Potential role of mitochondrial damage and S9 mixture including metabolic enzymes in ZnO nanoparticles-induced oxidative stress and genotoxicity in Chinese hamster lung (CHL/IU) cells.

The present study was designed to examine genotoxicity induced by 10-40 nm zinc oxide (ZnO) nanoparticles using the in vitro system. The frequency of micronuclei was significantly increased in a dose-dependent manner when cultured Chinese hamster lung (CHL/IU) cells were exposed to ZnO nanoparticles for 24, 48 and 72 h in the continuous treatment method. The maximal frequency of micronuclei was observed in exposure of CHL/IU cells to ZnO nanoparticles at a concentration of 125 µM. The frequency of micronuclei was profoundly enhanced when CHL/IU cells were exposed to ZnO nanoparticles in the presence vs. absence of S9 mixture including metabolic enzymes in the short-term treatment method, demonstrating an increase in the formation of micronuclei by S9 mixture. The maximal frequency of micronuclei was seen in exposure of CHL/IU cells to ZnO nanoparticles at a concentration of 140 µM. Similar results were obtained in chromosome aberrations, particularly structural aberrations. Surprisingly, administration of the superoxide radical scavenger, tempol, completely abolished an increase in the frequency of micronuclei in the presence or absence of S9 mixture, indicating a central role of superoxide radical in the formation of micronuclei. Indeed, reactive oxygen species (ROS) generation was elevated by simultaneous incubation of S9 mixture and ZnO nanoparticles and by exposure of CHL/IU cells to ZnO nanoparticles in the presence or absence of S9 mixture. An electron microscopic examination revealed mitochondrial damage in CHL/IU cells exposed to ZnO nanoparticles, indicating the participation of mitochondrial dysfunction in ROS generation in this setting. These observations suggest that ZnO nanoparticles evoke genotoxicity through superoxide radical-induced oxidative stress derived from mitochondrial damage in CHL/IU cells. S9 mixture appears to contribute to a further increase in genotoxicity through the generation of superoxide radical by metabolic activation of ZnO nanoparticles.

Three Combined Treatments, a Novel HDAC Inhibitor OBP-801/YM753, 5-Fluorouracil, and Paclitaxel, Induce G2 Phase Arrest Through the p38 Pathway in Human Ovarian Cancer Cells.

Ovarian cancer is the most lethal disease among gynecological malignancies. More effective therapy is required to counter high recurrence rates and chemotherapy resistance. We investigated the efficacy and molecular mechanisms of three combined treatments (TCTs)-a novel histone deacetylase (HDAC) inhibitor OBP-801/YM753, 5-fluorouracil (5-FU), and paclitaxel (PTX)-in human ovarian cancer SKOV-3 and OVCAR-3 cells. The inhibition of cell growth was stronger with TCTs than with each single agent and with two combined treatments. The TCTs significantly induce G2 phase arrest in both cell lines. We then analyzed the molecular mechanisms and found that the TCTs increased the phosphorylation of p38 (Thr180/Tyr182), decreased the expression of CDC25C, and increased the phosphorylation of CDC2 (Tyr15), an inactive form of CDC2. To examine the responsibilities of the p38 pathway for G2 phase arrest induced by the TCTs, we employed the p38 inhibitor SB203580. SB203580 inhibited G2 phase arrest, suppression of CDC25C, and phosphorylation of CDC2 (Tyr15) induced by the TCTs. These results suggest that the TCTs can induce G2 phase arrest through activation of the p38 signaling pathway. We therefore believe that this combination is promising as a novel therapeutic strategy against ovarian cancer.

[Corrigendum] Brassinin induces G1 phase arrest through increase of p21 and p27 by inhibition of the phosphatidylinositol 3-kinase signaling pathway in human colon cancer cells.

After the publication of the article, the authors noted that they had made the following errors. In Fig. 5B, total PDK1 is incorrect in the published paper. The corrected version of Fig. 5B is shown below. Also in Fig. 6B, the DMSO-treated group and the brassinin (400 µM)-treated group are incorrect, and the corrected Fig. 6B is shown below. The corrected figures demonstrate the same findings as the original figures. These corrections do not alter the interpretation of the results and conclusions. [the original article was published in the International Journal of Oncology 40: 816-824, 2012 DOI: 10.3892/ijo.2011.1246].

The alkaloid emetine sensitizes ovarian carcinoma cells to cisplatin through downregulation of bcl-xL.

Cisplatin and its platinum derivatives are first-line chemotherapeutic agents in the treatment of ovarian cancer. However, chemoresistance is the leading cause of therapeutic failure and is responsible for the poor overall survival rate. Here, we describe that emetine, a natural alkaloid used as an anti-amoebiasis drug, sensitized ovarian carcinoma cells to apoptosis induced by cisplatin. The single administration of cisplatin or emetine had a weak effect on cell death. However, co-treatment of cisplatin and emetine remarkably induced apoptosis and reduced the colony formation of ovarian carcinoma cells. Moreover, we showed that apoptosis induced by the combination of cisplatin and emetine was dependent on the activation of caspases -3, -7 and -8. As to the mechanism, downregulation of bcl-xL by emetine was shown to be responsible for enhancing the sensitivity of ovarian cancer cells to cisplatin. These findings suggest that the combination of cisplatin and emetine might be a promising treatment for ovarian cancer.

Combination of a novel HDAC inhibitor OBP-801/YM753 and a PI3K inhibitor LY294002 synergistically induces apoptosis in human endometrial carcinoma cells due to increase of Bim with accumulation of ROS.

OBJECTIVE: In most endometrial carcinoma, it has been observed that the PI3K/Akt pathway is abnormally accelerated in association with mutations in PIK3CA and PTEN. The present study aimed to examine the combined effect of a novel histone deacetylase (HDAC) inhibitor OBP-801/YM753 and a PI3K inhibitor LY294002 against human endometrial carcinoma cells. METHODS: The effects of OBP-801/YM753 and LY294002 on the growth of human endometrial carcinoma HEC-1A cells were examined using WST-8 and colony formation assays. The distribution of the cell cycle or apoptosis was analyzed by flow cytometry. The accumulation of intracellular reactive oxygen species (ROS) was measured with a 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) dye. The expression of apoptosis-related proteins was investigated by Western blotting. Mice engrafted with 1×10(8) HEC-1A cells were treated with OBP-801/YM753, LY294002 or the combination, and tumor volumes were measured. RESULTS: The combination of OBP-801/YM753 and LY294002 significantly inhibited the cell growth on comparison with each agent alone and synergistically increased apoptosis with the induction of Bim, a well-known apoptosis inducer. Additionally, the apoptosis induced by the combination was shown to be dependent on intracellular ROS accumulation and Bim induction. Moreover, the apoptosis-inducing effect of OBP-801/YM753 with LY294002 was more potent than that of SAHA with LY294002. Combined treatment with OBP-801/YM753 and LY294002 significantly suppressed tumor growth compared to the control in vivo. CONCLUSIONS: The combination of OBP-801/YM753 and LY294002 is effective on the inhibition of the growth of HEC-1A cells, and we suggest that this combination is promising a novel therapeutic strategy for endometrial carcinoma.

Dehydrozingerone, a structural analogue of curcumin, induces cell-cycle arrest at the G2/M phase and accumulates intracellular ROS in HT-29 human colon cancer cells.

Dehydrozingerone (1) is a pungent constituent present in the rhizomes of ginger (Zingiber officinale) and belongs structurally to the vanillyl ketone class. It is a representative of half the chemical structure of curcumin (2), which is an antioxidative yellow pigment obtained from the rhizomes of turmeric (Curcuma longa). Numerous studies have suggested that 2 is a promising phytochemical for the inhibition of malignant tumors, including colon cancer. On the other hand, there have been few studies on the potential antineoplastic properties of 1, and its mode of action based on a molecular mechanism is little known. Therefore, the antiproliferative effects of 1 were evaluated against HT-29 human colon cancer cells, and it was found that 1 dose-dependently inhibited growth at the G2/M phase with up-regulation of p21. Dehydrozingerone additionally led to the accumulation of intracellular ROS, although most radical scavengers could not clearly repress the cell-cycle arrest at the G2/M phase. Furthermore, two synthetic isomers of 1 (iso-dehydrozingerone, 3, and ortho-dehydrozingerone, 4) were also examined. On comparing of their activities, accumulation of intracellular ROS was found to be interrelated with growth-inhibitory effects. These results suggest that analogues of 1 may be potential chemotherapeutic agents for colon cancer.

p53 inactivation upregulates p73 expression through E2F-1 mediated transcription.

While p73 overexpression has been associated with increased apoptosis in cancer tissues, p73 overexpressing tumors appear to be of high grade malignancy. Why this putative tumor suppressor is overexpressed in cancer cells and what the function of overexpressed p73 is in breast cancers are critical questions to be addressed. By investigating the effect of p53 inactivation on p73 expression, we found that both protein and mRNA levels of TAp73 were increased in MCF-7/p53siRNA cells, MCF-7/p53mt135 cells and HCT-116/p53-/- cells, as compared to wild type control, suggesting that p53 inactivation by various forms upregulates p73. We showed that p53 knockdown induced p73 was mainly regulated at the transcriptional level. However, although p53 has a putative binding site in the TAp73 promoter, deletion of this binding site did not affect p53 knockdown mediated activation of TAp73 promoter. Chromatin immuno-precipitation (ChIP) data demonstrated that loss of p53 results in enhanced occupancy of E2F-1 in the TAp73 promoter. The responsive sequence of p53 inactivation mediated p73 upregulation was mapped to the proximal promoter region of the TAp73 gene. To test the role of E2F-1 in p53 inactivation mediated regulation of p73 transcription, we found that p53 knockdown enhanced E2F-1 dependent p73 transcription, and mutations in E2F-1 binding sites in the TAp73 promoter abrogated p53 knockdown mediated activation of TAp73 promoter. Moreover, we demonstrated that p21 is a mediator of p53-E2F crosstalk in the regulation of p73 transcription. We concluded that p53 knockdown/inactivation may upregulate TAp73 expression through E2F-1 mediated transcriptional regulation. p53 inactivation mediated upregulation of p73 suggests an intrinsic rescuing mechanism in response to p53 mutation/inactivation. These findings support further analysis of the correlation between p53 status and p73 expression and its prognostic/predictive significance in human cancers.

Brassinin induces G1 phase arrest through increase of p21 and p27 by inhibition of the phosphatidylinositol 3-kinase signaling pathway in human colon cancer cells.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is activated in a broad spectrum of human cancers, including colon cancer. The natural product brassinin is a type of indole compound derived from cruciferous vegetables, and has been shown to have anti-proliferative effects against cancer for both in vivo and in vitro models. Here, we show for the first time that brassinin inhibits cell growth in human colon cancer cells by arresting the cell cycle at the G1 phase via inhibition of the PI3K signaling pathway. Brassinin increased the expression of p21 and p27, resulting in hypophosphorylation of the retinoblastoma gene (RB). Knockdown of p21 or p27 by each siRNA significantly repressed G1 phase arrest induced by brassinin. The increase of p21 and p27 was associated with inhibition of the PI3K signaling pathway. In addition, exogenous expression of constitutively active Akt represses the cell cycle arrest at G1 phase induced by brassinin. These results suggest the possibility that brassinin inhibits the PI3K signaling pathway and upregulates the expression of p21 and p27, thereby inducing G1 phase arrest.

Polymorphisms in promoter sequences of the p15 ( INK4B ) and PTEN genes of normal Japanese individuals.

Gene promoter regions of p15(INK4B), a cyclin-dependent kinase inhibitor, and phosphatase and tensin homolog (PTEN), a dual-function protein and lipid phosphatase, interact with regulatory factors for gene transcription and methylation. Normal individuals exhibit sequence polymorphisms in these regulatory genes. We isolated genomic DNA from whole blood of healthy Japanese individuals and sequenced promoter regions of the p15 ( INK4B ) and PTEN genes. We also examined the influence of polymorphisms on promoter activity in several cell lines. We identified polymorphisms at positions -699, -394, and -242 and an insertion at position -320 in the p15 ( INK4B ) gene and a polymorphism at position -1142 in the PTEN gene. Reporter gene analysis revealed that these polymorphisms influenced transcriptional regulation in their cell lines. Our results indicate for the first time that promoter sequences of the p15 ( INK4B ) and PTEN genes differ among normal Japanese individuals and that promoter polymorphisms can influence gene transcription.

Histone deacetylase inhibitors and 15-deoxy-Delta12,14-prostaglandin J2 synergistically induce apoptosis.

PURPOSE: The clinically relevant histone deacetylase inhibitors (HDI) valproic acid (VPA) and suberoylanilide hydroxamic acid exert variable antitumor activities but increase therapeutic efficacy when combined with other agents. The natural endogenous ligand of peroxisome proliferator-activated receptor gamma 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a potent antineoplastic agent. Therefore, we investigated whether these HDIs in combination with 15d-PGJ(2) could show synergistic antitumor activity in colon cancer DLD-1 cells. EXPERIMENTAL DESIGN: Cell viability was determined using a Cell Counting Kit-8 assay. Apoptosis and reactive oxygen species (ROS) generation were determined using flow cytometry analysis. Western blotting and real-time reverse transcription-PCR analysis were carried out to investigate the expression of apoptosis-related molecules. Mice bearing DLD-1 xenograft were divided into four groups (n = 5) and injected everyday (i.p.) with diluent, VPA (100 mg/kg), 15d-PGJ(2) (5 mg/kg), or a combination for 25 days. RESULTS: HDI/15d-PGJ(2) cotreatments synergistically induced cell death through caspase-dependent apoptosis in DLD-1 cells. Moreover, HDIs/15d-PGJ(2) caused histone deacetylase inhibition, leading to subsequent ROS generation and endoplasmic reticulum stress to decrease the expression of antiapoptotic molecules Bcl-X(L) and XIAP and to increase that of proapoptotic molecules CAAT/enhancer binding protein homologous protein and death receptor 5. Additionally, VPA/15d-PGJ(2) cotreatment induced ROS-dependent apoptosis in other malignant tumor cells and was more effective than a VPA or 15d-PGJ(2) monotherapy in vivo. CONCLUSIONS: Cotreatments with the clinically relevant HDIs and the endogenous peroxisome proliferator-activated receptor gamma ligand 15d-PGJ(2) are promising for the treatment of a broad spectrum of malignant tumors.

Cucurbitacin B induces G2 arrest and apoptosis via a reactive oxygen species-dependent mechanism in human colon adenocarcinoma SW480 cells.

Cucurbitacin B (cucB) is a triterpenoid constituent of Cucurbitaceae vegetables and a promising phytochemical for cancer prevention. However, the mechanism of anti-tumor activity of cucB remains unknown, especially in colon cancers. Here, we demonstrate for the first time that cucB inhibited growth of human colon cancer SW480 cells through a reactive oxygen species (ROS)-dependent mechanism. CucB induced G(2) phase arrest and apoptosis in a dose-dependent manner. At the molecular level, cucB reduced the expression of cyclin B1 and cdc25C proteins and activated caspases in SW480 cells. On the other hand, the state of phosphorylation of signaling transducer and activator of transcription 3 (STAT3) was unchanged. We found that cucB increased intracellular ROS levels, and N-acetylcysteine, a well-known antioxidant, reduced the changes in expression of the molecules, and suppressed both G(2) arrest and apoptosis. These results suggested that cucB induced G(2) arrest and apoptosis through a STAT3-independent but ROS-dependent mechanism in SW480 cells.

A combination of indol-3-carbinol and genistein synergistically induces apoptosis in human colon cancer HT-29 cells by inhibiting Akt phosphorylation and progression of autophagy.

BACKGROUND: The chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies. RESULTS: Here we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not. CONCLUSION: Although in vivo study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.

Identification of JTP-70902, a p15(INK4b)-inductive compound, as a novel MEK1/2 inhibitor.

The INK4 family members p16(INK4a) and p15(INK4b) negatively regulate cell cycle progression by inhibition of cyclin-dependent kinase (CDK) 4/6. Loss of p16(INK4a) functional activity is frequently observed in tumor cells, and is thought to be one of the primary causes of carcinogenesis. In contrast, despite the biochemical similarity to p16(INK4a), the frequency of defects in p15(INK4b) was found to be lower than in p16(INK4a), suggesting that p15(INK4b)-inductive agents may be useful for tumor suppression. Here we report the discovery of a novel pyrido-pyrimidine derivative, JTP-70902, which exhibits p15(INK4b)-inducing activity in p16(INK4a)-inactivated human colon cancer HT-29 cells. JTP-70902 also induced another CDK-inhibitor, p27(KIP1), and downregulated the expression of c-Myc and cyclin D1, resulting in G(1) cell cycle arrest. MEK1/2 was identified by compound-immobilized affinity chromatography as the molecular target of JTP-70902, and this was further confirmed by the inhibitory activity of JTP-70902 against MEK1/2 in kinase assays. JTP-70902 suppressed the growth of most colorectal and some other cancer cell lines in vitro, and showed antitumor activity in an HT-29 xenograft model. However, JTP-70902 did not inhibit the growth of COLO320 DM cells; in these, constitutive extracellular signal-regulated kinase phosphorylation was not detected, and neither p15(INK4b) nor p27(KIP1) induction was observed. Moreover, p15(INK4b)-deficient mouse embryonic fibroblasts were found to be more resistant to the growth-inhibitory effect of JTP-70902 than wild-type mouse embryonic fibroblasts. These findings suggest that JTP-70902 restores CDK inhibitor-mediated cell cycle control by inhibiting MEK1/2 and exerts a potent antitumor effect.

ZD1839 induces p15INK4b and causes G1 arrest by inhibiting the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.

Inactivation of the retinoblastoma protein pathway is the most common abnormality in malignant tumors. We therefore tried to detect agents that induce the cyclin-dependent kinase inhibitor p15(INK4b) and found that ZD1839 (gefitinib, Iressa) could up-regulate p15(INK4b) expression. ZD1839 has been shown to inhibit cell cycle progression through inhibition of signaling pathways such as phosphatidylinositol 3'-kinase-Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascades. However, the mechanism responsible for the differential sensitivity of the signaling pathways to ZD1839 remains unclear. We here showed that ZD1839 up-regulated p15(INK4b), resulting in retinoblastoma hypophosphorylation and G(1) arrest in human immortalized keratinocyte HaCaT cells. p15(INK4b) induction was caused by MAPK/ERK kinase inhibitor (PD98059), but not by Akt inhibitor (SH-6, Akt-III). Moreover, mouse embryo fibroblasts lacking p15(INK4b) were resistant to the growth inhibitory effects of ZD1839 compared with wild-type mouse embryo fibroblasts. Additionally, the status of ERK phosphorylation was related to the antiproliferative activity of ZD1839 in human colon cancer HT-29 and Colo320DM cell lines. Our results suggest that induction of p15(INK4b) by inhibition of the MAPK/ERK pathway is associated with the antiproliferative effects of ZD1839.

Oct-1 is involved in the transcriptional repression of the p15(INK4b) gene.

p15(INK4b) functions as a tumor suppressor and implicated in cellular senescence. Here, we show that the Oct-1 binding site in the human p15(INK4b) gene promoter functions as a silencer. Oct-1 specifically interacts with this binding site in vitro and in vivo and SMRT and HDAC1 are present in the p15(INK4b) proximal promoter region. Moreover, mouse embryo fibroblasts (MEFs) lacking Oct-1 have shown significantly increased levels of p15(INK4b) protein compared to their normal counterparts. Treatment with a histone deacetylase (HDAC) inhibitor has activated the expression of p15(INK4b) in wild-type MEFs but has no effect in MEFs lacking Oct-1, suggesting that Oct-1 represses p15(INK4b) gene expression in an HDAC-dependent manner. Finally, we show that the expression of Oct-1 protein significantly decreases, whereas p15(INK4b) protein significantly increases with the cellular aging process. Taken together, these results suggest that Oct-1 is an important transcriptional repressor for p15(INK4b) gene and the transcriptional repression of the p15(INK4b) gene by Oct-1 may be one of the regulatory mechanisms of cellular senescence.

A novel synthetic drug, LB-18, closely related to lembehyne-A derived from a marine sponge, induces caspase-independent cell death to human neuroblastoma cells.

Neuroblastoma is a common solid tumor of children that arises from the sympathetic nervous system. Much work has consequently focused on the possibility of inducing marked cell death in neuroblastoma, and the new effective drugs are required. We have newly synthesized LB-18, closely related to lembehyne A (LB-A), a polyacetylene derived from a kind of marine sponge. LB-A has been shown to induce p21/WAF1 and causes G1 phase arrest in mouse neuroblastoma Neuro2A cells; however, we show here that LB-18 causes cell death in human neuroblastoma KP-N-TK cells in a dose-dependent manner. TUNEL assay and flow cytometric analysis showed that the cell death caused by LB-18 was associated with the DNA damage but the pan-caspase inhibitor, zVAD-fmk, could not prevent the cell death. Western blot analysis and cleavage of the caspase-3 or -7 substrate assay showed that LB-18 could not activate caspases 3, 7, 8 and 9. These results suggest that LB-18 causes caspase-independent cell death in human neuroblastoma cells. In the future, LB-18 may be useful for cancer therapeutics, especially for neuroblastoma.

Lysocellin, a metabolite of the novel drug 'alopestatin', induces G1 arrest and prevents cytotoxicity induced by etoposide.

We report here that lysocellin, a polyether antibiotic from a streptomycete, induces G1 phase arrest in human osteosarcoma MG63 cells. Lysocellin up-regulates p21WAF1/Cip1 and down-regulates cyclin D1 at the mRNA level. In addition, cyclin D1 is down-regulated by the proteasome-dependent signal pathway in MG63 cells. In drug combination studies, we found that lysocellin treatment weakened the cytotoxic activity of etoposide in MG63 cells using a colony-formation assay. To study the in vivo efficacy of lysocellin, we isolated a novel compound related to lysocellin from the same streptomycete, and found that the novel drug is converted to lysocellin in vivo and decreases etoposide-induced alopecia in a neonatal rat model. We raise the possibility that this novel drug, named 'alopestatin', may be a promising agent against alopecia.

Quercetin induces gadd45 expression through a p53-independent pathway.

Quercetin, a kind of flavonoid, is found in edible fruits and vegetables and has anti-tumorigenic activity. However, the mechanism of activity has not been elucidated. We show for the first time that gadd45 is a molecular target of quercetin, which inhibits growth of human cervical cancer HeLa cells. Apoptosis was detected in HeLa cells treated with quercetin. At the concentration inducing apoptosis, quercetin also increased gadd45 expression at the mRNA and protein level, however, the 5'-promoter region of the gadd45 gene was not activated by quercetin. Since gadd45 is known to be a downstream gene of the tumor suppressor p53, we examined whether or not quercetin regulates gadd45 induction via a p53 pathway. Quercetin did not activate transcription through p53-binding sites in HeLa cells, although it up-regulated gadd45 in p53-inactivated tumor cells. These results indicate that quercetin induces gadd45 expression in a p53-independent manner.

Inhibitory effects of cancer cell proliferation by novel histone deacetylase inhibitors involve p21/WAF1 induction and G2/M arrest.

Two compounds were synthesized which have a structural component other than those of our new series histone deacetylase (HDAC) inhibitors to determine the structure-activity relationship. It was also examined whether the inhibitory effects on cancer cell proliferation by HDAC inhibitors involve p21/WAF1 induction and G(1) or G(2)/M arrest in p53-mutated MG63 human osteosarcoma cells as do other HDAC inhibitors. It was demonstrated that inhibitors with the 2-naphthylcarbonyl group and hydroxamic acid at both termimal sides as well as the phenylene component at the center of molecule markedly induce the p21/WAF1 protein by stimulating p21/WAF1 gene promoter activity. Furthermore, cell cycle analysis revealed that these compounds arrest MG63 cells in the G(2)/M phase.

Apigenin induces cell cycle arrest and p21/WAF1 expression in a p53-independent pathway.

Apigenin, a common dietary flavonoid, has been shown to induce cell growth-inhibition and cell cycle arrest in many cancer cell lines. One important effect of apigenin is to increase the stability of the tumor suppressor p53 in normal cells. Therefore, apigenin is expected to play a large role in cancer prevention by modifying the effects of p53 protein. However, the mechanisms of apigenin's effects on p53-mutant cancer cells have not been revealed yet. We assessed the influence of apigenin on cell growth and the cell cycle in p53-mutant cell lines. Treatment with apigenin resulted in growth-inhibition and G2/M phase arrest in two p53-mutant cancer cell lines, HT-29 and MG63. These effects were associated with a marked increase in the protein expression of p21/WAF1. We have shown that p21/WAF1 mRNA expression was also markedly increased by treatment with apigenin in a dose- and time-dependent manner. However, we could not detect p21/WAF1 promoter activity following treatment with apigenin. Similarly, promoter activity from pG13-Luc, a p53-responsive promoter plasmid, was not activated by treatment with apigenin with or without p53 protein expression. These results suggest that there is a p53-independent pathway for apigenin in p53-mutant cell lines, which induces p21/WAF1 expression and growth-inhibition. Apigenin may be a useful chemopreventive agent not only in wild-type p53 status, but also in cancer with mutant p53.