Geldanamycin induces G2 arrest in U87MG glioblastoma cells through downregulation of Cdc2 and cyclin B1

Cell cycle progression requires precise expression and activation of several cyclins and cyclin-dependent kinases. Geldanamycin (GA) affects cell cycle progression in various kinds of cells. We analyzed GA-induced cell cycle regulation in glioblastoma cells. GA-induced G2 or M arrest in glioblastoma cells in a cell line-dependent manner. GA decreased the expression of Cdc2 and cyclin B1 in U87MG cells. And phosphorylated Cdc2 decreased along with Cdc2 in the GA-treated cells. This cell line showed G2 arrest after GA treatment. In contrast, GA failed to down-regulate these cell cycle regulators in U251MG cells. In U251MG cells, the cell cycle was arrested at M phase in addition to G2 by GA. Next, we analyzed the mechanism of the GA-induced regulation of Cdc2 and cyclin B1 in U87MG cells. Cdc2 and cyclin B1 were ubiquitinated by GA. MG132 abrogated the GA-induced decrease of Cdc2 and cyclin B1 indicating that these proteins were degraded by proteasomes. In conclusion, GA controls the stability of Cdc2 and cyclin B1 in glioblastomas cell species-dependently. Cdc2 and cyclin B1 might be responsible for the different responses of glioblastoma cell lines to GA.     

Difference of cell cycle arrests induced by lidamycin in human breast cancer cells

Lidamycin (LDM) is a member of the enediyne antibiotic family. It is undergoing phase I clinical trials in China as a potential chemotherapeutic agent. In the present study, we investigated the mechanism by which LDM induced cell cycle arrest in human breast cancer cells. The results showed that LDM induced G1 arrest in p53 wild-type MCF-7 cells at low concentrations, and caused both G1 and G2/M arrests at higher concentrations. In contrast, LDM induced only G2/M arrest in p53-mutant MCF-7/DOX cells. Western blotting analysis indicated that LDM-induced G1 and G2/M arrests in MCF-7 cells were associated with an increase of p53 and p21, and a decrease of phosphorylated retinoblastoma tumor suppressor protein, cyclin-dependent kinase (Cdk), Cdc2 and cyclin B1 protein levels. However, LDM-induced G2/M arrest in MCF-7/DOX cells was correlated with the reduction of cyclin B1 expression. Further study indicated that the downregulation of cyclin B1 by LDM in MCF-7 cells was associated with decreasing cyclin B1 mRNA levels and promoting protein degradation, whereas it was only due to inducing cyclin B1 protein degradation in MCF-7/DOX cells. In addition, activation of checkpoint kinases Chk1 or Chk2 maybe contributed to LDM-induced cell cycle arrest. Taken together, we provide the first evidence that LDM induces different cell cycle arrests in human breast cancer cells, which are dependent on drug concentration and p53 status. These findings are helpful in understanding the molecular anti-cancer mechanisms of LDM and support its clinical trials.     

Physalis angulata induced G2/M phase arrest in human breast cancer cells.

Physalis angulata (PA) is employed in herbal medicine around the world. It is used to treat diabetes, hepatitis, asthma and malaria in Taiwan. We have evaluated PA as a cancer chemopreventive agent in vitro by studying the role of PA in regulation of proliferation, cell cycle and apoptosis in human breast cancer cell lines. PA inhibited cell proliferation and induced G2/M arrest and apoptosis in human breast cancer MAD-MB 231 and MCF-7 cell lines. In this study, under treatment with various concentrations of PA in MDA-MB 231 cell line, we checked mRNA levels for cyclin A and cyclin B1 and the protein levels of cyclin A and cyclin B1, Cdc2 (cyclin-dependent kinases), p21(waf1/cip1) and P27(Kip1) (cyclin-dependent kinase inhibitors), Cdc25C, Chk2 and Wee1 kinase (cyclin-dependent kinase relative factors) in cell cycle G2/M phase. From those results, we determined that PA arrests MDA-MB 231 cells at the G2/M phase by (i) inhibiting synthesis or stability of mRNA and their downstream protein levels of cyclin A and cyclin B1, (ii) increasing p21(waf1/cip1) and P27(kip1) levels, (iii) increasing Chk2, thus causing an increase in Cdc25C phosphorylation/inactivation and inducing a decrease in Cdc2 levels and an increase in Wee1 level. According to the results obtained, PA appears to possess anticarcinogenic properties; these results suggest that the effect of PA on the levels of phosphorylated/inactivated Cdc25C are mediated by Chk2 activation, at least in part, via p21(waf1/cip1) and P27(kip1) cyclin-dependent kinase inhibitors pathway to arrest cells at G2/M phase in breast cancer carcinoma cells.     

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。     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDB, in chronic myeloid leukemia (HL-60) cells.

(Z)-2-(6-(Thieanisyl-2-yl)hexa-3-en-1,5-diynyl)benzenamine (THDB), an enediyne compound, was identified in our laboratory as a novel antineoplastic agent with broad spectrum of antitumor activities against many human cancer cells. THDB was found to inhibit the growth of HL-60 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in HL-60 cells following 48 h exposure to THDB. Analysis of the cell cycle regulatory proteins demonstrated that THDB did not change the steady-state levels of cyclin B1, cyclin E, Cdk1 and Cdc25C, but decreased the protein levels of Cdk2 and cyclin A. THDB also caused a marked increase in apoptosis, as characterized by DNA fragmentation (DNA ladder and sub G1 formation), and poly (ADP-ribose) polymerase (PARP) cleavage, which was associated with activation of caspase-3, caspase-8 and caspase-9. Moreover, the THDB-induced apoptosis was significantly attenuated in the presence of specific inhibitors of caspase-3, -8 and -9. These molecular alterations provide an insight into THDB-caused growth inhibition, G2/M arrest and apoptotic death of HL-60 cells.     

Oroxylin A induces G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells

We reported previously that oroxylin A, a natural product isolated from Scutellariae Radix, was a potent apoptosis inducer of human hepatoma HepG2 cells. In this study, cell-cycle arrest of BGC-823 human gastric carcinoma cells caused by oroxylin A has been investigated. Based on our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of oroxylin A caused an irreversible arrest in the G2/M phase of the cell cycle. Western blot analysis demonstrated that oroxylin A-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in cdc2/p34, cyclin B1 and cyclin A expression. In addition, oroxylin A-treated cells decreased the expression of Cdk7, which was responsible for the low expression of M phase promoting factor (cyclin B1/Cdc2). The results suggested that oroxylin A induced G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells.     

Kotomolide A arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and the initiation of mitochondrial system in human non-small cell lung cancer A549 cells

This study first investigates the anticancer effect of kotomolide A (KTA) in human non-small cell lung cancer cells, A549. KTA has exhibited effective cell growth inhibition by inducing cancer cells to undergo G2/M phase arrest and apoptosis. Blockade of cell cycle was associated with increased the activation of ataxia telangiectasia-mutated (ATM). Activation of ATM by KTA phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute-2 (MDM2) interaction. In addition, KTA-mediated G2/M phase arrest also was associated with the decrease in the amounts of cyclinB1, cyclinA, Cdc2 and Cdc25C and increase in the phosphorylation of Chk2, Cdc25C and Cdc2. Specific ATM inhibitor, caffeine, significantly decreased KTA-mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. KTA treatment triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in mitochondrial membrane potential loss and caspase-9 activation. Taken together, these results suggest a critical role for ATM and p53 in KTA-induced G2/M arrest and apoptosis of human non-small cell lung cancer cells.     

Induction of G2/M phase arrest and apoptosis by a novel indoloquinoline derivative,IQDMA, in K562 cells

The indoloquinoline, IQDMA (N′‐(11H‐indolo[3,2‐c]quinolin‐6‐yl)‐N,N‐dimethylethane‐1,2‐diamine), was identified as a novel antineoplastic agent with broad spectrum of antitumor activities against several human cancer cells. IQDMA‐induced G2/M arrest was accompanied by up‐regulation of the cyclin‐dependent kinase inhibitors (CDKIs), p21 and p27, and down‐regulation of Cdk1and Cdk2. IQDMA had no effect on the levels of cyclin A, cyclin B1, cyclin D3, or Cdc25C. IQDMA also increased apoptosis, as characterized by apoptotic body formation, increase of the sub G₁ population and poly (ADP‐ribose) polymerase (PARP) cleavage. Further mechanistic analysis demonstrated that IQDMA upregulated FasL protein expression, and kinetic studies showed the sequential activation of caspases‐8, ‐3, and ‐9. Both caspase‐8 and caspase‐3 inhibitors, but not a caspase‐9‐specific inhibitor, suppressed IQDMA‐induced cell death. These molecular alterations provide an insight into IQDMA‐caused growth inhibition, G2/M arrest, and apoptotic death of K562 cells. Drug Dev. Res. 67:743–751, 2006. © 2006 Wiley‐Liss, Inc.     

Effects of genistein on cell proliferation and cell cycle arrest in nonneoplastic human mammary epithelial cells: involvement of Cdc2, p21(waf/cip1), p27(kip1), and Cdc25C expression

Genistein, a soy isoflavone, has been reported to inhibit the multiplication of numerous neoplastic cells, including those in the breast. However, there is limited information on the effect of genistein on nonneoplastic human breast cells. In the present studies, genistein inhibited proliferation of, and DNA synthesis in, the nonneoplastic human mammary epithelial cell line MCF-10F with an IC(50) of approximately 19-22 microM, and caused a reversible G2/M block in cell cycle progression. Genistein treatment (45 microM) increased the phosphorylation of Cdc2 by 3-fold, decreased the activity of Cdc2 by 70% after 8 hr, and by 24 hr reduced the expression of Cdc2 by 70%. In addition, genistein enhanced the expression of the cell cycle inhibitor p21(waf/cip1) by 10- to 15-fold, increased p21(waf/cip1) association with Cdc2 by 2-fold, and increased the expression of the tumor suppressor p53 by 2.8-fold. Genistein did not alter the expression of p27(kip1) significantly. Furthermore, genistein inhibited the expression of the cell cycle-associated phosphatase Cdc25C by 80%. From these results, we conclude that genistein inhibits the growth of nonneoplastic MCF-10F human breast cells by preventing the G2/M phase transition, induces the expression of the cell cycle inhibitor p21(waf/cip1) as well as its interaction with Cdc2, and inhibits the activity of Cdc2 in a phosphorylation-related manner. Down-regulation of the cell cycle-associated phosphatase Cdc25C combined with up-regulation of p21(waf/cip1) expression appear to be important mechanisms by which genistein decreases Cdc2 kinase activity and causes G2 cell cycle arrest.     

Genistein arrests hepatoma cells at G2/M phase: involvement of ATM activation and upregulation of p21waf1/cip1 and Wee1

Genistein, a soy isoflavone, has a wide range of biological actions that suggest it may be of use in cancer prevention. We have recently reported that it arrests hepatoma cells at G2/M phase and inhibits Cdc2 kinase activity. In the present study, we examined the signaling pathway by which genistein modulates Cdc2 kinase activity in HepG2 cells and leads to G2/M arrest, and found that it caused an increase in both Cdc2 phosphorylation and expression of the Cdc2-active kinase, Wee1. Genistein also enhanced the expression of the cell cycle inhibitor, p21waf1/cip1, which interacts with Cdc2. Furthermore, phosphorylation/inactivation of Cdc25C phosphatase, which dephosphorylates/activates Cdc2, was increased. Genistein enhanced the activity of the checkpoint kinase, Chk2, which phosphorylates/inactivates Cdc25C, induced accumulation of p53, and activated the ataxia-telangiectasia-mutated (ATM) gene. Caffeine, an ATM kinase inhibitor, inhibited these effects of genistein on Chk2, p53, and p21waf1/cip1. These findings suggest that the effect of genistein on G2/M arrest in HepG2 cells is partly due to ATM-dependent Chk2 activation, an increase in Cdc2 phosphorylation/inactivation as a result of induction of Wee1 expression, and a decrease in Cdc2 activity as a result of induction of p21waf1/cip1 expression.     

The inhibitory effect of CIL-102 on the growth of human astrocytoma cells is mediated by the generation of reactive oxygen species and induction of ERK1/2 MAPK

CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone) is the major active agent of the alkaloid derivative of Camptotheca acuminata, with multiple pharmacological activities, including anticancer effects and promotion of apoptosis. The mechanism by which CIL-102 inhibits growth remains poorly understood in human astrocytoma cells. Herein, we investigated the molecular mechanisms by which CIL-102 affects the generation of reactive oxygen species (ROS) and cell cycle G2/M arrest in glioma cells. Treatment of U87 cells with 1.0 μM CIL-102 resulted in phosphorylation of extracellular signal-related kinase (ERK1/2), downregulation of cell cycle-related proteins (cyclin A, cyclin B, cyclin D1, and cdk1), and phosphorylation of cdk1Tyr¹⁵ and Cdc25cSer²¹⁶. Furthermore, treatment with the ERK1/2 inhibitor PD98059 abolished CIL-102-induced Cdc25cSer²¹⁶ expression and reversed CIL-102-inhibited cdk1 activation. In addition, N-acetyl cysteine (NAC), an ROS scavenger, blocked cell cycle G2/M arrest and phosphorylation of ERK1/2 and Cdc25cSer²¹⁶ in U87 cells. CIL-102-mediated ERK1/2 and ROS production, and cell cycle arrest were blocked by treatment with specific inhibitors. In conclusion, we have identified a novel CIL-102-inhibited proliferation in U87 cells by activating the ERK1/2 and Cdc25cSer²¹⁶ cell cycle-related proteins and inducing ROS production; this might be a new mechanism in human astrocytoma cells.     

Involvement of c-jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by cardiotoxin III (Naja naja atra) in K562 leukemia cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, may have a potentiality as a structural template for rational drug design in killing cancer cells. Treatment of K562 cells with 0.3 microM of CTX III resulted in G2/M phase cell cycle arrest that was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin A, cyclin B1, Cdk2 and Cdc25C. In contrast to no effect on the phosphorylation of ERK, p38 MAPK and Akt, an activation of JNK was noted when K562 cells were exposed to CTX III. CTX III-mediated G2/M phase arrest and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125, but not by ERK and p38MAPK inhibitors. Further investigation showed that the specific JNK inhibitor, SP600125, reduced the activation of caspase-3, caspase-9, and reversed the decline in the expression of cyclin B1. Taken together, our data show for the first time that JNK, but not ERK, p38MAPK or Akt signaling, plays an important role in CTX III-mediated G2/M arrest and apoptosis in K562 cancer cells.     

6-Mercaptopurine (6-MP) induces cell cycle arrest and apoptosis of neural progenitor cells in the developing fetal rat brain

6-Mercaptopurine (6-MP), an analogue of hypoxanthine, is used in the therapy of acute lymphoblastic leukemia and causes fetal neurotoxicity. To clarify the mechanisms of 6-MP-induced fetal neurotoxicity leading to the cell cycle arrest and apoptosis of neural progenitor cells, pregnant rats were treated with 50 mg/kg 6-MP on embryonic day (E) 13, and the fetal telencephalons were examined at 12 to 72 h (h) after treatment. Flow-cytometric analysis confirmed an accumulation of cells at G2/M, S, and sub-G1 (apoptotic cells) phases from 24 to 72 h. The number of phosphorylated histone H3-positive cells (mitotic cells) decreased from 36 to 72 h, and the phosphorylated (active) form of p53 protein, which is a mediator of apoptosis and cell cycle arrest, increased from 24 to 48 h. An executor of p53-mediated cell cycle arrest, p21, showed intense overexpression at both the mRNA and protein levels from 24 to 72 h. Cdc25A protein, which is needed for the progression of S phase, decreased at 36 and 48 h. In addition, phosphorylated cdc2 protein, which is an inactive form of cdc2 necessary for G2/M progression, increased from 24 to 48 h. These results suggest that 6-MP induced G2/M arrest, delayed S-phase progression, and finally induced apoptosis of neural progenitor cells mediated by p53 in the fetal rat telencephalon.     

Role of PDE3A in regulation of cell cycle progression in mouse vascular smooth muscle cells and oocytes: implications in cardiovascular diseases and infertility

Phosphodiesterase-3 (PDE3) is a major cAMP-hydrolyzing PDE in vascular smooth muscle cells (VSMCs) and oocytes. The exact role and contribution of the two PDE3 isoforms, PDE3A and PDE3B, in VSMC growth regulation and oocyte maturation was examined using PDE3A (3A) and PDE3B (3B) knockout (KO) mouse models. PDE3A-deficient VSMCs exhibit marked reduction in mitogen-induced cell growth due to cell cycle arrest at G?-G? phase, which resulted from dysregulation of cAMP/protein kinase A (PKA)-activated and mitogen-activated protein kinase (MAPK)-signaling pathways, as well as from alterations in key cell cycle regulatory proteins. Similarly, PDE3A-deficient oocytes exhibit cell cycle arrest at G?/M phase because increased cAMP/PKA signaling in KO oocytes most likely inhibits Cdc25B-catalyzed dephosphorylation/activation of Cdc2 (maturation promoting factor (MPF)), a key regulator of G?/M transition.     

Opposite roles of ERK and p38 mitogen-activated protein kinases in cadmium-induced genotoxicity and mitotic arrest

The roles of extracellular signal-regulated kinase (ERK) and p38 mitogen-activation protein kinase (MAPK) in guarding genome stability and regulating cell cycle progression were explored in CL3 human lung adenocarcinoma cells treated with cadmium (Cd), a human carcinogen. Exposing asynchronous cells to CdCl(2) for 2 h (45% viability) caused irreversible mitotic arrest. Exposing early-G(2) cells to Cd markedly delayed mitotic exit and subsequently induced sub-G(1) populations; however, this did not alter the levels of Cdc2 and cyclin B1. These results suggest that Cd elicits mitotic arrest without affecting the progression of G(2) to mitosis. Using counterflow centrifugal elutriation and flow cytometry analysis, CL3 cells synchronized at G(1)-, S-, and G(2)/M-phases were collected and treated with CdCl(2). G(2)/M was the most sensitive cell cycle phase to Cd for the induction of ERK and p38 MAPK activities, cytotoxicity, apoptosis, micronucleus, and intracellular peroxide; despite that similar Cd accumulation was observed in G(1)-, S-, and G(2)/M-cells. Co-treatment early-G(2) cells with Cd and SB202190, an inhibitor of p38 MAPK, significantly decreased the induction of micronucleus, mitotic arrest, and apoptosis. Conversely, PD98059, an inhibitor of the ERK upstream activators MKK1/2, enhanced micronucleus and apoptosis in Cd-treated early-G(2) cells. Together, the results suggest that intracellular peroxide may participate in the activation of ERK and p38 MAPK by Cd; also, the activated-p38 MAPK may contribute to mitotic arrest and genome instability, whereas the activated-ERK may help to maintain genome integrity and survival.     

Helioxanthin suppresses the cross talk of COX‐2/PGE2 and EGFR/ERK pathway to inhibit Arecoline‐induced Oral Cancer Cell (T28) proliferation and blocks tumor growth in xenografted nude mice

Helioxanthin, an active compound from Taiwania cryptomerioides Hayata, has been shown to have various biological activities. However, their anticancer effect in oral squamous cell carcinoma has not been well established yet. Helioxanthin inhibited the proliferation of oral squamous cell carcinoma cells in a dose‐dependent manner by inducing G2/M phase arrest. Similarly, helioxanthin inhibited cyclooxygenase‐2, (COX‐2), phosphorylated EGFR, and extracellular‐signal‐regulated kinases (ERK) protein level and further reduced the nuclear accumulation of phosphorylated epidermal growth factor receptor (pEGFR) and activator protein‐1(AP‐1) family protein, c‐fos. Moreover, helioxanthin at the dose of 20 and 30 mg kg^?1 for 15 days reduced the tumor growth in animal model. This study demonstrated that Helioxanthin exerts its anticancer activity against oral cancer cells by downregulating EGFR/ERK/c‐fos signaling pathway to inhibit COX‐2 level and by activating cyclin‐dependent kinase inhibitor (p27) to further induce G2/M cell cycle arrest. This helioxanthin may serve as a novel candidate for oral cancer prevention. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2045–2056, 2016.     

2(±)-7,8,3′,4′,5′-pentamethoxyflavan induces G2/M phase arrest and apoptosis in HL60 cells

2(±)-7,8,3′,4′,5′-pentamethoxyflavan (PMF), a synthetic flavan racemate, showed growth inhibitory effect on various kinds of tumor cells. The present study is to investigate the molecular mechanisms of action of PMF in human leukemia HL60 cells. Anti-proliferative effect of PMF on HL60 cells was associated with G2/M cell cycle arrest, which was mediated by regulating the expression of Cdc25C, cyclin A and p21 proteins and inhibiting the phosphorylation of Cdc2 at Thr161. PMF also induced apoptosis of HL60 cells via death receptor and mitochondria apoptotic pathways, which was characterized by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase, caspase-3, caspase-8 and caspase-9, changes of Bcl-2 and Bax expression, cytochrome c release from mitochondria and a decrease in the mitochondrial membrane potential (MMP). These data suggest that PMF produces anti-tumor effect via induction of G2/M cell cycle arrest and apoptosis.     

Mechanism of cell cycle arrest caused by histone deacetylase inhibitors in human carcinoma cells

Inhibitors of histone deacetylase (HDAC) block cell cycle progression at G1 in many cell types. We investigated the mechanism by which trichostatin A (TSA), a specific inhibitor of HDAC, induces G1 arrest in human cervix carcinoma HeLa cells. TSA treatment induced histone hyperacetylation followed by growth arrest in G as well as hypophosphorylation of pRb. The Cdk4 kinase activity was essentially unchanged during the TSA-induced G1 arrest. On the other hand, the arrest was accompanied by down-regulation of kinase activity of Cdk2, although the total protein levels of Cdk2 and its activator Cdc25A were unaffected. Upon TSA treatment, amounts of cyclin E and the CDK inhibitor p21WAF1/Cip1 were markedly increased, while that of cyclin A was reduced. The induction of p21 and down-regulation of cyclin A correlated well with the decreased Cdk2 activity and cell cycle arrest. Furthermore, gel filtration chromatography showed the association of p21 with the cyclin E-Cdk2 complex, suggesting that the activation of Cdk2 by the enhanced expression of cyclin E is blocked by the increased p21. The elevated expression of p2 is also observed in cells treated with trapoxin and FR901228, structurally unrelated histone deacetylase inhibitors. A human colorectal carcinoma cell line lacking both alleles of the p21 gene (p21-/-) was resistant to TSA several times more than the parental line (p21+/+). These results suggest that the suppression of Cdk2 kinase activity due to p21 overexpression play a critical role in HDAC inhibitor-induced growth inhibition.     

A new structural class of proteasome inhibitors identified by microbial screening using yeast-based assay

A yeast-based growth interference assay was developed utilizing a yeast strain in which expression of Xenopus cyclin A1 was induced to elevate cell division cycle 28 (Cdc28) kinase activity. Since the hyperactivation of Cdc28 kinase in yeast results in a growth-arrest phenotype, compounds which could rescue the cyclin A1-induced growth arrest might be potential new, antitumor drug candidates acting on the cyclin-dependent, kinase-mediated, cell cycle regulation pathway. In the course of our microbial screening program, the new Streptomyces metabolites, belactosins, were identified. As reported previously, belactosin A induced cell cycle arrest at G2/M phase in human cancer cells. However, the molecular mechanism of action was unknown. We herein demonstrate the proteasome inhibition by belactosin A. Belactosin A did not inhibit yeast Cdc28 kinase and human cyclin-dependent kinase in vitro. On the other hand, it inhibited the chymotrypsin-like activity of the rabbit 20S proteasome. From the initial SAR studies, we identified a hydrophobic belactosin A derivative, KF33955, which exhibited a 100-fold greater growth-inhibitory activity against HeLa S3 cells than belactosin A, presumably due to its higher cell permeability. The biochemical analysis using KF33955 suggested that the proteasome inhibitory activity of KF33955 were irreversible and required the beta-lactone moiety to inhibit the proteasome. KF33955 increased the intracellular levels of protein ubiquitination in NIH3T3 cells. In addition, KF33955 treatment resulted in the accumulation of known proteasome substrates in HeLa S3 cells. These results identify belactosin A as a useful lead compound to target proteasome for the treatment of disease whose etiology is dependent on the unregulated ubiquitin-proteasome pathway.     

Pirarubicin inhibits multidrug-resistant osteosarcoma cell proliferation through induction of G2/M phase cell cycle arrest

Aim:

Pirarubicin (THP) is recently found to be effective in treating patients with advanced, relapsed or recurrent high-grade osteosarcoma. In this study, the effects of THP on the multidrug-resistant (MDR) osteosarcoma cells were assessed, and the underlying mechanisms for the disruption of cell cycle kinetics by THP were explored.

Methods:

Human osteosarcoma cell line MG63 and human MDR osteosarcoma cell line MG63/DOX were tested. The cytotoxicity of drugs was examined using a cell proliferation assay with the Cell Counting Kit-8 (CCK-8). The distribution of cells across the cell cycle was determined with flow cytometry. The expression of cell cycle-regulated genes cyclin B1 and Cdc2 (CDK1), and the phosphorylated Cdc2 and Cdc25C was examined using Western blot analyses.

Results:

MG63/DOX cells were highly resistant to doxorubicin (ADM) and gemcitabine (GEM), but were sensitive or lowly resistant to THP, methotrexate (MTX) and cisplatin (DDP). Treatment of MG63/DOX cells with THP (200–1000 ng/mL) inhibited the cell proliferation in time- and concentration-dependent manners. THP (50–500 ng/mL) induced MG63/DOX cell cycle arrest at the G₂/M phase in time- and concentration-dependent manners. Furthermore, the treatment of MG63/DOX cells with THP (200–1000 ng/mL) downregulated cyclin B1 expression, and decreased the phosphorylated Cdc2 at Thr¹⁶¹. Conversely, the treatment increased the phosphorylated Cdc2 at Thr¹⁴/Tyr¹⁵ and Cdc25C at Ser²¹⁶, which led to a decrease in Cdc2-cyclin B1 activity.

Conclusion:

The cytotoxicity of THP to MG63/DOX cells may be in part due to its ability to arrest cell cycle progression at the G₂/M phase, which supports the use of THP for managing patients with MDR osteosarcoma.