NO66 overexpression rescues ethanol-induced cell apoptosis in human AC16 cardiomyocytes by suppressing PTEN and activating the PI3K/Akt signaling

2020 ◽  
Vol 52 (10) ◽  
pp. 1093-1101
Author(s):  
Hanyu Deng ◽  
Bo Yu ◽  
Yang Yu ◽  
Ge Tian ◽  
Liu Yang
Keyword(s):  
Cell Apoptosis ◽  
Cell Injury ◽  
Membrane Damage ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Alcohol Exposure ◽  
Ethanol Exposure ◽  
Cell Counting ◽  
Pi3k Inhibitor Ly294002 ◽  
Induced Apoptosis

Abstract Previously, Nucleolar protein 66 (NO66) was reported to be closely associated with alcohol exposure-induced injury. However, the role of NO66 in alcohol-induced cytotoxicity remains unclear. In this study, we explored the potential effect and mechanism of NO66 on ethanol-induced apoptosis in human AC16 cardiomyocytes. The AC16 cell lines with NO66 and phosphatase and tensin homolog (PTEN) overexpression were constructed. Cell counting kit-8 (CCK-8), lactate dehydrogenase (LDH) assay, Annexin V-FITC/PI staining, and flow cytometry were used to evaluate the cell viability, membrane damage, and apoptosis, respectively. Quantitative real-time PCR (qRT-PCR) and western blot analysis were applied to measure mRNA and protein expression. The results showed that acute ethanol exposure markedly augmented cytotoxicity and reduced NO66 level in AC16 cardiomyocytes. Overexpression of NO66 partially reversed ethanol-induced apoptosis. NO66 upregulation reversed the decrease in phosphorylation of protein kinase B (Akt) and B-cell lymphoma-2/Bcl-2-associated x (Bcl-2/Bax) ratio and the increase in PTEN, p53, and caspase-3 activity induced by ethanol treatment. Meanwhile, the application of PI3K inhibitor (LY294002) and PTEN overexpression attenuated the inhibition efficiency of NO66 on cell apoptosis. In addition, PTEN overexpression weakened the effect of NO66 on PI3K/Akt activation, without affecting the level of NO66. Our data suggested that NO66 overexpression might play an anti-apoptotic role in ethanol-induced cell injury via reducing PTEN and upregulating the PI3K/Akt pathway.

scholarly journals Dehydroxyhispolon Methyl Ether, A Hispolon Derivative, Inhibits WNT/β-Catenin Signaling to Elicit Human Colorectal Carcinoma Cell Apoptosis

2020 ◽  
Vol 21 (22) ◽  
pp. 8839
Author(s):  
Hueng-Chuen Fan ◽  
Ya-Chu Hsieh ◽  
Li-Hsuan Li ◽  
Ching-Chin Chang ◽  
Karolína Janoušková ◽  
...  
Keyword(s):  
Methyl Ether ◽  
Cell Apoptosis ◽  
Ectopic Expression ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Human Colon ◽  
Underlying Mechanism ◽  
Parp Cleavage ◽  
Signaling Axis ◽  
Induced Apoptosis

Colorectal cancer (CRC) is the fourth leading cause of cancer mortality worldwide. Aberrant activation of WNT/β-catenin signaling present in the vast majority of CRC cases is indispensable for CRC initiation and progression, and thus is a promising target for CRC therapeutics. Hispolon is a fungal-derived polyphenol with a pronounced anticancer effect. Several hispolon derivatives, including dehydroxyhispolon methyl ether (DHME), have been chemically synthesized for developing lead molecules with stronger anticancer activity. Herein, a DHME-elicited anti-CRC effect with the underlying mechanism is reported for the first time. Specifically, DHME was found to be more cytotoxic than hispolon against a panel of human CRC cell lines, while exerting limited toxicity to normal human colon cell line CCD 841 CoN. Additionally, the cytotoxic effect of DHME appeared to rely on inducing apoptosis. This notion was evidenced by DHME-elicited upregulation of poly (ADP-ribose) polymerase (PARP) cleavage and a cell population positively stained by annexin V, alongside the downregulation of antiapoptotic B-cell lymphoma 2 (BCL-2), whereas the blockade of apoptosis by the pan-caspase inhibitor z-VAD-fmk attenuated DHME-induced cytotoxicity. Further mechanistic inquiry revealed the inhibitory action of DHME on β-catenin-mediated, T-cell factor (TCF)-dependent transcription activity, suggesting that DHME thwarted the aberrantly active WNT/β-catenin signaling in CRC cells. Notably, ectopic expression of a dominant–active β-catenin mutant (∆N90-β-catenin) abolished DHME-induced apoptosis while also restoring BCL-2 expression. Collectively, we identified DHME as a selective proapoptotic agent against CRC cells, exerting more potent cytotoxicity than hispolon, and provoking CRC cell apoptosis via suppression of the WNT/β-catenin signaling axis.

scholarly journals Effect of Shuangdan Mingmu capsule, a Chinese herbal formula, on oxidative stress-induced apoptosis of pericytes through PARP/GAPDH pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fujiao Nie ◽  
Jiazhao Yan ◽  
Yanjun Ling ◽  
Zhengrong Liu ◽  
Chaojun Fu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Oxidative Damage ◽  
Damage Model ◽  
B Cell Lymphoma ◽  
Cell Counting ◽  
Network Pharmacology ◽  
Diabetic Patients ◽  
Induced Apoptosis

Abstract Background Diabetic retinopathy (DR) has become a worldwide concern because of the rising prevalence rate of diabetes mellitus (DM). Despite much energy has been committed to DR research, it remains a difficulty for diabetic patients all over the world. Since apoptosis of retinal microvascular pericytes (RMPs) is the early characteristic of DR, this study aimed to reveal the mechanism of Shuangdan Mingmu (SDMM) capsule, a Chinese patent medicine, on oxidative stress-induced apoptosis of pericytes implicated with poly (ADP-ribose) polymerase (PARP) / glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pathway. Methods Network pharmacology approach was performed to predict biofunction of components of SDMM capsule dissolved in plasma on DR. Both PARP1 and GAPDH were found involved in the hub network of protein-protein interaction (PPI) of potential targets and were found to take part in many bioprocesses, including responding to the regulation of reactive oxygen species (ROS) metabolic process, apoptotic signaling pathway, and response to oxygen levels through enrichment analysis. Therefore, in vitro research was carried out to validate the prediction. Human RMPs cultured with media containing 0.5 mM hydrogen oxide (H2O2) for 4 h was performed as an oxidative-damage model. Different concentrations of SDMM capsule, PARP1 inhibitor, PARP1 activation, and GAPDH inhibitor were used to intervene the oxidative-damage model with N-Acetyl-L-cysteine (NAC) as a contrast. Flow cytometry was performed to determine the apoptosis rate of cells and the expression of ROS. Cell counting kit 8 (CCK8) was used to determine the activity of pericytes. Moreover, nitric oxide (NO) concentration of cells supernatant and expression of endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), B cell lymphoma 2 (BCL2), vascular endothelial growth factor (VEGF), endothelin 1 (ET1), PARP1, and GAPDH were tested through RT-qPCR, western blot (WB), or immunocytochemistry (ICC). Results Overproduction of ROS, high apoptotic rate, and attenuated activity of pericytes were observed after cells were incubated with media containing 0.5 mM H2O2. Moreover, downregulation of SOD, NO, BCL2, and GAPDH, and upregulation of VEGFA, ET1, and PARP1 were discovered after cells were exposed to 0.5 mM H2O2 in this study, which could be improved by PARP1 inhibitor and SDMM capsule in a dose-dependent way, whereas worsened by PARP1 activation and GAPDH inhibitor. Conclusions SDMM capsule may attenuate oxidative stress-induced apoptosis of pericytes through downregulating PARP expression and upregulating GAPDH expression.

scholarly journals SNHG3 cooperates with ELAVL2 to modulate cell apoptosis and extracellular matrix accumulation by stabilizing SNAI2 in human trabecular meshwork cells under oxidative stress

2020 ◽  
Author(s):  
Sizhen Li ◽  
Qingsong Yang ◽  
Zixiu Zhou ◽  
Min Fu ◽  
Xiaodong Yang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Extracellular Matrix ◽  
Intraocular Pressure ◽  
Oxidative Damage ◽  
Trabecular Meshwork ◽  
Cell Apoptosis ◽  
Cell Injury ◽  
Quantitative Polymerase Chain Reaction ◽  
Subcellular Fractionation ◽  
Cell Counting

Abstract Background: Glaucoma is the main reason for irreversible blindness, and pathological increased intraocular pressure is the leading risk factor for glaucoma. It is reported that trabecular meshwork cell injury is closely associated with the elevated intraocular pressure. The current study aimed to investigate the role of SNHG3 in human trabecular meshwork (HTM) cells under oxidative stress. Methods: A series of experiments including real-time quantitative polymerase chain reaction (RT-qPCR), subcellular fractionation assay, western blot analysis, cell counting kit-8 (CCK-8) assay, RNA pull down, flow cytometry analysis, and RIP assay were employed to explore the biological function and regulatory mechanism of SNHG3 in HTM cells under oxidative stress.Results: First, we observed that H2O2 induced SNHG3 upregulation in HTM cells. Then, we found that SNHG3 silencing alleviated H2O2-induced oxidative damage in HTM cells. Moreover, SNAI2 knockdown alleviated the oxidative damage induced by H2O2 in HTM cells. Mechanistically, SNHG3 bound with ELAVL2 to stabilize SNAI2. Finally, SNAI2 overexpression counteracted the effect of SNHG3 silencing on H2O2-induced HTM cells. Conclusion: Our results demonstrated that SNHG3 cooperated with ELAVL2 to modulate cell apoptosis and extracellular matrix (ECM) accumulation by stabilizing SNAI2 in HTM cells under oxidative stress.

miR-30e-5p regulates autophagy and apoptosis by targeting Beclin1 involved in contrast-induced acute kidney injury

2021 ◽  
Vol 28 ◽  
Author(s):  
Xiaoqin Liu ◽  
Qingzhao Li ◽  
Lixin Sun ◽  
Limei Chen ◽  
Yue Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Kidney Injury ◽  
Cell Apoptosis ◽  
Cell Injury ◽  
Cell Model ◽  
Kidney Injury ◽  
Cell Vitality ◽  
A Cell ◽  
Renal Tubular ◽  
Induced Apoptosis

Aims: This study aims to verify if miR-30e-5p targets Beclin1 (BECN1), a key regulator of autophagy, and investigate the function of miR-30e-5p and Beclin1 through mediating autophagy and apoptosis in contrast-induced acute kidney injury (CI-AKI). Methods: Human renal tubular epithelial HK-2 cells were treated with Urografin to construct a cell model of CI-AKI. Real-time reverse transcription–polymerase chain reaction was used to detect gene expression. The dual-luciferase reporting assay and endogenous validation were used to verify targeting and regulating function. The expressions of protein were detected using Western blot. Cell proliferation was detected using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Cell apoptosis was detected using terminal-deoxynucleoitidyl transferase mediated nick end labeling assay, and autophagy was detected using transmission electron microscopy. Results: HK-2 cells exposed to Urografin for 2 h induced a significant increase in miR-30e-5p. miR-30e-5p had a targeting effect on Beclin1. Moreover, Urografin exposure can enhance cell apoptosis by increasing caspase 3 gene expression and inhibiting autophagy, which was induced by decreased Beclin1 expression regulated by miR-30e-5p, thereby resulting in renal cell injury. Downregulation of miR-30e-5p or upregulation of Beclin1 restored cell vitality by promoting autophagy and suppressing apoptosis in Urografin-treated cells. Conclusions: Urografin increased the expression of miR-30e-5p in HK-2 cells and thus decreased Beclin1 levels to inhibit autophagy, but induced apoptosis, which may be the mechanism for CI-AKI.

scholarly journals T-2 Toxin Exposure Induces Apoptosis in TM3 Cells by Inhibiting Mammalian Target of Rapamycin/Serine/Threonine Protein Kinase(mTORC2/AKT) to Promote Ca2+Production

2018 ◽  
Vol 19 (11) ◽  
pp. 3360 ◽  
Author(s):  
Ji Wang ◽  
Chenglin Yang ◽  
Zhihang Yuan ◽  
Jine Yi ◽  
Jing Wu
Keyword(s):  
Cell Injury ◽  
Mammalian Target Of Rapamycin ◽  
Annexin V ◽  
Target Of Rapamycin ◽  
Dependent Manner ◽  
Toxin Exposure ◽  
Concentration Changes ◽  
Vitro Model ◽  
Induced Apoptosis

Although mTOR (the mammalian target of rapamycin) can regulate intracellular free Ca2+concentration in normal cultured podocytes, it remains elusive as to how mTORC2/AKT-mediated Ca2+participates in the process of T-2 toxin-induced apoptosis. The potential signaling responsible for intracellular Ca2+ concentration changes was investigated using immunoblot assays in an in vitro model of TM3 cell injury induced by T-2 toxin. Changes in Ca2+ were assessed using the Ca2+-sensitive fluorescent indictor dye Fura 2-AM. The cytotoxicity of TM3 cells was assessed with an MTT bioassay, and apoptosis was measured using Annexin V-FITC staining. Following T-2 toxin treatment, the growth of cells, phospho-mTORSer2481, phospho-mTORSer2448, and phospho-AktSer473 were significantly decreased in a time-dependent manner, whereas Ca2+ and apoptosis were increased. T-2 toxin-induced apoptosis was prevented by BAPTA-AM (a Ca2+chelator) and MHY1485 (an mTOR activator), and the application of mTOR activator MHY1485 also prevented the increase of intracellular free Ca2+concentration in TM3 cells. Our results strongly suggest that T-2 toxin exposure induces apoptosis in TM3 cells by inhibiting mTORC2/AKT to promote Ca2+ production.

scholarly journals Dihydrochalcone Derivative Induces Breast Cancer Cell Apoptosis via Intrinsic, Extrinsic, and ER Stress Pathways but Abolishes EGFR/MAPK Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-18 ◽  
Author(s):  
Wasitta Rachakhom ◽  
Patompong Khaw-on ◽  
Wilart Pompimon ◽  
Ratana Banjerdpongchai
Keyword(s):  
Breast Cancer ◽  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Mapk Pathway ◽  
Annexin V ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Mcf 7

Dihydrochalcone derivatives are active compounds that have been purified from the Thai medicinal plant Cyathostemma argenteum. The objectives of this study were to investigate the effects of two dihydrochalcone derivatives on human breast cancer MDA-MB-231 and MCF-7 cell proliferation and to study the relevant mechanisms involved. The two dihydrochalcone derivatives are 4′,6′-dihydroxy-2′,4-dimethoxy-5′-(2″-hydroxybenzyl)dihydrochalcone (compound 1) and calomelanone (2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, compound 2), both of which induced cytotoxicity toward both cell lines in a dose-dependent manner by using MTT assay. Treatment with both derivatives induced apoptosis as determined by annexin V-FITC/propidium iodide employing flow cytometry. The reduction of mitochondrial transmembrane potential (staining with 3,3′-dihexyloxacarbocyanine iodide, DiOC6, employing a flow cytometer) was established in the compound 1-treated cells. Compound 1 induced caspase-3, caspase-8, and caspase-9 activities in both cell lines, as has been determined by specific colorimetric substrates and a spectrophotometric microplate reader which indicated the involvement of both the extrinsic and intrinsic pathways. Calcium ion levels in mitochondrial and cytosolic compartments increased in compound 1-treated cells as detected by Rhod-2AM and Fluo-3AM intensity, respectively, indicating the involvement of the endoplasmic reticulum (ER) stress pathway. Compound 1 induced cell cycle arrest via enhanced atm and atr expressions and by upregulating proapoptotic proteins, namely, Bim, Bad, and tBid. Moreover, compound 1 significantly inhibited the EGFR/MAPK signaling pathway. In conclusion, compound 1 induced MDA-MB-231 and MCF-7 cell apoptosis via intrinsic, extrinsic, and ER stress pathways, whereas it ameliorated the EGFR/MAPK pathway in the MCF-7 cell line. Consequently, it is believed that compound 1 could be effectively developed for cancer treatments.

scholarly journals Endoplasmic reticulum stress regulates cell injury in lipopolysaccharide-induced nerve cells

2020 ◽  
Vol 48 (9) ◽  
pp. 030006052094976
Author(s):  
Min Li ◽  
Ying Zhang ◽  
Jixing Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Viability ◽  
Er Stress ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Cell Injury ◽  
Cell Counting ◽  
Tunel Staining ◽  
Dependent Manner ◽  
Stress Markers

Objective Sepsis-associated encephalopathy (SAE) is a common complication of sepsis, and excessive endoplasmic reticulum (ER) stress is closely correlated with the cell injury caused by sepsis. This study aimed to analyze the possible role of ER stress in SAE cell models. Methods PC12 and MES23.5 cells were treated with increasing concentrations of lipopolysaccharides (LPS). The Cell Counting Kit-8 assay was used to detect cell viability and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to assess cell apoptosis. In addition, the protein expression levels of ER stress markers [GRP78, CHOP, inositol-requiring enzyme 1 (IRE1), and PKR-like ER kinase (PERK)] and apoptosis-related proteins (Bax, Bcl-2, caspase-3, and cleaved caspase-3) were analyzed using western blotting. Results LPS treatment activated ER stress markers in both the PC12 and MES23.5 cells. The overexpression of GRP78 significantly reduced cell viability and enhanced cell apoptosis in a time-dependent manner. An ER stress inhibitor, 4-PBA, significantly enhanced cell viability and inhibited the cell apoptosis induced by LPS. Therefore, an enhanced unfolded protein response (UPR) and UPR suppression may regulate cell apoptosis. Conclusions UPR was shown to be involved in regulating LPS-induced neuron injury. UPR could be a potential therapeutic target in SAE.

scholarly journals 20(S)-Ginsenoside Rg3 Promotes HeLa Cell Apoptosis by Regulating Autophagy

Molecules ◽  
2019 ◽  
Vol 24 (20) ◽  
pp. 3655 ◽  
Author(s):  
Bian ◽  
Zhao ◽  
Li ◽  
Lu ◽  
Wang ◽  
...  
Keyword(s):  
Hela Cell ◽  
Cell Apoptosis ◽  
Cell Counting ◽  
Ginsenoside Rg3 ◽  
Species Determination ◽  
Protein Levels ◽  
Autophagy Inhibitor ◽  
Autophagy Induction ◽  
Apoptotic Pathways ◽  
Induced Apoptosis

20(S)-Ginsenoside Rg3 (GRg3) has various bioactivities including anti-cancer effects and inhibition of autophagy. However, no reports have investigated the appearance of autophagy or the connection between autophagy and apoptosis in HeLa cells treated with 20(S)-GRg3. Cell viability was measured by CCK-8 (cell counting kit-8) assays. Apoptosis and the cell cycle were analyzed by Hoechst 33342 staining and flow cytometry. Apoptotic pathways were examined by ROS (reactive oxygen species) determination and rhodamine 123 assays. Western blot analysis was used to determine changes in protein levels. Autophagy induction was monitored by acidic vesicular organelle staining and EGFP-LC3 transfection. 20(S)-GRg3 inhibited autophagy of cells in a starved state, making it impossible for cells to maintain a steady state through autophagy, and then induced apoptosis. 20(S)-GRg3 blocked the late stage of autophagy (fusion of lysosomes and degradation of autophagic lysosomes), including a decrease in acidic vesicular organelle fluorescence, increased LC3 I–II conversion, accumulation of EGFP-LC3 fluorescence, GFP-mRFP-LC3 red-green fluorescence ratio, degradation of the substrate p62, and loss of the balance between autophagy and apoptosis, which induced apoptosis. ROS increased, the mitochondrial membrane potential decreased, apoptotic inducer AIF was released from mitochondria, and nuclear transfer occurred, triggering a series of subsequent apoptotic events. Autophagy inducer rapamycin inhibited the apoptosis induced by 20(S)-GRg3, whereas autophagy inhibitor BA1 promoted apoptosis induced by 20(S)-GRg3. Therefore, 20(S)-GRg3 promoted HeLa cell apoptosis by regulating autophagy. In the autophagic state, 20(S)-GRg3 can be used as a novel autophagy inhibitor in synergy with tumor-blocking therapies such as chemotherapy, which supports its application in the medical field.

scholarly journals Acetaldehyde accelerates HCV-induced impairment of innate immunity by suppressing methylation reactions in liver cells

2015 ◽  
Vol 309 (7) ◽  
pp. G566-G577 ◽  
Author(s):  
Murali Ganesan ◽  
Jinjin Zhang ◽  
Tatiana Bronich ◽  
Larisa I. Poluektova ◽  
Terrence M. Donohue ◽  
...  
Keyword(s):  
Alcohol Exposure ◽  
Liver Cells ◽  
Ethanol Exposure ◽  
Ethanol Metabolism ◽  
In Vivo Studies ◽  
Temporary Increase ◽  
Hcv Rna ◽  
Induced Apoptosis ◽  
Induced Defects

Alcohol exposure worsens the course and outcomes of hepatitis C virus (HCV) infection. Activation of protective antiviral genes is induced by IFN-α signaling, which is altered in liver cells by either HCV or ethanol exposure. However, the mechanisms of the combined effects of HCV and ethanol metabolism in IFN-α signaling modulation are not well elucidated. Here, we explored a possibility that ethanol metabolism potentiates HCV-mediated dysregulation of IFN-α signaling in liver cells via impairment of methylation reactions. HCV-infected Huh7.5 CYP2E1+ cells and human hepatocytes were exposed to acetaldehyde (Ach)-generating system (AGS) and stimulated with IFN-α to activate IFN-sensitive genes (ISG) via the Jak-STAT-1 pathway. We observed significant suppression of signaling events by Ach. Ach exposure decreased STAT-1 methylation via activation of protein phosphatase 2A and increased the protein inhibitor of activated STAT-1 (PIAS-1)-STAT-1 complex formation in both HCV+ and HCV− cells, preventing ISG activation. Treatment with a promethylating agent, betaine, attenuated all examined Ach-induced defects. Ethanol metabolism-induced changes in ISGs are methylation related and confirmed by in vivo studies on HCV+ transgenic mice. HCV- and Ach-induced impairment of IFN signaling temporarily increased HCV RNA levels followed by apoptosis of heavily infected cells. We concluded that Ach potentiates the suppressive effects of HCV on activation of ISGs attributable to methylation-dependent dysregulation of IFN-α signaling. A temporary increase in HCV RNA sensitizes the liver cells to Ach-induced apoptosis. Betaine reverses the inhibitory effects of Ach on IFN signaling and thus can be used for treatment of HCV+ alcohol-abusing patients.

Fenretinide (4HPR) Inhibits Growth of Myeloma Cells in Their Microenvironment and Is a Potent Inhibitor of Angiogenesis and Osteoclastogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3480-3480
Author(s):  
Xin Li ◽  
Wen Ling ◽  
Rinku Saha ◽  
Paul Perkins ◽  
Angela Pennisi ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Mtt Assay ◽  
Cell Apoptosis ◽  
Mononuclear Cells ◽  
Annexin V ◽  
Dependent Manner ◽  
Angiogenic Potential ◽  
Inhibition Of Angiogenesis ◽  
Induced Apoptosis

Abstract Fenretinide (4HPR) is a relatively safe neoclassical retinoid analog that inhibits growth of various tumors through increased intracellular ceramide and ROS, induction of tumor cell apoptosis and inhibition of angiogenesis. 4HPR has been successfully tested as a chemopreventive and chemotherapeutic agent in clinical trials on various malignancies. In contrast to retinoic acid, 4HPR induces cell apoptosis rather than differentiation and shows synergistic responses with chemotherapeutic drugs in different tumor cell types. The biological effect and therapeutic value in multiple myeloma (MM) has not been investigated. The aim of this study was to investigate the anti-MM effect and mechanism of action of 4HPR using 3 stroma-dependent and 2 stroma-independent MM cell lines established in our laboratory, CD138-selected primary MM cells and co-culture systems of these cells with human osteoclasts and mesenchymal stem cells (MSCs) as previously described (Yaccoby et al., Cancer Res 2004). MM cell apoptosis detected by annexin V flow cytometry and TUNNEL, tumor growth by MTT assay, changes in caspase 3, 8 and 9 activity using Western blotting and ROS production by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay. 4HPR inhibits growth of all tested MM cells in a dose- and time-dependent manner. The IC50 after 48 hrs in serum-containing media was 10 μM using MTT assay. 4HPR (3 μM) increased percent of apoptotic MM cells by 2.5±0.4 folds (p<0.01). Co-culture of these cell lines with osteoclasts only partially protected MM cells from the proapoptotic effect of this drug. Furthermore, 4HPR also induced apoptosis of primary CD138-selected MM cells co-cultured with osteoclasts or MSCs, and inhibited growth of bortezomib-resistant MM cell lines. In contrast, 4HPR had only minimal cytotoxic effect on blood mononuclear cells and MSCs. The proapoptotic effect of 4HPR involved increased level of ROS by 2.55±0.67 folds in MM cells (p<0.01). We also detected reduced levels of procaspase and increased cleaved caspase 8, 9 and 3 within 24 hrs of incubation with this drug. Sphingosine-1 phosphate (S1P) partially protected MM cells from 4HPR-induced apoptosis suggesting that, as reported for other tumors, anti-MM mechanism of this drug involved increased intracellular ceramide. 4HPR significantly inhibited tube formation by HUVEC in a matrigel assay (p<0.0001), confirming its anti-angiogenic potential. This drug also effectively prevented formation of multinucleated osteoclasts in culture of human osteoclast precursors with RANKL and M-CSF (p<0.0001). Furthermore, mature osteoclasts viability as assessed by MTT assays was reduced following incubation with 3 μM 4HPR (p<0.0001). We conclude that 4HPR is a potent anti-MM agent, affecting growth of MM cells in their microenvironment directly through induction of apoptosis in mechanisms involving ROS, caspase and possibly ceramide, and indirectly through inhibition of angiogenesis and osteoclastogenesis. Our data also suggests that S1P, which is highly produced by activated platelets, is an important survival factor for MM cells. Study is underway to test anti-MM efficacy of 4HPR in the SCID-hu model for primary myeloma.

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