New insights on neurodegeneration triggered by iron accumulation: Intersections with neutral lipid metabolism, ferroptosis, and motor impairment.

Brain iron accumulation constitutes a pathognomonic indicator in several neurodegenerative disorders. Metal accumulation associated with dopaminergic neuronal death has been documented in Parkinson's disease. Through the use of in vivo and in vitro models, we demonstrated that lipid dysregulation manifests as a neuronal and glial response during iron overload. In this study, we show that cholesterol content and triacylglycerol (TAG) hydrolysis were strongly elevated in mice midbrain. Lipid cacostasis was concomitant with the loss of dopaminergic neurons, astrogliosis and elevated expression of α-synuclein. Exacerbated lipid peroxidation and markers of ferroptosis were evident in the midbrain from mice challenged with iron overload. An imbalance in the activity of lipolytic and acylation enzymes was identified, favoring neutral lipid hydrolysis, and consequently reducing TAG and cholesteryl ester levels. Notably, these observed alterations were accompanied by motor impairment in iron-treated mice. In addition, neuronal and glial cultures along with their secretomes were used to gain further insight into the mechanism underlying TAG hydrolysis and cholesterol accumulation as cellular responses to iron accumulation. We demonstrated that TAG hydrolysis in neurons is triggered by astrocyte secretomes. Moreover, we found that the ferroptosis inhibitor, ferrostatin-1, effectively prevents cholesterol accumulation both in neurons and astrocytes. Taken together, these results indicate that lipid disturbances occur in iron-overloaded mice as a consequence of iron-induced oxidative stress and depend on neuron-glia crosstalk. Our findings suggest that developing therapies aimed at restoring lipid homeostasis may lead to specific treatment for neurodegeneration associated with ferroptosis and brain iron accumulation.

Structural analysis and biological activities of C25-amino and C25-nitro vitamin D analogs.

Intense synthetic efforts have been directed towards the development of noncalcemic analogs of 1,25-dihydroxyvitamin D3. We describe here the structural analysis and biological evaluation of two derivatives of 1,25-dihydroxyvitamin D3 with modifications limited to the replacement of the 25-hydroxyl group by a 25-amino or 25-nitro groups. Both compounds are agonists of the vitamin D receptor. They mediate biological effects similar to 1,25-dihydroxyvitamin D3, the 25-amino derivative being the most potent one while being less calcemic than 1,25-dihydroxyvitamin D3. The in vivo properties of the compounds make them of potential therapeutic value.

Structure-Activity Relationship Study of an Alkynylphosphonate and Vynilphosphonate Analogues of Calcitriol.

BACKGROUND: 1α,25-dihydroxy vitamin D3 (calcitriol) shows potent growth-inhibitory properties on different cancer cell lines, but its hypercalcemic effects have severely hampered its therapeutic application. Therefore, it is important to develop synthetic calcitriol analogues that retain or even increase its antitumoral effects and lack hypercalcemic activity. Based on previous evidence of the potent antitumor effects of the synthetic alkynylphosphonate EM1 analogue, we have now synthesized a derivative called SG. OBJECTIVE: The aim of the present work is to evaluate the calcemic activity and the antitumor effect of SG, comparing these effects with those exerted by calcitriol and with those previously published for EM1. In addition, we propose to analyze by in silico studies, the chemical structure-biological function relationship of these molecules. METHODS: We performed the synthesis of vinylphosphonate SG analogue; in vitro assays on different cancer cell lines; in vivo assays on mice; and in silico assays applying computational molecular modeling. RESULTS: The SG compound lacks hypercalcemic activity, similar to the parent compound EM1. However, the antitumor activity was blunted, as no antiproliferative or anti-migratory effects were observed. By in silico assays, we demonstrated that SG analogue has a lower affinity for the VDRligand- binding domain than the EM1 compound due to lack of interaction with the important residues His305 and His397. CONCLUSION: These results demonstrate that the chemical modification in the lateral side chain of the SG analogue affects the antitumoral activity observed previously for EM1 but does not affect the calcemic activity. These results contribute to the rational design and synthesis of novel calcitriol analogues.

Vitamin D receptor exhibits different pharmacodynamic features in tumoral and normal microenvironments: A molecular modeling study.

The vitamin D receptor (VDR) constitutes a promising therapeutic target for the treatment of cancer. Unfortunately, its natural agonist calcitriol does not have clinical utility due to its potential to induce hypercalcemic effects at the concentrations required to display antitumoral activity. For this reason, the search for new calcitriol analogues with adequate therapeutic profiles has been actively pursued by the scientific community. We have previously reported the obtaining and the biological activity evaluation of new calcitriol analogues by modification of its sidechain, which exhibited relevant antiproliferative and selectivity profiles against tumoral and normal cells. In this work we conducted molecular modeling studies (i.e. molecular docking, molecular dynamics, constant pH molecular dynamics (CpHMD) and free energy of binding analysis) to elucidate at an atomistic level the molecular basis related to the potential of the new calcitriol analogues to achieve selectivity between tumoral and normal cells. Two histidine residues (His305 and His397) were found to exhibit a particular tautomeric configuration that produces the observed bioactivity. Also, different acid-based properties were observed for His305 and His307 with His305 showing an increased acidity (pKa 5.2) compared to His397 (pKa 6.8) and to the typical histidine residue. This behavior favored the pharmacodynamic interaction of the calcitriol analogues exhibiting selectivity for tumoral cells when VDR was modeled at the more acidic tumoral environment (pH ≅ 6) compared to the case when VDR was modeled at pH 7.4 (normal cell environment). On the other hand, non-selective compounds, including calcitriol, exhibited a similar interaction pattern with VDR when the receptor was modeled at both pH conditions. The results presented constitute the first evidence on the properties of the VDR receptor in different physicochemical environments and thus represent a significant contribution to the in silico screening and design of new calcitriol analogues.

Heme-Oxygenase-1.

This Forum issue "Heme Oxygenase" (HO) includes original research articles and reviews that are aimed at understanding the role of HO-1 in several pathophysiological conditions, specially addressing those involving inflammation and oxidative damage. Overall, the seven contributions of this Forum highlight the dual role that HO-1 displays in cells and tissues, and address the molecular and cellular mechanisms through which HO-1 participates in the pathophysiology of the metabolic syndrome, obesity, cancer, and neurodegenerative, neurodevelopmental, and inflammatory bowel diseases. Indeed, one of the reviews thoroughly describes evidence of the anti-inflammatory properties of HO-1 in gut homeostasis, with potential to attenuate inflammatory bowel diseases. Three other reviews show the mostly beneficial effect of HO-1 expression in the attenuation of metabolic syndrome, obesity, cardiovascular disease, and diabetic cardiomyopathy. Contrariwise, one of the original articles show the overexpression of HO-1 in astroglia, models neurodegenerative (Parkinson-like) or neurodevelopmental (Schizophrenia-like) behaviors in mice, depending on the timing of expression of HO-1 during lifespan. The other original research communication demonstrates the role of HO-1 on the tropism of prostate cancer cells to bone, thus showing the involvement of this protein in the communication between bone and cancer cells. Finally, the Forum issue includes a review that elaborates on the classic and ultimate knowledge of HO-1 transcriptional regulation as well as the mechanisms of alternative splicing and post-transcriptional regulation of Hmox1 gene expression that have been little explored. Antioxid. Redox Signal. 32, 1239-1242.

Novel calcitriol analogue with an oxolane group: In vitro, in vivo, and in silico studies.

The active form of vitamin D3 , calcitriol, is a potent antiproliferative compound. However, when effective antitumor doses of calcitriol are used, hypercalcemic effects are observed, thus blocking its therapeutic application. To overcome this problem, structural analogues have been designed with the aim of retaining or even increasing the antitumor effects while decreasing its calcemic activity. This report aims at gaining insights into the structure-activity relationships of the novel oxolane-containing analogue, AM-27, recently synthesized. We herein demonstrate that this compound has antiproliferative and antimigratory effects in squamous cell carcinoma, glioblastoma, and breast cancer cell lines. Analyses of the mechanisms underlying the AM-27 effects on cell viability revealed induction of apoptosis by the analogue. Importantly, nonmalignant cell lines were little or not affected by the compound. In addition, the analogue did not produce hypercalcemia in mice. Also, in silico studies involving docking and molecular dynamics techniques showed that AM-27 is able to bind to the human vitamin D receptor with a higher affinity than the natural ligand calcitriol, a feature that is mostly derived from an electrostatic interaction pattern. Altogether, the proapoptotic effect observed in cancer cells, the lack of calcemic activity in mice, and the differential effects in normal cells suggest the potential of AM-27 as a therapeutic compound for cancer treatment.

The alkynylphosphonate analogue of calcitriol EM1 has potent anti-metastatic effects in breast cancer.

The active form of vitamin D3, calcitriol, plays a major role in maintaining calcium/phosphate homeostasis. In addition, it is a potent antiproliferative and prodifferentiating agent. However, when effective antitumor doses of calcitriol are employed, hypercalcemic effects are observed, thus precluding its therapeutic application. To overcome this problem, structural analogues have been designed with the aim at retaining or even increasing the antitumor effects while decreasing its calcemic activity. This report shows the biological evaluation of an alkynylphosphonate vitamin D less-calcemic analogue in a murine model of breast cancer. We demonstrate that this compound has potent anti-metastatic effects through its action over cellular migration and invasion likely mediated through the up-regulation of E-cadherin expression. Based on the current in vitro and in vivo results, EM1 is a promising candidate as a therapeutic agent in breast cancer.

Inhibition of p300 suppresses growth of breast cancer. Role of p300 subcellular localization.

There is evidence that p300, a transcriptional co-factor and a lysine acetyl-transferase, could play a role both as an oncoprotein and as a tumor suppressor, although little is known regarding its role in breast cancer (BC). First we investigated the role p300 has on BC by performing pharmacological inhibition of p300 acetyl-transferase function and analyzing the effects on cell count, migration and invasion in LM3 murine breast cancer cell line and on tumor progression in a syngeneic murine model. We subsequently studied p300 protein expression in human BC biopsies and evaluated its correlation with clinical and histopathological parameters of the patients. We observed that inhibition of p300 induced apoptosis and reduced migration and invasion in cultured LM3 cells. Furthermore, a significant reduction in tumor burden, number of lung metastases and number of tumors invading the abdominal cavity was observed in a syngeneic tumor model of LM3 following treatment with the p300 inhibitor. This reduction in tumor burden was accompanied by a decrease in the mitotic index and Ki-67 levels and an increase in Bax expression. Moreover, the analysis of p300 expression in human BC samples showed that p300 immunoreactivity is significantly higher in the cancerous tissues than in the non-malignant mammary tissues and in the histologically normal adjacent tissues. Interestingly, p300 was observed in the cytoplasm, and the rate of cytoplasmic p300 was higher in BC than in non-tumor tissues. Importantly, we found that cytoplasmic localization of p300 is associated with a longer overall survival time of the patients. In conclusion, we demonstrated that inhibition of the acetylase function of p300 reduces both cell count and invasion in LM3 cells, and decreases tumor progression in the animal model. In addition, we show that the presence of p300 in the cytoplasm correlates with increased survival of patients suggesting that its nuclear localization is necessary for the pro-tumoral effects.

Vitamin D receptor expression is associated with improved overall survival in human glioblastoma multiforme.

Vitamin D and its analogs have been shown to display anti-proliferative effects in a wide variety of cancer types including glioblastoma multiforme (GBM). These anticancer effects are mediated by its active metabolite, 1α, 25-dihydroxyvitamin D3 (calcitriol) acting mainly through vitamin D receptor (VDR) signaling. In addition to its involvement in calcitriol action, VDR has also been demonstrated to be useful as a prognostic factor for some types of cancer. However, to our knowledge, there are no studies evaluating the expression of VDR protein and its association with outcome in gliomas. Therefore, we investigated VDR expression by using immunohistochemical analysis in human glioma tissue microarrays, and analyzed the association between VDR expression and clinico-pathological parameters. We further investigated the effects of genetic and pharmacologic modulation of VDR on survival and migration of glioma cell lines. Our data demonstrate that VDR is increased in tumor tissues when compared with VDR in non-malignant brains, and that VDR expression is associated with an improved outcome in patients with GBM. We also show that both genetic and pharmacologic modulation of VDR modulates GBM cellular migration and survival and that VDR is necessary for calcitriol-mediated effects on migration. Altogether these results provide some limited evidence supporting a role for VDR in glioma progression.

Heme oxygenase-1 expression in human gliomas and its correlation with poor prognosis in patients with astrocytoma.

In human glioma tumors, heme oxygenase-1 (HO-1) has been shown to be upregulated both when compared with normal brain tissues and also during oligodendroglioma progression. The cell types that express HO-1 have been shown to be mainly macrophages/microglia and T cells. However, many other reports also demonstrated that cell lines derived from glioma tumors and astrocytes express HO-1 after the occurrence of a wide variety of cell injuries and stressors. In addition, the significance of HO-1 upregulation in glioma had not, so far, been addressed. We therefore aimed at investigating the expression and significance of HO-1 in human glial tumors. For this purpose, we performed a wide screening of HO-1 expression in gliomas by using tissue microarrays containing astrocytomas, oligodendrogliomas, mixed tumors, and normal brain tissues. We subsequently correlated protein expression with patient clinicopathological data. We found differences in HO-1 positivity rates between non-malignant brain (22 %) and gliomas (54%, p = 0.01). HO-1 was expressed by tumor cells and showed cytoplasmic localization, although 19% of tumor samples also depicted nuclear staining. Importantly, a significant decrease in the overall survival time of grade II and III astrocytoma patients with HO-1 expression was observed. This result was validated at the mRNA level in a cohort of 105 samples. However, no association of HO-1 nuclear localization with patient survival was detected. In vitro experiments aimed at investigating the role of HO-1 in glioma progression showed that HO-1 modulates glioma cell proliferation, but has no effects on cellular migration. In conclusion, our results corroborate the higher frequency of HO-1 protein expression in gliomas than in normal brain, demonstrate that HO-1 is expressed by glial malignant cells, and show an association of HO-1 expression with patients' shorter survival time.

Mammary differentiation induces expression of Tristetraprolin, a tumor suppressor AU-rich mRNA-binding protein.

Tristetraprolin (TTP) is a RNA-binding protein that inhibits the expression of pro-inflammatory cytokines and invasiveness-associated genes. TTP levels are decreased in many different cancer types and it has been proposed that this protein could be used as a prognostic factor in breast cancer. Here, using publicly available DNA microarray datasets, "serial analysis of gene expression" libraries and qRT-PCR analysis, we determined that TTP mRNA is present in normal breast cells and its levels are significantly decreased in all breast cancer subtypes. In addition, by immunostaining, we found that TTP expression is higher in normal breast tissue and benign lesions than in infiltrating carcinomas. Among these, lower grade tumors showed increased TTP expression compared to higher grade cancers. Therefore, these data indicate that TTP protein levels would provide a better negative correlation with breast cancer invasiveness than TTP transcript levels. In mice, we found that TTP mRNA and protein expression is also diminished in mammary tumors. Interestingly, a strong positive association of TTP expression and mammary differentiation was identified in normal and tumor cells. In fact, TTP expression is highly increased during lactation, showing good correlation with various mammary differentiation factors. TTP expression was also induced in mammary HC11 cells treated with lactogenic hormones, mainly by prolactin, through Stat5A activation. The effect of this hormone was highly dependent on mammary differentiation status, as prolactin was unable to elicit a similar response in proliferating or neoplastic mammary cells. In summary, these studies show that TTP expression is strongly linked to the mammary differentiation program in human and mice, suggesting that this protein might play specific and relevant roles in the normal physiology of the gland.

Synthesis and biological evaluation of a new vitamin D2 analogue.

A new vitamin D(2) analogue was synthesized using the Julia-Kocienski olefination. It has antiproliferative effects on cell lines from squamous cell carcinomas of colon and head and neck, but is also as hypercalcaemic as calcitriol in vivo.

Nuclear localization of heme oxygenase-1 is associated with tumor progression of head and neck squamous cell carcinomas.

The expression of heme oxygenase-1 (HO-1) was shown to be increased in multiple tumors compared with their surrounding healthy tissues and was also observed to be up-regulated in oral squamous cell carcinomas (OSCC). However, conflicting results were obtained and little information is available regarding HO-1 significance in head and neck squamous cell carcinoma (HNSCC). Therefore, the aim of the present study was to perform a wide screening of HO-1 expression in a large collection of human primary HNSCCs and to correlate the results with clinical and pathological parameters. For this purpose, we investigated the expression of this protein by immunohistochemistry (IHC) in tissue microarrays (TMAs) of HNSCC and in an independent cohort of paraffin-embedded tumor specimens. HO-1 expression was further validated by real-time qPCR performed on selected laser capture-microdissected (LCM) oral tissue samples. Both the number of HO-1-positive samples and HO-1 immunoreactivity in the cancerous tissues were significantly higher than those in the non-tumor tissues. These results were confirmed at the mRNA level. Interestingly, HO-1 localization was observed in the nucleus, and the rate of nuclear HO-1 in HNSCC was higher than that in non-malignant tissues. Nuclear HO-1 was observed in HNSCC cell lines and increased even further following hemin treatment. Analysis of HO-1 expression and sub-cellular localization in a mouse model of squamous cell carcinoma (SCC) and in human HNSCC revealed that nuclear HO-1 increases with tumor progression. Taken together, these results demonstrate that HO-1 is up-regulated in HNSCC and that nuclear localization of HO-1 is associated with malignant progression in this tumor type.

Expression of heme oxygenase-1 in non-small cell lung cancer (NSCLC) and its correlation with clinical data.

While changes in heme oxygenase (HO-1) in lung cancer have already been reported, conflicting results were obtained for enzyme expression in human lung cancer specimens. Therefore, the aim of this work was to study HO-1 expression in a large collection of human lung cancer samples. For this purpose, we analyzed the expression of HO-1 in an organized tissue microarray (TMA) and investigated its correlation with clinicopathological data. Ninety-six percent of tumor samples were positive for HO-1, and the expression of HO-1 was significantly higher in cancerous than in non-cancerous tissues. Importantly, HO-1 expression correlated with advanced stages and lymph node involvement. Additionally, quantitative RT-PCR in 18 pairs of human lung carcinomas and their adjacent non-malignant tissues was performed. Our results demonstrate that HO-1 protein is upregulated in epithelial malignant cells in NSCLC and its expression is associated with higher stages of the disease. Additionally, different subcellular localization is observed between tumor and adjacent non-malignant tissues.

Novel alkynylphosphonate analogue of calcitriol with potent antiproliferative effects in cancer cells and lack of calcemic activity.

Here, we describe the design and synthesis of diethyl [(5Z,7E)-(1S,3R)-1,3-dihydroxy-9,10-secochola-5,7,10(19)-trien-23-in-24-yl] phosphonate (compound 10), which combines the low calcemic properties of phosphonates with the decreased metabolic inactivation due to the presence of a triple bond in C-24 and studied its in vitro effects on several cancer cell lines and its in vivo effects on blood calcium levels. We demonstrate that this compound is a potent antiproliferative vitamin D analogue, showing lack of calcemic effects in vivo.

The expression of sphingosine kinase-1 in head and neck carcinoma.

Sphingosine kinase-1 (SPHK1) modulates the proliferation, apoptosis and differentiation of keratinocytes through the regulation of ceramide and sphingosine-1-phosphate levels. However, studies on the expression of SPHK1 in human head and neck squamous cell carcinoma (HNSCC) specimens are lacking. Therefore, the aim of the present work was to evaluate SPHK1 expression in human primary HNSCCs and to correlate the results with clinical and anatomopathological parameters. We investigated the expression of this protein by immunohistochemistry performed in tissue microarrays of HNSCC and in an independent cohort of 37 paraffin-embedded specimens. SPHK1 expression was further validated by real-time PCR performed on laser capture-microdissected tissue samples. The positive rate of SPHK1 protein in the cancerous tissues was significantly higher (74%) than that in the nontumor oral tissues (23%), and malignant tissues showed stronger immunoreactivity for SPHK1 than normal matching samples. These results were confirmed by real-time PCR quantification of SPHK1 mRNA. Interestingly, the positive expression of SPHK1 was associated with shorter patient survival time (Kaplan-Meier survival curves) and with the loss of p21 expression. Taken together, these results demonstrate that SPHK1 is upregulated in HNSCC and provide clues of the role SPHK1 might play in tumor progression.

Intracellular distribution of p300 and its differential recruitment to aggresomes in breast cancer.

It has been recently suggested that p300 cytoplasmic redistribution and degradation are important for controlling the availability and activity of the protein as a transcriptional coactivator. As a step towards determining the functional relevance of p300 intracellular redistribution in mammary cancer, we aimed at studying p300 localization in two different animal models of mammary carcinoma as well as in human primary breast carcinoma samples. Analysis of p300 protein levels showed stronger expression in tumor epithelia than in normal mammary gland. Cytoplasmic localization of p300 was observed in malignant cells. Furthermore, cytoplasmic p300 was found in tumor epithelia whereas nuclear localization was observed in normal mammary glands in both animal models and in non-malignant adjacent areas of human breast cancer specimens. Interestingly, proteasomal inhibition induced p300 redistribution to perinuclear inclusion bodies in tumor but not in normal mammary gland-derived cells. These inclusions were confirmed to be aggresomes by doing immunofluorescence for ubiquitin, vimentin and 20S proteasomal subunit. Taken together, these findings show that both the localization of p300 and the recruitment to aggresomes differ between mammary tumors and normal mammary glands, and suggest that the formation of these inclusions could be a potential target for therapeutic intervention.

Stratification of sphingosine kinase-1 expression and activity in rat kidney.

Sphingosine-1-phosphate, the product of sphingosine kinase (SK) activity, is a sphingolipid metabolite that regulates cell growth, survival and migration. It is also known to affect diuresis, natriuresis and renovascular contraction in rats, although the mechanisms through which it affects these processes are not known. No previous report has addressed the differences among the kidney zones regarding endogenous SK expression and activity. Therefore, we examined SK1 distribution and activity in the various kidney zones: cortex, medulla and papilla. We found that SK1 expression does not correlate with enzyme activity. Study of the expression showed that the enzyme is highly expressed in cortex, followed by medulla and papilla. However, medulla had the highest enzyme activity. In all kidney zones, SK1 expression was mainly cytosolic. Regarding enzyme activity, whereas we found no difference between cytosol, membrane and nucleus in renal medulla, the membrane-bound enzyme presented the highest activity in cortex and papilla. SK1 distribution observed by immunohistochemical staining showed higher expression in cortical proximal convoluted epithelial cells. In medulla, immunostaining was observed as patches of staining, whereas in papilla, positive immunostaining was exclusively restricted to collecting duct cells. We also evaluated the effects of bradykinin and angiotensin II on SK1 activity.

Phenoxodiol, a novel isoflavone, induces G1 arrest by specific loss in cyclin-dependent kinase 2 activity by p53-independent induction of p21WAF1/CIP1.

Phenoxodiol, an isoflavone derivative of genistein with unknown mechanism of action, is currently being evaluated in early human cancer clinical trials. To determine the mechanism of antiproliferative effects of phenoxodiol, we examined its effects in a battery of human cell lines. Although we observed caspase-dependent apoptosis in HN12 cells as early as 24 hours after exposure, clonogenic death occurred only after 48-hour exposure despite caspase blockade by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (ZVAD)-fmk. Moreover, clear evidence of cell death as determined by nuclear morphology and plasmatic membrane damage occur despite ZVAD, suggesting that another mechanism besides caspase-dependent apoptosis is required for clonogenic death induced by phenoxodiol. In search for other potential antiproliferative effects, we assessed the effects of phenoxodiol in the cell cycle progression of human carcinoma cell lines. A significant G(1)-S arrest was observed by 12 hours of exposure in HN12 cell lines at concentrations > or =5 microg/mL. Cell cycle arrest occurred several hours (approximately 12 hours) before induction of apoptosis. Analysis of in vitro purified cyclin-dependent kinase (cdk) activity showed that phenoxodiol did not inhibit cdk activity. In contrast, cellular cdk2 activity obtained from HN12 cell lines exposed to phenoxodiol for 12 hours decreased by 60%, whereas cdk6 activity remained unaltered, suggesting that the loss of cdk2 activity was specific. Loss in cdk2 activity was preceded by the accumulation of the endogenous cdk inhibitor p21(WAF1). To assess the role of p21(WAF1) induction by phenoxodiol, we used HCT116 isogenic cell lines and showed that phenoxodiol induced G(1) arrest together with p21(WAF1) expression in wild-type clones. In contrast, p21(-/-) variants failed to show G(1) arrest. Finally, induction of p21 by phenoxodiol is p53 independent, as phenoxodiol induced p21 in HCT116 lacking p53. These data therefore indicate that phenoxodiol promotes G(1)-S arrest by the specific loss in cdk2 activity due to p53-independent p21(WAF1) induction. This novel feature of phenoxodiol may have clinical implications, as the majority of human malignancies have aberrations in cell cycle progression regulation.

UCN-01-induced cell cycle arrest requires the transcriptional induction of p21(waf1/cip1) by activation of mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway.

The small molecule UCN-01 is a cyclin-dependent kinase (CDK) modulator shown to have antiproliferative effects against several in vitro and in vivo cancer models currently being tested in human clinical trials. Although UCN-01 may inhibit several serine-threonine kinases, the exact mechanism by which it promotes cell cycle arrest is still unclear. We have reported previously that UCN-01 promotes G(1)-S cell cycle arrest in a battery of head and neck squamous cancer cell lines. The arrest is accompanied by an increase in both p21(waf1/cip1) and p27(kip1) CDK inhibitors leading to loss in G(1) CDK activity. In this report, we explore the role and the mechanism for the induction of these endogenous CDK inhibitors. We observed that p21 was required for the cell cycle effects of UCN-01, as HCT116 lacking p21 (HCT116 p21(-/-)) was refractory to the cell cycle effects of UCN-01. Moreover, UCN-01 promoted the accumulation of p21 at the mRNA level in the p53-deficient HaCaT cells without increase in the p21 mRNA half-life, suggesting that UCN-01 induced p21 at the transcriptional level. To study UCN-01 transcriptional activation of p21, we used several p21(waf1/cip1) promoter-driven luciferase reporter plasmids and observed that UCN-01 activated the full-length p21(waf1/cip1) promoter and a construct lacking p53 binding sites. The minimal promoter region required for UCN-01 (from -110 bp to the transcription start site) was the same minimal p21(waf1/cip1) promoter region required for Ras enhancement of p21(waf1/cip1) transcription. Neither protein kinase C nor PDK1/AKT pathways were relevant for the induction of p21 by UCN-01. In contrast, the activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase mitogen-activated protein kinase pathways was required for p21 induction as UCN-01 activated this pathway, and genetic or chemical MEK inhibitors blunted p21 accumulation. These results demonstrated for the first time that p21 is required for UCN-01 cell cycle arrest. Moreover, we showed that the accumulation of p21 is transcriptional via activation of the MEK pathway. This novel mechanism, by which UCN-01 exerts its antiproliferative effect, represents a promising strategy to be exploited in future clinical trials.