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Objective:To investigate the synergistic antitumor effect of pyrotinib in combination with 5-fluorouracil(5-Fu)on human epidermal growth factor receptor 2(HER2)positive breast cancer cells and its underlying molecular mechanism. Methods:HER2 positive breast cancer cells were screened by Western blotting.HER2 positive SKBR-3 and BT474 cells were treated with pyrotinib and 5-Fu individually or in combination for the following experiments.MTT assay was used to assess the effect of different drugs on the proliferation of the treated cells,and the combination index(CI)values were calculated using Combidrug software.Colony formation assay was used to evaluate the effect of different drugs on the colony-forming ability of the treated cells.FCM assay was used to analyze the effect of different drugs on the apoptosis rate and cell cycle of the treated cells.Western blotting was used to examine the effect of different drugs on the expression levels of proteins in the proliferation-and apoptosis-related signaling pathways.SKBR-3-cell-based tumor xenograft model was established using BALB/c nude mice.After treatment with pyrotinib and 5-Fu individually or in combination,the growth profiles of the xenograft tumors were recorded and the expression levels of proteins in the proliferation-and apoptosis-related signaling pathways were examined in the tumor tissues. Results:HER2 positive breast cancer cell lines SKBR-3 and BT474 were selected for further experiments after screening.The proliferation SKBR-3 and BT474 cells could be inhibited after treatment with pyrotinib and 5-Fu individually or in combination(all P＜0.05).Compared with pyrotinib or 5-Fu single drug treatment,pyrotinib in combination with 5-Fu had higher inhibition rate on the proliferation of SKBR-3 and BT474 cells with a Cl value of＜1,indicating the synergistic effect of pyrotinib and 5-Fu.In addition,in contrast to pyrotinib or 5-Fu single drug treatment,there was a further decrease in the number of colonies formed,increase in apoptosis rate,and increase in the percentage of G0/G,cells in SKBR-3 and BT474 cells after treatment with pyrotinib in combination with 5-Fu(all P＜0.01).Animal experiment results showed that the growth rate of xenograft tumors in mice treated with pyrotinib in combination with 5-Fu was significantly slower than that of the single-drug treated mice(P＜0.05).Western blotting analysis showed that the expression levels of HER2,HER4,AKT and phosphorylated ERK were significantly decreased after treatment with pyrotinib in combination with 5-Fu both in vitro and in vivo(all P＜0.01),indicative of the blockage of proliferation-related signaling pathways.Meanwhile,analysis of the apoptosis-related proteins revealed a decrease in the expression levels of caspase 3,poly ADP-ribose polymerase(PARP),and Bcl-2(all P＜0.01),while an increase in the expression levels of cleaved-caspase 3,cleaved-PARP,and p21(all P＜0.01). Conclusion:Pyrotinib and 5-Fu had synergistical antitumor effect on HER2 positive breast cancer cells,and the underlying mechanism may be related to the blockage of proliferation-associated signaling pathways and the induction of apoptosis and cell cycle arrest.
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Objective To investigate the expression and clinical significance of CXCR3/CXCL10in human cancer.Methods CXCR3 and CXCL10 were detected in 60 paraffinic tissues of patients with primary breast cancer,20 of mammary fibroma and 20 of mastopathy by immunohistochemistry S-P method and two stage method.Results The expression of CXCR3 (40/60,66.7％ ) and CXCL10 (45/60,75％)in breast cancer was higher than that in mastopathy [CXCR3(8/20,40％ )x2 =4.44,P =0.035 ;CXCL10( 10/20,50％ )x2 =4.36,P =0.037)].The expression of CXCR3 was related to status of axillary lymph node metastasis,clinical stage and the expression of HER-2 (x2 =4.15,P =0.042; x2 =7.74,P =0.021 ;x2 =4.27,P =0.039).The expression of CXCR3 had positive relationship to the number axillary lymph node metastasis( rs =0.375,P =0.003 ),clinical stage ( rs =0.451,P =0.000).Conclusions CXCR3 may be related to the progression and metastasis of breast cancer,and it may be used as a marker of breast cancer prognosis.
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Objective To asses the value of detecting bacterial DNAs in rat's blood with PCR for early diagnosis of colonic anastemotic leakage.Methods 48 healthy female Wistar rats were random divided into three groups: Group A(n=8,sham operation group),Group B(n=20,colonic anastomosis group),Group C(n=20,colonic anastomotic leakage group).Group B and C rats underwent standardized colon resection 3cm away from the ileocecal junction 10cm,Group B rats were done with a complete anastomosis(end-to-end single layer anastomosis with 0# silk sutures) while Group C rats were done with an anastomosis leaving a 5mm opening in colonic anterior wall.lml and 3ml venous blood samples were collected from Group A,B and C.DNAs were extracted from these blood samples and PCR techniques were used to amplify lacZ genes from Escherichia coli and 16S ribosomal RNA genes(16SrRNA genes) 3 days after operation.The data were analyzed by chi square test.Specimens of the experimental intestine were HE stained for pathological studies.Results The positive ratios of expressing lacZ genes in peripheral blood(PB) with PCR in Group C were significantly higher than that in Group B(P<0.05),hut there were no differences between the two groups in expressing 16SrRNA genes(P>0.05).Conclusions It could be a useful way to detect lacZ genes of Escherichia coli from PB by PCR but not 16SrRNA genes for diagnosis of colonic anastomotic leakages.
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0.05),but the positive ratio of 16SrRNA genes expression in PB in Group C and Group E was significantly higher than that in group B and Group D respectively(P0.05).Conclusions(1)Detecting 16SrRNA genes from PB with PCR has certain significance for early diagnosis of jejunal anastomotic leakage and ileal anastomotic leakage,(2)PCR might be a useful tool for early diagnosis of jejunal anastomotic leakages and ileal anastomotic leakages by detecting lacZ genes or 16SrRNA genes from ascites.