Release behaviour and toxicity evaluation of levodopa from carboxylated single-walled carbon nanotubes

This work explores the potential use of commercially obtained, carboxylated, single-walled carbon nanotubes (SWCNT–COOH) as nanocarriers for the antiparkinson drug, levodopa (LD). The resulting nanohybrid was characterized using materials characterization methods including Fourier transform infrared spectroscopy, Raman spectroscopy, elemental analysis, UV–vis spectroscopy and scanning electron microscopy. The results showed that SWCNT–COOH were able to form supramolecular complexes with LD via a π–π stacking interaction and exhibited favourable, slow, sustained-release characteristics as a drug carrier with a release period over more than 20 h. The results obtained from the drug release studies of LD at different pH values showed that the LD-loaded nanohybrid is pH activated. The release kinetics of LD from SWCNT–COOH were well-described by a pseudo-second-order kinetic model. A cytotoxicity assay of the synthesized nanohybrid was also carried out in PC12 cell lines (a widely used, in vitro Parkinson’s model for neurotoxicity studies) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in order to investigate their possible effects on normal neuronal cells in vitro. It was found that the synthesized nanohybrid did not compromise the cell viability and the PC12 cells remained stable throughout the experiments up to 72 h after treatment

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 β-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

Induction of cell cycle arrest and apoptosis by copper complex Cu(SBCM)₂ towards oestrogen-receptor positive MCF-7 breast cancer cells

Copper complexes have the potential to be developed as targeted therapy for cancer because cancer cells take up larger amounts of copper than normal cells. Copper complex Cu(SBCM)2 has been reported to induce cell cycle arrest and apoptosis towards triple-negative breast cancer cells. Nevertheless, its effect towards other breast cancer subtypes has not been explored. Therefore, the present study was conducted to investigate the effect of Cu(SBCM)₂ towards oestrogen-receptor positive MCF-7 breast cancer cells. Growth inhibition of Cu(SBCM)₂ towards MCF-7 and human non-cancerous MCF-10A breast cells was determined by MTT assay. Morphological changes of Cu(SBCM)2-treated-MCF-7 cells were observed under an inverted microscope. Annexin V/PI apoptosis assay and cell cycle analysis were evaluated by flow cytometry. The expression of wild-type p53 protein was evaluated by Western blot analysis. The intracellular ROS levels of MCF-7 treated with Cu(SBCM)₂ were detected using DCFH-DA under a fluorescence microscope. The cells were then co-treated with Cu(SBCM)₂ and antioxidants to evaluate the involvement of ROS in the cytotoxicity of Cu(SBCM)2. Docking studies of Cu(SBCM)2 with DNA, DNA topoisomerase I, and human ribonucleotide reductase were also performed. The growth of MCF-7 cells was inhibited by Cu(SBCM)2 in a dose-dependent manner with less toxicity towards MCF-10A cells. It was found that Cu(SBCM)₂ induced G2/M cell cycle arrest and apoptosis in MCF-7 cells, possibly via a p53 pathway. Induction of intracellular ROS was not detected in MCF-7 cells. Interestingly, antioxidants enhance the cytotoxicity of Cu(SBCM)2 towards MCF-7 cells. DNA topoisomerase I may be the most likely target that accounts for the cytotoxicity of Cu(SBCM)₂

In Vitro Cytotoxicity and in Vivo Antitumour Properties of Kenaf Seed Oil Towards Leukaemia

The current treatments for leukaemia such as chemotherapy and radiation therapy have prolonged the survival rate. However, the adverse effects of these treatments are difficult to handle. Thus, there is a need to seek for other remedies, such as the use of natural products. Natural products such as plants play an important role in the current cancer treatment. The advantage of using plant-derived anticancer agents is that the produced adverse effects are lesser as compared to the synthetic drugs. Kenaf (Hibiscus cannabinus) seed oil (KSO) is a rich source of bioactive phytochemicals with high anti-oxidative and cancer chemopreventive properties. Nevertheless, the anti-leukaemia properties of KSO have yet been investigated. This study investigated the anti-leukaemia properties of KSO in vitro and in vivo. KSO was extracted by supercritical carbon dioxide fluid extractor (SFE), and evaluated for cytotoxic properties on leukaemia (HL-60, WEHI-3B and K562) and normal (3T3) cells by MTT assay with concentrations ranging from 50 to 800 μg/ml for 72 hours. The morphological changes of KSO-treated leukaemia cells were observed under an inverted light microscope and a fluorescence microscope. The cell cycle profile of KSO-treated leukaemia cells was analysed by flow cytometry. For in vivo, acute toxicity and anti-leukaemia properties of KSO were determined. Male BALB/c mice were injected intraperitoneally with WEHI-3B cells and administered orally with KSO at the dose of 0.5, 1.0 and 1.5 g/kg for 14 days. Upon completion, the blood of the mice was examined for the expression of cell surface marker of T cell (CD3), B cell (CD19), monocyte and granulocyte (CD11b) by staining with anti-CD3-FITC, anti-CD19-PE and anti-CD11b-PE antibodies, respectively. The livers and spleens were isolated, weighed and photographed. The spleens were processed for histopathological analysis. The yield of KSO by SFE ranged from 11 to 13% (w/w). KSO was found to be cytotoxic towards all the leukaemia cells in a dose-dependent manner with no effects on 3T3 cells even at the highest concentration employed (800 μg/ml). Oil from SFE at 600 bar 40 ºC (KSO V600/40) was the most cytotoxic towards HL-60, WEHI-3B and K562 cells as compared to other extractions (KSO V600/60, KSO V600/80 and Soxhlet) with the 50% inhibition concentration (IC50) values of 178.78±10.52, 189.43±11.63 and 213.33±15.45 μg/ml, respectively. KSO V600/40-treated leukaemia cells exhibited typical characteristics of apoptosis such as cellular shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. Cell cycle analysis revealed that KSO V600/40 at IC50 value induced G1 phase cell cycle arrest and significantly increased (p<0.05) the sub-G1 apoptotic population in the leukaemia cells. For in vivo, acute toxicity study revealed that KSO V600/40 did not cause any mortality in the healthy normal mice even at the highest dose (5.0 g/kg), suggesting that KSO is non-toxic by oral route. Treatment with KSO V600/40 at 1.0 and 1.5 g/kg increased the population of T cells, but decreased the population of immature monocytes and granulocytes in the blood of WEHI-3B injected BALB/c mice (WEHI-3B/BALB/c mice). Spleen and liver weight of WEHI-3B/BALB/c mice decreased after the treatment with KSO V600/40. Moreover, infiltration of leukaemic cells into the splenic red pulp reduced after the treatment, indicating that KSO V600/40 reduced the severity of leukaemia in WEHI-3B/BALB/c mice. In conclusion, KSO V600/40 showed cytotoxic effect via the induction of G1 phase cell cycle arrest and apoptosis in the leukaemia cells, and reduced the severity of leukaemia in WEHI-3B/BALB/c mice

Induction of apoptosis and the signalling pathways involved by dillenia suffruticosa dichloromethane root extract in mcf-7 and mda-mb-231 breast cancer cells

Dillenia suffruticosa has been used traditionally to treat cancerous growth. Previous study reported that dichloromethane extract of D. suffruticosa root (DCM-DS) was the most cytotoxic towards breast cancer cells. The present study investigated the mode of cell death and the signalling pathways involved in MCF-7 and MDA-MB-231 breast cancer cells treated with DCM-DS. DCM-DS was obtained by sequential solvent extraction. 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 AnnexinV/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) level were performed by using flow cytometry. The cells were co-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 compounds in DCM-DS were isolated by various chromatography techniques. The structure of the compounds was elucidated by using nuclear magnetic resonance analysis. DCM-DS was cytotoxic to the MCF-7 and MDA-MB-231 cells in a time-and dose-dependent manner. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 and MDA-MB-231 cells, respectively. DCMDS induced apoptosis and oxidative stress in these two cell lines. Treatment with α- tocopherol reduced the cytotoxicity of DCM-DS at 50 µg/mL in the cells, suggesting that DCM-DS induced lipid peroxidation to destroy the cancer cells. Therefore, DCMDS can be employed as a pro-oxidant agent to treat breast cancer. The induction of apoptosis in MCF-7 and MDA-MB-231 cells by DCM-DS is possibly due to the activation of pro-apoptotic JNK1 and down-regulation of anti-apoptotic ERK1 and AKT1, which in turn down-regulates anti-apoptotic BCL-2 to increase the BAX/BCL-2 ratio to initiate the mitochondrial apoptotic pathway. The induction of cell cycle arrest in MCF-7 and MDA-MB-231 cells is possibly via p53/p21-dependent and p53- independent but p21-dependent pathway, respectively. A total of seven triterpene compounds were isolated. Betulinic acid (BA) appears to be the major and most cytotoxic compound in DCM-DS. Therefore, BA could be used as a mean for standardisation of herbal product from D. suffruticosa. In conclusion, the data suggest the potential application of DCM-DS in the treatment of breast cancer

Kenaf seed oil from supercritical carbon dioxide fluid extraction shows cytotoxic effects towards various cancer cell lines

Hibiscus cannabinus (Kenaf) from the family of Malvaceae is a valuable fiber source and medicinal plant. It has long been prescribed as traditional folk medicine in Africa and India to treat various diseases. Nevertheless, little research has been carried out on the potentials of this plant as treatment for cancer. This study was designed to determine the cytotoxicity of kenaf seed oil from two varieties (Quiping 3 and V36) extracted by supercritical carbon dioxide fluid extraction (SFE) with different combinations of pressure (bars) and temperature (°C) towards breast cancer (MCF-7, MDA-MB-231, 4T1), cervical cancer (HeLa), lung cancer (A549) and leukemic (MOLT-4) cell lines. Even though kenaf seed oil from both varieties were cytotoxic to all the cancer cells, kenaf seed oil variety V36 extracted by SFE at 600 bars 40°C (V600/40) was the strongest towards MOLT-4 and MDA-MB-231, with the IC50 values of 153.26 and 483.35 μg/ml, respectively. MOLT-4 and MDA-MB-231 cells treated with V600/40 exhibited typical characteristics of apoptosis such as blebbing, chromatin condensation and nuclear fragmentation as viewed under an inverted light microscope and a fluorescence microscope. In conclusion, V600/40 was the most cytotoxic towards the MOLT-4 and MDA-MB-231 cells in a dosedependent manner possibly via the induction of apoptosis

Kenaf seed oil from supercritical carbon dioxide fluid extraction induced G1 phase cell cycle arrest and apoptosis in leukemia cells

This study was designed to determine the cytotoxic effects of kenaf seed oil (Hibiscus cannabinus) variety V36 extracted using supercritical carbon dioxide fluid extraction (SFE) with different combinations of pressure (bars) and temperature (°C). Extracted oils were tested on human promyelocytic HL-60, murine myelomonocytic WEHI-3B and human chronic myelogenous K562 leukemic cell lines. The yield of kenaf seed oil extracted by SFE ranged from 11 to 13% (w/w). Oils were found to be cytotoxic towards all the leukemia cell lines in a dose-dependent manner with no effects on normal cells (3T3). Oil from SFE at 600 bar 40°C (V600/40) was more cytotoxic towards HL-60, WEHI-3B and K562 when compared with other kenaf seed oils (extracted with different parameters) with the IC50 values of 178.78±10.52, 189.43±11.63 and 213.33±15.45 μg/ml, respectively. V600/40-treated leukemia cells exhibited typical characteristics of apoptosis such as nuclear fragmentation, chromatin condensation, nuclear margination, membrane blebbing and cellular shrinkage, as viewed under inverted light microscope and fluorescence microscope. Cell cycle analysis using flow cytometry revealed that, V600/40 induced G1 phase cell cycle arrest and significantly increased (P < 0.05) the sub- G1 apoptotic population in the leukemia cells. In conclusion, kenaf seed oil V600/40 induced apoptosis via G1 phase cell cycle arrest in HL-60, WEHI-3B and K562 leukemia cell lines

Development of erythropoietin receptor-targeted drug delivery system against breast cancer using tamoxifen-loade nanostructured lipid carriers

Tamoxifen (TAM) has been used in the treatment of breast cancers and is supplemented with erythropoietin (EPO) to alleviate the cancer-related anemia. The purported deleterious effects caused by the use of EPO with chemotherapeutic agents in the treatment of cancer-related anemia vary across studies and remain controversial. The use of nanoparticles as a drug delivery system has the potential to improve the specificity of anticancer drugs. In this study, we simultaneously incorporated two pharmacological active ingredients in one nanocarrier to develop EPO-conjugated TAM-loaded lipid nanoparticles (EPO-TAMNLC), a targeted delivery system, to enhance the cytotoxic activity while reducing the side effects of the ingredients. The effect of temperature in modulating the thermodynamic parameters associated with the binding of EPO and TAMNLC was assessed using isothermal titration calorimetry, while the unfolding of EPO structure was determined using fluorescence-quenching approach. The association efficiency of EPO and TAMNLC was 55.43%. Unlike binding of albumin to TAMNLC, the binding of EPO to TAMNLC occurred through endothermic and entropy-driven reaction. The EPO-TAMNLC formulation was stable because of the hydrophobic interaction and the high free energy, suggesting the spontaneity of the interactions between EPO and TAMNLC. The EPO-TAMNLC enhanced the in vitro cytotoxicity of TAM to MCF-7 cells. The EPO surface-functionalized TAMNLC could sequentially deliver EPO and TAM as well as improving site-specific delivery of these therapeutic compounds