Nadine Kretschmer

Naphthoquinones from Onosma paniculata Induce Cell-Cycle Arrest and Apoptosis in Melanoma Cells

Activity-guided fractionation of a petroleum ether-soluble extract of the roots of Onosma paniculata, which has been shown to affect the cell cycle and to induce apoptosis in melanoma cells, led to the isolation of several shikonin derivatives, namely, β-hydroxyisovalerylshikonin (1), acetylshikonin (2), dimethylacrylshikonin (3), and a mixture of α-methylbutyrylshikonin and isovalerylshikonin (4+5). All compounds exhibited strong cytotoxicity against eight cancer cell lines and MRC-5 lung fibroblasts, with 3 found to possess the most potent cytotoxicity toward four melanoma cell lines (SBcl2, WM35, WM9, and WM164). Furthermore, 3 and the mixture of 4+5 were found to interfere with cell-cycle progression in these cell lines and led to an increasing number of cells in the subG1 region as well as to caspase-3/7 activation, indicating apoptotic cell death.

Influence of seasonal variation on Thymus longicaulis C. Presl chemical composition and its antioxidant and anti-inflammatory properties.

Thymus longicaulis C. Presl. (Lamiaceae) is a small aromatic perennial herb typical of the Illyric-Mediterranean flora, traditionally used as remedy for cold, flu, cough, nephritis and abdominal pain. In order to carry out a thorough chemical and biological screening of the plant and to explore phenophases influence on its polyphenol content, samples of the plant were collected at different phases during its life cycle (July/October 2012 and January/April 2013). Each sample, previously extracted using a hydroalcoholic solution, was phytochemically analyzed for its metabolic constitution applying LC-DAD-ESI-MS/MS techniques. Although identified metabolites were differently concentrated at the various collection times, T. longicaulis leaf extracts were mainly constituted by low molecular weight phenols, and flavonoids. Rosmarinic acid was found as the main metabolite in Oct12 sample. Chemopreventive efficacy of the investigated extracts, by means of their anti-inflammatory, cytotoxic and antioxidant activities, was assessed. To this purpose, each extract underwent an extensive screening towards five human cell lines: CCRF-CEM (leukemia); U251 (glioblastoma); MDA-MB-231 (breast cancer); HCT-116 (colon cancer) and MRC-5 (lung fibroblasts) through XTT [2,3bis(2-metoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H tetrazolium hydroxide] test. The ability of the extracts to counteract cyclooxygenase-2 (COX-2) expression was also evaluated by COX-2 expression assay in human THP-1 monocyte-derived macrophages. COX-2 inhibition could represent a valuable anticancer strategy as it is associated with carcinogenesis and over-expressed in a variety of human malignancies. Oct12 extract, which was particularly rich in rosmarinic acid and methylapigenin, exhibited a strong antioxidant and anti-inflammatory effectiveness.

A petrol ether extract of the roots of Onosma paniculatum induces cell death in a caspase dependent manner

AIM OF THE STUDY Traditional Chinese medicine (TCM) has become very popular in Western countries during the last years. Zicao, a remedy of TCM, has been traditionally used to treat cancer, and, its main constituents, naphthoquinones, have been reported to possess antitumor activity (Chen et al., 2002; Papageorgiou et al., 1999). Here, we prepared extracts of different polarities of Onosma paniculatum Bur. & Franch., a plant which is amongst others used as Zicao, but, much less investigated. The extracts were analyzed concerning their growth inhibitory and apoptosis-inducing activity in various tumor cells. MATERIALS AND METHODS Cell viability was measured by XTT viability and a growth inhibition assay. Effects on the cell cycle and caspase-3 were determined by flow cytometry. RESULTS From three different extracts, a petrol ether extract showed significant growth inhibitory effect, cell cycle influence and caspase-3 dependent induction of apoptosis which was time and dose dependent. CONCLUSION To further determine the activity and mechanism of action of the petrol ether extract, we would like to isolate and identify the active principle and investigate the effects in more detail.

Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells.

Leukemia remains life-threatening despite remarkable advances in chemotherapy. The poor prognosis and drug resistance are challenging treatment. Novel drugs are urgently needed. Shikonin, a natural naphthoquinone, has been previously shown by us to be particularly effective towards various leukemia cell lines compared to solid tumors. However, the underlying mechanisms are still poorly understood. Here, we investigated shikonin and 14 derivatives on U937 leukemia cells. Four derivatives (isobutyrylshikonin, 2-methylbutyrylshikonin, isovalerylshikonin and β,β-dimethylacrylshikonin) were more active than shikonin. AnnexinV-PI analysis revealed that shikonins induced apoptosis. Cell cycle G1/S check point regulation and the transcription factor c-MYC, which plays a vital role in cell cycle regulation and proliferation, were identified as the most commonly down-regulated mechanisms upon treatment with shikonins in mRNA microarray hybridizations. Western blotting and DNA-binding assays confirmed the inhibition of c-MYC expression and transcriptional activity by shikonins. Reduction of c-MYC expression was closely associated with deregulated ERK, JNK MAPK and AKT activity, indicating their involvement in shikonin-triggered c-MYC inactivation. Molecular docking studies revealed that shikonin and its derivatives bind to the same DNA-binding domain of c-MYC as the known c-MYC inhibitors 10058-F4 and 10074-G5. This finding indicates that shikonins bind to c-MYC. The effect of shikonin on U937 cells was confirmed in other leukemia cell lines (Jurkat, Molt4, CCRF-CEM, and multidrug-resistant CEM/ADR5000), where shikonin also inhibited c-MYC expression and influenced phosphorylation of AKT, ERK1/2, and SAPK/JNK. In summary, inhibition of c-MYC and related pathways represents a novel mechanism of shikonin and its derivatives to explain their anti-leukemic activity.

Effect of costunolide and dehydrocostus lactone on cell cycle, apoptosis, and ABC transporter expression in human soft tissue sarcoma cells.

Human soft tissue sarcomas represent a rare group of malignant tumours that frequently exhibit chemotherapeutic resistance and increased metastatic potential following unsuccessful treatment. In this study, we investigated the effects of costunolide and dehydrocostus lactone, which have been isolated from Saussurea lappa using activity-guided isolation, on three soft tissue sarcoma cell lines of various origins. The effects on cell proliferation, cell cycle distribution, apoptosis induction, and ABC transporter expression were analysed. Both compounds inhibited cell viability dose- and time-dependently. IC50 values ranged from 6.2 µg/mL to 9.8 µg/mL. Cells treated with costunolide showed no changes in cell cycle, little in caspase 3/7 activity, and low levels of cleaved caspase-3 after 24 and 48 h. Dehydrocostus lactone caused a significant reduction of cells in the G1 phase and an increase of cells in the S and G2/M phase. Moreover, it led to enhanced caspase 3/7 activity, cleaved caspase-3, and cleaved PARP indicating apoptosis induction. In addition, the influence of costunolide and dehydrocostus lactone on the expression of ATP binding cassette transporters related to multidrug resistance (ABCB1/MDR1, ABCC1/MRP1, and ABCG2/BCRP1) was examined using real-time RT-PCR. The expressions of ABCB1/MDR1 and ABCG2/BCRP1 in liposarcoma and synovial sarcoma cells were significantly downregulated by dehydrocostus lactone. Our data demonstrate for the first time that dehydrocostus lactone affects cell viability, cell cycle distribution and ABC transporter expression in soft tissue sarcoma cell lines. Furthermore, it led to caspase 3/7 activity as well as caspase-3 and PARP cleavage, which are indicators of apoptosis. Therefore, this compound may be a promising lead candidate for the development of therapeutic agents against drug-resistant tumours.

Sesquiterpene Lactones Downregulate G2/M Cell Cycle Regulator Proteins and Affect the Invasive Potential of Human Soft Tissue Sarcoma Cells

Soft tissue sarcomas (STS) represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. Many studies have demonstrated the great potential of plant-derived agents in the treatment of various malignant entities. The present study investigates the effects of the sesquiterpene lactones costunolide and dehydrocostus lactone on cell cycle, MMP expression, and invasive potential of three human STS cell lines of various origins. Both compounds reduced cell proliferation in a time- and dose-dependent manner. Dehydrocostus lactone significantly inhibited cell proliferation, arrested the cells at the G2/M interface and caused a decrease in the expression of the cyclin-dependent kinase CDK2 and the cyclin-dependent kinase inhibitor p27Kip1. In addition, accumulation of cells at the G2/M phase transition interface resulted in a significant decrease in cdc2 (CDK1) together with cyclin B1. Costunolide had no effect on the cell cycle. Based on the fact that STS tend to form daughter cell nests and metastasize, the expression levels of matrix metalloproteinases (MMPs), which play a crucial role in extracellular matrix degradation and metastasis, were investigated by Luminex® technology and real-time RT-PCR. In the presence of costunolide, MMP-2 and -9 levels were significantly increased in SW-982 and TE-671 cells. Dehydrocostus lactone treatment significantly reduced MMP-2 and -9 expression in TE-671 cells, but increased MMP-9 level in SW-982 cells. In addition, the invasion potential was significantly reduced after treatment with both sesquiterpene lactones as investigated by the HTS FluoroBlock™ insert system.

Shikonin and its derivatives inhibit the epidermal growth factor receptor signaling and synergistically kill glioblastoma cells in combination with erlotinib

Overexpression and mutation of the epidermal growth factor receptor (EGFR) gene play a causal role in tumorigenesis and resistance to treatment of glioblastoma (GBM). EGFR inhibitors such as erlotinib are currently used for the treatment of GBM; however, their efficacy has been limited due to drug resistance. New treatment strategies are therefore urgently needed. Shikonin, a natural naphthoquinone, induces both apoptosis and necroptosis in human glioma cells, but the effectiveness of erlotinib‐shikonin combination treatment as well as the underlying molecular mechanisms is unknown yet. In this study, we investigated erlotinib in combination with shikonin and 14 shikonin derivatives in parental U87MG and transfected U87MG.ΔEGFR GBM cells. Most of the shikonin derivatives revealed strong cytotoxicity. Shikonin together with five other derivatives, namely deoxyshikonin, isobutyrylshikonin, acetylshikonin, β,β‐dimethylacrylshikonin and acetylalkannin showed synergistic cytotoxicity toward U87MG.ΔEGFR in combination with erlotinib. Moreover, the combined cytotoxic effect of shikonin and erlotinib was further confirmed with another three EGFR‐expressing cell lines, BS153, A431 and DK‐MG. Shikonin not only dose‐dependently inhibited EGFR phosphorylation and decreased phosphorylation of EGFR downstream molecules, including AKT, P44/42MAPK and PLCγ1, but also together with erlotinib synergistically inhibited ΔEGFR phosphorylation in U87MG.ΔEGFR cells as determined by Loewe additivity and Bliss independence drug interaction models. These results suggest that the combination of erlotinib with shikonin or its derivatives might be a potential strategy to overcome drug resistance to erlotinib.

Influence of harvest season on chemical composition and bioactivity of wild rue plant hydroalcoholic extracts.

The rue (Ruta graveolens) copiousness in rural areas of the Campania Region based a thorough chemical and biological investigation aimed at exploring the seasonal variability of phenol constituents in rue leaves and its influence on their antioxidant, cytotoxic and anti-inflammatory capabilities. To this purpose, hydroalcoholic extracts were prepared from plant samples seasonally collected. LC-ESI-MS/MS techniques were employed to analyze qualitatively and quantitatively the seasonal rue phenol content, whereas different chemical antioxidant assays (by DPPH, ABTS, Fe(3+) RP, ORAC, and FCR methods) and XTT redox metabolic activity assay were performed to screen the seasonal phenol complex-related antioxidant and cytotoxic power. The ability of the rue leaf extracts to counteract cyclooxygenase-2 (COX-2) expression was also evaluated. Data obtained highlighted that the adopted extraction procedure markedly pauperized the furanocoumarin content in all the prepared rue extracts. Flavonol glycosides, along with the flavone acacetin and two sinapic acid derivatives were the main constituents of the spring harvest-derived extract, which exerted the highest antioxidant capability in cell-free systems and was capable to inhibit COX-2 synthesis by 44% comparably to dexamethasone, used as positive control. Data provide new insights for developing a proper management of rue plants for new safe industrial purposes in herbal medicine field.

Synthesis of Tetrahydrohonokiol Derivates and Their Evaluation for Cytotoxic Activity against CCRF-CEM Leukemia, U251 Glioblastoma and HCT-116 Colon Cancer Cells

Biphenyl neolignans such as honokiol and magnolol, which are the major active constituents of the Asian medicinal plant Magnolia officinalis, are known to exert a multitude of pharmacological and biological activities. Among these, cytotoxic and tumor growth inhibitory activity against various tumour cell lines are well-documented. To further elucidate the cytotoxic effects of honokiol derivatives, derivatizations were performed using tetrahydrohonokiol as a scaffold. The derivatizations comprised the introduction of functional groups, e.g., nitro and amino groups, as well as alkylation. This way, 18 derivatives, of which 13 were previously undescribed compounds, were evaluated against CCRF-CEM leukemia cells, U251 glioblastoma and HCT-116 colon cancer cells. The results revealed no significant cytotoxic effects in any of the three tested cell lines at a test concentration of 10 µM.

25-O-acetyl-23,24-dihydro-cucurbitacin F induces cell cycle G2/M arrest and apoptosis in human soft tissue sarcoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE Quisqualis indica is used in traditional Chinese medicine to treat cancer and related syndromes and also known for its anthelminthic effects. AIM OF THE STUDY Soft tissue sarcomas represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. In this study, we evaluated the cytotoxic, apoptosis inducing and cell cycle arresting effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F which has been isolated from leaves and twigs of Q. indica. MATERIAL AND METHODS The present study investigates the effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F (1) on cell viability, cell cycle distribution, and apoptotic induction of three human sarcoma cell lines of various origins by using the CellTiter 96(®) AQueous One Solution Cell Proliferation Assay, flow cytometrical experiments, real-time RT-PCR, Western blotting, and the Caspase-Glo(®) 3/7 Assay RESULTS We could show that 1 reduced cell viability in a dose-dependent manner and arrested the cells at the G2/M interface. The accumulation of cells at the G2/M phase resulted in a significant decrease of the cell cycle checkpoint regulators cyclin B1, cyclin A, CDK1, and CDK2. Interestingly, 1 inhibited survivin expression significantly, which functions as a key regulator of mitosis and programmed cell death, and is overexpressed in many tumor types including sarcomas. Moreover, 1 induced apoptosis in liposarcoma and rhabdomyosarcoma cells caspase-3 dependently. CONCLUSION Our data strongly support 1 as a very interesting target for further investigation and development of novel therapeutics in sarcoma research.