Polymorphonuclear granulocytes induce antibody-dependent apoptosis in human breast cancer cells

Recent studies in HER-2/neu-targeted immunotherapy demonstrated that polymorphonuclear neutrophils (PMN) mediated Ab-dependent cellular cytotoxicity against HER-2/neu-positive breast cancer cell lines. However, the mechanism of cell death remained unclear. We used several assays to analyze the induction of apoptosis in the breast cancer cell line SK-BR-3 via PMN-dependent Ab-dependent cellular cytotoxicity. In the presence of the HER-2/neu Ab 520C9 and PMN from healthy donors, apoptosis occurred as detected by annexin V binding and disappearance of euploid SK-BR-3 nuclei, which can be differentiated from PMN nuclei by their increased DNA contents. Apoptosis induction was observed with E:T cell ratios as low as 10:1. Laser scanning fluorescence microscopy of TUNEL tumor cells or staining for cleaved cytokeratin-18 further confirmed apoptosis of the SK-BR-3 breast cancer cells. Killing via 520C9 was dependent on the interaction with FcR on PMN, because 1) F(ab')(2) fragments of 520C9 mediated no cytotoxicity, 2) target cell death was influenced by a biallelic polymorphism of FcgammaRIIa on the effector cells, and 3) a bispecific Ab against HER-2/neu and the IgA receptor (FcalphaRI) expressed on effector cells significantly induced apoptosis. Thus, PMN induce Ab-dependent apoptosis against human breast cancer cells targeted with HER-2/neu-directed mAbs or FcR directed bispecific Abs.     

Lipids isolated from bone induce the migration of human breast cancer cells

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.     

Xanthones induce cell-cycle arrest and apoptosis in human colon cancer DLD-1 cells

We investigated the antiproliferative effects of four structurally similar prenylated xanthones, alpha-mangostin, beta-mangostin, gamma-mangostin, and methoxy-beta-mangostin, in human colon cancer DLD-1 cells. These xanthones differ in the number of hydroxyl and methoxy groups. Except for methoxy-beta-mangostin, the other three xanthones strongly inhibited cell growth at 20 microM and their antitumor efficacy was correlated with the number of hydroxyl groups. Hoechst 33342 nuclear staining and nucleosomal DNA-gel electrophoresis revealed that the antiproliferative effects of alpha- and gamma-mangostin, but not that of beta-mangostin, were associated with apoptosis. It was also shown that their antiproliferative effects were associated with cell-cycle arrest by affecting the expression of cyclins, cdc2, and p27; G1 arrest was by alpha-mangostin and beta-mangostin, and S arrest by gamma-mangostin. These findings provide a relevant basis for the development of xanthones as an agent for cancer prevention and combination therapy with anti-cancer drugs.     

Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2)

Previous studies have shown that proteins extracted from Zebrafish embryo share some cytostatic characteristics in cancer cells. Our study was conducted to ascertain the biological properties of this protein network. Cancer cell growth and apoptosis were studied in Caco2 cells treated with embryonic extracts. Cell proliferation was significantly inhibited in a dose-dependent manner. Cell-cycle analysis in treated cells revealed a marked accumulation in the G₂/M phase preceding induction of apoptosis. Embryo proteins induced a significant reduction in FLIP levels, and increased caspase-3 and caspase-8 activity as well as the apoptotic rate. Increased phosphorylated pRb values were obtained in treated Caco2 cells: the modified balance in pRb phosphorylation was associated with an increase in E2F1 values and c-Myc over-expression. Our data support previous reports of an apoptotic enhancing effect displayed by embryo extracts, mainly through the pRb/E2F1 apoptotic pathway, which thus suggests that Zebrafish embryo proteins have complex anti-cancer properties.     

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.     

Cytotoxic constituents from Celastrus paniculatus induce apoptosis and autophagy in breast cancer cells

Celastrus paniculatus is a traditional medicinal plant with diverse pharmacological activities. To identify its bioactive constituents, three new β-dihydroagarofuranoid sesquiterpenes were isolated from the whole plant, of which the major constituent is (1α,2α,8β,9β)-1,8-bis(acetyloxy)-2,9-bis(benzoyloxy)-14-hydroxy-β-dihydroagarofuran. It was assessed for its antiproliferative activity, and it suppressed the viability of MCF-7 breast cancer cells with an IC₅₀ of 17 ± 1 μM. This growth inhibition was, in part, attributable to apoptosis. Moreover, this drug treatment led to LC3B-II accumulation, indicative of autophagy. Western blot analysis established its ability to target a broad range of signaling effectors related to survival and cell cycle progression, including Akt, NF-κB, p53, and MAP kinases. In addition, flow cytometry analysis indicates increased reactive oxygen species production in response to this compound. Taken together, these findings suggest a pleiotropic mode of mechanism that underlies the antiproliferative activity of this compound in MCF-7 breast cancer cells.     

Purification and characterization of beta-N-acetylhexosaminidase from the ascidian, Halocynthia roretzi

beta-N-Acetylhexosaminidase [EC 3.2.1.30] was purified 820-fold from the viscera of Halocynthia roretzi by Sephadex G-200 gel filtration and chromatography on columns of DEAE-Sephadex and CM-Sephadex. The final preparation was sufficiently free from alpha-N-acetylglucosaminidase, alpha-N-acetylgalactosaminidase, alpha- and beta-glucosidases, alpha- and beta-galactosidases, alpha- and beta-mannosidases, and alpha-L-fucosidase, and gave one protein band on disc gel electrophoresis. Two different molecular weight forms which depended upon the pH were observed on Sephadex gel filtration. At pH 7.0, a species with a molecular weight of 170,000 was observed, whereas at pH 4.5, an enzyme of 330,000 daltons was seen. The enzyme was active at pH 4.5 but inactive at pH 7.0. The optimum pH and the Km were pH 4.2 and 1.9 mM for p-nitrophenyl beta-N-acetylglucosaminide and pH 4.0 and 0.9 mM for p-nitrophenyl beta-N-acetylgalactosaminide. The terminal beta-N-acetylhexosamine of glycolipids such as globoside I, GM2, and asialo GM2 was cleaved by the ascidian beta-N-acetylhexosaminidase though GM2 was less susceptible to the enzyme.     

Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.     

Chitooligosaccharides induce apoptosis in human myeloid leukemia HL-60 cells

In this study we propose a novel anticancer agent using hetero-chitooligosaccharide (hetero-COS). To examine the possibility of the hetero-COS as a anticancer agent, we prepared nine kinds of hetero-COS with relatively higher molecular weights (90, 75 and 50-COS I, 5-10kDa), medium molecular weights (90, 75 and 50-COS II, 1-5kDa), and lower molecular weights (90, 75 and 50-III, below 1kDa), and their anticancer properties were investigated on HL-60 cells using flow cytometry and morphological analysis. The results obtained indicate that 90-COS III, which is relatively higher degree of deacetylation and lower molecular weights, showed the highest anticancer activity, and the data showed the anticancer property of the hetero-COSs depended on their degree of deacetylation values and molecular weight.     

Induction of apoptosis in DLD-1 human colon cancer cells by peridinin isolated from the dinoflagellate, Heterocapsa triquetra

Peridinin, which is uniquely present in dinoflagellates, is one of the most abundant carotenoids found in nature. We evaluated the apoptotic effect of peridinin on DLD-1 human colorectal cancer cells. Peridinin significantly reduced the cell viability in a dose-dependent manner (0-20 microM) and induced apoptosis by activating both caspase-8 and caspase-9. Our findings could be important for the high-performance utilization of marine bioproducts.     

Endomorphins, endogenous opioid peptides, induce apoptosis in human leukemia HL-60 cells

Opioids play a role in the apoptosis machinery. We studied the induction of apoptosis in endomorphin 1 (EM1) and endomorphin 2 (EM2), 2 newly isolated endogenous mu-opioid receptor agonists. These endomorphins were able to reduce the viability of cultured HL-60 cells. The antiproliferative properties of endomorphins appeared to be attributable to their induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 indicated down-regulation, but the Bax, Fas, and FasL expression showed up-regulation as compared with the untreated control cells. These data support the idea that endomorphins induce apoptosis in HL-60 cells through the activation of the Bcl-2-Bax and the Fas-FasL pathway. We suggest that endomorphins may play an important role in the regulation of tumor cell death.     

Normal breast epithelial cells induce apoptosis of breast cancer cells via Fas signaling

Fas/Fas ligand (Fas L) death pathway is an important mediator of apoptosis. Deregulation of Fas pathway is reported to be involved in the immune escape of breast cancer and the resistance to anti-cancer drugs. In this study, we demonstrated that conditioned medium by normal breast epithelial cells (NBEC-CM) induced apoptosis of MCF-7 and T-47D Fas-sensitive cells but had no effect on MDA-MB-231 Fas-resistant cells. Inhibition of PI3 kinase or NF-kappaB by specific inhibitors or transient transfections restored the sensitivity of MDA-MB-231 cells to NBEC-induced apoptosis. Moreover, the constitutive activation of NF-kappaB was controlled by PI3 kinase because inhibition of PI3 kinase reduced NF-kappaB activity. Inducible activation of NF-kappaB rendered MCF-7 cells resistant to NBEC-CM- and Fas agonist antibody-triggered apoptosis. Therefore, constitutive or inducible activation of PI3 kinase and/or NF-kappaB in breast cancer cells rendered them resistant to NBEC-triggered apoptosis. In addition, Fas neutralizing antibody and dominant negative Fas abolished NBEC-triggered apoptosis. Western blot and confocal microscopy analysis showed an increase of membrane Fas/Fas L when cells were induced into apoptotis by NBEC-CM. Taken together, these data show that NBEC induced apoptosis in breast cancer cells via Fas signaling.     

Indomethacin and juglone inhibit inflammatory molecules to induce apoptosis in colon cancer cells

Colorectal cancer (CRC) is the third most common fatal cancer. Indomethacin, a nonsteroidal anti‐inflammatory drug, is known to reduce the occurrence of CRC. This study evaluated the potential anticolon cancer effects of juglone (5‐hydroxy‐1,4‐naphthoquinone) in combination with indomethacin. Human colon adenocarcinoma cells (HT29) were subjected to treatment with indomethacin, juglone, and a combination of both. Morphological analysis, cell cycle regulation, and dual staining using acridine orange and ethidium bromide in control and treated cells revealed the apoptotic potential of these compounds. Bcl2 and inflammatory molecules (tumor necrosis factor‐α, nuclear factor kappa B, and Cox‐2) were found to be decreased with a concomitant increase in the expression of proapoptotic molecules (Bad, Bax, cytochrome c, and PUMA) as a result of the molecular regulation of Wnt, Notch, and peroxisome proliferator‐activated receptor‐γ signaling. Treatment with juglone was not as effective as with indomethacin; however, a combination of both was shown to be more effective, suggesting that juglone may be considered for therapeutic intervention of colon cancer.     

Mechanism of taurine-induced apoptosis in human colon cancer cells

Taurine （Tau） has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anti-cancer effect is not well understood. PUMA （p53-upregulated modulator of apoptosis） plays an important role in the process of apoptosis induction in a variety of human tumor ceils in both p53- dependent and -independent manners. However, whether PUMA is involved in the process of Tau-induced apoptosis in cancer cells has not been well studied. In the present study, we treated human colorectal cancer cells HT-29 （mutant p53） and LoVo （wild-type p53） with different concentrations of Tau, which led to the repression of cell proliferation and induction of apoptosis in both cell fines. Meanwhile, we also observed the increased expression of PUMA and high Bax/Bcl-2 ratios. To determine the role of PUMA in Tau-induced apoptosis, we used small interfering RNA interference to suppress PUMA expression. As a result, apoptosis was decreased in response to Tau treatment. All these results indicated that PUMA plays a critical role in Tauinduced apoptosis pathway in human colorectal cancer ceils. Demonstration of the molecular mechanism involved in the anti-tumor effect of Tau may be useful in the therapeutic target selection for p53-deficient colorectal cancer.     

Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi

Ficolins are animal lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding lectin (MBL) activates the complement lectin pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated approximately 40 kDa (p40) and approximately 50 kDa (p50) N-acetylglucosamine-binding lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizes N-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4-52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph lectin activity and the correlation of different functions with binding specificity.     

Medicarpin and millepurpan,two flavonoids isolated from Medicago sativa,induce apoptosis and overcome multidrug resistance in leukemia P388 cells

BackgroundHigh consumption of flavonoids has been associated with a decrease risk of cancer. Alfalfa (Medicago sativa) leaves have been widely used in traditional medicine and is currently used as a dietary supplement because of their high nutrient content. We previously reported the cytotoxic activity of alfalfa leaf extracts against several sensitive and multidrug resistant tumor cell lines.Hypothesis/purposeWe aimed to determine whether medicarpin and millepurpan, two isoflavonoids isolated from alfalfa leaves, may have pro-apoptotic effects against drug-sensitive (P388) and multidrug resistant P388 leukemia cells (P388/DOX).Study design/methodsCells were incubated with medicarpin or millepurpan for the appropriate time. Cell viability was assessed by the MTT assay. DNA fragmentation was analyzed by agarose gel electrophoresis. Cell cycle analysis was realized by flow cytometry technics. Caspases 3 and 9 activities were measured using Promega caspACE assay kits. Proteins and genes expression were visualized respectively by western-blot using specific antibodies and RT-PCR assay.ResultsP-glycoprotein-expressing P388/DOX cells did not show resistance to medicarpin (IC₅₀ ≈ 90 µM for P388 and P388/DOX cells) and millepurpan (IC₅₀ = 54 µM and 69 µM for P388 and P388/DOX cells, respectively). Treatment with medicarpin or millepurpan triggered apoptosis in sensitive as well as multidrug resistant P388 cells. These effects were mediated through the mitochondrial pathway by modifying the balance pro/anti-apoptotic proteins. While 3 µM doxorubicin alone could not induce cell death in P388/DOX cells, concomitant treatment with doxorubicin and subtoxic concentration of medicarpin or millepurpan restored the pro-apoptotic cascade. Each compound increased sensitivity of P388/DOX cells to doxorubicin whereas they had no effect in sensitive P388 cells. Vinblastine cytotoxicity was also enhanced in P388/DOX cells (IC₅₀ = 210 nM to 23 and 25 nM with medicarpin and millepurpan, respectively). This improved sensitivity was mediated by an increased uptake of doxorubicin in P388/DOX cells expressing P-gp. P-gp expression was not altered by exposure to medicarpin and millepurpan.ConclusionThese data indicate that medicarpin and millepurpan possess pro-apoptotic properties and potentiate the cytotoxicity of chemotherapy drugs in multidrug resistant P388 leukemia cells by modulating P-gp-mediated efflux of drugs. These flavonoids may be used as chemopreventive agents or as sensitizer to enhance cytotoxicity of chemotherapy drugs in multidrug resistant cancer cells.     

T-oligos inhibit growth and induce apoptosis in human ovarian cancer cells

Ovarian cancer remains a leading cause of death among women worldwide, and current treatment regimens for advanced disease are inadequate. Oligonucleotides with sequence homology to telomeres (called T-oligos) have been shown to mimic DNA damage responses in cells and induce cytotoxic effects in certain tumor cell lines. We studied the effects of 2 distinct 16 mer T-oligos in 4 human ovarian epithelial carcinoma cell lines. A T-oligo with perfect homology to the telomere overhang region demonstrated some cytotoxic activity in half of the cell lines. A G-rich T-oligo derivative showed more potency and broader cytotoxic activity in these lines than the parental T-oligo. Activation of apoptotic pathways in ovarian cancer cells by exposure to the T-oligo was demonstrated by multiple independent assays. T-oligo was shown to have additive, or more than additive, activity in combination with 2 different histone deacetylase drugs currently in clinical testing. T-oligos may therefore provide a new and tumor-targeted approach to ovarian cancers.     

Coumarin polysulfides inhibit cell growth and induce apoptosis in HCT116 colon cancer cells

Coumarins and coumarin derivatives as well as diallyl polysulfides are well known as anticancer drugs. In order to find new drugs with anticancer activities, we combined coumarins with polysulfides in the form of di-coumarin polysulfides. These novel compounds were tested in the HCT116 colorectal cancer cell line. It turned out that they reduced cell viability of cancer cells in a time and concentration dependent manner. Cells tested with these coumarin polysulfides accumulate in the G(2)/M phase of the cell cycle and finally they go into apoptosis. A decrease in bcl-2 level, and increase in the level of bax, cytochrome c release into the cytosol, cleavage of caspase 3/7and PARP suggested that coumarin polysulfides induced the intrinsic pathway of apoptosis. Comparison of these new coumarin compounds with the well known diallyl polysulfides revealed that the coumarin disulfides were more active than the corresponding diallyl disulfides. The activities of the coumarin tetrasulfides and the corresponding diallyl tetrasulfides are similar. The novel coumarin compounds regulated the phosphatase activity of the cell cycle regulating cdc25 family members, indicating that these phosphatases are implicated in the induction of cell cycle arrest and possibly in apoptosis induction as well. In addition, coumarin polysulfides also down-regulated the level of cdc25C, which also contributed to the arrest in the G(2)-phase of the cell cycle.     

Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi

Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B. To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members of the leukocyte integrin family in mammals. Using degenerate primers, we isolated two integrin alpha subunits (alpha(Hr1) and alpha(Hr2)) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of alpha(Hr1) was determined from cDNA clones. alpha(Hr1) contains an I domain, the inserted domain characteristic of a subset of mammalian alpha subunits, including the leukocyte integrin family. A phylogenetic tree constructed for the alpha subunits also supports the ancestral position of alpha(Hr1) in the monophyletic cluster of I domain-containing alpha integrins. The alpha(Hr1) gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-alpha(Hr1) Ab showed that alpha(Hr1) subunits exist on the surface of a subpopulation of phagocytic hemocytes. Furthermore, anti-alpha(Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes. These observations strongly suggest that alpha(Hr1) constitutes an integrin molecule on the hemocytes of H. roretzi that functions as an ancestral form of CR3 and CR4 and mediates phagocytosis in the primitive complement system of the ascidian.