The promoting effects of Grin2d expression in tumorigenesis and the aggressiveness of esophageal cancer.

Grin2d is an ionotropic NMDA receptor, a subunit of glutamate-dependent, and a facilitator of cellular calcium influx in neuronal tissue. In this study, we found that Grin2d expression was higher in esophageal cancer than in normal mucosa at both the mRNA and protein level using RT-PCR, bioinformatics analysis, and western blotting (p<0.05). Grin2d mRNA expression was positively correlated with old age, white race, heavy weight, distal location, adenocarcinoma, cancer with Barrett's lesion, or high-grade columnar dysplasia (p<0.05). The differential genes associated with Grin2d mRNA were involved in fat digestion and absorption, cholesterol metabolism, lipid transfer, lipoproteins, synaptic membranes, and ABC transporters (p<0.05). The Grin2d-related genes were classified into the following categories: metabolism of glycerolipids, galactose, and O-glycan, cell adhesion binding, actin binding, cadherin binding, the Hippo signaling pathway, cell-cell junctions, desmosomes, DNA-transcription activator binding, and skin development and differentiation (p<0.05). Grin2d immunoreactivity was positively correlated with distal metastasis and unfavorable overall survival in esophageal cancer (p<0.05). Grin2d overexpression promoted proliferation, migration, and invasion in esophageal cancer cells but blocked apoptosis (p<0.05) and increased the expression of PI3K, Akt and p-mTOR. Grin2d knockout caused the opposite effects. These findings indicated that upregulated Grin2d expression played an important role in esophageal carcinogenesis via the PI3K/Akt/mTOR pathway and might be a biological marker for aggressive tumor behavior and poor prognosis. Its silencing might represent a targeted therapy approach against esophageal cancer.

FAM64A aggravates proliferation, invasion, lipid droplet formation, and chemoresistance in gastric cancer: A biomarker for aggressiveness and a gene therapy target.

FAM64A is a mitogen-induced regulator of the metaphase and anaphase transition. Here, we found that FAM64A messenger RNA (mRNA) and protein expression levels were higher in gastric cancer tissue than in normal mucosa (p < .05). FAM64A methylation was negatively correlated with FAM64A mRNA expression (p < .05). The differentially expressed genes of FAM64A were mainly involved in digestion, potassium transporting or exchanging ATPase, contractile fibers, endopeptidase, and pancreatic secretion (p < .05). The FAM64A-related genes were principally categorized into ubiquitin-mediated proteolysis, cell cycle, chromosome segregation and mitosis, microtubule binding and organization, metabolism of amino acids, cytokine receptors, lipid droplet, central nervous system, and collagen trimer (p < .05). FAM64A protein expression was lower in normal gastric mucosa than intestinal metaplasia, adenoma, and primary cancer (p < .05), negatively correlated with older age, T stage, lymphatic and venous invasion, tumor, node, metastasis stage, and dedifferentiation (p < .05), and associated with a favorable overall survival of gastric cancer patients. FAM64A overexpression promoted proliferation, antiapoptosis, migration, invasion, and epithelial-mesenchymal transition via the EGFR/Akt/mTOR/NF-κB, while the opposite effect was observed for FAM64A knockdown. FAM64A also induced chemoresistance directly or indirectly through lipid droplet formation via ING5. These results suggested that upregulation of FAM64A expression might induce aggressive phenotypes, leading to gastric carcinogenesis and its subsequent progression. Thus, FAM64A could be regarded as a prognosis biomarker and a target for gene therapy.

The effects of REG4 expression on chemoresistance of ovarian cancer.

Although ovarian cancer usually responds well to platinum- and taxane-based first-line chemotherapy, most patients develop recurrence and chemoresistance. Regenerating gene 4 (REG4) is a secretory protein involved in cell differentiation and proliferation. We found higher REG4 expression in ovarian cancer than in normal tissues (p < .05). Regenerating gene 4 expression was negatively associated with overall, progression-free or post-progression survival rates of patients with ovarian cancer receiving platinum or paclitaxel treatment (p < .05) according to a Kaplan-Meier plotter. Regenerating gene 4 overexpression resulted in either cisplatin or paclitaxel resistance, and apoptosis resistance in CAOV3 ovarian cancer cells (p < .05). REG4-transfected ovarian cancer cells showed stronger migration and invasion treated with cisplatin or paclitaxel (p < .05). Additionally, cisplatin or paclitaxel exposure led to the overexpression of phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, phosphorylated mammalian target of rapamycin (p-mTOR), glutathione S-transferase-π, survivin, and B-cell lymphoma 2 in REG4 transfectants compared with control cells (p < .05). These findings suggested that REG4 expression was up-regulated in ovarian cancer, and associated with poor survival and chemotherapy resistance. REG4 promoted the occurrence, development, and chemotherapy resistance of ovarian cancer by regulating cell proliferation, apoptosis, migration, and invasion, and PI3K/Akt/m-TOR signalling pathways. IMPACT STATEMENTWhat is already known on this subject? REG4 mRNA expression is up-regulated in many digestive cancers. High REG4 expression was associated with an adverse prognosis, high tumour and nodal stages, poor differentiation, and hepatic and peritoneal metastases of digestive cancers. REG4 expression conferred cancer cells with increased resistance to chemoradiotherapy, especially 5-FU-based treatment, by activating the MAPK/Erk/Bim signalling pathway.What do the results of this study add? REG4 was highly expressed in ovarian cancer. The expression of p-PI3K, p-AKT, p-mTOR, GST-π, survivin, and Bcl-2 was increased in REG4-overexpressing cells. High REG4 expression was significantly associated with inferior OS, PFS, and PPS rates in patients with ovarian cancer receiving platinum chemotherapy. REG4 mediated cisplatin and paclitaxel resistance in CAOV3 ovarian cancer cells. The percentage of apoptotic cells was markedly lower in REG4-transfected compared to mock-transfected cells after cisplatin or paclitaxel treatment.What are the implications of these findings for clinical practice and/or further research? This study aimed to evaluate the prognostic significance of REG4 expression in ovarian cancer treated with platinum and paclitaxel, to explore REG4 chemoresistance mechanisms to platinum and paclitaxel, and to provide a scientific experimental basis for the clinical treatment and outcome evaluation of ovarian cancer. In order to provide comprehensive clinical treatment of ovarian cancer, it is helpful to improve our understanding of multi-drug resistance and identify new cancer diagnostic biomarkers.

The complete chloroplast genome sequence of Phalaenopsis wilsonii Rolfe, a vulnerable wild moth orchid species (Orchidaceae).

Phalaenopsis wilsonii Rolfe is a vulnerable wild moth orchid species with important horticultural value. The complete chloroplast genome sequence of P. wilsonii was generated by de novo assembly using whole genome next-generation sequencing to provide genomic data for further conservation genetics, phylogeny and molecular breeding in Phalaenopsis. The complete plastome of P. wilsonii is 145,096 bp in length, containing two inverted repeats (IR) regions (24,787 bp), a large single-copy (LSC) region (84,688 bp), and a small single-copy (SSC) region (10,834 bp). The chloroplast genome encoded 119 unique genes, including 73 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The overall GC content of the whole genome is 36.9%. Phylogenetic analysis indicated P. wilsonii was closely related to P. lowii.

Two isopentenyl resorcinols from Peperomia tetraphylla.

Two isopentenyl resorcinols, peperobtusin B and peperobtusin C, have been isolated from Peperomia tetraphylla. Their structures were determined on the basis of spectroscopic methods, especially 1H NMR, 13C NMR, 2D NMR, and HR-TOF-MS. Two compounds were evaluated for cytostatic activity against G2, A 549, Hela and HCT 116 cells, but cytostatic activity of both compounds is weak.

Two new secolignans from the roots of Urtica fissa.

Two new secolignans, 3,4-trans-3-hydroxymethyl-4-[bis(4-hydroxy-3- methoxyphenyl)methyl]butyrolactone (1) and 3,4-trans-3-hydroxymethyl-4- [bis(3,4-dimethoxyphenyl)methyl]butyrolactone (2) have been isolated from the roots of Urtica fissa E.Pritz. Their structures were determined on the basis of spectroscopic methods, especially 1H NMR, 13C NMR, 2D NMR, and HR-ESI-MS. The inhibitory effects on N1 and N2, two subtypes of neuraminidases (NAs), of these two compounds were assayed.

A new acylated flavonoid glycoside from the flowers of Camellia nitidissima and its effect on the induction of apoptosis in human lymphoma U937 cells.

From the EtOH extract of the flowers of Camellia nitidissima Chi, a new acylated flavonoid glycoside, quercetin 7-O-(6"-O-E-caffeoyl)-ß-D-glucopyranoside (1), has been isolated, together with three known flavonoids: quercetin (2), quercetin 3-O-ß-D-glucopyranoside (3), and quercetin 7-O-ß-D-glucopyranoside (4). Their structures were elucidated on the basis of spectroscopic analysis. Compound 1 was shown to inhibit proliferation and to induce apoptosis of human lymphoma U937 cells.

Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone, a radiolytic product of palmitic acid, in human lymphoma U937 cells.

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-L: -cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca(2+)](i) involved in apoptosis are induced by 2-DCB and PA in U937 cells.

Three new secolignan glycosides from Urtica fissa E. Pritz.

Three new secolignan glycosides {3,4-trans-4-[bis(3,4-dimethoxyphenyl)methyl]-2-oxotetrahydrafuran-3-yl}methyl-O-ß-glucopyranoside (1), {3,4-trans-4-[(3-methoxy-4-hydroxyphenyl)(3,4-dimethoxyphenyl)methyl]-2-oxotetrahydrafuran-3-yl}methyl-O-ß-glucopyranoside (2) and {3,4-cis-4-[(3-methoxy-4-hydroxyphenyl)(3,4-dimethoxyphenyl)methyl]-2-oxotetrahydrafuran-3-yl}methyl-O-ß-glucopyranoside (3) were isolated from the roots of Urtica fissa E. Pritz. Their structures were identified by spectral methods including 1D NMR, 2D NMR and HR-EI-MS.

Enhancement of hyperthermia-induced apoptosis by sanazole in human lymphoma U937 cells.

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. The aim of the present study was to investigate whether sanazole enhances apoptosis induced by hyperthermia at 44 degrees C for 20 min in human lymphoma U937 cells. Sanazole alone induced continuous increase in the intracellular superoxide generation in a time-dependent manner and transient increase in the peroxide formation, which further were enhanced at 1 hour after HT treatment. Moreover, when the cells were treated first with 10 mM sanazole for 40 min, exposed to HT at 44 degrees C for 20 min and the cells were further treated with the drug at 37 degrees C for 6 h, a significant enhancement of HT-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. Studying the apoptotic pathways involved in this enhancement, we found that loss of the mitochondrial membrane potential, release of cytochrome c from mitochondria to cytosol, and activation of caspase-3 and caspase-8 was enhanced significantly in the U937 cells after the combined treatment. Moreover, this combination enhanced activation of Bid, and down regulation of Hsp70. In addition, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), and externalization of Fas were observed immediately after sanazole and HT treatment. Our data indicate that sanazole can enhance the hyperthermia induced-apoptosis through the Fas-caspase-8- and [Ca(2+)](i)-dependent apoptotic pathways. In addition, the down regulation of Hsp70 contributed to this enhancement.

Design, synthesis, and biological evaluation of artificial macrosphelides in the search for new apoptosis-inducing agents.

Various artificial macrosphelides were designed and synthesized, including ring-enlarged analogues and epothilone-hybrid compounds. Syntheses were accomplished in an efficient manner by using a ring-closing metathesis (RCM) strategy in a key macrocyclization step. Biological evaluation of these new macrosphelide-based derivatives revealed that several epothilone hybrids, in which a thiazole-containing side chain was incorporated, exhibited potent apoptosis-inducing activity toward human lymphoma cells. These activities were considerably enhanced relative to those of natural macrosphelide compounds. Structure-activity relationship studies revealed that the "ene-dicarbonyl" substructure is apparently essential for bioactivity.

Enhancement of radiation-induced apoptosis of human lymphoma U937 cells by sanazole.

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. In this study, we determined whether sanazole enhances the radiation-induced apoptosis of human lymphoma U937 cells. Our results revealed that, compared with 10 mM sanazole or radiation alone, the combination of both resulted in a significant enhancement of apoptosis after 6 h, which was evaluated on the basis of DNA fragmentation, morphological changes, and phosphatidylserine externalization. Sanazole alone enhanced intracellular superoxide and hydrogen peroxide formation, which further increased when the cells were irradiated. Significant enhancement of Fas externalization, loss of mitochondrial membrane potential (MMP), and activation of caspase-3 and caspase-8 were observed after the combined treatment. Moreover, this combination could also enhance Bid activation, reduction of Hsp70 expression level and release of cytochrome c from the mitochondria to the cytosol. An immediate increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the combined treatment. These results suggest that the intracellular superoxide and peroxide generated by sanazole might be involved in the enhancement of radiation-induced apoptosis, and that these effects are associated with modulation of the Fas-mitochondria-caspase-dependent pathway, an increase in [Ca(2+)](i), and a decrease in the Hsp70 expression levels.

Enhancement of sodium butyrate-induced cell death and apoptosis by X-irradiation in the human colorectal cancer cell line HCT 116.

In this study, we aimed at evaluating the possible enhancing effect exerted by the combined use of sodium butyrate (SB) and X-rays on eradicating the human colorectal cancer cell line HCT 116 containing wild-type p53. We assessed the effect of this combination on the molecular pathways leading to cell death. HCT 116 cells were subjected to SB (1 mM) treatment followed by X-irradiation (5 Gy), and the effects on cell death, cell proliferation and cell cycle were examined. We also analyzed the apoptosis-indicating protein expression, mitochondrial membrane potential and intracellular superoxide formation. Treatment with SB alone significantly induced cell cycle arrest and apoptosis, whereas X-irradiation showed no effect on cell death despite its ability to block cell proliferation. Growth arrest and cell death were enhanced in the combined treatment groups. A marked reduction in the growth rate of the combined-treatment group was observed compared to that of the single-treatment groups. The apoptotic mitochondrial pathway was significantly enhanced with the combined use of the two agents. It was observed to be involved in the increased expression levels of p53 and p21, as well as in the release of cytochrome c and the alteration of the balance of anti- and pro-apoptotic Bcl-2 family proteins. Enhanced superoxide formation was also observed. However, the death receptor pathway was found to play no role in this phenomenon. These results suggest that X-irradiation promotes cell killing in synergy with SB treatment. Thus, the combined treatment led to a mutual potentiation of the killing effects of each agent.

Enhancement of sodium butyrate-induced cell death by hyperthermia in HCT 116 human colorectal cancer cells.

AIM: The possible enhancing effect of the combined use of sodium butyrate (SB) and hyperthermia to kill HCT 116 cells was evaluated. MATERIALS AND METHODS: HCT 116 cells were subjected to SB (1 mM) treatment followed by hyperthermia (44 degrees C 60 min) and the effects on cell death, cell proliferation and the cell cycle were examined. Apoptosis-indicating protein expressions and intracellular superoxide formation were also analysed. RESULTS: A marked reduction in the growth rate of the combined-treatment group was observed compared to those of the single-treatment groups. This involved the increased expression of p53 and p21, the alteration of the balance of anti- and proapoptotic Bcl-2 family proteins and enhanced superoxide formation. However, the death receptor pathway played no role. CONCLUSION: Hyperthermia synergistically promoted cell death induced by SB. Thus, the combined treatment led to mutual potentiation of the killing effects of each agent.

Apoptosis induced by an alkylated purine, 6-dimethylaminopurine, and changes in gene expression in human lymphoma U937 cells.

Apoptosis induced by an alkylated purine, 6-dimethylaminopurine (6-DMAP), was investigated to explore the p53-independent pathway in a human lymphoma U937 cell line. Here, it was discovered that the apoptosis was induced by 6-DMAP in a dose- and time-dependent manner and treatment for 16 h at a concentration of 5 mM induced apparent DNA fragmentation, phosphatidylserine externalization and lowering of the mitochondrial membrane potential, which are typical markers of apoptosis. Western blotting revealed reduced expression in Bcl-XL, increased expression in Bax and release of cytochrome c. These were associated with activation of caspase-3. The 6-DMAP-induced apoptosis was preceded by an increase in the intracellular calcium ion concentration ([Ca2+]i), indicating the involvement of intracellular Ca2+ ions in apoptosis. Two independent sets of cDNA microarray systems were used to examine changes in gene expression. Of the 3,893 and 886 genes analyzed, 32 and 13 genes were identified as down-regulated in cells treated with 6-DMAP for 3 h. No up-regulated gene was found. Real-time PCR revealed a significant decrease in mRNA of proliferating cell nuclear antigen, insulin-induced gene 1, serine proteinase inhibitor 2 and v-myc. Pathway analysis also revealed the interaction of genes on down-regulation. These findings suggest that intracellular calcium ions and mitochondrial caspase-dependent pathways play major roles in 6-DMAP-induced apoptosis, and protein kinase inhibition by the agent causes massive down-regulation of all genes relating to cell proliferation and progression of the cell cycle to induce apoptosis.

Enhancement of hyperthermia-induced apoptosis by a new synthesized class of benzocycloalkene compounds.

The aim of this study was to examine whether, a new synthesized class of benzocycloalkene derivatives (BCs), enhances apoptosis induced by hyperthermia. The combined effects of hyperthermia (44 degrees C, 20 min) and BCs on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (BC1 approximately 9), the combined treatment of 10 muM BC2 or BC4 and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia. And enhancement of hyperthermia-induced apoptosis by BC2 or BC4 in a dose-dependent manner was observed. When the cells were treated first with BC2 or BC4 at a nontoxic concentration of 20 muM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. Flow cytometry revealed an increase of intracellular superoxide due to BC2 or BC4, which was further increased when hyperthermia was combined. Mitochondrial membrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by hyperthermia, was enhanced by BC2 or BC4. An increase in the intracellular Ca2+ concentration [Ca2+](i), externalization of Fas, and decrease in Hsp70 were observed following the combined treatment. These results indicate that the intracellular superoxide generated by BC2 or BC4 is involved in the enhancement of apoptosis through Fas-mitochondria caspase and [Ca2+](i)-dependent pathways, and a decrease in Hsp70 also contributed to the enhancement of apoptosis.

Rapid and transient intracellular oxidative stress due to novel macrosphelides trigger apoptosis via Fas/caspase-8-dependent pathway in human lymphoma U937 cells.

The ability of the derivatives of macrosphelides (MS) core (simplified 16-membered core structure of natural MS) to induce apoptosis in human lymphoma U937 cells was investigated. Of the five compounds examined, MS core with ketones at 8 and 14 positions (MS5) showed the highest potency to induce apoptosis, while another, MS3 with one ketone, was minimal potent. MS5 was found to induce apoptosis in the U937 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis. MS5 treated cells showed increase in intracellular reactive oxygen species (ROS), glutathione depletion, Bid activation and lipid peroxidation. Pretreatment of cells with pancaspase inhibitor resulted in the complete inhibition of MS5-induced apoptosis. N-Acetyl-l-cysteine (NAC) pretreatment resulted in the increase in glutathione concentration, reduction of intracellular ROS, complete inhibition of DNA fragmentation, mitochondrial membrane potential (MMP) collapse, Fas externalization and caspase-8 activation. Furthermore, MS5-induced oxidative stress also triggered transient increase in intracellular calcium ion ([Ca2+]i) concentration which was completely inhibited by NAC. Pretreatment with an intracellular Ca2+ chelator, BAPTA-AM reduced MS5-induced DNA fragmentation and caspase-8 activation while it has marginal effects on MMP collapse. Taken together our present data showed that a rapid increase in intracellular ROS by MS5 triggers apoptosis via the Fas/caspase-8-mediated mitochondrial pathway suggesting that the presence of diketone makes the compound more potent to induce apoptosis. These characteristics of MS5 will make it useful for therapeutic applications of targeted apoptosis.

Enhancement of hyperthermia-induced apoptosis by a new synthesized class of furan-fused tetracyclic compounds.

The combined effects of hyperthermia (44 degrees C, 20 min) or X-rays (10 Gy) and a new class of furan-fused tetracyclic synthesized compounds (DFs), on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (DF1 approximately 6), the combined treatment of 10 microM DF with TIPS (triisopropylsilyloxy) (Designated #3 DF3) and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia but no enhancement was observed if it was combined with X-rays. Enhancement of hyperthermia-induced apoptosis by DF3 in a dose-dependent manner was observed. When the cells were treated first with DF3 at a nontoxic concentration of 20 microM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by hyperthermia, was enhanced by DF3. Mitochondrial transmembrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. Externalization of Fas was observed following the combined treatment. Flow cytometry revealed rapid and sustained increase of intracellular superoxide due to DF3, and showed subsequent and transient increase in the formation of intracellular hydrogen peroxide (H(2)O(2)), which was further increased when hyperthermia was combined. These results indicate that the intracellular superoxide and H(2)O(2) generated by DF3 enhance the hyperthermia-induced apoptosis via the Fas-mediated mitochondrial caspase-dependent pathway.