Whole genome gene expression changes and hematological effects of rikkunshito in patients with advanced non-small cell lung cancer receiving first line chemotherapy.

It has been demonstrated that the traditional Chinese medicine rikkunshito, ameliorates anorexia in several types of human cancer and attenuates lung injury by inhibiting neutrophil infiltration. The current study investigated the clinical and hematological effects of rikkunshito and its underlying mechanisms of action in the treatment of advanced non-small cell lung cancer (NSCLC). The Illumina microarray BeadChip was used to analyze the whole-genome expression profiles of peripheral blood mononuclear cells in 17 patients with advanced NSCLC. These patients were randomized to receive combination chemotherapy (cisplatin and gemcitabine) with (n=9, CTH+R group) or without (n=8, CTH group) rikkunshito. The primary endpoint was the treatment response and the categories of the scales of anorexia, nausea, vomiting and fatigue; secondary endpoints included the hematological effect and whole genome gene expression changes. The results of the current study indicated that there were no significant differences in clinical outcomes, including treatment response and toxicity events, between the two groups. Median one-year overall survival (OS) was 12 months in the CTH group and 11 months in the CTH+R group (P=0.058 by log-rank test), while old age (>60 years old) was the only independent factor associated with one-year OS (hazard ratio 1.095, 95% confidence interval, 1.09-1.189, P=0.030). Patients in the CTH+R group experienced significantly greater maximum decreases in both white cell count (P=0.034) and absolute neutrophil count (P=0.030) from the baseline. A total of 111 genes associated with neutrophil apoptosis, the cell-killing ability of neutrophils, natural killer cell activation and B cell proliferation were up-regulated following rikkunshito treatment. A total of 48 genes associated with neutrophil migration, coagulation, thrombosis and type I interferon signaling were down-regulated following rikkunshito treatment. Rikkunshito may therefore affect the blood neutrophil count when used with combination chemotherapy in patients with NSCLC, potentially by down-regulating prostaglandin-endoperoxidase synthase 1, MPL, AMICA1 and junctional adhesion molecule 3, while up-regulating elastase, neutrophil expressed, proteinase 3, cathepsin G and cluster of differentiation 24.

Risk factors of multidrug-resistant Acinetobacter baumannii recurrence after successful eradication in ventilated patients.

BACKGROUND: Clinically, multidrug-resistant Acinetobacter baumannii (MDR-AB) recurrence is found in some patients although identified as successfully eradicated. We aim to discover the characteristics of patients with MDR-AB recurrence in the respiratory tract. METHODS: We retrospectively collected 106 chronic respiratory failure patients with MDR-AB harvest in pulmonary secretion culture. RESULTS: MDR-AB was successfully eradicated in 69 patients. Diabetes mellitus (p = 0.030, odds ratio [OR]: 2.7, 95% confidence interval [CI]: 1.1-6.4) and acute respiratory distress syndrome (p = 0.001, OR = 4.8, 95% CI: 1.8-12.7) reduce the MDR-AB eradication rate. Besides, a classification of colonization or infection was made beyond the 69 MDR-AB eradicated patients. In the colonization group, diabetes mellitus (p = 0.009; OR = 5.1, 95% CI: 1.5-17.6) is the only independent factor to increase the recurrence rate. Glycated hemoglobin level is also analyzed for each group to investigate diabetes control effect, but no significant difference found. CONCLUSIONS: Diabetes mellitus is a risk factor of MDR-AB recurrence among MDR-AB-colonized patients; the impact of localized pneumonia patch in MDR-AB-infected patients requires further study to be clarified.

Co-upregulation of Toll-like receptors 2 and 6 on peripheral blood cells in patients with obstructive sleep apnea.

PURPOSE: Toll-like receptor (TLR) 2 can heterodimerise with TLR6 to detect diacylated lipoproteins. Hypoxia inducible factor-1 α co-ordinates selective induction of TLR2 and TLR6 during persistent hypoxia. We hypothesized that TLR 2/6 co-expression may be upregulated by chronic intermittent hypoxia with re-oxygenation (IHR) in obstructive sleep apnea (OSA). METHODS: TLR2/6 expressions on blood immune cells were measured in 144 patients with sleep-disordered breathing (SDB), including primary snoring (PS, n = 24), moderate to severe OSA (MSO, n = 60), very severe OSA (VSO, n = 36), and very severe OSA on continuous positive airway pressure (CPAP) treatment (VSOC, n = 24). An in vitro IHR experiment was also undertaken. RESULTS: Patients in both the MSO and VSO groups had increased TLR2/6 co-expression on CD16(+) neutrophil than those in the PS group. Patients in the VSOC group had reduced TLR2/6 co-expression on neutrophil than those in either the MSO or VSO group. Blood absolute neutrophil count was positively but weakly correlated with TLR2/6 co-expression on neutrophil. TLR2/6 co-expressions on both CD14(+) monocyte and CD3(+)CD4(+)T helper cell, and TLR2 expressions on both monocyte and T helper cell in SDB patients with low Minimum SaO2 (≦70%) were all higher than those with high Minimum SaO2. In vitro IHR for 1-4 days resulted in TLR2/6 co-upregulation on both neutrophil and monocyte. CONCLUSIONS: OSA patients had increased TLR2/6 co-expressions on blood immune cells, which were related to their immune cell counts and could be reversed with CPAP treatment. In vitro IHR could induce TLR2/6 co-upregulation.

Aberrant Toll-like receptor 2 promoter methylation in blood cells from patients with pulmonary tuberculosis.

OBJECTIVES: Toll-like receptor 2 (TLR2) is a major mediator of innate immunity against tuberculosis (TB). This study aimed to determine if TLR2 promoter DNA methylation is associated with pulmonary TB. METHODS: The DNA methylation levels of 20 CpG sites over the TLR2 promoter region and TLR2 gene/protein expressions of immune cells of the blood were examined in 99 sputum culture-positive pulmonary TB patients and 77 healthy subjects (HS). RESULTS: TB patients had higher methylation levels over five CpG sites (3, 7, 9, 13, and 18), lower TLR2 gene expression, lower TLR2 expression on monocyte, higher TLR2 expression on NK cell, and higher serum TNF-α/IFN-γ levels than HS after adjusting for confounding factors. Patients with a high bacillary load had lower methylation levels at CpG-15, -17, and -20. Patients with drug-resistant TB had higher CpG-18 methylation levels and lower TLR2 expression on NK cell. Patients with far advanced lesion on chest radiograph had higher serum TNF-α level and higher TLR2 expression on NK cell. Patients with a high TLR2 expression on NK cell had lower one-year survival. CpG-18 methylation level, TLR2 expressions on monocyte/NK cell, and TNF-α/IFN-γ levels were all reversed to normal after 6-month anti-TB treatment. CONCLUSIONS: Aberrant methylation of certain CpG sites over TLR2 promoter region is associated with active pulmonary TB or its phenotypes, probably through the down-regulation of TLR2 expression.

Euphol from Euphorbia tirucalli selectively inhibits human gastric cancer cell growth through the induction of ERK1/2-mediated apoptosis.

Gastric cancer is one of the most common malignancies worldwide, and the main cause of cancer-related death in Asia. The present study assessed the anticancer effects of euphol, a triterpene alcohol with anti-inflammatory and antiviral activities on human gastric cancer cells. Euphol showed higher cytotoxicity activity against human gastric CS12 cancer cells than against noncancer CSN cells. In addition, it up-regulated the pro-apoptotic protein BAX and down-regulated the prosurvival protein Bcl-2, causing mitochondrial dysfunction, possibly by caspase-3 activation. The anti-proliferative effects of euphol were associated with the increased p27(kip1) levels and decreased cyclin B1 levels. Inhibition of ERK1/2 activation by PD98059 reversed euphol-induced pro-apoptotic protein expression and cell death. Taken together, these findings suggest that euphol selectively induced gastric cancer cells apoptosis by modulation of ERK signaling, and could thus be of value for cancer therapy.

Pregnenolone derivatives as potential anticancer agents.

Pregnenolone (1) was used as a template to develop new anticancer compounds. Ring-D modification of 1 resulted in the synthesis of benzylidenes 2-17, pyrazolines 18-76, pyrazoles 85-91, hydrazones 77-84, and oximes 92-107 derivatives. The structure of compound 107 was also deduced through single crystal X-ray diffraction studies. The inclusion of furanyl and pyridyl rings to pregnenolone skeleton increases the cytotoxicity of all compounds significantly. Among benzylidene derivatives, only heterocyclic enone 8 (IC50=0.74 µM/mL against HepG2), and 17 (IC50=4.49 µM/mL against HepG2, IC50=5.01 µM/mL against MDA-MB-230 cancer cell line) exhibited a significant activity. The cytotoxicity data of pyrazoline derivatives 18-76 revealed that only furanyl bearing pyrazolines 40, 42-44, 48, and 49 exhibited significant activities. While all (O-carboxymethyl) oximes, hydazones, and pyrazoles derivatives of pregnenolone did not show any significant activity against both the cell lines. Thus the furanyl bearing enone 8 (IC50=0.74 µM/mL against HepG2), and its pyrazoline derivative 48 (IC50=0.91 µM/mL against MDA-MB-230 cancer cell lines) were identified as the most active compounds in all derivatives of pregnenolone.

A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species.

The protoapigenone analogue WYC02-9, a novel synthetic flavonoid, has been shown to act against a variety of experimental tumors. However, its effects on prostate cancer and its mechanism of action are unknown. Thus, WYC02-9 was investigated for its cytotoxicity against DU145 prostate cancer cells, as was the underlying mechanisms by which WYC02-9 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). WYC02-9 inhibited the cell growth of three prostate cancer cell lines, especially DU145 cells. In DU145 cells, WYC02-9 increased the generation of intracellular ROS, followed by induction of DNA damage and activation of the ATM-p53-H2A.X pathway and checkpoint-related signals Chk1/Chk2, which led to increased numbers of cells in the S and G2/M phases of the cell cycle. Furthermore, WYC02-9 induced apoptotic cell death through mitochondrial membrane potential decrease and activation of caspase-9, caspase-3, and PARP. The above effects were all prevented by the ROS scavenger N-acetylcysteine. Administration of WYC02-9 in a nude mouse DU145 xenograft model further identified the anti-cancer activity of WYC02-9. These findings therefore suggest that WYC02-9-induced DNA damage and mitochondria-dependent cell apoptosis in DU145 cells are mediated via ROS generation.

Structure and biological evaluation of novel cytotoxic sterol glycosides from the marine red alga Peyssonnelia sp.

Bioactivity-guided fractionation of the extract from a Fijian red alga Peyssonnelia sp. led to the isolation of two novel sterol glycosides 19-O-ß-d-glucopyranosyl-19-hydroxy-cholest-4-en-3-one (1) and 19-O-ß-d-N-acetyl-2-aminoglucopyranosyl-19-hydroxy-cholest-4-en-3-one (2), and two known alkaloids indole-3-carboxaldehyde (3) and 3-(hydroxyacetyl)indole (4). Their structures were characterized by 1D and 2D NMR and mass spectral analysis. The sterol glycosides inhibited cancer cell growth with mean IC50 values (for 11 human cancer cell lines) of 1.63 and 1.41µM for 1 and 2, respectively. The most sensitive cancer cell lines were MDA-MB-468 (breast) and A549 (lung), with IC50's in of 0.71-0.97µM for 1 and 2. Modification of the sterol glycoside structures revealed that the α,ß-unsaturated ketone at C-3 and oxygenation at C-19 of 1 and 2 are crucial for anticancer activity, whereas the glucosidic group was not essential but contributed to enhanced activity against the most sensitive cell lines.

Bioactive bromophycolides R-U from the Fijian red alga Callophycus serratus.

Four new bromophycolides, R-U (1-4), were isolated from the Fijian red alga Callophycus serratus and were identified by 1D and 2D NMR and mass spectroscopic analyses. These compounds expand the known structural variety of diterpene-benzoate macrolides and exhibited modest cytotoxicity toward selected human cancer cell lines. Bromophycolide S (2) also showed submicromolar activity against the human malaria parasite Plasmodium falciparum.

New cytotoxic lupane triterpenes from Perrottetia arisanensis.

Eight new lupane triterpenes, including 7beta-cis-coumaroylbetulinic acid (1), 7beta-trans-coumaroylbetulinic acid (2), 7beta-cis-coumaroyl-3-epi-betulinic acid (3), 7beta-trans-coumaroyl-3-epi-betulinic acid (4), 7beta-cis-coumaroylbetulonic acid (5), 7beta-trans-coumaroylbetulonic acid (6), 7beta-hydroxybetulinaldehyde (7) and 28-norlup-20(29)-ene-3alpha,17beta-diol (8), together with fifteen known compounds were isolated from the bioactive methanol extract of the stems of Perrottetia arisanensis. The structures of the new compounds were elucidated by spectroscopic and HR-ESI-MS analysis. All new compounds were evaluated for their cytotoxicity against six human cancer cell lines. Among them, lupane triterpene coumaroyl esters 1-6 showed moderate cytotoxicity with IC (50) values ranging from 3.75 to 21.29 microM. This is the first report for lupane triterpenes with a phenylpropane moiety substituted at C-7.

Antimalarial bromophycolides J-Q from the Fijian red alga Callophycus serratus.

Bromophycolides J-Q (1-8) were isolated from extracts of the Fijian red alga Callophycus serratus and identified with 1D and 2D NMR spectroscopy and mass spectral analyses. These diterpene-benzoate macrolides represent two novel carbon skeletons and add to the 10 previously reported bromophycolides (9-18) from this alga. Among these 18 bromophycolides, several exhibited activities in the low micromolar range against the human malaria parasite Plasmodium falciparum.

Acasiane A and B and farnesirane A and B, diterpene derivatives from the roots of Acacia farnesiana.

Four new diterpenes, acasiane A ( 1), acasiane B ( 2), farnesirane A ( 3), and farnesirane B ( 4), along with three known diterpenes ( 5 - 7), two triterpenes ( 8 and 9), and eight flavonoids ( 10 - 17) were isolated from the roots of Acacia farnesiana. The structures and relative configurations of these compounds were determined by various spectroscopic and x-ray analyses. All isolated compounds were evaluated for their in vitro cytotoxic activities against six human cancer cell lines (Hep G2, Hep 3B, MDA-MB-231, MCF-7, A549, and Ca9 - 22) with the MTT method. Betulinic acid ( 8) displayed moderate cytotoxicity (1.70 - 5.74 microg/mL) towards five human cancer cell lines and the flavonoids had slight effects. In addition, 8, diosmetin ( 13), and 3',4',5-trihydroxy-7-methoxyflavone ( 15) slightly inhibited superoxide anion generation or elastase release by human neutrophils, indicating moderate anti-inflammatory activities.

Lupane-type triterpenoids from Microtropis fokienensis and Perrottetia arisanensis and the apoptotic effect of 28-hydroxy-3-oxo-lup-20(29)-en-30-al.

Seven new lupane triterpenoids were isolated from bioactive methanol extracts of Microtropis fokienensis (1- 4) and Perrottetia arisanensis (4-7), along with 18 known compounds. The structures of the new compounds were elucidated on the basis of spectroscopic data analysis. All triterpenoids were evaluated for their in vitro cytotoxicity toward seven human cancer cell lines. Compound 8 (28-hydroxy-3-oxo-lup-20(29)-en-30-al) was among the most cytotoxic substances obtained and was found to induce apoptosis of human leukemia HL60 cells and mediate cleavage of PARP and up-regulation of Bax proteins.

5-Hydroxymethyl-2-furfural, a clinical trials agent for sickle cell anemia, and its mono/di-glucosides from classically processed steamed Rehmanniae Radix.

Rehmanniae Radix (Di Huang) is one of the most important traditional Chinese medicines (TCM), and is used for multiple therapeutic purposes. In our investigation of the chemical constituents of Rehmanniae Radix, steamed roots were prepared by the classical processing method. Reversed-phase HPLC of the 50% MeOH extract of steamed Rehmanniae Radix yielded three 5-hydroxymethylfurfural derivatives. The new furfural disaccharide 5-(alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyloxymethyl)-2-furancarboxaldehyde (1) was isolated and characterized, together with its known aglycone 5-hydroxymethyl-2-furfural (3), which is currently in sickle cell anemia Phase I clinical trials, and its corresponding monosaccharide 5-(alpha-D-glucopyranosyloxymethyl)-2-furancarboxaldehyde (2), which was isolated as a natural product for the first time. The presence of these three compounds, particularly 3, which were not found in the unprocessed extract of Rehmanniae Radix, could substantiate the traditional medicinal use of steamed Rehmanniae Radix.

Antitumor agents. 261. 20(S)-protopanaxadiol and 20(s)-protopanaxatriol as antiangiogenic agents and total assignment of (1)H NMR spectra.

Angiogenesis is a critical step in tumor progression and involves several steps including endothelial cell (EC) proliferation, migration, and matrix remodeling. We investigated the antiangiogenic effects of 20( S)-protopanaxadiol ( 1) and 20( S)-protopanaxatriol ( 2), the sapogenins of two major ginseng saponins, in an angiogenesis model of human umbilical vein endothelial cells (HUVECs). These compounds inhibited the proliferative activity of HUVECs in a dose-dependent manner and have potential as anticancer drug candidates. In addition, we report the complete and unambiguous assignment of (1)H NMR spectra of 1 and 2, based on analyses of 2D NMR spectra including COSY, NOESY, HSQC, and HMBC. This report is the first to completely assign the (1)H NMR signals of 2, together with correction of data for 1 from prior reports.

Separation and characterization of active flavonolignans of Silybum marianum by liquid chromatography connected with hybrid ion-trap and time-of-flight mass spectrometry (LC-MS/IT-TOF).

Silychristins A (1) and B (2), silydianin (3), silybins A (4) and B (5), and isosilybins A (6) and B (7) are major bioactive flavonolignans in silymarin, a herbal remedy derived from the milk thistle Silybum marianum. In this study, the seven major active flavonolignans including the diastereomers 1/ 2, 4/ 5, and 6/ 7 were completely separated using semi-micro-high performance liquid chromatography (HPLC) with a Nucleosil 100-3 C 18 HD column and a MeOH/water/formic acid mobile phase system. The collision-induced dissociation (CID) MS/MS and MS (3) spectra of these flavonolignans were studied systematically using hybrid ion-trap and time-of-flight (IT-TOF) mass spectrometry. Efficient differentiation between the seven flavonolignans (1- 7) was possible based on comparison of the resultant CID-MS/MS or MS (3) spectra. Each characteristic MS/MS or MS (3) fragmentation pattern was elucidated with high-resolution mass spectra by IT-TOF. The results with the present methodology show that liquid chromatography-mass spectrometry IT-TOF (LC-MS/IT-TOF) can be useful for general screening of active natural products from plant extracts and for the specific quality control of silymarin.

Chest radiographic presentation in patients with dengue hemorrhagic Fever.

There has been no previously reported case series study regarding chest radiographic (CXR) presentations in dengue hemorrhagic fever (DHF) patients. We retrospectively studied 363 DHF patients from June to December 2002 in southern Taiwan, and a total of 468 CXRs were obtained and reviewed. More than 50% of these showed abnormalities after the 3rd day, with infiltration only and small pleural effusion as the major findings. Progressive changes during the first week and improvements during the second week were observed in these abnormal CXRs. The CXR presentation was also significantly correlated with laboratory findings (white blood cell count, platelet levels, activated partial thromboplastin time, and alanine aminotransferase and albumin levels), as well as the clinical course (renal insufficiency, liver function impairment, upper gastrointestinal bleeding, combination bacterial infection, and duration of admission) and outcome (mortality). The CXR may therefore be a modality for evaluating the clinical course of DHF and should be made during first week after the onset of illness.

First total synthesis of protoapigenone and its analogues as potent cytotoxic agents.

Protoapigenone (1), isolated from Thelypteris torresiana, previously showed significant cytotoxic activity against five human cancer cell lines. In a continued structure-activity relationship study, the first total synthesis and modification of 1 were achieved. All synthesized compounds and related intermediates were evaluated for cytotoxic activity against five human cancer cell lines, HepG2, Hep3B, MDA-MB-231, MCF-7, and A549. Among them, 24 showed 2.2-14.2-fold greater cytotoxicity than 1 and naphthyl A-ring analogues remarkably enhanced the activity.

Acute respiratory failure in adult patients with dengue virus infection.

To investigate clinical course and outcome of dengue with acute respiratory failure (ARF), and to identify related risk factors for acquiring ARF in dengue, we retrospectively studied 11 dengue patients with ARF. From June to December 2002, a total of 606 adult patients were diagnosed as having dengue. Eleven (1.8%) of 606 dengue patients had complications of ARF. The main causes of ARF were sepsis (n = 6, 54.5%) and upper gastrointestinal (UGI) bleeding (n = 3, 27.3%). The mortality rate was 72.7% (n = 8). Additionally, univariate analysis showed that age, dyspnea, cough, prothrombin time, activated partial thromboplastin time, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine, albumin, renal insufficiency, acute renal failure, acute hepatic failure, UGI bleeding, and combination bacterial infection were significantly predictive variables associated with dengue patients with ARF.