Curcumin induces apoptosis in human leukemic cell lines through an IFIT2-dependent pathway

Curcumin, the primary bioactive component isolated from turmeric, has been shown to possess variety of biologic functions including anti-cancer activity. However, molecular mechanisms in different cancer cells are various. In the present study, we demonstrated that curcumin induced G2/M cell cycle arrest and apoptosis by increasing the expression levels of cleaved caspase-3, cleaved PARP and decreasing the expression of BCL^?2 in U937 human leukemic cells but not in K562 cells. We found some interferon induced genes, especially interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), were significantly upregulated when treated with curcumin in U937 cells by gene expression chip array, and further confirmed that the expression of IFIT2 was obviously higher in U937 than that in K562 cells by Western blot assay. In addition, inhibiting the expression of IFIT2 by shRNA in U937 rescued curcumin-induced apoptosis and exogenous overexpression of IFIT2 by lentiviral transduction or treating with IFNγ in K562 cells enhanced anti-cancer activity of curcumin. These results indicated for the first time that curcumin induced leukemic cell apoptosis via an IFIT2-dependent signaling pathways. The present study identified a novel mechanism underlying the antitumor effects of curcumin, and may provide a theoretical basis for curcumin combined with interferon in the cancer therapeutics.     

A conditioned medium from a human liposarcoma-derived cell line induces p53-dependent apoptosis in several tumor cell lines

A novel cell line, named LSA, has been obtained, stabilized, and characterized from a human liposarcoma. These cells have morphological and biochemical features strongly resembling the adipocytes and were able to grow in the Ham's F12 medium, in presence or absence of FCS. A conditioned medium (LSA-CM) was obtained by growing the LSA cells in the F12 medium in the absence of FCS. LSA-CM had cytostatic and cytotoxic effects (apoptosis and necrosis) associated with down-regulation of c-myc and upregulation of p53 in several human cell lines (breast, lung, glioblastoma, etc. ). The MCF-7 and glioblastoma cells were killed by LSA-CM in 5-6 days, whereas the same cells were killed by LSA-CM co-incubated with low doses of cisplatin in 30 h. LSA-CM peri-tumoral injections for 15 days in Balb-c-fc3H mice affected by mammary tumors, resulted in the rapid disruption of tumors and absence of metastases. In contrast, in the untreated animals the tumor masses were 4 times larger than initial lesions, and numerous metastases were found in the lungs. The toxicity analysis of LSA-CM, performed on three different animal species, showed that LSA-CM is absolutely free of acute, subacute, and subchronic toxicity. The possible use of LSA-CM/cisplatin for cancer treatment is discussed.     

Fetal fetuin selectively induces apoptosis in cancer cell lines and shows anti-cancer activity in tumor animal models

An apoptosis-inducing protein with molecular weight of 60 kDa has been purified from fetal bovine serum. The N-terminal amino acid sequence of this protein (e.g. I-P-L-D-P-V-A-G-Y-K) reveals that it is bovine fetuin, a fetal protein functioning to control embryogenesis. The apoptosis-inducing activity of fetuin is totally dependent on zinc. Depletion of zinc ion from fetuin or substitution of zinc ion by barium ion completely abolished the apoptosis-inducing activity of fetuin. Interestingly, while the fetuin isolated from fetal serum selectively induces apoptosis in cancer without affecting normal cells, the fetuin isolated from mature serum is completely inactive. This suggests that the biological activity of fetuin is under developmental regulation. In vivo, tumor animal model studies showed that fetuin enhanced survival by up to 141% in P388 leukemia animal model in mice. Fetuin was also found to inhibit prostate cancer formation in a PC-3 prostate cancer model in mice.     

Roles of AKT1 and AKT2 in non-small cell lung cancer cell survival, growth, and migration

Although AKT/protein kinase B is constitutively active in non‐small cell lung cancer (NSCLC) cells and is an attractive target for enhancing the cytotoxicity of therapeutic agents, the distinct roles of the AKT isoforms in NSCLC are largely unknown. In the present study, we investigated the roles of AKT1 and AKT2 in NSCLC cells using RNAi. The siRNA targeting of AKT1 or AKT2 effectively decreased protein levels of AKT1 and AKT2, respectively, in A549 and H460 cells. Cisplatin treatment of these cells increased apoptotic cell death compared with control. The siRNA‐induced knockdown of AKT1 in H460 cells significantly decreased basal MEK/ERK1/2 activity, resulting in nuclear factor‐κB activation, whereas knockdown of AKT2 resulted in anti‐apoptotic Bcl‐2 family protein MCL‐1 (MCL‐1) cleavage, the collapse of mitochondrial membrane potential, cytochrome c release, and activation of the caspase cascade. Consequently, both siRNA treatments enhanced the chemosensitivity of H460 cells to cisplatin. However, neither AKT1 nor AKT2 siRNA treatment had any effect of p27 expression, and although both treatments tended to induced G₂/M phase arrest, the effect was not statistically significant. Treatment with AKT1 siRNA markedly decreased colony formation growth and migration, but AKT2 siRNA had no significant effects on these parameters. These data suggest that AKT1 and AKT2 both contribute to cell survival, albeit via different mechanisms, and that the effects on cell growth and migration are predominantly regulated by AKT1. These findings may aid in refining targeted strategies for the inhibition of AKT isoforms towards the sensitization of NSCLC cells to therapeutic agents. (Cancer Sci 2011; 102: 1822–1828)     

Mutational analysis of AKT1, AKT2 and AKT3 genes in common human carcinomas

OBJECTIVE: Mounting evidence indicates that alterations in AKT proteins play an important role in the pathogenesis of cancer. The objective of this study was to see whether common human carcinomas harbor AKT mutations that might contribute to the development of cancer. METHODS: We performed mutational analysis of the kinase domains of AKT1-AKT3 by a single-strand conformation polymorphism assay in 294 carcinoma tissues from the stomach, lung, colon and breast. RESULTS: Overall, we detected three somatic mutations in AKT2, but no mutations in AKT1 or AKT3 in the 294 cancer tissues. The AKT2 mutations were detected in 1 of 51 gastric carcinomas (2.0%) and 2 of 79 lung carcinomas (2.5%). AKT2 mutations consisted of one missense mutation and 2 splice site mutations in the intron. We simultaneously analyzed somatic mutations in EGFR, ERBB2, K-RAS, PIK3CA and BRAF genes in the 3 samples with the AKT2 mutations, and found a lung adenocarcinoma with the AKT2 missense mutation harbored an EGFR mutation. CONCLUSION: This study demonstrated that somatic mutations in the kinase domain of AKT2 occur in a small fraction of common human cancers, and suggested that alterations in the AKT2-mediated signaling pathway by AKT2 mutation could contribute to the development of some cases of human cancers.     

SMC3 knockdown triggers genomic instability and p53-dependent apoptosis in human and zebrafish cells

Background  

The structural maintenance of chromosome 3 (SMC3) protein is a constituent of a number of nuclear multimeric protein complexes that are involved in DNA recombination and repair in addition to chromosomal segregation. Overexpression of SMC3 activates a tumorigenic cascade through which mammalian cells acquire a transformed phenotype. This has led us to examine in depth how SMC3 level affects cell growth and genomic stability. In this paper the effect of SMC3 knockdown has been investigated.     

Perillyl alcohol selectively induces G0/G1 arrest and apoptosis in Bcr/Abl-transformed myeloid cell lines.

The Bcr/Abl tyrosine kinase that is expressed from the Philadelphia chromosome protects leukemia cells from apoptosis caused by removal of growth factors or by cytotoxic agents and ionizing irradiation. This resistance to apoptosis is associated with a Bcr/Abl-mediated G2/M delay. Therefore, inhibiting Bcr/Abl signaling pathways should block the ability of the Bcr/Abl kinase to protect cells from apoptosis. The monoterpenes, limonene and perillyl alcohol (POH) are new anticancer agents that selectively induce apoptosis in neoplastic cells of a variety of rodent carcinoma models. Since the potential antitumor activities of monoterpenes overlap with signaling pathways affected by the Bcr/Abl kinase, POH and limonene were tested for antileukemia activity. POH, but not limonene selectively induced G0/G1 arrest followed by apoptosis in Bcr/Abl-transformed, but not nontransformed FDC.P1 and 32D myeloid cell lines. In contrast to their greater sensitivity to POH, Bcr/Abl-transformed cells were more resistant than nontransformed cells to several chemotherapy agents and ionizing irradiation. Since in Bcr/Abl-transformed cells, POH induces apoptosis associated with G0/G1 arrest, POH must activate an apoptotic pathway that is not protected by the Bcr/Abl-induced G2/M delay. Monoterpenes may represent novel agents for treating Ph+ leukemias.     

Iododeoxyuridine incorporation and radiosensitization in three human tumor cell lines.

Iododeoxyuridine is a halogenated pyrimidine and non-hypoxic cell radiosensitizer currently being used in clinical trials. The amount of radiosensitization by IdUrd is related to the amount of incorporation of the drug into a cell's DNA. These experiments were carried out in three human tumor cell lines (lung, glioma, and melanoma) in monolayer culture exposed to concentrations of IdUrd from 0.1-10 microM for one and three cell cycles before irradiation to determine incorporation and sensitization as a function of drug exposure. Except for the lung cell line, which required greater than 1 microM IdUrd, these cells demonstrate radiosensitization when exposed to 0.1 microM or greater of IdUrd. Maximum sensitization occurred at 10 microM IdUrd for all the cell lines at three cell cycles. The percent thymidine replacement by IdUrd increased with increasing concentrations, but was cell line dependent. Maximum percent replacement occurred at 10 microM at three cell cycles for all the cell lines: lung = 22.4%, glioma = 32.0%, and melanoma = 39.1%. The relationships between percent thymidine replacement and sensitization are not identical across these human tumor cell lines. If IdUrd is going to be a successful radiosensitizer in clinical trials, sustained plasma levels of 10 microM or greater for at least three cell cycles should be achieved during irradiation. This may be best accomplished with repeated short exposures to IdUrd (three cell cycles or approximately 4 days in these cell lines) every 1-2 weeks during radiation. Measurements of thymidine replacement in a tumor biopsy should be attempted prior to radiation to develop a predictive assay for radiosensitization.     

胞外环境Ca^2＋变化诱导人肿瘤细胞凋亡

目的：观察胞外环境Ca^2 变化对人肿瘤细胞的直接诱导凋亡作用。方法：通过EGTA螯合胞外Ca^2 或添加CaCl2改变胞外环境Ca^2 。用PI和Hoechst33342荧光双染观察细胞核形态，用流式细胞仪检测凋亡百分率。分别借助荧光探针Fluo-3和Rh123检测胞内Ca^2 和线粒体膜电位（△ψm)变化。用环孢菌素A（CsA)阻断PT孔，研究其在凋亡中的作用。结果：胞外环均Ca^2 升高或降低均诱导人胃癌MGC-803和喉癌Hep细胞凋亡，恢复胞外Ca^2 阻断凋亡。随胞外Ca^2 变化胞内Ca^2 相应升高或降低，但线粒体△ψm均表现为急剧下降。CsA对MGC-803细胞凋亡无明显抑制作用，轻微促进Hep细胞凋亡。结论：胞外环境Ca^2 变化引起胸内Ca^2 相应变化和线粒体△ψm下降并通过PT孔非依赖性途径诱导肿瘤细胞凋亡。     

胞外环境Ca~(2 )变化诱导人肿瘤细胞凋亡

目的 :观察胞外环境Ca2 变化对人肿瘤细胞的直接诱导凋亡作用。方法 :通过EGTA螯合胞外Ca2 或添加CaCl2 改变胞外环境Ca2 。用PI和Hoechst3334 2荧光双染观察细胞核形态 ,用流式细胞仪检测凋亡百分率。分别借助荧光探针Fluo 3和Rh12 3检测胞内Ca2 和线粒体膜电位 (ΔΨm)变化。用环孢菌素A(CsA)阻断PT孔 ,研究其在凋亡中的作用。结果 :胞外环境Ca2 升高或降低均诱导人胃癌MGC 80 3和喉癌Hep细胞凋亡 ,恢复胞外Ca2 阻断凋亡。随胞外Ca2 变化胞内Ca2 相应升高或降低 ,但线粒体ΔΨm均表现为急剧下降。CsA对MGC 80 3细胞凋亡无明显抑制作用 ,轻微促进Hep细胞凋亡。结论 :胞外环境Ca2 变化引起胞内Ca2 相应变化和线粒体ΔΨm下降并通过PT孔非依赖性途径诱导肿瘤细胞凋亡。     

Verotoxin-1 induction of apoptosis in Gb3-expressing human glioma cell lines

The aim of this study was to examine the cytotoxicity and mechanism of apoptosis induction of verotoxin-1 (VT-1) in human glioma cell lines. VT-1 is a member of the shiga-toxin family expressed by some serotypes of Escherichia coli and Shigella dysenteriae. Shiga-toxins have been shown to induce apoptosis by binding to its membrane receptor Gb3. The human glioma cell lines SF-767, U-343 MG, and U-251 MG were studied together with BT4C, a rat glioma cell line. Cells were first screened for Gb3 expression by flow cytometry. Fluorescein diacetate was used to determine cell viability after VT-1 and irradiation exposure and apoptosis was studied by TUNEL staining, a mitochondrial membrane potential assay, and caspase activity assays. SF-767 and U-343 MG cells were found to express Gb3 and were also sensitive to VT-1-induced cytotoxicity, whereas nonGb3-expressing U-251 MG and BT4C glioma cells were not. VT-1 depolarized the mitochondrial membrane and activated caspase-9 and -3 of SF-767 and U-343 MG cells. VT-1 exposure for 72 h resulted in approx. 60 and 90% TUNEL-stained cells, respectively. D, L-Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) an inhibitor of glucosylceramide synthesis was used to block Gb3 synthesis. Two mumol/L PPMP for 72 h abolished SF-767 and U-343 MG expression of Gb3 and made the cells completely resistant to VT-1 induced apoptosis. Key components of MAP kinase signalling pathways that control BAX and mitochondrial function were investigated. VT-1 induced JNK phosphorylation in both cell lines, suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. Immunohistochemistry of cryostat section from glioma biopsies demonstrated expression of Gb3 was in the vascular endothelial cells as well as tumor cells, but not in astrocytes. The high specificity and apoptosis inducing properties of verotoxin-1 indicates that the toxin may be a potential anti-neoplastic agent for Gb3-expressing gliomas.     

Arginase activity in human breast cancer cell lines: N(omega)-hydroxy-L-arginine selectively inhibits cell proliferation and induces apoptosis in MDA-MB-468 cells

L-Arginine is the common substrate for two enzymes, arginase and nitric oxide synthase (NOS). Arginase converts L-arginine to L-ornithine, which is the precursor of polyamines, which are essential components of cell proliferation. NOS converts L-arginine to produce NO, which inhibits proliferation of many cell lines. Various human breast cancer cell lines were initially screened for the presence of arginase and NOS. Two cell lines, BT-474 and MDA-MB-468, were found to have relatively high arginase activity and very low NOS activity. Another cell line, ZR-75-30, had the highest NOS activity and comparatively low arginase activity. The basal proliferation rates of MDA-MB-468 and BT-474 were found to be higher than the ZR-75-30 cell line. N-Hydroxy-L-arginine (NOHA), a stable intermediate product formed during conversion of L-arginine to NO, inhibited proliferation of the high arginase-expressing MDA-MB-468 cells and induced apoptosis after 48 h. NOHA arrested these cells in the S phase, increased the expression of p21, and reduced spermine content. These effects of NOHA were not observed in the ZR-75-30 cell line, which expresses high NOS and relatively low arginase. The effects of NOHA were antagonized in the presence of L-ornithine (500 microM), which suggests that in MDA-MB-468 cell line, the arginase pathway is very important for cell proliferation. Inhibition of the arginase pathway led to depletion of intracellular spermine and apoptosis as observed by terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling assay and induction of caspase 3. In contrast, the ZR-75-30 cell line maintained its viability and its L-ornithine and spermine levels in the presence of NOHA. We conclude that NOHA has antiproliferative and apoptotic actions on arginase-expressing human breast cancer cells that are independent of NO.     

TNF-alpha-mediated apoptosis in normal human prostate epithelial cells and tumor cell lines

In this study we compared the role of TNF-alpha in the regulation of growth and apoptosis in normal human prostate epithelial cells (NP) and prostate tumor cell lines PC3 and LNCap. The NP and PC3 cells were resistant whereas the LNCap cell line was highly sensitive to TNF-alpha induced growth arrest and apoptosis. The resistance of NP and PC3 cells was mediated via an NF-kB survival pathway as treatment of resistant cells with TNF-alpha was accompanied by phosphorylation of I-kBalpha and translocation of NF-kB to the nucleus. TNF-alpha did not induce phosphorylation of I-kB in the sensitive LNCap cells. The sensitivity of LNCap cells involved a cysteine protease cascade as Z-VAD-CH2 F reversed the sensitivity of LNCap cells and induced resistance to TNF-alpha. The differences in susceptibilities to TNF-alpha induced apoptosis of NP and certain prostate tumor cells offer intriguing possibilities for the treatment of prostate cancer without affecting the normal prostate tissue.     

Induction of apoptosis by morphine in human tumor cell lines in vitro

Most previous studies of the induction of tumor cell apoptosis by morphine have been conducted with concentrations very much higher than those used clinically. An investigation of the ability of morphine to induce apoptosis at its clinical concentration (10(-8) M) was therefore undertaken. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, induction of early apoptosis and necrosis by fluorescence-activated cell sorter (FACS) analysis with Annexin V and propidium iodide (PI), activation of caspase -2, -3, -8 and -9 by cleavage of specific substrates, DNA fragmentation by agarose gel electrophoresis, radical intensity and O2- scavenging activity by ESR spectroscopy. Millimolar concentrations of morphine showed higher cytotoxicity against human tumor cell lines (HL-60, A549, MCF7) than against normal human cells (HGF, HPC, HPLF). The clinical concentration of morphine produced early apoptotic markers in HL-60 and A549 cells whereas it induced higher numbers of necrotic cells in MCF7 cells, both in a naloxone-sensitive manner. The clinical concentration of morphine failed to activate any caspase species and induced only trace amounts of internucleosomal DNA fragmentation, in contrast to cytotoxic concentrations of morphine. Morphine, with a C-3 hydroxyl group, showed higher cytotoxicity and O2- scavenging activity than codeine, in which the hydroxyl group at C-3 was replaced with a methoxy group, suggesting the involvement of a radical-mediated reaction. The present report may offer new strategies for treatment and prevention of cancer using a clinical concentration of morphine not only as an anti-nociceptive, but also as an apoptosis or necrosis inducer.