Antitumor effect of dendritic cell loaded ex vivo and in vivo with tumor-associated antigens in lung cancer model.

Various ex vivo or in vivo loading protocols have been developed or evaluated for the delivery of tumor antigens to dendritic cells (DCs). We compared the antitumor effect of mature DCs electroporation-pulsed (EP/mDC) ex vivo with tumor cell lysate and immature DCs (iDCs) injected into the tumor apoptosed by ionizing radiation (IR/iDC) in lung cancer model. DCs were generated from bone marrow of C57BL/6 mice. Ionizing radiation (IR) was applied at a dose of 10 Gy to the tumor on the right thigh. iDCs were intratumorally injected into the irradiated tumor and EP/mDC was injected subcutaneously in the right flank. DC injection induced strong tumor-specific immunity against Lewis lung carcinoma, as compared with the tumor-bearing control and IR only treated mice. The growth of a distant tumor on the right and left flank was inhibited by IR/iDC and EP/mDC. Particularly, IR/iDC resulted in a more significant inhibition of tumor growth and prolonged survival time. It was related to increase of tumor-specific interferon-gamma, cytotoxicity, and decrease of regulatory T-cells. The results indicate that DCs electroporation-pulsed with tumor cell lysate induce a potent antitumor effect, but that iDCs intratumoral injected into the irradiated tumor induce a more potent antitumor effect.

Analytical performance of a new one-step quantitative prostate-specific antigen assay, the FREND™ PSA Plus.

BACKGROUND: We evaluated the analytical performance of a new one-step rapid quantitative sandwich immunoassay for total prostate-specific antigen (tPSA), the FREND™ PSA Plus (FREND PSA) (NanoEnTek Inc., Seoul, Korea). METHODS: The imprecision, linearity, hook effect, detection limit (LoD), and interference were evaluated and trueness verification and matrix validation were performed. For method comparison, 79 patient specimens were analyzed with FREND PSA and two comparative tPSA assays (Architect® total PSA and cobas® total PSA assay). RESULTS: Total CVs of the imprecision for low (0.208 ng/mL), medium (4.051 ng/mL), and high PSA levels (5.469 ng/mL) were 15.9%, 6.4%, and 9.1%, respectively. Linearity was observed from 1.01 to 19.15 ng/mL and the hook phenomenon was absent up to 171.48 ng/mL. The LoD was 0.094 ng/mL. The regression equations between FREND (y) and Architect or cobas were as follows: y=0.0133+1.054x (r=0.973), y=-0.2144+1.066x (r=0.977), respectively. Differences between FREND PSA and the comparative methods at a medical decision level of 4.0 ng/mL were less than the optimum specification bias (9.3%). The percentage biases from the trueness verification and interference test were less than the desirable specifications for bias (18.7%). The plasma tPSA level measured with lithium heparin or K2EDTA was comparable to that in the serum. CONCLUSIONS: The FREND PSA provided reliable analytical performance and test results in comparison to two widely used tPSA assays. It is a simple and rapid test for tPSA and can be applied in point-of-care testing.

Enhanced maturation and function of dendritic cells using hydrogel coated plate and antigen electroporation.

Dendritic cells (DCs) are potent antigen-presenting cells that can be matured in vitro from immature dendritic cells (iDCs) in the presence of several biological agents such as cytokine cocktail, CD40L, TNF-a and antigen loading, which are necessary and achieved using various protocols, such as lipofection, passive pulse or electroporation. However, these DCs maturation protocols may cause with a significant loss of cells because of cellular attachment and spreading during culturing. Some biomaterials that influence adhesion and development of cells have been used in cell culture techniques, and it was thought that they might be applied on the culture of DCs. In this study, we used polyHEMA, which is a hydrogel coating biomaterial that prevents DCs from adherence, and investigated whether hydrogel coating affects the maturation of iDCs. The efficiency in the generation of mDCs was improved through hydrogel coating procedure and a dendritic cell maturation marker, CD83, was significantly increased in hydrogel-coated culture condition. The antigen-loaded mDCs from electroporation were further expressed the CD83. The mDCs generated in the hydrogel-coated culture condition showed more, longer and thicker dendrites, and produced more amounts of cytokines such as IL-12 and IFN-γ. Therefore, it was suggested that the hydrogel-coated culture condition could improve function of mDCs. Cheol-Hun Son and Jae-Ho Bae contributed equally to this work.

Inhibiting invasion into human bladder carcinoma 5637 cells with diallyl trisulfide by inhibiting matrix metalloproteinase activities and tightening tight junctions.

Diallyl trisulfide (DATS), an organosulfur compound in garlic, possesses pronounced anti-cancer potential. However, the anti-invasive mechanism of this compound in human bladder carcinoma is not fully understood. In this study, we evaluated the anti-invasive effects of DATS on a human bladder carcinoma (5637) cell line and investigated the underlying mechanism. The results indicated that DATS suppressed migration and invasion of 5637 cells by reducing the activities and expression of matrix metalloproteinase (MMP)-2 and MMP-9 at both the protein and mRNA levels. DATS treatment up-regulated expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in 5637 cells. The inhibitory effects of DATS on invasiveness were associated with an increase in transepithelial electrical resistance and repression of the levels of claudin family members. Although further studies are needed, our data demonstrate that DATS exhibits anti-invasive effects in 5637 cells by down-regulating the activity of tight junctions and MMPs. DATS may have future utility in clinical applications for treating bladder cancer.

Flavonoids from Orostachys japonicus A. Berger inhibit the invasion of LnCaP prostate carcinoma cells by inactivating Akt and modulating tight junctions.

Tight junctions (TJs) are a mode of cell-to-cell adhesion in epithelial or endothelial cells, and serve as a physical barrier to maintenance of homeostasis in body by controlling paracellular transport. Claudins are the most important molecules of the TJs, but paradoxically these proteins are frequently over-expressed in cancers and their overexpression is implicated in the invasive potential of cancer. Hence, we investigated the effects of flavonoids extracted from Orostachys japonicus A. Berger (FEOJ) on TJs and the expression of claudins as well as cancer invasion along with in LnCaP human prostate cancer. FEOJ suppressed cancer cell motility and invasiveness at the concentrations where FEOJ did not show anti-proliferative activity. FEOJ increased transepithelial electrical resistance (TER) associated with tightening TJs, and suppressed expression of claudin proteins. Furthermore, FEOJ suppressed the activities of MMP-2 and -9 in a dose-dependent manner, which came from the activation of tissue inhibitor of metalloproteinases (TIMPs) by FEOJ. FEOJ suppressed migration and invasion by suppressing PI3K/Akt signaling pathway. Taken together, this study suggest that FEOJ suppresses cancer migration and invasion by tightening TJs through the suppression of claudin expression, and by suppressing MMPs in LnCaP human prostate cancer cells, which at least in part results from the suppression of PI3K/Akt signaling pathway.

Apoptosis induction of human prostate carcinoma DU145 cells by diallyl disulfide via modulation of JNK and PI3K/AKT signaling pathways.

Diallyl disulfide (DADS), a sulfur compound derived from garlic, has various biological properties, such as anticancer, antiangiogenic and anti-inflammatory effects. However, the mechanisms of action underlying the compound's anticancer activity have not been fully elucidated. In this study, the apoptotic effects of DADS were investigated in DU145 human prostate carcinoma cells. Our results showed that DADS markedly inhibited the growth of the DU145 cells by induction of apoptosis. Apoptosis was accompanied by modulation of Bcl-2 and inhibitor of apoptosis protein (IAP) family proteins, depolarization of the mitochondrial membrane potential (MMP, ΔΨm) and proteolytic activation of caspases. We also found that the expression of death-receptor 4 (DR4) and Fas ligand (FasL) proteins was increased and that the level of intact Bid proteins was down-regulated by DADS. Moreover, treatment with DADS induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular-signal regulating kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK). A specific JNK inhibitor, SP600125, significantly blocked DADS-induced-apoptosis, whereas inhibitors of the ERK (PD98059) and p38 MAPK (SB203580) had no effect. The induction of apoptosis was also accompanied by inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt and the PI3K inhibitor LY29004 significantly increased DADS-induced cell death. These findings provide evidence demonstrating that the proapoptotic effect of DADS is mediated through the activation of JNK and the inhibition of the PI3K/Akt signaling pathway in DU145 cells.

Induction of apoptosis in human leukemia U937 cells by anthocyanins through down-regulation of Bcl-2 and activation of caspases.

Anthocyanins are a class of flavonoids, widely spread throughout the plant kingdom, that exhibit important anti-oxidant and anti-inflammatory actions as well as chemotherapeutic effects. However, little is known concerning the molecular mechanisms by which these activities are exerted. In this study, we investigated the anthocyanins isolated from Vitis coignetiae Pulliat for their potential anti-proliferative and apoptotic effects on human leukemia U937 cells. It was found that these anthocyanins inhibit cell viability and induce apoptotic cell death of U937 cells in a dose-dependent manner, as measured by hemocytometer counts, by alteration in the mitochondrial membrane potential, by increases in sub-G1 populations and by DNA ladder formation. Apoptosis of U937 cells by anthocyanins was associated with modulation of expression of Bcl-2 and IAP family members. Consequently, anthocyanin treatment induced proteolytic activation of caspase-3, -8 and -9, and a concomitant degradation of poly(ADP-ribose) polymerase. However, anthocyanin-induced growth inhibition and apoptosis were significantly attenuated in Bcl-2 overexpressing U937 cells. Furthermore, z-DEVD-fmk, a caspase-3 specific inhibitor, blocked apoptosis and increased the survival of anthocyanin-treated U937 cells. Taken together, these results show that Bcl-2 and caspases are key regulators of apoptosis in response to anthocyanins in human leukemia U937 cells.

Platycodin D induces apoptosis and decreases telomerase activity in human leukemia cells.

Platycodin D (PD) is a major constituent of triterpene saponins found in the root of Platycodon grandiflorum. Recent studies have demonstrated that PD is a potentially interesting candidate for use in cancer chemotherapy. However, the molecular mechanisms responsible for PD-induced telomerase inhibition remain to be poorly known. In this study, we examined the effects of PD treatment on telomerase activity in different human leukemia cell lines. At concentrations between 10 and 20 microM, PD exerted a dose-dependent direct cytotoxic effect and inhibition of telomerase activity via downregulation of hTERT expression. Because c-Myc and Sp1 are known to directly regulate transcription of hTERT, we also evaluated the expression and DNA binding activity of these proteins. PD treatment reduced c-Myc and Sp1 protein levels and DNA binding activities in a dose-dependent manner. We also observed that PD treatment downregulates the activation of Akt, thereby reducing the phosphorylation and nuclear translocation of hTERT. We conclude that PD has direct cytotoxic effect on human leukemia cells and suppresses telomerase activity through transcriptional and posttranslational suppression of hTERT.

Induction of apoptosis by pectenotoxin-2 is mediated with the induction of DR4/DR5, Egr-1 and NAG-1, activation of caspases and modulation of the Bcl-2 family in p53-deficient Hep3B hepatocellular carcinoma cells.

The tumor suppressor protein p53 restricts proliferation in response to DNA damage or the deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival and in some settings promote genomic instability and resistance to certain anti-cancer drugs. It is very important to identify chemotherapeutic agents that activate in a p53-independent manner for the development of treatments for p53-deficient tumors. Pectenotoxin-2 (PTX-2), isolated from marine sponges has been reported to display significant cytotoxicity to p53-deficient cancer cell lines. In this study, we compared the anti-cancer activity of PTX-2 in order to further test the status of p53 using two well-known hepatocarcinoma cell lines, p53-deficient Hep3B and p53-wild-type HepG2. MTT assay indicated that Hep3B cells were highly susceptible, whereas HepG2 cells were more resistant to this compound which was connected with the induction of apoptotic cell death in p53-deficient Hep3B cells, though not in HepG2 cells. The apoptosis induced by PTX-2 in Hep3B cells was associated with the down-regulation of anti-apoptotic Bcl-2 members (Bcl-2 and Bcl-xL) and IAP family proteins, the up-regulation of pro-apoptotic Bax protein and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (DR4/DR5) and mitochondrial dysfunction. PTX-2 activated caspases (caspase-3, -8 and -9) and the blockade of caspase-3 activity by the caspase-3 inhibitor prevented the PTX-2-induced apoptosis in Hep3B cells. Additionally, the transcription factor early growth response-1 (Egr-1) gene was transcriptionally activated and the levels of non-steroidal anti-inflammatory drugs (NSAID)-activated gene-1 (NAG-1) protein were also elevated in PTX-2-treated Hep3B cells. Although further studies are needed to prove that an increased expression of Egr-1 by PTX-2 directly leads to NAG-1 induction and then apoptosis induction in p53-deficient Hep3B cells, the results of this study suggest that PTX-2 may be a good candidate for the development of a potential anti-tumorigenic agent in p53-deficient tumors.

Streptochlorin isolated from Streptomyces sp. Induces apoptosis in human hepatocarcinoma cells through a reactive oxygen species-mediated mitochondrial pathway.

Streptochlorin is a small molecule isolated from marine Streptomyces sp. that is known to have antiangiogenic and anticancer properties. In this study, we examined the effects of this compound on reactive oxygen species (ROS) production and the association of these effects with apoptotic tumor cell death, using a human hepatocarcinoma Hep3B cell line. The results of this study demonstrated that streptochlorin mediates ROS production, and that this mediation is followed by a decrease in the mitochondrial membrane potential (MMP, m), activation of caspase-3, and downregulation of antiapoptotic Bcl-2 protein. The quenching of ROS generation by N-acetyl-L-cysteine administration, a scavenger of ROS, reversed the streptochlorin-induced apoptosis effects via inhibition of ROS production, MMP collapse, and the subsequent activation of caspase-3. These observations clearly indicate that ROS are involved in the early molecular events in the streptochlorin-induced apoptotic pathway. Taken together, our data imply that streptochlorin-induced ROS is a key mediator of MMP collapse, which leads to the caspase-3 activation, culminating in apoptosis.

Combination treatment with decitabine and ionizing radiation enhances tumor cells susceptibility of T cells.

Decitabine has been found to have anti-metabolic and anti-tumor activities in various tumor cells. Recently, the use of decitabine in combination with other conventional therapies reportedly resulted in improved anti-tumor activity against various tumors. Ionizing radiation (IR) is widely used as a cancer treatment. Decitabine and IR improve immunogenicity and susceptibility of tumor cells to immune cells by up-regulating the expression of various molecules such as major histocompatibility complex (MHC) class I; natural-killer group 2, member D (NKG2D) ligands; and co-stimulatory molecules. However, the effects of combining decitabine and IR therapies are largely unknown. Our results indicate that decitabine or IR treatment upregulates MHC class I, along with various co-stimulatory molecules in target tumor cells. Furthermore, decitabine and IR combination treatment further upregulates MHC class I, along with the co-stimulatory molecules, when compared to the effect of each treatment alone. Importantly, decitabine treatment further enhanced T cell-mediated cytotoxicity and release of IFN- γ against target tumor cells which is induced by IR. Interestingly, decitabine did not affect NKG2D ligand expression or NK cell-mediated cytotoxicity in target tumor cells. These observations suggest that decitabine may be used as a useful immunomodulator to sensitize tumor cells in combination with other tumor therapies.

Combination effect of regulatory T-cell depletion and ionizing radiation in mouse models of lung and colon cancer.

PURPOSE: To investigate the potential of low-dose cyclophosphamide (LD-CTX) and anti-CD25 antibody to prevent activation of regulatory T cells (Tregs) during radiation therapy. METHODS AND MATERIALS: We used LD-CTX and anti-CD25 monoclonal antibody as a means to inhibit Tregs and improve the therapeutic effect of radiation in a mouse model of lung and colon cancer. Mice were irradiated on the tumor mass of the right leg and treated with LD-CTX and anti-CD25 antibody once per week for 3 weeks. RESULTS: Combined treatment of LD-CTX or anti-CD25 antibody with radiation significantly decreased Tregs in the spleen and tumor compared with control and irradiation only in both lung and colon cancer. Combinatorial treatments resulted in a significant increase in the effector T cells, longer survival rate, and suppressed irradiated and distal nonirradiated tumor growth. Specifically, the combinatorial treatment of LD-CTX with radiation resulted in outstanding regression of local and distant tumors in colon cancer, and almost all mice in this group survived until the end of the study. CONCLUSIONS: Our results suggest that Treg depletion strategies may enhance radiation-mediated antitumor immunity and further improve outcomes after radiation therapy.

Enhanced dendritic cell-based immunotherapy using low-dose cyclophosphamide and CD25-targeted antibody for transplanted Lewis lung carcinoma cells.

Regulatory T cells (Tregs) is one of the main obstacles to the success of cancer immunotherapy. The effect of dendritic cell (DC)-based immunotherapy can be attenuated by immune suppressive functions of Tregs. We used a CD25-targeted antibody and low-dose cyclophosphamide (CTX) as immunomodulators to increase the antitumor effect of intratumoral injection of immature DCs into the irradiated tumor cells (IR/iDC). CTX or CD25-targeted antibody alone showed a significant reduction in the number of Tregs within the tumor microenvironment. When they are combined with IR/iDC, the number of Tregs was further reduced. Although IR/IDC showed strong antitumor effects such as reduction in tumor growth, increase in Th1 immune response, and improvement of survival, the therapeutic effect was further improved by combining treatments with immunomodulators. CTX and CD25-targeted antibody showed no significant difference in tumor growth when combined with IR/iDC, but CTX further increased the number of interferon (IFN)-γ-secreting T cells, cytotoxicity, and survival rate. Although irradiation induced depletion of T lymphocytes, administration of DCs recovered this depletion. Particularly, the lymphocytes were more significantly increased when CTX and IR/iDC were combined. Low-dose CTX has already been used as an immunomodulator in clinical trials, and it offers several advantages, including convenience, low-cost, and familiarity to clinicians. However, CD25-targeted antibody cannot only deplete Tregs, but also may affect IL-2-dependent effector T lymphocytes. Therefore, CTX is an effective means to inhibit Tregs, and an effective immunomodulatory agent for multimodality therapy such as combination treatment of conventional cancer therapy and immunotherapy.

Diallyl trisulfide-induced apoptosis of bladder cancer cells is caspase-dependent and regulated by PI3K/Akt and JNK pathways.

Diallyl trisulfide (DATS) is one of the major organosulfur components of garlic (Allium sativum L.), which inhibits the proliferation of various cancer cells, but the exact mechanisms of this action in human bladder cancer cells still remain largely unresolved. In this study, we investigated how DATS induces apoptosis in T24 human bladder cancer cells in vitro. Treatment of T24 cells with DATS resulted in potent anti-proliferative activity. Additionally, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, DATS reduced the expression of anti-apoptotic Bcl-2 and Bcl-xL, and inhibitor of apoptosis protein family proteins, but the expression of pro-apoptotic Bax and death receptor-related proteins was increased compared with the controls. DATS also activated caspase-8 and -9, the respective initiator caspases of the extrinsic and the intrinsic apoptotic pathways. The increase in mitochondrial membrane depolarization was correlated with activation of effector caspase-3 and cleavage of poly-ADP-ribose polymerase, a vital substrate of activated caspase-3. Blockage of caspase activation through treatment with a pan-caspase inhibitor consistently inhibited apoptosis and abrogated growth inhibition in DATS-treated T24 cells. The study further investigated the roles of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways with respect to the apoptotic effect of DATS, and showed that DATS deactivates Akt. Additionally, DATS activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38 MAPK, in T24 cells. Unlike ERK, JNK inhibitors reversed DATS-induced apoptosis and growth inhibition; however, inhibition of PI3K/Akt notably enhanced the apoptotic action of DATS. The results suggest that the pro-apoptotic activity of DATS is probably regulated by a caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways, which is mediated through the blocking of PI3K/Akt and the activation of the JNK pathway.

CTLA-4 blockade enhances antitumor immunity of intratumoral injection of immature dendritic cells into irradiated tumor in a mouse colon cancer model.

Dendritic cells (DCs)-based cancer immunotherapy has been used various strategies to inhibit immune suppressive mechanisms. CD25 antibodies and cyclophosphamide are well-studied immunomodulators through inhibition of regulatory T cells (Treg) and a blockade the immune-checkpoint molecule, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) was recently targeted for immunomodulation. We used anti-CTLA-4 antibody, which is known to induce effective antitumor immunity by facilitating tumor-specific T-cell activation and suppressing Treg cells, as useful immunomodulator to provide a potentiating effect in the intratumoral injection of immature DCs (iDCs) into the irradiated tumor (IR/iDC). Ionizing radiation (IR) was applied at a dose of 10 Gy to the tumor on the right thigh of mice. Then, iDCs were intratumorally injected into the irradiated tumor. Anti-CTLA-4 antibody (100 µg/mouse) was administered intraperitoneally to mice on the same day with every iDCs injection. The growth of distant tumors was inhibited by IR/iDC and this effect was significantly augmented by combination treatment of anti-CTLA-4 antibody. Furthermore, the survival rate of tumor-bearing mice improved more by the combination treatment of anti-CTLA-4 antibody and IR/iDC compared with other groups. It was related to the increased tumor-specific interferon-γ-secreting T cells and CTL activity. Therefore, our results demonstrated that immunomodulator such as anti-CTLA-4 antibody enhances antitumor immunity of intratumoral injection of iDCs into irradiated tumor and suggested a new strategy to get more clinical benefits for cancer treatment.

Decitabine, a DNA methyltransferases inhibitor, induces cell cycle arrest at G2/M phase through p53-independent pathway in human cancer cells.

Decitabine (5-aza-2'-deoxycytidine), an inhibitor of DNA methyltransferases, has a wide range of anti-metabolic and anti-cancer activities. Decitabine also induces cell cycle arrest at G2/M phase and apoptosis in human cancer cells. However, the cellular and molecular mechanisms of this cell cycle arrest are poorly understood. In the present study, we investigated the roles of the tumor suppressor p53 and the cyclin-dependent kinase (Cdk) inhibitor p21 following decitabine-induced G2/M arrest in human cancer cells. DNA flow cytometric analyses indicated that decitabine induced a G2/M arrest in AGS gastric and A549 lung carcinoma cell lines, which have wild type p53. Western blot analyses using whole cell lysates from AGS cells demonstrated that decitabine treatment did not change the steady-state level of Cdks and Cdk inhibitor p27, but it partially inhibited expression of cyclin A, cyclin B1, and Cdc25C proteins. However, similar results were found using the A549 cell line, where decitabine induced a dramatic up-regulation of both p53 and p21 expression, and the increased levels of p21 were associated with increased binding of p21 with Cdks, cyclin A, and cyclin B1. Knockdown of p53 by small interfering RNA (siRNA) markedly abolished p53 induction by decitabine in AGS cells, yet p53 siRNA had no attenuating effect on p21 induction. In addition, depletion of p21 expression with siRNA, but not p53, significantly attenuated decitabine-induced G2/M arrest. We also observed that decitabine strongly induced G2/M arrest associated with p21 induction in both p53 allele-null (-/-) HCT116 and wild type p53 (+/+) HCT116 cell lines. Therefore, our data indicated that p21 plays a crucial role in decitabine-induced G2/M arrest and operates in a p53-independent manner.

Anti-invasive effects of decitabine, a DNA methyltransferase inhibitor, through tightening of tight junctions and inhibition of matrix metalloproteinase activities in AGS human gastric carcinoma cells.

The DNA methyltransferase inhibitor decitabine, 5-Aza-2'-deoxycytidine, possesses anti-metabolic and anticancer activities in various cancer cells. However, the biochemical mechanisms underlying decitabine-induced inhibition of invasiveness and metastasis have not been thoroughly studied. In this study, we investigated the effect of decitabine on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in AGS human gastric cancer cells. Our data indicated that the inhibitory effects of decitabine on cell motility and invasiveness were associated with increased tightness of the TJ, which was demonstrated by an increase in transepithelial electrical resistance (TER). Immunoblotting results indicated that decitabine repressed the levels of the claudin proteins, major components of TJs that play a key role in the control and selectivity of paracellular transport. Furthermore, matrix metalloproteinase (MMP)-2 and -9 activity in the AGS cells was dose-dependently inhibited by treatment with decitabine, and this was correlated with a decrease in mRNA and protein expression. In addition, these effects were related to inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in AGS cells. In conclusion, this study suggests that TJs and MMPs are critical targets of decitabine-induced inhibition of invasiveness in AGS human gastric cancer cells.

Apoptotic and anti-metastatic effects of the whole skin of Venenum bufonis in A549 human lung cancer cells.

In the present study, the effects of the whole skin of Venenum bufonis on apoptotic and anti-invasive activity in A549 human lung cancer cells were investigated. Treatment with extract of the whole skin of V. bufonis (SVB) resulted in a significant decrease in cell growth of A549 cells, depending on dosage, which was associated with apoptosis induction, as proved by chromatin condensation and accumulation of apoptotic fraction. SVB treatment induced expression of death receptor-related proteins, such as death receptor 4, which further triggered activation of caspase-8 and cleavage of Bid. In addition, the increase in apoptosis by SVB treatment was correlated with dysfunction of mitochondria, activation of caspase-9 and -3, downregulation of IAP family proteins, such as XIAP, cIAP-1 and cIAP-2, and concomitant degradation of activated caspase-3-specific target proteins, such as poly (ADP-ribose) polymerase and ß-catenin proteins. However, z-DEVD-fmk, a caspase-3-specific inhibitor, blocked SVB-induced apoptosis and increased the survival rate of SVB-treated cells, indicating that activation of caspase-3 plays a key role in SVB-induced apoptosis. In addition, within concentrations that were not cytotoxic to A549 cells, SVB induced marked inhibition of cell motility and invasiveness. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 in AGS cells were dose-dependently inhibited by treatment with SVB, and this was also correlated with a decrease in expression of their mRNA and proteins, and upregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 mRNA expression. Further studies are needed; however, the results indicated that SVB induces apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways. Our data also demonstrated that MMPs are critical targets of SVB-induced anti-invasiveness in A549 cells.

Decitabine, a DNA methyltransferase inhibitor, induces apoptosis in human leukemia cells through intracellular reactive oxygen species generation.

The DNA methyltransferase inhibitor decitabine, 5-aza-2'-deoxycytidine, has been found to exert anti-metabolic and anticancer activities when tested against various cultured cancer cells. Furthermore, decitabine has been found to play critical roles in cell cycle arrest and apoptosis in various cancer cell lines; however, these roles are not well understood. In this study, we investigated decitabine for its potential anti-proliferative and apoptotic effects in human leukemia cell lines U937 and HL60. Our results indicated that treatment with decitabine resulted in significantly inhibited cell growth in a concentration- and time-dependent manner by the induction of apoptosis. Decitabine-induced apoptosis in U937 and HL60 cells was correlated with the downregulation of anti-apoptotic Bcl-2, XIAP, cIAP-1 and cIAP-2 protein levels, the cleavage of Bid proteins, the activation of caspases and the collapse of mitochondrial membrane potential (MMP). However, apoptosis induced by decitabine was attenuated by caspase inhibitors, indicating an important role for caspases in decitabine responses. The data further demonstrated that decitabine increased intracellular reactive oxygen species (ROS) generation. Moreover, N-acetyl-L-cysteine, a widely used ROS scavenger, effectively blocked the decitabine-induced apoptotic effects via inhibition of ROS production and MMP collapse. These observations clearly indicate that decitabine-induced ROS in human leukemia cells are key mediators of MMP collapse, which leads to apoptosis induction followed by caspase activation.

Improvement of antitumor effect of intratumoral injection of immature dendritic cells into irradiated tumor by cyclophosphamide in mouse colon cancer model.

Recently, chemotherapy and radiotherapy are known to directly affect some immunosuppressive barriers within a tumor microenviroment. We used cyclophosphamide (CTX), which is known to enhance the immune response by suppressing CD4+CD25+ regulatory T cells (Treg cells) when used at a low dose, as a chemotherapeutic agent to provide a synergic effect in the irradiation and dendritic cells (DC) combination therapy. Some previous studies observed that a single-dose CTX treatment significantly reduced the number of Treg cells in 3-5 days, however, the reduced Treg cells increased rapidly after 5 days. To overcome the disadvantages of a single-dose CTX, we used 30 mg/kg dose of CTX, which was treated intraperitoneally to mice 3 days before every immature DC (iDC) injection (known as "metronomic schedule CTX"). Irradiation was applied at a dose of 10 Gy to the tumor on the right thigh by a linear accelerator. Then, iDC was intratumorally injected into the irradiated tumor site. Growth of a distant tumor on the right and left flank was suppressed by an injection of iDC into the irradiated tumor, and this effect was increased by the metronomic schedule CTX. Also, combinations treated with the metronomic schedule CTX and ionizing radiation (IR)/iDC, showed the longest survival time compared with other groups. This antitumor immune response of IR/iDC was improved by metronomic schedule CTX and this result was associated with decreasing the proportion of CD4+CD25+ Treg cells and increasing the number of tumor-specific interferon-γ-secreting T cells. Our results demonstrated that metronomic schedule CTX improves the antitumor effect of immunization with an injection of DC s into the irradiated tumor.