Volasertib suppresses tumor growth and potentiates the activity of cisplatin in cervical cancer

Volasertib (BI 6727), a highly selective and potent inhibitor of PLK1, has shown broad antitumor activities in the preclinical and clinical studies for the treatment of several types of cancers. However, the anticancer effect of volasertib on cervical cancer cells is still unknown. In the present study, we show that volasertib can markedly induce cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the decreased protein expressions of PLK1 substrates survivin and wee1 in human cervical cancer cells. Furthermore, volasertib also enhances the intracellular reactive oxidative species (ROS) levels, and pretreated with ROS scavenger N-acety-L-cysteine totally blocks ROS generation but partly reverses volasertib-induced apoptosis. In addition, volasertib significantly potentiates the activity of cisplatin to inhibit the growth of cervical cancer in vitro and in vivo. In brief, volasertib suppresses tumor growth and potentiates the activity of cisplatin in cervical cancer, suggesting the combination of volasertib and cisplatin may be a promising strategy for the treatment of patients with cervical cancer.     

15-Deoxy-Δ<Superscript>12,14</Superscript>-prostaglandin J<Subscript>2</Subscript> inhibits G<Subscript>2</Subscript>-M phase progression in human breast cancer cells via the down-regulation of cyclin B1 and survivin expression

The cyclopentenone prostaglandin 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) exerts a growth inhibitory effect on cancer cells, and this effect is linked to the induction of apoptosis or cell cycle arrest. Induction of apoptosis by 15d-PGJ₂ is associated with the down-regulation of anti-apoptotic proteins. G₀-G₁→S phase progression is inhibited by 15d-PGJ₂ via the degradation of cyclin D1. In this study, we further investigated the mechanism by which 15d-PGJ₂ inhibits cancer cell growth by using the breast cancer cell lines MCF-7 and T-47D. Treatment with 20 μM 15d-PGJ₂ for 72 h completely blocked the growth in both cell lines. However, the proportions of apoptotic MCF-7 and T-47D cells were 21.1% and 40.9%, respectively, indicating that the induction of apoptosis did not appear to fully account for growth inhibition by 15d-PGJ₂. Cell cycle analysis using cells synchronized at the G₀-G₁ or S phase revealed that 15d-PGJ₂ blocked not only G₀-G₁→S phase progression but also G₂-M phase progression. The expression of both cyclins D1 and B1 was decreased by 15d-PGJ₂. Furthermore, 15d-PGJ₂ inhibited aurora-B kinase activity, which coincided with the down-regulation of survivin. Thus, 15d-PGJ₂ induced cell cycle arrest at the G₂-M phase via inhibition of cyclin B1 expression and aurora-B kinase activity. We conclude that survivin may be an important target for 15d-PGJ₂, and its down-regulation may lead to a decrease in aurora-B kinase activity. Naoki Tsuji substantially contributed to this work and should also be considered a first author.     

姜黄素对脑胶质瘤的抑制作用及其机制研究

目的：研究姜黄素（curcumin）对脑胶质瘤的治疗作用,揭示姜黄素抗脑胶质瘤作用的分子机制。方法：U251胶质瘤细胞培养和药物处理;药物敏感性试验检测姜黄素抑制胶质瘤细胞体外生长作用;流式细胞仪检测细胞周期;DNA梯度（DNA ladder）凝胶电泳检测细胞凋亡;Western blot分析蛋白表达;利用t检验或one-way ANOVA分析统计学差异。结果：低剂量姜黄素对U251细胞没有明显的生长抑制作用,但高剂量作用下,生长抑制率达35%,与阴性对照相比差异显著（P〈0.05）;10μmol/L的姜黄素作用24h后细胞中ING4蛋白表达水平显著增加,p21waf/cipl和p53蛋白表达明显上调。结论：本研究发现姜黄素以p53依赖方式诱导脑瘤细胞发生细胞周期阻滞,在此过程中ING4表达上调,提示ING4可能通过增加与p53的结合,上调p21,发挥细胞周期阻滞作用。     

Quinacrine has anticancer activity in breast cancer cells through inhibition of topoisomerase activity

The small molecule Quinacrine (QC, a derivative of 9-aminoacridine), an anti-malaria drug, displays activity against cancer cell lines and can simultaneously suppress nuclear factor-κB (NF-κB) and activate p53 signaling. In this study, we investigated the anticancer mechanism underlying these drug activities in breast cancer cell lines. QC caused a dose-dependent decrease of both anchorage dependent and independent growth of breast cancer cells (MCF-7 and MDA-MB-231) without affecting normal breast epithelial cells (MCF-10A), as evident from clonogenic cell survival, [3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide] viability, wound healing and soft agar growth. QC activated the proapoptotic marker Bax, PARP cleavage, p53 and its downstream target, p21 (Cip1/Waf1) and downregulated the antiapoptotic marker Bcl-xL and relative luciferase activity of NF-κB in MCF-7 cells. Results of DAPI nuclear staining and FACS analysis show that QC increased apoptosis in a dose-dependent manner. QC caused apoptosis by increasing the cell population in S-phase and simultaneously decreasing the G1 and G2/M populations. A dose-dependent increase of DNA damage as measured by the comet assay was seen in MCF-7 cells after exposure to QC. With regards to the mechanism of DNA damage, we found that QC inhibited topoisomerase activity in MCF-7 cells by increasing the unwinding of supercoiled DNA. Collectively, the results demonstrate that QC has efficient anticancer potential against breast cancer cells via not only an induction of p53 and p21 but also an induction of S phase arrest, DNA damage and inhibition of topoisomerase activity.     

The bacterial cytolethal distending toxin (CDT) triggers a G2 cell cycle checkpoint in mammalian cells without preliminary induction of DNA strand breaks

The bacterial cytolethal distending toxin (CDT) was previously shown to arrest the tumor-derived HeLa cell line in the G2-phase of the cell cycle through inactivation of CDK1, a cyclin-dependent kinase whose state of activation determines entry into mitosis. We have analysed the effects induced in HeLa cells by CDT, in comparison to those induced by etoposide, a prototype anti-tumoral agent that triggers a G2 cell cycle checkpoint by inducing DNA damage. Both CDT and etoposide inhibit cell proliferation and induces the formation of enlarged mononucleated cells blocked in G2. In both cases, CDK1 from arrested cells could be reactivated both in vitro by dephosphorylation by recombinant Cdc25B phosphatase and in vivo by caffeine. However, the cell cycle arrest triggered by CDT, unlike etoposide, did not originate from DNA strand breaks as demonstrated in the single cell gel electrophoresis assay and by the absence of slowing down of S phase in synchronized cells. Together with additional observations on synchronized HeLa cells, our results suggest that CDT triggers a G2 cell cycle checkpoint that is initiated during DNA replication and that is independent of DNA damage.     

Isoliquiritigenin induces G2 and M phase arrest by inducing DNA damage and by inhibiting the metaphase/anaphase transition

Isoliquiritigenin, a natural flavonoid found in licorice, shallots, and bean sprouts, has been demonstrated to inhibit proliferation and to induce apoptosis in a variety of human cancer cells. We attempted to ascertain the underlying mechanism by which isoliquiritigenin induced cell cycle arrest and cytotoxicity in HeLa human cervical cancer cells. Isoliquiritigenin treatment arrested cells in both G2 and M phase. The cells arrested in interphase (G2) showed markers for DNA damage including the formation of γ-H2AX foci and the phosphorylation of ATM and Chk2, whereas the cells arrested in M phase evidenced separate poles and mitotic metaphase-like spindles with partially unaligned chromosomes. The induction of DNA damage and blockade at the metaphase/anaphase transition implied that isoliquiritigenin might function as a topoisomerase II poison, which was further demonstrated via an in vitro topoisomerase II inhibition assay. These results show that isoliquiritigenin inhibits topoiosmerase II activity, and the resultant DNA damage and arrest in mitotic metaphase-like stage contributes to the antiproliferative effects of isoliquiritigenin.     

Down-regulation of survivin by ultraviolet C radiation is dependent on p53 and results in G(2)-M arrest in A549 cells

Deregulation of survivin expression is implicated in tumorigenesis. To examine the regulation of survivin expression in response to DNA damage, we exposed A549 human lung cancer cells to ultraviolet C (UVC) radiation, which induces DNA single-strand breakage. UVC irradiation induced G(2)-M arrest that was accompanied by accumulation of p53 and subsequent down-regulation of survivin. Depletion of p53 by RNA interference prevented the UVC-induced down-regulation of survivin. Furthermore, depletion of survivin resulted in G(2)-M arrest, suggesting that down-regulation of survivin by p53 contributes to the p53-dependent G(2)-M checkpoint triggered by DNA damage.     

RNA干扰survivin基因对卵巢癌细胞生长凋亡及药物敏感性的影响

目的 探讨下调survivin基因对卵巢癌顺铂(DDP)耐药细胞株SKOV3/DDP细胞生长、凋亡及药物敏感性的影响.方法 构建survivin基因的小干扰RNA(siRNA)表达载体pSilencer-survivin,用脂质体方法转染SKOV3/DDP细胞株,另设未转染组和转染pSilencer-control组作为对照.显微镜下观察转染前后细胞的变化,逆转录聚合酶链反应(RT-PCR)和Western blot分别检测survivinmRNA及蛋白的表达,二苯基溴化四氮唑蓝(MTT)法检测细胞生长情况,流式细胞仪检测细胞凋亡及周期的变化.各组细胞加入DDP,检测其对DDP药物敏感性的影响.结果 survivin siRNA可明显下调SKOV3/DDP细胞中survivin mRNA及蛋白表达水平.SKOV3/DDP细胞生长受到明显抑制,生长曲线低平.转染48 h后,转染pSilencer-survivin组细胞凋亡率为19.1%,明显高于末转染组(2.6%)和转染pSilencer-control组(3.5%),G1/G0期细胞所占的比例增高,而G2/M期细胞所占的比例降低.转染pSilencer-survivin组细胞的IC50明显降低,为1.16 μg/mi.结论 survivin siRNA可下调SKOV3/DDP细胞中survivin的表达,使细胞生长减慢,凋亡增加,对DDP的药物敏感性增强.     

Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells

CHK1 and CHK2 function as effectors of cell cycle checkpoint arrest following DNA damage. Small molecule inhibitors of CHK proteins are under clinical evaluation in combination with chemotherapeutic agents known to induce DNA damage. We examined whether CHK inhibitors could be effective as single agents in malignant cells with inherent DNA damage because of deregulated expression of the oncogene c-Myc. Eμ-myc lymphoma cells showed a dramatic increase in the extent of DNA damage and DNA damage response (DDR) signalling within 1 h of treatment with CHK1 inhibitors followed by caspase-dependent apoptosis and cell death. In p53 wild-type/ARF null Eμ-myc lymphoma cells, apoptotic cell death was preceded by accumulation of DNA damage and the amount of DNA damage correlated with the extent of cell death. This effect was not observed in normal B cells indicating that DNA damage accumulation following CHK inhibition was specific to Eμ-myc lymphoma cells that exhibit inherent DNA damage because of MYC-induced replication stress. Similar results were obtained with another structurally distinct CHK-inhibitor. Eμ-myc p53 null lymphoma cells were more sensitive to a dual CHK1/CHK2 inhibitor than to a CHK1-specific inhibitor. In all cases, the level of DNA damage following treatment was the most consistent indicator of drug sensitivity. Our results suggest that CHK inhibitors would be beneficial therapeutic agents in MYC-driven cancers. We propose that inhibitors of CHK can act in a synthetically lethal manner in cancers with replication stress as a result of these cancers being reliant on CHK proteins for an effective DDR and cell survival.     

siRNA抑制survivin基因周期的影响表达对HeLa细胞

目的设计靶向survivin基因的干涉片段,构建真核表达载体pSUPER／survivin并转染宫颈癌HeLa细胞,探讨siRNA抑制survivin表达对联合1射线宫颈癌HeLa细胞周期的影响。方法化学合成干涉引物,退火获得survivin基因的干涉片段,连入pSUPER载体并测序。将测序正确的pSUPER／survivin载体转染HeLa细胞株,RT—PCR法和流式细胞术检测HeLa细胞中survivin基因的表达,流式细胞术检测转染联合＇射线照射后各组细胞的细胞周期数据。结果本实验成功的构建了pSUPER／survivin载体。转染pSUPER／survivin的HeLa细胞其survivin基因表达水平明显低于未转染的HeLa细胞及转染空载体pSUPER的HeLa细胞。1射线照射后,转染pSUPER／survivin的HeLa细胞发生了S期细胞比例下降,及G2／M期阻滞。结论成功的构建了pSUPER／survivin载体,siRNA干涉survivin可以作为提高放射敏感性的潜在分子靶点,通过减少S期细胞比例,促进肿瘤细胞凋亡,通过增加G2／M期阻滞增加放射线的杀伤。     

Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.     

Induction of p53-independent Apoptosis Associated with G2M Arrest Following DNA Damage in Human Colon Cancer Cell Lines

The tumor suppressor p53 protein induces apoptosis in response to various kinds of DNA damage in normal cells, but it is still unclear whether or not apoptosis induced by DNA damage correlates with the p53 status in tumor cells. We determined the status of p53 by functional analysis of separated alleles in yeast in five human colon cancer cell lines, SW-480, SW-620, DLD-1, COLO320 and LS174T and investigated whether p53 is necessary for apoptosis and cell cycle arrest after treatment of the cells with a DNA-damaging agent, etoposide (VP-16), or γ-irradiation. Of these cell lines, only LS174T expresses a functional p53. Apoptosis was detected in SW-480 and COLO320 cell lines, but not in the other cell lines, including LS174T cell line with a normal p53 function. Furthermore, cell cycle analysis revealed accumulation in the G2M phase preceding induction of apoptosis in SW-480 and COLO320 cells, but not in the other cells. These results suggest that apoptotic induction by DNA damage is not necessarily related to p53 status and that induction of p53-independent apoptosis following DNA damage may correlate with G2M arrest in the cell cycle, at least in the colon cancer cell lines used in this study.     

Mode of action of thiocoraline, a natural marine compound with anti-tumour activity.

Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase alpha at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase alpha activity.     

Sevoflurane inhibits proliferation, induces apoptosis, and blocks cell cycle progression of lung carcinoma cells

Purpose: Sevoflurane, an inhalational anesthetic, is used extensively during lung cancer surgery. However, the effect of sevoflurane on growth of lung carcinoma cells remains unclear. The purpose of this study is to investigate effects on proliferation, apoptosis, and cell cycling in the A549 human lung adenocarcinoma cell line. Methods: A549 cells were treated with 1.7%, 3.4%, and 5.1 % sevoflurane for 2, 4, and 6 hours. Cell proliferation was evaluated by the MTT assay and colony formation assay. Apoptosis and cell cycle was analyzed by flow cytometry. Expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, Bcl-2, Bax, caspase-3, cyclin A, cyclin B1, and cdc2 was measured by Western blotting. Results: Sgnificant inhibition of cell proliferation and induction of apoptosis were found in A549 cells after sevoflurane treatment. Simultaneously, expression of XIAP and survivin was surpressed, while that of caspase-3 increased significantly, but Bcl-2 and Bax were not altered. Sevoflurane caused cell cycle arrest at the G2/M phase. At the same time, data revealed that cyclin A, cyclin B1, and cdc2 expression was down-regulated after sevoflurane treatment. Conclusion: This study demonstrated that sevoflurane inhibited proliferation, and induced apoptosis in human lung adenocarcinoma A549 cells, associated with down-regulated expression of XIAP and suvivin, and activating caspase-3.     

Role of cell cycle in mediating sensitivity to radiotherapy

Multiple pathways are involved in maintaining the genetic integrity of a cell after its exposure to ionizing radiation. Although repair mechanisms such as homologous recombination and nonhomologous end-joining are important mammalian responses to double-strand DNA damage, cell cycle regulation is perhaps the most important determinant of ionizing radiation sensitivity. A common cellular response to DNA-damaging agents is the activation of cell cycle checkpoints. The DNA damage induced by ionizing radiation initiates signals that can ultimately activate either temporary checkpoints that permit time for genetic repair or irreversible growth arrest that results in cell death (necrosis or apoptosis). Such checkpoint activation constitutes an integrated response that involves sensor (RAD, BRCA, NBS1), transducer (ATM, CHK), and effector (p53, p21, CDK) genes. One of the key proteins in the checkpoint pathways is the tumor suppressor gene p53, which coordinates DNA repair with cell cycle progression and apoptosis. Specifically, in addition to other mediators of the checkpoint response (CHK kinases, p21), p53 mediates the two major DNA damage-dependent cellular checkpoints, one at the G(1)-S transition and the other at the G(2)-M transition, although the influence on the former process is more direct and significant. The cell cycle phase also determines a cell's relative radiosensitivity, with cells being most radiosensitive in the G(2)-M phase, less sensitive in the G(1) phase, and least sensitive during the latter part of the S phase. This understanding has, therefore, led to the realization that one way in which chemotherapy and fractionated radiotherapy may work better is by partial synchronization of cells in the most radiosensitive phase of the cell cycle. We describe how cell cycle and DNA damage checkpoint control relates to exposure to ionizing radiation.     

FR901228, a novel histone deacetylase inhibitor, induces cell cycle arrest and subsequent apoptosis in refractory human pancreatic cancer cells

Histone deacetylase (HDAC) inhibitors have antiproliferative activity against human cancer cells via cell cycle arrest, differentiation, and apoptosis. However, no report has focused on the apoptotic potential of HDAC inhibitors in refractory human pancreatic cancer. This study was designed to examine the apoptotic potential of FR901228, a novel HDAC inhibitor, in five human pancreatic cancer cell lines: Capan-1, BxPC-3, HPAF, Panc-1, and MIAPaCa-2. FR901228 markedly inhibited the proliferation of all five cell lines (IC50: 1-500 nM), with the greatest effect in MIAPaCa-2 cells. Treatment of each cell line with FR901228 (10-100 nM) caused cell cycle arrest at the G1 or G2/M phase and subsequent apoptosis. FR901228 induced expression of hyperacetylated histone H3 after 3 h of treatment and overexpression of p21Waf-1 after 6 h. In addition, FR901228 induced apoptosis by activating caspase-3, which led to cleavage of p21Waf-1 into a 15-kDa breakdown product and drove cancer cells from cell cycle arrest into apoptosis. FR901228 also decreased the protein level of survivin dramatically. Our results show that FR901228 markedly inhibits the growth of pancreatic cancer cells, not only through cell cycle arrest, but also through subsequent apoptosis; this was accompanied by caspase-3 activation, survivin degradation, and p21Waf-1 cleavage. FR901228 may prove clinically useful as an agent for refractory pancreatic cancers.     

Cotylenin A, a new differentiation inducer, and rapamycin cooperatively inhibit growth of cancer cells through induction of cyclin G2

Cotylenin A, a plant growth regulator, and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), are potent inducers of differentiation of myeloid leukemia cells. Recently, we found that cotylenin A and rapamycin effectively inhibited the proliferation of several human breast cancer cell lines including MCF-7. Herein, we demonstrate that cotylenin A and rapamycin rapidly and markedly induced the cyclin G2 gene expression in several cancer cells including MCF-7 cells. The growth arrest of the MCF-7 cells at the G1 phase, induced by the treatment with cotylenin A and rapamycin or the culture in low serum medium, markedly induced the cyclin G2 gene expression. Anticancer drugs including doxorubicin, etoposide and 5-fluorouracil also induced cyclin G2 expression during induction of growth arrest of the MCF-7 cell at the G1 phase or G2/M phase. Ectopically inducible cyclin G2 expression potently inhibited the proliferation of MCF-7 cells. Furthermore, cyclin G2 knockdown induced by cyclin G2 small interfering RNA markedly reduced the potency of cotylenin A plus rapamycin to induce growth inhibition. Taken together, our results suggest that cotylenin A and rapamycin induce inhibition of cancer cell growth through the induction of cyclin G2. ( Cancer Sci 2008; 99: 1693–1698)     

Phenethyl isothiocyanate (PEITC) promotes G2/M phase arrest via p53 expression and induces apoptosis through caspase- and mitochondria-dependent signaling pathways in human prostate cancer DU 145 cells

Phenethyl isothiocyanate (PEITC), one of many compounds found in cruciferous vegetables, has been reported as a potential anticancer agent. In earlier studies, PEITC was shown to inhibit cell growth and induction of apoptosis in many cancer cell lines. However, no report has shown whether PEITC can induce apoptosis in human prostate cancer cells. Herein, we aimed to determine whether PEITC has anticancer activity in DU 145 human prostate cancer cells. As a result, we found that PEITC induced a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G(2)/M phase of DU 145 cells. PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G(2)/M phase arrest was mediated by the increase of p53 and WEE1 and it reduced the level of CDC25C protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results also showed that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. This study showed that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.