Yin Sim Tor

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 β-sitosterol-3-O-β-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.

Acute and subacute toxicity profiles of thymoquinone-loaded nanostructured lipid carrier in BALB/c mice

Background Thymoquinone (TQ), the predominant active lipophilic component in Nigella sativa seed oil, has a variety of pharmacological properties such as anticancer activities. However, translation of TQ to clinical phase is still not possible due to its hydrophobic properties. This problem can be solved by encapsulating it in nanoformulations to enhance its pharmacological properties. In our previous study, TQ has been successfully encapsulated in a nanostructured lipid carrier (hereinafter referred to as TQNLC) with excellent physiochemical properties such as high encapsulation efficiency, high drug-loading capacity, particle diameter less than 100 nm, and stability up to 2 years. In vitro studies also proved that TQNLC exhibited antiproliferative activity toward breast and cervical cancer cell lines. However, no toxicity profile related to this formulation has been reported. In this study, we determine and compare the in vivo toxicity of both TQNLC and TQ. Materials and methods The in vivo toxicity (acute and subacute toxicity) study was carried out by oral administration of TQNLC and TQ to BALB/c mice. Animal survival, body weight, organ weight-to-body weight ratio, hematological profile, biochemistry profile, and histopathological changes were analyzed. Results In acute toxicity, TQ that is loaded in nanostructured lipid carrier (NLC) was found to be less toxic than pure TQ. It can be concluded that encapsulation of TQ in lipid carrier minimizes the toxicity of the compound. In the subacute toxicity study, oral administration of 100 mg/kg of TQNLC and TQ did not cause mortality to either male or female but resulted in toxicity to the liver. It is postulated that long-term consumption of TQNLC and TQ may cause toxicity to the liver but not to the extent of altering the functions of the organ. For both treatments, the no observed adverse effect level (NOAEL) was found to be 10 mg/kg/d for mice in both sexes. Conclusion For long-term oral consumption, TQ and TQNLC at a dose of 10 mg/kg is safe in mice and does not exert any toxic effect. The results provide safety information of TQNLC, which would further help researchers in clinical use.

Dillenia suffruticosa dichloromethane root extract induced apoptosis towards MDA-MB-231 triple-negative breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE Dillenia suffruticosa is traditionally used for treatment of cancerous growth including breast cancer in Malaysia. AIM OF THE STUDY Dillenia suffruticosa is a well-known medicinal plant in Malaysia for the treatment of cancer. Nevertheless, no study has been reported the cytotoxicity of this plant towards MDA-MB-231 triple-negative breast cancer cells. The present study was designed to investigate the mode of cell death and signalling pathways of MDA-MB-231 cells treated with dichloromethane Dillenia suffruticosa root extract (DCM-DS). METHODS Extraction of Dillenia suffruticosa root was performed by the use of sequential solvent procedure. The cytotoxicity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using an inverted light microscope and flow cytometry analysis using Annexin-V/PI. Cell cycle analysis and measurement of reactive oxygen species level were performed by using flow cytometry. The cells were treated with DCM-DS and antioxidants α-tocopherol or ascorbic acid to evaluate the involvement of ROS in the cytotoxicity of DCM-DS. Effect of DCM-DS on the expression of antioxidant, apoptotic, growth, survival genes and proteins were analysed by using GeXP-based multiplex system and Western blot, respectively. The cytotoxicity of compounds isolated from DCM-DS was evaluated towards MDA-MB-231 cells using MTT assay. RESULTS DCM-DS induced apoptosis, G2/M phase cell cycle arrest and oxidative stress in MDA-MB-231 cells. The induction of apoptosis in MDA-MB-231 cells by DCM-DS is possibly due to the activation of pro-apoptotic JNK1 and down-regulation of anti-apoptotic ERK1, which in turn down-regulates anti-apoptotic BCL-2 to increase the BAX/BCL-2 ratio to initiate the mitochondrial apoptotic pathway. The cell cycle arrest in DCM-DS-treated MDA-MB-231 cells is possibly via p53-independent but p21-dependent pathway. A total of 3 triterpene compounds were isolated from DCM-DS. Betulinic acid appears to be the most major and most cytotoxic compound in DCM-DS. CONCLUSION The data suggest the potential application of DCM-DS in the treatment of triple-negative breast cancer.

Chemopreventive Properties and Toxicity of Kelulut Honey in Sprague Dawley Rats Induced with Azoxymethane

Ethnopharmacological Relevance. Colon cancer has been a major problem worldwide. Kelulut honey (KH) is produced by the stingless bees from Trigona species and has strong antioxidant activities that could be one of the potential chemopreventive agents from natural resources. Aim of This Study. This study investigated the chemopreventive properties and toxicity of KH in Sprague Dawley rats induced with azoxymethane (AOM). Material and Method. Twenty-four male Sprague Dawley rats aged 5 weeks were divided into 4 groups: (G1) untreated group not induced with AOM, (G2) untreated group induced with AOM, (G3) treated group induced with AOM, and (G4) treated group not induced with AOM. Injection of AOM (15 mg/kg) was via intraperitoneal route once a week for two subsequent weeks. The treatment groups were given oral administration of KH (1183 mg/kg body weight) twice daily for 8 weeks. Results. Treatment with KH significantly reduced the total number of aberrant crypt foci (ACF) and aberrant crypts (AC) and crypt multiplicity. KH was not toxic to the animals since the level of blood profile parameters, liver enzymes, and kidney functions was in normal range. Conclusions. The current finding shows that KH has chemopreventive properties in rats induced with colorectal cancer and also was found not toxic towards the animals.

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.