined the expression of NKG2DLs, PD-L1 and PD-L2 in lung cancer cells after treatment of TGF-ß and a TGF-ß inhibitor, Galunisertib (LY2157299). RESULTS: TGF-ß reduced the level of surface proteins of five NKG2DLs without altered transcription levels in lung cancer cells. Galunisertib reversed the effect of TGF-ß on the expression of NKG2DLs. Since MMP inhibitors, MMPi III and MMP2 inhibitor I, restored the reduced expression of NKG2DLs after treatment of TGF-ß, it was thought that TGF-ß induced the expression of MMP2 which facilitated the shedding of the NKG2DLs in cancer cells. However, the expression of PD-L1, L2 were not changed by treatment with TGF-ß or Galunisertib. CONCLUSIONS: Therefore, inhibition of TGF-ß might reverse the immunosuppressive status on immune cells and restore NK cell mediated anticancer immune responses by upregulation of NKG2DLs in cancer cells.

Differential Effects of Histone Deacetylases on the Expression of NKG2D Ligands and NK Cell-Mediated Anticancer Immunity in Lung Cancer Cells.

Histone acetylation is an epigenetic mechanism that regulates the expression of various genes, such as natural killer group 2, member D (NKG2D) ligands. These NKG2D ligands are the key molecules that activate immune cells expressing the NKG2D receptor. It has been observed that cancer cells overexpress histone deacetylases (HDACs) and show reduced acetylation of nuclear histones. Furthermore, HDAC inhibitors are known to upregulate the expression of NKG2D ligands. Humans have 18 known HDAC enzymes that are divided into four classes. At present, it is not clear which types of HDAC are involved in the expression of NKG2D ligands. We hypothesized that specific types of HDAC genes might be responsible for altering the expression of NKG2D ligands. In this study, we monitored the expression of NKG2D ligands and major histocompatibility complex (MHC) class I molecules in lung cancer cells which were treated with six selective HDAC inhibitors and specific small interfering RNAs (siRNAs). We observed that treatment with FK228, which is a selective HDAC1/2 inhibitor, also known as Romidepsin, induced NKG2D ligand expression at the transcriptional and proteomic levels in two different lung cancer cell lines. It also caused an increase in the susceptibility of NCI-H23 cells to NK cells. Silencing HDAC1 or HDAC2 using specific siRNAs increased NKG2D ligand expression. In conclusion, it appears that HDAC1 and HDAC2 might be the key molecules regulating the expression of NKG2D ligands. These results imply that specifically inhibiting HDAC1 and HDAC2 could induce the expression of NKG2D ligands and improve the NK cell-mediated anti-cancer immunity.

An inhibitor of programmed death ligand 1 enhances natural killer cell-mediated immunity against malignant melanoma cells.

Since ionizing radiation has showed the dramatic effect to kill the cancer cells through direct DNA damage as well as triggering anti-cancer immune responses including induction of NKG2D ligands, it has used for long time to treat many cancer patients. However, it has been known that radiotherapy might promote the remnant cancer cells to escape immune system and metastasis. One of the suggested ways of immune evasion is induction of a ligand for programmed death-1 (PD-L1) in head and neck cancer, bladder cancer and lung cancer cells which engages the receptor, programmed death-1 (PD-1) in immune cells. PD-1/PD-L1 axis transduces the inhibitory signal and suppresses the adaptive immunity. However, their role in innate immunity remains poorly understood. Therefore, we investigated whether ionizing radiation could change the expression of PD-L1 in malignant melanoma cells and the receptor, programmed death-1 (PD-1), in NK-92 cells. Surface PD-L1 levels on melanoma cells were increased by ionizing radiation in a dose-independent manner but the level of PD-L1 was not changed significantly in NK-92 cells. Radiation-induced PD-L1 suppressed the activity of the NK-92 cells against melanoma cells despite of upregulation of NKG2D ligands. Furthermore, activated NK cells had high level of PD-1 and could not kill PD-L1+ melanoma cells effectively. When we used PD-L1 inhibitor or silenced PD-L1 gene, inhibited PD-1/PD-L1 axis reversed the activity of the suppressed NK cells. Through these results, we supposed that PD-1/PD-L1 blockade could enhance the immune responses of NK cells against melanoma cells after radiotherapy and might overcome the PD-L1 mediated radioresistance of cancer cells.

Potentiation of TRAIL­induced cell death by nonsteroidal anti­inflammatory drug in human hepatocellular carcinoma cells through the ER stress­dependent autophagy pathway.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed primary liver malignancy. The limited success with relapse of the disease in HCC therapy is frequently associated with the acquired resistance to anticancer drugs. To develop a strategy and design for overcoming the resistance of HCC cells to TNF­related apoptosis inducing ligand (TRAIL)­induced cell death, we evaluated the efficacy of a non­steroidal anti­inflammatory drug (NSAID) in combination with TRAIL against TRAIL­resistant HCC cells expressing a high level of CD44. We revealed by MTT and western blotting, respectively, that celecoxib (CCB), an NSAID, and 2,5­dimethyl celecoxib (DMC), a non­cyclooxygenase (COX)­2 inhibitor analog of CCB, were able to sensitize TRAIL­resistant HCC cells to TRAIL, implicating a COX­independent mechanism. CCB dose­dependently enhanced LC3­II and reduced p62 levels through AMPK activation and inhibition of the Akt/mTOR pathway and upregulated expression of ATF4/CHOP, leading to activation of endoplasmic reticulum (ER) stress­dependent autophagy. The TRAIL sensitization capacity of CCB in TRAIL­resistant HCC cells was abrogated by an ER stress inhibitor. In addition, we also revealed by flow cytometry and western blotting, respectively, that accelerated downregulation of TRAIL­mediated c­FLIP expression, DR5 activation and CD44 degradation/downregulation by NSAID resulted in activation of caspases and poly(ADP­ribose) polymerase (PARP), leading to the sensitization of TRAIL­resistant HCC cells to TRAIL and thereby reversal of TRAIL resistance. From these results, we propose that NSAID in combination with TRAIL may improve the antitumor activity of TRAIL in TRAIL­resistant HCC, and this approach may serve as a novel strategy that maximizes the therapeutic efficacy of TRAIL for clinical application.

17ß-Estradiol Inhibits Lysophosphatidylcholine-Induced Apoptosis in Cultured Vascular Smooth Muscle Cells.

OBJECTIVES: Coronary heart disease (CHD) risk increases in women after menopause, but menopausal hormone therapy (MHT) helps prevent CHD if started early after menopause. To explore the mechanism underlying the direct vascular actions of estrogen, the effects of 17ß-estradiol (E2) on apoptosis of vascular smooth muscle cells (VSMCs) induced with lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, were investigated in the present study. METHODS: VSMCs were isolated from rat aortas. Apoptosis and protein expression of caspases were assessed using propidium iodide staining and Western blot analysis, respectively. Intracellular formation of reactive oxygen species (ROS) was examined using dichlorofluorescein diacetate, a cell-permeable oxidation-sensitive probe, and quantitated with flow cytometry. Nuclear factor-κB (NF-κB) activation was determined after transfection with a reporter plasmid containing the luciferase reporter gene. RESULTS: After pre-treatment for 24 hours, 17ß-E2 suppressed lysoPC-induced (15 µM) apoptotic cell death in a dose-dependent manner with statistical significance at near physiological concentration. 17ß-E2 (10⁻6 M) also increased protein levels of caspase-9 and -8 precursors and decreased the active form of caspase-3. Western blot analysis using subcellular fractions showed that 17ß-E2 decreased mitochondrial Bax levels and concomitantly increased cytosolic Bax expression. Furthermore, intracellular production of ROS and NF-κB-mediated transcriptional activity were reduced with 17ß-E2. In addition, estrogen effects on apoptosis were partially blocked by ICI 182,780, a specific estrogen receptor antagonist. CONCLUSIONS: In cultured VSMCs treated with lysoPC, 17ß-E2 reduced apoptotic cell death by down-regulating both extrinsic and intrinsic apoptosis pathways, contributing to the preventive action of MHT against CHD.

Effects of 17ß-Estradiol on the Plasminogen Activator System in Vascular Smooth Muscle Cells Treated with Lysophophatidylcholine.

OBJECTIVES: When administered soon after menopause, hormone therapy can prevent coronary heart diseases in women. To explore the mechanism underlying the cardioprotective actions of estrogen, we investigated the effects of 17ß-estradiol (17ß-E2) on the plasminogen activator system using cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs were isolated from rat aortas. Protein expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated using Western blotting and enzyme-linked immunosorbent assay, respectively. The enzyme activity of PAI-1 in a conditioned medium was assessed via reverse fibrin overlay zymography and that of t-PA was assessed via fibrin overlay zymography. Gene expression was quantified using real-time reverse transcription-polymerase chain reaction. RESULTS: Following pre-treatment for 24 hours, 17ß-E2 suppressed both protein expression and enzyme activity of PAI-1 stimulated by lysophosphatidylcholine (lysoPC) in a significant and dose-dependent manner at a near physiological concentration. Moreover, 17ß-E2 (10⁻7 M) inhibited PAI-1 gene expression, and ICI 182,780-a specific estrogen receptor antagonist-blocked the effects of 17ß-E2 on the PAI-1 protein. 17ß-E2 did not affect t-PA secretion but significantly enhanced free t-PA activity through reduced binding to PAI-1. Furthermore, 17ß-E2 suppressed intracellular reactive oxygen species production and nuclear factor-κB-mediated transcription. CONCLUSIONS: In VSMCs stimulated with lysoPC, 17ß-E2 reduced PAI-1 expression through a non-receptor-mediated mechanism via antioxidant activity as well as a receptor-mediated mechanism; however, it did not alter t-PA secretion. Of note, 17ß-E2 suppressed PAI-1 activity and concurrently enhanced t-PA activity, suggesting a beneficial influence on fibrinolysis.

Prognostic Role of Zinc Finger Homeobox 4 in Ovarian Serous Cystadenocarcinoma.

Introduction: The zinc finger homeobox 4 (ZFHX4) protein is a crucial molecular regulator of tumor-initiating stem cell-like functions. Objective: This study aimed to determine the role of ZFHX4 in the progression of ovarian serous cystadenocarcinoma (OSC). Methods: Differential gene expression ZFHX4 among low-stage (stages I and II), high-stage (stages III and IV), low-grade (grades I and II), and high-grade (grades III and IV) OSC patients was identified using four independent cohorts from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). We compared ZFHX4 expression as a prognostic factor using Kaplan-Meier survival curves, multivariate analysis, the time-dependent area under the curve (AUC) of Uno's C-index, and the AUC of the receiver operating characteristics at 4 years post diagnosis. Results: ZFHX4 gene expression in high-stage tumors is significantly higher than in low-stage tumors (TCGA, p = 0.007; GSE9891, p = 0.001). A Kaplan-Meier analysis revealed that elevated expression of ZFHX4 was associated with a poor prognosis in OSC patients for all cohorts, regardless of stage and grade (TCGA, p = 1e-04; GSE9891, p = 0.0044; GSE13876, p = 0.00078; GSE26712, p = 0.039). Analysis of C-indices and the area under the receiver operating characteristic curve further supported this result (C-index: TCGA, 0.599; GSE9891, 0.642; GSE13876, 0.585; GSE26712, 0.597). Moreover, univariate and multivariate Cox hazards analyses confirmed the prognostic significance of ZFHX4 levels. Conclusion: Collectively, these findings suggest that ZFHX4 is a prognostic factor for OSC.

GABRQ expression is a potential prognostic marker for patients with clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. Novel biomarkers of ccRCC may provide crucial information on tumor features and prognosis. The present study aimed to determine whether the expression of γ-aminobutyric acid (GABA) A receptor subunit θ (GABRQ) could serve as a novel prognostic marker of ccRCC. GABA is the main inhibitory neurotransmitter in the brain that activates the receptor GABAA, which is comprised of three subunit isoforms: GABRA3, GABRB3 and GABRQ. A recent study reported that GABRQ is involved in the initiation and progression of hepatocellular carcinoma; however, the role of GABRQ in ccRCC remains unknown. In the present study, clinical and transcriptomic data were obtained from cohorts of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). Differential GABRQ expression levels among early (TI and II), late (TIII and IV), nonmetastatic (M0) and metastatic (M1, primary tumor) stages of ccRCC samples were then identified. Furthermore, the use of GABRQ as a prognostic gene was analyzed using Uno's C-index based on the time-dependent area under the curve (AUC), the AUC of the receiver operating characteristic curve at 5 years, the Kaplan-Meier survival curve and multivariate analysis. The survival curve analysis revealed that low GABRQ mRNA expression was significantly associated with a poor prognosis of ccRCC (P<0.001 and P=0.0012 for TCGA and ICGC data, respectively). In addition, analyses of the C-index and AUC values further supported this discriminatory power. Furthermore, the prognostic value of GABRQ mRNA expression was confirmed by multivariate Cox regression analysis. Taken together, these results suggested that GABRQ mRNA expression may be considered as a novel prognostic biomarker of ccRCC.

Prognostic role of the beta-2 adrenergic receptor in clear cell renal cell carcinoma.

The beta-2 adrenergic receptor (ADRB2) regulates the proliferation, apoptosis, angiogenesis, migration, and metastasis of cancer cells. However, its function in the progression of clear cell renal cell carcinoma (ccRCC) is unknown. Here, we report that ADRB2 can be a novel prognostic factor for patients with ccRCC. The differential expression of ADRB2 in low-stage (stages I and II), high-stage (stages III and IV), low-grade (grades I and II), and high-grade (grades III and IV) ccRCC was identified in cohorts of patients from The Cancer Genome Atlas and the International Cancer Genome Consortium. We evaluated ADRB2 expression as a prognostic factor using the Kaplan-Meier survival curve, multivariate analysis, time-dependent area under the curve (AUC) of Uno's C-index, and AUC of the receiver operating characteristics (ROC) at five years. Kaplan-Meier analysis revealed that reduced ADRB2 expression is associated with poor prognosis in ccRCC patients. Analysis of C-indices and AUC-ROC further confirmed this result. Moreover, multivariate analysis confirmed the prognostic significance of ADRB2 expression. Collectively, these findings suggest that ADRB2 is a potential prognostic factor for ccRCC.

SIRT1 is dispensable for maturation of hematopoietic stem cell in the bone marrow niche.

Sirtuin 1 (SIRT1) is a histone deacetylase implicated in stem cell homeostasis. Conditional Sirt1 deletion in the hematopoietic stem and progenitor system promotes hematopoietic stem and progenitor cell (HSPC) expansion under stress conditions. In addition, SIRT1 activators modulate the capacity and HSPC numbers in the bone marrow (BM). To investigate the role of SIRT1 in the BM niche, a conditional Sirt1 deletion in the BM niche was generated in a mouse model for the present study. Multicolor flow cytometric analyses were performed to determine HSC cell populations. Using 5-fluorouracil-induced proliferative stress, a survival curve was produced. In the present study, Sirt1 deletion in the BM niche demonstrated that the production of mature blood cells, lineage distribution within hematopoietic organs and frequencies of HSPC populations were comparable to those of controls. Additionally, Sirt1 deletion in the BM niche did not perturb HSC maturation under stress induced by transplantation. Therefore, these observations suggest that SIRT1 serves a dispensable role in HSC maturation in the BM niche.

Upregulation of Myc promotes the evasion of NK cell­mediated immunity through suppression of NKG2D ligands in K562 cells.

c­Myc is a characteristic oncogene with dual functions in cell proliferation and apoptosis. Since the overexpression of the c­Myc proto­oncogene is a common event in the development and growth of various human types of cancer, the present study investigated whether oncogenic c­Myc can alter natural killer (NK) cell­mediated immunity through the expression of associated genes, using PCR, western blotting and flow cytometry assays. Furthermore, whether c­Myc could influence the expression levels of natural killer group 2 member D (NKG2D) ligands, which are well known NK activation molecules, as well as NK cell­mediated immunity, was investigated. c­Myc was inhibited by 10058­F4 treatment and small interfering RNA transfection. Upregulation of c­Myc was achieved by transfection with a pCMV6­myc vector. The inhibition of c­Myc increased MHC class I polyeptide­related sequence B and UL16 binding protein 1 expressions among NKG2D ligands, and the overexpression of c­Myc suppressed the expression of all NKG2D ligands, except MHC class I polyeptide­related sequence A. Furthermore, the alteration of c­Myc activity altered the susceptibility of K562 cells to NK cells. These results suggested that the overexpression of c­Myc may contribute to the immune escape of cancer cells and cell proliferation. Combined treatment with NK­based cancer immunotherapy and inhibition of c­Myc may achieve improved therapeutic results.

Comparisons of cancer-associated fibroblasts in the intratumoral stroma and invasive front in colorectal cancer.

The aim of this study was to evaluate the cytomorphologic maturity and molecular activation of cancer-associated fibroblasts (CAFs) in the intratumoral stroma and invasive front in colorectal cancer and understand how they affect cancer invasion and long-term oncological outcomes.The cytomorphologic maturity of and α-smooth muscle actin (α-SMA), fibroblast activation protein α (FAPα), and fibroblast-specific protein 1 (FSP-1) expression in CAFs in the intratumoral stroma (CAF) and the invasive front (CAF) of colorectal cancer tissues were compared (n = 147). The correlations between CAF maturation, molecular activity markers, and cancer invasion were evaluated by network analysis. Overall survival and systemic recurrence were analyzed to assess the oncological effects of CAF properties.The cytomorphologic maturation rate was comparable between CAF and CAF. The presence of mature CAFs was related to epidermal growth factor receptor overexpression in cancer cells. Expression rates of α-SMA (96.6%-98.0%) and FAPα (18.6%-22.9%) were similar between CAF and CAF. FSP-1 expression was more frequent in CAF than in CAF (66.4% vs 58.2%, P = .038). There was a significant decrease in FSP-1 expression in CAF and CAF in higher stages. The infiltrating growth pattern of the tumor was more frequent in the immature CAF. In colorectal cancer with perineural invasion and lymph node metastasis, FSP-1 expression in CAF was significantly lower. On multivariate analysis using the Cox proportional hazards model, immature CAF was found to be an independent prognostic factor of overall survival. In non-metastatic (stage I-III) colorectal cancer patients, CAF maturity was not a prognostic factor for systemic recurrence.Cytomorphologic maturity and molecular activation markers were similar between CAFs in the intratumoral stroma and invasive front of colorectal cancer.

Prognostic Role of TMED3 in Clear Cell Renal Cell Carcinoma: A Retrospective Multi-Cohort Analysis.

Transmembrane p24 trafficking protein 3 (TMED3) is a metastatic suppressor in colon cancer and hepatocellular carcinoma. However, its function in the progression of clear cell renal cell carcinoma (ccRCC) is unknown. Here, we report that TMED3 could be a new prognostic marker for ccRCC. Patient data were extracted from cohorts in the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). Differential expression of TMED3 was observed between the low stage (Stage I and II) and high stage (Stage III and IV) patients in the TCGA and ICGC cohorts and between the low grade (Grade I and II) and high grade (Grade III and IV) patients in the TCGA cohort. Further, we evaluated TMED3 expression as a prognostic gene using Kaplan-Meier survival analysis, multivariate analysis, the time-dependent area under the curve (AUC) of Uno's C-index, and the AUC of the receiver operating characteristics at 5 years. The Kaplan-Meier analysis revealed that TMED3 overexpression was associated with poor prognosis for ccRCC patients. Analysis of the C-indices and area under the receiver operating characteristic curve further supported this. Multivariate analysis confirmed the prognostic significance of TMED3 expression levels (P = 0.005 and 0.006 for TCGA and ICGC, respectively). Taken together, these findings demonstrate that TMED3 is a potential prognostic factor for ccRCC.

Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation.

Recently, novel therapeutic strategies have been designed with the aim of killing cancer stem-like cells (CSCs), and considerable interest has been generated in the development of specific therapies that target stemness-related marker of CSCs. In this study, nonsteroidal anti-inflammatory drugs (NSAIDs) significantly potentiated Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-mediated cytotoxicity through apoptotic and autophagic cell death induction, but COX-2-inhibitory function was not required for NSAID-induced autophagy in CD44-overexpressing human chronic myeloid leukemia K562 (CD44highK562) cells. Importantly, we found that treatment with NSAIDs resulted in a dose-dependent increase in LC3-II level and decrease in p62 level and simultaneous reduction in multiple stemness-related markers including CD44, Oct4, c-Myc, and mutant p53 (mutp53) in CD44highK562 cells, suggesting that NSAIDs could induce autophagy, which might mediate degradation of stemness-related marker proteins. Activation of AMPK and inhibition of Akt/mTOR/p70S6K/4EBP1 participated in NSAID-induced autophagy in CD44highK562 cells. In addition, treatment of CD44highK562 cells with NSAIDs inhibited expression of HSF1/Hsps, which resulted in suppression of 17-AAG-induced activation of Hsp70, leading to reversal of 17-AAG resistance and sensitization of CD44highK562 cells to 17-AAG by NSAIDs. In conclusion, combining NSAIDs with Hsp90 inhibitor may offer one of the most promising strategies for eradication of CD44-overexpressing CSCs.

Cancer-Associated Fibroblasts and Desmoplastic Reactions Related to Cancer Invasiveness in Patients With Colorectal Cancer.

PURPOSE: We evaluated the relationship of cancer-associated fibroblasts (CAFs) and desmoplastic reactions with cancer invasiveness and long-term outcomes in patients with colorectal cancer (CRC). METHODS: Histologic evaluation of mature CAFs and desmoplasia was performed by observing the collagen fiber structure and fibroblast cytomorphology in the intratumoral stroma and invasive front of CRC tissues. Cancer-cell invasiveness was evaluated using lymphatic invasion, vascular invasion, perineural invasion, tumor budding, and tumor growth patterns. Overall survival and systemic recurrence were analyzed. A network analysis was performed between CAF maturation, desmoplastic reaction, and cancer invasiveness. RESULTS: The proportions of mature CAFs in the intratumoral stroma and the invasive front were 57.6% and 60.3%, respectively. Epidermal growth factor receptor (EGFR) overexpression was significantly higher in the mature CAFs in the invasive front as compared to immature CAFs. Lymphatic invasion increased as the number of mature fibroblasts in the intratumoral stroma increased. Tumor budding was observed in almost half of both mature and immature stroma samples and occurred more frequently in infiltrating tumors. On network analysis, well-connected islands were identified that was associated with EGFR overexpression, CAF maturation, and infiltrating tumor growth patterns leading to tumor budding. CONCLUSION: The maturity of CAFs and desmoplastic reactions were associated with cancer invasion. However, the cytomorphologic characteristics of CAFs were insufficient as an independent prognostic factor for patients with CRC.

Sensitization of multidrug-resistant cancer cells to Hsp90 inhibitors by NSAIDs-induced apoptotic and autophagic cell death.

NSAIDs (non-steroidal anti-inflammatory drugs) have potential use as anticancer agents, either alone or in combination with other cancer therapies. We found that NSAIDs including celecoxib (CCB) and ibuprofen (IBU) significantly potentiated the cytotoxicity of Hsp90 inhibitors in human multidrug-resistant (MDR) cells expressing high levels of mutant p53 (mutp53) protein and P-glycoprotein (P-gp), and reversed Hsp90 inhibitor resistance caused by activation of heat shock factor 1 (HSF1) and by up-regulation of heat shock proteins (Hsps) and P-gp. Inhibition of Akt/mTOR and STAT3 pathways by CCB induced autophagy, which promoted the degradation of mutp53, one of Hsp90 client proteins, and subsequently down-regulated HSF1/Hsps and P-gp. Inhibition of autophagy prevented mutp53 degradation and CCB-induced apoptosis, and inhibition of caspase-3-mediated apoptotic pathway by Z-DEVD-FMK did not completely block CCB-induced cell death in MDR cells, suggesting that autophagic and apoptotic cell death may contribute to CCB-induced cytotoxicity in MDR cells. Furthermore, CCB and IBU suppressed Hsp90 inhibitor-induced HSF1/Hsp70/P-gp activity and mutp53 expression in MDR cells. Our results suggest that NSAIDs can be used as potential Hsp90 inhibitor chemosensitizers and reverse resistance of MDR cells to Hsp90 inhibitors via induction of apoptosis and autophagy. These results might enable the use of lower, less toxic doses of Hsp90 inhibitors and facilitate the design of practically applicable, novel combination therapy for the treatment of MDR cancer.

Expansion of cytotoxic natural killer cells using irradiated autologous peripheral blood mononuclear cells and anti-CD16 antibody.

Natural killer (NK) cells are considered a promising strategy for cancer treatment. Various methods for large-scale NK cell expansion have been developed, but they should guarantee that no viable cells are mixed with the expanded NK cells because most methods involve cancer cells or genetically modified cells as feeder cells. We used an anti-CD16 monoclonal antibody (mAb) and irradiated autologous peripheral blood mononuclear cells (PBMCs) (IrAPs) to provide a suitable environment (activating receptor-ligand interactions) for the NK cell expansion. This method more potently expanded NK cells, and the final product was composed of highly purified NK cells with lesser T-cell contamination. The expanded NK cells showed greater upregulation of various activation receptors, CD107a, and secreted larger amounts of interferon gamma. IrAPs expressed NKG2D ligands and CD48, and coengagement of CD16 with NKG2D and 2B4 caused potent NK cell activation and proliferation. The expanded NK cells were cytotoxic toward various cancer cells in vitro and in vivo. Moreover, irradiation or a chemotherapeutic drug further enhanced this antitumor effect. Therefore, we developed an effective in vitro culture method for large-scale expansion of highly purified cytotoxic NK cells with potent antitumor activity using IrAPs instead of cancer cell-based feeder cells.

Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-ß signaling.

Cancer upregulated gene 2 (CUG2) enhances cell migration and invasion, but the underlying mechanism has not been revealed. Herein, CUG2 decreased the expression of E-cadherin and increased the expression of N-cadherin and vimentin, characteristics of the epithelial-mesenchymal transition (EMT). A CUG2 deletion mutant, lacking interaction with nucleophosmin 1 (NPM1), or suppression of NPM1 reduced wound healing and cell invasion, indicating that CUG2-mediated EMT requires NPM1. CUG2 enhanced activation of Smad2/3 and expression of Snail and Twist, while the CUG2 silence decreased these TGF-ß signaling pathways, leading to suppression of EMT. NPM silence also inhibited the CUG2-induced TGF-ß signaling. These results suggest that TGF-ß signaling is involved in CUG2-induced EMT. Treatment with EW-7197, a novel inhibitor of TGF-ß signaling, diminished CUG2-mediated EMT and inhibition of Akt, ERK, JNK, and p38 MAPK, non-canonical TGF-ß signaling molecules, also decreased expression of Smad2/3, Snail and Twist, leading to inhibition of EMT. The results confirm that TGF-ß signaling is essential for CUG2-mediated EMT. Interestingly, TGF-ß enhanced CUG2 expression. We further found that both CUG2-induced TGF-ß production and TGF-ß-induced CUG2 up-regulation required a physical interaction between Sp1 and Smad2/3 in the CUG2 and TGF-ß promoter, as demonstrated by a promoter reporter assay, immunoprecipitation, and ChIP assay. These results indicated close crosstalk between CUG2 and TGF-ß. Conversely, suppression of CUG2 or NPM1 did not completely inhibit TGF-ß-induced EMT, indicating that the effect of TGF-ß on EMT is dominant over the effect of CUG2 on EMT. Collectively, our findings suggest that CUG2 induces the EMT via TGF-ß signaling.

Potential Role of CD133 Expression in the Susceptibility of Human Liver Cancer Stem-Like Cells to TRAIL.

Hepatocellular carcinoma (HCC) is one of the most common malignancies, with a poor prognosis and high recurrence rate. In the present study, we identified CD133, one of the markers of cancer stem cells, as a novel molecular target of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In four human HCC cell lines established from primary HCC tumors, we found that CD133-high human liver cancer stem-like cells (CD133hi) derived from the SNU-475 cell line were highly susceptible to TRAIL compared to other HCC cell lines with a small population of CD133. CD133hi SNU-475 cells showed upregulation of TRAIL receptor DR5 and stemness-related genes such as c-Myc and ABC transporters compared to their CD133-low (CD133lo) cells. Hypersensitivity of CD133hi cells to TRAIL was associated with c-Myc-mediated upregulation of DR5 and downregulation of c-FLIPL in the cells. Knockdown of CD133 expression in CD133hi cells resulted in the downregulation of c-Myc, and depletion of c-Myc caused a decrease in the cell surface expression of DR5 and an increase in the expression of c-FLIPL and, consequently, attenuated TRAIL-induced cytotoxicity and apoptosis of CD133hi cells. These results suggest that TRAIL may provide a new strategy for CD133hi CSCs of HCC-targeted therapies and, potentially, for therapies of other CD133-expressing types of cancer.

Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1.

The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp.