Glioblastoma is a lethal neoplasm resistant to conventional radiotherapy and chemotherapy. Natural born killer (NBK), also known as Bcl-2-interacting killer (BIK), is a death-promoting Bcl-2 family protein sharing with Bcl-2 only the Bcl homology 3 (BH3) domain. We here report that an adenoviral vector encoding NBK (Ad-NBK) uniformly induces cell death in 12 human malignant glioma cell lines. Ad-NBK-induced cell death involves neither quantitative mitochondrial cytochrome c release nor caspase 8, 9, 7, or 3 processing and is unaffected by the viral caspase inhibitor, cytokine response modifier A (CRM-A), or selective caspase 8 or 9 inhibitors. In contrast, Ad-NBK-induced cell death is inhibited by the broad-range caspase inhibitor, zVAD-fmk, or by adenoviral gene transfer of the X-linked inhibitor of apoptosis protein (XIAP). Further, Ad-NBK-induced cell death is inhibited by Bcl-2 or Bcl-xL gene transfer. Interestingly, Bcl-2- and Bcl-xL-transfected glioma cells, which are partially protected from Ad-NBK-induced cell death, accumulate much higher levels of NBK than are ever observed in control-infected cells. This indicates that complex formation with Bcl-2 or Bcl-xL sequesters NBK in an inactive form and that free NBK, rather than an NBK-mediated depletion of free antiapoptotic Bcl-2 family proteins, is the proximate mediator of Ad-NBK-induced cell death. Conversely, proteasome inhibition-mediated accumulation of NBK strongly enhances Ad-NBK-induced cell death. Finally, Ad-NBK-infected LN-229 glioma cells are not tumorigenic in nude mice. Thus Ad-NBK triggers an XIAP- and zVAD-fmk-sensitive cell death pathway in glioma cells with potential therapeutic value, provided that NBK expression can be selectively targeted to cancer cells.

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik(+) clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik(+) clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik(+) clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik(+) clones.

We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills brain cancer cells. OSU toxicity was enhanced by the HSP90 inhibitor 17-N-Allylamino-17-demethoxygeldanamycin (17AAG) that correlated with reduced expression of ERBB1 and ERBB2. Inhibition of the extrinsic apoptosis pathway blocked the interaction between 17AAG and OSU. OSU toxicity was enhanced by the inhibitor of ERBB1/2/4, lapatinib. Knock down of ERBB1/2/4 in a cell line specific fashion promoted OSU toxicity. Combined exposure of cells to lapatinib and OSU resulted in reduced AKT and ERK1/2 activity; expression of activated forms of AKT and to a lesser extent MEK1 protected cells from the lethal effects of the drug combination. Knock down of PTEN suppressed, and expression of PTEN enhanced, the lethal interaction between OSU and lapatinib. Downstream of PTEN, inhibition of mTOR recapitulated the effects of lapatinib. Knock down of CD95, NOXA, PUMA, BIK or AIF, suppressed lapatinib and OSU toxicity. Knock down of MCL-1 enhanced, and overexpression of MCL-1 suppressed, drug combination lethality. Lapatinib and OSU interacted in vivo to suppress the growth of established tumors. Collectively our data argue that the inhibition of ERBB receptor function represents a useful way to enhance OSU lethality in brain tumor cells.

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, BimEL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.

OBJECTIVE

We evaluated the antitumor effects of docetaxel (Sigma®) and histone deacetylase inhibitors in hormone refractory prostate cancer cells, and analyzed the mechanism by which combination treatment induced cell death.

METHODS

We used LNCaP, DU145 and PC3 cells (ATCC®) to evaluate the in vitro apoptotic effects of histone deacetylase inhibitors and their combinations with docetaxel as well as the molecular mechanisms. The DU145 xenograft model was used to evaluate the in vivo efficacy of PXD101 combined with docetaxel.

RESULTS

Suberoylanilide hydroxamic acid or PXD101 inhibited the growth of hormone dependent LNCaP cells, and hormone independent DU145 and PC3 cells. It increased sub-G1 population and activated caspase-8, 9 and 3, indicating apoptosis induction. Pretreating DU145 cells with docetaxel followed by histone deacetylase inhibitors showed significant synergistic cytotoxicity compared with that of simultaneous co-treatment or reverse sequential treatment. Pretreatment with docetaxel followed by histone deacetylase inhibitors increased the apoptotic sub-G1 population, caspase activation and tubulin acetylation compared with that of docetaxel alone. Combination treatment decreased Mcl-1 and Bcl-xl, and increased t-Bid, Bik and Bim. Combined docetaxel and PXD101 reduced tumor size with efficacy equivalent to that of a double dose of docetaxel alone in the DU145 xenograft model.

CONCLUSIONS

These preclinical results indicate that the sequential combination of docetaxel and histone deacetylase inhibitors led to a synergistic increase in the death of hormone refractory prostate cancer cells via intrinsic and extrinsic apoptotic pathways by modulating Bcl-2 family proteins and tubulin in vitro and in vivo. Results suggest that this combination may be a new therapeutic modality in patients with hormone refractory prostate cancer.

In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response.

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X(L), Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.

Transforming growth factor-beta (TGF-beta) is central to the wound repair processes that follow local trauma and inflammation. In order to mimic the early events of wound-healing, we studied the effects of TGF-beta on mitogen-stimulated peripheral blood cells. TGF-beta added at the initiation of mitogenesis did not significantly alter T-cell activation, proliferation, CD45 isoform switching, or activation-induced cell death. By contrast, TGF-beta added 72 hr post-activation (or later) enhanced the cumulative increase in apoptotic T cells. TGF-beta had no effect on mitogen-induced up-regulation of Fas (CD95) or Fas ligand and did not enhance killing of the Fas-sensitive Jurkat cell line by activated T cells. Furthermore, TGF-beta had no direct effect on levels of mRNA for members of the bcl family (bcl-X, bfl-1, bik, bak, bax, bcl-2 and mcl-1). These findings suggest that TGF-beta does not directly induce apoptosis via the Fas system or by direct effects on bcl proteins. However, interleukin-2, which can 'rescue' lymphocytes from spontaneous apoptosis due to cytokine deprivation, abolished the pro-apoptotic effects of TGF-beta on post-activated T cells, thus demonstrating that TGF-beta increases the cytokine-dependence of T cells for survival. We propose a novel role for TGF-beta in the suppression of inflammation by promoting the elimination of post-activated T cells once the initiating stimulus has been resolved.

A 5.1 kb cDNA encoding an inward rectifier K+ channel (BIK) was isolated from a bovine aortic endothelial cell library. The cDNA codes for a 427-amino-acid protein with two putative transmembrane regions. Sequence analysis reveals that BIK is a member of the Kir2.1 family of inward rectifier K+ channels. Expression in Xenopus oocytes showed that BIK is a K+-specific strong inward rectifier channel that is sensitive to extracellular Ba2+, Cs+, and a variety of anti-arrhythmic agents. Northern analysis revealed that endothelial cells express a 5.5 kb BIK mRNA that is sensitive to shear stress.

Piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) is a polyphenol that is found in grapes, red wine, Rheum undulatum, and the seeds of Euphorbia lagascae. It has been previously reported that piceatannol inhibits the proliferation of a variety of cancer cell types. In the present study, we assessed the effects of piceatannol on the growth of androgen-insensitive DU145 prostate cancer cells at concentrations of 1-10 micromol/L. Piceatannol reduced the viable numbers and increased the numbers of apoptotic DU145 cells in a dose-dependent manner. Western blot analysis revealed that piceatannol increased the protein levels of cleaved caspase-8, -9, -7, and -3 and cleaved poly(ADP-ribose) polymerase (PARP). Piceatannol increased mitochondrial membrane permeability and cytochrome c release from the mitochondria to the cytosol. Piceatannol induced an increase in the levels of truncated Bid, Bax, Bik, Bok, and Fas but caused a decrease in the levels of Mcl-1 and Bcl-xL. Caspase-8 and -9 inhibitors mitigated piceatannol-induced apoptosis. The caspase-8 inhibitor suppressed the piceatannol-induced cleavage of Bid, caspase-3, and PARP. These results indicate that piceatannol induces apoptosis via the activation of the death receptor and mitochondrial-dependent pathways in prostate cancer cells.

Micro(mi)RNAs play important and varied roles in tumorigenesis; however, the full repertoire of miRNAs that affect cancer cell growth is not known. In this study, an miRNA library was screened to identify those that affect the growth of A549 tumor cells. Among 300 miRNAs, miR-28-5p, -323-5p, -510-5p, -552-3p, and -608 were the most effective in inhibiting cell growth. More specifically, overexpressing miR-28-5p, -323-5p, and -510-5p induced G1 arrest, as determined by flow cytometry, whereas that of miR-608 induced cell death in a caspase-dependent manner. Moreover, several genes involved in apoptosis and cell cycle progression were downregulated upon overexpression of each of the five miRNAs, with the functional targets of miR-552-3p and miR-608 confirmed by microarray, quantitative real-time PCR, and luciferase reporter assay. In miR-608-transfected cells, B cell lymphoma 2-like 1 (BCL2L1), D-type cyclin 1 (CCND1), CCND3, cytochrome b5 reductase 3 (CYB5R3), phosphoinositide 3-kinase regulatory subunit 2 (PIK3R2), specificity protein 1 (SP1), and phosphorylated Akt were all downregulated, while Bcl-2-interacting killer (BIK) was upregulated. Moreover, miR-608 was determined to have a suppressive function on tumor growth in an NCI-H460 xenograft model. These findings provide insights into the roles of five miRNAs in growth inhibition and their potential function as cancer therapeutics.

Better knowledge of peripheral B lymphocyte homeostasis is needed to address the human hypogammaglobulinemia diseases. A defect in the Bcmd gene shortens the B cell life span and causes B cell deficiency in A/WySnJ mice. Previous genetic mapping placed Bcmd near Srebf2 on chromosome 15. Inspection of the human chromosome 22 syntenic region identified the proapoptotic Bik gene as a candidate. Two mapping methods placed the homologous mouse gene, Blk, near Srebf2. The Blk genomic structure was highly homologous to BIK: Sequence analysis ruled out coding region mutations, but Blk transcripts were overly abundant in sorted A/WySnJ T1 B cells. Moreover, enriched transitional B cells showed a cell-autonomous defect leading to excessive apoptosis. Thus, Bcmd may be a direct mutation in Blk, or in a gene involved in Blk regulation, such that excess expression pushes the A/WySnJ transitional B cells past the apoptosis checkpoint to cell death.

We studied the effects, either combined or alone, of lectin from Korean mistletoe (Viscum album var. coloratum agglutinin, VCA) and doxorubicin (DOX) in MCF-7 (estrogen receptor-positive) and MDA-MB231 (estrogen receptor-negative) human breast cancer cells. When VCA and DOX were combined, a strong synergistic effect was shown in cell growth inhibition, compared to VCA or DOX treatment alone. In quantitative apoptosis studies analyzed by flow cytometry, a combination of two agents showed an increase in apoptosis in both cells, compared to agents alone. Also, pro-apoptotic proteins including Bax, Bik, and Puma were increased in both cells, and the survival factor Bcl-2 was inhibited in MCF-7 cells when drugs were combined. Furthermore, VCA combined with DOX mediated S phase arrest, accompanied with a decrease of cell number at G0/G1 phase. This suggests that VCA and DOX combination may possibly lead to a novel strategy for the treatment of breast cancer.

BACKGROUND

Targeting the ubiquitin-proteasome pathway is a promising approach for anticancer strategies. Recently, we found Bik accumulation in cancer cell lines after they were treated with bortezomib. However, recent evidence indicates that proteasome inhibitors may also induce the accumulation of anti-apoptotic Bcl-2 family members. The current study was designed to analyze the levels of several anti-apoptotic members of Bcl-2 family in different human cancer cell lines after they were treated with proteasome inhibitors.

METHODS

Different human cancer cell lines were treated with proteasome inhibitors. Western blot were used to investigate the expression of Mcl-1 and activation of mitochondrial apoptotic signaling. Cell viability was investigated using SRB assay, and induction of apoptosis was measured using flow cytometry.

RESULTS

We found elevated Mcl-1 level in human colon cancer cell lines DLD1, LOVO, SW620, and HCT116; human ovarian cancer cell line SKOV3; and human lung cancer cell line H1299, but not in human breast cancer cell line MCF7 after they were treated with bortezomib. This dramatic Mcl-1 accumulation was also observed when cells were treated with other two proteasome inhibitors, MG132 and calpain inhibitor I (ALLN). Moreover, our results showed Mcl-1 accumulation was caused by stabilization of the protein against degradation. Reducing Mcl-1 accumulation by Mcl-1 siRNA reduced Mcl-1 accumulation and enhanced proteasome inhibitor-induced cell death and apoptosis, as evidenced by the increased cleavage of caspase-9, caspase-3, and poly (ADP-ribose) polymerase.

CONCLUSIONS

Our results showed that it was not only Bik but also Mcl-1 accumulation during the treatment of proteasome inhibitors, and combining proteasome inhibitors with Mcl-1 siRNA would enhance the ultimate anticancer effect suggesting this combination might be a more effective strategy for cancer therapy.

Curcuma phaeocaulis Valeton is a commonly prescribed Chinese medical herb for tumor therapy. In this study, an extract of Curcuma phaeocaulis Valeton referred as Cpv was prepared and its anti-tumor effect was evaluated with MCF-7 and MDA-MB-231 cells. Curcuma phaeocaulis Valeton power was extracted with ethanol and the main components of the extract (Cpv) were analyzed with HPLC. The effect of Cpv on MCF-7 cells proliferation, intracellular reactive oxygen species (ROS) formation, mitochondrial membrane potential (ΔΨm), apoptosis, apoptotic related proteins, MDA-MB-231 cell migration, and integrins expression were determined. Furthermore, the effect of Cpv on some key signal transduction molecules was also investigated. Furanodienone, germacrone and furanodiene were identified as the main components of Cpv. Cpv treatment significantly inhibited cell proliferation, increased LDH release, induced intracellular ROS formation, and decreased ΔΨm in a dose-dependent manner in MCF-7 cells. Cpv induced apoptosis without affecting cell migration. Cpv increased protein expression of Bax, PARP, cleaved PARP, caspase-3, 7, JNK1, p-p42/44MAPK, NF-κB, IKKα, IKKβ, decreased protein expression of Bcl-2, Bcl-xL, Bim, Bik, Bad, integrin β5, p42/44MAPK without affecting integrin α5, β1, and p38MAPK protein expression. We concluded that Cpv inhibited MCF-7 cells proliferation by inducing apoptosis mediated by increasing ROS formation, decreasing ΔΨm, regulating Bcl-2 family proteins expression, and activating caspases. Cpv treatment also modulated several signaling transduction pathways. These results might provide some molecular basis for the anti-tumor activity of Curcuma phaeocaulis Valeton.

In the p53-deficient human B lymphoma Namalwa cell line that quickly undergoes apoptosis after DNA topoisomerase I inhibitor (camptothecin, CPT) treatment, we observed rapid and slight induction of the pro-apoptotic BH3-only Bik, Bim-EL, Bim-L and Bim-S proteins. In contrast, the expression levels of Bad and multidomain Bax-alpha and Bak remained mostly unchanged after CPT treatment. However, multiple pro-apoptotic proteins, including Bax-alpha, Bak, Bik, Bim-EL and Bim-L, translocated rapidly to the mitochondria after CPT treatment. Gel filtration chromatography experiments demonstrated that somes of the pro-apoptotic proteins assemble themselves into high molecular weight protein complexes. The protein composition of these oligomers was further analyzed by co-immunoprecipitation experiments performed on highly purified mitochondrial fractions, which revealed the formation of Bax/Bak, Bax/VDAC1, Bak/VDAC1, Bim/VDAC1 and Bim/Bcl-2 complexes after DNA damage induction. Thus, it appeared that induction, mitochondrial translocation and assembly in multimeric protein complexes of several pro-apoptotic members of the Bcl-2 family correlated with the rapid activation of apoptosis in a p53-independent pathway after CPT-mediated DNA strand breaks.

Hepatocellular carcinoma (HCC), the third leading cause of cancer death in the world, is the most general type of primary liver cancer. Although current treatment modalities, such as liver transplantation, resection, percutaneous ablation, transarterial embolization, chemotherapy and radiotherapy are potentially curative, these methods are not universally applicable to all of HCC patients, especially for those with poor prognosis in which no effective remedy is available. Therefore, development of novel therapeutic approach for the treatment of HCC is urgently needed. In the current study, we developed a promising HCC-targeted gene therapy vector driven by liver cancer-specific α-fetoprotein promoter/enhancer coupled to an established platform technology. The activity of this expression vector is comparable with or even higher than that of strong cytomegalovirus (CMV) promoter and exhibits strong promoter activity in liver cancer cells/tumors, but has nearly no or very low activity in normal cells/organs in vitro and in orthotopic animal models in vivo. Its cancer specificity exceeds that of the CMV promoter, which expresses non-specifically in both normal and tumor cells. In addition, targeted expression of a therapeutic BikDD, a mutant of proapoptotic gene Bik effectively and preferentially killed liver cancer cells, but not normal cells and significantly repressed growth of HCC tumors, and prolonged survival in multiple xenograft and syngeneic orthotopic mouse models of HCC through intravenous systemic gene delivery. Importantly, systemic administration of BikDD by our expression vector exerted no systemically acute toxicity compared with CMV-BikDD in mice. Taken together, this study elucidates a relatively safe and highly effective and specific systemic gene therapy strategy for liver cancer, and is worthy of further development for future clinical trials.

OBJECTIVE

Human hepatoma cells have been reported to be resistant to Fas-mediated apoptosis. Sodium butyrate (SB) induced apoptosis of several cancer cells. We investigated the effects of SB on Fas-mediated apoptosis of hepatoma cells.

METHODS

In hepatoma cells (HuH-6, HuH-7, Hep-G2, and PLC/PRF/5), susceptibility to Fas-mediated apoptosis and Fas expression were assessed. Caspase-3 activation and cell cycle progression were evaluated in HuH-6. A cDNA microarray assay was performed to screen the changes in the expression of mRNAs.

RESULTS

Pretreatment with SB caused an enhancement of the sensitivity to anti-Fas-mediated cytotoxicity, though it did not increase the expression of Fas. The cDNA microarray assay revealed up-regulation of pro-apoptotic Bik, Bak, Bid and c-Jun N-terminal protein kinase-1, and down-regulation of anti-apoptotic Bag-1 and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitor protein. In some molecules, expression of the proteins was confirmed by Western blotting. An increase in truncated-Bid accompanying the reduction in Bid was also observed.

CONCLUSIONS

SB enhances the susceptibility of hepatoma cells to anti-Fas-mediated cytotoxicity by altering the mRNA and protein expression and/or the activation status of proteins that could be involved in the Fas signaling pathway. SB may have an important role in the elimination of hepatoma cells.

For successful treatment of malignant B-cells it is crucial to understand intrinsic survival requirements in relation to their normal progenitors. Long-lived humoral immunity as well as most B-cell malignancies, originate in the germinal center (GC). Murine GC B-cells depend on pro-survival protein MCL-1, but not BCL-XL. In contrast, naive and memory B-cells depend on BCL-2, but not BCL-XL or MCL-1. For human B-cell subsets, the functional relationships among BCL-2 members are unclear, and also if and how they shift after malignant transformation. We here dissect these aspects in human tonsil and primary leukemia (CLL) cells by single and combined treatment with novel, highly specific BH3-mimetics. We found that MCL-1 expression in GC B-cells is regulated post-translationally and its importance is highlighted by preferential binding to pro-apoptotic BIM. In contrast, BCL-XL is transcriptionally induced and binds solely to weak sensitizer BIK, potentially explaining why BCL-XL is not required for GC B-cell survival. Using novel BH3-mimetics, we found that naive and memory B-cells depend on BCL-2, GC cells predominantly on MCL-1, whereas plasma cells need both BCL-XL and MCL-1 for survival. CLL cells switch from highly sensitive for BCL-2 inhibition to resistant after CD40-stimulation. However, combined inhibition of BCL-2, plus BCL-XL or MCL-1 effectively kills these cells, thus exposing a weakness that may be therapeutically useful. These general principles offer important clues for designing treatment strategies for B-cell malignancies.

Multiple myeloma (MM) is a plasma-cell (PC) malignancy that is heterogeneous in its clinical presentation and prognosis. Monoclonal gammopathy of undetermined significance (MGUS) consistently preceded development of MM. The presence of primary IgH translocations and the universal overexpression of cyclin D genes led to a molecular classification of MM patients into different disease subtypes. Since Bcl-2 family proteins determine cell fate, we analyzed a publicly available Affymetrix gene expression of 44 MGUS and 414 newly diagnosed MM patients to investigate (1) the global change of Bcl-2 family members in MM versus MGUS (2) whether the four major subtypes defined as hyperdiploid, CyclinD1, MAF, and MMSET, display specific apoptotic machineries. We showed that among the main anti-apoptotic members (Bcl-2, Bcl-xL, and Mcl-1), Mcl-1 up-regulation discriminated MM from MGUS, in agreement with the prominent role of Mcl-1 in PC differentiation. Surprisingly, the expression of multi-domain pro-apoptotic Bak and Bax were increased during the progression of MGUS to MM. The combined profile of Bcl-2, Bcl-xL, and Mcl-1 was sufficient to distinguish MM molecular groups. While specific pro-apoptotic members expression was observed for each MM subtypes, CyclinD1 subgroup, was identified as a particular entity characterized by a low expression of BH3-only (Puma, Bik, and Bad) and multi-domain pro-apoptotic members (Bax and Bak). Our analysis supports the notion that MM heterogeneity is extended to the differential expression of the Bcl-2 family content in each MM subgroup. The influence of Bcl-2 family profile in the survival of the different patient groups will be further discussed to establish the potential consequences for therapeutic interventions. Finally, the use of distinct pro-survival members in the different steps of immune responses to antigen raises also the question of whether the different Bcl-2 anti-apoptotic profile could reflect a different origin of MM cells.

OBJECTIVE

Gain-of-function mutations of enhancer of Zeste homolog 2 (EZH2) occur frequently in diffuse large B-cell lymphomas and in follicular lymphomas. However, the frequency of EZH2 mutation in Chinese follicular lymphomas and the potential targets affected by this mutation are unknown.

METHODS

We determined EZH2 codon 641 mutations in Chinese follicular lymphomas (n = 124) and compared them with Western follicular lymphomas (n = 70) using a sensitive pyrosequencing assay. Gene expression profiling (GEP) was performed to determine differential gene expression between the mutated versus unmutated subgroups, and selected genes were validated using immunohistochemistry.

RESULTS

Our results showed similar frequencies of EZH2 codon 641 mutations in Chinese and Western follicular lymphoma cohorts (16.9% vs. 18.6%, χ(2) test, P = 0.773), including all five reported mutation variants. We observed significant association of EZH2 mutation with low morphologic grade follicular lymphomas (grade 1-2, 23.6% vs. grade 3, 7.7%, χ(2) test, P = 0.02). EZH2 mutations also showed significant association with BCL2 rearrangement in the Chinese cohort (26.8% vs. 8.8%, χ(2) test, P = 0.008) and combined cohorts (26.3% vs. 9.1%, χ(2) test, P = 0.002). GEP analysis identified several genes, including TCF4, FOXP1, TCL1A, BIK, and RASSF6P, with significantly lower mRNA expression (P < 0.01) in mutated cases, and the potential target TCL1A showed consistent results at the protein level.

CONCLUSIONS

Similar prevalence of EZH2 mutation in two ethnic groups suggests shared pathogenetic mechanisms. The much lower frequency of EZH2 mutation in cases without BCL2 translocation suggests a different pattern of evolution of this subtype of follicular lymphoma. GEP studies showed a set of differentially expressed genes and suggested that EZH2 mutation may help to lock the tumor cells at the germinal center stage of differentiation.

To ensure the success of systemic gene therapy, it is critical to enhance the tumor specificity and activity of the promoter. In the current study, we determined that topoisomerase IIalpha promoter is selectively activated in breast cancer cells. An element containing an inverted CCAAT box (ICB) was shown to be responsible for the breast cancer specificity. When the ICB-harboring topoisomerase IIalpha minimal promoter was linked with an enhancer sequence from the cytomegalovirus immediate early gene promoter (CMV promoter), this composite promoter, CT90, exhibited activity comparable to or higher than the CMV promoter in breast cancer cells in vitro and in vivo, yet expresses much lower activity in normal cell lines and normal organs than the CMV promoter. A CT90-driven construct expressing BikDD, a potent proapoptotic gene, was shown to selectively kill breast cancer cells in vitro, and to suppress mammary tumor development in an animal model of intravenously administrated, liposome-delivered gene therapy. Expression of BikDD was readily detectable in the tumors but not in the normal organs (such as heart) of CT90-BikDD-treated animals. The results indicate that liposomal CT90-BikDD is an effective systemic breast cancer-targeting gene therapy.

Nodal-signaling is required for specification of mesoderm, endoderm, establishing left-right asymmetry, and craniofacial development. Wdr68 is a WD40-repeat domain-containing protein recently shown to be required for endothelin-1 (edn1) expression and subsequent lower jaw development. Previous reports detected the Wdr68 protein in multiprotein complexes containing mammalian members of the dual-specificity tyrosine-regulated kinase (dyrk) family. Here we describe the characterization of the zebrafish dyrk1b homolog. We report the detection of a physical interaction between Dyrk1b and Wdr68. We also found perturbations of nodal signaling in dyrk1b antisense morpholino knockdown (dyrk1b-MO) animals. Specifically, we found reduced expression of lft1 and lft2 (lft1/2) during gastrulation and a near complete loss of the later asymmetric lft1/2 expression domains. Although wdr68-MO animals did not display lft1/2 expression defects during gastrulation, they displayed a near complete loss of the later asymmetric lft1/2 expression domains. While expression of ndr1 was not substantially effected during gastrulation, ndr2 expression was moderately reduced in dyrk1b-MO animals. Analysis of additional downstream components of the nodal signaling pathway in dyrk1b-MO animals revealed modestly expanded expression of the dorsal axial mesoderm marker gsc while the pan-mesodermal marker bik was largely unaffected. The endodermal markers cas and sox17 were also moderately reduced in dyrk1b-MO animals. Notably, and similar to defects previously reported for wdr68 mutant animals, we also found reduced expression of the pharyngeal pouch marker edn1 in dyrk1b-MO animals. Taken together, these data reveal a role for dyrk1b in endoderm formation and craniofacial patterning in the zebrafish.

Endocrine sensitivity, assessed by the expression of estrogen receptor (ER), has long been the predict factor to guide therapeutic decisions. Tamoxifen has been the most successful hormonal treatment in endocrine-sensitive breast cancer. However, in estrogen-insensitive cancer tamoxifen showed less effectiveness than in estrogen-sensitive cancer. It is interesting to develop new drugs against both hormone-sensitive and insensitive tumor. In this present study we examined anticancer effects of evodiamine extracted from the Chinese herb, Evodiae fructus, in estrogen-dependent and -independent human breast cancer cells, MCF-7 and MDA-MB-231 cells, respectively. Evodiamine inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a concentration-dependent manner with concentration of 1×10(-6) and 1×10(-5) M. Evodiamine also induced apoptosis via up-regulation of caspase 7 activation, PARP cleavage (Bik and Bax expression). The expression of ER α and β in protein and mRNA levels was down-regulated by evodiamine according to data from immunoblotting and RT-PCR analysis. Overall, our results indicate that evodiamine mediates degradation of ER and induces caspase-dependent pathway leading to inhibit proliferation of breast cancer cell lines. It suggests that evodiamine may in part mediate through ER-inhibitory pathway to inhibit breast cancer cell proliferation.