Fluvastatin exerts an antitumor effect in vemurafenib-resistant melanoma cells.

Although vemurafenib has been shown to improve the overall survival of patients with metastatic melanoma harboring the BRAF V600E mutation, its efficacy is often hampered by drug resistance acquired within a relatively short period through several distinct mechanisms. In the present study, we investigated the effect of fluvastatin as a possible strategy to overcome such acquired resistance using a cultured cell line model. We established vemurafenib-resistant (VR) cells from three BRAF (V600E)-mutated melanoma lines (C32, HMY-1, and SK-MEL-28) and evaluated the mechanism of acquired resistance of VR cells by water-soluble tetrazolium salts assay, western blot, real-time quantitative PCR, and immunofluorescent microscopy. The efficacy of the combination of growth inhibitory effect of vemurafenib and fluvastatin on respective parental and VR cells were assessed by calculating combination index and western blot. IC50 values of three VR cells were ~5-100-fold higher than those for the respective parental cells. The VR cells derived from HMY-1 and SK-MEL-28 showed constitutive activation of AKT kinase, and the specific AKT inhibitor MK-2208 or the PI3K inhibitor wortmannin increased the cellular sensitivity to vemurafenib. Intriguingly, application of a statin-related drug, fluvastatin, also resulted in a synergistic increase of sensitivity to vemurafenib in the VR cells (combination index: 0.73-0.86) probably by alleviating constitutive AKT activation, whereas the same treatment did not notably alter the vemurafenib sensitivity of the parental cells. Our results suggest the possible usefulness of statin-related drugs for overcoming vemurafenib resistance acquired through constitutive activation of the PI3K-AKT axis.

Improvement in platelet count after 3rd-line and 4th-line eradication therapy for Helicobacter pylori in patients with immune thrombocytopenia.

Case 1: A 78-year-old woman was diagnosed with H. pylori positive gastritis at a previous hospital in April 2012 and received 3rd-line H. pylori eradication therapy, which ended in failure. She was referred to our department due to oral hemorrhage, petechiae involving all four extremities, and thrombocytopenia in January 2016. She was hospitalized with a diagnosis of ITP and received inpatient treatment. While receiving outpatient prednisolone (PSL) treatment, we administered 4th-line eradication therapy in March. Her platelet levels have since returned to normal, and PSL treatment has been discontinued. She is currently followed without treatment. Case 2: A 65-year-old woman was diagnosed with ITP at a previous hospital in June 2013 and received 2nd-line eradication therapy, which ended in failure. Thereafter, PSL treatment was continued but she was later referred to our department in March 2016. Since 3rd-line eradication therapy was successful, her platelet count normalized and PSL treatment has been discontinued. She is currently followed without treatment. Based on our observations in these two cases, third-line H. pylori eradication therapy is potentially effective in ITP patients.

Nucleus accumbens associated 1 is recruited within the promyelocytic leukemia nuclear body through SUMO modification.

Nucleus accumbens associated 1 (NACC1) is a cancer-associated BTB/POZ (pox virus and zinc finger/bric-a-brac tramtrack broad complex) gene, and is involved in several cellular functions in neurons, cancer and stem cells. Some of the BTB/POZ proteins associated with cancer biology are SUMOylated, which appears to play an important role in transcription regulation. We show that NACC1 is SUMOylated on a phylogenetically conserved lysine (K167) out of three consensus SUMOylation motif sites. Amino acid substitution in the SIM sequence (SIM/M) within the BTB/POZ domain partially reduced K167 SUMOylation activity of NACC1. Overexpression of GFP-NACC1 fusion protein leads to formation of discrete nuclear foci similar to promyelocytic leukemia nuclear bodies (PML-NB), which colocalized with SUMO paralogues (SUMO1/2/3). Both NACC1 nuclear body formation and colocalization with SUMO paralogues were completely suppressed in the GFP-NACC1-SIM/M mutant, whereas they were partially maintained in the NACC1 K167R mutant. Confocal immunofluorescence analysis showed that endogenous and exogenous NACC1 proteins colocalized with endogenous PML protein. A pull-down assay revealed that the consensus motifs of the SUMO acceptor site at K167 and the SIM within the BTB/POZ domain were both necessary for efficient binding to PML protein. Our study demonstrates that NACC1 can be modified by SUMO paralogues, and cooperates with PML protein.

Immunohistochemistry for histone h3 lysine 9 methyltransferase and demethylase proteins in human melanomas.

Methylation and demethylation of histone H3 lysine 9 (H3K9) play a role in the transcriptional regulation of several cancer-related genes and are closely associated with malignant tumor behavior. A novel study has recently demonstrated that SETDB1, a member of the H3K9 methyltransferases, accelerates tumor formation significantly in a zebrafish melanoma model. However, the expression of H3K9 methyltransferases including SETDB1 and demethylases has not been systematically examined in samples of human melanoma. Here, we used immunohistochemistry to examine the expression of the H3K9 methyltransferases, EHMT2 and SETDB1, and a H3K9 demethylase, LSD1, in 67 patients with melanoma. Overexpression of EHMT2, SETDB1, and LSD1 was observed in 14 (21%), 38 (57%), and 53 (79%) of the 67 patients, respectively. A significant relationship was observed between overexpression of EHMT2 or SETDB1 and aggressive tumor behavior such as lymph node metastasis and/or distant metastasis (P < 0.05), whereas no significant relationship was evident for LSD1 immunoreactivity. Univariate log-rank tests demonstrated that patients with melanoma overexpressing EHMT2 had a poorer outcome (P < 0.001), whereas overexpression of SETDB1 or LSD1 had no prognostic impact. These results suggest that overexpression of EHMT2 might be a prognostic marker in patients with melanoma.

BCL2 and BCLxL are key determinants of resistance to antitubulin chemotherapeutics in melanoma cells.

Malignant melanoma is refractory to various chemotherapeutics including antitubulin agents such as paclitaxel. Previous studies have suggested a link between ßIII-tubulin overexpression and paclitaxel resistance through alterations in the properties of the mitotic spindle. We found that paclitaxel treatment induced temporary mitotic arrest in 7 melanoma cell lines irrespective of the ßIII-tubulin level, suggesting that ßIII-tubulin had no significant influence on spindle properties. On the other hand, the amount of BCL2, an anti-apoptotic protein, was well correlated with paclitaxel resistance. Treatment of the paclitaxel-resistant cell lines with ABT-737, an inhibitor of BCL2 and BCLxL, or simultaneous knock-down of BCL2 and BCLxL dramatically increased the cells' sensitivity, while knock-down of MCL1, another member of the BCL2 family, had only a minimal effect. Our results suggest that the paclitaxel sensitivity of melanoma cells is attributable to apoptosis susceptibility rather than a change in spindle properties and that BCL2 and BCLxL play a pivotal role in the former.

Different susceptibility to insulin resistance and fatty liver depending on the combination of TNF-α C-857T and adiponectin G+276T gene polymorphisms in Japanese subjects with type 2 diabetes.

The C-857T promoter polymorphism of TNF-α gene is associated with obese type 2 diabetes, while the adiponectin G+276T gene polymorphism in intron 2 may influence the fat accumulation in the liver. In this study, we examined effects of these polymorphisms on clinical markers of insulin resistance and fatty liver (a liver/spleen CT ratio < 0.9). These polymorphisms were determined in 342 Japanese subjects with type 2 diabetes. The liver/spleen CT ratio was lower in the subjects with the adiponectin +276G/G genotype than that in the subjects with the +276T allele (P < 0.05), indicating that fat accumulation in the liver is associated with the +276G/G genotype. Multiple comparisons among the 4 combinations of each polymorphism of the TNF-α and adiponectin genes revealed a significant difference in the liver/spleen CT ratio (P < 0.05) among the 4 groups, indicating that the gene combinations influence the degree of fat accumulation in the liver. The subjects carrying the TNF-α -857T allele (C/T or T/T genotype) and the adiponectin +276G/G genotype had greater risks for fatty liver and insulin resistance that was evaluated by higher levels of fasting insulin and homeostasis model assessment of insulin resistance, as compared with the other groups. Therefore, Japanese subjects with the TNF-α -857T allele and the adiponectin +276G/G genotype may be more susceptible to insulin resistance and fatty liver. The present study provides the evidence for the interaction between TNF-α and adiponectin genes in the insulin resistance and fatty liver in Japanese subjects with type 2 diabetes.

DUSP4 Inactivation Leads to Reduced Extracellular Signal‒Regulated Kinase Activity through Upregulation of DUSP6 in Melanoma Cells.

A subset of dual-specificity phosphatases is a major negative regulator of MAPKs, and their involvement in tumorigenesis remains controversial. Among them, DUSP4 is reported to preferentially dephosphorylate extracellular signal‒regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase over p38. In this study, we aimed to identify a possible role of DUSP4 in melanoma genesis. An examination of large-scale public data on gene expression and dependency revealed a considerably high DUSP4 expression and dependency of the melanoma cell lines compared with those of other tumor cell lines, which was not apparent for the other 24 dual-specificity phosphatases genes encoded in the human genome. Using two melanoma lines, we confirmed that DUSP4 depletion impaired cell growth without notably inducing apoptosis. Interestingly, immunoblotting and kinase translocation reporter data revealed that DUSP4 depletion induces a decrease in ERK1/2 phosphorylation but barely affects c-Jun N-terminal kinase phosphorylation, suggesting that neither ERK nor c-Jun N-terminal kinase is a direct target of DUSP4 in our experimental setting. Notably, DUSP4 depletion led to an increase in DUSP6 level, possibly through a post-transcriptional process, and DUSP6 knockout almost eliminated the DUSP4-depletion effect on cell growth and ERK activity. Our findings suggest that DUSP4 plays a role in maintaining a high ERK1/2 activity by negatively regulating DUSP6 and thus contributes to the survival and growth of melanoma cells.

Expression profiles of nestin in vascular smooth muscle cells in vivo and in vitro.

Nestin is an intermediate filament protein expressed in neural and mesenchymal stem cells. Here, we investigated the expression of nestin in vascular smooth muscle cells (VSMCs) in vivo and in vitro. In the developing arteries, medial VSMCs were found to express nestin; its expression was prominent in embryos but was down-regulated after birth (3-6 weeks) in a region-dependent manner; its expression was abolished in the adult. Thus, the expression of nestin is specific to developing VSMCs. In primary VMSC cultures, nestin expression was induced by serum, but was independent of cell-cycle progression. Signaling analyses revealed that the serum-induced nestin expression depended on the extracellular signal-regulated kinase (ERK) and protein kinase B (PKB)(Akt) pathways, via the platelet derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Nestin expression was closely related to the up-regulation and activation of Sp1 and Sp3. Among major serum growth factors and cytokines, PDGF-BB was the most potent inducer of nestin expression. Nestin was also up-regulated in arteries undergoing vascular remodeling following balloon injury. Its expression was particularly strong in the cells lining the lumen of the neointima, suggesting a possible correlation between nestin expression and the progression of vascular remodeling.

Immunohistochemical study of HER2 and TUBB3 proteins in extramammary Paget disease.

Metastatic extramammary Paget disease (EMPD) is a potentially fatal malignancy for which effective chemotherapy and good biomarkers are desirable for management. We investigated the status of human epidermal growth factor receptor (HER2) and neuronal ß-tubulin isotype (class III ß-tubulin; TUBB3), whose overexpression is a factor involved in resistance of tumor cells to taxane derivatives) in 32 patients with EMPD. HER2 status was evaluated by immunohistochemistry followed by fluorescence in situ hybridization, and TUBB3 status was evaluated by immunohistochemistry. On the basis of the US Food and Drug Administration-approved criteria, 20 (63%) of the 32 EMPD tumors were found to overexpress HER2. Positive immunoreactivity for TUBB3 was observed in 7 (22%) of the 32 patients. Although some clinicopathologic variables (nodule formation, depth of tumor cells, presence of lymph node metastasis, and serum carcinoembryonic antigen level) were significantly associated with disease outcome (P < 0.05), HER2 gain or aberrant TUBB3 expression showed no significant correlation. However, the higher incidence of HER2 gain and the relatively lower incidence of aberrant TUBB3 expression suggested that HER2-targeted immunotherapy combined with taxane derivatives is warranted for metastatic EMPD, and that HER2 and TUBB3 status might be a good biomarker for determining the most appropriate therapeutic modality.

Detection of human papillomavirus in a urothelial carcinoma mimicking urethral caruncle.

Primary carcinoma of the female urethra is an uncommon diagnosis, accounting for less than 0.02% of all carcinomas in women. Urothelial carcinomas occupying the distal urethra in young females are considered to be extremely rare. Here we report what we believe to be the sixth case of primary urothelial carcinoma in the published English-language literature. The patient, a 26-year-old woman, presented with a distal urethral lesion that resembled a caruncle, but which was proved to be a urothelial carcinoma on histopathological examination of the resected specimen. Human papillomavirus type 51 DNA was detected in the tumor by polymerase chain reaction and in situ hybridization. These findings suggest that human papillomavirus might be involved in a subset of urothelial carcinomas of the urethra.

DNA hypomethylation at the CpG island is involved in aberrant expression of the L1 cell adhesion molecule gene in colorectal cancer.

The L1 cell adhesion molecule (L1CAM) has been identified as a target gene of beta-catenin-TCF signaling in colorectal cancer (CRC) and associated with aggressive tumor behavior such as invasion and metastasis. We investigated the methylation status at the L1CAM gene promoter and/or L1CAM mRNA/protein expression in 4 CRC cell lines and 71 primary CRCs. Aberrant L1CAM expression was immuno histochemically observed in 31 (43.7%) of 71 cases, and correlated with advanced stage and presence of lymph node and distant metastases (P<0.05). Treatment with a demethylating agent induced L1CAM mRNA/protein expression in two cell lines lacking L1CAM expression. Bisulfite-modified genome sequencing suggested that DNA methylation status at core promoter and putative TCF-binding sites within the L1CAM promoter was correlated with L1CAM mRNA/protein expression in 4 CRC cell lines. Using the crypt isolation followed by bisulfite-modified genome sequencing and methylation-specific PCR methods, we confirmed that the DNA hypomethylation at core promoter and putative TCF-binding sites was well correlated with the aberrant L1CAM protein expression in primary CRC samples. These results suggest that DNA hypomethylation at the L1CAM CpG islands might induce L1CAM aberrant expression and contribute to the acquisition of aggressive tumor behavior in CRC.

Differential microRNA expression between bone marrow side population cells and hepatocytes in adult mice.

Hematopoietic stem cells (HSCs) can differentiate into many kinds of parenchymal cells populating several organs. Nevertheless, the differentiation mechanism of HSCs toward hepatocytes remains poorly understood. To identify specific microRNAs (miRNAs) contributing to the mechanism, we investigated the differential expression of miRNAs between side population (SP) cells of bone marrow and hepatocytes in adult mice. We used a miRNA microarray followed by stem-loop-mediated reverse transcription real-time PCR to identify 12 SP-specific and 2 hepatocyte-specific miRNAs. Of these, 3 (miR-451, -150 and -223) were strongly expressed (>10-fold relative enrichment) in SP cells. Two of these miRNAs (miR-451 and -223) were strongly associated with the hematic cell lineage but not with progenitor characteristics. Two-thirds (6/9) of the miRNAs that were moderately expressed in SP cells in comparison with hepatocytes were also up-regulated in potential hepatic stem cells (HSCEs). The single miRNA (miRNA-127) that was up-regulated in SP cells compared with lineage-positive bone marrow cells might be an SP marker, since it was markedly down-regulated in HSCEs. These results suggest that SP cells and HSCE share a common profile of miRNA expression and that miRNA-127 may contribute to the maintenance of a quiescent state in SP cells.

Spontaneous [Ca2+]i oscillations in G1/S phase-synchronized cells.

Ca(2+) signaling controls a wide range of cellular functions such as division, fertilization, apoptosis and necrosis. Specifically, calcium signaling is thought to play a crucial role in driving cells through the different stages of the cell-division cycle. In most cells, however, this fact is far from being established. Few studies have examined this question from a different perspective: whether cells exhibit some characteristic cell cycle-dependent intracellular calcium-signaling patterns. This approach is effective in discerning the causal relationship between Ca(2+) signaling and the cell cycle. Through synchronization of the cell cycle, flow cytometry and confocal scanning microscopic intracellular calcium ion concentration ([Ca(2+)](i)) imaging, the present study shows that the G1/S phase transition is uniquely characterized by spontaneous [Ca(2+)](i) oscillations that last for up to 40 min. Most likely, these oscillations emanate from the [Ca(2+)](i) signaling that accompanies DNA replication as the cell prepares for the next division cycle. These temporal signals further affirm the significance of Ca(2+) in the cell cycle.

Correction: Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

[This corrects the article DOI: 10.1371/journal.pone.0209296.].

Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines.

Alterations of several microRNA (miRNA) have been linked to cancer development and its biology. To search for unique miRNA that might play a role in the development of anaplastic thyroid carcinoma (ATC), we examined the expression of multiple miRNA and their functional effects on target genes in human thyroid carcinoma cell lines. We quantitatively evaluated the expression of multiple miRNA in 10 ATC and five papillary thyroid carcinoma (PTC) cell lines, as well as primary tumors from 11 thyroid carcinoma patients (three ATC and eight PTC), using the stem-loop-mediated reverse transcription real-time polymerase chain reaction method. We also examined the target gene specificity of unique miRNA that showed differences in expression between ATC and PTC cell lines. One miRNA, miR-138, was significantly downregulated in ATC cell lines in comparison with PTC (P < 0.01). Eleven miRNA (including miR-138) potentially targeting the human telomerase reverse transcriptase (hTERT) gene were totally downregulated in both ATC and PTC cell lines in comparison with normal thyroid tissues. A tendency for an inverse correlation between miR-138 and hTERT protein expression was observed in the thyroid cancer cell lines, although this failed to reach significance (r = -0.392, P = 0.148). We demonstrated that overexpression of miR-138 induced a reduction in hTERT protein expression, and confirmed target specificity between miR-138 and the hTERT 3'-untranslated region by luciferase reporter assay. These results suggest that loss of miR-138 expression may partially contribute to the gain of hTERT protein expression in ATC, and that further multiple miRNA targeting hTERT mRNA might be involved in the development of thyroid carcinoma.

Epigenetic modification is involved in aberrant expression of class III beta-tubulin, TUBB3, in ovarian cancer cells.

Aberrant expression of class III beta-tubulin, TUBB3, has been reported to be one of the important mechanisms responsible for taxane resistance in diverse human malignancies. We investigated aberrant TUBB3 expression and its epigenetic modification in 66 primary tumors and 3 cell lines (OVCAR-3, JHOC-5 and JHOC-8) of ovarian cancers. Overexpression of TUBB3 protein was observed in 56 (85%) of the 66 ovarian cancers, and was significantly associated with aggressive tumor behavior (advanced stage, presence of ascites, suboptimal cytoreduction at surgery and presence of lymph node metastasis) (P<0.05). Responses to treatment with a demethylating agent (5-aza-2'-deoxycytidine, 5-Aza-CdR) and a histone deacetylase inhibitor (4-phenylbutyric acid, PBA) differed among the ovarian cancer cell lines. In 2 cell lines with weak expression of TUBB3 protein (OVCAR-3 and JHOC-8), TUBB3 induction was independently induced by treatment with 5-Aza-CdR (JHOC-8) or PBA (OVCAR-3), while neither agent markedly altered TUBB3 mRNA/protein expression in a strongly TUBB3-expressing cell line (JHOC-5). A CpG island within intron 1 was hypermethylated in 1 cell line (JHOC-8) that expressed TUBB3 weakly and required 5-Aza-CdR treatment for gene expression. A CpG island of another cell line showing faint expression of TUBB3 protein (OVCAR-3), in which a significant gain of TUBB3 expression was induced by treatment with PBA but not with 5-Aza-CdR, was hypomethylated, similarly to a cell line (JHOC-5) showing constitutive expression of TUBB3. We evaluated methylation status in this region in 14 primary tumors using methylation-specific PCR, but there was no significant relationship with TUBB3 immunoreactivity. These findings suggest that aberrant expression of TUBB3 protein might be associated with aggressive behavior of ovarian cancers, and that epigenetic modulation (DNA methylation and chromatin acetylation) might be partly involved in TUBB3 expression.

Angiotensin II and epidermal growth factor receptor cross-talk mediated by a disintegrin and metalloprotease accelerates tumor cell proliferation of hepatocellular carcinoma cell lines.

AIM: The cross-talk pathway between angiotensin II (AngII) and the epidermal growth factor receptor (EGFR) mediated by epidermal growth factor (EGF)-like ligands cleaved by a disintegrin and metalloprotease (ADAM) has been elucidated in several cell types. Even though the liver is a representative angiotensinogen-producing organ, such cross-talk has never been elucidated in hepatocellular carcinomas (HCCs). We investigated whether AngII exerted a mitogenic effect on HCC cell lines through the AngII-EGFR cross-talk pathway. METHODS: We determined the expression and/or phosphorylation status of AngII receptor type 1 (AGTR1), ADAM9, ADAM17, ERK1/2, STAT3, AKT and EGFR in five HCC cell lines using Western blotting. Proliferation and invasion activities were measured by ATP and Matrigel invasion assays, respectively. RESULTS: AGTR1 was expressed ubiquitously in HCC cell lines. EGFR expression in HepG2 was relatively weaker than that in the remaining HCC cell lines. The phosphorylation status of EGFR, ERK1/2, STAT3 and AKT was upregulated by AngII treatment in two EGFR-overexpressing cell lines (Huh7 and PLC/PRF/5), but not in HepG2 (showing weak EGFR expression). AngII stimulation significantly accelerated proliferation and invasion activities in Huh7 and PLC/PRF/5, and was inhibited by pretreatment with an ADAM inhibitor. A selective AGTR1 blocker significantly repressed proliferation activity in both cell lines, but did not significantly repress the invasion activity. Both chemical agents and neutralizing antibodies against ADAMs (ADAM9 and ADAM17) and EGF-like ligands suppressed EGFR transactivation and/or subsequent phosphorylation of ERK1/2, STAT3 and AKT. CONCLUSION: These results suggest that AngII-EGFR cross-talk signaling mediated by ADAMs is involved in the proliferation and invasion activities of several HCC cell lines.

Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

Cultured cells easily develop resistance to kinesin-5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin-12/KIF15, or by acquiring mutations in the N-terminal motor domain of kinesin-5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S-trityl L-cysteine (STLC), one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U-2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10-fold higher than those of the respective parental lines. Depletion of KIF15 largely eliminated the acquired K5I resistance in both cases, consistent with the proposed notion that KIF15 is indispensable for it. In contrast to the KIF11-independent property of the cells derived from HOS, those derived from U-2OS still required KIF11 for their growth and, intriguingly, expressed a C-terminal truncated variant of KIF11 resulting from a frame shift mutation (S1017fs). All of the isolated clones harbored the same mutation, suggesting its clonal expansion in the cell population due to the growth advantage during chronic STLC treatment. Transgenic expression of KIF11S1017fs in the parental U-2OS cells, as well as in HeLa cells, conferred a moderate but reproducible STLC resistance, probably owing to STLC-resistant localization of the mutant KIF11 on mitotic spindle. Our observations indicate that both KIF15 and the C-terminal-truncated KIF11 contributes to the STLC resistance of the U-2OS derived cells.

Carnosic acid, an inducer of NAD(P)H quinone oxidoreductase 1, enhances the cytotoxicity of ß-lapachone in melanoma cell lines.

NAD(P)H quinone oxidoreductase 1 (NQO1)-dependent antitumor drugs such as ß-lapachone (ß-lap) are attractive candidates for cancer chemotherapy because several tumors exhibit higher expression of NQO1 than adjacent tissues. Although the association between NQO1 and ß-lap has been elucidated, the effects of a NQO1-inducer and ß-lap used in combination remain to be clarified. It has previously been reported that melanoma cell lines have detectable levels of NQO1 expression and are sensitive to NQO1-dependent drugs such as 17-allylamino-17-demethoxygeldanamycin. The present study was conducted to investigate the involvement of NQO1 in ß-lap-mediated toxicity and the utility of combination treatment with a NQO1-inducer and ß-lap in malignant melanoma cell lines. Decreased expression or inhibition of NQO1 caused these cell lines to become less sensitive to ß-lap, indicating a requirement of NQO1 activity for ß-lap-mediated toxicity. Of note was that carnosic acid (CA), a compound extracted from rosemary, was able to induce further expression of NQO1 through NF-E2 related factor 2 (NRF2) stabilization, thus significantly enhancing the cytotoxicity of ß-lap in all of the melanoma cell lines tested. Taken together, the data presented in the current study indicated that the NRF2-NQO1 axis may have potential value as a therapeutic target in malignant melanoma to improve the rate of clinical response to NQO1-dependent antitumor drugs.

Deficiency of protein-L-isoaspartate (D-aspartate) O-methyl-transferase expression under endoplasmic reticulum stress promotes epithelial mesenchymal transition in lung adenocarcinoma.

A prognostic association between the novel chaperone protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) and lung adenocarcinoma has recently been reported. Here, we evaluated the functional roles of PIMT in the progression of lung adenocarcinoma. PIMT expression was detectable in 6 lung adenocarcinoma cell lines: A549, H441, H460, H1650, Calu 1, and Calu 6 cell lines. In A549 and H441 cells, knockdown by PIMT using silencing RNA of PIMT (si-PIMT) and/or small hairpin-RNA (sh-PIMT) induced a decrease in the expression of E-cadherin with an increase in vimentin expression, indicating that the epithelial to mesenchymal transition (EMT) was induced. Cell mobility, including migration and invasion capability, was increased in sh-PIMT A549 stable and si-PIMT H441 cells compared to in control cells. Endoplasmic reticulum (ER) stress, such as Thapsigargin (Tg) stress and hypoxia, induced EMT in A549 cells but not in other cell types, with an increase in GRP78 expression, whereas overexpression of PIMT reduced the EMT and cell invasion under stress conditions. The expression of hypoxia inducible factor-1 alpha (HIF1α) and Twist increased in sh-PIMT A549 and si-PIMT H441 cells, and Tg stress increased HIF1α expression levels in A549 cells in a dose-dependent manner. Moreover, LW6, an HIF1α inhibitor, reduced EMT, cancer invasion, and the levels of Twist in sh-PIMT A549 cells. Our results indicate that deficiency of supplemental PIMT expression under ER stress facilitates EMT and cell invasion in some cell types of lung adenocarcinoma.