Abstract

Background: Many current anti-cancer drugs used to treat breast cancer mediate tumor cell death through the induction of apoptosis. Cancer cells, however, often acquire multidrug-resistance following prolonged exposure to chemotherapeutics. Consequently, molecular pathways involved in tumor cell proliferation have become potential targets for pharmacological intervention. Ceramides are tumor suppressor lipids naturally found in the cell membrane, and are central molecules in the sphingolipid signalling pathway.

Methods: Our lab has targeted the ceramide signaling pathway for potential pharmacological intervention in the treatment of breast cancer. Previously, we have shown that certain ceramide analogs have therapeutic potential in the treatment of chemosensitive and multidrug-resistant breast cancers. Using the most active analog from our previous studies as the lead compound, new analogs containing a flavone moiety were designed and synthesized. In general, flavone derivatives often show interesting pharmacological properties, and compounds based on these molecules have been found useful in many different therapeutic areas including anti-tumor, anti-coagulants, and anti-HIV therapy.

Results: Synthesis and biological evaluation of five new flavonoid ceramide analogs are reported here. These compounds were also shown to be self-fluorescent, which can be useful when investigating their distribution and action in cancer cells.

Conclusion: Four out of the five flavone ceramide analogs in this study showed significant anti-proliferation activities in the three cell lines studied, MDA-MB-232, MCF-7, and MCF-7TN-R; some showing varying degrees of selectivity. The mechanisms involved in cell proliferation inhibition are complicated and further studies are needed.

Keywords: Breast Cancer; Ceramides; Flavones; Fluorescence

Introduction

Accounting for 12.5% of new cancer cases annually, breast cancer is the most common cancer world-wide [1]. Each year over 250,000 women and over 2,500 men in the United States are diagnosed with breast cancer [1,2]. Breast cancer risks and outcomes are not the same for all, and are highly linked to genetics and environmental causes, as well as health disparities [2]. Although Caucasian women in the US have a higher diagnosis rate for breast cancer, African Americans and other minorities suffer from lower survival rates than Caucasians, partly due to their lower rates of clinical breast cancer screening including mammography and access to cutting-edge treatments [2]. The World Health Organization (WHO) also reports breast cancer death inequities across the world, with high-income countries showing high survival rates (about 90%) and low-income countries such as South Africa showing low survival rates (about 40%) [3].

Even though chemotherapy continues to be the most common method of treatment for breast cancers, after prolonged exposure to chemotherapeutic drugs, cancer cells often develop multidrugresistance, making treatment more difficult [2,4]. Developing more effective and less systemically toxic treatments targeting drug-resistant breast cancers is thus of outmost importance.

Due to the crucial role of ceramides in cell death regulation, hundreds of anti-cancer ceramide analogs have been synthesized and investigated in recent years [4,5]. Ceramide analogs can be designed to target drug-resistant as well as drug-sensitive breast cancer cells [5-10]. Our previous work and that of many other research groups have shown that certain ceramide analogs preferentially inhibit the growth of chemo-resistant cancer cells in comparison to regular cancer cells [11-13].

In this study, the carbon chain length and functional groups of the backbone in the target compounds were kept the same as our previously reported analogs, to maintain optimal lipid solubility and facility of passage through membranes [11-15].The main structural variation was the introduction of a flavone moiety on the carbonyl of the side-chain amide functional group. Since certain flavone derivatives have been shown to have anti-cancer properties [16,17], we hypothesized that introducing a flavone moiety into the ceramide structure will impact its interactions with ceramide downstream targets, and potentially inhibit cell proliferation. In our initial studies, these flavonoid ceramide analogs were found to be self-fluorescent. This property can be used to follow the molecules’ movements and actions in the cells in vitro and further in-vivo [18,19]. Previously a number of fluorescent ceramide analogs were synthesized containing an additional fluorescent moiety or dye attachment [18-20]; however, this is the first time to our knowledge that the ceramide analog itself has been designed to have self-fluorescence properties.

The five target ceramide analogs reported here are structural isomers, and any differences observed in their activities should be due to the orientation of the flavone moiety leading to variations in shape and intermolecular interactions with the surrounding environment. Additional information regarding one of the five analogs was reported previously [6].

Materials and Methods

Syntheses

All flavone alcohol starting material were purchased from Indofine Chemical Company (Hillsborough, PA). The coupling reagents, bases, and solvents were purchased from Sigma-Aldrich (St. Louis, MO).

Compound 4 is used as a precursor for the syntheses of target ceramide analogs. We have optimized the conditions to perform its synthesis on a 10 g scale [15](Figure 1). Compound 1, an acid with a boc-protected amine group (1.0 eq), was treated with DCC (N,N’-dicyclohexylcarbodiimide, 1.1 eq), HOBt (1-hydroxyl benzotriazole, 1.1 eq), N-methyl morpholine (3.0 eq), and amine 2 (1.1 eq). Dichloromethane (DCM) was used as the solvent. The crude product was purified by recrystallization using ethyl acetate: hexane (2:1) to get the amide 3 (84% yield). Compound 3 was dissolved in DCM and treated with TFA (trifluoroacetic acid) to perform boc-deprotection to get compound 4, which was purified by recrystallization using ethyl acetate [18].

Figure 1: Synthesis scheme for compound 4.

Flavone-acids were synthesized using commercially available flavone alcohols or phenols as shown in Figure 2. The flavone alcohol or phenol 5 was dissolved in acetone in the presence of potassium carbonate (base) and reacted with bromoethyl acetate to get the corresponding ester intermediate 6. Compound 6 was purified by column chromatography before hydrolyzation with potassium hydroxide (base) in methanol to get the corresponding carboxylic acid, compound 7.

Figure 2: Synthesis scheme for the intermediate esters and acids.

The acid intermediate 7 was coupled with compound 4 using the coupling reagents EDCI (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and HOBt in the presence of DIPEA (N,N- diisopropylethylamine) as a base and DCM as the solvent (Figure 3). The final flavone ceramide analog 8 was purified using Combiflash chromatography (hexane : ethyl acetate as the solvent).

Figure 3. Last synthesis step leading to the final products.

The target compounds (Figure 4) were successfully synthesized with yields ranging from 10-71%. Since very small amounts of each of the analogs were needed for these studies, even the lower reaction yields were acceptable.

Figure 4: Structures of the target ceramide analogs, and the percent yield of each synthesis.

Bioassays

• Cell lines: In vitro studies were performed on the estrogen receptor (ER) positive chemo-sensitive MCF-7 breast cancer cell line (American Type Culture Collection (ATCC); catalog number HTB-22), as well as two drug-resistant breast cancer model systems, MDA-MB-231 (ATCC; catalog number HTB-26) and MCF-7TN-R. MDA-MB-231 cells are highly metastatic and triple-negative, lacking estrogen receptor (ER) and progesterone receptor (PR) expression, as well as human epidermal growth factor receptor 2 (HER2) amplification. MCF-7TN-R is a chemo-resistant breast cancer cell line derived from the chemo-sensitive breast cancer cell line MCF-7. MCF-7 cells were treated with the tumor necrosis factor alpha (TNF-α) until they acquired resistance to TNF-αinduced cell death. MCF-7TN-R cells are highly aggressive, and are also triple (ER/PR/Her2) negative. MCF-7TN-R cells used in this study were a gift from the Tulane University Medical Center.

Here, we studied the anti-cancer properties of the synthesized analogs, focusing on cell proliferation across these three cell lines.

• Cell Culture: MDA-MB-231, MCF-7,and MCF-7TN-R cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen; catalog number 11995-065) enriched with 10% fetal bovine serum (Gibco; catalog number 10437-028) and 1% antibiotic (antibiotic and antimycotic; Gibco; catalog number 15240-062). Cells were cultured in 75cm² tissue culture flasks in 37^oC humidified atmosphere of 5% CO₂ and 95% air.
• MTT Assays: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assays were performed on the newly synthesized compounds according to the manufacturer protocol (Life Technologies; catalog number M6494) to assess cell proliferation based on cell metabolic activity. Cells from each cell line were plated on 96-well plates at 15,000 cells/well in 200 µL of DMEM media in triplicates. Cells were incubated and given 24 hours to adhere to plates before removing the media. Cells were then treated with gradient concentrations of ceramide analogs in DMEM, or with dimethyl sulfoxide (DMSO) in DMEM as control, and incubated for an additional 48-hour period to allow proliferation. 20 µL of the MTT reagent was added to each well, followed by incubation for 2 hours. Media was carefully aspirated, and 150 µL of DMSO was added to dissolve the crystals. The absorbance of the plate was read using a Biotek Synergy H1 Microplate Reader at 550 nm. 

Statistical Analyses: Graph Pad Prism software (Graph-Pad Software, Inc.) was used for calculating the IC₅₀ values.

Docking Studies

Increased intracellular ceramide levels have been shown to increase apoptotic activity. Exogenous ceramides can be designed to cause inhibition of ceramide-metabolizing enzymes, such as ceramidase, leading to increased intercellular ceramide levels and apoptosis [21]. Ceramidase metabolizes ceramides into sphingosine, and is considered a regulator of cellular autophagy and drug-resistance in cancer cells [21]. To study the interactions of the five flavone ceramide analogs with the enzyme ceramidase in order to determine whether their anti-proliferation activities are due to the inhibition of this enzyme, their 3D structures were built using Molecular Operating Environment (MOE) from ChemComp Group. The X-ray crystal structure of ceramidase was obtained from the Protein Data Bank (2ZXC.pdb). Initial geometric optimizations of the ligands were carried out using the standard MMFF94 force field, with a 0.001 kcal/mol energy gradient convergence criterion and a distance-dependent dielectric constant employing Gasteiger and Marsili charges. Additional geometric optimizations were performed using the semi-empirical method molecular orbital package (MOPAC). Docking was performed using ‘dock’ module of the MOE software. The best docking pose was determined with visual inspection, considering the docking scores. Figure 5 shows the binding modes of the five analogs in the active site of the enzyme ceramidase. Figure 6 depicts the ligand interactions between our lead compound from previous studies, (S,E)-3-hydroxy-2-(2-hydroxybenzylidene)-aminoN-tetradecylpropanamide (analog 315) [15], and the flavone ceramide analogs with the ceramidase amino acid residues.