ABSTRACT
Objective:To evaluate the relationship between spinal cord cytoplasmic activation/proliferation-associated protein-1 (Caprin-1)-mediated expression of calmodulin-dependent protein kinase Ⅱ alpha(CaMKⅡα) and pain perception in mice.Methods:Nestin CreERT2 mice and Caprin-1 flox/flox mice were hybridized to obtain Nestin CreERT2; Caprin-1 flox/flox homozygous mice. The mice were divided into 2 groups ( n=5 each) using a random number table method: Nestin CreERT2; Caprin-1 flox/floxsolventcontrol group (SC group) and Caprin-1 gene knockout group (KO group). Tamoxifen 100 mg/kg was intraperitoneally injected for 5 consecutive days to generate inducible knockout of the Caprin-1 gene in KO group. The mechanical paw withdrawal threshold was measured at day 1 before and day 5 after the end of administration. Then the mice were sacrificed, and L 4-6 segments of the spinal cord were removed for determination of the expression of Caprin-1 and CaMKⅡα protein and mRNA (by Western blot or real-time quantitative polymerase chain reaction) and co-expression of Caprin-1 with CaMKⅡα (by immunofluorescent double staining). Results:Compared with SC group, the mechanical paw withdrawal threshold was significantly increased at 5 days after the end of tamoxifen administration, the expression of Caprin-1 and CaMKⅡα protein and mRNA was down-regulated ( P<0.05), and the co-localization expression of Caprin-1 and CaMKⅡα in the spinal dorsal horn was down-regulated in KO group. Conclusions:Caprin-1 is involved in the development of pain perception by promoting CaMKⅡα expression.
ABSTRACT
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.
ABSTRACT
Objective To investigate the role of spinal cord chemokine CXC ligand13(CXCL13) in the formation of rat bone cancer pain(BCP).Methods Twenty healthy female SD rats weighing 160-200 g were divided into four groups(n=5):sham operation group(S),BCP group(BP),small interference RNA(siRNA) negative control(NC-siRNA) group (NC) and CXCL13-siRNA group(CS).Normal saline was given by tibial medullary cavity injection in the S group.The tibial BCP model was established by tibial medullary cavity injection of equivalent Walker-256 breast cancer cells in the group BP,NC and CS.NC-siRNA lentivirus and CXCL13-siRNA lentivirus were injected intrathecally in the group NC and CS respectively.The mechanical pain threshold was measured on 1 d before model construction and on postoperative 7,9,14,21 d.The rats were killed after pain threshold measurement.The spinal cord and tibial tissue were taken.The co-expression of spinal CXCL13,microglia specific marker Iba-1 and neuron specific neucleoprotein NeuN was determined by using the immunofluorescence double standard staining,and expressions of CXCL13 and ionized calcium binding adaptor molecule-1 (Iba-1) protein and mRNA in spinal cord were detected by Western blot and RT-PCR;the HE staining microscopy was adopted to observe the tibial bone structure destroy situation.Results Compared with group S,the mechanical pain threshold in theBP group and NC group was decreased on 7-21 d after inoculation,CXCL13 expression in neuron was significantly increased and microglia was obviously activated,the expression of CXCL13 and Iba-1 protein and mRNA was significantly elevated (P＜0.05);compared with the NC group,the mechanical pain threshold on 9-21 d after model construction in the CS group was significantly increased,CXCL13 expression in neurons was significantly decreased,microglia activation was decreased and expression of CXCL13 and Iba-1 protein and mRNA was significantly decreased(P＜0.05);HE staining showed that the model groups appeared the tumor growth in bone marrow cavity,moreover which was eroded outwards and destroyed bone cortex,but no abnormality was found in the S group.Conclusion Spinal cord CXCL13 is involved in the BCP formation in rats by activating microglia.