Salvadora persica leaves: phytochemical profile and in vitro-inhibitory activity on inflammatory mediators implicated in periodontal disease.

CONTEXT: Virtually all parts of Salvadora persica L. (Salvadoraceae) are used in traditional medicine. The twigs and leaves are used for oral health, but leaves are far less investigated. OBJECTIVE: This study assesses the oral health-promoting potential of S. persica leaves with emphasis on anti-inflammatory and antiproliferative effects and provides an in depth-characterization of their metabolite profile. MATERIALS AND METHODS: Hot-water and methanolic S. persica leaf extracts (1, 10, and 100 µg/mL) and their major constituents (5, 10, and 50 µM), were subjected to cellular assays on IL-8 and TNFα release in LPS-stimulated human neutrophils, NO-release in LPS/IFNγ stimulated mouse macrophages, and proliferation of HNO97 human tongue carcinoma cells. Metabolite profiling was performed by UHPLC-HRMS analysis. Major constituents were isolated and structurally elucidated. RESULTS AND DISCUSSION: Both extracts showed pronounced anti-inflammatory activity in LPS-stimulated neutrophils. Major identified compound classes were flavonoid glycosides, the glucosinolate glucotropaeolin, phenyl- and benzylglycoside sulfates, and megastigmane glycosylsulfates, the latter ones identified for the first time in S. persica. Glucotropaeolin strongly inhibited the release of IL-8 and TNF-α (13.3 ± 2.0 and 22.7 ± 2.6% of the release of stimulated control cells at 50 µM), while some flavonoids and 3-(3'-O-sulfo-ß-d-glucopyranosyloxy)-7,8-dihydro-ß-ionone, a newly isolated megastigmane glycosylsulfate, were moderately active. Benzylisothiocyanate, which is likely formed from glucotropaeolin during traditional application of S. persica, showed considerable antiproliferative activity (IC50 in HNO97 cells: 10.19 ± 0.72 µM) besides strongly inhibiting IL-8 and TNFα release. CONCLUSIONS: Glucotropaeolin and benzylisothiocyanate are likely implicated in the oral health-promoting effects of S. persica leaves. The chemistry and pharmacology of the newly identified megastigmane glycosylsulfates should be further evaluated.

The Biological Assessment of Shikonin and ß,ß-dimethylacrylshikonin Using a Cellular Myxofibrosarcoma Tumor Heterogeneity Model.

Myxofibrosarcoma (MFS) is a subtype of soft tissue sarcoma of connective tissue, which is characterized by large intra-tumor heterogeneity. Therapy includes surgical resection. Additional chemotherapy is of limited effect. In this study, we demonstrated the potent anticancer activity of shikonin derivatives in our MFS cellular model of tumor heterogeneity for developing a new therapeutic approach. The impact of shikonin and ß,ß-dimethylacrylshikonin (DMAS) on viability, apoptotic induction, MAPK phosphorylation, and DNA damage response were analyzed by means of two human MFS cell lines, MUG-Myx2a and MUG-Myx2b, derived from a singular tumor tissue specimen. MFS cells showed a dose-dependent inhibition of cell viability and a significant induction of apoptosis. Treatment with shikonin derivatives caused an inhibition of pSTAT3 and an increase in pAKT, pERK, pJNK, and pp38. DMAS and shikonin inhibited the activation of the two master upstream regulators of the DNA damage response, ATR and ATM. MUG-Myx2b, which contains an additional PTEN mutation, was more sensitive in some targets. These data demonstrate the significant antitumorigenic effect of shikonin derivatives in MFS and highlight the importance of intra-tumor heterogeneity in treatment planning.

Cytotoxicity of Carvotacetones from Sphaeranthus africanus Against Cancer Cells and Their Potential to Induce Apoptosis.

Three carvotacetones (1 - 3: ) isolated from Sphaeranthus africanus were screened in 60 cancer cell lines at the National Cancer Institute (NCI) within the Developmental Therapeutics Program (DTP). At the concentration of 10-5 M, compound 1: (3,5-diangeloyloxy-7-hydroxycarvotacetone) turned out to be the most active compound against ACHN and UO-31 renal cancer cell lines with growth percent values of - 100% (all cells dead). Compound 2: (3-angeloyloxy-5-[2″,3″-epoxy-2″-methylbutanoyloxy]-7-hydroxycarvotacetone) showed strong effects in SK-MEL-5 melanoma and ACHN renal cancer cells with inhibition values of 93% and 97%, respectively. Compound 3: (3-angeloyloxy-5-[3″-chloro-2″-hydroxy-2″-methylbutanoyloxy]-7-hydroxy-carvotacetone) exhibited a quite strong effect on renal cancer cells with a growth inhibitory effect of 96% against ACHN and UO-31 cells. When treated with five different concentrations of 1: (1 × 10-8, 1 × 10-7, 1 × 10-6, 1 × 10-5, and 1 × 10-4 M), HOP-92 cells were found to be most sensitive with GI50, TGI, and LC50 values of 0.17, 0.40, and 0.96 µM, respectively. When using the ApoTox-Glo triplex assay to evaluate the apoptosis inducing effects of seven carvotacetones isolated from S. africanus in CCRF-CEM cells, compounds 1:  - 6: increased caspase-3/7 activity with 1, 2: , and 4: (3-angeloyloxy-5,7-dihydroxycarvotacetone) exhibiting the highest activitiy, indicating induction of caspase-dependent apoptosis.

Discrimination of Zicao Samples Based on DNA Barcoding and HPTLC Fingerprints, and Identification of (22E)-Ergosta-4,6,8(14),22-tetraen-3-one As a Marker Compound.

The unambiguous identification of plant material is a prerequisite of rational phytotherapy. Misidentification can even cause serious health problems, as in the case of the Chinese medicinal herb Zicao. Commercial material labelled "Zicao" may be derived from the roots of Arnebia euchroma (ruan zicao), Lithospermum erythrorhizon (ying zicao), or Onosma paniculata (dian zicao). All of these roots contain shikonin derivatives as main bioactive constituents, but ying zicao and dian zicao contain also hepatotoxic pyrrolizidine alkaloids in high amounts. Therefore, the use of A. euchroma with a very low pyrrolizidine alkaloid content is desirable. Confusions of the species occur quite often, indicating an urgent need for an unambiguous identification method. Discrimination of 23 zicao samples has been achieved by analyses of the nuclear internal transcribed spacer ITS2 and trnL-F intergenic spacer of the chloroplast DNA. Data were analyzed using Bioedit, ClustalX, Mega 11 and BLAST. Results indicate that ITS2 barcoding can accurately distinguish Arnebia euchroma from their adulterants. Subsequently, an HPTLC method has been developed allowing a chemical discrimination of the most widely used species. (22E)-Ergosta-4,6,8(14),22-tetraen-3-one has been identified as characteristic marker compound, allowing an unambiguous discrimination of A. euchroma and L. erythrorhizon.

Shikonin derivatives cause apoptosis and cell cycle arrest in human chondrosarcoma cells via death receptors and MAPK regulation.

BACKGROUND: Although chondrosarcoma is the second most common primary malignant bone tumor, treatment options are limited due to its extensive resistance to a chemo- and radiation therapy. Since shikonin has shown potent anticancer activity in various types of cancer cells, it represents a promising compound for the development of a new therapeutic approach. METHODS: The dose-relationships of shikonin and its derivatives acetylshikonin and cyclopropylshikonin on two human chondrosarcoma cell lines were measured using the CellTiter-Glo®. The changes in the cell cycle were presented by flow cytometry. Protein phosphorylation and expression apoptotic markers, MAPKs and their downstream targets were analyzed using western blotting and gene expression were evaluated using RT-qPCR. RESULTS: Chondrosarcoma cells showed a dose-dependent inhibition of cell viability after treatment with shikonin and its derivatives, with the strongest effect for shikonin and IC50 values of 1.3 ± 0.2 µM. Flow cytometric measurements revealed a G2/M arrest of the cells after treatment. Protein and gene expression analysis demonstrated a dose-dependent downregulation of survivin and XIAP, and an upregulation of Noxa, γH2AX, cleaved caspase-8, -9, -3, and -PARP. Furthermore, the expression of various death receptors was modulated. As MAPK signaling pathways play a key role in tumor biology, their phosphorylation pattern and their corresponding downstream gene regulation were analyzed. Treatment with shikonin derivatives caused an inhibition of pSTAT3 and an increase of pAKT and the MAPKs pERK, pJNK, and pp38 in a dose-dependent manner. CONCLUSIONS: These data demonstrated the significant anti-tumorigenic effect of shikonin derivatives in chondrosarcoma and encourage further research.

SK119, a Novel Shikonin Derivative, Leads to Apoptosis in Melanoma Cell Lines and Exhibits Synergistic Effects with Vemurafenib and Cobimetinib.

Melanoma is a complex and heterogenous disease, displays the deadliest form of skin cancer, and accounts for approx. 80% of all skin cancer deaths. In this study, we reported on the synthesis and pharmacological effects of a novel shikonin derivative (SK119), which is active in a nano-molar range and exhibits several promising in vitro effects in different human melanoma cells. SK119 was synthesized from shikonin as part of our search for novel, promising shikonin derivatives. It was screened against a panel of melanoma and non-tumorigenic cell lines using XTT viability assays. Moreover, we studied its pharmacological effects using apoptosis and Western blot experiments. Finally, it was combined with current clinically used melanoma therapeutics. SK119 exhibited IC50 values in a nano-molar range, induced apoptosis and led to a dose-dependent increase in the expression and protein phosphorylation of HSP27 and HSP90 in WM9 and MUG-Mel 2 cells. Combinatorial treatment, which is highly recommended in melanoma, revealed the synergistic effects of SK119 with vemurafenib and cobimetinib. SK119 treatment changed the expression levels of apoptosis genes and death receptor expression and exhibited synergistic effects with vemurafenib and cobimetinib in human melanoma cells. Further research indicates a promising potential in melanoma therapy.

Structural Diversity of Complex Phloroglucinol Derivatives from Eucalyptus Species.

Several species of the genus Eucalyptus are used in many traditional medicine systems for the treatment of respiratory tract infections, colds, flu, sore throats, and bronchitis. The genus Eucalyptus (Myrtaceae) is a well-known natural source of bioactive phloroglucinols. These polyphenolic compounds bear an aromatic phenyl ring with three hydroxy groups (1,3,5-trihydroxybenzene) which have been exhibiting a variety of biological activities such as antimicrobial, anticancer, anti-allergic, anti-inflammatory, and antioxidant activities. This review summarizes the literature published from 1997 until the end of 2021 and addresses the structure diversity of phloroglucinols isolated from Eucalyptus species and their biological activities. Phloroglucinol-terpene adducts are the main class of compounds that have been reported in this genus.

Shikonin Derivatives Inhibit Inflammation Processes and Modulate MAPK Signaling in Human Healthy and Osteoarthritis Chondrocytes.

Osteoarthritis (OA) is the most common joint disorder and is characterized by the degeneration of articular cartilage. To develop new therapeutic approaches, we investigated the effect of shikonin derivatives on inflammation, MMP expression, and the regulation of MAPK signaling in human healthy (HC) and OA chondrocytes (pCH-OA). Viability was analyzed using the CellTiter-Glo® Assay. Inflammatory processes were investigated using a proteome profiler™ assay. Furthermore, we analyzed the effects of the shikonin derivatives by protein expression analysis of the phosphorylation pattern and the corresponding downstream gene regulation using RT-qPCR. Both HC and pCH-OA showed a dose-dependent decrease in viability after treatment. The strongest effects were found for shikonin with IC50 values of 1.2 ± 0.1 µM. Shikonin counteracts the inflammatory response by massively reducing the expression of the pro-inflammatory mediators. The phosphorylation level of ERK changed slightly. pJNK and pp38 showed a significant increase, and the downstream targets c/EBPs and MEF2c may play a role in the cartilage homeostasis. STAT3 phosphorylation decreased significantly and has a chondroprotective function through the regulation of cyclin D1 and Sox9. Our results demonstrate for the first time that shikonin derivatives have extensive effects on the inflammatory processes, MAPKs, and IL6/STAT3 downstream regulation in healthy and OA chondrocytes.

MUG Mel3 Cell Lines Reflect Heterogeneity in Melanoma and Represent a Robust Model for Melanoma in Pregnancy.

Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options-especially during pregnancy.

Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives.

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGloTM Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma.

Preparation of new 1,3-dibenzyl tetrahydropyridinylidene ammonium salts and their antimicrobial and anticellular activities.

New 1,3 dibenzyl -tetrahydropyridinylidene ammonium salts have been prepared from unsubstituted or N-benzylated tetrahydropyridinylidene ammonium salts. The antiplasmodial and antitrypanosomal activities as well as their cytotoxic effects were determined using microplate assays. In addition, their activities against two gram positive and two gram negative bacteria strains and a yeast strain were examined. Furthermore, anticancer effects against two cell lines were investigated. Physicochemical parameters were calculated and structure-activity-relationships discussed. One compound showed antiplasmodial activity against a multiresistant strain of Plasmodium falciparum in subnanomolar concentration. Antitrypanosomal activities were detected in low nanomolar concentrations. A single compound was active against grampositive and gramnegative bacteria, as well as yeast. One compound inhibited the growth of a HCT cell line in low concentration.

Naphthoquinone Derivatives Isolated from Plants: Recent Advances in Biological Activity.

Naturally occurring naphthoquinones (NQs) comprising highly reactive small molecules are the subject of increasing attention due to their promising biological activities such as antioxidant, antimicrobial, apoptosis-inducing activities, and especially anticancer activity. Lapachol, lapachone, and napabucasin belong to the NQs and are in phase II clinical trials for the treatment of many cancers. This review aims to provide a comprehensive and updated overview on the biological activities of several new NQs isolated from different species of plants reported from January 2013 to January 2020, their potential therapeutic applications and their clinical significance.

Periplocin mediates TRAIL-induced apoptosis and cell cycle arrest in human myxofibrosarcoma cells via the ERK/p38/JNK pathway.

BACKGROUND: Periploca sepium is traditionally used in Chinese medicine to treat particularly rheumatic disorders and as a tonic. Periplocin was found as the most cytotoxic compound of its root bark and induced death receptor mediated apoptosis in liposarcoma cells. Sarcomas are a rare type of cancer with only a few treatment options. The five-year survival rate of advanced tumors is low. PURPOSE: In this study, we investigated the effects of periplocin in two myxofibrosarcoma (MFS)cell lines, MUG-Myx2a and MUG-Myx2b, which are subclones of the same tumor and reflect the tumor´s heterogeneity, and in T60 primary myxofibrosarcoma cells. METHODS: The xCELLigence system and the CellTiter 96® AQueous assay were used for studying cell viability. FACS and Western blot experiments were used to investigate the effects of periplocin on apoptosis induction, cell cycle distribution, and the expression of cleaved PARP, caspase 3, p53, phospho-histone γH2AX, ERK/phospho ERK, p38/phospho p38, and, finally, JNK/phospho JNK. Additionally, the expression of the apoptotic markers Bim, NOXA, Bak, Bcl-2, Bcl-xl, and the death receptors IGFR, FADD, TRADD, TNFR1A, TRAIL-R1, and TRAIL-R2 were evaluated using reversed real-time PCR. RESULTS: Periplocin decreased dose-dependently the viability of all MFS cell lines and was more effective than the standard chemotherapeutic doxorubicin. It arrested the cells in the G2/M phase and led to caspase activation. Moreover, periplocin increased the mRNA expression of NOXA, Bak, Bcl-2, and death receptors such as TRAIL-R1 and TRAIL-R2 and the protein expression of ERK/phospho ERK, p38/phospho p38, and JNK/phospho JNK. In all cases, differences in the effects in the different subclones were observed. CONCLUSION: Periplocin showed promising effects in MFS cells. The higher effectiveness compared to doxorubicin is an important aspect for further research with regard as a treatment option. The different effects of periplocin in the two subclones showed the great importance of intratumoral heterogeneity in MFS therapy.

ß,ß-Dimethylacrylshikonin Induces Apoptosis in Melanoma Cell Lines by NOXA Upregulation.

Melanoma is the most aggressive form of skin cancer, with high metastasis rates and poor prognosis. Survival rates and possible therapies depend on the state of the tumor and its mutational profile. BRAF and NRAS are the most frequent driver mutations. Currently, there is no efficient therapy for NRAS-mutated or late-stage melanoma. In this study, the therapeutic potential of ß,ß-dimethylacrylshikonin (DMAS) was investigated on melanoma. The influence of DMAS was determined in five different melanoma cell lines with different mutational profiles. The effects of this compound on cell viability, apoptosis, and gene and protein expression were examined. The results obtained were validated in vivo. DMAS significantly reduced the viability of several melanoma cell lines in a concentration- and time-dependent manner. Furthermore, DMAS induced caspase-3-dependent apoptosis via NOXA upregulation, as confirmed by NOXA knockdown experiments. This is the first time that NOXA-dependent apoptosis was shown with respect to a shikonin derivative and melanoma. Additionally, tumor regression and necrosis under DMAS treatment were demonstrated in vivo. Importantly, BRAF as well as NRAS-mutated metastatic human melanoma cell lines were treated successfully in vitro and in vivo. Taken together, DMAS showed promising results and is worthy of further study.

C13 Megastigmane Derivatives From Epipremnum pinnatum: ß-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-κB Signaling.

In order to identify active constituents and to gain some information regarding their mode of action, extracts from leaves of Epipremnum pinnatum were tested for their ability to inhibit inflammatory gene expression in endothelial- and monocyte-like cells (HUVECtert and THP-1, respectively). Bioactivity-guided fractionation using expression of PTGS2 (COX-2) mRNA as a readout resulted in the isolation of two C13 megastigmane glycosides, gusanlungionoside C (1) and citroside A (3), and the phenylalcohol glycoside phenylmethyl-2-O-(6-O-rhamnosyl)-ß-D-galactopyranoside (2). Further analysis identified six additional megastigmane glycosides and the aglycones ß-damascenone (10), megastigmatrienone (11), 3-hydroxy-ß-damascenone (12), and 3-oxo-7,8-dihydro-α-ionol (13). Pharmacological analysis demonstrated that 10 inhibits LPS-stimulated induction of mRNAs encoding for proinflammatory cytokines and leukocyte adhesion molecules, such as TNF-α, IL-1ß, IL-8, COX-2, E-selectin, ICAM-1, and VCAM-1 in HUVECtert and THP-1 cells. 10 inhibited induction of inflammatory genes in HUVECtert and THP-1 cells treated with different agonists, such as TNF-α, IL-1ß, and LPS. In addition to mRNA, also the upregulation of inflammatory proteins was inhibited by 10 as demonstrated by immune assays for cell surface E-selectin and secreted TNF-α. Finally, using a luciferase reporter construct, it was shown, that 10 inhibits NF-κB-dependent transcription. Therefore, we hypothesize that inhibition of NF-κB by ß-damascenone (10) may represent one of the mechanisms underlying the in vitro anti-inflammatory activity of Epipremnum pinnatum extracts.

Anti-inflammatory and antiproliferative compounds from Sphaeranthus africanus.

BACKGROUND: Sphaeranthus africanus has been used in traditional Vietnamese medicine to treat sore throat, and to relieve pain and swelling. However, the anti-inflammatory activity of this plant had not yet been investigated. Previously, we isolated five carvotacetones (1-5) from this plant that displayed cytotoxicity against several cancer cell lines. PURPOSE: The objective of this study was to isolate further constituents from S. africanus and to investigate the anti-inflammatory activity of all constituents. Furthermore, the anti-proliferative activity of the newly isolated compounds was evaluated. STUDY DESIGN AND METHODS: Compounds were isolated from the upper parts of S. africanus by chromatographic methods. Structures were determined using spectroscopic techniques, like NMR and MS. All nine compounds isolated from S. africanus were evaluated for inhibitory activity against COX-1 and COX-2 isoenzymes in-vitro, COX-2 mRNA expression and influence on NO production. The anti-proliferative activities of newly isolated compounds (6-9) were evaluated by XTT viability assay with four cancer cell lines, namely CCRF-CEM, MDA-MB-231, HCT-116, and U-251 cells. RESULTS: Two diastereomeric carvotacetones (3-angeloyloxy-5-[2″S,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (6) and 3-angeloyloxy-5-[2″R,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (7), asperglaucide (8) and chrysoplenol D (9) were isolated from S. africanus. COX-1 and COX-2 assays of compounds 1-9 revealed that compounds 1 and 2 possess potent and selective COX-2 inhibitory activity with IC50 values of 3.6 and 0.5 µM, respectively. COX-2 gene expression assay showed that some carvotacetones exhibited inhibitory effects on COX-2 gene expression in THP-1 macrophages. Compound 4 is the most active compound inhibiting the synthesis of COX-2 by 55% at 2.06 µM. In the iNOS assay, all seven carvotacetones inhibited NO production in BV2 and RAW cell lines with IC50 values ranging from 0.2 to 2.9 µM. Compound 4 showed potent inhibitory activity with IC50 values of 0.2 µM in both BV2 and RAW cell lines. Molecular docking studies revealed the binding orientations of 1 and 2 in the active sites of COX-2. XTT assay of the newly isolated compounds revealed that the two isomeric carvotacetones (6-7) exhibited considerable anti-proliferative activity against four cancer cell lines (CCRF-CEM, MDA-MB-231, HCT-116, U-251) with IC50 values ranging from 1.23 to 8 µM. CONCLUSION: For the first-time, the diastereomeric carvotacetones (6-7) were isolated as separate compounds, and their anti-proliferative activity was determined. Selective COX-2 inhibitory, COX-2 mRNA expression and NO production inhibitory activities by some of the major constituents of S. africanus supports the traditional medical application of this plant for the treatment of inflammation-related disorders.

Synthesis of Jacaranone-Derived Nitrogenous Cyclohexadienones and Their Antiproliferative and Antiprotozoal Activities.

The cytotoxic and antiprotozoal activities of the phytoquinoide, jacaranone, and related compounds have been an ongoing topic in recent drug discovery. Starting from the natural product-derived cyclohexadienone scaffold, a series of nitrogen-containing derivatives were synthesized and subsequently evaluated for their antiproliferative and antiprotozoal activity. Anticancer potency was analyzed using different types of cancer cell lines: MDA-MB-231 breast cancer, CCRF-CEM leukemia, HCT-116 colon cancer, U251 glioblastoma, and, in addition, non-tumorigenic MRC-5 lung fibroblasts. Antiproliferative activities at micromolar concentrations could be shown. Antiprotozoal activity was assessed against Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense STIB900. For all compounds, selectivity indices (SI) were calculated based on assessed cytotoxicity towards L6 cells. In addition, the structure-activity-relationships and physicochemical parameters of these compounds are discussed.

Periplocin, the most anti-proliferative constituent of Periploca sepium, specifically kills liposarcoma cells by death receptor mediated apoptosis.

BACKGROUND: During a screening of Chinese plants traditionally used for the treatment of cancer and related diseases, extracts of the root bark of Periploca sepium Bunge showed strong cytotoxic activity. PURPOSE: Isolate and identify cytotoxic compounds from P. sepium and investigate the effects and mechanism of action on different cancer cell lines. METHODS: Extracts obtained with solvents of different polarities of the root bark of P. sepium were tested for their anti-proliferative effects. The most active extract was subjected to activity-guided fractionation using different chromatographic methods. The most active compound was further investigated on sarcoma cell lines regarding its effects concerning apoptosis, DNA damage and death receptor expression. RESULTS: We isolated the cardiac glycosides periplocin, glucosyl divostroside, periplogenin, periplocymarin and periplocoside M with periplocin exhibiting the lowest IC50 value against leukemia and liposarcoma cells. Liposarcomas are rare tumors within the heterogeneous group of soft tissue sarcomas and respond poorly to conventional treatments. Periplocin led to growth inhibition and apoptosis induction by changing the expression of death receptors and inducing DNA double strand breaks in SW-872 cells. CONCLUSION: Periplocin displays a promising mechanism of action in sarcoma cells because altering the death receptor expression is an interesting target in sarcoma treatment especially to overcome TRAIL resistance.

Synthesis of Novel Shikonin Derivatives and Pharmacological Effects of Cyclopropylacetylshikonin on Melanoma Cells.

Despite much research in the last centuries, treatment of malignant melanoma is still challenging because of its mostly unnoticeable metastatic spreading and aggressive growth rate. Therefore, the discovery of novel drug leads is an important goal. In a previous study, we have isolated several shikonin derivatives from the roots of Onosma paniculata Bureau & Franchet (Boraginaceae) which evolved as promising anticancer candidates. ß,ß-Dimethylacrylshikonin (1) was the most cytotoxic derivative and exhibited strong tumor growth inhibitory activity, in particular, towards melanoma cells. In this study, we synthesized eighteen novel shikonin derivatives in order to obtain compounds which exhibit a higher cytotoxicity than 1. We investigated their cytotoxic potential against various melanoma cell lines and juvenile skin fibroblasts. The most active compound was (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopropylacetate (cyclopropylacetylshikonin) (6). It revealed significant stronger tumor growth inhibitory activity towards two melanoma cell lines derived from metastatic lesions (WM164 and MUG-Mel2). Further investigations have shown that 6 induced apoptosis caspase-dependently, increased the protein levels of cleaved PARP, and led to double-stranded DNA breaks as shown by phosphorylation of H2AX. Cell membrane damage and cell cycle arrest were not observed.

Comparative Gene Expression Analysis in WM164 Melanoma Cells Revealed That ß-ß-Dimethylacrylshikonin Leads to ROS Generation, Loss of Mitochondrial Membrane Potential, and Autophagy Induction.

Skin cancer is currently diagnosed as one in every three cancers. Melanoma, the most aggressive form of skin cancer, is responsible for 79% of skin cancer deaths and the incidence is rising faster than in any other solid tumor type. Previously, we have demonstrated that dimethylacrylshikonin (DMAS), isolated from the roots of Onosma paniculata (Boraginaceae), exhibited the lowest IC50 values against different tumor types out of several isolated shikonin derivatives. DMAS was especially cytotoxic towards melanoma cells and led to apoptosis and cell cycle arrest. In this study, we performed a comprehensive gene expression study to investigate the mechanism of action in more detail. Gene expression signature was compared to vehicle-treated WM164 control cells after 24 h of DMAS treatment; where 1192 distinct mRNAs could be identified as expressed in all replicates and 89 were at least 2-fold differentially expressed. DMAS favored catabolic processes and led in particular to p62 increase which is involved in cell growth, survival, and autophagy. More in-depth experiments revealed that DMAS led to autophagy, ROS generation, and loss of mitochondrial membrane potential in different melanoma cells. It has been reported that the induction of an autophagic cell death represents a highly effective approach in melanoma therapy.