Effect Of Ketogenic Diet On Neuroblastoma In Balb/c-nu Mouse Model

Objective:In this study,we intend to investigate the effect of ketogenic diet(KD)on tumor growth of neuroblastoma(NB)in BALB/c-nu mouse models.Methods:Xenografts were established in 5-week-old female BALB/c-nu mice by subcutaneous injection of neuroblastoma cell line(SH-SY5Y).When tumor volume reached 250.000 mm~3,mice were randomized to four groups(SD,KD,CP+SD,CP+KD,n=8).The length(a)and the short diameter(b)of the tumor were measured by caliper and tumor volume was calculated according to the formula(axb~2)/2.Body weight,blood glucose,ketone body(?-Hydroxybutyrate)levels and hepatic steatosis were monitored as well.The described indices were carried every three days.The levels of cell proliferation protein Ki67 and autophagic proteins(Beclin1,LC3A/B and P62)were analyzed by Immunohistochemistry(IHC).And the levels of apoptosis proteins(Caspase3,Caspase8)and the mentioned autophagic proteins were detected by Western Blot.Meanwhile,tumor slices were used for autophagosome by transmission electron microscopy(TEM).Results:1.(1)On the 25th day post cell inoculation,the overall survival rates of host in SD,KD,CP+SD and CP+KD group were 0%,75%,87.5%and 100%separately.By contrast with SD group,KD,CP+SD and CP+KD group could significantly prolong the overall survival of host(X~2=19.69,P<0.001).(2)The tumor growth of KD and CP+KD group was delayed compared to that of SD group.On day 19,22 post tumor cell injection,tumor volume of KD group was smaller than that of SD group respectively(P<0.05);on day 19,22 post tumor cell injection,tumor volume of CP+SD group was much smaller than that of SD group respectively(P<0.01);on day 16,19,22 post tumor cell injection,tumor volume of CP+KD group was much smaller than that of SD group respectively(P<0.01).2.(1)There was no significant difference on body weight between SD and KD group for the measured time(P>0.05).(2)Mean blood glucose of SD group was higher than that of KD and CP+KD group on the day 13 post tumor cell injection(P<0.05),which was not for the other measured time(P>0.05).(3)The mean blood ketone of the CP+KD and KD group were higher than those in the SD group(P<0.05),while the glucose ketone index(GKI)of the CP+KD and KD group were lower than those in the SD group separately(P<0.05).(4)Hepatic steatosis scores of the KD and CP+KD group were higher than those of SD group(P<0.05).3.(1)The expression of Ki67 protein was strongly positive(+++)in SD group,and moderately positive(++)in KD,CP+SD and CP+KD group.(2)The apoptotic protein Cleaved-caspase3,Full-length caspase3 and Caspase8were expressed in all groups.4.(1)Autophagosomes in KD group were more than those in SD group by TEM.(2)Comparing to SD group,the protein levels of Beclin1 and LC3A/B were up-regulated in KD group(P<0.05),while P62 was inhibited(P<0.01).It was basically in line with the corresponding results of IHC.Conclusion:KD could exert a robust anti-tumor effect for NB,and its possible mechanism is related to autophagy;KD is relatively safe,which have little effect on body weight and blood glucose level in tumor-bearing mice;KD is expected to become a new supplementary therapy for NB.