Study Of The Anti-pancreatic Cancer Role And Molecular Mechanisms Of Tomatidine

Pancreatic cancer is one of the common malignant tumors in digestive system.Due to its occult incidence,difficulty in early diagnosis,less chance of radical surgery and limited effect of conventional chemotherapy,the prognosis of pancreatic cancer is still very poor,and the 5-year survival rate is less than 10%.Therefore,more and more researches aim to find new therapeutic targets and develop new anticancer drugs for pancreatic cancer,in order to improve the therapeutic effect and prognosis of pancreatic cancer.Natural products are considered as feasible sources to find new anti-pancreatic cancer drugs because of its advantages of high safety,less adverse reactions,extensive sources and multiple targets.Among them,alkaloids,as a kind of important nitrogen-containing basic compounds widely existing in plants,have been used as candidate sources of anticancer drugs due to their good anti-tumor effects.Tomatidine is a kind of steroidal alkaloid extracted from Solanaceae plants,has been shown to have good inhibitory effect on a variety of cancers.However,the effect of tomatidine on pancreatic cancer has not been reported.Therefore,the present study aims to explore the inhibitory effect of tomatidine on pancreatic cancer and its possible molecular mechanism,so as to provide experimental evidence for the anti-pancreatic cancer role of tomatidine.This study focuses on the effects of tomatidine in inhibiting the proliferation,migration and invasion of pancreatic cancer and is divided into the following two parts:(1)Tomatidine suppresses tumor growth by regulating NDRG1 in pancreatic cancer;(2)Tomatidine inhibits pancreatic cancer cell migration and invasion through the inhibition of TGF-β/Smad signaling and epithelial-mesenchymal transition.Part I Tomatidine suppresses tumor growth by regulating NDRG1 in pancreatic cancer ObjectiveTo explore the effect of tomatidine on the growth and proliferation of pancreatic cancer cells and its possible mechanism.MethodsThe effects of tomatidine on the proliferation of pancreatic cancer cells were studied by MTT assays,colony formation assays and Ed U assays.The effects of tomatidine on apoptosis and cell cycle of pancreatic cancer cells were studied by flow cytometry.The effect of tomatidine on pancreatic cancer in vivo was verified by subcutaneous tumorigenesis experiment in nude mice.By sequencing the transcriptome of pancreatic cancer cells after tomatidine treatment,the differentially expressed gene were found.Exploring its role in pancreatic cancer cells by using small interfering RNA knockdown and plasmid overexpress the differentially expressed gene.QRT-PCR,Western-Blot and cell function related experiments were used to explore the mechanism of tomatidine regulating the differentially expressed gene to inhibit the growth of pancreatic cancer.ResultsMTT assays,colony formation assays and Ed U assays indicated that tomatidine could significantly inhibit the growth and proliferation of pancreatic cancer cells in vitro.Flow cytometry indicated that pancreatic cancer cell apoptosis increased,G0/G1 phase block after tomatidine treatment.Western-Blot showed that the expression levels of apoptosis and cell cycle related regulatory proteins changed.Moreover,subcutaneous tumorigenesis experiment in nude mice suggested that tomatidine had a good inhibitory effect on pancreatic cancer in vivo.Transcriptome sequencing found that the expression of NDRG1 was up-regulated after tomatidine treatment.QRT-PCR and Western-Blot experiments validation showed that tomatidine could increase the expression of NDRG1.Cell functional experiments indicated that knockdown of NDRG1 could promote the growth of pancreatic cancer,and overexpression of NDRG1 could inhibit the growth of pancreatic cancer.CCK8 assays indicated that NDRG1 knockdown could counteract the inhibitory effect of tomatidine on the proliferation of pancreatic cancer.Western-Blot suggested that tomatidine could inhibit the activation of AKT signaling pathway and inhibit the growth of pancreatic cancer by up-regulating the expression of NDRG1.ConclusionsTomatidine can inhibit the growth of pancreatic cancer by inducing the up-regulation of NDRG1 and inhibiting AKT signaling pathway.Part II Tomatidine inhibits pancreatic cancer cell migration and invasion through the inhibition of TGF-β/Smad signaling and epithelial-mesenchymal transitionObjective To explore the effect of tomatidine on the migration and invasion of pancreatic cancer cells,to reveal the possible molecular mechanism through the TGF-β/Smad signaling and epithelial-mesenchymal transition.Methods Pancreatic cancer cells were treated with non-cytotoxic doses.Scratch-wound healing assays and Transwell assays were used to detect the effects of tomatidine on the migration and invasion of pancreatic cancer cells;Western-Blot method was used to detect the expression levels of TGF-β/Smad signaling and epithelial-mesenchymal transition related markers.Results Scratch-wound healing assays and Transwell assays showed that tomatidine had anti-migration and anti-invasion effects on pancreatic cancer CFPAC-1 and MIA Pa Ca-2 cells.Western-Blot showed that tomatidine can inhibit epithelial-mesenchymal transition by up-regulating the expression of E-cadherin and down-regulating the expression of N-cadherin and Vimentin.Tomatidine could inhibit the TGF-β/Smad signaling pathway by down-regulating the expression of p-Smad2 and p-Smad3.Moreover,tomatidine could reverse the migration and invasion effect of TGF-β1 on pancreatic cancer cells.Conclusions Tomatidine can inhibit the migration and invasion of pancreatic cancer cells;the mechanism may be to inhibit TGF-β/Smad signaling and epithelial-mesenchymal transition.