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AN-7, a prodrug of butyric acid, induced histone hyperacetylation and differentiation and inhibited proliferation of human prostate 22Rv1 cancer cells in vitro and in vivo. In nude mice implanted with these cells, 50 mg/kg AN-7 given orally thrice a week led to inhibition of tumor growth and metastasis, tumor regression in >25% of animals and increased survival. Median time to the experimental end point (tumor volume 2 cm3 or death) in the untreated was 52 days, and average tumor volume was 0.8 +/- 0.18 cm3. At the same time, 94.4% of AN-7-treated mice survived and had average tumor volumes of 0.37 +/- 0.1 cm3. PSA expression was a useful marker for 22Rv1 lung metastasis detection. Sizeable metastases positively stained for PSA and limited air gaps were found in lungs of untreated mice. In animals treated with AN-7, lung morphology appeared normal. Primary tumors of treated animals were highly positive for PSA and had an elevated level of p21 and the proapoptotic protein Bax. Sections taken from AN-7-treated animals, examined under an electron microscope, exhibited condensed chromatin and apoptotic bodies. PSA serum levels were higher in untreated compared to treated animals and correlated with tumor volume. Since prolonged oral administration with 50 mg/kg or a single oral dose of 1.2 g/kg AN-7 did not cause adverse effects and the former exhibited significant anticancer activity, AN-7 is likely to display a high therapeutic index and may be beneficial for prostate cancer patients.

The stimulation of protoporphyrin (PP) biosynthesis in BI6 melanoma cells in order to facilitate photodynamic cell killing was studied. Biosynthesis and accumulation of PP in the melanoma cells was increased from 8 to 15 pmol/mg protein by the use of dimethyl-sulfoxide (DMSO), a differentiation-inducer. Treatment of the cells with the porphyrogenic agent alh/l-isopropyl-acetamide (AIA) stimulated an additional PP increase. The most remarkable enhancement of intracellular PP was achieved by the supplementation of 5-aminolevulinic acid (5-ALA) to the growth medium following the addition of DMSO and AIA during the induction phase. The intracellular concentration of PP exceeded 21 950 pmol/mg protein following combined stimulation by DMSO/AIA and 5-ALA. The porphyrins produced in the incubated cells, in serum-depleted medium, consisted of 95% PP; 88% of it was recovered from the cells and only 7% was excreted into the medium. Photosensitization of the B16 melanoma cells containing high PP concentrations was effective even at low light doses. Potassium (K) efflux was the first measurable sign of cell damage determined by X-ray microanalysis (XRMA) following fast liquid-nitrogen fixation. During a I min interval, 70% of cellular K was lost. After 5 min illumination, complete cell destruction was detected by scanning electron microscopy (SEM) and XRMA. The photodam-aged cells showed influx of Na, CI and Ca ions accompanying the immediate K losses. Ultrastructural cell damage was manifested by disintegration of the outer membrane. Total cell death of B16 melanoma cells was achieved by chemical induction of endogenous PP and photosensitization.

The effect of protoporphyrin on erythroid, myeloid and lymphoid leukemic cells and their destruction induced by the photoactivated porphyrin was studied. Friend erythroleukemic cells (FL) and myelocytic leukemic cells (ML) accumulated protoporphyrin in a cap or patch-like pattern observed by fluorescence microscopy. Photoactivated protoporphyrin induced the appearance of “holes” on the cell membrane demonstrated by scanning electron microscopy. On the other hand, Burkitt lymphoma (BL) and mastocytoma (MS) cells accumulated porphyrin intracellularly around the nuclear envelope and as circular profiles, respectively. Photoactivated protoporphyrin induced development of multiple blebs on the cell membrane, and even complete cell destruction. Cytotoxicity of protoporphyrin at short-term incubation periods was determined by [3H]thymidine and [3H]uridine incorporation. Protoporphyrin, unexposed to light, reduced the incorporation of both precursors only to a moderate extent. On the other hand, porphyrin-treated cells exposed to light showed complete inhibition of RNA and DNA synthesis. Long-term exposure of ML and BL cells to porphyrin in the dark induced a nearly 50 % inhibition of RNA and DNA synthesis. Although the cytotoxic effect of protoporphyrin in the dark was lower than that of photoactivated porphyrin, this may possess a potential activity in vivo even without illumination.

The Friend erythroleukemia cell line was used to study the binding and biological properties of the photosensitizer hematoporphyrin derivative (HPD) on a differentiating system. In addition, the effect of cholesterol hemisuccinate (CHS) enrichment of cell membranes on HPD activity was tested on the same cell system. Differentiation of Friend erythroleukemia cells (FLC) was induced with dimethyl sulfoxide (DMSO) and resulted in a decreased cell volume and an increased rate of hemoglobin synthesis as a function of the duration of DMSO treatment. Differentiated cells seem to bind less porphyrin than their undifferentiated counterparts. Thus, cells treated for 6 days with DMSO bound 30-40% less dye than an identical number of untreated FLC. In contrast, a similar inhibition of both DNA and protein synthesis by photoactivated HPD was evident in either DMSO-treated or untreated FLC. Enrichment of cell membranes with CHS led to the same degree of protection from the damaging activity of the photoactivated dye in both differentiated and undifferentiated FLC. The decreased binding of HPD to DMSO-treated FLC is most likely a result of a reduction in cell volume of differentiated cells and is not related to an intrinsic property of the differentiation process.