Photocytotoxic effect of pseudohypericin versus hypericin.

Pseudohypericin and hypericin, the major photosensitizing constituents of Hypericum perforatum, are believed to cause hypericism. Since hypericin has been proposed as a photosensitizer for photodynamic cancer therapy, the photocytotoxicity of its congener pseudohypericin has been investigated. The presence of foetal calf serum (FCS) or albumin extensively inhibits the photocytotoxic effect of pseudohypericin against A431 tumour cells, and is associated with a large decrease in cellular uptake of the compound. These results suggest that pseudohypericin, in contrast to hypericin, interacts strongly with constituents of FCS, lowering its interaction with cells. Since pseudohypericin is two to three times more abundant in Hypericum than hypericin and the bioavailabilities of pseudohypericin and hypericin after oral administration are similar, these results suggest that hypericin, and not pseudohypericin, is likely to be the constituent responsible for hypericism. Moreover, the dramatic decrease of photosensitizing activity of pseudohypericin in the presence of serum may restrict its applicability in clinical situations.

Cytotoxicity and antiproliferative effect of hypericin and derivatives after photosensitization.

The toxicity on three human tumor cell lines (A431, HeLa and MCF7) of five phenanthroperylenequinones (hypericin and derivatives) and two perylenequinones (cercosporin and calphostin C) was investigated after photosensitization (4 J/cm2). Furthermore, the antiproliferative effect on HeLa cells was studied for the phenanthroperylenequinones. Hypericin, 2,5-dibromohypericin, 2,5,9,12-tetrabromohypericin and perylenequinones displayed a potent cytotoxic and antiproliferative effect in the nanomolar range. Hypericin dicarboxylic acid exhibited no photoactivity. In general, the antiproliferative activity correlated well with the photocytotoxicity. However, the nonphotocytotoxic compound hexamethylhypericin showed potent antiproliferative activity in the nanomolar range, probably exerting its action by protein kinase C inhibition. Without light irradiation, no cytotoxic and antiproliferative effect was observed for any photocytotoxic phenanthroperylenequinone compound. Furthermore, confocal laser microscopy revealed that the subcellular localization in A431 cells was similar for the photoactive compounds; the photosensitizers were mainly concentrated in the perinuclear region, probably corresponding with the Golgi apparatus and the endoplasmic reticulum. In addition, the accumulation of the photosensitizers in HeLa cells was investigated. All compounds except hypericin dicarboxylic acid were found to concentrate to a large extent in the cells. The compound 2,5,9,12-tetrabromohypericin seemed intrinsically more effective than hypericin since the intracellular concentration of the bromoderivative was a magnitude of order lower than that of hypericin although both compounds showed similar photobiological activity.

Differential cytotoxic effects induced after photosensitization by hypericin.

The cytotoxic effects of the natural photosensitizing agent hypericin were evaluated. A dramatic difference in the sensitivity of several different human and mouse cell lines towards photoactivated hypericin (4 J cm-2) was demonstrated using a neutral red assay (e.g. A431, IC50 = 0.14 +/- 0.02 microM; HeLa, IC50 = 0.32 +/- 0.05 microM, MCF7, IC50 = 1.84 +/- 0.22 microM). Dark cytotoxicity was absent, even at high hypericin concentration (25 microM). The differential phototoxicity of hypericin did not correlate with the expression of the epidermal growth factor (EGF) receptor nor with the expression of the P170 glycoprotein in the cell. The reduction of the intracellular glutathione content did not enhance further the cytotoxic effects of photoactivated hypericin, as investigated with the A431, HeLa and MCF7 cells. Conversely, using confocal laser microscopy, it was shown that the phototoxicity correlated well with the hypericin cellular uptake.