scholarly journals Oxymatrine Induces Apoptosis in Human Myeloma Cells By Cell Cycle Arrest and Activation of Caspase-Dependent Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5727-5727
Author(s):  
Wenjun Wu ◽  
Cai Wu ◽  
Fuming Zi ◽  
Yi Li ◽  
Li Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Growth Inhibition ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Conflicts Of Interest ◽  
Dependent Manner ◽  
Rt Pcr

Abstract Background : Multiple myeloma (MM) is a B cell malignant hematologic cancer. Despite the introduction of new drugs and improvement of chemotherapy, MM is still an incurable disease. Oxymatrine (OMT), the active ingredients of traditional Chinese herbal medicine sophora, has been reported to have antitumor activity. This study was to estimate the therapeutic efficacy of OMT in MM. Methods: The growth inhibition of myeloma cell lines (RPMI8226, U266, ARP-1) or primary cells by OMT was assessed by MTT assay. Apoptosis of MM cells was examined by annexin V-FITC using flow cytometry analysis. DNA content was analyzed by flow cytometry. RT-PCR and western-blot analysis were used to assess the expression of Bcl-2 family proteins and the IAP family proteins. Western blotting was also used to elucidate the signaling pathway that may mediate OMT-induced apoptosis of MM cells. Results: OMT treatment resulted in cell growth inhibition and apoptosis in primary MM cells and all tested MM cell lines in a dose-dependent manner (P <0.05). To elucidate OMT -induced MM cell apoptosis, MM cell lines were treated with or without OMT for 24h and assessed for caspase activation and signaling pathway by Western blotting. The results showed the cleavage of PARP, caspase-3, and caspase-9, and p-AKT were down-regulated after OMT treatment. The mRNA expression of survivin and HIAP by RT-PCR was down-regulated. OMT treatment at 5mM for 48h resulted in increased G-phase cells and decreased S-phase cells in MM cell lines (P <0.05). Cell cycle repressor P21 protein was up-regulated while CDK4, CDK6 and CyclinD1 expression was down-regulated. Our finding also showed a synergistic anti-MM activity of OMT and dexamethasone or adriamycin at a low does (CI<1). In addition, LC3-II expression was significantly increased both in RPMI8226 and U266 cells after treatment with OMT. However, treatment with different doses of OMT and 5 mM autophagy inhibitor 3-MA, significant increased cell apoptosis (P <0.05). Conclusion: Our findings demonstrate the anti-MM activity of OMT and indicate that OMT alone or together with other MM chemotherapeutics may be a prospective treatment for MM. Disclosures No relevant conflicts of interest to declare.

Here Is the Sub-Title:Apoptosis on Multiple Myeloma U266 Cell by Phosphorylation c-Jun of Brucine

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5112-5112
Author(s):  
Yanping Ma ◽  
Zhihua Li ◽  
Yihua Wang ◽  
Jing Feng
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Expression Change ◽  
U266 Cell ◽  
Apoptotic Effect

Abstract Abstract 5112 Objective: To investigate the Mechanism of the apoptotic effect of brucine on human multiple myeloma. Method: U266 cells (5×104) were plated in the presence or absence of the brucine (0, 0.05, 0.1, 0.2, 0.4 mg/ml) in 96 well culture plates for 24–72 h. The anti-proliferative response of brucine was assessed by MTT assay. The analysis of cell cycle of U266 cell with or without brucine was mesured by flow cytometry. The expression change of c-Jun after joining brucine, brucine and JNK specific inhibitor SP600125 was detected using RT-PCR. Results The apoptotic effect of brucine show a dose and time dependent manner. Cell cycle analysis using flow cytometry revealed accumulation of cells at sub-G0/G1 phase. The apoptosis rate separately were (4.137±0.01)%, (10.55±0.03)%, (12.31±0.04)%, (27.67±0.08)%, (29.67±0.09)% (p<0.01). Detecting c-Jun gene expression respectively after joining brucine, brucine and JNK specific inhibitor SP600125 by RT-PCR. The gray scale values were (0.7961±0.007),(0.4683±0.003). Conclusions Within the 0.4mg/ml concentration of brucine can induce apoptosis in U266 cells. Brucine by JNK signaling pathway through phosphorylation of c-Jun induced apoptosis in U266 cells. Disclosures: No relevant conflicts of interest to declare.

PI3K a Selective Inhibitor Induces Growth Inhibition In Mantle Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1812-1812
Author(s):  
Yixin Zhou ◽  
Linhua Jin ◽  
Stefania Pittaluga ◽  
Mark Raffeld ◽  
Takashi Miida ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Pi3k Inhibitor ◽  
Dependent Manner ◽  
Pi3k Inhibitor Ly294002 ◽  
Pi3k Inhibitors ◽  
Cycle Arrest

Abstract Abstract 1812 Deregulation of the phosphatidylinositol 3-kinase (PI3K)-mediated signaling plays an important role in the development of cell proliferation of mantle cell lymphoma (MCL). The PI3K pathway activation in MCL has been shown to result from constitutive B cell receptor (BCR) activation which is directly mediated by the Class IA PI3K p110 isoforms (a, β, and d). However, their relative contribution in MCL is not fully understood. In this study, the activity and molecular mechanisms of isoform-selective PI3K inhibitors which target different isoforms of the p110-kDa subunit has been investigated. We utilized the isoform-selective PI3K inhibitors; PI3-Ka inhibitor IV (p110a), TGX115 (p110b), IC87114 (p110d) and the non-specific PI3K inhibitor LY294002 (all inhibitors were purchased commercially). The p110a and p110d but not p110b isoform protein expression was detected in all tested MCL cell lines (Granta 519, JVM-2, Z138, Jeko-1, MINO). PI3-Ka inhibitor IV as well as non-specific PI3K inhibitor LY294002 induced cell growth inhibition with dose-dependent manner (IC50 at 48 hrs; PI3-Ka inhibitor IV: 17.5 μM for Granta 519, 14.3 μM for Jeko-1, 16.5 μM for Z138, LY294002: 14.8 μM for Granta 519, 19.4 μM for Jeko-1, 15.0 μM for Z138, MTT test). However, neither IC87114 nor TGX115 showed significant cell growth inhibition up to 40mM. Low dose of PI3-Ka inhibitor IV (5 μM) or LY294002 (5 μM) induced G0/G1 cell cycle arrest (increase of G0/G1 phase: PI3-Ka inhibitor IV 17.9 % for Granta 519, 28.2 % for Jeko-1, LY294002 19.3 % for Granta 519, 14.5 % for Jeko-1), and the higher dose (10 μM) increased apoptosis(specific apoptosis: PI3-Ka inhibitor IV 10.8 % for Granta 519, 15.3 % for Jeko-1, LY294002 13.6 % for Granta 519, 19.6 % for Jeko-1). No induction of cell cycle arrest/apoptosis by IC87114 or TGX115 treatment was observed. We then tried to assess the inhibition of PI3K/Akt signaling activation by p110a and p110d inhibitors. PI3-Ka inhibitor IV (10 μM) completely diminished phosphorylated (p-) Akt in all cell lines analyzed. Further investigation with 1–10 μM PI3-Ka inhibitor IV or IC87114 in Granta 519 and Jeko-1 cells declared that 1 μM PI3-Ka inhibitor IV almost diminished p-Akt and p-S6rp in both cells. The phosphorylation level of other PI3K/Akt signaling downstream substrates, GSK3-b and 4E-BP1, were down-regulated in dose dependent manner. Recently, GSK3-b kinase has been shown to negatively regulate cell cycle progression through Cyclin D1 repression in MCL. We observed that PI3-Ka inhibitor IV decreased Cyclin D1 expression and active pRb which are responsible for G0/G1 cell cycle arrest. The treatment with IC87114 (10 μM) performed moderate decrease of p-Akt, p-S6rp, and p-4E-BP, while no change in the levels of p-GSK3-b, Cyclin D1, or p-pRb was observed in both Granta 519 and Jeko-1 cells. We also tested whether the combination of PI3-Ka inhibitor IV or IC87114 with the proteasome inhibitor bortezomib induces synergistic cytotoxicity in MCL. No synergistic anti-proliferative effect was observed in any of the MCL cell lines analyzed. These findings demonstrate that p110a may be the responsible Class IA PI3K isoform for the development of MCL cell proliferation, and p110a isoform-selective PI3K inhibitor but not p110d or p110b inhibitors may provide a better therapeutic index relative to pan-PI3K inhibitors. Disclosures: No relevant conflicts of interest to declare.

Membrane Lipid Biosynthesis As a Potential Therapeutic Target in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1836-1836
Author(s):  
Carolyne Bardeleben ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Kennedy Pathway ◽  
Phosphatidylcholine Synthesis

Abstract Abstract 1836 Phosphatidylcholine (PC) is the most prominent phospholipid in mammalian endoplasmic reticulum (ER) membranes. The rate-limiting step in PC synthesis through the Kennedy pathway is the conversion of phosphocholine + cytidine triphosphate (CTP) to cytidine diphosphocholine, (CDP)-choline, via the enzyme CTP:phosphocholine cytidylyltransferase (CCT) (see figure). Multiple myeloma (MM) cells may be particularly dependent on this biosynthetic reaction because of their high consistent level of ER stress and requirement to continuously replenish ER membranes. Indeed, CCT-null mice have a defect in differentiation of B lymphocytes to plasma cells and deficiencies in Ig synthesis. To test whether this pathway remains critical in survival of malignant MM cells, we exposed MM cell lines to an inhibitor shown to inhibit CCT activity, HexPC. HexPC induced apoptosis in all MM cell lines in a concentration- and time-dependent manner. The addition of lysophosphatidylcholine (LPC), presumably converted to PC independently of the Kennedy pathway, completely rescued MM cell apoptosis. In contrast, similar concentrations of LPC in the same cell lines could not rescue apoptosis induced by bortezomib. An additional intervention to inhibit phosphatidylcholine synthesis, namely inducing pyrimidine starvation, also resulted in MM cell apoptosis and down-regulation of CDP-choline levels. Apoptosis of MM cells induced by HexPC was associated with induction of ER stress as shown by enhanced phosphorylation of IRE1 and eIF-2alpha. This ER stress was also prevented when LPC was added to HexPC although LPC could not prevent similar ER stress induced by bortezomib. These results underscore the importance of this phosphatidylcholine synthesis pathway in MM cells and provide new targets for future therapy. Disclosures: No relevant conflicts of interest to declare.

Cyclin-Dependent Kinase 4/6 Inhibitor Abemaciclib Exerts Dose-Dependent Cytostatic and Cytocidal Effects on Multiple Myeloma Cells Via Autophagy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4478-4478 ◽  
Author(s):  
Noriyoshi Iriyama ◽  
Hirotsugu Hino ◽  
Shota Moriya ◽  
Masaki Hiramoto ◽  
Yoshihiro Hatta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Annexin V ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Dose Dependent

Abstract Background:Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow. D-type cyclins (CCNDs), an important family of cell cycle regulators, are thought to be implicated in multiple myeloma (MM) development because CCNDs are commonly expressed in myeloma cells. CCND is known to positively regulate the cell cycle from G1 to S-phase initiation by binding to cyclin-dependent kinase (CDK) 4/6, resulting in potentiation of myeloma cell growth. These findings suggest a possible role for CDK4/6-targeting therapy in MM, yet the details remain incompletely understood. In this regard, we investigated the biological activity of abemaciclib, a potent, highly selective CDK4/6 inhibitor, in myeloma cell lines, to elucidate the mechanisms underlying the involvement of the CCND-CDK4/6 complex in cell cycle regulation and survival. Methods:The effects of abemaciclib on myeloma cells were investigated using three myeloma cell lines, KMS12-PE (CCND1-positive and CCND2-negative), RPMI8226 (CCND1-negative and CCND2-positive), and IM-9 (both CCND1- and CCND2-positive). Cell growth was assessed by trypan blue exclusion assay. Cell cycle analysis was performed using propidium iodide (PI) and apoptosis was measured using annexin V/PI staining via flow cytometry. Cell cycle regulated proteins, including p21 and p27, and phosphorylated proteins, including STAT1, STAT3, ERK, JNK, p38, and AKT, were evaluated using a phospho-flow method. Autophagy was assessed using CYTO-ID via flow cytometry. PARP cleavage was investigated via western blotting. Clarithromycin, an antibiotic agent belonging to the macrolide class, was used as an autophagy inhibitor. Results:Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all the cell lines evaluated, with significant differences seen at a concentration of 320 nM. Annexin V/PI staining revealed that 1 μM abemaciclib showed little or no effect on apoptosis, but 3.2 μM abemaciclib induced apparent myeloma cell apoptosis, with an increase in both the early and late apoptotic fractions. Therefore, 1 and 3.2 μM of abemaciclib were used in subsequent experiments for the assessment of cell growth and apoptosis, respectively. Cell cycle analyses revealed that 1 μM abemaciclib increased the fraction of cells in G0/G1 phase and decreased the fraction in S-G2/M phase. Furthermore, this effect was associated with the upregulation of p21 and p27 in the evaluated myeloma cells. PARP cleavage was observed in KMS12-PE cells treated with 3.2 μM abemaciclib, but not 1 μM, suggesting a close connection between the degree of PARP cleavage and apoptosis in myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner. However, no apparent inhibitory effect on the autophagy-related phosphorylated proteins STAT1 (Y701), STAT3 (Y705), ERK (T202/Y204), JNK (T183/Y185), p38 (T180/Y182), or AKT (Y315) was observed in myeloma cells treated with 3.2 μM abemaciclib. To investigate the role of abemaciclib-induced autophagy on myeloma cell apoptosis, we further assessed the apoptotic effect of 3.2 μM abemaciclib or 50 μg/mL clarithromycin, alone or in combination. Clarithromycin did not induce apoptosis of myeloma cells. Importantly, clarithromycin treatment in combination with abemaciclib attenuated the apoptotic effect of abemaciclib. Discussion & Conclusions: Although the underlying mechanisms conferring the level of CCND expression are known to differ greatly (e.g., CCND translocation, hyperdiploidy, or activation of upstream pathways of CCND transcription), the results of the current study indicate that the CCND-CDK4/6 complex is closely involved in myeloma cell growth and survival regardless of the CCND family member present. In addition, we demonstrate that abemaciclib exerts multiple effects, such as myeloma cell apoptosis, via the PARP pathway or autophagy, as well as cell cycle regulation. Because abemaciclib in combination with clarithromycin inhibits myeloma cell apoptosis, the autophagy induced by abemaciclib is considered to have a critical role in the induction of apoptosis, so-called "autophagic cell death." These results provide novel insights into a possible therapeutic approach using abemaciclib to target CDK4/6 in patients with MM, and offer new possibilities for combination therapy with CDK4/6 inhibitors and autophagy regulators. Disclosures Iriyama: Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Hatta:Novartis Pharma: Honoraria.

scholarly journals TMEM60 Promotes the Proliferation and Migration and Inhibits the Apoptosis of Glioma through Modulating AKT Signaling

2022 ◽  
Vol 2022 ◽  
pp. 1-11
Author(s):  
Jingwen Wu ◽  
Xinghua Tang ◽  
Xuejuan Yu ◽  
Xiaoli Zhang ◽  
Wenjun Yang ◽  
...  
Keyword(s):  
Cell Viability ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Glioma Cell ◽  
Akt Signaling ◽  
Migration And Invasion ◽  
Akt Signaling Pathway ◽  
Glioma Cell Lines

Glioma is a highly fatal malignancy with aggressive proliferation, migration, and invasion metastasis due to aberrant genetic regulation. This work aimed to determine the function of transmembrane protein 60 (TMEM60) during glioma development. The level of TMEM60 in glioma tissues and normal tissues and its correlation with glioma prognosis were checked in The Cancer Genome Atlas (TCGA) database. The levels of TMEM60 in glioma cell lines and normal astrocytes were determined by quantitative real-time PCR and western blotting assay. TMEM60 knockdown and overexpression were conducted, followed by detection of cell viability, migration, invasion, and apoptosis. CCK-8 and colony formation assay were adopted to detect cell viability proliferation. Transwell assay was performed to measure cell migration and invasion. Cell apoptosis was evaluated by flow cytometry. The alternation of key proteins in the PI3K/Akt signaling pathway was measured by western blotting. TMEM60 expression was significantly higher in glioma tissues than that in the healthy control and was correlated with poor overall survival of patients. The protein and mRNA levels of TMEM60 were both elevated in glioma cell lines in comparison with the normal cell lines. Elevated level of TMEM60 led to enhanced proliferation, migration, and invasion and suppressed cell apoptosis. TMEM60 promoted the activation of PI3K/Akt signaling. Our data suggested that TMEM60 plays an oncogenic role in glioma progression via activating the PI3K/Akt signaling pathway.

scholarly journals IMP3 accelerates the progression of prostate cancer through inhibiting PTEN expression in a SMURF1-dependent way

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Xiang Zhang ◽  
Dawei Wang ◽  
Boke Liu ◽  
Xingwei Jin ◽  
Xianjin Wang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Viability ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Expression Level ◽  
Mtor Signaling Pathway ◽  
Expression Levels ◽  
Cancer Tissues

Abstract Background Insulin-like growth factor 2 (IGF2) messenger RNA binding protein 3 (IMP3) has been testified to be overexpressed in prostate cancer and strongly related to patients’ poor prognosis. However, the functions of IMP3 and the underlying mechanisms in prostate cancer still remain unknown. Therefore, the current study was carried out to reveal the role and molecular mechanism of IMP3 in prostate cancer progression. Methods The expression levels of IMP3 in prostate cancer tissues and cells were detected by immunohistochemistry (IHC), western blotting and RT-PCR. CCK-8, clone formation, flow cytometry and in vivo tumor formation assays were used to determine cell growth, clone formation apoptosis and tumorigenesis, respectively. The effect of IMP3 on the expression levels of the key proteins in PI3K/AKT/mTOR signaling pathway, including PIP2, PIP3, p-AKT, AKT, p-mTOR, mTOR, PTEN and BAD activation of was determined by western blotting. IP (Immunoprecipitation) assay was used to evaluate the effects of IMP3 and SMURF1 (SMAD specific E3 ubiquitin protein ligase 1) on the ubiquitination of PTEN protein. Results IMP3 expression level was significantly increased in prostate cancer tissues and cell lines (LNCap, PC3 and DU145) as compared with the paracancerous normal tissues and cells (RWPE-1), respectively. High expression of IMP3 apparently promoted cell viability, tumorigenesis and inhibited cell apoptosis in prostate cancer LNCap, DU145 and PC3 cell lines. In mechanism, IMP3 upregulation significantly increased the phosphorylation levels of AKT and mTOR, and elevated PIP3 expression level, while induced significant reductions in the expression levels of BAD, PTEN and PIP2. And, IMP3 overexpression increased SMURF1 expression, which facilitated PTEN ubiquitination. In addition, SMURF1 overexpression enhanced prostate cancer cell viability and inhibited cell apoptosis. Silence of SMURF1 rescued the enhancements in cell proliferation and tumorigenesis and the inhibition in cell apoptosis rates induced by IMP3 in prostate cancer DU145 and LNCap cells. Conclusion This study reveals that IMP3 is overdressed in prostate cancer, which accelerates the progression of prostate cancer through activating PI3K/AKT/mTOR signaling pathway via increasing SMURF1-mediated PTEN ubiquitination.

Propranolol induced apoptosis and autophagy via the ROS/JNK signaling pathway in human ovarian cancer

2020 ◽  
Author(s):  
Shujun Zhao ◽  
Suzhen Fan ◽  
Yanyu Shi ◽  
Hongyan Ren ◽  
Hanqing Hong ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Human Ovarian Cancer ◽  
Dependent Manner ◽  
Flow Cytometry Analysis ◽  
Jnk Signaling ◽  
Jnk Signaling Pathway ◽  
Induced Apoptosis

Abstract Background: Propranolol has a significant anti-cancer effect on various cancers. The present study aimed to investigate the underlying mechanism behind the therapeutic effect of Propranolol on the ovarian cancer.Materials and methods: The effect of Propranolol on cell viability was examined by MTT analysis. Cellular apoptosis was evaluated by flow cytometry analysis. Autophagy was defined by autophagosome observed by confocal microscopy after infected with GFP-LC3 adenovirus. In addition, the expression of marker proteins involved in cell apoptosis, autophagy, and ROS/JNK signaling pathway were estimated by Western Blotting assay.Results: Propranolol significantly reduced the viability of human ovarian cancer cell lines SKOV-3 and A2780 in a dose- and time-dependent manner. Flow cytometry analysis revealed that Propranolol induced the cell cycle arrest at G2/M phase and resulted in apoptosis. Moreover, autophagy inhibitor 3-MA markedly enhanced the Propranolol-induced apoptosis. In addition, reactive oxygen species (ROS) was demonstrated dramatically increased after Propranolol treatment and Propranolol activated the phosphorylation of JNK. What is more, p38 inhibitor SB203580 and JNK inhibitor SP600125 attenuated the upregulated expression of LC3-II and cleaved-caspase-3 by the effect of Propranolol. ROS exclusive inhibitor antioxidant N-acetyl cysteine (NAC) weaken the phosphorylation of JNK proteins induced by Propranolol.Conclusions：In summary, our results suggested that Propranolol induced cell apoptosis and protective autophagy through the ROS/JNK signaling pathway in human ovarian cancer cells.

Characterization of Targets of Plitidepsin In JAK2V617F-Mutated Cells From Myeloproliferative Neoplasms

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4093-4093
Author(s):  
Paola Guglielmelli ◽  
Costanza Bogani ◽  
Niccolò Bartalucci ◽  
Lisa Pieri ◽  
Alessandro Pancrazzi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Western Blotting ◽  
Phase Ii ◽  
Protein Level ◽  
Gene Expression Analysis ◽  
Myeloproliferative Neoplasms ◽  
Receptor Interaction

Abstract Abstract 4093 Background: In a previous study (Verrucci M et al, J. Cell Physiol, 2010, Epub May10) we reported that the cyclic depsipeptide Aplidin®, a potent cytotoxic agent currently in phase II/III clinical trials for solid and hematologic neoplasia, displayed activity in a murine model of myelofibrosis, the GATA1-low mice. In fact, Aplidin improved the proliferation of Gata1-low hematopoietic cells, corrected abnormal microvessel density and reduced bone marrow fibrosis. These effects were largely attributed to improved maturation of megakaryocytes, as suggested by the increased platelet count in thrombocytopenic Gata1-low mice. It has been previously shown that a low expression of p27 in tumor cells might correlate with their response to Aplidin; of interest, GATA1-low megakaryocytes expressed reduced levels of p27. Overall, these results suggested that Aplidin could have the potential to alter the course of myelofibrosis-like disease in Gata1-low mice and could be useful for the treatment of myelofibrosis. Aims: to evaluate activity and targets of Aplidin in cellular models of myeloproliferative neoplasms (MPN). Methods: We measured the effect of Aplidin on the proliferation of JAK2V617F-mutated cell lines (UKE-1, HEL, SET2) and of primary cells from MPN patients in liquid cultures and semisolid medium, the rate of apoptosis using Annexin V flow cytometry analysis, and the cell cycle by propidium iodide staining. Expression of mRNA was quantified by real-time PCR, while protein level was measured by western blotting. Gene expression profiling was accomplished with Agilent Whole Human Genome Oligo Microarrays (44K). Results: Aplidin reduced the proliferation of all human V617F-mutated cell lines in the low nanomolar range, with IC50 from 0.5+/−0.03nM for UKE-1 to 1.5+/−0.05nM for HEL cells. Aplidin increased the proportion of cells in the G1/G0 phase of cell cycle, up to a mean of 80+/−5% from 60+/−3% in control cells (P<0.01). Also the proportion of apoptotic cells dose-dependently increased from 20+/−3% in control cultures to 50+/−6% at 10 nM in UKE-1 cells. After 24 h of incubation with Aplidin, the level of p27 mRNA was dose-dependently increased in all cell lines evaluated, while the BCR/ABL mutated K562 cells, whose proliferative rate was inhibited at higher Aplidin concentration, did not show significant changes of p27 mRNA. By western blotting analysis, level of p27 was overtly increased in UKE-1 cells treated with 5 and 10 nM Aplidin for 24h, and at the highest concentration we also observed reduction of phosphorylated STAT5. On the other hand, there was no change in JAK2, pJAK2, STAT5, STAT3, pSTAT3, Akt, pAkt. We then measured the level of p27 mRNA in peripheral blood cells obtained from patients with polycythemia vera (PV) or primary myelofibrosis (PMF). We found that PMF patients had significantly reduced p27 mRNA and protein level compared to controls and PV patients (P<.001 for both). After incubation with 1nM Aplidin there was a 2.5+/−2.0-fold increase in the p27 mRNA level and protein in cells of PMF patients while it was substantially unaffected in controls or PV patients. Gene expression analysis was performed in SET2 and UKE-1 cells that had been treated with 3nM of Aplidin for 24 hr. Aplidin treatment of SET2 cell line resulted in the up-regulation (UR) of 268 probsets and down-regulation (DR) of 247 (in total, about 1.5% of 40,961 genes called as “present”); corresponding figures in UKE-1 cells were 261 for UR (0.6%) and 364 (0.9%) for DR genes. MAPK signalling, TGF-beta signaling pathway, Cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), neuroactive ligand-receptor interaction, T cell receptor signaling, calcium signaling pathway, and regulation of actin cytoskeleton were the most involved pathways. Conclusions: Current results suggest that Aplidin has growth inhibitory activity and induces apoptosis in MPN cells. Response to Aplidin involves increasing p27 level, similar to findings in myelofibrotic GATA1-low mice; furthermore, we have identified low p27 expression as a characteristic of PMF cells compared to normal and PV cells. Finally, gene expression analysis allowed to identify a set of genes and involved pathways undergoing differential regulation in response to Aplidin. These information could be of value also for analyzing the response to Aplidin of PMF patients enrolled in an ongoing Phase II clinical trial. Disclosures: Aracil: PharmaMar: Employment. Vannucchi:PharmaMar: Research Funding.

Effects of 5-Bromotetrandrine and Daunorubicin on the Expression of Survivin In K562/AO2 Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4947-4947
Author(s):  
Bao-An Chen ◽  
Xiao-hui Cai ◽  
Jun Wang ◽  
Chong Gao ◽  
Jia-hua Ding ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mtt Assay ◽  
Conflicts Of Interest ◽  
Acquired Resistance ◽  
Leukemic Cells ◽  
Control Group ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Typical Experiment

Abstract Abstract 4947 Objective: The aim of this study was to investigate the expression of survivin and the apoptosis induced by DNR and BrTet in the leukemic cells K562/A02. Methods: In a typical experiment, the K562/AO2 cells were treated with daunorubicin (DNR), 5-bromotetrandrine (BrTet), or DNR and BrTet for 48 hours, and the cells treated without any drugs were used as control group. Cell proliferation was analyzed by MTT assay. Cells apoptosis and the concentration of DNR within the cells were measured by Flow cytometry (FCM). The expressions of mRNA and protein of survivin were determined by semi-quantitative reverse transcription PCR (RT-PCR) and Western blot, respectively. Results: The results of MTT assay indicated that DNR and BrTet were both able to inhibit the proliferation of K562/AO2 cells in dose-dependent manner. The fresh evidence from flow cytometry showed that a higher apoptosis rate could be induced and a higer concentration of DNR could be detected in K562/AO2 cells by DNR and BrTet as compared with those by DNR or BrTet in the same concentrations(P<0.01). RT-PCR revealed that the expression of survivin mRNA, a higer expression in K562/AO2 cells with acquired resistance to adriamycin than that in parent K-562 cells, decreased in the DNR and BrTet group (P<0.05), but there was no obvious change in other groups(P>0.05). Western bolt demonstrated that the expression of survivin protein was much lower in the DNR and BrTet group(P<0.05). Conclusion: BrTet could increase the concentration of DNR and reverse the multidrug resistance(MDR) in the K562/AO2 cells. Survivin may play an important role in apoptosis induced by DNR. Survivin could be a target for the treatment of MDR in haematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Inhibitory Effect of Hydroxysafflor Yellow B on the Proliferation of Human Breast Cancer MCF-7 Cells

2019 ◽  
Vol 14 (2) ◽  
pp. 187-197 ◽  
Author(s):  
Chuanjun Qu ◽  
Weiwei Zhu ◽  
Kaijie Dong ◽  
Zhaohai Pan ◽  
Ying Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Apoptosis ◽  
Human Breast Cancer ◽  
S Phase ◽  
Dependent Manner ◽  
Human Breast ◽  
Protein Levels ◽  
Mcf 7

Background:A recent patent has been issued for hydroxysafflor yellow A (HSYA) as a drug to prevent blood circulation disorders. Hydroxysafflor yellow B (HSYB), an isomer of HSYA with antioxidative effects, has been isolated from the florets of Carthamus tinctorius. The effects of HSYB on the proliferation of cancer cells and its mechanism of action have not been investigated.Objective:The aims of this study were to investigate the anti-cancer effects and the molecular mechanism of HSYB for breast cancer MCF-7 cells.Methods:MTT assays and colony formation assays were used to assess the survival and proliferation of MCF-7 cells, respectively. Hoechst 33258 and flow cytometry were used to measure cell apoptosis and flow cytometry to determine effects on the cell cycle. Western blots were used to measure protein levels.Results:Treatment with HSYB reduced survival and proliferation of human breast cancer MCF-7 cells in a dose-dependent manner. Furthermore, HSYB arrested the MCF-7 cell cycle at the S phase and downregulated cyclin D1, cyclin E, and CDK2. Compared with a control group, HSYB suppressed the protein levels of p-PI3K, PI3K, AKT, and p-AKT in MCF-7 cells. In addition, HSYB decreased the levels of Bcl- 2, increased the levels of Bax, cleaved caspase-3 and caspase-9, and subsequently induced MCF-7 cell apoptosis.Conclusion:These data demonstrate that HSYB arrests the MCF-7 cell cycle at the S phase and induces cell apoptosis. Patent US20170246228 indicates that HSYB can be potentially used for the prevention and treatment of human breast cancer.

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