Curcumin (diferuloylmethane) inhibits cell proliferation, induces apoptosis, and decreases hormone levels and secretion in pituitary tumor cells

Prolactinomas are the most prevalent functional pituitary adenomas. Dopamine D2 receptor (D2R) agonists, such as bromocriptine are the first line of therapy; however, drug intolerance/resistance to D2R agonists exists. Apart from D2R agonists, there is no established medical therapy for prolactinomas; therefore, identifying novel therapeutics is warranted. Curcumin, a commonly used food additive in South Asian cooking, inhibits proliferation of several tumor cell lines; however, its effect on pituitary tumor cell proliferation has not been determined. Our objectives were to: 1) determine whether curcumin inhibits proliferation of pituitary tumor cell lines; 2) identify the signaling intermediaries that mediate the effect of curcumin; 3) examine whether curcumin inhibited pituitary hormone production and release; and 4) examine whether curcumin could enhance the growth-inhibitory effect of bromocriptine. Using rat lactotroph cell lines, GH3 and MMQ cells, we report that curcumin had a robust dose and time-dependent inhibitory effect on GH3 and MMQ cell proliferation. Inhibitory effects of curcumin persisted, even on removal of curcumin, and curcumin also blocked colony formation ability of pituitary tumor cells. The growth-inhibitory effect of curcumin was accompanied by decreased expression of cyclin D3 and ser 780 phosphorylation of retinoblastoma protein. In addition, curcumin also induced apoptosis in both GH3 and MMQ cells. Furthermore, curcumin suppresses intracellular levels and release of both prolactin and GH. Finally, we show that low concentrations of curcumin enhanced the growth-inhibitory effect of bromocriptine on MMQ cell proliferation. Taken together we demonstrate that curcumin inhibits pituitary tumor cell proliferation, induces apoptosis, and decreases hormone production and release, and thus, we propose developing curcumin as a novel therapeutic tool in the management of prolactinomas.     

Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-kappa B in human endothelial cells

OBJECTIVE: Nuclear factor-kappa B (NF-kappa B) plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various vascular diseases. Although oxygen free-radicals are known to activate NF-kappa B, the signaling pathway of oxygen free radical-induced NF-kappa B activation remains largely unclear. Thus, this study was performed to examine the possible involvement of protein kinase C (PKC) in the oxygen free radical-induced NF-kappa B activation in human umbilical vein endothelial cells (HUVECs). METHODS: Superoxide anion was generated by xanthine and xanthine oxidase. An electrophoretic mobility shift assay (EMSA) was performed using a kappa B-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against I kappa B alpha, phosphorylated by I kappa B alpha kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-kappa B luciferase reporter gene assay was also performed. RESULTS: The treatment of the cells with superoxide anion for 60 min increased the NF-kappa B/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of I kappa B alpha within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of I kappa B alpha. Pretreatment of the cells with calphostin C (100-400 nmol/l) and chelerythrine chloride (5-10 mumol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-kappa B activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-kappa B activation to a basal level. Superoxide anion induced the luciferase reporter gene and this induction was completely inhibited by calphostin C (200 nmol/l) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron). CONCLUSION: These results suggest that PKC is involved in the activation of NF-kappa B by superoxide anion in human endothelial cells.     

Baicalein, a component of Scutellaria radix from Huang-Lian-Jie-Du-Tang (HLJDT), leads to suppression of proliferation and induction of apoptosis in human myeloma cells

In the search for a more effective adjuvant therapy to treat multiple myeloma (MM), we investigated the effects of the traditional Chinese herbal medicines Huang-Lian-Jie-Du-Tang (HLJDT), Gui-Zhi-Fu-Ling-Wan (GZFLW), and Huang-Lian-Tang (HLT) on the proliferation and apoptosis of myeloma cells. HLJDT inhibited the proliferation of myeloma cell lines and the survival of primary myeloma cells, especially MPC-1- immature myeloma cells, and induced apoptosis in myeloma cell lines via a mitochondria-mediated pathway by reducing mitochondrial membrane potential and activating caspase-9 and caspase-3. Further experiments confirmed that Scutellaria radix was responsible for the suppressive effect of HLJDT on myeloma cell proliferation, and the baicalein in Scutellaria radix showed strong growth inhibition and induction of apoptosis in comparison with baicalin or wogonin. Baicalein as well as baicalin suppressed the survival in vitro of MPC-1- immature myeloma cells rather than MPC-1+ myeloma cells from myeloma patients. Baicalein inhibited the phosphorylation of IkB-alpha, which was followed by decreased expression of the IL-6 and XIAP genes and activation of caspase-9 and caspase-3. Therefore, HLJDT and Scutellaria radix have an antiproliferative effect on myeloma cells, especially MPC-1- immature myeloma cells, and baicalein may be responsible for the suppressive effect of Scutellaria radix by blocking IkB-alpha degradation.     

Human myeloma cells stimulate the receptor activator of nuclear factor-kappa B ligand (RANKL) in T lymphocytes: a potential role in multiple myeloma bone disease

The biologic mechanisms involved in the pathogenesis of multiple myeloma (MM) bone disease are not completely understood. Recent evidence suggests that T cells may regulate bone resorption through the cross-talk between the critical osteoclastogenetic factor, receptor activator of nuclear factor-kappaB ligand (RANKL), and interferon gamma (IFN-gamma) that strongly suppresses osteoclastogenesis. Using a coculture transwell system we found that human myeloma cell lines (HMCLs) increased the expression and secretion of RANKL in activated T lymphocytes and similarly purified MM cells stimulated RANKL production in autologous T lymphocytes. In addition, either anti-interleukin 6 (anti-IL-6) or anti-IL-7 antibody inhibited HMCL-induced RANKL overexpression. Consistently, we demonstrated that HMCLs and fresh MM cells express IL-7 mRNA and secrete IL-7 in the presence of IL-6 and that bone marrow (BM) IL-7 levels were significantly higher in patients with MM. Moreover, we found that the release of IFN-gamma by T lymphocytes was reduced in presence of both HMCLs and purified MM cells. Furthermore, in a stromal cell-free system, osteoclastogenesis was stimulated by conditioned medium of T cells cocultured with HMCLs and inhibited by recombinant human osteoprotegerin (OPG; 100 ng/mL to 1 microg/mL). Finally, RANKL mRNA was up-regulated in BM T lymphocytes of MM patients with severe osteolytic lesions, suggesting that T cells could be involved at least in part in MM-induced osteolysis through the RANKL overexpression.     

Enediyne lidamycin induces apoptosis in human multiple myeloma cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase

In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, on two human multiple myeloma (MM) cell lines, U266 and SKO-007, were evaluated. In MTS assay, LDM showed much more potent cytotoxicity than conventional anti-MM agents to both cell lines. The IC₅₀ values of LDM for the U266 and SKO-007 cells were 0.0575 ± 0.0015 and 0.1585 ± 0.0166 nM, respectively, much lower than those of adriamycin, dexamethasone, and vincristine. Mechanistically, LDM triggered MM cells apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in LDM-induced cell death. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed the LDM-induced apoptosis through decreasing the level of cleaved PARP and caspase-3/7. Interestingly, phosphorylation of extracellular signal-related kinase was increased by LDM; conversely, MEK inhibitor synergistically enhanced LDM-induced cytotoxicity and apoptosis in MM cells. The results demonstrated that LDM suppresses MM cell growth through the activation of p38 MAPK and JNK, with the potential to be developed as a chemotherapeutic agent for MM.     

Protease inhibitors differentially regulate tumor necrosis factor- induced apoptosis, nuclear factor-kappa B activation, cytotoxicity, and differentiation

We investigated the effect of various protease inhibitors on several tumor necrosis factor (TNF)-mediated cellular responses. Treatment of a human myelogenous leukemia cell line, ML-1a, with TNF in the presence of cycloheximide triggers endonucleolytic activity and apoptotic cell death within 90 minutes. The general serine protease inhibitor diisopropyl fluorophosphate (DFP) and the chymotrypsin-like protease inhibitor N-tosyl-L-lysyl chloromethyl ketone (TPCK) completely abrogated TNF-induced DNA fragmentation and the formation of apoptotic bodies. However, 13 other protease inhibitors, including serine protease inhibitors, did not. The addition of TPCK to cells 30 minutes after TNF treatment completely inhibited the cytokine action, indicating that TPCK-sensitive proteases are not involved in the early stages of signal transduction. TNF is cytotoxic and induces differentiation in ML-1a cells after a 3-day incubation. TPCK had no effect on the TNF-induced cytotoxicity and differentiation, indicating that TPCK-sensitive proteases are specific for DNA fragmentation. TPCK also blocked TNF-induced activation of nuclear factor (NF)-kappa B. The dose-response and the time-course of the inhibitor, however, indicated that the site of action of TPCK for NF-kappa B activation and for DNA fragmentation are quite distinct. Therefore, we conclude that TNF activates two distinct TPCK-sensitive pathways, one leading to apoptosis and the other to NF-kappa B activation.     

Dysregulated Fas and Bcl-2 expression leading to enhanced apoptosis in T cells of multiple myeloma patients

We have previously reported the presence of activated (HLA-DR+) T cells in multiple myeloma (MM) patients. These cells produce high amounts of interleukin (IL)-2 and interferon (IFN)-gamma and generate a potent antiplasma cell activity after appropriate in vitro stimulation, but they are unable in vivo to hold in check the disease. Activated T cells are highly susceptible to apoptosis, a form of programmed cell death involved in the modulation of immune responses and regulated by molecules such as Fas (CD95) and bcl-2. The aim of this study was to determine the expression of Fas and bcl-2 antigens and the susceptibility to apoptosis in T cells of MM patients. Fas+ cells were significantly higher, whereas bcl-2+ cells were significantly lower in MM patients than in the controls. MM patients with the highest number of HLA-DR+ T cells showed the highest Fas and the lowest bcl-2 expression. Two-color cytofluorometric analysis confirmed in individual cells that HLA-DR+ T cells coexpressed Fas and lacked bcl-2. Susceptibility to apoptosis was then investigated to evaluate the consequence of dysregulated Fas and bcl-2 expression. The percentage of apoptotic cells after incubation in medium alone (spontaneous apoptosis) or in the presence of methylprednisolone (MP) or anti-Fas monoclonal antibody (triggered apoptosis) was significantly higher in MM and mainly restricted to HLA-DR+ T cells. Spontaneous apoptotosis was reverted by exogenous IL-2. In conclusion, MM T cells have a dysregulated expression of Fas and bcl-2 antigens that is associated with an enhanced susceptibility to apoptosis. These data may unravel a novel mechanism by which activated MM T cells are weakened in their ability to exert an effective antitumor activity in vivo.     

Bone morphogenetic protein-4 inhibits proliferation and induces apoptosis of multiple myeloma cells

Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. Here it is shown that BMP-4 inhibited DNA synthesis in a dose-dependent manner in 3 IL-6-dependent multiple myeloma (MM) cell lines (OH-2, IH-1, and ANBL-6). In contrast, no effect on DNA synthesis was observed in 3 IL-6-independent MM cell lines (JJN-3, U266, and RPMI 8226). BMP-4 induced cell cycle growth arrest in the G(0)/G(1) phase in OH-2 and ANBL-6 cells but not in IH-1 cells. BMP-4 induced apoptosis in OH-2 and IH-1 cells, but not significantly in ANBL-6 cells. Furthermore, BMP-4 induced apoptosis in freshly isolated MM cells from 4 of 13 patients. In the OH-2 and ANBL-6 cell lines and in a patient sample, immunoblotting showed that BMP-4 down-regulated IL-6-induced tyrosine phosphorylation of Stat3, suggesting a mechanism for the apparent antagonism between IL-6 and BMP-4. BMP-4 or analogues may be attractive therapeutic agents in MM because of possible beneficial effects on both tumor burden and bone disease.     

Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-kappaB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells

Whether resveratrol, a component of red grapes, berries, and peanuts, could suppress the proliferation of multiple myeloma (MM) cells by interfering with NF-kappaB and STAT3 pathways, was investigated. Resveratrol inhibited the proliferation of human multiple myeloma cell lines regardless of whether they were sensitive or resistant to the conventional chemotherapy agents. This stilbene also potentiated the apoptotic effects of bortezomib and thalidomide. Resveratrol induced apoptosis as indicated by accumulation of sub-G(1) population, increase in Bax release, and activation of caspase-3. This correlated with down-regulation of various proliferative and antiapoptotic gene products, including cyclin D1, cIAP-2, XIAP, survivin, Bcl-2, Bcl-xL, Bfl-1/A1, and TRAF2. In addition, resveratrol down-regulated the constitutive activation of AKT. These effects of resveratrol are mediated through suppression of constitutively active NF-kappaB through inhibition of IkappaBalpha kinase and the phosphorylation of IkappaBalpha and of p65. Resveratrol inhibited both the constitutive and the interleukin 6-induced activation of STAT3. When we examined CD138(+) plasma cells from patients with MM, resveratrol inhibited constitutive activation of both NF-kappaB and STAT3, leading to down-regulation of cell proliferation and potentiation of apoptosis induced by bortezomib and thalidomide. These mechanistic findings suggest that resveratrol may have a potential in the treatment of multiple myeloma.     

The novel inhibitor of histone deacetylase resminostat (RAS2410) inhibits proliferation and induces apoptosis in multiple myeloma (MM) cells

Inhibition of histone deacetylase (HDAC) is a promising mechanism for novel, anti‐myeloma agents. We investigated the effects of the novel HDAC inhibitor resminostat on multiple myeloma (MM) cells in vitro. Resminostat is a potent inhibitor of HDACs 1, 3 and 6 [50% inhibitory concentration (IC₅₀) = 43–72 nmol/l] representing HDAC classes I and II and induces hyperacetylation of histone H4 in MM cells. Low micromolar concentrations of resminostat abrogated cell growth and strongly induced apoptosis (IC₅₀ = 2·5–3 μmol/l in 3 out of 4 MM cell lines) in MM cell lines as well as primary MM cells. At 1 μmol/l, resminostat inhibited proliferation and induced G0/G1 cell cycle arrest in 3 out of 4 MM cell lines accompanied with decreased levels of cyclin D1, cdc25a, Cdk4 and pRb as well as upregulation of p21. Resminostat decreased phosphorylation of 4E‐BP1 and p70S6k indicating an interference with Akt pathway signalling. Treatment with resminostat resulted in increased protein levels of Bim and Bax and decreased levels of Bcl‐xL. Caspases 3, 8 and 9 were activated by resminostat. Furthermore, synergistic effects were observed for combinations of resminostat with melphalan and the proteasome inhibitors bortezomib and S‐2209. In conclusion, we have identified potent anti‐myeloma activity for this novel HDAC inhibitor.     

Protein kinase A-independent inhibition of proliferation and induction of apoptosis in human thyroid cancer cells by 8-Cl-adenosine

PURPOSE: Protein kinase A (PKA) affects cell proliferation in many cell types and is a potential target for cancer treatment. PKA activity is stimulated by cAMP and cAMP analogs. One such substance, 8-Cl-cAMP, and its metabolite 8-Cl-adenosine (8-Cl-ADO) are known inhibitors of cancer cell proliferation; however, their mechanism of action is controversial. We have investigated the antiproliferative effects of 8-Cl-cAMP and 8-CL-ADO on human thyroid cancer cells and determined PKA's involvement. EXPERIMENTAL DESIGN: We employed proliferation and apoptosis assays and PKA activity and cell cycle analysis to understand the effect of 8-Cl-ADO and 8-Cl-cAMP on human thyroid cancer and HeLa cell lines. RESULTS: 8-Cl-ADO inhibited proliferation of all cells, an effect that lasted for at least 4 d. Proliferation was also inhibited by 8-Cl-cAMP, but this inhibition was reduced by 3-isobutyl-1-methylxanthine; both drugs stimulated apoptosis, and 3-isobutyl-1-methylxanthine drastically reduced 8-Cl-cAMP-induced cell death. 8-Cl-ADO induced cell accumulation in G1/S or G2/M cell cycle phases and differentially altered PKA activity and subunit levels. PKA stimulation or inhibition and adenosine receptor agonists or antagonists did not significantly affect proliferation. CONCLUSIONS: 8-Cl-ADO and 8-Cl-cAMP inhibit proliferation, induce cell cycle phase accumulation, and stimulate apoptosis in thyroid cancer cells. The effect of 8-Cl-cAMP is likely due to its metabolite 8-Cl-ADO, and PKA does not appear to have direct involvement in the inhibition of proliferation by 8-Cl-ADO. 8-Cl-ADO may be a useful therapeutic agent to be explored in aggressive thyroid cancer.     

Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells

Aims/hypothesis The incretin hormone glucagon-like peptide-1 augments islet cell mass in vivo by increasing proliferation and decreasing apoptosis of the beta cells. However, the signalling pathways that mediate these effects are mostly unknown. Using a clonal rat pancreatic beta cell line (INS-1), we examined the role of protein kinase B in mediating beta-cell growth and survival stimulated by glucagon-like peptide-1.Methods Immunoblot analysis was used to detect active (phospho-) and total protein kinase B. Proliferation was assessed using ³H-thymidine incorporation, while apoptosis was quantitated using 4-6-diamidino-2-phenylindole staining and APO percentage apoptosis assay. Kinase-dead and wild-type protein kinase B was introduced into cells using adenoviral vectors.Results Glucagon-like peptide-1 rapidly activated protein kinase B in INS-1 cells (by 2.7±0.7-fold, p<0.05). This effect was completely abrogated by inhibition, with wortmannin, of the upstream activator of protein kinase B, phosphatidylinositol-3-kinase. Glucagon-like peptide-1 also stimulated INS-1 cell proliferation in a dose-dependent manner (by 1.8±0.5-fold at 10^–7 mol/l, p<0.01), and inhibited staurosporine-induced apoptosis (by 69±12%, p<0.05). Both of these effects were also prevented by wortmannin treatment. Ablation of protein kinase B by adenovirus-mediated overexpression of the kinase-dead form of protein kinase B prevented protein kinase B phosphorylation and completely abrogated both cellular proliferation (p<0.05) and protection from drug-induced cellular death (p<0.01) induced by glucagon-like peptide-1.Conclusions/interpretation These results identify protein kinase B as an essential mediator linking the glucagon-like peptide-1 signal to the intracellular machinery that modulates beta-cell growth and survival.Abbreviations AdV adenovirus - DAPI 4-6-diamidino-2-phenylindole - GFP enhanced green fluorescent protein - GIP glucose-dependent insulinotropic peptide - GLP glucagon-like peptide - GPCR G protein-coupled receptor - GSK3/ glycogen synthase kinase-3/ - KD kinase-dead - MAPK mitogen-activated protein kinase - PI3-K phosphatidylinositol-3-kinase - PKB protein kinase B - PTHrP parathyroid hormone-related peptide - WT wild-typeQ. Wang and L. Li contributed equally to this article     

Synergistic effects of rmhTRAIL and 17-AAG on the proliferation and apoptosis of multiple myeloma cells

ABSTRACT

Objective: This study aimed to investigate synergistic effects of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) and heat-shock protein 90 (HSP90) inhibitor (geldanamycin derivative 17 -allylamino- 17-demethoxy -geldanamycin, 17-AAG) on the proliferation and apoptosis of multiple myeloma (MM) cells.

Methods: MTT assays evaluated inhibitory effects of rmhTRAIL and 17-AAG in different concentrations and treatment durations on the proliferation of RPMI8226 and U266 cells. The half maximal inhibitory concentration was calculated using OriginPro7.5. Synergistic effects of rmhTRAIL and 17-AAG on apoptosis of MM cells were detected using flow cytometry at 24 and 48?h post-treatment. To evaluate synergistic effects of rmhTRAIL and 17-AAG, the Q-value was calculated using King's formula.

Results: rmhTRAIL exhibited significant inhibitory effects on the proliferation of RPMI8226 cells in a dose- and time-dependent manner (>50%), whereas U266 cells were not sensitive to rmhTRAIL (<50%). 17-AAG inhibited the proliferation of RPMI8226 and U266 cells in a dose-dependent manner (>80%). Significant synergistic effects of rmhTRAIL and 17-AAG on the proliferation of RPMI8226 cells were revealed (Q-value?>?1.15), whereas synergistic effects were not evident on the proliferation of U266 cells (Q-value?<?1.15). rmhTRAIL and 17-AAG exhibited significant synergistic effects on apoptosis of RPMI8226 and U266 cells (Q-value?>?1.15).

Conclusion: The combined application of rmhTRAIL and 17-AAG revealed favorable synergistic effects in the treatment of MM.     

Combining prenylation inhibitors causes synergistic cytotoxicity, apoptosis and disruption of RAS-to-MAP kinase signalling in multiple myeloma cells

The high incidence of activating RAS mutations, coupled with accumulating evidence linking RAS to multiple myeloma (MM) pathogenesis, indicate that novel therapies utilising inhibitors of RAS prenylation and signalling may be successful in the management of this disease. While preclinical studies investigating prenylation inhibitors, such as lovastatin, farnesyltransferase inhibitors (FTI) and geranylgeranyltransferase inhibitors (GGTI), have been promising, recent phase I/II clinical trials with FTI R115777 were disappointing, suggesting resistance to FTI monotherapy. To address this issue, the effects of FTI, GGTI and lovastatin alone and in combination were analysed in MM cell lines and primary cells. FTI treatment blocked H-RAS processing, but was ineffective at inhibiting K- and N-RAS prenylation because of alternative geranylgeranylation of these isoforms. However, combinations of FTI and GGTI or lovastatin were found to synergistically inhibit MM cell proliferation, migration, K- and N-RAS processing, RAS-to-mitogen-activated protein kinase signalling and to induce apoptosis. In contrast to FTI, lovastatin and some GGTI were found to cause intracellular accumulation of Rho proteins. Our results suggest that clinical efficacy of prenylation inhibitors in MM are limited by alternative prenylation of several small G-proteins, such as RhoB, K- and N-RAS. Furthermore, strategies combining FTI with GGTI or statins may provide greater efficacy in MM treatment.