K252a inhibits proliferation of glioma cells by blocking platelet-derived growth factor signal transduction.

Growth factors are known to regulate glioma proliferation. The glioma cell lines U87 and T98G were examined for evidence of an autocrine stimulatory loop involving the neurotrophin family of growth factors. Although neurotrophin-3 and TrkC RNA were detected by reverse transcription-PCR, there was no evidence of significant interaction between neurotrophin-3 and its cognate receptor TrkC. The microbial alkaloid K252a has been described to inhibit both Trk tyrosine kinase activity and neuroblastoma cell proliferation. K252a inhibited proliferation in U87 (IC50 = 1170 nM) and T98G (IC50 = 529 nM) but induced apoptosis in U87 cells only. At concentrations of 500 nM to 1 microM, K252a blocked only platelet-derived growth factor (PDGF)-mediated receptor autophosphorylation. These results suggest that an autocrine loop involving PDGF is functional and important for maintaining tumor growth. There is no evidence to support the existence of a neurotrophin-mediated autocrine loop. K252a, through inhibition of PDGF signal transduction, may be a novel therapeutic agent in the treatment of human gliomas.

4-Aminopyridine causes apoptosis and blocks an outward rectifier K+ channel in malignant astrocytoma cell lines.

Among the ion channels and pumps activated by growth factor stimulation, K+ channels have been implicated in the growth and proliferation of several cancer cell lines. The role of these channels in central nervous system tumors, however, has not been described. This study used the malignant astrocytoma cell lines U87 and A172. 4-Aminopyridine (4-AP) inhibition of proliferation was dose dependent, and assessment using a TUNEL in situ assay revealed that apoptosis occurred in U87 cells with wild-type p53 but not in A172 cells with mutant p53 (24-hr incubation with mM 4-AP). In patch clamp experiments, we identified two types of K+ currents in both cell lines, a charybdotoxin-sensitive Ca2(+)-activated K+ channel and a 4-AP-sensitive outward rectifier K+ current. The outward rectifier current was blocked by 4-AP in a dose-dependent manner, with half-maximal block occurring at 3.9 mM. The blocking effect of 4 mM 4-AP was noticeable at potentials as low as -65 mV and was statistically significant at -60 mV and above, suggesting that 4-AP-sensitive current is active at physiological potentials. By contrast, charybdotoxin (1 microM) and tetraethylammonium. Cl (2 mM) blocked the Ca2(+)-activated K+ channel in both cell lines but had no appreciable effect on cell growth. Our findings reveal that 4-AP inhibits proliferation and the outward rectifier K+ channel in both U87 and A172 cells. More studies are needed, however, to describe the mechanism by which K+ channels influence proliferation and induce apoptosis.

Adenosine diphosphate potentiates the inhibition of norepinephrine-induced relaxation by 5-hydroxytryptamine in canine coronary arteries.

Vasoactive substances released from aggregating platelets inhibit beta-adrenergic neurotransmission in coronary arteries. Studies were carried out on the effects of two such vasoactive substances on canine coronary arteries, at concentrations equivalent to that released by platelets under physiological conditions. 5-Hydroxytryptamine (5 X 10(-7) M) reduced the sensitivity of coronary artery ring segments to the beta-adrenergic relaxing effects of norepinephrine. Adenosine diphosphate (3 X 10(-6) M) further reduced the sensitivity to norepinephrine caused by 5-hydroxytryptamine, while the nucleotide alone had no significant effect. 5-Hydroxytryptamine and adenosine diphosphate acted selectively on the norepinephrine-induced relaxation; whereas the relaxatory response of the vessel to nitroprusside, a direct muscle relaxant, was unaffected. 5-Hydroxytryptamine caused contraction of the tissue, but this opposing response did not account for the inhibition of the norepinephrine-induced relaxation observed in the presence of the indoleamine. The decreases in sensitivity to norepinephrine caused by 5-hydroxytryptamine and adenosine diphosphate were prevented by the serotonin receptor antagonist, methiothepin. The potentiation by adenosine diphosphate of the 5-hydroxytryptamine-induced shift in the relaxation caused by norepinephrine was blocked by the purine receptor antagonist, 8-(p-sulfophenyl)-theophylline. Neither adenosine nor alpha,beta-methylene adenosine diphosphate potentiated the action of 5-hydroxytryptamine, suggesting that phosphate hydrolysis of the nucleotide is required for the action of adenosine diphosphate. These results suggest that adenosine diphosphate potentiates the inhibitory effect of 5-hydroxytryptamine on the beta-adrenergic response of coronary arteries exposed to vasoactive substances released from platelets.