Preclinical and clinical development of the proteasome inhibitor bortezomib in cancer treatment.

The proteasome is a ubiquitous enzyme complex that plays a critical role in the degradation of many proteins involved in cell cycle regulation, apoptosis and angiogenesis. Since these pathways are fundamental for cell survival and proliferation, particularly in cancer cells, the inhibition of proteasome is an attractive potential anticancer therapy. The present review will focus on the proteasome inhibitor bortezomib (Velcade, formerly PS-341; Millennium Pharmaceuticals, Inc., Cambridge, MA, USA). Bortezomib is an extremely potent and selective proteasome inhibitor that shows strong activity in in vitro and in vivo laboratory studies against many solid and hematologic tumor types. Moreover, bortezomib, mainly by inhibition of the NF-kappaB pathway, has a chemosensitizing effect when administered together with other antitumoral drugs. Based on these results, bortezomib entered clinical phase I trials, alone or in combination with chemotherapy, that showed good tolerance at doses that achieved a desired degree of proteasome inhibition. Phase II studies showed high response rates in refractory multiple myeloma patients, which led to the accelerated approval of bortezomib by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for this indication. A phase III trial comparing bortezomib with dexamethasone in refractory/ relapsed multiple myeloma patients had to be halted due to a survival advantage in the bortezomib arm. Additional studies are focusing on the potential benefit of bortezomib in newly diagnosed multiple myeloma patients. In other solid and hematological malignancies, phase II studies with bortezomib alone or in combination with other agents are ongoing. Encouraging results, particularly in lung cancer and lymphoma, have been observed. The critical molecules or genes responsible for tumor sensitivity to bortezomib continue to be evaluated using novel technologies.

Vascular gene discovery using laser capture microdissection of human blood vessels and quantitative PCR.

Recent approaches to candidate gene identification and cellular localization have included RNA derived from complex whole tissue profiling on cDNA microarrays followed by in situ hybridization with riboprobes. In this study, the Arcturus PixCell II laser capture microdissection (LCM) system, an argon-based laser-assisted method for the isolation of specific cell types from heterogeneous tissue samples, was used to microdissect the tunica media from normal human arteries and veins (n = 5 in each group). Total RNA was extracted from the sum of 10,000 shots for each blood vessel using the Strataprep MicroKit. RNA was reverse-transcribed, and the resulting cDNA was analyzed using the Applied Biosystems 7700 quantitative PCR system (Q-PCR). Control genes, such as the L-type calcium channel, PECAM (CD-31), and beta-2 microglobulin, were used to assess sample quality and purity. Of 10 laser-captured media samples, five (50%) showed a gene profile that indicated high-quality RNA (abundance of housekeeping genes) and smooth muscle cell enrichment (low levels of PECAM and high levels of the L-type calcium channel). We conclude that the application of the LCM technique to collect smooth muscle cell RNA from the tunica media of human blood vessels can assist in the validation of gene expression and potentially expedite the identification of novel, regulated genes present within vascular smooth muscle.

Fish oil dietary supplementation for prevention of indomethacin induced gastric and small bowel toxicity in healthy volunteers.

OBJECTIVE: To determine if N-3 fatty acid (fish oil) dietary supplements could favorably alter indomethacin induced gastric and small bowel toxicity related to use of nonsteroidal antiinflammatory drugs (NSAID). METHODS: Healthy volunteers consumed 8 g of N-3 fatty acids for 16 weeks, while controls consumed corn oil. Subjects ingested indomethacin 50 mg tid between Weeks 12 and 16. Upper gastrointestinal (GI) endoscopy with biopsies and Cr-EDTA swallows were performed at Week 12 and again at Week 16. Biopsy specimens were graded for inflammation and endoscopic scores were recorded. RESULTS: No significant differences were seen between groups in any study variable, although the direction of change favored the fish oil subjects for inflammation scores in both the stomach and duodenum (fish oil subjects -0.50 +/- 1.2 stomach, -0.28 +/- 0.97 duodenum; and corn oil subjects +0.10 +/- 0.84 stomach, +0.20 +/- 0.79 duodenum; p = 0.086). Direction of change in stomach inflammation showed a trend favoring fish oil (p = 0.056 by chi square). CONCLUSION: Although no significant differences were seen between groups, the changes observed in inflammation suggest a possible benefit of fish oil in the amelioration of NSAID induced GI inflammation.

Evolving delivery systems for clinical laboratories: the Albany Medical Center Regional Laboratory Network.

The Albany Medical Center is a not-for-profit institution consisting of the Albany Medical College and the Albany Medical Center Hospital. Before 1989, the college's pathology department was not affiliated with the clinical laboratories of the hospital and no regional laboratory program existed. Subsequent to 1989, with the recruitment of a new chairman of the department of pathology, the department and the clinical laboratories of the hospital merged to create the Department of Pathology and laboratory Medicine. The strategic plan for the redesign included goals to respond to new opportunities and seek new contracts in the community, to enhance the efficiency within the clinical laboratories, and to increase the volume of anatomic pathology specimens. The newly formed department decided to explore the opportunities to work with hospitals, physician groups, and insurers in the area to create a regional laboratory network. This article describes how changes were implemented, including some of the problems that were encountered. An assessment of the result evaluates the success of the network and discusses its future direction.

Fish oil, atherogenesis, and thrombogenesis.

Marine fish consumption is known to reduce mortality from ischemic heart disease. The use of fish oil as a dietary supplement, however, is not universally recommended. In large doses, fish oil reduces plasma cholesterol and triacylglycerol but increases low density lipoprotein (LDL) levels and the potential for free radical generation and bleeding. Moderate marine fish consumption is known to reduce mortality without altering commonly measured variables, i.e., plasma cholesterol levels, in vitro platelet aggregation, and bleeding times. In swine, we observed that monocyte adhesions and platelet clumps over the lesion surface of proximal left anterior descending (LAD) coronary arteries are markedly reduced when an atherogenic diet was supplemented with cod-liver oil, even when the cholesterol levels were equalized with the untreated group. These findings suggest that fish oil is hypothrombogenic. We developed an in vitro assay to delineate the mechanism whereby fish oil reduced monocyte-endothelial cell interactions in vivo. The effects of supplementing the culture medium with different fatty acids on adhesions between lipopolysaccharide (LPS) stimulated swine aortic endothelial cells (SAEC) and the human monocyte-like cell line, U937, was investigated in a 10 minute adhesion assay at 37 degrees C. Exposure of SAEC for 6 hours to media containing 50-200 microMs eicosapentaenoic (EPA), stearic, oleic, linoleic, and arachidonic acid, respectively, revealed that only EPA reduced U937-SAEC adhesion. Exposure of U937 to EPA also reduced adhesions. EPA was not effective when added to the SAEC more than 2 hours after they were stimulated with LPS. Exposure of human umbilical vein endothelial cells (HUVEC) to EPA reduced the expression of VCAM-1, ELAM-1, and ICAM-1 after 5 hours of stimulation with LPS. These results suggest that EPA may functionally impair the induction/expression of adhesion molecules.

Dietary fish oil reduces microthrombi over atherosclerotic lesions in hyperlipidemic swine even in the absence of plasma cholesterol reduction.

We have investigated in swine the effect of fish oil additives to a butter-cholesterol hyperlipidemic diet (BT) on atherogenesis and thrombogenesis when average plasma cholesterol levels were kept similar in fish oil-treated and untreated BT groups. The studies included evaluation of lesion sizes and cell numbers, counts of adherent monocytes over lesions, and counts of platelet clumps (microthrombi) over lesions either attached directly to endothelium or to adherent monocytes. Anatomic sites studied for lesion development were the left anterior descending coronary artery (LAD), the distal 1/5 of the abdominal aorta, and a proximal portion of the thoracic aorta. Counts of attached monocytes and platelet clumps were made by scanning electron microscopy only for the LAD and expressed per mm2 of surface. The most striking new result was in regard to the platelet clumps. These were reduced by the fish oil from 996 +/- 295/mm2 in the untreated BT group to 313 +/- 59 and 364 +/- 105 in BT+cod liver oil and BT+menhaden oil groups, respectively. Most of the platelet clumps were adherent to attached monocytes in all groups and the number of attached monocytes were greatly reduced by the fish oil additive. Thus there were close relationships among platelet clumps, monocytes, and lesion endothelium. Numbers of attachments over nonlesion endothelium were much less than those over lesions in all dietary groups. The most surprising result was the lack of retardation of lesion growth by the fish oil additives in spite of the reduction in attached monocytes and platelet clumps. In previous studies where the high plasma cholesterol levels in the BT swine had been modestly reduced (about 25%) there had been a marked retardation of lesion growth. The current result suggests that plasma cholesterol is the major factor controlling lesion growth in this model through under milder conditions and longer observation periods other factors might become apparent.

Comparison of effects of fish oil and corn oil supplements on hyperlipidemic diet induced atherogenesis in swine.

The addition of a fish oil supplement rich in n - 3 unsaturated fatty acids to a high cholesterol, high saturated fat (BT) diet for swine has been shown previously to result in modest lowering of plasma cholesterol levels and in marked retardation of atherogenesis. It has been suggested that the effect was due to the change in polyunsaturated (PUFA) to saturated fatty acid ratios (P/S) and that a supplement of PUFA of the n - 6 series might have the same effect as the fish oil. We have tested this hypothesis in swine fed an atherogenic diet by comparing the effect of a fish oil supplement producing a P/S ratio of 0.28 to that of corn oil in the same amount producing a ratio of 0.46. The P/S ratio of the atherogenic diet without supplements was 0.16. Thirteen young male Yorkshire swine were fed either BT alone (n = 4), BT + cod liver oil (n = 4) or BT + corn oil (n = 5) for 6 months and then killed for quantitative studies of atherosclerosis in the aortas and coronary arteries including lesion areas, number of lesion cells, and number of monocytes attached to endothelium. Plasma cholesterol levels were determined periodically and lipoproteins were separated terminally by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. The fish oil supplement resulted in a 30% reduction in time-weighted average plasma cholesterol levels, and a marked shift in terminal lipoprotein patterns from predominantly apo B and E containing ones to predominantly apo B only ones. Atherogenesis was reduced by the fish oil supplement as judged by several morphometric criteria including size of lesions, number of lesion cells, and number of monocytes attached to lesion endothelium. The corn oil supplement produced no significant reductions in any of these variables from those in swine fed the atherogenic BT diet without the supplement. We conclude that the n - 3 fatty acid rich fish oil supplemented diet retarded atherogenesis, but that this effect was not shared by the corn oil supplemented diet which had an even higher P/S ratio.

Reductions in serum thromboxane, prostacyclin, and leukotriene B4 levels in swine fed a fish oil supplement to an atherogenic diet.

We have reported previously that fish oil rich in omega-3 fatty acids added to a butter-cholesterol atherogenic diet for swine resulted in marked retardation of the atherosclerotic process which many regard as largely an inflammatory response to injury by excessive lipids in the intima. In this report on the same swine we present serum levels of several eicosanoids derived from arachidonic acid via the cyclooxygenase and lipoxygenase pathways. The study involves six swine fed a high fat, high cholesterol diet (BT group) for 4 months, six swine fed the same diet but with 30 ml/day fish oil added (BT + FO), and five swine fed a low fat, low cholesterol mash diet (MA). The serum eicosanoids were measured by radioimmunoassay. Thromboxane B2 levels (ng/dl: means +/- SEM) were 543 +/- 49 for MA, 231 +/- 12 for BT, and 105 +/- 20 for BT + FO, and all differences were statistically highly significant, 6-Keto PGF1 alpha (a relatively stable prostacyclin metabolite) levels were 249 +/- 31 for MA, 184 +/- 12 for BT, and 101 +/- 10 for BT + FO, and all differences were significant. Leukotriene B4 levels at 4 months were 151 +/- 25 for MA, 112 +/- 11 for BT, and 84 +/- 11 for BT + FO. BT + FO was significantly different from both MA and BT, but BT was not significantly different from MA. Leukotriene C4 levels were not significantly different among the three groups. Of special interest was the effect of the BT diet without the FO additive in reducing several eicosanoid levels compared to MA values. The affected eicosanoid levels were reduced still further by the fish oil additive, indicating its ability to inhibit both the cyclooxygenase and the lipoxygenase pathways. The relation of the fish oil-induced inhibition to the observed retardation of atherogenesis is not as yet clear but there are several theoretical possibilities, including reduction in recruitment of monocytes and in proliferation of smooth muscle cells.

N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide-induced bladder cancer in mice: augmentation by sutures through the bladder wall.

The augmenting effect of chronic inflammation on bladder cancer was studied in female Swiss mice treated with N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT), 0.15% by weight of diet for 20 weeks. To produce chronic inflammation one silk suture and one catgut suture were placed through the bladder wall of 63 mice to receive FANFT and 18 to receive normal diet. In remaining mice (no. = 62) the bladder was simply touched without suture insertion. Thirty-two animals treated with FANFT and 33 treated with FANFT + sutures also received supplements to their drinking water throughout the experiment of the sulfhydryl-reducing agent N-acetylcysteine (500 mg./kg. body weight/day). Seven weeks following FANFT treatment, bladder cancers developed in 12% of mice with sutures and FANFT and in 19% of those with sutures, FANFT, and N-acetylcysteine. No cancers developed in mice receiving FANFT alone or FANFT with N-acetylcysteine and none in mice treated with sutures only. Placement of silk sutures through the bladder wall of mice augments FANFT-induced bladder cancer. N-acetylcysteine at these doses does not influence the incidence.

Vanadate supplements and 1,2-dimethylhydrazine induced colon cancer in mice: increased thymidine incorporation without enhanced carcinogenesis.

Because vanadate ion is a potent mitogen and accumulates in the gut of rodents fed vanadate supplements, effects of ammonium metavanadate in drinking water (10 ppm or 20 ppm) were studied on the development of large bowel neoplasms in mice treated with 1,2-dimethylhydrazine (DMH) (20 mg kg-1 weekly for 20 weeks). In the colon at 30 weeks DMH treatment caused a 14% increase in RNA content, an 18% increase in DNA content, and 33% deeper crypts. Vanadate at either 10 ppm or 20 ppm decreased RNA content by approximately 11%. Although vanadate increased thymidine incorporation 210% to 550% compared with controls, it had no influence on the attack rate, incidence, or histological type of tumours induced by DMH.

Augmentation of 1,2-dimethylhydrazine-induced colon cancer by experimental colitis in mice: role of dietary vitamin E.

Because ulcerative colitis predisposes to colonic cancer, for determination of the effect of colitis on experimental colon carcinogenesis, rectal instillations of peptides that attract and activate neutrophils were used to induce colitis in CD-1 (ICR) BR mice receiving 20 weekly injections of the carcinogen 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8]. From week 4 through week 15 of DMH injections, twice-weekly enemas of formyl-norleucyl-leucyl-phenylalanine were given to DMH-treated mice. The effect of the antioxidant vitamin E in the diet (1,750 IU/kg diet) was studied in another group of mice treated with DMH and having colitis. Four weeks after DMH was discontinued, cancer occurred in 9 of 28 (32%) animals with DMH plus control enemas, in 22 of 29 (76%) animals with DMH plus colitis (P = .001), and in 16 of 28 (57%) animals with DMH plus colitis plus supplemental vitamin E (P = .11 compared with the group with DMH and colitis). Colitis enhances DMH-induced colonic carcinogenesis.

Effects of alpha-difluoromethylornithine on the growth of experimental Wilms' tumor and renal adenocarcinoma.

Because polyamines are essential for cellular growth and differentiation, and because human renal carcinomas have spermidine levels that are higher than those in normal renal tissue, effects of 2-difluoromethylornithine (DFMO) on the growth of experimental renal tumors were investigated. DFMO is a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme controlling polyamine biosynthesis. DFMO (2%) in drinking water was administered to BALB/c mice with intrarenal transplants of a renal adenocarcinoma cell suspension and to Wistar/Furth rats with s.c. transplants of a Wilms' tumor. At 28 days, renal carcinomas in DFMO-fed mice weighed 72% less than those in control animals (p less than 0.001). Wilms' tumor weight was not affected by DFMO feeding. DFMO caused 72 to 75% inactivation of ornithine decarboxylase activity and reduced putrescine levels in renal carcinoma and Wilms' tumor, reduced spermidine levels in Wilms' tumor, and apparently raised spermine levels in the latter as a consequence. DNA content was not affected by DFMO feeding. The mean number of lung metastases in DFMO-fed, renal carcinoma-bearing mice was 0.1 and in controls was 1.4 (p less than 0.001). DFMO feeding increased survival of mice bearing renal carcinomas by 3.0 +/- 0.8 (S.E.) days (p less than 0.05), i.e., from 30.5 +/- 0.8 days to 33.5 +/- 1.2 days. DFMO did not affect the growth of Wilms' tumor; however, in renal adenocarcinoma, it reduced growth, prevented lung metastases, and increased survival.