Anti-Leukemic Effects of Typhonium Flagelliforme on Human Lymphoblastoid Cells (CEMss) and Murine Leukemic (WEHI-3) Model

To date, there has been no literature reported on the mechanism of Typhonium flagelliforme and its effects on leukemia. Hence, the anti-leukemia effect of Typhonium flagelliforme was investigated in vitro and in vivo leukemic model. Extraction and fractionation using organic solvents were applied to obtain fractions from T. flagelliforme and subsequently, chemical analysis was done using GC-MS. In vitro cytotoxic effects of extracts and fractions were tested in several human cancer cell lines including leukemia (CEMss cells) using MTT assay. Various microscopy techniques were used to study morphological changes occurring during treatment. The Annexin V assay, TUNEL assay, cell cycle analysis and DNA laddering were employed to detect apoptosis. Colourimetric assays for caspase-3 and 9, immunoblot analysis for cytochrome c, BcL-2, PARP, FasL and β-actin were analysed. The in vivo model of leukemia was induced in male BALB/c mice using WEHI-3 cells. The DCM extract of the plant tuber was used for treatment at various doses. Amongst 8 plant extracts investigated, the dichloromethane (DCM) extracts of T. flagelliforme tuber demonstrated low and significant anti proliferative effect against both CEMss (6.5±0.4 μg/ml) and WEHI-3 cells (24.0±5.2 μg/ml) (p<0.05). Further fractionation of the DCM tuber extract resulted into 12 fractions. Seven of these 12 fractions showed significant cytotoxicity against CEMss, in which the DCM/F7, DCM/F11 and DCM/F12 fractions showed highest anti-cancer activities of 3.0, 5.0 and 6.2 μg/ml respectively. Further studies of these fractions towards non cancerous Peripheral Blood Lymphocytes (PBL) exhibited significant selectivity of DCM/F7 compared to other fractions. Phytochemical analysis using GC-MS revealed that the DCM/F7 fraction contains linoleic acid (51.20%), n-hexadecanoic acid (17.89%), 9-hexadecanoic acid (6.99%) and Stigmasta-5,22-dien-3-ol (6.06%). Cytological observations exhibited chromatin condensation, cell shrinkage, abnormalities of cristae, membrane blebbing, cytoplasmic extrusions and formation of apoptotic bodies, further confirmed using AO/PI, SEM and TEM analysis. The Annexin V and TUNEL assay revealed apoptotic induction in CEMss cells exposed to the DCM/F7 in a time-dependent manner, whilst DNA fragmentation of CEMss cells were detected using 1.0% agarose gel electrophoresis. The DCM/F7 fraction significantly (p<0.05) stimulated both caspases 3 and 9 activities. The immunoblot results revealed that DCM/F7 caused the release of mitochondrial cytochrome c and cleaved 116 kDa PARP into 85 kDa fragments. The Bcl-2 protein was found to decrease during treatment. Meanwhile, FasL did not exhibit up or down regulation on treatment. Cell cycle analysis revealed that there is significant (p<0.05) G1 phase arrest in a time-depended manner. The DCM extract of T. flagelliforme tuber in vivo markedly inhibited the proliferation of WEHI-3 in male BALB/c mice as evidenced by reduction in the percentage of immature monocytes as well as granulocytes, liver weight, spleen weight and histopathological profiles of H&E stained spleen tissue. The DCM tuber extract of T. flagelliforme significantly decreased the spleen tumor size, which had dose-dependent effects. Sections of spleen tissue of the BALB/c mice treated with the extract. Treatment at 800 mg/kg dose showed evidence of apoptosis in comparison to the control groups. Collectively, results presented in this study demonstrate that T. flagelliforme, a local herbal medicinal plant in Malaysia inhibited the proliferation of leukemia in vitro selectively, leading to the programmed cell death, which was later confirmed to lead through mitochondrial pathways. Moreover, in vivo study on an orthotopic BALB/c mice model clearly shows that, T. flagelliforme tuber extract has inhibited the proliferation of leukemia via the induction of apoptosis

β-mangostin suppresses LA-7 cells proliferation in vitro and in vivo: involvement of antioxidant enzyme modulation; suppression of matrix metalloproteinase and α6β4 integrin signalling pathways

β-mangostin (βM) was isolated from Cratoxylum arborescens to investigate anti-breast cancer effect in vitro and in vivo. βM exhibited an inhibitory effect on the growth of LA-7 cells in vitro with apoptosis formation. In the animal model, βM treatment was found to be effective in improving the tissue antioxidant enzymes such as superoxide dismutase and catalase activity (P < 0.05). βM treatment clearly exhibited apoptosis in mammary tumour tissues, and it was associated with regulation of PCNA and p53. The cDNA microarray gene expression followed by qRT-PCR based validation demonstrated that βM could mediate tumour reduction and prevent metastasis by reduction of MMP-9, MMP-13, and MMP-27. Moreover, the reduction of both 14-3-3β and ITGB4 genes indicated the involvement of α6β4 integrin signalling pathway. These findings showed that β-mangostin is a promising compound candidate as an anti-tumour agent against breast cancer

Apoptosis effect of girinimbine isolated from Murraya koenigii on lung cancer cells in vitro

Murraya koenigii Spreng has been traditionally claimed as a remedy for cancer. The current study investigated the anticancer effects of girinimbine, a carbazole alkaloid isolated from Murraya koenigii Spreng, on A549 lung cancer cells in relation to apoptotic mechanistic pathway. Girinimbine was isolated from Murraya koenigii Spreng. The antiproliferative activity was assayed using MTT and the apoptosis detection was done by annexin V and lysosomal stability assays. Multiparameter cytotoxicity assays were performed to investigate the change in mitochondrial membrane potential and cytochrome c translocation. ROS, caspase, and human apoptosis proteome profiler assays were done to investigate the apoptotic mechanism of cell death. The MTT assay revealed that the girinimbine induces cell death with an IC50 of 19.01 μM. A significant induction of early phase of apoptosis was shown by annexin V and lysosomal stability assays. After 24 h treatment with 19.01 μM of girinimbine, decrease in the nuclear area and increase in mitochondrial membrane potential and plasma membrane permeability were readily visible. Moreover the translocation of cytochrome c also was observed. Girinimbine mediates its antiproliferative and apoptotic effects through up- and downregulation of apoptotic and antiapoptotic proteins. There was a significant involvement of both intrinsic and extrinsic pathways. Moreover, the upregulation of p53 as well as the cell proliferation repressor proteins, p27 and p21, and the significant role of insulin/IGF-1 signaling were also identified. Moreover the caspases 3 and 8 were found to be significantly activated. Our results taken together indicated that girinimbine may be a potential agent for anticancer drug development

The establishment and use of an in vivo animal model for cervical intra-epithelial neoplasia.

Cervical cancer is the second most common cancer of female reproductive tracts. In developing countries, cervical carcinoma is the leading cause of cancer fatality in women Despite attempts to lower the fatality rate, very few in vivo models are in place to investigate this cancer. We therefore are able to develop an in vivo animal model that is suitabie to conduct such study. In our attempt to secure an in vivo animal model for cervical cancer, the carcinogenic property of diethylstilboestrol (DES) was exploited to establish a model for Cervical Intruepithelial Neoplasia or carcinoma (CIN). Female-Balb/C mice were injected with several dosages of DES (i.p) during pregnancy at day 13-18. Female offspring were reared and sacrificed at age of 48-54 days and the cervix tissues taken for histological evaluation using H and E. The progression of the cancer and hence, disease state is monitored by measuring serum IL-6 using an ELISA kit. Proliferative cell nuclear antigen (PCNA) expressions were studied by implying immunohistochemical techniques. All parameters with regards to CIN were compared to a control group of treating the cancer using a used drug, cisplatin, used preferentially to treat cervical cancer in humans. The results of this study revealed that a significant difference in serum IL-6 concentration between DES-treated group and control groups (p<.05). CIN histological related lesions was noticed to be prominently dominant in DES-treated animals whilst these lesions were absent in control groups. In addition to that PCNA index in DES-treated animal was found to be a significant different compared to control group. The above findings indicate that DES could be utilized and further exploited as cervical carcinogenesis initiator in animal models to screen and study new potential anti-cervical cancer compounds in vivo

β Mangostin suppress LPS-induced inflammatory response in RAW 264.7 macrophages in vitro and carrageenan-induced peritonitis in vivo

Ethnopharmacological relevance: The fruit hull of Garcinia mangostana Linn. has been used in traditional medicine for treatment of various inflammatory diseases. Hence, this study aims to investigate the in vitro and in vivo anti-inflammatory effect of β mangostin (βM), a major compound present in Garcinia mangostana. Materials and methods: The in silico analysis of inflammatory mediators such as cyclooxygenase (COX) and nuclear factor-kappa B (NF-kB) were performed via molecular docking. Further evaluation of anti-inflammatory effect was conducted in lipopolysaccharide (LPS) induced RAW 264.7 macrophages. Suppression of activated NF-kB was analyzed by high content screening. βM triggered inhibition of COX-1 and COX-2 in vitro were studied using biochemical kit. The in vivo model used in this study was carrageenan-induced peritonitis model, where reduction in carrageenan-induced peritonitis is measured by leukocyte migration and vascular permeability. In addition, the evaluation of βM׳s effect on carrageenan induced TNF-α and IL-1β release on peritoneal fluid was also carried out. Results: Treatment with βM could inhibit the LPS-induced NO production but not the viability of RAW 264.7. Similarly, βM inhibited PGE2 production and the cytokines: TNF-α and IL-6. The COX catalyzed prostaglandin biosynthesis assay had showed selective COX-2 inhibition with a 53.0±6.01% inhibition at 20 µg/ml. Apart from this, βM was capable in repressing translocation of NF-kB into the nucleus. These results were concurrent with molecular docking which revealed COX-2 selectivity and NF-kB inhibition. The in vivo analysis showed that after four hours of peritonitis, βM was unable to reduce vascular permeability, yet could decrease the total leukocyte migration; particularly, neutrophils. Meanwhile, dexamethasone 0.5 mg/kg, successfully reduced vascular permeability. The levels of TNF-α and IL-1β in peritoneal fluid was reduced significantly by βM treatment. Conclusion: The current study supports the traditional use of Garcinia mangostana fruit hull for treatment of inflammatory conditions. In addition, it is clear that the anti-inflammatory efficacy of this plant is not limited to the presence of α and γ, but β also with significant activity

Anticancer activity of natural compound (zerumbone) extracted from Zingiber zerumbet in human HeLa cervical cancer cells

A natural compound, zerumbone was extracted, isolated and purified from the rhizomes of edible plant Zingiber zerumbet using methanol extraction and Column Chromatography (CC) method. The isolated and purified zerumbone crystals were subjected to High Performance Liquid Chromatography (HPLC), Liquid Chromatography Mass Spectrometry (LCMS) and 13C NMR and 1H NMR analysis to confirm the purity, molecular weight and molecular structure. The study investigated the purified zerumbone crystals for its anti-cancer properties on human cervical cancer cell line (HeLa). Cisplatin, was used as a positive control in this study. The cytotoxicity of zerumbone and cisplatin were investigated using the MTT assay and caspases-3 was estimated with colorimetric assay in zerumbone treated HeLa cells. Morphological analysis showed that there were changes observed on HeLa cancer cells after treatment with zerumbone and cisplatin. The MTT assay results demonstrated that the IC50 value ( ± SEM) of zerumbone was determined to be 11.3 μM (2.5 μg mL-1) whilst the IC50 value of cisplatin was at 7.5 μM (1.6 μg mL-1). Prominent growth retardation was identified to the HeLa cancer cells, after treatment with both compounds, while caspase-3 was observed to be significantly increased in zerumbone treated cells as compared to untreated control cells. This study showed promising avenues towards zerumbone to be developed as a new chemo-natural drug for treatment of cervical cancer

Attenuation of cisplatin-induced hepatotoxicity in rats using zerumbone.

Zerumbone is a natural compound isolated from the fresh rhizomes of Zingiber zerumbet. This bioactive compound has shown a chemo-preventive, anti-inflammatory and free radical scavenging activities. This study examines, the effect of zerumbone on the extent of tissue damage in Cisplatin-induced hepatotoxicity in rats. The rats received a single dose injection of 45 mg kg-1 Cisplatin. Other groups of rats received zerumbone (100 and 200 mg kg-1), corn oil or the vehicle (DMSO) intraperitoneally for 4 days prior to Cisplatin injections. All animals were decapitated 16 h after Cisplatin injection. Trunk blood was collected and analyzed for alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase and gama-glutamyl transferase. Liver tissue was kept for the quantification of malondialdehyde and glutathione levels. Histopathological investigations were carried out and severity of lesions was scored to obtain quantitative data. This study revealed that zerumbone reduced the extent of liver damage and preserved liver functions as proved by microscopic observations and lesion scoring. Increase in liver MDA levels with a simultaneous reduction in GSH in the Cisplatin 45 mg kg-1 group was attenuated by zerumbone treatment (p<0.05). Zerumbone is beneficial in Cisplatin-induced liver dysfunction and organ damage in rats via prevention of lipid peroxidation and preservation of antioxidant glutathione

Investigations of antioxidant and antibacterial activities of Typhonium flagelliforme (Lodd.) Blume leaves.

The antioxidant and antibacterial activity of different extracts from of Typhonium flagelliforme (L.) Blume leave (family: Araceae) commonly called 'Rodent Tuber' was assessed towards different antioxidant models as well as in selected bacteria. None of the extracts showed significant activity against the selected strains. The only exception is hexane extract (2.0±0.15 mm diameter) against Pseudomonas aeruginosa. The positive control, Streptomycin had shown zone of inhibition of 20±1.5, 20±1.3, 23±1.5 and 23±1.0 mm in Methicillin Resistant Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella choleraesuis and Bacillus subtilis, respectively. All the extracts were subjected to screening for their possible antioxidant activity. Two complementary test systems, namely DPPH free radical scavenging and total phenolic compounds, were used for the analysis. The results showed that the inhibitory activity of Dichloromethane (60.7±3.2%) and Methanol (60.1±2.3%) extracts were comparatively commendable inhibition capacity when compared to the positive control BHT (95.3±1.3%). The total phenolic content of Methanol extracts (5.69±0.15 GAE mg g-1 extract) was superior to all other extracts, followed by dichloromethane and ethyl acetate. Considering all the results collectively T. flagelliforme appears to be a promising plant demonstrating antioxidant activity that requires further investigation

β-Mangostin induces p53-dependent G2/M cell cycle arrest and apoptosis through ROS mediated mitochondrial pathway and NfkB suppression in MCF-7 cells

β-Mangostin (βM) was isolated from Cratoxylum arborescens to investigate its anti-cancer effect in MCF-7 cells. βM induced apoptosis by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol. The release of caspase-9 and -7 and consequently cleaved PARP leading to apoptotic was observed upon treatment. Reduction of both bid and caspase 8 and the up regulation of Fas showed the involvement of the extrinsic pathway. Significantly up regulated GADD45A and HRK genes were observed upon treatment, with concomitant inhibition of NF-kB to nucleus. The protein array had demonstrated the expression of HSP 70, HSP 60, XIAP, Survivin, p53 and Bax. Moreover, βM had showed p53-dependent G2/M cell cycle arrest by down regulation of cdc2 and PCNA. Together, the results demonstrated that the βM induced anti-proliferative effect, leading to G2/M phase cell cycle arrest and apoptosis through both the extrinsic and mitochondrial pathways with the involvement of the multiple pro and anti-apoptosis and NF-kB signalling pathways

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