scholarly journals Bortezomib-Induced Apoptosis in Myeloma Cell Via Oxidation of Peroxiredoxin III and Thioredoxin 2

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2081-2081
Author(s):  
Yeung-Chul Mun ◽  
Jee-Young Ahn ◽  
Seung Hye Kang ◽  
Eun-Sun Yoo ◽  
Kyoung Eun Lee ◽  
...  
Keyword(s):  
Western Blot ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Reduced Form ◽  
Ros Generation ◽  
Down Regulation ◽  
Western Blot Assay ◽  
Myeloma Cells ◽  
Mitochondrial Ros ◽  
Induced Apoptosis

Abstract Backgrounds : Bortezomib (BTZ) is the first generation proteosome inhibitor providing excellent response in newly diagnosed multiple myeloma. BTZ treatment increases oxidative stress in myeloma cells. However the roles of antioxidant enzymes during upregulation of ROS and the mechanisms of resistance from BTZ treatment are unclear. The aims of current study are to elucidate that the changes of redox enzyme could have a important roles of anti-myeloma effect during BTZ treatment. Methods : MM.1S, MM.1R, and RPMI8226, the human myeloma cell lines, were treated with BTZ to induce apoptosis. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS) respectively. Sulfinic acid, (SO2) form of preoxiredoxin (Prx) was studied by western blot assay using Prx SO2 form-specific antibody. Monomer/dimer assay for subtypes of Prx and thioredoxin (Trx) was performed by western blot using non-reducing gel. To evaluate the effect of down regulation on sulfiredoxin (Srx), myeloma cells were transfected with small interfering RNA (siRNA) followed by Western blot analysis. Results : Mitochondrial over cytosolic ROS of MM cells was increased significantly after 19 hour of BTZ (2.5 nM). Apoptosis of MM cell after BTZ treatment was increased in concordance with mitochondrial ROS increment of MM cells. N-acetylcystein (NAC) reversed BTZ-induced mitochondrial ROS elevation and apoptosis of MM cells as well. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during BTZ-induced MM cell apoptosis. Monomer, indicated active and reduced form, of Prx III was decreased and dimer, indicated inactive and oxidized form, of Prx III was increased in MM cells after treatment of BTZ among Prxs. Similarly, monomer of Trx 2, mitochondrial Trx was decreased in MM cells after BTZ treatment. However, increment of cysteine SO2 Prx III was not observed. Meanwhile, Srx, reducing enzyme of SO2 Prx, was induced in MM cells after BTZ treatment. Down regulation of Srx by siRNA did not promote ROS generation or apoptosis in BTZ-treated MM cells, otherwise. Conclusions : Our results showed inactivation of Prx III by multimer formation as hyperoxidation is found during BTZ-induced mitochondrial ROS generation and apoptosis in MM cells. This Prx III oxidation was due to down regulation of reduced Trx 2 in BTZ-treated MM cells. To design of raising ROS stress and down regulation of anti-oxidants(ie, Trx), as a treatment-strategies may be worthwhile to potentiate BTZ-induced apoptosis in MM cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of Autophagy with 3-Methyladenine Potentiates Bortezomib-Induced Apoptosis of Myeloma Cell Via up Regulation of Mitochondrial Reactive Oxygen Species

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4472-4472
Author(s):  
Yeung-Chul Mun ◽  
Jee Young Ahn ◽  
Eun Sun Yoo ◽  
Kyoung Eun Lee ◽  
Eunmi Nam ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Apoptosis ◽  
Conflicts Of Interest ◽  
Thioredoxin Reductase ◽  
Myeloma Cell ◽  
Cellular Level ◽  
Redox Enzymes ◽  
Myeloma Cells ◽  
Mitochondrial Ros ◽  
Induced Apoptosis

Abstract Backgrounds : Autophagy plays an important role in the pathogenesis of multiple myeloma. Autophagy usually acts on a pro-survival mechanism, and cooperates with the ubiquitin proteasome system in the homeostasis of myeloma cells by degrading excessive and misfolded proteins for energy recycling. Therefore, the inhibition of autophagy could effectively induce death and could synergize with proteasome inhibitors in myeloma cells. In this study, we investigated the synergistic mechanism of apoptosis with bortezomib (BTZ) and autophagy inhibitors in the MM cells. Methods : We evaluated the change of autophagy, apoptosis, mitochondrial ROS and cellular level of redox enzymes, including peroxiredoxin (Prx), thioredoxin (Trx), thioredoxin reductase (Trx-R) using human myeloma cell lines, MM.1S and MM.1R during BTZ treatment. To study the status of oxidation for redox enzyme, sulfinic acid (SO2) or multimeric form of Prx, Trx and Trx-R by western blot were measured using non-reducing or reducing gel. Results : Mitochondrial over cytosolic ROS of MM cells was increased significantly after 24 hour of BTZ (2.5 nM) treatment. Apoptosis of MM cell after BTZ treatment was increased in concordance with mitochondrial ROS increment of MM cells. N-acetylcystein (NAC) reversed BTZ-induced mitochondrial ROS elevation and apoptosis of MM cells as well. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during BTZ-induced MM cell apoptosis. LC3-II expression was elevated along with increment of mitochondrial ROS and apoptosis of MM cells after BTZ treatment. Oxidation of PRX4 and TRX2 was observed during BTZ-induced apoptosis of MM cells. After treatment of NAC, LC3-II and PRX4/TRX2 expression was reversed. Inhibition of autophagy with 3-Methyladenine (3-MA) not only resulted in a further increase in BTZ-induced apoptosis but also induced mitochondrial ROS in MM cells. Conclusions : Autophagy is induced during BTZ treatment in MM cells. Our experiment showed crosstalk between autophagy and mitochondrial ROS and its redox enzymes during BTZ-induced MM cell apoptosis. Inhibition of autophagy with 3-MA potentiates BTZ-induced apoptosis of MM cells via up regulation of mitochondrial ROS. Our results provide new perspective on the cellular mechanism of action of BTZ and support the synergism of BTZ with autophagy inhibitor for an improved therapy of the multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Induction of CRBN(Cereblon) mRNA Expression by Baicalein

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5025-5025
Author(s):  
Zi Ma ◽  
Rui bo Zhang ◽  
Li He ◽  
Chao ping Xu ◽  
Ding xin Zhou ◽  
...  
Keyword(s):  
Maintenance Therapy ◽  
Mrna Expression ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Novel Strategy ◽  
Induced Apoptosis ◽  
Cell Inhibition ◽  
Effective Drugs

Abstract Abstract 5025 The CRBN gene that encodes the cereblon protein is found on the short arm at position p26. 3 of human chromosome 3. Cereblon is a primary target of thalidomide teratogenicity and required for the anti-myeloma activity of lenalidomide and pomalidomide. CRBN depletion myeloma cells become highly resistant to both lenalidomide and pomalidomide. Baicalein, a component of Scutellaria radix from HLJDT, not only suppressed proliferation and induced apoptosis of myeloma cells by down-regulating interleukin −6(IL-6) and XIAP gene expression, but also inhibited the signaling cascades mediated by IL-6 and facilitated myeloma cell inhibition induced by dexamethasone. In clinic, we found that treatment of thlidomide- or lenalidomide-resistant myeloma patients by applying Huang-Lian-Jie-Du-Tang (HLJDT) can induce hematological remission. The precise molecular mechanism of HLJDT exerts its anti-tumor effects remains unclear. Here, by RT-PCR, we demonstrated that treatment of U266 cells and primary myeloma cells with 20μM baicalein can induce CRBN mRNA expression in time-dependent manner. As lenalidomide and thlidomide are effective drugs for maintenance therapy with the advantage of oral administration. It was particularly active in patients with higher cereblon expression. Thus, the combination of HLJDT with thlidomide or lenalidomide may be a novel strategy of maintenance therapy for myeloma patients. Disclosures: No relevant conflicts of interest to declare.

Induction of Cyclin D1 Gene in Myeloma Cells Down-Regulates the Expression of Cyclin D2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3411-3411
Author(s):  
Yoshiaki Kuroda ◽  
Akira Sakai ◽  
Naohiro Tsuyama ◽  
Mami Mizuno ◽  
Nanae Nakaju ◽  
...  
Keyword(s):  
Western Blot ◽  
Cyclin D1 ◽  
D1 Protein ◽  
Myeloma Cell ◽  
Cyclin D3 ◽  
Proliferation Index ◽  
Cyclin D2 ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Cyclin D1 Protein

Abstract Cyclin D is dysregulated in at least two-thirds of multiple myeloma (MM) tumors. In addition, recent reports showed that the dysregulation of cyclin D1 is frequent in the absence of a t(11;14) translocation in MM. However, as we also reported (Int J Oncol, 2004), there appears to be no obvious correlation between the expression of cyclin D1 and the proliferation index (PI) or Ki67 expression. Therefore, we thought that the down-regulation of cyclin D2 might offset the expression of cyclin D1 in myeloma cells with cyclin D1 overexpression in cDNA microarray, since primary myeloma cells or myeloma cell lines express cyclin D3 ubiquitously. Here we transfected cyclin D1 gene into a myeloma cell line (RPMI8226), originally not expressing cyclin D1, using a retrovirus-mediated gene transfer system. In this method we inserted a 1.1 kb fragment containing the open reading frame of cyclin D1 removing from a tet-cyclin D1 plasmid (kindly provided by Dr. Reed SI, MCB, 1994) into a retrovirus vector (pQCXIP). First, we analyzed the expression of cyclin D1 in the bulk culture of cyclin D1 transfectant. We detected the expression of cyclin D1 by western blot, and found that the limited numbers of transfectant expressed cyclin D1 protein by immunohistocytochemical staining. Subsequently, we separated the two types of cyclin D1 transfectant by limiting dilution. Both transfectants showed the expression of cyclin D1 mRNA in RT-PCR, however, one of the two did not show the expression of cyclin D1 protein in western blot and immunohistocytochemical staining. Interestingly, we clearly detected the down-regulation of cyclin D2 mRNA in the transfectant with cyclin D1 protein expression by RQ-PCR. Furthermore, we detected an increase of cells in S phase in the transfectant with cyclin D1 protein by flow cytometry. Unlike in the study of Lamb J et al. (Cell, 2003), we could not observe the induction of IL-6 by the transfection of cyclin D1 gene. Although the mechanism of the impairment of cyclin D1 translation is unclear, here we suggest that the lack of correlation between the expression of cyclin D1 and PI might be due to the impairment of cyclin D1 translation or the offset of the expression of cyclin D1 by the down-regulation of cyclin D2. We are now analyzing the effects of velcade and IMiDs on these transfectants, since we suspect that these differences would affect the response to chemotherapy for MM. Furthermore, we are going to analyze the difference of gene expression between these transfectants using cDNA microaray. Therefore, these transfectants could be useful materials to analyze the cyclin D1 dysregulation in myeloma cells.

Inhibition of Heat Shock Factor 1 (HSF1) Is More Effective At Sensitizing Myeloma Cells to Bortezomib Than Inhibition of Individual HSF1 Targets.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2953-2953
Author(s):  
Shardule P Shah ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
Multiple Myeloma ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Conflicts Of Interest ◽  
The United States ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Shock Response ◽  
Induced Apoptosis

Abstract Abstract 2953 Multiple myeloma is a plasma cell disorder with an average incidence of 21,000 new cases per year in the United States. Recent advances in therapeutic approaches such as the use of proteasome inhibitors have resulted in a significant increase in the overall survival of myeloma patients. Myeloma cells maintain many of the characteristics of normal plasma cells, including constitutive immunoglobulin production and secretion, therefore management of ER stress plays a role in myeloma cell sensitivity to proteasome inhibition. However, myeloma cells also upregulate protective genes in response to the proteotoxic stress that can limit the therapeutic response. Previous groups have published on the importance of the heat shock response and the heat shock protein (HSP) family, supporting preclinical and clinical exploration of HSP inhibition in myeloma. Our group had interest in regulation of the HSP response and has evaluated the master regulator HSF1 as a potential therapeutic target. We found that siRNA-mediated silencing of HSF1 enhances bortezomib-induced apoptosis in a myeloma cell line. To define the effectors of the heat shock response important in regulating bortezomib response, we determined which heat shock response genes are induced by bortezomib in an HSF1-dependent manner. From a realtime PCR array of 84 HSP family genes, we found 21 genes that were induced greater than 2-fold by bortezomib. Of these 21 genes, 10 genes showed >50% reduction in HSF1-silenced cells. 7/10 genes were confirmed by independent qRT-PCR and western blot analysis. These genes include: CRYAB (alpha-crystallin B chain), DNAJB1 (HSP40 subfamily B), HSPA1A (HSP70-1A), HSPA1B (HSP70-1B), HSPB1 (HSP27), HSPH1 (HSP105/110), and HSP90AB1 (HSP90b1). To begin to determine which of these genes was important for the HSF1-dependent protective response we silenced the 7 genes individually and subsequently treated the cells with bortezomib. Surprisingly only 1 of the 7 genes silenced individually, DNAJB1, had an observable effect on bortezomib-induced death. However DNAJB1 silencing does not account for all the HSF1 activity as the increase in cell death due to bortezomib is only 48% of that observed with HSF1 silencing. Thus targeting HSF1 is more effective at sensitizing multiple myeloma cells to bortezomib-induced apoptosis than targeting individual HSPs. Moreover these data suggest that HSP90 inhibitors are functioning by inhibiting at least two members of this family to be effective as single agents. Therefore, while clinical trials for individual HSP and HSP in combination with bortezomib are being conducted, a more effective strategy for apoptosis induction is achieved through inhibition of HSP regulators such as HSF1 in combination with bortezomib. These results provide support for investigating HSP regulation in response to PI to increase the efficacy of myeloma therapy. Disclosures: No relevant conflicts of interest to declare.

The Role of Peroxiredoxin III and Sulfiredoxin for Mitochondrial ROS Production to Arsenic Trioxide in Acute Promyelocytic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5217-5217
Author(s):  
Yeung-Chul Mun ◽  
Jee-Young Ahn ◽  
Eun-Sun Yoo ◽  
Kyoung Min Cho ◽  
Kyoung Eun Lee ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Caspase 3 ◽  
Promyelocytic Leukemia ◽  
Ros Generation ◽  
Down Regulation ◽  
Nb4 Cells ◽  
Mitochondrial Ros ◽  
Induced Apoptosis

Abstract Backgrounds: The Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia (APL), but in some cases, APL cells are resistant to ATO treatment. ATO exerts its effect mainly raising oxidative stress. However, not only the mechanisms of reactive oxygen species (ROS) generation by ATO but involvement of redox enzymes including peroxiredoxin (PRX) during ATO-induced apoptosis and its resistance remain elusive. Recently, Rhee et al had reported that PRX III and sufiredoxin together protect mice from pyrazole-induced oxidative liver injury was found (Antioxid & Redox Signal, 2012:17:1351-1361). Aims of current study are to elucidate that the changes of redox enzyme could be a mechanism of anti-leukemia effect in APL-derived NB4 cells during ATO treatment and to find ways to potentiate the anti-leukemic effects of ATO on APL cells. Methods: NB4, one of the human acute promyelocytic leukemia cell lines, was treated with 0~10 μM arsenic trioxide to induce apoptosis for 16-48 hours in RPMI-1640 medium supplemented with 10% FBS in CO2humidified atmosphere at 37°C. Apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) with flow cytometry. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red was used to detect cellular and mitochondrial ROS. SO2 form for PRX I, PRX II, and PRX III was detected by western blot assay using PRX SO2 form-specific antibody. Sulfiredoxin (SRX) and caspase 3, 9 were also detected by western blot analysis. To evaluate the effect of SRX depletion, NB4 cells were transfected with small interfering RNA (siRNA). Results: Intracellular ROS of NB4 cells was increased significantly after 16 hour of ATO treatment but decreased after 24 hour of ATO treatment. Mitochondrial ROS of NB4 cells was increased significantly after 39 hour of ATO treatment. Apoptosis of NB4 cell after ATO treatment was increased as time elapsed (24% on 16hr, 26% on 24hr, 48% on 39hr, and 60% on 48hr). Increased cysteine sulfinic acid (Cys–SO2H) PRX III, inactive and oxidized form, was observed as a hyperoxidation reaction in NB4 cells after ATO treatment in concordance with mitochondrial ROS increment of NB4 cells. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during NB4 cell apoptosis by ATO treatment. Meanwhile, SRX expression was increased in NB4 cells after ATO treatment. Down regulation of SRX by siRNA promoted ROS generation and apoptosis in ATO-treated NB4 cells. Conclusions: Our data showed inactivation of PRX III by Cys–SO2H formation as hyperoxidation is developed during ATO-induced mitochondrial ROS generation and apoptosis process in APL cells. In addition, ATO promotes expression of SRX, which is known as reducing enzyme of Cys–SO2H PRX and which leads to down regulation of ROS accumulation in APL cells. These findings might be due to protective effect of SRX from ATO on mitochondrial oxidative stress. These findings suggest ATO-induced anti-leukemic activity could be down regulated by an enhancing PRX III reduction after ATO-induced SRX activation. Currently, the effect of down regulation of SRX by siRNA are being investigated to amplify the apoptosis in ATO-treated NB4 cells. Our study may provide the insights for finding novel targets in the development of new therapies, which potentiate ATO-induced apoptosis in APL cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Resistin Induces Multidrug Resistance in Myeloma By Inhibiting Cell Death and Upregulating ABC Transporter Expression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5656-5656
Author(s):  
Jianan Pang ◽  
Qiaofa Shi ◽  
Zhiqiang Liu ◽  
Jin He ◽  
Huan Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protective Effect ◽  
Abc Transporters ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Long Term Outcome ◽  
Myeloma Cells ◽  
Chemotherapy Drugs ◽  
Response To Chemotherapy ◽  
Induced Apoptosis

Abstract Chemoresistance is a major hurdle in multiple myeloma. Most patients are prone to develop resistance to a wide spectrum of anticancer agents, significantly hampers the patients' long term outcome. Many studies point to bone marrow microenvironment as an important player in myeloma chemoresistance, in which marrow stromal cells and stromal-secreted soluble factors are shown to promote myeloma cell growth and survival. Our previous study has demonstrated that marrow-derived adipocytes protect myeloma cells against chemotherapy-induced apoptosis through adipocyte-secreted adipokines, one of such is leptin. However, the level of leptin expression in myeloma patients is not significantly changed, indicating the involvement of additional adipokines in this process. Interestingly, in a clinical study, an elevation of the adipokine resistin in the serum of myeloma patients after thalidomide treatment were observed as compared with that in patients before treatment, suggesting a potential role of this adipokine in response to chemotherapy. As a 12.5-kDa hormone that is mainly secreted by adipocytes and also secreted by other cells, resistin has a function in production of inflammatory cytokines that are important for cancer development. We thus hypothesized that resistin protects myeloma cells against chemotherapy. In our experiments, human myeloma cell lines and primary myeloma cells isolated from patient bone marrow aspirates were cultured in medium with addition of the recombinant human resistin and chemotherapy drugs melphalan or bortezomib for 24 hours. Cells without resistin served as a control. After cultures, an annexin-V binding assay for assessing apoptosis, western blot analysis for assessing cleavage of caspases and phosphorylation of signaling kinases, and the eFluxx-ID Gold uptake assay for examining ABC transporters activity were performed. In the animal study, myeloma-bearing SCID mice were treated with or without resistin and/or melphalan. Our results showed that resistin treatment reduced melphalan- or bortezomib-induced apoptosis both in vitro and in vivo. This protective effect has been further confirmed by the reduced cleavage of caspase-9, caspase-3, and poly (ADP-ribose) polymerase in myeloma cells. Mechanistic studies showed that culturing myeloma cells with resistin upregulated expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL and downregulated expression of the pro-apoptotic protein Bax via the NF-kB and the PI3K/Akt signaling pathways. Addition of resistin also reduced the intracellular accumulation of eFluxx-ID gold fluorescence in myeloma cells ARP-1 and MM.1S, when compared to that in cells without resistin. In addition, resistin significantly increased the mRNA and protein expression of ATP-binding cassette (ABC) transporters in myeloma cells by downregulating the expression of DNA methyltransferase 1 and 3a, and CpG methylation in the promoters of ABC transporters. Thus, our study demonstrates that resistin is a novel factor contributing to myeloma chemoresistance, and also implicates that disruption of its protective effect can be a potential strategy to improve current chemotherapy in patients and prolong survival. Disclosures No relevant conflicts of interest to declare.

PKC412 Induces Apoptosis in Human Multiple Myeloma Cell Lines and Primary Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1587-1587
Author(s):  
Janelle Sharkey ◽  
Tiffany Khong ◽  
Andrew Spencer
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Western Blot ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell ◽  
Nfkb Activation ◽  
Human Multiple Myeloma ◽  
Induced Apoptosis

Abstract This study was conducted to explore the effect and mode of action of the protein kinase C inhibitor PKC412 on proliferation and apoptosis in human multiple myeloma cell lines (HMCL) and primary myeloma cells. MTS assay was used to measure cell proliferation of 6 HMCLs and to examine the effect of co-treatment of HMCLs with PKC412, Bortezomib or the NFkB inhibitor, SN50. PKC412-induced apoptosis was evaluated by western blot (PARP cleavage) and annexin-V-FITC / propidium iodide (PI) flow cytometry. Similarly, the level of PKC412-induced apoptosis of HMCLs co-treated with the JNK inhibitor SP600125 was also determined. PKC412 and/or Bortezomib-induced primary myeloma cell death after 72hr treatment was measured by flow cytometry by identifying CD38+/CD138+/PI positive and negative cells. PKC activity after PKC412 treatment was assessed using the Peptag PKC functional assay and expression of PKC isoforms by HMCLs was determined by western blot. c-fos expression was evaluated by both RT-PCR and western blot. Expression of c-jun and phospho-c-jun was evaluated by western blot. NFkB activation was determined using the NFkB transcriptional assay kit. Sensitivity to PKC412 varied but some activity was seen against all HMCLs after 72hr treatment at doses of 0.5μM and above. PARP cleavage was observed in all HMCLs tested at both 24 and 72hr following treatment. PKC activity of PKC412 treated HMCLs was decreased in all HMCLs tested but no correlation between PKC isoform expression and sensitivity to PKC412 was found. However, a correlation between sensitivity to PKC412 and the presence of either an activating ras or FGFR3 mutation in HMCLs was observed. When HMCLs were pre-treated with PKC412, the PMA-induced increase in c-fos mRNA was inhibited, and PKC412 down-regulated baseline c-fos mRNA expression over time. PKC412 also down-regulated c-fos protein expression in nuclear extracts of HMCLs. c-jun phosphorylation was up-regulated for 24hr after PKC412 treatment and this increase in expression and phosphorylation was inhibited by co-treatment with the JNK inhibitor SP600125. Co-treatment of the HMCL NCI-H929 with PKC412 and SP600125 partially abrogated PKC412-induced apoptosis, indicating that it is partially JNK dependent. Increased NFkB activation was observed in PKC412 treated HMCLs with the level of increase correlating with PKC412 sensitivity. Enhanced killing of HMCL was seen when NFkB activation was inhibited with either Bortezomib or SN50, suggesting the increase in activation of NFkB may be a PKC412-induced survival response. This effect was also seen when primary myeloma cells where co-treated with PKC412 and Bortezomib for 72hr. The sequence in which compounds where added when treating both primary myeloma cells and the HMCL NCI-H929 with PKC412 and Bortezomib was important, with greater killing seen when cells were treated with PKC412 for 6hr before the addition of Bortezomib. Our results demonstrate that PKC412 induces apoptosis in HMCLs which is partially dependent on JNK. Furthermore PKC412 is more effective when used in combination with NFkB inhibition. The further evaluation of PKC412 in the treatment of multiple myeloma is justified.

Disruption of the Crosstalk CXCL12/CXCR4 Chemotactic Pathway To Enhance Melphalan-Induced Apoptosis in Multiple Myeloma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3445-3445
Author(s):  
Ayman A. Saad ◽  
James Fortney ◽  
Lin Wang ◽  
Heather O’Leary ◽  
Laura Gibson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Western Blot ◽  
Cell Lines ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Apoptotic Markers ◽  
Induced Apoptosis ◽  
B1 Cells

Abstract INTRODUCTION: Multiple myeloma cells display functional CXCR4 chemokine receptor that stimulates the migration of these cells toward their natural ligand, CXCL12 (stromal-derived factor, SDF-1a). CXCL12 is secreted by bone marrow stroma. Consistent with their CXCR4 expression, myeloma cells home to the marrow microenvironment, where adhesive interactions promote growth, survival, and confer cell adhesion-mediated drug resistance. METHODS U266-B1 cells (ATCC myeloma cell line) were pre-treated with recombinant CXCL12 for 30 minutes prior to the addition of melphalan for up to 72 hours. Both melphalan and CXCL12 were added at 24 hour intervals. Cell lines alone and cell lines with only melphalan or only CXCL12 were used as controls. We have also tested the influence of adding AMD3100, a reversible inhibitor of CXCR4, on myeloma cell survival. U266-B1 cells in media alone were pre-treated with AMD3100 for 24 hours prior to treatment with melphalan for 16 hours. Cell viability following treatment was quantified by flow cytometry assay using Annexin-V-FITC staining. Western blot analysis was used to quantify the apoptotic activity of the cell lines using 4 apoptotic markers: PARP (poly ADP-ribose polymerase), caspase-3, Bcl-2, and Mcl-1. RESULTS: Recombinant CXCL12 conferred a protective effect to myeloma cell lines during melphalan treatment. This effect was more pronounced at 72 hours of treatment. Western blot analysis showed diminished expression of the apoptotic markers, cleaved PARP (poly ADP-ribose polymerase) and active caspase-3 in the melphalan-treated cell lines with prior exposure to CXCL12. Additionally, pretreatment with AMD3100 resulted in enhanced apoptosis following melphalan treatment. CONCLUSION: Our data showed that CXCL12, a naturally occurring cytokine secreted by bone marrow stromal cells confers a protective effect on myeloma cells against apoptosis. Disruption of this effect by AMD3100 resulted in enhanced melphalan-induced apoptosis of myeloma cells. We are continuing to study this effect with the potential future utility of AMD3100 as a melphalan chemo-sensitizer in the treatment of multiple myeloma.

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

Blood ◽  
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.

scholarly journals Gambogic acid protects LPS-induced apoptosis and inflammation in a cell model of neonatal pneumonia through the regulation of TrkA/Akt signaling pathway

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xu Gao ◽  
Jingya Dai ◽  
Guifang Li ◽  
Xinya Dai
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Cell Model ◽  
Akt Signaling ◽  
Gambogic Acid ◽  
Akt Signaling Pathway ◽  
Western Blot Assay ◽  
A Cell ◽  
Induced Apoptosis ◽  
Apoptosis And Inflammation

Abstract Objective In this work, we investigated the effects of gambogic acid (GA) on lipopolysaccharide (LPS)-induced apoptosis and inflammation in a cell model of neonatal pneumonia. Method Human WI-38 cells were maintained in vitro and incubated with various concentrations of GA to examine WI-38 survival. GA-preincubated WI-38 cells were then treated with LPS to investigate the protective effects of GA on LPS-induced death, apoptosis and inflammation. Western blot assay was utilized to analyze the effect of GA on tropomyosin receptor kinase A (TrkA) signaling pathway in LPS-treated WI-38 cells. In addition, human AKT serine/threonine kinase 1 (Akt) gene was knocked down in WI-38 cells to further investigate the associated genetic mechanisms of GA in protecting LPS-induced inflammation and apoptosis. Results Pre-incubating WI-38 cells with low and medium concentrations GA protected LPS-induced cell death, apoptosis and inflammatory protein productions of IL-6 and MCP-1. Using western blot assay, it was demonstrated that GA promoted TrkA phosphorylation and Akt activation in LPS-treated WI-38 cells. Knocking down Akt gene in WI-38 cells showed that GA-associated protections against LPS-induced apoptosis and inflammation were significantly reduced. Conclusions GA protected LPS-induced apoptosis and inflammation, possibly through the activations of TrkA and Akt signaling pathway. This work may broaden our understanding on the molecular mechanisms of human neonatal pneumonia.

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