Inhibition of cellular growth and induction of apoptosis in pituitary adenoma cell lines by the protein kinase C inhibitor hypericin: potential therapeutic application

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✓ Protein kinase C (PKC) is an enzyme involved in the regulation of cellular growth, proliferation, and differentiation in a number of tissues including the anterior pituitary, in which it is also believed to play a role in hormone secretion. Protein kinase C activity and expression have been found to be greater in adenomatous pituitary cells than in normal human and rat pituitary cells and higher in invasive pituitary tumor cells than in noninvasive ones. Inhibition of PKC activity has been shown in a variety of tumor cells to inhibit growth in a dose-related fashion. The purpose of the current study was to determine whether hypericin, a potent inhibitor of PKC activity that may be administered clinically, alters the growth and proliferation in established pituitary adenoma lines and to determine if inhibition of PKC activity induces apoptosis, as reported in some other tumor cell types. Two established pituitary adenoma cell lines, AtT-20 and GH4C1, were treated with hypericin in tissue culture for defined periods following passage. Inhibition of growth was found to be dose dependent in all three cell lines in low micromolar concentrations of hypericin, as determined by viable cell counts, methylthiotetrazole assay, and [3H]thymidine uptake studies. Concentrations of hypericin as low as 100 nM also induced apoptosis in these established lines, whereas treatment of normal human fibroblasts with a concentration of 10 µM failed to induce apoptosis. The potential use of hypericin in the therapy of pituitary adenomas warrants additional in vitro investigations with the aim of later moving toward therapeutic trials in selected patients in whom surgical or medical therapy has failed.

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Address reprint requests to: William T. Couldwell, M.D., Ph.D., Trinity Medical Center, One Burdick Expressway West, Minot, North Dakota 58701.

© AANS, except where prohibited by US copyright law.

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    Graph displaying hypericin inhibition of protein kinase C (PKC) activity in the established pituitary adenoma cell line AtT-20. A marked inhibition of total PKC activity occurs in AtT-20 cells following incubation with 10 µM hypericin. The PKC activity units are expressed as nanomoles of adenosine triphosphate transferred per minute per milligram of protein.9 Measurements of total PKC activity obtained as early as 2 hours after treatment indicate significantly decreased PKC activity in the treated cultures. All values represent the mean of triplicate treated cultures plus the standard error of the mean. Asterisks indicate values significantly different from controls (one-way ANOVA, Duncan's multiple comparison; *p < 0.05).

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    Graphs showing hypericin inhibition of pituitary adenoma cell growth. Results of a methylthiotetrazole (MTT) assay indicate a dose-related decrease in the absorbance of AtT-20 cells (correlating with viable cell numbers) following administration of hypericin for a period of 3 (upper left) or 6 (upper center) days. The inhibition of growth was facilitated slightly by the longer incubation period. Similarly, the absorbance of GH4C1 cells decreased in a dose-related manner in the presence of hypericin for 4 days (upper right). All values represent the mean of quintuplicate wells + the standard error of the mean (SEM). Similar to the results of the MTT assay, cell viability, as determined by the trypan blue dye exclusion test, was decreased in cell cultures treated with increasing concentrations of hypericin in both AtT-20 (lower left) and GH4C1 cell lines (lower center). All values represent the mean of triplicate cultures + SEM. To complement the cell viability assays, [3H]thymidine uptake was determined in AtT-20 cells following incubation with hypericin for 72 hours (lower right). Thymidine uptake was inhibited significantly at doses of 1 µM hypericin and greater for this time period. All values represent the mean of quadruplicate wells + SEM. For all plots in Fig. 2, asterisks indicate values that are significantly different from controls (one-way ANOVA, Duncan's multiple comparison; *p< 0.05, **p < 0.01).

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    Ultraviolet fluorescence photographs obtained after agarose gel electrophoresis showing that hypericin induces apoptosis in established pituitary adenoma cell lines. Treatment of pituitary adenoma cells with hypericin and DNA isolation are described in Materials and Methods. The DNA that was isolated from the established adenoma cell lines AtT-20 and GH4C1 in the absence of hypericin (Lane Ø) and in the presence of varying concentrations of hypericin (values given in micromolar concentrations) were electrophoresed in 1.2% agarose and stained with ethidium bromide (upper and center gels). Note the presence of oligonucleosome-sized fragments, which produce a classic ladder pattern in the lanes containing DNA from treated cells at doses of 0.1 µM and higher (arrowheads). The human fibroblast control cell culture demonstrated no evidence of DNA fragmentation after treatment with 10 µM hypericin on either gel (Lane FIB). The sizes of oligonucleosomal fragments were determined by comparison to DNA size markers in Lane M. The DNA isolated from line AtT-20 at various time periods after addition of 10 µM hypericin indicates the presence of apoptosis after 24 and 72 hours of treatment (lower gel). No evident DNA fragmentation was seen at the 6-hour time period.

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