Antineoplastic Properties by Proapoptotic Mechanisms Induction of Inula viscosa and Its Sesquiterpene Lactones Tomentosin and Inuviscolide.

Cancer is a complex disease including approximately 200 different entities that can potentially affect all body tissues. Among the conventional treatments, radiotherapy and chemotherapy are most often applied to different types of cancers. Despite substantial advances in the development of innovative antineoplastic drugs, cancer remains one of the most significant causes of death, worldwide. The principal pitfall of successful cancer treatment is the intrinsic or acquired resistance to therapeutic agents. The development of more effective or synergistic therapeutic approaches to improve patient outcomes and minimize toxicity has become an urgent issue. Inula viscosa is widely distributed throughout Europe, Africa, and Asia. Used as a medicinal plant in different countries, I. viscosa has been characterized for its complex chemical composition in order to identify the bioactive compounds responsible for its biological activities, including anticancer effects. Sesquiterpene lactones (SLs) are natural, biologically active products that have attracted considerable attention due to their biological activities. SLs are alkylating agents that form covalent adducts with free cysteine residues within enzymes and key proteins favoring cancer cell cytotoxicity. They are effective inducers of apoptosis in several cancer cell types through different molecular mechanisms. This review focuses on recent advances in the cytotoxic effects of I. viscosa and SLs in the treatment of neoplastic diseases, with a special emphasis on their proapoptotic molecular mechanisms.

Tomentosin induces apoptosis in pancreatic cancer cells through increasing reactive oxygen species and decreasing mitochondrial membrane potential.

The aim of this study was to determine possible anticancer effect of tomentosin, a natural sesquiterpene lactone, on pancreatic cancer cells. The cytotoxic effect of tomentosin was determined by XTT analysis. Colony formation and apoptosis analyzes were performed, Reactive oxygen species (ROS) level and change in mitochondrial membrane potential (MMP) were evaluated in control and tomentosin-treated cells. The effect of tomentosin on expression levels of apoptosis-related genes was determined by qRT-PCR and Caspase-3 and Caspase-9 proteins were analyzed by western blot. And, the effect of tomentosin on migration and invasion of cells were evaluated. The IC50 dose of tomentosin was found to be 31.11 µM in PANC-1 cells and 33.93 µM in MIA PaCa-2 cells for 48 h. And, treatment of tomentosin at IC50 dose suppressed the colony forming capacity of cells. While tomentosin increased apoptosis rate and ROS production, an decrease was observed in MMP. Tomentosin affected expression level of apoptosis-related genes and increased Caspase-3 and Caspase-9 protein levels. After tomentosin treatment, cell migration and invasion were suppressed. As a result, this study reveals that tomentosin has anticancer effects on pancreatic cancer cells, and therefore it predicts that tomentosin can be evaluated as an effective agent against pancreatic cancer.

Antioxidant Activities and Mechanisms of Tomentosin in Human Keratinocytes.

Tomentosin, one of natural sesquiterpene lactones sourced from Inula viscosa L., exerts therapeutic effects in various cell types. Here, we investigated the antioxidant activities and the underlying action mechanisms of tomentosin in HaCaT cells (a human keratinocyte cell line). Specifically, we examined the involvement of tomentosin in aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. Treatment with tomentosin for up to 60 min triggered the production of reactive oxygen species (ROS), whereas treatment for 4 h or longer decreased ROS production. Tomentosin treatment also induced the nuclear translocation of Nrf2 and upregulated the expression of Nrf2 and its target genes. These data indicate that tomentosin induces ROS production at an early stage which activates the Nrf2 pathway by disrupting the Nrf2-Keap1 complex. However, at a later stage, ROS levels were reduced by tomentosin-induced upregulation of antioxidant genes. In addition, tomentosin induced the phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 MAPK and c-Jun N-terminal kinase (JNK). SB203580 (a p38 MAPK inhibitor) and SP600125 (a JNK inhibitor) attenuated the tomentosin-induced phosphorylation of Nrf2, suggesting that JNK and p38 MAPK signaling pathways can contribute to the tomentosin-induced Nrf2 activation through phosphorylation of Nrf2. Furthermore, N-acetyl-L-cysteine (NAC) treatment blocked both tomentosin-induced production of ROS and the nuclear translocation of Nrf2. These data suggest that tomentosin-induced Nrf2 signaling is mediated both by tomentosin-induced ROS production and the activation of p38 MAPK and JNK. Moreover, tomentosin inhibited the AhR signaling pathway, as evidenced by the suppression of xenobiotic-response element (XRE) reporter activity and the translocation of AhR into nucleus induced by urban pollutants, especially benzo[a]pyrene. These findings suggest that tomentosin can ameliorate skin damage induced by environmental pollutants.

Design, Synthesis, and Biological Evaluation of Novel Tomentosin Derivatives in NMDA-Induced Excitotoxicity.

N-methyl-D-aspartate (NMDA) receptor stimulation may lead to excitotoxicity, which triggers neuronal death in brain disorders. In addition to current clinical therapeutic approaches, treatment strategies by phytochemicals or their derivatives are under investigation for neurodegenerative diseases. In the present study, novel amino and 1,2,3-triazole derivatives of tomentosin were prepared and tested for their protective and anti-apoptotic effects in NMDA-induced excitotoxicity. Amino-tomentosin derivatives were generated through a diastereoselective conjugate addition of several secondary amines to the α-methylene-γ-butyrolactone function, while the 1,2,3-triazolo-tomentosin was prepared by a regioselective Michael-type addition carried out in the presence of trimethylsilyl azide (TMSN3) and the α-methylene-γ-lactone function. The intermediate key thus obtained underwent 1,3-dipolar Huisgen cycloaddition using a wide range of terminal alkynes. The possible effects of the derivatives on cell viability and free-radical production following NMDA treatment were measured by Water-Soluble Tetrazolium Salts (WST-1) and Dichlorofluorescein Diacetate (DCF-DA) assays, respectively. The alterations in apoptosis-related proteins were examined by Western blot technique. Our study provides evidence that synthesized triazolo- and amino-tomentosin derivatives show neuroprotective effects by increasing cellular viability, decreasing ROS production, and increasing the Bcl-2/Bax ratio in NMDA-induced excitotoxicity. The findings highlight particularly 2e, 2g, and 6d as potential regulators and neuroprotective agents in NMDA overactivation.

A new specific method for isolation of tomentosin with a high yield from Inula viscosa (L.) and determination of its bioactivities.

INTRODUCTION: Tomentosin, the characteristic component of Inula viscosa (L.) is an important sesquiterpene lactone with anticarcinogenic effects. Methods of obtaining pure tomentosin are not sufficient for anticancer drug research. OBJECTIVES: This study aims to develop a specific method to isolate tomentosin from I. viscosa with high yield. It also aims to investigate the inhibitory effects of tomentosin on human carbonic anhydrase I (hCAI), human carbonic anhydrase II (hCAII), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-glucosidase, and α-amylase enzymes. MATERIAL AND METHODS: Tomentosin was purified by a specific column chromatography method. The content of tomentosin in dichloromethane, dichloromethane by Soxhlet method, ethanol and ethanol by Soxhlet method extracts of I. viscosa was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Half maximal inhibitory concentration (IC50 ) and inhibition constant (Ki ) values were calculated to determine in vitro enzyme inhibition effects. RESULTS: Tomentosin was isolated in high yield (0.64%). The IC50 and Ki values for tomentosin were calculated as 5.00 ± 0.19 (r = 0.9688) and 4.62 ± 0.10 µM for hCAI, 5.40 ± 0.26 (r = 0.9677) and 5.22 ± 0.31 µM for hCAII, 6.75 ± 0.208 (r = 0.9891) and 3.75 ± 0.27 µM for AChE, 6.67 ± 0.307 (r = 0.9820) and 0.51 ± 0.11 µM for BChE, 26.61 ± 0.236 (r = 0.9815) and 2.61 ± 0.71 µM for α-glucosidase and 26.89 ± 1.54 µM (r = 0.9670) for α-amylase, respectively. CONCLUSION: Tomentosin was isolated in high yield from the paste-like extract of I. viscosa compared to the positive controls, it was determined that tomentosin was weakly effective against hCAI, hCAII, AChE and BChE, but thoroughly effective against α-glucosidase and α-amylase. These results suggested that tomentosin has α-glucosidase and α-amylase inhibitor potential.

Tomentosin a Sesquiterpene Lactone Induces Antiproliferative and Proapoptotic Effects in Human Burkitt Lymphoma by Deregulation of Anti- and Pro-Apoptotic Genes.

(1) Tomentosin is the most representative sesquiterpene lactone extracted by I. viscosa. Recently, it has gained particular attention in therapeutic oncologic fields due to its anti-tumor properties. (2) In this study, the potential anticancer features of tomentosin were evaluated on human Burkitt's lymphoma (BL) cell line, treated with increasing tomentosin concentration for cytotoxicity screening. (3) Our data showed that both cell cycle arrest and cell apoptosis induction are responsible of the antiproliferative effects of tomentosin and may end in the inhibition of BL cell viability. Moreover, a microarray gene expression profile was performed to assess differentially expressed genes contributing to tomentosin activity. Seventy-five genes deregulated by tomentosin have been identified. Downregulated genes are enriched in immune-system pathways, and PI3K/AKT and JAK/STAT pathways which favor proliferation and growth processes. Importantly, different deregulated genes identified in tomentosin-treated BL cells are prevalent in molecular pathways known to lead to cellular death, specifically by apoptosis. Tomentosin-treatment in BL cells induces the downregulation of antiapoptotic genes such as BCL2A1 and CDKN1A and upregulation of the proapoptotic PMAIP1 gene. (4) Overall, our results suggest that tomentosin could be taken into consideration as a potential natural product with limited toxicity and relevant anti-tumoral activity in the therapeutic options available to BL patients.

Clarifying the molecular mechanism of tomentosin­induced antiproliferative and proapoptotic effects in human multiple myeloma via gene expression profile and genetic interaction network analysis.

Multiple myeloma (MM) is an aggressive B cell malignancy. Substantial progress has been made in the therapeutic context for patients with MM, however it still represents an incurable disease due to drug resistance and recurrence. Development of more effective or synergistic therapeutic approaches undoubtedly represents an unmet clinical need. Tomentosin is a bioactive natural sesquiterpene lactone extracted by various plants with therapeutic properties, including anti­neoplastic effects. In the present study, the potential antitumor activity of tomentosin was evaluated on the human RPMI­8226 cell line, treated with increasing tomentosin concentration for cytotoxicity screening. The data suggested that both cell cycle arrest and cell apoptosis could explain the antiproliferative effects of tomentosin and may result in the inhibition of RPMI­8226 cell viability. To assess differentially expressed genes contributing to tomentosin activity and identify its mechanism of action, a microarray gene expression profile was performed, identifying 126 genes deregulated by tomentosin. To address the systems biology and identify how tomentosin deregulates gene expression in MM from a systems perspective, all deregulated genes were submitted to enrichment and molecular network analysis. The Protein­Protein Interaction (PPI) network analysis showed that tomentosin in human MM induced the downregulation of genes involved in several pathways known to lead immune­system processes, such as cytokine­cytokine receptor interaction, chemokine or NF­κB signaling pathway, as well as genes involved in pathways playing a central role in cellular neoplastic processes, such as growth, proliferation, migration, invasion and apoptosis. Tomentosin also induced endoplasmic reticulum stress via upregulation of cyclic AMP­dependent transcription factor ATF­4 and DNA damage­inducible transcript 3 protein genes, suggesting that in the presence of tomentosin the protective unfolded protein response signaling may induce cell apoptosis. The functional connections analysis executed using the Connectivity Map tool, suggested that the effects of tomentosin on RPMI­8226 cells might be similar to those exerted by heat shock proteins inhibitors. Taken together, these data suggested that tomentosin may be a potential drug candidate for the treatment of MM.

Tomentosin inhibits cell proliferation and induces apoptosis in MOLT-4 leukemia cancer cells through the inhibition of mTOR/PI3K/Akt signaling pathway.

Leukemia is amongst the cancers accountable for substantial mortality around the world. Tomentosin is a bioactive compound with a pharmacological significance, and its anticancer property against human leukemia MOLT-4 cell line has never been reported. Hence, the objective of this study was to explore the anticancer activity of tomentosin in MOLT-4 human leukemia cells. In the current investigation, the cytotoxic effects of tomentosin ensuing potent toxicity (IC50 : 10 µM) in MOLT-4 cells after incubation at 24 h have been presented. Furthermore, tomentosin triggered intracellular reactive oxygen species production and showed the induction of intrinsic/mitochondrial pathways in treated MOLT-4 cells, revealing a significant cytotoxicity activity. Also, fluorescent microscopic studies using acridine orange/ethidium bromide and propidium iodide staining confirmed the occurrence of apoptosis in tomentosin-treated MOLT-4 cells. Quantitative reverse transcription polymerase chain reaction presented a negative regulation of cyclin D1 and BcL-2 expression and a positive regulated BAX and caspase-3 messenger RNA expression in tomentosin-treated MOLT-4 cells. Tomentosin further inhibited the inflammatory transcription factors such as nuclear factor κB (NF-κB), tumor necrosis factor α, interleukin 1ß (IL-1ß), and IL-6. Additionally, inhibition of the m-TOR/PI3K/AKT protein expression by tomentosin in MOLT-4 cells was confirmed. Overall, these findings lead to a conclusion that tomentosin induces apoptosis in MOLT-4 cells through caspase-facilitated proapoptotic pathway, and inhibition of the NF-κB-stimulated Bcl-2 facilitated the antiapoptotic pathway.

Induction of cell cycle arrest and apoptosis by tomentosin in hepatocellular carcinoma HepG2 and Huh7 cells.

Tomentosin, a sesquiterpene lactone, is known to possess various biological activities. However, its anticarcinogenic activity against human hepatocellular carcinoma (HCC) cells has not been investigated in detail. Thus, this study aimed to elucidate the cytotoxic mechanism of tomentosin in human HCC cell lines HepG2 and Huh7. WST-1, cell counting, and colony formation assay results showed that treatment with tomentosin decreased the viability and suppressed the proliferation rate of HepG2 and Huh7 cells in a dose- and time-dependent manner. Cell cycle analysis revealed increased population of cells at the SubG1 and G2/M stage, and decreased population of cells at the G0/1 stage in HepG2 and Huh7 cells treated with tomentosin. Annexin V/propidium iodide double staining and TUNEL assay results showed increased apoptotic cell population and DNA fragmentation in HepG2 and Huh7 cells treated with tomentosin. Western blotting analysis results showed that tomentosin treatment significantly increased the expression level of Bax, Bim (short form), cleaved PARP1, FOXO3, p53, pSer15p53, pSer20p53, pSer46p53, p21, and p27, but decreased the expression of Bcl2, caspase3, caspase7, caspase9, cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclinB1, cyclinD1, cyclinD2, cyclinD3, and cyclinE in a dose-dependent manner. Taken together, this study revealed that tomentosin, which acted through cell cycle arrest and apoptosis, may be a useful therapeutic option against HCC.

Tomentosin inhibit cerebral ischemia/reperfusion induced inflammatory response via TLR4/ NLRP3 signalling pathway - in vivo and in vitro studies.

Stoke is a global threat, leading to 50 % of deaths worldwide and it causes permanent disability to about 5 million individuals globally each year. In this study, we assessed the potency of tomentosin to inhibit the neuroinflammation in in vivo and in vitro models. The Sprague Dawley rats were pretreated with 25 mg/kg bodyweight (b.wt) and 50 mg/kg b.wt of tomentosin for seven days followed by induction of cerebral ischemic reperfusion. The brain edema and cerebral infractions were analyzed. The levels of antioxidants and the interleukins were measured by standard methods. The NLRP3 signaling proteins expression was evaluated using qPCR analysis. In vitro studies were performed in SH-SY5Y-cells pretreated with tomentosin and subjected to OGD-R treatment. Our results depicts tomentosin scavenges the free radicals, enhances antioxidant system, inhibits the NLRP3 signaling. In vitro results substantiates with in vivo results. To conclude, our in vivo and in vitro results confirm tomentosin may be potent alternative for existing antistroke drugs.

Tomentosin induces apoptotic pathway by blocking inflammatory mediators via modulation of cell proteins in AGS gastric cancer cell line.

In this study, we investigated the in vitro effect of tomentosin on cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, reactive oxygen species by 2',7'-dichlorofluorescein diacetate staining assay, apoptosis (AO/EtBr, propidium iodide, and 4',6-diamidino-2-phenylindole staining, mitochondrial membrane potential), cell adherent, cell migration, inflammation, apoptosis, and oxidative stress from gastric cancer cells (GCCs) AGS. Upon their relative cell proliferative, inflammatory, and apoptotic molecular markers were analyzed by using the enzyme-linked immunosorbent assay and Western blot analysis method. Treatment with tomentosin (IC50 = 20 µM) significantly inhibited cell proliferation and oxidative stress-induced anti-cell proliferative (proliferating cell nuclear antigen and cyclin-D1) also regulated expression, drastically diminished tumor necrosis factor-α, nuclear factor-κB, interleukin-6, and interleukin-1ß expression levels, significantly upregulated Bcl-2 and Bax expression. Thus, this tomentosin can significantly reduce GCC proliferation via cytotoxicity which is stimulated apoptosis markers via morphology staining changes and inhibitory inflammatory markers. The tomentosin-induced oxidative stress may be involved to stimulate apoptotic mechanisms via mitochondria-mediated signaling by the inhibition of inflammation. Taken together, our findings suggest a possible future use of chemotherapeutic agents for pharmacological benefits and as an anti-cancer treatment option.

Tomentosin Displays Anti-Carcinogenic Effect in Human Osteosarcoma MG-63 Cells via the Induction of Intracellular Reactive Oxygen Species.

Tomentosin is a natural sesquiterpene lactone extracted from various plants and is widely used as a medicine because it exhibits essential therapeutic properties. In this study, we investigated the anti-carcinogenic effects of tomentosin in human osteosarcoma MG-63 cells by performing cell migration/viability/proliferation, apoptosis, and reactive oxygen species (ROS) analysis assays. MG-63 cells were treated with various doses of tomentosin. After treatment with tomentosin, MG-63 cells were analyzed using the MTT assay, colony formation assay, cell counting assay, wound healing assay, Boyden chamber assay, zymography assay, cell cycle analysis, FITC Annexin V apoptosis assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, western blot analysis, and ROS detection analysis. Our results indicated that tomentosin decreased cell viability and migration ability in MG-63 cells. Moreover, tomentosin induced apoptosis, cell cycle arrest, DNA damage, and ROS production in MG-63 cells. Furthermore, tomentosin-induced intracellular ROS decreased cell viability and induced apoptosis, cell cycle arrest, and DNA damage in MG-63 cells. Taken together, our results suggested that tomentosin exerted anti-carcinogenic effects in MG-63 cells by induction of intracellular ROS.

Allergenic sesquiterpene lactones from cushion bush (Leucophyta brownii Cass.): new and old sensitizers in a shrub-turned-a-pot plant.

BACKGROUND: The Australian cushion bush (Leucophyta brownii) of the Compositae family of plants has become a popular pot and container plant. The plant produces the sesquiterpene lactone allergen calocephalin. OBJECTIVES: To assess the sensitizing potential of sesquiterpene lactones from cushion bush. PATIENTS/MATERIALS/METHODS: Eleven Compositae-sensitive patients were patch tested with seven sesquiterpene lactones isolated from cushion bush. RESULTS: Six of seven sesquiterpene lactones elicited positive reactions in 4 of 11 patients. CONCLUSIONS: The well-known sesquiterpene lactone pseudoivalin and its derivative pseudoivalin acetate, as well as calocephalin and tomentosin, were confirmed to be sensitizers, whereas leucophytalin A and 4α-hydroxy-5αH,10αH-1,11(13)-guaidien-8ß,12-olide were shown to be allergenic for the first time. The patch test reaction patterns seem to follow the chemical patterns, which may eventually make it possible to trace primary sensitizers and advise patients more precisely.

Tomentosin Induces Telomere Shortening and Caspase-Dependant Apoptosis in Cervical Cancer Cells.

Tomentosin, a natural sesquiterpene lactone purified from of Inula viscosa L., was investigated for its anti-proliferative, telomere shortening, and apoptotic effects on human cervical cancer HeLa and SiHa cell lines. Tomentosin was found to inhibit the growth of SiHa and HeLa cell lines in dose and time-dependent manner (IC50 values of 7.10 ± 0.78 µM and 5.87 ± 0.36 µM, respectively after 96 h of treatment). As evidenced by TTAGGG telomere length assay, tomentosin target specifically the telomeric overhang lengthening. This was confirmed by the evaluation of the cytotoxic effects of tomentosin in the foetal fibroblast Wi38 and JW10 cells which were derived from Wi38 and express hTERT, the telomerase catalytic subunit. We found that JW10 cells are 4.7-fold more sensitive to tomentosin which argues for telomere as its specific target. Furthermore, we found that tomentosin mediate this cytotoxic effect by inducing apoptosis and cell cycle arrest at G2/M phase. Morphological features of treated cells, as evidenced by Hoechst 33324 staining, revealed that the cytotoxic effect was due to induction of apoptosis. This was accompanied by pro-caspase-3 cleavage, an increase in caspase-3 activity and a cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, tomentosin induced a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species (ROS), accompanied by a decrease in Bcl-2 expression. This indicates that tomentosin-induced apoptosis may involve a mitochondria-mediated signaling pathway. This study provides the first evidence that tomentosin targets telomere machinery and induces apoptosis in cervical cancer cells. The molecular mechanism underlying tomentosin-induced apoptosis may involve a mitochondria-mediated signaling pathway. J. Cell. Biochem. 118: 1689-1698, 2017. © 2016 Wiley Periodicals, Inc.