Nimbolide inhibits pancreatic cancer growth and metastasis through ROS-mediated apoptosis and inhibition of epithelial-to-mesenchymal transition

The mortality and morbidity rates of pancreatic cancer are high because of its extremely invasive and metastatic nature. Its lack of symptoms, late diagnosis and chemo–resistance and the ineffective treatment modalities warrant the development of new chemo–therapeutic agents for pancreatic cancer. Agents from medicinal plants have demonstrated therapeutic benefits in various human cancers. Nimbolide, an active molecule isolated from Azadirachta indica, has been reported to exhibit several medicinal properties. This study assessed the anticancer properties of nimbolide against pancreatic cancer. Our data reveal that nimbolide induces excessive generation of reactive oxygen species (ROS), thereby regulating both apoptosis and autophagy in pancreatic cancer cells. Experiments with the autophagy inhibitors 3-methyladenine and chloroquine diphosphate salt and the apoptosis inhibitor z-VAD-fmk demonstrated that nimbolide-mediated ROS generation inhibited proliferation (through reduced PI3K/AKT/mTOR and ERK signaling) and metastasis (through decreased EMT, invasion, migration and colony forming abilities) via mitochondrial-mediated apoptotic cell death but not via autophagy. In vivo experiments also demonstrated that nimbolide was effective in inhibiting pancreatic cancer growth and metastasis. Overall, our data suggest that nimbolide can serve as a potential chemo–therapeutic agent for pancreatic cancer.


Matrigel invasion assay
The invading ability of the pancreatic cancer cells was assessed using a total of 5X10 4 cells in the upper chamber of a transwell polycarbonate membrane coated with 1 mg/ml of Matrigel.
Nimbolide was applied using FBS-free media in the appropriate wells for 48 h along with untreated control cells in the upper chamber for an appropriate comparison. Then, 600 µl of growth media with 10% FBS was added as a chemo-attractant in the lower chamber. Cells that invaded the lower chamber were fixed with 0.2% crystal violet in 5% formalin, and then, images from six randomly selected fields were captured using a Nikon Eclipse TS 100 microscope with a magnification of 20X 1 . Experiments were repeated three times.

Soft agar colony-formation assay
Pancreatic cancer cells were seeded on 60-mm dishes with a top layer of 0.7% agar at densities of 2×10 4 cells per dish and a bottom layer of 1% agar. The cells were incubated in medium with or without nimbolide (IC 50 concentrations). Every 2 days the medium was changed for up to 16 days for MIAPaCa-2, 27 days for PANC-1, and 30 days for HPAC. The cells were incubated at 37ºC and stained with 0.2% crystal violet in 5% formalin solution. Colonies containing 15 cells or more were counted manually, and images were captured using Nikon SMZ 1500 at 10 and 40X magnifications 1 .

ROS generation assay
Intracellular ROS generation was assessed using the ROS assay kit. A total of 1X10 4 cells were

IHC for key apoptotic, proliferative, EMT and autophagy markers in tumor xenograft tissues
Paraffin-embedded tumor tissue sections were used to study the levels and localization of pAKT, E-cadherin, LC3A/B, p62, Bax, and cleaved Caspase 3 using IHC staining. After incubation at 58ºC for 2 h, the slides were deparaffinized and rehydrated in alcohol of serial dilutions oscillating from 100, 95, 70, 50, and 30% ending in a distilled water bath for 5 min. Epitope retrieval was achieved with heating at 95ºC for 15 min with trilogy (Cell Marque, Rocklin, CA, USA). After blocking, the tissues were incubated with their respective primary antibodies and followed by ultra Marque polyscan HRP labeling (Cell Marque, Rocklin, CA, USA). Once stained with chromogen and hematoxylin, the slides were dehydrated with increasing dilutions of ethanol (30, 50, 70, 95, and 100%) ending with a xylene bath. Finally, the sections were sealed with mounting media (Surgipath Medical Industries, Richmond, IL, USA), and the images were obtained using a Nikon Microscope-ECLIPSE 50i at 40X magnification as described 1,4 .

Hematoxylin and eosin Staining
Three-to four-micron sections were cut and placed on a positively charged slide. These embedded sections were deparaffinized and dehydrated gradually followed by H&E staining.
H&E-stained pancreatic sections of both control and nimbolide treatment groups were analyzed for metastatic lesions in the brain, lung and liver. Histology images of tumor, brain, lung and liver were captured using a Nikon Microscope-ECLIPSE 50i at 10X magnification. 5