Ji-Hua Dong

Downregulation of XIAP expression induces apoptosis and enhances chemotherapeutic sensitivity in human gastric cancer cells.

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the inhibitor of apoptosis protein (IAP) gene family in terms of its ability to inhibit caspases and suppress apoptosis. Recent evidence has suggested that XIAP is a key determinant in chemoresistance of cancer cells. To explore a novel approach for ameliorating chemotherapy of gastric cancer, the antisense expression vector for the XIAP gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). This transfer resulted in significant downregulation of XIAP expression, decreased in vitro cell viabilities, and induced apoptosis. In transferred cells, inactive caspase-3 precursors were cleaved into the active subunits (p20 and p17) during apoptosis induced by downregulation of XIAP. The inhibitory effects of cisplatin and mitomycin C on the growth of XIAP downregulated cancer cells were significantly enhanced. In addition, this process occurred only in wild-type p53 (MKN-45), but not in mutant-type p53 (MKN-28) gastric cancer cells. The data presented suggest that downregulation of XIAP via antisense RNA can lead to apoptosis of gastric cancer cells in vitro, correlating with cellular p53 status and activation of caspase-3. This finding could lead to a potential strategy for improving the efficiency of therapies for gastric cancer.

Small RNA interference-mediated gene silencing of heparanase abolishes the invasion, metastasis and angiogenesis of gastric cancer cells.

BackgroundHeparanase facilitates the invasion and metastasis of cancer cells, and is over-expressed in many kinds of malignancies. Our studies indicated that heparanase was frequently expressed in advanced gastric cancers. The aim of this study is to determine whether silencing of heparanase expression can abolish the malignant characteristics of gastric cancer cells.MethodsThree heparanase-specific small interfering RNA (siRNAs) were designed, synthesized, and transfected into cultured gastric cancer cell line SGC-7901. Heparanase expression was measured by RT-PCR, real-time quantitative PCR and Western blot. Cell proliferation was detected by MTT colorimetry and colony formation assay. The in vitro invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells.ResultsTransfection of siRNA against 1496-1514 bp of encoding regions resulted in reduced expression of heparanase, which started at 24 hrs and lasted for 120 hrs post-transfection. The siRNA-mediated silencing of heparanase suppressed the cellular proliferation of SGC-7901 cells. In addition, the in vitro invasion and metastasis of cancer cells were attenuated after knock-down of heparanase. Moreover, transfection of heparanase-specific siRNA attenuated the in vitro angiogenesis of cancer cells in a dose-dependent manner.ConclusionsThese results demonstrated that gene silencing of heparanase can efficiently abolish the proliferation, invasion, metastasis and angiogenesis of human gastric cancer cells in vitro, suggesting that heparanase-specific siRNA is of potential values as a novel therapeutic agent for human gastric cancer.

Apoptosis-inducing effects of curcumin derivatives in human bladder cancer cells

Our aim was to prepare curcumin derivatives and study their apoptosis-inducing effects on bladder cancer cells in order to establish a basis for targeted chemotherapy of cancer. n-Maleoyl-L-valine-curcumin (NVC) and n-maleoyl-glycine-curcumin (NGC) were chemically synthesized. Intracellular esterase activity of the human bladder cancer EJ cell line and renal tubular epithelial (HKC) cells was examined by 6-carboxyfluorescein diacetate fluorometry. After incubation with NVC or NGC for 6–24 h, cell viability was detected by MTT colorimetry. Cell apoptosis and apoptotic rates were measured by acridine orange/ethidium bromide staining, TUNEL labeling and flow cytometry. Intracellular caspase-3 activities were determined by spectrophotometry. The esterase activity within EJ cells was 10.2-fold higher than that of HKC cells, which was abolished by bis-p-nitrophenylphosphate, an esterase inhibitor, resulting in decreases in NVC- and NGC-mediated cell viability arrest. For EJ cells, the IC50 values of NVC (20.1 μmol/l) and NGC (18.7 μmol/l) were close to curcumin (16.5 μmol/l). Meanwhile, their IC50 values on HKC cells were, respectively, 4.06- and 3.23-fold higher than curcumin. Moreover, NVC and NGC induced apoptosis of EJ cells by 10.13–23.36 and 12.42–28.56%, respectively. Administration of these two derivatives resulted in decreased apoptosis of HKC cells compared with curcumin. The caspase-3 activities of EJ cells, but not of HKC cells, were 5.21- and 5.63-fold enhanced by NVC and NGC, respectively. Thus, novel esterase-sensitive curcumin derivatives were synthesized, which induced extensive apoptosis of bladder cancer EJ cells, but not normal cells.

Methyl jasmonate downregulates expression of proliferating cell nuclear antigen and induces apoptosis in human neuroblastoma cell lines.

Recent evidence indicates that methyl jasmonate, a plant stress hormone, exhibits anticancer activity on human cancer cells. Whether methyl jasmonate could inhibit the growth of human neuroblastoma cells still, however, remains largely unknown. In this study, administration of methyl jasmonate to cultured neuroblastoma cell lines, SK-N-SH and BE(2)-C, resulted in a decrease of cell viability in a dose-dependent and time-dependent manner as demonstrated by MTT colorimetry and colony formation assay. The results from RT-PCR indicated that the expression of proliferating cell nuclear antigen, but not of cyclin D1, was downregulated by methyl jasmonate. Accordingly, the cell cycle of methyl jasmonate-treated neuroblastoma cells was arrested at the G0/G1 phase. Moreover, incubation of SK-N-SH and BE(2)-C cells with methyl jasmonate resulted in characteristic changes of apoptosis, as demonstrated by acridine orange–ethidium bromide (AO/EB) staining, Hoechst 33258 staining and flow cytometry. Moreover, methyl jasmonate decreased the expression of the X-linked inhibitor of apoptosis protein and survivin, critical members of the inhibitors of apoptosis protein family, in neuroblastoma cells. These findings indicate that methyl jasmonate suppresses the growth of cultured human neuroblastoma cells associated with downregulation of proliferating cell nuclear antigen, and induces apoptosis accompanied by downregulation of the X-linked inhibitor of apoptosis protein and survivin, which lays the groundwork for further investigation into the mechanisms of methyl jasmonate-mediated anticancer activities.

Natural jasmonates of different structures suppress the growth of human neuroblastoma cell line SH-SY5Y and its mechanisms

AbstractAim:Recent evidence has indicated that members of natural jasmonates, a family of plant stress hormones, exhibit anticancer activity. The current study was undertaken to investigate the effects of jasmonates on the in vitro growth of human neuroblastomas, one of the most common solid tumors in children.Methods:Cellular proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetry and colony formation assay. Apoptosis was detected by Hoechst 33258 staining and flow cytometry. Western blotting was applied to assay gene expression.Results:The administration of natural jasmonates, methyl jasmonate, cis-jasmone, and jasmonic acid to cultured neuro-blastoma cell line SH-SY5Y, resulted in a decrease of cell proliferation in a dose-and time-dependent manner. However, the in vitro growth of cultured human embryonic kidney (HEK) cell line HEK 293 was not affected by jasmonates. The cell cycles of jasmonate-treated SH-SY5Y cells were arrested at the G2/M phase. The incubation of SH-SY5Y cells with jasmonates resulted in characteristic changes of apoptosis. The anticancer activities of natural jasmonates on SH-SY5Y cells are as follows: methyl jasmonate>cis-jasmone>jasmonic acid. In addition, the expressions of proliferating cell nuclear antigen and N-myc were downregulated by methyl jasmonate. Moreover, methyl jasmonate decreased the expression of the X-linked inhibitor of apoptosis protein and survivin, critical members of inhibitors of the apoptosis protein family, in SH-SY5Y cells.Conclusion:Jasmonates suppress the growth of human neuroblastoma cell line SH-SY5Y via inhibiting cell proliferation and inducing apoptosis, which lays the groundwork for further investigation into the anticancer activities and its mechanisms of natural jasmonates on human neuroblastomas.

Nitrofen suppresses cell proliferation and promotes mitochondria-mediated apoptosis in type II pneumocytes

AbstractAim:To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia.Methods:After administration of nitrofen to cultured type IIA549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide colorimetry, colony formation assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot.Results:Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompanied by downregulation of PCNA. As a result, the DNA synthesis of nitrofentreated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover, nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Ala-Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-xL, but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 μmol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells.Conclusion:Nitrofen suppresses the proliferation of cultured type II pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involving the activation of p38-MAPK.

Influence of silencing TRAF6 with shRNA on LPS/TLR4 signaling in vitro.

SummaryThis study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-α, IL-1β and TGF-β1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibit NF-κB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-β1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-α, IL-1β and TGF-β1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibit NF-κB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-β1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

Selection of optimal antisense accessible sites of uroplakin II mRNA for bladder urothelium

SummaryThe optimal antisense accessible sites (AAS) of uroplakin II (UP II) mRNA, a specific gene expressed in bladder urothelium, were selected in order to provide a novel method for targeted therapy of transitional cell carcinoma (TCC) of bladder. The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcripted total UP II cRNA, then digested by RNase H. After primer extension and autoradiography, the AAS of UP II were selected. The RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODN) were synthesized. With the AS-ODN0 designed only by RNADraw as a control, the AS-ODNs were transferred into UP II highly-expressing cell line RT4. The cellular expression of UP II mRNA was detected by RT-PCR and Western blot. Twelve AAS of UP II mRNA were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 558–577, 552–571, 217–236 and 97–116 bp of UP II mRNA respectively. After transfection with the corresponding AS-ODN (AS-ODN1, AS-ODN2, AS-ODN3 and AS-ODN4) for 18 h, the UP II mRNA levels in RT4 cells were reduced by 29.3%, 82.7%, 71.3% and 70.9%, while UP II protein levels were decreased by 20.2%, 78.5%, 65.2% and 64.4% respectively, which were significantly higher than those of AS-ODN0 (14.3%, 12.1% respectively) (P<0.01). The AAS of UP II mRNA was effectively selected in vitro by random oligonucletide library/RNase H cleavage method in combination with computer software analysis, which had important reference values for further studying biological functions of UP II gene and targeted therapeutic strategy for TCC of bladder.The optimal antisense accessible sites (AAS) of uroplakin II (UP II) mRNA, a specific gene expressed in bladder urothelium, were selected in order to provide a novel method for targeted therapy of transitional cell carcinoma (TCC) of bladder. The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcripted total UP II cRNA, then digested by RNase H. After primer extension and autoradiography, the AAS of UP II were selected. The RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODN) were synthesized. With the AS-ODN0 designed only by RNADraw as a control, the AS-ODNs were transferred into UP II highly-expressing cell line RT4. The cellular expression of UP II mRNA was detected by RT-PCR and Western blot. Twelve AAS of UP II mRNA were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 558–577, 552–571, 217–236 and 97–116 bp of UP II mRNA respectively. After transfection with the corresponding AS-ODN (AS-ODN1, AS-ODN2, AS-ODN3 and AS-ODN4) for 18 h, the UP II mRNA levels in RT4 cells were reduced by 29.3%, 82.7%, 71.3% and 70.9%, while UP II protein levels were decreased by 20.2%, 78.5%, 65.2% and 64.4% respectively, which were significantly higher than those of AS-ODN0 (14.3%, 12.1% respectively) (P<0.01). The AAS of UP II mRNA was effectively selected in vitro by random oligonucletide library/RNase H cleavage method in combination with computer software analysis, which had important reference values for further studying biological functions of UP II gene and targeted therapeutic strategy for TCC of bladder.