伊马替尼对人卵巢癌细胞SKOV-3增殖及顺铂、紫杉醇敏感性的影响

目的：探讨伊马替尼对人卵巢癌细胞SKOV-3增殖和对顺铂、紫杉醇敏感性的影响及其作用机制。方法：四甲基偶氮唑蓝（MTT）快速比色法检测细胞增殖率、Hoechst33342染色检测细胞凋亡;采用免疫荧光测定伊马替尼处理前后人卵巢癌细胞株SKOV-3 p-PDGFRβ的表达情况。结果：伊马替尼可以诱导SKOV-3细胞凋亡,对其增殖抑制作用与对照组相比差异有统计学意义（P〈0.05）,并显著提高了SKOV-3细胞对顺铂、紫杉醇的敏感性（P〈0.05）;PDGF-BB可促进PDGFRβ发生磷酸化,伊马替尼在体外阻断了SKOV-3细胞p-PDGFRβ的表达,p-PDGFRβ可能是伊马替尼的主要作用靶点。结论：伊马替尼对SKOV-3细胞有明显杀伤作用,并显著增强该细胞株对顺铂、紫杉醇的敏感性;伊马替尼可在体外阻断SKOV-3细胞PDGFRβ的磷酸化,这可能是其影响细胞增殖的主要途径之一。     

The Effect of the MDM2-p53 Loop on the Sensitivity of Ovarian Cancer Cells to Cisplatin

OBJECTIVE To explore whether MDM2 transfection can alter the MDM2-p53 autoregulatory feedback loop so as to change the sensitivity of ovarian cancer cell lines to cisplatin. METHODS The ovarian cancer cell line A2780 expressing wild-type P53 and the ovarian cancer cell line SKOV-3 with the p53 null type were stably transfected with pCMV-MDM2 or pCMV as a control. The blocked expression of P53 was determined by Western blots. Cytotoxicity was assessed using the MTT assay and the trypan blue exclusion assay. Flow cytometry was used to detect changes in the cell cycle and removal of platinum -DNA adducts was measured by atomic absorption spec-troscopy. RESULTS (1) Parental A2780 and A2780-V cells (IC50= 15.14±1.39 μmol) have similar cisplatin sensitivities, whereas sensitivity to cisplatin in A2780-M cells (IC50=7.98±1.32 μmol) was 2 to 3 fold greater (P=0.001). The trypan blue exclusion assay demonstrated that cisplatin killed a higher percentage of A2780-M cells compared to A2780-V cells. There was no significant change following MDM2 transfection in SKOV-3 cells. (2) After cisplatin treatment, A2780-M cells showed a pronounced S-phase arrest, however, A2780 cells with the intact wild-type P53, arrested primarily at the G2/M transition. (3) Platinum uptake was similar for all of the A2780 cell lines after ciaplatin treatment, but the removal of plat-inum-DNA adducts was reduced in the A2780-M cells compared with A2780-V cells. CONCLUSION MDM2 increases cisplatin cytotoxicity in ovarian cancer cells by blocking the expression of p53 through the MDM2-p53 autoregulatory feedback loop.     

Trail activity in human ovarian cancer cells: potentiation of the action of cytotoxic drugs

The ability of the TRAIL ligand to induce cell killing in three ovarian cancer cell lines was investigated using a glutathione-S-transferase (GST)-TRAIL fusion protein. One of the three lines was sensitive to TRAIL, which induced cell killing in a range of concentrations similar to those necessary to kill the TRAIL-sensitive leukaemic cell line Jurkat. The relative mRNA expression of the four TRAIL receptors did not explain the different sensitivities of the three ovarian cancer cell lines to TRAIL treatment. The TRAIL-sensitive IGROV-1 cell line expressed slightly lower levels of the anti-apoptotic protein FLIP than the two TRAIL-insensitive cell lines (A2780 and SKOV-3). Nevertheless, although TRAIL did not significantly reduce cell growth in the A2780 and SKOV-3 cells it did enhance the activity of paclitaxel and cisplatin (DDP), the two most widely used drugs for the treatment of ovarian cancer, increasing their ability to induce apoptosis. The use of TRAIL in combination with classical anticancer agents might thus boost the apoptotic response, improving the activity of DDP and paclitaxel in ovarian cancer.     

Selenite cytotoxicity in drug resistant and nonresistant human ovarian tumor cells.

Our previous studies on selenite cytotoxicity led us to hypothesize that drug resistant tumor cells with high intracellular glutathione will exhibit a high degree of sensitivity to selenite. To examine this we studied the effects of selenite on drug resistant human ovarian tumor (NIH:OVCAR-3) cells in three assays of cytotoxicity: proliferation; cell viability (trypan blue exclusion); and attachment to a solid matrix. The cells were sensitive to low levels of selenite: concentrations as low as 5 microM inhibited cell proliferation and attachment; and viability was decreased by concentrations as low as 20 microM. In each of these assays the NIH:OVCAR-3 cells were more sensitive to selenite than were drug sensitive human ovarian tumor (A2780) cells. These results suggest the potential for the utilization of selenite in the treatment of some drug resistant tumors.     

Cytotoxicity of phenolic acid phenethyl esters on oral cancer cells

Many phenolic acid phenethyl esters possess diverse biological effects including anti-cancer activity. A series of 14 derivatives were synthesized for the evaluation of their cytotoxic effect on oral cancer cells. These derivatives were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and trypan blue dye exclusion assay on the growth of oral squamous cell carcinoma (SAS), oral epidermoid carcinoma-Meng 1 (OEC-M1), and normal human oral fibroblast (NHOF) cells, respectively. Caffeic acid phenethyl esters, 3a (CAPE), and 3b, 3c, and 3d showed cytotoxic effects on the SAS and OEC-M1 cell lines, but not the NHOF cell line at a 5-100 microM dose range. Flow cytometric analysis showed that 3c caused OEC-M1 cell arrest at G2/M phase. Such differential effects on representative cancer and normal cells suggested these compounds might be useful in oral cancer chemotherapy.     

紫杉醇脂质体对卵巢癌细胞生长抑制作用的实验研究

目的分析紫杉醇脂质体对卵巢癌SKOV-3细胞的生长抑制作用,观察药物作用的时间-浓度关系,探究药物作用机制。方法应用不同剂量紫杉醇脂质体对SKOV-3细胞进行处理,通过MTT检测细胞存活率,倒置显微镜观察细胞形态,流式细胞术检测细胞凋亡作用及其对细胞周期的阻滞状态,Western blot分析Bcl-2基因蛋白表达情况。利用SPSS 13.0对数据进行统计学分析。结果紫杉醇脂质体对SKOV-3细胞有明显的剂量-时间依赖效应。紫杉醇与紫杉醇脂质体均可将SKOV-3细胞阻滞在G2/M期,且随药物浓度增加,凋亡细胞所占比例逐渐增加。通过Western blot方法发现,紫杉醇脂质体作用后SKOV-3细胞Bcl-2蛋白表达明显下降,Bax蛋白表达明显上调,Bax/Bal-2比例明显上调。结论紫杉醇以脂质体为转运载体后,并未改变其对卵巢癌SKOV-3细胞的抑制作用及作用周期,具有较好的临床应用前景。     

1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death

We investigated the capacity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans-retinoic acid (ATRA) to sensitize three breast cancer cell lines to the cell killing effects of paclitaxel (Taxol) and Adriamycin, two chemotherapeutic agents commonly used in the treatment of breast cancer. In tissue culture colony assays, 1,25(OH)2D3 and ATRA were synergistic in inhibiting the clonogenicity of MCF-7 and T-47D cells that expressed estrogen receptor; vitamin D receptor; retinoic acid receptors (RARs) alpha, beta, and gamma; and retinoid X receptors alpha, beta, and gamma but were not additive in MDA-MB-231 cells that lacked expression of estrogen receptor, RARalpha, and RARbeta. The hormones used individually or in combination induced up to 40-50% cell death by a trypan blue exclusion assay in a dose-dependent manner up to concentrations of 10(-7) M in MCF-7 and T-47D cells, more modestly in MDA-MB-231 cells, and not at all in MCF-10 and MCF-12 nontransformed mammary epithelial cells. Pretreating the cancer cell lines with 1,25(OH)2D3 and ATRA individually or in combination for 3 days prior to a 1-h incubation with paclitaxel or Adriamycin decreased the ED50 for inhibition of colony formation or for cell death by trypan blue by up to 2 logs for paclitaxel and up to 1 log for Adriamycin in all three cell lines but had no effect on chemotherapy-induced MCF-12 cell death. The effects of the hormones were synergistic with those of the chemotherapy agents in all of the breast cancer cell lines, generally at the higher concentrations. Cell death took place by apoptosis. To determine one potential reason for the greater potentiation of the effects of paclitaxel than those of Adriamycin, we determined the effects of preincubation of MCF-7 cells on paclitaxel-induced phosphorylation of Bcl-2. Pretreatment of MCF-7 cells with either 1,25(OH)2D3 or ATRA increased the phosphorylation of Bcl-2 by variable concentrations of paclitaxel. These data suggest that pretreatment of breast cancer with 1,25(OH)2D3 or ATRA lowers the threshold for cell killing by chemotherapy agents and may provide a novel treatment option for this disease.     

索拉非尼对人卵巢癌SKOV-3细胞凋亡的影响

目的探讨索拉非尼对人卵巢癌SKOV-3细胞凋亡的影响。方法采用MTT法,检测不同浓度索拉非尼(1.5、3.0、6.0、12.0、24.0μmol/L)作用人卵巢癌SKOV-3细胞24、48、72 h的抑制率;Hoechst染色法观察药物处理后细胞凋亡情况;应用索拉非尼与紫杉醇不同给药方式作用于SKOV-3细胞,流式细胞仪检测药物处理后细胞周期和凋亡率变化。结果索拉非尼对SKOV-3细胞增殖具有明显抑制作用,与对照组比较差异有统计学意义(P<0.05),并且呈剂量和时间依赖性;索拉非尼主要使SKOV-3细胞周期阻滞在S期,与紫杉醇联合给药后S期及G2/M期均有延长,且凋亡率较高(P<0.05)。结论索拉非尼对SKOV-3细胞增殖有显著抑制作用,且呈剂量和时间依赖性,紫杉醇序于索拉非尼给药时细胞凋亡率更高。     

甲基转移酶抑制剂5-Aza-dC对FANCF基因表达的调控在卵巢癌治疗中的作用

目的:探讨甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-aza-2’-deoxycytidine,5-Aza-dC)对卵巢癌细胞系OVCAR3中FANCF(Fanconi anemia complementation group F)基因表达的影响,及其对化疗药物紫杉醇敏感性的影响,以了解5-Aza-dC的抗肿瘤效应,寻找卵巢癌治疗的新靶点.方法:采用5-Aza-dC处理FANCF基因甲基化的卵巢癌细胞OVCAR3和FANCF基因非甲基化的卵巢癌细胞A2780.应用甲基化特异性PCR(methylation specific-PCR,MSP)、RT-PCR和蛋白质印迹法分析这2种细胞经5-Aza-dC处理前后FANCF基因的甲基化状态以及FANCF mRNA和蛋白的表达情况.MTT法检测卵巢癌细胞的增殖情况.FCM检测紫杉醇对卵巢癌细胞凋亡的影响.建立OVCAR3和A2780细胞裸鼠移植瘤模型,观察5-Aza-dC对裸鼠移植瘤生长的影响,并采用免疫组织化学法检测移植瘤中FANCF蛋白的表达情况.结果:体外实验显示,5-Aza-dC处理后,OVCAR3细胞中FANCF基因发生去甲基化,FANCF mRNA和蛋白的表达水平增加,细胞生长缓慢.5-Aza-dC处理组OVCAR3细胞裸鼠移植瘤体积较未处理组明显缩小,质量也明显降低,FANCF蛋白表达增加;而A2780细胞裸鼠移植瘤组中未观察到上述变化.结论:甲基转移酶抑制剂5-Aza-dC可能通过抑制卵巢癌细胞增殖而成为潜在的卵巢癌治疗药物,但同时也会增加紫杉醇的耐药风险.     

两位点短发卡状RNA诱导AKT2基因沉默在卵巢癌细胞紫杉醇敏感性中的应用研究

目的：探讨AKT2基因在肿瘤细胞对紫杉醇敏感性中的作用。方法：分别构建2个针对同一AKT2基因不同位点的短发卡状RNA（shRNA）载体，转染人卵巢癌细胞A2780、SKOV3，荧光显微镜观察细胞内荧光蛋白的表达及转染效率，运用RT—PCR、蛋白质免疫印迹（western blotting）方法比较单独转染的抑制瘤和共转染对AKT2表达的沉默效率；抑制AKT2表达后，流式细胞仪（FACS）检测肿瘤细胞对紫杉醇的敏感性。结果：构建的2种shRNA表达载体均可抑制AKT2mRNA和蛋白的表达，共转染的抑制率明显高于分别单独转染的抑制率（P〈0．05）；FACS结果显示，共转染沉默AKT2基因后，细胞凋亡率明显增加（P〈0．05）。结论：共转染针对AKT2基因不同位点的2个shRNA真核表达载体，对AKT2基因的抑制效率高于单独转染1个载体；抑制卵巢癌细胞中AKT2基因表达，能增强其对紫杉醇的敏感性。     

沉默信号转导和转录活化因子（STAT3）对卵巢癌细胞SKOV-3中肌糖蛋白（TN-C）表达的影响

目的:探讨肌糖蛋白C（TN-C）对卵巢癌细胞SKOV-3细胞增殖及凋亡的影响,并研究SKOV-3中沉默信号转导和转录活化因子（STAT3）对TN-C表达的调控。方法:用不同浓度的重组TN-C蛋白（0、50、100和200ng/ml）处理人卵巢癌SKOV-3细胞48h后,采用MTT法检测细胞增殖速率;以血清饥饿诱导细胞凋亡,同时给予TN-C刺激,Annexin V/PI双染法检测SKOV-3细胞凋亡。构建STAT3过表达载体或特异性siRNA 序列并转染SKOV-3,建立SKOV-3STAT3（+）或SKOV-3STAT3（-）细胞,Western blotting检测不同SKOV-3细胞核中STAT3、p-STAT3的水平变化以及细胞TN-C水平的变化。结果:MTT及Annexin V/PI双染法结果显示TN-C呈剂量依赖性促进SKOV-3细胞增殖,同时抑制血清饥饿诱导的SKOV-3细胞大量凋亡。SKOV-3细胞核中p-STAT3与细胞TN-C水平正相关,抑制p-STAT3或减少STAT3的表达都导致TN-C水平的下降。结论:TN-C在卵巢癌的转移和侵袭中有重要的作用,STAT3可调节卵巢癌细胞中TN-C的表达水平,提示二者可联合作为临床上卵巢癌治疗的潜在靶点。     

Silencing survivin results in synergy between methylseleninic acid and paclitaxel against skov3 ovarian cancer cells

This study evaluates methylseleninic acid (MSeA) improvement of paclitaxel efficacy against human ovarian cancer (skov3) with regard to survivin expression. MSeA and paclitaxel alone and in concurrent or sequential combination treatments were tested. Cell growth/death was evaluated using SRB, trypan blue, colony formation and ELISA assays. Cells were transfected with survivin shRNA and survivin's expression was measured using RT-PCR and Western blots. Drugs interaction was further evaluated using isobologram analyses. Different treatments with MSeA did not enhance paclitaxel's efficacy in the wild type skov3. Silencing survivin had no effect on MSeA or paclitaxel efficacy when used alone or in concurrent combination. After sequential combination treatment, synergy and significant induction of apoptosis were observed in cells transfected with survivin shRNA. However, antagonism and minimal induction of apoptosis were observed in empty or scramble survivin shRNA transfected cells. In conclusion, these data suggest that synergy between MSeA and paclitaxel in skov3 is associated with silencing survivin expression.     

"OA02" peptide facilitates the precise targeting of paclitaxel-loaded micellar nanoparticles to ovarian cancer in vivo

Micellar nanoparticles based on linear polyethylene glycol (PEG) block dendritic cholic acids (CA) copolymers (telodendrimers), for the targeted delivery of chemotherapeutic drugs in the treatment of cancers, are reported. The micellar nanoparticles have been decorated with a high-affinity "OA02" peptide against α-3 integrin receptor to improve the tumor-targeting specificity which is overexpressed on the surface of ovarian cancer cells. "Click chemistry" was used to conjugate alkyne-containing OA02 peptide to the azide group at the distal terminus of the PEG chain in a representative PEG(5k)-CA(8) telodendrimer (micelle-forming unit). The conjugation of OA02 peptide had negligible influence on the physicochemical properties of PEG(5k)-CA(8) nanoparticles and as hypothesized, OA02 peptide dramatically enhanced the uptake efficiency of PEG(5k)-CA(8) nanoparticles (NP) in SKOV-3 and ES-2 ovarian cancer cells via receptor-mediated endocytosis, but not in α-3 integrin-negative K562 leukemia cells. When loaded with paclitaxel, OA02-NPs had significantly higher in vitro cytotoxicity against both SKOV-3 and ES-2 ovarian cancer cells as compared with nontargeted nanoparticles. Furthermore, the in vivo biodistribution study showed OA02 peptide greatly facilitated tumor localization and the intracellular uptake of PEG(5k)-CA(8) nanoparticles into ovarian cancer cells as validated in SKOV3-luc tumor-bearing mice. Finally, paclitaxel (PTX)-loaded OA02-NPs exhibited superior antitumor efficacy and lower systemic toxicity profile in nude mice bearing SKOV-3 tumor xenografts, when compared with equivalent doses of nontargeted PTX-NPs as well as clinical paclitaxel formulation (Taxol). Therefore, OA02-targeted telodendrimers loaded with paclitaxel have great potential as a new therapeutic approach for patients with ovarian cancer.     

低能超声对人肺癌细胞的体外杀伤作用

目的 探讨低能超声对人肺癌细胞的影响。方法 用低能量连续波超声处理小细胞肺癌细胞系ＳＭ、腺癌细胞系Ａ２及人肺纤维母细胞ＦＢ,用胎盘蓝染料排斥试验及克隆存活试验判定效果,结果 低能量（０．８Ｗ／ｃｍ＾２）超声３分钟分各种人肺癌细胞及人肺纤维母细胞有杀伤作用（Ｐ〈０．０５）,１０分钟可杀死全部细胞（Ｐ〈０．００１）,细胞浓度越高,杀伤作用越低,超声１０分钟后含细胞悬液的试管内温度未见显著上升,声强０．８Ｗ／ｃｍ＾２的超声使阿霉对肺癌细胞ＳＭ的杀伤作用增加１００倍（Ｐ〈０．００１）,结论 低能连续波超声可杀伤肺癌细胞,并能显著增加阿霉素对肺癌细胞的细胞毒性。     

枸橼酸他莫昔芬对人卵巢癌细胞增殖的影响

目的:观察枸橼酸他莫昔芬(Tamoxifen Citrate)对体外培养的人卵巢癌细胞SKOV-3增殖的影响.方法:采用四甲基偶氮唑蓝(MTT)比色法检测不同浓度(0、1、5、10、12、14、15、16、18、20nmol/L)的枸橼酸他莫昔芬在不同的时间点(24、48、72h)对SKOV-3细胞增殖的影响;采用流式细胞技术(FCM)分析不同浓度的枸橼酸他莫昔芬处理细胞24h和48h后对细胞凋亡的影响.结果:枸橼酸他莫昔芬对SKOV-3细胞的增殖具有明显的抑制作用,与对照组相比具有显著性差异(P＜0.05),并呈现剂量和时间依赖性.枸橼酸他莫昔芬能够诱导细胞凋亡,且凋亡率随着枸橼酸他莫昔芬浓度和处理时间的增加而升高(P＜0.01).结论:枸橼酸他莫昔芬可显著抑制SKOV-3细胞的增殖,其作用可能与其诱导细胞凋亡有关.     

Inhibition of CDK4 sensitizes multidrug resistant ovarian cancer cells to paclitaxel by increasing apoptosiss

Purpose

Overexpression of cyclin-dependent kinase (CDK) 4 has been observed in a variety of cancers and has been found to contribute to tumor cell growth and proliferation. However, the effect of inhibition of CDK4 in ovarian cancer is unknown. We investigated the therapeutic effect of the CDK4 inhibitor palbociclib in combination with paclitaxel in ovarian cancer cells.

Methods

Cell viabilities were determined by MTT assay after exposure to different dosages of palbociclib and/or paclitaxel. Western blot, immunofluorescence, and Calcein AM assays were conducted to determine the mechanisms underlying the cytotoxic effects of palbociclib in combination with paclitaxel. CDK4 siRNA was used to validate the outcome of targeting CDK4 by palbociclib in ovarian cancer cells.

Results

We found that combinations of palbociclib and paclitaxel significantly enhanced drug sensitivity in both Rb-positive (SKOV3TR) and Rb-negative (OVCAR8TR) ovarian cancer-derived cells. When combined with paclitaxel, palbociclib induced apoptosis in both SKOV3TR and OVCAR8TR cells. We also found that palbociclib inhibited the activity of P-glycoprotein (Pgp), and that siRNA-mediated CDK4 knockdown sensitized multidrug resistant (MDR) SKOV3TR and OVCAR8TR cells to paclitaxel.

Conclusions

Inhibition of CDK4 by palbociclib can enhance paclitaxel sensitivity in both Rb-positive and Rb-negative MDR ovarian cancer cells by increasing apoptosis. CDK4 may serve as a promising target in the treatment of ovarian cancer.

     

Hyaluronic acid�Cpaclitaxel: effects of intraperitoneal administration against CD44(+) human ovarian cancer xenografts

Purpose

Hyaluronan (HA)-receptors (mainly CD44 and RHAMM) are overexpressed in a wide variety of cancers including ovarian tumors, and HA-bioconjugates have been developed to enhance selective entry of cytotoxic drugs into HA receptor-expressing cancerous cells. Here, we evaluated the potential application of a new HA-paclitaxel bioconjugate, ONCOFID-P, for intraperitoneal (IP) treatment of ovarian cancer.

Methods

In vitro cytotoxic effect of ONCOFID-P was first assessed on CD44(+) OVCAR-3 and SKOV-3 human ovarian cancer cell lines. Studies were performed in female Balb/c athymic mice IP implanted with OVCAR-3 or SKOV-3 and treated with IP ONCOFID-P, and IP and intravenous (IV) free paclitaxel, at their maximum tolerated dose (MTD 168, 80 and 80?mg/kg, total dose, respectively). The potential detrimental effect of the IP ONCOFID-P and IP free paclitaxel on hematopoiesis was also assessed on peripheral blood, bone marrow and spleen.

Results

Results show that ONCOFID-P cytotoxicity against both OVCAR-3 and SKOV-3 cell lines was somewhat less effective than free paclitaxel. Conversely, in in vivo experiments, IP treatment with ONCOFID-P was overall more effective than IV and IP free paclitaxel in inhibiting intra-abdominal tumor dissemination, abrogating ascites, prolonging survival and curing mice. ONCOFID-P and IP free paclitaxel were equivalent in terms of myelotoxicity, although the former was administered at a two-fold higher dose.

Conclusions

Present data strongly support the development of ONCOFID-P for locoregional treatment of ovarian cancer.     

Inhibition of the growth of malignant mouse lymphoid cells by selenodiglutathione and selenodicysteine

Intraperitoneal injections of selenodiglutathione (GSSeSG) or selenodicysteine (CySSeSCy) in mice, previously inoculated with tumor cells, inhibited tumor growth and increased the life span of treated as compared with untreated control mice. Determination of cell number at different times after injection of GSSeSG or CySSeSCy indicated that less tumor cells were present in the ascites fluid of these mice than in control mice.The trypan blue dye exclusion method performed 1 day after the injection of GSSeSG showed a high percentage of dead cells (84–99%).