Quantitative Analysis of Isopimpinellin from Ammi majus L. Fruits and Evaluation of Its Biological Effect on Selected Human Tumor Cells.

Ammi majus L. (Apiaceae) is a medicinal plant with a well-documented history in phytotherapy. The aim of the present work was to isolate isopimpinellin (5,8-methoxypsoralen; IsoP) from the fruit of this plant and evaluate its biological activity against selected tumor cell lines. The methanol extract obtained with the use of an accelerated solvent extraction (ASE) method was the most suitable for the quantitative analysis of coumarins in the A. majus fruit matrix. The coumarin content was estimated by RP-HPLC/DAD, and the amount of IsoP was found to be 404.14 mg/100 g dry wt., constituting 24.56% of the total coumarin fraction (1.65 g/100 g). This, along with the presence of xanthotoxin (368.04 mg/100 g, 22.36%) and bergapten (253.05 mg/100 g, 15.38%), confirmed A. majus fruits as an excellent source of these compounds. IsoP was isolated (99.8% purity) by combined liquid chromatography/centrifugal partition chromatography (LC/CPC) and tested for the first time on its antiproliferative activity against human colorectal adenocarcinoma (HT29, SW620), osteosarcoma (Saos-2, HOS), and multiple myeloma (RPMI8226, U266) cell lines. MTT assay results (96 h incubation) demonstrated a dose- and cell line-dependent decrease in cell proliferation/viability, with the strongest effect of IsoP against the Saos-2 cell line (IC50; 42.59 µM), medium effect against U266, HT-29, and RPMI8226 (IC50 = 84.14, 95.53, and 105.0 µM, respectively), and very weak activity against invasive HOS (IC50; 321.6 µM) and SW620 (IC50; 711.30 µM) cells, as well as normal human skin fibroblasts (HSFs), with IC50; 410.7 µM. The mechanistic study on the Saos-2 cell line showed that IsoP was able to reduce DNA synthesis and trigger apoptosis via caspase-3 activation. In general, IsoP was found to have more potency towards cancerous cells (except for HOS and SW620) than against healthy cells. The Selective Index (SI) was determined, underlining the higher selectivity of IsoP towards cancer cells compared to healthy cells (SI = 9.62 against Saos-2). All these results suggest that IsoP might be a promising molecule in the chemo-prevention and treatment of primary osteosarcoma.

Anti-cancer potential of selenium-chitosan-polyethylene glycol-carvacrol nanocomposites in multiple myeloma U266 cells.

Multiple myeloma (MM) is an incurable cancer that is characterized by malignant plasma cell proliferation. Approximately 10% of all blood cancers are MM, and there is no standard curative therapy. In this work, we intended to synthesize, characterize, and assess the anticancer effects of selenium/chitosan/polyethylene glycol-carvacrol nanocomposites (SCP-Car-NCs) on MM U266 cells in vitro. Various characterization techniques were used to characterize the synthesized SCP-Car-NCs. Several in vitro free radical scavenging experiments were conducted to test the ability of synthesized SCP-Car-NCs to scavenge the different free radicals. The cytotoxicity of SCP-Car-NCs was assessed on Vero and U266 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. By using various fluorescence staining techniques, the amount of reactive oxygen species (ROS) generation, MMP, and apoptosis were measured. Using commercial test kits, the levels of oxidative stress and apoptotic biomarkers in control and treated U266 cells were assessed. The highest peak in the UV spectral analysis was found to be at 271 nm, demonstrating the development of SCP-Car-NCs. Fourier transform infrared analysis showed that the synthesized SCP-Car-NCs contained a variety of stretching and bonding. The X-ray diffraction study confirmed the crystallinity of SCP-Car-NCs. The dynamic light scattering analysis showed that the SCP-Car-NCs had an average size of 171 nm. The different free radicals, such as the 2,2-diphenyl-1-picrylhydrazyl, hydroxyl, and peroxyl radicals, were significantly scavenged by the SCP-Car-NCs. According to the MTT assay results, the SCP-Car-NCs decreased the viability of U266 cells while having no impact on the proliferation of Vero cells. The SCP-Car-NCs significantly boosted ROS production, decreased the MMP level, and promoted apoptosis, as evidenced by the fluorescence staining experiments. In U266 cells treated with SCP-Car-NCs, the level of thiobarbituric acid reactive substances increased while superoxide dismutases and glutathione levels were reduced. In the SCP-Car-NCs treated U266 cells, it was found that the Bax, caspase-3, and -9 activities had increased while the Bcl-2 level had decreased. In conclusion, our findings show that SCP-Car-NCs treatment reduced the viability and increased apoptosis in the U266 cells, providing a new insight on SCP-Car-NCs' potential for usage in the future to treat MM.

Evaluation of the Biological Effect of Non-UV-Activated Bergapten on Selected Human Tumor Cells and the Insight into the Molecular Mechanism of Its Action.

There is some evidence that non-photoactivated psoralens may be active against breast and colon tumor cells. Therefore, we evaluated the antiproliferative, proapoptotic, and anti-migrative effect of 5-methoxypsoralen (5-MOP) isolated from Peucedanum tauricum MB fruits in human colorectal adenocarcinoma (HT-29 and SW620), osteosarcoma (Saos-2 and HOS), and multiple myeloma (RPMI8226 and U266). Dose- and cell-line-dependent effects of 5-MOP on viability and proliferation were observed, with the strongest inhibitory effect against Saos-2 and a moderate effect against the HOS, HT-29, and SW620 cells. Multiple myeloma showed low sensitivity. The high viability of human normal cell cultures (HSF and hFOB) in a wide range of 5-MOP concentrations tested (6.25-100 µM) was confirmed. Moreover, the migration of treated Saos-2, SW620, and HT-29 cell lines was impaired, as indicated via a wound healing assay. Flow cytometry analysis conducted on Saos-2 cells revealed the ability of 5-MOP to block the cell cycle in the G2 phase and trigger apoptosis, which was accompanied by a loss of mitochondrial membrane potential, caspases (-9 and -3) activation, the altered expression of the Bax and Bcl-2 proteins, and decreased AKT phosphorylation. This is the first report evaluating the antiproliferative and antimigratory impact of non-UV-activated bergapten on the abovementioned (except for HT-29) tumor cells, which provides new data on the potential role of 5-MOP in inhibiting the growth of various types of therapeutic-resistant cancers.

Effects of In Vitro Digestion on Anti-α-Amylase and Cytotoxic Potentials of Sargassum spp.

This is the first study to examine the effects of in vitro digestion on biological activities of Sargassum spp., a broadly known brown seaweed for therapeutic potential. Three fractions (F1-F3) were obtained from hexane extract by column chromatography. Under in vitro simulated digestion, the anti-α-amylase capacity of F1 in oral and intestinal phases increases, while it significantly decreases in the gastric phase. The α-amylase inhibition of F2 promotes throughout all digestive stages while the activity of F3 significantly reduces. The cytotoxic activity of F1 against U266 cell-line accelerates over the oral, gastric, and intestinal stages. The fractions F2 and F3 exhibited the declined cytotoxic potentialities in oral and gastric phases, but they were strengthened under intestinal condition. Palmitic acid and fucosterol may play an active role in antidiabetic and cytotoxic activity against multiple myeloma U266 cell line of Sargassum spp. However, the involvement of other phytochemicals in the seaweed should be further investigated.

Antioxidant Potential of Four Species of Natural Product and Therapeutic Strategies for Cancer through Suppression of Viability in the Human Multiple Myeloma Cell Line U266.

OBJECTIVE: This research aimed to evaluate the protective effects of bioactive compounds such as phenolic acids, flavonoids, and tannins present in four species extracted with methanol. METHODS: The total phenolic content of the methanolic extracts was measured spectrophotometrically. The effect of the extracts on cell viability in U266 cells was measured. The effects of extracts on free radical scavenging were assessed by the DPPH test and FRAP assay. Antibacterial effects of the natural products in this report were investigated by using the disc diffusion method. RESULTS: Our results clearly demonstrated that the methanolic extracts were characterized by a high amount of phenolic compounds. It has been speculated that ME-TA and ME-TAl exhibit a significant (P < 0.05) and dose-dependent antiradical potential. The exposure of cells to high doses of extracts almost completely suppressed cell growth in vitro. ME-TA and ME-TAl showed significant cytotoxic effects at a concentration of 100 µg/mL in the U266 cell line. ME-TAl and ME-CF inhibited the growth of B. subtilis and S. aureus, respectively, to the same extent as 10 µg/µL of chloramphenicol at a concentration of 1 mg/mL. CONCLUSION: Overall, these results suggest that plants used in traditional medicine have a novel application as free radical scavengers, bacterial inhibitors and tumor suppressors.

2,4-Dihydroxy-3'-methoxy-4'-ethoxychalcone suppresses cell proliferation and induces apoptosis of multiple myeloma via the PI3K/akt/mTOR signaling pathway.

Context: Caragana pruinosa Kom. (Fabaceae), a commonly used folk medicine, has been found to possess antitumor effects. However, the antiproliferative effect of 2,4-dihydroxy-3'-methoxy-4'-ethoxychalcone (DMEC) derived from C. pruinosa against multiple myeloma (MM) has never been investigated. Objective: This study systematically evaluates the antiproliferative effect of DMEC against MM cells. Materials and methods: The antiproliferative effect of DMEC (1, 2, 4, 8, 16, 32, and 64 µM) on MM cells lines, including RPMI8226, MM.1S, and U266, was examined using Cell counting kit-8 (CCK-8) assay after 24 h incubation. The proapoptotic effect of DMEC (20 µM) was determined using fluorescent microscope and flow cytometer, and its possible underlying mechanisms were further studied by using western blotting analysis. Results: The half maximal inhibitory concentrations (IC50) of DMEC on RPMI8226, MM.1S, and U266 cells were calculated as 25.97, 18.36, and 15.02 µM, respectively. The inhibitory effect of DMEC on MM cells was related to mitochondria-mediated apoptosis via upregulation of the cleaved-caspase-3 (C-3), cleaved-caspase-9 (C-9), Bad, and cytochrome C (Cyto C), but downregulation of the Bcl-2 and poly ADP-ribose polymerase (PARP). Furthermore, DMEC (5, 10, and 20 µM) reduced the expression of phosphatidylinositol-3-kinase (PI3K), phosphorylated (p)-protein kinase B (Akt), and p-mammalian target of rapamycin (p-mTOR), which were further evidenced by pretreatment with IGF-1, a PI3K activator. Conclusion: Collectively, our results indicate that the DMEC could be treated as a new candidate for treatment of multiple myeloma in the future. Also, an in vivo study is warranted in the future.

Alkaline Phosphatase Activity of Normal and Malignant Human Lymphocytes.

Bone marrow aspirates form patients with multiple myeloma were obtained and lymphocytes were isolated using density gradient centrifugation. The APase activity of the samples ranged from 12 to 88 nmol/0.2 × 106 cells as compared to the very low activity present in normal human peripheral blood lymphocytes PBL. Neither the unstimulated nor the mitogen stimulated lymphocytes from peripheral blood of normal humans showed appreciable APase activity. The lymphocytes from bone marrow of multiple myeloma patients as well as myeloma cell lines-RPMI 8226 and U266 B1 express APase activity constitutively. The results of the present investigation are discussed in the light of existing literature.

Suppressive effect of ethanol extract from mango (Mangifera indica L.) peel on IgE production in vitro and in vivo.

Immunoglobulin E (IgE) is involved in the onset of allergic reaction, and the suppression of IgE production leads to alleviation of allergic symptoms. We found that mango peel ethanol extract (MPE) significantly suppresses IgE production by human myeloma cell line U266 cells, suggesting that MPE has an anti-allergic effect by inhibiting the production of IgE. Although mangiferin is contained in mango, which suppresses IgE production by U266 cells, it was not contained in MPE. We investigated the suppressive effect of MPE in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model mice. The elevation of serum IgE level was significantly suppressed by oral administration of MPE. Intake of MPE also suppressed the expression level of IL-4 in the DNFB-challenged ears, suggesting that MPE suppresses the IL-4-mediated maturation into IgE-producing cells. Our findings indicate that MPE has a potential to alleviate the increase in serum IgE level that is feature of type I allergy.

Latent cytomegalovirus infection enhances anti-tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans.

Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression.

Acute exercise preferentially redeploys NK-cells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells.

NK-cells undergo a "licensing" process as they develop into fully-functional cells capable of efficiently killing targets. NK-cell differentiation is accompanied by an increased surface expression of inhibitory killer immunoglobulin-like receptor (KIR) molecules, which is positively associated with cytotoxicity against the HLA-deficient K562 cell line. NK-cells are rapidly redeployed between the blood and tissues in response to acute exercise, but it is not known if exercise evokes a preferential trafficking of differentiated NK-cells or impacts NK-cell cytotoxic activity (NKCA) against HLA-expressing target cells. Sixteen healthy cyclists performed three 30-min bouts of cycling exercise at -5%, +5%, and +15% of lactate threshold. Blood samples obtained before, immediately after, and 1h after exercise were used to enumerate NK-cells and their subsets, and determine NKCA and degranulating subsets (CD107+) against cell lines of multiple myeloma (U266 and RPMI-8226), lymphoma (721.221 and 221 AEH), and leukemia (K562) origin by 4 and 10-color flow cytometry, respectively. Exercise evoked a stepwise redeployment of NK-cell subsets in accordance with differentiation status [highly-differentiated (KIR+/NKG2A-) >medium-differentiated (KIR+/NKG2A+)>low-differentiated (KIR-/NKG2A+)] that was consistent across all exercise intensities. NKCA per cell increased ∼1.6-fold against U266 and 221 AEH targets 1h post-exercise and was associated with a decreased proportion of NK-cells expressing the inhibitory receptor CD158b and increased proportion of NK-cells expressing the activating receptor NKG2C, respectively. We conclude that exercise evokes a preferential redeployment of NK-cell subsets with a high differentiation phenotype and augments cytotoxicity against HLA-expressing target cells. Exercise may serve as a simple strategy to enrich the blood compartment of highly cytotoxic NK-cell subsets that can be harvested for clinical use.

Reactive oxygen species-mediated activation of AMP-activated protein kinase and c-Jun N-terminal kinase plays a critical role in beta-sitosterol-induced apoptosis in multiple myeloma U266 cells.

Although beta-sitosterol has been well known to have anti-tumor activity in liver, lung, colon, stomach, breast and prostate cancers via cell cycle arrest and apoptosis induction, the underlying mechanism of anti-cancer effect of beta-sitosterol in multiple myeloma cells was never elucidated until now. Thus, in the present study, the role of reactive oxygen species (ROS) in association with AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK) pathways was demonstrated in beta-sitosterol-treated multiple myeloma U266 cells. Beta-sitosterol exerted cytotoxicity, increased sub-G1 apoptotic population and activated caspase-9 and -3, cleaved poly (ADP-ribose) polymerase (PARP) followed by decrease in mitochondrial potential in U266 cells. Beta-sitosterol promoted ROS production, activated AMPK, acetyl-CoA carboxylase (ACC) and JNK in U266 cells. Also, beta-sitosterol attenuated the phosphorylation of AKT, mammalian target of rapamycin and S6K, and the expression of cyclooxygenase-2 and VEGF in U266 cells. Conversely, AMPK inhibitor compound C and JNK inhibitor SP600125 suppressed apoptosis induced by beta-sitosterol in U266 cells. Furthermore, ROS scavenger N-acetyl L-cysteine attenuated beta-sitosterol-mediated sub-G1 accumulation, PARP cleavage, JNK and AMPK activation in U266 cells. Overall, these findings for the first time suggest that ROS-mediated activation of cancer metabolism-related genes such as AMPK and JNK plays an important role in beta-sitosterol-induced apoptosis in U266 multiple myeloma cells.

Effect of human bone marrow mesenchymal stem cell-derived microvesicles on the apoptosis of the multiple myeloma cell line U266.

BACKGROUND: Microvesicles are membraned particles produced by different types of cells recently investigated for anticancer purposes. The current study aimed to investigate the effects of human bone marrow mesenchymal stem cell-derived microvesicles (BMSC-MVs) on the multiple myeloma cell line U266. BMSC-MVs were isolated from BMSCs via ultracentrifugation and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). U266 cells were treated with 15, 30, 60, and 120 µg/mL BMSC-MVs for three and seven days and the effects of treatment in terms of viability, cytotoxicity, and DNA damage were investigated via the MTT assay, lactate dehydrogenase (LDH) assay, and 8­hydroxy-2'-deoxyguanosine (8­OHdG) measurement, respectively. Moreover, the apoptosis rate of the U266 cells treated with 60 µg/mL BMSC-MVs was also assessed seven days following treatment via flow cytometry. Ultimately, the expression level of BCL2, BAX, and CCND1 by the U266 cells was examined seven days following treatment with 60 µg/mL BMSC-MVs using qRT-PCR. RESULTS: BMSC-MVs had an average size of ~ 410 nm. According to the MTT and LDH assays, BMSC-MV treatment reduced the U266 cell viability and mediated cytotoxic effects against them, respectively. Moreover, elevated 8­OHdG levels following BMSC-MV treatment demonstrated a dose-dependent increase of DNA damage in the treated cells. BMSC-MV-treated U266 cells also exhibited an increased apoptosis rate after seven days of treatment. The expression level of BCL2 and CCND1 decreased in the treated cells whereas the BAX expression demonstrated an incremental pattern. CONCLUSIONS: Our findings accentuate the therapeutic benefit of BMSC-MVs against the multiple myeloma cell line U266 and demonstrate how microvesicles could be of therapeutic advantage. Future in vivo studies could further corroborate these findings.

Alkali-soluble pectin suppresses IgE production in human myeloma cell line in vitro.

We found that strawberry (Fragaria x ananassa) extract has an IgE production suppressive activity and its oral administration improved skin manifestation in atopic dermatitis model mice. In present study, we identified an active substance using the IgE-producing human myeloma cell line U266. Gel filtration experiment indicated that the IgE suppressor was more than 6 kDa in molecular size. In addition, its pectinase treatment inhibited the activity, suggesting that the active substance in strawberry extract is pectin. Among solutions of water-(WP), hexametaphosphate-(HXP), acid-(HP) and alkali soluble pectin (OHP) extracted from strawberry, only OHP suppressed IgE production, and their suppressive activity was cancelled by pectinase treatment. In addition, OHP extracted from apple also inhibited IgE production. Furthermore, OHP also suppressed IgE production and did not affect IgG and IgM production in human peripheral blood mononuclear cells in an in vitro immunization condition. From these results, we concluded that OHP was an IgE suppressor in strawberry extract.

Blocking MET receptor signaling in multiple myeloma cells in vitro and in vivo.

BACKGROUND: Numerous studies have shown a role of the hepatocyte growth factor (HGF) as a ligand for the MET receptor in promoting aggressiveness in myeloma cells. OBJECTIVES: The aim of this study was to confirm the presence of the MET receptor in myeloma cell lines, to establish a stable lentiviral construct directed against MET receptor mRNA and then to evaluate the effect of blocking MET receptor expression both in vitro and in vivo. MATERIAL AND METHODS: The U266 and INA6 cells were transduced using a lentiviral vector carrying siRNA to achieve the reduction of MET receptor expression. The ocular sinus of NOD/SCID mice was injected with wt-U266, shMET-U266 and shLacZ-U266 cells. RESULTS: MET receptor expression was demonstrated in all tested myeloma cell lines. Blocking the HGF/MET axis did not affect the growth of transduced U266 and INA6 cell lines. The inoculation of NOD/SCID mice with myeloma cells with reduced expression of MET led to increased survival of the animals. CONCLUSIONS: MET receptor expression was constituently expressed in all tested myeloma cell lines. A lentiviral construct can effectively reduce the expression of the MET receptor in myeloma cells. Further studies are necessary to evaluate the effect of the reduction of MET receptor expression in multiple myeloma, focusing on animal models with a larger test group size.

Immunosuppressive effect of extracts from leaves of Fraxinus Mandshurica Rupr.

Plants provide a rich resource of medicinal material for research and development of new medicine. To discover new compounds as Immunosuppressant from plants, we evaluated the immunosuppressive effect of different fractions and particularly one compound (Calceolarioside A) that were extracted from the leaves of Fraxinus Mandshurica Rupr. The fractions and the compound were tested on the ability to reduce Immunoglobulin E (IgE) secretion by human U266 multiple myeloma cells (U266 cells) and to reduce interleukin-2 (IL-2) secretion by mouse spleen cells. Our results showed that both the butanol extract fraction and the compound of Calceolarioside A inhibited the IgE and IL-2 production in U266 cells and mouse spleen cells respectively, and no cytotoxicity was observed within the effective dose range. These results suggest that Calceolarioside A could potentially serve as an immunosuppressant.

Regulation of STAT3 and NF-κB activations by S-nitrosylation in multiple myeloma.

Numerous reports suggest that aberrant activations of STAT3 and NF-κB promote survival and proliferation of multiple myeloma (MM) cells. In the present report, we demonstrate that a synthetic S-nitrosothiol compound, S-nitroso-N-acetylcysteine (SNAC), inhibits proliferation and survival of multiple MM cells via S-nitrosylation-dependent inhibition of STAT3 and NF-κB. In human MM cells (e.g. U266, H929, and IM-9 cells), SNAC treatment increased S-nitrosylation of STAT3 and NF-κB and inhibited their activities. Consequently, SNAC treatment resulted in MM cell cycle arrest at G1/S check point and inhibited their proliferation. SNAC also decreased the expression of cell survival factors and increased the activities of caspases, thus increased sensitivity of MM cells to melphalan, a chemotherapeutic agent for MM. In U266 xenografted mice, SNAC treatment decreased the activity of STAT3 and reduced the growth of human CD138 positive cells (U266 cells) in the bone marrow and also reduced their production of human IgE into the serum. Taken together, these data document the S-nitrosylation mediated inhibition of MM cell proliferation and cell survival via inhibition of STAT3 and NF-κB pathways and its efficacy in animal model of MM.

NF-Kß Activation in U266 Cells on Mesenchymal Stem Cells.

Purpose: Mesenchymal Stem Cells (MSCs) are one of the essential members of Bone Marrow (BM) microenvironment and the cells affect normal and malignant cells in BM milieu. One of the most important hematological malignancies is Multiple Myeloma (MM). Numerous studies reported various effects of MSCs on myeloma cells. MSCs initiate various signaling pathways in myeloma cells, particularly NF-kß. NF-kß signaling pathway plays pivotal role in the survival, proliferation and resistance of myeloma cells to the anticancer drugs, therefore this pathway can be said to be a vital target for cancer therapy. This study examined the relationship between U266 cells and MSCs. Methods: U266 cells were cultured with Umbilical Cord Blood derived-MSCs (UCB-MSCs) and Conditioned Medium (C.M). Effect of UCB-MSCs and C.M on proliferation rate and CD54 expression of U266 cells were examined with MTT assay and Flowcytometry respectively. Furthermore, expression of CXCL1, PECAM-1, JUNB, CCL2, CD44, CCL4, IL-6, and IL-8 were analyzed by Real Time-PCR (RT-PCR). Moreover, status of p65 protein in NF-kß pathway assessed by western blotting. Results: Our findings confirm that UCB-MSCs support U266 cells proliferation and they increase CD54 expression. In addition, we demonstrate that UCB-MSCs alter the expression of CCL4, IL-6, IL-8, CXCL1 and the levels of phosphorylated p65 in U266 cells. Conclusion: Our study provides a novel sight to the role of MSCs in the activation of NF-kß signaling pathway. So, NF-kß signaling pathway will be targeted in future therapies against MM.

Bortezomib modulates CHIT1 and YKL40 in monocyte-derived osteoclast and in myeloma cells.

Osteolytic bone disease is a common manifestation of multiple myeloma (MM) that leads to progressive skeleton destruction and is the most severe cause of morbidity in MM patients. It results from increased osteolytic activity and decrease osteoblastic function. Activation of mammalian chitinases chitotriosidase (CHIT1) and YKL40 is associated with osteoclast (OCs) differentiation and bone digestion. In the current study, we investigated the effect of two Bortezomib's concentration (2.5 and 5 nM) on osteoclastogenesis by analyzing regulation of chitinase expression. OCs exposition to bortezomib (BO) was able to inhibit the expression of different OCs markers such as RANK, CTSK, TRAP, and MMP9. In addition BO-treatment reduced CHIT1 enzymatic activity and both CHIT1 and YKL40 mRNA expression levels and cytoplasmatic and secreted protein. Moreover, immunofluorescence evaluation of mature OCs showed that BO was able to translocate YKL40 into the nucleus, while CHIT1 remained into the cytoplasm. Since MM cell lines such as U266, SKM-M1 and MM1 showed high levels of CHIT1 activity, we analyzed bone resorption ability of U266 using dentin disk assay resorption pits. Silencing chitinase proteins in U266 cell line with specific small interfering RNA, resulted in pits number reduction on dentine disks. In conclusion, we showed that BO decreases osteoclastogenesis and reduces bone resorption in OCs and U266 cell line by modulating the chitinases CHIT1 and YKL40. These results indicate that chitinases may be a therapeutic target for bone disease in MM patients.

Evaluation of the change in sphingolipids in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 treated with arsenic trioxide.

Arsenic trioxide (As2O3) has been found to display anticancer activity against many types of tumors and has been developed into an anticancer drug in clinical treatments. Sphingolipids are membrane lipids that participate in many signal transduction pathways. In this paper, the changes in sphingolipids of the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803 treated with arsenic trioxide were investigated using an HPLC-ESI-MS/MS method. Analytes were separated by an XBridge BEH C8 column used for Cer, HexCer, LacCer and SM chromatographic separation, and a Capcell PAK MG II C18 column was used for Sph, dhSph, S1P and dhS1P chromatographic separation and gradient elution with acetonitrile-water containing 0.1% formic acid as a mobile phase. A tandem mass spectrometer QTrap in SRM mode was employed in combination with RPLC as a detector for quantitative analysis. The ceramide/sphingolipid internal standard (IS) mixture was used to quantify the levels of sphingolipids. The distributions of sphingolipids were found to be different in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803. Ceramide (Cer), hexosylceramide (HexCer) and dihexosylceramide (Hex2Cer) levels in U266 cell line are higher than those in MGC-803 cell line. Additionally, sphingomyelin (SM), sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (dhS1P) levels in the MGC-803 cell line are higher than those in the U266 cell line. When treated with arsenic trioxide (1-5µM iAs(III)(As(III) ions)), the levels of Hex2Cer in the human multiple myeloma cell line U266 decreased, and the levels of S1P and dhS1P in the human gastric cancer cell line MGC-803 decreased. The decrease of Hex2Cer, S1P and dhS1P in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 were observed when the concentration of iAs(III) is 1.0µM. Therefore, arsenic trioxide exhibits anti-cancer activity by altering the sphingolipid pathway in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803.