Background Breast cancer is considered as an increasing major life-threatening concern among the malignancies encountered globally in females. C-3 (secondary), C-14 (allylic) and C-19 (main) on the basic structural skeleton were reported to be responsible for its biological activities [8, 9]. In recent years several studies have indicated that andrographolide also possess antitumor activity [10, 11]. Breast malignancy is a major life-threatening concern among the malignancies encountered in females and ranks second as a cause of death . Apoptosis is usually a programmed cell death which occurs due to the activation of certain evolutionarily conserved intracellular functions. Many naturally occurring phytochemicals were reported to possess anti-tumor effect thus inducing apoptosis of malignancy cells. Curcumin from turmeric, epigallocatechin gallete from green tea, resveratrol from grape seed extract and quercetin from fruits are some examples of chemopreventive brokers derived from herb that induce apoptosis with some being in clinical intervention trials [13, 14]. Earlier reports based on the pharmacological properties of andrographolide, especially on its Dye 937 antitumorogenic activity through numerous mechanisms, such as, inhibiting cell cycle progression, reducing invasiveness of malignancy cells or inducing apoptosis through different cell-death mechanism in different carcinoma cells [10, 15] prompted us to evaluate the possible induction of apoptosis by andrographolide on breast cancer cell collection. With this background, this study was designed to evaluate in vitro anticancer activity of andrographolide in a breast cancer cell collection, MDA-MB-231 which is usually highly invasive, proliferative, estrogen receptor (ER) unfavorable and harbors mutated p53. Dye 937 Although, earlier studies with other breast cancer cells made up of functional ER and wild type p53 showed cell growth inhibition and apoptosis induced by andrographolide [16, 17], reports on the effect on this particular triple unfavorable breast malignancy (TNBC) cell collection are scanty. Dye 937 Therefore, it is advantageous to investigate the inhibitory and/or apoptosis inducing effect of andrographolide on MDA-MB-231 as this cell collection is Dye 937 clinically harder to treat . Malignancy cells harboring mutated p53 is usually exhibited as more resistant to certain anticancer drugs because mutated p53 no longer renders the tumor suppressing abilities of the wild type, rather it often contributes to the Rabbit Polyclonal to FPRL2 oncogenic characteristics . Furthermore, metastatis-derived MDA-MB-231 breast cancer cell collection is not hormone sensitive (ER unfavorable). Blocking the Estrogen receptor in these cells will not serve the purpose of inhibiting malignancy. Thus MDA-MB-231 cells are more resistant to drug therapy in comparison to other breast malignancy cells like MCF-7. For instance, while resveratrol inhibits cell proliferation and activity in both MCF-7 and MDA-MB-231 cells, it was able to induce apoptosis in MCF-7 cells only . In the present study, attempts have been made to elucidate the molecular mechanism by which andrographolide renders its inhibitory effects on cell proliferation, cell cycle, expression levels of pro- and anti-apoptotic proteins and finally towards apoptosis in this clinically unique cell collection. Our results show that andrographolide can inhibit the cellular growth of MDA-MB-231 by causing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Additionally, andrographolide was analyzed by LC-MS/MS method to determine its pharmacokinetic characteristics in the plasma of BALB/c mice and these pharmacokinetic results are important for further study of the clinical applications of andrographolide. Methods Materials and reagents Andrographolide was procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in DMSO and kept at 4?C at a concentration of 50?mM. AnnexinV-FITC Apoptosis Detection Kit was purchased from BD Pharmingen (Pharmingen, USA). Caspases fluoremetric assay kit was purchased from Chemicon International Corporation (USA). Ac-DEVD-CHO (caspase-3 inhibitor), and Ac-LEHD-CHO (caspase-9 inhibitor) were from Calbiochem (La Jolla, USA). Main antibodies (Bcl-2, Bcl-xL, Bax, Apaf-1, cytochrome usage of regular pet drinking water and diet plan. In vitro cytotoxicity assay The result of andrographolide on cell viability was assessed by MTT assay following technique by Mosmann . Quickly, the cells (1??105 cells per ml) were seeded within a 96 well micro titer dish (100?l per good) with replications. Treatment was executed for 24 and 48?h with different concentrations (0, 5, 7.5, 15, 30, 45, 60, 75 and 100?M) of andrographolide. After incubation, 20?l of 5?mg/ml MTT share solution was put into each very well and incubated for 4?h in 37?C. The attained formazan crystals had been solubilized with DMSO as well as the absorbance was assessed at 570?nm utilizing a microplate audience (SpectraMax M5, Molecular Gadgets, USA). Cell viability (%) provides been shown being a ratio.