Clausenidin Induces Caspase 8-Dependent Apoptosis and Suppresses Production of VEGF in Liver Cancer Cells

Clausena excavata Burm f. is used by traditional healers to treat cancer patients in South East Asia. The use of the plant and its compounds is based on Asian folklore with little or no scientific evidence supporting the therapeutic efficacy The current study aimed to determine the effect of pure clausenidin isolated from C. excavata on caspase-8-induced cell death as well as angiogenesis in the HepG2 hepatocellular carcinoma cell line. Caspase-8 and extrinsic death receptor protein expression was determined using spectrophotometry and protein profile arrays, respectively. Ultrastructural analysis of clausenidin-treated cells was conducted using transmission electron microscopy. In addition, anti-angiogenic effects of clausenidin were investigated by Western blot analysis. Clausenidin significantly (p<0.05) increased the activity of caspase-8 and expression of protein components of the death inducing signaling complex (DISC) in HepG2 cells. Ultrastructural analysis of the clausenidin-treated HepG2 cells revealed morphological abnormalities typical of apoptosis. Furthermore, clausenidin significantly (p<0.05) decreased the expression of vascular endothelial growth factor (VEGF). Therefore, clausenidin is a potential anti-angiogenic agent which may induce apoptosis of hepatocellular carcinoma cells.

Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells.

Uridine-cytidine kinase 2 is an enzyme that is overexpressed in abnormal cell growth and its implication is considered a hallmark of cancer. Due to the selective expression of UCK2 in cancer cells, a selective inhibition of this key enzyme necessitates the discovery of its potential inhibitors for cancer chemotherapy. The present study was carried out to demonstrate the potentials of natural phytochemicals from the rhizome of Alpinia mutica to inhibit UCK2 useful for colorectal cancer. Here, we employed the used of in vitro to investigate the effectiveness of natural UCK2 inhibitors to cause HT-29 cell death. Extracts, flavokawain B, and alpinetin compound from the rhizome of Alpinia mutica was used in the study. The study demonstrated that the expression of UCK2 mRNA were substantially reduced in treated HT-29 cells. In addition, downregulation in expression of 18S ribosomal RNA was also observed in all treated HT-29 cells. This was confirmed by fluorescence imaging to measure the level of expression of 18S ribosomal RNA in live cell images. The study suggests the possibility of MDM2 protein was downregulated and its suppression subsequently activates the expression of p53 during inhibition of UCK2 enzyme. The expression of p53 is directly linked to a blockage of cell cycle progression at G0/G1 phase and upregulates Bax, cytochrome c, and caspase 3 while Bcl2 was deregulated. In this respect, apoptosis induction and DNA fragmentation were observed in treated HT-29 cells. Initial results from in vitro studies have shown the ability of the bioactive compounds of flavokawain B and alpinetin to target UCK2 enzyme specifically, inducing cell cycle arrest and subsequently leading to cancer cell death, possibly through interfering the MDM2-p53 signalling pathway. These phenomena have proven that the bioactive compounds could be useful for future therapeutic use in colon cancer.

Clausenidin from Clausena excavata induces apoptosis in hepG2 cells via the mitochondrial pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Clausena excavata Burm.f. is used locally in folk medicine for the treatment of cancer in South East Asia. AIM OF THE STUDY: To determine the mechanism of action of pure clausenidin crystals in the induction of hepatocellular carcinoma (hepG2) cells apoptosis. MATERIALS AND METHODS: Pure clausenidin was isolated from Clausena excavata Burm.f. and characterized using 1H and 13C NMR spectra. Clausenidin-induced cytotoxicity was determined by MTT assay. The morphology of hepG2 after treatment with clausenidin was determined by fluorescence and Scanning Electron Microscopy. The effect of clausenidin on the apoptotic genes and proteins were determined by real-time qPCR and protein array profiling, respectively. The involvement of the mitochondria in clausenidin-induced apoptosis was investigated using MMP, caspase 3 and 9 assays. RESULTS: Clausenidin induced significant (p<0.05) and dose-dependent apoptosis of hepG2 cells. Cell cycle assay showed that clausenidin induced a G2/M phase arrest, caused mitochondrial membrane depolarization and significantly (p<0.05) increased expression of caspases 3 and 9, which suggest the involvement of the mitochondria in the apoptotic signals. In addition, clausenidin caused decreased expression of the anti-apoptotic protein, Bcl 2 and increased expression of the pro-apoptotic protein, Bax. This finding was confirmed by the downregulation of Bcl-2 gene and upregulation of the Bax gene in the treated hepG2 cells. CONCLUSION: Clausenidin extracted from Clausena excavata Burm.f. is an anti-hepG2 cell compound as shown by its ability to induce apoptosis through the mitochondrial pathway of apoptosis. Clausenidin can potentially be developed into an anticancer compound.

Artonin E and Structural Analogs from Artocarpus Species Abrogates Estrogen Receptor Signaling in Breast Cancer.

The increasing rate of mortality ensued from breast cancer has encouraged research into safer and efficient therapy. The human Estrogen receptor α has been implicated in the majority of reported breast cancer cases. Molecular docking employing Glide, Schrodinger suite 2015, was used to study the binding affinities of small molecules from the Artocarpus species after their drug-like properties were ascertained. The structure of the ligand-binding domain of human Estrogen receptor α was retrieved from Protein Data Bank while the structures of compounds were collected from PubChem database. The binding interactions of the studied compounds were reported as well as their glide scores. The best glide scored ligand, was Artonin E with a score of -12.72 Kcal when compared to other studied phytomolecules and it evoked growth inhibition of an estrogen receptor positive breast cancer cells in submicromolar concentration (3.8-6.9 µM) in comparison to a reference standard Tamoxifen (18.9-24.1 µM) within the tested time point (24-72 h). The studied ligands, which had good interactions with the target receptor, were also drug-like when compared with 95% of orally available drugs with the exception of Artoelastin, whose predicted physicochemical properties rendered it less drug-like. The in silico physicochemical properties, docking interactions and growth inhibition of the best glide scorer are indications of the anti-breast cancer relevance of the studied molecules.

Dentatin from Clausena excavata Induces Apoptosis in HepG2 Cells via Mitochondrial Mediated Signaling.

Hepatocellular carcinoma (HCC) is a primary liver cancer with high global incidence and mortality rates. Current candidate drugs to treat HCC remain lacking and those in use possess undesirable side effects. In this investigation, the antiproliferative effects of dentatin (DTN), a natural coumarin, were evaluated on HepG2 cells and DTN's probable preliminary molecular mechanisms in apoptosis induction were further investigated. DTN significantly (p<0.05) suppressed proliferation of HepG2 cells with an IC50 value of 12.0 µg/mL, without affecting human normal liver cells, WRL-68 (IC50>50 µg/mL) causing G0/G1 cell cycle arrest via apoptosis induction. Caspase colorimetric assays showed markedly increased levels of caspase-3 and caspase-9 activities throughout the treatment period. Western blotting of treated HepG2 cells revealed inhibition of NF-κB that triggers the mitochondrial-mediated apoptotic signaling pathway by up-regulating cytoplasmic cytochrome c and Bax, and down-regulating Bcl-2 and Bcl-xL. The current findings suggest DTN has the potential to be developed further as an anticancer compound targeting human HCC.

Phytochemicals from Mangifera pajang Kosterm and their biological activities.

BACKGROUND: Mangifera pajang Kosterm is a plant species from the mango family (Anacardiaceae). The fruits are edible and have been reported to have high antioxidant content. However, the detailed phytochemical studies of the plant have not been reported previously. This study investigates the phytochemicals and biological activities of different parts of Mangifera pajang. METHODS: The plant samples were extracted with solvents of different polarity to obtain the crude extracts. The isolated compounds were characterized using spectroscopic methods. The extracts and isolated compounds were subjected to cytotoxicity tests using human breast cancer (MCF-7), human cervical cancer (HeLa) and human colon cancer (HT-29) cells. The free radical scavenging activity test was conducted using the DPPH assay. Antimicrobial activity tests were carried out by using the disc diffusion method. RESULTS: Phytochemical investigation on the kernel, stem bark and leaves of Mangifera pajang led to the isolation of methyl gallate (1), mixture of benzaldehyde (2) and benzyl alcohol (3), mangiferonic acid (4), 3ß-hydroxy-cycloart-24-ene-26-oic acid (5), 3ß,23-dihydroxy-cycloart-24-ene-26-oic acid (6), lupeol(7) lupenone(8), ß-sitosterol(9), stigmasterol(10), trans-sobrerol(11) and quercitrin (12). Crude ethyl acetate and methanol extracts from the kernel indicated strong cytotoxic activity towards MCF-7 and HeLa cells with IC50 values of less than 10 µg/mL, while petroleum ether, chloroform and ethyl acetate extracts of the stem bark showed strong to moderate activity against MCF-7, HeLa and HT-29 cancer cell lines with IC50 values ranging from 5 to 30 µg/mL. As for the antimicrobial assays, only the ethyl acetate and methanol extracts from the kernel displayed some inhibition against the microbes in the antibacterial assays. The kernel extracts showed highest free radical scavenging activity with IC50 values of less than 10 µg/mL, while the ethyl acetate and methanol extracts of leaves displayed only weak activity in the DPPH assays. CONCLUSIONS: Phytochemical investigations on various parts of Mangifera pajang have identified terpenoids and a flavonol derivative as major constituents. Bioassay studies have indicated that the crude extracts and isolated compounds have potential as naturally-derived anticancer and antimicrobial agents, besides possess high free radical scavenging activity.

Phytochemicals from Kaempferia angustifolia Rosc. and their cytotoxic and antimicrobial activities.

Phytochemical investigation on rhizomes of Kaempferia angustifolia has afforded a new abietene diterpene, kaempfolienol (1) along with crotepoxide (2), boesenboxide (3), 2'-hydroxy-4,4',6'-trimethoxychalcone (4), zeylenol (5), 6-methylzeylenol (6), (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3ß-ol (7), sucrose, ß-sitosterol, and its glycoside (8). The structures of the compounds were elucidated on the basis of spectroscopic methods (IR, MS, and NMR). Isolation of 6-methylzeylenol (6), (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3ß-ol (7), and ß-sitosterol-3-O-ß-D-glucopyranoside (8) from this plant species has never been reported previously. The spectroscopic data of (7) is firstly described in this paper. Cytotoxic screening indicated that most of the pure compounds tested showed significant activity with (4) showing the most potent activity against HL-60 (human promyelocytic leukemia) and MCF-7 (human breast cancer) cell lines. However, all extracts and most of the pure compounds tested were found to be inactive against HT-29 (human colon cancer) and HeLa (human cervical cancer) cell lines. Similarly, none of the extracts or compounds showed activity in the antimicrobial testing.

Biomedical properties of a natural dietary plant metabolite, zerumbone, in cancer therapy and chemoprevention trials.

Zerumbone (ZER) is a naturally occurring dietary compound, present in many natural foods consumed today. The compound derived from several plant species of the Zingiberaceae family that has been found to possess multiple biomedical properties, such as antiproliferative, antioxidant, anti-inflammatory, and anticancer activities. However, evidence of efficacy is sparse, pointing to the need for a more systematic review for assessing scientific evidence to support therapeutic claims made for ZER and to identify future research needs. This review provides an updated overview of in vitro and in vivo investigations of ZER, its cancer chemopreventive properties, and mechanisms of action. Therapeutic effects of ZER were found to be scientifically plausible and could be explained partially by in vivo and in vitro pharmacological activities. Much of the research outlined in this paper will serve as a foundation to explain ZER anticancer bioactivity, which will open the door for the development of strategies in the treatment of malignancies using ZER.

Inclusion Complex of Zerumbone with Hydroxypropyl- ß -Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio.

Zerumbone (ZER) isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl- ß -cyclodextrin (HP ß CD) to enhance ZER's solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HP ß CD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HP ß CD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HP ß CD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.

A Phenylbutenoid Dimer, cis-3-(3',4'-Dimethoxyphenyl)-4-[(E)-3''',4'''-Dimethoxystyryl] Cyclohex-1-ene, Exhibits Apoptogenic Properties in T-Acute Lymphoblastic Leukemia Cells via Induction of p53-Independent Mitochondrial Signalling Pathway.

The current study was designed to evaluate the in vitro cytotoxicity effect of a phenylbutenoid dimer, cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-3 (‴) ,4 (‴) -dimethoxystyryl]cyclohex-1-ene (ZC-B11) isolated from the rhizome of Zingiber cassumunar on various cancer cell line, and normal human blood mononuclear cells, and to further investigate the involvement of apoptosis-related proteins that leads, to the probable pathway in which apoptosis is triggered. Cytotoxicity test using MTT assay showed selective inhibition of ZC-B11 towards T-acute lymphoblastic leukemia cells, CEMss, with an IC50 value of 7.11 ± 0.240 µ g/mL, which did not reveal cytotoxic effects towards normal human blood mononuclear cells (IC50 > 50 µ g/mL). Morphology assessments demonstrated distinctive morphological changes corresponding to a typical apoptosis. ZC-B11 also arrested cell cycle progression at S phase and causes DNA fragmentation in CEMss cells. Decline of mitochondrial membrane potential was also determined qualitatively. In the apoptosis-related protein determination, ZC-B11 was found to significantly upregulate Bax, caspase 3/7, caspase 9, cytochrome c, and SMAC and downregulate Bcl-2, HSP70, and XIAP, but did not affect caspase 8, p53, and BID. These results demonstrated for the first time the apoptogenic property of ZC-B11 on CEMss cell line, leading to the programmed cell death via intrinsic mitochondrial pathway of apoptosis induction.

Evaluation of biological activities of Alpinia mutica Roxb. and its chemical constituents.

Phytochemicals investigation on rhizomes of Alpinia mutica has afforded five compounds namely 5,6-dehydrokawain (1), flavokawin B (2), pinostrobin (3) and pinocembrin (4) together with ß-sitosterol (5). All crude extracts of the plant demonstrated strong cytotoxicity against CEMss (human T4 lymphoblastoid) cancer cells with IC50 values less than 19 µg/mL, while flavokawin B (2) was the most cytotoxic isolate with IC50 value 1.86±0.37 µg/mL. Most of the crude extracts and isolated compounds showed weak activity in antimicrobial and diphenylpicrylhydrazyl (DPPH) radical scavenging activity tests.

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.

Dentatin Induces Apoptosis in Prostate Cancer Cells via Bcl-2, Bcl-xL, Survivin Downregulation, Caspase-9, -3/7 Activation, and NF-κB Inhibition.

This study was set to investigate antiproliferative potential of dentatin (a natural coumarin isolated from Clausena excavata Burm. F) against prostate cancer and to delineate the underlying mechanism of action. Treatment with dentatin dose-dependently inhibited cell growth of PC-3 and LNCaP prostate cancer cell lines, whereas it showed less cytotoxic effects on normal prostate epithelial cell line (RWPE-1). The inhibitory effect of dentatin on prostate cancer cell growth was due to induction of apoptosis as evidenced by Annexin V staining and cell shrinkage. We found that dentatin-mediated accumulation of reactive oxygen species (ROS) and downregulated expression levels of antiapoptotic molecules (Bcl-2, Bcl-xL, and Survivin), leading to disruption of mitochondrial membrane potential (MMP), cell membrane permeability, and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-9, -3/7 activities, and subsequent DNA fragmentation. In addition, we found that dentatin inhibited TNF-α-induced nuclear translocation of p65, suggesting dentatin as a potential NF-κB inhibitor. Thus, we suggest that dentatin may have therapeutic value in prostate cancer treatment worthy of further development.

The growth suppressing effects of girinimbine on HepG2 involve induction of apoptosis and cell cycle arrest.

Murraya koenigii is an edible herb widely used in folk medicine. Here we report that girinimbine, a carbazole alkaloid isolated from this plant, inhibited the growth and induced apoptosis in human hepatocellular carcinoma, HepG2 cells. The MTT and LDH assay results showed that girinimbine decreased cell viability and increased cytotoxicity in a dose-and time-dependent manner selectively. Girinimbine-treated HepG2 cells showed typical morphological features of apoptosis, as observed from normal inverted microscopy and Hoechst 33342 assay. Furthermore, girinimbine treatment resulted in DNA fragmentation and elevated levels of caspase-3 in HepG2 cells. Girinimbine treatment also displayed a time-dependent accumulation of the Sub-G(0)/G(1) peak (hypodiploid) and caused G(0)/G(1)-phase arrest. Together, these results demonstrated for the first time that girinimbine could effectively induce programmed cell death in HepG2 cells and suggests the importance of conducting further investigations in preclinical human hepatocellular carcinoma models, especially on in vivo efficacy, to promote girinimbine for use as an anticancer agent against hepatocellular carcinoma.

The methanolic extract of Boesenbergia rotunda (L.) Mansf. and its major compound pinostrobin induces anti-ulcerogenic property in vivo: possible involvement of indirect antioxidant action.

ETHNOPHARMACOLOGICAL RELEVANCE: Boesenbergia rotunda (L) Mansf. has been used for the treatment of gastrointestinal disorders including peptic ulcer. In the current study we aimed to investiagte the anti-ulcer activities of methanolic extract of B. rotunda (MEBR) and its main active compound, pinostrobin on ethanol-induced ulcer in rats. The possible involevement of lipid peroxidation, nitric oxide, cyclooxygenases and free radical scavenging mechanisms also has been investigated. MATERIALS AND METHODS: Pinostrobin was isolated form the rhizomes of B. rotunda. Ulcer index, gastric juice acidity, mucus content, gross and histological gastric lesions and thiobarbituric acid reactive substances (TBARS) were evaluated in ethanol-induced ulcer in vivo. The effect of pinostrobin into lipopolysaccharide/interferon-γ stimulated rodent cells, COX-1 and COX-2 activities were done in vitro. RESULTS: Pre-treatment with MEBR, pinostrobin or omeprazole protected the gastric mucosa as seen by reduction in ulcer area and mucosal content, reduced or absence of submucosal edema and leucocytes infiltration. Pinostrobin significantly (p<0.05) lowered the elevated TBARS level into gasteric homogenate. Pinostrobin did not produced significant in vitro inhibition of NO from LPS/IFN-γ activated rodent cells without affecting the viability of these cells. Further, the compound did bot revleaed inhibitory effects on both COX- 1& 2 enzymes. The antioxidant assays also exhibited non significance in vitro. CONCLUSION: Thus it can be concluded that MEBR possesses anti-ulcer activity, which could be attributed to indirect anti-oxidant mechanism of pinostrobin but not to the intervention with nitric oxide and COX inflammation pathways.

Antiapoptotic and Antioxidant Properties of Orthosiphon stamineus Benth (Cat's Whiskers): Intervention in the Bcl-2-Mediated Apoptotic Pathway.

Antiapoptotic and antioxidant activities of aqueous-methanolic extract (CAME) of Orthosiphonstamineus Benth(OS), and its hexane (HF), chloroform (CF), n-butanol (NBF), ethyl acetate (EAF) and water (WF) fractions were investigated. Antioxidant properties were evaluated using the assays of Folin-Ciocalteu, aluminiumtrichloride, ß-carotene bleaching and DPPH. The role of OS against hydrogen peroxide induced apoptosis on MDA-M231 epithelial cells was examined using MTT assay, phase contrast microscope, colorimetric assay of caspase-3, western blot and quantitative real-time PCR. Results showed that EAF showed the highest total phenolic content followed by CAME, NBF, WF, CF and HF, respectively. Flavonoid content was in the order of the CF > EAF > HF > CAME > NBF > WF. The IC(50) values on DPPH assay for different extract/fractions were 126.2 ± 23, 31.25 ± 1.2, 15.25 ± 2.3, 13.56 ± 1.9, 23.0 ± 3.2, and 16.66 ± 1.5 µg/ml for HF, CF, EAF, NBF, WF and CAME, respectively. OSreduced the oxidation of ß-carotene by hydroperoxides. Cell death was dose-dependently inhibited by pretreatment with OS. Caspase-3 and distinct morphological features suggest the anti-apoptotic activities of OS. This plant not only increased the expression of Bcl-2, but also decreased Bax expression, and ultimately reduced H(2)O(2)-induced apoptosis. The current results showed that phenolics may provide health and nutritional benefits.

Eleucine indica Possesses Antioxidant, Antibacterial and Cytotoxic Properties.

The use of evidence-based complementary and alternative medicine is increasing rapidly. Eleucine indica (EI) is traditionally used in ailments associated with liver and kidneys. The therapeutic benefit of the medicinal plants is often attributed to their antioxidant properties. Therefore, the aim of this study was to screen the hexane, dicholoromethane, ethyl acetate (EA) and methanol extracts (MeTH) of EI for their antioxidant, antibacterial and anti-cancer effects using total phenolic contents (TPCs) and DPPH, disc diffusion method and MTT cytotoxicity assays, respectively. The MeTH was showed to have the highest TPC and scavenging activity (77.7%) on DPPH assay, followed by EA (64.5%), hexane (47.19%) and DCM (40.83%) extracts, whereas the MeTH showed no inhibitory effect on all tested bacteria strains. However, the EA extract exhibited a broad spectrum antibacterial activity against all tested bacteria except Bacillus subtilis, in which this bacterium was found to be resistant to all EI extracts. Meanwhile, hexane extract was demonstrated to have a remarkable antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, while the dicholoromethane extract did not exhibit significant activity against P. aeruginosa. None of the extracts showed significant cytotoxic activity towards MCF-7, HT-29 and CEM-SS human cancer cell lines after 72 h incubation time (IC(50) > 30 µg/ml). These results demonstrate that the extract prepared from the EI possesses antioxidant activity in vitro in addition to antibacterial properties. Further investigations are needed to verify the antioxidant effects in vitro and in vivo.

Typhonium flagelliforme induces apoptosis in CEMss cells via activation of caspase-9, PARP cleavage and cytochrome c release: its activation coupled with G0/G1 phase cell cycle arrest.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Typhonium flagelliforme (TF), commonly known as 'rodent tuber' in Malaysia, is often used as traditional remedy for cancer, including leukemia. AIM OF THE STUDY: We had previously identified morphologically that the linoleic acid rich fraction (DCM/F7) from the tubers of this plant induces selective anti-proliferative effects and apoptosis in CEMss cells. In this present study, we subjected the same DCM/F7 fraction to cell based activity analyses in order to determine the possible mechanism of cell death in leukemic CEMss cells in vitro. MATERIALS AND METHODS: Extraction of Typhonium flagelliforme tuber has done and fractionation has been done by vacuum liquid column chromatography. The anti-proliferative activity was assayed using MTT and the apoptosis detection was done by Annexin V and DNA laddering assay. Colorimetric caspase assay and immunoblot analysis were employed to detect the expression of protein associated with cell death. Cell cycle analysis was done using flow cytometry. RESULTS: We found that the cancer inhibitory effect of the DCM/F7 fraction in CEMss cells was 3 ± 0.08 µg/ml (IC(50)). An early apoptotic induction in CEMss cells was observed by Annexin V assay, which showed a clear dose-dependent DNA fragmentation being observed in gel electrophoresis at 10 and 20 µg/ml. The DCM/F7 fraction at 3 µg/ml significantly arrested CEMss cells at G0/G1 phase (p<0.05). A constant but increasing pattern-related Sub-G0/G1 index was observed between 12 and 72 h treatment. In relation to this, we further investigated the biochemical events leading to cell death and found that the DCM/F7 fraction increased the cellular levels of caspase-3 and -9 on treated cells. Our results indicated that cytochrome c from mitochondria into the cytosol increased gradually as the DCM/F7 concentration increases, which later lead to the subsequent cleavage of PARP in to 85kDa fragments. On the contrary, Bcl-2 protein was found to decrease concomitantly during treatment. CONCLUSIONS: Collectively, results presented in this study demonstrated that the DCM/F7 fraction inhibited the proliferation of leukemia cells, leading to the programmed cell death, which was confirmed to be through the mitochondrial pathway.

Typhonium flagelliforme inhibits the proliferation of murine leukemia WEHI-3 cells in vitro and induces apoptosis in vivo.

Typhonium flagelliforme (TF) is a tropical plant, traditionally used by the ethnic population of Malaysia for the cure of various cancers. This plant had shown to induce antiproliferative effect as well as apoptosis in cancer cells. However, there is no available information to address that TF affects murine leukemia cells in vitro and in vivo. Here, we investigated in vitro and in vivo effects of TF on murine leukemia WEHI-3 cells. It was found that the growth of leukemia cells in vitro was inhibited by the various extracts of TF. Among these fractions, the dichloromethane (DCM) tuber extracts of TF showed the lowest IC(50) (24.0 ± 5.2 µg/ml) and had demonstrated apoptogenic effect when observed under fluorescent microscope. We investigated the in vivo effects of DCM tuber extracts of TF on murine leukemia cells, and the results showed that the counts of immature granulocytes and monocytes were significantly decreased in peripheral blood of BALB/c leukemia mice after the oral administration of DCM tuber extracts of TF for 28 days with three doses (200, 400 and 800 mg/kg). These results were confirmed by observing the spleen histopathology and morphology of enlarged spleen and liver in leukemia mice when compared with the control. Furthermore, the cell death mechanism in the spleen tissue of treated mice was found via apoptosis.

Potential chemoprevention of diethylnitrosamine-initiated and 2-acetylaminofluorene-promoted hepatocarcinogenesis by zerumbone from the rhizomes of the subtropical ginger (Zingiber zerumbet).

Zerumbone (ZER), a monosesquiterpene found in the subtropical ginger (Zingiber zerumbet Smith), possesses antiproliferative properties to several cancer cells lines, including the cervical, skin and colon cancers. In this study, the antitumourigenic effects of ZER were assessed in rats induced to develop liver cancer with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and dietary 2-acetylaminofluorene (AAF) (0.02%). The rats also received intraperitoneal ZER injections at 15, 30 or 60 mg/kg body wt. twice a week for 11 weeks, beginning week four post-DEN injection. The hepatocytes of positive control (DEN/AAF) rats were smaller with larger hyperchromatic nuclei than normal, showing cytoplasmic granulation and intracytoplasmic violaceous material, which were characteristics of hepatocarcinogenesis. Histopathological evaluations showed that ZER protects the rat liver from the carcinogenic effects of DEN and AAF. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (AP) and alpha-fetoprotein (AFP) were significantly lower (P<0.05) in ZER-treated than untreated rats with liver cancer. The liver malondialdehyde (MDA) concentrations significantly (P<0.05) increased in the untreated DEN/AAF rats indicating hepatic lipid peroxidation. There was also significant (P<0.05) reduction in the hepatic tissue glutathione (GSH) concentrations. The liver sections of untreated DEN/AAF rats also showed abundant proliferating cell nuclear antigen (PCNA), while in ZER-treated rats the expression of this antigen was significantly (P<0.05) lowered. By the TUNEL assay, there were significantly (P<0.05) higher numbers of apoptotic cells in DEN/AAF rats treated with ZER than those untreated. Zerumbone treatment had also increased Bax and decreased Bcl-2 protein expression in the livers of DEN/AAF rats, which suggested increased apoptosis. Even after 11 weeks of ZER treatment, there was no evidence of abnormality in the liver of normal rats. This study suggests that ZER reduces oxidative stress, inhibits proliferation, induces mitochondria-regulated apoptosis, thus minimising DEN/AAF-induced carcinogenesis in rat liver. Therefore, ZER has great potential in the treatment of liver cancers.