Chemical constituents of Callistemon citrinus from Egypt and their antiausterity activity against PANC-1 human pancreatic cancer cell line.

Human pancreatic cancer is resistant to almost all conventional chemotherapeutic agents. It is known to proliferate aggressively within hypovascular tumor microenvironment by exhibiting remarkable tolerance to nutrition starvation,  a phenomenon termed as "austerity". Search for the new agents that eliminate the tolerance of cancer cells to nutrition starvation is a promising strategy in anticancer drug discovery. In this study, two new meroterpenoids named callistrilones O and P (1 and 2) together with eight known triterpenes (3-10) were isolated from the active dichloromethane extract of Callistemon citrinus leaves. The structure elucidation of the new compounds was achieved by HRFABMS, 1D, 2D NMR, and ECD quantum calculations. All isolated compounds were tested for their preferential cytotoxicity against PANC-1 human pancreatic cancer cells. Among these, callistrilone O (1) exhibited the most potent preferential cytotoxicity with a PC50 value of 0.3 nM, the strongest activity with over 2000 times potent than the positive control arctigenin. Callistrilone O (1) induced dramatic alterations in PANC-1 cell morphology leading to cell death under nutrient-deprived conditions. Compound 1 also inhibited PANC-1 cell migration and -PANC-1 colony formation under the nutrient-rich condition.

Highly Potent Antiausterity Agents from Callistemon citrinus and Their Mechanism of Action against the PANC-1 Human Pancreatic Cancer Cell Line.

Human pancreatic cancer cells display remarkable tolerance to nutrition starvation that help them to survive in a hypovascular tumor microenvironment, a phenomenon known as "austerity". The elucidation of agents countering this tolerance is an established antiausterity strategy in anticancer drug discovery. In this study, a Callistemon citrinus leaf extract inhibited the viability of PANC-1 human pancreatic cancer cells preferentially under nutrient-deprived medium (NDM) with a PC50 value of 7.4 µg/mL. Workup of this extract resulted in the isolation of three new meroterpenoids, callistrilones L-N (1-3), together with 14 known compounds (4-17). The structure elucidation of the new compounds was achieved by HRFABMS and by NMR and ECD spectroscopic analysis. The new compounds showed highly potent preferential cytotoxicity against PANC-1 cells with PC50 values ranging from 10 to 65 nM in NDM. Of these, callistrilone L (1) inhibited PANC-1 cell migration and colony formation in a normal nutrient-rich condition. Callistrilone L (1) also strongly suppressed the migration of PANC-1 cells in real time. Mechanistically, 1 was found to inhibit the Akt/mTOR and autophagy activation pathway. Callistrilone L (1) and related meroterpenoids are promising leads for anticancer drug development based on the antiausterity strategy used in this work.

Discovery of potential antiausterity agents from the Japanese cypress Chamaecyparis obtusa.

The chloroform extract of the Japanese cypress Chamaecyparis obtusa was found to kill PANC-1 human pancreatic cancer cells preferentially in the nutrient-deprived medium without causing toxicity in the nutrient rich condition. Phytochemical investigation on this extract led to the isolation of a new sesquiterpene (1), together with the six sesquiterpenes (2-7) and a lignan (8). The isolated compounds were tested for their preferential cytotoxicity activity against five different human pancreatic cancer cell lines [PANC-1, MIA PaCa2, CAPAN-1, PSN-1, and KLM-1] by utilizing an antiausterity strategy. Among them, α-cadinol (2) was identified as the most active constituent. α-Cadinol (2) was found to inhibit the activation of Akt/mTOR pathway, and the hyperactivation of autophagy leading to preferential PANC-1 cell death during nutrient-starvation.

Highly oxygenated antiausterity agents from the leaves of Uvaria dac.

From the chloroform extract of the leaves of Uvaria dac, four new highly-oxygenated cyclohexene derivatives named uvaridacols I-L (1-4) were isolated together with nine previously reported compounds (5-13). Their structures were determined based on the extensive NMR spectroscopic data and circular dichroism spectroscopic analysis. Among the new compounds, uvaridacol L (4) displayed strong preferential cytotoxicity in the nutrient deprived medium against five different tested pancreatic cancer cell lines, PANC-1 (PC50, 20.1µM), PSN-1 (PC50, 9.7µM), MIA PaCa-2 (PC50, 29.1µM), Capan-1 (73.0µM) and KLM-1 (25.9µM).

A New Cassane-type Diterpene from the Seed of Caesalpinia sappan.

Phytochemical investigation of the CH2Cl2 extract of the Vietnamese medicinal plant Caesalpinia sappan Linn resulted in the isolation of a new cassane-type diterpene named tomocin I (1). Its chemical structure was determined by NMR spectroscopic and mass spectrometric analysis.

Evaluation of synthetic coumarins for antiausterity cytotoxicity against pancreatic cancers.

A series of functionalized coumarins were synthesized and evaluated for their capacity to inhibit the resistance to starvation of pancreatic cancer cells. This form of cytotoxicity, termed 'antiausterity' activity, was evaluated using a preferential cytotoxicity assay that compared cell survival in nutrient poor and nutrient rich conditions. Six of the seventeen compounds showed weak antiausterity activity against PANC-1. Compound 34 was active against PANC-1, MIA PaCa-2, and Capan-1 cancer cell lines. All of the compounds tested were simplified structural analogs of previously reported natural product leads. Six of the compounds, including 34, contain functionalized triazoles as novel potential bioisosteres of the side chain of the natural product angelmarin. Overall, the analogs were found to have low antiausterity activity relative to the corresponding natural products.

Cassane diterpenes from the seed kernels of Caesalpinia sappan.

Eight structurally diverse cassane diterpenes named tomocins A-H were isolated from the seed kernels of Vietnamese Caesalpinia sappan Linn. Their structures were determined by extensive NMR and CD spectroscopic analysis. Among the isolated compounds, tomocin A, phanginin A, F, and H exhibited mild preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived condition without causing toxicity in normal nutrient-rich conditions.

Attempt to synthesize 2,3,5,4'-tetrahydroxystilbene derived from 2,3,5,4'-tetrahydroxystilbene-2-O-ß-glucoside (THSG).

An attempt to synthesize aglycone 1 derived from 2,3,5,4'-tetrahydroxystilbene-2-O-ß-glucoside (THSG) via the Wittig reaction and Mizoroki-Heck reaction is described. In the Wittig protocol, 2,3,5,4'-tetramethoxystilbene 2 was obtained. Additionally, a palladium-catalyzed Mizoroki-Heck reaction strategy yielded 2-aryl-2,3-dihydrobenzofuran 13 instead of derivative 12 in good yield.

Geranyl dihydrochalcones from Artocarpus altilis and their antiausteric activity.

Human pancreatic cancer cell lines have 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 is a novel antiausterity strategy in anticancer drug discovery. In this study, the methanolic extract of the leaves of Artocarpus altilis showed 100 % preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions at a concentration of 50 µg/mL. Further investigation of this extract led to the isolation of eight new geranylated dihydrochalcones named sakenins A-H (1-8) together with four known compounds (9-12). Among them, sakenins F (6) and H (8) were identified as potent preferentially cytotoxic candidates with PC50 values of 8.0 µM and 11.1 µM, respectively.

(+)-Grandifloracin, an antiausterity agent, induces autophagic PANC-1 pancreatic cancer cell death.

Human pancreatic tumors are known to be highly resistant to nutrient starvation, and this prolongs their survival in the hypovascular (austere) tumor microenvironment. Agents that retard this tolerance to nutrient starvation represent a novel antiausterity strategy in anticancer drug discovery. (+)-Grandifloracin (GF), isolated from Uvaria dac, has shown preferential toxicity to PANC-1 human pancreatic cancer cells under nutrient starvation, with a PC50 value of 14.5 µM. However, the underlying mechanism is not clear. In this study, GF was found to preferentially induce PANC-1 cell death in a nutrient-deprived medium via hyperactivation of autophagy, as evidenced by a dramatic upregulation of microtubule-associated protein 1 light chain 3. No change was observed in expression of the caspase-3 and Bcl-2 apoptosis marker proteins. GF was also found to strongly inhibit the activation of Akt, a key regulator of cancer cell survival and proliferation. Because pancreatic tumors are highly resistant to current therapies that induce apoptosis, the alternative cell death mechanism exhibited by GF provides a novel therapeutic insight into antiausterity drug candidates.

Discovery of 2-pyridineformamide thiosemicarbazones as potent antiausterity agents.

Series of 2-pyridineformamide thiosemicarbazones were synthesized. Their preferential cytotoxicity in nutrient deprived medium (NDM) was evaluated using PANC-1 human pancreatic cancer cells by employing an antiausterity strategy. 2-Pyridineformamide thiosemicarbazones induced apoptosis and exhibited preferential cytotoxic activity toward PANC-1 cells in NDM, with potencies in the submicromolar range. These compounds are potential candidates for the development of therapeutics against pancreatic cancer.

Cleistanthane diterpenes from the seed of Caesalpinia sappan and their antiausterity activity against PANC-1 human pancreatic cancer cell line.

Three new cleistanthane diterpenes named tomocinon (1), tomocinol A (2), and tomocinol B (3), were isolated from the EtOAc extract of the seed of Caesalpinia sappan. Their structures were determined by extensive NMR spectroscopic analysis. The absolute stereochemistry of tomocinon (1) has been established by CD spectroscopic analysis. Cleistanthane diterpenes (1-3) represents the novel class of antiausterity agents having preferential cytotoxicity against PANC-1 human pancreatic cancer cell line under nutrient deprived condition with PC50 value of 34.7 µM, 42.4 µM and 39.4 µM, respectively.

Uvaridacols E-H, highly oxygenated antiausterity agents from Uvaria dac.

Chemical investigation of the stems of Uvaria dac yielded four new highly oxygenated cyclohexene derivatives named uvaridacols E-H (1-4). Their structures were established through NMR and circular dichroism spectroscopic analysis. Uvaridacols E (1), F (2), and H (4) displayed weak preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions in a concentration-dependent manner, without causing toxicity in normal nutrient-rich conditions.

Antiausterity agents from Uvaria dac and their preferential cytotoxic activity against human pancreatic cancer cell lines in a nutrient-deprived condition.

Human pancreatic cancer cell lines are known for their inherent tolerance to nutrition starvation, which enables them to survive under a hypovascular (austerity) tumor microenvironment. The search for agents that preferentially retard the survival of cancer cells under low nutrition conditions (antiausterity agent) is a novel approach to anticancer drug discovery. In this study, it was found that a dichloromethane extract of the stem of Uvaria dac preferentially inhibited PANC-1 human pancreatic cancer cells survival under nutrition-deprived conditions at a concentration of 10 µg/mL. Workup of this bioactive extract led to the discovery of (+)-grandifloracin (8) as a potent antiausterity agent as evaluated in a panel of four human pancreatic cancer cell lines, PANC-1 (PC(50), 14.5 µM), PSN-1 (PC(50), 32.6 µM), MIA PaCa-2 (PC(50), 17.5 µM), and KLM-1 (32.7 µM). (+)-Grandifloracin (8) has been isolated from a natural source for the first time. Its absolute stereochemistry was established by single-crystal X-ray crystallography and circular dichroism spectroscopic analysis. In addition to this, seven other new highly oxygenated cyclohexene derivatives, named uvaridacanes A (1) and B (2), uvaridacols A-D (3, 4, 6, 7), and uvaridapoxide A (5), were also isolated and structurally characterized.

Tyrosyl-DNA phosphodiesterase 1 inhibitor from an anamorphic fungus.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an enzyme that catalyzes hydrolysis of 3'-phosphotyrosyl bonds and is involved in repair of irreversible topoisomerase I (Top1)-DNA covalent complexes. Tdp1 inhibitors are regarded as potential cancer therapeutics in combination with Top1 inhibitors, which are currently used to treat human cancers. While screening for Tdp1 inhibitors, we discovered a novel compound, JBIR-21 (1), from the culture of an anamorphic fungus, RF-13305. The structure of 1 was established by extensive NMR and MS analyses. Compound 1 showed inhibitory activity against Tdp1 (IC(50) value, 18 µM) and cytotoxic activity against cancer cell lines (IC(50) values, 3.5-13 µM). Compound 1 also exhibited antitumor activity in a mouse xenograft model without adverse effects.

Induction of non-apoptotic cell death by Odontioda Marie Noel 'Velano' extracts in human oral squamous cell carcinoma cell lines.

BACKGROUND: We recently reported that the MeOH extract from bulbs of Odontioda Marie Noel 'Velano' exhibited diverse biological activities but most of the activity was concentrated into the EtOAc layer separated by sequential organic solvent extractions. In the present study, the EtOAc layer was subjected to silica-gel column chromatography for further separation into five fractions, and the cytotoxicity and apoptosis-inducing activity of the fractions against human normal oral and tumor cells was further investigated. MATERIALS AND METHODS: Cytotoxic activity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined by the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to the one of tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3/-7 activation was monitored by cleavage of substrates either spectrophotometrically or by western blot analysis. RESULTS: Among five fractions, the most hydrophobic fraction (Fr. 1) showed the highest cytotoxicity against all cell lines tested, followed by Fr. 2 >Fr. 3 >Fr. 4 >Fr. 5, in order of increasing polarity. Fr. 2 had the highest tumor-specificity, followed by Fr. 3, Fr. 4, Fr. 1 and Fr. 5. Fr. 1 induced caspase-3 activation more potently in promyelocytic leukemia HL-60 cells, than in oral squamous cell carcinoma (OSCC) HSC-2 cells, whereas it did not induce internucleosomal DNA fragmentation in either of these cell lines. CONCLUSION: The present study suggests that hydrophobic substances in the EtOAc extract of Odontioda Marie Noel 'Velano' exhibit tumor-specific cytotoxicity without inducing apoptosis in the HSC-2 OSCC cell line.

Induction of non-apoptotic cell death in human oral squamous cell carcinoma cell lines by Rhinacanthus nasutus extract.

BACKGROUND: We recently reported that the MeOH extract of aerial parts and root of Rhinacanthus nasutus showed diverse biological activity, with most activity being concentrated into the EtOAc layer separated by sequential organic solvent extractions. In the present study, the EtOAc extracts were further separated by silica-gel column chromatography into five fractions (Frs. 1-5), and their cytotoxicity and apoptosis-inducing activity investigated. MATERIALS AND METHODS: Cytotoxic activity was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined from the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to the one for tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis. Caspase-3/-7 activation was monitored by cleavage of substrates either spectrophotometrically or by western blot analysis. RESULTS: Among five fractions of the EtOAc extract, Fr. 1, eluted with CHCl(3)-MeOH (50:1), showed the highest tumor specificity (TS=3.3) as compared with other fractions eluted at higher concentrations of MeOH in CHCl(3) (TS=1.0-2.8). Fr. 1 did not induce internucleosomal DNA fragmentation or induced only marginal level of caspase-3 activity in either human promyelocytic leukemia HL-60 cells and human oral squamous cell carcinoma (OSCC) cell lines HSC-2. CONCLUSION: The present study suggests that hydrophobic substances of EtOAc extract show tumor specific cytotoxicity by inducing little or no apoptosis.

Analysis of the biological activity of a novel 24-membered macrolide JBIR-19 in Saccharomyces cerevisiae by the morphological imaging program CalMorph.

To investigate the biological activity of a novel 24-membered macrolide compound, JBIR-19, isolated from the culture broth of the entomopathogenic fungus Metarhizium sp. fE61, morphological changes in yeast cells were examined using the automated image-processing program CalMorph. Principal components analysis was used to elucidate dynamic changes in the phenotypes, revealing two independent effects of JBIR-19 in yeast cells: bud elongation and increased size of the actin region. Using a fitness assay, we identified the genes required for robust growth in the presence of JBIR-19. Among these were CCW12, YLR111W, and DHH1, which are also involved in abnormal bud morphology. Based on these results and others, we predict intracellular targets of JBIR-19 and its functional interactions.

New biological activity of Rhinacanthus nasutus extracts.

BACKGROUND: The MeOH extracts from aerial part and roots of Rhinacanthus nasutus were investigated for new biological activities. MATERIALS AND METHODS: The MeOH extract of the root was stepwise separated by organic solvents into n-hexane, EtOAc, n-BuOH and H(2)O layer fractions. Cytotoxic activity against human tumor and normal cells was determined by the MTT method. Nitric oxide (NO) was determined by the Griess method. Osteoclastogenesis was monitored by tartrate-resistant acid phosphatase (TRAP) activity. RESULTS: The MeOH extract of the root showed much higher tumor-specific cytotoxicity than that of the aerial part. The EtOAc fraction of the root showed the highest tumor-specific cytotoxicity, followed by the n-BuOH, n-hexane and H(2)O fractions. None of the four fractions protected the cells from the cytotoxicity of UV irradiation. The n-BuOH fraction not only stimulated NO production by mouse macrophage-like RAW264.7 cells, but also inhibited the lipopolysaccharide (LPS)-stimulated NO production. The EtOAc fraction inhibited the receptor activator for nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis of the RAW264.7 cells most potently, followed by the n-hexane, n-BuOH and H(2)O fractions. The n-BuOH fraction slightly, but significantly stimulated osteoclastogenesis. CONCLUSION: Antitumor and macrophage/osteoclast-modulating substances are enriched in EtOAc and n-BuOH fractions of MeOH extract of R. nasutus roots.

Diverse biological activity of Odontioda Marie Noel 'Velano' extracts.

BACKGROUND: Several pharmacologically active substances have been isolated from orchid plants other than Odontioda Marie Noel 'Velano'. Whether or not MeOH extract fractions from O. Marie Noel 'Velano' bulbs exert various biological activities was investigated. MATERIALS AND METHODS: The MeOH extract was stepwise separated by organic solvents into n-hexane, EtOAc, n-BuOH and H(2)O layer fractions. Cytotoxic activity against human tumor and normal cells was determined by the MTT method. Nitric oxide (NO) was determined by the Griess method. Osteoclastogenesis was monitored by tartrate-resistant acid phosphatase (TRAP) activity. RESULT: The EtOAc fraction showed the highest tumor-specific cytotoxicity, followed by the n-hexane and other fractions. The EtOAc and n-BuOH fractions protected the cells from the cytotoxicity induced by UV irradiation. The EtOAc and n-hexane fractions inhibited NO production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like cells. The EtOAc fraction most strongly inhibited the receptor activator for nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, followed by the n-BuOH, n-hexane and H(2)O fractions. CONCLUSION: Most of the biological activities tested were concentrated in the EtOAc fraction, and separation from cytotoxic substances is needed to identify the active principle(s).