Kenaf (Hibiscus cannabinus) from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β -sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO) was from supercritical carbon dioxide extraction fluid (SFE) at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29) and mouse embryonic fibroblast (NIH/3T3) cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining.
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 μ M. A significant induction of early phase of apoptosis was shown by annexin V and lysosomal stability assays. After 24 h treatment with 19.01 μ M 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.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells, without causing significant toxicity in normal tissues. We previously reported that galactoxyloglucan (PST001) possesses significant antitumor and immunomodulatory properties. However, the exact mechanism in mediating this anticancer effect is unknown. This study, for the first time, indicated that PST001 sensitizes non-small cell lung cancer (A549) and nasopharyngeal (KB) cells to TRAIL-mediated apoptosis. In vitro studies suggested that PST001 induced apoptosis primarily via death receptors and predominantly activated caspases belonging to the extrinsic apoptotic cascade. Microarray profiling of PST001 treated A549 and KB cells showed the suppression of survivin (BIRC5) and anti-apoptotic Bcl-2, as well as increased cytochrome C. TaqMan low density array analysis of A549 cells also confirmed that the induction of apoptosis by the polysaccharide occurred through the TRAIL-DR4/DR5 pathways. This was finally confirmed by in silico analysis, which revealed that PST001 binds to TRAIL-DR4/DR5 complexes more strongly than TNF and Fas ligand-receptor complexes. In summary, our results suggest the potential of PST001 to be developed as an anticancer agent that not only preserves innate biological activity of TRAIL, but also sensitizes cancer cells to TRAIL-mediated apoptosis.
para‑Phenylenediamine (p‑PD) is a potential carcinogen, and widely used in marketed hair dye formulations. In the present study, the role of the protein tyrosine kinase (PTK)/Ras/Raf/c‑Jun N‑terminal kinase (JNK) and phosphoinositide 3‑kinase (PI3k)/protein kinase B (Akt) pathways on the growth of NRK‑52E cells was investigated. The results demonstrated that p‑PD reduced cell viability in a dose‑dependent manner. The cell death due to apoptosis was confirmed by cell cycle analysis and an Annexin‑V‑fluorescein isothiocyanate binding assay. Subsequent to staining with 2',7'‑dichlorofluorescin diacetate, the treated cells demonstrated a significant increase in reactive oxygen species (ROS) generation compared with the controls. The effects of p‑PD on the signalling pathways were analysed by western blotting. p‑PD‑treated cells exhibited an upregulated phospho‑stress‑activated protein kinase/JNK protein expression level and downregulated Ras and Raf protein expression levels; however, Akt, Bcl‑2, Bcl‑XL and Bad protein expression levels were not significantly altered compared with the control. In conclusion, p‑PD induced apoptosis by a PTK/Ras/Raf/JNK‑dependent pathway and was independent of the PI3K/Akt pathway in NRK‑52E cells.
Alpha-tomatine (α-tomatine) is the major saponin in tomato (Lycopersicon esculentum). This study investigates the chemopreventive potential of α-tomatine on androgen-independent human prostatic adenocarcinoma PC-3 cells.
Acute myeloid leukemia (AML) is one of the most frequent types of leukemia which mostly affects adult people. Resistance to therapeutic drugs is considered as a major clinical concern resulting in a weaker response to chemotherapy, disease relapse and decreased survival rate. Survivin, a member of Inhibitor of Apoptosis Proteins (IAPs), is associated with drug resistance and inhibition of apoptotic mechanisms in numerous hematological malignancies. In the present study, we examined the combined effect of etoposide and siRNA-mediated silencing of survivin on U-937 acute myeloid leukemia cells. The AML cells were transfected with survivin specific siRNA and gene knockdown was confirmed by quantitative real time PCR and western blotting. Subsequently, U-937 cells were assessed for response to etoposide treatment and apoptosis rate was measured with flowcytometery. The cytotoxic effects in siRNA-etoposide group were measured and compared to etoposide single therapy group. Survivin siRNA effectively knocked down the mRNA and protein levels of survivin, which led to lower cell proliferation and enhanced apoptosis. Furthermore, combined treatment of etoposide and survivin siRNA synergistically increased the cell toxic effects of etoposide and its ability to induce apoptosis.
Cervical cancer accounts for the second most frequent cancer and also third leading cause of cancer mortality (15%) among women worldwide. The major problems of chemotherapeutic treatment in cervical cancer are non-specific cytotoxicity and drug resistance. Plant-derived products, known as natural therapies, have been used for thousands of years in cancer treatment with a very low number of side effects. Allium atroviolaceum is a species in the genus Allium and Liliaceae family, which could prove to have beneficial effects on cancer treatment, although there is a lack of corresponding attention. The methanolic extract from the A.atroviolaceum flower displayed marked anticancer activity on HeLa human cervix carcinoma cells with much lower cytotoxic effects on normal cells (3T3). The A.atroviolaceum extract induced apoptosis, confirmed by cell cycle arrest at the sub-G0 (apoptosis) phase, characteristic morphological changes, evident DNA fragmentation, observed by fluorescent microscope, and early and late apoptosis detection by Annexin V. Furthermore, down-regulation of Bcl-2 and activation of caspase-9 and -3 strongly indicated that the mitochondrial pathway was involved in the apoptosis signal pathway. Moreover, combination of A.atroviolaceum extract with doxorubicin revealed a significant reduction of IC50and led to a synergistic effect. In summary, A.atroviolaceum displayed a significant anti-tumour effect through apoptosis induction in HeLa cells, suggesting that the A.atroviolaceum flower might have therapeutic potential against cervix carcinoma.
Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00 ± 0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00 ± 0.52 μg/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug.
Mesenchymal stem cells (MSCs) are emerging as vehicles for anti-tumor cytotherapy; however, investigation on its efficacy to target a specific cancer stem cell (CSC) population in non-small cell lung cancer (NSCLC) is lacking. Using assays to evaluate cell proliferation, apoptosis, and gene expression, we investigated the efficacy of MSCs expressing tumour necrosis factor (TNF)-related apoptosis inducing ligand (MSC-TRAIL) to target and destroy CD133+ (prominin-1 positive) NSCLC-derived CSCs. Characterization of TRAIL death receptor 5 (DR5) revealed that it was highly expressed in the CD133+ CSCs of both H460 and H2170 cell lines. The human MSC-TRAIL generated in the study maintained its multipotent characteristics, and caused significant tumor cell inhibition in NSCLC-derived CSCs in a co-culture. The MSC-TRAIL induced an increase in annexin V expression, an indicator of apoptosis in H460 and H2170 derived CD133+ CSCs. Through investigation of mitochondria membrane potential, we found that MSC-TRAIL was capable of inducing intrinsic apoptosis to the CSCs. Using pathway-specific gene expression profiling, we uncovered candidate genes such as NFKB1, BAG3, MCL1, GADD45A, and HRK in CD133+ CSCs, which, if targeted, might increase the sensitivity of NSCLC to MSC-TRAIL-mediated inhibition. As such, our findings add credibility to the utilization of MSC-TRAIL for the treatment of NSCLC through targeting of CD133+ CSCs.
In the biomedical field, there is growing interest in using human stem cell-derived neurons as in vitro models for pharmacological and toxicological screening of bioactive compounds extracted from natural products. Lignosus rhinocerus (Tiger Milk Mushroom) is used by indigenous communities in Malaysia as a traditional medicine to treat various diseases. The sclerotium of L. rhinocerus has been reported to have medicinal properties, including various bioactivities such as neuritogenic, anti-inflammatory, and anticancer effects. This study aims to investigate the neuroprotective activities of L. rhinocerus sclerotial extracts. Human embryonic stem cell (hESC)-derived neural lineages exposed to the synthetic glucocorticoid, dexamethasone (DEX), were used as the in vitro models. Excess glucocorticoids have been shown to adversely affect fetal brain development and impair differentiation of neural progenitor cells. Screening of different L. rhinocerus sclerotial extracts and DEX on the hESC-derived neural lineages was conducted using cell viability and neurite outgrowth assays. The neuroprotective effects of L. rhinocerus sclerotial extracts against DEX were further evaluated using apoptosis assays and Western blot analysis. Hot aqueous and methanol extracts of L. rhinocerus sclerotium promoted neurite outgrowth of hESC-derived neural stem cells (NSCs) with negligible cytotoxicity. Treatment with DEX decreased viability of NSCs by inducing apoptosis. Coincubation of L. rhinocerus methanol extract with DEX attenuated the DEX-induced apoptosis and reduction in phospho-Akt (pAkt) level in NSCs. These results suggest the involvement of Akt signaling in the neuroprotection of L. rhinocerus methanol extract against DEX-induced apoptosis in NSCs. Methanol extract of L. rhinocerus sclerotium exhibited potential neuroprotective activities against DEX-induced toxicity in hESC-derived NSCs. This study thus validates the use of human stem cell-derived neural lineages as potential in vitro models for screening of natural products with neuroprotective properties.
Manilkara zapota (L.) P. Royen, called sapodilla, or locally known as ciku, belongs to the family Sapotaceae. We found that Manilkara zapota leaf water extract has cytotoxic effect against human hepatocellular carcinoma (HepG2) cell line in our earlier study. Therefore, this study aimed to explore the anticancer properties of Manilkara zapota leaf water extract in HepG2 cells. We also aimed to unravel yet undiscovered mechanisms and identified several expressed genes whose functions in cytotoxicity activity of Manilkara zapota leaf water extract in HepG2 cells have not been well-studied. The apoptosis and intracellular reactive oxygen species (ROS) activities were analyzed using Annexin V-propidium iodide staining and dichlorodihydrofluorescein diacetate, respectively, by NovoCyte Flow Cytometer. Bax and Bcl-2 expression were assessed using Enzyme-Linked Immunosorbent Assay. The associated molecular pathways were evaluated by quantitative real-time PCR. Overall analyses revealed that Manilkara zapota leaf water extract can increase percentage of early apoptotic cells, induce the formation of ROS, upregulate c-Jun N-terminal kinase 1 (JNK1) and inducible nitric oxide synthase (iNOS), and reduce Akt1 and vascular endothelial growth factor A (VEGFA) transcriptional activities. Our data suggest that Manilkara zapota leaf water extract can suppress the growth of HepG2 cells via modulation of ERK1/2/Akt1/JNK1 transcriptional expression.
In this study, we determined the molecular mechanism of γ-tocotrienol (GTT) in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs). Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal) and promoted G(0)/G(1) cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P < 0.05). GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P < 0.05). Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P < 0.05) in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.
Boesenbergia rotunda (Roxb.) Schlecht (family zingiberaceae) is a rhizomatous herb that is distributed from north-eastern India to south-east Asia, especially in Indonesia, Thailand and Malaysia. Previous research has shown that the crude extract of this plant has cytotoxic properties. The current study examines the cytotoxic properties of boesenbergin A isolated from Boesenbergia rotunda.
BACKGROUND: The ethiopathogenesis of increased apoptosis of lymphocytes in systemic lupus erythematosus (SLE) is still incompletely understood but anti-C1q autoantibodies have been shown to induce apoptosis in lymphocytes from healthy donors and certain cell lines.
AIM: This study was undertaken to investigate the relationship between peripheral lymphocyte apoptosis and serum levels of anti-C1q autoantibodies in SLE patients.
METHODS: The sera of 124 patients with SLE involving 62 active SLE and 62 inactive SLE, fulfilling America College of Rheumatology (ACR) classification criteria for SLE (1997) were incubated with peripheral blood lymphocytes of healthy donors. The results were compared with 124 sex- and age-matched normal controls. Apoptotic lymphocytes (AL) were detected by flow cytometry using annexin V and propidium iodide binding. Anti-C1q autoantibodies were detected by an enzyme-linked immunoassay kit for all SLE patients.
RESULTS: Results demonstrated that the percentage of AL in the peripheral blood of active SLE patients was significantly higher (n = 62, 34.95 ± 12.78%) than that of the inactive SLE patients (n = 62, 30.69 ± 10.13%, P = 0.042, 95%CI = 0.16-8.36) and normal controls (n = 124, 27.92 ± 10.22%, P = 0.001, 95%CI = 3.33-10.73). The percentage of AL significantly correlated with serum levels of anti-C1q autoantibodies in the active SLE patients (r = 0.263, P = 0.039) but not in the inactive SLE patients (r = 0.170, P = 0.185).
CONCLUSION: The results of this study suggest that increased serum levels of anti-C1q autoantibodies are responsible for apoptosis and may play a pathogenic role in SLE patients, especially in active disease.
KEYWORDS: anti-C1q; apoptosis; flowcytometry; systemic lupus erythematosus
Study site: Medical outpatient clinic and medical wards, Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia
Vitamin C is generally thought to enhance immunity and is widely taken as a supplement especially during cancer treatment. Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial oestrogen receptor beta in MCF-7 cells and induces apoptosis by activation of pro-caspase-8 followed by downstream events, including an increase in reactive oxygen species and the release of pro-apoptotic factors from the mitochondria. In addition to that, TAM binds with high affinity to the microsomal anti-oestrogen-binding site and inhibits cholesterol esterification at therapeutic doses. This study aimed to investigate the role of vitamin C in TAM-mediated apoptosis. Cells were loaded with vitamin C by exposure to dehydroascorbic acid, thereby circumventing in vitro artefacts associated with the poor transport and pro-oxidant effects of ascorbic acid. Pre-treatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by acridine-orange/propidium iodide (AO/PI) and Annexin V assay after treatment with TAM. Vitamin C dose-dependently protected cancer cells against lipid peroxidation caused by TAM treatment. By real-time PCR analysis, an impressive increase in FasL and tumour necrosis factor-α (TNF-α) mRNA was detected after TAM treatment. In addition, a decrease in mitochondrial transmembrane potential was observed. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.