MAOB expression correlates with a favourable prognosis in prostate cancer, and its genetic variants are associated with the metastasis of the disease.

Monoamine oxidase B (MAOB), a neurotransmitter-degrading enzyme, was reported to reveal conflicting roles in various cancers. However, the functional role of MAOB and impacts of its genetic variants on prostate cancer (PCa) is unknown. Herein, we genotyped four loci of MAOB single-nucleotide polymorphisms (SNPs), including rs1799836 (A/G), rs3027452 (G/A), rs6651806 (A/C) and rs6324 (G/A) in 702 PCa Taiwanese patients. We discovered that PCa patients carrying the MAOB rs6324 A-allele exhibited an increased risk of having a high initial prostate-specific antigen (iPSA) level (>10 ng/mL). Additionally, patients with the rs3027452 A-allele had a higher risk of developing distal metastasis, particularly in the subpopulation with high iPSA levels. In a subpopulation without postoperative biochemical recurrence, patients carrying the rs1799836 G-allele had a higher risk of developing lymph node metastasis and recurrence compared to those carrying the A-allele. Furthermore, genotype screening in PCa cell lines revealed that cells carrying the rs1799836 G-allele expressed lower MAOB levels than those carrying the A-allele. Functionally, overexpression and knockdown of MAOB in PCa cells respectively suppressed and enhanced cell motility and proliferation. In clinical observations, correlations of lower MAOB expression levels with higher Gleason scores, advanced clinical T stages, tumour metastasis, and poorer prognosis in PCa patients were noted. Our findings suggest that MAOB may act as a suppressor of PCa progression, and the rs3027452 and rs1799836 genetic variants of MAOB are linked to PCa metastasis within the Taiwanese population.

Activin A downregulates the CD69-MT2A axis via p38MAPK to induce erythroid differentiation that sensitizes BCR-ABL-positive cells to imatinib.

Induction of differentiation sensitizes chronic myeloid leukemia (CML) cells to the BCR-ABL inhibitor imatinib by mechanisms that remain unknown. We previously identified the BCR-ABL downstream effector CD69 which inhibits imatinib-induced CML cell differentiation. Herein, we found that the erythroid differentiation inducers activin A and aclacinomycin A induced expression of erythroid markers (α-globin, ζ-globin, GATA-1, and glycophorin A) and simultaneously reduced CD69 levels in K562 CML cells. Blockade of p38MAPK by SB203580 and shRNA eliminated the inhibitory effect of activin A on the promoter, mRNA, and protein levels and positive cell population of CD69. CD69 overexpression inhibited activin A-induced erythroid marker expression. Pretreatment of K562 cells with activin A to induce differentiation followed by a subtoxic concentration of imatinib caused growth inhibition and apoptosis that was reduced by CD69 overexpression. Activin A also reduced the expression of CD69's potential downstream molecule metallothionein 2A (MT2A) via p38MAPK. MT2A-knockdown reduced CD69 inhibition of activin A-induced erythroid marker expression. Furthermore, MT2A-knockdown reduced CD69 inhibition of activin A-imatinib sequential treatment-mediated growth inhibition and apoptosis in K562 and BCR-ABL-expressing CD34+ cells. These results suggest that CD69 inhibits activin A induction of erythroid differentiation-mediated CML cell sensitivity to imatinib via MT2A. Therefore, activin A induction of erythroid differentiation sensitizes BCR-ABL-positive cells to imatinib by downregulating the erythroid differentiation suppressors CD69 and MT2A.

Activation of PERK in ET-1- and thrombin-induced pulmonary fibroblast differentiation: Inhibitory effects of curcumin.

In the present study, we investigated the role of PKR-like endoplasmic reticular kinase (PERK), an endoplasmic reticulum (ER) stress kinase, in endothelin 1 (ET-1)- and thrombin-induced pulmonary fibrosis (PF), and the preventive effects of curcumin (CUR). Using the human embryonic WI-38 lung fibroblast cell line, ET-1 and thrombin induced the expression of ER stress-related proteins (CCAAT-enhancer-binding protein homologous protein, PERK, and binding immunoglobulin protein), a profibrogenic factor (cellular communication network factor 2 [CCN2]), and differentiation markers including α-smooth muscle actin (α-SMA), collagen I (Col I), and Col IV. Knockdown of PERK expression via small interfering RNA (siRNA) significantly reduced the increases in CCN2, α-SMA, Col I, and Col IV proteins in WI-38 cells according to western blot analysis and immunohistochemistry (IHC). Activation of c-Jun N-terminal kinase (JNK) was observed in ET-1- and thrombin-treated WI-38 cells, and the addition of a JNK inhibitor (SP) suppressed the induction of the indicated proteins by ET-1 and thrombin. Thapsigargin (TG), an ER stress inducer, elevated expressions of PERK and ER stress-related proteins with increased differentiation of WI-38 cells. Knockdown of PERK by siRNA or the PERK inhibitor glycogen synthesis kinase reduced expressions of the differentiation markers, α-SMA and Col IV, in WI-38 cells. CUR concentration-dependently inhibited ET-1- or thrombin-induced CCN2, α-SMA, and vimentin proteins with decreased levels of phosphorylated mitogen-activated protein kinase and PERK in WI-38 cells. An in vivo bleomycin-induced PF study showed that an intraperitoneal injection of CUR (30 mg/kg) reduced expressions of α-SMA, CCN2, Col IV, and vimentin in lung tissues via IHC staining using specific antibodies. This study is the first to demonstrate that PERK activation contributes to pulmonary fibroblast differentiation elicited by ET-1 or thrombin, and the inhibitory activity of CUR against PF is demonstrated herein.

Jagged1-expressing adenovirus-infected dendritic cells induce expansion of Foxp3+ regulatory T cells and alleviate T helper type 2-mediated allergic asthma in mice.

Dendritic cells (DCs) are professional antigen-presenting cells that play a key role in directing T-cell responses. Regulatory T (Treg) cells possess an immunosuppressive ability to inhibit effector T-cell responses, and Notch ligand Jagged1 (Jag1) is implicated in Treg cell differentiation. In this study, we evaluated whether bone marrow-derived DCs genetically engineered to express Jag1 (Jag1-DCs) would affect the maturation and function of DCs in vitro and further investigated the immunoregulatory ability of Jag1-DCs to manipulate T helper type 2 (Th2) -mediated allergic asthma in mice. We produced Jag1-DCs by adenoviral transduction. Overexpression of Jag1 by ovalbumin (OVA) -stimulated Jag1-DCs exhibited increased expression of programmed cell death ligand 1 (PD-L1) and OX40L molecules. Subsequently, co-culture of these OVA-pulsed Jag1-DCs with allogeneic or syngeneic CD4+ T cells promoted the generation of Foxp3+ Treg cells, and blocking PD-L1 using specific antibodies partially reduced Treg cell expansion. Furthermore, adoptive transfer of OVA-pulsed Jag1-DCs to mice with OVA-induced asthma reduced allergen-specific immunoglobulin E production, airway hyperresponsiveness, airway inflammation, and secretion of Th2-type cytokines (interleukin-4, interleukin-5, and interleukin-13). Notably, an increased number of Foxp3+ Treg cells associated with enhanced levels of transforming growth factor-ß production was observed in Jag1-DC-treated mice. These data indicate that transgenic expression of Jag1 by DCs promotes induction of Foxp3+ Treg cells, which ameliorated Th2-mediated allergic asthma in mice. Our study supports an attractive strategy to artificially generate immunoregulatory DCs and provides a novel approach for manipulating Th2 cell-driven deleterious immune diseases.

Application of Electric Cell-Substrate Impedance Sensing to Investigate the Cytotoxic Effects of Andrographolide on U-87 MG Glioblastoma Cell Migration and Apoptosis.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In recent studies, the efficacy of suberoylanilide hydroxamic acid (SAHA) has been investigated for GBM. We explored the effects of two exploratory compounds, the histone deacetylase SAHA and the natural product andrographolide, on Uppsala 87 Malignant Glioma (U-87 MG) cell migration and viability in comparison with the clinically used therapeutic agent temozolomide (TMZ). We used the electric cell-substrate impedance sensing (ECIS) system to monitor the migration of U-87 MG cells after treatment with various concentrations of these compounds. Moreover, we used the Alamar blue assay and western blotting to observe the concentration-dependent changes in the viability and apoptosis of U-87 MG cells. Our results demonstrated that both SAHA and andrographolide (10-300 µM) significantly inhibited GBM cell migration in a concentration-dependent manner, and 10 µM SAHA and 56 µM andrographolide demonstrated remarkable inhibitory effects on U-87 MG migration. Western blotting indicated that compared with TMZ, both SAHA and andrographolide induced higher expression levels of apoptosis-related proteins, such as caspase-3, BAX, and PARP in U-87 MG cells. Furthermore, all three drugs downregulated the expression of the antiapoptotic protein Bcl-2. In conclusion, SAHA and andrographolide showed exceptional results in inhibiting cell migration and motility. The ECIS wound healing assay is a powerful technique to identify and screen potential therapeutic agents that can inhibit cancer cell migration.

CD69 partially inhibits apoptosis and erythroid differentiation via CD24, and their knockdown increase imatinib sensitivity in BCR-ABL-positive cells.

Chronic myeloid leukemia (CML) is caused by a constitutively active BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKIs) imatinib and its derivatives represent a breakthrough for CML therapy, but the use of TKI alone is ineffective for many CML patients. CD69, an early activation marker of lymphocytes, participates in immune and inflammatory responses. Previous studies revealed that BCR-ABL upregulates CD69 expression; however, the role of CD69 in CML cells is unknown. Here, we demonstrate that BCR-ABL induced CD69 promoter activity and mRNA and protein expression via the NF-κB pathway. CD69 knockdown partially increased apoptosis and expression of erythroid differentiation markers, α-globin, ζ-globin, and glycophorin A, and increased imatinib sensitivity in K562 and KU812 CML cells. Gene microarray analysis and quantitative real-time PCR verified that CD24, an oncogenic gene, downregulated in K562 cells upon CD69 knockdown. CD69 overexpression increased, whereas CD69 knockdown inhibited CD24 promoter activity and mRNA and protein levels. CD24 knockdown also partially increased apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells, whereas its overexpression inhibited the effects of CD69 knockdown on apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells. Imatinib-induced apoptosis and erythroid differentiation were also inhibited by CD69 or CD24 overexpression in BCR-ABL-expressing CML cell lines and CD34+ cells. Taken together, CD24 is a downstream effector of CD69. CD69 and CD24 partially inhibit apoptosis and erythroid differentiation in CML cells; thus, they may be potential targets to increase imatinib sensitivity.

Nilotinib induction of melanogenesis via reactive oxygen species-dependent JNK activation in B16F0 mouse melanoma cells.

Nilotinib (AMN), a second-generation tyrosine kinase inhibitor, induces apoptosis in various cancer cells, and our recent study showed that AMN effectively reduced the viability of human ovarian cancer cells via mitochondrion-dependent apoptosis. The effect of AMN in the melanogenesis of melanoma cells is still unclear. In the present study, we found that the addition of AMN but not imatinib (STI) significantly increased the darkness of B16F0 melanoma cells, and the absorptive value increased with the concentration of AMN. A decrease in the viability of B16F0 cells by AMN was detected in a concentration-dependent manner, accompanied by increased DNA ladders, hypodiploid cells and cleavage of the caspase-3 protein. An in vitro tyrosinase (TYR) activity assay showed that increased TYR activity by AMN was detected in a concentration-dependent manner; however, induction of TYR activity by STI at a concentration of 40 µmol/L was observed. Increased intracellular peroxide by AMN was detected in B16F0 cells, and application of the antioxidant, N-acetylcysteine (NAC), significantly reduced AMN-induced peroxide production which also reduced the darkness of B16F0 cells. Additionally, AMN induced c-Jun N-terminal kinase (JNK) protein phosphorylation in B16F0 cells, which was inhibited by the addition of NAC. AMN-induced melanogenesis of B16F0 cells was significantly inhibited by the addition of NAC and the JNK inhibitor, SP600125 (SP). Data of Western blotting showed that increased protein levels of melanogenesis-related enzymes of tyrosinase-related protein-1 (TRP1), TRP2 and TYR were observed in AMN-treated B16F0 cells which were inhibited by the addition of NAC and SP. Evidence is provided supporting AMN effectively inducing the melanogenesis of B16F0 melanoma cells via reactive oxygen species-dependent JNK activation.

MPT0B169 and MPT0B002, New Tubulin Inhibitors, Induce Growth Inhibition, G2/M Cell Cycle Arrest, and Apoptosis in Human Colorectal Cancer Cells.

We previously synthesized new tubulin inhibitors, MPT0B169 and MPT0B002, which induced growth inhibition and apoptosis in leukemia cells. However, their effects on solid tumor cells have not been determined. In this study, we investigated the effects of MPT0B169 and MPT0B002 on glioblastoma, breast, lung, and colorectal cancer (CRC) cell lines. A cell viability analysis showed that MPT0B169 and MPT0B002 were more effective in inhibiting the proliferation of COLO205 and HT29 CRC cells than U87MG and GBM8401 glioblastoma, MCF-7 and MDA-MB-231 breast cancer, and A549 lung cancer cells. MPT0B169 and MPT0B002 inhibited growth of COLO205 and HT29 cells in dose- and time-dependent manners. A colony-formation assay confirmed the growth inhibitory effects of MPT0B169 and MPT0B002 on COLO205 and HT29 cells. MPT0B169 and MPT0B002 disrupted tubulin polymerization and arrested the cell cycle at the G2/M phase, with a concomitant increase of the cyclin B1 level. MPT0B169 and MPT0B002 induced apoptosis, accompanied by induction of the intrinsic apoptotic pathway, as shown by a reduction in the caspase-9 level and increases in cleaved caspase-3 and cleaved PARP. These results suggest that MPT0B169 and MPT0B002, new tubulin inhibitors, induced growth inhibition, G2/M arrest, and apoptosis in COLO205 and HT29 cells, and they could potentially be anticancer agents for CRC cells.

MPT0B002, a novel microtubule inhibitor, downregulates T315I mutant Bcr-Abl and induces apoptosis of imatinib-resistant chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy caused by the constitutive activation of Bcr-Abl tyrosine kinase. The Bcr-Abl inhibitor imatinib and other second-generation tyrosine kinase inhibitors such as dasatinib and nilotinib have remarkable efficacy in CML treatment. However, gene mutation-mediated drug resistance remains a critical problem. Among point mutations, the Bcr-Abl T315I mutation confers resistance to these Bcr-Abl inhibitors. Previously, we have synthesized the compound (1-methyl-1H-indol-5-yl)-(3,4,5-trimethoxy-phenyl)-methanone (MPT0B002) as a novel microtubule inhibitor. In this study, we evaluated its effects on the proliferation, cell cycle, and apoptosis of K562 CML cells and BaF3 cells expressing either wild-type Bcr-Abl (BaF3/p210) or T315I-mutated Bcr-Abl (BaF3/T315I). MPT0B002 inhibited cell viability in a dose-dependent manner in these cells but did not affect the proliferation of human umbilical vein endothelial cells. It disrupted tubulin polymerization and arrested cell cycle at the G2/M phase. Treatment with MPT0B002 induced apoptosis, and this induction was associated with increased levels of cleaved caspase-3 and cleaved PARP. Furthermore, MPT0B002 can downregulate both Bcr-Abl and Bcr-Abl-T315I mRNA expressions and protein levels and the downstream signaling pathways. Taken together, our findings suggest that MPT0B002 may be considered a promising compound to downregulate not only wild type Bcr-Abl but also the T315I mutant to overcome Bcr-Abl-T315I mutation-mediated resistance in CML cells.

A novel tubulin polymerization inhibitor, MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells.

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.

MPT0B169, a novel tubulin inhibitor, induces apoptosis in taxol-resistant acute myeloid leukemia cells through mitochondrial dysfunction and Mcl-1 downregulation.

Acute myeloid leukemia (AML) is a hematological malignant disorder. AML cells are not susceptible to chemotherapeutic drugs because of their multidrug resistance (MDR). Antitubulin agents are currently employed in cancer treatments; however, drug resistance results in treatment failures because of MDR1 expressing cancer cells. We previously synthesized a new tubulin inhibitor, 2-dimethylamino-N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-acetamide (MPT0B169), which inhibits AML cell proliferation by arresting cell cycle at the G2/M phase. In this study, we explored the effect of MPT0B169 on apoptosis in AML HL60 and NB4 cells and MDR1-mediated taxol-resistant HL60/TaxR cells and the underlying mechanism. MPT0B169 induced concentration- and time-dependent apoptosis in these cancer cells, as observed through annexin V/propidium iodide double staining and flow cytometry. Furthermore, DNA fragmentation analysis confirmed MPT0B169-induced apoptosis. MPT0B169 induced a loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, cleavage and activation of caspase-9 and caspase-3, and consequently cleavage of poly (ADP ribose) polymerase. Western blot analysis showed that MPT0B169 markedly reduced Mcl-1 (an antiapoptotic protein) levels; however, it caused no changes in Bcl-2 or BAX (a proapoptotic protein). Knockdown of Mcl-1 using small interfering RNA (siRNA) slightly induced growth inhibition and apoptosis in the HL60 and HL60/TaxR cells. Further investigation revealed that Mcl-1 siRNA enhanced the sensitivity of HL60 and HL60/TaxR cells to MPT0B169-induced growth inhibition and apoptosis. Together, these results demonstrated that MPT0B169-induced apoptosis in nonresistant and MDR1-mediated taxol-resistant AML cells through Mcl-1 downregulation and a mitochondria-mediated pathway. MPT0B169 can overcome MDR1-mediated drug resistance in AML cells.

Activin A induction of erythroid differentiation sensitizes K562 chronic myeloid leukemia cells to a subtoxic concentration of imatinib.

Chronic myeloid leukemia (CML) is a hematopoietic stem/progenitor cell disorder in which Bcr-Abl oncoprotein inhibits cell differentiation. Differentiation induction is considered an alternative strategy for treating CML. Activin A, a member of the transforming growth factor-ß superfamily, induces erythroid differentiation of CML cells through the p38 MAPK pathway. In this study, treatment of the K562 CML stem/progenitor cell line with activin A followed by a subtoxic concentration of the Bcr-Abl inhibitor imatinib strongly induced growth inhibition and apoptosis compared with simultaneous treatment with activin A and imatinib. Imatinib-induced growth inhibition and apoptosis following activin A pretreatment were dose- and time-dependent. Imatinib-induced growth inhibition and apoptosis were also dependent on the pretreatment dose of activin A. More than 90% of the activin A-induced increases in glycophorin A-positive cells were sensitive to imatinib. However, only some of original glycophorin A-positive cells in the activin A treatment group were sensitive to imatinib. Sequential treatment with activin A and imatinib decreased Bcr-Abl, procaspase-3, Mcl-1, and Bcl-xL and also induced cleavage of procaspase-3/poly(ADP-ribose)polymerase. The reduction of erythroid differentiation in p38 MAPK dominant-negative mutants or by short hairpin RNA knockdown of p38 MAPK decreased the growth inhibition and apoptosis mediated by sequential treatment with activin A and imatinib. Furthermore, the same inhibition level of multidrug resistance 1 expression was observed in cells treated with activin A alone, treated sequentially with activin A and imatinib, or treated simultaneously with activin A and imatinib. The p38 MAPK inhibitor SB-203580 can restore activin A-inhibited multidrug resistance 1 expression. Taken together, our results suggest that a subtoxic concentration of imatinib could exhibit strong cytotoxicity against erythroid-differentiated K562 CML cells.

Interferon-γ suppresses activin A/NF-E2 induction of erythroid gene expression through the NF-κB/c-Jun pathway.

Interferon (IFN)-γ is a proinflammatory cytokine that is linked to erythropoiesis inhibition and may contribute to anemia. However, the mechanism of IFN-γ-inhibited erythropoiesis is unknown. Activin A, a member of the transforming growth factor (TGF)-ß superfamily, induces the erythropoiesis of hematopoietic progenitor cells. In this study, a luciferase reporter assay showed that IFN-γ suppressed activin A-induced ζ-globin promoter activation in K562 erythroblast cells in a dose-dependent manner. Activin A reversed the suppressive effect of IFN-γ on the luciferase activity of ζ-globin promoter in a dose-dependent manner. IFN-γ also suppressed the activation of activin A-induced α-globin promoter. IFN-γ reduced the mRNA expression of α-globin, ζ-globin, NF-E2p45, and GATA-1 induced by activin A. The results also showed that IFN-γ induced c-Jun expression when NF-κBp65 and c-Jun bound to two AP-1-binding sites on the c-Jun promoter. The luciferase activity of α-globin and ζ-globin promoters were enhanced by wild-type c-Jun and eliminated by dominant-negative (DN) c-Jun. The suppressive effects of IFN-γ on the mRNA expression of α-globin and ζ-globin were absent in cells expressing DN c-Jun. The ability of NF-E2 to enhance activin A-induced ζ-globin promoter activation decreased when c-Jun was present, and IFN-γ treatment further enhanced the decreasing effect of c-Jun. Chromatin immunoprecipitation revealed that NF-E2p45 bound to the upstream regulatory element (HS-40) of the α-globin gene cluster in response to activin A, whereas c-Jun eliminated this binding. These results suggest that IFN-γ modulates NF-κB/c-Jun to antagonize activin A-mediated NF-E2 transcriptional activity on globin gene expression.

MPT0B169, a new tubulin inhibitor, inhibits cell growth and induces G2/M arrest in nonresistant and paclitaxel-resistant cancer cells.

The polymerization of tubulin molecules forms microtubules which are considered an attractive target for cancer treatment. Herein, we synthesized a new tubulin inhibitor, MPT0B169 (2-dimethylamino-N-[1-(4-methoxy-benzenesulfonyl)-2,​3-dihydro-1H-indol-7-yl]-acetamide) and demonstrated its action in leukemia cell lines HL60 and NB4 and lymphoma cell line U937. We found that MPT0B169 prevented tubulin assembly by binding the colchicine-binding site of tubulin in vitro. MPT0B169 also induced tubulin depolymerization in vivo. MPT0B169 inhibited the growth of HL60, NB4, and U937 cells in dose- and time-dependent manners. It also inhibited the growth of paclitaxel-resistant cancer cells. In addition, MPT0B169 caused G2/M cell cycle arrest in nonresistant and paclitaxel-resistant cancer cells, with a concomitant increase in cyclin B1 levels and cyclin-dependent kinase 1 (CDK1) phosphorylation. These results suggest that MPT0B169, a tubulin inhibitor, inhibits cell growth and induces G2/M cell cycle arrest of cancer cells through the disruption of tubulin polymerization.

Diverse effects of type II collagen on osteogenic and adipogenic differentiation of mesenchymal stem cells.

Type II collagen is known to modulate chondrogenesis of mesenchymal stem cells (MSCs). In this study, MSCs from human bone marrow aspirates were used to study the modulating effects of type II collagen on MSC differentiation during the early stages of osteogenesis and adipogenesis. With osteogenic induction, MSCs cultured on the type II collagen-coated surface showed an enhanced calcium deposition level with increasing mRNA expressions of RUNX2, osteocalcin, and alkaline phosphatase. A synthetic integrin binding peptide, which specifically interacts with the I-domain of α(1)ß(1)/α(2)ß(1) integrins significantly blocks the mineralization-enhancing effect of type II collagen. MSCs attached on the type II collagen-coated plates exhibited expanded cell morphology with increasing spreading area, and the pretreatment of cells with integrin α(1)ß(1) or α(2)ß(1)-blocking antibody reduced the effect. The phosphorylation levels of FAK, ERK, and JNK significantly increased in the MSCs that attached on the type II collagen-coated plates. On the contrary, the mineralization-enhancing effect of type II collagen was diminished by JNK and MEK inhibitors. Furthermore, type II collagen blocked the adipogenic differentiation of MSCs, and this effect is rescued by JNK and MEK inhibitors. In conclusion, type II collagen facilitates osteogenesis and suppresses adipogenesis during early stage MSC differentiation. Such effects are integrin binding-mediated and conducted through FAK-JNK and/or FAK-ERK signaling cascades. These results inspire a novel strategy encompassing type II collagen in bone tissue engineering.

Proanthocyanidins-loaded complex coacervates-based drug delivery attenuates oral squamous cell carcinoma cells metastatic potential through down-regulating the Akt signaling pathway.

Oral cancer, constituted up to 90% by squamous cell carcinomas, is a significant health burden globally. Grape seed proanthocyanidins (PA) have been suggested as a potential chemopreventive agent for oral cancer. However, their efficacy can be restricted due to the low bioavailability and bioaccessibility. Inspired by sandcastle worm adhesive, we adapted the concept of complex coacervation to generate a new type of drug delivery platform. Complex coacervates are a dense liquid phase formed by the associative separation of a mixture of oppositely charged polyelectrolytes, can serve as a drug delivery platform to protect labile cargo. In this study, we developed a complex coacervates-based delivery of PA. The release kinetics was measured, and anticancer effects were determined in two human tongue squamous cell carcinoma cell lines. The results showed that complex coacervate successfully formed and able to encapsulate PA. Additionally, PA were steadily released from the system in a pH-dependent manner. The drug delivery system could significantly inhibit the cell proliferation, migration, and invasion of cancer cells. Moreover, it could markedly reduce the expression of certain matrix metalloproteinases (MMP-2, 9, and 13) crucial to metastatic processes. We also found that suppression of protein kinase B (Akt) pathway might be the underlying mechanism for these anticancer activities. Taken together, complex coacervates-based delivery of PA can act as an effective anticancer approach for oral cancer therapy.

Selective activation of specific PKC isoforms dictating the fate of CD14(+) monocytes towards differentiation or apoptosis.

In this study, phorbol-12-myristate-13-acetate (PMA) at low concentrations (<10 nM; L-PMA) induces the differentiation of CD14(+) monocytes into monocyte-derived macrophages (MDMs) while PMA at high concentrations (>100 nM; H-PMA) causes the apoptosis of these cells. The pre-treatment with Go6976 (a PKC-α/ß(1) selective inhibitor), not anilinemonoindolylmaleimide [a PKC-ß inhibitor (PKC-ß inh.)], significantly (P < 0.05) reduces the L-PMA-induced generation of MDMs in the cultured CD14(+) monocytes. On the other hand, either of the above two PKC inhibitors is capable of suppressing the H-PMA-induced apoptosis of CD14(+) monocytes. However, only the inclusion of PKC-ß inh., not Go6976, prevents the cells from serum deprivation-induced cell apoptosis. Although the membrane translocation of conventional PKC-α, ß(1), and ß(2) isoforms was observed in the H-PMA-treated CD14(+) monocytes, only PKC-ß(2) exhibits a mitochondrial translocation activity among those PKCs responsive to H-PMA treatment. Moreover, the activation of DEVD-dependent caspases (DEVDase) was also detected in the H-PMA-treated CD14(+) monocytes, indicating the involvement of a caspase-dependent signaling pathway in the H-PMA-induced cell apoptosis of CD14(+) monocytes. Together with our previous findings that the selective activation of PKC-α or PKC-ß(1) induces the differentiation of CD14(+) monocytes into MDMs or dendritic cells (MoDCs), respectively, the results in this study further demonstrate that PKC-ß(2) activation is responsible for relaying the apoptotic signal to intrinsic mitochondria-dependent caspase signaling cascades in the CD14(+) monocytes. It is likely that the selective activation of specific PKC isoforms provides a new strategy to manipulate the differential cell fate commitment of multipotent CD14(+) monocytes towards apoptosis or differentiation into MDMs, MoDCs, and other cell types.

Activin A induction of erythroid differentiation through MKK6-p38alpha/p38beta pathway is inhibited by follistatin.

Activin A is a member of the transforming growth factor (TGF)-beta superfamily that regulates cell proliferation and differentiation. Using the p38 inhibitor SB203580, our previous studies demonstrated that p38 was involved in activin A-mediated hemoglobin (Hb) synthesis in K562 cells. SB203580 is an inhibitor of p38alpha and p38beta isoforms. In this study, we show that p38alpha and p38beta mRNA were expressed in K562 cells and that activin A activated the kinase activities of these isoforms. To investigate the roles of p38alpha and p38beta isoforms in activin A-mediated erythroid differentiation, we generated stable clones that over-expressed the dominant negative p38 isoforms p38alpha(AF) and p38beta(AF) in K562 cells. The expressions of either p38alpha(AF) or p38beta(AF) reduced activin A-induced p38 activation, Hb synthesis, and zeta-globin promoter activity. Similarly, down-regulation of either p38alpha or p38beta by isoform-specific siRNAs also reduced activin A-induced zeta-globin promoter activity. Co-expressions of p38alpha(AF) and p38beta(AF), together, greatly inhibited the transcription activity of the zeta-globin promoter. Conversely, expression of mitogen-activated protein kinase kinase (MKK) 6b(E), a constitutive activator of p38, significantly activated zeta-globin promoter. Co-expressions of either p38alpha or p38beta with MKK6b had a similar activation of zeta-globin promoter. Activin A induction of erythroid differentiation was inhibited by follistatin. Activin A-induced phosphorylation of MKK6 and p38 was also inhibited by follistatin. Moreover, over-expression of MKK6b(E) reverted follistatin inhibition of activin A-induced zeta-globin promoter activity. These results demonstrate that activin A induces erythroid differentiation of K562 cells through activation of MKK6-p38alpha/p38beta pathway and follistatin inhibits those effects.

Histone deacetylase inhibitor MPT0B291 suppresses Glioma Growth in vitro and in vivo partially through acetylation of p53.

Background: Histone deacetylase (HDAC) inhibitors have emerged as a new class of anti-tumor agents for various types of tumors, including glioblastoma. Methods and results: We found that a novel HDAC inhibitor, MPT0B291, significantly reduced the cell viability and increased cell death of human and rat glioma cell lines, but not in normal astrocytes. We also demonstrated that MPT0B291 suppressed proliferation by inducing G1 phase cell cycle arrest and increased apoptosis in human and rat glioma cell lines by flow cytometry and immunocytochemistry. We further investigated the anti-tumor effects of MPT0B291 in xenograft (mouse) and allograft (rat) models. The IVIS200 images and histological analysis indicated MPT0B291 (25 mg/kg, p. o.) reduced tumor volume. Mechanistically, MPT0B291 increased phosphorylation and acetylation/activation of p53 and increased mRNA levels of the apoptosis related genes PUMA, Bax, and Apaf1 as well as increased protein level of PUMA, Apaf1 in C6 cell line. The expression of cell cycle related gene p21 was also increased and Cdk2, Cdk4 were decreased by MPT0B291. Conclusion: Our study highlights the anti-tumor efficacy of a novel compound MPT0B291 on glioma growth.

c-Jun blocks cell differentiation but not growth inhibition or apoptosis of chronic myelogenous leukemia cells induced by STI571 and by histone deacetylase inhibitors.

The constitutively active Bcr-Abl tyrosine kinase plays a crucial role in chronic myelogenous leukemia (CML) pathogenesis. The Bcr-Abl protein induces the upregulation of proto-oncogene c-Jun, which is involved in Bcr-Abl transforming activity in Bcr-Abl positive cells. Recent studies reported that c-Jun inhibited hemoglobin synthesis in human CML cell line K562. However, c-Jun also plays a critical role in cell proliferation and apoptosis. In this study, we investigated the physiological roles of c-Jun in cell proliferation, apoptosis and erythroid differentiation of K562 cells. Firstly, we generated K562 cell lines stably overexpressing c-Jun. These clones have the same proliferation rate as the parental cell line in general culture medium. Endogenous c-Jun expression was analyzed to determine the effective concentration of STI571 for inhibiting Bcr-Abl signaling. Western blots show that STI571 inhibited c-Jun expression in a dose-dependent manner, reaching a maximum inhibition at 1 microM. STI571 could inhibit c-Jun expression in K562 cells, but not in c-Jun-overexpression cells. c-Jun did not alter growth inhibition and apoptotic induction by STI571 treatment, but inhibited STI571-induced erythroid differentiation. Moreover, c-Jun did not alter growth inhibition and apoptotic induction by histone deacetylase (HDAC) inhibitors (apicidin, sodium butyrate, and MS275) treatment, but inhibited HDAC inhibitors-induced erythroid differentiation. These results suggest that c-Jun may modulate anticancer drugs-induced cell differentiation but not growth inhibition and apoptosis in CML cells.