Mechanism Of Apoptosis Induction Research Articles

Mechanisms of activity of the TORC1 inhibitor everolimus in Waldenstrom macroglobulinemia.

The TORC1 inhibitor everolimus has previously shown significant activity as a single agent in hematologic malignancies, with reported responses of 30% to 70% in Waldenstrom macroglobulinemia. However, the specific mechanisms by which this class of mTOR inhibitors exerts anti-Waldenstrom macroglobulinemia activity have not been fully investigated. We therefore sought to dissect the mechanisms of everolimus-dependent modulation of Waldenstrom macroglobulinemia cell survival. We confirmed that everolimus targets mTOR in patients treated with everolimus and responding to therapy. We evaluated the effect of everolimus on proliferation and survival of primary Waldenstrom macroglobulinemia cells, as well as of other IgM-secreting lymphoma cell lines. Everolimus-dependent mechanisms of induced apoptosis and its effect on Waldenstrom macroglobulinemia cells in the context of bone marrow microenvironment have been also evaluated. miRNA-155 loss-of-function studies were conducted. Moreover, the combinatory effect of bortezomib and rituximab has been tested. We showed that everolimus targeted mTOR downstream signaling pathways, ex vivo, in patients responding to everolimus treatment. Everolimus induced toxicity in primary Waldenstrom macroglobulinemia cells, as well as in other IgM-secreting lymphoma cells, supported by cell-cycle arrest and caspase-dependent and -independent induction of apoptosis. Importantly, everolimus targeted Waldenstrom macroglobulinemia cells even in the context of bone marrow milieu, where it affected migration, adhesion, and angiogenesis. Everolimus-dependent anti-Waldenstrom macroglobulinemia activity was partially driven by miRNA-155. Moreover, everolimus synergized with bortezomib and rituximab in targeting Waldenstrom macroglobulinemia cells, as shown by synergistic inhibition of p65/ and p50/NF-κB activities. These findings provide a better understanding of the mechanisms that are responsible for everolimus-induced anti-Waldenstrom macroglobulinemia activity.

Nilotinib shows prolonged intracellular accumulation upon pulse-exposure: a novel mechanism for induction of apoptosis in CML cells

Nilotinib shows prolonged intracellular accumulation upon pulse-exposure: a novel mechanism for induction of apoptosis in CML cells

The effects of baicalein on canine osteosarcoma cell proliferation and death

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti-inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage-independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response.

Apoptotic effect of coix polysaccharides on A549 lung cancer cells in vitro

背景与目的薏仁是禾木科植物薏米的干燥成熟种仁，是常用的中药，又是普遍、常吃的食物。本研究旨在探讨薏仁多糖诱导A549细胞凋亡的作用。方法本研究通过水提醇沉法提取粗的薏仁多糖，再通过透析、离子交换柱层析等方法得到薏仁多糖组份（CP-1）。采用MTT测定A549细胞的存活率；扫描电镜观察细胞形态；流式细胞仪检测细胞周期；RT-PCR测定caspase-3和caspase-9两种基因相对表达量；单细胞凝胶电泳检测细胞DNA变化。结果MTT测得薏仁多糖对A549细胞存活率有明显的生长抑制作用。扫描电子显微镜观察到测试组A549细胞产生凋亡小体，流式细胞仪分析薏仁多糖处理导致A549细胞S期阻滞，并产生凋亡峰。RT-PCR结果表明与对照组比较，测试组caspase-3和caspase-9表达量上升。通过DNA彗星实验薏仁多糖可延长尾距，表明DNA断裂破碎。结论研究结果表明薏仁多糖组分可有效地诱发A549癌细胞凋亡。

Mechanism of apoptosis induction by Hedyotic diffusa on human glioma cells

Objective To investigate the possible mechanism of apoptosis induction by hedyotic diffusa (HD) on human glioma U87 cells.Methods U87 cells were cultured for 24 h in the medium containing HD with different concentrations (0,100,200 ml/L).The apoptosis of U87 cells and collapse of mitochondrial membrane potential were examined by flow cytometry.The protein expression levels of bcl-2 and bax were detected by using immunoblot.Results The apoptosis rate in 100 and 200 ml/L HD groups was (17.31 ± 1.36)％ and (41.23 ± 0.72)％ respectively,significantly higher than that in 0 ml/L HD group (P ＜0.01).The JC-1-green-bright rate of cells treated with 100 and 200 ml/L HD was (11.90 ± 0.20) ％ and (34.17 ± 2.79) ％ respectively,significantly higher than untreated U87 cells (P ＜ 0.05).The protein expression level of bax was increased and bcl-2-to-bax ratio was reduced dose-dependently.Conclusion HD induced apoptosis of U87 cells via mitochondrion pathway. Key words: Hedyotic diffusa; Glioma; Apoptosis; Mitochondrion

Nitrogen-containing bisphosphonates induce apoptosis of hematopoietic tumor cells via inhibition of Ras signaling pathways and Bim-mediated activation of the intrinsic apoptotic pathway

Nitrogen-containing bisphosphonates (N-BPs) induce apoptosis in tumor cells by inhibiting the prenylation of small G-proteins. However, the details of the apoptosis-inducing mechanism remain obscure. The present study showed that the induction of apoptosis by N-BPs in hematopoietic tumor cells is mediated by mitochondrial apoptotic signaling pathways, which are activated by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. Furthermore, N-BPs decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK) and mTOR via suppression of Ras prenylation and enhanced Bim expression. The present results indicated that N-BPs induce apoptosis by decreasing the mitochondrial transmembrane potential, increasing the activation of caspase-9 and caspase-3, and enhancing Bim expression through inhibition of the Ras/MEK/ERK and Ras/mTOR pathways. The accumulation of N-BPs in bones suggests that they may act more effectively on tumors that have spread to bones or on Ras-variable tumors. This is the first study to show that the specific molecular pathways of N-BP-induced apoptosis.

Mechanisms of apoptosis induction by simultaneous inhibition of PI3K and FLT3-ITD in AML cells in the hypoxic bone marrow microenvironment

We investigated the antileukemia effects and molecular mechanisms of apoptosis induction by simultaneous blockade of PI3K and mutant FLT3 in AML cells grown under hypoxia in co-cultures with bone marrow stromal cells. Combined treatment with selective class I PI3K inhibitor GDC-0941 and sorafenib reversed the protective effects of bone marrow stromal cells on FLT3-mutant AML cells in hypoxia, which was associated with downregulation of Pim-1 and Mcl-1 expression levels. These findings suggest that combined inhibition of PI3K and FLT3-ITD may constitute a targeted approach to eradicating chemoresistant AML cells sequestered in hypoxic bone marrow niches.

Withaferin A-induced apoptosis in human breast cancer cells is associated with suppression of inhibitor of apoptosis family protein expression

The present study provides novel insight into the mechanism of apoptosis induction by withaferin A (WA), which is a bioactive constituent of an Ayurvedic medicine plant (Withania somnifera). Exposure of MDA-MB-231 and MCF-7 human breast cancer cells to WA resulted in suppression of XIAP, cIAP-2, and Survivin protein levels. The WA-induced apoptosis was significantly attenuated by ectopic expression of XIAP, Survivin, and cIAP-2 in both cells. However, the WA-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with suppression of Survivin protein level only. These results indicate important contribution of Survivin suppression in WA-induced apoptosis.

P066 Signaling molecules critical for apoptosis induction by TNF and TRAIL

Apoptosis is essential for the maintenance of the immune system, and elimination of cells infected with pathogens and tumor cells. Death receptors (DRs) including TNFR1, Fas, DR3, TRAILRs and DR6 are responsible to transduce apoptosis signals. To elucidate the signal pathways mediated by TNF-related cytokines is important for the treatment of cancers and immune diseases. DAP3 is critical for DR-mediated signals, although the molecular mechanisms for induction of apoptosis by DAP3 are poorly understood. We found a molecule, designated as death ligand signal enhancer (DELE), involved in TNF- α or TRAIL-mediated signals. n addition, we identified CLIPR-59 as a novel adaptor protein for TNFR1. Inhibition of CLIPR-59 expression levels prevented apoptosis induction and caspase activation in TNF- α signaling. We examined the function of CLIPR-59 for the regulation of its binding proteins in TNF- α -induced apoptosis signals. We examined the function of DELE on DR-mediated apoptosis using established cell lines, expressing DELE in TNF- α or TRAIL-mediated signals. Next, we examined the effect of knockdown of DELE expression on these signals in HeLa cells. Cell viability and caspase activation were analyzed in these cells. On the other hand, we examined the role of CLIPR-59 in apoptosis induced by TNF- α . Effects of RNAi-mediated knockdown of CLIPR-59 on apoptosis stimulated by TNF- α were assessed by a cell viability assay.In addition, by Caspase-Glo assay using a luminogenic Caspase-3/7 substrate, we evaluated the importance for CLIPR-59 in caspase activation. Furthermore, we identified the molecules RIP1 and CYLD which bind to CLIPR-59 and analyzed the function of these molecules in TNF- α -mediated signals. Expression of DELE enhanced apoptosis induction by the stimulation of TNF- α and TRAIL. In addition, knockdown of DELE expression rescued the HeLa cells from DR-mediated apoptosis induction. Moreover, activation of caspase-3, caspase-8 and caspase-9 induced by stimulation of TNF- α , anti-Fas or TRAIL was significantly inhibited by the knockdown of DELE expression. CLIPR-59 regulated TNF- α -induced ubiquitination of RIP1 by its association with CYLD, a de-ubiquitinating enzyme. This ubiquitination of RIP1 resulted in the formation of Complex-II and subsequently promoting Caspase-8 activation to induce apoptosis by TNF- α . We identified a novel DAP3-binding protein, DELE to regulate the apoptosis induction by the stimulation of TNF- α and TRAIL. In addition, activation of caspase-3, caspase-8 and caspase-9 induced by stimulation of TNF- α , anti-Fas or TRAIL was induced by DELE. In addition, we identified CLIPR-59, which associated with TNFR1 and regulated TNF- α -induced ubiquitination of RIP1 by its association with CYLD, a de-ubiquitinating enzyme. CLIPR-59-mediated ubiquitination of RIP1 resulted in the activation of Caspase-8 to induce apoptosis by TNF- α .

CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

CD137 (4-1BB, TNFRSF9), a member of the tumor necrosis factor (TNF) receptor family, is a potent T cell co-stimulatory molecule. CD137 ligand (CD137L) is expressed by antigen presenting cells (APC) as a transmembrane protein and transmits activating signals into APC. In this study we investigated the effects of CD137L signaling in microglia, the resident APC in the central nervous system. In vitro, the murine microglia cell lines BV-2 and N9, as well as primary murine microglia responded with activation as evidenced by adherence and secretion of proinflammatory cytokines, MMP-9, and soluble intercellular adhesion molecule (ICAM). CD137L signaling is also important for microglia activation in vivo, since CD137L-deficient mice exhibited profoundly less microglia activation during experimental autoimmune encephalomyelitis (EAE) which is a well-established murine model for neuroinflammation and human multiple sclerosis (MS). Also CD137 is expressed in the CNS of mice during EAE. Activated microglia has been reported to mediate the destruction of axonal myelin sheaths and cause the death of oligodendrocytes, the main pathogenic mechanisms in EAE and MS. Corresponding to the lower microglia activation there were also fewer apoptotic oligodendrocytes in the CNS of CD137L-deficient mice. In vitro co-culture confirmed that CD137L-activated microglia induces apoptosis in oligodendrocytes, and identified reactive oxygen species as the mechanism of apoptosis induction. These data demonstrate activating effects of CD137L signaling to microglia, and show for the first time that the CD137 receptor/ligand system may be a mediator of neuroinflammatory and neurodegenerative disease, by activating microglia which in turn kill oligodendrocytes.

Apoptosis signaling in influenza virus propagation, innate host defense, and lung injury

Programmed cell death is a crucial cellular response frequently observed in IV-infected tissue. This article reviews the current knowledge on the molecular virus-host interactions that induce apoptosis pathways in an IV-infected cell and the functional implications of these cellular signaling events on viral propagation at distinct steps during the viral replication cycle. Furthermore, it summarizes the role of IV-induced apoptosis pathways in equilibrating the host's antiviral immune response between effective viral clearance and development of severe apoptotic lung injury.

Fludarabine nucleoside induces accumulations of p53, p63 and p73 in the nuclei of human B-lymphoid cell lines, with cytosolic and mitochondrial increases in p53

Human Raji cells treated with fludarabine nucleoside (2-FaraA, 3 μM) undergo apoptosis with accumulation of p53 in the nuclei as multiple phosphorylated isoforms and C-terminal truncated derivatives. Changes induced by 2-FaraA in the levels of p53, p63 and p73 in the nuclear, cytosolic and mitochondrial fractions have been determined in four human B-lymphoid cell lines that are TP53-functional (Raji and IM9) and TP53-mutated (MEC1 and U266). The B-lymphoid cell lines were treated with 2-FaraA (3 μM, 24 h, 48 h) and viability determined. Protein extracts of subcellular fractions from 2-FaraA-treated cells were analysed by 1D and 2D electrophoresis; multiple phosphorylated isoforms and truncated derivatives were identified by Western blots for p53, p63 and p73. p53 and p63 were present in all three fractions, while p73 was only detected in nuclei. After treatment with 2-FaraA, nuclear p53, p63 and p73 accumulated as multiple phosphorylated isoforms and truncated derivatives. The association of p63 with mitochondria in human cells is novel. Comprehensive information on the subcellular distributions and responses of p53, p63 and p73 to 2-FaraA provides additional insight into mechanisms for induction of apoptosis in the treatment of B-lymphoproliferative disorders with fludarabine.

Astrocytes Secrete Exosomes Enriched with Proapoptotic Ceramide and Prostate Apoptosis Response 4 (PAR-4): POTENTIAL MECHANISM OF APOPTOSIS INDUCTION IN ALZHEIMER DISEASE (AD)

Amyloid protein is well known to induce neuronal cell death, whereas only little is known about its effect on astrocytes. We found that amyloid peptides activated caspase 3 and induced apoptosis in primary cultured astrocytes, which was prevented by caspase 3 inhibition. Apoptosis was also prevented by shRNA-mediated down-regulation of PAR-4, a protein sensitizing cells to the sphingolipid ceramide. Consistent with a potentially proapoptotic effect of PAR-4 and ceramide, astrocytes surrounding amyloid plaques in brain sections of the 5xFAD mouse (and Alzheimer disease patient brain) showed caspase 3 activation and were apoptotic when co-expressing PAR-4 and ceramide. Apoptosis was not observed in astrocytes with deficient neutral sphingomyelinase 2 (nSMase2), indicating that ceramide generated by nSMase2 is critical for amyloid-induced apoptosis. Antibodies against PAR-4 and ceramide prevented amyloid-induced apoptosis in vitro and in vivo, suggesting that apoptosis was mediated by exogenous PAR-4 and ceramide, potentially associated with secreted lipid vesicles. This was confirmed by the analysis of lipid vesicles from conditioned medium showing that amyloid peptide induced the secretion of PAR-4 and C18 ceramide-enriched exosomes. Exosomes were not secreted by nSMase2-deficient astrocytes, indicating that ceramide generated by nSMase2 is critical for exosome secretion. Consistent with the ceramide composition in amyloid-induced exosomes, exogenously added C18 ceramide restored PAR-4-containing exosome secretion in nSMase2-deficient astrocytes. Moreover, isolated PAR-4/ceramide-enriched exosomes were taken up by astrocytes and induced apoptosis in the absence of amyloid peptide. Taken together, we report a novel mechanism of apoptosis induction by PAR-4/ceramide-enriched exosomes, which may critically contribute to Alzheimer disease.

Abstract 3995: C-terminal fragment of prostate apoptosis response-4 (Par-4) generated by caspase-3 cleavage induces apoptosis in cancer cells

Abstract Prostate apoptosis response-4 (Par-4) is a ubiquitously expressed pro-apoptotic tumor suppressor protein. Previously, we have shown that Par-4 undergoes caspase-3 mediated cleavage in normal and cancer cells. In the present study, we demonstrated that caspase-3 generated cleaved fragment of Par-4 displays apoptotic activity. Hela cells were transiently transfected with empty or Myc tagged C-terminal fragment of Par-4. The results from immunofluorescence studies revealed that the cleaved fragment of Par-4 is predominantly localized in the nuclear compartment of the cells. Further, cells expressing C-terminal Par-4 fragment underwent significant apoptosis as compared to empty vector transfected cells as measured by flow cytometry using annexin V/PI staining. Next, we tested whether C-terminal fragment of Par-4 follows similar mechanism of apoptosis induction as the full length Par-4 by the inhibition of protein kinase C (PKC)/NF-κB pathway. Western blot analysis revealed that the overexpression of C-terminal Par-4 fragment reduced the protein levels of Bcl-2 and FLIP as compared to empty vector transfected cells. Overexpression of the C-terminal Par-4 fragment also reduced the levels of phosphorylated IαBβ, however, there was no change in the levels of total IαBβ. Interestingly, C-terminal fragment of Par-4 prevented the nuclear mobilization of NF-κB (p65) compared to empty vector transfected cells. On the other hand, NF-κB (p50) protein levels were found to be decreased in cells expressing the C-terminal Par-4 fragment suggesting that C-terminal fragment of Par-4 possesses pro-apoptotic activity and regulates apoptotic induction by inhibiting the nuclear localization and activity of NF-κB in a manner similar to wild type full length Par-4. To conclude, our data suggest that caspase-3 mediated cleavage of Par-4 results in nuclear localization of C-terminal Par-4 fragment which possesses pro-apoptotic activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3995. doi:1538-7445.AM2012-3995

Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone, a radiolytic product of palmitic acid, in human lymphoma U937 cells

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-L: -cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca(2+)](i) involved in apoptosis are induced by 2-DCB and PA in U937 cells.

Cytotoxic oleane-type triterpene saponins from Glochidion eriocarpum

The anticancer activity of ten compounds from the aerial parts of Glochidion eriocarpum were evaluated on two human cancer cell lines, HL-60 and HCT-116. Compounds 1-4 displayed highly potent cytotoxic activity on the HCT-116 cancer cell line with IC(50) values ranging of 0.41∼1.16 μM. Compounds 1-4 significantly inhibited the HL-60 cell line with IC(50) values ranging of 4.51∼6.33 μM. These results suggested that the benzoyl group at the C-22 position in oleane-type triterpene saponins was essential for cytotoxicity towards tumor cells. Moreover, compounds 2 and 3 showed more potent cytotoxicity than compounds 1 and 4 against HL-60 and HCT-116 cells. With respect to the mechanism underlying cytotoxicity, compounds 1-4 increased chromatin condensation, a typical apoptotic characteristic in HL-60 and HCT-116 cells. In the mechanism of apoptosis induction, compounds 1-4 reduced Bcl-2 expression, whereas the expression of Bax was increased compared to controls in HCT-116 cells. In addition, compounds 1-4 decreased the level of procaspase-3. The cleavage of poly (ADP-ribose) polymerase (PARP), a vital substrate of effector caspase, was observed in HCT-116 cells. Furthermore, the induction of apoptosis was also accompanied by an activation of extracellular signal-regulated kinase (ERK) and p38 kinase in HCT-116 cells. These findings provide evidence demonstrating that the pro-apoptotic effects of compounds 1-4 are mediated through the activation of ERK and p38 in HCT-116 cells.

Пошук та ідентифікація молекулярних мішеней дії ангуциклінового антибіотика ландоміцину Е у злоякісних клітинах людини

Study of cellular and molecular mechanisms of anticancer activity of novel perspective angucycline antibiotic landomycin E was carried out. It was revealed that this compound uses mechanisms of apoptosis induction in tumor cells that differ from such mechanisms used by structurally related drug doxorubicin which is widely applied in clinics. In particular, landomycin E blocks cells in G1 phase of cell cycle, and this action is mediated by the reactive oxygen species. Such G1-specificity of landomycin E action of can be of great importance in chemotherapy, since it is known that cancer stem cells which are responsible for recurrent tumors, are staying mainly in G0/G1 phase, and, thus, are insensitive to most anticancer drugs. Application of Western-blot analysis for studying potential intracellular targets of the landomycin E action allowed identifying procaspase-7 as a targeted effector apoptotic protein. There are no other cases of direct activation of this enzyme by the anticancer drugs described in literature till now. Thus, such a unique mechanism of anticancer activity might allow landomycin E to kill tumor cells effectively even in the absence of other key enzymes involved in apoptosis, such as caspase-3. Taking into consideration high antineoplastic potential of landomycin E and unique mechanism of its action, this antibiotic could be recommended as anticancer drug of second line defense at treatment of cancer patients. Keywords: anticancer drugs, tumor drug resistance, landomycin E, apoptosis, cell cycle.

Anti-Cancer Activities of Quercetin: Mechanisms of Apoptosis Induction

This review centers on the anti-cancer activities of the well-known flavonoid-quercetin. In this review, we firstly discuss the pharmacokinetic characters of quercetin. Secondly, we focus on its anti-cancer properties. Then, we give attention to the different mechanisms involved in the apoptosis induction activities of quercetin.

Anticancer effect and apoptosis induction by quercetin in the human lung cancer cell line A-549

The aim of the present study was to investigate the anticancer effect of quercetin (QC) in the human lung cancer cell line A-549 and further study the mechanism of apoptosis induction by QC. Low differentiation potential A-549 human lung cancer cells were treated with QC at different doses and for different times, and the growth inhibitory rates were detected by MTT assay. Apoptosis induced by QC in A-549 cells was observed by Annexin V/PI double staining and flow cytometric assay. The relative tumor growth ratio of the treated/control tumors (T/C) (%) was chosen to represent the tumor growth inhibition of A-549 cell nude mouse xenografts by QC. Apoptosis of the nude mouse xenografts was observed by Annexin V/PI double staining and flow cytometric assay and DNA fragmentation assay. To further determine the molecular mechanism of apoptosis induced by QC, changes in the expression of bcl-2 and bax genes were detected by RT-PCR. Following incubation with QC, the cell growth of the low differentiation potential A-549 human lung cancer cells was dramatically inhibited in a dose-dependent manner. After the cells were exposed to QC for 24, 48 and 72 h, the IC50 value was 1.02 ± 0.05, 1.41 ± 0.20 and 1.14 ± 0.19 µmol/l, respectively. Apoptosis in the A-549 cells induced by QC was noted. The apoptotic subpopulation of A-549 cells was approximately 12.96 and 24.58%, respectively, when cells were incubated with 1.2 µmol/l QC for 48 and 72 h. T/C (%) of A-549 nude mouse xenografts was 44.3, when the nude mice were treated with QC (8 mg/kg). Meanwhile, apoptosis induced by QC was observed in the A-549 nude mouse xenografts. Increased expression of the bax gene and decreased expression of the bc1-2 gene were noted using RT-PCR. Our results provide further evidence of the growth inhibition of the A-549 human lung adenocarcinoma cancer cell line by QC. This effect is associated with the induction of apoptosis in A-549 cells and the molecular mechanism may be related to the reduction in expression of the apoptosis-regulating gene bcl-2, and increase in expression of the apoptosis-regulating gene bax. These results were also confirmed in vivo.

Anti-Myeloma Activity of LDC-000067, a Novel Selective Inhibitor of Cyclin Dependent Kinase 9: A Targeted Approach

Abstract 5110 Background:Cyclin-dependent kinase (CDK) inhibitors are reported to induce apoptosis in myeloma cells (Gojo et al., 2002; MacCallum et al., 2005). Here we investigated pro-apoptotic and anti-proliferative effects of LDC-000067, a novel selective CDK9-inhibitor, in three multiple myeloma (MM) cell lines (L-363; RPMI-8226; U-266). In addition, we studied consequences of selective CDK 9-inhibition regarding transcriptional activity (RNA-polymerase II phosphorylation) and related anti-apoptotic pathways (Mcl-1 and NFkB). Methods:L-363, RPMI-8226 and U-266 cells were exposed to either DMSO (vehicle control) or serial dilutions of LDC-000067 [0.5–20μM]. Apoptosis was quantified by flow cytometry analyses (FITC Annexin V / propidium iodide staining) after 24 hours in corresponding samples. To further investigate mechanisms of induced apoptosis by LDC-000067 we examined corresponding protein extracts by immunoblot analyses. Results:We found that LDC-000067 showed anti-myeloma activity in all myeloma cell lines tested. The proportion of induced apoptosis by exposure to LDC-000067 was equipotent (RPMI-8226) or higher (L-363 and U-266) compared to Seliciclib, a selective CDK 2-, 7- and 9-inhibitor. The IC50 for LDC-000067 was 4.0 μmol/L for L-363, 6.9 μmol/L for U-266 and 11.5 μmol/L for RPMI-8226. LDC-000067 inhibited phosphorylation of RNA polymerase II at Ser2 of the carboxyl-terminal domain in all MM lines tested. In addition, exposure to LDC-000067 resulted in a rapid decline of the protein levels of Mcl-1, known as an anti-apoptotic protein in MM. Furthermore, it is reported that in 40% of MM cell lines and 17% of MM patients the NFkB pathway is constitutionally activated by mutations (Demchenko et al., 2010) and inhibition of the NFkB activity causes induction of apoptosis (Hongyu et al., 2001). In our study we showed that LDC-000067 potently inhibited the NFkB pathway. Immunoblot analyses revealed a decreased phosphorylation and degradation of the NFkB inhibitor IkBa and an inhibition of phosphorylation of the NFkB subunit RelA (p65). Moreover, our data demonstrated that exposure to LDC-000067 caused a significant reduction of the protein levels of cyclin D1 and cyclin D2 strongly correlated to cell cycle arrest. Conclusion:In conclusion, our data corroborate that LDC-000067, a novel selective CDK 9-inhibitor, shows anti-myeloma activity by targeting three key pathways in the molecular pathogenesis of MM: firstly, it induces consecutive decline of Mcl-1 protein and inhibition of RNA polymerase II phosphorylation; secondly, it inhibits constitutively activated NFkB pathway; and thirdly, it reduces levels of D-type cyclins. Disclosures:No relevant conflicts of interest to declare.