GLI inhibitors overcome Erlotinib resistance in human pancreatic cancer cells by modulating E-cadherin

Abstract

Inhibition of hedgehog (Hh) signalling pathway, including its end effector GLI1, can reverse epithelial-to-mesenchymal transition (EMT) which plays an important role in drug resistance of pancreatic cancer cells to Erlotinib (ETB). This study investigated the effect of GLI inhibitors Forskolin (FSK), GANT-61 (GNT), and Arsenic trioxide (ATX) on suppressing the resistance of pancreatic cancer cells to ETB. The effect of GLI inhibitors was evaluated by measuring mRNA expression levels of EMT factors using quantitative RT-PCR. Immunocytochemistry and flow cytometry were used to assess E-cadherin (E-Cad) and GLI1 protein levels. MTT and apoptosis assays were used to evaluate the synergistic effects for the combination treatment of each GLI inhibitor with ETB. Pancreatic cancer cells PANC-1 treated by GNT showed the highest significant reduction in mRNA levels of GLI1 and other EMT pathway genes. Moreover, GNT was able to upregulate E-Cad and downregulate GLI1 proteins, more than FSK, while ATX had no effect. Apoptosis levels of PANC-1 cells following treatment with LD30 concentrations of FSK, GNT, or ATX, showed 57%, 62% and 67%, respectively, in comparison to ETB (∼48%). Importantly, combination treatments of ETB with either FSK, GNT, or ATX demonstrated a significant increase in apoptotic cells reaching 61% (ETB + FSK), 80% (ETB + GNT) or 88% (ETB + ATX). FSK did not have much effect on the drug resistance of PANC-1 cells to ETB. However, GNT, but more effectively ATX, were able to reduce the drug resistance of this cell line to ETB.

Keywords:

• GANT-61
• Forskolin
• arsenic trioxide
• Erlotinib
• pancreatic cancer

Acknowledgements

This work was supported by a grant from Cell and Molecular Research Center of Yasuj University of Medical Science, Yasuj, Iran and L-CNRS and Lebanese University (KZ).

Notes on contributors

Amir Ghanbari graduated in PhD course of anatomical sciences. He is working at the department of anatomy in Yasuj University of Medical Sciences. He is doing research in Neurosciences and neural stem cells specially oligodendrogensis.

Zeinab Cheraghzadeh Graduated in anatomy sciences from Yasuj University of Medical Sciences. Her research was about involvement of developmental pathway in cancer.

Reza Mahmoudi obtained PhD from anatomy and embryology from Tehran University in 2005. He is working as professor at Yasuj University of Medical Science. He has more than 24 years of teaching and research experience. His research interest are includes assisted reproductive technology, stem cell and cancer.

Dr. Kazem Zibara obtained his PhD from Lyon-1 University in 1999 with high distinction. During his thesis, he cloned 2 new genes implicated in cardiovascular diseases and he also did single, double and triple knock outs of a number of adhesion molecules in mice. His first Post-doc in France was on the identification of new genes implicated in hyperglycemia and Diabetes Type II. During his second Post-doc at the Thrombosis Research Institute in London, he set-up a “functional Genomics” Lab and worked on the identification of new genes implicated in vascular lesions using microarrays. His third Post-doc was at Cancer Research UK in London, where he developed his expertise in cell sorting, transcriptome expression profiling using Affymetrix chips, stem cell biology, and engraftment of HSCs in immune-deficient mice. After his return to Lebanon, he developed an in vivo stem cell lab. Dr Zibara holds two patents in addition to five international awards. He was co-founder of 2 companies, Arteria and CliniGenetics, in the area of drug discovery in the cardiovascular and diabetes fields. He is currently a full professor at the LU and director of Research. His current research interests span Cancer Biology, Metastasis, AML, Traumatic Brain Injury and Multiple sclerosis.

Dr. Ebrahim Hosseini obtained his PhD from Tarbiat-Modares University in 2015. His thesis was in cancer virotherapy field, he constructed an adoneovirus armed with MDA-RGD modified gene. Now he is working as an assistance professor of medical genetic in Yasuj University of Medical Science. His research is mainly focused on cancer biotherapy and some of human genetic disease including tyrosinemia and hearing loss.

Disclosure statement

No potential conflict of interest was reported by the authors.