Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines

Sulforaphane, an isothiocyanate found in cruciferous vegetables, has been shown to induce phase 2 detoxication enzymes and inhibit the growth of chemically induced mammary tumors in rats, although the exact mechanisms of action of sulforaphane are not understood. In this study, we evaluated the effects of sulforaphane on cell growth and death in several human breast cancer cell lines and examined the hypothesis that sulforaphane acts as a histone deacetylase (HDAC) inhibitor in these cell lines. Sulforaphane treatment inhibited cell growth, induced a G(2)-M cell cycle block, increased expression of cyclin B1, and induced oligonucleosomal DNA fragmentation in the four human breast cancer cell lines examined, MDA-MB-231, MDA-MB-468, MCF-7, and T47D cells. Activation of apoptosis by sulforaphane in MDA-MB-231 cells seemed to be initiated through induction of Fas ligand, which resulted in activation of caspase-8, caspase-3, and poly(ADP-ribose) polymerase, whereas apoptosis in the other breast cancer cell lines was initiated by decreased Bcl-2 expression, release of cytochrome c into the cytosol, activation of caspase-9 and caspase-3, but not caspase-8, and poly(ADP-ribose) polymerase cleavage. Sulforaphane inhibited HDAC activity and decreased the expression of estrogen receptor-alpha, epidermal growth factor receptor, and human epidermal growth factor receptor-2 in each cell line, although no change in the acetylation of H3 or H4 was seen. These data suggest that sulforaphane inhibits cell growth, activates apoptosis, inhibits HDAC activity, and decreases the expression of key proteins involved in breast cancer proliferation in human breast cancer cells. These results support testing sulforaphane in vivo and warrant future studies examining the clinical potential of sulforaphane in human breast cancer.     

Sonoporation induces apoptosis and cell cycle arrest in human promyelocytic leukemia cells

Despite being a transient biophysical phenomenon, sonoporation is known to disturb the homeostasis of living cells. This work presents new evidence on how sonoporation may lead to antiproliferation effects including cell-cycle arrest and apoptosis through disrupting various cell signaling pathways. Our findings were obtained from sonoporation experiments conducted on HL-60 human promyelocytic leukemia cells (with 1% v/v microbubbles; 1 MHz ultrasound; 0.3 or 0.5MPa peak negative pressure; 10% duty cycle; 1 kHz pulse repetition frequency; 1 min exposure period). Membrane resealing in these sonoporated cells was first verified using scanning electron microscopy. Time-lapse flow cytometry analysis of cellular deoxyribonucleic acid (DNA) contents was then performed at four post-sonoporation time points (4 h, 8 h, 12 h and 24 h). Results indicate that an increasing trend in the apoptotic cell population can be observed for at least 12 h after sonoporation, whilst viable sonoporated cells are found to temporarily accumulate in the G₂/M (gap-2/mitosis) phase of the cell cycle. Further analysis using western blotting reveals that sonoporation-induced apoptosis involves cleavage of poly adenosine diphosphate ribose polymerase (PARP) proteins: a pro-apoptotic hallmark related to loss of DNA repair functionality. Also, mitochondrial signaling seems to have taken part in triggering this cellular event as the expression of two complementary regulators for mitochondrial release of pro-apoptotic molecules, Bcl-2 (B-cell lymphoma 2) and Bax (Bcl-2-associated X), are seen to be imbalanced in sonoporated cells. Furthermore, sonoporation is found to induce cell-cycle arrest through perturbing the expression of various cyclin and Cdk (cyclin-dependent kinase) checkpoint proteins that play an enabling role in cell-cycle progression. These bioeffects should be taken into account when using sonoporation for therapeutic purposes.     

Poncirus trifoliata fruit induces apoptosis in human promyelocytic leukemia cells

Background: Substances inducing apoptosis have shown efficacy in the treatment of cancers. Poncirus trifoliata (L.) Raf. (Rutaceae) fruits (PTF) has been used for the treatment of various cancers among Korean Oriental Medical doctors. Methods: PTF-induced cytotoxicity of human leukemia HL-60 cells was monitored by the MTT assay. The apoptosis was determined by (a) apoptotic morphology in microscopy; (b) DNA fragmentation in electrophoresis and FACS analysis; and (c) activation of caspase-3 and poly-ADP-ribose polymerase (PARP) cleavage assay. Results: The cytotoxic activity of PTF in HL-60 cells was increased in a concentration- and time-dependent manner. PTF caused the cell shrinkage, cell membrane blebbing, apoptotic body and DNA fragmentation. PTF-induced apoptosis is accompanied by the activation of caspase-3 and the specific proteolytic cleavage of PARP. However, PTF did not show cytotoxicity in normal peripheral blood mononuclear cells. Conclusions: Our novel finding provides evidence that PTF could be a candidate as an anti-leukemic agent through apoptosis of cancer cells.     

Plumbagin induces ROS-mediated apoptosis and cell cycle arrest and inhibits EMT in human cervical carcinoma cells

Plumbagin, an important phytochemical from the roots of the medicinal plant Plumbago zeylanica L. has shown many biological activities. The roots of this plant have been in use in the Indian system of medicine for more than twenty five centuries for treatments of various ailments. It has shown anticancer activities, however, the anticancer and anti-metastatic effects of plumbagin are largely unknown against cervical cancer cells. Herein, we investigated the molecular alterations associated with plumbagin-mediated inhibition of growth, survival and epithelial to mesenchymal transition of human cervical cancer SiHa and HeLa cells. Plumbagin (1–4 μM) caused a significant decrease in the cell viability and increased the cell death in SiHa and Hela cells after 24 and 48 h. Plumbagin also caused strong G2/M and S-G2/M phase cell cycle arrest in SiHa and HeLa cells, respectively which was accompanied by a decrease in the expression of cyclin and CDK levels. The expression levels of both mRNAs and proteins of cyclin B1, A and E2 and CDK 1 and 2 decreased after 24 and 48 h. Plumbagin strongly induced apoptosis along with increased ratio of Bax : Bcl2 and cleavage of caspase 3, 9, and PARP. Plumbagin caused a significant increase in reactive oxygen species generation which mediated cell death as it was attenuated by pre-treatment with N-acetyl cysteine. Additionally, we also report for the first time that plumbagin possesses an anti-metastatic effect at non-cytotoxic doses that was accompanied by the modulation of MMP-2, 9, E-cadherin, N-cadherin, β-catenin and vimentin. Taken together, our findings suggest that plumbagin has strong anticancer and anti-metastatic effects against human cervical cancer cells.

Proposed mechanism of action of plumbagin in human cervical carcinoma SiHa and HeLa cells.     

Lobaplatin suppresses proliferation and induces apoptosis in the human colorectal carcinoma cell Line LOVO in vitro

Lobaplatin, as the third-generation platinum antineoplastic agent, showed promising antineoplastic effects in variety of preclinical test tumor models. We investigated the inhibition effect of lobaplatin on the colorectal carcinoma cell line LOVO in vitro, and explored its mechanism of action. The MTT assay was used to determine the inhibitory effect and inhibition ratio of lobaplatin on LOVO at various lobaplatin concentrations (500 μM, 1000 μM, 2000 μM). Apoptosis was detected by terminal deoxynucleotide transferase-mediated dUTP nickend labelling (TUNEL). The cell cycle and apoptotic rate were analyzed by flow cytometry (FCM) and the expression of caspase-3,8,9 in cells was detected by chromometry. The results of MTT assay showed that proliferation of LOVO cells was inhibited by lobaplatin in a concentration-dependent manner. Apoptosis was detected in LOVO cells by TUNEL. The FCM assay indicated that lobaplatin altered the cell cycle and induced apoptosis of the LOVO cells when treated for 24 h, the percentages of cells in the S phase transition were increased, whereas the percentages of cells in the G₂ transition were decreased. The expressions of caspase-389 is higher than the control group after LOVO cells were treated by lobaplatin. Lobaplatin can inhibit the proliferation of colorectal carcinoma cell line LOVO by inducing apoptosis in vitro. The mechanism may be related to the “S” cycle arrest in cell cycle distribution and the up-regulated expression of caspase-8 and caspase-9 which up-regulated the expression of caspase-3.     

RASSF10 is epigenetically inactivated and induces apoptosis in lung cancer cell lines

Ras-association domain family 10 (RASSF10), the latest member of the RASSF family with Ras effector function, has been frequently inactivated by aberrant promoter hypermethylation in several human cancers. However, its role in lung cancer has remained unclear. In this study, we investigated the methylation status of RASSF10 by combined bisulfate restriction analysis (COBRA) and examined its preliminary function in lung cancer cell lines. RASSF10 was methylated in four out of six lung cancer cell lines, including NCI-H157, NCI-460, SPCA-1 and NCI-H446. Treatment with a DNA methylation inhibitor, 5-aza-2′-deoxycytiding (5-aza-DC), restored RASSF10 mRNA expression and the restoration of RASSF10 increased cell apoptosis in a dose dependent manner, whereas knockdown of RASSF10 improved cell proliferation ability and inhibited cell apoptosis rate significantly. Immunofluorescence revealed that RASSF10 protein was located in the cell membrane. Taken together, our data for the first time demonstrates the frequent epigenetic inactivation of RASSF10 in lung cancer cell lines. RASSF10 induces cell apoptosis and might function as a tumor suppressor gene in lung cancer.     

Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo

In this study, we investigated the functional role of Akt and c-jun-NH(2)-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reduction in apigenin-induced caspase activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied by inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens.     

卡介苗对人鼻咽癌CNE-2Z细胞周期及凋亡的影响

目的 探讨卡介苗(BCG)对鼻咽癌CNE-2Z细胞周期及凋亡的影响.方法 碘化丙啶染色法检测细胞周期,Annexin V-FITC检测细胞凋亡,Western blotting检测周期相关蛋白表达.结果 BCG作用不同时间后,G1期细胞百分率由未处理细胞(对照组)的(47.1±0.9)%升高至72 h作用组的(65.3±1.3)%,S期细胞百分率由对照组的(39.2±0.7)%降低至72 h作用组的(17.6±0.5)%.不同处理时间组和对照组均未检出明显细胞凋亡.CyclinD1蛋白表达随着BCG作用时间的延长逐渐减弱.结论 BCG体外诱导鼻咽癌CNE-2Z细胞CyclinD1低表达,促使细胞周期发生G1/S期阻滞.     

The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD184352 (CI-1040) selectively induces apoptosis in malignant schwannoma cell lines

Type 1 neurofibromatosis (NF1) is a common autosomal dominant disorder that results in neuroectodermal tumors. The NF1 tumor-suppressor gene encodes neurofibromin, which includes a GTPase-activating domain for Ras inactivation. Affinity purification showed N-Ras to be the predominant activated isoform of Ras in two independent neurofibrosarcoma cell lines from NF1 patients (lines ST88-14 and NF90-8). These NF1 cells also demonstrated increased constitutive activity of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) mitogen-activated protein (MAP) kinases compared with a sporadic malignant schwannoma cell line that maintains neurofibromin expression (STS-26T). Thus, MAP kinase kinase (MEK) inhibitors may be a rational approach to NF1 therapy. The MEK inhibitors PD98059 [2'-amino-3'-methoxyflavone], PD184352 (also called CI-1040) [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide], and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] all produced concentration-dependent suppression of the proliferation of the three cell lines. Individual MEK inhibitors had similar effects in all three cell lines. However, only the antiproliferative effects of PD184352 correlated closely with the elimination of ERK1,2 MAP kinase activities. PD98059 was primarily cytostatic, whereas U0126 and PD184352 were cytotoxic. Only PD184352 induced apoptosis in all three lines, as indicated by morphology, activation of DEVDase, procaspase-3 cleavage, and the appearance of populations having sub-G(0)/G(1) DNA contents. The differential effects of the MEK inhibitors on cell survival were not dependent on p53 status or effects on the ERK5 pathway. PD184352 was also proapoptotic to primary rat Schwann cells. Hence, although PD184352 effectively killed neurofibrosarcoma cells, its effects on normal Schwann cells may limit its usefulness in the clinic.     

Bovine seminal RNase induces apoptosis in normal proliferating lymphocytes

Bovine seminal ribonuclease is a member of the RISBAses (ribonucleases with special biological actions) family. It exerts specific anti-tumor, embryotoxic, aspermatogenic and immunosuppressive activities. The cytotoxic effect of bovine seminal ribonuclease on tumor cells is accompanied by the induction of apoptosis. We provide ultrastructural and flow cytometry evidence of apoptotic death following bovine seminal ribonuclease treatment, in normal cells and phytohemagglutinin-stimulated lymphocytes. Transmission and scanning electron microscopy, which were fully supported by flow cytometry data, showed typical features of apoptosis, such as decreased cell size, chromatin condensation, fragmentation in micronuclei, and the presence of apoptotic bodies.     

Novel taspine derivative 12k inhibits cell growth and induces apoptosis in lung cell carcinoma

Taspine is an active compound in anticancer agent development. 12k was synthesized with taspine as lead compound bearing biphenyl scaffold and showed potent anticancer activity. Here, we investigated the effect of taspine derivative 12k on A549 lung cells. We showed that 12k not only decreased significantly A549 cell viability, A549 cell colony formation but also impaired A549 cell migration. Moreover, 12k treatment blocked cell cycle progression by increasing cell number in S phase to 42.80% for 6 μmol/L vs. 28.86% for control while decreasing cell number in G1 phase. Accordingly, this was associated with an increase protein expression of cyclin E and a decrease protein expression of cyclin D1, cyclin B1 and its associated CDK1 (cdc2). Meanwhile, we found that 12k induced A549 cell apoptosis, which was closely associated with the effect of the Bcl-2 family. Increase of Bad, Bak and Bax expression levels, decrease of Bcl-2 and Mcl-1 expression levels were observed. SiRNA knockdown of c-myc in A549 cells significantly attenuated tumor inhibition effects of 12k. In conclusion, our results demonstrate that 12k has an inhibitory effect on growth of A549 cell by inducing cell cycle arrest and apoptosis.     

抗氧化剂吡咯烷二硫代氨基甲酸盐对宫颈癌SiHa细胞增殖和凋亡的影响

目的:探讨抗氧化剂吡略烷二硫代氨基甲酸盐(PDTC)的体外抗宫颈癌作用机制.方法:体外培养SiHa细胞,PDTC处理后采用WST-8方法检测SiHa细胞的增殖活性:Hoechst 33258染色、Annexin V-FITC/PI双标法细胞凋亡试剂盒检测细胞凋亡;凝胶迁移变动分析(EMSA)检测SiHa细胞核内核转录因子-kB(NF-kB)活性.结果:PDTC能有效抑制SiHa细胞增殖和诱导SiHa细胞凋亡;染色可见典型的凋亡细胞形态;各组间早期凋亡率比较差异有显著性(P＜0.01),100 μmol/L PDTC晚期凋亡率较其他组明显升高(P＜0.01):PDTC可抑制SiHa细胞核内NF-kB活性,其抑制强度与作用24 h细胞生长抑制率呈直线正相关(r=0.931,P=0.035),与作用48 h早期凋亡率呈曲线正相关(r=1.000,P=0.011).结论:PDTC抑制SiHa细胞增殖与其诱导细胞凋亡作用有关,而致凋亡作用可能与其抑制NF-kB信号转导通路,启动凋亡程序有关.     

Lobaplatin induces apoptosis and arrests cell cycle progression in human cholangiocarcinoma cell line RBE

The aim of this study was to determine the anticancer effects of lobaplatin in cholangiocarcinoma (CCA) RBE cells. We also explored the mechanism of action of lobaplatin by analyzing its influence on apoptosis and cell cycle. Our findings have shown that lobaplatin inhibits cell proliferations in human CCA cells with an IC50 value of approximately 5.26 ± 0.63 μg/mL. Flowcytometry assay confirmed that lobaplatin affected CCA cell survival by blocking cell cycle progression and inducing apoptosis. Lobaplatin treatment reduced Cyclin D1, CDK4, and CDK6 expression, which led to the blocking of G0/G1 transition. In addition, lobaplatin increased p53, Bax expression, caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage, and reduced Bcl-2 expression, which contributed to the apoptosis of CCA cells. Lobaplatin showed a good anti-tumour activity in in vitro models of human CCA cells. These results indicate that Lobaplatin, as the third-generation platinum antineoplastic agent, could be an effective chemotherapeutic agent in human CCA treatment through induction apoptosis and cell cycle arrest.     

双去甲氧基姜黄素抑制肝癌细胞增殖并诱导凋亡的机制

目的探讨双去甲氧基姜黄素(BDMC)对肝癌细胞的作用及其机制。方法不同BDMC(0、20、40、80μmol/L)干预肝癌细胞株HEPG2 72 h,MTT评价细胞增殖,Tunel分析细胞凋亡,ELISA检测活性氧(ROS)水平和p-STAT3水平,Western blot检测AMPK。采用ROS抑制剂N-乙酰-L半胱氨酸(NAC 2.5 mmol/L)处理30 min后BDMC再干预24 h,评价细胞凋亡能力。结果 BDMC可诱导肝癌细胞凋亡,增加ROS生成和AMPK磷酸化水平,抑制p-STAT3表达。抑制ROS生成可抑制BDMC诱导HEPG2凋亡的能力。结论 BDMC通过ROS/AMPK/STAT3信号通路诱导肝癌细胞凋亡。     

Tamoxifen inhibits proliferation and induces apoptosis in human hepatocellular carcinoma cell line HepG2 via down-regulation of survivin expression

Tamoxifen has been used in patients with hepatocellular carcinoma (HCC). However, its inhibitory mechanism remains unknown. In current study, we evaluated the effect of tamoxifen on the estrogen receptor-α-negative HCC cell proliferation, apoptosis and expression of survivin which had been known to play an important role in promotion of cellular proliferation as well as inhibition of apoptosis in cancer cells. HepG2 cells were incubated with tamoxifen (0.1, 1, 10, or 20 μM) for up to 72 h. Cell proliferation was assessed, flow cytometric analysis was performed, and survivin expression was detected. Our results are showed as follows. Ten or 20 μM tamoxifen induced a reduction of cell proliferation. Basically reduction of proliferation was related to an increase in the fraction of G0/1-phase. When tamoxifen was administrated at higher concentration (20 μM), the increase of the relative apoptosis appeared with a delay, augmenting the effect of tamoxifen on cell proliferation. When apoptosis was induced, a significant depression of survivin expression preceded. In conclusion, the tamoxifen decreasing cell proliferation and induction of apoptosis of HepG2 cells depends on drug concentration, which is due to cytostatic and cytocide effects, the latter may be mediated by a down-regulation of survivin expression.     

5-氟尿嘧啶诱导人大肠癌多药耐药细胞株及细胞凋亡的实验研究

目的:研究5-氟尿嘧啶(5-FU)诱导LOVO细胞凋亡,探讨人大肠癌多药耐药机制。方法:用噻唑蓝(MTT)法、光镜及电镜、DNA凝胶电泳和原位DNA末端转移酶标记法,分别对耐药细胞株LOVO/5-FU细胞及敏感细胞株LOVO细胞进行5-FU诱导细胞凋亡的实验研究。结果:LOVO/5-FU细胞存活率明显高于LOVO细胞,光镜和电镜观察凋亡形态学改变,凋亡细胞DNA凝胶电泳可以出现梯形(ladder)凋亡带,LOVO细胞原位DNA末端转移酶标记阳性率高于LOVO/5-FU。结论:(1)5-FU诱导人大肠癌LOVO细胞凋亡,有明显的时间和剂量效应。(2)在相同条件下,耐药细胞株LOVO/5-FU与敏感细胞株LOVO相比,前者凋亡受到抑制。(3)细胞凋亡对研究大肠癌LOVO细胞多药耐药机制,克服化疗产生的多药耐药及逆转多药耐药具有重要的临床价值。     

香加皮杠柳苷抑制Stat5信号通路诱导SMMC-7721细胞凋亡的实验研究

目的 研究香加皮提取物杠柳苷对Stat5信号通路的影响,探讨其诱导肿瘤细胞凋亡的分子机制.方法 MTT比色法检测细胞增殖活性,流式细胞术观察肿瘤细胞的周期变化和凋亡,western blot法检测杠柳苷作用前后人肝癌细胞株SMMC-7721细胞内Stat5蛋白质表达.结果 杠柳苷明显抑制SMMC-7721细胞的增殖,使细胞周期停滞在G2/M期,并能够诱导SMMC-7721细胞凋亡.杠柳苷作用后,细胞核内的Stat5蛋白量降低而胞浆中的量未见明显改变.结论 香加皮杠柳苷能够抑制Stat5信号转导通路,进而抑制SMMC-7721细胞的增殖并诱导其凋亡.     

Four new human renal cell carcinoma cell lines expressing globo-series gangliosides

Clinicopathological studies revealed that monosialosyl galactosyl globoside (MSGG) and disialosyl galactosyl globoside (DSGG) expressed by renal cell carcinoma (RCC) are one of the biochemical indicator related to the metastatic potential. The present study examines the characteristics of four new human RCC cell lines and compares the expression of MSGG and DSGG among them using TLC immunostaining and flow cytometry. TOS-1 and TOS-2 were derived from metastatic subcutaneous tissues. TOS-3 and TOS-3LN were derived from the primary lesion and from metastatic lymph nodes respectively. Monolayer culture, light microscopy and electron microscopy of these cells showed that these cell lines were derived from RCC. TLC immunostaining and flow cytometric analysis revealed increased levels of MSGG in TOS-2 and TOS-3LN, and increased DSGG in TOS-1 and TOS-3LN. These cell lines would be useful for functional studies of globo-series ganglioside expressed by RCC.     

The binding of some human antiendothelial cell antibodies induces endothelial cell apoptosis.

The pathogenic role of antiendothelial cell antibodies (AECA) remains unclear. They are frequently associated with antibodies to anionic phospholipids (PL), such as phosphatidylserine (PS), which is difficult to reconcile with the distribution of PL molecular species within the plasma membrane. Since it is already known that PS is transferred to the outer face of the membrane as a preclude to apoptosis, the possibility exists that apoptosis is initiated by AECA. AECA-positive/anti-PL antibody-negative sera from eight patients with systemic sclerosis (SS) and 21 control patients were evaluated. Endothelial cells (EC) were incubated with AECA and the exposure of PS was established through the binding of annexin V. Hypoploid cell enumeration, DNA fragmentation, and optical and ultrastructural analyses of EC were used to confirm apoptosis. Incubation of EC with AECA derived from six of eight patients with SS led to the expression of PS on the surface of the cells. This phenomenon was significantly more frequent in SS (P < 0.04) than in control diseases. The redistribution of plasma membrane PS preceded other events associated with apoptosis: hypoploidy, DNA fragmentation, and morphology characteristic for apoptosis. Apoptosis-inducing AECA did not recognize the Fas receptor. We conclude that AECA may be pathogenic by inducing apoptosis.