Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells

Ethnopharmacological relevance

Ziziphus extracts have been used in Traditional Chinese Medicine for the treatment of cancer.

Aim of the study

In the present study we have investigated the effects of Ziziphus jujube extracts (ZEs) on breast cancer.

Materials and methods

We evaluated the effects of increasing concentrations of ZEs on ERα positive MCF-7 and ERα negative SKBR3 breast cancer cell proliferation using MTT assays. Apoptosis was analyzed by evaluating the involvement of some pro-apoptotic proteins, including Bax, Bad, Bid and PARP cleavage by immunoblotting analysis. Moreover, the effects of ZEs treatment on apoptosis were tested by both DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. By using chromatographic techniques, we identified the constituents of the effective extracts.

Results

ZE1, ZE2, and ZE4 exerted significant antiproliferative effects on estrogen receptor alpha (ERα) positive MCF-7 (IC₅₀ values of 14.42, 7.64, 1.69 μg/mL) and ERα negative SKBR3 (IC₅₀ values of 14.06, 6.21, 3.70 μg/mL) human breast cancer cells. Remarkably, ZEs did not affect cell viability of both normal human fibroblasts BJ1-hTERT and nonmalignant breast epithelial MCF-10A cells. Treatment with ZEs induced cell death by apoptosis in both malignant breast cells. We found that the most effective extracts ZE2 and ZE4 shared a number of triterpenic acids, already known for their anticancer activities.

Conclusions

Our data provide a rational base for the use of Ziziphus extracts in the treatment of breast cancer in Traditional Chinese Medicine.     

Silibinin prevents TPA-induced MMP-9 expression by down-regulation of COX-2 in human breast cancer cells

Ethnopharmacological relevance

The expression of matrix metalloproteinase-9 (MMP-9) and cyclooxygenase-2 (COX-2) are pivotal steps in breast cancer pathogenesis. In a previous study, we reported that silibinin suppresses TPA-induced MMP-9 expression through the Raf/MEK/ERK pathway.

Aims of the study

Herein we determined the co-relationship between MMP-9 and COX-2, as well as the effect of silibinin on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 and COX-2 expression in the human breast cancer cells, MCF-7 and MDA-MB231.

Methods

The toxicity of silibinin was evaluated by Quick Cell Proliferation Assay Kit II. MMP-9 and COX-2 expression were analyzed by Zymography and Western blotting, respectively. Adenoviral constitutively active (CA)-MEK was used to activate MEK/ERK pathway.

Results

The expression of MMP-9 and COX-2 in response to TPA was increased, whereas TPA-induced MMP-9 and COX-2 expression was decreased by silibinin. Our results showed that TPA-induced MMP-9 expression was inhibited by celecoxib in a dose-dependent fashion, but not MMP-1-expression. Both MMP-9 and COX-2 expression were significantly increased by CA-MEK overexpression. In contrast, TPA-induced MMP-9 and COX-2 expression was decreased by UO126 (MEK1/2 inhibitor).

Conclusion

Silibinin down-regulates TPA-induced MMP-9 expression through inhibition of COX-2 expression in breast cancer cells.     

Effect of Artemisia species on cellular proliferation and apoptosis in human breast cancer cells via estrogen receptor-related pathway

Objective

To investigate the mechanism underlying the anticancer effect of Artemisia species through the inhibition of cell growth and induction of apoptosis in breast carcinoma cells.

Methods

To evaluate the anticancer activity of methanol extracts of eight Artemisia species (Artemisia stolonifera, Artemisia selengensis, Artemisia japonica, Artemisia Montana, Artemisia capillaris, Artemisia sylvatica, Artemisia keiskeana, and Artemisia scoparia), we first investigated the proliferation of estrogen receptor (ER)-positive MCF-7 breast carcinoma cells exposed to 5 or 200 g/mL for 72 h. Apoptosis induction was assessed by an Annexin V binding assay in cells exposed to extracts at a high concentration (200 g/mL). To verify the mechanism of apoptosis, ER expression and its related signaling was investigated using an immunoblot assay under the same conditions.

Results

MCF-7 cells showed the strongest antiproliferative response to the tested extracts. However, a biphasic effect was observed: the extracts inhibited proliferation at high concentrations whereas they stimulated it at low ones. ER expression was similarly modulated by the extracts. However, all of the extracts induced apoptosis at a high concentration (200 g/mL). Compared to the control level, exposure to the extracts resulted in a remarkable increase in the shift of cell populations.

Conclusion

The present study suggests that the tested Artemisia species exerted their anticancer effects through the induction of apoptosis via an ER-related pathway.     

Cytotoxic activity of 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda against human cancer cells

Ethnopharmacological relevance

Eulophia nuda L. (Orchidaceae) is a medicinally important terrestrial orchid used for the treatment of tumours and various health problems by the local healers throughout the Western Ghats region in Maharashtra (India).

Aim of the study

To isolate the active molecule from Eulophia nuda and to study its cytotoxic potential against human cancer cells.

Materials and methods

The crude methanolic extract of Eulophia nuda tubers was fractionated by stepwise gradient of the solvents—chloroform–methanol to isolate the pure compound. Isolated pure compound was assessed for its cytotoxic potential against human breast cancer cell lines, MCF-7 and MDA-MB-231 using MTT assay. Structure elucidation of the isolated active compound was carried out by extensive spectroscopic analysis including ¹H NMR, ¹³C NMR, NOESY, COSY, LC–MS and IR.

Results

The isolated active molecule was identified as phenanthrene derivative 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol. This compound showed good antiproliferative activity against human breast cancer cell lines MCF-7 (91%) and MDA-MB-231 (85%) at 1000 μg/ml concentration.

Conclusion

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda tubers showed good growth suppressive effect against human cancer cell lines MCF-7 and MDA-MB-231 making it a potential biomolecule against human cancer.     

Cyto-/genotoxic effects of the ethanol extract of Chan Su,a traditional Chinese medicine,in human cancer cell lines

Ethnopharmacological relevance

Chan Su, an ethanolic extract from skin and parotid venom glands of the Bufo bufo gargarizans Cantor, is widely used as a traditional Chinese medicine for cancer therapy. Although the anti-cancer properties of Chan Su have been investigated, no information exists regarding whether Chan Su has genotoxic effects in cancer cells. The aim of the present study was to examine the cyto-/genotoxic effect of Chan Su in human breast carcinoma (MCF-7 cells), human lung carcinoma (A-549 cells), human T cell leukemia (Jurkat T cells), and normal human lymphocytes.

Materials and methods

Effects on the viability of MCF-7, A-549, Jurkat T cells, and normal lymphocytes were evaluated by Trypan blue exclusion assays. The DNA content in the sub-G1 region was detected by propidium iodide (PI) staining and flow cytometry. The genotoxicity of Chan Su was assessed by single-cell gel electrophoresis (comet assay) and the cytokinesis-block micronucleus assay (CBMN assay).

Results

Chan Su significantly inhibited the viability of MCF-7, A-549, and Jurkat T cells dose dependently, but had no effect on normal human lymphocytes. Apoptotic death of the cancer cells was evident after treatment. Chan Su also induced genotoxicity in a dose-dependent manner, as indicated by the comet and cytokinesis-block micronucleus assays.

Conclusions

These findings suggest that Chan Su can induce apoptotic death of, and exert genotoxic effects on, MCF-7, A-549, and Jurkat T cells.     

Cytotoxicity of Chinese motherwort (YiMuCao) aqueous ethanol extract is non-apoptotic and estrogen receptor independent on human breast cancer cells

Background

Motherwort has been used as medicinal herb for many years in both China and Europe. In particular, Chinese motherwort has been commonly used to treat disorders of mammary gland in Chinese traditional medicine (TCM). Chinese motherwort aqueous extract (MAE) was previously reported to have anti-cancer activity in breast cancer cells with low potency (IC50s in a range of 8–40 mg/mL). However, treatment of motherwort ethanol extract in vivo markedly suppressed the development of uterine adenomyosis and mammary cancers in mice. Therefore, anti-cancer activity of Chinese motherwort, especially in a form of ethanol extract, needs to be confirmed further at cellular level.

Materials and methods

Aerial part of Chinese motherwort (Leonurus japonicus Houtt) dry powder is extracted with 70% ethanol and the chemical components were characterized with HPLC finger print as well as mass spectrometry. Cytotoxicity of the motherwort aqueous ethanol extract (MAEE) was analyzed with MTT assay on ER negative MDA-MB-231 and ER positive MCF-7 human breast cancer cell lines. Hoechst 33342 staining and flow cytometry were used to verify whether the cell death induced by MAEE is apoptosis in nature. Cell cycle status of MAEE treated cells were analyzed with flow cytometry.

Results

Our results showed that MAEE caused cell death in a dose-dependent and time-dependent fashion in both ER positive and negative breast cancer cells. Morphology, Hoechst 33342 staining and flow cytometry evidence all indicated the cell death is not in an apoptotic nature. Furthermore, low concentrations of MAEE caused cell cycle arrest at G2/M phase.

Conclusions

These data suggest that Chinese motherwort aqueous ethanol extract may effectively inhibit the proliferation of breast cancer cells through mechanisms of both cytotoxicity and cell cycle arrest. The cellular effects of MAEE are non-apoptotic and ER independent on breast cancer cells.     

In vitro anti-breast cancer activity of ethanolic extract of Wrightia tomentosa: Role of pro-apoptotic effects of oleanolic acid and urosolic acid

Ethnopharmacological relevance

Wrightia tomentosa Roem. & Schult. (Apocynaceae) is known in the traditional medicine for anti-cancer activity along with other broad indications like snake and scorpion bites, renal complications, menstrual disorders etc. However, the anti-cancer activity of this plant or its constituents has never been studied systematically in any cancer types so far.

Aim of the study

To evaluate the anti-cancer activities of the ethanolic extract of W. tomentosa and identified constituent active molecule(s) against breast cancer.

Material and methods

Powdered leaves of W. tomentosa were extracted with ethanol. The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa were tested for its anti-proliferative and pro-apoptotic effects in breast cancer cells MCF-7 and MDA-MB-231.

Results

The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa inhibited the proliferation of human breast cancer cell lines, MCF-7 and MDA-MB-231. The fraction F-4 obtained from hexane fraction inhibited proliferation of MCF-7 and MDA-MB-231 cells in concentration and time dependent manner with IC₅₀ of 50 μg/ml and 30 μg/ml for 24 h, 28 μg/ml and 22 μg/ml for 48 h and 25 μg/ml and 20 μg/ml for 72 h respectively. The fraction F-4 induced G1 cell cycle arrest, reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and subsequent apoptosis. Apoptosis is indicated in terms of increased Bax/Bcl-2 ratio, enhanced Annexin-V positivity, caspase 8 activation and DNA fragmentation. The active molecule isolated from fraction F-4, oleanolic acid and urosolic acid inhibited cell proliferation of MCF-7 and MDA-MB-231 cells at IC₅₀ value of 7.5 μM and 7.0 μM respectively, whereas there is devoid of significant cell inhibiting activity in non-cancer originated cells, HEK-293. In both MCF-7 and MDA-MB-231, oleanolic acid and urosolic acid induced cell cycle arrest and apoptosis as indicated by significant increase in Annexin-V positive apoptotic cell counts.

Conclusion

Our results suggest that W. tomentosa extracts has significant anti-cancer activity against breast cancer cells due to induction of apoptosis pathway. Olenolic and urosolic acid are important constituent molecules in the extract responsible for anti-cancer activity of W. tomentosa.     

Estrogenic and serotonergic butenolides from the leaves of Piper hispidum Swingle (Piperaceae)

Ethnopharmacological relevance

Our previous work has demonstrated that several plants in the Piperaceae family are commonly used by the Q’eqchi Maya of Livingston, Guatemala to treat amenorrhea, dysmenorrhea, and pain. Extracts of Piper hispidum Swingle (Piperaceae), bound to the estrogen (ER) and serotonin (5-HT7) receptors.

Aim of the study

To investigate the estrogenic and serotonergic activities of Piper hispidum extracts in functionalized assays, identify the active chemical constituents in the leaf extract, and test these compounds as agonists or antagonists of ER and 5-HT7.

Materials and methods

The effects of the Piper hispidum leaf extracts were investigated in estrogen reporter gene and endogenous gene assays in MCF-7 cells to determine if the extracts acted as an estrogen agonist or antagonist. In addition, the active compounds were isolated using ER- and 5-HT7 receptor bioassay-guided fractionation. The structures of the purified compounds were identified using high-resolution LC–MS and NMR spectroscopic methods. The ER- and 5-HT7-agonist effects of the purified chemical constituents were tested in a 2ERE-reporter gene assay in MCF-7 cells and in serotonin binding and functionalized assays.

Results

Three butenolides including one new compound (1) were isolated from the leaves of Piper hispidum, and their structures were determined. Compound 1 bound to the serotonin receptor 5-HT₇ with IC₅₀ values of 16.1 and 8.3 μM, respectively, and using GTP shift assays, Compound 1 was found to be a partial agonist of the 5-HT₇ receptor. The Piper hispidum leaf extracts, as well as Compounds 2 and 3 enhanced the expression of estrogen responsive reporter and endogenous genes in MCF-7 cells, demonstrating estrogen agonist effects.

Conclusions

Extracts of Piper hispidum act as agonists of the ER and 5-HT₇ receptors. Compound 1, a new natural product, identified as 9,10-methylenedioxy-5,6-Z-fadyenolide, was isolated as the 5-HT₇ agonist. Compounds 2 and 3 are reported for the first time in Piper hispidum, and identified as the estrogen agonists. No inhibition of CYP450 was observed for any of these compounds in concentrations up to 1 μM. These activities are consistent with the Q’eqchi traditional use of the plant for the treatment of disorders associated with the female reproductive cycle.     

Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: A bioassay-guided approach

Ethnopharmacological relevance

Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer.

Materials and methods

To investigate the apoptosis mechanism, we isolated dentatin (DTN) from this plant using a bioassay-guided approach. DTN-induced cytotoxicity was observed with the MTT assay. Acridine orange/propidium iodide staining was used to detect cells in early apoptosis and high content screening (HCS) to observe nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Apoptosis was confirmed with a clonogenic assay, DNA laddering and caspase 3/7 and 9 assays. Reactive oxygen species (ROS) formation, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. The involvement of nuclear factor-kappa B (NF-κB) was analysed with the HCS assay.

Results

A significant increase in chromatin condensation in the cell nucleus was observed by fluorescence analysis. Apoptosis was confirmed by the reduced number of colonies in the clonogenic assay and the increased number of cellular DNA breaks in treated cells observed as a DNA ladder. Treatment of MCF-7 cells with DTN encouraged apoptosis with cell death-transducing signals that reduced MMP by down-regulation of Bcl-2 and up-regulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase 9 followed by the executioner caspase 3/7. DTN treatment significantly arrested MCF-7 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated. Moreover, DTN significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus.

Conclusion

Together, the results demonstrated that the DTN isolated from Clausena excavata inhibited the proliferation of MCF-7 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway with involvement of the NF-κB signalling pathway.     

An in vitro study on antiproliferative and antiestrogenic effects of Boerhaavia diffusa L. extracts

Ethnopharmacological relevance

Boerhaavia diffusa L. (Nyctinaceae) is a plant of tropical region used in Indian traditional medicine for the treatment of human ailments including abdominal tumor, jaundice, dyspepsia, menstrual disorders, etc. This plant also has antilymphoproliferative, antimetastatic and immunomodulatory effects.

Aim of the study

This study aimed to assess the antiproliferative and antiestrogenic properties of methanol extract of Boerhaavia diffusa (BME) in MCF-7 breast cancer cell lines.

Materials and methods

The effective concentration range of BME on cell viability was analyzed using MTT assay. Hydroxylapatite assay (HAP) was carried out to confirm the competitive binding of BME to the estrogen receptor (ER). The effect of BME on the expression of a selected estrogen responsive gene pS2 was analyzed by RT-PCR. The ability of BME to alter the cell cycle phases and distributions were studied using FACS analysis.

Results

Treatment with varying concentrations of BME (20–320 μg/mL) resulted in moderate to very strong growth inhibition in MCF-7 cell lines. BME competed with [³H]-estradiol for binding to ER with IC₅₀ value of 320 ± 25 μg/mL. RT-PCR analysis revealed that BME reduced the mRNA expression of pS2 indicating the antiestrogenic action of BME. BME treatment for 48 h resulted in a remarkable increase in the number of MCF-7 cells in the G0-G1 fraction from 69.1% to 75.8%, with a reciprocal decrease of cells in all other phases indicating cell cycle arrest at G0-G1 phase.

Conclusions

The results demonstrate that Boerhaavia diffusa possess antiproliferative and antiestrogenic properties and suggest that it may have therapeutic potential in estrogen dependent breast cancers.     

Water extract of Rheum officinale Baill. induces apoptosis in human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines

Ethnopharmacological relevance

Rheum officinale Baill. (Da Huang) is one of the herbs commonly used in traditional Chinese medicine formulae against cancer. The traditional decoction is similar to the water extract used in the present study.

Aim of the study

The water extract of Da Huang was investigated to see if it possesses anticancer effects through apoptotic pathways.

Materials and methods

Human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines were treated with different concentrations of Da Huang water extract at different time intervals. Growth inhibition was detected by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and colony formation assays; apoptosis was detected by cell morphologic analysis, DNA fragmentation analysis and COMET assay.

Results

Da Huang water extract was found to have significant growth inhibitory effects on both A549 and MCF-7 cell lines with IC₅₀ values 620 ± 12.7 and 515 ± 10.1 μg/ml, respectively. Growth inhibitory effects were dose- and time-dependent. A significant decrease in cell number, DNA fragmentation and single DNA strand breakages were observed in the Da Huang water extract treated A549 and MCF-7 cells.

Conclusions

This suggests that the water extract of Da Huang exerts potential anticancer activity through growth inhibition and apoptosis on MCF-7 and A549 cells lines.     

Paris saponin VII from trillium tschonoskii reverses multidrug resistance of adriamycin-resistant MCF-7/ADR cells via P-glycoprotein inhibition and apoptosis augmentation

Ethnopharmacological relevance

Saponins of several herbs are known to induce apoptosis in some cancer cells and are proposed to be promising modulators of drug resistance. In the present study, we extracted Paris saponin VII (PS VII), a kind of saponin, from Trillium tschonoskii Maxim. and observed its effect on adriamycin-resistant breast cancer cells.

Materials and methods

An adriamycin-resistant human breast cancer cell line, MCF-7/ADR cells were exposed to different concentrations of PS VII (0–100 μmol/L). Then, flow cytometric assays and a human apoptosis array were used to detect apoptotic cells and apoptosis related protein expression. P-glycoprotein levels and intracellular rhodamine 123 (RH-123) accumulations were measured to evaluate the expression and activity of P-glycoprotein.

Results

PS VII dose dependently suppressed cell viability as well as triggered apoptosis and modulated drug resistance of MCF-7/ADR cells. Further results showed that PS VII treatment in MCF-7/ADR cells led to increased TNFR1, TRAIL R1/DR4, TRAIL R2/DR5, and FADD expression, and activation of PARP, caspase-8, and 3. In parallel to the alterations, P-glycoprotein expression and activity were also reduced.

Conclusion

These findings showed that PS VII might be an effective tumouristatic agent for the treatment of MDR breast cancer.     

In vitro estrogenic activity of Asplenium trichomanes L. extracts and isolated compounds

Ethnopharmacological relevance

Asplenium trichomanes was used as an expectorant, anti-cough remedy, laxative, emmenagogue, abortifacient and for irregular menses.

Aim of the study

To investigate the in vitro estrogenic activity of Asplenium trichomanes extracts and isolated compounds and their ability to activate ERα and ERβ.

Materials and methods

Leaves infusion (IF), decoction (DC) and methanol extract (ME) were prepared. MCF7/EREluc cell line which expresses endogenous ERα, and SK-NBE cells transiently transfected with the estrogen receptors (ERα and ERβ) were used for the estrogenic activity assays. Phytochemical investigations were performed (CC, HPLC, etc.) and structure of isolated compounds were achieved on the basis of 1D and 2D NMR techniques and HR-MS spectrometry.

Results

IF and ME were active in our MCF7 model; selectivity for the ERβ receptor was observed in the SK-NBE test. Two new phenol derivatives, 4-vinyl-phenol-1-O-[α-l-rhamno(1 → 6)-β-d-glucopyranosyde] (1) and kaempferol-3-O-α-[2′acetyl]-arabinofuranosyl-7-O-α-l-rhamnopyranoside (2) were isolated with six known compounds (3–8). Compounds 2–4, 7 and 8 showed selectivity for the activation of the ERβ receptor although with a moderate activity compared with 17-β-estradiol.

Conclusion

Further investigations about the estrogenic effects of this plant are needed but our data can, at least in part, explain some of its traditional use as emmeagogue.     

Hempseed oil induces reactive oxygen species- and C/EBP homologous protein-mediated apoptosis in MH7A human rheumatoid arthritis fibroblast-like synovial cells

Ethnopharmacological relevance

The medicinal efficacy of hempseed (Cannabis sativa L.), which is rich in polyunsaturated fatty acids, in atopic dermatitis, inflammation, and rheumatoid arthritis (RA) has been suggested for centuries. Hempseed has been used as a treatment for these diseases in Korean and Chinese folk medicine. The aim of the study is to investigate the effects of hempseed oil (HO) on MH7A human RA fibroblast-like synovial cells.

Materials and methods

MH7A cells were used to study the anti-rheumatoid effects of hempseed (Cannabis sativa L., cv. Cheungsam/Cannabaceae) oil by investigating cell viability, apoptosis, lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress-induced apoptosis.

Results

HO treatment reduced the survival rate of MH7A cells and promoted apoptotic cell death in a time- and dose-dependent manner. Both lipid accumulation and the level of intracellular reactive oxygen species (ROS) increased in HO-treated MH7A cells. Co-treatment with the antioxidant Tiron effectively abrogated the cytotoxic effects of HO; the ROS level was reduced, cell viability was recovered, and apoptotic cell death was significantly diminished. Moreover, HO-treated cells exhibited increased expression of the major ER stress markers, glucose-regulated protein 78 and C/EBP homologous protein (CHOP). The siRNA-mediated knockdown of CHOP prevented HO-induced apoptosis.

Conclusions

Our results suggest that HO treatment induced lipid accumulation, ROS production, CHOP expression, and apoptosis in MH7A cells, and that CHOP functions as an anti-rheumatoid factor downstream of HO in MH7A cells.     

Bee venom suppresses PMA-mediated MMP-9 gene activation via JNK/p38 and NF-κB-dependent mechanisms

Ethnopharmacological relevance

Bee venom has been used for the treatment of inflammatory diseases such as rheumatoid arthritis and for the relief of pain in traditional oriental medicine.

Aim of the study

The purpose of this study is to elucidate the effects of bee venom on MMP-9 expression and determine possible mechanisms by which bee venom relieves or prevents the expression of MMP-9 during invasion and metastasis of breast cancer cells. We examined the expression and activity of MMP-9 and possible signaling pathway affected in PMA-induced MCF-7 cells.

Material and methods

Bee venom was obtained from the National Institute of Agricultural Science and Technology of Korea. Matrigel invasion assay, wound-healing assay, zymography assay, western blot assay, electrophoretic mobility shift assay and luciferase gene assay were used for assessment.

Results

Bee venom inhibited cell invasion and migration, and also suppressed MMP-9 activity and expression, processes related to tumor invasion and metastasis, in PMA-induced MCF-7 cells. Bee venom specifically suppressed the phosphorylation of p38/JNK and at the same time, suppressed the protein expression, DNA binding and promoter activity of NF-κB. The levels of phosphorylated ERK1/2 and c-Jun did not change. We also investigated MMP-9 inhibition by melittin, apamin and PLA₂, representative single component of bee venom. We confirmed that PMA-induced MMP-9 activity was significantly decreased by melittin, but not by apamin and phospholipase A₂. These data demonstrated that the expression of MMP-9 was abolished by melittin, the main component of bee venom.

Conclusion

Bee venom inhibits PMA-induced MMP-9 expression and activity by inhibition of NF-κB via p38 MAPK and JNK signaling pathways in MCF-7 cells. These results indicate that bee venom can be a potential anti-metastatic and anti-invasive agent. This useful effect may lead to future clinical research on the anti-cancer properties of bee venom.     

Cytotoxic and antibacterial triterpenoids derivatives from Clematis ganpiniana

Ethnopharmacological relevance

The roots and rhizomes of Clematis are commonly used as an analgesic, abirritative, antibacterial, antiphlogistic, anticancer and diuretic agent. The Naxi people traditionally used Clematis ganpiniana's (Lévl. Et Vant.) as a diuretic agent, an anti-inflammatory and anticancer remedy.

Aim of study

To investigate the cytotoxic and antibacterial components from Clematis ganpiniana.

Materials and methods

The aboveground part of Clematis ganpiniana was isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods and comparison with published data. Their cytotoxic, apoptosis and antibacterial activities of purified components were also performed.

Results

By bioassay-guided fractionation techniques and chemical characterization, four triterpene glycosides were isolated and their cytotoxicity against cancer cells and antibacterial activity were tested. They showed significant inhibitory activities against MCF-7, MDA-MB-231 with IC50 value of 0.7–16.5 μg/ml, and significant apoptosis for MCF-7 and MDA-MB-231. Moreover, compound 4 showed weak wide-spectrum antibacterial activity.

Conclusion

These results provide promising baseline information for the potential use of Clematis ganpiniana as well as some of the isolated compounds in the treatment of cancer and infectious disease.     

Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata mycelia are mediated by down-regulation of the expressions of four tumor-related genes in human non-small cell lung carcinoma A549 cell

Aim of the study

Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan which is believed to be effective in preventing diseases. Recent reports demonstrate that Antrodia camphorata products induce the apoptosis of various kinds of tumor cells. In this study we determined the inhibitory effects of alcohol extract and individual fractions of alcohol extract on the proliferation of human non-small cell lung carcinoma A549 cell and clarified the mechanism underlying the anti-cancer activities.

Materials and methods

Alcohol extracts of Antrodia camphorata mycelia were prepared by the serial extraction with the solvents with increasing polarity and fractionated using HPLC. Cell viability was determined by MTT assay. Apoptosis detection was carried out by subG₁ analysis and annexin V/propidium iodide staining using flow cytometry. The impacts of HPLC fractions on the expression levels of apoptosis- and cancer-related proteins were evaluated by western blotting.

Results

Three HPLC fractions, fractions 5–7, had robust inhibition of human A549 cells and among them fraction 6 (Fr-6) possessed the most potent effectiveness. Apoptotic assay showed that Fr-6-induced human A549 cell apoptosis by triggering the mitochondrial pathway and endothelium reticulum (ER) stress. Immunoblotting results demonstrated that Fr-6 possibly activated ER stress by lowering the expression level of calpain 1/2 small subunit and Fr-6-mediated decrease in cell proliferation might attribute to the suppressive effect on the Erk 1/2 pathway, which arose from Fr-6-derived low galectin-1 expression. Furthermore Fr-6 could diminish Rho GDP dissociation inhibitor α (RhoGDI-α) expression and subsequently activated c-Jun NH₂-terminal kinase (JNK) pathway, which is linked to cell apoptosis. Fr-6 also could decrease the production level of eukaryotic translation initiation factor 5A, which is a potential cancer intervention target.

Conclusion

These results suggested that the anti-cancer activity of Antrodia camphorata might be due to multiple active metabolites, which work together to induce cell apoptosis via various pathways.