Kam Lau and Dr. Heidi Vollmer-Snarr, Chemsitry and Biochemistry Department

Introduction

Cancer is the second leading cause of death in the United States. According to the American Cancer Society, about half of all men and one-third of all women in the US will develop cancer during their lifetimes. Today, millions of people are living with cancer or have had cancer.1 Even though there is still no ultimate treatment for cancer, chemotherapy, radiation therapy, and surgery are common existing treatments for different types of cancers. New kinds of anti-cancer agents, developed in our laboratory, called amino-retinoid compounds also show promise in treating cancer. Currently, retinoid compounds and germcitabine (a chemotherapy drug) are used in combination therapies for the treatment of cancer.2 The main problem is how to balance between efficacy and toxicity when using retinoid compounds and chemotherapeutic compounds against cancer.

Research suggests that the amino-retinoid (AR) compound, A2E (a pyridinium bis-retinoid fluorophore, Shown in figure 1), found in high concentrations in retinal pigment epithelial (RPE) cell lipofuscin (undigested cell waste), causes cellular damage in RPE cells, including DNA damage and apoptosis which finally result in age-related macular degeneration. Studies also show that A2E is not significantly toxic at low concentrations. However, once A2E is photo-oxidized (Figure 2), it becomes relatively cytotoxic. A2E is selectively photo-oxidized with blue light (λmax = 430nm). Studies in our laboratory demonstrate that cancer cells illustrate the same behavior when exposed to A2E and photo-oxidized A2E. The photo-oxidation products of A2E are strong electrophiles that react readily with the numerous cellular nucleophiles in a variety of cancer cells, which results in cellular death. Peripheral damage to healthy cells can be avoided by the targeted irradiation of cancer cells. Using biomimetic synthesis of

amino-retinoid compounds (figs. 3 and 4), different cytotoxic amino-retinoid compounds can be prepared which may serve as potential anti-cancer agents. To further test the cyctoxicity of these novel AR compounds, we are in the process of labeling them with folic acid, which should result in the formation of cancer targeting cytotoxic compounds as many cancer cells have more folic acid receptors than normal cells.3

Recently in our laboratory (collaboration with Moran Eye Center at University of Utah), two AR compounds, A2-Serotonin (shown in figure 2) and A2-Tyramine, were suspected to be found in AMD patient’s eyes with very similar molecular weight and UV absorbance compared to other AR compounds. To gain a better understanding of the relationship between AR compounds and AMD, it is important to know what types of AR compounds are present in the human eye. With MS+ and HPLC/UV analyses, we can compare these two AR compounds (A2-Serotonin and A2-Tyramine) synthesized in the biomimetic pathways with the two AR like compounds found in the AMD patient’s eyes. This comparison will aid in identification of the different AR compounds present in the eye which may lead to AMD. In addition, we will continue to investigate the cyctotoxicity of AR compounds towards cancer cells.

Materials and Methods

Biomimetic synthesis of A2E. Two equivalents of all-trans-retinal and one equivalent of ethanolamine in ethanol were stirred in the presence of acetic acid (1eq) at room temperature in the dark for 48 hours. The residue was filtered, concentrated, and purified by silica gel column chromatography. The column was eluted with MeOH:CH2Cl2 (5:95) up to MeOH: CH2Cl2: Trifluoroacetic acid (TFA) (10:90:0.0075) to give A2E (15-20% yield). The purity of A2E was characterized by NMR and HPLC.

Cytotoxcity Cell Assays. The CRL-1932 kidney cancer cells were grown in sterile flasks to about 70-80% confluency, and 100 µl of kidney cancer cells in media were plated onto a 96 well plate (5000 cells in each well, estimated by trypan blue cell counts). The kidney cancer cells were incubated with 0, 5, 10, and 15 µM of A2E for 24 hours. The selected wells were irradiated inside the incubator using a blue light (λmax= 430 nm, light flux per area =136.3 Js-1cm-2) for 20-30 minutes. After another 24 hours, the cell viabilities were determined by Trypan blue cell counts.

Results

Biomimetic synthesis of A2E. As a result of several reactions, over 100 mg of A2E (~90% purity) was purified using silica gel chromatography. The purity of A2E was determined by both NMR and HPLC analysis (see below).

Total chemical synthesis. Intermediate 3,
tributylstannanyl-prop-2-en-ol (see figure 3), which is the product from the first step in the synthesis of pyridine bis-retinoids, was synthesized and purified. Compounds 8, 10, 11, and 12 were also synthesized and purified in gram scales by silica gel chromatography with different ratios of ethyl acetate and hexane as eluting solvents for purification.

Folate bis-retinoid bioconjugates. Using HPLC analysis, the possible UV absorbance of A2E-folic acid conjugates in DCC condition has shown below. However, the area of Wavelength at 430 nm A2E-folic acid peak is relatively small (only about 3%). Using Yamaguchi conditions, no A2E-folic acid like compound was detected by HPLC analysis.

Cytotoxcity Cell Assays. The kidney cancer cell viabilities were calculated by trypan blue cell counts. In graph 1, the Percent cell death was determined as compared to non-irradiated cells with equivalent concentrations of A2E.

Discussion

In our laboratory, we have successfully made amino-retinoid (AR) compounds using biosynthetic routes. However, these syntheses usually result in low yields (10-20%). In order to facilitate easier purification and higher yields, new total chemical synthetic pathways for AR compounds were developed in our laboratory. Currently, we are in the final two steps of the synthesis. The characterization of above intermediate compounds shown in figure 3 will be performed by standard techniques such as NMR and MS.
During HPLC analysis, it shows that the A2E-folic acid conjugate is forming. However, the % yield is relatively low (only about 3%). With different solvent systems such as dichloromethane, DMSO, THF, and benzene, the % yield is still not improving much. It might be the case that A2E is lipid soluble and folic acid is hydrophilic. The different solubility properties between A2E and folic acid might contribute to the instability of the A2E-folic acid conjugate. Also, the linkage between A2E and folic acid is quite short, so the steric effect might also play a role in the difficulty of linking these compounds. By using other AR compounds with longer linkers, it might be easier to connect the pyridinium compound with folic acid.

The cytotoxicity assays show that 15 µM of photo-oxidized A2E causes 96% kidney cancer cell damage compare to equivalent concentration of non-irradiated A2E. Our ultimate goal is to label A2E or other AR compounds with folic acid which results in cancer targeting cytotoxic compounds as many cancer cells have more folic acid receptors than normal cells. The accuracy of our trypan blue cell count is not very reliable because of relatively large standard deviations. In the future, the CellTiter 96® Aqueous Cell Proliferation Assay will be used. This protocol is performed by using MTS/PMS solution to measure the metabolism of the different cancer cells. After adding MTS/PMS solution, each well can be read at 490 nm on a plate reader. Also, our CRL-1932 kidney cancer cell line seems quite sensitive to DMSO. The amount of DMSO should be minimized while dissolving different AR compounds in this solvent.

Conclusion

Once the novel amino-retinoid compounds and their folic acid conjugates are synthesized and characterized, the potential of AR compounds and their folic acid conjugates as anti-cancer agents will be further understood.