Marine-Inspired Bis-indoles Possessing Antiproliferative Activity against Breast Cancer; Design, Synthesis, and Biological Evaluation.

Diverse indoles and bis-indoles extracted from marine sources have been identified as promising anticancer leads. Herein, we designed and synthesized novel bis-indole series 7a–f and 9a–h as Topsentin and Nortopsentin analogs. Our design is based on replacing the heterocyclic spacer in the natural leads by a more flexible hydrazide linker while sparing the two peripheral indole rings. All the synthesized bis-indoles were examined for their antiproliferative action against human breast cancer (MCF-7 and MDA-MB-231) cell lines. The most potent congeners 7e and 9a against MCF-7 cells (IC50 = 0.44 ± 0.01 and 1.28 ± 0.04 μM, respectively) induced apoptosis in MCF-7 cells (23.7-, and 16.8-fold increase in the total apoptosis percentage) as evident by the externalization of plasma membrane phosphatidylserine detected by Annexin V-FITC/PI assay. This evidence was supported by the Bax/Bcl-2 ratio augmentation (18.65- and 11.1-fold compared to control) with a concomitant increase in the level of caspase-3 (11.7- and 9.5-fold) and p53 (15.4- and 11.75-fold). Both compounds arrested the cell cycle mainly in the G2/M phase. Furthermore, 7e and 9a displayed good selectivity toward tumor cells (S.I. = 38.7 and 18.3), upon testing of their cytotoxicity toward non-tumorigenic breast MCF-10A cells. Finally, compounds 7a, 7b, 7d, 7e, and 9a were examined for their plausible CDK2 inhibitory action. The obtained results (% inhibition range: 16%–58%) unveiled incompetence of the target bis-indoles to inhibit CDK2 significantly. Collectively, these results suggested that herein reported bis-indoles are good lead compounds for further optimization and development as potential efficient anti-breast cancer drugs.


Anti-proliferative action toward human breast cell lines
The two examined human breast cancer cell lines (MCF-7 and Breast MDA-MB-231), and non-tumorigenic human breast epithelial cell line (MCF-10A) have been obtained from American Type Culture Collection (ATCC). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (GIBCO), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37 o C in humidified atmosphere containing 5% CO2. Cells at a concentration of 0.50 x 10 6 were grown in a 25 cm 2 flask in 5 ml of culture medium.
The anti-proliferative activity of the tested compounds was measured in vitro using the Sulfo-Rhodamine-B stain (SRB) assay. Briefly, Cells were inoculated in 96-well microtiter plate (5X10 4 cells/ well) for 24 h before treatment with the tested compounds to allow attachment of cell to the wall of the plate. Tested compounds were dissolved in DMSO at 1 mg/ml immediately before use and diluted to the appropriate volume just before addition to the cell culture. Different concentrations of tested compounds, doxorubicin and sorafenib were added to the cells (three wells were prepared for each individual dose). Cells were incubated with the compounds for 48 h at 37°C and in atmosphere of 5% CO2. After 48 h cells were fixed, washed, and stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader. The relation between percent of surviving fraction and drug concentration is plotted to get the survival curve for each cell line.
The concentration required for 50% inhibition of cell viability (IC50) was calculated.

Cell Cycle Analysis
Breast cancer MCF-7 cells were treated with bis-indoles 7e and 9a for 24 h (at their

ELISA Immunoassay
The levels of the pro-apoptotic markers (Bax, caspase-3 and p53) as well as the antiapoptotic marker Bcl-2 were determined using ELISA colorimetric kits per the manufacturer's instructions. Breast cancer MCF-7 cells were cultured as a monolayer in T-25 flasks and were seeded to attain 30% confluency prior to treatment. Cells were then treated separately with bis-indoles 7e and 9a at their IC50 concentrations for 48 h.
At the end of treatment, cells were collected via trypsinization and centrifuged at 10,000 rpm. The pellet was then rinsed with PBS and lysed in RIPA lysis buffer at 4 °C for 45 min, then centrifuged at 14,000 rpm for 20 min to remove the cellular debris. Lysates were then collected and stored at −80 °C for later protein determination using Pierce BCA Protein Assay Kit according to manufacturer's recommendations.
The cell lysate was diluted 10 times, and 100 μL (50 mg protein) was added to the wells of four separate microtiter plates for the four ELISA kits that were pre-coated with primary antibodies specific to Bax, Bcl-2, caspase-3 and p53 proteins, respectively. A secondary biotin-linked antibody specific to the protein captured by the primary antibody was further added to bind the captured protein, forming a "sandwich" of specific antibodies around the desired protein in the cell lysate. The streptavidin-HRP complex was then used to bind the biotin-linked secondary antibody through its streptavidin portion. The HRP domain reacted with the added TMB substrate to form a colored product that measured at 450 nm by a plate reader ChroMate-4300 after the reaction was terminated via the addition of stop solution.

Annexin V-FITC Apoptosis Assay
Phosphatidylserine externalization was assayed using Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, USA) according to the manufacturer's instructions. Breast cancer MCF-7 cells were cultured to a monolayer then treated with bis-indoles 7e and 9a at their IC50 concentration. Briefly, cells were then harvested via trypsinization, and rinsed twice in PBS followed by binding buffer. Moreover, cells were re-suspended in 100 μL of binding buffer with the addition of 1 μL of FITC-Annexin V followed by an incubation period of 30 min at 4 °C. Cells were then rinsed in binding buffer and resuspended in 150 μL of binding buffer with the addition of 1 μL of DAPI (1 μg/μL in PBS). Cells were then analyzed using the flow cytometer BD FACS Canto II and the results were interpreted with FlowJo7.6.4 software (Tree Star, Ashland, OR, USA).