New Cytotoxic Premyrsinane-Type Diterpenes from Euphorbia aleppica Against Breast Cancer Cells.

Euphorbia is used in traditional medicine to remove warts, possibly due to its cytotoxic or antiviral effects. This study investigated its phytochemistry and bioactive compounds. Euphorbia aleppica from the Euphorbiaceae family was collected from Kuhdasht, Lorestan, Iran. Plant material was dried and ground. Extraction was performed by maceration using a dichloromethane-acetone solvent. After removing fatty contents, fractionation was done by open column chromatography. Based on the initial H-NMR spectra, fractions containing diterpenoid compounds were identified. The Sephadex column and HPLC performed isolation. The HPLC was done with a regular YMC silica column using a hexane: Ethyl acetate (70: 30) solvent. The selected sub-fractions were identified by one and two-dimensional corelative NMR spectra. Accurate mass spectra confirmed the molecular formula of the obtained structures. Cytotoxicity was assessed using a standard MTT assay against breast cancer cells. The NMR and mass analysis identified compound 1 as a newly described and compound 2 as a pre-defined compound as 3, 7, 15ß-triacetyl-5α-tigliate-13(17)-α-epoxy-14-oxopremyrsinane and 3, 7, 14, 15, 17-pentaacetyl-5-tigliate-13(17)-epoxypremyrsinane, respectively. Compound 1 showed moderate cytotoxicity, and compound 2 exhibited a potent cytotoxic effect dose-dependently against MCF-7 and MDA-MB 231 breast cancer cells, probably because of 14-O-acetyl and 17-O-acetylated hemiacetal groups.

Phytochemical analysis of Artemisia kopetdaghensis: Sesquiterpene lactones with proapoptotic activity against prostate cancer cells.

Phytochemical investigation of the aerial parts of Artemisia kopetdaghensis resulted in the isolation and characterization of three undescribed eudesmane-type sesquiterpene lactones, persianolide A, 4-epi-persianolide A, and 3α,4-epoxypersianolide A, together with three previously described eudesmane-type sesquiterpene lactones, 11-epi-artapshin, 1ß,8α-dihydroxy-11α,13-dihydrobalchanin, and 1ß-hydroxy-11-epi-colartin. The abundantly obtained 11-epi-artapshin was oxidized to undescribed 11α,13-dihydroeudesma-12,6α-olide-1,8-dione and 8ß-hydroxy-11α,13-dihydroeudesma-12,6α-olide-1-one and acetylated to the undescribed 1,8-O-diacetyl-11α,13-dihydroeudesma-12,6α-olide. Structures were elucidated based on extensive spectral data analyses, including 1D and 2D NMR and HRESIMS. The absolute configuration was determined using calculated and experimental ECD spectral data. Compounds were subsequently subjected to the MTT assay to evaluate their cytotoxicity against prostate cancer cells (DU-145 and LNCaP). Related factors associated with the sequence of apoptosis were tested by ELISA, western blotting, and biochemical assay. Results suggested that 11-epi-artapshin hinders the growth of DU-145 cells through mitochondria-mediated apoptosis initiated by stimulation of ROS build-up, ΔΨm depletion, regulation of the Bax/Bcl-2 ratio, and activation of caspase 3, respectively.

Identification of New Hsp90 Inhibitors: Structure Based Virtual Screening, Molecular Dynamic Simulation, Synthesis and Biological Evaluation.

BACKGROUND: Heat shock protein90 (Hsp90) is overexpressed in tumor cells, thus the inhibition of the Hsp90 ATPase activity would be a meaningfully effective strategy in cancer therapy. OBJECTIVE: The present work was aimed at four steps: designing new Hsp90 inhibitors as anti-cancer by a virtual screening study; synthesize designed compounds; biological evaluation of them and finally molecular dynamic (MD) simulations of best compounds. METHODS: A virtual screening study was performed on a library (100 compounds) of the ZINC database with benzimidazole scaffold; then an extracted compound and two derivatives were synthesized. The anti-proliferative and ATPase inhibitory activities of these compounds were evaluated by MTT and ATPase inhibition assays, respectively. The western blot analysis was performed to the evaluation of the expression level of Hsp70 and Her2 proteins. Finally, 200 ns molecular dynamic simulation was carried out to confirm the stability of the strongest synthesized compound in Hsp90 active site. RESULTS: ZINC00173501 compound with an aminobenzimidazole scaffold was chosen by the virtual screening study. ZINC00173501 compound and two of its derivatives were synthesized. ATPase inhibitory activity of three synthesized compounds shown that ZINC00173501 compound was the most potent inhibitor (IC50= 8.6 µM) with the anti-proliferative activity 14.41 µM, 19.07 µM and more than 100 µM against MCF-7, HeLa and HUVEC cell lines, respectively. The high level of Hsp70 expression and low level of Her2 expression confirmed ZINC00173501 as an Hsp90 inhibitor. Finally, molecular dynamics simulation showed that ZINC00173501 was stable in Hsp90 active site during 200 ns simulation. CONCLUSION: The biological evaluation results show that 2-aminobenzimidazole scaffold could be suggested as a lead for inhibition of Hsp90.

15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) inhibits proliferation and induces cell cycle arrest and apoptosis in ovarian cancer cells.

AIM: 15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) isolated from Juniperus foetidissima, has been recently identified as an antiproliferative agent; however, the molecular basis of antiproliferative effects of HLCA remains unknown. To investigate it, the current study has emphasized the hypothesis that HLCA induced cell death is a consequence of intracellular reactive oxygen species (ROS) production followed by cell cycle arrest and apoptosis. MAIN METHODS: Human ovarian OVCAR-3 and Caov-4 cells were treated with various concentrations of HLCA (48 h) and the measurement of intracellular ROS was considered. Then, the potential of HLCA in promoting apoptosis was investigated via flow cytometry, western blot, and caspase activity assay. Also, the inhibitory effect of HLCA on the cell cycle was evaluated using flow cytometry and western blot analysis. KEY FINDINGS: We found intracellular (ROS) accumulation in HLCA-treated cells. Subsequent observation of the increment in pro-apoptotic Bax as well as the decrement in antiapoptotic Bcl2 revealed that the HLCA-induced cytotoxicity may be triggered by the intrinsic pathway of apoptosis. Our subsequent experiments suggested that caspase-9 and -3 were activated and led the cells to apoptosis during the process. Cell cycle disruption at the G1 phase via down-regulation of cyclin D1 and Cyclin-dependent kinase 4 (CDK4) was another proved mechanism by which HLCA exerts its antiproliferative effects on the ovarian cell lines, OVCAR-3 and Caov-4, especially at relatively lower concentrations. SIGNIFICANCE: This is the first study that reveals the apoptotic effects of HLCA, suggesting its therapeutic potential as an effective anti-tumor agent. However, further in vivo studies are required to confirm these effects.

Kopetdaghinanes, pro-apoptotic hemiacetialic cyclomyrsinanes from Euphorbia kopetdaghi.

Euphorbia kopetdaghi grows wild in the Northeast parts of Iran. Phytochemical study of its aerial parts led to the isolation of two undescribed cyclomyrsinol macrocyclic diterpenes with a new tetrahydrofuran oxidation pattern containing a hemiacetal group named: kopetdaghinane A and B. The structure of the isolated compounds was elucidated by extensive spectroscopic methods. Cytotoxic activity of kopetdaghinane A was evaluated using standard MTT assay against MCF-7 breast cancer and OVCAR-3 ovary cells. HUVEC cells were used as a normal cell line for calculation of the selectivity index. The MTT showed cyclomyrsinol diterpene has a significant cytotoxic effect with good selectivity indexes against both cell lines but with more selectivity against MCF-7 cells. Apoptosis induction by cyclomyrsinol treatment was confirmed by annexin V-FITC/PI staining, and caspase-6 activation. Western blot analysis showed that the expression of Bcl-2 was noticeably decreased in response to kopetdaghinane A treatment, while the expression of Bax protein was increased. Moreover, the apoptotic effect of cyclomyrsinol was shown to be related to ROS production, and loss of mitochondrial membrane potential (ΔΨm). Taken together, these results showed that kopetdaghinane A inhibits the growth of MCF-7 breast cancer cells through the activation of the mitochondrial apoptotic pathway and may be considered as an investigational compound in breast cancer preclinical study.

Centaurea cyanus extracted 13-O-acetylsolstitialin A decrease Bax/Bcl-2 ratio and expression of cyclin D1/Cdk-4 to induce apoptosis and cell cycle arrest in MCF-7 and MDA-MB-231 breast cancer cell lines.

Natural products are considered recently as one of the source for production of efficient therapeutical agents for breast cancer treatment. In this study, a sesquiterpene lactone, 13-O-acetylsolstitialin A (13ASA), isolated from Centaurea cyanus, showed cytotoxic activities against MCF-7 and MDA-MB-231 breast cancer cell lines using standard 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. To find the mechanism of action of cytotoxicity, annexin V/propidium iodide (PI) staining was performed for evaluation of apoptosis. This process was further confirmed by immunoblotting of anti- and proapoptotic, Bcl-2 and Bax, proteins. Cell cycle arrest was evaluated by measurement of fluorescence intensity of PI dye and further confirmed by immunoblotting of Cdk-4 and cyclin D1. Mitochondrial transmembrane potential (ΔΨm) and generation of reactive oxygen species (ROS) were measured using the JC-1 and DCFDA fluorescence probes, respectively. These experiments showed that 13ASA is a potent cytotoxic agent, which activates apoptosis-mediated cell death. In response to this compound, Bax/Bcl-2 ratio was noticeably increased in MCF-7 and MDA-MB-231 cells. Moreover, 13ASA induced cell cycle arrest at subG1 and G1 phases by decreasing protein levels of cyclin D1 and Cdk-4. It was done possibly through the decrease of ΔΨm and increase of ROS levels which induce apoptosis. In conclusion, this study mentioned that 13ASA inhibit the growth of MCF-7 and MDA-MB-231 breast cancer cell lines through the induction of cell cycle arrest, which triggers apoptotic pathways. 13ASA can be considered as a susceptible compound for further investigation in breast cancer study.

Pimpinelol, a novel atypical Sesquiterpene lactone from Pimpinella haussknechtii fruits with evaluation of endoplasmic reticulum stress in breast cancer cells.

Pimpinella haussknechtii is an annual native plant grows in west of Iran. Phytochemical study of the fruits of P. haussknechtii led to the isolation of a novel irregular sesquiterpene lactone with a new skeletone and oxidation pattern named: pimpinelol. The structure of the isolated compound was elucidated by extensive spectroscopic methods. Cytotoxic activity of Pimpinelol was evaluated using standard MTT assay against breast cancer cells. Induced endoplasmic reticulum stress and related gene expressions were evaluated with fluorescence microscopy analysis and real time PCR, respectively. These findings confirmed that Pimpinelol concentrations dependently increased protein aggregation and the mRNA expression of ATF-4, CHOP, GADD34 and TRIB3 in MCF-7 breast cancer cell line. According to the obtain results, we demonstrated for the first time that Pimpinelol decreased breast cancer cell viability by inducing ER stress.

New 6(17)-epoxylathyrane diterpene: aellinane from Euphorbia aellenii induces apoptosis via mitochondrial pathway in ovarian cancer cell line.

Euphorbia species have been used in traditional medicine in many countries for the treatment of cancer. This article aims to evaluate the capability of a new lathyrane diterpene isolated from Euphorbia aellenii to induce apoptosis in the Caov-4 cell line to determine the underlying mechanism of its anticancer effects. A new 6(17)-epoxylathyrane diterpenes: aellinane from Euphorbia aellenii was evaluated for viability of Caov-4 cells by MTT method. Apoptosis induction by lathyrane diterpene was confirmed by annexin V-FITC/PI staining, and caspase-6 activation. The Bcl2 and Bax protein content were detected by Western blot analysis. Finally, we employed the fluorescent ROS detection kit and fluorochrome JC-1 to determine ROS levels and loss of mitochondria membrane potential (ΔΨm) in Caov-4 cells, respectively. The results show that lathyrane diterpene has significant cytotoxic effect against Caov-4 cells. The IC50 value was 45 µM. Annexin V/propidium iodide (PI) staining and caspase-6 activity assay confirmed that lathyrane diterpene is able to induce apoptosis in Caov-4 cells. The results also demonstrate that lathyrane diterpene up-regulated Bax and down-regulated Bcl-2 proteins. Moreover, apoptotic effect of lathyrane diterpene was also related to ROS production and loss of mitochondrial membrane potential (ΔΨm). This study demonstrated that lathyrane diterpene has profound activity against Caov-4 cells. Analysis of apoptosis-related proteins revealed that lathyrane diterpene triggered the mitochondrion-mediated apoptosis pathway, which led to the loss of mitochondrial membrane potential (ΔΨm) and activation of caspase-6. Therefore, we believe that lathyrane diterpene might be a promising natural compound in ovarian cancer therapy.

Induction of G2/M phase arrest and apoptosis by a new tetrahydroingenol diterpenoid from Euphorbia erythradenia Bioss. in melanoma cancer cells.

In the current study, a new tetrahydroingenol diterpene isolated from Euphorbia erythradenia, 7,13-diacetyl-5-angeloyl-20-nicotinyl-3-propionyl-1,2,6,7-tetrahydroingenol (DANPT), were tested for the molecular mechanism of its anti-cancer activity in two human melanoma cancer cell lines, A375 and HMCB. DANPT was found cytotoxic against A375 and HMCB cells with IC50 value of 15.37±2.6µM and 15.62±1.89µM, respectively. Flow cytometric analysis showed that DANPT halted the A375 and HMCB cells in G2/M phase and induced apoptosis in a dose-dependent manner. Cell cycle arrest was associated with down-regulation of cyclin B and Cdk-1 and subsequent up-regulation of p53 and p21. Moreover, DANPT induced Bax and inhibited Bcl-2 expression, which results in increasing Bax/Bcl-2 ratio and activation of caspase-3. Furthermore, the apoptotic effect of DANPT was also related to ROS production and loss of mitochondrial membrane potential (ΔYm). Overall, our results suggest that DANPT can inhibit proliferation of human melanoma cancer cells by promoting apoptosis and inducing cell cycle arrest and therefore, it can be a promising natural agent for the treatment of melanoma cancer.

The effect of ω-fatty acids on the expression of phospholipase A2 group 2A in human gastric cancer patients.

BACKGROUND: Studies show that polyunsaturated fatty acids (PUFAs) may have an inhibitory role in carcinogenesis. It was previously shown that PLA2 group 2A (PLA2G2A) messenger RNA (mRNA) expression is associated with less frequent metastasis and longer survival in gastric adenocarcinoma. This study intends to investigate the effect of PUFAs on the expression of PLA2G2A in patients with gastric cancer. MATERIALS AND METHODS: Thirty-four patients with gastric cancer (GC) were randomly divided into two groups. The first group received cisplatin medication. The second group received cisplatin medication and supplements of ω-fatty acids for three courses. The total RNA was extracted from the tissues and cDNA was synthesized. The gene expression of PLA2G2A was evaluated by the real-time polymerase chain reaction (PCR) method. To confirm the changes in gene expression, frozen section was utilized. The frozen tissue samples were sectioned and stained using the immunohistochemistry technique. RESULTS: After chemotherapy and chemotherapy plus supplement, the relative mean of PLA2G2A gene expression increased 1.5 ± 0.5-fold and 7.4 ± 2.6-fold, respectively (P = 0.006). The relative mean of gene expression in patients who received cisplatin and ω-fatty acids supplement increased more significantly (7.5 ± 3.3-fold) than in patients who received only cisplatin (P = 0.016). CONCLUSION: It was found that PUFAs increased the gene and protein expression of PLA2G2A in gastric cancer. Concerning the fact that studies reveal protective function of PLA2G2A in gastric cancer, it is suggested that increased expression of PLA2G2A is helpful. Furthermore, PUFAs can be considered as a useful therapeutic supplement for patients with gastric cancer.

Bioactivity-guided isolation of new antiproliferative compounds from Juniperus foetidissima Willd.

Based on a literature survey on cytotoxic medicinal plants, Juniper species were identified as interesting source of antitumor compounds. Using bioassay-guided fractionation against Caov-4 cancer cells on acetone extract of leaves and branchlets of Juniperus foetidissima led to the isolation of a new 3H-benzofuaran-2-one: 4-methyl-3-methoxy-3H-benzofuaran-2-one (1), a new sesquiterpene: 4,9(α)-dihydroxy-nardosin-6-en (2) and an already known labdane-type diterpene: 15-hydroxy-8(17),13(E)-labdadiene-19-carboxilic acid (3). Compounds 1-3 exhibited cytotoxic effects, with moderate cytotoxicity against the EJ-138 bladder and CAOV-4 ovary cancer cell lines.

Herbal Extracts Induce Dermal Papilla Cell Proliferation of Human Hair Follicles.

BACKGROUND: The number of people suffering from balding or hair thinning is increasing, despite the advances in various medical therapies. Therefore, it is highly important to develop new therapies to inhibit balding and increase hair proliferation. OBJECTIVE: We investigated the effects of herbal extracts commonly used for improving balding in traditional medicine to identify potential agents for hair proliferation. METHODS: The expression levels of 5α-reductase isoforms (type I and II) were analyzed using quantitative real-time reverse transcription polymerase chain reaction in the human follicular dermal papilla cells (DPCs). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylteterazolium bromide and bromodeoxyuridine tests were used to evaluate the cell proliferation effect of herbal extracts in DPCs. The expression levels of extracellular signal-regulated kinase (ERK), Akt, cyclin D1, cyclin-dependent kinase 4 (Cdk4), B-cell lymphoma (Bcl-2) and Bcl-2-associated X protein (Bax) were measured using western blot analysis. RESULTS: The 5α-reductase isoform mRNAs and proteins were detected in the cultured DPCs, and the expression level of 5α-R2 in DPCs in the presence of the herbal extracts was gradually decreased. Herbal extracts were found to significantly increase the proliferation of human DPCs at concentrations ranging from 1.5% to 4.5%. These results show that the herbal extracts tested affected the protein expressions of ERK, Akt, cyclin D1, Cdk4, Bcl-2, and Bax in DPCs. CONCLUSION: These results suggest that herbal extracts exert positive effects on hair proliferation via ERK, Akt, cyclin D1, and Cdk4 signaling in DPCs; they also suggest that herbal extracts could be a great alternative therapy for increasing hair proliferation.

Molecular mechanism of apoptosis induction by Gaillardin, a sesquiterpene lactone, in breast cancer cell lines : Gaillardin-induced apoptosis in breast cancer cell lines.

Medicinal plant extracts have been widely used for cancer treatment. Gaillardin is a natural sesquiterpene lactone that has recently been reported to have anticancer properties. The ability to induce apoptosis is an important property of a candidate anticancer drug, which discriminates between anticancer drugs and toxic compounds. The current study was therefore carried out to address the issue if Gaillardin is able to induce apoptosis in the breast cancer cell lines MCF-7 and MDA-MB-468 and to determine the underlying mechanism of its anticancer effects. Apoptosis induction by Gaillardin treatment was confirmed by annexin V-FITC/PI staining, and caspase-3,-6, and-9 activation. Using Western blot analysis, we found that Gaillardin upregulated the pro-apoptotic protein Bax and p53 and downregulated the anti-apoptotic protein Bcl-2. Moreover, the apoptotic effect of Gaillardin was also related to ROS production and loss of mitochondrial membrane potential (ΔΨm). Taken together, these results demonstrate that Gaillardin can inhibit proliferation of breast cancer cells via inducing mitochondrial apoptotic pathway and therefore, might be a promising molecule in cancer chemoprevention or chemotherapy.

Unusual ingenoids from Euphorbia erythradenia Bioss. with pro-apoptotic effects.

Dichloromethane-acetone extract of Euphorbia erythradenia Bioss., a spurge endemic to the Iran, afforded four novel tetrahydroingenol diterpenes, one new myrsinane type diterpene, and two known triterpenes. Tetrahydroingenoids are novel compounds not only for the double bond reduction but also for their unique hydroxylation pattern at C-11 and C-13. The structures of these compounds were elucidated by spectroscopic methods, and especially 2D NMR measurements. The biological effects of the compounds were done by the MTT and Annexin V-FITC & PI staining assays on different cancer cell lines. The results obtained on EJ-138, CAOV-4, and OVCAR-3 cell lines suggested that tetrahydroingenanes inhibit cell proliferation through apoptosis in cancer cell lines. In conclusion, the new pattern of hydrogenation and hydroxylation of these compounds compared to other ingenoids, along with their apoptosis inducing properties on cancer cells, makes them of great interest for more investigation.

Combination of herbal extracts and platelet-rich plasma induced dermal papilla cell proliferation: involvement of ERK and Akt pathways.

BACKGROUND AND AIMS: Recently, platelet-rich plasma (PRP) has attracted attention in various medical fields, including plastic surgery, treatment for problematic wounds, and dermatology. Specifically, PRP has been tested during hair transplantation to reduce swelling and pain and to increase hair density. We examined the effects of PRP and herbal extracts combination in order to identify potential stimulants of hair growth. METHODS: PRP was prepared using the double-spin method and applied to dermal papilla cells (DPCs). MTT viability test and BrdU cell proliferation assay were used to study the effect of herbal extracts and PRP on proliferation of DPCs. To understand the mechanisms of herbal extracts and PRP involved in the regulation of hair growth, we evaluated signaling pathways and measured the expressions of ERK and Akt, by Western blot. RESULTS: Combination of herbal extracts and PRP was found to induce significant proliferation of human DPCs at concentrations ranging from 1.5% to 4.5%. The present study shows that herbal extracts and PRP affect the expressions of extracellular signal-regulated kinase (ERK) and Akt in DPCs. CONCLUSION: In this study, we have shown that combination of herbal extracts and PRP plays an active role in promoting the proliferation of human dermal papilla (DP) cells via the regulation of ERK and Akt proteins, and this may be applicable to the future development of herbal extracts and PRP combination therapeutics to enhance hair growth.