Iberin induces cell cycle arrest and apoptosis in human neuroblastoma cells

Epidemiological studies have indicated that increased consumption of cruciferous vegetables is associated with a statistically significant reduction in the risk for cancers. The major bioactive agent in these vegetables is a class of sulfur-containing glycosides called glucosinolates. Isothiocyanates, derivatives of glucosinolates, have been shown to possess anticancer properties in a variety of tumor cell lines. In this study, we evaluated the antigrowth, cell cycle modulation and proapoptotic effects of isothiocyanate iberin in human neuroblastoma cells. Treatment of neuroblastoma cells with iberin resulted in a dose- and time-dependent inhibition of growth, increased cytotoxicity, and G1 or G2 cell cycle arrest depending upon cell type. The iberin-induced cell cycle arrest in neuroblastoma cells was associated with inhibition of expression of Cdk2, Cdk4, and Cdk6 proteins. Fluorescence microscopic analysis of DNA-staining patterns with DAPI revealed an increase in apoptotic cell death in iberin-treated cells as compared with control cells. FLICA staining showed that iberin induced apoptosis, and this apoptotic induction was found to be associated with the activation of caspase-9, caspase-3, and PARP. These findings suggest that the anticancer efficacy of iberin is mediated via induction of cell cycle arrest and apoptosis in human neuroblastoma cells and has strong potential for development as a therapeutic agent against cancer.     

Activation-induced cell death of human melanoma specific cytotoxic T lymphocytes is mediated by apoptosis-inducing factor

Activation-induced cell death (AICD) of T cells can be an impediment towards achieving a robust and long-lived cytolytic T lymphocyte (CTL) response from active specific immunization or after adoptive cell transfer in cancer immunotherapy. The mechanism of AICD in primary CTL, however, remains poorly understood. It is widely believed that AICD is driven by signals from death receptors (DR) and that the cell death takes place in a caspase-dependent manner, although it has been shown that AICD of T cells can be induced by internal triggers and that death takes place in a caspase-independent manner. We show here that AICD in human melanoma epitope-specific primary CTL involves selective mitochondrio-nuclear translocation of the apoptosis inducing factor (AIF) without cytochrome c release, caspase-3 and caspase-8 activation, and results from large-scale DNA fragmentation. The c-jun-N terminal kinase (JNK) inhibitor, SP600125, blocks the mitochondrio-nuclear translocation of AIF and prevents AICD in these CTL. These findings suggest that the AICD in human melanoma epitope specific primary CTL is mediated by mitochondrial AIF release and JNK is involved in regulation of this death process.     

雷公藤内酯醇诱导人淋巴细胞凋亡的作用与细胞周期的关系

目的：通过观察雷公藤仙酯醇诱导淋巴细胞发生凋亡与细胞周期的关系。探讨该化合物导致细胞凋亡作用的机制。方法;以人外周血Ｔ细胞为研究对象,选择流式细胞分析术,ＤＮＡ凝胶电泳及ＤＮＡ片段测定等作为检测细胞凋亡的方法。结果;雷公藤内酯醇只能造成活化的Ｔ细胞发生细胞凋亡,且与雷公藤内酯醇的浓度正相关;雷公藤内酯醇诱导活化Ｔ细胞发生细胞凋亡的作用与细胞活化的程度密切相关;     

The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 are not essential in T cell anergy

Recent evidence suggests that the cyclin-dependent kinase (Cdk) inhibitors p27Kip1 and p21Cip1 are important factors in T cell anergy, but it has remained unclear whether anergy can be induced in their absence. We therefore induced anergy by stimulation of purified T cells from wild-type, p21Cip1-/-, and p27Kip1-/- mice with anti-CD3 antibodies. Anergic wild-type T cells were arrested in the G1 phase of the cell cycle with a high p27Kip1 protein level and low Cdk2 activity. In p27-/- and p21-/- T cells, the pattern of protein expression was preserved, but Cdk2 activity was increased. To confirm the in vivo relevance of these data, anergy was induced by repeated injection of mice with staphylococcal enterotoxin B (SEB), which leads to partial deletion of the responsive Vbeta8+ T cell population and anergy in the remaining T cells. p21-/- mice and wild-type mice reacted similarly to this treatment. p27-/- mice showed reduced deletion of SEB-responsive T cells, but persisting T cells were anergic. These data indicate that other cell cycle regulators contribute to the cell cycle arrest of anergic T cells, as neither Cdk inhibitor is required for the induction of anergy.     

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri(L.) Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.     

The role of CD95 in the regulation of peripheral T-cell apoptosis

Summary: Apoptosis of activated peripheral T cells during the termination phase of an immune response is critical to maintain T‐cell homeostasis. Activated T cells can be removed by two mechanisms: activation‐induced cell death (AICD) and death by neglect. AICD is triggered by death receptors, whereas death by neglect is induced by cytokine withdrawal. CD95 (APO‐1/Fas) belongs to the subfamily of death receptors and plays a major role in AICD. In this review, we focus on the molecular mechanisms of AICD, in particular those involving the CD95 system. Moreover, we discuss the relative contribution of AICD and death by neglect to terminate a T‐cell immune response. In order to become fully activated, T cells require a second signal provided by antigen‐presenting cells. We discuss how these costimulatory signals counteract pro‐apoptotic signals and, finally, which signals might protect T cells from death to generate a pool of memory T cells.     

Protection of T cells from activation-induced cell death by Fas+ B cells

Naive CD4+ T cells proliferate strongly in response to stimulation by superantigens such as staphylococcal enterotoxin B (SEB). However, when these same cells revert to a resting phenotype and are subjected to restimulation with either SEB or anti-CD3, the majority of these SEB-responsive cells undergo Fas ligand (FasL)-mediated activation-induced cell death (AICD). We investigated the impact of Fas expression on T cell AICD by utilizing B cell stimulators that lacked functional FasL and either expressed or did not express the Fas receptor. Our results indicate that B cells play an important role in modulating the level of T cell AICD via the Fas/FasL pathway. Activated B cells expressing high levels of Fas receptor can redirect the FasL expressed by T cells primed to undergo AICD away from the T cells and prevent the induction of AICD in these cells. Furthermore, B cells stimulated through both the CD40 receptor and membrane IgM appear to mediate a stronger protective effect on T cells by virtue of their resistance to FasL-mediated cytolysis. These observations suggest a mechanism by which normal B cell and T cell responses to foreign antigen are maintained, while responses to self antigen are not.     

A recombined protein (rSj16) derived from Schistosoma japonicum induces cell cycle arrest and apoptosis of murine myeloid leukemia cells

rSj16, a recombined protein from Schistosoma japonicum, has been identified as an anti-inflammatory molecule. In this study, we demonstrated that rSj16 strongly suppressed the growth of murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. rSj16 induced apoptosis by increasing the proportion of sub-G1 apoptotic cells as well as causing cell cycle arrest at the G0/G1 phase. The expressions of cyclin D1, D2, D3, and E, and Cdk 2, 4, and 6 genes in WEHI-3B JCS cells were significantly down-regulated at 24 h as measured by real-time PCR. Furthermore, apoptosis induced by rSj16 was confirmed by 4,6-diamidino-2-phenylindole nuclear staining assay and annexin V/propidium iodide double staining. A reduction of the mitochondrial membrane potential indicated an active involvement of mitochondria in the apoptosis process. rSj16 treatment induced an increase in the activity of caspase 3, 6, and 9, and expression of pro-apoptotic Bax. Meanwhile, the decreased expression of anti-apoptotic Bcl-2 was observed after rSj16 treatment. Taken together, our results implied that rSj16 can inhibit proliferation by inducing G0/G1 cell cycle arrest and apoptosis of murine myeloid leukemia cells via activation of the caspase-mediated mechanism by regulating the expression of Bcl-2 family.     

Differential involvement of p38 MAP kinase pathway and Bax translocation in the mitochondria-mediated cell death in TCR- and dexamethasone-stimulated thymocytes

Mitochondria play a central role in many apoptotic reactions. Although mitochondrial apoptotic changes and caspase activation have been demonstrated in the apoptotic thymocytes, cell death signal through mitochondria in TCR-stimulated thymocytes has not been fully understood. In this study, we show that TCR stimulation induced disruption of mitochondrial transmembrane potential (Delta Psi(m)), the cytochrome c release from mitochondira, capase-3 activation, and the cell death of thymocytes. Bongkrekic acid, an inhibitor of Delta Psi(m) disruption, blocked the cytochrome c release from mitochondria and the following caspase-3-mediated cell death. Furthermore, a pro-apoptotic Bcl-2 family protein, Bax, but not Bad or Bid, was translocated from cytosol to mitochondria in TCR-stimulated thymocytes. This translocation and the following apoptotic changes were inhibited by SB203580, a p38 kinase inhibitor, in a specific manner. These results suggest that activated p38 kinase pathway by TCR stimulation induces translocation of Bax to mitochondria, causing Delta Psi(m) disruption, and the release of cytochrome c, which finally induces caspase-3-mediated apoptosis in thymocytes.     

HCV core/gC1qR interaction arrests T cell cycle progression through stabilization of the cell cycle inhibitor p27Kip1

Hepatitis C virus (HCV) is efficient in the establishment of persistent infection. We have previously shown that HCV core protein inhibits T cell proliferation through its interaction with the complement receptor, gC1qR. Here we show that HCV core-induced inhibition of T cell proliferation involves a G(0)/G(1) cell cycle arrest, which is reversible upon addition of anti-gC1qR antibody. Correspondingly, the expression of cyclin-dependent kinases (Cdk) 2/4 and cyclin E/D, as well as subsequent phosphorylation of retinoblastoma (pRb), is reduced in core-treated T cells in response to mitogenic stimulation. Remarkably, degradation of p27(Kip1), a negative regulator of both Cdk4/cyclin D and Cdk2/cyclin E complexes, is significantly diminished in T cells treated with HCV core upon mitogenic stimulation. These data indicate that the stability of p27(Kip1) by HCV core is associated with blocking activated T cells for the G(1) to S phase transition and inhibiting T cell proliferation.     

Diallyl trisulfide selectively causes Bax- and Bak-mediated apoptosis in human lung cancer cells

Garlic-derived organosulfur compounds (OSCs) are highly effective in affording protection against chemically induced pulmonary carcinogenesis in animal models. We now demonstrate that garlic constituent diallyl trisulfide (DATS) suppresses viability of cultured human lung cancer cell lines H358 (anon-small cell lung cancer cell line) and H460 (a large cell lung cancer cell line) by causing G2-M phase cell cycle arrest and apoptotic cell death. On the other hand, a normal human bronchial epithelial cell line BEAS-2B was significantly more resistant to growth inhibition and apoptosis induction by DATS compared with lung cancer cells. We also found that even a subtle change in the OSC structure could have a significant impact on its biological activity. For example, DATS was significantly more effective than either diallyl sulfide or diallyl disulfide against proliferation of lung cancer cells. The DATS-mediated G2-M phase cell cycle arrest was explained by down-regulation of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C protein expression leading to accumulation of Tyr15 phosphorylated (inactive) Cdk1. The DATS-induced apoptosis correlated with induction of pro-apoptotic proteins Bax, Bak and BID, and a decrease in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL in lung cancer cells but not in BEAS-2B. Knockdown of Bax and Bak proteins conferred significant protection against DATS-induced apoptotic cytoplasmic histone-associated DNA fragmentation. On the other hand, BID protein was dispensable for DATS-induced apoptosis. In conclusion, the present study indicates that Bax and Bak proteins are critical targets of DATS-induced apoptosis in human lung cancer cells.     

Opioid analgesic-induced apoptosis and caspase-independent cell death in human lung carcinoma A549 cells

We characterized anticancer effects of opioid analgesics that are clinically used for cancer patients for pain relief. Treatment with 100 microM buprenorphine, a representative analgesic, induced cell death of human carcinomas, such as A549 (squamous epithelial cell of lung cancer), MCF-7 (breast cancer) and N417 (small cell of lung cancer), but not in KATO III (gastric cancer) cells as evaluated by alamar blue assay. Among 18 clinically utilized and related analgesics, buprenorphine and loperamide showed potent inhibition of cell viability. However, these anti-cancer effects were not affected by opioid receptor antagonists nor by pertussis toxin. Buprenorphine-induced cell death occurred as early as 1 h after the addition, and its T1/2 of cell viability inhibition was 3 h. The cell death manifested the characteristics of apoptosis, such as DNA-laddering and nuclear fragmentation, which were sensitive to a caspase inhibitor, Z-Asp-CH2-DCB. The nuclear fragmentation was independent of cell cycle phase specificity. The activity of caspase-3-like protease which is known to be closely related to apoptotic DNA laddering was markedly enhanced by buprenorphine. However, the inhibition of cell viability by buprenorphine was not affected by the caspase inhibitor. These findings suggest that some opioid analgesics induce typical apoptotic features sensitive to the caspase inhibitor, while also inhibition of cell viability insensitive to the inhibitor.     

Effect of amentoflavone, a phenolic component from Biophytum sensitivum, on cell cycling and apoptosis of B16F-10 melanoma cells

Flavanoids are polyphenolic compounds that are found in fruits and vegetables and have diverse, beneficial biochemical and antioxidant effects. The objective of this study was to assess the effect of amentoflavone, a biflavanoid isolated from Biophytum sensitivum, on cell cycling distribution and apoptosis in B16F-10 melanoma cells. Treatment of B16F-10 melanoma cells with amentoflavone (10 μg/mL) increased cells in the sub-G0/G1 phase accompanied by a decrease in G0/G1 phase cells in a time-dependent manner. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of amentoflavone-treated B16F-10 melanoma cells confirmed that the cells were undergoing apoptosis. Amentoflavone was found to significantly inhibit B16F-10 melanoma-induced solid tumor development in C57BL/6 mice. The increase in apoptotic cells in paraffin sections obtained from amentoflavone-treated animals indicates that the reduction may be mediated through induction of apoptosis. Murine cell cycle-regulating genes, such as p21 and p27, and apoptosis-regulating genes, such as Bax and caspase-9, were found to be upregulated, whereas cyclin D1 and Bid were downregulated in amentoflavone-treated cells. These results demonstrate that amentoflavone can induce apoptosis via inhibiting progression of cells from G0/G1 to S phase and regulating genes involved in cell cycle regulation and apoptotic intrinsic pathways.     

Hepatitis C virus NS5A protein protects against TNF-alpha mediated apoptotic cell death

Hepatitis C virus (HCV) often causes a prolonged and persistent infection which may lead to hepatocellular carcinoma. We have previously reported that the nonstructural 5A (NS5A) protein of HCV promotes cell growth [Ghosh, A.K., Steele, R., Meyer, K., Ray, R., Ray, R.B., 1999. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J. Gen. Virol. 80, 1179-1183]. In this study, we investigated the role of HCV NS5A (genotype 1a, strain H) in TNF-alpha induced apoptotic cell death. HepG2 cells expressing NS5A exhibited an inhibitory role in relation to TNF-alpha mediated apoptotic cell death. The NS5A protein blocked the activation of caspase-3 and inhibited proteolytic cleavage of the death substrate poly (ADP-ribose) polymerase in TNF-alpha induced cells. Together, these results suggest that HCV NS5A protein protects against TNF-alpha mediated apoptotic cell death.     

HPV16 E1--E4 protein is phosphorylated by Cdk2/cyclin A and relocalizes this complex to the cytoplasm

The human papillomavirus type 16 E1--E4 protein is expressed abundantly in cells supporting viral DNA amplification, but its expression is lost during malignant progression. In cell culture, 16E1--E4 causes G2 cell cycle arrest by associating with and preventing the nuclear entry of Cdk1/cyclin B1 complexes. Here, we show that 16E1--E4 is also able to associate with cyclin A and Cdk2 during the G2 phase of the cell cycle. Only a weak association was apparent during S-phase, and progression through S-phase appeared unaffected. As with cyclin B1, the interaction of 16E1--E4 with cyclin A is dependent on residues T22/T23 and results in the accumulation of cyclin A in the cytoplasm where it colocalizes with 16E1--E4. 16E1--E4 serine 32 was found to be phosphorylated by Cdk2/cyclin A. We hypothesize that the interaction of 16E1--E4 with cyclin A may serve to increase the efficiency with which 16E1--E4 is able to prevent mitotic entry.     

Comparison of MHC antigen expression on PHA- and MLC-induced T cell lines with that on T and B lymphoblastoid cell lines by cell cycle dependency

Although it is well known that the expression of major histocompatibility complex (MHC) antigens on the surface of lymphoblastoid cell lines are cell cycle dependent, the way in which the MHC antigen expression on activated T cells varies with cell cycle phase has not previously been described. Using 11 lymphoblastoid cell lines from malignant and nonmalignant tissues (B cells, T cells, and myeloid cells) and five activated T cell lines (two cell lines activated by phytohemagglutinin and three alloreactive T cell clones), MHC antigen expression was quantitatively studied by dual-beam flow cytometry. Correlated measurements of surface antigen quantity (immunofluorescence), DNA content (Hoechst 33342), and cell size (light scatter), uninfluenced by induction synchrony and cell fixation, were performed. The data indicate that cell surface antigen quantity and cell surface area demonstrate specific values at each phase of the cell cycle when the cells are in logarithmic growth. Examining cells in logarithmic growth, it was confirmed, for all lymphoblastoid cell lines, that the quantity of MHC antigens on G2 (S + G2 + M) cells was greater than that on G1 cells. In addition, it was found, by analyzing antigen quantity and surface area, that class I antigen density in the G2 phase is 17% less than that in the G1 phase in leukemic T cell lines, and that both class I and class II antigen densities in the G2 phase were 21% less than that in the G1 phase in lymphoblastoid B cell lines. In activated T cells, class I antigen density in the G2 phase was 11% less than that in the G1 phase, while class II antigen density in the G2 phase was 12% greater than that in the G1 phase. We describe four important observations in this report. In both G1 and G2 phases, activated T cells express: quantitatively fewer class I antigens than lymphoblastoid B cell lines; similar quantity of class I antigens as that of leukemic T cell lines; and similar quantity of class II antigens as that of lymphoblastoid B cell lines. Also, class II antigens are expressed in greater density in the G2 phase than in the G1 phase in activated T cells. In contrast, lymphoblastoid B cell lines express greater density of class II antigens in the G1 phase than in the G2 phase of the cell cycle. These findings differ from previous reports, suggesting that G1 phase cells may have a more significant role than G2 phase cells as target cells for MHC restricted cytotoxic cells.     

6]-Gingerol induces cell cycle arrest and cell death of mutant p53-expressing pancreatic cancer cells

[6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild- type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21cip1, was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53- expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53- expressing cells by inducing temporal growth arrest.     

Activated CD8(+) T cells from aged mice exhibit decreased activation-induced cell death

To uncouple the defects of activation and apoptosis of T cells from aged mice, we used anti-CD3 plus IL-2 stimulation to induce an activation response and analyzed the subsequent activation-induced cell death (AICD) response of T cells from 16-month-old mice. The results herein demonstrate that T cells from 16-month-old mice could be activated by anti-CD3-induced activation signals but exhibited distinct phenotypic and functional features compared to young (2-month-old) mice. These include a decrease in AICD, a delayed entry into the cell cycle, and a decreased telomerase activity. The decreased AICD of T cells from 16-month-old mice is associated with a decreased expression of Fas and Fas ligand (FasL), decreased susceptibility to anti-Fas-induced apoptosis, and an increased expansion of a CD8(+) T-cell population. Prior to activation, these T cells exhibit a phenotype that is CD44(hi)CD62L(hi). After stimulation, these T cells produced high levels of the pro-inflammatory cytokine, IFN-gamma, and developed an increased population of IFN-gamma(+)IFN-gamma R(-) T cells. Our results suggest that there is a dysregulation in T-cell homeostasis in aged mice associated with a decrease in AICD of CD8(+) T cells.     

Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division

Cells expressing indoleamine 2,3‐dioxygenase (IDO), an enzyme which catabolizes tryptophan, prevent T‐cell proliferation in vitro, suppress maternal antifetal immunity during pregnancy and inhibit T‐cell‐mediated responses to tumour‐associated antigens. To examine the mechanistic basis of these phenomena we activated naïve murine T cells in chemically defined tryptophan‐free media. Under these conditions T cells expressed CD25 and CD69 and progressed through the first 12 hr of G0/G1 phase but did not express CD71, cyclin D3, cdk4, begin DNA synthesis, or differentiate into cytotoxic effector cells. In addition, activated T cells with their growth arrested by tryptophan deprivation exhibited enhanced tendencies to die via apoptosis when exposed to anti‐Fas antibodies. Apoptosis was inhibited by caspase inhibitor and was not observed when T cells originated from Fas‐deficient mice. These findings suggest that T cells activated in the absence of free tryptophan entered the cell cycle but cell cycle progression ceased in mid‐G1 phase and T cells became susceptible to death via apoptosis, in part though Fas‐mediated signalling. Thus, mature antigen‐presenting cells expressing IDO and Fas‐ligand may induce antigen‐specific T‐cell tolerance by blocking T‐cell cycle progression and by rapid induction of T‐cell activation induced cell death in local tissue microenvironments.