Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines.

Epidemiologic studies have suggested that nutrition plays an important role in carcinogenesis and that 30% of cancer morbidity and mortality can potentially be prevented with proper adjustment of diets. Resveratrol, a polyphenol present in red wines and a variety of human foods, has recently been reported to exhibit chemopreventive properties when tested in a mouse skin cancer model system. In this study, we investigated the effects of resveratrol on growth, induction of apoptosis, and modulation of prostate-specific gene expression using cultured prostate cancer cells that mimic the initial (hormone-sensitive) and advanced (hormone-refractory) stages of prostate carcinoma. Androgen-responsive LNCaP and androgen-nonresponsive DU-145, PC-3, and JCA-1 human prostate cancer cells were cultured with different concentrations of resveratrol (2. 5 x 10(-5)-10(-7) M). Cell growth, cell cycle distribution, and apoptosis were determined. Addition of 2.5 x 10(-5) M resveratrol led to a substantial decrease in growth of LNCaP and in PC-3 and DU-145 cells, but only had a modest inhibitory effect on proliferation of JCA-1 cells. Flow cytometric analysis showed resveratrol to partially disrupt G1/S transition in all three androgen-nonresponsive cell lines, but had no effect in the androgen-responsive LNCaP cells. In difference to the androgen-nonresponsive prostate cancer cells however, resveratrol causes a significant percentage of LNCaP cells to undergo apoptosis and significantly lowers both intracellular and secreted prostate-specific antigen (PSA) levels without affecting the expression of the androgen receptor (AR). These results suggest that resveratrol negatively modulates prostate cancer cell growth, by affecting mitogenesis as well as inducing apoptosis, in a prostate cell-type-specific manner. Resveratrol also regulates PSA gene expression by an AR-independent mechanism.     

Anti-cancer effect of Cassia auriculata leaf extract in vitro through cell cycle arrest and induction of apoptosis in human breast and larynx cancer cell lines

The in vitro anti-cancer effect of Cassia auriculata leaf extract (CALE) was evaluated in human breast adenocarcinoma MCF-7 and human larynx carcinoma Hep-2 cell lines. CALE preferentially inhibited the growth of both the cell lines in a dose-dependent manner with IC₅₀ values of 400 and 500 μg for MCF-7 and Hep-2 cells, respectively. The results showed the anti-cancer action is due to nuclear fragmentation and condensation, associated with the appearance of A₀ peak in cell cycle analysis that is indicative of apoptosis. In addition, CALE treated MCF-7 and Hep-2 cells had decreased expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein, eventually leading a decrease in the Bcl-2/Bax ratio. These results demonstrated that CALE inhibits the proliferation of MCF-7 and Hep-2 cells through induction of apoptosis, making CALE a candidate as new anti-cancer drug.     

Effects of liposome-entrapped annamycin in human breast cancer cells: interference with cell cycle progression and induction of apoptosis

The effects of liposome-encapsulated annamycin (L-Ann) were investigated in two human breast cancer cell lines, MCF7 and MDA-MB-435. For comparative purposes, doxorubicin (Dx) was used throughout the study. A 4-hour treatment with L-Ann was significantly more active in MDA-MB-435 than in MCF7 cells (IC(50) values of 0.03 and 0.08 microg/ml, respectively), whereas Dx was equally active in the two cell lines (IC(50) 0.12 microg/ml). L-Ann induced an accumulation of cells in G2M phases which was dose-dependent in MDA-MB-435 but not in MCF7 cells. Dx also caused a dose-dependent increase of G2M cell fraction in MDA-MB-435 cells, whereas a G2M cell accumulation was observed only after treatment with the highest Dx concentration in MCF7 cells. G2M phase cell accumulations induced in MCF7 cells by L-Ann or Dx were accompanied by a decrease in cdc2 kinase activity and in cyclin B1 and cdc2 expression. Conversely, in MDA-MB-435 cells exposed to L-Ann or Dx, cdc2 kinase activity, cyclin B1 and cdc2 expression increased in parallel to the increase in the number of cells accumulated in the G2M phase. L-Ann and Dx induced apoptosis in MDA-MB-435 but not in MCF7 cells. In MDA-MB-435 cells exposed to L-Ann or Dx, no change was observed in the expression of bax, but there was a p53-independent increase in p21(waf1) expression. In MCF7 cells, treatment with L-Ann or Dx induced an increase in p53 expression with a consequent transactivation of p21(waf1) and bax. Our results indicate that L-Ann is more cytotoxic than Dx in breast cancer cells and is able to induce apoptosis through p53-independent mechanisms.     

Characteristics of apoptosis induction in human breast cancer cells treated with a ceramidase inhibitor

Cancer is a complex disease with high mortality rates. Breast cancer is one of the most fatal diseases both for men and woman. Despite the positive developments on cancer treatment, a successful treatment agent/method has not been developed, yet. Recently, cancer research has been involved in sphingolipid metabolism. The key molecule here is ceramide. Ceramides mediate growth suppress, apoptosis and aging regulation. Ceramidases metabolize ceramide and decrease its level in cells and cause escape the death. Inhibition of ceramidases as new targets for cancer treatment is shown in the literature. Herein, we found that d-erythro-MAPP and its nanoparticle formulation, reduce the viability of MCF-7 cells in a dose-dependent manner with IC₅₀ value of 4.4 µM, and 15.6 µM, respectively. Confocal and transmission electron microscopy results revealed apoptotic morphological and ultrastructural changes for both agents. Apoptosis and cell cycle arrest were supported by annexin-V, mitochondrial membrane potential changings and cell cycle analysis, respectively.     

Rapid induction of apoptosis in human gastric cancer cell lines by sorbitol

Most solid tumors, including gastric cancers, respond poorly to non-surgical treatments which are expected to induce an apoptosis-dependent involution. We hypothesize that the apoptotic machinery in solid tumors is either defective or in a suppressed condition. Overcoming the ineffective induction of apoptosis may improve the responsiveness of solid tumors to non-surgical treatments. Recently, sorbitol, a kind of hexose, has been found to be an effective inducer of apoptosis in HEp-2 cells. Therefore, it is of particular interest to examine the effect of sorbitol-treatment on gastric cancer cells. In the present study, we selected 4 gastric cancer cell lines which have been reported to exhibit different abilities in regard to apoptosis induction, and examined the effect of sorbitol-treatment on apoptosis induction. Within 3 hr after sorbitol-treatment, apoptosis was induced comparably in all cell lines examined. Cell death in MKN-1, MKN-28 or MKN-74 proceeded in a biphasic manner, while cell death in KATO-III was monophasic. The cell death partially depended on caspase activity. Treatments with sorbitol in combination with 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly suppressed the apoptotic cell death, suggesting a role of protein kinase-C-dependent process. To our knowledge, this is the most rapid induction of apoptosis in human gastric cancer cells reported to date.     

Molecular mechanism of apoptosis induction by Gaillardin,a sesquiterpene lactone,in breast cancer cell lines

Medicinal plant extracts have been widely used for cancer treatment. Gaillardin is a natural sesquiterpene lactone that has recently been reported to have anticancer properties. The ability to induce apoptosis is an important property of a candidate anticancer drug, which discriminates between anticancer drugs and toxic compounds. The current study was therefore carried out to address the issue if Gaillardin is able to induce apoptosis in the breast cancer cell lines MCF-7 and MDA-MB-468 and to determine the underlying mechanism of its anticancer effects. Apoptosis induction by Gaillardin treatment was confirmed by annexin V–FITC/PI staining, and caspase-3,-6, and-9 activation. Using Western blot analysis, we found that Gaillardin upregulated the pro-apoptotic protein Bax and p53 and downregulated the anti-apoptotic protein Bcl-2. Moreover, the apoptotic effect of Gaillardin was also related to ROS production and loss of mitochondrial membrane potential (ΔΨm). Taken together, these results demonstrate that Gaillardin can inhibit proliferation of breast cancer cells via inducing mitochondrial apoptotic pathway and therefore, might be a promising molecule in cancer chemoprevention or chemotherapy.     

Brassinosteroids cause cell cycle arrest and apoptosis of human breast cancer cells

Brassinosteroids (BRs) are plant hormones that appear to be ubiquitous in both lower and higher plants. Recently, we published the first evidence that some natural BRs induce cell growth inhibitory responses in several human cancer cell lines without affecting normal non-tumor cell growth (BJ fibroblasts). The aim of the study presented here was to examine the mechanism of the antiproliferative activity of the natural BRs 28-homocastasterone (28-homoCS) and 24-epibrassinolide (24-epiBL) in human hormone-sensitive and -insensitive (MCF-7 and MDA-MB-468, respectively) breast cancer cell lines. The effects of 6, 12 and 24 h treatments with 28-homoCS and 24-epiBL on cancer cells were surveyed using flow cytometry, Western blotting, TUNEL assays and immunofluorescence analyses. The studied BRs inhibited cell growth and induced blocks in the G₁ cell cycle phase. ER-α immunoreactivity was uniformly present in the nuclei of control MCF-7 cells, while cytoplasmic speckles of ER-α immunofluorescence appeared in BR-treated cells (IC₅₀, 24 h). ER-β was relocated to the nuclei following 28-homoCS treatment and found predominantly at the periphery of the nuclei in 24-epiBL-treated cells after 24 h of treatment. These changes were also accompanied by down-regulation of the ERs following BR treatment. In addition, BR application to breast cancer cells resulted in G₁ phase arrest. Furthermore, TUNEL staining and double staining with propidium iodide and acridine orange demonstrated the BR-mediated induction of apoptosis in both cell lines, although changes in the expression of apoptosis-related proteins were modulated differently by the BRs in each cell line. The studied BRs seem to exert potent growth inhibitory effects via interactions with the cell cycle machinery, and they could be highly valuable leads for agents for managing breast cancer.     

salvador Promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines

The number of cells in an organism is determined by regulating both cell proliferation and cell death. Relatively few mechanisms have been identified that can modulate both of these processes. In a screen for Drosophila mutations that result in tissue overgrowth, we identified salvador (sav), a gene that promotes both cell cycle exit and cell death. Elevated Cyclin E and DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts (or LATS) protein kinase. The human ortholog of salvador (hWW45) is mutated in three cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis.     

2'-Phosphodiesterase activity in human cell lines treated or untreated with human interferon

2'-Phosphodiesterase activity was investigated, by measuring either the disappearance of (2',5')oligo(adenylate) or the release of 5'AMP, in several human cell lines (RSa, IFr, HEC-1, WGAr and HeLa) possessing different sensitivities to interferon, and treated or untreated with human interferon. In various cell lines whose (2'-5')oligo(adenylate) synthetase was normally induced by interferon treatment, both kinetic studies and measurements at different enzyme concentrations indicated that 2'-phosphodiesterase activity remained unchanged after interferon treatment. This observation was confirmed over a broad range of substrate concentrations (1-25000 nM). The activity of 2'-phosphodiesterase was dependent on Mg(OAc)2. Our results indicate that in various human cell lines the modulation of (2'-5')oligo(adenylate) metabolism by interferon does not involve an increase of 2'-phosphodiesterase activity.     

Neuropeptides bombesin and calcitonin induce resistance to etoposide induced apoptosis in prostate cancer cell lines

BACKGROUND: Neuroendocrine differentiation in prostatic carcinoma has been related to regulation of proliferation and metastatic potential and correlated with prognosis. More than 80% of prostate carcinomas initially respond to androgen ablation, but most relapse, due to the heterogeneous presence of androgen-dependent and independent clones. The pathways of cellular proliferation and apoptosis are inexorably linked to minimize the occurrence of neoplasia, and disfunction of apoptosis is proposed as a pathogenic process in malignant tumors. Androgen-dependent prostatic cancer cells undergo apoptosis after androgen deprivation, but not androgen-independent ones due to a defect in the initiation step. Anyway, they retain the basic cellular machinery to undergo apoptosis. We suggest a possible role of neuroendocrine differentiation in the onset and regulation of apoptosis in prostatic neoplasia. METHODS: LNCaP, PC-3 and DU 145 prostatic cancer cell lines were induced to undergo apoptosis after treatment with etoposide alone or plus androgen ablation. We tested the role of neuropeptides bombesin and calcitonin at modulating etoposide induced apoptosis. RESULTS: Etoposide-induced apoptosis in all cancer cell lines was achieved. In LNCaP androgen ablation was also required. Apoptosis is prevented in all three lines when bombesin was added. Calcitonin addition prevents apoptosis in PC-3, LNCaP and in an etoposide dose-dependent way in DU 145. CONCLUSION: Neuropeptides bombesin and calcitonin can modulate the apoptotic response of prostate cancer cells by inducing resistance to etoposide-induced apoptosis, suggesting that neuropeptides can be used as a target of therapeutical approach in prostatic carcinoma.     

PAP modulations in Daudi cells and Molt-3 cells treated with etoposide are mutually associated with morphological evidence of apoptosis

Daudi (B-cell line) and Molt-3 (T-cell line) cells provide a model for the study of apoptosis, the induction of which is often accompanied by concominant modulations of proteins involved in mRNA maturation. One of these proteins is poly(A) polymerase (PAP), which is responsible for mRNA cleavage and polyadenylation. A number of recent reports also suggest involvement of mRNA maturation and stability in the induction of specific pathways of cell apoptosis. In this study we identified PAP activity levels and isoform modulations in two different cell lines (Daudi and Molt-3) and related them to DNA fragmentation (a hallmark of apoptosis) and cell cycle phase specificity in terms of the temporal sequence of events and the time that elapsed between administration of the apoptosis inducer (the widely used anticancer drug etoposide) and the observed effects. Treatment of both cell lines with 20 microg/mL etoposide induced apoptosis after four hours in Molt-3 cells and only after 24 hours in Daudi cells, as revealed by two independent methods. In Daudi cells the PAP activity levels and isoforms were downregulated prior to deltapsim reduction, DNA fragmentation and the morphological changes of the nucleus, whereas in Molt-3 cells no PAP activity and isoform modulations were observed prior to the early hallmarks of apoptosis.     

Interaction of recombinant human tumor necrosis factor and etoposide in human lung cancer cell lines

Studies have suggested that recombinant tumor necrosis factor-alpha (TNF-alpha) may potentiate the killing of murine tumor cells by drugs targeted at DNA topoisomerase II. We have examined the combined cytotoxic effects of the topoisomerase-targeted drug etoposide and TNF in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines using clonogenic assays and a novel flow cytometry technique relying on differential uptake of fluorescein diacetate (FDA) and propidium iodide (PI) by viable and nonviable cells. Good correlation of IC50 determinations for etoposide were noted between clonogenic assays and the FDA/PI technique for both classic and variant SCLC cell lines. The effects of etoposide on the classic SCLC line H209 were potentiated by TNF with a decrease in the IC50 from 3.3 microM to 1.0 microM as determined by FDA/PI. Tumor necrosis factor alone had little effect on the growth or cloning efficiency of H209 cells. Tumor necrosis factor alone stimulated the growth and cloning of variant SCLC line N417, but the cytotoxicity of etoposide was not potentiated by TNF in N417 cells. Tumor necrosis factor alone inhibited the growth and cloning of the NSCLC line H125 but exerted a marked protective effect against higher concentrations of etoposide. It appears that the interaction of TNF with etoposide varies between cell lines and between subclasses of human lung cancer.     

Inhibition of breast cancer xenografts in a mouse model and the induction of apoptosis in multiple breast cancer cell lines by lactoferricin B peptide

Breast cancer has a diverse aetiology characterized by the heterogeneous expression of hormone receptors and signalling molecules, resulting in varied sensitivity to chemotherapy. The adverse side effects of chemotherapy coupled with the development of drug resistance have prompted the exploration of natural products to combat cancer. Lactoferricin B (LfcinB) is a natural peptide derived from bovine lactoferrin that exhibits anticancer properties. LfcinB was evaluated in vitro for its inhibitory effects on cell lines representing different categories of breast cancer and in vivo for its suppressive effects on tumour xenografts in NOD-SCID mice. The different breast cancer cell lines exhibited varied levels of sensitivity to apoptosis induced by LfcinB in the order of SKBR3>MDA-MB-231>MDA-MB-468>MCF7, while the normal breast epithelial cells MCF-10A were not sensitive to LfcinB. The peptide also inhibited the invasion of the MDA-MB-231 and MDA-MB-468 cell lines. In the mouse xenograft model, intratumoural injections of LfcinB significantly reduced tumour growth rate and tumour size, as depicted by live imaging of the mice using in vivo imaging systems (IVIS). Harvested tumour volume and weight were significantly reduced by LfcinB treatment. LfcinB, therefore, is a promising and safe candidate that can be considered for the treatment of breast cancer.     

Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines

Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.     

BRCA1 facilitates stress-induced apoptosis in breast and ovarian cancer cell lines

The BRCA1 tumor suppressor gene has previously been implicated in induction of high levels of apoptosis in osteocarcinoma cell lines. Overexpression of BRCA1 was shown to induce an apoptotic signaling pathway involving the c-Jun N-terminal kinase (JNK), but the signaling steps upstream and downstream of JNK were not delineated. To better understand the role of BRCA1 in apoptosis, we examined the effect of wild-type and C-terminal-truncated dominant negative BRCA1 on breast and ovarian cancer cell lines subjected to a number of different pro-apoptotic stimuli, including growth factor withdrawal, substratum detachment, ionizing radiation, and treatment with anticancer agents. All of these treatments were found to induce substantial levels of apoptosis in the presence of wild-type BRCA1, whereas dominant negative BRCA1 truncation mutants diminished the apoptotic response. Subsequent mapping of the apoptotic pathway induced by growth factor withdrawal demonstrated that BRCA1 enhanced signaling through a pathway that sequentially involved H-Ras, MEKK4, JNK, Fas ligand/Fas interactions, and caspase-9 activation. In addition, the pathway functioned independently of the p53 tumor suppressor. These data suggest that BRCA1 is an important modulator of the response to cellular stress and that loss of this apoptotic potential due to BRCA1 mutations may contribute to tumor development.     

Silencing Sirtuin 6 induces cell cycle arrest and apoptosis in non-small cell lung cancer cell lines

Sirtuins (SIRT1–7), are NAD-dependent deacetylases and ADP-ribosyl transferases, plays a major part in carcinogenesis. The previous report suggests that in cancer, sirtuins gained tremendous interest and critical regulators of the unusual processes. In carcinogenesis, sirtuins possess either tumor suppressor or promoter. However, in lung cancer condition the studies of sirtuins are less studied. Hence, this designed study investigates the impact of multifaceted sirtuins in NSCLC cells. We evaluated the mRNA and protein expressions of sirtuins by RTPCR and western blot. We found SIRT6 significantly overexpressed in NCI-H520, A549, and NCI-H460 compared with the normal BEAS-2B cell line. Silencing of SIRT6 by siRNA in NSCLC cells caused activation of p53/p21 mediated inhibition of cell proliferation leading to arrest in cell cycle and apoptosis induction. Our results implied that SIRT6 is a tumor promoter in NSCLC development, progression, and regulation. The silencing of SIRT6 to be a novel therapy for lung cancer.     

2-(3,5-Dihydroxyphenyl)-6-hydroxybenzothiazole arrests cell growth and cell cycle and induces apoptosis in breast cancer cell lines

2-Arylbenzothiazoles are an important class of bicyclic privileged substructures present in various natural or synthetic compounds that have been shown to possess anticancer, antifungal, antibacterial, anti-inflammatory, and antiallergic activities. This study examined the antiproliferative properties of 2-(3,5-dihydroxyphenyl)-6-hydroxybenzothiazole (DH) and its molecular mechanism of action in human breast cancer MDA-MB-231 cells. DH inhibits the growth of MDA-MB-231 cells with an IC(50) value of 25 μM in a dose/time-dependent manner as measured by the microculture tetrazolium method. Cell cycle analysis by flow cytometry showed that DH-induced growth arrest could be associated to apoptosis in MDA-MB-231 cells.     

Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions.

Many arachidonic acid metabolites function in growth signaling for epithelial cells, and we previously reported the expression of the major arachidonic acid enzymes in human breast cancer cell lines. To evaluate the role of the 5-lipoxygenase (5-LO) pathway on breast cancer growth regulation, we exposed cells to insulinlike growth factor-1 or transferrin, which increased the levels of the 5-LO metabolite, 5(S)-hydrooxyeicosa-6E,8C,11Z,14Z-tetraenoic acid (5-HETE), by radioimmunoassay and high-performance liquid chromatography. Addition of 5-HETE to breast cancer cells resulted in growth stimulation, whereas selective biochemical inhibitors of 5-LO reduced the levels of 5-HETE and related metabolites. Application of 5-LO or 5-LO activating protein-directed inhibitors, but not a cyclooxygenase inhibitor, reduced growth, increased apoptosis, down-regulated bcl-2, up-regulated bax, and increased G1 arrest. Exposure of breast cancer cells to a 5-LO inhibitor up-regulated peroxisome proliferator-activated receptor (PPAR)a and PPARg expression, and these same cells were growth inhibited when exposed to relevant PPAR agonists. These results suggest that disruption of the 5-LO signaling pathway mediates growth arrest and apoptosis in breast cancer cells. Additional experiments suggest that this involves the interplay of several factors, including the loss of growth stimulation by 5-LO products, the induction of PPARg, and the potential activation of PPARg by interactions with shunted endoperoxides.