Study On The Anti-tumor Effect Of Vitamin C Combining With Hyperbaric Oxygen

ObjectiveTumor is one of the most important health problems in the world,and many tumors have no effective treatment methods at present.At present,surgical treatment,radiotherapy and chemotherapy are three traditional Anti-tumor methods.The traditional treatment methods are limited by side effects,curative effect and complication.With the development of the tumor molecular classification,targeted drugs are gradually replacing traditional chemotherapeutic drugs as a hot field in oncology research.Compared with traditional chemotherapeutic drugs,targeted drugs have the advantages of high targeting,small side effects and effective improvement of patients’quality of life.Tumor drug target screening and targeting drug search have gradually become a hot field in oncology research.In the 70’s,Linus Pauling,the two Nobel laureate,had suggested that vitamin C（VC）could have therapeutic effects on tumours,but subsequent large-scale clinical trials did not support the hypothesis.Therefore,high dose VC anti-tumor therapy has been controversial.In recent years,domestic and international studies have shown that VC can inhibit or assist in the treatment of various tumors.Yun J found that^[1,2],KRAS or BRAF mutation in colorectal cancer cells with high expression of glucose transporter,further study found that GLUT1 on the oxidation of vitamin C（dehydroascorbate,DHA）has the role of transporters in the extracellular DHA translocation from intracellular influx of intracellular DHA the easy decomposition,formed at the same time VC lead to the increase of ROS（reactive oxygen species）,GSH（glutathione）and NADPH（nicotinamide adenine dinucleotide phosphate）consume large amounts of reducing substances,which consumes NAD+（nicotinamide adenine dinucleotide）,the activity of GAPDH（glyceraldehyde-3-phosphate dehydrogenase）was inhibited,depletion of ATP,eventually leading to cell death.Yun J et al^[1]continued to experiment with colorectal cancer cell lines containing the same gene:without adding reducing agent under the condition of VC in the cell culture medium was oxidized to DHA,the GSH or GLUT1inhibitor STF reduction agent were added to the two groups of cells in the experimental group,two cells VC intake significantly reduce the removal of third groups;GLUT1 cell line after joining VC,found VC intake also decreased significantly.Considering the enhanced expression of GLUT1 in the mutant gene cells,the GLUT1 in the normal cell lines of the control group increased to the same level as that in the mutant group,and the intake of VC did not increase significantly.Previous studies on oral VC can not achieve the goal of anti-tumor in vivo.Intravenous infusion of VC can break the restriction of oral blood concentration and make blood VC level 100-500 times higher than that of oral intake.It is this super high concentration of VC that is the key to attack cancer cell ability.If VC can be a new tumor targeting drug,it will be the gospel for cancer patients.Based on the above studies,we further explored（1）the expression of GLUT1 in various tumor cells and whether GLUT1 could transfer VC to cells in other tumor cells?（2）If GLUT1 is overexpressed or DHA concentration increases,will DHA intake increase?（3）Using HBO to increase oxidative stress in tumor microenvironment will increase the anti-tumor effect by increasing DHA?On the basis of the cell experiment in vitro,by constructing the nude mouse model,the effectiveness of VC combined with HBO is first explored to provide a theoretical basis for the clinical VC combined with HBO for the treatment of tumor.MethodsPart one detection of cell proliferation by vitamin C1、We used qRT-PCR technology to preliminarily detect and analyze the GLUT1related coding genes of 13 different tumor 33 cell lines.According to the screening results,we chose GLUT1 high expression and GLUT1 low expression tumor cells for follow-up control experiment.2、Select GLUT1 high expression tumor cells,add different concentrations of reduced VC、DHA,We use MTT method to detect cell proliferation activity after 12,24and 48h,and select the appropriate VC concentration and action time for subsequent experiments.3、Using the screened VC and DHA with reasonable concentration,the tumor cells with high GLUT1 expression and low GLUT1 expression were processed respectively,and the control group was set up.The proliferative activity of cells was detected by MTT.4、Morphological changes were observed under inverted microscope,and Giemsa staining was used to observe the changes of cell membrane,cytoplasm and nucleus.5、GLUT1 lentivirus transfected GLUT1 overexpressed tumor cells,down regulated the expression of GLUT1 mRNA,and then divided them into control group,VC group,DHA group was further verified,MTT assay was used to detect cell proliferation activity.Part two The establishment of tumor model of tumor bearing miceThe cell concentration of the subcutaneous transplanted tumor of tumor bearing mice was 1*10⁷ml^-1 with PBS,and 0.2ml/only was inoculated on the back of nude mice with GLUT1 high expression of tumor cell suspension.A total of 25 nude mice were inoculated with transplanted tumor.They were randomly divided into three groups according to the size of transplanted tumor,5 in each group:the control group（group control,n=5）,the hyperbaric oxygen group（HBO group,n=5）,the reduced VC group（VC group,n=5）,HBO group combined VC group（HBO+VC group,n=5）.Records were observed daily appetite,activity and tumorigenicity,respectively in 1,2,3,4 weeks to measure tumor length diameter（a,b）,the changes of tumor volume is calculated according to the formula ab²/2,after 4 weeks to break the neck of the mice were killed,the complete separation of tumor tissue,tumor weight.The statistical software SPSS 16was used to analyze the experimental data,and（P<0.05）was significant for the difference.Results1、33 tumor cell lines could detect the expression of GLUT1,but the level of expression was significantly different.In order to further study the correlation between VC and tumor,we chose GLUT1 high expression lung cancer A549 cells and GLUT1low expression lung cancer H1299 cells as the control subjects.2、The proliferation activity of lung cancer A549 cells was detected by MTT method.The results showed that VC and DHA inhibited the proliferation of A549 cells.In a certain range,the inhibition rate of cell proliferation increased with the increase of VC dose.The same concentration,the same time,compared with the control group,DHA has significant inhibitory effect on lung cancer cell A549（P=0.09）,DHA group compared with the reduced VC,the proliferation of A549 cells was significantly inhibited（P=0.03）;the same concentration and the same time,compared with the control group,DHA,the prototype of VC had no obvious inhibition on the proliferation of H1299 cells（P>0.05）,compared with the prototype of VC,significantly inhibited the proliferation of DHA cells（P>0.05）.3、After the use of lentivirus infection in lung cancer A549 cells,the expression of GLUT1mRNA in siR-GLUT1 A549 cells was down regulated by 80%.In the control group,the VC group was also treated with DHA group.Compared with the control group,DHA and VC group had no obvious inhibition on the proliferation of siR-Glut1A549cells.Compared with the VC,DHA had no significant difference in inhibiting the proliferation of siR-Glut1A549 tumor cells.4、In vivo,from first weeks to the prototype of VC mice,tumor volume growth than before,the control group,HBO tumor volume increased significantly;reduced the tumor volume in VC group,the prototype of VC combined with HBO group of tumor volume was slightly increased,increased less than the former two groups.There was a significant difference in the volume and tumor weight between the combined group and the control group（P<0.05）.There was no significant difference in the weight and volume between the combined group and the VC tumor group（P>0.05）.Conclusion1、All the tumor cells expressed GLUT1,but the expression level was different.We successfully screened the GLUT1 high expression of lung cancer A549 and GLUT1 low expression of lung cancer H1299 tumor cells.2、In lung cancer A549,GLUT1 transport DHA into cells,GLUT1 high expression or DHA increase,DHA intake increased.3、Compared with the original VC,the combination of VC and HBO has the trend of synergistic antitumor,but it has no statistical significance.