The analysis of biochemical markers of MCF-7 cells apoptosis induced by recombinant analog of lactaptin

Earlier we isolated and characterized human milk pro-apoptotic peptide - lactaptin and generated its engineered analog - RL2. It was shown that both lactaptin and RL2 are capable to induce apoptotic death of MCF-7 cells. In this report we have analyzed biochemical markers of RL2 induced MCF-7 apoptosis. The activation of initiator and effector caspases as well as mitochondrial membrane potential and cytoplasm membrane changes were analyzed using flow cytometry and Western-blot methods. We have found that RL2 induced apoptotic death of MCF-7 cells was accompanied by PS exposure on the plasma membrane surface. It also was shown that RL2 has induced dissipation of mitochondrial membrane potential and resulted in activation of initiator caspases 8, 9 and effector caspase 7.     

Analysis of biochemical markers of MCF-7 cell apoptosis induced by a recombinant analogue of lactaptin

Earlier, a pro-apoptotic peptide lactaptin was isolated from human milk and characterized and its recombinant analogue RL2 was generated. Both peptides were demonstrated to be capable of apoptotic cell death induction in human breast adenocarcinoma MCF-7 cells. In the work, biochemical markers of RL2-induced MCF-7 apoptosis are analyzed. Activation of initiator and effector caspases, as well as apoptotic changes in mitochondrial membrane potential and cytoplasm membrane composition in cells treated with RL2, were analyzed using flow cytometry and Western blot techniques. MCF-7 cell death induced by RL2 was found to be associated with phosphatidylserine exposure on the surface of the plasma membrane. Also, RL2 was demonstrated to induce dissipation of the mitochondrial membrane potential and activation of initiator caspases 8 and 9 and an effector caspase 7.     

Lactaptin Induces p53-Independent Cell Death Associated with Features of Apoptosis and Autophagy and Delays Growth of Breast Cancer Cells in Mouse Xenografts

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs.     

Penetration of the peptide lactaptin into human cancer cells

Lactaptin, a human milk protein with a molecular weight of 8.6 kDa, is a fragment of human ?casein, which has cytotoxic activity toward mammalian cancer cells in vitro. RL2 is a recombinant analogue of lactaptin, which induces the apoptosis of human cancer cells in culture and suppresses the tumor growth in vivo. It has been shown earlier that RL2 penetrates into both human cancer and nonmalignized cells and binds to cytoskeletal structures. In this process, it induces the apoptosis of cancer cells and does not diminish the viability of normal cells. The mechanism of the penetration of RL2 into human cancer cells has been studied by flow cytometry and fluorescence microscopy using the inhibitors of different endocytosis pathways. It has been shown that RL2 penetrates into cells partly through lipid raft-mediated dynamin-independent pinocytosis and partly through direct penetration across the plasma cell membrane. An analysis of the primary structure of RL2 and the mechanism of its penetration into the cell suggests that it can be assigned to the class of cell-penetrating peptides.     

A novel pro-apoptotic effector lactaptin inhibits tumor growth in mice models

Lactaptin, a human milk-derived protein, induces apoptosis in cultured tumor cells. We designed a recombinant analog of lactaptin (RL2) and tested its antitumor activity. The sensitivity of hepatocarcinoma A-1 (HA-1), Lewis lung carcinoma, and Ehrlich carcinoma to RL2 were tested to determine the most reliable in vitro animal model. HA-1 cells, which had the highest sensitivity to RL2, were transplanted into A/Sn mice to investigate RL2 antitumor activity in vivo. Investigation of the molecular effects of RL2 shows that RL2 induces apoptotic transformation of HA-1 cells in vitro: phosphatidylserine translocation from inner side of the lipid bilayer to the outer one and dissipation of the mitochondrial membrane potential. Repetitive injections of RL2 (5–50 mg/kg) for 3–5 days effectively inhibited ascites and solid tumor transplant growth when administered intravenously or intraperitoneally, without obvious side effects. The solid tumor inhibitory effect of RL2 (5 i.v. injections, cumulative dose 125 mg/kg) was comparable with that of cyclophosphamide at a therapeutic dose (5 i.v. injections, cumulative dose 150 mg/kg). In combination therapy with cyclophosphamide, RL2 had an additive antitumor effect for ascites-producing tumors. Histomorphometric analysis indicated a three-fold reduction of spontaneous metastases in the liver of RL2-treated mice with solid tumor transplants in comparison with control animals. Repeated RL2 treatment substantially prolonged the lifespan of mice with intravenously injected tumor cells. Recombinant analog of lactaptin effectively induced apoptosis of tumor cells in vitro and suppressed the growth of sensitive tumors and metastases in vivo.     

Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells

Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 μg/mL of oleuropein or 50 μg/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G₁ to S phase transition manifested by the increase of cell number in G₀/G₁ phase.     

Novel thiosemicarbazides induced apoptosis in human MCF-7 breast cancer cells via JNK signaling

Abstract

In this study, novel thiosemicarbazides and 1,3,4-oxadiazoles were synthesized and evaluated for their anticancer effects on human MCF-7 breast cancer cell lines. Among the synthesized derivatives studied, compound 2-(3-(4-chlorophenyl)-3-hydroxybutanoyl)-N-phenylhydrazinecarbothioamide 4c showed the highest cytotoxicity against MCF-7 breast cancer cells as it reduced cell viability to approximately 15% compared to approximately 25% in normal breast epithelial cells. Therefore, we focused on 4c for further investigations. Our data showed that 4c induced apoptosis in MCF-7 cells which was further confirmed by TUNEL assay. Western blotting analysis showed that compound 4c up-regulated the pro-survival proteins Bax, Bad and ERK1/2, while it down-regulated anti-apoptotic proteins Bcl-2, Akt and STAT-3. Additionally, 4c induced phosphorylation of SAPK/JNK in MCF-7 cells. Pretreatment of MCF-7 cells with 10?µM of JNK inhibitor significantly reduced 4c-induced apoptosis. Molecular docking results suggested that compound 4c showed a binding pattern close to the pattern observed in the structure of the lead fragment bound to JNK1. Collectively, the data of current study suggested that the thiosemicarbazide 4c might trigger apoptosis in human MCF-7 cells by targeting JNK signaling.     

BCL-2 antisense and cisplatin combination treatment of MCF-7 breast cancer cells with or without functional p53

Summary Chemotherapy has been used for treatment of breast cancer but with limited success. We characterized the effects of bcl-2 antisense and cisplatin combination therapy in two human isogenic breast carcinoma cells p53(+)MCF-7 and p53(−)MCF-7/E6. The transferrin-facilitated lipofection strategy we have developed yielded same transfection efficiency in both cells. Bcl-2 antisense delivered with this strategy significantly induced more cell death, apoptosis, and cytochrome c release in MCF-7/E6 than in MCF-7, but did not affect Fas level in both cells and activated caspase-8 equally. Cisplatin exerted same effects on cell viability and apoptosis in both cells, but released smaller amounts of cytochrome c while activated more caspase-8 in MCF-7/E6. The combination treatment yielded greater effects on cell viability, apoptosis, cytochrome c release, and caspase-8 activation than individual treatments in both cells although p53(−) cells were more sensitive. The potentiated activation of caspase-8 in the combination treatment suggested that caspase-8-mediated (but cytochrome c-independent) apoptotic pathway is the major contributor of the enhanced cell killing. Thus, bcl-2 antisense delivered with transferrin-facilitated lipofection can achieve the efficacy of killing breast cancer cells and sensitizing them to chemotherapy. Bcl-2 antisense and cisplatin combination treatment is a potentially useful therapeutic strategy for breast cancer irrespective of p53 status. Hesham Basma and Hesham El-Refaey contributed equally     

Novel quinuclidinone derivatives induced apoptosis in human breast cancer via targeting p53

Small molecules that can target human cancers have been highly sought to increase the anticancer efficacy, the present work describes the design and synthesis of novel series of five quinuclidinone derivatives (2a-2e). Their anticancer activities were investigated against breast cancer cells MCF-7, MDA-MB-231 breast cancer cells harboring mutant p53 and normal breast counterpart MCF-12a. Derivative 2e reduced proliferation of MCF-7 and MCF-12a while it has no effect on MDA-MB-231. Derivative 2e induced apoptosis in MCF-7 cells which is further confirmed by TUNEL assay and it reduced the percentage of cell in G2/M phase as confirmed by increased expression of cyclin B and reduced expression of cyclin D1. Derivative 2e reduced expression levels of Mdm2, Akt and ERK1/2 by and increased expression level of p53. Moreover, the apoptosis induction by 2e was also inhibited by PFT-α as evidenced by non-significant induction of apoptosis after treatment of MCF-7 cells with both derivative 2e and PFT-α. In addition, docking study reveals that derivative 2e has a binding pattern close to the pattern observed in the structure of the lead fragment 5,6-dimethoxy-2-methylbenzothiazole bound to T-p53C-Y220C. The above findings demonstrate that derivative 2e induces apoptosis in MCF-7 cells via targeting p53 which merits further development.     

Bisphenol A prevents MCF-7 breast cell apoptosis via the inhibition of progesterone receptor transactivation

2,2-Bis(4-hydroxyphenyl)propane (bisphenol A; BPA) is an environmental endocrine-disrupting chemical. It mimics the effects of estrogen at multiple levels by activating estrogen receptors (ERs); however, BPA also affects the proliferation of human breast cancer cells independent of ERs. Although BPA inhibits progesterone (P4) signaling, the toxicological significance of its effects remain unknown. Tripartite motif-containing 22 (TRIM22) has been identified as a P4-responsive and apoptosis-related gene. Nevertheless, it has not yet been established whether exogenous chemicals change TRIM22 gene levels. Therefore, the present study investigated the effects of BPA on P4 signaling and TRIM22 and TP53 expression in human breast carcinoma MCF-7 cells. In MCF-7 cells incubated with various concentrations of P4, TRIM22 messenger RNA (mRNA) levels increased in a dose-dependent manner. P4 induced apoptosis and decreased viability in MCF-7 cells. The knockdown of TRIM22 abolished P4-induced decreases in cell viability and P4-induced apoptosis. P4 increased TP53 mRNA expression and p53 knockdown decrease the basal level of TRIM22 and P4 increased TRIM22 mRNA expression independent of p53 expression. BPA attenuated P4-induced increases in the ratio of cell apoptosis in a concentration-dependent manner, and the P4-induced decreases in cell viability was abolished in the presence of 100 nM and higher BPA concentrations. Furthermore, BPA inhibited P4-induced TRIM22 and TP53 expression. In conclusion, BPA inhibited P4-induced apoptosis in MCF-7 cells via the inhibition of P4 receptor transactivation. TRIM22 gene has potential as a biomarker for investigating the disruption of P4 signaling by chemicals.     

Arthpyrone L,a New Pyridone Alkaloid from a Deep-Sea Arthrinium sp., Inhibits Proliferation of MG63 Osteosarcoma Cells by Inducing G0/G1 Arrest and Apoptosis

Fractionation of the ethanol extract of a marine fungus, Arthrinium sp., afforded a new pyridone alkaloid (arthpyrone L ( 1 )), the structure with absolute configuration of which was established by comprehensive spectroscopic analyses. In vitro cell viability assays revealed that compound 1 showed antiproliferative effects toward human A549 (lung), MG63, U2OS (bone), MCF-7 and MDA-MB-231 (breast) cancer cells. MG63 cell lines were chosen for further biological evaluations and presented apoptosis and cell cycle arrest (G0/G1 phase) upon treatment of 1 . Subsequent mechanism studies demonstrated that the growth inhibition of 1 against MG63 cells was via activation of caspase-modulated apoptotic pathway and inhibition of PI3K/Akt pathway.     

(6E,10E) Isopolycerasoidol and (6E,10E) Isopolycerasoidol Methyl Ester,Prenylated Benzopyran Derivatives from Pseuduvaria monticola Induce Mitochondrial-Mediated Apoptosis in Human Breast Adenocarcinoma Cells

Phytochemicals from Pseuduvaria species have been reported to display a wide range of biological activities. In the present study, a known benzopyran derivative, (6E,10E) isopolycerasoidol (1), and a new benzopyran derivative, (6E,10E) isopolycerasoidol methyl ester (2), were isolated from a methanol extract of Pseuduvaria monticola leaves. The structures of the isolated compounds were elucidated by spectroscopic methods including 1D and 2D NMR, IR, UV, and LCMS-QTOF, and by comparison with previously published data. The anti-proliferative and cytotoxic effects of these compounds on human breast cancer cell-lines (MCF-7 and MDA-MB-231) and a human normal breast epithelial cell line (MCF-10A) were investigated. MTT results revealed both (1) and (2) were efficient in reducing cell viability of breast cancer cells. Flow cytometry analysis demonstrated that (1) and (2) induced cell death via apoptosis, as demonstrated by an increase in phosphotidylserine exposure. Both compounds elevated ROS production, leading to reduced mitochondrial membrane potential and increased plasma membrane permeability in breast cancer cells. These effects occurred concomitantly with a dose-dependent activation of caspase 3/7 and 9, a down-regulation of the anti-apoptotic gene BCL2 and the accumulation of p38 MAPK in the nucleus. Taken together, our data demonstrate that (1) and (2) induce intrinsic mitochondrial-mediated apoptosis in human breast cancer cells, which provides the first pharmacological evidence for their future development as anticancer agents.     

Salubrinal-Mediated Upregulation of eIF2α Phosphorylation Increases Doxorubicin Sensitivity in MCF-7/ADR Cells

Eukaryotic translation initiation factor 2 alpha (eIF2α), which is a component of the eukaryotic translation initiation complex, functions in cell death and survival under various stress conditions. In this study, we investigated the roles of eIF2α phosphorylation in cell death using the breast cancer cell lines MCF-7 and MCF-7/ADR. MCF-7/ADR cells are MCF-7-driven cells that have acquired resistance to doxorubicin (ADR). Treatment of doxorubicin reduced the viability and induced apoptosis in both cell lines, although susceptibility to the drug was very different. Treatment with doxorubicin induced phosphorylation of eIF2α in MCF-7 cells but not in MCF-7/ADR cells. Basal expression levels of Growth Arrest and DNA Damage 34 (GADD34), a regulator of eIF2α, were higher in MCF-7/ADR cells compared to MCF-7 cells. Indeed, treatment with salubrinal, an inhibitor of GADD34, resulted in the upregulation of eIF2α phosphorylation and enhanced doxorubicin-mediated apoptosis in MCF-7/ADR cells. However, MCF-7 cells did not show such synergic effects. These results suggest that dephosphorylation of eIF2α by GADD34 plays an important role in doxorubicin resistance in MCF-7/ADR cells.     

Dryofragin, a phloroglucinol derivative, induces apoptosis in human breast cancer MCF-7 cells through ROS-mediated mitochondrial pathway

Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott. In this study, the anticancer activity of dryofragin on human breast cancer MCF-7 cells was investigated. Dryofragin inhibited the growth of MCF-7 cells in a time and concentration-dependent manner. The cell viability was measured using MTT assay. After treatment with dryofragin for 72, 48 and 24h, the IC(50) values were 27.26, 37.51 and 76.10μM, respectively. Further analyses of DNA fragmentation and Annexin V-PI double-labeling indicated an induction of apoptosis. Dryofragin-treatment MCF-7 cells had a significantly accumulation of reactive oxygen species (ROS), as well as an increased percentage of cells with mitochondrial membrane potential (MMP) disruption. These phenomena were blocked by pretreatment for 2h of MCF-7 cells with the antioxidant compound N-acetyl-l-cysteine (NAC, 5mM). These results speak for the involvement of a ROS-mediated mitochondria-dependent pathway in dryofragin-induced apoptosis. Western blot results showed that dryofragin inhibited Bcl-2 and induced Bax expression which led to an activation of caspases-9 and -3 in the cytosol, and further cleavage of poly ADP-ribose polymerase (PARP) in the nucleus, then induced cell apoptosis. In conclusion, the present study provides evidence that dryofragin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway.     

In situ observation of a cell adhesion and metabolism using surface infrared spectroscopy

In this study, we report on an in situ monitoring system of living cultured cells using infrared absorption spectroscopy in the geometry of multiple internal reflections (MIR-IRAS). In order to observe living cultured cells, the temperature in the sample chamber of a FT-IR spectrometer was maintained at 37 °C and a humidified gas mixture containing 5% CO₂ was introduced into the sample chamber. Human breast cell line MCF-7 cultured on Si MIR prisms were placed in the sample chamber and infrared spectra of MCF-7 cells were collected for 5 h. It was found that the adhesion and metabolism of MCF-7 cells could be monitored by the absorption intensity of amide-II protein band (1,545 cm⁻¹) and also by the absorption intensities of CH _x bands (2,700–3,100 cm⁻¹). These results suggest that our system is useful for a nondestructive and non-label monitoring of cell viability. Our method based on infrared absorption spectroscopy has a potential for bioscreening application.     

Silencing of the <Emphasis Type="Italic">CCNB1, Her2</Emphasis>, and <Emphasis Type="Italic">PKC</Emphasis> genes by small interfering RNA differently retards the division of different human cancer cell lines

Deregulation of genes encoding proteins responsible for cell cycle control frequently accompanies cell malignization and switches the cell program from differentiation and apoptosis to uncontrollable proliferation. We used siRNAs targeted to HER2, protein kinase C (PKC), and cyclin B1 (CCNB1) mRNAs to evaluate the therapeutic potential of the suppression of genes coding for key cell cycle regulators in different human cancer cells. The CCNB1, HER2, or PKC mRNA levels were efficiently reduced within 48 h after transfection with siCycB1, siHER2 or siPKC, respectively. Silencing of HER2, PKC, and CCNB1 substantially reduced the growth rates of all cell lines under study except HL-60 but did not affect cell death or apoptosis. The most pronounced inhibition of cell division was induced by siCycB1 in SK-N-MC cells and by siPKC in MCF-7 cells. We conclude that the selected siRNAs inhibit tumor cell division, and the investigated genes can be promising targets in cancer treatment.     

Cytotoxic effect of sanguiin H-6 on MCF-7 and MDA-MB-231 human breast carcinoma cells

Sanguiin H-6 is a dimer of casuarictin linked by a bond between the gallic acid residue and one of the hexahydroxydiphenic acid units. It is an effective compound extracted from Rubus coreanus. It has an anticancer effect against several human cancer cells; however, its effect on breast cancer cells has not been clearly demonstrated. Thus, we aimed to investigate the anticancer effect and mechanism of action of sanguiin H-6 against two human breast carcinoma cell lines (MCF-7 and MDA-MB-231). We found that sanguiin H-6 significantly reduced cell viability in a concentration-dependent manner. It also increased the rates at which MCF-7 and MDA-MB-231 cells underwent apoptosis. Furthermore, sanguiin H-6 induced the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase, which resulted in apoptosis. However, cleavage of caspase-9 was only detectable in MCF-7 cells. In addition, sanguiin H-6 increased the ratio of Bax to Bcl-2 in both MCF-7 and MDA-MB-231 cells. These findings suggest that sanguiin H-6 is a potent therapeutic agent against breast cancer cells. In addition, it exerts its anticancer effect in an estrogen-receptor-independent manner.     

In vitro anti-proliferative activities of ellagic acid

The potential cytotoxic and anti-proliferative activities of ellagic acid (a naturally occurring bioactive compound in berries, grapes, and nuts) was evaluated using human umbilical vein endothelial cells (HUVEC), normal human lung fibroblast cells HEL 299, Caco-2 colon, MCF-7 breast, Hs 578T breast, and DU 145 human prostatic cancer cells. Ellagic acid at concentration in the range 10-100 micromol/L did not affect the viability of normal fibroblast cells during a 24-hour incubation. An increase in adenosine triphosphate (ATP) bioluminescence of approximately 18-21% was observed in normal cells incubated with ellagic acid. In contrast, ellagic acid at 1-100 micromol/L dose-dependently inhibited HUVEC tube formation and proliferation on a reconstituted extracellular matrix and showed strong anti-proliferative activity against the colon, breast, and prostatic cancer cell lines investigated. The most sensitive cells were the Caco-2, and the most resistant were the breast cancer cells. Ellagic acid induced cancer cell death by apoptosis as shown by the microscopic examination of cell gross morphology. Ellagic acid induced reduced cancer cell viability as shown by decreased ATP levels of the cancer cells. After 24 hours incubation of 100 micromol/L of ellagic acid with Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells, ellagic acid suppressed fetal bovine serum (FBS) stimulation of cell migration. The apoptosis induction was accompanied by a decreased in the levels of pro-matrix metalloproteinase-2 (pro-MMP-2 or gelatinase A), pro-matrix metalloproteinase-9 (pro-MMP-9 or gelatinase B), and vascular endothelial growth factor (VEGF(165)) in conditioned media. The results suggest that ellagic acid expressed a selective cytotoxicity and anti-proliferative activity, and induced apoptosis in Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells without any toxic effect on the viability of normal human lung fibroblast cells. It was also observed that the mechanism of apoptosis induction in ellagic acid-treated cancer cells was associated with decreased ATP production, which is crucial for the viability of cancer cells.     

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21^Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.