Crown Ether Host-Rotaxanes as Cytotoxic Agents

Highly toxic bacterial ionophores are commonly used in veterinary medicine, but their therapeutic index is too narrow for human usage. With the goal of developing ionophores with a broader therapeutic index, we constructed highly derivatized synthetic ionophores. The toxicities of crown ether host-rotaxanes (CEHRs) against the SKOV-3 cell line were measured. The effect of Mg²⁺ or Ca²⁺ on toxicity was explored because changes in the intracellular concentration of these cations can cause cell death through apoptosis. We found that Boc-CEHR is highly toxic and Arg-CEHR is slightly less toxic with IC₅₀ values of 0.5 and 6 μM, respectively, in standard growth medium. Increasing the concentration of Ca²⁺ resulted in greater toxicity of the CEHRs, whereas increasing the concentration of Mg²⁺ was less effective on reducing IC₅₀. Cell death occurs mainly through apoptosis. Although preliminary, these results suggest that the CEHRs deliver Ca²⁺ and perhaps Mg²⁺ into cells inducing apoptosis

Low doses of BPA have an adverse effect on centrosome numbers in prostate cancer cells.

<p>The cell lines NPrEC, RWPE1, LNCaP, C4-2, 22Rv1, and PC3 were treated with medium containing 10% CSS plus 0, 0.01 nM, 0.1 nM, 1 nM, 10 nM and 100 nM BPA for 72 h. Cells were fixed with 100% cold methanol and immunostained for centrosomes and nuclei. The number of centrosomes per cell was scored by fluorescence microscopy. The results are shown as an average determined from five separate experiments. The scatter plot was generated of the percentage of cells with an abnormal number of centrosomes in response to BPA. Analyses was performed using a fixed effect model for each cell line. <i>Post hoc</i> comparisons of means were adjusted using Bonferroni's tests. The fold change is the percentage of cells with abnormal centrosomes at 0.1 nM BPA/the percentage of cells with abnormal centrosomes at 0 nM BPA.</p

Cells grown in the absence and presence of 0.1-independent growth.

<p>Representative pictures of colonies after 2 weeks of incubation in agar. C4-2 cells in the presence of 0.1 nM BPA formed larger colonies (B, B′, 100–1200 µm diameter) compared with those grown in the absence of BPA (A, A′, 50–400 µm diameter).</p

Scatter plots of LnBPA.

<p>Urine BPA levels are associated with PCa. The log-transformed BPA is referred to as LnBPA. Values in graph are mean ± SD of LnBPA. (A) Urine BPA levels are higher in PCa patients than in non-PCa patients. Means of LnBPA  = 1.75±1.97 in PCa (blue, n = 27) vs. 0.35±2.14 in non-PCa (red, n = 33), <i>p</i> = 0.012. (B) LnBPA in PCa vs. LnBPA in non-PCa, stratified by age = 65. Urine BPA levels are significantly higher in young PCa patients than in the respective non-PCa patients only in the age group <65 years old; <i>p</i> = 0.006. (C) Linear regression analyses of Serum PSA vs. LnBPA in patients <65 years old only (n = 30). Blue solid squares represent PCa patients; red inverse-circles represent non-PCa patients. Blue and red solid lines represent their regression lines, respectively. (D) Comparison of the geometric mean of BPA in PCa and non-PCa groups. The geometric mean (Geo) is defined as the exponential of the mean of LnBPA. Values are geometric means (95% CI) of BPA in unit of µg/g creatinine.</p

Summary of baseline characteristics (n = 60).

<p>*Numerical variables are summarized using mean ± standard deviation (SD). Categorical variables are summarized using frequency (in %).</p>†<p><i>p</i> values are calculated from t-tests.</p><p>#Serum PSA significantly rose during follow-up.</p

Fold change in the percentage of cells with centrosomal amplification in presence of 100

<p>*Fold change is defined as % of cells with abnormal centrosomes at 0.1 nM BPA/% cells with abnormal centrosomes in untreated cells.</p>†<p><i>Post hoc</i> comparisons were performed under a fixed effect model and adjusted using Bonferroni's methods. Only the p-values of comparing NPrEC-1 to other cell lines are presented. Other comparisons between the cell lines were not statistically different.</p

Anchorage- independent growth in the presence and absence of BPA.

<p>*<i>p</i> values were computed using t-tests.</p

Summary of LnBPA (log-transformed BPA) values and cancer-related characteristics (n = 27) for PCa patients.

<p>Summary of LnBPA (log-transformed BPA) values and cancer-related characteristics (n = 27) for PCa patients.</p

Ca²⁺ Selective Host Rotaxane Is Highly Toxic Against Prostate Cancer Cells

New therapies are needed to eradicate androgen resistant, prostate cancer. Prostate cancer usually metastasizes to bone where the concentration of calcium is high, making Ca²⁺ a promising toxin. Ionophores can deliver metal cations into cells, but are currently too toxic for human use. We synthesized a new rotaxane (CEHR2) that contains a benzyl 15-crown-5 ether as a blocking group to efficiently bind Ca²⁺. CEHR2 transfers Ca²⁺ from an aqueous solution into CHCl₃ to greater extent than alkali metal cations and Mg²⁺. It also transfers Ca²⁺ to a greater extent than CEHR1, which is a rotaxane with an 18-crown-6 ether as a blocking group. CEHR2 was more toxic against the prostate cancer cell lines PC-3, 22Rv1, and C4-2 than CEHR1. This project demonstrates that crown ether rotaxanes can be designed to bind a targeted metal cation, and this selective cation association can result in enhanced toxicity