The Preliminarily Study Of Anti-tumor Effect And Its Molecular Mechanisms Of Calculus Bovis Cultivated By Glucuronidase

ObjectiveTo detect the anti-tumor and immunomodulatory effect of calculus bovis cultivated byglucuronidase(CBCG)on S180tumor-bearing mice, and preliminarily explore its molecularmechanisms. The synergy and attenuation action of CBCG on cyclophosphamide(CTX)are also investigated in this study.MethodsS180tumor-bearing mice were randomly divided into seven groups: Model group(given bezoar solvent,ig), CBCG high, medium and low dose group (2g/kg,1g/kg,0.5g/kg,ig), calculus bovis(NCB) Group (1g/kg,ig), CTX group (0.02g/kg,ip), the combinationgroup (CTX0.02g/kg,ip+CBCG1g/kg, ig). Measured the largest and the shortest diameterseveryday during the medication, and draw the tumor growth curve diagram. After10daystreatment, blood were obtained to evaluate the complete blood counts, then the serum wereseparated by centrifugation. The contents of serum TNF-α and IL-2was detected byEnzyme Linked Immunosorbent Assay Kit. The mice were sacrificed after blood taken, thetumors were excised and weighed, the tumor inhibition rate was calculated. The partial ofthe tumors was collected for pathological sectioning with hematoxylin–eosin (HE) stain.The expressions of pten, AKT, BAX, BCL-2, p53, which related to PI3K/AKTpathway,are detected by Western blot. ALL thymus and spleens of mice were collected todetect visceral index of thymus and spleen, respectively.Taken mice spleen lymphocytes aseffector cells, sarcoma S180cells as target cells, determined mice spleen CTL killingactivity by MTT-based assay.Results1. In vivo anti-tumor effects of CBCG in S180-bearing miceThe results illustrated that tumor weights and volumes of the mice in model group increased rapidly during the10days. In contrast, treatment of calculus bovis and CTXeffectively suppression the tumor growth（P<0.001）and CBCG displayed a dose-dependentmanner. Histopathological analysis of tumor tissues showed that tumor cells arrangedclosely with irregular shape and disequilibrium nuclear-cytoplasmic ratio in model group,as well as chromosomes abnormity. However, in treated groups, tumor cells appeared to beinduced to apoptosis and necrosis by calculus bovis and CTX. The nucleus was broken butencapsulated by intact membrane, containing membrane blebbing, formation of apoptoticbodies. For some necrosis places in CBCG high dose group, CTX group and combinationgroup, the tumor cells died following with the nucleus atrophy and the disruption or thedissolution. The apoptosis degree of NCB group was more than CBCG middle but lessthan high group.2. The regulation of immune system in S180-bearing miceAs shown in results, high-dose CBCG and NCB obviously improved the weight ofspleen and thymus, shown a dose-dependent manner in S180-bearing mice. CBCG middleand low group did not change obviously, compared with the model. A great weight loss ofthymus and spleen in CTX group could be observed, which accounted for theimmunosuppressive side effect by CTX during the therapy for mice. The thymus andspleen indices in the combination-treated mice were significantly higher than the CTXgroup (P<0.05). According to the results, calculus bovis treatment elevated level of IL-2and lower the level TNF-α in tumor-bearing mice in a dose-dependent manner. Level ofIL-2, were significant reduction in tumor-bearing mice in CTX group as compared withmodel mice in serum. The CTL activity between groups were not significant.3. The molecular mechanisms of anti-tumor effect of CBCG in S180-bearing miceThe expression of anti-apoptotic BCL-2, P-AKT(Thr308)、P-AKT(Ser473) expressionwere inhibited in a dose-dependent manner in treatment mice, whereas the levels of BAX,PTEN, P53were increased. Compared with the model group, the BCL-2/BAX ratio of thehigh-dose CBCG group, NCB group, CTX group and the combination group wereobviously lower (P<0.001). The BCL-2/BAX ratio of combination group was furtherdeclined than CTX, the difference was statistically significant (P<0.01). These resultssuggested that CBCG could induce apoptosis in mice by shifting the BAX/BCL-2ratio.4. The synergy and attenuation action of CBCG on CTXCompared to the CTX group, the counts of the haemoglobin and white blood cellswere elevated in high-dose CBCG, NCB and combination groups, the differences werestatistically significant. The results showed that CBCG could improve the immuno- suppression caused by CTX.ConclusionsThese results suggest that CBCG produced the anti-tumor effect and immunomo-dulatory in S180tumor-bearing mice, the possible molecular mechanism may be related tothe PI3K/AKT/apoptosis pathway.