PR-924, a selective inhibitor of the immunoproteasome subunit LMP-7, blocks multiple myeloma cell growth both in vitro and in vivo.

PR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase-8, caspase-9, BID, PARP and cytochrome-c release. In vivo administration of PR-924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM.

Pharmacodynamic and efficacy studies of the novel proteasome inhibitor NPI-0052 (marizomib) in a human plasmacytoma xenograft murine model.

Our previous study showed that the novel proteasome inhibitor NPI-0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib (Velcade, Takeda, Boston, MA, USA) therapies. In vivo studies using human MM-xenografts demonstrated that NPI-0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for an ongoing phase-1 clinical trial of NPI-0052 in relapsed/refractory MM patients. Here we performed pharmacodynamic (PD) studies of NPI-0052 using human MM xenograft murine model. Our results showed that NPI-0052: (i) rapidly left the vascular compartment in an active form after intravenous (i.v.) administration, (ii) inhibited 20S proteasome chymotrypsin-like (CT-L, beta5), trypsin-like (T-L, beta2), and caspase-like (C-L, beta1) activities in extra-vascular tumours, packed whole blood (PWB), lung, liver, spleen, and kidney, but not brain and (iii) triggered a more sustained (>24 h) proteasome inhibition in tumours and PWB than in other organs (<24 h). Tissue distribution analysis of radiolabeled compound (3H-NPI-0052) in mice demonstrated that NPI-0052 left the vascular space and entered organs as the parent compound. Importantly, treatment of MM.1S-bearing mice with NPI-0052 showed reduced tumour growth without significant toxicity, which was associated with prolonged inhibition of proteasome activity in tumours and PWB but not normal tissues.

Gastrointestinal plasmacytoma that caused anemia in a patient with multiple myeloma.

BACKGROUND: A 70-year-old white male diagnosed with IgA lambda multiple myeloma, who had been treated with two cycles of melphalan and prednisone, was evaluated for persistent anemia. He had required more than 15 U of packed red blood cells within a 2-month period for his anemia, despite recombinant erythropoietin treatment, and his hemoglobin level was persistently below 9 g/dl. INVESTIGATIONS: Physical examination and laboratory tests, which included a red blood cell mean corpuscular volume, platelet counts, coagulation studies, a peripheral blood smear, lactate dehydrogenase level, haptoglobin and bilirubin level, vitamin B12 and folate level, serum iron studies, bone marrow biopsy and immunophenotyping. Additionally, Congo red staining of the subcutaneous fat aspirate and a bone marrow biopsy were carried out, as well as esophagogastroduodenoscopy with gastric and duodenal biopsies. DIAGNOSIS: Gastrointestinal plasmacytoma. MANAGEMENT: Control of underlying disease (multiple myeloma) with 2 cycles of treatment with melphalan and prednisone followed by high-dose pulse dexamethasone chemotherapy as outlined by the oncologist. PPI therapy was continued and NSAIDs were avoided. The patient died because of infectious complications with subsequent multi-organ failure while awaiting work up for autologous stem cell transplantation.

Prostaglandin E2 induces IL-23 production in bone marrow-derived dendritic cells.

Interleukin-23, a recently described cytokine produced by activated antigen-presenting cells, including dendritic cells, is a p19/p40 heterodimer. The p40 subunit is shared with IL-12, the major Th1-driving cytokine, while p19 is distantly related to IL-12 p35. IL-23 has pro-inflammatory actions, inducing IL-17 secretion from activated CD4+ T cells, and stimulating the proliferation of memory CD4+ T cells. Here, we examined the effects of PGE2, a well-known immunomodulator, on the production of IL-23 by bone marrow- derived dendritic cells (BM-DCs). Our results indicate that PGE2 increases the production of functional IL-23 from immature BM-DCs in a time- and dose-dependent manner. PGE2 induces both the expression of p19 and p40, without affecting p35 expression. The effect of PGE2 is mediated through the specific receptors EP2/4 and is mimicked by cAMP-inducing agents, such as forskolin and dbcAMP. Although PGE2 also induces IL-1beta and IL-6 expression in non-stimulated DCs, the stimulatory effect of PGE2 on IL-23 production is not mediated through IL-1beta or IL-6. GM-CSF, the pro-inflammatory cytokine required for the generation of BM-DCs, amplifies the IL-23 inducing activity of PGE2 in a synergistic manner. Recent studies described both pro- and anti-inflammatory effects of PGE2, and our results suggest an additional mechanism for its pro-inflammatory role, particularly significant for autoimmune diseases, such as rheumatoid arthritis.

Selenium deficiency abrogates inflammation-dependent plasma cell tumors in mice.

The role of the micronutrient, selenium, in human cancers associated with chronic inflammations and persistent infections is poorly understood. Peritoneal plasmacytomas (PCTs) in strain BALB/c (C), the premier experimental model of inflammation-dependent plasma cell transformation in mice, may afford an opportunity to gain additional insights into the significance of selenium in neoplastic development. Here, we report that selenium-depleted C mice (n = 32) maintained on a torula-based low-selenium diet (5-8 micro g of selenium/kg) were totally refractory to pristane induction of PCT. In contrast, 11 of 26 (42.3%) control mice maintained on a selenium adequate torula diet (300 micro g of selenium/kg) and 15 of 40 (37.5%) control mice fed standard Purina chow (440 micro g of selenium/kg) developed PCT by 275 days postpristane. Abrogation of PCT was caused in part by the striking inhibition of the formation of the inflammatory tissue in which PCT develop (pristane granuloma). This was associated with the reduced responsiveness of selenium-deficient inflammatory cells (monocytes and neutrophils) to chemoattractants, such as thioredoxin and chemokines. Selenium-deficient C mice exhibited little evidence of disturbed redox homeostasis and increased mutant frequency of a transgenic lacZ reporter gene in vivo. These findings implicate selenium, via the selenoproteins, in the promotion of inflammation-induced PCT and suggest that small drug inhibitors of selenoproteins might be useful for preventing human cancers linked with chronic inflammations and persistent infections.

Toxic proteins from European mistletoe (Viscum album L.): increase of intracellular IL-4 but decrease of IFN-gamma in apoptotic cells.

BACKGROUND: Mistletoe lectins (ML), the major biologically active components of mistletoe extracts, which are used for adjuvant cancer therapy, induce apoptosis in lymphocytes and tumor cells. In addition, ML at toxic concentrations induce the release of cytokines, but it remains unclear as to whether dying or activated cells are responsible. MATERIALS AND METHODS: By flow cytometry, expression of IFN-gamma, IL-4, apoptosis marker Apo2.7 and anti-apoptotic Bcl-2 proteins were analyzed in response to ML or viscotoxins (VT) in PBMC from controls and plasmocytoma cells (U-266). RESULTS: While ML inhibited PMA/Ca-ionophore/monensin co-stimulated IFN-gamma production, they increased IL-4 expression in CD8+ and CD4+ T-cells. Thereby, IL-4 was mainly expressed in apoptotic cells with a low level of Bcl-2 proteins. In contrast, the cell membrane permeabilising VT induced complete loss of Bcl-2 proteins but did not stimulate IL-4 production within 24 hours, indicating that IL-4 expression is related to apoptosis but not to necrosis. CONCLUSION: Despite the role of IL-4 during activation of type2 T-helper cells, IL-4 expression may play an important yet undefined role during apoptosis of normal and tumor cells.

Extramedullary plasmacytoma of anorectum: case report.

A young anxious looking male presented with a referral diagnosis of recurred bilateral ischiorectal abscesses. Clinical examination and investigations showed an advanced lesion of the anorectum. Histology revealed an extramedullary plasmacytoma of the anorectum to which the patient succumbed and died within two weeks of presentation.

The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation.

IP-10 is a member of the chemokine family of cytokines and is induced in a variety of cells in response to interferon gamma and lipopolysaccharide. The self-aggregation common to many chemokines, including IP-10, has hindered the identification of a specific IP-10 receptor. Using an IP-10 alkaline phosphatase fusion protein that fortuitously blocks this self-aggregation, we have identified an IP-10 binding site on a variety of cells including endothelial, epithelial, and hematopoietic cells. This binding site has a Kd of 25 nM, is inhibited by recombinant murine or human IP-10, and is dependent on the presence of cell surface heparan sulfate proteoglycans (HSPG). This conclusion is based on the findings that IP-10 binding to cells is: (a) inhibited by heparin and heparan sulfate; (b) sensitive to a 1 M NaCl wash; (c) eliminated by treatment with heparinase and trypsin; and (d) absent on mutant CHO cells that do not express cell surface HSPG. Platelet factor 4 (PF4), but not IL-8, monocyte chemoattractant protein-1, RANTES, monocyte inflammatory protein (MIP)-1 alpha, or MIP-1 beta, can compete effectively with IP-10 for binding to the cell surface. Furthermore, IP-10 shares with PF4 the ability to inhibit endothelial cell proliferation (IC50 = 150 nM). These studies demonstrate specificity in the interaction of chemokines and HSPG, and they define IP-10 and PF4 as a distinct subset of chemokines sharing an HSPG-binding site and angiostatic properties.

Control of a murine plasmacytoma with doxorubicin-cisplatin: dependence on circadian stage of treatment.

In anticipation of the development of clinical chronotherapy and in order to pick clinical test times for doxorubicin and cisplatin trials, two large studies were performed on rats bearing a transplanted plasmacytoma. The circadian timing of each of two anticancer drugs given at precisely equal dose intensities was expected to improve therapeutic benefit over conventionally given (time-unqualified) treatment. In each chronotherapeutic study, maximal benefit and minimal toxic effects were found when cisplatin was administered in the middle to latter part of the daily activity (dark) span, while doxorubicin was administered near the end of the daily resting (light) span for these nocturnally active rodents living on a 12-hour-12-hour or 8-hour-16-hour light-dark schedule. This was true whether doxorubicin or cisplatin was given first and whether there was a lag of only a few hours or a few days between the administration of these two agents.

A simple method for efficiently establishing 8-azaguanine-resistant mutant human leukemia and myeloma cell lines.

A simple and convenient method for efficiently establishing 8-azaguanine-resistant mutant leukemia and myeloma cell lines (for example, the T cell lines Jurkat and CCRF-CEM, human myeloid/macrophage-like cell lines HL60 and U937, Burkitt lymphoma line Raji and the human myeloma line RPMI 8226), is described. The method relies on culturing the cell lines in RPMI 1640 medium containing 8-azaguanine and supplemented with 15% heat-inactivated fetal calf serum and large amounts of amino acids and vitamins, and removes the necessity for pretreatment with mutagenic reagents such as ethyl methylsulfonate or X-irradiation. The possibility of obtaining mutant cell lines using the method described here is about 15 times greater than using media without high levels of amino acids and vitamins. Hybridomas produced between mitogen-activated human peripheral blood lymphocytes and an 8-azaguanine-resistant Jurkat mutant cell line (established by this method) were shown to produce soluble T cell-derived macrophage activating factor (MAF)-like material.

Wavelength and light-dose dependence in tumour phototherapy with haematoporphyrin derivative.

Red light (c. 630 nm) is almost universally used in tumour phototherapy as it is the most penetrating of the porphyrin excitation wavebands. However, measurements of tumour attenuation of light of different wavelengths and of the excitation spectrum of haematoporphyrin derivative in vitro suggested that green light might be more efficient than red in destroying thin tumours. Experimentally, we confirmed this for tumours up to approximately 1.2 mm thick, a depth exceeding that of most carcinomas-in-situ. The superiority of green light over red in terms of the illumination time required to produce equivalent depths of necrosis may extend to greater depths (3-4 mm) if the former is produced by an argon laser and the latter by an argon-pumped dye laser. The relation between depth of necrosis Zn and light dose D is shown to be Zn = sigma gamma-1 1n(D/theta gamma) where sigma gamma is the attenuation coefficient for light at wavelength gamma and theta gamma the threshold light dose for producing necrosis at that wavelength. This logarithmic relationship suggests that it may be difficult to eradicate large tumours merely by increasing the light dose, and indicates the need for other approaches.

Inhibition of plasmacytoma development in BALB/c mice by indomethacin.

Indomethacin given continuously in the drinking water (20 micrograms/ml) to BALB/cAn pi mice during the latent period of pristane-induced plasmacytoma development dramatically reduced the plasmacytoma incidence from 34.9 to 2.2%. Additionally, indomethacin given from day 0 to 120 or begun as late as 60 d after a single injection of 1.0 ml pristane was also highly effective in reducing the development of plasmacytomas. Indomethacin treatment did not prevent the formation of a peritoneal inflammatory exudate or peritoneal oil granulomatous tissue, although it had a mild inhibitory effect on the intensity of the cellular inflammation, particularly after extensive treatment of greater than 100 d. Indomethacin treatment reduced the incidence of arthritis by 50%. A major effect of indomethacin treatment was a reduction in the appearance of microscopic plasmacytomas that appear in the oil granuloma before plasmacytomas can be detected by routine sampling of the peritoneal exudate. Between days 116 and 181, 16 of 20 mice given 0.5 ml pristane were found to have foci of plasmacytoma cells, while only 2 of 20 indomethacin-treated mice had foci-containing plasmacytoma cells. The number of mice with microscopic foci in the pristane-treated group greatly exceeded the expected incidence of plasmacytomas (22%) at this dose of pristane. The growth of primary plasmacytomas in transplant that is dependent on the pristane-conditioned peritoneal environment was not inhibited by indomethacin treatment. The role of indomethacin in inhibiting plasmacytoma development was not established; two possibilities are that it inhibits production of mutagenic and tissue destructive oxidants by inflammatory cells, and it inhibits prostaglandin synthesis and intracellular production of oxidant biproducts.

Dichloro[1,2-bis(4-hydroxyphenyl)ethylenediamine]platinum(II) complexes: an approach to develop compounds with a specific effect on the hormone-dependent mammary carcinoma.

Stereoisomeric dichloro [1,2-bis(4-hydroxyphenyl)ethylenediamine]platinum(II) complexes (meso-3a, (+/-)-3b, (+)-3c, (-)-3d) and their N,N'-dibutyl derivatives (meso-4a, (+/-)-4b, (+)-4c, (-)-4d) were synthesized and tested on antitumor activity. The most active compound, 3d, shows a modest inhibition of the [3H]estradiol receptor interaction and causes a marked effect on the growth of the hormone-dependent human MCF 7 breast cancer cell line. It is also active on the hormone-independent human MDA-MB 231 breast cancer cell line, on the ADJ/PC6 plasmacytoma of the Balb/C mouse, and on the L 5222 leukemia of the BD IX rat. Apparently the inhibition of the MCF 7 cell line is not mediated by the estrogen receptor system. Histopathological studies on 3d revealed very low toxicity.

The tumor-inhibiting effect of isomeric dichloro(diphenylethylenediamine)platinum(II) complexes.

Ring unsubstituted dichloro(diphenylethylenediamine)platinum(II) complexes show a dependence of their antitumor activity on the configuration and position of phenyl rings in ethylenediamine ligand. Dichloro(1,1-diphenylethylenediamine)platinum(II) (1d) and meso-dichloro(1,2-diphenylethylenediamine)-platinum(II) (meso-2d) have a weaker effect on the human breast-cancer cell line MDA-MB 231 and on rat leukemia L 5222 than (+/-)-dichloro(1,2-diphenylethylenediamine)platinum(II)((+)-2d) and its enantiomers (+)-2d and (-)-2d which cause marked and comparable inhibition of both tumors; (+/-)-2d is also active on ADJ/PC 6 plasmacytoma of the mouse and on cisplatin-, daunomycin-, and cisplatin/daunomycin-resistant Ehrlich ascites tumors of the mouse. The differences in activity of the diastereomers (+/-)-2d and meso-2d, for which distinct influences on the DNA secondary structure can be demonstrated CD spectroscopically may be explained by a steric hindrance of the drug-DNA interaction.

Activity of a novel anthracenyl bishydrazone, 9,10-anthracenedicarboxyaldehyde Bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride, against experimental tumors in mice.

9,10-Anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride (CL 216942; bisantrene hydrochloride; NSC 337766), a member of a new chemical class of compounds with antineoplastic properties, has been evaluated for antitumor activity in experimental murine tumor systems. The compound produced significant increases in life span (LS) and long-term survivors among mice bearing transplantable leukemias and solid tumors. Optimal treatment regimens resulted in an ILS of greater than 173 and 151% in mice with P388 and L1210 leukemia, respectively, an ILS of greater than 85% in mice with Lieberman plasma cell tumor, and an ILS of greater than 200, 150, and 63%, respectively, in mice with B16 melanoma, colon tumor 26, and Ridgway osteogenic sarcoma. An adriamycin-resistant subline of P388 leukemia showed complete cross-resistance to CL of 216942. The compound was active when administered by the i.p., i.v., and s.c. routes, but p.o. activity was not observed. Significant schedule dependency was not observed when the drug was administered once daily for 9 days, once every 4 days, or as a single dose, but single doses typically produced the best effects. CL 216942 was a potent inhibitor of DNA and RNA synthesis in L5178Y lymphoma cells cultured in vitro, and preliminary studies indicated the drug was a DNA-intercalating agent. The drug was cytotoxic for rapidly proliferating and nonproliferating (G0) human colon carcinoma WiDR cells in vitro.U

Cell kinetic analysis of human tumor stem cells.

The application of the tumor stem cell assay to the study of human tumor cell kinetics has the potential for major advances in our knowledge of proliferative characteristics of clonogenic human tumor cells. From simultaneous evaluation of in vitro drug sensitivity, in vitro doubling time, and the thymidine suicide index, plus flow cytometry, cytogenetic analysis, and assessment of differentiation markers, substantial insights into the basic biology of tumor cell growth may be attained. As discussed in other chapters, using modifications of the two-layer system, one can assess both local cell-mediated and humoral factors that might influence in vitro kinetics and drug sensitivity. The next few years should see the acquisition and integration of valuable new information that should allow more rational approaches to the treatment of tumors by taking full advantage of information on both kinetics and drug sensitivity of clonogenic human tumor cells.

[Solitary plasmacytoma of the hypothalamus]. / Solitarnaia plazmotsitoma gipotalamusa.

A rare case of intracranial extra osseal isolated plasmocytoma in a girl of 18 is presented. The tumour localized in the hypothalamus and extended into the intracranial parts of the optic nerves and chiasma opticum. It consisted of mature and immature plasma cells and contained paraamyloid depositions. The disease had a progredient course accompanied by the disorders of vision, endocrine-metabolic disturbances and hyperglobulinemia.

The influence of hypoxia and acidity on the hyperthermic response of malignant cells in vitro.

Colony formation of JB-1-E tumor cells was studied after hyperthermic treatment (42.5 degrees C) at a pH of 6.4 or 7.2 under hypoxic and euoxic conditions. At a pH of 7.2 and normal oxygen tension, there was a moderate decrease in colony formation with increasing duration of hyperthermic treatment (To = 65 min.). This effect was slightly enhanced under hypoxic conditions (To = 36 min.). The hyperthermic effect was enhanced to a considerably greater degree when treatment was performed at a pH of 6.4 (To = 19 min.), with no observable difference between hypoxia and euoxia. These findings indicate that environmental acidity is a determining factor in the hyperthermic effect. The hypoxic effect at a pH of 7.2 is probably due to a slight decrease in the intracellular pH caused by increased production of lactic acid.