Fig 6 - available from: Apoptosis
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VX-11e in combination with voreloxin increased p21 and reduced survivin and NF-κB p105/p50 protein levels. a MOLM-14, b K562, c REH and d MOLT-4 cells were incubated for 24 h with VX-11e (VX) and voreloxin (VOR) alone or in combination. The expression of p21, survivin, p50 and p105 proteins was detected by Western blot. β-actin was used as a loading control. Quantification of the proteins was performed by densitometric analysis of the blots and normalized to the internal loading control. Each value is the mean ± SD of three independent experiments. *(p < 0.05), **(p < 0.01) versus control; # (p < 0.05), ## (p < 0.01) versus VX-11e; $ (p < 0.05), $$ (p < 0.01) versus voreloxin
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ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To eva...
Citations
... We sought to understand the function of ERK2-mediated phosphorylation of TOP2B during transcriptional activation via inhibition of the catalytic activity of ERK2. VX-11e (VX11) is a small chemical inhibitor, specific to ERK2 76,77 . HEK293 cells were treated with 100 nM VX11 for 3 h. ...
Transcription of stress-inducible genes requires synchronized and robust activation, which is critical for organismal survival and homeostasis. The function of mitogen-activated protein kinase (MAPK) signaling pathway is involved in the activation of immediate early genes (IEGs), including EGR1 and FOS , for cell growth ¹⁻³ . In addition, recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation in IEGs ⁴⁻⁶ . However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation remains unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicated that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Inhibition of ERK2 kinase activity or ERK2 knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, the cryo-EM structure of the TOP2B- EGR1 transcription start site-etoposide complex demonstrated breakage and dramatic bending of the double-stranded DNA, suggesting the mechanism of TOP2B-mediated transcriptional activation. Taken together, this study suggests that the activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions during transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be important for the dissociation step.
... For this reason, suppressing Nrf2 expression through various inhibitory pathways such as siRNA technology and natural compounds (TPL, Wogonin, chaetominine) could help decrease drug resistance in CML. The PBMC of patients with CLL had higher levels of Nrf2 than normal blood samples [203] CLL In vitro Blood samples (PBMC) Increased ROR1 expression in CLL cells increases APRIL and BAFF-R expression, leading to the recruitment and accumulation of the p62 protein, which triggers several separate signaling pathways such as Nrf2 [204] ALL In silico -The Nrf2 inhibitory pathway and activation of this factor are disrupted in patients with ALL [205] ALL In vitro REH, MOLT-4 Inhibition of MAPK/ERK and PI3K/AKT pathways reduced Nrf2/NF-κΒ and drug resistance The combination of MAPK/ERK pathway inhibitors plus topoisomerase II inhibitor treatment synergistically increased the production of ROS and caused apoptosis in leukemic cells [193,207,208] ...
... Gajda et al. demonstrated that inhibiting the MAPK/ERK and PI3K/AKT pathways reduces Nrf2/NF-κΒ levels and prevents drug resistance in REH and MOLT-4 ALL cell lines [193]. Furthermore, a combination of MAPK/ERK pathway inhibitors plus topoisomerase II inhibitor treatment synergistically increased the production of ROS and caused apoptosis in leukemic cells [207,208]. ...
NF-E2-related factor 2 (Nrf2) transcription factor has contradictory roles in cancer, which can act as a tumor suppressor or a proto-oncogene in different cell conditions (depending on the cell type and the conditions of the cell environment). Nrf2 pathway regulates several cellular processes, including signaling, energy metabolism, autophagy, inflammation, redox homeostasis, and antioxidant regulation. As a result, it plays a crucial role in cell survival. Conversely, Nrf2 protects cancerous cells from apoptosis and increases proliferation, angiogenesis, and metastasis. It promotes resistance to chemotherapy and radiotherapy in various solid tumors and hematological malignancies, so we want to elucidate the role of Nrf2 in cancer and the positive point of its targeting. Also, in the past few years, many studies have shown that Nrf2 protects cancer cells, especially leukemic cells, from the effects of chemotherapeutic drugs. The present paper summarizes these studies to scrutinize whether targeting Nrf2 combined with chemotherapy would be a therapeutic approach for leukemia treatment. Also, we discussed how Nrf2 and NF-κB work together to control the cellular redox pathway. The role of these two factors in inflammation (antagonistic) and leukemia (synergistic) is also summarized.
... VX-11e is a potent and selective ERK2 inhibitor that reduces tumor growth, proliferation and viability in a variety of cancer cell lines. VX-11e affects G0/G1 cell cycle arrest and induces high expression of p21 cell cycle inhibitors [40]. Our study showed that WHSC1L1 was indirectly linked to the regulation of the G0 to G1 transition. ...
Nuclear receptor-binding SET domain protein (NSD), a histone methyltransferase, is known to play an important role in cancer pathogenesis. The WHSC1L1 (Wolf-Hirschhorn syndrome candidate 1-like 1) gene, encoding NSD3, is highly expressed in breast cancer, but its role in the development of breast cancer is still unknown. The purpose of this study was to analyze the survival rates and immune responses of breast cancer patients with high WHSC1L1 expression and to validate the results using gradient boosting machine (GBM) in breast cancer. We investigated the clinicopathologic parameters, proportions of immune cells, pathway networks and in vitro drug responses according to WHSC1L1 expression in 456, 1500 and 776 breast cancer patients from the Hanyang University Guri Hospital, METABRIC and TCGA, respectively. High WHSC1L1 expression was associated with poor prognosis, decreased CD8+ T cells and high CD274 expression (encoding PD-L1). In the pathway networks, WHSC1L1 was indirectly linked to the regulation of the lymphocyte apoptotic process. The GBM model with WHSC1L1 showed improved prognostic performance compared with the model without WHSC1L1. We found that VX-11e, CZC24832, LY2109761, oxaliplatin and erlotinib were effective in inhibiting breast cancer cell lines with high WHSC1L1 expression. High WHSC1L1 expression could play potential roles in the progression of breast cancer and targeting WHSC1L1 could be a potential strategy for the treatment of breast cancer.
... Accordingly, a synergistic effect was observed for ERK2 inhibitor in combination with megestrol in T-cell prolymphocytic leukemia cells (17). In our previous studies, we also demonstrated that MEK1/2 and ERK2 inhibitors in combination with voreloxin increased antiproliferative and pro-apoptotic effects in leukemia cells (10,18). Recently, it was reported that combination of MEK and STAT3 inhibitors enhanced therapeutic response in pancreatic ductal adenocarcinoma (19). ...
... In contrast, it was shown that taxol increased p21 expression and induced growth arrest and apoptosis in breast cancer cells (27). Similarly, we have shown previously that combination of VX-11e with voreloxin increased the level of p21 protein in REH cells, leading to apoptosis (18). ...
... In this study we observed a significant reduction in the survivin level in MOLT-4 cells treated with both VX-11e and STA-21. Similarly, we found a decrease in survivin level in leukemia cell lines after combined treatment with VX-11e and voreloxin (18). ...
Background/aim:
Deregulated activation of signaling through the RAS/RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/RAF/MEK/ERK) and signal transducer and activator of transcription (STAT) pathways is involved in numerous hematological malignancies, making it an attractive therapeutic target. This study aimed to assess the effect of the combination of ERK2 inhibitor VX-11e and STAT3 inhibitor STA-21 on acute lymphoblastic leukemia cell lines REH and MOLT-4.
Materials and methods:
REH and MOLT-4 cell lines were cultured with each drug alone and in combination. Cell viability, ERK activity, cell cycle distribution, apoptosis and oxidative stress induction were assessed by flow cytometry. Protein levels of STAT3, phospho-STAT3, protein tyrosine phosphatase 4A3 (PTP4A3), survivin, p53 and p21 were determined by western blotting.
Results:
VX-11e in combination with STA-21 significantly inhibited cell viability, induced G0/G1 cell-cycle arrest, enhanced production of reactive oxygen species, and induced apoptosis. These effects were associated with an increased level of p21 protein in REH cells and with reduced levels of phopho-STAT3, survivin and PTP4A3 proteins in MOLT-4 cells.
Conclusion:
Our findings provide a rationale for combined inhibition of RAS/RAF/MEK/ERK and STAT3 pathways in order to enhance anticancer effects against acute lymphoblastic leukemia cells.
... AZD034, a novel selective ERK1/2 inhibitor, has been found to inhibit the growth of human lung cancer cell lines with KRAS mutations [5]. In our previous studies, we have demonstrated that combined treatment of MAPK/ERK pathway inhibitors with topoisomerase II inhibitor is highly synergistic and enhances reactive oxygen species (ROS) production, leading to apoptosis in leukemia cells [6,7]. ZSTK474, a PI3K pan-inhibitor, has been shown to decrease cell survival and induce apoptosis in nelarabinetreated T-lymphoblastic acute leukemia (T-ALL) cells [8]. ...
... These results suggest that the treatment of ALL REH and MOLT-4 cells with studied inhibitors affects the reduction of NF-κB protein level, which in turn decreases antioxidant protein levels, such as NRF2/HO-1 and TrxR, and leads to the excessive generation of ROS. These findings are in accordance with our previous study, in which the reduction of NF-κB protein level and an increase in ROS production was observed after treatment with ERK2 inhibitor VX-11e and voreloxin in leukemia cell lines [7]. Furthermore, in the present study, we showed that combined treatment with AZD0364 and ZSTK474 reduced GSH/GSSG ratios in both REH and MOLT-4 cells, which may also lead to overproduction of ROS and induction of apoptosis. ...
... In the present study, survivin levels were significantly decreased in REH and MOLT-4 cells after combined treatment with AZD0364 and ZSTK474. Previously, we and others have shown that the synergistic pro-apoptotic effect of MAPK/ERK inhibitors combined with other drugs was accompanied by reduced expression of survivin [7,50]. Moreover, it has been demonstrated that NRF2 regulates survivin expression in endometrial cancer cells [51]. ...
The mitogen-activated protein kinase (MAPK)/extracellular signal kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal transduction pathways have been implicated in the pathogenesis of leukemia. The aim of this study was to investigate the effect of the combination of ERK1/2 inhibitor AZD0364 and PI3K inhibitor ZSTK474 on acute lymphoblastic leukemia (ALL) REH, MOLT-4, acute myeloid leukemia (AML) MOLM-14, and chronic myeloid leukemia (CML) K562 cell lines. To evaluate the interactions of the drugs, cells were treated for 48 h with AZD0364 or ZSTK474 alone and in combination at fixed ratios. The combinatorial effects of both inhibitors were synergistic over a wide range of concentrations in REH, MOLT-4, and MOLM-14 cell lines. However, in K562 cells, the effects were found to be antagonistic. Furthermore, AZD0364 and ZSTK474 significantly decreased both ERK1/2 and AKT activation in REH, MOLT-4, and MOLM-14 cells. The results showed that incubation with both AZD0364 and ZSTK474 inhibited cell viability, increased reactive oxygen species (ROS) production, and induced apoptosis in leukemia cells. We observed that combined treatment with AZD0364 and ZSTK474 affected nuclear factor-κB (NF-κB) and antioxidant protein levels: NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), thioredoxin (Trx), thioredoxin reductase (TrxR), and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio. These effects were accompanied with decreased antiapoptotic survivin protein level. However, distinct cell line dependent effects were observed. In conclusion, the combination of AZD0364 and ZSTK474 can exert a synergistic anticancer effect in ALL and AML cells, which is associated with the induction of oxidative stress and the involvement of cellular antioxidant defense mechanisms.
The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (–30 to +20) that has the strongest affinity to TOP2B within –423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.
The development of effective therapy is required for glioblastoma multiforme (GBM) patients because of the resistance action to temozolomide (TMZ). Bromelain (BRO) is a plant-derived therapeutic used against inflammation. The objective of this study is to appraise the effect of the BRO + TMZ combination on U87MG and LN229 cells and investigate its mechanism. The BRO + TMZ treatment resulted in synergistic effects that restricted cell growth and inhibited the colony formation of GBM cells. The combined treatment led to the initiation of early apoptosis resulting in the overexpression of BAX/caspase-3 (CASP3) and decreased transcript level of BCL-2. The BRO + TMZ induced the arrest in G0/G1 phase and decreased the migration mediated by the dysregulation of MMP-2 and MMP-9. The BRO and TMZ combination treatment was observed to be more effective than the individual compound to inhibit cell growth in human GBM cell lines.
Necroptosis of chondrocytes contributes to the progression of osteoarthritis (OA). Recent studies have shown that VX-11e, an ERK inhibitor, exhibited a contrasting expression pattern to RIP3, the key protein of necroptosis. However, its effect on OA remains to be determined. Therefore, we investigated whether VX-11e affected the loss of articular cartilage and subchondral bone during OA.In in vivo experiments, a mouse OA model induced by medial meniscus instability (destabilization of the medial meniscus [DMM]) was used. In in vitro experiments, interleukin-1β (IL-1β) was used to simulate the inflammatory microenvironment of chondrocytes, and RANKL was used to induce osteoclast differentiation. Histological analysis, cell viability experiments, high-density cell culture experiments, immunofluorescence assay, western blot assay, quantitative PCR, and molecular docking experiments were conducted to determine the protective effect of VX-11e on articular cartilage during OA. We also performed histological analysis, tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring formation test, quantitative PCR, and western blot assay to study the effect of VX-11e on subchondral bone during OA progression.We found that after the medial meniscus was severed, the articular cartilage of the mice showed pathological changes, accompanied with the loss of subchondral bone. However, an intraperitoneal injection of VX-11e protected the cartilage and subchondral bone of the mouse knee joint. The results of in vitro experiments showed that VX-11e promoted the anabolism of the extracellular matrix of chondrocytes by inhibiting the expression and phosphorylation of RIP3 and MLKL. VX-11e also inhibited RANKL-induced osteoclast differentiation by inhibiting the ERK/RSK signaling pathway, but not the NF-κB pathway. Overall, VX-11e inhibited the loss of articular cartilage and subchondral bone during OA by regulating the RIP1/RIP3/MLKL and MAPK signaling pathways.
