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.     

Studies on stroke-prone spontaneously hypertensive rats (SHRSP) fed a high-fat and high-cholesterol diet--effects of salt intake on serum lipoprotein and apolipoprotein metabolism

Recently, food intake in Japan has been characterized by an increase in fat intake, especially animal-fat intake and the maintenance of excess salt (sodium chloride) intake. It is generally accepted that the increase in fat intake is closely related to atherosclerosis, and excess salt intake is a high risk factor for the development of hypertension and cerebrovascular lesions. So far, in almost all reports, the increase in fat intake and excess salt intake have been studied independently, and there have been few reports on the combined effects of these two factors. Taking the above things into consideration, it would seem to be very interesting to investigate the effect of excess salt intake on lipid metabolism. In this paper, we studied the effects of excess salt intake on lipoprotein and apolipoprotein metabolisms, using stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Kyo: Wistar rats (WKY) as model animals. The results obtained were as follows: A significant increase in the concentration of serum total cholesterol (TC) was observed in SHRSP and WKY, when the rats were given a regular diet (CE-2, Clea Japan Inc.) and 1% sodium chloride solution (1% NaCl) as drinking water for 4 weeks. This was accompanied by a tendency toward increases in the concentrations of serum apolipoproteins in both strains. These results suggest that excess salt intake could accelerate the production of serum total lipoproteins in SHRSP and WKY, when the rats are fed a regular diet. Next, 1% NaCl and a high-fat and high-cholesterol diet (HFC diet) were simultaneously given to SHRSP and WKY for 6 weeks. The effects of simultaneous administration on lipoprotein and apolipoprotein metabolisms were compared with those of HFC feeding. One percent NaCl did not markedly affect hypercholesterolemia in WKY, while it induced more marked hypercholesterolemia in SHRSP that was associated with extreme elevations of serum TC and the atherogenic index (A.I.). This deleterious effect of 1% NaCl in SHRSP was due to drastic elevations of cholesterol contents in the very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) fractions. This was also associated with marked increases in apo B contents in the VLDL, IDL and LDL fractions and significant increases in apo E contents in the VLDL and IDL fractions. These results indicate that 1% NaCl induced much larger increases in serum atherogenic beta-lipoproteins in SHRSP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural characterization of the tau-immunoreactive tubules in the oligodendroglial perikarya and their inner loop processes in progressive supranuclear palsy

Coiled bodies and interfascicular threads are conspicuous white matter abnormalities of brains of patients with progressive supranuclear palsy (PSP). Both structures are argyrophilic and immunoreactive for the microtubule-binding protein tau. This report concerns the ultrastructural localization of interfascicular threads and their relationship to coiled bodies in five PSP patients. We showed for the first time that abnormal tubules with a 13- to 15-nm diameter and fuzzy outer contours were the common structures of coiled bodies in the oligodendroglial perikarya and of interfascicular threads. Moreover, the tubules were immunolabeled by anti-tau antibodies. The abnormal tau-positive tubules of interfascicular threads were located in the inner loop of the myelin sheath. Our study further indicated that the thread-like structures in the white matter comprised, at least in part, oligodendroglial processes, and that they were also present in gray matter. We consider that the formation of coiled bodies in the perikarya and of interfascicular threads represents a common cytoskeletal abnormality of the oligodendroglia of PSP patients. Moreover, even though the white matter alterations of PSP resemble those of corticobasal degeneration, there are certain ultrastructural differences in the abnormal oligodendroglial tubules of the two diseases. Received: 4 October 1996 / Accepted: 6 December 1996     

Detection of nephritogenic antigen from the Lewis rat renal tubular basement membrane

Immunopathogenicity of trypsin-solubilized or non-solubilized renal tubular basement membrane (TBM) of the Lewis (LEW) rat was investigated. Autoimmune tubulointerstitial nephritis (TIN) was induced in BALB/c mice by immunization with trypsin-solubilized LEW rat TBM, while immunization with non-solubilized TBM did not produce the disease. Based on this preliminary experiment we studied the characterization of immunogenic and nephritogenic TBM antigen of the LEW rat. TIN was characterized by severe mononuclear cell infiltrates with multi-nucleated giant cells in the interstitium, tubular destruction and intensive IgG and C3 deposits along the TBM. Anti-TBM antisera and eluate from the nephritic mouse kidneys reacted with the TBM of normal LEW rat kidney by immunofluorescence. LEW rat TBM was also detected immunofluorescently by using antisera from BALB/c mice immunized with autologous trypsin-solubilized TBM. A competitive inhibition test revealed a higher titer of anti-TBM antibody in the eluate than in the adsorption-treated antisera per microgram IgG. Immunoblotting showed one reactive band with a molecular weight of 45,000 daltons, and the blotting patterns in tryptic TBM of the Brown Norway (BN) and LEW rats appeared similar. Amino acid analysis of nephritogenic LEW rat tryptic TBM showed that it contained no hydroxyproline and hydroxylysine, suggesting that this TBM preparation was not collagenous. These findings suggest that tryptic digestion contributes to the release of nephritogenic antigen from the LEW rat TBM and that this antigen system might participate in the immune system involved in the anti-TBM associated TIN that is well known to be induced by non-digested TBM of TBM antigen positive animals.     

An infertile man with a 45X karyotype

We describe an infertile man with normal secondary sexual characteristics and a 45X karyotype. Physical examination showed bilateral soft small testes and the semen contained no sperm. Serum testosterone, estrogen and gonadotropin levels were within normal range. Biopsy of the testis showed germinal aplasia.     

Clinical and pathological evaluations of methotrexate, vinblastine, Adriamycin and cisplatin chemotherapy for advanced urothelial cancers

We have treated advanced transitional-cell carcinoma of the urothelial tract with methotrexate, vinblastine, Adriamycin, and cisplatin (M-VAC) chemotherapy since July of 1985. We analyzed the effect of that chemotherapy in 26 patients with advanced urothelial cancer who were treated in our hospital and followed up. They were divided into two groups. Group 1 consisted of 15 patients with distant metastases. In all, 11 of them received M-VAC as adjuvant chemotherapy for metastatic lesions after surgical removal of the primary lesion, and the remaining 4 patients were not operable since they had very advanced-stage tumors; they received only M-VAC chemotherapy. Group 2 contained 11 patients who received M-VAC neo-adjuvant chemotherapy. In group 1, the overall response rate was 57.1% and the mean duration of response was 12.6 months. In the 11 patients who had received M-VAC as adjuvant therapy after surgical removal of the primary tumor, the mean duration of response was 14.1 months. After M-VAC chemotherapy, six patients underwent surgical resection of metastatic lesions and restaging was done pathologically in these cases. The clinical response coincided with the pathological response in all six cases. In group 2,5 of 11 patients experienced histological downstaging of the resected bladder. M-VAC chemotherapy combined with surgical resection of residual tumors has proved to be an effective option against advanced urothelial cancer.Presented at the 4th International Conference on Treatment of Urinary Tract Tumors with Adriamycin/Farmorubicin, 16–17 November 1990, Osaka, Japan