Silencing of X-linked inhibitor of apoptosis decreases resistance to cisplatin and paclitaxel but not gemcitabine in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a highly lethal malignancy that often becomes resistant to chemotherapy. The effect of silencing the X-linked inhibitor of apoptosis gene (XIAP) on resistance to cisplatin, paclitaxel and gemcitabine was studied in the NSCLC cell lines A549 and H460. Transfection of these cells with small interfering RNA (siRNA) for XIAP blocked overexpression of the gene, suppressed cell proliferation, increased apoptosis and increased the cells' sensitivity to cisplatin and paclitaxel by preventing the binding of XIAP to caspase3 and increasing the activity of this enzyme. There was no significant difference in resistance to gemcitabine between XIAP-silenced cells and non-transfected cells. Changes in chemoresistance were independent of the activity of caspase-9. Silencing XIAP with siRNA can decrease chemoresistance in NSCLC and may have a potential role in the treatment of this disease.     

硼替佐米联合低浓度阿糖胞苷诱导U937细胞凋亡的研究

本研究探讨蛋白酶体抑制剂硼替佐米联合低浓度阿糖胞苷(Ara-C)对U937细胞株的作用及机制。通过细胞计数,细胞形态学检查,流式细胞术和Western blot方法检测硼替佐米(10 nmol/L)和(或)Ara-C(50 nmol/L)处理前后U937细胞的增殖抑制和凋亡及其机制。结果显示,硼替佐米和Ara-C单药均能抑制U937细胞增殖,两药联合的抑制作用更加明显,细胞增殖抑制率在24和48 h分别达(55.00±2.81)%和(70.02±3.33)%;硼替佐米联合低浓度Ara-C能协同诱导U937细胞凋亡,促使线粒体跨膜电位下降,阳性细胞率达(38.70±1.54)%。两药联合应用还能协同诱导caspase-9,-8,-3的活化。结论:硼替佐米联合低浓度Ara-C主要通过线粒体途径、可能还通过死亡受体途径诱导U937细胞凋亡。     

Caspase-2 functions upstream of mitochondria in endoplasmic reticulum stress-induced apoptosis by bortezomib in human myeloma cells

Multiple myeloma is an incurable plasma cell malignancy. The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in multiple myeloma cells; however, the mechanism by which this compound acts remains unknown. Here, we, using immunoblotting analysis, observed that the expression of BiP, CHOP, and XBP-1 is up-regulated in bortezomib-induced apoptosis in human multiple myeloma cell lines NCI-H929 and RPMI-8226/S, strongly suggesting that endoplasmic reticulum (ER) stress response or the unfolded protein response (UPR), a signaling pathway activated by the accumulation of unfolded proteins within ER, is initiated. In the meantime, we also showed that bortezomib inhibited classic ER stressor brefeldin A-induced up-regulation of prosurvival UPR components BiP and XBP-1, resulting in increased induction of apoptosis in multiple myeloma cell lines, raising the possibility that bortezomib induces apoptosis of multiple myeloma cells by means of evoking the severe ER stress but disrupting the prosurvival UPR required. Using caspase inhibitors and a RNA interference approach, we finally confirmed that bortezomib-triggered apoptosis in multiple myeloma cells is dependent on caspase-2 activation, which is associated with ER stress and required for release of cytochrome c, breakdown of mitochondrial transmembrane potential, and its downstream caspase-9 activation. Taken together, these data strongly suggest that caspase-2 can serve as a proximal caspase that functions upstream of mitochondrial signaling during ER stress-induced apoptosis by bortezomib in multiple myeloma cells.     

Bortezomib-induced survival signals and genes in human proximal tubular cells

Bortezomib has been introduced recently in the therapy of multiple myeloma (MM), a disease that is frequently associated with progressive renal failure. Because bortezomib-based therapy has been reported to lead to a rapid recovery of kidney function in patients with MM, we decided to study its direct effects in proximal tubular epithelial cells (PTCs) compared with glomerular mesangial cells (GMCs). After 24 h of stimulation, 50 nM bortezomib led to a 6.37-fold induction of apoptosis and markedly activated caspase-9 and -3 in GMCs but not in PTCs. In PTCs but not in GMCs, bortezomib led to a strong time-dependent degradation of IkappaB-alpha and to a long-lasting phosphorylation of both NF-kappaBp65 and extracellular signal-regulated kinase 1/2. Microarray analysis in bortezomib-treated PTCs revealed a time-dependent predominance of antiapoptotic genes compared with proapoptotic genes. Bortezomib (50 nM) induced heat shock protein (Hsp) 70 mRNA and protein levels in PTCs, whereas basal and bortezomib-stimulated Hsp70 protein expression was much weaker in GMCs. Moreover, bortezomib induced Bcl-2-associated athanogene (BAG) 3 mRNA and protein expression but inhibited BAG5 mRNA levels in PTCs. These data suggest that the reduced susceptibility of PTCs to bortezomib-induced cell apoptosis is because of cell type-specific effects of this compound on apoptosis/survival genes and pathways. The concept of bortezomib representing a blocker of both NF-kappaB activation and cell survival should be carefully examined in particular renal cell types.     

Characterization of cell death events induced by anti-neoplastic drugs cisplatin, paclitaxel and 5-fluorouracil on human hepatoma cell lines: Possible mechanisms of cell resistance.

Two different hepatoma cell lines were incubated for 48h with chemotherapeutic drugs cisplatin, paclitaxel and 5-FU to determine their ability to induce cytotoxicity and DNA fragmentation as well as to modify the expression of some cell death-related genes that could be involved in the resistance to therapy. We observed that cisplatin and paclitaxel induced cytotoxicity, but significant differences between both cell lines, were found only in the case of paclitaxel. At 48h, apoptosis was clearly present in Hep3B cells treated with cisplatin and HepG2 cells treated with paclitaxel. 5-FU induced cytotoxicity in both cell lines but only at higher concentrations than the other two drugs, triggering apoptosis and necrosis in HepG2 cells and only necrosis in Hep3B. When a time course was performed for the first 8h of treatment to elucidate the initial mechanism of cell death responsible for DNA fragmentation, we observed that 5-FU in Hep3B, and cisplatin in both cell lines, induces primary necrosis, whereas at the concentration tested here, paclitaxel clearly triggers apoptosis in both cell lines. HepG2 cells were weakly sensitive to 5-FU in the first 8h of treatment, so the primary mechanism of cell death was not clear, but results seem to indicate that it could be apoptosis. At 48h, Bax was not up-regulated with any of the treatments, whereas cisplatin was able to induce Bcl-xL down-regulation in both cell lines. Treatment with 5-FU also down-regulated Bcl-xL in HepG2 cells. We also measured variations in the expression of survivin, an inhibitor of apoptosis that has also been involved in mitototic catastrophe. Hep3B cells seem to show an increase in protein levels with all treatments. Exposure to paclitaxel resulted in the highest effect. In the case of HepG2 cells, there was a decrease in survivin expression when cells were treated with 5FU and paclitaxel, both treatments showing complete loss of the protein. Using an antibody that recognizes unprocessed caspase-3, we observed that the enzyme was assumingly activated in HepG2 cells treated with 5FU and paclitaxel, but only weakly after treatment with cisplatin. Hep3B cells did not show activation since the levels of the pro-enzyme remained the same as that in the control. In conclusion, the three drugs tested in this study could induce cell death, with paclitaxel being more effective inducing apoptosis. 5FU was only effective at high doses and its mechanism seems to be primarily related to necrosis in Hep3B and probably apoptosis in HepG2. Cisplatin mechanism of cell death is probably mediated by the decrease in anti-apoptotic protein Bcl-xL whereas paclitaxel and 5FU are decreasing the apoptosis inhibitor survivin. According to pro-enzyme levels, caspase-3 was only activated in HepG2 cells, whereas in the case of Hep3B cells the mechanisms of toxicity appear to be caspase-3-independent at the time and concentrations tested in this study. The resistance of Hep3B cells to death induced by chemotherapy could be related to an increase in the expression of IAP survivin, which can decrease cell response to the treatment or even switch the type of death from apoptosis to another kind, making therapy less efficient.     

Down-regulation of Bcl-2 is associated with cisplatin resistance in human small cell lung cancer H69 cells

Overexpression of the anti-apoptotic protein Bcl-2 has been associated with several malignancies, including small cell lung cancer (SCLC). In the present study, we have investigated if Bcl-2 contributes to the emergence of cisplatin resistance in SCLC H69 cells. The ability of cisplatin to induce apoptosis was decreased in H69 cells that acquired resistance to cisplatin (H69/CP). The level of Bcl-2 was, however, substantially reduced in H69/CP cells compared to parental H69 cells. There was little change in Bcl-2 content in H69 cells that were resistant to etoposide (VP-16) or Taxol. Bcl-2 was constitutively phosphorylated at serine 70 in H69 cells but not in H69/CP cells and cisplatin had little effect on Bcl-2 phosphorylation. The level of procaspase-3 was elevated in H69/CP cells but the ability of cisplatin to induce mitochondrial depolarization, caspase-9 activation, and poly(ADP-ribose) polymerase (PARP) cleavage was compromised in H69/CP cells. The level of the anti-apoptotic protein Bcl-x(L) and the pro-apoptotic protein Bax was slightly reduced in H69/CP cells but the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family proteins was not sufficient to explain cellular resistance to cisplatin. These results suggest that the acquisition of cisplatin resistance by H69 cells was not due to an increase in the level/phosphorylation status of the anti-apoptotic protein Bcl-2.     

三氧化二砷联合硼替佐米诱导多发性骨髓瘤RPMI8226细胞株凋亡及相关机制的研究

目的本研究旨在探讨三氧化二砷与硼替佐米联合应用对多发性骨髓瘤(Multiple myeloma,MM)细胞株RPMI8226细胞凋亡的影响及其相关机制。方法 CCK-8法检测三氧化二砷与硼替佐米联合应用对RPMI8226细胞增殖的抑制作用;采用流式细胞术检测两种药物诱导RPMI8226细胞凋亡情况及各药物组处理前后的细胞表面死亡受体4(death receptor 4,DR4)和死亡受体5(death receptor 5,DR5)表达的变化;Western blot检测各药物组Bcl-2、caspase-3、caspase-8、caspase-9和PARP蛋白表达水平的变化。结果三氧化二砷与硼替佐米联合组对RPMI8226细胞生长抑制明显高于三氧化二砷单药组,细胞凋亡率均较三氧化二砷单药组明显增多,细胞表面DR4和DR5表达明显增高。与三氧化二砷或硼替佐咪单药组相比较,联合用药组RPMI8226细胞Bcl-2表达下调明显,而caspase-3,caspase-8和caspase-9表达上调明显。结论硼替佐米可以增加RPMI8226细胞对三氧化二砷诱导凋亡的敏感性,这可能与Bcl-2的表达下调及caspase-3、caspase-8、caspase-9和PARP蛋白的表达上调有关。     

西达本胺对人B淋巴瘤细胞株的作用及其机制研究

本研究旨在探讨组蛋白去乙酰化酶抑制剂(HDACI)西达本胺对3种B淋巴瘤细胞株Raji(Burkitt淋巴瘤)、Maver及Z-138(套细胞淋巴瘤)的抑制增殖和诱导凋亡的作用及其机制。以不同浓度的西达本胺及不同时间作用于体外培养的3种B淋巴瘤细胞,采用CCK-8法检测细胞的增殖;流式细胞术检测细胞的凋亡及线粒体膜电位;Western blot方法检测细胞内组蛋白H3、H4乙酰化水平及caspase-3蛋白的表达。结果表明,西达本胺可抑制这3种B淋巴瘤细胞的增殖,其抑制作用具有一定的时间和浓度依赖性,尤其能较早较快地抑制Z-138细胞的增殖;另外,西达本胺还可诱导3种B淋巴瘤细胞发生凋亡并引起细胞线粒体膜电位下降,Maver及Z-138细胞对西达本胺较Raji细胞更为敏感;西达本胺可以提高细胞内组蛋白H3、H4乙酰化水平及Maver和Z-138细胞的caspase-3活性。结论:西达本胺能够抑制B淋巴瘤细胞株的增殖,诱导细胞凋亡,其机制可能与西达本胺上调组蛋白H3、H4乙酰化水平,触发线粒体凋亡途径及活化caspase-3有关。     

The antiangiogenic agent Neovastat (AE-941) induces endothelial cell apoptosis

Neovastat (AE-941), a naturally occurring multifunctional antiangiogenic agent, has been shown to inhibit key components of the angiogenic process, including matrix metalloproteinases and vascular endothelial growth factor-mediated signaling events. In this study, we report the presence of a proapoptotic activity within this compound. Neovastat treatment of bovine aortic endothelial cells caused cell death with characteristics of apoptosis, including chromatin condensation and DNA fragmentation. Neovastat markedly induced caspase-3, caspase-8, and caspase-9 activities, at similar levels to those measured in cells treated with tumor necrosis factor-alpha. Activation of caspases by Neovastat appears to be essential for its proapoptotic effects because all apoptotic features were blocked by zVAD-fmk, a broad-spectrum caspase inhibitor. The activation of caspases was correlated with the cleavage of the nuclear substrate poly(ADP-ribose) polymerase, and by a concomitant release of cytochrome c from mitochondria to the cytoplasm. Neovastat-induced apoptosis appears to be specific to endothelial cells because treatment of other cell types such as U-87, COS-7, NIH-3T3, and SW1353 did not result in increased caspase-3 activity. These results demonstrate that Neovastat contains a proapoptotic factor that specifically induces the activation of caspases in endothelial cells and the resulting apoptosis of these cells.     

KD5170, a novel mercaptoketone-based histone deacetylase inhibitor, exerts antimyeloma effects by DNA damage and mitochondrial signaling

Histone deacetylase inhibitors have emerged as promising anticancer drugs. Using an unbiased ultrahigh throughput screening system, a novel mercaptoketone-based histone deacetylase inhibitor series was identified that was optimized to the lead compound, KD5170. KD5170 inhibited the proliferation of myeloma cell lines and the viability of CD138(+) primary myeloma cells by induction of apoptosis, accompanied by an increase of acetylation of histones and activation of caspase-3, caspase-8, and caspase-9. Treatment with KD5170 caused a loss of mitochondrial membrane potential resulting in release of apoptogenic factors such as cytochrome c, Smac, and apoptosis-inducing factor. Furthermore, KD5170 induced oxidative stress and oxidative DNA damage in myeloma cells as evidenced by the up-regulation of heme oxygenase-1 and H2A.X phosphorylation. Combination of KD5170 with proteasome inhibitor bortezomib or tumor necrosis factor-related apoptosis-inducing ligand synergistically enhanced the antimyeloma activity. We further found that resistance of myeloma cells to KD5170 was associated with activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway under treatment with KD5170. Pretreatment with the mitogen-activated protein kinase inhibitor U0126 restored sensitivity to KD5170, suggesting that the combination of KD5170 with U0126 could overcome drug resistance. Growth of myeloma tumor xenografts in KD5170-treated nude mice was significantly inhibited and survival was prolonged. Histone acetylation was increased in spleen and tumor tissues of animals treated with KD5170. Our data indicate that KD5170 has potent antimyeloma activity in vitro and in vivo, which is mediated by DNA damage and mitochondrial signaling and subsequent induction of apoptosis.     

Influence of SiRNA targeting survivin on chemosensitivity of H460/cDDP lung cancer cells

We investigated the difference in survivin expression between a multidrug-resistant lung cancer cell line (H460/cDDP) and its parental counterpart (H460) and the influence of siRNA targeting survivin on the chemosensitivity of H460/cDDP. SiRNA targeting survivin was transfected into H460/cDDP cells using a liposome approach. Survivin mRNA and protein expression were significantly higher in H460/cDDP than H460 cells. The median inhibitory concentrations (IC(50)s) for cisplatin and paclitaxol in vitro against H460/cDDP cells were significantly lower in cells treated with survivin-specific siRNA than in control cells. Apoptosis and cleaved caspase-3 expression were analysed using annexin V and Western blotting, respectively, and showed a significant increase in apoptosis after treatment with the chemotherapeutic agents plus specific siRNA. Specific siRNA sensitized H460/cDDP cells to both cisplatin and paclitaxol. Thus, survivin appears to participate in the multidrug resistance mechanism of H460/cDDP cells and siRNA targeting survivin has the potential to increase the sensitivity of drug-resistant cancer cells to anticancer drugs.     

Emodin azide methyl anthraquinone derivative triggers mitochondrial-dependent cell apoptosis involving in caspase-8-mediated Bid cleavage

AMAD, an emodin azide methyl anthraquinone derivative, was extracted from the nature giant knotweed rhizome of traditional Chinese herbs. Here, we investigated the anticancer activities and signaling pathways implicated in AMAD-induced apoptosis in human breast cancer cell lines MDA-MB-453 and human lung adenocarcinoma Calu-3 cells. AMAD was found to have a potent cytotoxic effect on both cell lines. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. Moreover, this apoptotic induction was associated with a collapse of the mitochondrial membrane potential and activated caspases (cysteine aspartase) cascade involving in caspase-8, caspase-9, caspase-3, and poly(ADP-ribose) polymerase cleavage in a concentration-dependent manner. It was noteworthy that AMAD also effectively cleaved Bid, a BH3 domain-containing proapoptotic Bcl-2 family member, and induced the subsequent release of cytochrome c from mitochondria into the cytosol. Furthermore, suppression of caspase-8 activity with Z-IETD-FMK partially inhibited release of cytochrome c and Bid cleavage induced by AMAD, whereas exposure to Z-LETD-FMK, a caspase-9 inhibitor, had no effect. Additionally, there was significant change in other mitochondrial membrane proteins triggered by AMAD, such as Bcl-xl and Bad. It was intriguing that AMAD decreased the generation of reactive oxygen species in both cell lines. DNA-binding assay exhibited apoptosis induced by AMAD was not involved in intercalating to DNA. Taken together, these data suggested that AMAD induced apoptosis via a mitochondrial pathway involving caspase-8/Bid activation in both cell lines.     

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.     

Oncogene AF1q enhances doxorubicin-induced apoptosis through BAD-mediated mitochondrial apoptotic pathway

AF1q is an oncogenic factor involved in leukemia development, thyroid tumorigenesis, and breast cancer metastasis. In the present study, AF1q was found to be down-regulated in a doxorubicin-resistant subline of human squamous carcinoma A431 cells. Knockdown of AF1q decreased the apoptosis induced by doxorubicin, Taxol, gamma-radiation, IFN-alpha, and IFN-gamma in A431 cells. On the other hand, overexpression of AF1q increased the doxorubicin-induced apoptosis in A431 cells as well as in HepG2 and HL60 cells. Both exogenous and ectopic expression of AF1q in A431 cells increased the mRNA and protein levels of BAD, a proapoptotic BCL-2 family protein. Gene silencing of BAD by small interfering RNA suppressed the AF1q enhancement of apoptosis, suggesting that BAD is downstream of AF1q in regulation of apoptosis. Furthermore, AF1q enhanced the mitochondrial membrane depolarization, mitochondrial cytochrome c release, and activation of caspase-9 and caspase-3 on doxorubicin treatment. Collectively, AF1q increases doxorubicin-induced apoptosis in cells through activation of BAD-mediated apoptotic pathway. The study provides the first evidence that AF1q plays a critical role in the regulation of apoptosis and drug resistance.     

IRE1α-TRAF2-ASK1 pathway is involved in CSTMP-induced apoptosis and ER stress in human non-small cell lung cancer A549 cells

BackgroundCSTMP, a Tetramethylpyrazine (TMP) analogue, is designed and synthesized based on the pharmacophores of TMP and resveratrol. Recent studies showed that CSTMP had strong protective effects in endothelial cells apoptosis by its anti-oxidant activity. However, the pharmacological function of CSTMP in cancer have not been elucidated to date. The objective of this study was to investigate the anti-cancer effect of CSTMP against human non-small cell lung cancer (NSCLC) A549 cells and the underlying mechanisms.MethodsThe cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Caspases activity was determined spectrophotometricaly at 405 nm using a microtiter plate reader. Western blot and real-time PCR was used to assess the protein and mRNA expression. Immunoprecipitation was used to examine the protein–protein interactions.ResultsCSTMP inhibited the proliferation and induced cell cycle arrest and apoptosis of A549 cells. Caspase3, 8, 9 and PARP-1 activation, and Bax/Bcl-2 ratio analyses demonstrated that the anti-cancer effect of CSTMP in A549 cells was mediated by promoting caspase- and mitochondria-dependent apoptosis. Furthermore, CSTMP induced ER stress in A549 cells as evidenced by elevated levels of GRP78, GRP94, CHOP, IRE1α, TRAF2, p-ASK1 and p-JNK, activation of caspase12 and 4, and enhanced formation of an IRE1α-TRAF2-ASK1 complex. Knockdown of IRE1α by siRNA suppressed activation of IRE1α, TRAF2, p-ASK1 and p-JNK in CSTMP treated A549 cells. In addition, the effects of CSTMP on the formation of an IRE1α-TRAF2-ASK1 complex, caspase- and mitochondria-dependent apoptosis were also reversed by IRE1α siRNA in A549 cells.ConclusionsCollectively, we showed that CSTMP induced apoptosis of A549 cells were through IRE1α-TRAF2-ASK1 complex-mediated ER stress, JNK activation, and mitochondrial dysfunction. These insights on this novel compound CSTMP may provide a novel anti-cancer candidate for the treatment of NSCLC.     

Amphiregulin Promotes BAX Inhibition and Resistance to Gefitinib in Non-small-cell Lung Cancers

Molecular resistance mechanisms affecting the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small-cell lung cancer (NSCLC) cells are not fully understood. Amphiregulin (Areg) overexpression has been proposed to predict NSCLC resistance to gefitinib and we have established that Areg-overexpressing H358 NSCLC cells resist apoptosis. Here, we demonstrate that Areg prevents gefitinib-induced apoptosis in NSCLC cells. We show that H358 cells are resistant to gefitinib in contrast to H322 cells, which do not overexpress Areg. Inhibition of Areg expression by small-interfering RNAs (siRNAs) restores gefitinib sensitivity in H358 cells, whereas addition of recombinant Areg confers resistance in H322 cells. Areg knockdown overcomes resistance to gefitinib and induced apoptosis in NSCLC H358 cells in vitro and in vivo. Under gefitinib treatment, Areg decreases the expression of the proapoptotic protein BAX, inhibits its conformational change and its mitochondrial translocation. Thus, in the presence of Areg, gefitinib-mediated apoptosis is reduced because BAX is sequestered in the cytoplasm. This suggests that treatments using epidermal growth factor receptor (EGFR) inhibitors may be poorly efficient in patients with elevated levels of Areg. These findings indicate the need for inhibition of Areg to enhance the efficiency of the EGFR inhibitors in patients suffering NSCLC.     

Bcl-2 regulatory pathway is functional in chronic lymphocytic leukemia

BACKGROUND: Chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal, malignant CD5(+), CD23(+) B cells. In vivo, these cells have an antiapoptotic phenotype (high levels of Bcl-2 and low levels of proapoptotic Bcl-2 family proteins, such as Bax). Abnormal B cells accumulate due to altered apoptosis regulation rather than to increased proliferation. However, it is unclear whether there are inherent Bcl-2 apoptotic pathway defects. With in vitro culture, these B cells rapidly apoptosis. METHODS: To investigate apoptosis regulation, Bcl-2, Bax, mitochondrial membrane potential, annexin V, and caspase activation were simultaneous monitored in individual cells during in vitro apoptosis. RESULTS: With in vitro culture, 30% to 50% of B cells were apoptotic at 24 h compared with fewer than 10% of T cells. Apoptotic B cells showed dramatic Bax upregulation and slight Bcl-2 decreases accompanied by decreased mitochondrial membrane potential and increased activated caspase-3 protein levels. Caspase-3 and caspase-9 activities were increased 18- to 51-fold and 6- to 11-fold, respectively, after 24 h of culture. Caspase-8 showed limited or no activation (less than fourfold). CONCLUSIONS: These data show that in vitro apoptosis of CLL B cells occurs through a well-characterized Bcl-2 regulatory pathway consistent with that pathway being functional. Further, these cells' antiapoptotic phenotype is dependent on the in vivo environment, potentially involving paracrine/autocrine interactions.     

Acetylation suppresses the proapoptotic activity of GD3 ganglioside

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.