Selective induction of DNA damage,G2 abrogation,and mitochondrial apoptosis by leaf extract of traditional medicinal plant <Emphasis Type="Italic">Wrightia arborea</Emphasis> in K562 cells

Plants have proved to be an important source of anti-cancer drugs. Wrightia arborea, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. This study evaluates the antiproliferative/apoptotic potential of Wrightia arborea leaf extracts, prepared in different organic solvents, on cancer cell lines. MTT assay, light and fluorescence microscopy, flow cytometry, DNA laddering, alkaline comet assay, and western blotting were some of the techniques used for evaluation. Combinations of camptothecin, either with CHK1 inhibitor-PD407824 or with W. arborea leaf extract, were deployed to determine the G2 abrogating potential of the extract. The chloroform extract (WAC) selectively killed K562 cells, without affecting cancerous MCF-7, Hep G2 cells, and normal human peripheral blood lymphocytes. Cell death was characterized by observation of apoptotic bodies, increased Ca2+ and ROS, phosphatidyl serine externalization, mitochondrial membrane depolarization, DNA laddering, increased sub-G1 population, and altered expression of caspase 3, ?9, and PARP. WAC also induced DNA damage, alterations in key G2/M phase protein expression, cell cycle perturbation, and potent G2 abrogation. The present study showed that W. arborea leaf extract, WAC, is capable of selectively killing leukemic cancer cells leaving normal lymphocytes unaffected. Our results indicate that this is effectuated through DNA damage and G2 abrogation leading to mitochondrial apoptosis. Taken together, this report contributes toward a better understanding of the anticancer properties of this traditional medicinal plant extract possessing valuable bioactive constituents which can serve as a bioresource for promising complimentary/alternative/chemopreventive therapeutics.     

Characterization of the antiproliferative activity of Xylopia aethiopica

Background

Xylopia aethiopica, a plant found throughout West Africa, has both nutritional and medicinal uses. The present study aims to characterize the effects of extracts of this plant on cancer cells.

Results

We report that X. aethiopica extract prepared with 70% ethanol has antiproliferative activity against a panel of cancer cell lines. The IC50 was estimated at 12 ??g/ml against HCT116 colon cancer cells, 7.5 ??g/ml and > 25 ??g/ml against U937 and KG1a leukemia cells, respectively. Upon fractionation of the extract by HPLC, the active fraction induced DNA damage, cell cycle arrest in G1 phase and apoptotic cell death. By using NMR and mass spectrometry, we determined the structure of the active natural product in the HPLC fraction as ent-15-oxokaur-16-en-19-oic acid.

Conclusion

The main cytotoxic and DNA-damaging compound in ethanolic extracts of Xylopia aethiopica is ent-15-oxokaur-16-en-19-oic acid.     

Validation of the antiproliferative effects of <Emphasis Type="Italic">Euphorbia tirucalli</Emphasis> extracts in breast cancer cell lines

Medicinal plant extracts have recently attracted attention of modern medical science research due to their non-lethal activity. Currently, up to 50% of the world drugs including chemotherapeutic drugs such as taxol and camptothecin are derived from natural products. Euphorbia tirucalli has a long history of usage as traditional medicine in Africa and has been widely used in the treatment of different cancers. In this study, we explore the medical properties of E. tirucalli extracts in breast cancer development. To achieve this, stems of E. tirucalli were dried, crushed and extracted with butanol, hexane or methanol (based on 1 g of dry substance in 10 mL of a solvent). The dried extracts were re-dissolved in DMSO and investigated. Composition of each extract was analyzed using liquid chromatography-mass spectroscopy (LC-MS). Extracts were found to contain different types of secondary metabolites mainly terpenes and flavonoids. Breast cancer cell lines (MCF-7 and MDA-MB 231) were treated with various concentrations of the extracts for up to 48 h. Cell viability, cell cycle, apoptosis and gene expression were analysed. In cells, extracts were found to inhibit cell proliferation in a concentration and cell type dependent manner. Analysis of the cause of antiproliferation revealed that most cells were arrested at the G0/G1 phase by p21 overexpression. In general, most pro-apoptotic genes like Bax and caspase-8 were significantly up-regulated in cells treated with plant extracts. These results suggest that the extracts might induce cell cycle arrest at G0/G1 with p21 attributing to this molecular mechanism.     

G2/M cell cycle arrest and induction of apoptosis by a stilbenoid, 3,4,5-trimethoxy-4'-bromo-cis-stilbene, in human lung cancer cells

Stilbenoids, including resveratrol (3,5,4'-trihydroxy-trans-stilbene) which is a naturally occurring phytoalexin abundant in grapes and several plants, have been shown to be active in inhibiting proliferation and inducing apoptosis in human cancer cell lines. Using resveratrol as the prototype, we have synthesized various analogs and evaluated their growth inhibitory effects in cultured human cancer cells. In the present study, we show that one of the stilbenoids, 3,4,5-trimethoxy-4'-bromo-cis-stilbene (BCS), was more effective than its corresponding trans-isomer and resveratrol on the inhibition of cancer cell growth. Prompted by the strong growth inhibitory activity of BCS (IC50; 0.03 microM) compared to its trans-isomer (IC50; 6.36 microM) and resveratrol (IC50; 33.0 microM) in cultured human lung cancer cells (A549), we investigated its mechanism of action. BCS induced arrest at the G2/M phase cell cycle in the early time and subsequently increased in the sub-G1 phase DNA contents in a time-dependent manner, indicating induction of apoptosis. Morphological observation with round-up shape and DNA fragmentation was also revealed the apoptotic phenomena. BCS treatment elevated the expression levels of the pro-apoptotic protein p53, the cyclin-dependent kinase inhibitor p21, and the release of cytochrome c in the cytosol. The down-regulation of checkpoint protein cyclin B1 by BCS was well correlated with the cell cycle arrest at G2/M. These data suggest the potential of BCS to serve as a cancer chemotherapeutic or chemopreventive agent by virtue of arresting the cell cycle and induction of apoptosis of human lung cancer cells.     

Differential effects of soybean and fenugreek extracts on the growth of MCF-7 cells

The effect of aqueous and ethanol extracts of soybean and fenugreek on the growth of MCF-7 cells, an estrogen receptor positive breast cancer cell line, has been examined in this study. Soybean is well known for the presence of phytoestrogens and fenugreek is reported to have medicinal use including anticancer properties. In a dose dependent manner soybean aqueous and ethanol extract promoted the growth and DNA synthesis in MCF-7 cells. On the contrary ethanol extract of fenugreek decreased the cell viability and induced early apoptotic changes such as flipping of phosphatidylserine and decrease of mitochondrial membrane potential. Degradation of cellular DNA into fragments comprising multiples of approximately 180-200 base pair was also observed. Cell cycle analysis by flow cytometry showed the presence of a subG1 apoptotic population which was more prominent at higher concentrations along with cell cycle arrest at G2/M phase. Our experiments show that while the soybean extract acts as a promoter of MCF-7 cell growth, the fenugreek extract induces apoptosis.     

Inhibition of human tumor cell proliferation by novel anthraquinones from daylilies

Daylilies (Hemerocallis) are used medicinally in eastern Asia and extracts of the plant had been shown to inhibit cell proliferation and induce cancer cells to undergo differentiation. In our studies of the constituents of Hemerocallis fulva var. 'Kwanzo' roots, we isolated a series of new [kwanzoquinones A (1), B (2), C (4), D (5), E (6), F (7), G (9)] and known [2-hydroxychrysophanol (3) and rhein (8)] anthraquinones. These compounds were tested in order to determine their potential roles as cancer cell growth inhibitors. Kwanzoquinones A-C and E, kwanzoquinone A and B monoacetates (1a and 2a), 2-hydroxychrysophanol, and rhein inhibited the proliferation of human breast, CNS, colon, and lung cancer cells with GI50 values between 1.8 to 21.1 microg/mL. However, upon exposure of the cancer cells to the GI50 concentrations of the bioactive anthraquinones, most of the cancer cell lines exhibited higher than anticipated levels of cell viability. Co-incubation of the anthraquinones with vitamins C and E increased the viability of breast cancer cells. In contrast, vitamins C and E potentiated the cytotoxic effects of the anthraquinones against the colon cancer cells. None of the anthraquinones inhibited the activity of topoisomerase.     

p53 Regulates Cellular Responses to Environmental Carcinogen Benzo[a]pyrene-7,8-diol-9,10-epoxide in Human Lung Cancer Cells

The p53 tumor suppressor is a mutational target of environmental carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). We now demonstrate that p53 plays an important role in regulation of cellular responses to BPDE. Exposure of p53-null H1299 human lung cancer cells to BPDE resulted in S and G2 phase cell cycle arrest, but not mitotic block, which correlated with induction of cyclin B1 protein expression, down-modulation of cell division cycle 25C (Cdc25C) and Cdc25B protein levels, and hyperphosphorylation of Cdc25C (S216), cyclin-dependent kinase 1 (Cdk1; Y15), checkpoint kinase 1 (Chk1; S317 and S345) and Chk2 (T68). The BPDE-induced S phase block, but not the G2/M phase arrest, was significantly attenuated by knockdown of Chk1 protein level. The BPDE-mediated accumulation of sub-diploid fraction (apoptotic cells) was significantly decreased in H1299 cells transiently transfected with both Chk1 and Chk2 specific siRNAs. The H460 human lung cancer cell line (wild-type p53) was relatively more sensitive to BPDE-mediated growth inhibition and enrichment of sub-diploid apoptotic fraction compared with H1299 cells. The BPDE exposure failed to activate either S or G2 phase checkpoint in H460 cells. Instead, the BPDE-treated H460 cells exhibited a nearly 8-fold increase in sub-diploid apoptotic cells that was accompanied by phosphorylation of p53 at multiple sites. Knockdown of p53 protein level in H460 cells attenuated BPDE-induced apoptosis but enforced activation of S and G2 phase checkpoints. In conclusion, the present study points towards an important role of p53 in regulation of cellular responses to BPDE in human lung cancer cells.     

Resveratrol, a natural phenolic compound, inhibits cell proliferation and prevents oxidative DNA damage

Resveratrol (3,4',5-trihydroxystilbene) is a naturally occurring phenolic compound which is present at high levels in wine and has been recently proposed as a potential cancer chemopreventive and chemoterapeutic agent. In this study, we evaluated the antiproliferative activity of resveratrol on a panel of cell lines of various histogenetic origin, including normal rat fibroblasts and mouse mammary epithelial cells compared to human breast, colon and prostate cancer cells. The concentration of resveratrol inhibiting cell growth by 50% (IC(50)) ranged from about 20 to 100 microM. At such concentration, we were unable to detect a significant increase in the apoptotic index in most of the cell lines analyzed.We also studied the effects of resveratrol on cell cycle distribution. The most striking effect was a reduction in the percentage of cells in the G2/M phase which was most frequently associated with an increase of cells in the S phase of the cell cycle. We also found that resveratrol is able to prevent the increase in reactive oxygen species (ROS) following exposure to oxidative agents (i.e. tobacco-smoke condensate (TAR) and H(2)O(2)). Resveratrol also reduced nuclear DNA fragmentation, as assessed by single cell gel electrophoresis (comet test). Taken together our results suggest that resveratrol can act as an antimutagenic/anticarcinogenic agent by preventing oxidative DNA damage which plays a pivotal role in the carcinogenic activity of many genotoxic agents.     

A potential antioxidant resource: Endophytic fungi from medicinal plants

Medicinal plants and their endophytes are important resources for discovery of natural products. Several previous studies have found a positive correlation between total antioxidant capacity (TAC) and total phenolic content (TPC) of many medicinal plant extracts. However, no information is available on whether such a relationship also exists in their endophytic fungal metabolites. We investigated the relationship between TAC and TPC for 292 morphologically distinct endophytic fungi isolated from 29 traditional Chinese medicinal plants. The antioxidant capacities of the endophytic fungal cultures were significantly correlated with their total phenolic contents, suggesting that phenolics were also the major antioxidant constituents of the endophytes. Some of the endophytes were found to produce metabolites possessing strong antioxidant activities. Several bioactive constituents from the fungal cultures and host plant extracts were identified. This investigation reveals that the metabolites produced by a wide diversity of endophytic fungi in culture can be a potential source of novel natural antioxidants.     

Antioxidant and anticancer activity of extract from Betula platyphylla var. japonica

The antioxidant and anticancer properties of a medicinal plant, Betula platyphylla var. japonica were investigated. The total methanol extract of B. platyphylla var. japonica had protective effects against hydrogen peroxide (H2O2) in the Chinese hamster lung fibroblast (V79-4) cell line and induced apoptotic cell death in human promyelocytic leukemia (HL-60) cells, a cancer cell line. B. platyphylla var. japonica extract significantly increased cell viability against H2O2. The extract also showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 2.4 microg/ml) and lipid peroxidation inhibitory activity (IC50 below 4.0 microg/ml). Furthermore, B. platyphylla var. japonica extract reduced the number of V79-4 cells arrested in G2/M in response to H2O2 treatment and increased the activities of several cellular antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. Treatment with B. platyphylla var. japonica extract induced cytotoxicity and apoptosis in HL-60 cells, as shown by nucleosomal DNA fragmentation, increases in the subdiploid cell population, and fluorescence microscopy. B. platyphylla var. japonica extract gradually increased the expression of pro-apoptotic Bax and led to the activation of caspase-3 and cleavage of PARP. These findings suggest that B. platyphylla var. japonica exhibits potential antioxidant and anticancer properties.     

Molecular perspective and anticancer activity of medicinal plants

To evaluate phytochemical constituents from the methanolic extracts of medicinal plants Aloe castellorum and Aloe pseudorubroviolacea. The cytotoxic activity of Aloe castellorum and Aloe pseudorubroviolacea leaf extracts against Human colon cancer cell line (HCT-116) was also assessed. The two medicinal plant extracts having significant cytotoxic activity, meanwhile the methanolic extract of Aloe castellorum shows higher cytotoxic activity than Aloe pseudorubroviolacea extract. The Aloe castellorum shows remarkable activity against respective cell line than control. The characteristic chemical constituents of Aloe castellorum and Aloe pseudorubroviolacea leaf extracts were recognized from Gas chromatography and Mass spectrometry (GC–MS) technique. The molecular docking studies also support the cytotoxic activity.     

Effects of dihydroxy gymnemic triacetate (DGT) on expression of apoptosis associated proteins in human prostate cancer cell lines (PC3)

Abstract

Prostate cancer is the most common malignancies among men. The present study is aimed at the investigation of dihydroxy gymnemic triacetate (DGT) from Gymnema sylvestre on mitochondrial apoptotic pathway and cell cycle arrest. Treatment of DGT resulted in a dose-dependent inhibition of growth of PC-3 cells. The cell cycle arrest was observed at the G2/M phase and accumulation of apoptotic cells was observed in DGT-treated prostate cancer cell lines. The occurrence of apoptosis in these cells was observed by DNA fragmentation. These events were associated with increased levels of pro-apoptotic proteins Bax, Bad and reduced levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1. DGT also induces the activation of caspase-9 and caspase-3. The above results, clearly, suggest that DGT induces apoptosis by the intrinsic pathways which could be very useful for the treatment of prostate cancer.     

Comparative Quantitative Study of Ardisiacrispin A in Extracts from Ardisia crenata Sims Varieties and Their Cytotoxic Activities

Ardisia crenata Sims (Primulaceae) occurs in natural habitats in two varieties, bearing red or white fruits. While roots of the red-berried ardisia are valued as a medicinal product, the pharmacological activity of which is attributed to triterpene saponins, including ardisiacrispin A, data on the white-berried variety are scarce. A TLC-densitometric method was developed and validated to estimate the levels of saponins, calculated as ardisiacrispin A, in different plant parts in both varieties. Their content amounted to 22.17±4.75 and 25.72±1.46 mg/g d.w. in roots, and 2.64±0.74 and 3.43±0.70 mg/g d.w. in fruits of red-berried and white-berried ardisia, respectively. Assessment of cytotoxicity of ardisiacrispin A and A. crenata extracts on a panel of human cancer cell lines revealed a similar effect of root extracts from both varieties, with the highest potency against melanoma WM793 and colon cancer Caco2. Thus, roots of the white-berried variety may be treated as a substitute for red-berried ardisia and serve as an alternative source for the acquisition of plant material rich in bioactive saponins.     

Proliferative inhibition, cell-cycle dysregulation, and induction of apoptosis by ursolic acid in human non-small cell lung cancer A549 cells

Ursolic acid (UA) is a pentacyclic triterpene compound isolated from many types of medicinal plants and is present in human diet. It has been reported to possess a wide range of pharmacological properties, and is one of the most promising chemopreventive agents for cancer. Here, we report that UA inhibits the cell proliferation of human lung cancer cell line A549 and provide a molecular understanding of this effect. The results showed that UA blocked cell cycle progression in the G1 phase that was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner cdk2, 4, and 6 with concomitant induction of p21/WAF1. This accumulation of p21/WAF1 might be through a p53-dependent manner. Further, UA treatment also resulted in the triggering of apoptosis as determined by DNA fragmentation assay. This effect was found to correlate with the up-regulation of Fas/APO-1, Fas ligand, and Bax, and down-regulation of NF-kappaB, Bcl-2, and Bcl-XL. Taken together, our study indicated that UA might be a potential chemopreventive agent for lung cancer.     

Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines

In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70–90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2–3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast and colorectal cancers.     

Knockdown of response gene to complement 32 (RGC32) induces apoptosis and inhibits cell growth,migration, and invasion in human lung cancer cells

Response gene to complement 32 (RGC32) is a novel protein originally identified as a cell cycle activator and has been demonstrated to be overexpressed in a variety of human malignancies, including lung cancer. However, the potential role of RGC32 in lung cancer initiation and progression remains to be elucidated. In the present study, RNA interference mediated by plasmid expressing RGC32 short-hairpin RNA (shRNA) was utilized to knockdown RGC32 expression in human lung cancer LTE cells. We found that the mRNA and protein expression levels of RGC32 were significantly decreased in RGC32-specific shRNA-transfected cells in comparison with the untransfected and control shRNA-transfected cells. Furthermore, knockdown of RGC32 dramatically reduced cell proliferation, colony formation, and invasion and migration capacities of LTE cells in vitro. Specific down-regulation of RGC32 caused G0/G1 cell cycle arrest and eventual apoptosis. Meanwhile, Western blot analysis indicated that cells with stably knockdown of RGC32 showed decreased expression levels of Cyclin D1, Cyclin E, Bcl-2, matrix metalloproteinase (MMP)-2, and MMP-9, but increased expression levels of activate caspase-3, Bax, and cleaved poly (ADP-ribose) polymerase (PARP) in comparison with control shRNA-transfected cells. Taken together, our data suggest that RGC32 is involved in tumorigenesis of human lung cancer and may serve as a promising therapeutic target for lung cancer.     

The in vitro antibiofilm activity of selected culinary herbs and medicinal plants against Listeria monocytogenes

Aims:  The antibiofilm activity of extracts obtained from selected herbs, spices, beverages and commercially important medicinal plants was investigated on Listeria monocytogenes .
Methods and Results:  The growth and development of the biofilm was assessed using the crystal violet (CV) assay. The respiratory activity was assessed using the 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. The majority of extracts tested prevented cell adhesion to the polyvinyl chloride (PVC) surface. Seven of the 15 extracts reduced biofilm adhesion of both the clinical and the type strains by at least 50%. In contrast, inhibition of a preformed biofilm was more difficult to achieve, with only three extracts ( Rosmarinus officinalis, Mentha piperita and Melaleuca alternifolia ) inhibiting the growth of both strains by at least 50%.
Conclusions:  Although most extracts were able to reduce initial cell attachment, inhibition of growth in a preformed biofilm was more difficult to achieve.
Significance and Impact of the Study:  The ability to reduce biofilm biomass as shown by several plant extracts warrants further investigation to explore the use of natural products in antibiofilm adhesion.     

Vernodalidimer L,a sesquiterpene lactone dimer from Vernonia extensa and anti-tumor effects of vernodalin,vernolepin, and vernolide on HepG2 liver cancer cells

Vernonia extensa, known as “Phim Phai Lin” in Thai, is distributed in most regions of Thailand. The plant has been used in Ayurveda and traditionally used to treat malaria and cancer, and possesses several sesquiterpene lactones. This study aimed to investigate and identify the active constituents by bioactivity-based analysis, as well as to evaluate the cytotoxic activity of V. extensa by MTT or XTT assays in ten cancer cell lines (Liver HepG2 and S102; Bile duct HuCCA-1; Leukemia HL-60 and MOLT-3; Lung A549 and H69AR; Breast MDA-MB-231 and T47D; Cervical HeLa). Bioactivity-guided fractionation and semi-preparative HPLC purification were used to separate the bioactive constituents. Apoptosis-inducing activity and cell cycle inhibitory effect of selected active compounds were determined on HepG2 cells by flow cytometric analysis. Bioactivity-guided fractionation of the CH₂Cl₂ extract and chemical investigation of the cytotoxic fractions led to the isolation of a new sesquiterpenoid pseudo-dimer named vernodalidimer L, together with eight known sesquiterpenoids from the aerial part of V. extensa. The structures of the isolates were elucidated based on spectroscopic analysis, including 1D and 2D NMR and HRMS. Vernolide has potent broad-spectrum cytotoxicity with IC₅₀ values in the range of 0.91–13.84 μM, against all ten cancer cell lines. The annexin-V flow cytometric analysis showed that vernodalin, vernolepin, and vernolide induced apoptosis on HepG2 cells in a dose dependent manner and these effects correlated with G2/M phase cell cycle arrest. Our results indicated that vernodalin, vernolepin, and vernolide have potential to be used as lead compounds in the development of a therapeutic natural product for treatment of liver cancer.