Drug Release Profiles of Mitomycin C Encapsulated Quantum Dots-Chitosan Nanocarrier System for the Possible Treatment of Non-Muscle Invasive Bladder Cancer.

Nanotechnology-based drug delivery systems are an emerging technology for the targeted delivery of chemotherapeutic agents in cancer therapy with low/no toxicity to the non-cancer cells. With that view, the present work reports the synthesis, characterization, and testing of Mn:ZnS quantum dots (QDs) conjugated chitosan (CS)-based nanocarrier system encapsulated with Mitomycin C (MMC) drug. This fabricated nanocarrier, MMC@CS-Mn:ZnS, has been tested thoroughly for the drug loading capacity, drug encapsulation efficiency, and release properties at a fixed wavelength (358 nm) using a UV-Vis spectrophotometer. Followed by the physicochemical characterization, the cumulative drug release profiling data of MMC@CS-Mn:ZnS nanocarrier (at pH of 6.5, 6.8, 7.2, and 7.5) were investigated to have the highest release of 56.48% at pH 6.8, followed by 50.22%, 30.88%, and 10.75% at pH 7.2, 6.5, and 7.5, respectively. Additionally, the drug release studies were fitted to five different pharmacokinetic models including pesudo-first-order, pseudo-second-order, Higuchi, Hixson-Crowell, and Korsmeyers-Peppas models. From the analysis, the cumulative MMC release suits the Higuchi model well, revealing the diffusion-controlled mechanism involving the correlation of cumulative drug release proportional to the function square root of time at equilibrium, with the correlation coefficient values (R2) of 0.9849, 0.9604, 0.9783, and 0.7989 for drug release at pH 6.5, 6.8, 7.2, and 7.5, respectively. Based on the overall results analysis, the formulated nanocarrier system of MMC synergistically envisages the efficient delivery of chemotherapeutic agents to the target cancerous sites, able to sustain it for a longer time, etc. Consequently, the developed nanocarrier system has the capacity to improve the drug loading efficacy in combating the reoccurrence and progression of cancer in non-muscle invasive bladder diseases.

Radioprotective Effects of Kelulut Honey in Zebrafish Model.

Large doses of ionizing radiation can damage human tissues. Therefore, there is a need to investigate the radiation effects as well as identify effective and non-toxic radioprotectors. This study evaluated the radioprotective effects of Kelulut honey (KH) from stingless bee (Trigona sp.) on zebrafish (Danio rerio) embryos. Viable zebrafish embryos at 24 hpf were dechorionated and divided into four groups, namely untreated and non-irradiated, untreated and irradiated, KH pre-treatment and amifostine pre-treatment. The embryos were first treated with KH (8 mg/mL) or amifostine (4 mM) before irradiation at doses of 11 Gy to 20 Gy using gamma ray source, caesium-137 (137Cs). Lethality and abnormality analysis were performed on all of the embryos in the study. Immunohistochemistry assay was also performed using selected proteins, namely γ-H2AX and caspase-3, to investigate DNA damages and incidences of apoptosis. KH was found to reduce coagulation effects at up to 20 Gy in the lethality analysis. The embryos developed combinations of abnormality, namely microphthalmia (M), body curvature and microphthalmia (BM), body curvature with microphthalmia and microcephaly (BMC), microphthalmia and pericardial oedema (MO), pericardial oedema (O), microphthalmia with microcephaly and pericardial oedema (MCO) and all of the abnormalities (AA). There were more abnormalities developed from 24 to 72 h (h) post-irradiation in all groups. At 96 h post-irradiation, KH was identified to reduce body curvature effect in the irradiated embryos (up to 16 Gy). γ-H2AX and caspase-3 intensities in the embryos pre-treated with KH were also found to be lower than the untreated group at gamma irradiation doses of 11 Gy to 20 Gy and 11 Gy to 19 Gy, respectively. KH was proven to increase the survival rate of zebrafish embryos and exhibited protection against organ-specific abnormality. KH was also found to possess cellular protective mechanism by reducing DNA damage and apoptosis proteins expression.

Enhanced anti-mammary gland cancer activities of tamoxifen-loaded erythropoietin-coated drug delivery system.

Nanomedicine is an emerging area in the medical field, particularly in the treatment of cancers. Nanostructured lipid carrier (NLC) was shown to be a good nanoparticulated carrier for the delivery of tamoxifen (TAM). In this study, the tamoxifen-loaded erythropoietin-coated nanostructured lipid carriers (EPO-TAMNLC) were developed to enhance the anti-cancer properties and targetability of TAM, using EPO as the homing ligand for EPO receptors (EpoRs) on breast cancer tissue cells. Tamoxifen-loaded NLC (TAMNLC) was used for comparison. The LA7 cells and LA7 cell-induced rat mammary gland tumor were used as models in the study. Immunocytochemistry staining showed that LA7 cells express estrogen receptors (ERs) and EpoRs. EPO-TAMNLC and TAMNLC significantly (p<0.05) inhibited proliferation of LA7 in dose- and time-dependent manner. EPO-TAMNLC induced apoptosis and G0/G1 cell cycle arrest of LA7 cells. Both drug delivery systems showed anti-mammary gland tumor properties. At an intravenous dose of 5 mg kg-1 body weight, EPO-TAMNLC and TAMNLC were not toxic to rats, suggesting that both are safe therapeutic compounds. In conclusion, EPO-TAMNLC is not only a unique drug delivery system because of the dual drug-loading feature, but also potentially highly specific in the targeting of breast cancer tissues positive for ERs and EpoRs. The incorporation of TAM into NLC with and without EPO coat had significantly (p<0.05) improved specificity and safety of the drug carriers in the treatment of mammary gland tumors.

Migration and Proliferation Effects of Thymoquinone-Loaded Nanostructured Lipid Carrier (TQ-NLC) and Thymoquinone (TQ) on In Vitro Wound Healing Models.

Wound healing is a regulated biological event that involves several processes including infiltrating leukocyte subtypes and resident cells. Impaired wound healing is one of the major problems in diabetic patients due to the abnormal physiological changes of tissues and cells in major processes. Thymoquinone, a bioactive compound found in Nigella sativa has been demonstrated to possess antidiabetic, anti-inflammatory, and antioxidant effects. Today, the rapidly progressing nanotechnology sets a new alternative carrier to enhance and favour the speed of healing process. In order to overcome its low bioavailability, TQ is loaded into a colloidal drug carrier known as a nanostructured lipid carrier (NLC). This study aimed to determine the effect of TQ-NLC and TQ on cell proliferation and migration, mode of cell death, and the antioxidant levels in normal and diabetic cell models, 3T3 and 3T3-L1. Cytotoxicity of TQ-NLC and TQ was determined by MTT assay. The IC10 values obtained for 3T3-L1 treated with TQ-NLC and TQ for 24 hours were 4.7 ± 3.3 and 5.3 ± 0.6 µM, respectively. As for 3T3, the IC10 values obtained for TQ-NLC and TQ at 24 hours were 4.3 ± 0.17 and 3.9 ± 2.05 µM, respectively. TQ-NLC was observed to increase the number of 3T3 and 3T3-L1 healthy cells (87-95%) and gradually decrease early apoptotic cells in time- and dose-dependant manner compared with TQ. In the proliferation and migration assay, 3T3-L1 treated with TQ-NLC showed higher proliferation and migration rate (p < 0.05) compared with TQ. TQ-NLC also acted as an antioxidant by reducing the ROS levels in both cells after injury at concentration as low as 3 µM. Thus, this study demonstrated that TQ-NLC has better proliferation and migration as well as antioxidant effect compared with TQ especially on 3T3-L1 which confirms its ability as a good antidiabetic and antioxidant agent.

Induction of apoptosis and G2/M arrest by ampelopsin E from Dryobalanops towards triple negative breast cancer cells, MDA-MB-231.

BACKGROUND: Several compounds isolated from Dryobalanops have been reported to exhibit cytotoxic effects to several cancer cell lines. This study investigated the cytotoxic effects, cell cycle arrest and mode of cell death in ampelopsin E-treated triple negative cells, MDA-MB-231. METHODS: Cytotoxicity of ampelopsin E, ampelopsin F, flexuosol A, laevifonol, Malaysianol A, Malaysianol D and nepalensinol E isolated from Dryobalanops towards human colon cancer HT-29, breast cancer MDA-MB-231 and MCF-7, alveolar carcinoma HeLa and mouse embryonic fibroblast NIH/3 T3 cells were determined by MTT assay. The cells were treated with the compounds (0.94-30 µM) for 72 h. The mode of cell death was evaluated by using an inverted light microscope and annexin V/PI analysis. Cell cycle analysis was performed by using a flow cytometer. RESULTS: Data showed that ampelopsin E was most cytotoxic toward MDA-MB-231 with the IC50 (50 % inhibition of cell viability compared to control) of 14.5 ± 0.71 µM at 72 h. Cell shrinkage, membrane blebbing and formation apoptotic bodies characteristic of apoptosis were observed following treatment with ampelopsin E. The annexin V/PI flow cytometric analysis further confirmed that ampelopsin E induced apoptosis in MDA-MB-231 cells. Cell cycle analysis revealed that ampelopsin E induced G2/M phase cell cycle arrest in the cells. CONCLUSION: Ampelopsin E induced apoptosis and cell cycle arrest in MDA-MB-231 cells. Therefore, ampelopsin E has the potential to be developed into an anticancer agent for treatment of triple negative breast cancer.

Endoplasmic reticulum stress-induced apoptotic pathway and mitochondrial dysregulation in HeLa cells treated with dichloromethane extract of Dillenia suffruticosa.

Ethyl acetate and dichloromethane extract of Dillenia suffruticosa (EADS and DCMDS, respectively) can be a potential anticancer agent. The effects of EADS and DCMDS on the growth of HeLa cervical cancer cells and the expression of apoptotic-related proteins had been investigated in vitro. Cytotoxicity of the extracts toward the cells was determined by 5-diphenyltetrazolium bromide assay, the effects on cell cycle progression and the mode of cell death were analyzed by flow cytometry technique, while the effects on apoptotic-related genes and proteins were evaluated by quantitative real-time polymerase chain reaction, and Western blot and enzyme-linked immunosorbent assay, respectively. Treatment with DCMDS inhibited (P < 0.05) proliferation and induced apoptosis in HeLa cells. The expression of cyclin B1 was downregulated that led to G2/M arrest in the cells after treatment with DCMDA. In summary, DCMDS induced apoptosis in HeLa cells via endoplasmic reticulum stress-induced apoptotic pathway and dysregulation of mitochondria. The data suggest the potential application of DCMDS in the treatment of cervical cancer.

Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile.

Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and ß-sitosterol-3-O-ß-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 µg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.

Induction of cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 cells by Dillenia suffruticosa root extract via multiple signalling pathways.

BACKGROUND: Dillenia suffruticosa root dichloromethane extract (DCM-DS) has been reported to exhibit strong cytotoxicity towards breast cancer cells. The present study was designed to investigate the cell cycle profile, mode of cell death and signalling pathways of DCM-DS-treated human caspase-3 deficient MCF-7 breast cancer cells. METHODS: Dillenia suffruticosa root was extracted by sequential solvent extraction. The anti-proliferative activity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using inverted light microscope and Annexin-V/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry. MCF-7 cells were co-treated with antioxidants α-tocopherol and ascorbic acid to evaluate whether the cell death was mainly due to oxidative stress. GeXP-based multiplex system was employed to investigate the expression of apoptotic, growth and survival genes in MCF-7 cells. Western blot analysis was performed to confirm the expression of the genes. RESULTS: DCM-DS was cytotoxic to the MCF-7 cells in a time-and dose-dependent manner. The IC50 values of DCM-DS at 24, 48 and 72 hours were 20.3 ± 2.8, 17.8 ± 1.5 and 15.5 ± 0.5 µg/mL, respectively. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 cells at low concentration (12.5 and 25 µg/mL) and high concentration (50 µg/mL), respectively. Although Annexin-V/PI-flow cytometry analysis has confirmed that DCM-DS induced apoptosis in MCF-7 cells, the distinct characteristics of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies were not observed under microscope. DCM-DS induced formation of ROS in MCF-7 cells. Nevertheless, co-treatment with antioxidants did not attenuate the cell death at low concentration of DCM-DS. The pro-apoptotic gene JNK was up-regulated whereby anti-apoptotic genes AKT1 and ERK1/2 were down-regulated in a dose-dependent manner. Western blot analysis has confirmed that DCM-DS significantly up-regulated the expression of pro-apoptotic JNK1, pJNK and down-regulated anti-apoptotic AKT1, ERK1 in MCF-7 cells. CONCLUSION: DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via multiple signalling pathways. It shows the potential of DCM-DS to be developed to target the cancer cells with mutant caspase-3.

Ardisia crispa roots inhibit cyclooxygenase and suppress angiogenesis.

BACKGROUND: In our previous studies conducted on Ardisia crispa roots, it was shown that Ardisia crispa root inhibited inflammation-induced angiogenesis in vivo. The present study was conducted to identify whether the anti-angiogenic properties of Ardisia crispa roots was partly due to either cyclooxygenase (COX) or/and lipoxygenase (LOX) activity inhibition in separate in vitro studies. METHODS: Benzoquinonoid fraction (BQ) was isolated from hexane extract by column chromatography, and later analyzed by using gas chromatography-mass spectrometry (GC-MS). Anti-angiogenic effect was studied on mouse sponge implantation assay. Ardisia crispa ethanolic rich fraction (ACRH), quinone-rich fraction (QRF) and BQ were screened for COX assay to evaluate their selectivity towards two isoforms (COX-1 and COX-2), The experiment on soy lipoxygenase (LOX) inhibitory assay was also performed to determine the inhibitory effect of ACRH, QRF and BQ on soy LOX. RESULTS: BQ was confirmed to consist of 2-methoxy-6-undecyl-1,4-benzoquinone, when compared with previous data. Antiangiogenesis study exhibited a reduction of mean vascular density (MVD) in both ACRH and QRF, compared to control. In vitro study showed that both ACRH and QRF inhibited both COX-1 and COX-2, despite COX-2 inhibition being slightly higher than COX-1 in BQ. On the other hand, both ACRH and QRF were shown to have poor LOX inhibitory activity, but not BQ. CONCLUSIONS: In conclusion, ACRH and QRF might possibly exhibit its anti-angiogenic effect by inhibiting cyclooxygenase. However, both of them were shown to possess poor LOX inhibitory activity. On the other hand, BQ displayed selectivity to COX-2 inhibitory property as well as LOX inhibitory effect.

Induction of apoptosis through oxidative stress-related pathways in MCF-7, human breast cancer cells, by ethyl acetate extract of Dillenia suffruticosa.

BACKGROUND: Breast cancer is one of the most dreading types of cancer among women. Herbal medicine has becoming a potential source of treatment for breast cancer. Herbal plant Dillenia suffruticosa (Griff) Martelli under the family Dilleniaceae has been traditionally used to treat cancerous growth. In this study, the anticancer effect of ethyl acetate extract of D. suffruticosa (EADs) was examined on human breast adenocarcinoma cell line MCF-7 and the molecular pathway involved was elucidated. METHODS: EADs was obtained from the root of D. suffruticosa by using sequential solvent extraction. Cytotoxicity was determined by using MTT assay, mode of cell death by cell cycle analysis and apoptosis induction by Annexin-FITC/PI assay. Morphology changes in cells were observed under inverted light microscope. Involvement of selected genes in the oxidative stress-mediated signaling pathway was explored using multiplex gene expression analysis. RESULTS: The treatment of EADs caused cytotoxicity to MCF-7 cells in a dose- and time-dependent manner at 24, 48 and 72 hours with IC50 of 76 ± 2.3, 58 ± 0.7 and 39 ± 3.6 µg/mL, respectively. The IC50 of tamoxifen-treated MCF-7 cells was 8 ± 0.5 µg/mL. Induction of apoptosis by EADs was dose- and time- dependent. EADs induced non-phase specific cell cycle arrest at different concentration and time point. The multiplex mRNA expression study indicated that EADs-induced apoptosis was accompanied by upregulation of the expression of SOD1, SOD2, NF-κB, p53, p38 MAPK, and catalase, but downregulation of Akt1. CONCLUSION: It is suggested that EADs induced apoptosis in MCF-7 cells by modulating numerous genes which are involved in oxidative stress pathway. Therefore, EADs has the potential to act as an effective intervention against breast cancer cells.

Dillenia Suffruticosa extract inhibits proliferation of human breast cancer cell lines (MCF-7 and MDA-MB-231) via induction of G2/M arrest and apoptosis.

The present research was designed to evaluate the anticancer properties of Dillenia suffruticosa extract. Our focus was on the mode of cell death and cell cycle arrest induced in breast cancer cells by the active fractions (designated as D/F4, D/F5 and EA/P2) derived from chromatographic fractionation of D. suffruticosa extracts. The results showed that the active fractions are more cytotoxic towards MCF-7 (estrogen positive breast cancer cells) and MDA-MB-231 (estrogen negative breast cancer cells) as compared to other selected cancer cell lines that included HeLa, A459 and CaOV3. The induction of cell death through apoptosis by the active fractions on the breast cancer cells was confirmed by Annexin V-FITC and PI staining. Cell cycle analysis revealed that D/F4 and EA/P2 induced G2/M phase cell cycle arrest in MCF-7 cells. On the other hand, MDA-MB-231 cells treated with D/F4 and D/F5 accumulated in the sub-G1 phase without cell cycle arrest, suggesting the induction of cell death through apoptosis. The data suggest that the active fractions of D. suffruticosa extract eliminated breast cancer cells through induction of apoptosis and cell cycle arrest. The reason why MCF-7 was more sensitive towards the treatment than MDA-MB-231 remains unclear. This warrants further work, especially on the role of hormones in response towards cytotoxic agents. In addition, more studies on the mechanisms underlying the induction of apoptosis and cell cycle arrest by the plant extract also need to be carried out.

Isolation of a quinone-rich fraction from Ardisia crispa roots and its attenuating effects on murine skin tumorigenesis.

Ardisia crispa (Family: Myrsinaceae) is an evergreen, fruiting shrub that has been traditionally used as folklore medicine. Despite a scarcity of research publications, we have succeeded in showing suppressive effects on murine skin papillomagenesis. In extension, the present research was aimed at determining the effect of a quinone-rich fraction (QRF) isolated from the same root hexane extract on both initiation and promotion stages of carcinogenesis, at the selected dose of 30 mg/kg. Mice (groups I-IV) were initiated with a single dose of 7,12-dimethylbenz(α)anthracene (DMBA, 100 µg/100 µl) followed by repeated promotion of croton oil (1%) twice weekly for 20 weeks. In addition, group I (anti-initiation) received QRF 7 days before and after DMBA; group II (anti-promotion) received QRF 30 minutes before each croton oil application; group III (anti-initiation/ promotion) was treated with QRF as a combination of group I and II. A further two groups served as vehicle control (group V) and treated control (group VI). As carcinogen control, group IV showed the highest tumor volume (8.79±5.44) and tumor burden (3.60±1.17). Comparatively, group III revealed only 20% of tumor incidence, tumor burden (3.00±1.00) and tumor volume (2.40±1.12), which were significantly different from group IV. Group II also showed significant reduction of tumor volume (3.11), tumor burden (3.00) and tumor incidence (11.11%), along with prominent increase of latency period of tumor formation (week 12). Group I, nonetheless, demonstrated marked increment of tumor incidence by 40% with prompted latency period of tumor formation (week 7). No tumor formation was observed in groups V and VI. This study provided clear evidence of inhibitory effects of QRF during promotion period which was in agreement with our previous findings. The mechanism(s) underlying such effects have yet to be elucidated.

The improvement of in vivo model (Balb/c mice) for cervical carcinogenesis using diethylstilbestrol (DES).

Cervical cancer is the most common gynecological cancer and one of the major causes of female cancer-related death worldwide particularly in developing countries. Thus far, there are a few in vivo models have been developed in investigating this type of cancer. In this study, we induced cervical cancer in Balb/c mice by exploiting the carcinogenic property of diestylstilbestrol (DES). The Balb/c pregnant mice were given subcutaneous (SC) injection of 67µg/kg body weight of DES on GD 13, and the mice gave birth approximately at gestation day 19-22. Female offspring were reared and the body weight was recorded once weekly. The female offspring were sacrificed at age of 5 months. Upon termination, blood was collected in a plain tube via cardiac puncture and the reproductive tracts were collected and weighed. The reproductive tract sections were stained using H&E for observation of pathological changes. The progression of disease state was monitored by measuring the level of serum interleukin (IL-6) using the Mouse IL-6 ELISA Assay Kit (BD OptEIA™, USA). All parameters were compared with Not-induced group. The outcome of this study demonstrated a significant difference in body weight gain, reproductive organ weight, diameter of cervix and the level of serum IL-6 in the Induced group as compared to the Not-induced group (P<0.05). Histopathological findings revealed the presence of adenosis only in the Induced group. It shows that DES could be employed as an agent to induce cervical carcinogenesis in animal model. In addition to that, new potential anti-cancer agents from various sources could be further evaluated using this technique.

Dillenia suffruticosa exhibited antioxidant and cytotoxic activity through induction of apoptosis and G2/M cell cycle arrest.

ETHNOPHARMACOLOGICAL RELEVANCE: Dillenia suffruticosa (Family: Dilleniaceae) locally known as Simpoh air has been reported to be used traditionally to treat cancerous growth. Therefore, the present study was attempted to investigate the antioxidant and cytotoxic properties of different parts (root, flower, fruit and leaf) of D. suffruticosa extracts. METHODS AND MATERIALS: In this study, direct solvent extraction (aqueous and methanol) from different parts of D. suffruticosa (root, flower, fruit and leaf) were carried out. Antioxidant activities of D. suffruticosa extract were determined by using DPPH, ABTS FRAP and ß-carotene bleaching assays. Cytotoxicity and cell cycle arrest of the active extract were determined using MTT assay and flow cytometer, respectively. Sequential solvent extraction (hexane, DCM, EtOAc, and MeOH) were also carried out in root of D. suffruticosa to further evaluate the antioxidant and cytotoxic activity of the different solvent extracts. RESULTS: Methanol (MeOH) root extract showed the highest TPC, antioxidant and cytotoxic activities (especially towards HeLa) compared to others (P<0.05). Based on the results, sequential solvent extraction (hexane, DCM, EtOAc and MeOH) was carried out in the roots of D. suffruticosa. MeOH extract exhibited the highest antioxidant activities among others and significantly correlated (P<0.05) with TPC, suggesting the important contribution of phenolic compounds to its antioxidant activity. On the other hand, the DCM and EtOAc exhibited higher cytotoxic activity to selected cancer cells (HeLa, MCF-7, MDA-MB-231, A549 and HT29) compared to others. In short, there is no established correlation between antioxidant and cytotoxic activities of D. suffruticosa extracts indicating that an agent with high antioxidant activities will not necessarily possesses good cytotoxic activities in return. Qualitative phytochemical screening of D. suffruticosa extracts suggested the presence of saponins, triterpenes, sterols, and polyphenolic compounds which are believed to contribute to the cytotoxic activities. CONCLUSION: It is suggested that the cytotoxicity of the active extracts in HeLa was due to the induction of apoptosis and cell cycle arrest at G2/M.

The hexane fraction of Ardisia crispa Thunb. A. DC. roots inhibits inflammation-induced angiogenesis.

BACKGROUND: Ardisia crispa (Myrsinaceae) is used in traditional Malay medicine to treat various ailments associated with inflammation, including rheumatism. The plant's hexane fraction was previously shown to inhibit several diseases associated with inflammation. As there is a strong correlation between inflammation and angiogenesis, we conducted the present study to investigate the anti-angiogenic effects of the plant's roots in animal models of inflammation-induced angiogenesis. METHODS: We first performed phytochemical screening and high-performance liquid chromatography (HPLC) fingerprinting of the hexane fraction of Ardisia crispa roots ethanolic extract (ACRH) and its quinone-rich fraction (QRF). The anti-inflammatory properties of ACRH and QRF were tested using the Miles vascular permeability assay and the murine air pouch granuloma model following oral administration at various doses. RESULTS: Preliminary phytochemical screening of ACRH revealed the presence of flavonoids, triterpenes, and tannins. The QRF was separated from ACRH (38.38% w/w) by column chromatography, and was isolated to yield a benzoquinonoid compound. The ACRH and QRF were quantified by HPLC. The LD(50) value of ACRH was 617.02 mg/kg. In the Miles vascular permeability assay, the lowest dose of ACRH (10 mg/kg) and all doses of QRF significantly reduced vascular endothelial growth factor (VEGF)-induced hyperpermeability, when compared with the vehicle control. In the murine air pouch granuloma model, ACRH and QRF both displayed significant and dose-dependent anti-inflammatory effects, without granuloma weight. ACRH and QRF significantly reduced the vascular index, but not granuloma tissue weight. CONCLUSIONS: In conclusion, both ACRH and QRF showed potential anti-inflammatory properties in a model of inflammation-induced angiogenesis model, demonstrating their potential anti-angiogenic properties.

Vanillin differentially affects azoxymethane-injected rat colon carcinogenesis and gene expression.

Vanillin is the substance responsible for the flavor and smell of vanilla, a widely used flavoring agent. Previous studies reported that vanillin is a good antimutagen and anticarcinogen. However, there are also some contradicting findings showing that vanillin was a comutagen and cocarcinogen. This study investigated whether vanillin is an anticarcinogen or a cocarcinogen in rats induced with azoxymethane (AOM). Rats induced with AOM will develop aberrant crypt foci (ACF). AOM-challenged rats were treated with vanillin orally and intraperitoneally at low and high concentrations and ACF density, multiplicity, and distribution were observed. The gene expression of 14 colorectal cancer-related genes was also studied. Results showed that vanillin consumed orally had no effect on ACF. However, high concentrations (300 mg/kg body weight) of vanillin administered through intraperitoneal injection could increase ACF density and ACF multiplicity. ACF were mainly found in the distal colon rather than in the mid-section and proximal colon. The expression of colorectal cancer biomarkers, protooncogenes, recombinational repair, mismatch repair, and cell cycle arrest, and tumor suppressor gene expression were also affected by vanillin. Vanillin was not cocarcinogenic when consumed orally. However, it was cocarcinogenic when being administered intraperitoneally at high concentration. Hence, the use of vanillin in food should be safe but might have cocarcinogenic potential when it is used in high concentration for therapeutic purposes.

Kenaf seed supercritical fluid extract reduces aberrant crypt foci formation in azoxymethane-induced rats.

Kenaf (Hibiscus cannabinus) a plant of the family Malvaceae, is a valuable fiber plant native to India and Africa. Kenaf seeds contain alpha-linolenic acid, phytosterol such as ß-sitosterol, vitamin E and other antioxidants with chemopreventive properties. In the present study we examined the hypothesis that kenaf seed 'supercritical fluid extract' (SFE) extract could suppress the early colon carcinogenesis in vivo by virtue of its bioactive compounds. To accomplish this goal, 60 male rats were randomly assigned to 5 groups which were (1) negative control group [not induced with azoxymethane (AOM)]; (2) positive control group (induced with AOM but received no treatment); (3) group treated with 500 mg/kg kenaf seed SFE extract; (4) group treated with 1000 mg/kg kenaf seed SFE extract; (5) group treated with 1500 mg/kg kenaf seed SFE extract. At 7 weeks of age, all rats except the negative control group received 15 mg/kg of AOM injection subcutaneously once a week for 2 weeks. Rats were euthanized at 13 weeks of the experiment. Number of ACF (mean±SD) ranged from 84.4±4.43 to 179.5±12.78 in group 2, 3, 4, 5. ACF reductions compared with the untreated group were 45.3, 51.4 and 53.1% in rats fed with 500, 1000 and 1500 mg/kg body weight, respectively. There were no significant differences in weight gain among groups. Our finding indicates that kenaf seed SFE extract reduced AOM-induced ACF in Sprague-Dawley male rats.

Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein.

Thymoquinone (TQ), the active constituent of Nigella sativa or black cumin exhibited cytotoxic effects in several cancer cell lines. In this study, the cytotoxicity of TQ in human cervical squamous carcinoma cells (SiHa) was investigated. TQ was cytotoxic towards SiHa cells with IC50 values of 10.67 ± 0.12 and 9.33 ± 0.19 µg/mL as determined by MTT assay and trypan blue dye exclusion test, respectively, after 72 h of incubation. TQ was more cytotoxic towards SiHa cells compared to cisplatin. Interestingly, TQ was less cytotoxic towards the normal cells (3T3-L1 and Vero). Cell cycle analysis performed by flowcytometer showed a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Apoptosis induction by TQ was further confirmed by Annexin V/PI and AO/PI staining. Significant elevation of p53 and down-regulation of the anti-apoptotic Bcl-2 protein was found in the treated cells, without any changes in the expression of the pro-apoptotic Bax protein. In conclusion, thymoquinone from N. sativa was more potent than cisplatin in elimination of SiHa cells via apoptosis with down-regulation of Bcl-2 protein.

Toxicology study of vanillin on rats via oral and intra-peritoneal administration.

Vanillin is useful as anti-sickle cell anemia, anti-mutagen and anti-bacteria agent. However, vanillin must be administered at high concentration and cannot be oxidized by the upper gastrointestinal track of patients to be medically effective. In this study, we assessed the toxic effect of vanillin when administered in an un-oxidized form at high concentrations (150 and 300 mg/kg) via oral and intra-peritoneal injection. It was found that 300 mg/kg vanillin injection caused the rats to be unconscious without exerting any toxic effect on blood cells, kidney and liver. Besides, it showed blood protective property. Further analysis with GenomeLab GeXP genetic system on brain tissues showed that the expression of most xenobiotic metabolism, cell progression, tumor suppressor, DNA damage and inflammation genes were maintained at normal level. However, the expression of a few xenobiotic metabolism, cell cycle arrest and apoptosis genes were up-regulated by 5% ethanol injection. Nevertheless, when 5% ethanol was injected with the presence of vanillin, the expression was back to normal level. It is postulated that vanillin might have neuro-protective property. In conclusion, vanillin is not toxic at high concentration in both oral and intra-peritoneal injection and could provide blood and brain protective properties.

Apoptosis and cell cycle arrest of human colorectal cancer cell line HT-29 induced by vanillin.

BACKGROUND: Vanillin is responsible for the flavor and smell of vanilla, a widely used flavoring agent. Previous studies showed that vanillin could enhance the repair of mutations and thus function as an anti-mutagen. However, its role in cancer, a disease that is closely related to mutation has not yet been fully elucidated. METHODS: Hence, this study investigated the cytolytic and cytostatic properties of vanillin against HT-29, a human colorectal cancer cell line. Methods used including cell viability assay, acridine orange (AO)-ethidium bromide (EB) double staining cell morphological analysis, Cell cycle analysis, annexin V-propidium iodide apoptosis test and 5-bromo-2-deoxyuridine (BrdU)-labeling cell proliferation assay. RESULTS: Results showed that apoptosis was induced by vanillin and the IC(50) for HT-29 and NIH/3T3 normal cell lines were 400 microg/ml and 1000 microg/ml, respectively. Different concentrations of vanillin arrest cell cycle at different checkpoints. 5-Bromo-2-deoxyuridine-labeling cell proliferation assay showed that G0/G1 arrest was achieved at lower concentration of vanillin (200 microg/ml) while cell cycle analysis by flow cytometer showed that G2/M arrest occurs at higher concentration of vanillin (1000 microg/ml). CONCLUSION: Cytolytic and cytostatic effects shown by vanillin showed that it could be a useful colorectal cancer preventive agent. Further in vivo study should be carried out to confirm that similar effects could happen in animals.