Activation of ERK1/2 by MOS and TPL2 leads to dasatinib resistance in chronic myeloid leukaemia cells.

The development of BCR::ABL1 tyrosine kinase inhibitors (TKIs), such as dasatinib, has dramatically improved survival in cases of chronic myeloid leukaemia (CML). However, the development of resistance to BCR::ABL1 TKIs is a clinical problem. BCR::ABL1 TKI resistance is known to have BCR::ABL1-dependent or BCR::ABL1-independent mechanisms, but the mechanism of BCR::ABL1 independence is not well understood. In the present study, we investigated the mechanism of BCR::ABL1-independent dasatinib resistance. The expression and activation level of genes or proteins were evaluated using array CGH, real time PCR, or western blot analysis. Gene expression was modulated using siRNA-mediated knockdown. Cell survival was assessed by using trypan blue dye method. We found that dasatinib-resistant K562/DR and KU812/DR cells did not harbour a BCR::ABL1 mutation but had elevated expression and/or activation of MOS, TPL2 and ERK1/2. In addition, MOS siRNA, TPL2 siRNA and trametinib resensitized dasatinib-resistant cells to dasatinib. Moreover, expression levels of MOS in dasatinib non-responder patients with CML were higher than those in dasatinib responders, and the expression of TPL2 tended to increase in dasatinib non-responder patients compared with that in responder patients. Our results indicate that activation of ERK1/2 by elevated MOS and TPL2 expression is involved in dasatinib resistance, and inhibition of these proteins overcomes dasatinib resistance. Therefore, MOS, TPL2 and ERK1/2 inhibitors may be therapeutically useful for treating BCR::ABL1-independent dasatinib-resistant CML.

Laparoscopic hemostasis for abdominal brunt massive hemorrhage due to endometriosis.

In the gynecological literature, a limited number of studies have reported intraperitoneal bleeding due to abdominal blunt trauma. In this report, we describe a rare case of massive intraabdominal hemorrhage from the uterine artery triggered by a fall injury without apparent abdominal bruising in the presence of severe endometriosis and a uterine fibroid. A 28-year-old woman who fell from a railway platform was transported to an emergency hospital. Although she did not sustain abdominal bruising and initially had no abdominal symptoms, she complained of gradually worsening abdominal pain. Abdominal CT identified intraabdominal massive hematoma, and emergency exploratory laparoscopy revealed active bleeding from the right uterine artery eroded by endometriosis, which was treated with laparoscopic electrocoagulation. The cause of the intraabdominal bleeding was associated with avulsion of the endometriosis adhesion between the right perimetrium and the right uterine artery due to inertial forces of the uterus during the fall injury. A uterine fibroid discovered during laparoscopy was suspected to strengthen the inertial forces of the uterus. In the case of hemoperitoneum after trauma, gynecological sources of bleeding must be kept in mind, especially for patients with a known history of fibroids or endometriosis.

Perifosine enhances the potential antitumor effect of 5-fluorourasil and oxaliplatin in colon cancer cells harboring the PIK3CA mutation.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation in colon cancer contributes to the poor prognosis of the disease and chemoresistance of tumors. New therapies are needed; however, the lack of knowledge of the mechanism of chemoresistance has hindered progress. In this study, we investigated the mechanism of the reduced sensitivity of colon cancer cells to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP), and the effects of perifosine, an Akt inhibitor that enhances the cytotoxicity of 5-FU and L-OHP in colon cancer cells harboring the PIK3CA mutation. The use of 5-FU or L-OHP alone or in combination induced significant death of Caco-2 cells (PIK3CA wild type), but only weakly decreased the viability of DLD-1 and SW948 cells harboring the PIK3CA mutation. The use of 5-FU and L-OHP, either alone or in combination, strongly suppressed Akt activation, Survivin, Bcl-2, and Bcl-xL expression, and enhanced Puma, phospho-p53, and p53 expression in Caco-2 cells than in DLD-1 cells. In addition, perifosine enhanced the cytotoxicity of the 5-FU and L-OHP combination, inhibited Akt activation and the expression of Survivin, Bcl-2, and Bcl-xL, and increased the expression of Puma, phospho-p53, and p53 in DLD-1 cells. These results indicate that PIK3CA mutation contributes to reduced sensitivity to 5-FU and L-OHP via Akt activation in colon cancer cells. Perifosine increases the efficacy of 5-FU and L-OHP by suppressing Akt activation. Thus, the use of an Akt inhibitor in combination with 5-FU and L-OHP may be beneficial in colon cancer with cells harboring the PIK3CA mutation.

Activation of Serum/Glucocorticoid Regulated Kinase 1/Nuclear Factor-κB Pathway Are Correlated with Low Sensitivity to Bortezomib and Ixazomib in Resistant Multiple Myeloma Cells.

Multiple myeloma (MM) is an incurable malignancy often associated with primary and acquired resistance to therapeutic agents, such as proteasome inhibitors. However, the mechanisms underlying the proteasome inhibitor resistance are poorly understood. Here, we elucidate the mechanism of primary resistance to bortezomib and ixazomib in the MM cell lines, KMS-20, KMS-26, and KMS-28BM. We find that low bortezomib and ixazomib concentrations induce cell death in KMS-26 and KMS-28BM cells. However, high bortezomib and ixazomib concentrations induce cell death only in KMS-20 cells. During Gene Expression Omnibus analysis, KMS-20 cells exhibit high levels of expression of various genes, including anti-phospho-fibroblast growth factor receptor 1 (FGFR1), chemokine receptor type (CCR2), and serum and glucocorticoid regulated kinase (SGK)1. The SGK1 inhibitor enhances the cytotoxic effects of bortezomib and ixazomib; however, FGFR1 and CCR2 inhibitors do not show such effect in KMS-20 cells. Moreover, SGK1 activation induces the phosphorylation of NF-κB p65, and an NF-κB inhibitor enhances the sensitivity of KMS-20 cells to bortezomib and ixazomib. Additionally, high levels of expression of SGK1 and NF-κB p65 is associated with a low sensitivity to bortezomib and a poor prognosis in MM patients. These results indicate that the activation of the SGK1/NF-κB pathway correlates with a low sensitivity to bortezomib and ixazomib, and a combination of bortezomib and ixazomib with an SGK1 or NF-κB inhibitor may be involved in the treatment of MM via activation of the SGK1/NF-κB pathway.

The HGF/Met/NF-κB Pathway Regulates RANKL Expression in Osteoblasts and Bone Marrow Stromal Cells.

Multiple myeloma (MM)-induced bone disease occurs through hyperactivation of osteoclasts by several factors secreted by MM cells. MM cell-secreted factors induce osteoclast differentiation and activation via direct and indirect actions including enhanced expression of receptor activator of nuclear factor κB ligand (RANKL) in osteoblasts and bone marrow stromal cells (BMSCs). Hepatocyte growth factor (HGF) is elevated in MM patients and is associated with MM-induced bone disease, although the mechanism by which HGF promotes bone disease remains unclear. In the present study, we demonstrated that HGF induces RANKL expression in osteoblasts and BMSCs, and investigated the mechanism of induction. We found that HGF and MM cell supernatants induced RANKL expression in ST2 cells, MC3T3-E1 cells, and mouse BMSCs. In addition, HGF increased phosphorylation of Met and nuclear factor κB (NF-κB) in ST2 cells, MC3T3-E1 cells, or mouse BMSCs. Moreover, Met and NF-κB inhibitors suppressed HGF-induced RANKL expression in ST2 cells, MC3T3-E1 cells, and mouse BMSCs. These results indicated that HGF promotes RANKL expression in osteoblasts and BMSCs via the Met/NF-κB signaling pathway, and Met and NF-κB inhibitors suppressed HGF-induced RANKL expression. Our findings suggest that Met and NF-κB inhibitors are potentially useful in mitigating MM-induced bone disease in patients expressing high levels of HGF.

Overactivation of Akt Contributes to MEK Inhibitor Primary and Acquired Resistance in Colorectal Cancer Cells.

RAS and BRAF-mutated colorectal cancers are associated with resistance to chemotherapy and poor prognosis, highlighting the need for new therapeutic strategies. Although these cancers sometimes respond to mitogen activated protein kinase kinase (MEK) inhibitor treatment, they often acquire resistance via mechanisms, which are poorly understood. Here, we investigated the mechanism of MEK inhibitor resistance in primary- and acquired-resistant cells. Cell viability was examined using the trypan blue dye exclusion assay. Protein expression was analyzed by western blotting. Somatic mutations in colorectal cancer cells were investigated using the polymerase chain reaction array. PD0325901 and trametinib induced cell death in LoVo and Colo-205 cells but not in DLD-1 and HT-29 cells, which have a PIK3CA mutation constitutively activating Akt and NF-κB. Treatment with PD0325901 and trametinib suppressed ERK1/2 activation in all four cell lines but only induced Akt and NF-κB activation in DLD-1 and HT-29 cells. Inhibition of Akt but not NF-κB, overcame MEK inhibitor resistance in DLD-1 and HT-29 cells. Acquired-resistant LoVo/PR, Colo-205/PR and LoVo/TR cells have constitutively active Akt due to a M1043V mutation in the kinase activation loop of PIK3CA and Akt inhibitor resensitized these cells to MEK inhibitor. These results demonstrate that the overactivation of Akt plays a critical role in MEK inhibitor primary and acquired resistance and implicate combined Akt/MEK inhibition as a potentially useful treatment for RAS/BRAF-mutated colorectal cancer.

Hyperactivation of Nrf2 in early tubular development induces nephrogenic diabetes insipidus.

NF-E2-related factor-2 (Nrf2) regulates cellular responses to oxidative and electrophilic stress. Loss of Keap1 increases Nrf2 protein levels, and Keap1-null mice die of oesophageal hyperkeratosis because of Nrf2 hyperactivation. Here we show that deletion of oesophageal Nrf2 in Keap1-null mice allows survival until adulthood, but the animals develop polyuria with low osmolality and bilateral hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced aquaporin 2 levels in the kidney. Renal tubular deletion of Keap1 promotes nephrogenic diabetes insipidus features, confirming that Nrf2 activation in developing tubular cells causes a water reabsorption defect. These findings suggest that Nrf2 activity should be tightly controlled during development in order to maintain renal homeostasis. In addition, tissue-specific ablation of Nrf2 in Keap1-null mice might create useful animal models to uncover novel physiological functions of Nrf2.

[Effects of combined addition of atovaquone and lithium on the in vitro cell growth of the pathogenic yeast Candida albicans].

Atovaquone, an analog of ubiquinone, binds tightly to the ubiquinol oxidation site (Qo site) of parasite cytochrome bc(1) complex to inhibit electron transport at concentrations far lower than those at which the mammalian system is affected. The mode of action is thought similar to that of myxothiazol. To treat Pneumocystis jirovecii and Plasmodium falciparum infections, atovaquone has been used worldwide whereas it is unapproved in Japan. Since the pathogenic Candida species fungi seem resistant to atovaquone, this drug is not clinically available for candidosis, particularly deep mycosis. We examined the effects of atovaquone on cellular respiration and in vitro growth of C. albicans to explore a new therapeutic possibility for fungal infections. Atovaquone strongly inhibited glucose-dependent cellular respiration similarly to antimycin A, stigmatellin, and myxothiazol, specific bc(1) complex inhibitors. However, atovaquone suppressed glucose-dependent cell growth to a much lesser extent versus the comparator agents. When added alone, lithium exerted slight growth inhibition. The combined addition of lithium with atovaquone showed a significant increase in inhibition of growth. Although the way lithium acts synergistically with atovaquone remains to be elucidated, our results suggest a new therapeutic possibility of this combination for the treatment of candidosis.