Induction of apoptosis by eugenol in human breast cancer cells

In the present study, potential anticancer effect of eugenol on inhibition of cell proliferation and induction of apoptosis in human MCF-7 breast cancer cells was investigated. Induction of cell death by eugenol was evaluated following MTT assay and monitoring lactate dehydrogenase released into the culture medium for cell viability and cytotoxicity, giemsa staining for morphological alterations, fluorescence microscopy analysis of cells using ethidium bromide and acridine orange and quantitation of DNA fragments for induction of apoptosis. Effect of eugenol on intracellular redox status of the human breast cancer cells was assessed by determining the level of glutathione and lipid peroxidation products (TBARS). Eugenol treatment inhibited the growth and proliferation of human MCF-7 breast cancer cells through induction of cell death, which was dose and time dependent. Microscopic examination of eugenol treated cells showed cell shrinkage, membrane blebbing and apoptotic body formation. Further, eugenol treatment also depleted the level of intracellular glutathione and increased the level of lipid peroxidation. The dose dependent increase in the percentage of apoptotic cells and DNA fragments suggested that apoptosis was involved in eugenol induced cell death and apoptosis might have played a role in the chemopreventive action of eugenol.     

The cytotoxic nature of Acanthopanax sessiliflorus stem bark extracts in human breast cancer cells

Acanthopanax sessiliflorus, a small woody shrub has traditionally been referred to have anticancer activity, but it has not been scientifically explored so far. Therefore, to investigate the anticancer effects of A. sessiliflorus stem bark extracts (ASSBE), MDA-MB-231 and MCF-7 human breast cancer cells were treated with one of its bioactive fractions, n-hexane (ASSBE-nHF). Cytotoxicity (24 h) was determined by MTT assay and antiproliferative effect was assessed by counting cell numbers after 72 h treatment using hemocytometer. The role of ASSBE-nHF on apoptosis was analysed by annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation pattern and immunoblotting of apoptosis markers. For the assay of enhanced production of ROS and mitochondrial membrane depolarization, specific stains such as DCFH-DA and JC-1 were used, respectively. To understand the mode of action of ASSBE-nHF on MCF-7 cells, cells were pre-treated with antioxidant, n-acetylcysteine. The hexane fraction of ASSBE showed maximum activity towards human breast cancer cells compared to other two fractions at a minimal concentration of 50 μg/ml. The annexin V-FITC/PI, Hoechst 33342 staining, DNA fragmentation and immunoblotting assays showed that ASSBE-nHF induces non-apoptotic cell death in MCF-7 and MDA-MB-231 cells. ASSBE-nHF significantly increased the production of ROS and decreased the mitochondrial membrane potential (MMP) in MCF-7 cells. Similarly, it decreased the MMP in MDA-MB-231 cells, but had no effect on ROS production. Further, the cytotoxic effect of ASSBE-nHF in MCF-7 cells was not significantly reversed even in the presence of n-acetylcysteine, an antioxidant. These findings revealed that ASSBE-nHF induces non-apoptotic cell death via mitochondria associated with both ROS dependent and independent pathways in human breast cancer cells.     

Dieckol exerts anticancer activity in human osteosarcoma (MG-63) cells through the inhibition of PI3K/AKT/mTOR signaling pathway

BackgroundOsteosarcoma (OS) is the most common malignant bone cancer with more metastasis and increased occurrence in children and teen-agers and being responsible for more number of morbidity and mortality worldwide.ObjectiveThe current exploration was planned study the in vitro anticancer actions of dieckol against human OS MG-63 cells via PI3K/AKT/mTOR signaling inhibition.MethodologyThe cytotoxicity of dieckol was scrutinized by MTT assay. Effects of dieckol on the ROS accumulation, apoptotic cell death, and MMP level in the MG-63 cells were studied by respective fluorescence staining assays. The levels of proliferative, inflammatory, and apoptotic markers in the dieckol treated MG-63 cells were scrutinized by marker specific kits. The expressions of PI3K, AKT, and mTOR was assayed by RT-PCR.ResultsThe MTT assay revealed that the dieckol dose dependently prevented MG-63 cells viability and the IC50 was found at 15 µM. Dieckol treatment effectively reduced the MMP level and improved the ROS generation and apoptosis in MG-63 cells. Dieckol also regulated the proliferative (cyclin D1), inflammatory (COX-2, IL-6, TNF-α, and NF-κB), and apoptotic (caspase-3, Bax, Bcl-2) markers in the MG-63 cells. The PI3K/AKT/mTOR signaling in the MG-63 cells were effectively inhibited by the dieckol treatment.ConclusionIn conclusion, our findings from this study recommends that the dieckol could be a talented anticancer candidate for the OS management in the future.     

Rhaponticin suppresses osteosarcoma through the inhibition of PI3K-Akt-mTOR pathway

Osteosarcoma is the frequent pediatric bone cancer where pediatric osteosarcoma incidences are more than 10% within the population. Most of the patients with osteosarcoma fall within the age of 15–30 years. Therefore, in this research, we examined the anticancer effect of Rhaponticin against the human osteosarcoma (MG-63) cells. The cytotoxicity of Rhaponticin on the MC3T3-E1 and MG-63 cells was examined through the MTT assay. The intracellular ROS accumulation, cell nuclear morphological alterations, apoptotic cell death and nuclear damages, and MMP status of Rhaponticin administered MG-63 cells were inspected by fluorescent staining techniques. The cell migration was assessed through scratch assay. The mRNA expressions of PI3K-Akt-mTOR signaling proteins were studied by RT-PCR analysis. Rhaponticin showed potent cytotoxicity, substantially inhibited the MG-63 cell growth, and displayed morphological alterations. However, rhaponticin did not affect the MC3T3-E1 cell viability. Rhaponticin administered MG-63 cells demonstrated augmented intracellular ROS accretion, weakened MMP, increased nuclear damages, and increased apoptosis. Rhaponticin effectively down-regulated the PI3K-Akt-mTOR signaling cascade in the MG-63 cells. These outcomes proved that the Rhaponticin can be a hopeful chemotherapeutic agent in the future to treat human osteosarcoma.     

Prodigiosin-induced apoptosis in human colon cancer cells

Prodigiosin is a red pigment produced by various bacteria including Serratia marcescens. Colorectal cancer is one of the most frequent malignancies and one of the most frequent causes of cancer death in the Western world. Its treatment is far from satisfactory and the challenge to oncologists is to find novel chemical entities with less toxicity and greater effectiveness than those used in current chemotherapy. Here we characterize the apoptotic action of prodigiosin in colon cancer cells. DLD-1 and SW-620 human colon adenocarcinoma cells, NRK and Swiss-3T3 nonmalignant cells were assayed by the MTT assay, fragmentation pattern of DNA, Hoechst 33342 staining and study of PARP cleavage by Western blot, in order to characterize the prodigiosin-induced apoptosis. Prodigiosin was purified and its structure was confirmed. Metastatic SW-620 cells were more sensitive to prodigiosin (IC50: 275 nM) than DLD-1. We did not observe a significant decrease in the viability of NRK cells. We confirmed that prodigiosin induces apoptosis in both cancer cell lines by the characteristic DNA laddering pattern and condensed nuclei or apoptotic bodies identified by fluorescence microscopy. These results indicate that prodigiosin induces apoptosis in colon cancer cells.     

Estrogen-induced resistance to osteoblast apoptosis is associated with increased hsp27 expression

Estrogen has been shown to protect osteoblastic cells from apoptosis. Similarly, estrogen treatment preceding heat shock elevates heat shock protein 27 (hsp27) expression and increases thermoresistance in the murine estrogen receptor-transformed SMER14 osteoblastic cell line. Forced expression of hsp27 expression in other cell lines limits apoptosis. The purpose of this study was to examine the effects of estrogen on staurosporine-induced apoptosis in the context of hsp27 expression. Cell viability was measured by the MTT assay. Early apoptotic events were examined by fluorescent microscopy by using FITC-conjugated Annexin V staining. TUNEL labeling was used to compare the number of apoptotic nuclei following staurosporine treatment of estrogen pretreated or untreated cells. Estrogen treatment increased SMER14 cell viability, but not ROS17/2.8 cell viability, in the presence of staurosporine. Estrogen treatment also reduced annexin V staining and DNA fragmentation. Similar treatment increased SMER14 cell hsp27 levels. The concurrent reduction in induced apoptosis suggests a possible estrogenic mechanism for increasing and/or maintaining the number of viable osteoblasts in bone.     

Induction of apoptosis in human cancer cells by a Bacillus lipopeptide bacillomycin D

A newly isolated and characterized Bacillus amyloliquefaciens strain fiply 3A has been found to produce an extracellular cyclic lipopeptide which structurally resembled bacillomycin D, earlier reported to be produced by Bacillus subtilis. The lipopeptide showed a dose dependent killing of three different human cancer cell lines viz. A549 (alveolar adenocarcinoma), A498 (renal carcinoma) and HCT-15 (colon adenocarcinoma), while not affecting the normal cell line L-132 (pulmonary epithelial cells) when analyzed using MTT assay and FACS analysis. Staining the cells with H₂-DCFDA showed an increase in reactive oxygen species (ROS) formation in the lipopeptide treated cell population. Hoechst 33342 staining of nuclei further indicated apoptosis as a major mechanism of cell death in lipopeptide treated cells and the typical symptoms of apoptosis including cell shrinkage, nuclear condensation and fragmentation of nuclei were observed. Lipopeptide treatment induced extensive DNA damage in the treated cells, which was indicated by a TUNEL assay. Flow cytometric analysis exhibited lipopeptide concentration dependent apoptosis which was further confirmed during clonogenic assay of the lipopeptide treated cells.     

Early detection of staurosporine-induced apoptosis by comet and annexin V assays

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction. Accepted: 2 June 1999     

Amphotericin B potentiates the anticancer activity of doxorubicin on the MCF-7 breast cancer cells

Despite the improvements in cancer treatment, breast cancer still remains the second most common cause of death from cancer in women. Doxorubicin (DOXO) is widely used for cancer treatment. However, drug resistance limits the treatment outcome. Here, we investigated the toxicity of DOXO in combination with an antifungal agent amphotericin B (AmB) against the MCF-7 breast cancer cell line. The cell viability was measured using MTT assay. The apoptosis was studied by caspase-8 and caspase-9 activity measurements and DNA fragmentation was investigated by TUNEL assay. The combination of two drugs significantly increased the apoptotic index and the caspase-8 and caspase-9 activities in comparison to DOXO-treated cells. Our finding showed that pre-treatment of MCF-7 cells with AmB synergistically exerted the anticancer effect of DOXO through the caspase-dependent apoptosis manner.     

Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans

The antifungal activity of Nerol (NEL) against Candida albicans, a pathogenic fungus, has a minimum inhibitory concentration (MIC) of 4.4 mM that causes noteworthy candidacidal activity through an apoptosis-like mechanism. Calcium (Ca²⁺) levels and reactive oxygen species (ROS) production, which are the major causes of apoptosis, were determined in C. albicans cells treated with NEL and were found to increase, which related to mitochondrial dysfunction and disruption. A series of characteristic changes of apoptosis caused by NEL, including mitochondrial membrane depolarization, cytochrome c (cyt c) release, and metacaspase activation were examined using a flow cytometer and Western blot. The results showed that an increase in intracellular Ca²⁺ and ROS led to dramatically decreased mitochondrial membrane potential (MMP); cyt c was also released from the mitochondria to the cytosol. Other early apoptotic features were also observed with the metacaspase activation. Finally, the morphological changes of the cells were observed, including phosphatidylserine (PS) externalization, nuclear condensation, and DNA fragmentation through Annexin V-FITC and PI double staining, TUNEL assay, and DAPI staining. The results supported the hypothesis that NEL was involved in the apoptosis of C. albicans cells not only at the early stages, but also at the late stages. In summary, NEL can trigger mitochondrial dysfunction and disruption via elevation of Ca²⁺ and ROS leading to apoptosis in C. albicans. This research on the mechanism of cell death triggered by NEL against C. albicans has important significance for providing a novel treatment of C. albicans infections.     

Induction of yeast apoptosis by an antimicrobial peptide, Papiliocin

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, we found that Papiliocin induced the accumulation of reactive oxygen species (ROS) and hydroxyl radicals known to be important regulators of apoptosis in Candida albicans. To examine the relationship between the accumulation of ROS and the induction of apoptosis, we investigated the apoptotic effects of Papiliocin using apoptotic markers. Cells treated with Papiliocin showed a series of cellular changes normally seen in cells undergoing apoptosis: plasma membrane translocation of phosphatidylserine from the inner to the outer membrane leaflet, measured by Annexin V staining, dissipation of the mitochondrial membrane potential, observed by DiOC₆(3) staining; and the presence of active metacaspases, measured using the CaspACE FITC-VAD-FMK, as early apoptotic events. In addition, DNA condensation and fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. Therefore, these results suggest that Papiliocin leads to apoptosis in C. albicans via ROS accumulation.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Phosphoinositide 3-OH kinase/protein kinase B inhibits apoptotic cell death induced by reactive oxygen species in Saccharomyces cerevisiae

Apoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.     

Protective effects of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon: removal of O(6)-methylguanine DNA adducts, p53 expression, inducible nitric oxide synthase downregulation and apoptotic induction

Previous studies have shown that dietary micronutrient vanadium can protect neoplastic development induced by chemical carcinogens. Current investigation is an attempt to evaluate the role of vanadium (4.27 micro mol/l) in inhibiting 1,2 dimethyhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. We investigated the effect of vanadium against the formation of DMH-induced O(6)-methylguanine (O(6)-Meg) DNA adduct, a potent cytotoxic and mutagenic agent for colon cancer. Supplementation of vanadium significantly reduced the hepatic (P<0.05), and colonic (at three sequential time points; ANOVA, F=4.96, P<0.05) O(6)-Meg DNA adduct levels in rats, indicating vanadium's potency in limiting the initiation event of colon carcinogenesis. Removal of initiated and damaged precancerous cells by apoptosis can prevent tumorigenesis and further malignancy. DNA fragmentation study revealed the vanadium-mediated apoptotic induction in colon tumors. The increased value of apoptotic index (AI) (62.27%; P<0.01) in subsequent TUNEL assay further confirmed the apoptosis induction by vanadium. This paralleled the nuclear immunoexpression of p53. A significant positive correlation between p53 immunoexpression and AI (P=0.0026, r=0.83, r(2)=0.69) links its association with vanadium-mediated apoptotic induction. Vanadium treatment also abated the mRNA expression of iNOS (54.03%), reflecting its protective effect against nitric oxide-mediated genotoxicity and colon tumorigenesis. These studies cumulatively provide strong evidence for the inhibitory actions of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon.     

Fucoxanthin induced apoptotic cell death in oral squamous carcinoma (KB) cells

Fucoxanthin (Fx) is an active compound commonly found in the many types of seaweed with numerous biological activities. The main goal of this investigation is to explore the effect of Fx against the cell proliferation, apoptotic induction and oxidative stress in the oral squamous (KB) cell line. Cytotoxicity of Fx was determined by MTT assay. The intracellular ROS production, mitochondrial membrane potential (MMP) and apoptosis induction in KB cells were examined through DCFH-DA, Rhodamine-123 and DAPI, and dual staining techniques. Effect of Fx on the antioxidant enzymes and lipid peroxidation in the KB cells was studied through the standard procedures. Fx treated KB cells showed morphological changes and reduced cell survival, which is exhibited by the cytotoxic activity of 50 µM/ml (IC50) Fx against the KB cells. The Fx treatment considerably induced the apoptotosis cells (EB/AO) and decreased the MMP (Rh-123) in KB cells. Further, it was pointed out that there was an increased lipid peroxidation (LPO) with decreased antioxidants (CAT, SOD and GSH). These results concluded that Fx has the cytotoxic effect against KB cells and has the potential to induce the apoptosis via increased oxidative stress. Hence, the Fx can be a promising agent for the treatment of oral cancer and it may lead to the development of cancer therapeutics.     

Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway

Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-l-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.     

Delayed reproductive death and ROS levels in the progeny of irradiated melanoma cells

The cell death and survival of proliferating (clonogenic) cells were investigated in two human melanoma cell lines to assess the optimal conditions for preparation of apoptotic bodies from melanoma cells. After 50 J/m2 UVB+UVC the maximal levels of apoptotic cells assayed by Trypan blue staining, nucleosomal DNA fragmentation, MTT, and TUNEL tests were observed within 2-3 d of radiation. In 100 Gy gamma-irradiated cultures these apoptosis indicators were delayed for up to 3 weeks. In addition, clonogenic cells were observed only in exponentially growing cultures irradiated with UV at high cell density but not in gamma-irradiated cultures. The response of melanoma cultures after high UV radiation doses contrasted to the response in lethally gamma-irradiated cultures. UV-irradiated melanoma cultures were recovered within two weeks. Most of the clonogenic cells in the recovered colonies contained micronuclei. ROS levels determined by DCF fluorescence and a modified MTT test were also normalized obviously due to the extensive antioxidant defense system of melanoma cells. UV radiation of tumor cells might be the preferential method for preparation of apoptotic bodies. The presence of clonogenic cells in the suspension of apoptotic bodies from melanoma cells used for pulsing of dendritic cells with tumor antigens might compromise this protocol for preparation of cell vaccines.     

Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using Annexin V and propidium iodide versus the neutral comet assay

BACKGROUND: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. METHODS: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. RESULTS: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. CONCLUSIONS: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway.     

Anti-Cancer Effect and the Underlying Mechanisms of Gypenosides on Human Colorectal Cancer SW-480 Cells

Background

Gypenosides (Gyp), the main components from Gynostemma pentaphyllum Makino, are widely used in traditional Chinese medicine. The present study aimed to investigate the anti-cancer effect and the underlying mechanisms of Gyp on human colorectal cancer cells SW-480.

Materials and Methods

The inhibitory effect of Gyp on SW-480 cells was evaluated by MTT assay. Apoptotic cell death was detected by nuclear Hoechst 33342 staining and DNA fragmentation analysis. Apoptosis was analyzed using Annexin V-PE/7-amino-actinomycin D staining. Cell membrane integrity was evaluated with flow cytometry following PI staining. Changes of mitochondrial membrane potential (Δψ _m) were detected through flow cytometry analysis of rhodamine 123 (Rh123). The role of reactive oxygen species (ROS) in Gyp induced cell death was investigated by intracellular ROS generation and general ROS scavenger. Wound-healing assay was carried out to investigate Gyp-inhibited migration of SW-480 cells in vitro. Additionally, the alterations in F-actin microfilaments were analyzed by FITC-labeled phalloidin toxin staining and the morphological changes were evaluated under scanning electron microscope (SEM).

Results

After the Gyp treatment, the plasma membrane permeability of SW-480 cell was increased, Δψ _m was decreased significantly, the level of intracellular ROS level was increased, DNA fragmentation and apoptotic morphology were observed. Cells treated with Gyp exert serious microfilament network collapse as well as the significant decrease in the number of microvilli. Gyp induced the changes of cell viability, cell migration, intracellular ROS generation and nuclear morphology were alleviated obviously by NAC.

Conclusion

The results in this study implied that ROS play an important role in Gyp induced cell toxicity and apoptosis, and the mitochondria damage may be upstream of ROS generation post Gyp treatment. The findings of the present study provide new evidences for anti-tumor mechanisms by which Gyp induces apoptosis in vitro.     

Antiproliferative and apoptosis-induction studies of 5-hydroxy 3′,4′,7-trimethoxyflavone in human breast cancer cells MCF-7: an in vitro and in silico approach

The aim of this study was to find the efficacy of 5-hydroxy 3′,4′,7-trimethoxyflavone (HTMF), a flavonoid compound isolated from the medicinal plant Lippia nodiflora, in inhibiting the proliferation and inducing apoptosis in human breast cancer cell line MCF-7. The anti-proliferative effect of the compound HTMF was confirmed using MTT cytotoxicity assay. Increased apoptotic induction by HTMF was demonstrated by acridine orange/ethidium bromide (AO/EtBr) and Hoechst 33258 staining studies. The phosphatidylserine translocation, an early feature of apoptosis and DNA damage were revealed through AnnexinV-Cy3 staining and comet assay. Moreover, the significant elevation of cellular ROS was observed in the treated cells, as measured by 2,7-diacetyl dichlorofluorescein (DCFH-DA). The mRNA expression studies also supported the effectiveness of HTMF by shifting the Bax:Bcl-2 ratio. The treatment of MCF-7 cells with HTMF encouraged apoptosis through the modulation of apoptotic markers, such as p53, Bcl-2, Bax, and cleaved PARP. In silico molecular docking and dynamics studies with MDM2-p53 protein revealed that HTMF was more potent compound that could inhibit the binding of MDM2 with p53 and, therefore, could trigger apoptosis in cancer cell. Overall, this study brings up scientific evidence for the efficacy of HTMF against MCF-7 breast cancer cells.