Cells In G1 Stage Research Articles

Effect of Denticleless E3 Ubiquitin Protein Ligase on Proliferation and Colone Formation of Multiple Myeloma Cells

Objective To determine the effect of denticleless E3 ubiquitin protein ligase(DTL)on the proliferation and clone formation of multiple myeloma(MM)cells and investigate the related mechanism. Methods Mononuclear cells were extracted from 34 MM patients.Mononuclear cells harvested from 14 healthy volunteers were used as controls.Quantitative polymerase chain reaction was used to detect the change of DTL at mRNA level.Furthermore,12 MM patients and 2 controls were selected,in whom the change of DTL at protein level was detected by Western blot.Human MM cell line RPMI8226 was divided into control(CON)group and DTL-short hairpin RNA(DTL-shRNA)group,which was infected with the CON and DTL-shRNA virus,respectively,for 48 hours.The infection efficiency was detected by using flow cytometry,the knock-down efficiencies at mRNA and protein levels were detected by quantitative polymerase chain reaction and Western blot,the change of cell counts in the next 0,24,48,72,96 hours were measured with CCK8 assay.The CON and DTL-shRNA cells were cultured in semisolid medium.Ten days later,inverted phase microscopy was used to measure the number of colones that contain more than 50 cells,annexin V/propidium iodide double staining to detect apoptosis,and propidium iodide staning to detect cell cycle.Finally,Western blot was empoyed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)in nuclear factor-κB(NF-κB)pathway and electrophoretic mobility shift assay(EMSA)to detect the NF-κB transcriptional ability. Results The DTL expression was(1.00±0.12)and(9.36±3.71),respectively in the bone marrow mononuclear cells of healthy volunteers and in the CD138+cells of MM patients(t=3.65,P=0.0024).DTL was also highly expressed in MM CD138+positive cells at protein level.After RPMI8226 was infected by CON and DTL-shRNA virus for 48 hours,green fluorescent protein-positive cells accounted for more than 90%.The relative expression of DTL was(1.00±0.01)and(0.21±0.04)(t=33.19,P<0.0001)at mRNA level and(0.52±0.13)and(0.11±0.02)at protein level(t=5.399,P=0.0057).CCK8 revealed that CON and DTL-shRNA cells proliferated by(1.00±0.03)vs.(1.00±0.02),(2.19±0.28)vs.(1.47±0.13),(3.50±0.14)vs.(2.24±0.19),(5.43±0.41)vs.(3.08±0.14),(7.42±0.17)vs.(4.29±013)after 0,24,48,72,and 96 hours(F=24.58,P=0.001).The number of colone containing more than 50 cells was in 76±4 in CON group and 0 in DTL-shRNA group(P<0.01).The proportion of G1 stage cells was(28.61±8.64)% in CON group and(57.25±10.37)% in DTL-shRNA group(t=3.675,P=0.0213).The proportion of annexin V+in CON and DTL-shRNA groups was(3.21±0.89)% vs.(34.71±18.68)%(t=2.895,P=0.0443).After RPMI8226 was infected with CON or DTL-shRNA virus for 48 hours,the relative expression of phosphorylation P65 was(1.52±0.14)vs.(0.82±0.11)(t=6.81,P=0.0024),the P65 relative expression was(0.25±0.04)vs.(0.24±0.08)(t=0.19,P=0.85),the CON and DTL-shRNA phosphorylation-IκBα relative expression was(0.19±0.03)vs.(0.13±0.02)(t=2.882,P=0.0449),and the IκBα was(0.22±0.05)vs.(1.01±0.06)(t=17.52,P<0.0001).Detection of the transcriptional ability of DTL-shRNA NF-κB by EMSA further confirmed the down-regulation of DTL suppressed the NF-κB transcriptional ability. Conclusions DTL is highly expressed in MM cells,and down-regulation of DTL suppresses the cell proliferation,inhibit the colony formation,and induce cell apoptosis and cell cycle arrest.The effect of DTL on the biological functions of MM cells is related to the change of NF-κB pathway.

MiR-122-5p inhibits the proliferation of melanoma cells by targeting NOP14

To investigate the expression profile of miR-122-5p in melanoma tissues and the effect of miR-122-5p on the proliferation, cell cycle and apoptosis of human melanoma cell lines SK-MEL-110 and A375. The expression profiles of miR-122-5p in melanoma and pigmented nevus tissues were detected using real-time fluorescence quantitative PCR (qRT-PCR). SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor or negative control inhibitor (NC) I were examined for miR-122- 5p expression using qRT-PCR and changes in cell proliferation, cell cycle and apoptosis using MTT assay or flow cytometry. NOP14 mRNA and protein expressions in the cells were detected using qRT- PCR and Western blotting, respectively. Luciferase reporter assay was used to confirm the identity of NOP14 as the direct target of miR-122-5p. The relative expression of miR-122-5p in human pigmented nevus tissues and melanoma tissues was 1.23±0.270 and 7.65 ± 1.37, respectively. The relative expression of miR-122-5p in SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor was 0.21 ± 0.08 and 0.17 ± 0.05, respectively. miR-122-5p inhibitor obviously inhibited the cell proliferation and increased the percentage of cells in G1 stage in both SK-MEL-110 and A-375 cells, but did not cause obvious changes in the apoptosis of the two cells. miR-122-5p inhibitor did not significantly affect the expression level of NOP14 mRNA, but obviously increased the expression level of NOP14 protein. Luciferase reporter assay revealed a significantly lower luciferase activity in cells co-transfected with miR-122-5p mimics and wild-type psi-CHECK2-3'UTR plasmid than in the cells cotransfected with NC and wild-type psi-CHECK2-3'UTR plasmid (0.21 ± 0.14 vs 0.56 ± 0.1, P &lt; 0.01). miR-122-5p expression is upregulated in melanoma tissues, indicating its involvement in the development of melanoma. miR-122-5p inhibits the proliferation of SK-MEL-110 and A-375 cells possibly by affecting the cycle through NOP14.

Neuroprotective Effects of Cerebral Ischemic Preconditioning in a Rat Middle Cerebral Artery Occlusion Model: The Role of the Notch Signaling Pathway.

Cerebral ischemia-reperfusion (I/R) injury is a major problem worldwide. The Notch signaling pathway plays an important role in neural progenitor cell differentiation and in the inflammatory response after central nervous system injury. This study evaluated whether the neuroprotective effect of cerebral ischemic preconditioning (cIPC) is mediated by the preactivation of the Notch signaling pathway. A rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and glucose deprivation/reoxygenation (OGD/R) cell model were constructed to detect the neuroprotective effects of cIPC. In in vivo experiments, cIPC reduces the neurological functional deficit, cerebral infarction, and cellular apoptosis in the hippocampus induced by middle cerebral artery occlusion/reperfusion (MCAO/R), thus indicating that cIPC can improve neurologic function. Moreover, cIPC can reveal the expression peak of Jagged1, Notch1, NICD, and Hes1 protein, thereby indicating that cIPC can preactivate Notch signaling. However, cIPC-induced improvements in neurologic function are compromised by the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-1-alanyl)-S-phenylglycine t-butyl ester (DAPT). In in vitro experiments, OGD preconditioning (OGDPC) can clearly upregulate Notch1 expression in the OGD/R-treated neuron and neural stem cell. Notch1 pre-overexpression can decrease neuron death and apoptosis under OGD/R treatment. Notch1 pre-overexpression can decrease the percentage of G1 stage cells and increase the percentage of S stage cells in OGD/R-treated neural stem cell. Furthermore, Notch1 pre-knockdown has the opposite effect on cell survival, apoptosis, and cycle in both OGD/R-treated neuron and neural stem cell. In conclusion, our results demonstrate that the neuroprotective effects of cIPC in a rat MCAO/R model are mediated by the preactivation of the Notch signaling pathway.

Vinegar processing attenuates toxicity on IEC-6 cells caused by chloroform extraction of Daphne genkwa

To screen the toxic polar fractions of Daphne genkwa, compare the toxicity of D. genkwa on crypts epithelial cells IEC-6 before and after vinegar processing, and preliminarily investigate the mechanism of D. genkwa vinegar processing on toxicity reducing. The proliferation of IEC-6 cells was observed by MTT. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), lactate dehydrogenase (LDH), as well as the enzyme activity of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase were determined in IEC-6 cells to evaluate the oxidative damages degree of IEC-6 cells. The apoptosis and cell cycle were analyzed by Flow Cytometry. The results showed that the dichloromethane extraction was the toxic polar fraction of D. genkwa, and after vinegar processing, the toxicity of dichloromethane fraction was significantly reduced (P<0.01). As compared with the blank control group, the dichloromethane fraction of D. genkwa can obviously decrease the levels of SOD, Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase (P<0.01) and content of GSH, but increase the level of LDH and MDA in cell supernatant (P<0.01). Besides, it obviously increased the early and late apoptotic rate of IEC-6 cells, obviously decreased the proportion of G₁stage cells, increased the ratio of S stage cells and M stage cells (P<0.01). After vinegar processing, as compared with D. genkwa groups of various doses, it can significantly increase the levels of SOD, Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase (P<0.01) and content of GSH, decrease the level of LDH, MDA(P<0.01), significantly decrease the early and late apoptosis rate of IEC-6 cells (P<0.01), increase the proportion of G₁stage cells, and decrease the ratio of S stage cells and M stage cells (P<0.01). Vinegar processing can reduce the toxicity of dichloromethane fraction of D. genkwa, and its mechanism may be associated with improving the activity of antioxidant enzymes and permeability in IEC-6 cells, and decreasing the oxidative damage.

RETRACTED: Inhibition of PI3K/AKt/mTOR signaling pathway protects against d-galactosamine/lipopolysaccharide-induced acute liver failure by chaperone-mediated autophagy in rats

This study aims to investigate the effects of PI3K/AKt/mTOR signaling pathway on the proliferation and apoptosis in acute liver failure (ALF) by chaperone mediated autophagy (CMA). The hepatocytes extracted from both normal rats and rats with ALF were assigned to control, acute injury, P13K agonist, and P13K inhibitor groups. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used as part of this investigation to detect the expression of PI3K/AKt/mTOR signaling pathway related-proteins (PI3K, AKt, mTOR), apoptosis related-proteins (Fas, Bax, Bcl-2), chaperone-mediated autophagy (CMA) marker proteins (LAMP-2A, HSC 70), p-PI3K, p-AKt, p-4E-BPI, and p-S6K. An MTT assay was used for analysis of cell proliferation after transfection. Flow cytometry is performed to detect the cell apoptosis. In comparison to the normal group, the model group showed enhanced positive rate of PI3K, AKt, mTOR, increased expression levels of PI3K, AKt, mTOR, Fas, Bax, p-PI3K, p-AKt, p-4E-BPI and p-S6K, reduced expression levels of Bcl-2, LAMP-2A and HSC 70. The results in vitro experiment: compared with the acute injury group, the PI3K agonist group showed elevated expression levels of PI3K, AKt, mTOR, Fas, Bax, p-PI3K, p-AKt, p-4E-BPI and p-S6K, decreased expression levels of Bcl-2, LAMP-2A and HSC 70, inhibited cell proliferation, more arrested cells in G1 stage, and promoted cell apoptosis. Opposing this, the P13K inhibitor group exhibited an opposite trend. In conclusion, inhibition of the PI3K/AKt/mTOR signaling pathway plays a protective role in ALF by promoting CMA expression, which could arrest cell proliferation and promote cell apoptosis.

Nucleus transfer efficiency of ear fibroblast cells isolated from Bama miniature pigs at various ages.

Somatic cell nucleus transfer (SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer (NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts (P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells (85% to 91%) than those at 6 and 12 months (66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group (8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

Resistance reverse effect of anemoside B4 and tetrandrine on oxaliplatin-resistant human colon cancer cells and the mechanism

Background and purpose: Oxaliplatin(L-OHP) is one of the most commonly used chemotherapy drugs in colorectal cancer and L-OHP-resistance is very common in colorectal cancer therapy. This research was to discuss the reversal effect in L-OHP-resistant human colon cancer cell line Lo Vo/L-OHP by anemoside B4(AB4) and tetrandrine(Tet) and to clarify their molecular mechanism. Methods: Lo Vo/L-OHP cells were treated for 48 h by AB4 and Tet at different concentrations to get non-toxic dose. Drug sensitivity was measured by MTT. After the treatment, the cell cycle and apoptosis of the cells were detected. Expression of P-gp m RNA, z DHHC9 m RNA and SMAD4 m RNA were detected by RT-PCR. Expression of P-gp, z DHHC9 and SMAD4 protein were detected by Western blot. Results: The IC50 of Lo Vo/L-OHP for L-OHP was(112.5±23.6) μg/m L, and the IC50 decreased to(62.8±21.4) μg/m L(P0.05) and(58.9±26.3) μg/m L(P0.05) after the treatment with 0.71 μg/m L AB4 and 0.45 μg/m L Tet(non-toxic dose) separately. The cell cycle experiment showed that the cells in G1 stage decreased and in S stage increased but with no statistical significance(P0.05). The cell apoptosis experiment showed that the apoptotic rate increased after the treatment with AB4 and Tet with non-toxic dose but with no statistical significance(P0.05). The RT-PCR experiment showed that the expressions of P-gp m RNA and z DHHC9 m RNA were increased and SMAD4 m RNA was decreased in Lo Vo/L-OHP cells compared with in Lo Vo cells(P0.05), while it was found that P-gp m RNA in Lo Vo/L-OHP cells was decreased after the treatment with AB4 at non-toxic dose(P0.05). Western blot experiment showed the protein of P-gp in Lo Vo/L-OHP cells was decreased after treatment with AB4(P0.05), which was accordant to the PCR result. The expression of z DHHC9 protein in Lo Vo/L-OHP cells was decreased in Lo Vo/L-OHP cells after treatment with Tet(P0.05). Conclusion: AB4 and Tet have some reversal effect on resistant to L-OHP in Lo Vo/L-OHP cells. The molecular mechanism of the resistance reverse effect was related to down-regulation of P-gp for AB4 and down-regulation of z DHHC9 for Tet.

Effects of melatonin combined with Cis-platinum or methotrexate on the proliferation of osteosarcoma cell line SaOS-2.

To evaluate the effects of melatonin (Mel) combined with cis-platinum (DDP) or methotrexate (MTX) on the proliferation of osteosarcoma cell line SaOS-2, and to explore whether Mel combined with DDP or MTX could play a synergistic antitumor effect. SaOS-2 was treated with Mel alone or Mel combined with DDP or MTX. Cell counting kit-8 assay was used to measure the cell activities. Combination index(CI) value was used to evaluate the combined effects: CI<1 indicating synergetic effect, CI=1 additive, and CI>1 antagonistic.Flow cytometry was used to analyze cell cycle distribution and cell apoptosis. After treated with Mel (0.5,1,2,4,5 mmol/L), DDP (6.67, 16.67, 33.33, 66.66 μmol/L) or MTX(0.1, 0.5, 1, 2, 4 mmol/L)alone,SaOS-2 cell activities decreased in a dose-dependent manner (all P<0.05). The activities of SaOS-2 cell treated with both Mel (1 mmol/L) and DDP or MTX were significantly lower than that of DDP or MTX alone (all P<0.05).CI values of cells exposed to 1 mmol/L Mel plus 6.67, 16.67, 33.33, and 66.66 μmol/L DDP were 1.18, 1.21, 1.09, and 0.84, respectively,and CI values of cells exposed to 1 mmol/L Mel plus 0.1, 0.5, 1, 2, and 4 mmol/L MTX were 0.88, 0.88 ,0.83, 0.78, and 0.81, respectively. The G1-stage cells were increased and the S-stage cells were reduced when the cells were treated with Mel (1 mmol/L) alone or combined with MTX (0.5 mmol/L) (P<0.05). The S-stage cells were increased when the cells treated with MTX (0.5 mmol/L) (P<0.05). The apoptotic cells were increased when they treated with Mel (1 mmol/L) alone or combined with DDP (16.67 μmol/L) or MTX (0.5 mmol/L) (P<0.05). When the cells were treated with Mel combined with DDP or MTX, the apoptotic cells were more than that of DDP or Mel alone(P<0.05). Mel can inhibit SaOS-2 cells activity,block the cell cycle at G1-stage,and induce apoptosis. Mel has an antagonistic effect with lower concentration of DDP but a synergistic effect with MTX or higher concentration of DDP.

Synthesis and pharmacological evaluation of novel bisindole derivatives bearing oximes moiety: Identification of novel proapoptotic agents

In an effort to develop potent anti-cancer chemopreventive agents, a novel series of bisindole derivatives bearing oxime moiety were synthesized. Structures of all compounds were characterized by NMR and HRMS. Anti-proliferative activities for all of these compounds were investigated by the method of MTT assay on 7 human cancer lines and the normal cell lines (HUVEC). Most of them showed a noteworthy anti-cancer activity in vitro, the half maximal inhibitory concentration (IC50) value is 4.31 μM of 4e against T24. The results from Hoechst 33258 and acridine orange/propidium iodide staining as well as annexinV-FITC assays provided evidence for an apoptotic cell death. The further mechanisms of compound 4e-induced apoptosis in T24 cells demonstrated that compound 4e induced the productions of ROS, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. Moreover, the cell cycle analysis and western-blot analysis indicated that compound 4e effectively arrested T24 cells in G1 stage and possibly has an effect on cell cycle regulatory proteins particularly cyclin D1.

Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF) at 5mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50μM or 1mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5mM NaF together with radiation (1Gy) induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

Synthesis and biological evaluation of novel aniline-derived asiatic acid derivatives as potential anticancer agents

Asiatic acid (AA) derivatives 4 and 5 modified at the C-11 and C-28 positions were designed and synthesized, their structures were confirmed using HRMS, 1H NMR and 13C NMR. In vitro antitumor activities of all compounds against MGC-803, NCI-H460, HepG2, Hela and 7404 cancer cell lines were evaluated and compared with commercial anticancer drug 5-fluorouracil (5-FU), employing standard MTT assay. The new compounds 5a–5t showed stronger anti-proliferative activity than AA, especially compound 5b was found to be the best inhibition activity on HepG2 cell line. In addition, the mechanism of compound 5b was preliminarily investigated by acridine orange/ethidium bromide staining, Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining, flow cytometric, qRT-PCR (quantitative real-time PCR) and Western blot. Compound 5b induced the productions of ROS, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. Moreover, the cell cycle analysis showed that compound 5b mainly arrested HepG2 cells in G1 stage.

Synthesis and antitumor properties of novel alizarin analogs

Series of novel hybrids of alizarin and diamide scaffold (3a–3h, 7a–7h) were designed and synthesized. In vitro antitumor activities of all compounds against HepG-2, CNE, Spca-2, Hct-116, and MGC-803 cell lines were evaluated, and employing standard MTT assay compared with commercial anticancer drug 5-fluorouracil (5-FU). Compounds 7b, 7c, 7d, and 7e showed relatively high cytotoxicity. Especially, compound 7c exhibited the best cytotoxicity against CNE cells with IC50 9.08 µM, which was even stronger than that of 5-FU. All the synthesized compounds exhibited low cytotoxicity against HUVEC cells. The action mechanism of representative compound 7c was preliminarily investigated by flow cytometry, which indicated that the compound can induce cell apoptosis in CNE cells. Cell cycle analysis showed that compound 7c mainly arrested CNE cells in G1 stage. In addition, the binding properties of a model analog 7c to DNA were investigated by different methods (fluorescence, CD spectroscopy), and the results indicated that 7c showed a moderate preference for binding ct-DNA.

Synthesis and antitumor activities of novel rhein α-aminophosphonates conjugates

Several rhein α-aminophosphonates conjugates (5a–5q) were synthesized and evaluated for in vitro cytotoxicity against HepG-2, CNE, Spca-2, Hela and Hct-116 cell lines. Some compounds showed relatively high cytotoxicity. Especially, compound 5i exhibited the strongest cytotoxicity against Hct-116 cells (IC50 was 5.32μM). All the synthesized compounds exhibited low cytotoxicity against HUVEC cells. The mechanism of compound 5i was preliminarily investigated by Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining and flow cytometry, which indicated that the compound 5i induced apoptosis in Hct-116 cancer cells. Cell cycle analysis showed that these compound 5i mainly arrested Hct-116 cells in G1 stage. The effects of 5i on the activation of caspases expression indicated that 5i might induce apoptosis via the membrane death receptor pathways. In addition, the binding properties of a model analog 5i to DNA were investigated by methods (UV–vis, fluorescence, CD spectroscopy and FRET-melting) in compare with that of rhein. Results indicated that 5i showed moderate ability to interact ct-DNA.

Synthesis and antitumor activities of novel α-aminophosphonates dehydroabietic acid derivatives

A series of novel α-aminophosphonate derivatives containing DHA structure were designed and synthesized as antitumor agents. In vitro antitumor activities of these compounds against the NCI-H460 (human lung cancer cell), A549 (human lung adenocarcinoma cell), HepG2 (human liver cancer cell) and SKOV3 (human ovarian cancer cell) human cancer cell lines were evaluated and compared with commercial anticancer drug 5-fluorouracil (5-FU), employing standard MTT assay. The pharmacological screening results revealed that many compounds exhibited moderate to high levels of antitumor activities against the tested cancer cell lines and that most demonstrated more potent inhibitory activities compared with the commercial anticancer drug 5-FU. The action mechanism of representative compound 7c was preliminarily investigated by acridine orange/ethidium bromide staining, Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining and flow cytometry, which indicated that the compound can induce cell apoptosis in NCI-H460 cells. Cell cycle analysis showed that compound 7c mainly arrested NCI-H460 cells in G1 stage.

Cell-selective gene silencing in prostate cancer LNCap cells using prostate-specific membrane antigen promoter and enhancerin vitroandin vivo

RNAi (RNA interference) has been widely used to silence specific genes. However, RNAi may also cause off-target silencing and elicit non-specific side effects. To achieve cell-specific gene silencing, a cell-selective promoter has to be used to drive RNAi expression. Furthermore, different terminators of cell-selective promoters may cause different silencing efficacies. In order to explore the best promoter and terminator combination and prove the cell-selective gene silencing effect of PSMAe/p (prostate-specific membrane antigen enhancer/promoter), we first constructed three plasmids by using PSMAe/p and three different terminators [poly(A), minipoly(A) and poly(U)] to explore the cell-selective driving ability of PSMAe/p by targeting EGFP (enhanced green fluorescent protein) in LNCaP, PC-3, EJ and HEK-293 (human embryonic kidney) cells. Then we chose NS (nucleostemin), an important endogenous gene of prostate cancer, and constructed the NS-targeting shRNA (small-hairpin RNA) expression plasmid by using PSMAe/p-poly(A) combination. Cell proliferation, cell cycle and early apoptosis in vitro and xenograft tumour growth in BALB/c nude mice in vivo were detected after NS knockdown. Results showed that PSMAe/p can drive EGFP silencing in LNCaP, not in PC-3, EJ and HEK-293 cells and PSMAe/p-poly(A) combination achieved the best silencing efficacy. Then PSMAe/p-shNS-poly(A) drives NS knockdown in LNCaP cells, not in PC-3, EJ and HEK-293 cells. Furthermore, RNAi-mediated NS knockdown not only reduces cell proliferation rate, reduces the percentage of S-stage cells and increases the percentage of G1-stage cells and increases the early apoptosis ratio in LNCaP cells in vitro, but also inhibited the LNCaP xenograft tumour growth in BALB/c nude mice in vivo by intratumoural injection. In conclusion, we have demonstrated that PSMAe/p-poly(A) combination is a promising delivery system for targeted RNAi gene therapy of prostate cancer. We showed one effective antitumour strategy by targeting NS protein, an important target in prostate cancer, with PSMAe/p-shNS-poly(A). These results serve as an important step for developing novel strategies to treat prostate cancer.

Effect of miR-221 on the viability and apoptosis of hepatocellular carcinoma HepG2 cells

To investigate the effect of microRNA-221 (miR-221) on cell viability and apoptosis of hepatocellular carcinoma HepG2 cells. MiR-221 inhibitors and mimics were transfected into HepG2 cells. The expression of miR-221 was detected by real time quantitative RT-PCR. CellTiter-blue cell viability kit, Hoechst 33342/propidium iodide (PI) double staining assay, flow cytometry and Apo-ONE homogeneous caspase-3/7 kit were used to measure cell viability and apoptosis. MiR-221 inhibitors significantly inhibited the cell growth and miR-221 mimics increased cell viability 48 hours post-transfection measured by both CellTiter-blue cell viability kit and Hoechst 33342/PI assay (P is less than to 0.05). There was a positive correlation between these two assays (r = 0.993, P is less than to 0.01). With miR-221 mimics, the number of G1 stage cells (47.67%+/-1.53%) was significantly reduced as compared to the blank control (59.00%+/-1.00%) and the negative control (58.00%+/-1.00%, F = 81.77, P is less than to 0.01), and it was significantly raised in S stage (20.33%+/-1.15%) than in blank control (11.00%+/-1.00%) and negative control (12.00%+/-1.00%, F = 70.9, P is less than to 0.01) with flow cytometry analysis. More cell apoptosis and necrosis were significantly induced by miR-221 inhibitors 48 hours post-transfection detected by both Hoechst 33342/PI assay and flow cytometry PE Annexin V kit (P is less than to 0.05). The result from Apo-ONE homogeneous caspase-3/7 kit was consistent with the above two apoptotic assays, which showed that with miR-221 inhibitors, the activity of caspase-3/7 was significantly enhanced (P is less than to 0.05). MiR-221 contributes to the growth of hepatocellular carcinoma cells and miR-221 inhibition can induce cell apoptosis. miR-221 has the potential to become one of the new molecular targets for liver cancer therapy.

Effects of human cytomegalovirus on the cell cycle of duct epithelial cell cultures of human salivary gland in vitro and relative mechanism

To investigate the effects of human cytomegalovirus (HCMV) on the cell cycle of duct epithelial cell cultures of human salivary gland (HSG) in vitro and relative mechanism. HSG was cultured in vitro. Reverse transcriptase polymerase chain reaction (RT-PCR) and nest-RT-PCR were used respectively to investigate ie1/ie2 transcription in HSG infected by human cytomegalovirus(HCMV). The effects of HCMV on the cell cycle of HSG were studied by flow cytometry in vitro. The expression of cyclin D1 in HSG infected by HCMV was detected by Western blotting. HCMV iel/ie2 transcription could be detected in HSG infected by HCMV. HCMV arrested productively infected cells in G1 stage. And cyclin D1 was down-regulated in HCMV infected HSG. HCMV inhibits proliferation of HSG by affecting G1/S check point and down-regulating cyclin D1 in vitro.

OVCA1 inhibits the proliferation of epithelial ovarian cancer cells by decreasing cyclin D1 and increasing p16

OVCA1, a tumor suppressor gene, is deleted or lower expressed in about 80% of ovarian cancer. Over expression of OVCA1 in human ovarian cancer A2780 cells inhibits cell proliferation and arrests cells in G1 stage. However, the fact that the molecular mechanism of OVCA1 inhibits cell growth is presently elusive. Here we investigated the potential signaling pathway induced by over-expression of OVCA1. Our results show that over-expression of human OVCA1 in ovarian cancer cells A2780 leads to down-regulation of cyclin D1, and up-regulation of p16, but no effect on the expression of NF-κB. It indicates that OVCA1 could inhibit the proliferation of ovarian cancer cell A2780 by p16/cyclin D1 pathway, but not by NF-κB.

Expression of nucleostemin in prostate cancer and its effect on the proliferation of PC-3 cells

Nucleostemin is essential for the proliferation and survival of stem and cancer cells, but it is unknown whether this newly identified molecule is involved in prostate cancer pathogenesis. Total RNA and protein were extracted from prostate cancer tissues and PC-3, LNCap and DU145 cell lines. The nucleostemin mRNA and protein expression were measured by RT-PCR and Western blot. Immunohistochemistry was also used to detect the nucleostemin protein expression in prostate cancer tissues and PC-3 cells. A nucleostemin specific, short hairpin RNA, expression plasmid was used to transfect PC-3 cells. The changes of nucleostemin gene were detected and the proliferative capacity of the cells was determined. Nucleostemin was highly expressed in prostate cancer tissues and cell lines. Nucleostemin expression level in the silencer group PC-3 cells remarkably reduced. The proliferation rate of silencer group PC-3 cells decreased and the percentage of G1 stage cells increased. The neoplasm forming capacity in nude mice of the silencer group PC-3 cells decreased significantly. Nucleostemin is highly expressed in prostate cancer tissues and cell lines. The proliferative capacity of PC-3 cells is remarkably reduced after silencing nucleostemin gene expression.

P3-040: Sinomenine inhibits proliferation and apoptosis induction in lung cancer cells

Sinomenine, an alkaloid extract from the Chinese medicinal plant Sinomenium acutum, has a wide range of pharmacological actions. Here, we examined whether sinomenine possesses anti-lung cancer properties. The human A549 lung cancer cell line and the murine Lewis lung carcinoma cell line were used in this study. The cells were treated with sinomenine (0.1, 0.2, 0.4, 0.625, 1.25 or 2.5 mM) for 24, 48 or 72 h. Cellular proliferation was evaluated with a CCK-8 assay. Cell cycle phase distribution and apoptosis were examined by flow cytometry. Sinomenine treatment inhibited cellular proliferation (P<0.01) in a time and dose-dependent manner. Cell cycle analysis showed that the percentage of cells in G1 stage in the sinomenine-treated groups was significantly greater, while the percentage of cells in S stage was significantly less than in the control group. The apoptosis assay showed that the apoptotic cell rate in the sinomenine-treated groups was significantly elevated in a time and dose-dependent manner (P <0.05 vs. controls). Sinomenine can effectively inhibit proliferation of A549 and Lewis cancer cells. The cell cycle arrest and apoptosis induced by sinomenine treatment may underlie, at least in part, the purported anti-cancer effects of this extract. If so, sinomenine may be developed into an effective treatment against lung tumors.