Surfactin Induces Apoptosis and G<Subscript>2</Subscript>/M Arrest in Human Breast Cancer MCF-7 Cells Through Cell Cycle Factor Regulation

Surfactin, purified from Bacillus subtilis natto TK-1, inhibited proliferation of human breast cancer MCF-7 cells in a dose- and time-dependent manner, with IC₅₀ at 24, 48, and 72 h of 82.6, 27.3, and 14.8 μM, respectively. Surfactin-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change by acridine orange/ethidium bromide staining and Transferase-mediated dUTP Nick End-labeling assay. [Ca²⁺]i measurement revealed that surfactin induced a sustained increase in concentration of intracellular [Ca²⁺]i. Flow cytometric analysis also demonstrated that surfactin caused time-dependent apoptosis of MCF-7 cells through cell arrest at G₂/M phase. Western blot revealed that surfactin induced accumulation of the tumor suppressor p53 and cyclin kinase inhibitor p21^waf1/cip1, and inhibited the activity of the G₂-specific kinase, cyclin B1/p34^cdc2. Based on our findings, surfactin inhibited proliferation in MCF-7 cells by inducing apoptosis and the elevation of [Ca²⁺]i may play an important role in the apoptosis. The mechanism which surfactin caused G₂/M arrest seems to be through cell cycle factor regulation.     

Bcl-2 delays cell cycle through mitochondrial ATP and ROS

Bcl-2 inhibits cell proliferation by delaying G₀/G₁ to S phase entry. We tested the hypothesis that Bcl-2 regulates S phase entry through mitochondrial pathways. Existing evidence indicates mitochondrial adenosine tri-phosphate (ATP) and reactive oxygen species (ROS) are important signals in cell survival and cell death, however, the molecular details of how these 2 processes are linked remain unknown. In this study, 2 cell lines stably expressing Bcl-2, 3T3Bcl-2 and C3HBcl-2, and vector-alone PB controls were arrested in G₀/G₁ phase by serum starvation and contact inhibition, and ATP and ROS were measured during re-stimulation of cell cycle entry. Both ATP and ROS levels were decreased in G₀/G₁ arrested cells compared with normal growing cells. In addition, ROS levels were significant lower in synchronized Bcl-2 cells than those in PB controls. After re-stimulation, ATP levels increased with time, reaching peak value 1–3 hours ahead of S phase entry for both Bcl-2 cells and PB controls. Consistent with 2 hours of S phase delay, Bcl-2 cells reached ATP peaks 2 hours later than PB control, which suggests a rise in ATP levels is required for S phase entry. To examine the role of ATP and ROS in cell cycle regulation, ATP and ROS level were changed. We observed that elevation of ATP accelerated cell cycle progression in both PB and Bcl-2 cells, and decrease of ATP and ROS to the level equivalent to Bcl-2 cells delayed S phase entry in PB cells. Our results support the hypothesis that Bcl-2 protein regulates mitochondrial metabolism to produce less ATP and ROS, which contributes to S phase entry delay in Bcl-2 cells. These findings reveal a novel mechanistic basis for understanding the link between mitochondrial metabolism and tumor-suppressive function of Bcl-2.     

Cellular and biomolecular responses of human ovarian cancer cells to cytostatic dinuclear platinum(II) complexes

Polynuclear platinum(II) complexes represent a class of potential anticancer agents that have shown promising pharmacological properties in preclinical studies. The nature of cellular responses induced by these complexes, however, is poorly understood. In this research, the cellular responses of human ovarian cancer COC1 cells to dinuclear platinum(II) complexes {[cis-Pt(NH₃)₂Cl]₂L¹}(NO₃)₂ (1) and {[cis-Pt(NH₃)₂Cl]₂L²}(NO₃)₂ (2) (L¹ = α,α′-diamino-p-xylene, L² = 4,4′-methylenedianiline) has been studied using cisplatin as a reference. The effect of platinum complexes on the proliferation, death mode, mitochondrial membrane potential, and cell cycle progression has been examined by MTT assay and flow cytometry. The activation of cell cycle checkpoint kinases (CHK1/2), extracellular signal-regulated kinases (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) of the cells by the complexes has also been analyzed using phospho-specific flow cytometry. Complex 1 is more cytotoxic than complex 2 and cisplatin at most concentrations; complex 2 and cisplatin are comparably cytotoxic. These complexes kill the cells through an apoptotic or apoptosis-like pathway characterized by exposure of phosphatidylserine and dissipation of mitochondrial membrane potential. Complex 1 shows the strongest inductive effect on the morphological changes of the cells, followed by cisplatin and complex 2. Complexes 1 and 2 arrest the cell cycle in G2 or M phase, while cisplatin arrests the cell cycle in S phase. The influence of these complexes on CHK1/2, ERK1/2, and p38 MAPK varies with the dose of the drugs or reaction time. Activation of phospho-ERK1/2 and phospho-p38 MAPK by these complexes is closely related to the cytostatic activity. The results demonstrate that dinuclear platinum(II) complexes can induce some cellular responses different from those caused by cisplatin.     

Interleukin-17F attenuates H2O2-induced cell cycle arrest

Interleukin IL-17F was expressed in colon epithelial cells and showed multiple functions in colon tumorigenesis. However, the role of IL-17F in colon cancer cell cycle progression remains unclear. In this study, we analyzed the effects of IL-17F on oxidant-induced cell cycle shift in human colon cancer cells. IL-17F overexpressing and wildtype HCT116 cells were challenged with H₂O₂. Cell cycle distribution analysis showed IL-17F attenuated H₂O₂-induced G2/M phase arrest by inhibiting S to G2/M transition. We further checked expression levels of two critical cell cycle regulators p21 and p27. The results showed that IL-17F could inhibit H₂O₂ induced p27 up-regulation. Meanwhile, IL-17F could increase the phosphorylation of p38 after H₂O₂ treatment. The regulations of p27 level and p38 activity may contribute to the impaired G2/M phase arrest by IL-17F. Taken together, our findings extend IL-17F as an important factor in colon cancer development and provide new insight into the signaling pathway.     

Intracellular chloride regulates cell proliferation through the activation of stress‐activated protein kinases in MKN28 human gastric cancer cells

Recently, we reported that reduction of intracellular Cl^? concentration ([Cl^?]_i) inhibited proliferation of MKN28 gastric cancer cells by diminishing the transition rate from G₁ to S cell‐cycle phase through upregulation of p21, cyclin‐dependent kinase inhibitor, in a p53‐independent manner. However, it is still unknown how intracellular Cl^? regulates p21 expression level. In this study, we demonstrate that mitogen‐activated protein kinases (MAPKs) are involved in the p21 upregulation and cell‐cycle arrest induced by reduction of [Cl^?]_i. Culture of MKN28 cells in a low Cl^? medium significantly induced phosphorylation (activation) of MAPKs (ERK, p38, and JNK) and G₁/S cell‐cycle arrest. To clarify the involvement of MAPKs in p21 upregulation and cell growth inhibition in the low Cl^? medium, we studied effects of specific MAPKs inhibitors on p21 upregulation and G₁/S cell‐cycle arrest in MKN28 cells. Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl^? medium and rescued MKN28 cells from the low Cl^?‐induced G₁ cell‐cycle arrest, whereas treatment with an ERK inhibitor had no significant effect on p21 expression or the growth of MKN28 cells in the low Cl^? medium. These results strongly suggest that the intracellular Cl^? affects the cell proliferation via activation of p38 and/or JNK cascades through upregulation of the cyclin‐dependent kinase inhibitor (p21) in a p53‐independent manner in MKN28 cells. J. Cell. Physiol. 223:764–770, 2010. © 2010 Wiley‐Liss, Inc.     

Cell cycle propagation is driven by light–dark stimulation in a cultured symbiotic dinoflagellate isolated from corals

Endosymbiosis is an intriguing plant–animal interaction in the dinoflagellate–Cnidaria association. Throughout the life span of the majority of corals, the dinoflagellate Symbiodinium sp. is a common symbiont residing inside host gastrodermal cells. The mechanism of regulating the cell proliferation of host cells and their intracellular symbionts is critical for a stable endosymbiotic association. In the present study, the cell cycle of a cultured Symbiodinium sp. (clade B) isolated from the hermatypic coral Euphyllia glabrescens was investigated using flow cytometry. The results showed that the external light–dark (L:D) stimulation played a pivotal role in regulating the cell cycle process. The sequential light (40–100 μmol m⁻² s⁻¹ ~ 12 h) followed by dark (0 μmol m⁻² s⁻¹ ~ 12 h) treatment entrained a single cell cycle from the G₁ to the S phase, and then to the G₂/M phase, within 24 h. Blue light (~450 nm) alone mimicked regular white light, while lights of wavelengths in the red and infrared area of the spectrum had little or no effect in entraining the cell cycle. This diel pattern of the cell cycle was consistent with changes in cell motility, morphology, and photosynthetic efficiency (F _v /F _m ). Light treatment drove cells to enter the growing/DNA synthesis stage (i.e., G₁ to S to G₂/M), accompanied by increasing motility and photosynthetic efficiency. Inhibition of photosynthesis by 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea (DCMU) treatment blocked the cell proliferation process. Dark treatment was required for the mitotic division stage, where cells return from G₂/M to G₁. Two different pools of adenylyl cyclase (AC) activities were shown to be involved in the growing/DNA synthesis and mitotic division states, respectively. Communicated by Biology Editor Dr Michael Lesser     

Overexpression of peptidylarginine deiminase IV features in apoptosis of haematopoietic cells

Peptidylarginine deiminases (PADIs) convert peptidylarginine into citrulline via posttranslational modification. One member of the family, PADI4, plays an important role in immune cell differentiation and cell death. To elucidate the participation of PADI4 in haematopoietic cell death, we examine whether inducible overexpression of PADI4 enhances the apoptotic cell death. PADI4 reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells and human acute T leukemia Jurkat cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G1 appearance, loss of mitochondrial membrane potential (Δψ_m), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following PADI4 overexpression, cells arrest in G1 phase significantly before their entrance into apoptotic cell death. PADI4 increases tumor suppressor p53 and its downstream p21 to control cell cycle. In the detections of protein expression and kinase activity, all protein levels of cyclin-dependent kinases (CDKs) and cyclins are not reduced except cyclin D, however, CDK2 (G1 entry S phase) and CDK1 (G2 entry M phase) enzyme activities are inhibited by conditionally inducible PADI4. p53 also expands its other downstream Bax to induce cytochrome c release from mitochondria. According to these data, we suggest that PADI4 induces apoptosis mainly through cell cycle arrest and mitochondria-mediated pathway. Furthermore, p53 features in PADI4-induced apoptosis by increasing intracellular p21 to control cell cycle and by Bax accumulation to decline Bcl-2 function, destroy Δψ_m, release cytochrome c to cytoplasm and activate the caspase cascade.     

miR‐181c‐5p mediates simulated microgravity‐induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase

Impaired osteoblast proliferation plays fundamental roles in microgravity‐induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G₂ phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR‐181c‐5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3′UTR. Lastly, we demonstrated that inhibition of miR‐181c‐5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G₂ phase in primary mouse osteoblasts partially through the miR‐181c‐5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity‐induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.     

Gamma irradiation induced apoptotic changes in the chromatin structure of human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were synchronized by centrifugal elutriation prior to and after Co⁶⁰ γ-irradiation (4 Gy). Forward scatter flow cytometry used for size analysis revealed the increase of an early apoptotic cell population ranging from lower (0.05 C-value) to higher DNA content (∼1 C) as the cells progressed through the S phase. The increase in cellular DNA content expressed in C-values correlated with apoptotic chromatin changes manifested as many small apoptotic bodies in early S phase and larger but less numerous disintegrated apoptotic bodies in late S phase. Most significant changes after exposure to γ-irradiation took place in early S phase resulting in an increase of nuclear size by more than 50%. Cell fractions containing irradiated cells showed enhanced growth arrest at 2.4 C-value, which was accompanied by apoptosis. Apoptotic cell cycle arrest near to the G₁/G₀ checkpoint and apoptotic changes indicate that the radiation resistance of K562 cells is related to the bypass of the early stage of the p53 apoptotic pathway. Apoptotic changes in chromatin structure induced by γ-irradiation indicate that these injury-specific changes can be identified and distinguished from chromatin changes induced by UV radiation or heavy metals.     

Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110^Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34^cdc2 synthesis, and cyclin A synthesis. In control cultures, p110^Rb phosphorylation was detected within 24 hr of PHA stimulation; p34^cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c-fos, IL-2, and IL-4 mRNAs expression, and IL-2 receptor (p55) expression, were only marginally affected, if at all, in PHA-stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL-2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL-2/IL-2 receptor interaction but prior to p110^Rb phosphorylation and other key regulatory events signaling G₁/S transition. © 1993 Wiley-Liss, Inc.     

PKCα mediated induction of miR-101 in human hepatoma HepG2 cells

Background  

Protein Kinase C (PKC) is a serine/threonine kinase that involved in controlling of many cellular processes such as cell proliferation and differentiation. We have observed previously that TPA (12-O-tetradecanoylphorbol 13-acetate) induces cell cycle arrest in G0/G1 phase in human hepatoma HepG2 cells. However, is there any miRNA involved in PKCα mediated cell growth arrest is still unknown.     

The mechanisms of nadroparin‐mediated inhibition of proliferation of two human lung cancer cell lines

Objectives

Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines.

Materials and methods

A549 and CALU1 cells’ viability was assessed by MTT and trypan blue exclusion assays. Cell proliferation was assessed using 5‐bromo‐2‐deoxyuridine incorporation. Apoptosis and cell‐cycle distribution were analysed by flow cytometry; cyclin B1, Cdk1, p‐Cdk1 Cdc25C, p‐Cdc25C and p21 expressions were analysed by western blotting. mRNA levels were analysed by real time RT‐PCR.

Results

Nadroparin inhibited cell proliferation by 30% in both cell lines; it affected the cell cycle in A549, but not in CALU‐1 cells, inducing arrest in the G₂/M phase. Nadroparin in A549 culture inhibited cyclin B1, Cdk1, Cdc25C and p‐Cdc25C, while levels of p‐Cdk1 were elevated; p21 expression was not altered. Dalteparin caused a similar reduction in A549 cell population growth; however, it did not alter cyclin B1 expression as expected, based on previous reports. Fondaparinux caused minimal inhibition of A549 cell population growth and no effect on either cell cycle or cyclin B1 expression.

Conclusions

Nadroparin inhibited proliferation of A549 cells by inducing G₂/M phase cell‐cycle arrest that was dependent on the Cdc25C pathway, whereas CALU‐1 cell proliferation was halted by as yet not elucidated modes.     

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G₀/G₁ cell cycle arrest and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G₀/G₁ cell cycle phase arrest and increased levels of p27^kip1 in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G₀ state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.     

Analysis of radiation‐induced changes to human melanoma cultures using a mathematical model

Objectives: Tumour cells respond to ionizing radiation by cycle arrest, cell death or repair and possible regrowth. We have developed a dynamic mathematical model of the cell cycle to incorporate transition probabilities for entry into DNA replication and mitosis. In this study, we used the model to analyse effects of radiation on cultures of five human melanoma cell lines. Materials and methods: Cell lines were irradiated (9 Gy) prior to further culture and harvesting at multiple points up to 96 h later. Cells were fixed, stained with propidium iodide and analysed for G₁‐, S‐ and G₂/M‐phase cells by flow cytometry. Data for all time points were fitted to a mathematical model. To provide unique solutions, cultures were grown in the presence and absence of the mitotic poison paclitaxel, added to prevent cell division. Results: The model demonstrated that irradiation at 9 Gy induced G₂‐phase arrest in all lines for at least 96 h. Two cell lines with wild‐type p53 status additionally exhibited G₁‐phase arrest with recovery over 15 h, as well as evidence of cell loss. Resumption of cycling of surviving cells, as indicated by increases in G₁/S and G₂/M‐phase transitions, was broadly comparable with results of clonogenic assays. Conclusions: The results, combined with existing data from clonogenic survival assays, support the hypothesis that a dominant effect of radiation in these melanoma lines is the induction of long‐term cell cycle arrest.     

Ganoderic acid Me induces G1 arrest in wild-type p53 human tumor cells while G1/S transition arrest in p53-null cells

The mechanism of cell cycle arrest of tumor cells induced by ganoderic acid Me (GA-Me) is not understood. In this work, GA-Me was found to possess remarkable cytotoxicity on highly metastatic lung carcinoma 95-D cell line in both dose- and time-dependent manners. The effect of GA-Me on cell cycle arrest was found in 95-D, p53-null lung cancer cells H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? human colon cancer cells. To obtain an insight into the role of p53 in cell cycle arrest by GA-Me, 95-D, H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? cells were used for further investigation. GA-Me arrested cell cycle at G₁ phase in 95-D and HCT-116 p53^+/+ cells while S phase or G₁/S transition arrest in H1299 and HCT-116 p53^?/? cells. The results suggested that p53 may be a target of GA-Me, and it may be looked at as a new promising candidate for the treatment of carcinoma cells.     

Inhibitory role of TGIF in the As<Subscript>2</Subscript>O<Subscript>3</Subscript>-regulated p21<Superscript><Emphasis Type="Italic">WAF1/CIP1</Emphasis></Superscript> expression

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As₂O₃) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (−84/−64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As₂O₃-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As₂O₃ treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As₂O₃-inhibited p21 expression, and then blocks the cell cycle arrest.