Huei-Mei Huang

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.

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.

Paclitaxel (PTX) is a common regimen used to treat patients with ovarian cancer. Although approximately 60% of ovarian cancer patients exhibit a pathologic complete response (pCR), approximately 40% of patients appear to be insensitive to PTX adjuvant therapy. Thus, identifying a useful biomarker to predict pCR would be of great help to ovarian cancer patients who decide to receive PTX treatment. We found that FBXL7 was downregulated in OVSAHO (PTX-sensitive) but upregulated in KURAMOCHI (PTX-resistant) cells after PTX treatment at cytotoxic concentrations. Moreover, our data showed that the fold change of FBXL7 expression post-treatment with PTX was causally correlated with the 50% inhibitory concentrations (IC₅₀) of PTX in a panel of ovarian cancer cell lines. In assessments of progression-free survival probability, high levels of FBXL7 transcript strongly predicted a poor prognosis and unfavorable response to PTX-based chemotherapy in patients with ovarian cancer. The knockdown of FBXL7 predominantly enhanced the cytotoxic effectiveness of PTX on the PTX-resistant KURAMOCHI cells. FBXL7 may be a useful biomarker for predicting complete pathologic response in ovarian cancer patients who decide to receive post-operative PTX therapy.

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.

Summary 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.

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.

Chronic myeloid leukemia (CML) is a clonal disorder of hematopoietic stem/progenitor cells that is caused by the Bcr-Abl oncoprotein. Clinical resistance to the Bcr-Abl inhibitor imatinib is a critical problem in treating CML. This study investigated the antitumor effect and mechanism of MPT0B169, a new antitubulin agent, in K562 CML cells and their derived imatinib-resistant cells, IMR2 and IMR3. IMR2 and IMR3 cells showed complete resistance to imatinib-induced growth inhibition and apoptosis. Resistance involved ERK1/2 overactivation and MDR1 overexpression. MPT0B169 inhibited the growth of K562, IMR2, and IMR3 cells in a dose- and time-dependent manner. MPT0B169 substantially inhibited the mRNA and protein levels of Bcr-Abl, followed by its downstream pathways including Akt, ERK1/2, and STAT3 in these cells. MPT0B169 treatment resulted in a decrease in the polymer form of tubulin according to Western blot analysis. It triggered cell cycle arrest at the G2/M phase before apoptosis, which was related to the upregulation of the mitotic marker MPM2 and the cyclin B1 level, and a change in the phosphorylation of Cdk1. MPT0B169 induced apoptosis in nonresistant and imatinib-resistant cells via a mitochondrion-mediated caspase pathway. Further study showed that the agent led to a decrease in the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 and an increase in the apoptotic protein Bax. Taken together, our results suggest that MPT0B169 might be a promising agent for overcoming imatinib resistance in CML cells.