Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth,apoptosis, and gene expression in 3LL Lewis lung carcinoma cells

To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes because it lacks GM3 and SM3 but accumulates lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth (as measured by colony-forming ability in soft agar) of the SM3- reconstituted cells was almost completely lost, which supports our previous observation showing the suppression of tumorigenic potential in vivo and beta1 integrin gene expression induced by the introduction of cerebroside sulfotransferase gene (Kabayama et al. [2001] J. Biol. Chem., 276, 26777-26783). The GM3-reconstituted cells formed a significantly higher number of colonies in soft agar compared to mock-transfected cells and began to proliferate and become resistant to apoptosis when serum was depleted, indicating that endogenous GM3 is essential for maintaining these fundamental properties of malignant cells. We also found that serum-induced ERK1/2 activation was suppressed in the GM3-reconstituted cells, suggesting that anchorage-independent cell cycle initiation by endogenous GM3 is elicited through pathway(s) independent of ERK1/2 activation. The selective down-regulation of platelet-derived growth factor (PDGF)-dependent ERK1/2 activation in the GM3-reconstituted cells was due to the substantial decreases of PDGF alpha receptor mRNA and protein, but in the SM3-reconstituted cells PDGF alpha receptor expression was similar to mock cells. Thus, endogenously produced GM3 and SM3 differentially and distinctly regulate tumor-progression ability, that is, GM3 leads the transformed phenotype of J5 cells to promotion and SM3 to abrogation.     

Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.     

Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3

In chronic myeloid leukemia K562 cells, differentiation is also blocked because of low levels of ganglioside GM3, derived by the high expression of sialidase Neu3 active on GM3. In this article, we studied the effects of Neu3 silencing (40-70% and 63-93% decrease in protein content and activity, respectively) in these cells. The effects were as follows: (a) gangliosides GM3, GM1, and sialosylnorhexaosylceramide increased markedly; (b) cell growth and [(3)H]thymidine incorporation diminished relevantly; (c) as mRNA, cyclin D2, and Myc were much less expressed, whereas cyclin D1 was expressed more like its inhibitor p21; (d) as mRNA, pro-apoptotic proteins Bax and Bad increased with concurrent decrease and increase in the anti-apoptotic proteins Bcl-2 and Bcl-XL, respectively; (e) the apoptosis inducers etoposide and staurosporine were active on Neu3 silencing cells but not on mock cells; (f) as mRNA, the megakaryocytic markers CD10, CD44, CD41, and CD61 increased similar to the case of mock cells stimulated with PMA; (g) the signaling cascades mediated by PLC-beta2, PKC, RAF, ERK1/2, RSK90, and JNK were largely activated. The induction of a GM3-rich ganglioside pattern in K562 cells by treatment with brefeldin A elicited a phenotype similar to that of Neu3 silencing cells. In conclusion, upon Neu3 silencing, K562 cells show a decrease in proliferation, propensity to undergo apoptosis, and megakaryocytic differentiation.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

Adrenomedullin prevents apoptosis in prostate cancer cells

The 52-aminoacid peptide adrenomedullin (AM) is expressed in the normal and malignant prostate. We have previously shown that prostate cancer cells produce and secrete AM, which acts as an autocrine growth inhibitory factor. We have evaluated in the present study the role of AM in prostate cancer cell apoptosis, induced either by serum deprivation or treatment with the chemotherapeutic agent etoposide (which acts as an inhibitor of topoisomerase II). For this purpose we over-expressed AM in PC-3, DU 145 and LNCaP cells, which were transfected with an expression vector carrying AM. We also treated the parental cell lines with synthetic AM in normal culture conditions and in conditions of induced-apoptosis. After serum removal, AM prevented apoptosis in DU 145 and PC-3 cells, but not in LNCaP cells. When treated with etoposide, AM prevented apoptosis in PC-3 and LNCaP cells, but not in DU 145 cells. Cell cycle analysis demonstrated a significant decrease in the percentage of AM-overexpressing PC-3 cells in the subG0/G1 phase after treatment with etoposide, as compared to the percentage of mock-transfected PC-3 treated cells. Western blot showed that protein levels of phosphorylated ERK1/2 increased in parental PC-3 cells after treatment with etoposide. In PC-3 cells overexpressing AM, phosphorylated ERK1/2 basal levels were lower than basal levels of parental PC-3 cells, and treatment with etoposide did not result in such an increase. Etoposide produced a significant increase in cleaved PARP in parental PC-3 cells. However, PC-3 clones overexpressing AM that were treated with etoposide only showed a mild increase in fragmented PARP. The ratio Bcl-2/Bax was reduced in parental or mock-transfected PC-3 cells after treatment with etoposide. On the contrary, this ratio was not reduced in PC-3 clones with AM overexpression that were treated with etoposide. All these data demonstrate that AM plays a protective role against induced apoptosis in prostate cancer cells. These results may have important implications in prostate cancer resistance to chemotherapeutic agents.     

Augmentation of drug-induced cell death by ER protein BRI3BP

To determine the contribution of the endoplasmic reticulum (ER) to cell fate decision, we focused on BRI3-binding protein (BRI3BP) residing in this organelle. BRI3BP, when overexpressed, augmented the apoptosis of human embryonic kidney 293T cells challenged with drugs including the anti-cancer agent etoposide. In contrast, the knockdown of BRI3BP reduced the drug-triggered apoptosis. BRI3BP overexpression enhanced both mitochondrial cytochrome c release and caspase-3 activity in etoposide-treated cells. In response to etoposide, the ER reorganized into irregularly shaped lamellae in mock-transfected cells, whereas in BRI3BP-overexpressing cells, such reorganization was not observed. These observations suggest that BRI3BP is involved in the structural dynamics of the ER and affects mitochondrial viability. Taken together, BRI3BP, widely expressed in animal cell types, seems to possess a pro-apoptotic property and can potentiate drug-induced apoptosis.     

INrf2 (Keap1) targets Bcl-2 degradation and controls cellular apoptosis

Cytosolic inhibitor of Nrf2 (INrf2) is an adaptor protein that mediates ubiquitination/degradation of NF-E2-related factor 2 (Nrf2), a master regulator of cytoprotective gene expression. In this paper, we demonstrate that INrf2 degrades endogenous antiapoptotic B-cell CLL/lymphoma 2 (Bcl-2) protein and controls cellular apoptosis. The DGR domain of INrf2 interacts with the BH2 domain of Bcl-2 and facilitates INrf2:Cul3–Rbx1-mediated ubiquitination of Bcl-2 by the conjugation of ubiquitin molecules to lysine17 of Bcl-2. Further studies showed that INrf2 enhanced etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. Antioxidants antagonized Bcl-2:INrf2 interaction, led to the release and stabilization of Bcl-2, increased Bcl-2:Bax heterodimers and reduced apoptosis. Moreover, dysfunctional/mutant INrf2 in human lung cancer cells failed to degrade Bcl-2, resulting in decreased etoposide and UV/γ radiation-mediated DNA fragmentation. These data provide the first evidence of INrf2 control of Bcl-2 and apoptotic cell death, with implications in antioxidant protection, survival of cancer cells containing dysfunctional INrf2, and drug resistance.     

Loss of choleragen receptors and ganglioside upon differentiation of 3T3-L1 preadipocytes

3T3-L1 preadipocytes differentiate in culture into cells having the enzymatic and morphological characteristics of adipocytes. Differentiation is accompanied by a decrease in total cellular ganglioside content; the ganglioside level is 1.8 to 2.5-fold higher in undifferentiated than in differentiated cells. Gangliosides GM3 and GD1a constitute a majority of total cell gangliosides in both cell types, while ganglioside GM1, the putative choleragen receptor, constitutes less than 5%. Differentiation results in a 75 to 85% decrease in ganglioside GM1. An inverse correlation exists between the percentage of adipocytes in the cell population and: 1) total ganglioside and ganglioside GM1 content, and 2) surface ganglioside GM1 as estimated by choleragen binding or fluorescent staining of bound choleragen. Nondifferentiating 3T3-C2 control cells do not exhibit changes in total ganglioside, ganglioside GM1, or choleragen binding that are observed with 3T3-L1 cells.     

Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells

The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.     

Glycogen synthase kinase-3β regulates etoposide-induced apoptosis via Bcl-2 mediated caspase-3 activation in C3H10T1/2 cells

Glycogen synthase kinase-3β (GSK3β) controls the survival of osteoblasts during bone development through Wnt canonical signaling. GSK3β is a key factor for osteoblastogenesis, but relatively less is known regarding its role in osteoblast apoptosis. Genotoxic stress induced by etoposide promoted apoptotic signaling by GSK3β activation in C3H10T1/2 cells, a mouse mesenchymal cell line. Etoposide led to the time-dependent activation of GSK3β and caspase-3, which resulted in PARP cleavage. LiCl (a specific inhibitor) and siRNA (gene knock-down) of GSK3β prevented the effects of etoposide on apoptosis. Staurosporine also induced apoptosis in C3H10T1/2 cells, but LiCl could not rescue. Bcl-2 was decreased in the cells by exposure to etoposide. LiCl completely recovered Bcl-2 expression as shown by both the mRNA and the protein expression levels. In conclusion, etoposide-induced apoptosis in C3H10T1/2 cells is mediated by GSK3β, which leads to caspase-3 activation via decrease in Bcl-2 expression. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Smac调控Caspase-3对耐药肺腺癌细胞株生物活性及相关信号的研究

摘要 目的：探讨Smac基因调控Caspase-3表达对紫杉醇耐药肺腺癌细胞株生物活性及经典凋亡信号通路的作用机制。方法：取构建好的耐药A549细胞,将其分为A549细胞（LC）组、A549细胞+Smac-NC（SN）组、A549细胞+Smac抑制剂（SI）组、A549细胞+Smac激动剂（SM）组、A549细胞+Caspase-3-NC（CN）组、A549细胞+Caspase-3抑制剂（CI）组、A549细胞+Caspase-3激动剂（CM）组、A549细胞+Smac激动剂+Caspase-3激动剂（MM）组;Real-time PCR法检测正常肺上皮细胞及4种肺腺癌细胞系中Smac、Caspase-3表达水平,将阴性对照、Smac、Caspase-3类似物转染至紫杉醇耐药肺腺癌细胞株,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,免疫印迹法检测经典凋亡信号通路表达,并分析Smac与Caspase-3的相关性。结果：肺腺癌细胞系中的Smac、Caspase-3 mRNA表达量显著低于正常肺上皮细胞系BEAS-2B（P＜0.05）,其中A549的Smac、Caspase-3 mRNA值最小（P＜0.05）,因此选取其作为此次实验细胞;LC组与SN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与SN组相比,SI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低（P＜0.05）,增殖率、Bcl-2表达明显升高（P＜0.05）,与SI组相比,SM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;LC组与CN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与CN组相比,CI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低（P＜0.05）,增殖率、Bcl-2表达明显升高（P＜0.05）,与CI组相比,CM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;SM组与CM组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与CM组相比,MM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;Smac与Caspase-3呈现正相关（r=0.470,P=0.002）,组间具有显著差异。结论：Smac基因可显著改善紫杉醇耐药肺腺癌细胞株细胞生物活性,并激活经典凋亡信号通路,其作用机制可能与调控Caspase-3表达有关。     

Enhanced suppression of proliferation and migration in highly-metastatic lung cancer cells by combination of valproic acid and coumarin-3-carboxylic acid and its molecular mechanisms of action

Valproic acid (VPA) as a broad-spectrum inhibitor of histone deacetylase, has been used in cancer therapy. Recently, the combination of VPA with other anticancer agents has been considered as a useful and necessary strategy to inhibit tumor growth and progression. The coumarin derivates from natural plants have been shown to be the promising natural anticancer agents. However, no literature is available on the anticancer effects of the combination of VPA and coumarin-3-carboxylic acid (HCCA). Here we show that this combination significantly increases inhibitory effects against the proliferation and migration in highly-metastatic lung cancer cells by inducing apoptosis and cell cycle arrest as well as regulating related protein expressions. Our results indicate that this combination of VPA with HCCA not only enhances the protein levels of Bax, cytosolic cytochrome c, caspase-3 and PARP-1 but also reduces the protein expressions of Bcl-2, cyclin D1 and NF-κB as well as inhibits the phosphorylation and expressions of Akt, EGFR, VEGFR2 and c-Met in the cancer cells. Our results suggest that the combination of VPA with HCCA suppresses the proliferation and migration of lung cancer cells via EGFR/VEGFR2/c-Met-Akt-NF-κB signaling pathways; this combination may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of lung cancer.     

TIPE2 Inhibits Lung Cancer Growth Attributing to Promotion of Apoptosis by Regulating Some Apoptotic Molecules Expression

Recent studies found that TIPE2 was involved in cancer development. However, little is known about TIPE2 in lung cancer. Our study aims to clarify the role of TIPE2 in lung carcinogenesis. We examined the expression of TIPE2 in lung squamous cancer (LSC), small cell lung cancer and lung adenocarcinoma (AdC) tissues and found that TIPE2 expression was lost in small cell lung cancer, compared with adjacent non-tumor tissues. Overexpression of TIPE2 significantly inhibited the growth of lung cancer cell H446 in vitro and even suppressed tumor formation in vivo. Flow cytometry analysis found TIPE2 overexpression promoted apoptosis of H446. In TIPE2 over-expression cells, caspase-3, caspase-9, and Bax were significantly up-regulated while Bcl-2 was down-regulated. Moreover, coincident results were shown by immunohistochemistry in tumors from nude mice. TIPE2 inhibited the phosphorylation of Akt, while promoting the phosphorylation of P38, but had no effect on IκBα and ERK pathway. Taken together, TIPE2 promoted lung cancer cell apoptosis through affecting apoptosis-related molecules caspase-3, caspase-9, Bcl-2 and Bax, possibly via regulating P38 and Akt pathways, indicating that TIPE2 might be a novel marker for lung cancer diagnosis and therapy.     

Differential regulation of doxorubicin-induced mitochondrial dysfunction and apoptosis by Bcl-2 in mammary adenocarcinoma (MTLn3) cells

Various anticancer drugs cause mitochondrial perturbations in association with apoptosis. Here we investigated the involvement of caspase- and Bcl-2-dependent pathways in doxorubicin-induced mitochondrial perturbations and apoptosis. For this purpose, we set up a novel three-color flow cytometric assay using rhodamine 123, annexin V-allophycocyanin, and propidium iodide to assess the involvement of the mitochondria in apoptosis caused by doxorubicin in the breast cancer cell line MTLn3. Doxorubicin-induced apoptosis was preceded by up-regulation of CD95 and CD95L and a collapse of mitochondrial membrane potential (Deltapsi) occurring prior to phosphatidylserine externalization. This drop in Deltapsi was independent of caspase activity, since benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone did not inhibit it. Benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone also blocked activation of caspase-8, thus excluding an involvement of the death receptor pathway in Deltapsi dissipation. Furthermore, although overexpression of Bcl-2 in MTLn3 cells inhibited apoptosis, dissipation of Deltapsi was still observed. No decrease in Deltapsi was observed in cells undergoing etoposide-induced apoptosis. Immunofluorescent analysis of Deltapsi and cytochrome c localization on a cell-to-cell basis indicates that the collapse of Deltapsi and cytochrome c release are mutually independent in both normal and Bcl-2-overexpressing cells. Together, these data indicate that doxorubicin-induced dissipation of the mitochondrial membrane potential precedes phosphatidylserine externalization and is independent of a caspase- or Bcl-2-controlled checkpoint.     

C3-induced 3LL cell proliferation is mediated by C kinase

It has been demonstrated that the third component of complement (C3)(1) and its peptides increase normal and tumour cell proliferation. However, the signal cascade responsible for this phenomenon is still unknown. In this study, we elucidate some of the mechanisms involved in the signalling of C3 stimulation of cell proliferation. We have first investigated the in and out traffic of C3 peptides, then we have identified the subcellular localisation of internalised C3 and, finally, we have explored the role of protein phosphorylation in C3 traffic and in the proliferation of the Lewis lung carcinoma (3LL) cells. Our results indicate that traffic of C3 is not dependent on cytoskeletal integrity and requires protein kinase C-dependent phosphorylation. In addition, proliferation of 3LL cells stimulated by C3 depends on both C3 internalisation and protein-kinase C phosphorylation.