Histopathological factors affecting the extraction of high quality genomic DNA from tissue sections for next-generation sequencing.

To enable the widespread application of genomic medicine, the extraction of genomic DNA from thin sections of archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for next-generation sequencing (NGS) is often necessary. However, there are currently no guidelines available on which specific regions of the microtome sections to use for macrodissection with respect to the histopathological factors observed under microscopic examination. The aim of this study was to clarify the relationship between histopathological factors and DNA quality, and to standardize the macrodissection method for more efficient implementation of NGS. FFPE tissue specimens of 218 patients from the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics study were used to investigate the relationship between 15 histopathological factors and the quantitative ratio of double-stranded DNA (dsDNA) to total nucleic acids, as well as the ∆ crossing point value of each tissue specimen. Multivariate logistic regression analysis revealed that specimen storage of ≥3 years was negatively associated with dsDNA quality (P=0.0007, OR: 4.30, 95% CI: 1.85-10.04). In contrast, the presence of a mucus pool was positively associated with dsDNA quality (P=0.0308, OR: 0.23, 95% CI: 0.06-0.87). Metastatic tumors and longer specimen storage periods were significantly associated with lower ∆Cp values (P=0.0007, OR: 4.43, 95% CI: 1.87-10.49; and P=0.0003, OR: 5.51, 95% CI: 2.18-13.95, respectively). Therefore, macrodissection should not be performed on specimens exhibiting histopathological factors associated with poor DNA quality. In particular, the use of tissue blocks with a storage period of <3 years allows the extraction of genomic DNA suitable for NGS.

Change in plasma lactate concentration during arctigenin administration in a phase I clinical trial in patients with gemcitabine-refractory pancreatic cancer.

Arctigenin is evaluated for antitumor efficacy in patients with pancreatic cancer. It has an inhibitory activity on mitochondrial complex I.Therefore, plasma lactate level of patients after arctigenin administration was evaluated for biomarker of clinical response and/or adverse effect. Plasma lactate level in 15 patients enrolled in a Phase I clinical trial of GBS-01 rich in arctigenin was analyzed by colorimetric assay. Statistical analyses for association of plasma lactate and clinical responses, pharmacokinetics of arctigenin, and background factors of each patient by multivariate and univariate analyses.In about half of the patients, transient increase of lactate was observed. Correlation between plasma lactate level and pharmacokinetic parameters of arctigenin and its glucuronide conjugate, and clinical outcome was not detected. Regarding to the determinant of lactate level, only slight association with liver function test was detected. Plasma lactate level is primary determined by reutilization rather than production for antitumor effect and dose not serve as a biomarker. Arctigenin, inhibition of mitochondrial complex I, plasma lactate concentration, phase I clinical trial of GBS-01, Cori cycle.

Clinical significance of BRAF non-V600E mutations on the therapeutic effects of anti-EGFR monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer: the Biomarker Research for anti-EGFR monoclonal Antibodies by Comprehensive Cancer genomics (BREAC) study.

BACKGROUND: Patients with BRAFV600E-mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAFV600E (BRAFnon-V600E mutations) contribute to anti-EGFR antibody resistance. METHODS: This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. RESULTS: In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAFV600E (6.0%), and 7 patients with BRAFnon-V600E mutations (4.7%), respectively. The response rates in RAS, BRAFV600E, and BRAFnon-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAFnon-V600E mutations was 2.4 months, similar to that in RAS or BRAFV600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). CONCLUSIONS: Although BRAFnon-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAFnon-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

Constituents of the Rhizomes of Boesenbergia pandurata and Their Antiausterity Activities against the PANC-1 Human Pancreatic Cancer Line.

Human pancreatic cancer cell lines have a remarkable tolerance to nutrition starvation, which enables them to survive under a tumor microenvironment. The search for agents that preferentially inhibit the survival of cancer cells under low nutrient conditions represents a novel antiausterity strategy in anticancer drug discovery. In this investigation, a methanol extract of the rhizomes of Boesenbergia pandurata showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions, with a PC50 value of 6.6 µg/mL. Phytochemical investigation of this extract led to the isolation of 15 compounds, including eight new cyclohexene chalcones (1-8). The structures of the new compounds were elucidated by NMR spectroscopic data analysis. Among the isolated compounds obtained, isopanduratin A1 (14) and nicolaioidesin C (15) exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions, with PC50 values of 1.0 and 0.84 µM, respectively.

Antiausterity activity of arctigenin enantiomers: importance of (2R,3R)-absolute configuration.

From a MeOH extract of powdered roots of Wikstroemia indica, six dibenzyl-gamma-butyrolactone-type lignans with (2S,3S)-absolute configuration [(+)-arctigenin (1), (+)-matairesinol (2), (+)-trachelogenin (3), (+)-nortrachelogenin (4), (+)-hinokinin (5), and (+)-kusunokinin (6)] were isolated, whereas three dibenzyl-gamma-butyrolactone-type lignans with (2R,3R)-absolute configuration [(-)-arctigenin (1*), (-)-matairesinol (2*), (-)-trachelogenin (3*)] were isolated from Trachelospermum asiaticum. The in vitro preferential cytotoxic activity of the nine compounds was evaluated against human pancreatic PANC-1 cancer cells in nutrient-deprived medium (NDM), but none of the six lignans (1-6) with (2S,3S)-absolute configuration showed preferential cytotoxicity. On the other hand, three lignans (1*-3*) with (2R,3R)-absolute configuration exhibited preferential cytotoxicity in a concentration-dependent manner with PC50 values of 0.54, 6.82, and 5.85 microM, respectively. Furthermore, the effect of (-)- and (+)-arctigenin was evaluated against the activation of Akt, which is a key process in the tolerance to nutrition starvation. Interestingly, only (-)-arctigenin (1*) strongly suppressed the activation of Akt. These results indicate that the (2R,3R)-absolute configuration of (-)-enantiomers should be required for the preferential cytotoxicity through the inhibition of Akt activation.

Synthesis and evaluation of anticancer natural product analogues based on angelmarin: targeting the tolerance towards nutrient deprivation.

Inspired by nature: Angelmarin is an anticancer natural product with potent antiausterity activity, that is, selective cytotoxicity towards nutrient-deprived, resistant cancer cells. Through structure-activity relationship studies, three analogues were identified as lead compounds for the develpoment of molecular probes for the investigation of the mode of action and biological targets of the antiausterity compounds.

Identification of chrysoplenetin from Vitex negundo as a potential cytotoxic agent against PANC-1 and a panel of 39 human cancer cell lines (JFCR-39).

Human pancreatic cancer is known to be the most deadly disease with the lowest 5-year survival rate and is resistant to well known conventional chemotherapeutic drugs in clinical use. Screening of medicinal plants from Myanmar utilizing antiausterity strategy led to the identification of Vitex negundo as one of the medicinal plants having potent preferential cytotoxic activity against PANC-1 human pancreatic cancer cells. Bioactivity-guided phytochemical investigation led to the isolation of chrysoplenetin (1) and chrysosplenol D (2) as the active constituents with a PC(50) value of 3.4 µg/mL and 4.6 µg/mL, respectively, against PANC-1 cells. Both these compounds induced apoptosis-like morphological changes in PANC-1 cells. Chrysoplenetin was further tested against a panel of 39 human cancer cell lines (JFCR-39) at the Japanese Foundation for Cancer Research, and 25 cell lines belonging to lung, breast, CNS, colon, melanoma, ovarian, prostate cancer and stomach cancer cell lines were found to be highly sensitive to chrysoplenetin at a submicromolar range. In the JFCR-39 panel, lung NCI-H522, ovarian OVCAR-3 and prostate PC-3 cells were found to be most sensitive with GI(50) of 0.12, 0.18 and 0.17 µm, respectively. The COMPARE analysis suggested that the molecular mode of action of chrysoplenetin was unique compared with the existing anticancer drugs.

Novel anticancer agents, kayeassamins C-I from the flower of Kayea assamica of Myanmar.

A CHCl3-soluble fraction of 70% EtOH extract of the flower of Kayea assamica from Myanmar exhibited 100% preferential cytotoxicity (PC(100)) against human pancreatic cancer PANC-1 cells under nutrient-deprived conditions at 1 microg/mL. Bioassay-guided fractionation and isolation afforded nine new coumarins, kayeassamins A (8), B (9), and C-I (1-7), together with nine known coumarins (10-18). The structures of these compounds were identified by extensive spectroscopic techniques as well as by comparison with published data. Absolute configuration at C-1' of 1 was established as S-configuration by the modified Mosher method. All the isolates were evaluated for their in vitro preferential cytotoxicity using novel anti-austerity strategy. Among them, the novel coumarins, kayeassamins A (8), B (9), D (2), E (3), and G (5) exhibited the most potent preferential cytotoxicity (PC(100) 1 microM) in a concentration- and time-dependent manner and induced apoptosis-like morphological changes of PANC-1 cells within 24 h of treatment. Based on the observed cytotoxicity, structure-activity relationships have been established.

Novel anticancer agents, kayeassamins A and B from the flower of Kayea assamica of Myanmar.

The CHCl(3)-soluble fraction of 70% EtOH extract of the flower of Kayea assamica completely killed human pancreatic PANC-1 cancer cells preferentially under nutrient-deprived conditions at 1 microg/mL. Bioassay-guided fractionation and isolation afforded two novel compounds, kayeassamins A (1) and B (2). Their structures were elucidated using extensive spectroscopic methods and the modified Mosher method. Each compound showed 100% preferential cytotoxicity (PC(100)) against PANC-1 cells under nutrient-deprived conditions at 1 microM. Furthermore, both compounds inhibited the migration of PANC-1 cells in the wound closure assay.

Constituents of Brazilian red propolis and their preferential cytotoxic activity against human pancreatic PANC-1 cancer cell line in nutrient-deprived condition.

Human pancreatic cancer cells such as PANC-1 are known to exhibit marked tolerance to nutrition starvation that enables them to survive for prolonged period of time even under extremely nutrient-deprived conditions. Thus, elimination of this tolerance to nutrition starvation is regarded as a novel approach in anticancer drug development. In this study, the MeOH soluble extract of Brazilian red propolis was found to kill 100% PANC-1 cells preferentially in the nutrient-deprived condition at the concentration of 10 microg/mL. Further phytochemical investigation led to the isolation of 43 compounds including three new compounds, (6aS,11aS)-6a-ethoxymedicarpan (1), 2-(2',4'-dihydroxyphenyl)-3-methyl-6-methoxybenzofuran (2), and 2,6-dihydroxy-2-[(4-hydroxyphenyl)methyl]-3-benzofuranone (3). Among them, (6aR,11aR)-3,8-dihydroxy-9-methoxypterocarpan (21, DMPC) displayed the most potent 100% preferential cytotoxicity (PC(100)) at the concentration of 12.5 microM. Further study on the mode of cell death induced by DMPC against PANC-1 cells indicated that killing process was not accompanied by DNA fragmentation, rather through a nonapoptotic pathway accompanied by necrotic-type morphological changes.

Bioactive secondary metabolites from Boesenbergia pandurata of Myanmar and their preferential cytotoxicity against human pancreatic cancer PANC-1 cell line in nutrient-deprived medium.

The chloroform extract of rhizomes of Boesenbergia pandurata demonstrated marked preferential cytotoxicity against human pancreatic PANC-1 cancer cells in nutrient-deprived medium. Bioactivity-directed investigation of this extract yielded four new secondary metabolites, geranyl-2,4-dihydroxy-6-phenethylbenzoate ( 1), 2',4'-dihydroxy-3'-(1''-geranyl)-6'-methoxychalcone ( 2), (1' R,2' S,6' R)-2-hydroxyisopanduratin A ( 3), and (2 R)-8-geranylpinostrobin ( 4), and twenty known compounds ( 5- 24). Among the known compounds, (2 S)-6-geranylpinostrobin ( 5), (+/-)-6-methoxypanduratin A ( 6), and (2 S)-7,8-dihydro-5-hydroxy-2-methyl-2-(4''-methyl-3''-pentenyl)-8-phenyl-2 H,6 H-benzo[1,2- b:5,4- b']dipyran-6-one ( 7) were isolated for the first time from a natural source. The structures of these compounds were elucidated using extensive spectroscopic techniques including CD measurements. All the isolated compounds showed varying degrees of in vitro preferential cytotoxicity against PANC-1 cells. Nicolaioidesin B ( 11) and panduratin A ( 17) were most potent, each showing a PC 100 at 2.5 microM.

Diterpenes from "Pini Resina" and their preferential cytotoxic activity under nutrient-deprived condition.

In the course of our search for anticancer agents based on a novel anti-austerity strategy, we found that the 70 % EtOH extract of "Pini Resina" showed 100 % preferential cytotoxicity at the concentration of 50 microg/mL. Further bioassay-guided fractionation and purification led to the isolation of 15 compounds including one new compound 7-oxo-13 alpha-hydroxyabiet-8(14)-en-18-oic acid (1). Their structures were elucidated on the basis of spectroscopic analysis. Among the isolated compounds, methyl abieta-8,11,13-trien-18-oate (7) showed the most potent preferential cytotoxicity at 10 microg/mL under nutrient-deprived condition.

Identification of arctigenin as an antitumor agent having the ability to eliminate the tolerance of cancer cells to nutrient starvation.

Tumor cells generally proliferate rapidly and the demand for essential nutrients as well as oxygen always exceeds the supply due to the unregulated growth and the insufficient and inappropriate vascular supply. However, cancer cells show an inherent ability to tolerate extreme conditions, such as that characterized by low nutrient and oxygen supply, by modulating their energy metabolism. Thus, targeting nutrient-deprived cancer cells may be a novel strategy in anticancer drug development. Based on that, we established a novel screening method to discover anticancer agents that preferentially inhibit cancer cell viability under the nutrient-deprived condition. After screening 500 medicinal plant extracts used in Japanese Kampo medicine, we found that a CH(2)Cl(2)-soluble extract of Arctium lappa exhibited 100% preferential cytotoxicity under the nutrient-deprived condition at a concentration of 50 microg/mL with virtually no cytotoxicity under nutrient-rich condition. Further bioassay-guided fractionation and isolation led to the isolation of arctigenin as the primary compound responsible for such preferential cytotoxicity; the compound exhibited 100% preferential cytotoxicity against nutrient-deprived cells at a concentration of 0.01 microg/mL. Furthermore, arctigenin was also found to strongly suppress the PANC-1 tumor growth in nude mice, as well as the growth of several of the tested pancreatic cancer cell lines, suggesting the feasibility of this novel antiausterity approach in cancer therapy. Further investigation of the mechanism of action of arctigenin revealed that the compound blocked the activation of Akt induced by glucose starvation, which is a key process in the tolerance exhibited by cancer cells to glucose starvation.

Angelmarin, a novel anti-cancer agent able to eliminate the tolerance of cancer cells to nutrient starvation.

The CH(2)Cl(2)-soluble extract of Angelica pubescens was found to kill PANC-1 cancer cells preferentially under nutrition starvation at a concentration of 50 microg/ml, with virtually no cytotoxicity under nutrient-rich conditions. Further bioassay-guided fractionation and isolation led to the isolation of a novel compound named angelmarin as the primary compound responsible for the preferential cytotoxicity; the compound exhibited 100% preferential cytotoxicity against PANC-1 cells at a concentration of 0.01 microg/ml.