Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis

Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to ~5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy.


Chemicals and Reagents
All the chemicals used in the present study were purchased from Sigma Aldrich (St. Louis, MO, USA) or Sisco Research Laboratories (India). Antibodies were from Santa Cruz Biotechnology (USA), BD Biosciences (USA) and Cell Signaling Technology (USA). Purified quercetin and ellagic acid (Suppl. Fig. 1) were purchased from Sigma Aldrich (USA). Stock solutions of quercetin and ellagic acid were prepared in DMSO and stored at 4 o C till use. When required, dilutions were done in autoclaved miiliQ water.

Animal and ethics statement
Female Swiss albino mice weighing 18-22 g and 6-8 weeks old were purchased from central animal facility of IISc, India for the present study. The mice were maintained under controlled conditions of temperature and humidity with 12 h light and dark cycle. They were housed in polypropylene cages and standard pellet diet (Agro Corporation Pvt. Ltd. Bangalore, India) was provided along with water ad libitum. The standard pellet diet was composed of 21% protein, 4% crude fiber, 5% lipids, 3.4% glucose, 2% vitamins, 1% calcium, 0.6% phosphorus, 8% ash and 55% nitrogen-free extract (carbohydrates).

TUNEL assay
Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was performed on histological sections (5 µm) of quercetin treated tumor tissue and untreated tumor control after 30 th day of treatment for the detection of DNA strand breaks occurred during apoptosis. Sections were de-paraffinized and assay was performed by using the TUNEL assay kit (DNA fragmentation Detection Kit, Calbiochem, USA). Briefly, sections were washed in PBS (1X) and treated with proteinase K for permeabilization of the specimen. Labeling was done with dUTP at nicked DNA ends with the help of TdT enzyme at 37 o C for 1 h, 30 min followed by DAB staining. Methyl green was used as a counterstain. Sections were observed under light microscope (Carl Zeiss, Germany) and images were captured. Percentage of TUNEL positive nuclei were determined by counting a minimum of five different randomly selected sections and shown as bar diagram with SEM.

Immunohistochemistry (IHC)
Sections with thickness 5 µm from control tumor and quercetin treated tumor tissues embedded in paraffin wax were cut in rotary microtome and deparaffinized as described earlier 1,2 . Sections were then rehydrated and treated with 3% H 2 O 2 . Antigen retrieval was performed in 0.01% sodium citrate buffer followed by blocking (0.1% BSA and 10% FBS in PBST) for 1 h at room temperature. Following incubation in primary antibody (Ki-67, p53 and p-p53; overnight at

Confocal microscopy
Annexin V-FITC/PI stained Nalm6 cells were prepared as described above after treatment with quercetin (0, 20 and 50 µM, for 48 h). Cells were mounted on glass slides and images were captured under inverted Zeiss confocal laser-scanning microscope (Ziess Meta 510 LSM; Carl Zeiss, Jena, Germany).

Detection of intracellular ROS by flow cytometry
Reactive oxygen species (ROS) is produced by each cell at intrinsic level, which may get enhanced after treatment with some cytotoxic compounds.  Suppl. Figure 3 Suppl. Figure 4 A B Suppl. Figure 5 Suppl. Figure 6