| OBJECTIVEMultiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in the bone marrow and by recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections. The aim of this study was to investigate TLR initiated responses in MM cells including proliferation, anti-apoptosis and immune escape. TLR4 ecognizes lipopolysaccharide (LPS) from gram-negative bacteria. The TLR signaling pathway activates several different signaling elements, including nuclear factor?B (NF-?B) and extracellular signal-regulated kinase (ERK)/c-Jun-NH2-kinase (JNK)/p38, which regulate many immunologically relevant proteins. Coincidentally, many of these same signaling elements are also involved in tumorigenesis and tumor growth, suggesting that TLRs may affect tumor growth..The bone marrow microenvironment promotes cell growth and survival, and it is mediated by different cytokines, chemokines, and growth factors. STAT-3 is an oncogenic protein that is contributes to the pathogenesis of many cancers, including multiple myeloma (MM). So we detect the effect of broad panel of cytokines and growth factor on STAT3 phosphorylation in myeloma cells, we found U266 constitutively STAT3 actives, while no in MM.1S and ARP-1. Cytokines IL-6, IFN?, and IFN?stimulated phosphorylation of STAT3 and STAT5 in myeloma cells. MM BM environment is hypoxic and HIF alpha could be a potential therapeutic target in MM patients.MATERIALS AND METHODS1. TLRs mRNA expression detected by reverse transcriptase-polymerase chain reaction (RT-PCR), Flow cytometry PE-labeled to analysed TLR4 protein expression. Real-time polymerase chain reaction detected B7-H1?B7-H2?CD40?VEGF and TGF-?expression.2. Cell proliferation was measured by 3H-thymidine and MTS, Cell apoptosis was evaluated by flow cytometry with FITC-conjugated Annexin V and propidium iodide (BD Biosciences).3. IL-12,IL-6, and IL-18 secretion in culture supernatants was tested by human ELISA kit (R&D, Minneapolis, MN) according to the manufacturer's protocol.4. Western-blot analysis MAPK family ERK, JNK and p38 phosphorylation and NF-?B protein expression.5. Meso Scale Discovery and Western-blot detect p-STAT3, STAT3, p-STAT5, STAT5, HIF1?and HIF1?expression.RESULTS1. Toll-like receptors are expressed on human myeloma cell lines (HMCL) and primary myeloma cells:In this study, we first screened four commonly used HMCL MM.1R, MM.1S, ARP-1, and U266 for the expression of major TLRs including TLR 1-2,4-7 and 9 by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs We focused our attention on TLR4, which was highly expressed among all TLRs. Consistent with RT-PCR results, flow cytometry analysis revealed that TLR4 protein was also present on the surface of the HMCL We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells.2. Toll-like receptor-specific ligand increased proliferation and survival, and induced cell cycle change:We next asked if TLR4 was functional in MM cells. After incubation of cells with LPS, cell proliferation was measured by 3H-thymidine incorporation. Interestingly, after treatment with TLR4 ligand LPS at 1,2,4?g/ml for 48 hours (h), MM.1S, MM.1R and ARP-1 cells showed significantly increased proliferation, whereas U266 cells did not. As anti-apoptosis plays a major role for MM cells to escape from chemotherapy, we sought to determine the effect of TLR4 ligand on MM cell survival. We first looked at the apoptosis induced by adriamycin (ADM; 0.4?g/ml for 24 h) which is a effective drug for multiple myeloma. Pretreatment of MM.1S and ARP-1 cells with LPS partially protected the cells from ADM-induced apoptosis in vitro. In addition to proliferation and apoptosis, we also examined the effect of TLR4 ligand on cell cycle. We used MM.1S and ARP-1 HMCL which responded to LPS by increased proliferation, after treatment with LPS (2?g/ml) for 12,24 and 24 h, cells in the G0/G1 phase decreased and the percentages of S-phase cells increased.3. Toll-like receptor-specific ligand promoted proliferation via MAPKs:To explore whether MAPK pathways were implicated in LPS-induced MM.1S and ARP-1 proliferation, we investigated the role of ERK1/2, p38, and JNK MAPKs in LPS-stimulated MM.1S and ARP-1 cells by using specific inhibitors of MAPK pathways, such as SB203580 for p38 MAPK, U0126 for ERK1/2 and SP600125 for JNK. First, time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in MM.1S and ARP-1 cells. ERK1/2, p38, and JNK phosphorylation and NF-?B were significantly upregulated following LPS treatment, with the maximum phosphorylation occurring at 20,60, or 180 minutes (min), respectively, after stimulation. Pretreatment of the cells with these inhibitors inhibited LPS-induced phosphorylation of ERK1/2 and JNK, respectively. It is interesting to note that JNK inhibitors inhibited phosphorylation of JNK as well as p38. Moreover, ERK1/2 inhibitors inhibited phosphorylation of ERK 1/2 but increased phosphorylation of p38. These results suggest that ERK, JNK and p38 pathways were interconnected. We also examined cell proliferation following pretreatment with the specific inhibitors and stimulation with LPS. Our findings demonstrate that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling, suggesting that ERK and p38 pathways are involved in LPS-induced MM.1S cell proliferation. The kinase inhibitors did not induce apoptosis in myeloma cells.4. Toll-like receptor-specific ligand induced immunoregulatory factor secretion and facilitated evasion of MM.1S cells from immune surveillance:Toll-like receptors activate innate and adaptive immune responses, therefore we further studied the secretion of immunoregulatory factors IL-12 and IL-18 in the culture supernatants of MM cells treated with TLR4 ligand LPS. MM cells MM.1R, MM.1S, ARP-1, and U266 were stimulated by LPS (2?g/ml) for 48 h. and IL-12 and IL-18 secretion in the culture supernatants was determined by ELISA, we found that treatment with LPS increased the production of IL-18 in MM.1R, MM.1S and ARP-1 cells. All HMCL secreted IL-12 and LPS treatment had no effect on IL-12 secretion by the cells.5. With real-time PCR, we detected the expression of immunoregulatory molecules B7-H1, B7-H2, CD40, TGF-?and VEGF in MM.1S cells after treatment with TLR4 ligand LPS (2?g/ml). We found that B7-H1, B7-H2, CD40 mRNA expression was upregulated. Consistent with real-time PCR results, flow cytometry analysis revealed that B7-H1, B7-H2, and CD40 proteins were also present on the surface of the HMCL. To further examine whether the effect of LPS on IL-18 production was involved in immune escape, HMCL MM.1S cells were co-cultured with T cells from normal blood donors, and 3H-thymidine incorporation assay was used to detect T cell proliferation. T cell proliferation decreased when stimulatory HMCL cells were treated with LPS (2?g/ml) for 48 h. We next investigated if LPS stimulation affected the proliferation, expression of immunoregulatory factors B7-H1 mRNA, and 6. secretion of cytokine IL-18 by purified CD138+ myeloma cells from MM patients. Bone marrow plasma cells were isolated from two MM patients (MM1 and MM2). We found that LPS induced increased proliferation, and upregulated the expression of immunoregulatory factor B7-H1 mRNA and secretion of cytokine IL-1 by primary MM cells. In contrast, LPS did not affect IL-18 secretion by monocyte-derived DC derived from the peripheral blood of the same MM patients.6. WP1066 inhibit the proliferation of multiple myeloma cells:First, we tested the inhibit proliferation effect of inhibitors WP1066 on multiple myeloma cell lines MM.1S, U266 and ARP-1. For these, we incubated each of the three cell lines with indicated concentrations of the drug for 24,48 and 72hours and measured drug-induced growth inhibition of myeloma cells in vitro by MTT assays. The IC50 values for three cell lines measured by MTT assays after 24,48 and 72 hours of incubation with the drugs were in the range of 0.5-10?M. The IC50 of ARP-1 cell line was 3.25?M for 24h,2.74 for 48h and 1.67?M for 72h; the IC50 of MM.1S cell line was 3.77?M for 24h,3.20 for 48h and 2.32?M for 72h; and IC50 of U266 cell line was 8.81?M for 24h,4.61 for 48h and 2.15?M for 72h;. Then we also detect the inhibit proliferation effect of WP1066 on multiple myeloma primary CD138+ cells, and CD138- cells as control, we found WP1066 effected on CD138+ cells more than CD138- cells obviously,24h IC50 was 5.674?M for CD138+ cells, while it was more than 10?M for CD138- cell.7. WP1066 induced apoptosis of multiple myeloma cells and caused caspase-3 activation and PARP cleavage:we also analyzed the apoptosis effect of WP1066 on myeloma cells. We used WP1066 1,2,5?M treatment three myeloma cell line MM.1S, U266 and ARP-1 24h, we found WP1066 induced cells apoptosis at range from 34% to 68%. At the same time, we use WP1066 2.5,5 10?M effect cells 24 hours, MSD and western blot detect apoptosis related genes caspase-3 activation and PARP cleavage. We found WP1066 induced increased caspase-3 activation and PARP cleavage significantly.8. Effect of a broad panel of cytokines and growth factors on STAT3 and STAT5 phosphorylation in multiple myeloma cell lines:in order to detect the effect of broad panel of cytokines and growth factor on STAT3 phosphorylation in multiple myeloma cell lines, we first detected three myeloma cell lines MM.1S, U266 and ARP-1 about original STAT3 actives, we found in three cell lines, cell line U266 constitutively STAT3 actives, while no constitutively STAT3 actives in MM.1S and ARP-1 cell lines. So we select cell lines MM.1S and ARP-1, cells were serum starved overnight, then stimulated with EGF 50ng/ml, bFGF 20ng/ml, G-CSF 20ng/ml, Erythropoirtin 10 IU/ml, IFNa-2a 50000IU/ml, IFN?20ng/ml, IFN?20ng/ml, IL-2 20ng/ml, IL-4 20ng/ml, IL-6 50ng/ml, IL-7 20ng/ml, IL-8 20ng/ml, IL-10 50ng/ml, IL-12 20ng/ml, IL-15 20ng/ml, IL-16 20ng/ml, IL-20 20ng/ml, IL-22 20ng/ml respectively for 30 minutes at serum-free condition, we found cytokines IL-6, IFN?, and IFN?stimulated phosphorylation of STAT3 and STAT5 in myeloma cell line cells.9. WP1066 potently inhibited the IL-6 induced STAT3 phosphorylation in MM cell lines in dose and time dependent:we have confirmed cytokines also stimulated pSTAT3 and pSTAT5 expression at serum condition, so we further detected the effect of inhibitors AG490, CABE, WP1066 on cytokines-induced STAT3 phosphorylation, we decided the concentration 50,100 and 300?M for AG490 and 50,100?M for CABE, and 2.5,5, 10?M for WP1066, cells were exposure 5 hours with drug and 30min with IL-6, we found inhibitors AG490, CABE, WP1066 potently inhibited IL-6 induced STAT3 phosphorylation in MM.1S and ARP1 cell lines respectively at different concentration, About 300?M for AG490, 100?M for CABE and 5-10?M for WP1066. WP1066 also inhibited IL-6 induced STAT5 phosphorylation in MM.1S cell line at different concentration,300?M for AG490, 100?M for CABE and 5?M for WP1066. We also detected the effect of inhibitors AG490, CABE, WP1066 on cytokines-induced STAT3 phosphorylation at time course, we decided the concentration 300?M for AG490 and 100?M for CABE, and 5?M for WP1066, MM.1S cells were exposure 1,4,6 hours with drug and IL-6 30min, we found the drugs effect on IL-6 induced STAT3 phosphorylation in time dependent, the time point for maxium inhibit effect was 6 hours.10. WP1066 potently inhibited the IFNa induced STAT3 phosphorylation in MM cell lines:we have confirmed IFNa also stimulated STAT3 phosphorylation, so we further detected the effect of inhibitors AG490, CABE, WP1066 on IFN?-induced STAT3 phosphorylation in MM cell line ARP-1 and U266 cells, we decided the concentration 50,100 and 300?M for AG490 and CABE, and 2.5,5, 10?M for WP1066, cells were exposure 5 hours with drug and 30min with IFN?, we also found AG490, CABE,WP1066 potently inhibited IFNa induced STAT3 phosphorylation in ARP1 cell lines and inhibited consistitutively STAT3 actived in U266 cells at different concentration, and the inhibited effect for ARP-1 cell line was better than U266..11. WP1066 down regulated hypoxia-induced HIF-1?expression:hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in available oxygen in the cellular environment, specifically, to decreases in oxygen, or hypoxia. The HIF1?signaling cascade mediates the effects of hypoxia, the state of low oxygen concentration on the cell. Hypoxia often keeps cells from differentiating. However, hypoxia promotes the formation of blood vessels, and is important for the formation of a vascular system in embryos, and cancer tumors. HIF1?activity is involved in angiogenesis required for cancer tumor growth, so HIF1?inhibitors are under investigation for anti-cancer effects. Therefore we also detect the effect of WP1066 on HIF1?activity. First we check the original activation of HIF1?in myeloma cells, we found three myeloma cell lines MM.1S, U266 and ARP-1 expression low level HIFla at normal condition and cells HIF1?expression increased significantly at hypoxia condition, and the maximum effect of hypoxia on HIF1?was 8 hours, so we select hypoxia time and WP1066 drug time for 8 hours, we found that WP1066 potently inhibited hypoxia induced HIF1?expression in soluble lyses, and HIF1?expression increased in insoluble pallet.12. WP1066 down regulated STAT3 expression in dose and time dependent:we have found that WP1066 potently inhibited hypoxia induced HIF1?expression in soluble lyses, and HIF1?went into insoluble pallet, we also think about if WP1066 has similar effect on STAT3 expression and activation. So we use three myeloma cell lines MM.1S, U266 and ARP-1, WP1066 effect cells 24 hours and IL-6 effect cells 30 minutes before cells were collected and western blot detect pSTAT3 and STAT3 expression. We found WP1066 inhibited STAT3 phosphorylation in soluble similar as in insoluble. But we get similar result as HIF1?at the effect of WP1066 inhibited total STAT3, we found that WP1066 also inhibited total STAT3 expression with dose dependent manner and total STAT3 also went into insoluble pallet.We already found the drug effect on total STAT3 in dose dependent at 24 hours, we further wanted to know when the drug began to effect on the total STAT3, STATS and Jak2 within 24 hours, we decided the concentration 5?M for WP1066, ARP-1 and U266 cells were exposure 4,8,12 and 24 hours with drug, we found the drugs effect on STAT3, STAT5 in time dependent, the time point for start to inhibit effect was 8-12 hours,and the inhibited effect for Jak2 was not obviously.CONCLUSION1. Toll-like receptors are expressed on human myeloma cell lines (HMCL) and primary myeloma cells.2. Toll-like receptor-specific ligand increased proliferation and survival, and induced cell cycle change.3. Toll-like receptor-specific ligand LPS promoted proliferation via MAPKs.4. Toll-like receptor-specific ligand induced immunoregulatory factor secretion and facilitated evasion of MM.1S cells from immune surveillance.5. Cytokines IL-6, IFNa, and IFN?stimulated phosphorylation of STAT3 and STAT5 in myeloma cell line cells. WP1066 inhibit the proliferation of multiple myeloma cells. WP1066 induced apoptosis of multiple myeloma cells and caused caspase-3 activation and PARP cleavage.6. WP1066 potently inhibited the IL-6 induced STAT3 phosphorylation in MM cell lines in dose and time dependent. WP1066 potently inhibited the IFNa induced STAT3 phosphorylation in MM cell lines.7. WP1066 down regulated STAT3 expression in dose and time dependent and WP1066 down regulated hypoxia-induced HIF-la expression. |
PDF Full Text Request