Mdm2 Inhibitor Nutlin-3 Enhances the Cytotoxic Synergism Induced by the Combination of MEK1 Inhibitor and Arsenic Trioxide (ATO) in AML Cells.

Abstract We recently reported that PD184352 (PD) (Pfizer), a highly selective inhibitor of MEK1 phosphorylation and activation, sensitizes primary acute myelogenous leukemia (AML) to ATO-induced apoptosis via p73, a p53 paralogue, and Bad pro-apoptotic pathways activation (Blood107: 4549–4554, 2006). We also demonstrated that high doses of ATO (2μM) induced p53 accumulation (more than two-fold increase compared with control) in 11 out of 21 patients (52%), indicating a possible contribution of p53 pathway in apoptosis induction of dual treated blasts. TP53 mutations are quite rare in AML (5–10%) and MDM2 (murine double minute 2), its principal negative regulator, has been found to be frequently overexpressed in AML, a process that can actively enhance tumorigenic potential and resistance to apoptosis. The aim of this study was to investigate whether Nutlin-3, a potent and selective small-molecule MDM2 antagonist, can potentiate the apoptotic effect of the PD plus ATO combination in AML cells that retain a functional p53. Apoptosis was evaluated, after 24 and 48 hours of treatment, by measurement of sub-G1 DNA content, annexin V binding and mitochondrial transmembrane potential assays. We first analyzed the pharmacologic interactions between Nutlin-3, PD and ATO (0, 0.25, 0.5, 1, 2, 5, or 10 μM) using a fixed-ratio (1:1:1) experimental design in OCI-AML-3 cell line that has wild-type p53. We found that the three-drugs combination showed cytotoxic synergism stronger than PD plus ATO combination indicating that the inhibition of the p53-MDM2 interaction can positively influence the pro-apoptotic efficacy of dual-treated (PD plus ATO) cells: the averaged Combination Index (CI) values calculated from the ED50 (50% effective dose), ED75 and ED90, in Nutlin-3 plus PD plus ATO versus PD plus ATO treated cells were 0.36± 0.03 and 0.66± 0.02 respectively (CI values equal to 0.85 or less indicate synergism). In order to investigate the molecular effectors involved in Nutlin-3-PD-ATO or PD-ATO-induced apoptosis we first studied the kinetics (2h, 12h, 24h and 48h) of p53, p73 and phospho-Bad at Ser112 in OCI-AML-3. In the absence of Nutlin-3 ATO, even at high doses, did not promote a p53 accumulation whereas modulated the expression of the p73 gene by inducing the pro-apoptotic and anti-proliferative TAp73 and the anti-apoptotic and pro-proliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD reduced the level of ΔNp73 and blunted the ATO-mediated up-regulation of ΔNp73 thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. In the presence of Nutlin-3, p53 accumulated and the triple combination of Nutlin-3, PD and ATO enhanced the loss of mitochondrial membrane potential occurred in PD plus ATO treatment. Furthermore, pretreatment with MEK1 inhibitor strongly increased the expression of dephosphorylated Bad and blunted the ATO-mediated phosphorylation of Bad at Ser112, thereby activating its pro-apoptotic functions. These findings suggest that the pro-apoptotic p73 and Bad pathways, both involved in PD plus ATO efficacy, can be potentiated by the rescue of p53 pathway in AML cells that possess a functional p53 pathway.

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EXPERIMENTAL MODELING OF APOPTOSIS UNDER CONDITIONS OF STABLE STRONTIUM EXPOSURE

Medical Immunology (Russia) ◽

10.15789/1563-0625-2019-1-137-140 ◽

2019 ◽

Vol 21 (1) ◽

pp. 137-140

Author(s):

O. V. Dolgikh ◽

N. V. Zaitseva ◽

D. G. Dianova ◽

A. V. Krivtsov ◽

K. D. Starkova ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Programmed Cell Death ◽

Annexin V ◽

Fold Increase ◽

Apoptosis Markers ◽

Stable Strontium ◽

Cell Death Mechanisms ◽

Apoptotic Pathways ◽

And Control

Apoptosis is defined as a highly regulated form of programmed cell death with typical morphological and biochemical features. A variety of factors, including heavy metals, may influence the intensity of programmed cell death. The aim of the work was to simulate apoptosis in an in vitrosystem under the conditions of stable strontium exposure. The children’s population consuming drinking water with high strontium (Sr2+) content (n = 49) was observed. The level of lymphocyte apoptosis was determined with flow cytometry technique, by means of labeled annexin V-FITC conjugate (AnnV-FITC) and propidium iodide (PI) staining. AnnV-FITC+PI- cells were regarded as early apoptotic forms, whereas late apoptotic and/or necrotic cells were AnnV-FITC+PI+. The isolated leukocytes were incubated with Sr2+ at a concentration of 7.0 mg/l, the maximal permitted concentration (MPC) for water of aqueous objects, for 4 hours at 37 ºC. Expression of CD95 and p53 apoptosis markers was performed by flow cytometry using labeled monoclonal antibodies.In vitroexposure to strontium was associated with significantly decreased expression of apoptosisregulating factors, i.e., membrane marker CD95 and intracellular transcription protein p53, 1.56- and 1.68-fold, respectively. Meanwhile, we revealed a significantly (4.68-fold) decreased amounts of AnnV-FITC+PI--cells, as well as a statistically significant (1.35-fold) increase of the AnnV-FITC+PI+-cells. Moreover, the amounts of AnnV-FITC+ PI--lymphocytes in all samples were below the physiological ranges and control values. The number of samples with higher contents of AnnV-FITC+PI+-lymphocyte exceeding the established standards and control values, was 30.8%. Thus, it has been experimentally proven that strontium, at a concentration corresponding to MPC for water objects may significantly inhibit cell death along apoptotic pathways, with switching to necrotic cell death mechanisms, according to phosphatidylserine contents, as detected by annexin V binding test. The data have revealed an ability of strontium to have a significant effect upon the parameters of regulation and maintenance of cellular homeostasis, by influencing the apoptosis intensity, due to shifting a balance towards necrosis and reducing expression of apoptosis-regulating factors. The results of this study may be used in order to identify some marker indexes of immune disorders potentially induced by external influence of strontium upon human health under specific environmental factors.

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L-securinine inhibits cell growth and metastasis of human androgen-independent prostate cancer DU145 cells via regulating mitochondrial and AGTR1/MEK/ERK/STAT3/PAX2 apoptotic pathways

Bioscience Reports ◽

10.1042/bsr20190469 ◽

2019 ◽

Vol 39 (5) ◽

Cited By ~ 5

Author(s):

Dongxu Zhang ◽

Houxian Liu ◽

Binbin Yang ◽

Jiasheng Hu ◽

Yue Cheng

Keyword(s):

Prostate Cancer ◽

Protein Expression ◽

Annexin V ◽

Inhibitory Effect ◽

Apoptotic Pathway ◽

Growth Inhibitory ◽

Du145 Cells ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Androgen Independent

Abstract The present study aims to evaluate the anticancer effect of L-securinine on androgen-independent prostate cancer (AIPC) DU145 cells. L-securinine (2.5, 5, and 10 μM) treatment for 24, 48 and 72 h displayed strong growth inhibitory effect on DU145 cells in a concentration and time-dependent fashion but has less toxicity toward normal androgen-dependent LNCaP cells. Hoechst 332582 staining of DU145 cells and Annexin V-FITC/ PI dual-labeling followed by flow cytometry assay identified that this growth inhibition by L-securinine would be due to the induction of apoptosis. Moreover Transwell assay revealed that L-securinine significantly inhibited the cell migration/invasion ability of DU145 cells. Furthermore, results of western blotting showed that the involvement of mitochondrial apoptotic pathway in the L-securinine-induced apoptosis of DU145 cell, as evidenced by an increase in the protein expression of Bax, cleaved caspase-9, cleaved caspase-3, cytosolic cytochrome c, and cleaved PARP, together with a unchanged cleaved caspase-8 and decreased Bcl-2 protein expression. Also, L-securinine-induced antimetastatic activity in DU145 cells was associated with decreased protein expression of MMP-2 and MMP-9 and concurrent reduction of VEGF. In addition, further studies revealed that L-securinine may inhibit the protein expression of AGTR1, p-MEK1/2, p-ERK1/2, p-STAT3, PAX2, and p-PAX2, while the expression of ERK1/2, MEK1/2, and STAT3 protein retains intact. These findings suggest that L-securinine may be a promising chemopreventive agent against AIPC.

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Effet radiosensibilisateur de l'acide linoléique conjugué chez les cellules cancéreuses du sein MCF-7 et MDA-MB-231

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y04-003 ◽

2004 ◽

Vol 82 (2) ◽

pp. 94-102 ◽

Cited By ~ 1

Author(s):

Geneviève Drouin ◽

Annie Douillette ◽

Pierre Lacasse ◽

Benoit Paquette

Keyword(s):

Breast Cancer ◽

Linoleic Acid ◽

Cancer Cells ◽

Conjugated Linoleic Acid ◽

Breast Cancer Cells ◽

Fold Increase ◽

Breast Cancer Cell Lines ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Mcf 7

Apoptotic pathways in breast cancer cells are frequently altered, reducing the efficiency of radiotherapy. Conjugated linoleic acid (CLA), known to trigger apoptosis, was tested as radiosensitizer in breast cancer cells MCF-7 and MDA-MB-231. The CLA-mix, made up of the isomers CLA-9cis 11trans and CLA-10trans 12cis, was compared to three purified isomers, i.e., the CLA-9cis 11cis, CLA-9cis 11trans, and CLA-10trans 12cis. Using the apoptotic marker YO-PRO®-1, the CLA-9cis 11cis at 50 µmol/L turned out to be the best apoptotic inducer leading to a 10-fold increase in MCF-7 cells and a 2,5-fold increase in MDA-MB-231 cells, comparatively to the CLA-mix. Contrary to previous studies on colorectal and prostate cancer cells, CLA-10trans 12cis does not lead to an apoptotic response on breast cancer cell lines MCF-7 and MDA-MB-231. Our results also suggest that the main components of the CLA-mix (CLA-9cis 11trans and CLA-10trans 12cis) are not involved in the induction of apoptosis in the breast cancer cells studied. A dose of 5 Gy did not induce apoptosis in MCF-7 and MDA-MB-231 cells. The addition of CLA-9cis 11cis or CLA-mix has allowed us to observe a radiation-induced apoptosis, with the CLA-9cis 11cis being about 8-fold better than the CLA-mix. CLA-9cis 11cis turned out to be the best radiosensitizer, although the isomers CLA-9cis 11trans and CLA-10trans 12cis have also reduced the cell survival following irradiation, but using a mechanism not related to apoptosis. In conclusion, the radiosensitizing property of CLA-9cis 11cis supports its potential as an agent to improve radiotherapy against breast carcinoma.Key words: breast cancer, conjugated linoleic acid (CLA), radiotherapy, apoptosis.

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Targeting xIAP Is More Important Than Bcl-Xl in Lymphomas with Constitutive Activation of NF-kB.

Blood ◽

10.1182/blood.v108.11.2390.2390 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2390-2390

Author(s):

Yanjuan He ◽

Joan Cain ◽

Lee Ratner ◽

Leon Bernal-Mizrachi

Keyword(s):

Cell Lines ◽

Annexin V ◽

Fold Increase ◽

Daudi Cell ◽

Xiap Expression ◽

Apoptotic Effect ◽

Effective Doses ◽

Induction Of Apoptosis ◽

Apoptotic Pathways

Abstract Pathways resulting in resistance to apoptosis are essential to the process of lymphomagenesis. One such pathway, the nuclear factor-kB (NFkB), has been shown to be a key element in coordinating the anti-apoptotic effect of these malignancies. However the mechanisms used by which NFkB prevents apoptosis are not well understood. It has been suggested that NFkB inhibits activation of the intrinsic, extrinsic and common apoptotic pathways. Previous work in our lab using two different virally mediated lymphoma models (Tax/HTLV1 and LMP1/EBV driven tumors) has identified two candidates that could explain these results: X chromosome-linked inhibitor of apoptosis (xIAP) and BCL-xL. Although the current literature extensively demonstrates the role of BCL-xL in lymphomas, little is known about the importance of xIAP in these malignancies. To answer this question we tested the apoptotic effect of etoposide or tumor necrosis factor (TNF) after knocking down bcl-xL and xIAP expression in our lymphoma models (SC and Daudi cell lines) using a lentivirus expressing siRNAs. After 24 hours of treatment with etoposide and TNF, we measured apoptosis by flow cytometry using double staining with Annexin V-Alexa Fluorescense and propidium iodide. Interestingly, xIAP siRNA-expressing cell lines demonstrated 2–4 fold increase in the induction of apoptosis after treatment with etoposide as compared to a nearly 2 fold increase in those expressing Bcl-xL siRNA (see Table below). No synergism was seen after treatment with TNF. Based on this finding, we then tested a novel small molecule, homolog smac, (SHC, kindly provided by Dr. PG Harren) to determine the possible therapeutic effect of xIAP inhibitors. After titration, the two most effective doses were selected (25 μM and 50 μM) to treat Daudi cell lines for 24hrs, with either etoposide or TNF. At doses of 25 μM , we observed a 2 fold increase in the induction of apoptosis produced by etoposide compared to that seen in control (DMSO + etoposide) or SHC alone and no synergism with TNF confirming the siRNA data. More importantly, at doses of 50 μM, SHC alone demonstrated activity with a 5 fold increase in apoptosis and a nearly 10 fold increase as compared to control (DMSO) when etoposide was added. Overall, we have demonstrated that xIAP and bcl-xL are important in mediating NFkB-resistance to apoptosis. However, our findings suggested that xIAP is a more potent anti-apoptotic signal and opens the door for further drug development aimed at testing xIAP-inhibitors in lymphomas. Induction of Apoptosis in xIAP or Bcl-xL siRNA expressing cell lines siRNA/Compound Etoposide TNF Untreated xIAP 43.1 ± 17.6 17.04 ± 1.4 14.3 ± 2 SC Bcl-xL 18.39± 3.7 9.4 ± 0.22 12.5 ± 2.7 Luc/DMSO 14.9 ± 1.8 14.4 ± 5.6 14.03 ± 1.25 xIAP 9.2 ± 3.2 4.7 ± 0.48 4.6 ± 0.44 Bcl-xL 8.9 ± 0.5 5.3 ± 1.7 4.16 ± 0.4 Daudi Luc/DMSO 5.49 ± 1.71 4.28 ± 0.5 6.2 ± 0.9 SHC 25 μM 20.07 ± 4.8 12.8 ± 3.9 12.1 ± 3.2 SHC 50 μM 47.7 ± 14.55 38.3 ± 0.99 32.7 ± 8.99

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Marine-Inspired Bis-indoles Possessing Antiproliferative Activity against Breast Cancer; Design, Synthesis, and Biological Evaluation

Marine Drugs ◽

10.3390/md18040190 ◽

2020 ◽

Vol 18 (4) ◽

pp. 190 ◽

Cited By ~ 3

Author(s):

Wagdy M. Eldehna ◽

Ghada S. Hassan ◽

Sara T. Al-Rashood ◽

Hamad M. Alkahtani ◽

Abdulrahman A. Almehizia ◽

...

Keyword(s):

Breast Cancer ◽

Annexin V ◽

Fold Increase ◽

Biological Evaluation ◽

Design Synthesis ◽

Lead Compounds ◽

M Phase ◽

Indole Series ◽

Induced Apoptosis ◽

Mcf 7

Diverse indoles and bis-indoles extracted from marine sources have been identified as promising anticancer leads. Herein, we designed and synthesized novel bis-indole series 7a–f and 9a–h as Topsentin and Nortopsentin analogs. Our design is based on replacing the heterocyclic spacer in the natural leads by a more flexible hydrazide linker while sparing the two peripheral indole rings. All the synthesized bis-indoles were examined for their antiproliferative action against human breast cancer (MCF-7 and MDA-MB-231) cell lines. The most potent congeners 7e and 9a against MCF-7 cells (IC50 = 0.44 ± 0.01 and 1.28 ± 0.04 μM, respectively) induced apoptosis in MCF-7 cells (23.7-, and 16.8-fold increase in the total apoptosis percentage) as evident by the externalization of plasma membrane phosphatidylserine detected by Annexin V-FITC/PI assay. This evidence was supported by the Bax/Bcl-2 ratio augmentation (18.65- and 11.1-fold compared to control) with a concomitant increase in the level of caspase-3 (11.7- and 9.5-fold) and p53 (15.4- and 11.75-fold). Both compounds arrested the cell cycle mainly in the G2/M phase. Furthermore, 7e and 9a displayed good selectivity toward tumor cells (S.I. = 38.7 and 18.3), upon testing of their cytotoxicity toward non-tumorigenic breast MCF-10A cells. Finally, compounds 7a, 7b, 7d, 7e, and 9a were examined for their plausible CDK2 inhibitory action. The obtained results (% inhibition range: 16%–58%) unveiled incompetence of the target bis-indoles to inhibit CDK2 significantly. Collectively, these results suggested that herein reported bis-indoles are good lead compounds for further optimization and development as potential efficient anti-breast cancer drugs.

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Synergistic Activation of p53 Family of Proteins by the Combination of Nutlin 3a and SAHA in Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v114.22.1738.1738 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1738-1738 ◽

Cited By ~ 1

Author(s):

Gautam Borthakur ◽

Seshagiri Duvvuri ◽

Ismael J. Samudio ◽

Kensuke Kojima ◽

Marina Konopleva ◽

...

Keyword(s):

Cell Lines ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Synergistic Activity ◽

P53 Family ◽

P53 Pathway ◽

Protein Levels ◽

Acute Myelogenous ◽

Induced Apoptosis ◽

Transcriptional Target

Abstract Abstract 1738 Poster Board I-764 Background Nutlin 3a binds to murine double minute 2 (MDM2) a negative regulator of p53 and potently increases p53 protein levels by inhibiting MDM2 mediated ubiquitilation. Our group has shown activity of Nutlin 3a in acute myelogenous leukemia (AML) and an intact p53 pathway appeared to be necessary for this activity (Blood. 2005;106:3150-9). The lysine residues on p53 that are ubiquitilated by MDM2 are also sites for acetylation, a process that stabilizes p53 for efficient transcription of p53 target proteins. SAHA is a wide spectrum Histone de-acetylase inhibitor (HDACi) and is known to induce acetylation of non-histone proteins. We postulated that the combination of Nutlin 3a and SAHA will lead to increased acetylation of p53, enhanced transcription of pro-apoptotic targets of p53 and synergistic activity in AML. As Nutlin 3a has been reported to disrupt interaction of MDM2 with other p53 family members notably p73 (Oncogene. 2008;27:997-1003) and HDACi can induce pro-apoptotic p73 (Oncogene. 2004;23:4807-17), we further postulated that the combination may be effective in AML cells with defective p53 pathway. Methods and Results In this study we investigated the effects of Nutlin 3a/SAHA combination on AML cell lines. OCI-AML-3 (p53 wild-type) cells were pre-treated with Nutlin 3a for 6 hrs and treated with Nutlin 3a+SAHA for a further 48 hrs. SAHA and Nutlin 3a induced apoptosis synergistically even in low drug concentrations (2.5 μM Nutlin 3a and 0.5 μM SAHA). In OCI-AML-3 cells, combination treatment resulted in increased protein levels of p53, increased p53 acetylation at Lys373/Lys382and induction of pro-apoptotic p53 transcriptional target protein, Noxa.. Interestingly, synergistic apoptosis induction with Nutlin 3a+SAHA was seen in p53 null (HL-60) and mutant (NB4 and OCI-AML-2) cells after 72 hrs. In HL-60 cells, treatment with Nutlin 3a+SAHA, resulted in increase in protein levels of p73. Noxa is also a transcriptional target of p73 and the treatment with Nutlin 3a+SAHA combination upregulated protein levels of Noxa in HL60 cells. Experiments with “knock-down” of p73 and Noxa and combination of Nutlin 3a with other HDACi are in progress. Though SAHA is known to be active against cancer cell lines with P-glycoprotein (Pgp) expression, we plan to exclude the possibility of modulation of P-gp by Nutlin 3a contributing to this synergy. Co-culture with normal bone marrow mesenchymal stromal cells (MSCs) confers chemo-resistance to leukemia cells. Treatment with the combination of Nutlin 3a and SAHA for 72 hrs effectively induced apoptosis in OCI-AML-3 cells co-cultured with MSCs. Conclusion The combination of MDM2 inhibitor, Nutlin 3a and HDACi, SAHA show promising synergistic activity against AML cells irrespective of their p53 status and can potentially overcome the resistance mediated by the “microenvironment”. Disclosures Off Label Use: This is an in vitro study of SAHA (FDA approved) and the investigative agent Nutlin. No clinical data included..

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Molecular Mechanisms Mediating Antimyeloma Activity of An MDM2 Antagonist Nutlin.

Blood ◽

10.1182/blood.v114.22.3841.3841 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3841-3841 ◽

Cited By ~ 1

Author(s):

Manujendra N. Saha ◽

Jennifer Jayakar ◽

Connie Qi ◽

Donna E. Reece ◽

Hong Chang

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Molecular Mechanisms ◽

Target Genes ◽

Annexin V ◽

Downstream Targets ◽

Mdm2 Antagonist ◽

P53 Signaling ◽

P53 Activation ◽

Induced Apoptosis

Abstract Abstract 3841 Poster Board III-777 Background and aims p53 gene mutations or deletions are rare in multiple myeloma (MM). Thus, strategies to induce p53 activation in myeloma cells with wild type (wt) p53 may have therapeutic promise. However, p53 signaling and functional consequences are not clearly understood in MM. In this study we used a small molecule MDM2 antagonist; nutlin, to explore the molecular mechanisms associated with nutlin-mediated activation of the p53 signaling pathway resulting in cell cycle arrest and/or apoptosis. Methods MM1.S and H929 cell lines harboring wt p53, LP1 and U266 cell lines expressing mutant (mt) p53, and five primary samples from patients with MM were treated with different concentrations of nutlin or DMSO control. Nutlin-induced cells were assessed for cell viability, induction of p53 and its downstream targets, cell cycle analysis, apoptosis assays and gene expression profile at different time periods. Results Treatment of MM cells harboring wt p53 with nutlin led to a dose-dependent increase in the expression of p53 and its downstream targets, p21 and MDM2. After 24 hours incubation with 10 μM nutlin, p53 protein levels increased approximately 4 to 6-fold in MM1.S and H929 cell lines and in two primary MM samples. Induction of p53 downstream targets was strictly correlated with induction of p53. In contrast, nutlin did not modulate the expression of these proteins in the cells with mt p53. Proliferation of the cells was also affected by nutlin in MM cells harboring wt p53 but not mt p53. Seventy-two hours after incubation with 10 μM nutlin, the viability of the cells (MM cell lines and primary MM samples) with wt p53 declined to 30% as assessed by MTT assay. Nutlin caused up-regulation of several pro-apoptotic targets, PUMA, Bax, and Bak, and down-regulation of two anti-apoptotic targets, Bcl2 and survivin in MM1.S cells. Furthermore, nutlin effectively arrested cell cycle progression in wt p53 MM cells, depleting the cells in S-phase compartment and increasing the cells in G1 and G2/M phase compartment, indicating G1 and G2 arrest. Annexin-V staining and flow cytometry (FCM) studies showed that there was a dose- and time-dependent increase in annexin-V binding in MM1.S cells but not in LP1 or U266 cells. These results suggest that nutlin-induced apoptosis is p53-dependent. At 72 hours following treatment with 10 μM nutlin, annexin-V binding was increased from 22% (1.0 μM) to 80% in MM1.S cells. Western blot (WB) analysis of nutlin-induced cells revealed activation of both caspase-8 and caspase-9 followed by activation of caspase-3 suggesting the association of both extrinsic and intrinsic pathways of apoptosis. In addition, direct inhibition of endogenous survivin by siRNA further enhanced nutlin-induced apoptosis, as measured by WB analysis for activation of caspase-3 and FCM assay for annexin-V binding. Forty-eight hours after nutlin treatment, 60% of MM1.S cells transfected with survivin siRNA were annexin-V positive, whereas control siRNA-transfected cells were 30% annexin-V positive. Moreover, selective blocking of p53 transcription by a p53 inhibitor, pifithrin-α (25 μM), inhibits nutlin-induced up-regulation of p53-transcriptional target genes p21, PUMA, and Bax in MM1.S cells, suggesting a transcription-dependent apoptosis. Studies by confocal microscopy and WB analysis of the fractionated samples revealed accumulation of p53 in both nuclear and cytoplasmic fractions in nutlin-treated MM1.S cells. Since transcriptional activation of p53-target genes occurs in the nucleus, while cytoplasmic p53 mediates transcription-independent apoptosis, the accumulation of nuclear and cytoplasmic p53 suggests that activated p53 used both transcription-dependent and transcription-independent pathways to induce apoptosis in MM cells. Finally, the consequence of the p53 activation in MM was validated by gene expression profiling of MM1.S cells with or without nutlin stimulation, demonstrating up-regulation of p53 and its downstream targets p21, MDM2, and BAX. Conclusion Nongenotoxic activation of the p53 pathway by nutlin sensitized MM cells harboring wt p53 to transcription-dependent and transcription-independent apoptosis. Our studies provide the preclinical framework for the evaluation of nutlin as a novel therapeutic approach in the treatment of MM. Disclosures: No relevant conflicts of interest to declare.

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Kaempferide Targets Side Population, the Putative Cancer Stem Cell, In Myeloma and Induced Apoptosis In Dose-Dependant Manner

Blood ◽

10.1182/blood.v116.21.5029.5029 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5029-5029

Author(s):

Yen Siew Loh ◽

George Li ◽

Kei Fan ◽

Iyad Ahmed ◽

Basil Roufogalis ◽

...

Keyword(s):

Time Series ◽

Growth Inhibition ◽

Propidium Iodide ◽

Side Population ◽

Annexin V ◽

Myelogenous Leukemia ◽

Drug Transporter ◽

Incubation Experiment ◽

Anti Cancer ◽

Induced Apoptosis

Abstract Abstract 5029 Multiple myeloma (MM), cancer of the plasma cells, remains incurable despite advancement in therapeutic regiments. Studies showed that the ‘side population (SP)’, a subpopulation enriched with CSC in various cancers, has higher drug transporter activity than the bulk tumor; suggesting the reason why these cells survived despite post-chemotherapy. The increasing evidence of cancer stem cells (CSC) suggests that anti-cancer drugs targeting this subpopulation may lead to eradication of the root of cancer recurrence. In this study, we explored the capability of kaempferide (KFD), a flavanoid in propolis (bee glue) that can reverse drug transporter activity, to combat SP cells in myeloma. KFD was one of the compounds in Brazilian propolis that reduced SP percentage in myeloma cell lines. We report for the first time that KFD is able to induce apoptosis in the SP cells in myeloma. The ability of KFD to inhibit growth of unfractionated KMS-11 cells was first investigated. Parthenolide (PAR), a natural product shown to inhibit growth of putative CSC in acute and chronic myelogenous leukemia was included as control. Unfractionated cells were seeded at 20000 cells per well in a 96 well plate. After 24h of incubation with various concentration of KFD, PAR, and DMSO (vehicle control), MTS solution was added and absorbance at 490 nm was determined. The IC50 of KFD and PAR in KMS-11 cells was 26 μM and 5 μM with 95% confidence intervals between 17.7 to 33.3 μM and 4.0 to 5.8 μM respectively. This is shown in the dose-response curve in figure 1A. No significant growth inhibition was observed in DMSO (0.1% to 0.5 % v/v) treated cells. We then examined if the KFD causes apoptosis in the sorted-SP cells. Sorted-SP cells were treated with 26 μM KFD, 5 μM PAR, or 0.2% v/v DMSO as mentioned above. After 1, 3, and 6 hour of incubation, cells were harvested and stained with Annexin V and propidium iodide and then analyzed using FACS Calibur. Result showed that percentage of Annexin V+ apoptotic cells in KFD-treated cells increased in a time series manner (1, 3, 6 h) (figure 1B). Because KFD was reported to be capable to reverse the activity of drug efflux transporter, further studies to investigate the synergistic effect of KFD with conventional drugs to treat myeloma will be carried out. Alternative medicines employing natural products have become increasingly sought after when conventional drugs cause immense side effects. Component in propolis that was reported to have anti-cancer properties, has low toxicity at high concentration, and spare normal cells, appeals to be a potential compound to combat myeloma. Exploitation of KFD that has the dual effect to induce apoptosis in putative CSC in myeloma and reverse drug transporter activity offers great opportunity in cancer drug development and future clinical trials in patients with myeloma. A B Annexin V-FITC (FL-1) Propidium iodied (FL-3) Figure 1. (A) Growth inhibition by KFD and PAR after 24h of incubation. Experiment was repeated at least 3 times. (B) KFD induced apoptosis in KMS-11 in a time-series manner. Cells were treated with 26 μM KFD or 5 μM PAR for 1, 3, and 6 hour and was stained with Annexin V and propidium iodide. KFD PAR Figure 1. (A) Growth inhibition by KFD and PAR after 24h of incubation. Experiment was repeated at least 3 times. (B) KFD induced apoptosis in KMS-11 in a time-series manner. Cells were treated with 26 μM KFD or 5 μM PAR for 1, 3, and 6 hour and was stained with Annexin V and propidium iodide. . / KFD . / PAR Disclosures: No relevant conflicts of interest to declare.

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Effects of short-term infusion of lipid emulsions on pro-inflammatory cytokines and lymphocyte apoptosis in septic and non-septic rats

British Journal Of Nutrition ◽

10.1017/s0007114510005751 ◽

2011 ◽

Vol 106 (1) ◽

pp. 27-32 ◽

Cited By ~ 1

Author(s):

Patrick Scheiermann ◽

Juliane Ott ◽

Sandra Hoegl ◽

Matthias Hecker ◽

Christian Hofstetter ◽

...

Keyword(s):

Annexin V ◽

Observation Time ◽

Lipid Emulsions ◽

Immune Functions ◽

Splenic Lymphocytes ◽

Short Term ◽

Term Administration ◽

Free Arachidonic Acid ◽

Apoptotic Pathways ◽

Induced Apoptosis

Long-term administration of PUFA is known to modulate immune functions and apoptotic pathways depending on the respective amount of n-6 and n-3 fatty acids (FA). Data on short-term effects on apoptotic pathways are rare. Apoptosis of splenic lymphocytes is the hallmark of detrimental sepsis. Therefore, we aimed to compare the immediate effects of parenterally administered n-6-enriched soyabean oil (SO)- and n-3-enriched fish oil (FO)-based lipid emulsions after laparotomy (LAP; sham procedure) and after induction of acute, severe sepsis by caecal ligation and incision. After 390 min of observation time, plasma was analysed for IL-1β, IL-6 and NEFA. Apoptosis in splenic lymphocytes was quantified by Annexin-V expression. After LAP, infusion of both FO and SO did not change cytokine concentrations. Sepsis increased both cytokines. FO but not SO further augmented the rise. After LAP, SO increased NEFA, and both lipid emulsions reduced free arachidonic acid (AA). Sepsis resulted in a dramatic decrease in NEFA and AA. The drop in NEFA and AA was prevented by both SO and FO. In addition, FO resulted in an increased concentration of n-3 FA under both conditions. Infusion of both lipid emulsions induced apoptosis in splenic lymphocytes after LAP. Sepsis-induced apoptosis was not further enhanced by FO or SO. The present study shows that short-term administration of FO as opposed to SO caused pro-inflammatory effects during sepsis. Moreover, short-term administration of both SO and FO suffices to induce apoptosis in splenic lymphocytes. Finally, SO and FO do not further enhance sepsis-induced splenic apoptosis.

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Treatment of MGUS by Hesperetin, potentially Delaying/Blocking the Conversion of MGUS to Symptomatic Myeloma.

Blood ◽

10.1182/blood.v114.22.1885.1885 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1885-1885

Author(s):

Yoshitaka Kikukawa ◽

Hiroaki Mitsuya ◽

Hiroyuki Hata

Keyword(s):

Membrane Potential ◽

Western Blot ◽

Cell Lines ◽

Mitochondrial Membrane ◽

Annexin V ◽

Myeloma Cell ◽

M Protein ◽

Myeloma Cells ◽

Apoptotic Pathways ◽

Induced Apoptosis

Abstract Abstract 1885 Poster Board I-908 Treatment of MGUS by hesperetin, potentially delaying/blocking the conversion of MGUS to symptomatic myeloma Yoshitaka Kikukawa, Hiroaki Mitsuya, Hiroyuki Hata. Department of Hematology, Kumamoto Unversity Hospital INTRODUCTION: Although prognosis of multiple myeloma has recently been improved by novel therapeutic regimens, all currently available therapuetics target symptomatic myeloma only. We saw an MGUS patient, experiencing a gradual and continuous decrease of M-protein (IgA-k) from 2,080 mg/dl to 878 mg/dl over 3 years (Fig. 1). History examiantion revealed that the decrease of M-protein initiated when the patient started taking a dietary supplement containing hesperetin-7-glucoside, which is a glycosylated form of hesperetin designated to achive an approximately 104-fold increase of bioavailability comparing to aglycon structure (Hayashibara Biochemical Laboratories, Inc.). Because this compound is eventually metabolized to hesperetin, a possible anti-myeloma effect of hesperetin was examined in vitro. METHODS: Myeloma cells were obtained from myeloma patients and purified with CD138 magnetic beads. Anti-myeloma effects of test compounds on purified myeloma cells or myeloma cell lines were evaluated by WST-8 assay. Apoptosis of myeloma cells was quantified either by annexin V staining or the propidium iodide method, followed by flowcytomerty analysis or by morphological analysis of cytospin slides. Mitochondrial membrane potential was quantified using JC-1 staining Kit (Cayman Chemical Co.) and flow cytometry. Caspase activation and total ubiqutinated protein were analysed by western blotting. Proteasomal chymotrypsin-like activity was measured using 20S Proteasome Assay Kit (Enzo Life Sciences). RESULTS: Hesperetin showed inhibitory effects in a dose-dependent manner on the growth of 4 myeloma cell lines and freshly isolated myeloma cells. Two myeloma cell ines, RPMI8226 and 12PE, were utilized as representative cell lines for further analysis. Hesperetin induced annexin V/PI positive cells, morphological fragmentation of the nucleus of myeloma cells, and activation of caspase-3, 8 and 9, at a concentration around 500 microM, which is clinically achievable with peroral administration of glycosylated hesperetin, suggesting that the observed anti-myeloma effects by hesperitine through apoptotic pathways. Further analysis revealed that hesperetin disrupted mitochondrial membrane potential which leads to release of cytochrome c from mitochondria to cytoplasm as assessed by western blot analysis. Caspase-8 and 9 inhibitors(Z-IETD-fmk and Z-LEHD-fmk, respectively)did not inhibit the hesperetine-induced apoptosis, although they completely inhibited anti-Fas antibody-induced apoptosis, suggesting that the hesperitine-induced apoptosis is not dependent on death receptor signaling. A pan-caspase inhibitor, Z-VAD-fmk, completely blocked the hesperetin-induced apoptosis in 12PE cells, but only partially in RPMI8226 cells, suggesting that hesperetin also mediated caspase-independent apoptosis in RPMI8226 cells. Moreover, western blot showed that hesperetin treatment induced an accumlation of poly-ubiquitine proteins. Analysis of proteasome activity revealed that hesperetin at 500 microM exerted a moderate inhibition of proteasome activity with 72.6% of the inhibition exerted by bortezomib at 40 nM. CONCLUTIONS: This is the first report showing anti-myeloma effect of hesperetin. It showed anti-myeloma effects via caspase-dependent and independent apoptotic pathways and proteasome inhibition activity. Given a fact that hesperitin has been approved by Japanese Goverment as a safe supplementary compound, and the present MGUS case had been taking hesperitin over >3 years without any adverse events, this compound should be well torelated over a long period. If a delay of conversion from either MGUS or asymptomatic myeloma to symptomatic myeloma could be achieved, prognosis of MM could be improve. Based on these findings, an open-label, pilot clinical trial to test the efficacy of hespertin, enroling asymptomatic myeloma patients, has currently been underway (Approved by IRB of Kumamoto Univerisity Hospital). Disclosures: No relevant conflicts of interest to declare.

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