Oral simvastatin administration delays castration-resistant progression and reduces intratumoral steroidogenesis of LNCaP prostate cancer xenografts.

BACKGROUND: Growing evidence supports the idea that de novo steroidogenesis has an important role in prostate cancer's progression to the castration-resistant state following androgen deprivation therapy. Therefore, reducing the availability of cholesterol for use as a precursor in androgen synthesis may reduce proliferation and disease progression. METHODS: LNCaP xenograft-bearing mice were castrated and administered simvastatin via diet, and tumor volume and PSA concentration were monitored for 8 weeks post castration. Levels of serum and intratumoral androgens along with serum simvastatin and common toxicity markers were measured at end point. RESULTS: Reduced post-castration tumor growth rate in simvastatin-treated mice correlated with delayed time to castration-resistant progression, determined by two serum PSA doublings from post-castration nadir, when compared with xenografts in mice on control diet. At 8 weeks post castration, serum simvastatin levels were comparable to clinically relevant human doses with no evidence of overt muscle or liver toxicity. This suppressed post-castration tumor growth in the simvastatin diet group was correlated with reduced intratumoral testosterone and dihydrotestosterone levels. CONCLUSIONS: Reduced tumor growth and intratumoral androgen levels observed in simvastatin-treated, castrated mice harboring LNCaP xenograft suggests that suppressing de novo steroidogenesis can delay castration-resistant progression of this tumor model.

Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model.

OBJECTIVE: To evaluate the effect of clusterin overexpression on radiation-induced tumour growth rates and apoptosis in human prostate LNCaP cells, as prostate cancer cells are relatively resistant to radiation-induced apoptosis and local recurrences are common, but overexpression of the anti-apoptotic protein clusterin can accelerate progression to androgen-independence and to confer a chemoresistant phenotype in various prostate cancer models. MATERIALS AND METHODS: Western blot analysis and immunohistochemistry were used to compare clusterin expression levels in parental (P) and clusterin-transfected (T) LNCaP cells in vitro and in vivo. The effects of radiation on clusterin-expression in both parental LNCaP/P and clusterin-transfected LNCaP/T tumours were analysed by Northern blot analysis. The cellular response to radiation was determined up to 3 weeks after irradiation using tetrazolium and re-growth assays, and cell-cycle analysis by flow cytometry. RESULTS: Clusterin mRNA expression increased from undetectable to low levels in LNCaP/P tumours after radiation and more than three-fold in LNCaP/T tumours. Clusterin overexpression decreased the radiosensitivity in a time-dependent manner, reducing the extent of growth arrest and apoptosis by up to 54%. Re-growth assays showed that the improved survival rates of LNCaP/T cells after radiation did not change after 3 days, remaining constant over 3 weeks. CONCLUSIONS: These results identify clusterin as a promoter of cell survival that may help mediate resistance to radiation-induced apoptosis. Furthermore, clusterin overexpression seems to provide an extended protection against radiation-induced cell cycle arrest and apoptosis.

Assessment of toxicity of bis-platinum complexes in hypoxic and aerobic cells.

There is considerable interest in the bis-platinum series of complexes as potential chemotherapeutic agents, due to their activity in cisplatin-resistant lines and in various tumor types. Our interest in their hypoxic selectivity stems from the fact that cisplatin exhibits greater cytotoxicity in hypoxic than aerobic cells. Unlike nitroaromatics, quinones, tirapazamine and many other hypoxia selective agents, a 'bioreductive' moiety cannot explain these observations. We hypothesized that DNA-protein cross-links (D-P) might play a role in the mechanism. Bis-platinum complexes have variable cross-linking potentials, and their toxicities were assessed in air or hypoxia in CHO cells. Of the three classes examined, only those from the 2,2/cis,cis series show greater hypoxic selectivity than cisplatin. These have greater potential for cross-links than cisplatin, being potentially bifunctional at each platinum, with the two leaving groups (X) in the cis position, and with variable distance (n) between the platinum centers: cis-[(PtX2(NH3))2H2N(CH2)nNH2]. Cellular platinum accumulation and DNA binding were also measured, and like cisplatin, results are consistent with a more toxic lesion formed in hypoxia. Lower hypoxic selectivity in the UV20 cell line may reflect an inability to excise the relevant lesion. These results support the D-P hypothesis. Further support comes from a 1,1/trans,trans complex which does not form D-P and which exhibited the reverse behavior to 2,2/cis,cis or cisplatin, i.e. higher toxicity in aerobic than in hypoxic cells. This study examines the possibility of an additional mechanism of selection for hypoxic toxicity involving DNA-protein cross-links.

Response of V79 cells to low doses of X-rays and negative pi-mesons: clonogenic survival and DNA strand breaks.

Mammalian cells are hypersensitive to very low doses of X-rays (< 0.2 Gy), a response which is followed by increased radioresistance up to 1 Gy. Increased radioresistance is postulated to be a response to DNA damage, possibly single-strand breaks, and it appears to be a characteristic of low linear energy transfer (LET) radiation. Here we demonstrate a correspondence between the extent of the increased radioresistance and linear energy transfer of 250 kVp X-rays and plateau and Bragg peak negative pi-mesons. The results support our hypothesis since the size of the increased radioresistant response appears to correspond to the number of radiation induced single-strand breaks. Furthermore, since survival prior to the increased radioresistant response (< 0.2 Gy) was LET-independent, these data support the notion that the increased radioresistant response may dictate the overall survival response to higher doses. However, while these data provide further circumstantial evidence for the involvement of DNA strand breaks in the triggering of increased radioresistance, more direct conclusions cannot be made. The data are not accurate enough to detect structure in the single-strand break profiles, the production of single-strand breaks being apparently linear with dose.

Immunocytochemical labelling of aerobic and hypoxic mammalian cells using a platinated derivative of EF5.

The monoclonal antibody ELK3-51 was previously developed to detect adducts of the 2-nitroimidazole EF5. Direct immunofluorescence was used to detect adducts of EF5 or of a platinated derivative cis-[PtCl2(NH3)EF5] in SCCVII cells treated under aerobic or hypoxic conditions. Fluorescence measurements of these cells using both image and flow cytometric methods were compared, giving similar profiles. Platination significantly decreased immunofluorescence levels (approximately 4-fold less than EF5) after 3 h in hypoxia, but also increased levels after exposure in air (approximately 1.5 x) such that the hypoxic ratio decreased from approximately 50 to approximately 13. Platinated EF5 also showed significantly greater cytotoxicity than its parent in both aerobic and hypoxic cells. These results are consistent with targeting of EF5 to DNA, which was confirmed qualitatively by confocal microscopy.

Recognition of platinum-induced DNA damage by nuclear proteins: screening for mechanisms.

The association of high mobility group (HMG) proteins with cisplatin-damaged DNA has attracted considerable interest because of a possible relationship with drug-acquired resistance and the repair of DNA damage caused by this important chemotherapeutic agent. We have further characterized the binding of HMG proteins to cisplatin-damaged DNA using a modification of the damaged DNA affinity precipitation assay (DDAP) and proteins isolated from the nuclei of V79 cells. HMG proteins recognized cisplatin adducts only in double-stranded DNA sequences. Pre-treatment of cells with cisplatin (1 microM) prior to protein extraction decreased the apparent yield of HMG proteins. However, the pre-treatment of isolated protein did not prevent recognition of the DNA adducts. To investigate the possible role of HMG proteins in toxicity and resistance, we have extended the DDAP method to study other platinum agents, some of which are active in cisplatin-resistant cells. A comparison of trans- and cis-[PtCl2(NH3)quinoline] is presented as an example. HMG proteins recognized DNA damage caused by the cis, but not the trans isomer. However, the trans isomer is known to be significantly more toxic and is highly active in cisplatin-resistant cells, suggesting a mechanism of action different from cisplatin. Therefore, the toxicity of trans-[PtCl2(NH3)quinoline] appears to be unrelated to the recognition of damage by HMG proteins. The DDAP assay may provide an additional screen for new mechanisms of cytotoxic platinum agents.

Additive coclastogenicity of sodium selenite and caffeine in CHO cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

The clastogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster ovary (CHO) cells and its modulation by Na2SeO3 and caffeine were studied by metaphase analysis of chromosome aberrations (CA) as well as by measuring the formation and repair of single-strand (ss) DNA breaks employing hydroxylapatite chromatography. Treatment of CHO cells with MNNG (1.25 or 2.5 x 10-5M) for 3 h caused CA in 11 and 19% of metaphases scored, respectively. Pretreatment of cells with Na2SeO3 (1-5 micrograms/mL) or caffeine (0.2-2.0 mg/mL) for 2 h resulted in a 2-3.5-fold increase of CA frequency. Addition of both modulators during the mutagen exposure tended to cause a slight inhibition of clastogenic activity of MNNG (1.25 x 10(-5) M) or had no effect on CA number when MNNG was used at a concentration of 2.5 x 10(-5) M). Posttreatment of CHO cells with Na2SeO3 for 20 h after MNNG was ineffective in influencing the number of metaphases with CA, whereas, at these conditions, caffeine enhanced up to 6-7-fold the clastogenic activity of MNNG. Addition of both modulators during the whole experiment, 2 h pretreatment included, resulted in a further significant increase of CA frequency up to the total pulverization of chromosomes in all metaphases scored. The coclastogenic effect of caffeine was greater in this case. The enhancement of chromosome-damaging activity of MNNG by selenite and caffeine was better expressed when this carcinogen was applied at the higher concentration used. An additive coclastogenic effect was observed in CHO cells treated simultaneously with Na2SeO3 and caffeine plus MNNG.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiosensitizing and toxic properties of quinoline and nitroquinoline complexes of platinum [PtCl2(NH3)quinoline].

The assessment of drugs designed to be useful in the eradication of hypoxic (resistant) cells involves comparison of hypoxic and aerobic radiosensitization, cytotoxicities, as well as DNA binding and reduction potentials. Three pairs of isomers of quinoline complexes [amino dichloro quinoline platinum (II)] were studied in this context. For the cis 5- and 6-nitroquinoline complexes, the DNA binding and toxicity were higher with the 5-substituted ligand. Reduction potentials were similar (-260 and -280 mV). No selectivity for hypoxic toxicity was observed, but radiosensitizing ability by both complexes was greater in hypoxic (than oxic) Chinese hamster ovary (CHO) cells. The trans 5-nitroquinoline complex produced better sensitization in hypoxia than its cis isomer [enhancement ratio (ER) 1.7 at 10 microM versus 40 microM for cis]. However, this was accompanied by some aerobic sensitization. The trans isomer of the (unsubstituted) quinoline complex was considerably more toxic than its cis isomer. Neither showed selectivity for hypoxia, either as radiosensitizers or as cytotoxins, which may be attributable to the lack of a reducible (nitro) function. Four quinoline complexes showed high activity in cisplatin-resistant L1210 cells, with the lowest resistance factor being for the trans quinoline complex. Results suggest that trans complexes with one aromatic ring may have activity different from the cis geometry, which should be exploited with respect to cisplatin resistance and cross-resistance with radiation.

Radiosensitization by nickel lapachol.

The properties of interest in the radiosensitization of a metal complex, nickel lapachol, are compared with those of the 2-nitroimidazole, misonidazole. These very different compounds were found to be surprisingly similar in terms of their reduction potential (-370 mV), enhancement ratios for killing of hypoxic Chinese hamster ovary cells by X-irradiation, and enhancement of DNA breaks in hypoxia. For nitroimidazoles, the sensitization depends on 'electron affinity', reduction of the nitro group; for nickel lapachol it is the metal which is necessary for reduction, yet the sensitization efficiencies are remarkably close. However, the metal complex has additional activities (some sensitization in aerobic cells; increased sensitization with preincubation) which are as yet unexplained but are assumed to be related to the nature of the naphthoquinone ligand, rather than to the reduction of the metal.

The effect of differentiation on photosensitizer uptake by HL60 cells.

The capability of human promyelocytic leukemia cells HL60 to be induced to differentiate to various stages along the monocytic or myelocytic pathway was exploited for investigation of the uptake of selected photosensitizers by diverse types of cells of the same origin. The results showed that there was no substantial difference in photofrin uptake between noninduced HL60 cells, immature monocytes, immature neutrophils and cells differentiated along the eosinophilic pathway. In contrast, HL60 cells differentiated into macrophages (HL60 phi) exhibited markedly increased photofrin uptake, which was further enhanced by their pretreatment with bacterial lipopolysaccharide. Similar results were obtained with other photosensitizers tested: di- and tetrasulfonated aluminum phthalocyanines (A1PcS2 and A1PcS4), tetrasulfonated zinc phthalocyanine (ZnPcS4), tetraphenylporphine tetrasulfonate (TPPS4) and benzoporphyrin derivative monoacid (BPD). Despite marked differences in the state of self-aggregation and other chemical properties of these compounds, the degree of their preferential uptake by HL60 phi cells showed very little variation. In a typical experiment, the uptake of these photosensitizers by HL60 phi cells was four to five times higher than the uptake by noninduced HL60 cells. In addition to the fluorometric assay employed in most of the experiments, cellular concentration of A1PcS4 was determined by measurement of elementary aluminum using atomic absorption spectroscopy.

The effect of hypoxia on cytotoxicity, accumulation and DNA binding of cisplatin in Chinese hamster ovary cells.

The literature contains conflicting evidence regarding the hypoxic toxicity of cisplatin. We have found that in Chinese hamster ovary cells, cisplatin was up to 2.6 times more effective at reducing survival to 2% in hypoxic, compared with aerobic cells. Furthermore, using atomic absorption spectroscopy, it was determined that cells treated in hypoxia showed consistently higher accumulation (up to 1.5 times) and DNA binding (up to 1.7 times) than aerobic cells. Hypoxic cells also showed greater toxicity per platinum-DNA adduct than those treated in air, suggesting that increased binding of the drug, alone, cannot account for its increased hypoxic cytotoxicity. We suggest that the hypoxia selectivity of cisplatin involves several different mechanisms and possible explanations for these results are discussed.

Toxicity of [PtCl2(NH3)L] in hypoxia; L = misonidazole or metronidazole.

There is increasing interest in compounds which show selective toxicity to the resistant hypoxic portions of tumors. Cisplatin does not generally show preferential toxicity in hypoxic cells, as do nitroimidazoles. It is proposed that attachment of a nitroimidazole could add a degree of hypoxic selectivity to Pt agents. Platinum complexes containing one nitroimidazole ligand bind to DNA and show higher toxicity in hypoxic than aerobic CHO cells. Cis and trans isomers of complexes with misonidazole (a 2-nitroimidazole) and metronidazole (a 5-nitroimidazole) are compared with respect to binding to DNA (approximately the same), reduction potential (trans miso greater than cis miso greater than cis metro greater than trans metro), and toxicity (trans greater than cis miso, cis greater than trans metro, with trans miso approximately cis metro in hypoxia, despite significantly different reduction potentials). The effect of platination on nitroimidazole toxicity is not entirely explained by DNA binding and increased reduction potential. These compounds do not exhibit cross resistance with cisplatin in L1210 resistant cells. This factor, their selectivity for hypoxia, and preliminary results in vivo indicating potentiation of anti-tumor activity by the vasoactive compound, hydralazine, which increases tumor hypoxia, suggest further development of these compounds for use in tumors with resistant hypoxic portions.

Platinum complexes with one radiosensitizing ligand [PtCl2(NH3) (sensitizer)]: radiosensitization and toxicity studies in vitro.

Complexes of general formula [PtCl2(NH3)L] with one radiosensitizing ligand per platinum are compared with ligand L alone, complexes with two radiosensitizers per platinum [PtCl2L2], and their analogs with NH3 ligands, with respect to radiosensitizing properties and toxicity in CHO cells. Radiosensitizing ligands, L, were misonidazole, metronidazole, 4(5)-nitroimidazole, and 2-amino-5-nitrothiazole, and the ammine analogs were cis- and trans-DDP [diamminedichloroplatinum(II)] and the monoammine, K[PtCl3(NH3)]. Results are related to a previous study on plasmid DNA binding by these series. The toxicity of the mono series [PtCl2(NH3)L], attributable to DNA binding, is much higher than the corresponding bis complexes, [PtCl2L2]. For L = misonidazole, toxicity is similar to the monoammine, but higher in hypoxic than in aerobic cells. trans-[PtCl2(NH3)-(misonidazole)] is more toxic than the cis isomer. Except for L = 4(5)-nitroimidazole, the complexes [PtCl2(NH3)L] are more toxic than L in air and hypoxia. Hypoxic radiosensitization by the mono complexes is comparable to the monoammine and is not better than free sensitizers, again except for L = 4(5)-nitroimidazole. Significantly lower sensitization is observed in oxic cells. The bis complexes [PtCl2L2], which do not bind to DNA as well as the mono complexes, are less effective radiosensitizers and less toxic than the [PtCl2(NH3)L] series.

Assessment of DNA binding of platinum-radiosensitizer complexes by inhibition of restriction enzymes.

A simple and rapid method has been used to compare the binding of platinum complexes to DNA, in a relatively qualitative manner. A compound bound at or near the restriction site inhibits enzymatic cleavage of DNA; inhibition of BamHI and EcoRI activity by complexes was assessed in this study using linearized pSV2-gpt plasmid. Our particular interest was in DNA binding by complexes of platinum (Pt) with known organic radiosensitizers (RS), to determine whether the Pt was able to target the RS to the DNA. Although the Pt-RS complexes investigated themselves have moderate radiosensitizing ability (like the inorganic complexes, cis- or trans-diamminedichloroplatinum(II), c- or t-DDP) none of the Pt-RS inhibit to the same extent as c- or t-DDP. However, there appears to be some correlation between enhanced radiosensitization by Pt-RS over Pt(RS)2, with the degree of Pt binding (as assessed by our assay). Our results using isolated DNA suggest that not all complexes bind well (e.g. Pt with two RS ligands), but that in certain cases (e.g. Pt with only one RS), it is possible to target the drug to the DNA. An ammine or amine ligand may be required in order to target a radiosensitizer to DNA using platinum.

Ocular absorption and toxicity of a radiosensitizer and its effect on hypoxic cells.

The recurrence of retinoblastoma after radiation treatment may be related to hypoxic cell radioresistance. Radiosensitizing drugs acting on hypoxic cells without affecting the response of oxygenated cells could improve treatment of ocular tumors while minimizing complications of radiotherapy. A desmethyl derivative of misonidazole is as effective a radiosensitizer as misonidazole (as measured in vitro) and is better suited to ocular administration, since it is more soluble than misonidazole. We studied the ocular toxic effects of a desmethyl derivative of misonidazole after subconjunctival administration of 140 and 70 mg. The higher dose produced an intolerable ocular toxic effect, but at the lower dose, the toxic effect was moderate and reversible. We compared ocular pharmacokinetics of the desmethyl derivative of misonidazole after subconjunctival and intravenous (IV) injections. Subconjunctival administration yielded vitreous and anterior chamber concentrations of the radiosensitizer sufficient to produce a notable dose-modifying effect (as high as 1.8 in the anterior chamber and 1.25 in the vitreous at 70-mg doses). In contrast, even at doses of 140 mg, IV injections of the desmethyl derivative of misonidazole did not result in therapeutically useful ocular levels.