Cantharidin induces DNA damage and inhibits DNA repair‐associated protein levels in NCI‐H460 human lung cancer cells

Cantharidin is one of the major compounds from mylabris and it has cytotoxic effects in many different types of human cancer cells. Previously, we found that cantharidin induced cell death through cell cycle arrest and apoptosis induction in human lung cancer NCI‐H460 cells. However, cantharidin‐affected DNA damage, repair, and associated protein levels in NCI‐H460 cells have not been examined. In this study, we determined whether cantharidin induced DNA damage and condensation and altered levels of proteins in NCI‐H460 cells in vitro. Incubation of NCI‐H460 cells with 0, 2.5, 5, 10, and 15 μM of cantharidin caused a longer DNA migration smear (comet tail). Cantharidin also increased DNA condensation. These effects were dose‐dependent. Cantharidin (5, 10, and 15 μM) treatment of NCI‐H460 cells reduced protein levels of ataxia telangiectasia mutated (ATM), breast cancer 1, early onset (BRCA‐1), 14‐3‐3 proteins sigma (14‐3‐3σ), DNA‐dependent serine/threonine protein kinase (DNA‐PK), O⁶‐methylguanine‐DNA methyltransferase (MGMT), and mediator of DNA damage checkpoint protein 1 (MDC1). Protein translocation of p‐p53, p‐H2A.X (S140), and MDC1 from cytoplasm to nucleus was induced by cantharidin in NCI‐H460 cells. Taken together, this study showed that cantharidin caused DNA damage and inhibited levels of DNA repair‐associated proteins. These effects may contribute to cantharidin‐induced cell death in vitro. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1135–1143, 2015.     

Bisdemethoxycurcumin‐induced S phase arrest through the inhibition of cyclin A and E and induction of apoptosis via endoplasmic reticulum stress and mitochondria‐dependent pathways in human lung cancer NCI H460 cells

Curcuminoids are the major natural phenolic compounds found in the rhizome of many Curcuma species. Curcuminoids consist of a mixture of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Although numerous studies have shown that curcumin induced cell apoptosis in many human cancer cells, however, mechanisms of BDMC‐inhibited cell growth and ‐induced apoptosis in human lung cancer cells still remain unclear. Herein, we investigated the effect of BDMC on the cell death via the cell cycle arrest and induction of apoptosis in NCI H460 human lung cancer cells. Flow cytometry assay was used to measure viable cells, cell cycle distribution, the productions of reactive oxygen species (ROS) and Ca²⁺, mitochondrial membrane potential (ΔΨ_m) and caspase‐3, ‐8 and ‐9 activity. DNA damage and condension were assayed by Comet assay and DAPI staining, respectively. Western blotting was used to measure the changes of cell cycle and apoptosis associated protein expressions. Results indicated that BDMC significantly induced cell death through induced S phase arrest and induced apoptosis. Moreover, DMC induced DNA damage and condension, increased ROS and Ca²⁺ productions and decreased the levels of ΔΨ_m and promoted activities caspase‐3, ‐8, and ‐9. Western blotting results showed that BDMC inhibited Cdc25A, cyclin A and E for causing S phase arrest, furthermore, promoted the expression of AIF, Endo G and PARP and the levels of Fas ligand (Fas L) and Fas were also up‐regulated. Results also indicated that BDMC increased ER stress associated protein expression such as GRP78, GADD153, IRE1α, IRE1β, ATF‐6α, ATF‐6β, and caspase‐4. Taken together, we suggest that BDMC induced cell apoptosis through multiple signal pathways such as extrinsic, intrinsic and ES tress pathway. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1899–1908, 2016.     

Alpha‐phellandrene‐induced DNA damage and affect DNA repair protein expression in WEHI‐3 murine leukemia cells in vitro

Although there are few reports regarding α‐phellandrene (α‐PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α‐PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α‐PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α‐PA on total cell viability and the results indicated that α‐PA induced cell death. Comet assay and 4,6‐diamidino‐2‐phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α‐PA induced DNA damage and condensation in a concentration‐dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α‐PA induced DNA damage in WEHI‐3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α‐PA increased p‐p53, p‐H2A.X, 14‐3‐3‐σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA‐PK, and BRCA‐1. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1322–1330, 2015.     

Bufalin induces apoptosis in vitro and has Antitumor activity against human lung cancer xenografts in vivo

Bufalin has been shown to be effective against a variety of cancer cells, but its role in lung cancer has never been studied in an animal model. In this study, we evaluated bufalin effects in a human lung cancer cell line NCI‐H460 both in vitro and in vivo . Bufalin caused significant cytotoxicity in NCI‐H460 cells at a concentration as low as 1 μM. DNA condensation was observed in bufalin‐treated cells in a dose‐dependent manner. Mitochondrial membrane potential (ΔΨ_m) was reduced and reactive oxygen species (ROS) were increased in bufalin‐treated NCI‐H460 cells. Levels of several proapoptotic proteins such as Fas, Fas‐ligand, cytochrome c , apoptosis protease activating factor‐1, endonuclease G, caspase‐3 and caspase‐9 were increased after bufalin treatment. At the same time, anti‐apoptotic B‐cell lymphoma 2 protein levels were reduced. Bufalin decreased glucose regulated protein‐78 gene expression but increased growth arrest‐ and DNA damage‐inducible 153 gene expression. Bufalin injected intraperitoneally in a dose‐dependent manner reduced tumor size in BALB/C nu/nu mice implanted with NCI‐H460 cells. Bufalin injection did not produce significant drug‐related toxicity in experimental animals except at a high dose (0.4 mg kg^?1). In conclusion, low concentrations of bufalin can induce apoptosis in the human lung cancer cell line NCI‐H460 in vitro . Bufalin also reduced tumor size in mice injected with NCI‐H460 cells without significant drug‐related toxicity. These results indicate that bufalin may have potential to be developed as an agent for treating human non‐small cell lung cancer. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1305–1317, 2017.     

DNA damage response to the Mdm2 inhibitor Nutlin-3

Mdm2 inhibitors represent a promising class of p53 activating compounds that may be useful in cancer treatment and prevention. However, the consequences of pharmacological p53 activation are not entirely clear. We observed that Nutlin-3 triggered a DNA damage response in azoxymethane-induced mouse AJ02-NM₀ colon cancer cells, characterized by the phosphorylation of H2AX (at Ser-139) and p53 (at Ser-15). The DNA damage response was highest in cells showing robust p53 stabilization, it could be triggered by the active but not the inactive Nutlin-3 enantiomer, and it was also activated by another pharmacological Mdm2 inhibitor (Caylin-1). Quantification of γH2AX-positive cells following Nutlin-3 exposure showed that approximately 17% of cells in late S and G2/M were mounting a DNA damage response (compared to a ∼50% response to 5-fluorouracil). Nutlin-3 treatment caused the formation of double-strand DNA strand breaks, promoted the formation of micronuclei, accentuated strand breakage induced by doxorubicin and sensitized the mouse colon cancer cells to DNA break-inducing topoisomerase II inhibitors. Although the HCT116 colon cancer cells did not mount a significant DNA damage response following Nutlin-3 treatment, Nutlin-3 enhanced the DNA damage response to the nucleotide synthesis inhibitor hydroxyurea in a p53-dependent manner. Finally, p21 deletion also sensitized HCT116 cells to the Nutlin-3-induced DNA damage response, suggesting that cell cycle checkpoint abnormalities may promote this response. We propose that p53 activation by Mdm2 inhibitors can result in the slowing of double-stranded DNA repair. Although this effect may suppress illegitimate homologous recombination repair, it may also increase the risk of clastogenic events.     

Mangiferin inhibits lipopolysaccharide‐induced epithelial‐mesenchymal transition (EMT) and enhances the expression of tumor suppressor gene PER1 in non‐small cell lung cancer cells

Non‐small cell lung cancer (NSCLC) is often complicated by pulmonary infection, which affects treatment and prognosis. Bacterial lipopolysaccharide (LPS) is an effective stimulator of inflammatory cytokine production, and previous studies have reported that LPS promotes tumor invasion and metastasis. Mangiferin is a plant‐derived C‐glucosylxanthone with many biological activities, such as antioxidation and anti‐inflammation. This research mainly explored the mechanism of its antitumor activities on LPS‐induced A549, NCI‐H460, and NCI‐H520 NSCLC cells. We determined that mangiferin exhibits growth inhibiting activity against LPS‐induced NSCLC cells through the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) assay. In addition, mangiferin reversed the LPS‐induced downregulation of E‐cadherin (epithelial marker); conversely, it significantly inhibited the expression of raised vimentin (mesenchymal markers). Moreover, the ability of NSCLC cells to migrate, as evidenced by the wound healing and transwell migration assays, and the expression of CXCR4 increased by LPS were significantly repressed by mangiferin. In addition, mangiferin markedly mediated protein levels of PER1 and NLRP3 in LPS‐induced NSCLC cells and reduced the secretion of IL‐1β. These results indicate that mangiferin is not only a remarkable anti‐inflammatory compound but also an antitumor agent; thus, it has the potential for being developed into anti‐inflammatory and antitumor drugs in the future.     

Ser1778 of 53BP1 Plays a Role in DNA Double-strand Break Repairs

53BP1 is an important genome stability regulator, which protects cells against double-strand breaks. Following DNA damage, 53BP1 is rapidly recruited to sites of DNA breakage, along with other DNA damage response proteins, including γ-H2AX, MDC1, and BRCA1. The recruitment of 53BP1 requires a tandem Tudor fold which associates with methylated histones H3 and H4. It has already been determined that the majority of DNA damage response proteins are phosphorylated by ATM and/or ATR after DNA damage, and then recruited to the break sites. 53BP1 is also phosphorylated at several sites, like other proteins after DNA damage, but this phosphorylation is not critically relevant to recruitment or repair processes. In this study, we evaluated the functions of phosphor-53BP1 and the role of the BRCT domain of 53BP1 in DNA repair. From our data, we were able to detect differences in the phosphorylation patterns in Ser25 and Ser1778 of 53BP1 after neocarzinostatin-induced DNA damage. Furthermore, the foci formation patterns in both phosphorylation sites of 53BP1 also evidenced sizeable differences following DNA damage. From our results, we concluded that each phosphoryaltion site of 53BP1 performs different roles, and Ser1778 is more important than Ser25 in the process of DNA repair.     

Benzo(a)pyrene induces p73 mRNA expression and necrosis in human lung adenocarcinoma H1299 cells

p53 can mediate DNA damage‐induced apoptosis in various cell lines treated with Benzo(a)pyrene (BaP). However, the potential role of p73, one of the p53 family members, in BaP‐induced apoptotic cell death remains to be determined. In this study, normal fetal lung fibroblasts (MRC‐5) and human lung adenocarcinoma cells (H1299, p53‐null) were treated with BaP at concentrations of 8, 16, 32, 64, and 128 μM for 4 and 12 h. The oxidative stress status, extent of DNA damage, expression of p53, p73, mdm2, bcl‐2, and bax at the mRNA and protein levels, and the percentages of apoptosis and/or necrosis were assessed. In the two BaP‐treated cell lines, we observed increased malondialdehyde (MDA) formation and decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) activity at 4 h after the treatment; furthermore, at the time points of 4 and 12 h, we observed extremely high levels of DNA damage. In addition, at 4 h after the treatment, BaP had induced necrosis in MRC‐5 and H1299 cells, but it had inhibited apoptosis in MRC‐5 cells (P < 0.01 for all). Furthermore, in BaP‐treated H1299 cells, only the p73 mRNA level was up‐regulated. The results suggested that BaP‐induced DNA damage could trigger a shift from apoptotic cell death toward necrotic cell death and that necrotic cell death is independent of p53 and p73 in these cell lines. Future studies are needed to investigate the time course of changes in the type of BaP‐induced cell death in more cell lines. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

DNA-PK inhibition by M3814 enhances chemosensitivity in non-small cell lung cancer

A significant proportion of non-small cell lung cancer (NSCLC) patients experience accumulating chemotherapy-related adverse events, motivating the design of chemosensitizating strategies. The main cytotoxic damage induced by chemotherapeutic agents is DNA double-strand breaks (DSB). It is thus conceivable that DNA-dependent protein kinase (DNA-PK) inhibitors which attenuate DNA repair would enhance the anti-tumor effect of chemotherapy. The present study aims to systematically evaluate the efficacy and safety of a novel DNA-PK inhibitor M3814 in synergy with chemotherapies on NSCLC. We identified increased expression of DNA-PK in human NSCLC tissues which was associated with poor prognosis. M3814 potentiated the anti-tumor effect of paclitaxel and etoposide in A549, H460 and H1703 NSCLC cell lines. In the four combinations based on two NSCLC xenograft models and two chemotherapy, we also observed tumor regression at tolerated doses in vivo. Moreover, we identified a P53-dependent accelerated senescence response by M3814 following treatment with paclitaxel/etoposide. The present study provides a theoretical basis for the use of M3814 in combination with paclitaxel and etoposide in clinical practice, with hope to aid the optimization of NSCLC treatment.     

Protective effect of magnolol on TBHP-lnduced injury in h460 cells partially via a p53 dependent mechanism

The aim is to investigate the effect of Magnolol preserved H460 cells from an oxidative agent tert-butylhydroperoxide (TBHP)-induced cell death. Magnolol augmented cell survival ratio after TBHP challenged. The protective action of this drug was more efficacious than that of N-acetylcysteine (NAC) which is a putative antioxidant. DNA damage, detected by the comet assay, was diminished after treatment of Magnolol. The cells viability decreased after treatment with 0.15 mM TBHP for 24 h, accompanied by inducing apoptotic death of the cells. Cytotoxicity and apoptosis induced by TBHP were significantly inhibited or attenuated after pretreatment with 20 microM Magnolol. Magnolol contributes to the cells survival through downregulated the p53 phosphorylation and PTEN expression, and upregulated Akt phosphorylation. Taken together, Magnolol was effective against DNA single strand breaks (SSB) formation, cytotoxicity and lipid peroxidation induced by TBHP, and its effects on p53 phosphorylation, PTEN and Akt phosphorylation were due to its antioxidative function, and partially via a p53 dependent mechanism in this protective effects.     

Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways

Ouabain, a cardiotonic steroid and specific Na⁺/K⁺‐ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca²⁺, and mitochondrial membrane potential (ΔΨ_m) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca²⁺ production, but decreased the levels of ΔΨ_m in DU 145 cells. Ouabain also increased the activities of caspase‐3, ‐8, and ‐9. Western blotting was used for measuring the alterations of apoptosis‐associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATM^Ser1981, p‐H2A.X^Ser139, and p‐p53^Ser15) and ER‐stress‐associated proteins (Grp78, ATF6β, p‐PERK^Thr981, PERK, eIF2A, GADD153, CaMKIIβ, and caspase‐4) in time‐dependently. Furthermore, ouabain increased apoptosis‐associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro‐apoptotic protein Bax, increased apoptotic‐associated proteins, such as cytochrome c, AIF, Endo G, caspase‐3, ‐8, and ‐9, but reduced anti‐apoptotic protein Bcl‐2 and Bcl‐x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase‐dependent and mitochondria‐dependent pathways in human prostate cancer DU 145 cells.     

Digitoxin and a synthetic monosaccharide analog inhibit cell viability in lung cancer cells

Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung cancer (NCI-H460) cells remain unclear. Understanding how digitoxin or derivate analogs induce their cytotoxic effect below therapeutically relevant concentrations will help in designing and developing novel, safer and more effective anti-cancer drugs. In this study, NCI-H460 cells were treated with digitoxin and a synthetic analog D6-MA to determine their anti-cancer activity. Different concentrations of digitoxin and D6-MA were used and the subsequent changes in cell morphology, viability, cell cycle, and protein expressions were determined. Digitoxin and D6-MA induced dose-dependent apoptotic morphologic changes in NCI-H460 cells via caspase-9 cleavage, with D6-MA possessing 5-fold greater potency than digitoxin. In comparison, non-tumorigenic immortalized bronchial and small airway epithelial cells displayed significantly less apoptotic sensitivity compared to NCI-H460 cells suggesting that both digitoxin and D6-MA were selective for NSCLC. Furthermore, NCI-H460 cells arrested in G(2)/M phase following digitoxin and D6-MA treatment. Post-treatment evaluation of key G2/M checkpoint regulatory proteins identified down-regulation of cyclin B1/cdc2 complex and survivin. Additionally, Chk1/2 and p53 related proteins experienced down-regulation suggesting a p53-independent cell cycle arrest mechanism. In summary, digitoxin and D6-MA exert anti-cancer effects on NCI-H460 cells through apoptosis or cell cycle arrest, with D6-MA showing at least 5-fold greater potency relative to digitoxin.     

环磷酰胺衍生物SLXM-2对小鼠肝癌H_（22）细胞的DNA损伤作用

化合物SLXM-2是一种环磷酰胺衍生物,前期研究已经证实其具有良好的肿瘤抑制作用,并具有较低的毒副作用。但是其作用机制尤其是对细胞DNA的损伤作用仍不清楚。本研究旨在评价SLXM-2对肝癌H22腹水小鼠的生命延长作用与DNA损伤的关系,并探讨可能的分子机制。实验结果证实,SLXM-2能够显著提高肝癌H22腹水小鼠的生命延长率（P〈0.05）。进一步实验表明,SLXM-2能够造成肝癌H22细胞DNA损伤,显著上调γH2AX（Ser139）,p-Chk1（Ser296）,p-Chk2（Thr68）,p-p53（Ser15）,p-p53（Ser20）和p21的蛋白表达,并显著下调p-ATR（Ser428）和p-ATM（Ser1981）的表达（P〈0.05）。总之,SLXM-2对肝癌H22细胞具有显著的抑制作用,分子机制与其能够造成肿瘤细胞DNA损伤有关。     

Combining DNA damaging agents and checkpoint 1 inhibitors

During the cell cycle that leads to mitosis, checkpoints are activated in response to DNA damage. The checkpoints control the ability of cells to arrest the cell cycle allowing time to repair the DNA. In more than 50% of cancer cells, the G1 checkpoint is inactive due to mutations of p53. Therefore, the combination of a DNA damaging agent with a G2 checkpoint inhibitor should force selectively cancer cells into a premature and lethal mitosis. This approach which has recently drawn considerable interest is discussed in this paper.     

Classification of polycyclic aromatic hydrocarbons based on mutagenicity in lung tissue through DNA microarray

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants produced in the combustion of organic matter. Exposure to PAHs raises the risk of lung cancer and inflammatory and allergic disorders such as asthma. DNA microarray technologies have been applied to research on toxicogenomics in the recent years. To evaluate the mutagenicity of PAHs and constituents of environmental pollutants in lung tissue, including metabolic activation, human alveolar epithelial type II cells (A549) were treated with nonmutagenic PAH pyrene and with the mutagenic PAHs benzo‐[a]‐pyrene, 1‐nitropyrene, or 1,8‐dinitropyrene. Comparison of genome‐wide microarray expression profiles between a nonmutagenic and a mutagenic PAH‐treated group revealed that xenobiotic response genes such as CYP1B1 were commonly upregulated in two groups and that DNA damage induced genes, especially p53‐downstream genes such as p21 (CDKN1A) were upregulated only in the mutagenic PAH‐treated group. Pretreatment with cytochrome P450 inhibitor α‐naphthoflavone or p53 inhibitor pifithrin‐α inhibited the benzo‐[a]‐pyrene‐induced p21 expression. These data suggest that when PAHs enter the cells, lung epithelium induces PAH metabolic activating enzymes, and then the DNA damages‐recognition signal is converged with p53 downstream genes. This metabolic activation and DNA damage is induced in lung epithelium, and the mutagenicity of PAHs can be classified by DNA microarray expression profiles. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28:652–659, 2013.     

Gallic acid provokes DNA damage and suppresses DNA repair gene expression in human prostate cancer PC‐3 cells

Our earlier studies have demonstrated that gallic acid (GA) induced cytotoxic effects including induction of apoptosis and DNA damage and inhibited the cell migration and invasion in human cancer cells. However, GA‐affected DNA damage and repair gene expressions in human prostate cancer cells are still unclear. In this study, we investigated whether or not GA induces DNA damage and inhibits DNA repair gene expression in a human prostate cancer cell line (PC‐3). The results from flow cytometric assay indicated that GA decreased the percentage of viable PC‐3 cells in a dose‐ and time‐dependent manner. PC‐3 cells after exposure to different doses (50, 100, and 200 μM) of GA and various periods of time (12, 24, and 48 h) led to a longer DNA migration smear (comet tail) occurred based on the single cell gel electrophoresis (comet assay). These observations indicated that GA‐induced DNA damage in PC‐3 cells, which also confirmed by 4,6‐diamidino‐2‐phenylindole dihydrochloride staining and DNA agarose gel electrophoresis. Alternatively, results from real‐time polymerase chain reaction assay also indicated that GA inhibited ataxia telangiectasia mutated, ataxia‐telangiectasia and Rad3‐related, O⁶‐methylguanine‐DNA methyltransferase, DNA‐dependent serine/threonine protein kinase, and p53 mRNA expressions in PC‐3 cells. Taken together, the present study showed that GA caused DNA damage and inhibited DNA repair genes as well as both effects may be the critical factors for GA‐inhibited growth of PC‐3 cells in vitro. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 579–587, 2013.     

Novel checkpoint 1 inhibitors

Cell cycle ckeckpoints are activated in response to DNA damage. Their role consists in blocking the cell cycle to allow time for DNA repair. The activity of the G1 checkpoint is dependent on the p53 protein. In more than 50% of human tumor cells, the p53 gene is mutated. In the p53 mutated cells, the G1 checkpoint is lacking. In these cells, only the G2 checkpoint, although weaker than in healthy cells, provides cancer cells with the opportunity to repair the DNA after damage. Therefore, combining a G2 checkpoint inhibitor with a DNA damaging agent should force, selectively cancer cells, into a premature and lethal mitosis, due to an accumulation of DNA lesions. Among the regulators of the G2 checkpoint, Checkpoint 1 kinase (Chk1) plays a major role. A widespread interest has been recently devoted to the discovery of Chk1 inhibitors as potential useful compounds to enhance the antitumor efficiency of DNA damaging agents. This review article will summarize: (i) the chemical structures of the novel Chk1 inhibitors reported in the recent patents; (ii) their inhibitory activity towards Chk1; (iii) their effects on tumor cells in combination with DNA damaging agents; and (iv) the in vivo results on animal models.     

5-Methoxyflavanone induces cell cycle arrest at the G2/M phase, apoptosis and autophagy in HCT116 human colon cancer cells

Natural flavonoids have diverse pharmacological activities, including anti-oxidative, anti-inflammatory, and anti-cancer activities. In this study, we investigated the molecular mechanism underlying the action of 5-methoxyflavanone (5-MF) which has a strong bioavailability and metabolic stability. Our results show that 5-MF inhibited the growth and clonogenicity of HCT116 human colon cancer cells, and that it activated DNA damage responses, as revealed by the accumulation of p53 and the phosphorylation of DNA damage-sensitive proteins, including ataxia-telangiectasia mutated (ATM) at Ser1981, checkpoint kinase 2 (Chk2) at Thr68, and histone H2AX at Ser139. 5-MF-induced DNA damage was confirmed in a comet tail assay. We also found that 5-MF increased the cleavage of caspase-2 and -7, leading to the induction of apoptosis. Pretreatment with the ATM inhibitor KU55933 enhanced 5-MF-induced γ-H2AX formation and caspase-7 cleavage. HCT116 cells lacking p53 (p53^−/−) or p21 (p21^−/−) exhibited increased sensitivity to 5-MF compared to wild-type cells. 5-MF further induced autophagy via an ERK signaling pathway. Blockage of autophagy with the MEK inhibitor U0126 potentiated 5-MF-induced γ-H2AX formation and caspase-2 activation. These results suggest that a caspase-2 cascade mediates 5-MF-induced anti-tumor activity, while an ATM/Chk2/p53/p21 checkpoint pathway and ERK-mediated autophagy act as a survival program to block caspase-2-mediated apoptosis induced by 5-MF.     

Characterization of protein kinase chk1 essential for the cell cycle checkpoint after exposure of human head and neck carcinoma A253 cells to a novel topoisomerase I inhibitor BNP1350

Cellular topoisomerase I is an important target in cancer chemotherapy. A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity against human head and neck carcinoma A253 cells in vitro. As a basis for future clinical trials of BNP1350 in human head and neck carcinoma, in vitro studies were carried out to investigate its effect on DNA damage and cell cycle checkpoint response. The treatment of A253 cells with BNP1350 caused biphasic profiles of DNA fragmentation displayed from 0 to 48 h after 2-h exposure. Pulsed-field gel electrophoresis demonstrated that the first wave of DNA damage was mainly megabase DNA fragmentation, but the second wave of DNA damage was 50- to 300-kb DNA fragmentation in addition to megabase DNA damage. The cell cycle checkpoint response was characterized after exposure to 0.07 and 0.7 microM concentrations of BNP1350, the IC(50) and IC(90) values, respectively. After exposure to a low concentration of BNP1350 (IC(50)), A253 cells accumulated primarily in G(2) phase. In contrast, treatment with a high concentration of BNP1350 (IC(90)) resulted in S phase accumulation. The concentration-associated cell cycle perturbation by BNP1350 was correlated with different profiles of cell cycle-regulatory protein expression. When treated with the low concentration of BNP1350, cyclin B/cdc2 protein expression was up-regulated, whereas with the high concentration, no significant change was observed at 24 and 48 h. In addition, increased phosphorylation of a G(2) checkpoint kinase chk1 was observed when cells were treated with a low concentration of BNP1350, whereas only slight inhibition of chk1 activity was found in the cells treated with the higher concentration. Altered chk1 phosphorylation after DNA damage appears to be associated with specific phases of cell cycle arrest induced by BNP1350. Because A253 cells do not express the p53 protein, the drug-induced alterations of the G(2) checkpoint kinase chk1 are not p53-dependent.