Functional regulation of D-type cyclins by insulin-like growth factor-I and serum in multiple myeloma cells

D-type cyclin genes are universally dysregulated in multiple myeloma (MM), but the functional consequences are unclear as D-type cyclin gene expression does not correlate with proliferation or disease progression. We examined the protein expression and regulation of D-type cyclins and other cell cycle regulators in human myeloma cell lines and primary CD138(+) plasma cells (PCs). Cyclin D1, cyclin D2, cyclin dependent kinase (CDK) 4, CDK6, p27(Kip1) p18(INK4C) and retinoblastoma protein (pRb) were absent in normal PCs, heterogeneously expressed in primary MM cells and positively correlated with disease activity/progression. Cyclins D1 and D2 complexed with both CDK4 and CDK6, suggesting that both phosphorylate pRb in MM. Furthermore, cyclin D2 expressed via either t(14;16) or t(4;14) IgH translocations was functionally upregulated by fetal calf serum or insulin-like growth factor-I, leading to pRb phosphorylation and cell cycle entry/progression, and in some cases inversely correlated with p27(Kip1). However, pRb phosphorylation and cell cycle progression mediated by cyclin D1 expressed via t(11;14) was less dependent on exogenous stimuli. These data suggest that the presence or absence of specific IgH translocations underlying aberrant D-type cyclin expression may influence their response to mitogens in the bone marrow microenvironment. We showed for the first time that D-type cyclins are functionally regulated in MM, differentially responsive to exogenous growth factors and upregulated with disease progression.     

Multiple myeloma cells are protected against dexamethasone-induced apoptosis by insulin-like growth factors

Multiple myeloma cell lines express functional receptors for insulin-like growth factors (IGFs) and several cell types that make up the bone marrow microenvironment produce these cytokines. This suggests that IGFs may play a role in survival and/or expansion of the malignant clone within the marrow in patients with multiple myeloma. We tested the effects of these growth factors on myeloma cells challenged with dexamethasone. Dye exclusion and MTT assays demonstrated that both IGF-I and IGF-II protected the 8226 and dox-40 myeloma cell lines and three primary myeloma cultures from dexamethasone-induced cytotoxicity in a dose-dependent fashion. Morphologic studies of target cells and their nuclei as well as DNA electrophoresis confirmed the IGFs afforded protection against dexamethasone-induced apoptosis. Insulin also protected but was less impressive and required much higher concentrations. IGFs also protected against cycloheximide-induced apoptosis but were ineffective against serum starvation, topoisomerase II inhibitors, or anti-fas antibodies. IGF-induced protection against dexamethasone was not associated with any alteration in quantitative or qualitative expression of BCL-2, BAX or BCL-X proteins. These data indicate that insulin-like growth factors may play a role in maintenance of the malignant clone in patients with myeloma by protecting tumour cells from apoptotic death.     

Glucocorticoid receptor in multiple myeloma

Glucocorticoid receptor levels of myeloma cells were quantitated in 7 patients with multiple myeloma. In 4 patients, glucocorticoid receptor levels were less than 10 fmol/10(6) cells. In 3 patients, receptor levels were from 17.6 to 21.4 fmol/10(6) cells. We further examined the correlation between glucocorticoid receptor levels and effect of dexamethasone on 14C-thymidine incorporation and cell viability using 2 myeloma cell lines, OPM-1 and OPM-2, established from a patient with multiple myeloma. Glucocorticoid receptor levels of OPM-1 and OPM-2 were 8.5 fmol/10(6) cells and 63.2 fmol/10(6) cells, respectively. The sensitivity of OPM-1 to dexamethasone in these studies was lower than that of OPM-2. These results suggest the possibility that the low level of glucocorticoid receptor in myeloma cells may be important for predicting a poor response to glucocorticoid therapy.     

The pan-histone deacetylase inhibitor CR2408 disrupts cell cycle progression, diminishes proliferation and causes apoptosis in multiple myeloma cells

In view of the fact that histone deacetylases (HDACs) are promising targets for myeloma therapy, we investigated the effects of the HDAC inhibitor CR2408 on multiple myeloma (MM) cells in vitro. CR2408 is a direct pan-HDAC inhibitor and inhibits all known 11 HDACs with a 50% inhibitory concentration (IC(50) ) of 12 nmol/l (HDAC 6) to 520 nmol/l (HDAC 8). Correspondingly, CR2408 induces hyperacetylation of histone H4, inhibits cell growth and strongly induces apoptosis (IC(50) =0.1-0.5 μmol/l) in MM cell lines and primary MM cells. CR2408 leads to fragmentation of cells and induces an accumulation in the subG1 phase accompanied with moderately decreased levels of cyclin D1 and cdk4 and strongly decreased levels of cdc25a, pRb and p53. Interruption of the cell cycle is reflected by inhibition of cell proliferation and is accompanied by decreased phosphorylation of 4E-BP1 and p70S6k. Treatment with CR2408 results in increased protein levels of Bim and pJNK and downregulation of Bad and Bcl-xL and activation of Caspases 3, 8 and 9. Furthermore, as HDAC inhibitors have shown synergism with other drugs, these effects were investigated and synergism was observed for combinations of CR2408 with doxorubicin and bortezomib. In conclusion, we have identified potent anti-myeloma activity for this novel HDAC inhibitor that gives further insights into the biological sequelae of HDAC inhibition in MM.     

Choleretic actions of insulin-like growth factor-I,prednisolone, and ursodeoxycholic acid in rats

A recent study by our group demonstrated that a 1-week infusion of insulin-like growth factor-I increases bile flow volume and bile acid secretion in rats, suggesting it is important in in vivo choleresis. In the present study, the effect in rats of a single administration of insulin-like growth factor-I on bile flow volume was investigated and compared with the choleretic drugs prednisolone and ursodeoxycholic acid. A significant and long-lasting increase in bile flow volume was observed in rats treated with insulin-like growth factor-I or prednisolone. Ursodeoxycholic acid significantly, but transiently increased. Combined treatment using insulin-like growth factor-I with prednisolone or ursodeoxycholic acid additively increased bile flow volume. Overall, this study demonstrated that the stimulatory effect of insulin-like growth factor-I on bile flow volume is almost equally potent to that of prednisolone and ursodeoxycholic acid, indicating the possible therapeutic potential of insulin-like growth factor-I in cholestatic liver diseases.     

The synthetic furanonaphthoquinone induces growth arrest, apoptosis and differentiation in a variety of leukaemias and multiple myeloma cells

2-methyl-naphtho[2,3-b]furan-4,9-dione (FNQ3), a synthetic analogue of the quinone kigelinone, has demonstrated a real potential for use in the treatment of a variety of solid tumours. Unlike other quinones, such as mitomycin-C and adriamycin, the cytotoxicity of FNQ3 is often 10- to 14-fold more potent towards the tumour cells than their normal counterparts. We report, for the first time, that the drug had activity against a broad spectrum of leukaemias and multiple myeloma cells. It decreased the growth of acute myeloid leukaemia (AML) and multiple myeloma cell lines in a dose-dependent fashion (50% inhibitory concentration approximately 1.25 microg/ml against most of the leukaemia cell lines). This dose apparently initiated mitochondrial collapse as measured by depolarisation of the mitochondrial membrane. FNQ3 potentiated the differentiation of HL-60 myeloid cells in the presence of either 1alpha, 25(OH)(2) dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or all-trans-retinoic acid (ATRA). FNQ3 inhibited the proliferation of primary AML cells while inducing apoptosis. Eleven of 14 (79%) AML marrow samples had a prominent decrease in their clonogenic growth when cultured in the presence of the drug. In summary, this drug has growth inhibitory, apoptotic and differentiative effects against myeloid leukaemias and multiple myeloma cells. FNQ3 may represent a new therapeutic approach to these malignancies.     

The novel, orally bioavailable HSP90 inhibitor NVP-HSP990 induces cell cycle arrest and apoptosis in multiple myeloma cells and acts synergistically with melphalan by increased cleavage of caspases

Heat shock protein 90 (HSP90) binds and stabilizes numerous proteins and kinases essential for myeloma cell survival and proliferation. We and others have recently demonstrated that inhibition of HSP90 by small molecular mass inhibitors induces cell death in multiple myeloma (MM). However, some of the HSP90 inhibitors involved in early clinical trials have shown limited antitumor activity and unfavorable toxicity profiles. Here, we analyzed the effects of the novel, orally bioavailable HSP90 inhibitor NVP-HSP990 on MM cell proliferation and survival. The inhibitor led to a significant reduction in myeloma cell viability and induced G2 cell cycle arrest, degradation of caspase-8 and caspase-3, and induction of apoptosis. Inhibition of the HSP90 ATPase activity was accompanied by the degradation of MM phospho-Akt and phospho-ERK1/2 and upregulation of Hsp70. Exposure of MM cells to a combination of NVP-HSP990 and either melphalan or histone deacetylase (HDAC) inhibitors caused synergistic inhibition of viability, increased induction of apoptosis, and was able to overcome the primary resistance of the cell line RPMI-8226 to HSP90 inhibition. Combined incubation with melphalan and NVP-HSP990 led to synergistically increased cleavage of caspase-2, caspase-9, and caspase-3. These data demonstrate promising activity for NVP-HSP990 as single agent or combination treatment in MM and provide a rationale for clinical trials.     

Treatment with growth hormone and insulin-like growth factor-I in critical illness

The wider availability of recombinant human growth hormone and insulin-like growth factor-I has resulted in an investigation into the potential benefits of the pharmacological administration of these anabolic peptides in a variety of clinical conditions, characterized by an increase in catabolic rate. The initial studies were small, often uncontrolled open investigations, but investigators have more recently concentrated on larger, controlled multi-centre trials. Studies to date have included patients with cardiac failure, sepsis, burns, cancer cachexia, end-stage renal failure, trauma and AIDS, and those prior to or following major surgery. The authors have in general cautiously interpreted positive effects of treatment with growth hormone and insulin-like growth factor-I, either alone or in combination, on net protein balance, body composition, well-being and performance. Two large, randomized, placebo-controlled European multi-centre studies have recently detailed the effects of growth hormone treatment in critically ill intensive care patients. Major increases in mortality and morbidity were associated with growth hormone treatment. The mechanism(s) accounting for the increased mortality remain poorly understood. These negative findings have led to a decrease in the clinical use of growth hormone and in research activity in the area of anabolic treatment in human illness.     

The effect of acute and chronic insulin administration on insulin-like growth factor-I expression in the pituitary-intact and hypophysectomised rat

Summary Although growth hormone is known to be the main regulator of insulin-like growth factor-I, insulin has also been shown to play a role in regulating serum insulin-like growth factor I levels in diabetic animals. While this effect is thought to be due to correction of metabolic perturbations, some studies have suggest that insulin may have a direct effect on growth and/or insulin-like growth factor-I levels. We have examined the effects of acute and chronic insulin administration to non-diabetic, pituitary-intact and hypophysectomised rats. Rats were injected intraperitoneally with insulin as an acute bolus (10 U) or a chronic subcutanious infusion (low dose; 2.4 U/day, high dose; 12 U/day) over 5 days. Insulin-like growth factor-I mRNA was quantitated by Northern and slot blots of RNA from various tissues. A small (less than 2-fold) but significant increase (p<0.05) was seen in hepatic insulin-like growth factor-I mRNA abundance in pituitary-intact rats following acute insulin injection and chronic low dose insulin infusion. An increase in insulin-like growth factor-I mRNA levels was also seen in other tissues including diaphragm, lung, kidney and heart. A significant increase (p<0.05) in serum insulin-like growth factor-I levels was also observed 6 h after insulin injection. In contrast, in pituitary-intact rats which received high dose insulin infusion and were hypoglycaemic at the time of death, tissue levels of insulin-like growth factor-I mRNA were reduced compared to saline-treated control groups. Similarly in the hypophysectomised rats neither acute nor chronic insulin administration had any consistent effect on insulin-like growth factor-I mRNA abundance in any of the tissues examined. This data suggests that insulin has no direct effect in regulating insulin-like growth factor-I gene expression. The small effects demonstrable in pituitary-intact rats may result from a synergistic action of insulin with growth hormone or other pituitary factors.     

Therapeutic aspects of growth hormone and insulin-like growth factor-I treatment on visceral fat and insulin sensitivity in adults

Growth hormone (GH) is generally considered to exert anti-insulin actions, whereas insulin-like growth factor I (IGF-I) has insulin-like properties. Paradoxically, GH deficient adults and those with acromegaly are both predisposed to insulin resistance, but one cannot extrapolate from these pathological conditions to determine the normal metabolic roles of GH and IGF-I on glucose homeostasis. High doses of GH treatment have major effects on lipolysis, which plays a crucial role in promoting its anti-insulin effects, whereas IGF-I acts as an insulin sensitizer that does not exert any direct effect on lipolysis or lipogenesis. Under physiological conditions, the insulin-sensitizing effect of IGF-I is only evident after feeding when the bioavailability of circulating IGF-I is increased. In contrast, many studies in GH deficient adults have consistently shown that GH replacement improves the body composition profile although these studies differ considerably in terms of age, the presence or absence of multiple pituitary hormone deficiency, and whether GH deficiency was childhood or adult-onset. However, the improvement in body composition does not necessarily translate into improvements in insulin sensitivity presumably due to the anti-insulin effects of high doses of GH therapy. More recently, we have found that a very low dose GH therapy (0.1 mg/day) improved insulin sensitivity without affecting body composition in GH-deficient adults and in subjects with metabolic syndrome, and we postulate that these effects are mediated by its ability to increase free 'bioavailable' IGF-I without the induction of lipolysis. These results raise the possibility that this low GH dose may play a role in preventing the decline of beta-cell function and the development of type 2 diabetes in these "high risk" subjects.     

The circulating molecular weight forms of infused recombinant insulin-like growth factor-I and effects on glucose and fat metabolism in lambs

Summary We have investigated the relationship between the plasma distribution of infused recominant insulin-like growth factor-I across the insulin-like growth factor binding proteins and the resultant effects on glucose and fat metabolism. The studies were performed in 24-h fasted ram lambs which received primed constant infusions of ³H labelled glucose tracer. When isotopic equilibrium had been reached, the animals received 90-min infusions of human insulin-like growth factor-I at various doses (2.5, 20, 40 and 120 g· kg^–1·h^–1, n=3 for each dose). Total plasma insulin-like growth factor-I was significantly elevated by infusion at a rate of 40 g·kg^–1·h^–1 (from 185±14 g/l to 442±41 g/l, p<0.05) and 120g·kg^–1h^–1 (from 181±2 g/l to 953±39 g/1, p<0.005). The plasma concentrations of insulin-like growth factor-I not associated with binding proteins remained undetectable (<15 g/l) at the end of the 2.5 and 20 g·kg^–1·h^–1 doses, but were significantly elevated at the end of the 40 and 120 g·kg^–1·h^–1 infusions (to 71±14 g/l, p<0.05 and 176±55 g/l, p<0.01 respectively). The infused insulin-like growth factor-I associated primarily with 35–60 kilodalton binding proteins. Glucose kinetics were significantly altered only by the highest dose infusion, during which there was a fall in plasma glucose concentration from 3.5±0.2 mmol/l to 1.9±0.2 mmol/l (p<0.05). This was due to a 51% increase in the rate of glucose clearance. There was no significant change in the rate of glucose production. The plasma concentrations of glycerol and non-esterified fatty acid were not changed by any of the doses infused. We conclude that the hypoglycaemic action of infused recombinant insulin-like growth factor-I relates to a marked elevation of free insulin-like growth factor-I in the plasma, but that a threshold concentration of free insulin-like growth factor-I must be exceeded before this action is observed. The hypoglycaemic action of recominant insulin-like growth factor-I results primarily from an increase in glucose clearance while glucose metabolism was more sensitive than fat metabolism to infused recominant insulin-like growth factor-I. Both these actions contrast with those of insulin, and suggest that the acute metabolic effects of recombinant insulin-like growth factor-I are not mediated simply by cross-reaction with insulin receptors.     

A candidate targeting molecule of insulin-like growth factor-I receptor for gastrointestinal cancers

Advances in molecular research in cancer have brought new therapeutic strategies into clinical usage.One new group of targets is tyrosine kinase receptors,which can be treated by several strategies,including small molecule tyrosine kinase inhibitors(TKIs) and monoclonal antibodies(mAbs).Aberrant activation of growth factors/receptors and their signal pathways are required for malignant transformation and progression in gastrointestinal(GI) carcinomas.The concept of targeting specif ic carcinogenic receptors has been validated by successful clinical application of many new drugs.Type I insulin-like growth factor(IGF) receptor(IGF-IR) signaling potently stimulates tumor progression and cellular differentiation,and is a promising new molecular target in human malignancies.In this review,we focus on this promising therapeutic target,IGF-IR.The IGF/IGF-IR axis is an important modifier of tumor cell proliferation,survival,growth,and treatment sensitivity in many malignant diseases,including human GI cancers.Preclinical studies demonstrated that downregulation of IGF-IR signals reversed the neoplastic phenotype and sensitized cells to anticancer treatments.These results were mainly obtained through our strategy of adenoviruses expressing dominant negative IGF-IR(IGF-IR/dn) against gastrointestinal cancers,including esophagus,stomach,colon,and pancreas.We also summarize a variety of strategies to interrupt the IGFs/IGF-IR axis and their preclinical experiences.Several mAbs and TKIs targeting IGF-IR have entered clinical trials,and early results have suggested that these agents have generally acceptable safety profiles as single agents.We summarize the advantages and disadvantages of each strategy and discuss the merits/demerits of dual targeting of IGF-IR and other growth factor receptors,including Her2 and the insulin receptor,as well as other alternatives and possible drug combinations.Thus,IGF-IR might be a candidate for a molecular therapeutic target in human GI carcinomas.     

胃癌组织中胰岛素样生长因子受体的表达及意义

目的探讨胰岛素样生长因子Ⅰ、Ⅱ受体（IGF-ⅠR、IGF-ⅡR）表达与胃癌分型、分期、浸润和转移等生物学行为的关系。方法选择67例胃癌患者（胃癌组）手术标本,16例慢性萎缩性胃炎患者（CAG组）和13例慢性萎缩性胃炎伴重度异型增生患者（CAGD组）胃镜活检标本,采用免疫组化SP法检测各组IGF-ⅠR、IGF-ⅡR的表达,并与胃癌的生物学行为进行相关性分析。结果胃癌组IGF-ⅠR阳性表达率为64.2%,胃癌组和CAGD组IGF-ⅠR阳性表达率均明显高于CAG组（P〈0.01或〈0.05）;胃癌组IGF-ⅡR阳性表达率为59.7%,较CAG组明显升高（P〈0.01）。IGF-ⅠR阳性表达与胃癌的分化程度、浸润深度、肿瘤大小、淋巴结转移及TNM分期均密切相关（P均〈0.05）,IGF-ⅡR仅与胃癌的分化程度密切相关（P〈0.05）。结论 IGF-ⅠR、IGF-ⅡR的过表达均与胃癌的生物学行为有关,但IGF-ⅠR的关系更密切,两者联合检测可作为胃癌手术治疗及评估预后的参考指标。     

The peptide-semicarbazone S-2209, a representative of a new class of proteasome inhibitors, induces apoptosis and cell growth arrest in multiple myeloma cells

Bortezomib is the first approved member of a new class of anti-myeloma agents, the proteasome inhibitors. Further proteasome inhibitors are needed to optimise this promising treatment option. S-2209 [1-[1-{1-[(2,4-Dioxo-imidazolidin-1-ylimino)-methyl]-2-phenyl-ethylcarbamoyl}-2-(1H-indol-3-yl)-ethylcarbamoyl]-2-(1H-indol)] inhibits the chymotryptic activity of the human 20S proteasome (half maximal effective concentration, IC₅₀∼220 nmol/l) which was determined by a proteasome inhibition assay. A nuclear factor κB inhibition assay revealed a half maximal effective concentration (EC₅₀) of 0·9 μmol/l. The WST-1 growth assay showed inhibition of cell growth of all tested multiple myeloma (MM) cell lines with an IC₅₀ between 100 nmol/l and 600 nmol/l. Strong induction of apoptosis was seen in MM cells at nanomolar concentrations (IC₅₀∼300 nm) as well as in primary myeloma cells. No induction of apoptosis was detected in peripheral blood mononuclear cells from healthy humans. Upregulation of p53, activation of JNK protein, and downregulation of Mcl-1 was revealed. Despite the administration of 15 mg S-2209/kg/d in wistar rats, no toxicity with respect to body weight, hepatic enzymes, creatinine or haemoglobin was seen. Proteasome inhibition in white blood cells isolated from the treated rats was higher in the S-2209 treated animals in comparison with the control animals treated with 0·1 mg/kg/d bortezomib. S-2209 is active in myeloma cells and shows a favourable toxicity profile in first in-vivo studies. S-2209 is a promising agent for further clinical development.     

丙戊酸钠对多发性骨髓瘤细胞株KM3细胞增殖及其VEGF受体表达的影响

目的：研究丙戊酸钠（VPA）对多发性骨髓瘤细胞株KM3细胞增殖和凋亡的影响,探讨其抗多发性骨髓瘤细胞的分子生物学机制。方法：采用MTT法检测细胞增殖,流式细胞仪检测凋亡率,RT—PCR检测KM3细胞VEGFRmRNA的表达;采用免疫细胞化学法观察KM3细胞VEGFR、ac—H4蛋白的表达。结果：VPA可明显抑制KM3细胞增殖,且具有时间剂量依赖性（P〈0．05）;不同浓度VPA处理48h可以明显诱导细胞凋亡,具有剂量依赖性（P〈0．05）,VPA（0．5、1．0、2．0、4．0mmol／L）诱导总凋亡率分别为（11．77&#177;4．64）％、（22．13&#177;1．20）％、（23．95&#177;2．57）％和（42．72&#177;4．61）％;RT—PCR结果显示,KM3细胞仅表达VEGFR-1（flt—1）,且VPA能在mRNA水平抑制VEGFR-1的表达;免疫细胞化学结果显示,VPA（4mmol／L）作用48h后,KM3细胞中acH4吸光度值明显增加而VEGFR-1的吸光度明显降低（P〈0．05）。结论：VPA通过增加组蛋白乙酰化程度,下调骨髓瘤细胞表面VEGFR的表达,对KM3细胞的增殖起抑制作用。