E2F1-mediated DNA damage is implicated in 8-Cl-adenosine-induced chromosome missegregation and apoptosis in human lung cancer H1299 cells

Gambogic acid (GA) is the dry resin of Garcinia hanburyi (Guttiferae) with potent anti-tumor activity, various bioactivities, including detoxification, homeostasis, anti-inflammatory, and parasiticide, whereas the effect of this natural compound on cancer cells has not been clearly clarified. Here, we examined cellular cytotoxicity by cell viability assay and DNA fragmentation by DNA-ladder assay. Activation of different protein expressions were detected by western blot analyses. We first demonstrated that GA reduces the human SH-SY5Y neuroblastoma cell viability with IC₅₀ of 1.28 μM at 6 h which has less toxicity in fibroblast cells. However, lower concentration GA significantly downregulated the expression of anti-apoptotic protein including Bcl-2, Bcl-xL, and Mcl-1, which also dramatically activated cleaved caspase-9 and -3 in a dose- and time-dependent manner. Consequently, GA-induced cytotoxicity was not mediated by the Fas/FasL and PI3 K/AKT/GSK-3β signaling pathway. In addition, GA-induced cells showed damage morphology which had become cell rounding, neurite retraction, membrane blebbing and shrunken in a dose- and time-dependent manner that clearly indicates this morphological change might be due to the process of apoptosis which shows fragmented DNA. Therefore, the findings presented in this study demonstrate that apoptotic effects of GA on SH-SY5Y cells are mediated by intrinsic mitochondrion-dependent caspase pathway which suggests this natural compound might be effective as an anti-cancer agent for neuroblastoma malignancies.     

2-Methoxyestradiol induces G2/M arrest and apoptosis in prostate cancer

Few therapeutic treatment options are available for patients suffering from metastatic androgen-independent prostate cancer. We investigated the ability of the estrogen metabolite 2-methoxyestradiol to inhibit the proliferation of a variety of human prostate cancer cell lines in vitro and to inhibit the growth of androgen-independent prostate cancer in a transgenic mouse model in vivo. Our results showed that 2-methoxyestradiol is a powerful growth inhibitor of LNCaP, DU 145, PC-3, and ALVA-31 prostate cancer cells. Cell flow cytometry of 2-methoxyestradiol-treated DU 145 cells showed a marked accumulation of cells in the G2/M phase of the cell cycle and an increase in the sub-G1 fraction (apoptotic). In addition, staining for annexin V, changes in nuclear morphology, and inhibition of caspase activity support a role for apoptosis. More importantly, we showed that 2-methoxyestradiol inhibits prostate tumor progression in the Ggamma/T-15 transgenic mouse model of androgen-independent prostate cancer without toxic side effects. These results in cell culture and an animal model support investigations into the clinical use of 2-methoxyestradiol in patients with androgen-independent prostate cancer.     

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.     

E2F1 介导8-氯-腺苷引起的人肺癌细胞H1299的凋亡

8-氯-腺苷（8-Cl-adenosine,8-Cl-Ado）可诱导人非小细胞性肺癌细胞H1299发生凋亡,但其分子机制还没有阐明．首先用四唑盐（MTT）比色法检测了8-Cl-Ado 对H1299 细胞的生长抑制作用．进一步采用蛋白质免疫印迹法(Western blotting) 检测了8-Cl-Ado 处理H1299细胞后,procaspase-3 的激活情况以及E2F1的蛋白水平．通过用pcDNA-HA-E2F1表达载体和pSUPER-E2F1 RNA 干扰载体分别转染H1299 细胞,研究在E2F1 过表达和RNA 干扰（RNA interference, RNAi）两种情况下对凋亡的影响．实验结果表明,8-Cl-Ado可抑制H1299 细胞的生长,激活凋亡关键执行蛋白procaspase-3,升高E2F1 蛋白水平．当E2F1 过表达后,同时伴有procaspase-3 的激活,而E2F1 表达受到抑制后,与对照相比,8-Cl-Ado 引起的procaspase-3 的激活被明显抑制,说明E2F1 介导8-Cl-Ado 引起的人肺癌细胞H1299 的凋亡．     

Anthraquinone derivatives induce G2/M cell cycle arrest and apoptosis in NTUB1 cells

Thirteen anthraquinone derivatives 5-17 including two 3-(3-alkylaminopropoxy)-9,10-anthraquinone (NHA) derivatives 5 and 6, and 11 1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinone (MHA) derivatives 7-17 were synthesized, evaluated for cytotoxicities against two cancer cell lines, and assayed the generation of reactive oxygen species (ROS) in NTUB1 cells (a human bladder carcinoma cell line). Compound 9 bearing a pyrrolidinyl group induced the stronger cytotoxic effect than those of other synthesized NHA and MHA derivatives. Exposure of NTUB1 cells to 9, 13, and 17 for 24h significantly increased the production of ROS, respectively. Flow cytometric analysis exhibited that the exposure of NTUB1 cells to the selective 9 led to the G2/M phase arrest accompanied by an increase of apoptotic cell death after the incubation for 24h. Compound 9 induced up-regulation of cyclinB1 and p21 expressions. Biological results suggested that the induction of G2/M arrest, apoptosis, and cell death by 9 may associate with increased expression of p21 and cyclin B1, elevation of Bax and p53 levels, and generation of ROS in the cell. In conclusion, these series of compounds may be used as anticancer agents.     

Induction of G2/M cell cycle arrest and apoptosis by a benz[f]indole-4,9-dione analog in cultured human lung (A549) cancer cells

A synthetic benz[f]indole-4,9-dione analog, 2-amino-3-ethoxycarbonyl-N-methylbenz[f]indole-4,9-dione (SME-6), showed a potent growth inhibition of a panel of human cancer cell lines. The mechanism of action study revealed that the growth inhibitory effect of SME-6 was highly related to the induction of G2/M cell cycle arrest and apoptosis in human lung cancer cells (A549). These data may provide new information for understanding the mechanisms by benz[f]indole-4,9-diones-mediated antitumor activity.     

Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.     

A synthetic 2,3-diarylindole induces microtubule destabilization and G2/M cell cycle arrest in lung cancer cells

The anticancer potential of a synthetic 2,3-diarylindole (PCNT13) has been demonstrated in A549 lung cancer cells by inducing both apoptosis and autophagic cell death. In this report, we designed to connect a fluorophore to the compound via a hydrophilic linker for monitoring intracellular localization. The best position for linker attachment was identified from cytotoxicity and effect on cell morphology of newly synthesized PCNT13 derivatives bearing hydrophilic linker. Cytotoxicity and effect on cell morphology related to the parental compound were used to identify the optimum position for linker attachment in the PCNT13 chemical structure. The fluorophore-PCNT13 conjugate was found to localize in the cytoplasm. Microtubules were found to be one of the cytosolic target proteins of PCNT13, as the compound could inhibit tubulin polymerization in vitro. A molecular docking study revealed that PCNT13 binds at the colchicine binding site on the α/β-tubulin heterodimer. The effect of PCNT13 on microtubule dynamics caused cell cycle arrest in the G2/M phase as analyzed by flow cytometric analysis.     

Abrogation of G2/M arrest sensitizes curcumin-resistant hepatoma cells to apoptosis

In this study, we showed that curcumin treatment resulted in activation of Chk1-mediated G2 checkpoint, which was associated with the induction of G2/M arrest and the resistance of cancer cells to curcumin-induced apoptosis. Further investigation revealed that inhibition of Chk1 significantly abrogated G2/M arrest and sensitized curcumin-resistant cells to apoptosis via upregulation of Bad and in turn the loss of mitochondrial membrane potential. These results indicate that Chk1-mediated G2/M arrest may serve as a mechanism for curcumin resistance and Chk1 represents a potential target for the reversal of this resistance. Our findings should be helpful for clinical application of curcumin.     

Polycystin-1 induced apoptosis and cell cycle arrest in G0/G1 phase in cancer cells

Studies have shown that polycystin-1, encoded by PKD1, the major ADPKD, may have a central role in regulating both apoptosis and proliferation, which could prevent the malignant transformation of affected cells. However, as a putative tumor suppressor, direct studies on the possibility that polycystin-1 may play a role in cancer cells' biological properties have not yet been reported. We have demonstrated that the apoptosis of cancer cells was induced by overexpression of polycystin-1. After transfection with polycystin-1, three cancer cell lines, HepG2, A549, and SW480, showed significantly increased apoptosis compared with the respective control groups. This was accompanied by cell cycle arrest at G(0)/G(1) phase, whereas cell proliferation was not significantly affected. Overexpression of polycystin-1 induces apoptosis in cancer cells, at least partially, through Wnt and a caspase-dependent pathway.     

E2F1-dependent pathways are involved in amonafide analogue 7-d-induced DNA damage, G2/M arrest, and apoptosis in p53-deficient K562 cells

The E2F1 gene well known is its pivotal role in regulating the entry from G1 to S phase, while the salvage antitumoral pathway which implicates it, especially in the absence of p53, is not fully characterized. We therefore attempted to identify the up‐ and down‐stream events involved in the activation of the E2F1‐dependent pro‐apoptotic pathway. For this purpose, a amonafide analogue, 7‐d (2‐(3‐(2‐(Dimethylamino)ethylamino)propyl)‐6‐(dodecylamino)‐1H‐benzo[de]isoquinoline‐1,3(2H)‐dione) was screened, which exhibited high antitumor activity against p53‐deficient human Chronic Myelogenous Leukemia (CML) K562 cells. Analysis of flow cytometry and western blots of K562 cells treated with 7‐d revealed an appreciable G2/M cycle arrest and apoptosis in a dose and time‐dependent manner via p53‐independent pathway. A striking increase in “Comet tail” formation and γ‐H2AX expression showed that DNA double strand breaks (DSB) were caused by 7‐d treatment. ATM/ATR signaling was reported to connect E2F1 induction with apoptosis in response to DNA damage. Indeed, 7‐d‐induced G2/M arrest and apoptosis were antagonized by ATM/ATR signaling inhibitor, Caffeine, which suggested that ATM/ATR signaling was activated by 7‐d treatment. Furthermore, the increased expression of E2F1, p73, and Apaf‐1 and p73 dissociation from HDM2 was induced by 7‐d treatment, however, knockout of E2F1 expression reversed p73, Apaf‐1, and p21^Cip1/WAF1 expression, reactivated cell cycle progression, and inhibited 7‐d‐induced apoptosis. Altogether our results for the first time indicate that 7‐d mediates its growth inhibitory effects on CML p53‐deficient cells via the activation of an E2F1‐dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets p73, Apaf‐1, and p21^Cip1/WAF1. J. Cell. Biochem. 113: 3165–3177, 2012. © 2012 Wiley Periodicals, Inc.     

A resveratrol analog, phoyunbene B, induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells

Among the seven natural resveratrol analogs separated and identified from Pholidota yunnanensis R(OLFE), we found phoyunbene B (PYB, trans-3,4'-dihydroxy-2',3',5-trimethoxystilbene) was more effective in inhibiting the growth of HepG2 hepatocellular carcinoma cells than resveratrol. The inhibitory effect of PYB in HepG2 cells was due to its induction of G2/M cell cycle arrest and apoptosis. Induction of G2/M phase cell cycle arrest by PYB was associated with its up-regulation of Cyclin B1, while its induction of apoptosis was accompanied with its down-regulation of Bcl-2 and up-regulation of Bax. Our in vitro invasion/migration assays also showed that PYB could inhibit the invasion of hepatocellular carcinoma cells.     

Molecular mechanisms of Lycoris aurea agglutinin‐induced apoptosis and G2/M cell cycle arrest in human lung adenocarcinoma A549 cells,both in vitro and in vivo

Objectives

Lycoris is aurea agglutinin (LAA) has attracted rising attention due to its remarkable bioactivities. Here, we aimed at investigating its anti‐tumor activities.

Material and Methods

In vitro methods including MTT, cellular morphology observation, FCM and immunoblotting were performed. In vivo methods like detection of tumor volume, body weight and survival ratio, as well as TUNEL staining were performed.

Results and Conclusion

LAA triggers G₂/M phase cell cycle arrest via up‐regulating p21expression as well as down‐regulating cdk‐1cyclinA singling pathway, and induces apoptotic cell death through inhibiting PI3K‐Akt survival pathway in human lung adenocarcinoma A549 cells. While LAA has no significant cytotoxic effect toward normal human embryonic lung fibroblast HELF cells, and moreover, LAA could amplify the antineoplastic effects of cisplatin toward A549 cells. Lastly LAA also bears anti‐cancer and apoptosis‐inducing effects in vivo, and it could decrease the volume and weight of subcutaneous tumor mass obviously as well as expand lifespan of mice. These findings may provide a new perspective for elucidating the complicated molecular mechanisms of LAA‐induced cancer cell growth‐inhibition and death, providing a new opportunity of LAA as a potential candidate anti‐neoplastic drug for future cancer therapeutics.

     

Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis

We have recently shown that transient expression of 10-formyltetrahydrofolate dehydrogenase (FDH) strongly inhibits proliferation of several cancer cell lines and ultimately results in cell death. In the present studies using Tet-On system, we have generated a stable A549 lung carcinoma cell line capable of inducible FDH expression. Using this system, we were able to express FDH at different levels depending on concentration of the inducer, doxycycline, and we have observed that inhibition of proliferation depends on FDH intracellular levels. We have further shown that induction of FDH expression results in initiation of apoptosis beginning 24 h post-induction. Apoptotic cells revealed cleavage of poly-(ADP-ribose) polymerase and general caspase inhibitor zVAD-fmk protected cells against FDH-induced apoptosis. FDH-expressing cells showed accumulation of cells in G(0)-G(1) phase and a sharp decrease of cells in S phase. Accumulation of intracellular FDH was followed by accumulation of the tumor suppressor protein p53 and its downstream target p21. These results indicate that FDH antiproliferative effects on A549 cells include both G(1) cell cycle arrest and caspase-dependent apoptosis.     

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background

Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results

In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G₀/G₁ and Sub-G₁ cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G₀/G₁ phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions

The results demonstrated that HCT-induced G₀/G₁ phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells