Antitumor effect of KT6124, a novel derivative of protein kinase inhibitor K-252a and its mechanism of action

Summary Novel derivatives of K-252a, (8R*,9S*,11S*)-(–)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo [a,g]-cycloocta[cde]trinden-1-one, an inhibitor of protein kinases and calmodulin-dependent phosphodiesterase, were synthesized and evaluated for their antitumor activity in vitro and in vivo. Of ten derivatives tested, four were active against the P388 murine leukemia i. p.-i. p. system, although K-252a was inactive. Among these derivatives, KT6124 was selected for further biological evaluation studies because its efficacy was the highest. KT6124 was also active against sarcoma 180 and B16 melanoma. It exerted a relatively broad spectrum of antiproliferative activity against 20 human tumor cell lines in vitro. To determine the mechanism(s) of action underlying the antitumor activity of KT6124, we tested the drug for inhibition of protein kinases, including Ca²⁺-and phospholipid-dependent protein kinase (PKC), in intact A431 human epidermoid carcinoma cells in comparison with the PKC-inhibitory activity of K-252a. KT6124 did not antagonize the action of phorbol 12-myristate 13-acetate (PMA) in A431 cells, whereas K-252a did, suggesting that KT6124 may not act on protein kinases in the cells. The interaction of KT6124 with DNA in living cells was examined by the alkaline elution method. KT6124 apparantly exhibited DNA scission both dose-and time-dependently in the target cells. The DNA breakage was dependent on proteinase K treatment, suggesting its possible interaction with DNA-related enzyme(s). These results indicate that KT6124 exerts antitumor activity by acting on DNA or on DNA-related enzyme(s) in tumor cells rather than via the inhibition of protein kinases.     

Antigen presentation by a B-cell line transfected with cloned immunoglobulin heavy- and light-chain genes specific for a defined hapten.

The rearranged genes encoding immunoglobulin heavy (mu) and light (kappa) chains specific for the hapten 2,4,6-trinitrophenyl (Tnp) were introduced into a B-lymphoma line that bears surface IgG with an unknown specificity and expresses surface Ia molecules. A transformant expressing surface IgM specific for Tnp was obtained. The transformant was found to present Tnp-proteins to antigen (protein)-specific T cells far more efficiently than the parenteral B-lymphoma line. This artificial system, utilizing recombinant DNA technology and gene transfer, provides several approaches for the study of T-cell-B-cell interactions.     

Surgical experience of subacute pulmonary thromboembolism with severe pulmonary hypertension.

Surgical treatment for subacute pulmonary arterial thromboembolism has previously been considered to be inappropriate. We undertook a pulmonary arterial thrombectomy and removal of a floating right heart thrombus in a patient who had been symptomatic for over a month. The pulmonary arterial pressure, which had been equal to the systemic pressure preoperatively, decreased gradually and almost normalized one month postoperatively. Pulmonary perfusion scintigraphy revealed a dramatic improvement and the patient returned to normal life activities.     

Use of Glycopeptidolipid Core Antigen for Serodiagnosis of Mycobacterium avium Complex Pulmonary Disease in Immunocompetent Patients

We report the development of a serodiagnostic method for Mycobacterium avium complex (MAC) disease with an enzyme immunoassay (EIA) with the MAC-specific glycopeptidolipid (GPL) core as the antigen. In this study, we confirmed by EIA that the GPL core antibody was in the sera of immunocompetent patients with MAC disease. The EIA for quantifying the GPL core antibody was evaluated as a clinical tool for serodiagnosis of pulmonary MAC disease. A significant increase in GPL core antibodies (immunoglobulins G, A, and M) was detected in sera of patients with MAC pulmonary diseases when they were compared to patients who were colonized with MAC, patients with Mycobacterium kansasii disease or tuberculosis, and healthy subjects. The sensitivities and specificities of the GPL core-based EIA for diagnosis of MAC pulmonary disease were 72.6% and 92.2%, respectively, for IgG, 92.5% and 95.1%, respectively, for IgA, and 78.3% and 91.0%, respectively, for IgM. The best sensitivity and specificity were obtained by measuring immunoglobulin A antibodies against GPL core antigen. The level of GPL core antibodies reflected disease activity, since it decreased in cured MAC patients who had responded to chemotherapy. Measurement of serum antibodies against GPL core is useful for both diagnosis and assessment of disease activity in MAC disease of the lung.     

P-glycoprotein expression in soft-tissue sarcomas

Multidrug resistance (MDR) is an important problem in chemotheraphy for neoplastic disease. In humans, MDR is mainly mediated by P-glycoprotein (P-gp), a product of theMDR1 gene, which acts as a transmembrane protein pump and eliminates chemotherapeutic agents from the cells. Expression of P-gp was immunohistochemically studied by using two monoclonal antibodies, JSB-1 and C-219, on paraffin-embedded sections from 55 patients with soft-tissue sarcoma. The histological diagnosis of tumors was malignant fibrous histiocytoma in 24 cases, liposarcoma in 9, synovial sarcoma in 7, malignant neurogenic tumors in 6, leiomyosarcoma in 5, others in 4. The histological grade was determined on the basis of criteria previously proposed by us. Out of 55 cases, 34 (62%) were positive for P-gp expression. There was a significant difference in P-gp expression between high-grade (90%) and intermediate and low-grade tumors (46%) (P<0.005). Tumors expressing P-gp had a less favorable prognosis than P-gp-negative tumors in the high- and intermediate-grade tumors. The current study demonstrated that the estimation of P-gp expression could be used to select appropriate therapeutic modalities.Abbreviations MDR multidrug resistance - P-gp P-glyco-protein     

Ultrastructure of capillary permeability in human brain tumor: primary meningeal malignant melanoma

The tumor vessels of a primary meningeal malignant melanoma were studied by electron microscopy. There were numerous endothelial fenestrae and basal lamina abnormalities in the intrinsic tumor capillaries. They resembled the tumor vessels found in nonglial tumors, but were distinctly different from those seen in glial tumors with nonfenestrated capillaries. These findings were anticipated because leptomeninges have fenestrated capillaries.     

Effect of calcitonin on total body bone mineral contents of experimental osteoporotic rats determined by dual photon absorptiometry

Summary Total body bone mineral (TBBM) content in rats was measured by dual photon absorptiometry (DPA). TBBM showed significant increases over 4 weeks in control groups with significant bone loss over the same time in prednisolone-injected rats on low calcium feed. Daily injections of calcitonin significantly reduced loss of bone mass. Both prednisolone- and prednisolone-calcitonin-injected groups showed significantly elevated serum alkaline phosphatase with the prednisolone-calcitonin group also exhibiting elevated serum calcium and phosphate levels, confirming the impact of the experimental protocol. TBBM measured by DPA in all groups correlated well (r=0.928,P<0.001 n=20) with the total ash weight suggesting that the method reflects total skeletal mineral content in the small animal. TBBM measurement by DPA proves well-suited to monitoring bone mineral in a small animal experimental setting.     

Protective effect of prostaglandins D2, E1 and I2 against cerebral hypoxia/anoxia in mice

The protective effect of prostaglandins (PGs) against cerebral hypoxia/anoxia was investigated with a variety of experimental models in relation to their CNS depressant effects in mice. Furthermore, the effect of PGs on the changes of cerebral energy metabolites and cyclic nucleotide was examined in hypoxic mice. Mice were given s.c. doses of PGs 30 min before tests. Among the PGs tested, treatment with PGD2, PGE1 and PGI2 Na showed a consistent and dose-dependent protection against cerebral anoxia induced by all models studied: histotoxic anoxia by KCN, hypobaric hypoxia, normobaric hypoxia and decapitation-induced gasping. However, PGA1, PGA2, PGB1, PGB2, PGE2, PGF1 alpha, PGF2 alpha and 6-keto-PGF1 alpha at a dose of 3 mg/kg were without effect against normobaric hypoxia and gasping duration. The three PGs, i.e. PGD2, PGE1 and PGI2 which showed anti-hypoxic effects decreased locomotor activity and potentiated hexobarbital-induced sleep. On the other hand, PGE2, PGA1, PGA2 and PGB2 also caused a decrease in locomotor activity. Similarly, PGE2 and PGA1 caused a potentiation of hexobarbital-induced sleep, but interestingly they did not cause clear-cut increase in cerebral resistance to hypoxia, in contrast with the former three PGs. Thus general depression of CNS function appears not to be responsible for the PGD2-, PGE1- and PGI2-induced increase in cerebral resistance to hypoxia. The levels of Cr-P and ATP were significantly reduced and those of ADP and AMP were markedly elevated in hypoxic brain, resulting in a decrease in a calculated energy charge potential. The lactate level and lactate/pyruvate ratio increased and the glucose level decreased markedly.(ABSTRACT TRUNCATED AT 250 WORDS)