PPARγ-Mediated p21 Induction in Aerodigestive Preneoplastic Cell Lines.

INTRODUCTION: Oral leukoplakia is defined as a mucous membrane disorder characterized by white patches that cannot be scraped off. Leukoplakia is the most frequent, potentially premalignant oral mucosa disorder and a good candidate for chemopreventive therapies. Pioglitazone activates peroxisome proliferator-activated receptor gamma (PPARγ), which forms a complex with nuclear cofactors and regulates gene expression of a variety of cell-cycle proteins and is currently being tested preclinically and clinically in aerodigestive cancer prevention. METHODS: In the present study, we hypothesized that pioglitazone would decrease proliferation of human leukoplakia cells (MSK Leuk1) and transformed bronchial epithelial cells (BEAS-2B) through regulatory changes of G1 checkpoint protein regulators, p21 and cyclin-D1. MSK Leuk1 and BEAS-2B cells were treated with pioglitazone and assayed for cell proliferation and p21 transcriptional activity. RESULTS: We discovered pioglitazone significantly inhibited cell proliferation in a dose-dependent fashion. We also observed p21 protein induction after treatment with pioglitazone, which was preceded by measurable increases in p21 mRNA induction. CONCLUSIONS: We conclude the PPARγ activator, pioglitazone, can activate p21, which is associated with decreased proliferation in 2 aerodigestive preneoplastic cell lines. In addition, the p21 gene may be a potential hypothesis-driven biomarker in translational studies of pioglitazone as a chemoprevention agent for aerodigestive cancer.

LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels.

AIMS: This study reports the biological properties of LQFM030 in vivo, a molecular simplification of the compound nutlin-1. MAIN METHODS: Ehrlich ascites tumor (EAT)-bearing mice were treated intraperitoneally with LQFM030 (50, 75 or 150mg/kg) for 10days to determine changes in ascites tumor volume, body weight, cytotoxicity and angiogenesis. Moreover, flow cytometric expression of p53 and p21 proteins and caspase-3/7, -8 and -9 activation were investigated in EAT cells from mice treated. Acute oral systemic toxicity potential of LQFM030 in mice was also investigated using an alternative method. KEY FINDINGS: Treatment of EAT-bearing mice with LQFM030 resulted in a marked decline in tumor cell proliferation and the vascular endothelial growth factor (VEGF) levels along with enhanced survival of the mice. Apoptotic tumor cell death was detected through p53 and p21 modulation and increase of caspase-3/7, -8 and -9 activity. LQFM030 also showed orally well tolerated, being classified in the UN GHS category 5 (LD50>2000-5000mg/Kg). SIGNIFICANCE: LQFM030 seems to be a promising antitumor candidate for combinatory therapy with typical cytotoxic compounds, reducing the toxicity burden while allowing a superior anticancer activity. Moreover, these data also open new perspectives for LQFM030 as an antiangiogenic agent for treatment of diseases involving VEGF overexpression.

Endothelial cell senescence with aging in healthy humans: prevention by habitual exercise and relation to vascular endothelial function.

Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P < 0.05), respectively, in ECs obtained from antecubital veins of older sedentary (60 ± 1 yr, n = 12) versus young sedentary (22 ± 1 yr, n = 9) adults. These age-related differences were not present (all P > 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 (r = -0.49, P = 0.003), p21 (r = -0.38, P = 0.03), and p16 (r = -0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P < 0.05), respectively, in ECs sampled from brachial arteries of healthy older sedentary (63 ± 1 yr, n = 18) versus young sedentary (25 ± 1 yr, n = 9) adults; age-related changes in arterial EC p53 and p21 expression were not observed (P > 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age.NEW & NOTEWORTHY Our study provides novel evidence in humans of increased endothelial cell senescence with sedentary aging, which is associated with impaired vascular endothelial function. Furthermore, our data suggest an absence of age-related increases in endothelial cell senescence in older exercising adults, which is linked with preserved vascular endothelial function.

Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

The Role of Embryonic Stem Cell-expressed RAS (ERAS) in the Maintenance of Quiescent Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) were recently identified as liver-resident mesenchymal stem cells. HSCs are activated after liver injury and involved in pivotal processes, such as liver development, immunoregulation, regeneration, and also fibrogenesis. To date, several studies have reported candidate pathways that regulate the plasticity of HSCs during physiological and pathophysiological processes. Here we analyzed the expression changes and activity of the RAS family GTPases and thereby investigated the signaling networks of quiescent HSCs versus activated HSCs. For the first time, we report that embryonic stem cell-expressed RAS (ERAS) is specifically expressed in quiescent HSCs and down-regulated during HSC activation via promoter DNA methylation. Notably, in quiescent HSCs, the high level of ERAS protein correlates with the activation of AKT, STAT3, mTORC2, and HIPPO signaling pathways and inactivation of FOXO1 and YAP. Our data strongly indicate that in quiescent HSCs, ERAS targets AKT via two distinct pathways driven by PI3Kα/δ and mTORC2, whereas in activated HSCs, RAS signaling shifts to RAF-MEK-ERK. Thus, in contrast to the reported role of ERAS in tumor cells associated with cell proliferation, our findings indicate that ERAS is important to maintain quiescence in HSCs.

MicroRNA-143 replenishment re-sensitizes colorectal cancer cells harboring mutant, but not wild-type, KRAS to paclitaxel treatment.

Colorectal cancer (CRC) global incidence is one of the highest among cancers. The KRAS gene has been shown as a robust biomarker for poor prognosis and drug resistance. MicroRNA-143 (miR-143) and let-7 are families of tumor suppressor microRNAs that are often downregulated in CRC, especially with coexistent KRAS mutations. In order to evaluate if miR-143 and/or let-7b replenishment would re-sensitize CRC cells to paclitaxel treatment, we investigated in effect of miR-143 and let-7b replenishments on sensitivity to paclitaxel treatment in KRAS mutant LoVo and wild-type SW48 CRC cell lines. Our results showed that miR-143, but not let-7b, increased sensitization of KRAS mutant tumor cells to paclitaxel. Furthermore, transfection of miR-143, but not let-7b, mimic negatively regulated the expression of mutant but not wild-type KRAS. Combination of miR-143 mimic and paclitaxel induced the onset of apoptosis, and reverted in vitro metastatic properties (migration and invasion) in KRAS mutant tumor cells. MiR-143 thus can be used as a chemosensitizer for the treatment of KRAS mutant tumors and warrants further investigations in in vitro and pre-clinical in vivo models.

MicroRNAs: Modulators of the Ras Oncogenes in Oral Cancer.

Oral squamous cell carcinoma (OSCC) of the head and neck is one of the six most common cancers in the world. OSCC remains the most common cause of cancer deaths in Asian countries. Conventional treatments for OSCC have not improved the overall 5 years survival and therefore alternative therapeutic targets are often sought. Ras is one of the most frequently deregulated oncogenes in oral cancer. Direct targeting the ras has proven unrealistic and hence, exploring and understanding alternative pathways and/or molecules which regulate ras and its signaling that could pave the way for novel molecular targets and therapy for oral cancer. Recently, microRNAs (miRNAs) have been reported to regulate ras oncogenes in human cancers. In this article, we address the microRNA-mediated regulation of the ras oncogenes in oral cancer. We describe extensively the tumor suppressive and oncogenic roles of miRNAs in regulation of ras oncogenes in OSCC. We also discuss the role of miRNA-mediated ras regulation in therapeutic determination of oral cancer. Complete understanding of the miRNA regulation of ras oncogenes in oral cancer may facilitate to plan better strategies for diagnosis, molecular therapeutic targeting and the overall prognosis of this common and deadly cancer.

Kirenol, a compound from Herba Siegesbeckiae, induces apoptosis in human chronic myeloid leukemia K562 cells.

Kirenol is a biologically active substance isolated from Herba Siegesbeckiae. In the experiments, we explored a novel antitumor activity of kirenol. The data demonstrated that kirenol had strong cytotoxic effects to human chronic myeloid leukemia K562 cells, the 50% inhibitory concentration (IC50) for kirenol was 53.05 ig/ml, 18.19 pg/ml and 15.08 microg/ml for 24, 48 and 72 h, determined using the MTT assay. Further studies showed that kirenol treatment caused externalization of phosphatidylserine, accumulation of ROS (reactive oxygen species), alteration of mitochondrial membrane potential, release of cytochrome c, reduction in the level of the Bcl-2 protein and upregulation of Bax and tBid, kirenol induced cell apoptosis in a caspase-independent manner. Further studies indicated that kirenol treatment triggered the arrest of cell cycle S period which might resulted from the up-regulation of phosphorylation of p53 (Ser 6 and Ser 37) and expression of p21 protein. Our results indicated that kirenol possesses antitumor action on human chronic myeloid leukemia K562 cells in vitro. Kirenol may have therapeutic potential for the treatment of cancer that deserves further investigation.

The ZEB1 transcription factor acts in a negative feedback loop with miR200 downstream of Ras and Rb1 to regulate Bmi1 expression.

Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.

Changes in mRNA and protein expression in the renal cortex of male and female F344 rats treated with bromate.

Bromate (BrO3(-)), a by-product of ozonation of drinking water, induces nephrotoxicity in male rats at much lower doses than in female rats. This difference appears to be related to the development of α-2u-globulin nephropathy in males. To determine sex-dependent changes in mRNA and protein expression in the renal cortex attributable to α-2u-globulin nephropathy, we performed microarray and immunohistochemical analyses in proximal renal tubules of male and female F344 rats treated with KBrO3 for 28 days. Particular attention was paid to molecular biomarkers of renal tubular injury. Microarray analysis of male and female rats treated with BrO3(-) at low doses (125 mg/L KBrO3) displayed marked sex-dependent changes in renal gene expression. The greatest differences were seen in genes encoding for cellular differentiation, apoptosis, ion transport, and cell proliferation. Differences by sex were especially prominent for the cell cycle checkpoint gene p21, the renal injury protein Kim-1, and the kidney injury and cancer biomarker protein osteopontin. Dose-related nephrotoxicity, assessed by hematoxylin and eosin staining, was greater in males compared to female rats, as was cellular proliferation, assessed by bromodeoxyuridine staining. The fraction of proximal renal cells with elevated 8-oxodeoxyguanosine (8-OH-dG) was only increased at the high dose and did not differ by sex. Dose-dependent increases in the expression of osteopontin were detected immunohistochemically only in male rats and were localized in proximal tubule cells. Similarly, BrO3(-) treatment increased clusterin and Kim-1 staining in the proximal tubules; however, staining for these proteins did not differ appreciably between males and females. These data demonstrate both qualitative and quantitative differences in the response of male versus female kidneys to BrO3(-)-treatment. These sex-dependent effects likely contribute to renal carcinogenesis of BrO3(-) in the male rat.

miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1.

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, down-regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.

Relationship between expression of ras p21 oncoprotein and mutation status of the K-ras gene in sporadic colorectal cancer patients in Tunisia.

INTRODUCTION: The K-ras proto-oncogene encodes a protein (p21-ras) belonging to the family of GTP/GDP-binding proteins with GTPase activity. The activation of ras family genes plays an important role in colorectal tumorigenesis. Frequency of K-ras mutations and overexpression of the protein in colorectal cancer (CRC) vary between 14% and 50% and between 29% and 76%, respectively. AIMS: We investigated the clinicopathologic characteristics of patients with CRC and their relationship with point mutations of K-ras oncogene codons 12/13 and ras p21 expression. MATERIALS AND METHODS: K-ras codons 12 and 13 point mutations were examined by direct sequence analysis, whereas the ras p21 expression was evaluated using immunohistochemistry. RESULTS: Statistical analysis of immunohistochemical results showed that the expression of ras p21 was correlated with the advanced age of patients (P=0.0001), whereas loss of signal was associated with mucinous histotype (P=0.0001). Mutations in the K-ras gene were detected in 12 of the patients with CRC. Mutations in K-ras gene were found in 12 of 52 tumors (23.07%), and 7 mutations were G→A transitions (58.33% of all mutations), 4 were G→T transversions (33.33%), and only 1 was G→C transversion (8.33%). A total of 83.33% of the mutation occurred at codon 12 and 16.67% at codon 13. Moreover, K-ras mutations were associated with the sex of patients (P=0.017). CONCLUSIONS: Genetic K-ras alterations were rather low in the Tunisian population, but further study is necessary to unravel the molecular background of CRC.

Tumor suppressor p16INK4a controls oncogenic K-Ras function in human pancreatic cancer cells.

Pancreatic cancer is characterized by oncogenic activation of K-Ras and inactivation of the cell cycle inhibitor p16(INK4a) . We previously demonstrated that reintroduction of p16(INK4a) reversed anoikis resistance and clonogenicity of human pancreatic cancer cells, properties commonly attributed to the transforming potential of oncogenic K-Ras. Therefore, we aimed to determine the role of Ras after p16(INK4a) re-expression. Here, we show that restitution of p16(INK4a) in pancreatic cancer cell lines elicits a profound suppression of K-Ras activity. A more detailed analysis in p16(INK4a) reconstituted Capan-1 cells indicated selective reduction of both K-Ras activity and protein stability. Re-expression of K-Ras in p16(INK4a) restituted Capan-1 cells reversed the anoikis-sensitive phenotype and increased colony formation, indicating that K-Ras suppression was required for p16(INK4a) -mediated reversion of the transformed phenotype. Inducible expression of p16(INK4a) in DanG cells confirmed inhibition of K-Ras activity as well as an increase in anoikis susceptibility. Thus, our results delineate a novel functional interaction with defined biological consequences for the two most frequent alterations observed in pancreatic cancer.

Arsenic trioxide-induced apoptosis in TM4 Sertoli cells: the potential involvement of p21 expression and p53 phosphorylation.

Arsenic is a toxic metalloid that exists ubiquitously in the environment, and exhibits carcinogenicity. Conversely, arsenic trioxide (AsTO) has successfully been employed in the treatment of acute promyelocytic leukemia (APL). It has been shown that AsTO efficiently induces apoptosis in the malignant cells of APL in vitro. Although the mechanisms underlying AsTO-induced apoptosis in certain types of cancer cells, such as APL cells, have been delineated, the mechanism underlying AsTO-induced cell death in non-cancer cells remains unknown. In the present study, we examined AsTO-provoked cytotoxicity and cell death mechanism(s) in TM4 Sertoli cells. Exposure of these cells to AsTO generates reactive oxygen species and alters mitochondrial apoptosis, inducing cell death via both caspase-dependent and caspase-independent pathways. AsTO-induced apoptosis was concomitant with the downregulation of p53, phosphorylation of p53 at serine residues, and G2/M cell cycle arrest. Particularly, the interaction of p21 with caspase-3 proteins during AsTO treatment suggested an antiapoptotic role of p21 against genotoxic stresses in TM4 Sertoli cells. However, clinically relevant concentrations of AsTO failed to induce cell death in TM4 Sertoli cells, indicating that these cells could be resistant to cancer treatment. The results presented herein may not represent the actual effect of AsTO on Sertoli cells in vivo. Thus, further studies on the exposure effects of AsTO on the morphology and function of Sertoli cells in animal experiments will provide a more precise knowledge of AsTO cytotoxicity on male reproduction.

Bortezomib induces apoptosis in human neuroblastoma CHP126 cells.

Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in >60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression, and new treatment modalities or potent drugs are further needed. Bortezomib owns a substantial cytotoxicity through regulating degradation of protein associated with cell cycle control and tumor growth. The involvement of bortezomib in neuroblastoma is largely unkown. The aim of this study was to investigate the effects and mechanisms of bortezomib on human neuroblastoma CHP126 cells. Our results indicated that bortezomib inhibits proliferation of neuroblastoma cells in a time- and dose- dependent manner, and the concentration that caused 50% inhibition of CHP126 cells growth was 11.25 nM. Furthermore, bortezomib-induced proliferation inhibition results from massive cell death characterized by apoptosis. Besides, the NFkappaB pathway was not involved in bortezomib treatment in neuroblastoma CHP126 cells, bortezomib-driven apoptotic events were associated with promoting p21 and Bax expression and down-regulating Bcl-2 expression. Ultimately, caspase-3 was activated and the cleavage of PARP was induced. Above all, our data revealed that bortezomib triggered apoptosis by enhancing the caspase 3 activation and/or modulating the Bax/Bcl-2 balance, and also provided preliminary data for further researches of bortezomib on pediatric neuroblastoma.

Epigenetic regulation of the embryonic oncogene ERas in gastric cancer cells.

ERas is a recently identified oncogene that supports the tumorigenic growth of embryonic stem cells, it is constitutively active in the absence of mutation. ERas oncogene is expressed only in viviparity phase cells, but not in somatic cells because of epigenetic transcriptional silencing in the somatic phase. The aim of this study was to clarify the ERas expression and its epigenetic regulation in gastric cancer of somatic phase. Fifteen gastric cancer cell lines were used. ERas mRNA expression and its epigenetic regulation were examined by reverse transcription-polymerase chain reaction and bisulfite sequencing analysis. To identify a subset of cancer stem cells, termed 'side population' (SP) cells, flow cytometry analysis was performed. ERas is expressed in 8 of the 15 gastric cancer cell lines, but is silenced in the remaining 7 cancer cell lines and normal cell lines. Six of 7 cancer cell lines without ERas expression had promoter methylation, which correlated with silencing of ERas expression. ERas expression is re-activated following treatment with the DNA methyltransferase inhibitor 5-aza-CdR. The percentage of SP fraction of ERas-positive gastric cancer cells was significantly (p=0.024) higher (3.4+/-1.8%), in comparison to that of ERas-negative cells (1.6+/-0.4%). These findings suggested that the activating ERas oncogene might be associated with tumorigenic growth of somatic cells, and might be a putative molecule responsible for cancer stem cell-like characteristics in gastric cancer. Loss of methylation in the promoter of ERas might be one of mechanisms responsible for the re-expression of an embryonic oncogene in gastric cancer.

Effects of nitric oxide and antioxidants on advanced glycation end products-induced hypertrophic growth in human renal tubular cells.

The accumulation of advanced glycation end products (AGE) is a key mediator of renal tubular hypertrophy in diabetic nephropathy (DN). Reactive oxygen species and nitric oxide (NO) were involved in the progression of DN. In this study, the molecular mechanisms of NO and antioxidants responsible for inhibition of AGE-induced renal tubular hypertrophy were examined. We found that AGE (but not nonglycated bovine serum albumin) significantly suppressed the NO/cGMP/PKG signaling in human renal proximal tubular cells. NO donors S-nitroso-N-acetylpenicillamine (SNAP)/sodium nitroprusside (SNP) and antioxidants N-acetylcysteine (NAC)/taurine treatments significantly attenuated AGE-inhibited NO production, cGMP synthesis, and inducible NO synthase/cGMP-dependent protein kinase (PKG) activation. Moreover, AGE-induced extracellular signal-regulated kinase/c-Jun N-terminal kinase/p38 mitogen-activated protein kinase activation was markedly blocked by antireceptor for AGE (RAGE), SNAP, SNP, NAC, and taurine. The abilities of NO and antioxidants to inhibit AGE/RAGE-induced hypertrophic growth were verified by the observation that SNAP, SNP, NAC, and taurine inhibited fibronectin, p21(Waf1/Cip1), and RAGE expression. Therefore, antioxidants significantly attenuated AGE/RAGE-enhanced cellular hypertrophy partly through induction of the NO/cGMP/PKG signaling.

The relative activity of cisplatin, oxaliplatin and satraplatin in testicular germ cell tumour sensitive and resistant cell lines.

BACKGROUND: Germ cell tumours (GCT) can become resistant to cisplatin, which is associated with a relatively poor prognosis. Oxaliplatin and satraplatin have been developed to overcome cisplatin resistance in other cancers, but their effect in cisplatin resistant (cisR) GCTs is unclear. In this work we address this issue by comparing their efficacy in three paired sensitive and cisR GCT cell lines. METHODS: Three established cisplatin sensitive (cisS) and resistant cell line pairs were used (GCT27, GCT27r: SUSA, SUSAr: 833k, 833kr). Viability was assessed using a luciferase based ATP assay and EC(50) and EC(80) concentrations were calculated. Western blot analysis and flow cytometry was used for further assessment. RESULTS: Sensitivity to the three platinum compounds was broadly similar in the three cisS lines GCT cell lines (EC(50) = 0.27-0.51 microM for cisplatin, 0.52-0.79 microM for oxaliplatin, 0.31-1.26 microM for satraplatin). EC(50) values for cisplatin in the three cisR sub lines were 1.8- to 3.8-fold higher than in the sensitive parental lines. Cross resistance to satraplatin and oxaliplatin occurred in all three cisR cell lines (resistance factor 1.9-4.4), with the exception of oxaliplatin in the 833Kr (resistance factor 0.9). Differences in the effect of specific drugs on cell cycle distribution, p53, p21 and MDM2 were observed. CONCLUSIONS: These data suggest that satraplatin and oxaliplatin could theoretically be used in chemo-naive GCTs and support the further clinical evaluation of these agents in this setting. The mechanism of cross resistance to these drugs appears multifactorial.

Erucin, a new promising cancer chemopreventive agent from rocket salads, shows anti-proliferative activity on human lung carcinoma A549 cells.

Erucin (ER) is a dietary isothiocyanate present in cruciferous vegetables, such as rocket salads (Erucasativa Mill., Diplotaxis sp.), that has been recently considered a promising cancer chemopreventive phytochemical. Biological activity of ER was investigated on human lung adenocarcinoma A549 cells, analyzing its effects on molecular pathways involved in apoptosis and cell cycle arrest, such as PARP-1 cleavage, p53 and p21 protein expression. Our results show that ER affects the A549 cell proliferation, enhancing significantly p53 and p21 protein expression in a dose-dependent manner (p<0.001). PARP-1 cleavage occurs only after exposure to high concentrations of ER (50 microM), in accordance to previous studies showing similar bioactivity of other isothiocyanates (ITCs). Our study reports for the first time that the induction of p53, p21 and PARP-1 cleavage may participate in the anti-proliferative activity of ER in human lung adenocarcinoma A549 cells. Comparison of data with those obtained with the isothiocyanate sulforaphane (SF), structurally related to ER, underlines the strong relationship between structural analogy of ITCs and their biological activity. The ability of dietary compounds to modulate molecular mechanisms that affect cancer cell proliferation is certainly a key point of the cancer prevention potential by functional foods.

Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment.

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported (34) that chronic hemin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 muM for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at G0/G1 phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, hemin-induced HO-1 expression was further increased in the presence of SnPP. Hemin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of hemin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without hemin for 7 days. The inhibitory effect of hemin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin.