Effects Of Sulforaphane On Bladder Cancer Cells Migration And The Mechanism Through Targeting Energy Metabolism Rearrangement

Re-arranging the energy metabolism model to meet the ATP demand of tumor cells for rapid proliferation and metastasis has been regarded as one of the new features of cancer.Abnormally active aerobic glycolysis and de novo fatty acid synthesis are the most prominent features of tumor energy metabolism rearrangement.Bladder cancer（Bladder Cancer,BC）is one of the most common malignant tumors in the urinary system.Invasion and metastasis are the primary cause of death of bladder cancer.In the process of tumor cell invasion and metastasis,a large amount of energy supply is needed.The characteristics of energy metabolism are currently unclear.Sulforaphane（SFN）is found in a variety of cruciferous vegetables and is the most widely studied isothiocyanate with anticancer activity.The previous research results of the research group showed that SFN has a strong inhibitory effect on the metastasis of bladder cancer cells,but its in-depth systemic mechanism research is limited,especially the research data on energy metabolism regulation is still lacking.Based on the analysis of the characteristics of glycolysis,tricarboxylic acid cycle and pentose phosphate metabolism in patients with bladder cancer,this paper systematically studied the regulation and signal regulation of SFN on the glucose metabolism of bladder cancer cells through isolated cell and whole animal experiments.mechanism.The main results are as followings.The tumors and adjacent tissues of bladder cancer patients with different pathological stages were collected,and the changes in the levels of key metabolites in glycolysis,tricarboxylic acid cycle and pentose phosphate pathway were detected by GC-MS.ATP and various metabolites are highly expressed in tumor tissues,which indicates that glucose metabolism in bladder cancer patients is abnormally active.Subsequently,the metabolic pathway enrichment analysis was performed for the abnormally expressed metabolites.81 enriched metabolic signaling pathways were obtained,among which the most enriched is the"Warburg effect".Using q PCR to detect the m RNA levels of various glucose metabolizing enzymes,it was found that the gene expression of PFK-1,LDHA and PDH in bladder tumor tissue was abnormally increased（P<0.05）.Serum enzyme activity detection revealed that the activities of key rate-limiting enzymes HK2,LDHA,PDH and PKM2 in the patient’s body were abnormally increased.Subsequently,the analysis of the correlation between enzyme activity and TNM staging found that PKM2 was positively correlated with tumor TNM staging（r=0.4538,P=0.0103）;while PDH was negatively correlated（r=-0.3742,P=0.0381）.Then,we used UMUC3（pathological grade 1）and T24 cells（pathological grade 3）as in vitro models to study the regulatory mechanism of SFN from the perspectives of glycolysis and mitochondrial oxidative phosphorylation.SFN can strongly down regulate intracellular ATP level by inhibiting glycolysis and mitochondrial respiration in bladder cancer cells.SFN significantly inhibited the protein expression of HK2,PDH and LDHA in UMUC3 and T24 cells;The inhibitory effect of SFN on HK2 was the strongest in UMUC3 cells,which indicated that SFN had more obvious inhibitory effect on glycolysis of low-grade bladder cancer cells.Transient transfection of p^{EGFP n1-AKT1} plasmid can strongly reverse the suppression of HK2protein expression in T24 cells by SFN,which suggests that SFN regulates HK2 in bladder cancer cells through AKT1.In addition,SFN can also regulate the aerobic oxidation of bladder cancer cells by reducing the number of mitochondria in T24 cells and inhibiting the activity of mitochondrial complexes I,II,III and V.q PCR detection revealed that SFN can strongly down-regulate the gene expression of key metabolic enzymes ACC and FASN by inhibiting the m RNA level of SREBP1,thereby effectively regulating the fatty acid synthesis and metabolism of bladder cancer cells.In the BBN-induced mouse bladder cancer model,it was confirmed that SFN can further inhibit HK2 by regulating the expression of AKT1 in tumor tissues of bladder cancer mice;and it also has a strong inhibitory effect on the protein levels of key metabolic enzymes LDHA,PDH and PKM2.On the basis of clarifying that SFN can mainly regulate the activity of AKT1-HK2axis and mitochondrial complex of bladder cancer cells to inhibit glucose metabolism and energy supply,we further paid attention to the regulation mechanism of SFN on pseudopodia,a key part of cell metastasis.Scratch test and Transwell were used to detect cell migration and invasion ability,and it was found that SFN strongly downregulated the metastasis and invasion of bladder cancer cells in vitro.Observed by transmission electron microscope and scanning electron microscope,it is found that SFN can shed villi on the cell surface and severely destroy the morphology of bladder cancer cells.Phalloidin was used to label F-actin,the main component of cell pseudopodia,and the cytoskeleton morphology changes were observed based on laser confocal microscope.When AKT1 is overexpressed,SFN’s inhibition of pseudopodia formation regulator Arp2 is greatly weakened,and the damaging effect on cell pseudopodia disappears.The findings indicate that SFN can regulate cell metabolism and energy supply through AKT1,inhibit Arp2 expression,and block bladder cancer cells.Pseudopodia formation.AKT1 overexpression can also strongly reverse the inhibitory effect of SFN on T24 cell invasion and metastasis.In addition,SFN strongly inhibits the protein expression of cortactin and p-cortactin,revealing that regulating the activation and expression of cortactin is another major way for SFN to destroy the formation of pseudopodia in bladder cancer cells.Finally,a model of lung metastasis of bladder cancer cells was created by tail vein injection in nude mice,which confirmed that SFN can strongly down-regulate the gene expression of cortactin and Arp2 in bladder cancer mice;delay and inhibit the formation and metastasis of bladder tumors in the lungs.