Acute myeloid leukemia (AML) is the most lethal form of leukemia. Standard anti-AML treatment remains almost unchanged for decades. Tingenone (TG) and 22-hydroxytingenone (22-HTG) are quinonemethide ...triterpenes found in the Amazonian plant Salacia impressifolia (Celastraceae), with cytotoxic properties in different histological types of cancer cells. In the present work, we investigated the anti-AML action mechanism of TG and 22-HTG in the AML HL-60 cell line. Both compounds exhibited potent cytotoxicity in a panel of cancer cell lines. Mechanistic studies found that TG and 22-HTG reduced cell growth and caused the externalization of phosphatidylserine, the fragmentation of internucleosomal DNA and the loss of mitochondrial transmembrane potential in HL-60 cells. In addition, pre-incubation with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, prevented TG- and 22-HTG-induced apoptosis, indicating cell death by apoptosis via a caspase-dependent pathway. The analysis of the RNA transcripts of several genes indicated the interruption of the cellular antioxidant system, including the downregulation of thioredoxin, as a target for TG and 22-HTG. The application of N-acetyl-cysteine, an antioxidant, completely prevented apoptosis induced by TG and 22-HTG, indicating activation of the apoptosis pathway mediated by oxidative stress. Moreover, TG and 22-HTG induced DNA double-strand break and phosphorylation of JNK2 (T183/Y185) and p38α (T180/Y182), and co-incubation with SP 600125 (JNK/SAPK inhibitor) and PD 169316 (p38 MAPK inhibitor) partially prevented apoptosis induced by TG and 22-HTG. Together, these data indicate that TG and 22-HTG are new candidate for anti-AML therapy targeting thioredoxin.
•TG and 22-HTG induce caspase-mediated apoptotic cell death.•TG and 22-HTG target oxidative stress through downregulation of thioredoxin.•TG and 22-HTG cause DNA damage.•TG and 22-HTG cause JNK/p38-mediated apoptosis.
22β-hydroxytingenone (22-HTG) is a quinonemethide triterpene isolated from Salacia impressifolia (Miers) A. C. Smith (family Celastraceae), which has been used in traditional medicine to treat a ...variety of diseases, including dengue, renal infections, rheumatism and cancer. However, the anticancer effects of 22-HTG and the underlying molecular mechanisms in melanoma cells have not yet been elucidated.
The present study investigated apoptosis induction and antimetastatic potencial of 22-HTG in SK-MEL-28 human melanoma cells.
First, the in vitro cytotoxic activity of 22-HTG in cultured cancer cells was evaluated. Then, cell viability was determined using the trypan blue assay in melanoma cells (SK-MEL-28), which was followed by cell cycle, annexin V-FITC/propidium iodide assays (Annexin/PI), as well as assays to evaluate mitochondrial membrane potential, production of reactive oxygen species (ROS) using flow cytometry. Fluorescence microscopy using acridine orange/ethidium bromide (AO/BE) staining was also performed. RT-qPCR was carried out to evaluate the expression of BRAF, NRAS, and KRAS genes. The anti-invasiveness potential of 22-HTG was evaluated in a three-dimensional (3D) model of reconstructed human skin.
22-HTG reduced viability of SK-MEL-28 cells and caused morphological changes, as cell shrinkage, chromatin condensation, and nuclear fragmentation. Furthermore, 22-HTG caused apoptosis, which was demonstrated by increased staining with AO/BE and Annexin/PI. The apoptosis may have been caused by mitochondrial instability without the involvement of ROS production. The expression of BRAF, NRAS, and KRAS, which are important biomarkers in melanoma development, was reduced by the 22-HTG treatment. In the reconstructed skin model, 22-HTG was able to decrease the invasion capacity of melanoma cells in the dermis.
Our data indicate that 22-HTG has anti-tumorigenic properties against melanoma cells through the induction of cell cycle arrest, apoptosis and inhibition of invasiveness potential, as observed in the 3D model. As such, the results provide new insights for future work on the utilization of 22-HTG in malignant melanoma treatment.
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•Apoptosis was the death pathway of SK-MEL-28 cells treated with 22β-hydroxytingenone.•22β-hydroxytingenone promoted mitochondrial dysfunction without ROS generation.•22β-hydroxytingenone inhibited expression of BRAF, NRAS, and KRAS genes.•22β-hydroxytingenone reduced invasion of SK-MEL-28 cells in reconstructed skin model.
Salacia impressifolia (Miers) A. C. Smith (family Celastraceae) is a traditional medicinal plant found in the Amazon Rainforest known as “miraruíra”, "cipó-miraruíra" or “panu” and is traditionally ...used to treat dengue, flu, inflammation, pain, diabetes, male impotency, renal affections, rheumatism and cancer. Aim of the study: The aim of this study was to investigate in vitro and in vivo anti-leukemia activity of the stem bark of S. impressifolia in experimental models. Materials and methods: The in vitro cytotoxic activity of extracts, fractions and quinonemethide triterpenes (22-hydroxytingenone, tingenone and pristimerin) from the stem bark of S. impressifolia in cultured cancer cells was determined. The in vivo antitumor activity of the ethyl acetate extract (EAE) and of its fraction (FEAE.3) from the stem bark of S. impressifolia was assessed in C.B-17 severe combined immunodeficient (SCID) mice engrafted with human promyelocytic leukemia HL-60 cells. Results: The extract EAE, its fraction FEAE.3, and quinonemethide triterpenes exhibited potent cytotoxicity against cancer cell lines, including in vitro anti-leukemia activity against HL-60 and K-562 cells. Moreover, extract EAE and its fraction FEAE.3 inhibited the in vivo development of HL-60 cells engrafted in C.B-17 SCID mice. Tumor mass inhibition rates were measured as 40.4% and 81.5% for the extract EAE (20 mg/kg) and for its fraction FEAE.3 (20 mg/kg), respectively. Conclusions: Ethyl acetate extract and its fraction from the stem bark of S. impressifolia exhibit in vitro and in vivo anti-leukemia activity that can be attributed to their quinonemethide triterpenes. These data confirm the ethnopharmacological use of this species and may contribute to the development of a novel anticancer herbal medicine.
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Melanoma is a skin cancer with high invasive potential and high lethality. Considering that quinonemethide triterpenes are described as promising anticancer agents, the aim of this study was to ...evaluate the effect of 22β-hydroxytingenone (22-HTG) against human melanoma cells. Alamar blue assay was performed in order to evaluate its cytotoxic effect. Thus, subtoxic concentrations (1.0, 2.0, and 2.5 μM) were used to evaluate the effect of this compound on proliferation, migration, metabolism, and invasion. IC50 value against SK-MEL-28 cell line was 4.35, 3.72, and 3.29 μM after 24, 48, and 72 h of incubation, respectively. 22-HTG reduced proliferation, migration and invasion by melanoma cells, with decreased activity of metalloproteinases (MMP-2 and MMP-9). Futhermore, 22-HTG decreased expression of lactate dehydrogenase (LDHA), an enzyme associated with cell metabolism. Howerver, the small reduction in LDHA enzyme activity must have occurred by the cytotoxic effect of 22-HTG. According to the results, 22-HTG interferes with important characteristics of cancer, with anti-proliferative, and anti-invasive effect against melanoma cells. The data suggest that 22-HTG is an effective substance against melanoma cells and it should be considered as a potential anticancer agent.
•22-HTG has cytotoxic effect against human melanoma cells.•22-HTG reduced the proliferation of SK-MEL-28 cells at subtoxic concentrations.•22-HTG reduced migration and invasion of melanoma cells.•22-HTG decreases the activity of the MMP-2 and MMP-9 enzymes.
The effects of crude extracts, fractions and isolated compounds from Vitex polygama Cham. and Siphoneugena densiflora Berg were evaluated on the development of Spodoptera frugiperda JE Smith, a ...destructive insect pest of corn and several other crops. The extracts and fractions were incorporated into an artificial diet at 1 mg g⁻¹ and offered to the insect during its larval stage. Length and viability of larval and pupal stages as well as pupal weight were assessed. Isolated compounds were tested through superficial contamination of the diet at 0.1 mg g⁻¹. Weight and viability of ten-day-old larvae were determined. Methanolic and hydroalcoholic S. densiflora extracts caused 100% larval mortality, while leaf and fruit hydroalcoholic extracts from V. polygama were the most active. Among the isolated compounds, flavonoids presented the best insecticidal results, and tannins the best larval growth inhibition.
► The Gedunin compound is a natural product and has shown large biologic activity. ► The energy difference between conformers is low, indicating that both are stables. ► The minimum molecules are ...separated by low energy barriers. ► The interaction between conformers is strong (13.09
kcal/mol).
The Gedunin compound (C
28H
34O
6) is a natural product extracted from
Trichilia pallida that has shown a wide activity. The crystallographic structure shows two conformers in the asymmetric unit, which differ in a rotation of the furan group. To understand this molecular arrangement, the density functional calculations, Molecular Electrostatic Potential (MEP) and thermodynamic function calculation have been performed at the B3LYP/6-311++g(d,p) level. Both conformers were optimized and the agreement with the experimental structure was very good, making possible further theoretical analysis of the structure. The inter-conversion between two conformers depends on the energy barrier. This process is studied in the vacuum and shows two transition states with a low energetic barrier for a potential energy curve scanning rigid around furan group: 4.37
kcal/mol and 16.52
kcal/mol. As the first transition state has a notably lower energetic barrier, the preferred inter-conversion pathway between the conformers involves the first rather than the second transition state. Understanding this transition state in detail led us to perform its optimization, showing an energetic barrier around 3.66
kcal/mol. The negative free energy and low enthalpy confirm that the process is spontaneous and exothermic. The results show that this requirement makes the existence of the two conformers in the asymmetric unit possible. The structure of molecules in the asymmetric unit is better understood when the MEP is used on the interaction between molecules. For Gedunin, both molecules have shown MEP with well-defined regions, and this behavior contributes to the observed link between molecules and for the negative regions complementing positive regions of another molecule.
Dichloromethane (DIC) leaf and fruit extracts of Trichiliapallida Swartz were obtained for the isolation and identification of molecules with insecticidal activity against the tomato leafminer, Tuta ...absoluta (Meyrick). DIC leaf extracts of T. pallida yielded six compounds, the triterpenes 24-methylenecycloarta-3beta-ol (TRIT-1), 24-methylenecycloarta-3beta-26-diol (TRIT-2) and cycloarta-23-eno-3beta,25-diol (TRIT-3), the sterols 24-methylene-3,22-dihydroxycholesterol (EST-1), 24-methylenecholesterol (EST-2) and 24-methylene-3beta,4beta,22-trihydroxycholesterol (EST-3), while the fruit extract yielded the limonoid gedunine (LIM). These molecules were dissolved in acetone and sprayed at 0.1% on tomato leaflets infested with newly-hatched larvae. Larval mortality at day 5 and 9 after infestation, larval and pupal developmental time and survival, pupal weight and adult malformation were evaluated. TRIT-1, EST-1 and LIM were the most effective against T. absoluta due to larval development arrestment and reduced larval survivorship.
Dichloromethane (DIC) leaf and fruit extracts of Trichiliapallida Swartz were obtained for the isolation and identification of molecules with insecticidal activity against the tomato leafminer, Tuta ...absoluta (Meyrick). DIC leaf extracts of T. pallida yielded six compounds, the triterpenes 24-methylenecycloarta-3b-ol (TRIT-1), 24-methylenecycloarta-3b- 26-diol (TRIT-2) and cycloarta-23-eno-3b,25-diol (TRIT-3), the sterols 24- methylene-3,22-dihydroxycholesterol (EST-1), 24-methylenecholesterol (EST-2) and 24-methylene-3b,4b,22-trihydroxycholesterol (EST-3), while the fruit extract yielded the limonoid gedunine (LIM). These molecules were dissolved in acetone and sprayed at 0.1% on tomato leaflets infested with newly-hatched larvae. Larval mortality at day 5 and 9 after infestation, larval and pupal developmental time and survival, pupal weight and adult malformation were evaluated. TRIT-1, EST-1 and LIM were the most effective against T. absoluta due to larval development arrestment and reduced larval survivorship.
A partir do extrato em diclorometano (DIC) de folhas e frutos de Trichilia pallida Swartz, objetivou-se fazer o isolamento e identificação de substâncias com atividade inseticida sobre a ...traça-do-tomateiro, Tuta absoluta (Meyrick). Do extrato em DIC de folhas de T. pallida foram isolados os triterpenos 24-metilenocicloarta-3β-ol (TRIT-1), 24-metilenocicloarta-3β-26-diol (TRIT-2) e cicloarta-23-eno-3β,25-diol (TRIT-3), os esteróides 24-metileno-3,22-diidroxicolesterol (EST-1), 24-metilenocolesterol (EST-2) e 24-metileno-3β,4β,22-triidroxicolesterol (EST-3), além do limonóide gedunina (LIM) obtido de frutos da planta. As substâncias foram dissolvidas em acetona e pulverizadas a 0,1% em folíolos de tomateiro infestados com lagartas recém-eclodidas. Foram avaliadas a mortalidade de lagartas aos quinto e nono dias após a infestação, duração e sobrevivência das fases de larva e pupa, peso de pupas e porcentagem de adultos deformados. TRIT-1, EST-1 e LIM foram as moléculas que apresentaram maior atividade sobre T. absoluta, alongando o desenvolvimento larval e reduzindo a sobrevivência dessa fase.
Dichloromethane (DIC) leaf and fruit extracts of Trichiliapallida Swartz were obtained for the isolation and identification of molecules with insecticidal activity against the tomato leafminer, Tuta absoluta (Meyrick). DIC leaf extracts of T. pallida yielded six compounds, the triterpenes 24-methylenecycloarta-3β-ol (TRIT-1), 24-methylenecycloarta-3β-26-diol (TRIT-2) and cycloarta-23-eno-3β,25-diol (TRIT-3), the sterols 24-methylene-3,22-dihydroxycholesterol (EST-1), 24-methylenecholesterol (EST-2) and 24-methylene-3β,4β,22-trihydroxycholesterol (EST-3), while the fruit extract yielded the limonoid gedunine (LIM). These molecules were dissolved in acetone and sprayed at 0.1% on tomato leaflets infested with newly-hatched larvae. Larval mortality at day 5 and 9 after infestation, larval and pupal developmental time and survival, pupal weight and adult malformation were evaluated. TRIT-1, EST-1 and LIM were the most effective against T. absoluta due to larval development arrestment and reduced larval survivorship.
A partir de extratos aquosos e não aquosos de Trichilia pallida Swartz (Meliaceae), objetivou-se identificar frações com atividade inseticida sobre a traça-do-tomateiro, Tuta absoluta (Meyrick). Para ...tanto, extratos aquosos liofilizados (EAL) a 3%, de folhas e ramos da planta, foram ressuspendidos em água e aplicados, por meio de mini-atomizador, sobre folíolos de tomateiro infestados com lagartas recém-eclodidas. Com base nos resultados de mortalidades aos 5 e 10 dias após a infestação (DAI), considerou-se o EAL de folhas mais eficiente que o de ramos. Numa segunda etapa, foram obtidos por maceração, extratos de folhas em hexano (HEX), diclorometano (DIC) e metanol (MET), os quais foram avaliados a1 % (p/v) como descrito anteriormente, incluindo-se acetona e água como controles. Dentre os referidos extratos, aquele em DIC foi o mais promissor como fonte de substância(s) com atividade inseticida sobre lagartas de T. absoluta. Na seqüência, através de partição líquido-líquido do extrato DIC, obtiveram-se as frações em HEX, MET, acetato de etila (AET), n-butanol (NBU) e aquosa (AQ). Destas frações, a AET e MET a 0,15% foram consideradas as mais promissoras como fontes de substâncias com atividade inseticida sobre T. absoluta.
Organic and aqueous extracts of the Trichilia pallida Swartz (Meliaceae) were obtained for identification of fractions with insecticidal activity against the tomato leafminer Tuta absoluta (Meyrick). Leaf and twig freeze-dried aqueous extracts (FDA) were resuspended in water at 3% and sprayed, with the aid of a mini-atomizer, over tomato leaflets infested with newly-hatched larvae. Larval mortality at 5 and 10 days after infestation (DAI) were higher for leaf extracts. In a second set of experiments, leaf extracts at 1% (w/v) were obtained by maceration in hexane (HEX), dichloromethane (DIC) and methanol (MET), and tested as described above using acetone and water as control. DIC extracts were the most promising as a source of substances with insecticidal activity against larvae of T. absoluta. DIC leaf extracts were further processed and subjected to partition assays, producing fractions in HEX, MET, ethyl acetate (ETA), n-butanol (NBU) and water (WA). The 0.15% ETA and MET fractions obtained from the partition assay of DIC extracts showed the highest insecticidal activity against T. absoluta.
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