Guidelines for Handling of Cytological Specimens in Cancer Genomic Medicine.

Rapid advances are being made in cancer drug therapy. Since molecularly targeted therapy has been introduced, personalized medicine is being practiced, pathological tissue from malignant tumors obtained during routine practice is frequently used for genomic testing. Whereas cytological specimens fixed mainly in alcohol are considered to be more advantageous in terms of preservation of the nucleic acid quality and quantity. This article is aimed to share the information for the proper handling of cytological specimens in practice for genomic medicine based on the findings established in "Guidelines for Handling of Cytological Specimens in Cancer Genomic Medicine (in Japanese)" published by the Japanese Society of Clinical Cytology in 2021. The three-part practical guidelines are based on empirical data analyses; Part 1 describes general remarks on the use of cytological specimens in cancer genomic medicine, then Part 2 describes proper handling of cytological specimens, and Part 3 describes the empirical data related to handling of cytological specimens. The guidelines indicated proper handling of specimens in each fixation, preparation, and evaluation.

Cytotoxic triterpene and steroidal glycosides from the seeds of Digitalis purpurea and the synergistic cytotoxicity of steroidal glycosides and etoposide in SBC-3 cells.

The phytochemical investigations of the seeds of Digitalis purpurea have revealed their richness in cardenolide and pregnane glycosides exhibiting potent cytotoxicity; further chemical examinations of the D. purpurea seeds have achieved the isolation of six triterpene glycosides (1-6), six spirostanol glycosides (7-12), and three furostanol glycosides (13-15), including seven previously unidentified compounds (1-3, 10-12, and 14). Here, the structures of 1-3, 10-12, and 14 were determined via extensive spectroscopic analyses, including two-dimensional (2D) NMR; hydrolysis, followed by chromatographic and spectroscopic analyses; and X-ray crystallographic analysis. The cytotoxic activities of the isolated compounds (1-15) against SBC-3 small cell lung carcinoma and TIG-3 normal human diploid fibroblast cells were evaluated. Triterpene glycoside 3 and spirostanol glycoside 9 exhibited considerable cytotoxicity with IC50 values of 1.0 and 1.7 µM, respectively; they induced apoptotic cell death, which was accompanied by the activation of caspase-3 in SBC-3 cells. Spirostanol glycoside 7 exhibited cytotoxicity toward the SBC-3 cells (IC50 1.3 µM). Additionally, 7 at 0.1 and 1.0 µM synergistically enhanced the cytotoxicity of etoposide against SBC-3 cells; compound 7 induced the release of DAMPs; the release of HMGB1, the secretion of ATP, and the exposure of CALR in the SBC-3 cells. Furthermore, the combination of 7 and etoposide resulted in increasing the extracellular release of DAMPs. These data indicated that 7, as well as its combination with etoposide, might potentially cause immunogenic cell death.

Chemical Constituents of the Bulbs of Scilla peruviana and Their Pancreatic Lipase Inhibitory Activity.

Scilla species are used as medicinal plants and contain lanosterol-type triterpene glycosides. The phytochemical investigation of the bulbs of Scilla peruviana led to the isolation of 17 compounds, including three new rearranged pentacyclic-lanosterol glycosides (1-3) and two new homoisoflavanone glycosides (12 and 13). The structures of the undescribed compounds were determined by extensive spectroscopic analyses, including two-dimensional (2D) NMR. Among the triterpene glycosides, 2, 3, and 6 showed significant pancreatic lipase inhibitory activity in a concentration-dependent manner in vitro. The oral administration of scillascilloside D-2 (6) reduced serum triglyceride levels in a dose-dependent manner in soybean oil-loaded mice.

Daisaikoto Inhibits Pancreatic Lipase Activity and Decreases Serum Triglyceride Levels in Mice.

Daisaikoto Extract, a Kampo medicine listed in the Japanese pharmacopoeia 17th edition, is clinically used to treat obesity and related symptoms. Lipid metabolism is closely related to obesity, and pancreatic lipase inhibitors are therefore regarded as effective for the treatment of obesity. Although Daisaikoto has shown promise in the treatment of obesity, its mechanism of action has yet to be elucidated. In the present study, we found that Daisaikoto extract inhibits pancreatic lipase activity in a dose-dependent manner and decreases serum triglyceride levels in mice. To determine the crude drugs responsible for lipase inhibition, 8 variants of Daisaikoto extract without one crude drug were prepared and evaluated for lipase inhibitory activity. The lipase inhibitory activity of the Daisaikoto extract was reduced by excluding Scutellariae Radix (SR), which was found to inhibit lipase activity with an IC50 value of 1.70 mg/mL. In conclusion, Daisaikoto represents a natural medicine, in particular SR, capable of inhibiting pancreatic lipase and lipid absorption.

Cycloartane and Oleanane Glycosides from the Tubers of Eranthis cilicica.

Phytochemical analysis of the tubers of Eranthis cilicica was performed as part of our continuous study on the plants of the family Ranunculaceae, which resulted in the isolation of eleven new cycloartane glycosides (1⁻11) and one new oleanane glycoside (13), together with one known oleanane glycoside (12). The structures of the new compounds were determined by extensive spectroscopic analysis, including two-dimensional (2D) NMR, and enzymatic hydrolysis followed by either X-ray crystallographic or chromatographic analysis. The aglycone (1a) of 2 and its C-23 epimer (8a), and the oleanane glycosides (12 and 13) showed cytotoxic activity against HL-60 leukemia cells with IC50 values ranging from 10.6 µM to 101.6 µM. HL-60 cells were much more sensitive to 8a (IC50 14.8 µM) than 1a (IC50 101.1 µM), indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. Compound 12 was revealed so as to partially induce apoptotic cell death in HL-60 cells, as was evident from morphology of HL-60 cells treated with 12.

Steroidal Glycosides from Convallaria majalis Whole Plants and Their Cytotoxic Activity.

Phytochemical examination of Convallaria majalis (Liliaceae) whole plants yielded 15 steroidal glycosides (1-15), including nine new compounds (4-6, 10-15) with a lycotetrose unit. The structures of the new compounds were determined using two-dimensional Nuclear magnetic resonance (NMR) analyses and chemical methods. The isolated compounds were evaluated for cytotoxicity against HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, and HSC-4 and HSC-2 human oral squamous cell carcinoma cell lines. Of these, (25S)-spirost-5-en-3ß-yl O-ß-d-glucopyranosyl-(1→2)-O-[ß-d-xylopyranosyl-(1→3)]-O-ß-d-glucopyranosyl-(1→4)-ß-d-galactopyranoside (1) exhibited cytotoxic activity against HL-60, A549, HSC-4, and HSC-2 cells with IC50 values ranging from 0.96 to 3.15 µM. The corresponding furostanol glycoside of 1, (25S)-26-[(ß-d-glucopyranosyl)oxy]-22α-hydroxyfurost-5-en-3ß-yl O-ß-d-glucopyranosyl-(1→2)-O-[ß-d-xylopyranosyl-(1→3)]-O-ß-d-glucopyranosyl-(1→4)-ß-d-galactopyranoside (8), was cytotoxic to the adherent cell lines of A549, HSC-4, and HSC-2 cells with IC50 values of 2.97, 11.04, and 8.25 µM, respectively. The spirostanol lycotetroside (1) caused necrotic cell death in A549 cells in a dose-dependent manner. Alternatively, the furostanol lycotetroside (8) induced apoptotic cell death in A549 cells in a time-dependent manner, as was evident by morphological observations and flow cytometry analyses.

Structural Characterization of Cholestane Rhamnosides from Ornithogalum saundersiae Bulbs and Their Cytotoxic Activity against Cultured Tumor Cells.

Previous phytochemical studies of the bulbs of Ornithogalum saundersiae, an ornamental perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides, some of which were structurally unique and showed potent cytotoxic activity against cultured tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides (1-12) and seven known compounds (13-19). The structures of the new compounds (1-12) were identified via NMR-based structural characterization methods, and through a sequence of chemical transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of the isolated compounds (1-19) and the derivatives (1a and 6a) against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10-12, 16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent cytotoxicity with an IC50 value of 0.16 µM against HL-60 cells and induced apoptotic cell death via a mitochondrion-independent pathway.

Vetiverianines A, B, and C: Sesquiterpenoids from Vetiveria zizanioides Roots.

Three new sesquiterpenoids-vetiverianines A (1), B (2), and C (3)-and a known eudesmane sesquiterpenoid (4) were isolated from the roots of Vetiveria zizanioides. Vetiverianine A (1) has a unique carbon framework comprising a rigid tricyclic ring system. Vetiverianines B (2) and C (3) are new eremophilane sesquiterpenoids. The structures of sesquiterpenoids 1-3, including the absolute configurations, were determined by NMR spectroscopic, X-ray crystallography, and vibrational circular dichroism data analysis. Vetiverianine C (3) exhibited weak cytotoxic activity against HL-60 cells.

Prevalent and up-regulated vimentin expression in micropapillary components of lung adenocarcinomas and its adverse prognostic significance.

The factors conferring the increased malignancy on lung adenocarcinoma with micropapillary component (AC-MPC) remain to be elucidated. On proteomics based on 2-dimensional gel electrophoresis, 19 proteins differentially expressed by more than 1.5-fold between AC-MPC and conventional adenocarcinoma (CAC); in particular, vimentin, one of the proteins, was 3.5-fold up-regulated in AC-MPC. Subsequent semi-quantitative investigation by immunohistochemistry with large cohorts comprised 101 AC-MPC and 119 CAC, respectively, of different stages revealed that vimentin was expressed in MPC of 95 (94.1%) AC-MPC and the expression scores were higher than those of well- and moderately differentiated CAC, as well as the background non-MPC of the AC-MPC (P < 0.0001), but not significantly different from those of poorly differentiated CAC (P = 0.561). Even within the AC-MPC entity, higher vimentin expression was correlated with more frequent vascular invasion and more advanced node metastasis (P < 0.02), and multivariate analysis showed that high vimentin expression and worse node statuses were independent indicators of adverse prognosis (P < 0.048). In conclusion, vimentin expression is prevalent and markedly up-regulated in MPC, which might reflect the biological essence of poorer differentiation or dedifferentiation of MPC, and this might have a role in the acquisition and increase of invasiveness and consequent more malignant nature of MPC.

Cardenolide glycosides from the seeds of Digitalis purpurea exhibit carcinoma-specific cytotoxicity toward renal adenocarcinoma and hepatocellular carcinoma cells.

Four cardenolide glycosides, glucodigifucoside (2), 3'-O-acetylglucoevatromonoside (9), digitoxigenin 3-O-ß-D-glucopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 4)-3-O-acetyl-ß-D-digitoxopyranoside (11), and purpureaglycoside A (12), isolated from the seeds of Digitalis purpurea, exhibited potent cytotoxicity against human renal adenocarcinoma cell line ACHN. These compounds exhibited significantly lower IC50 values against ACHN than that against normal human renal proximal tubule-derived cell line HK-2. In particular, 2 exhibited the most potent and carcinoma-specific cytotoxicity, with a sixfold lower IC50 value against ACHN than that against HK-2. Measurement of cyclin-dependent kinase inhibitor levels revealed that upregulation of p21/Cip1 expression was involved in the carcinoma-specific cytotoxicity of 2. Further, compound 2 also exhibited the carcinoma-specific cytotoxicity toward hepatocellular carcinoma cell line.

Novel Steroidal Glycosides from the Bulbs of Lilium pumilum.

Examination of the bulbs of Lilium pumilum (Liliaceae) led to the isolation of four novel steroidal glycosides (1-4) with a 2,3,4-trisubstituted ß-d-glucopyranosyl unit. In 1 and 3, the α-L-arabinopyranosyl moiety is linked to C-3 of the inner trisubstituted ß-D-glucopyranosyl group and is present as an usual 4C1 conformation. In contrast, in 2 and 4, the α-L-arabinopyranosyl moiety, which is attached to C-4 of the inner trisubstituted ß-D-glucopyranosyl group, is present as a ¹C4 conformation. The structures of the new steroidal glycosides were determined based on the results of spectroscopic analyses, including two-dimensional (2D) NMR data and hydrolysis.

A rare type of sesquiterpene and ß-santalol derivatives from Santalum album and their cytotoxic activities.

A rare type of sesquiterpene with a spiro bicyclic system (1) and seven new (2-8) and four known (9-12) ß-santalol derivatives were isolated from the heartwood of Santalum album (Santalaceae). The structures of these new compounds were determined by analysis of extensive spectroscopic data. The isolated compounds and derivatives were evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, HSC-2 and HSC-4 human oral squamous cell carcinoma cell lines, and TIG-3 normal human diploid fibroblasts. cis-ß-Santalol (9) and ß-santaldiol (10) induced apoptotic cell death in HL-60 cells.

Search for new steroidal glycosides with anti-cancer potential from natural resources.

Chemical investigations of higher plants, with particular attention paid to their steroidal glycosides, present a promising approach for generating anti-cancer agents from natural products. We conducted a systematic phytochemical investigation of nine higher plants-whole plants and rhizomes of Convallaria majalis, whole plants of Agave utahensis, roots of Adonis amurensis, seeds of Adonis aestivalis, bulbs of Bessera elegans, bulbs of Fritillaria meleagris, seeds of Digitalis purpurea, underground parts of Yucca glauca, and bulbs of Lilium pumilum-which led to the discovery of novel steroidal glycosides. The structures of these new constituents were determined based on spectroscopic data and chemical transformations. The identification of the monosaccharides including their absolute configurations was carried out by direct HPLC analysis of their hydrolysates using an optical rotation detector. Cytotoxicity of the isolated steroidal glycosides was evaluated against various tumor cells (A549, ACHN, HepG-2, HL-60, HSC-2, HSC-3, HSC-4, HSG, and SBC-3) and normal cells (Fa2 N-4, HK-2, and TIG-3 cells). Certain steroidal glycosides exhibit selective cytotoxicity and synergistic effects, making them potential lead compounds for use as anti-cancer agents. We document the isolation of 139 steroidal glycosides from higher plants and assessment their cytotoxic activities.

New cardenolide glycosides from the seeds of Digitalis purpurea and their cytotoxic activity.

A chemical investigation of Digitalis purpurea seeds led to the isolation of three new cardenolide glycosides (1, 8 and 11), together with 12 known cardenolide glycosides (2-7, 9, 10 and 12-15). The structures of 1, 8 and 11 were determined by 1D and 2D NMR spectroscopic analyses and the results of an acid or enzymatic hydrolysis. The cytotoxic activity of the isolated compounds (1-15) against HL-60 leukemia cells was examined. Compounds 2, 9, 11 and 12 showed potent cytotoxicity against HL-60 cells with respective 50% inhibition concentration (IC50) values of 0.060, 0.069, 0.038, and 0.034 µM. Compounds 2, 9 and 11 also exhibited potent cytotoxic activity against HepG2 human liver cancer cells with respective IC50 values of 0.38, 0.79, and 0.71 µM. An investigation of the structure-activity relationship showed that the cytotoxic activity was reduced by the introduction of a hydroxy group at C-16 of the digitoxigenin aglycone, methylation of the C-3' hydroxy group at the fucopyranosyl moiety, and acetylation of the C-3' hydroxy group at the digitoxopyranoyl moiety.

Molecular Dynamics Simulation of the Complex of PDE5 and Evodiamine.

Alzheimer's disease is an irreversible neurological disorder for which there are no effective small molecule therapeutics. A phosphodiesterase 5 (PDE5) inhibitor is a candidate medicine for the treatment of Alzheimer's disease. Rutaecarpine, an indole alkaloid found in Euodiae Fructus, has inhibitory activity for PDE5. Euodiae Fructus contains more evodiamine than rutaecarpine. Therefore, we performed molecular dynamics simulations of the complex of PDE5 and evodiamine. The results showed that the PDE5 and (-)-evodiamine complexes were placed inside the reaction center compared to the case of PDE5 and (+)-evodiamine complex. The binding of (-)-evodiamine to PDE5 increased the root-mean-square deviation and radius of gyration of PDE5. In the PDE5 with (-)-evodiamine complex, the value of the root-mean-square fluctuation of the M-loop, which is thought to be important for activity, increased. This result suggests that (-)-evodiamine may have inhibitory activity.

Lanceolanone A, a new biflavanone, and a chalcone glucoside from the flower heads of Coreopsis lanceolata and their aldose reductase inhibitory activity and AMPK activation.

The MeOH extract of the flower heads of Coreopsis lanceolata L. (Asteraceae) exhibited aldose reductase (AR) inhibitory activity (IC50 8.36 µg/mL). Bioassay-guided fractionation of the extract resulted in the isolation of a new biflavanone-named Lanceolanone A (1) and a chalcone glucoside (6), along with 12 known compounds (2-5 and 7-14), of which 4, 7, 9, 10, and 12 were isolated from C. lanceolata for the first time. The structures of the new compounds (1 and 6) were determined by extensive spectroscopic analysis, including two-dimensional (2D) NMR, and ECD calculation method. Compounds 2, 4, 11, 13, and 14 exhibited AR inhibitory activities with IC50 values between 2.40 and 9.99 µM. Furthermore, 8-13 at 1.0 mM activated AMPK expression in HepG2 human hepatoma cells compared to the control.

The role of CCR7 in allergic airway inflammation induced by house dust mite exposure.

House dust mite (HDM), the most common allergen, activate both the IgE-associated and innate immune responses. To clarify the process of sensitization, we investigated the role of the CCL21, CCL19, and CCR7 axis in a mouse model of HDM-induced allergic asthma. HDM inhalation without systemic immunization resulted in a HDM-specific IgE response. CCR7-knockout (CCR7KO) mice exhibited greater airway inflammation and IgE responses compared to wild-type mice. We examined FoxP3 expression in these mice to clarify the contribution of regulatory cells to the responses. FoxP3 expression was higher in the lungs but not in the lymph nodes of CCR7KO mice compared to wild-type mice. In CCR7KO mice, FoxP3-positive cells were found in lung, but we observed higher release of IL-13, IL-5, TGF-ß, IL-17, and HMGB1 in bronchoalveolar lavage fluid. We demonstrate here that immuno-regulation through CCR7 expression in T cells plays a role in HDM-specific sensitization in the airway.

Suppressive effects of a pyrazole derivative of curcumin on airway inflammation and remodeling.

To advance the control of airway epithelial cell function and asthma, we investigated the effects of a new curcumin derivative, CNB001, which possesses improved pharmacological properties. Normal human bronchial epithelial (NHBE) cells were stimulated with synthetic double-stranded RNA, Poly(I:C). CNB001 significantly suppressed IL-6, TNF-α, and GM-CSF production by NHBE cells, and did so more effectively than did curcumin or dexamethasone (DEX). CNB001 significantly inhibited the decrease of E-cadherin mRNA expression and increase of vimentin mRNA expression observed in NHBE cells induced by a combination of TGF-ß1 and TNF-α, which are markers of airway remodeling. In NHBE cells stimulated by TGF-ß1, CNB001 significantly downregulated the level of active serine peptidase inhibitor clade E member (SERPINE) 1, which is also reported to be related to airway remodeling. Whereas DEX alone significantly increased the active SERPINE1 level, the combination of DEX and CNB001 significantly suppressed active SERPINE1. In addition, CNB001 significantly suppressed neutrophil infiltration, IL-6, TNF-α, IL-13 and active SERPINE1 production in bronchoalveolar lavage fluid of the murine asthma model, which was not observed in the case of DEX. In conclusion, the curcumin derivative, CNB001, is a promising candidate to treat asthma associated with neutrophilic airway inflammation and remodeling.

New cardenolides from the seeds of Adonis aestivalis.

Chemical investigation of the seeds of Adonis aestivalis has led to the isolation of a new cardenolide (3ß,5α,14ß,17ß-tetrahydroxycard-20,22-enolide) (1), two new glycosides (2, 3) of 1, and a new strophanthidin hexaglycoside (4), together with a known compound, strophanthidin 3-O-ß-D-glucopyranoside (5). The structures of 1-4 were determined by 1D- and 2D-NMR spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds (1-5) were examined for their cytotoxic activity against neoplastic HSC-2, HSC-3, HSC-4, and HL-60 cells, as well as HGF, HPLF, and HPC normal cell lines. Compounds 2, 4, and 5 were found to display selective cytotoxicity toward malignant tumor cell lines. Although the morphological observations of HL-60 and HSC-2 cell deaths by 2, 4, and 5 revealed changes characteristic of apoptosis, neither DNA degradation nor activation of caspase-3 was observed. Our findings demonstrated that 2, 4, and 5 may trigger caspase-3-independent apoptotic cell death in HL-60 and HSC-2 cells.

Characterization of morphological alterations in micropapillary adenocarcinoma of the lung using an established cell line.

Micropapillary adenocarcinoma of the lung is a type of cancer associated with a poor prognosis and is characterized by the presence of tumor cells with a ring­like glandular structure floating within alveolar spaces. In the present study, the association between its morphological, biochemical and immunohistochemical characteristics, and malignancy was investigated using the KU­Lu­MPPt3 cell line established from a patient with MIP adenocarcinoma. Two subpopulations of KU­Lu­MPPt3 cells, namely adhesive (AD) and clumpy and suspended (CS) cells, were prepared and subjected to DNA microarray, reverse transcription­quantitative PCR, western blot and immunostaining analyses. Protein expression patterns were compared between the cell types and their derived tissues using immunostaining. The results revealed similar protein expression patterns between the tumor cells found in the alveolar spaces and CS cells, which exhibited morphological characteristic of MIP adenocarcinoma. Based on the results of DNA microarray analysis, the present study then focused on Akt and focal adhesion kinase (FAK), which were markedly activated in the KU­Lu­MPPt3 CS and AD cells, respectively. Following KU­Lu­MPPt3 CS cell plating onto collagen­coated culture dishes, some cells exhibited a transformation of their morphology into KU­Lu­MPPt3 AD­like cells within a few days, and their Akt and FAK activities were similar to those of the AD cells. Additionally, the inhibition of Akt and FAK activities with Akt and FAK inhibitors reduced KU­Lu­MPPt3 CS cell adhesion and proliferation. Thus, the aforementioned results indicated that the phosphorylation of FAK and Akt may play a crucial role in the regulation of KU­Lu­MPPt3 CS cell adhesion and proliferation, respectively. Furthermore, the malignant potential of MIP adenocarcinoma may be attributed to these morphological and biochemical alterations in the KU­Lu­MPPt3 cells.