Werner helicase is required for proliferation and DNA damage repair in multiple myeloma.

BACKGROUND: Multiple myeloma (MM), characterized by extensive genomic instability and aberrant DNA damage repair, is a plasma cell malignancy due to the excessive proliferation of monoclonal antibody-producing plasma cells in the bone marrow. Despite the significant improvement in the survival of patients with the development of novel therapeutic agents, MM remains an incurable disease. Werner (WRN) helicase, a member of the RecQ helicase family that contributes to DNA replication, recombination, and repair, has been highlighted in cancer cell survival, yet the role and mechanism of WRN in MM remain unclear. METHODS AND RESULTS: Increased mRNA expression of WRN in newly diagnosed and relapsed CD138+ myeloma plasma cells than normal CD138+ plasma cells and their matched CD138- non-tumorigenic cells were detected by qPCR. Using NSC19630, a specific WRN helicase inhibitor, we further showed decreased cell viability, proliferation, and DNA repair and increased DNA damage and apoptosis in MM cells by MTT assay, cell cycle assay, apoptosis assay, and Western blotting. CONCLUSIONS: The results of the present study demonstrate that WRN is essential in MM cell viability, proliferation, and genomic stability, indicating its inhibition may enhance the efficacy of chemotherapy in MM.

Evaluation of protein levels of autophagy markers (Beclin 1 and SQSTM1/p62) and phosphorylation of cyclin E in the placenta of women with preeclampsia.

Preeclampsia is a severe multisystem disorder, and its pathophysiology is still not completely understood. Autophagy, a recycling process that maintains cellular homoeostasis during differentiation and development, is controversial regarding increased or decreased autophagic activity in preeclampsia. The aim of this study was to determine whether autophagy is increased in the placentas of women with preeclampsia by examining the protein levels of autophagy markers (Beclin 1 and SQSTM1/p62) and phosphorylation of cyclin E. For this purpose, placentas from preeclampsia (n=10) and control (n=10) pregnancies were included in this study. The protein expression of autophagy-related markers Beclin1, SQSTM1/p62 and phosphorylation status of cyclin E were detected by Western blot. Our data showed that the protein levels of both Beclin 1 and SQSTM1/p62 were significantly increased, while the phosphorylation level of cyclin E was significantly decreased in placentas with preeclampsia compared to those derived from controls. The results of this study suggest that the autophagic activity is perpetually increased in preeclampsia and cyclin E protein stabilisation might be involved in the induction of autophagy.

Deregulation of c-Src tyrosine kinase and its downstream targets in pre-eclamptic placenta.

AIM: Pre-eclampsia is a serious pregnancy disorder characterized by the new onset of hypertension and proteinuria in the second trimester of pregnancy. The determination of a key signaling regulatory mechanism involved in placental functions is critical to understanding the pathogenesis of pre-eclampsia. The aim of this study was to examine the activity of c-Src and its downstream targets, extracellular signal-regulated kinase 1/2, p38 and Jun N-terminal kinase, as well as nuclear factor (NF)-ĸB in placental tissues collected from women with pre-eclampsia. METHODS: Ten pre-eclamptic (PE) placentas and 10 control placentas were used in this study. The Western blot method was performed to evaluate the c-Src/ mitogen activated protein kinase/NF-ĸB signaling pathway in each group. RESULTS: c-Src phosphorylation at Tyr-416, used as a measure of c-Src activity, was significantly decreased in PE placentas relative to the control. Reduced c-Src activity resulted in the suppression of extracellular signal-regulated kinase 1/2 phosphorylation and a significant reduction in the phosphorylation of p38 and Jun N-terminal kinase in PE placentas. Moreover, IĸBα phosphorylation was significantly elevated, while NF-ĸB phosphorylation was suppressed in PE placentas. CONCLUSIONS: The c-Src/MAPK/NF-ĸB signaling pathway may contribute to the pathogenesis of pre-eclampsia.

HIV-1 integrase inhibitor raltegravir promotes DNA damage-induced apoptosis in multiple myeloma.

Raltegravir, the first integrase inhibitor approved for the treatment of HIV infection, has been implicated as a promising potential in cancer treatment. Therefore, the present study aimed to investigate the repurposing of raltegravir as an anticancer agent and its mechanism of action in multiple myeloma (MM). Human MM cell lines (RPMI-8226, NCI H929, and U266) and normal peripheral blood mononuclear cells (PBMCs) were cultured with different concentrations of raltegravir for 48 and 72 h. Cell viability and apoptosis were then measured by MTT and Annexin V/PI assays, respectively. Protein levels of cleaved PARP, Bcl-2, Beclin-1, and phosphorylation of histone H2AX were detected by Western blotting. In addition, the mRNA levels of V(D)J recombination and DNA repair genes were analyzed using qPCR. Raltegravir treatment for 72 h significantly decreased cell viability, increased apoptosis, and DNA damage in MM cells while having minimum toxicity on cell viability of normal PBMCs approximately from 200 nM (0.2 µM; p < .01 for U66 and p < .0001 for NCI H929 and RPMI 8226 cells). Furthermore, raltegravir treatment altered the mRNA levels of V(D)J recombination and DNA repair genes. We report for the first time that treatment with raltegravir is associated with decreased cell viability, apoptosis induction, DNA damage accumulation, and alteration of mRNA expression of genes involved in V(D)J recombination and DNA repair in MM cell lines, all of which show its potential for anti-myeloma effects. Hence, raltegravir may significantly impact the treatment of MM, and further studies are required to confirm its efficacy and mechanism of action in more detail in patient-derived myeloma cells and in-vivo models.

Targeting the anaphase-promoting complex/cyclosome (APC/C) enhanced antiproliferative and apoptotic response in bladder cancer.

Improving the chemotherapy sensitivity of bladder cancer is a current clinical challenge. It is critical to seek out effective combination therapies that include low doses of cisplatin due to its dose-limiting toxicity. This study aims to investigate the cytotoxic effects of the combination therapy including proTAME, a small molecule inhibitor, targeting Cdc-20 and to determine the expression levels of several APC/C pathway-related genes that may play a role in the chemotherapy response of RT-4 (bladder cancer) and ARPE-19 (normal epithelial) cells. The IC20 and IC50 values were determined by MTS assay. The expression levels of apoptosis-associated (Bax and Bcl-2) and APC/C-associated (Cdc-20, Cyclin-B1, Securin, and Cdh-1) genes were assessed by qRT-PCR. Cell colonization ability and apoptosis were examined by clonogenic survival experiment and Annexin V/PI staining, respectively. Low-dose combination therapy showed a superior inhibition effect on RT-4 cells by increasing cell death and inhibiting colony formation. Triple-agent combination therapy further increased the percentage of late apoptotic and necrotic cells compared to the doublet-therapy with gemcitabine and cisplatin. ProTAME-containing combination therapies resulted in an elevation in Bax/Bcl-2 ratio in RT-4 cells, while a significant decrease was observed in proTAME-treated ARPE-19 cells. Cdc-20 expression in proTAME combined treatment groups were found to be decreased compared to their control groups. Low-dose triple-agent combination induced cytotoxicity and apoptosis in RT-4 cells effectively. It is essential to evaluate the role of APC/C pathway-associated potential biomarkers as therapeutic targets and define new combination therapy regimens to achieve improved tolerability in bladder cancer patients in the future.

IL-33 induces ADAMTS5 expression and cell migration in glioblastoma multiforme.

Glioblastoma multiforme (GBM), characterized by a high rate of proliferation and migration capacity, is an incurable brain tumor in adults. Interleukin-33 (IL-33), a member of the IL-1 cytokine superfamily, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), a family of zinc dependent metalloproteinases, are known to have essential roles in GBM migration and invasion. Previous studies have separately revealed elevated expressions of IL-33 and ADAMTS5 in GBM; however, the interaction between IL-33 and ADAMTS5 in GBM remains unclear. Here, using publically available GlioVis and GEPIA programs, we showed that mRNA expressions of IL-33 and ADAMTS5 are significantly high in GBM cells, and a positive correlation between IL-33 and ADAMTS5 was also determined in these cells. In parallel with the mRNA data of IL-33 and ADAMTS5, by Western blot analysis, protein levels were found to be elevated in GBM tissues and increased gradually with the disease progression. Primary GBM cells and low-grade glioma cells were then treated with IL-33 to examine its stimulating effect on ADAMTS5 expression. Exposure to IL-33 raised ADAMTS5 protein levels in a dose-dependent manner. Finally, the wound-healing method was performed to confirm the impact of IL-33 on migration in primary GBM cells. IL-33 promoted migration of primary GBM cells three times higher than untreated GBM cells. Thus, the current study suggests for the first time that IL-33 might have a role in playing a part in GBM progression through induction of ADAMTS5 expression and promotion of migration in GBM cells.

Drug Targeting of Genomic Instability in Multiple Myeloma.

Genomic instability can be observed at both chromosomal and chromatin levels. Instability at the macro level includes centrosome abnormalities (CA) resulting in numerical as well as structural chromosomal changes, whereas instability at the micro level is characterized by defects in DNA repair pathways resulting in microsatellite instability (MIN) or mutations. Genomic instability occurs during carcinogenesis without impairing survival and growth, though the precise mechanisms remain unclear. Solid tumors arising from most cells of epithelial origin are characterized by genomic instability which renders them resistant to chemotherapy and radiotherapy. This instability is also observed in 25% of myeloma patients and has been shown to be highly prognostic, independently of the international staging system (ISS). However, a biomarker of aberrant DNA repair and loss of heterozygosity (LOH), was only observed at a frequency of 5% in newly diagnosed patients. Several new molecules targeting the pathways involved in genomic instability are under development and some have already entered clinical trials. Poly(ADP-ribose) polymerase-1 (PARP) inhibitors have been FDA-approved for the treatment of breast cancer type 1 susceptibility protein (BRCA1)-mutated metastatic breast cancer, as well as ovarian and lung cancer. Topoisomerase inhibitors and epigenetic histone modification-targeting inhibitors, such as HDAC (Histone Deacetylase) inhibitors which are novel agents that can target genomic instability. Several of the small molecule inhibitors targeting chromosomal level instability such as PARP, Akt, Aurora kinase, cyclin dependent kinase or spindle kinase inhibitors have been tested in mouse models and early phase I/II trials. ATM, ATR kinase inhibitors and DNA helicase inhibitors are also promising novel agents. However, most of these drugs are not effective as single agents but appear to act synergistically with DNA damaging agents such as radiotherapy, platinum derivatives, immunomodulators, and proteasome inhibitors. In this review, new drugs targeting genomic instability and their mechanisms of action will be discussed.