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Objective To investigate the effects of lobaplatin on proliferation and invasion of cervical cancer CaSki cells.Methods Human cervical cancer CaSki cells were randomly divided into blank control group,2,6 and 12 μg/ml lobaplatin groups by random number table method.The proliferations of the cells were detected by methyl thiazolyl tetrazolium (MTT).The morphological changes of the cells were observed by inverted microscope.The invasive abilities of the cells were detected by Transwell invasion test.The protein expressions of extracellular signal-regulated kinase (ERK) and phospho-extracellular signal-regulated kinase (p-ERK) were detected by Western blotting.Results The absorbance (A) values of blank group,2,6,12 μg/ml lobaplatin groups cultured for 24 h were 0.513 ± 0.023,0.428 ± 0.014,0.380 ± 0.012 and 0.300 ± 0.013 respectively,those of the cells cultured for 48 h were 0.831 ± 0.024,0.558 ± 0.019,0.415 ± 0.015 and 0.088 ±0.009 respectively,and those of the cells cultured for 72 h were 1.153 ±0.022,0.572 ± 0.023,0.201 ± 0.017 and 0.052 ± 0.014 respectively.The differences were statistically significant (F =12.922,P ＜ 0.001;F =10.192,P ＜ 0.001;F =11.192,P ＜ 0.001),and the differences between each two groups were statistically significant (all P ＜ 0.05).Under inverted microscope,the cells of the platinum groups were shrunken and round,the volume and quantity were reduced,the morphology was irregular,the gap was increased,and the changes were more obvious with the increase of the concentration and the culture time.The numbers of penetrating cells of the blank group,2,6,12 μg/ml lobaplatin groups were 87.27 ±9.38,71.02 ± 8.92,53.20 ± 10.02 and 21.02 ± 7.37 respectively.The difference was statistically significant (F =87.291,P ＜ 0.001),and the differences between each two groups were statistically significant (all P ＜ 0.05).The A values of ERK protein in the blank group,2,6,12 μ~ml lobaplatin groups (0.955 ± 0.021、0.953 ± 0.023、0.950 ± 0.020、0.951 ±0.022)showed no significant difference (F =2.033,P =0.783),but the A values of p-ERK protein in the four groups were 0.941 ±0.015,0.831 ±0.020,0.620 ±0.019 and 0.493 ±0.017 respectively,which showed significant difference (F =11.921,P ＜0.001),and the differences between each two groups were statistically significant (all P ＜ 0.05).Conclusion Lobaplatin can inhibit the proliferation and invasion of cervical cancer CaSki cells,which may be related to the inhibition of the expression of p-ERK protein.
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Objective:To investigate effects of Lidamycin (LDM,C-1027) on the proliferation and immunogenic transform of human Caski cervical cancer cells and to provide the basic experiment data and theoretical supports for establishment of the new immu-notherapy method mediated by LDM. Methods:MTT was used for the analysis of cell proliferation;apoptosis rate was analyzed by flow cytometry;Western blot was used to analyze the effect of LDM on the expression of Bax and Bcl-2 in Caski cells;the Flow cytometry was used to detect the expression of Calreticulin ( CRT ) on the cell surface. Results: Lidamycin inhibited proliferation of Caski cells significantly in the time-and dose-dependent manners;The apoptotic cell ratio induced by 5 μg/L Lidamycin was 11. 5% ,Comparing with the control group, Lidamycin treatment increased Bax but decreased Bcl-2 contents significantly within Caski cells, it also significantly increased the expression of CRT on the cell surface of Caski cells from 2. 31% to 67. 2%. Conclusion: Lidamycin has pharmacological activity in inhibiting proliferation of the human cervical Caski cells and the underlying mechanism is related with inducing the intrinsic mitochondrial pathway of apoptosis. In the same time,Lidamycin can increase the expression of CRT on the cell surface,so it may have the ability to promote the immunogenic apoptosis of tumor cells.
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Objective To construct rCB1 gene eukaryotic expression vector, detect its expression in the cell, and explore its influence on apoptosis in human cervical cancer CaSki cells.Methods The total RNA was extracted from rat brains.The rCB1gene was amplified by RT-PCR.The pcDNA3.1(+)-rCB1 was constructed by enzyme digestion, purifi-cation, bind the PCR purification products and pcDNA3.1 (+) DNA.The pcDNA3.1 (+)-rCB1 plasmid was transfect-ed into HEK293 and CaSki cells by liposomes.The expression and localization of rCB1 were detected by Western blot and immunofluorescence combined with confocal laser scanning microscopy.The apoptosis rate of CaSki cells was detected by flow cytometry.The expression of rCB1, Bcl-2, Bax and Bad was detected by Western blot and real-time fluorescence quantitative RT-PCR (qRT-PCR).Results The 5300 bp pcDNA3.1(+) and 1500 bp rCB1 were obtained after diges-ting the pcDNA3.1 ( +)-rCB1.The result of sequencing was in agreement with the sequence of rCB1 gene ( NM_012784.4 ) .The rCB1 expressed in the membrane and cytoplasm when pcDNA3.1 (+)-rCB1 plasmid was transfected into HEK293 cells.The apoptosis rate of rCB1 group was increased compared with the blank group when pcDNA3.1 (+)-rCB1 plasmid was transfected into CaSki cells (P<0.05).Compared with the blank group, rCB1 gene upregulated the expres-sion of Bax and Bad, and suppressed the expression of Bcl-2.The statistical difference was significant ( P <0.05). Conclusions The pCDNA3.1(+)-rCB1 eukaryotic expression vector is constructed successfully.It is found that rCB1 is expressed in membrane and cytoplasm of HEK293 cells.rCB1 can significantly promote the apoptosis in cervical cancer CaSki cells by up-regulating the expression of Bax and Bad, and down-regulating the expression of Bcl-2 as well.
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Objective: To observe the inductive effect of cytotoxic T lymphocytes (CTLs) against human cervical cancer cell line CaSki using therapeutic dendritic cells (DCs) vaccine in vitro . Methods:. Immature mouse DCs were isolated and cultured. The expressions of cell-surface CD40, CD86, major histocompatibility complex (MHC)-. and CD11c in immature DCs were detected by flow cytometry (FCM). Then the immature mouse DCs were infected with recombinant adenoviral vector carrying human papillomavirus (HPV )16 E 6/E 7 (pAd-E6/E7), and the CaSki cell lysate-loaded autologous DCs vaccine was prepared. The expression of green fluorescent protein in pAd-E6/E7-infected immature mouse DCs was observed under a laser scanning confocal microscope, and the expression of E6 protein was detected by Western blotting. DCs vaccine was used to induce specific CTLs, were subsequently co-cultured with CaSki cells. The killing effect of CTLs against CaSki cells was determined using cell counting kit8(CCK8) assay. Results: HPV16 E6/E7-specific DCs vaccine was successfully prepared. CTLs which induced by DCs vaccine exerted a killing effect on CaSki cells. This killing effect was higher in pAd-E6/E7-infected group than those in CaSki cell lysate-loaded group and the untreated control group (P<0.05). Conclusion: Genetically modified DC vaccine can successfully be prepared by infection with pAd-E6/E7, and it has a significant effect on triggering of specific CTLs against CaSki cells.
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Objective To investigate the effect of S100A8/A9 protein complex on the surface morphology and the F-actin network in human cervical carcinoma cell line,CasKi cells.Methods After being cultured with 20 ?g/ml S100A8/A9 protein complex,the cell skeleton of the CasKi cells were observed under a confocal scanning fluorescence microscope by staining the F-actin network.Atomic force microscopy(AFM) was employed to reveal the change of ultrastructure of the cell surface in vivo.ResultsAfter being cultured with the S100A8/A9 protein complex for 24 hours,the F-actin network disorder was revealed.Most of the F-actins distributed peripherally.The OD value of the F-actin decreased significantly from 92.42?5.16 to 57.67?3.70 after been treated with the S100A8/A9(t=5.268,P=0.000).The AFM showed a withdrawing morphology with reduced pseudopodia and destruction of stress fibers. Conclusion S100A8/A9 protein complex can change the ultrastructure of the surface of CasKi cells and its stress fibers by re-distributing of the F-actin in the cells.
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Objective:To prepare the vaccine of DC derived from human peripheral blood and transfected with HPV16E6 antigen gene, and to detect its morphological character,surface marker and immunological effect.Methods:DC-enriched populations were prepared from human peripheral blood mononuclear cell(PBMC) with the combination of rhGM-CSF,rhIL-4 and rhTNF-?. The plasmid containing HPV16E6 gene was transfected into DC with lipofectamine. The morphology of DC was observed dynamically, and the expression of surface markers of DC vaccine could be detected using immuno-cytochemical staining and flow cytometry. MTT assay was applied to detect the activity of CTL in vitro.Results:The transfected DC had typical morphologic and phenotypic characteristics, and expressed E6 protein 47.3%, CD80 82.5%, CD86 79.8% and CD83 85.7%. The killing activities of CTL to Caski cells induced by transfected DC were higher evidently than that of control groups(P