Activation of p38 mitogen-activated protein kinase by celecoxib oppositely regulates survivin and gamma-H2AX in human colorectal cancer cells

Cancer cells express survivin that facilitates tumorigenesis. Celecoxib has been shown to reduce human colorectal cancers. However, the role and regulation of survivin by celecoxib in colorectal carcinoma cells remain unclear. Treatment with 40-80 muM celecoxib for 24 h induced cytotoxicity and proliferation inhibition via a concentration-dependent manner in RKO colorectal carcinoma cells. Celecoxib blocked the survivin protein expression and increased the phosphorylation of H2AX at serine-193 (gamma-H2AX). The survivin gene knockdown by transfection with a survivin siRNA revealed that the loss of survivin correlated with the expression of gamma-H2AX. Meanwhile, celecoxib increased caspase-3 activation and apoptosis. Celecoxib activated the phosphorylation of p38 mitogen-activated protein (MAP) kinase. The phosphorylated proteins of p38 MAP kinase and gamma-H2AX were observed in the apoptotic cells. SB203580, a specific p38 MAP kinase inhibitor, protected the survivin protein expression and decreased the levels of gamma-H2AX and apoptosis in the celecoxib-exposed cells. The blockade of survivin expression increased the celecoxib-induced cytotoxicity; conversely, overexpression of survivin by transfection with a survivin-expressing vector raised the cancer cell proliferation and resisted the celecoxib-induced cell death. Our results provide for the first time that p38 MAP kinase participates in the down-regulation of survivin and subsequently induces the activation of gamma-H2AX for mediating apoptosis following treatment with celecoxib in human colorectal cancer cells.     

Inhibition of G1/S transition potentiates oxaliplatin-induced cell death in colon cancer cell lines

In a series of colorectal cancer cell lines, both necrosis and apoptosis were induced upon exposure to oxaliplatin, and enhanced by co-administration of the Hsp90 inhibitor 17-AAG. We analyzed the effects of these interventions on the cell cycle, and found that oxaliplatin treatment caused G1 and G2 arrest in HCT116 cells, and S-phase accumulation in two p53-deficient cell lines (HT29 and DLD1). Addition of 17-AAG enhanced cell cycle effects of oxaliplatin in HCT116, and induced G1 arrest and decrease in S-phase population in the other cell lines. Analysis of cell cycle proteins revealed that the major difference between the cell lines was that in HCT116, 17-AAG resulted in profound inhibition of expression and phosphorylation of late G1 proteins cyclin E and cdk2, with no effect on p21/WAF1 induction. Consistent with these, an HCT116 p53(-/-) line, lacking p21, showed resistance to oxaliplatin, failure to enter apoptosis, and an accumulation of cells in S-phase. Introduction of p21 in these cells caused reversal of that phenotype, including restoration of the G1 block and re-sensitization to oxaliplatin. Inhibition of G1/S progression using cdk2 inhibitor also enhanced oxaliplatin cytotoxicity. We conclude that in colon cancer cells with impaired p53 function, interventions directed to cycle arrest in G1 may potentiate oxaliplatin activity.     

Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels

Background and purpose:

Checkpoint kinase 2 (CHK2) is activated by DNA damage and can contribute to p53 stabilization, modulating growth arrest and/or apoptosis. We investigated the contribution of CHK2 to oxaliplatin-mediated toxicity in a colorectal cancer model.

Experimental approach:

We evaluated the ability of CHK2 small molecule inhibitors to potentiate oxaliplatin-induced toxicity. The role of CHK2 in oxaliplatin-induced apoptosis was investigated in HCT116 cells that were wild-type (WT) or KO for CHK2. Small molecule inhibitors of CHK2 were used in combination studies with oxaliplatin in this cell model.

Key results:

In oxaliplatin-treated CHK2 KO cells, accelerated apoptosis was accompanied by attenuated p53 stabilization and p21^WAF-1 up-regulation correlating with increased Bax expression, cytochrome c release and elevated caspase activity. The higher levels of apoptosis in CHK2 KO cells were restored to control (WT) levels when CHK2 was re-introduced. This ‘uncoupling’ of p53 stabilization and Bax up-regulation in CHK2 KO cells suggested oxaliplatin-induced apoptosis was due to a p53-independent response. Combination studies revealed that CHK2 inhibitor II or debromohymenialdisine antagonized the responses to oxaliplatin. This inhibitory effect correlated with decreases in apoptosis, p53 stabilization and DNA inter-strand cross-link formation, and was dependent on the presence (but not activity) of CHK2.

Conclusions and implications:

Combinations of CHK2 inhibitors with oxaliplatin should further sensitize cells to oxaliplatin treatment. However, these inhibitors produced an antagonistic effect on the response to oxaliplatin, which was reversed on the re-introduction of CHK2. These observations may have implications for the use of oxaliplatin in colorectal cancer therapy in combination with therapies targeting CHK2.     

上调NDRG1表达降低奥沙利铂耐药结肠癌细胞的存活率

目的观察上调NDRG1表达对结肠癌细胞及奥沙利铂耐药结肠癌细胞的毒性作用并探讨其机制。方法以重组真核表达质粒pEGFP-NDRG1-N3转染HCT 116及奥沙利铂耐药的OHP-HCT 116结肠癌细胞,以实时定量(qRT)-PCR法和Western blot法检测转染效率。MTT法检测奥沙利铂对不同结肠癌细胞的毒性及验证OHP-HCT 116细胞的耐药性。给予奥沙利铂干预转染pEGFP-NDRG1-N3的HCT 116细胞及OHP-HCT 116细胞,MTT法检测细胞存活率,流式细胞术检测细胞凋亡率,Western blot法检测凋亡蛋白Bcl-2及p53蛋白水平变化。结果不同浓度奥沙利铂干预下的3株结肠癌细胞中HCT 116细胞对奥沙利铂最敏感,OHP-HCT 116细胞对奥沙利铂耐药得到验证。以不同浓度梯度奥沙利铂干扰转染pEGFP-NDRG1-N3的HCT 116细胞及OHP-HCT 116细胞,与MOCK及siNC组比较,在HCT 116细胞及OHP-HCT 116细胞,转染pEGFP-NDRG1-N3细胞的存活率下降(P<0.05),凋亡率升高(P<0.05),Bcl-2表达降低(P<0.05),p53表达升高(P<0.05)。结论上调NDRG1表达通过调控p53表达改善结肠癌细胞奥沙利铂耐药,提高化疗敏感性。     

Podophyllotoxin Induces ROS-Mediated Apoptosis and Cell Cycle Arrest in Human Colorectal Cancer Cells via p38 MAPK Signaling

Podophyllotoxin (PT), a lignan compound from the roots and rhizomes of Podophyllum peltatum, has diverse pharmacological activities including anticancer effect in several types of cancer. The molecular mechanism of the anticancer effects of PT on colorectal cancer cells has not been reported yet. In this study, we sought to evaluate the anticancer effect of PT on human colorectal cancer HCT116 cells and identify the detailed molecular mechanism. PT inhibited the growth of cells and colony formation in a concentration-dependent manner and induced apoptosis as determined by the annexin V/7-aminoactinomycin D double staining assay. PT-induced apoptosis was accompanied by cell cycle arrest in the G2/M phase and an increase in the generation of reactive oxygen species (ROS). The effects of PT on the induction of ROS and apoptosis were prevented by pretreatment with N-acetyl-L-cysteine (NAC), indicating that an increase in ROS generation mediates the apoptosis of HCT116 cells induced by PT. Furthermore, Western blot analysis showed that PT upregulated the level of phospho (p)-p38 mitogen-activated protein kinase (MAPK). The treatment of SB203580, a p38 inhibitor, strongly prevented the apoptosis induced by PT, suggesting that PT-induced apoptosis involved the p38 MAPK signaling pathway. In addition, PT induced the loss of mitochondrial membrane potential and multi-caspase activation. The results suggested that PT induced cell cycle arrest in the G2/M phase and apoptosis through the p38 MAPK signaling pathway by upregulating ROS in HCT116 cells.     

奥沙利铂联合NK4基因对大肠癌HCT116细胞凋亡的影响

目的观察奥沙利铂联合NK4基因对HCT116细胞增殖、凋亡的影响。方法使用Effectene转染试剂,将pcDNA3．1-NK4质柱转染至大肠癌HCT116细胞中,常规条件下培养24h。不同奥沙利铂药物浓度处理HCT116细胞,CCK-8法测定细胞的OD值,分析在不同奥沙利铂药物浓度下对HCT116细胞毒性的影响;检测奥沙利铂最低药物浓度联合NK4基因对HCT116细胞凋亡的影响。结果当奥沙利铂药物浓度为12．5μg／mL时,显著抑制细胞的增殖;奥沙利铂（12．5μg／mL）联合NK4基因处理组与奥沙利铂处理组此较,显著诱导HCT116细胞的凋亡,差异具有统计学意义（P〈0．05）。结论与奥沙利铂组相比,奥沙利铂联合NK4基因后HCT116细胞的凋亡率显著升高。     

LncRNA-MALAT1通过miR-142-3p/TEAD1分子轴调控结直肠癌细胞增殖与凋亡#br#

目的　探讨长链非编码RNA-肺腺癌转移相关转录子1（LncRNA-MALAT1）对结直肠癌细胞增殖与凋亡的影响及相关作用机制。方法　通过Real-time PCR与Western blot实验检测结直肠癌细胞株与人正常结肠上皮细胞中LncRNA-MALAT1、miR-142-3p基因以及TEA结构域转录因子1（TEAD1）蛋白的表达;使用si-MALAT1转染HCT116细胞,在此基础上共转染miR-142-3p inhibitor,利用Real-time PCR与Western blot检测细胞中LncRNA-MALAT1与miR-142-3p基因以及TEAD1、Bax、Bcl-2与Cyclin D1蛋白的表达,通过CCK-8实验检测细胞的增殖水平,通过流式细胞术检测细胞的凋亡水平,通过双荧光素酶实验检测LncRNA-MALAT1与miR-142-3p以及miR-142-3p与TEAD1的结合。结果　与人正常结肠上皮细胞相比,结直肠癌细胞株中LncRNA-MALAT1基因与TEAD1蛋白呈高表达,miR-142-3p基因呈低表达（P＜0.05）;沉默LncRNA-MALAT1能够促进miR-142-3p基因与Bax蛋白表达,抑制LncRNA-MALAT1基因以及Bcl-2、Cyclin D1与TEAD1蛋白表达,抑制细胞增殖,并促进细胞凋亡;在此基础上沉默miR-142-3p能够对上述调控作用实现部分逆转;双荧光素酶实验结果显示,LncRNA-MALAT1与miR-142-3p以及miR-142-3p与TEAD1能够结合。结论　LncRNA-MALAT1能够结合miR-142-3p,促进miR-142-3p靶基因TEAD1表达,进而促进结直肠癌细胞增殖,抑制其细胞凋亡,促进结直肠癌的病理进程。     

Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation

Aim:

The phosphorylation of histone H2AX, a novel tumor suppressor protein, is involved in regulation of cancer cell apoptosis. The aim of this study was to examine whether H2AX phosphorylation was required for resveratrol-induced apoptosis of human chronic myelogenous leukemia (CML) cells in vitro.

Methods:

K562 cells were tested. Cell apoptosis was analyzed using flow cytometry, and the phosphorylation of H2AX and other signaling proteins was examined with Western blotting. To analyze the signaling pathways, the cells were transfected with lentiviral vectors encoding H2AX-wt or specific siRNAs.

Results:

Treatment of K562 cells with resveratrol (20–100 μmol/L) induced apoptosis and phosphorylation of H2AX at Ser139 in time- and dose-dependent manners, but reduced phosphorylation of histone H3 at Ser10. Resveratrol treatment activated two MAPK family members p38 and JNK, and blocked the activation of another MAPK family member ERK. Pretreatment with the p38 inhibitor SB202190 or the JNK inhibitor SP600125 dose-dependently reduced resveratrol-induced phosphorylation of H2AX, which were also observed when the cells were transfected with p38- or JNK-specific siRNAs. Overexpression of H2AX in K562 cells markedly increased resveratrol-induced apoptosis, whereas overexpression of H2AX-139m (Ser139 was mutated to block phosphorylation) inhibited resveratrol-induced apoptosis. K562 cells transfected with H2AX-specific siRNAs were resistant to resveratrol-induced apoptosis.

Conclusion:

H2AX phosphorylation at Ser139 in human CML cells, which is regulated by p38 and JNK, is essential for resveratrol-induced apoptosis.     

Gamma-H2AX - a novel biomarker for DNA double-strand breaks

When DNA damage, whether it is endogenous or exogenous, forms double stranded breaks (DSBs), it is always followed by the phosphorylation of the histone, H2AX. H2AX is a variant of the H2A protein family, which is a component of the histone octomer in nucleosomes. It is phosphorylated by kinases such as ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) in the PI3K pathway. This newly phosphorylated protein, gamma-H2AX, is the first step in recruiting and localizing DNA repair proteins. DSBs can be induced by mechanisms such as ionizing radiation or cytotoxic agents and subsequently, gamma-H2AX foci quickly form. These foci represent the DSBs in a 1:1 manner and can be used as a biomarker for damage. An antibody can be raised against gamma-H2AX which can therefore be visualized by immunofluorescence through secondary antibodies. The detection and visualization of gamma-H2AX by flow cytometry allow the assessment of DNA damage, related DNA damage proteins and DNA repair. Gamma-H2AX also has other applications in the detection of genomic damage caused by cytotoxic chemical agents and environmental and physical damage, especially in the context of cancer treatment and therapy.     

The BH3-only protein, PUMA, is involved in oxaliplatin-induced apoptosis in colon cancer cells

Oxaliplatin, the first line chemotherapeutic of colon cancer, induces damage to tumors via induction of apoptosis. PUMA (p53 up-regulate modulator of apoptosis) is an important pro-apoptotic member of Bcl-2 family and regulated mainly by p53. Here we investigated the role of PUMA in oxalipaltin-induced apoptosis and the potential mechanism. We showed that oxaliplatin-induced PUMA expression in a time- and dose-dependent manner and suppression of PUMA expression by stable transfecting anti-sense PUMA plasmid decreased oxaliplatin-induced apoptosis in colon cancer cells. By abrogating the function of p53, we further demonstrated that the induction was p53-independent. We also found that oxaliplatin could inactivate ERK and suppression of ERK activity by its specific inhibitor (PD98059), and dominant negative plasmid (DN-MEK1) enhanced the oxaliplatin-induced PUMA expression and apoptosis in a p53-independent manner. Taken together, our data suggest that PUMA plays an important role in oxaliplatin-induced apoptosis and the induction could be both p53-dependent and p53-independent. Moreover, PUMA expression and apoptosis in oxaliplatin-treated colon cancer cells could be regulated partly by ERK inactivation. Identification of the molecular components involved in regulating the cellular sensitivity to oxaliplatin may provide potential targets for development of novel compounds that may be useful in enhancement of oxaliplatin cytotoxicity in p53 deficient colon cancer.     

Comparative examination of anti-proliferative activities of (-)-epigallocatechin gallate and (--)-epigallocatechin against HCT116 colorectal carcinoma cells

We compared anti-proliferative activities of (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) against HCT116 colorectal carcinoma cells. These catechins inhibited cell growth to nearly the same extent at low cell confluency in plates. However, their inhibitory effect grew weaker as cell confluence increased, and this tendency was more conspicuous for EGC than for EGCG. Both EGCG and EGC activated the phosphorylation of the major MAPKs, ERK, JNK, and p38, in the HCT116 cells as in many other established human cancer cells though to different extents. Cell cycle analyses, DNA fragmentation assays, and TUNEL assays as well as Western blot assays suggested that these catechins inhibited cell growth through mitogen-activated protein kinase (MAPK)-mediated apoptosis rather than cell cycle regulation.     

Depletion of securin increases arsenite-induced chromosome instability and apoptosis via a p53-independent pathway.

Arsenic is a pathologic factor of cardiovascular diseases and cancers; nevertheless, it also acts as an anticancer agent effective on acute promyelocytic leukemia and multiple myeloma. Securin, a proposed proto-oncogene, regulates cell proliferation and tumorigenesis. However, roles of securin on the arsenic-induced cell cycle arrest and apoptosis remain unknown. In this study, the effects of sodium arsenite on the expression of securin in two tissue types of cell lines, the vascular endothelial and colorectal epithelial cells, were investigated. Arsenite (8-16 microM, 24 h) increased the cytotoxicity, apoptosis, and growth inhibition in both endothelial and epithelial cells. The levels of phospho-CDC2 (threonine-161), CDC2, and cyclin B1 proteins were decreased, and the G2/M fractions were increased by arsenite. Concomitantly, arsenite markedly diminished the securin protein expression and induced the abnormal sister chromatid separation. The depletion of securin proteins increased the induction of mitotic arrest, aberrant chromosome segregation, and apoptosis after arsenite treatment. p53, a tumor suppressor protein, balances the cell survival and apoptosis. Arsenite raised the levels of phospho-p53 (serine-15) and p53 (DO-1) proteins in both the securin-wild-type and -null cells. The p53-functional cells were more susceptible than the p53-mutational cells to arsenite on the cytotoxicity and apoptosis. Besides, arsenite decreased the levels of securin proteins to a similar degree in both the p53-functional and -mutational cells. Together, it is the first time to demonstrate that the inhibition of securin expression induced by arsenite increases the chromosomal instability and apoptosis via a p53-independent pathway.     

The pivotal role of intracellular calcium in oxaliplatin-induced inhibition of neurite outgrowth but not cell death in differentiated PC12 cells

The antineoplastic efficacy of oxaliplatin, a widely used anticancer drug, is restricted by its adverse effects such as peripheral neuropathy. Infusing a combination of calcium gluconate and magnesium sulfate (Ca/Mg) suppresses the acute neurotoxic side effects of oxaliplatin, although the mechanism is unclear. To elucidate the molecular mechanisms of oxaliplatin-induced neurotoxicity and the effects of Ca/Mg against this toxicity, we examined the effect of Ca/Mg on oxaliplatin-induced inhibition of neurite outgrowth in PC12 cells, a commonly used neuronal cell model. Oxaliplatin and oxalate suppressed nerve growth factor (NGF)-induced neurite outgrowth and reduced the NGF-mediated increase in the intracellular calcium concentration [Ca(2+)](i). A calcium-chelating agent, BAPTA/AM, also exhibited similar inhibitory effects on neurite outgrowth and [Ca(2+)](i). The addition of Ca/Mg attenuated these inhibitions induced by oxaliplatin and oxalate. The NGF-induced upregulation of growth-associated protein-43 (GAP-43) was suppressed by oxaliplatin and oxalate. Oxaliplatin, but not oxalate, suppressed NGF-stimulated extracellular signal-regulated kinase activation, and this inhibition was not affected by Ca/Mg. Ca/Mg did not modify the oxaliplatin-induced loss of cell viability or apoptosis in PC12 or HCT-116 cells, a human colorectal cancer cell line. These results suggest that the inhibition of neurite outgrowth but not tumor cell death induced by oxaliplatin is partly associated with reductions in [Ca(2+)](i) and GAP-43 expression, and this inhibition was suppressed by the addition of Ca/Mg. Therefore, it may be assumed that Ca/Mg is useful for protecting against oxaliplatin-induced neurotoxicity without reducing the antitumor activity of oxaliplatin.     

ROS在奥沙利铂诱导PUMA表达致肠癌细胞凋亡过程中的作用

目的探讨活性氧分子在奥沙利铂诱导凋亡相关基因PUMA表达致肠癌细胞凋亡过程中的作用及影响。方法采用过氧化氢做为体外氧化应激的模型,WesternBlot检测过氧化氢处理后PUMA蛋白的表达,Hoechst33258荧光染色法检测过氧化氢处理后肠癌细胞的凋亡,MTT法检测在联合应用奥沙利铂和抗氧化剂后肠癌细胞的增殖率。结果一定量的过氧化氢可以诱导肠癌细胞的凋亡及PUMA的表达;抑制PUMA的表达可以减小过氧化氢诱导的肠癌细胞的凋亡;抗氧化剂可以减弱奥沙利铂对肠癌细胞中PUMA表达的诱导;抗氧化剂可以增加奥沙利铂处理后肠癌细胞的增殖率。结论PUMA在氧化应激诱导的肠癌细胞的凋亡过程中具有重要作用;氧化应激在奥沙利铂诱导肠癌细胞凋亡的过程中具有一定作用;奥沙利铂可能部分通过增加活性氧分子的产生而诱导PUMA的表达,从而诱导肠癌细胞发生凋亡。     

Role of Bcl-xL/Beclin-1 in interplay between apoptosis and autophagy in oxaliplatin and bortezomib-induced cell death

Recent studies indicate that a complex relationship exists between autophagy and apoptosis. In this study we investigated a regulatory relationship between autophagy and apoptosis in colorectal cancer cells utilizing molecular and biochemical approaches. For this study, human colorectal carcinoma HCT116 and CX-1 cells were treated with two chemotherapeutic agents—oxaliplatin, which induces apoptosis, and bortezomib, which triggers both apoptosis and autophagy. A combinatorial treatment of oxaliplatin and bortezomib caused a synergistic induction of apoptosis which was mediated through an increase in caspase activation. The combinational treatment of oxaliplatin and bortezomib promoted the JNK-Bcl-xL-Bax pathway which modulated the synergistic effect through the mitochondria-dependent apoptotic pathway. JNK signaling led to Bcl-xL phosphorylation at serine 62, oligomerization of Bax, alteration of mitochondrial membrane potential, and subsequent cytochrome c release. Overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed cytochrome c release and synergistic death effect. Interestingly, Bcl-xL also affected autophagy through alteration of interaction with Beclin-1. Beclin-1 was dissociated from Bcl-xL and initiated autophagy during treatment with oxaliplatin and bortezomib. However, activated caspase 8 cleaved Beclin-1 and suppressed Beclin-1-associated autophagy and enhanced apoptosis. A combinatorial treatment of oxaliplatin and bortezomib-induced Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133AA/D149A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. In addition, the combinatorial treatment significantly inhibited colorectal cancer xenografts’ tumor growth. An understanding of the molecular mechanisms of crosstalk between apoptosis and autophagy will support the application of combinatorial treatment to colorectal cancer.     

甲磺酸阿帕替尼通过p53抑制结肠癌细胞HCT116增殖的作用及机制

摘要：目的 探讨阿帕替尼对人结肠癌细胞HCT116及敲除野生型p53的HCT116细胞抑制作用的差异并探讨 其机制。方法 以0、15、30、60 μmol/L阿帕替尼处理HCT116 p53+/+、HCT116 p53-/-细胞24、48 h,以CCK-8法检测阿 帕替尼对两株细胞增殖的抑制作用;流式细胞术（Annexin V/PI法）检测0、15、30 μmol/L阿帕替尼处理细胞24 h后凋 亡率变化;实时荧光定量PCR法和Western blot法检测0、15、30 μmol/L阿帕替尼处理细胞24 h后,p53、NF-κB p65、 Caspase-3 mRNA和蛋白表达变化。结果 CCK-8结果显示,阿帕替尼能抑制HCT116 p53+/+、HCT116 p53-/-细胞的增 殖,抑制作用具有剂量依赖性（P＜0.01）。流式细胞术结果显示,阿帕替尼干预后,HCT116 p53+/+、HCT116 p53-/-细胞 凋亡率升高（P＜0.01）。与HCT116 p53+/+细胞相比,阿帕替尼处理后HCT116 p53-/-细胞的凋亡率较低（P＜0.05）。阿 帕替尼处理后,HCT116 p53+/+、HCT116 p53-/-细胞中 p53、NF-κB p65、capsase-3 mRNA 和 Caspase-3 蛋白表达升高 （P＜0.05）。阿帕替尼干预后,HCT116 p53+/+细胞中 p53、NF-κB p65 蛋白表达下调而 HCT116 p53-/-细胞中 NF-κB p65蛋白表达上调（P＜0.01）。结论 阿帕替尼可能通过p53/NF-κB通路抑制HCT116 p53+/+、HCT116 p53-/-细胞增殖 并促进细胞凋亡,结肠癌细胞可能通过野生型p53对阿帕替尼产生耐药。     

The blockage of survivin and securin expression increases the cytochalasin B-induced cell death and growth inhibition in human cancer cells

Survivin and securin proteins are overexpressed in most cancer cells that have been shown to regulate mitotic progression. In this study, we investigated the roles of survivin and securin on cytochalasin B, a cytokinesis blocker mediating the cytotoxicity and cell growth inhibition in human cancer cells. The human lung carcinoma cell lines A549 and H1299 highly expressed survivin proteins in mitosis and concentrated on the midbodies during cytokinesis. Cytochalasin B significantly decreased cell survival, inhibited cell growth, increased the levels of G(2)/M fractions, and induced binuclei formation in lung carcinoma cells; however, the survivin proteins were concentration-dependently increased by 1 to 5 mug/ml cytochalasin B for 24 h. It is noteworthy that the expression of securin proteins was decreased in cytochalasin B-treated lung carcinoma cells. Transfection of 20 to 40 nM survivin siRNA for 48 h significantly induced the formation of multiple nuclei and apoptosis but decreased the levels of survivin and securin proteins in A549 cells. Cotreatment with survivin small interfering RNA (siRNA) and cytochalasin B increased the cytotoxicity and cell growth inhibition. In addition, the securin-null colorectal carcinoma cells were more susceptible to the cytotoxicity after cytochalasin B and survivin siRNA treatments than the securin-wild-type cells. As a whole, our results indicate that the inhibition of survivin and securin protein expression may increase the cell death and growth inhibition after cytochalasin B treatment in human cancer cells.     

尼卡地平对结肠癌细胞HCT116/L耐药的逆转作用研究

目的 探究尼卡地平逆转耐奥沙利铂结肠癌细胞HCT116/L耐药的疗效及其机制.方法 采用MTT法分别检测尼卡地平和奥沙利铂在HCT116/L和HCT116细胞上的药敏性,并检测尼卡地平能否逆转奥沙利铂耐药.采用流式细胞仪(FCM)检测HCT116/L细胞凋亡和细胞内罗丹明-123的浓度.结果 奥沙利铂在耐药细胞HCT1...     

西红花苷抑制NFATc3活性促进与肿瘤成纤维细胞共培养的结直肠癌细胞放射敏感性

目的 探究西红花苷对与肿瘤成纤维细胞（CAFs）共培养的结直肠癌细胞放射敏感性的影响,并探究分子机制。方法 通过实时荧光定量聚合酶链式反应（qRT-PCR）和蛋白质免疫印记（Western blotting）比较人正常结肠上皮细胞NCM460和结直肠癌细胞系SW480、SW620、HCT116、LOVO中活化T细胞核因子c3（NFATc3）表达差异。建立CAFs与HCT116细胞共培养体系,西红花苷处理细胞后经不同剂量（0、2、4、6、8 Gy）X射线照射,细胞克隆形成实验分析细胞存活分数（SF）。再将HCT116细胞分为对照组、6 Gy组、CAFs/HCT116＋6 Gy组、CAFs/HCT116＋西红花苷＋6 Gy组,进行对应处理后,MTT法检测各处理组HCT116细胞活性,流式细胞术检测各处理组HCT116细胞凋亡率,Hoechst 33258染色观察各处理组HCT116细胞凋亡形态,qRT-PCR和Western blotting测定各处理组HCT116细胞中NFATc3表达水平。结果 相较于人正常结肠上皮细胞NCM460,人结直肠癌细胞系SW480、SW620、HCT116、LOVO中的NFATc3 mRNA和蛋白相对表达量均显著上调（P＜0.05）。与HCT116细胞比较,与CAFs共培养的HCT116细胞经辐射后的SF显著增高（P＜0.05）;与CAFs共培养的HCT116细胞比较,与CAFs共培养的HCT116细胞经西红花苷处理再进行辐射的SF显著降低（P＜0.05）。与6 Gy组比较,CAFs/HCT116＋6 Gy组HCT116细胞存活率显著升高（P＜0.05）,细胞凋亡率显著下降（P＜0.05）,细胞内染色也较为均匀,未见致密浓染的凋亡状细胞,细胞中NFATc3 mRNA和蛋白相对表达量均显著上调（P＜0.05）;而与CAFs/HCT116＋6 Gy组比较,CAFs/HCT116＋西红花苷＋6 Gy组HCT116细胞存活率显著降低（P＜0.05）,细胞凋亡率显著升高（P＜0.05）,细胞中有较多致密浓染、碎裂荧光块,同时,细胞中NFATc3 mRNA和蛋白相对表达量显著下调（P＜0.05）。结论 西红花苷能够明显提高与CAFs共培养的结直肠癌HCT116细胞的放射敏感性,从而促进细胞发生凋亡,该作用可能与抑制NFATc3有关。     

gamma-H2AX as a therapeutic target for improving the efficacy of radiation therapy

Exposure to ionizing radiation (IR) results in the formation of DNA double strand breaks, resulting in the activation of phosphatidylinositol 3'-kinase-like kinases ATM, ATR and DNK-PKcs. A physiologically important downstream target is the minor histone H2A variant, H2AX, which is rapidly phosphorylated on Ser 139 of the carboxyl tail after IR. Recent work suggests that phosphorylated H2AX (gamma-H2AX) plays an important role in the recruitment and/or retention of DNA repair and checkpoint proteins such as BRCA1, MRE11/RAD50/NBS1 complex, MDC1 and 53BP1. H2AX-/- mouse embryonic fibroblasts are radiation sensitive and demonstrate deficits in repairing DNA damage compared to their wildtype counterparts. Cells treated with peptide inhibitors of gamma-H2AX demonstrate increased radiosensitivity following radiation compared with untreated irradiated cells. Analysis of the kinetics of gamma-H2AX clearance after IR or other DNA damaging agents reveals a correlation between increased gamma-H2AX persistence and unrepaired DNA damage and cell death. These data highlight the potential of post-translational modifications of chromatin as a therapeutic target for enhancing the efficacy of radiotherapy. Therapies that either block gamma-H2AX foci formation by inhibiting upstream kinase activity or that directly inhibit H2AX function may interfere with DNA damage repair processes and warrant further investigation as potential radiosensitizing agents. Agents that increase persistence of gamma-H2AX after IR are likely to increase unrepaired DNA damage.