| Pigeonpea (Cajanus cajan (L.) Millsp.) is one of grain legume crop grown in many developing countries in semi-arid tropics and subtropics. It plays an important role in human diet as a protein source, especially for vegetarians. Because it is a nontoxic, edible and high-value medicinal plant,pigeonpea is widely used in folk medicine for the prevention of various disorders, such as sickle cell anaemia, plasmodiosis, hepatic disorders, liver injury, and hypocholesterolemic effect. Cajanol is an isoflavanone from Pigeonpea [Cajanus cajan (L.) Millsp.]roots. As the most effective phytoalexin in pigeonpea, the bioactivities of cajanol have sparsely been elucidated with the exception of their antiplasmodial and antifungal activity. In the present study, the cytotoxic activity of cajanol towards cancer cells and mechanism of cell growth inhibition of Cajanol were investigated.The results were as follows:1. Growth inhibition was analyzed by MTT assay. It was found that Cajanol markedly inhibited proliferation of several tumor cell lines, including human colon cancer cells (HCT-8, IC50:19.59±1.07μg/mL), human prostate cancer cells (PC-3, IC50:19.37±0.94μg/mL), human lung adenocarcinoma cells(A549,IC50:19.92±1.10μg/mL), human ovarian cancer cells (HO-8910, IC50:18.00±0.55μg/mL), human leukemic lymphoblasts (CCRF-CEM, IC50: 18.36±1.18μg/mL), human breast cancer cells (MCF-7, IC50:17.08±0.69μg/mL), suggesting that MCF-7 cell line is the most sensitive. The IC50 of Cajanol towards african green monkey kidney (Vero) cells were 34.26±1.52μg/mL.2. Apoptosis is an active process that ultimately leads to the activation of endonucleases and cleavage of DNA into fragments of about 180-200 base pairs. Treatment of MCF-7 cells with cajanol resulted in the formation of a DNA ladder. Apoptosis-related DNA laddering was visible after treatment with 10 or 20μg/mL cajanol for 48 h, but not after treatment with 5μg/mL cajanol. Untreated control cells did not induce apoptosis.3. Hoechst 33258 staining showed that there were considerable morphological changes in the nuclear chromatin. Control MCF-7 cells that were not subjected to cajanol treatment did not exhibit chromatin condensation. The nuclei were stained in a less bright blue and the color was homogeneous. By contrast, after treatment with 20μg/mL cajanol for 48 h, we found that most of the cells exhibited very intense staining of condensed and fragmented chromatin, and some of them formed typical apoptotic bodies. Only a few nuclei still displayed normal morphology. These data clearly indicate that cajanol induces apoptosis in MCF-7 cells.4. Apoptotic population of MCF-7 cells was analyzed by flow cytometry using annexinV-FITC/PI. The apoptosis results showed that the apoptotic population increased in a concentration-dependent manner after the cells treated with Cajanol.5. The DNA content was measured by flow cytometry. As shown in concentration kinetic measurements, exposure to cajanol in a range from 5 to 20μg/mL caused an increase of the G2/M phase population. Hence, cajanol exerted growth-inhibitory effects via G2/M phase arrest in a concentration-dependent manner.6. We investigated Cajanol induce alterations in mitochondrial membrane potential of MCF-7 cells by flow cytometry. Flow cytometry analysis showed that the percentage of cells in low fluorescence produced a time-dependent increase, indicating that Cajanol caused depolarization ofΔΨm. These data showed that Cajanol induces apoptosis accompanied by the alterationsin the mitochondrial membrane potential.7. Generation of ROS upon cajanol treatment was measured by means of DCFH-DA and flow cytomery as an indicator of peroxides and superoxide accumulation. Upon challenge of MCF-7 cells for 48 h with cajanol, a concentration-dependent increase of ROS production was observed. Fluorescence intensities of the fluorogenic probe in the MCF-7 cell lines were higher for all drug concentrations tested than for control samples for all the tested drug concentrations. These results suggest that ROS generation represents an important mechanism contributing to the cytotoxicity of cajanol in tumor cells.8. Alteration of intracellular pH homeostasis in apoptosis was investigated by the confocal laser scanning microscope. The mean optical density was detected by consequent decrease in BCECF AM fluorescence, which showed that Cajanol induced intracellular acidification in MCF-7.9. Western blot analysis showed that cajanol inhibited the Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Cytochrome c released from mitochondria is associated with the activation of caspase-3 and caspase-9 cascade, active-caspase-3 was involved in PARP cleaving. All signal transduction pathway played a role in initiating apoptosis.To the best of our knowledge, the cytotoxic activity of cajanol towards cancer cells was studied for the first time in the present study. Our studies encourage the development of novel, efficient and less toxic plant derived molecules for cancer chemotherapy. |
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