Inhibition of NADH oxidase activity and growth of HeLa cells by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984) and response to epidermal growth factor

Right side-out plasma membrane vesicles isolated from HeLa cells exhibited an NADH oxidase activity at their external surfaces that was inhibited by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984). Intact HeLa cells (fresh or frozen) also exhibited an NADH oxidase activity at the external cell surface. The inhibition of this activity by LY181984 was enhanced by the addition of epidermal growth factor (EGF). The order of addition was critical. It was necessary that the LY181984 be followed by the EGF. If the EGF was administered first, the response to LY181984 was unaffected by EGF. Binding of [³H]LY181984 to HeLa cells also was enhanced by EGF. Growth experiments with HeLa cells revealed a similar pattern of response to EGF. The EC₅₀ of growth inhibition of LY181984 was about 100 μM. However, if the LY181984 was followed by addition of 10 nM EGF, the EC₅₀ for LY181984 was reduced to about 30 nM which now approximated the previously determined K_d of [³H]LY181984 binding of 30 nM and the EC₅₀ of 30 nM for inhibition of NADH oxidase activity by LY181984 by isolated vesicles of plasma membranes. The tumor-inactive sulfonylurea N-(methylphenylsulfonyl-N′-(phenyl)urea (LY181985) was ineffective in the inhibition of NADH oxidation and of growth with HeLa cells either in the presence or absence of EGF.     

Response of NADH oxidation and growth in K-562 cells to the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984)

Summary Growth of K-562 cells in culture is inhibited by the antitumor sulfonylureaLY181984 (N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea) with an ED₅₀ of about 30 M. LY181984 was shown previously to inhibit NADH oxidation by plasma membranes from HeLa cells and other sources and to influence mitochondrial oxidative phosphorylation. With K-562 cells, NADH oxidation by plasma membranes was transiently stimulated and then inhibited by LY181984. NADH oxidation by whole cells was transiently stimulated and then inhibited by 0.1 to 100 M LY181984 as well. Both the stimulations and inhibitions of activity were time-dependent. NADH oxidation by lower phase membranes depleted of plasma membranes by aqueous two-phase partition also was inhibited by micromolar and submicromolar concentrations of LY181984. Inhibition did not correlate with mitochondrial presence but rather with membranes that appeared to be fragments of the Golgi apparatus. The oxidation of NADH by whole cells and of plasma membranes that was inhibited by LY181984 was distinguished from mitochondrial NADH oxidation by resistance to inhibition by cyanide and by proceeding under oxygen-depleted conditions or an argon atmosphere. In contrast to the active antitumor agent LY181984, the inactive but chemically-related analog, LY181985 (N-(4-methylphenyl-sulfonyl)-N-(4-phenylurea), inhibited neither growth nor NADH oxidation with K-562 cells or cell fractions.     

Mechanism of killing of HeLa cells by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984)

Summary The antitumor sulfonylureas appear to inhibit both mitochondrial activity in susceptible human colon lines and to inhibit the oxidation of NADH by isolated plasma membrane vesicles from HeLa cells. The results reported here describe the morphological appearance of HeLa cells treated with the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984). The cells remain viable for several days although the rate of increase in cell number is slowed especially at high concentrations of the drug. Cells become smaller with normal nuclei or maintain a normal size but contain multiple or enlarged nuclei. The morphological observations suggest that the drug may somehow interfere with the ability of the cells to enlarge following cytokinesis. Between 72 and 96 h, the cells begin to die. Cell death is accompanied by a condensed and fragmented appearance of the nuclear DNA as revealed by fluorescence microscopy with 4,6-diamidino-2-phenylindole suggestive of apoptosis. Early transients in loss of pH control (4 min after sulfonylurea addition) and an increase in cytoplasmic calcium (4 h after sulfonylurea addition) were observed but were small and perhaps secondary to the mechanism responsible for the failure of the cells to grow and ensuing cell death.     

Golgi apparatus respond to the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984) in sulfonylurea-susceptible but not in resistant cell lines

Summary Cell lines susceptible or resistant to the active antitumor sulfonylurea [N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)-urea] (LY 181984) were treated with 100 M sulfonylurea for 1 or 3 h followed by monensin for 1 h. With cell lines where growth was inhibited by the active sulfonylurea, swollen Golgi apparatus cisternae following treatment were fewer and smaller than in untreated cells. Overall the volume of monensin-responsive trans cisternae was reduced by about 50% to 75% in cells lines where the antitumor sulfonylurea was growth inhibitory. The swelling response was unaffected by sulfonylurea in sulfonylurea-unresponsive cells. The antitumor-inactive sulfonylurea [N-(4-methylphenylsulfonyl)-N-(phenyl)urea] (LY 181985) was without effect on cisternal swelling with both susceptible and resistant cell lines. The results suggest a response of the trans Golgi apparatus to the active antitumor sulfonylurea that resulted in reduced acidification of the trans Golgi apparatus cisternae. This response appears to be restricted to susceptible cell lines where growth was inhibited by the active antitumor sulfonylurea but not in resistant cell lines where growth was unaffected by the active antitumor sulfonylurea.     

A Circulating Form of NADH Oxidase Activity Responsive to the Antitumor Sulfonylurea N-4-(methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984) Specific to Sera from Cancer Patients

Our laboratory has described a drug-responsive NADH oxidase activity of the external surface of the plasma membrane of HeLa and other cancer cells, but not from normal cells, that was shed into media conditioned by the growth of cancer cells such as HeLa. The shed form of the activity exhibited the same drug responsiveness as the plasma membrane-associated form. In this study, sera from tumor-bearing and control rats, cancer patients, normal volunteers, and patients with diseases other than cancer were collected and assayed for a cancer-specific form of NADH oxidase responsive to the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984). With sera from tumor-bearing rats and cancer patients, LY181984 added at a final concentration of 1 M either inhibited or stimulated the activity. With sera from control rats, normal volunteers, or patients with disorders other than cancer, the drug was without effect on the NADH oxidase activity of the sera. The activity altered by the antitumor sulfonylurea was present both in freshly collected sera and in sera stored frozen. Inhibition was half maximal at about 30 nM LY181984. The sulfonylurea-altered activity was found in sera of nearly 200 cancer patients including patients with solid cancers (e.g., breast, prostate, lung, ovarian) and with leukemias and lymphomas. We postulate that the serum presence of the antitumor sulfonylurea-responsive NADH oxidase represents an origin due to shedding from the patient's cancer. If so, the antitumor-responsive NADH oxidase would represent the first reported cell surface change universally associated with all forms of human cancer.     

Mannosyl-β(1-4)-N-acetylglucosaminyl-β(1-N)-urea,a compound isolated from the urine of patients with β-mannosidosis

A previously unknown substance, mannosyl-(1–4)-N-acetylglucosaminyl-(1-N)-urea, has been isolated from the urine of patients with -mannosidosis in addition to the main metabolite mannosyl-(1–4)-N-acetylglucosamine. Structural investigation was carried out by fast atom bombardment mass spectrometry and high-resolution¹H-nuclear magnetic resonance spectroscopy at 500 MHz. It was postulated that the occurrence of this carbohydrate-urea conjugate in urine results mainly from urine handling.