Differential modulation of zinc-stimulated p21(Cip/WAF1) and cyclin D1 induction by inhibition of PI3 kinase in HT-29 colorectal cancer cells

Activation of the extra cellular signal regulated kinase (ERK) pathway is involved in both proliferation and growth arrest of cells depending on intensity and duration of stimuli. In this study, we have elucidated differential regulation of the zinc-stimulated p21(CiP/WAF1) and cyclin D1 activation by inhibition of phosphoinositide 3-kinase (PI3K). In HT-29 colorectal cancer cells, the ERK activities were increased by zinc, which was accompanied by the induction of p21(Cip/WAF1) and cyclin D1. However, in the HT-29 cells pre-treated with PI3K inhibitor, LY294002, zinc induced further the p21(CiP/WAF) induction whereas abrogated cyclin D1 induction. In addition, the induction of p21(Cip/WAF1) expression completely inhibited the incorporation of bromodeoxyuridine (BrdU) into the nucleus, indicating that p21(CiP/WAF1) is an important indicator for ERK-dependent growth arrest. These studies suggest presence of an inter-related regulatory mechanism of cell proliferation by ERK and PI3K pathways.     

p21Cip/WAF1 activation is an important factor for the ERK pathway dependent anti-proliferation of colorectal cancer cells

p21Cip/WAF1, an important regulator of cell proliferation, is induced by both p53- and extracellular signal regulated kinase (ERK) pathways. The induction of p21Cip/WAF1 occurs by prolonged activation of the ERKs caused by extracellular stimuli, such as zinc. However, not all the cells appeared to respond to ERK pathway dependent p21Cip/WAF1 induction. Here we investigated the cause of such difference using colorectal cancer cells. p21Cip/WAF1 induction and concomitant reduction of bromodeoxyuridine (BrdU) incorporation were observed by zinc treatment within HT-29 and DLD-1. However, HCT-116 cells with high endogenous p21Cip/WAF1 levels did not show any additional increment of p21Cip/WAF1 levels by zinc treatment and did maintain high BrdU incorporation level. The p21Cip/WAF1 induction by zinc depended upon prolonged activation of extracellular signal regulated kinase (ERK) was not observed in HCT-116 cells. The percentage of BrdU positive cells was 50% higher in p21Cip/WAF1 -/- HCT-116 cells compared to p21Cip/WAF1 +/+ HCT- 116 cells, and no cells induced p21Cip/WAF1 incorporated BrdU in its nucleus, yet confirming the importance of p21Cip/WAF1 induction in anti- proliferation. These results again support that p21Cip/WAF1 induction is a determinant in the regulation of colonic proliferation by the ERK pathway.     

Induction of G2/M Cell Cycle Arrest via p38/p21Waf1/Cip1-Dependent Signaling Pathway Activation by Bavachinin in Non-Small-Cell Lung Cancer Cells

Lung cancer is the most commonly diagnosed malignant cancer in the world. Non-small-cell lung cancer (NSCLC) is the major category of lung cancer. Although effective therapies have been administered, for improving the NSCLC patient’s survival, the incident rate is still high. Therefore, searching for a good strategy for preventing NSCLC is urgent. Traditional Chinese medicine (TCM) are brilliant materials for cancer chemoprevention, because of their high biological safety and low cost. Bavachinin, which is an active flavanone of Proralea corylifolia L., possesses anti-inflammation, anti-angiogenesis, and anti-cancer activities. The present study’s aim was to evaluate the anti-cancer activity of bavachinin on NSCLC, and its regulating molecular mechanisms. The results exhibited that a dose-dependent decrease in the cell viability and colony formation capacity of three NSCLC cell lines, by bavachinin, were through G2/M cell cycle arrest induction. Meanwhile, the expression of the G2/M cell cycle regulators, such as cyclin B, p-cdc2^Y15, p-cdc2^T161, and p-wee1, was suppressed. With the dramatic up-regulation of the cyclin-dependent kinase inhibitor, p21^Waf1/Cip1, the expression and association of p21^Waf1/Cip1 with the cyclin B/cdc2 complex was observed. Silencing the p21^Waf1/Cip1 expression significantly rescued bavachinin-induced G2/M cell accumulation. Furthermore, the expression of p21^Waf1/Cip1 mRNA was up-regulated in bavachinin-treated NSCLC cells. In addition, MAPK and AKT signaling were activated in bavachinin-added NSCLC cells. Interestingly, bavachinin-induced p21^Waf1/Cip1 expression was repressed after restraint p38 MAPK activation. The inhibition of p38 MAPK activation reversed bavachinin-induced p21^Waf1/Cip1 mRNA expression and G2/M cell cycle arrest. Collectively, bavachinin-induced G2/M cell cycle arrest was through the p38 MAPK-mediated p21^Waf1/Cip1-dependent signaling pathway in the NSCLC cells.     

Role of Kupffer cells in thioacetamide-induced cell cycle dysfunction

It is well known that gadolinium chloride (GD) attenuates drug-induced hepatotoxicity by selectively inactivating Kupffer cells. In the present study the effect of GD in reference to cell cycle and postnecrotic liver regeneration induced by thioacetamide (TA) in rats was studied. Two months male rats, intraveously pretreated with a single dose of GD (0.1 mmol/Kg), were intraperitoneally injected with TA (6.6 mmol/Kg). Samples of blood and liver were obtained from rats at 0, 12, 24, 48, 72 and 96 h following TA intoxication. Parameters related to liver damage were determined in blood. In order to evaluate the mechanisms involved in the post-necrotic regenerative state, the levels of cyclin D and cyclin E as well as protein p27 and Proliferating Cell Nuclear Antigen (PCNA) were determined in liver extracts because of their roles in the control of cell cycle check-points. The results showed that GD significantly reduced the extent of necrosis. Noticeable changes were detected in the levels of cyclin D1, cyclin E, p27 and PCNA when compared to those induced by thioacetamide. Thus GD pre-treatment reduced TA-induced liver injury and accelerated the postnecrotic liver regeneration. These results demonstrate that Kupffer cells are involved in TA-induced liver and also in the postnecrotic proliferative liver states.     

Targeting glycosylation pathways and the cell cycle: sugar-dependent activity of butyrate-carbohydrate cancer prodrugs

Short-chain fatty acid (SCFA)-carbohydrate hybrid molecules that target both histone deacetylation and glycosylation pathways to achieve sugar-dependent activity against cancer cells are described in this article. Specifically, n-butyrate esters of N-acetyl-D-mannosamine (But4ManNAc, 1) induced apoptosis, whereas corresponding N-acetyl-D-glucosamine (But4GlcNAc, 2), D-mannose (But5Man, 3), or glycerol (tributryin, 4) derivatives only provided transient cell cycle arrest. Western blots, reporter gene assays, and cell cycle analysis established that n-butyrate, when delivered to cells via any carbohydrate scaffold, functioned as a histone deacetylase inhibitor (HDACi), upregulated p21WAF1/Cip1 expression, and inhibited proliferation. However, only 1, a compound that primed sialic acid biosynthesis and modulated the expression of a different set of genes compared to 3, ultimately killed the cells. These results demonstrate that the biological activity of butyrate can be tuned by sugars to improve its anticancer properties.     

Inhibitory Effects of Mistletoe Alkali on Salivary Adenoid Cystic Carcinoma Cells

Mistletoe alkali plays an important role in salivary adenoid cystic carcinoma(SACC) cell proliferation, apoptosis and invasion. Mistletoe alkali shows potent anticaner property. In this paper,immunocytochemical and immunofluorescence staining were employed to evaluate the expression levels of proliferating cell nuclear antigen(PCNA), Caspase 3, Caspase 8 and Caspase 9. Apoptosis was detected by acridine orange/ethidium bromide (AO/EB) staining, cell invasion ability was assessed by Boyden Chamber assay. Pretreatment with mistletoe alkali markedly decreased PCNA expression in SACC cells in a dose-dependent manner(P<0.001) and also led to increase the expression of Caspase 3, Caspase 8 and Caspase 9 in SACC cells compared with control group(P<0.001). Number of apoptotic cells increased dramatically in mistletoe alkali group(P<0.001). In Boyden Chamber assay, mistletoe alkali treatment could inhibit SACC cells to penetrate the artificial basement membrane compared with control group(P<0.01). Mistletoe alkali remarkably inhibited the proliferation and invasion of SACC cells and induced the apoptosis of SACC cells. These results provide an insight into the mechanisms of anticancer effects of mistletoe alkali, and highlight the potential clinical application of it.     

Total Synthesis and Biological Evaluation of Grassypeptolide A

Herein, we describe in full our investigations into the synthesis of grassypeptolide A ( 1 ) in 17 linear steps with an overall yield of 11.3 %. In particular, this work features the late‐stage introduction of sensitive bis(thiazoline) heterocycles and 31‐membered macrocyclization conducted at the sterically congested secondary amide site in superb conversion (72 % yield). Biological evaluation indicated that grassypeptolide A significantly inhibited cancer cell proliferation in a dose‐dependent manner. It induced cancer cell apoptosis, which was associated with increased cleavage of poly(ADP‐ribose) polymerase (PARP) and decreased expression of bcl‐2 and bcl‐xL. Furthermore, grassypeptolide A also caused cell cycle redistribution by increasing cells in the G1 phase and decreasing cells in the S and G2 phases. In addition, cell cycle arrest was correlated with downregulation of cyclin D and upregulation of p27 and p21.     

A new curcumin derivative, HBC, interferes with the cell cycle progression of colon cancer cells via antagonization of the Ca2+/calmodulin function

HBC (4-[3,5-Bis-[2-(4-hydroxy-3-methoxy-phenyl)-ethyl]-4,5-dihydro-pyrazol-1-yl]-benzoic acid) is a recently developed curcumin derivative which exhibits potent inhibitory activities against the proliferation of several tumor cell lines. In the present study, we identified Ca2+/calmodulin (Ca2+/CaM) as a direct target protein of HBC using phage display biopanning. Ca2+/CaM-expressing phages specifically bound to the immobilized HBC, and the binding was Ca2+ dependent. Moreover, flexible docking modeling demonstrated that HBC is compatible with the binding cavity for a known inhibitor, W7, in the C-terminal hydrophobic pocket of Ca2+/CaM. In biological systems, HBC induced prolonged phosphorylation of ERK1/2 and activated p21(WAF1) expression, resulting in the induction of G0/G1 cell cycle arrest in HCT15 colon cancer cells. These results suggest that HBC inhibits the cell cycle progression of colon cancer cells via antagonizing of Ca2+/CaM functions.     

CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34+ cells

A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34+ cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34+ cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34+ cells were compared with those in untreated CD34+ cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34+ cells. Here we describe an in vitro system in which CB CD34+ cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.     

Photodynamic Therapy Mediated by 5‐aminolevulinic Acid Promotes the Upregulation and Modifies the Intracellular Expression of Surveillance Proteins in Oral Squamous Cell Carcinoma

Expression of proteins related to cell surveillance has been described in tumors presenting resistance to photodynamic therapy (PDT). The aim of this study was to verify whether there was upregulation of proteins related to resistance in oral squamous cell carcinoma (OSCC) after PDT. OSCC was chemically induced in rats and treated after one cycle of PDT mediated by 5‐aminolevulinic acid (5‐ALA‐PDT). Immunolabeling of p‐NFκB, Bcl‐2, survivin, iNOS, p‐Akt, p‐mTOR and cyclin D1 was performed after the treatment. There was increased expression of Bcl‐2 (P = 0.008), iNOS (P = 0.020), p‐Akt (P = 0.020) and p‐mTOR (P = 0.010) by surviving neoplastic cells after PDT when compared to the control. In conclusion, after one cycle of 5‐ALA‐mediated PDT, Bcl‐2, p‐Akt, p‐mTOR and iNOS were upregulated in neoplastic cells of OSCC, suggesting an activation of antiapoptosis and cell proliferation pathways. This fact must be considered in the establishment of PDT protocols for OSCC treatment, mainly those in which PDT will be combined with chemotherapy drugs targeted at the studied proteins.     

Trichostatin A-mediated upregulation of p21(WAF1) contributes to osteoclast apoptosis

Histone deacetylase inhibitors (HDIs), a new class of anti-cancer agents, have been reported to suppress formation of osteoclast precursors and their fusion into multinucleated cells. However, little is known about the effect of HDIs on mature osteoclasts, which may have significance for their therapeutic use. Here, we demonstrate a novel action of HDIs on osteoclast apoptosis. Primary multinucleated mature osteoclasts were prepared from mouse bone marrow cells. Treatment of osteoclasts with the HDI trichostatin A (TSA) caused apoptosis, as confirmed by annexin V staining and caspase activation. TSA caused the upregulation of p21WAF1 in osteoclasts. To understand the role of p21(WAF1) upregulation in TSA-treated osteoclasts, shRNA against p21(WAF1)-containing lentivirus was introduced into osteoclasts. The suppression of p21(WAF1) decreased TSA-directed osteoclast apoptosis. Collectively, our results provide evidence that TSA causes osteoclast apoptosis, which involves, in part, TSA-induced upregulation of p21(WAF1), and strongly supports HDIs as potential therapeutic agents for excessive bone resorption.     

The effect of ferulic acid ethyl ester on leptin-induced proliferation and migration of aortic smooth muscle cells

Leptin is a peptide hormone, which has a central role in the regulation of body weight; it also exerts many potentially atherogenic effects. Ferulic acid ethyl ester (FAEE) has been approved for antioxidant properties. The aim of this study was to investigate whether FAEE can inhibit the atherogenic effects of leptin and the possible molecular mechanism of its action. Both of cell proliferation and migration were measured when the aortic smooth muscle cell (A10 cell) treated with leptin and/or FAEE. Phosphorylated p44/42MAPK, cell cycle-regulatory protein (for example, cyclin D1, p21, p27), β-catenin and matrix metalloproteinase-9 (MMP-9) proteins levels were also measured. Results demonstrated that leptin (10, 100 ng ml⁻¹) significantly increased the proliferation of cells and the phosphorylation of p44/42MAPK in A10 cells. The proliferative effect of leptin was significantly reduced by the pretreatment of U0126 (0.5 μM), a MEK inhibitor, in A10 cells. Meanwhile, leptin significantly increased the protein expression of cyclin D1, p21, β-catenin and decreased the expression of p27 in A10 cells. In addition, leptin (10 ng ml⁻¹) significantly increased the migration of A10 cells and the expression of MMP-9 protein. Above effects of leptin were significantly reduced by the pretreatment of FAEE (1 and 10 μM) in A10 cells. In conclusion, FAEE exerts multiple effects on leptin-induced cell proliferation and migration, including the inhibition of p44/42MAPK phosphorylation, cell cycle-regulatory proteins and MMP-9, thereby suggesting that FAEE may be a possible therapeutic approach to the inhibition of obese vascular disease.     

Ionizing radiation induces blockade of c-Jun N-terminal kinase-dependent cell death pathway in a manner correlated with p21(Cip/WAF1) induction in primary cultured normal human fibroblasts

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H(2)O(2). This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H(2)O(2) failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H(2)O(2) treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H(2)O(2), but elevated cellular levels of p21(Cip/WAF1), a p53 target that can suppress H(2)O(2)-induced cell death by blocking JNK activation. Consistently, H(2)O(2)-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.