Sorafenib And Glucose Restriction Synergistically Induce Hepatocellular Carcinoma Cell Death

Objective Based on our previous experimental results that glucose plays an important role in HCC cell survival,this study specifically aims to investigate the influence of the combined treatment of glucose restriction and sorafenib（sora）on HCC cell death in vitro and xenograft growth in vivo,to explore the mechanic roles of mitochondrial function and mitophagy;and to lay biochemical foundations for further researches in the treatment of Transcatheter hepatic arterial chemoembolization（TACE）combined with sora and clinical practice.Methods（1）Establishment of an in vitro model for the combined treatment of nutrient deficiency with sora:HCC cells were treated with different types of nutrient deficient mediumsin the presence or absence ofsora to determine which nutrient was most important for cell survival.Then the following experiments will be conducted using the selected sensitive medium plus sorafenib.（2）Cell growth and cell death:HCC cell survival and cell death were determined by PI（propidium iodide）exclusion assay,sub G1 assay,lactate dehydrogenase（LDH）assay and colony formation assay among different experimental groups.（3）Mitochondrion function:the production of general reactive oxygen species（ROS）and mitochondrial derived ROS were detected by H₂DCFDA（DCF）and Mito SOX Red（Mito SOX）staining respectively.Tetramethylrhodamine methyl（TMRM）and JC-1 were used to detect mitochondrial membrane potentials.The contents of adenosine triphosphate（ATP）in cells were directly detected by ATP assay kit.Metabolic measurement of oxygen consumption rate（OCR）was measured using the Seahorse metabolic assay.（4）Mitophagy:The contents of mitophagyrelated proteins in cells were measured by Western blotting analysis.The changes of mito DNA（mt DNA）staining and m Keima fluorescence were detected to determine the occurrence of mitophagy.Specific si RNAs were used to silence genes involved in mitophagy to identify the essential role of the protein.（5）Xenograft model analysis:After the inoculated HCC xenografts wereidentifiable withtumor volume higher than 50 mm³,all athymic mice were randomly divided into four groups and administered with:（i）vehicle control group（saline）,（ii）sora alone group（20mg/kg）,（iii）canagliflozin alone group（cana,30mg/kg）,or（iv）sora and canacombined group（sora20mg/kgplus cana 30mg/kg）by gavage once every other day.Mice were sacrificed and tumor nodules were collected after two weeks’administration.The tumor cell death was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling（TUNEL）staining and by hematein&eosin（HE）staining,while cell proliferation by Ki-67 immunohistochemical staining respectively.Results（1）HCC cells were treated with different types of nutrient deficient mediumsin the presence or absence of sora.The cell death results showed that only glucose restriction could synergize with sora to promote HCC cell death.The effect of sorafenib is specific because two other anti-angiogenesis drugs（Lenvatinib and Brivanib）could not sensitize glucose restriction induced cell death.In addition,the induced cell death could be protected by NAC,rather than other inhibitors including ferrostatin-1（ferroptosis inhibitor）,z-vad-FMK（caspase inhibitor）and necrotic-1（necrosis inhibitor）.（2）The combined treatment with sora and glucose restriction impaired mitochondrial function by reducing mitochondrial membrane potentials（as evidenced by the changes of TMRM red fluorescent intensity and JC-1 green fluorescence intensity）,decreasing ATP synthesis capacity and the corresponding mitochondrial oxygen consumption rates,and producing a large amount of ROS.（3）Sora alone increased modifications of phosphorylated poly-ubiquitin chain（phospho-poly-Ub）on mitochondrial proteins and decreased the expression of many inner mitochondrial membrane（IMM）or outer mitochondrial membrane（OMM）proteins in both time-and dose-dependent manners.Sora also reduced the mitochondrial DNA content and increased the red fluorescence of m Keima.The above results suggested the occurrence of mitophagy.However,all the changes were abolished whensora was combined with glucose restriction.Compared with the sora alone group,the combined treatment decreased phospho-poly-Ub modification,increased IMM and OMM protein expression,increased the content of mitochondrial DNA,and decreased the red fluorescence of m Keima.When PINK1,an important gene of mitophagy,was silenced,the aforementioned changes of phospho-poly-Ub modification,IMM and OMM protein expression were reversed.More importantly,a higher percentage of cell death was observed after treatment with sora in the PINK1-silenced cells.（4）Canagliflozin（cana）is one of clinical-available anti-diabetic drugs,which acts through inhibition of glucose transporter SGLT2.As same as glucose restriction,cana could synergize with sorafenib to induce HCC cell death.The combined treatment consistently inhibited the effect of sora alone inducedmitophagy changes（increased phospho-poly-Ub modification,decreased expression of IMM and OMM proteinsand the increased red fluorescence intensity of m Keima）.（5）The results of in vivo xenograft study showed that sora synergized with cana to inhibit markedly HCC xenograft growth.The TUNEL staining in the combination group was stronger than those in sora alone or cana alone groups.Similarly,the affected area withextensive cell deathas stained by H&E in the combined group was greater than those in the two single treatment groups.In contrast,ki-67 immunohistochemical staining was weaker in the combination group.Conclusions（1）Only glucose restriction and sora could synergistically induce HCC cells death.Theinduced cell death neither belonged to ferroptosis,apoptosis nor necrotic apoptosis.In contrast,it was ROS dependent,which could be prevented by NAC.（2）Soraspecifically damaged mitochondria,which was evidenced by the impairment of mitochondrial membrane potentials,increased production of ROS,but decreased ATP.This effect was accompanied by mitophagy,a protective mechanism to limit excessive mitochondria damages.However,the combined treatment of sora with glucose restriction inhibited the mitophagy pathway by inhibiting the activity of PINK1.This causedsevere mitochondrial damages,deficiencies in ATP levels,excessive ROS production and consequent extensive cell death.（3）The combined treatment of cana andsora could also promote HCC cell death.The combined treatment consistently inhibited the mitophagy induced by sora alone.Furthermore,co-administration of both sora and cana could inhibit the tumor growth of HCC xenograft,inhibit HCC cell proliferation ratesbut promote HCC cell death in vivo.