Anthraquinone modifier KA-4c triggers endoplasmic reticulum stress and targets ATF6 against breast cancer cells / 中国药理学通报

Aim To explore the mechanism of the effect of anthraquinone modifier KA-4c on breast cancer cells, and determine its action target by drug affinity reaction target stability technique (DARTS). Methods The cell viability was detected by MTT method. The effect of KA-4c on the morphology of breast cancer cells was studied by HE staining, ER-Tracker Red and electron microscope. The apoptosis rate of breast cancer cells induced by KA-4c was detected by flow cytometry. The expression of apoptotic protein was detected by Western blotting. DARTS and CETSA were used to determine the target of KA-4c. Results KA-4c had the most significant inhibitory effect on the proliferation of triple negative breast cancer MDA-MB231 cells, and could cause endoplasmic reticulum and mitochondrial vacuolation to damage the cells. The apoptosis rate and the expression of apoptosis-related proteins CHOP and caspase-7 increased with the increase of KA-4c concentration. DARTS results showed that KA-4c could activate endoplasmic reticulum protein processing signaling pathway, in which KA-4c bound to ATF6 protein and was resistant to protease hydrolysis. The results of CETSA experiments showed that KA-4c could enhance the expression of ATF6 protein in a concentration-dependent manner. Conclusions KA-4 triggers endoplasmic reticulum stress to induce apoptosis in breast cancer cells. ATF6 may be one of the targets of KA-4c.

Isolation and identification of side population cells in human lung adenocarcinoma cell line A549 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To isolate and characterize the side population cells (SP cells) in the lung adenocarcinomas cell line A549.</p><p><b>METHODS</b>The protein expression of ABCG2 in human lung adenocarcinoma cell line A549 was detected by immunohistochemistry. SP and NSP cells in the cell line A549 were isolated by FACS, and their differentiation was analysed. ABCG2 expression in the two cell subsets was detected by RT-PCR. The cell growth curves, cell division indexes, cell cycles, plate clone formation tests, migration and invasion assays, chemotherapeutic susceptibility tests, tests of the intracellular drug levels, and the tumor cell implantation experiments on nude mice were applied to study the biological properties of the two cell subsets. The expression of ABCG2 in the transplanted tumor in nude mice was detected by immunohistochemistry and RT-PCR.</p><p><b>RESULTS</b>The positive rate of ABCG2 expression in the A549 cells by immunohistochemistry was 2.13%. SP and NSP cells were isolated by FACS. The SP cells could produce both SP and NSP cells, while NSP cells only produced NSP cells. SP cells expressed ABCG2, but NSP cells did not. The proliferation and migration abilities of the two cell subsets were similar, but the invasion and tumorigenic ability of SP cells was significantly higher than that of NSP cells. The susceptibilities to DDP and its intracellular levels of the two cell subsets were similar, but the susceptibilities to 5-FU, VP16, NVB and GEM and their intracellular levels of NSP cells were significantly higher than those of the SP cells.</p><p><b>CONCLUSIONS</b>SP cells in the human lung adenocarcinomas cell line A549 is enriched with tumor stem cells. An effective way to get lung adenocarcinomas stem cells is to isolate SP cells by FACS.</p>

Inhibitory effects of antisense MMP-9 oligodeoxynucleotides on invasiveness and adherence of ovarian cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the inhibitory effects of antisense MMP-9 oligodeoxynucleotides on invasiveness and adhesion ability in vitro of ovarian cancer cells, and to investigate the mechanisms of action.</p><p><b>METHODS</b>MMP-9 antisense oligonucleotides were transfected by lipofectinmin into ovarian cancer cell line HO-8910PM cells expressing MMP-9 induced with fibronectin. RT-PCR, Western blot and gelatin zymography were used to detected MMP-9 expression of mRNA and protein and enzymatic activity. The ability of invasion and migration of ovarian cancer cells was assayed in Transwell cell culture chamber. Cell adhersion assay was carried out in a microculture well pre-coated with fibronectin.</p><p><b>RESULTS</b>MMP-9 expressions of mRNA and protein were significantly decreased in the antisense-transfected cells. Comparing with the control group, the inhibition rate was 34. 8% and 42. 5% , respectively (P <0. 05). Its gelatin enzymatic activity was inhibited. Matrigel invasion assay and Transwell migration assay revealed markedly reduction in invasion and migration for the antisense group. The inhibition rates were 22. 4% and 24. 8% , respectively. The adhesion ability was also reduced. The inhibition rates were 49. 8% and 38. 3% at 60 min and 90 min, respectively.</p><p><b>CONCLUSION</b>MMP-9 down-regulation can significantly inhibit the ability of invasion and attachment of ovarian cells in vitro. MMP-9 may play an important role in invasion and metastasis of ovarian cells and potentially be a molecular target of blocking invasion and metastasis of ovarian cancer.</p>

Establishment of human ovarian carcinoma cell lines with directional highly lymphatic metastasis and study of their biological characteristics / 中华妇产科杂志

Objective To establish a human ovarian carcinoma cell line with directional highly lymphatic metastasis and to study their biological characteristics.Methods The clone cells of ovarian carcinoma,SKOV3,were inoculated into the hind foot pad of nude mice.The cancer cells of lymph node metastatic foci were transplanted into nude mice again when the metastatic nude of mice were observed.After repetition of this procedure for 3 cycles,the metastatic rate and the metastatic paths were observed in nude mice of every passage.We used limited dilution method to separate and select colonial cells with directional highly lymphatic metastatic potentials from the lymphatic metastasis of human ovarian carcinoma cell line SKOV3.The ceils with biological characteristics were assayed by growth curve,HE staining,karyotype analysis,nude mice transplantation and immunohistochemistry,respectively.Results We established a series of cell lines from lymph node metastasis and designated them as SKOV3-PM1,SKOV3-PM2 and SKOV3-PM3 cell strain.When the cells of SKOV3-PM3 were injected into the hind foot pad of nude mice, they produced 100%(10/10)spontaneous lymphatic metastasis.The lymphatic metastatic rates(26/10) were stable and higher than the mother cell line(1/10,P

Inhibitory effects of RNA interference on expression of matrix metalloproteinase-9 gene and invasiveness and adhesion in ovarian cancer cells / 中华妇产科杂志

Objective To investigate the inhibitory effects of RNA interference(RNAi)on the expression of matrix metalloproteinase-9(MMP-9)gene and invasiveness and adhesion of ovarian cancer cells.Methods Four groups of different specific target sequence in coding region of MMP-9 and one non- specific sequence were chosen,which were Sitel,Site2,Site3,Site4 and Site5.Small interference RNA (siRNA)expression cassettes(SEC)were constructed by PCR and transfected into ovarian cancer HO- 8910PM cells.RT-PCR and western blot were used to detect mRNA and protein expression of MMP-9 gene; the abilities of invasion and adhesion were detected by Matrigel invasion assay and cell adhesion assay. Results The expression of MMP-9 was inhibited and the inhibitory effects of different sequence were varied.The mRNA expression was 0.64?0.06,0.47?0.07,0.55?0.10 in Sitel,Site2,Site3 group, and protein expression was 0.30?0.09,0.27?0.08,0.37?0.12,respectively.Site2 group had the most efficient inhibitory effect,followed by Sitel and Site3 groups.Cell growth curve revealed that cell growth was significantly inhibited in Site2 group.Invasiveness and adhesion were significantly reduced,the inhibitory rate on invasion in Site1,Site2,Site3 groups were 50.0%,50.0% and 37.5%,respectively;the inhibitory rate on adhesion in Site1,Site2,Site3,Site4 groups were 43.8%,48.8%,33.9%,24.2% at 60 min and 41.6%,40.2%,35.1%,16.0% at 90 min,respectively.Conclusions RNAi exists in ovarian HO-8910PM cells.MMP-9 siRNA can specifically down-regulate MMP-9 expression and lead to the inhibition of invasiveness and adhesion in ovarian cancer cells.