Fidarestat Restricts Hepatocellular Carcinoma Through Down Regulating AKR1B10 And Promoting Glycolysis Of NK Cells

Objective(s):Hepatocellular carcinoma(HCC)is the most common type of liver cancer.Compared with other organs,the liver provides a completely different microenvironment,coupled with the lack of biomarkers to identify advanced or early metastatic tumors,drug resistance and high intratumoral heterogeneity,which makes the treatment of liver cancer more difficult.Natural killer cells are an ideal tool for tumor immunotherapy.To improve the anti-tumor ability of NK cells is one of the ideal plans for the treatment of tumors.However,the differentiation,activation and function of NK cells could be influenced by tumor microenvironment.This effect may be achieved by regulating the metabolism of NK cells.Aldose reductase AKR1B10 is highly expressed in tumor tissues,and the aldose reductase inhibitor Fidarestat can directly inhibit the expression of aldose reductase AKR1B10.Fidarestat has been proved to be effective against some tumors.It is safe and without side effects.Based on this,we speculate that the expression of AKR1B10 in infiltrated NK cells is increased under the effect of tumor microenvironment.Therefore,the glycolysis and lactate metabolism and cytotoxicity of NK cells are affected.There are some questions still unclear:whether Fidarestat restricts HCC through regulating AKR1B10 expression and metabolism in NK cells.Therefore,in this paper,we tested the anti HCC function of Fidarestat,and tried to clarify the possible molecular mechanism of Fidarestat in the treatment of liver cancer.It will provide a theoretical basis for cancer treatment.Methods:In order to study the anti HCC effect of Fidarestat,we established a nude mouse model of transplanted tumor.After confirming the effective concentration of Fidarestat on different hepatoma cell lines,the body weight of nude mice was detected after giving the effective concentration of Fidarestat to determine the toxicity of the drug in vivo.To determine the anti HCC ability of the drug in nude mice transplanted hepatoma model,the size and weight of tumor formation in nude mice was detected,as well as the metastasis in lung tissues were detected by HE stain.Flow cytometry was used to analyze the number and proportion of immune cells in the tumor.The proportion of granzymeb,perforin,IFN-y and TNF-α positive cells in NK cells was detected by flow cytometry to analyze their killing activity.The number of tumor infiltrating NK cells were further confirmed by immunohistochemistry.The serum cytokine IL-2,IFN-y and TNF-α levels were detected by ELISA to determine the effect of Fidarestat on immune cells;NK cell specific antibody was used to mimic NK cells depletion,and the tumor size and weight of nude mice,as well as the metastasis of lung tissue were detected to determine that Fidarestat really regulates the anti-tumor activity of NK cells;NK cells were co-cultured with hepatoma cells in vitro and treated with Fidarestat.CCK-8,Transwell and scratch tests were used to detect the survival rate,invasion and migration ability of hepatoma cells,so as to clarify the effect of Fidarestat-NK cell system on the survival and characteristics of hepatoma cells in vitro;the glycolysis ability of NK cells and the expression level of AKR1B10 were detected by ECAR and WB,so as to determine that Fidarestat can affect NK cells metabolic and AKR1B10 expression.After AKR1B10 overexpression in NK cells,the glycolysis ability and lactic acid production level of NK cells was detected again,and the killing effect of NK cells on Huh7 cells was detected,as well as the survival rate,invasion and migration ability of hepatoma cells.It was confirmed that Fidarestat could inhibit the expression of AKR1B10 in NK cells and improve NK cell glycolysis to play an anti-tumor role.Finally,by detecting the expression of related pathway proteins,the drug can up-regulate the expression of PI3K/Akt/mTOR and MAPK axis and inhibit the expression of TGF-β,so as to clarify the anti HCC mechanism of Fidarestat.Results:We first confirmed that Huh7 was the most sensitive hepatoma cell line to Fidarestat,and 10μM Fidarestat is the lowest effective concentration(IC50 is about 7-8μM).Then in nude mice tumor model,we found that Fidarestat had anti HCC activity in a dose-dependent;at the same time,there was no significant inhibition on the body weight of tumor bearing nude mice,and there was no drug-related death,indicating that the direct toxicity of Fidarestat was weak.H&E observation on lung sections of nude mice bearing tumor showed that Fidarestat could significantly inhibit lung metastasis of nude mice bearing tumor.It is suggested that Fidarestat has good anti HCC activity in vivo.Subsequently,we analyzed the immune cells of tumor bearing nude mice and speculated that the anti-tumor activity of Fidarestat might through regulating NK cells function.It was found that the number of NK cells in mice tumor tissues treated with Fidarestat was significantly up-regulated in the Huh7 transplanted tumor model.The immunohistochemistry analysis of NK cells further showed that the proportion of NK cells in tumor tissues was significantly up-regulated after Fidarestat treated,suggesting that Fidarestat can recruit NK cells into tumor tissues.It is speculated that Fidarestat can exert anti-tumor activity by affecting NK cells.Then,flow cytometry showed that Fidarestat could up regulate the proportion of granzyme B,perforin,IFN-y and TNF-α positive NK cells,which indicated that Fidarestat might exert its anti HCC activity by enhancing killer cytokines secrete in NK cells.Subsequently,the serum levels of IL-2,IFN-y and TNF-α were detected.It was found that Fidarestat could significantly increase the levels of these cytokines.These results suggest that Fidarestat may exert its anti HCC activity by increasing the degranulation ability of NK cells and secreting inflammatory cytokines.Next,NK cells were co-cultured with normal hepatocyte/hepatoma cell lines using different effector target ratios.It was found that with the increase of the proportion of NK cells in different effect target ratios,the killing activity of NK cells against hepatoma cells could be enhanced after Fidarestat treated,which indicated that Fidarestat could affect the function of NK cells and enhance their killing activity.At the same time,Fidarestat did not increase the killing activity of NK cells against human normal liver cell line HL-7702.The results of cell invasion test showed that the NK cells treated with Fidarestat could inhibit the invasion of hepatoma cell line Huh7;scratch test also proved that the NK cells treated with Fidarestat could inhibit the migration of hepatoma cell line MHCC97L and Huh7.Therefore,Fidarestat can inhibit the activity of hepatoma cells and the invasion and migration of hepatoma cells by acting on NK cells in vitro.In order to further confirm whether Fidarestat can improve the anti HCC activity by affecting the activity of NK cells.The in vivo tumor transplantation experiment showed that Fidarestat could increase the infiltration of NK cells in tumor,and anti-NK1.1 could significantly inhibit the recruitment of NK cells to tumor tissue after NK cells exhaust.At the same time,consistent with previous experiments,Fidarestat can inhibit the proliferation of tumor cells,showing the decrease of tumor volume and weight,and inhibit the metastasis of tumor cells to the lung.It is consistent with the expectation that it can significantly weaken the efficacy of Fidarestat after NK cell depletion.Therefore,Fidarestat can inhibit the metastasis,invasion and activity of liver cancer cells in vivo and in vitro by affecting the function of NK cells,while it has little effect on normal liver cells.In the Huh7 tumor bearing nude mice model,after treated with Fidarestat,the primary NK cells were isolated to detect the glycolysis ability of NK cells.The glycolysis ability of NK cells treated with Fidarestat was up-regulated,and the production of lactic acid was promoted.Western blot showed that Fidarestat could down regulate the expression of AKR1B10 in NK cells.The expression of AKR1B10 protein in NK92 cells was detected in vitro.It was found that Fidarestat could down regulate the expression of AKR1B10 protein in NK92 cells,which was consistent with that in primary NK cells.The AKR1B10 overexpression plasmid was constructed and packaged as lentivirus for transduction.WB test showed that AKR1B10 expression in NK92 cells was significantly up-regulated after lentiviral transduction of AKR1B10;Fidarestat alone could inhibit the expression of AKR1B10,while overexpression of AKR1B10 could weaken the inhibitory effect of Fidarestat on AKR1B10;adding Fidarestat,it was found that Fidarestat could promote glycolysis and lactate production of NK cells;empty plasmid group had little effect on glycolysis and lactate production.However,the effect of Fidarestat on NK glycolysis and lactate production was weakened when AKR1B10 overexpression,suggesting that Fidarestat could promote glycolysis by inhibiting AKR1B10.When overexpression of AKR1B10,NK cells were co-cultured with Huh7 cells.It was found that the killing activity of NK92 cells to Huh7 cells was decreased after overexpression of AKR1B10.By the scratch test and invasion test,it was found that after overexpression of AKR1B10 and adding Fidarestat,Fidarestat could restrict Huh7 cells invasion and migration.Finally,Western blot was used to detect the changes of related pathways in NK cells treated with Fidarestat in vitro.It was found that Fidarestat could activate the expression of PI3K/Akt/mTOR and MAPK axises and inhibit the expression of TGF-β.These results indicate that Fidarestat interferes with the PI3K/Akt/mTOR molecular axis to affect the glycolysis and lactate production of NK cells and MAPK axis to affect the cell growth and differentiation of NK cells,and inhibits the TGF-βpathway to affect the metabolism and function of NK cells.Conclusion(s):Fidarestat can down regulate AKR1B10 in NK cells,promote the glycolysis of NK cells,and enhance the ability of NK cells to kill tumor cells.It can inhibit the growth and metastasis of tumor in nude mice in vivo.It can also increase the killing activity of NK cells to hepatoma cells,and inhibit the migration and invasion of hepatoma cells in vitro.At the same time,Fidarestat can activate the expression of PI3K/Akt/mTOR and MAPK axis and inhibit the expression of TGF-β.In general,Fidarestat can improve the disorder of glucose metabolism of NK cells by down regulating AKR1B10,and play an anti-hepatoma role.