Validation of a venous thromboembolism risk assessment model in gynecologic oncology.

OBJECTIVES: Gynecologic oncology patients undergoing surgery are at an increased risk for venous thromboembolism (VTE). We attempted to validate a VTE risk assessment model in gynecologic oncology patients. METHODS: All gynecologic oncology patients who underwent a laparotomy for the diagnosis or suspicion of gynecologic malignancy from 2004 to 2010 were included. Demographic, surgicopathologic, and complication data were collected. VTE was based on the symptomatic diagnosis. Data for the Caprini risk assessment model (RAM) was used to score and stratify patients on their risk for VTE. RESULTS: 1123 gynecologic oncology patients were included within this study. Ovarian cancer was the most common diagnosis (39%) with a median age of 56.1. All patients received SCDs with 40% receiving double prophylaxis. The overall incidence of VTE was 3.3%, with lower extremity deep venous thrombosis (DVT) n=17 and pulmonary embolism (PE) n=20. Complication rates were similar in each group. Based on the Caprini scoring model 92% of patients scored in the "Highest Risk" category. The Caprini RAM accurately predicted all 37 VTEs, all of which scored in the "Highest Risk" category. The percentage of patients that received double prophylaxis increased with time from 12% in 2004 to 63% in 2010. Importantly, 25 of the 37 VTEs (68%) did not receive double prophylaxis. CONCLUSIONS: The use of the Caprini RAM accurately predicted patients at the highest risk of experiencing VTE. Considering accurate identification of patients allows proper administration of double prophylaxis, we recommend the use of this scoring model preoperatively in patients undergoing surgery for gynecologic malignancies.

Myb overexpression overrides androgen depletion-induced cell cycle arrest and apoptosis in prostate cancer cells, and confers aggressive malignant traits: potential role in castration resistance.

Myb, a cellular progenitor of v-Myb oncogenes, is amplified in prostate cancer and exhibits greater amplification frequency in hormone-refractory disease. Here, we have investigated the functional significance of Myb in prostate cancer. Our studies demonstrate Myb expression in all prostate cancer cell lines (LNCaP, C4-2, PC3 and DU145) examined, whereas it is negligibly expressed in normal/benign prostate epithelial cells (RWPE1 and RWPE2). Notably, Myb is significantly upregulated, both at transcript (>60-fold) and protein (>15-fold) levels, in castration-resistant (C4-2) cells as compared with androgen-dependent (LNCaP) prostate cancer cells of the same genotypic lineage. Using loss and gain of function approaches, we demonstrate that Myb promotes and sustains cell cycle progression and survival under androgen-supplemented and -deprived conditions, respectively, through induction of cyclins (A1, D1 and E1), Bcl-xL and Bcl2 and downregulation of p27 and Bax. Interestingly, Myb overexpression is also associated with enhanced prostate-specific antigen expression. Furthermore, our data show a role of Myb in enhanced motility and invasion and decreased homotypic interactions of prostate cancer cells. Myb overexpression is also associated with actin reorganization leading to the formation of filopodia-like cellular protrusions. Immunoblot analyses demonstrate gain of mesenchymal and loss of epithelial markers and vice versa, in Myb-overexpressing LNCaP and -silenced C4-2 cells, respectively, indicating a role of Myb in epithelial to mesenchymal transition. Altogether, our studies provide first experimental evidence for a functional role of Myb in growth and malignant behavior of prostate cancer cells and suggest a novel mechanism for castration resistance.

Treatment of chemotherapy-induced anemia in ovarian cancer patients: does the use of erythropoiesis-stimulating agents worsen survival?

OBJECTIVE: Considering the paucity of data relating erythropoiesis-stimulating agent (ESA) use to ovarian cancer survival, our objective was to evaluate the effect of ESA as used for the treatment of chemotherapy-induced anemia (CIA) on survival in ovarian cancer patients. MATERIALS AND METHODS: A multi-institution retrospective chart review was performed on ovarian cancer patients. Data collection included patient demographic, surgicopathologic, chemotherapy, ESA, and survival data. Patients were stratified by ever-use of ESA and were compared using appropriate statistical methods. RESULTS: A total of 581 patients were eligible for analysis with 39% (n = 229) patients with ever-use of ESA (ESA-YES) and 61% (n = 352) never-use ESA (ESA-NO). Mean age was 60.4 years with most patients having stage IIIC (60%) of papillary serous histological diagnosis (64%) with an optimal cytoreduction (67%). Median follow-up for the cohort was 27 months. Both ESA-YES and ESA-NO groups were similar regarding age, body mass index, race, stage, histological diagnosis, and debulking status. Compared with the ESA-NO group, ESA-YES patients were significantly more likely to experience recurrence (56% vs 80%, P < 0.001) and death (46% vs 59%, P = 0.002). Kaplan-Meier curves demonstrated a significant reduction in progression-free survival for ESA-YES patients (16 vs 24 months, P < 0.001); however, overall survival was statistically similar between the 2 groups (38 vs 46 months, P = 0.10). When stratifying by ever experiencing a CIA, ESA-YES patients demonstrated a significantly worse progression-free survival (17 vs 24 months, P = 0.02) and overall survival (37 vs 146 months, P < 0.001). CONCLUSIONS: Our data evaluating the use of ESA as a treatment of CIA in ovarian cancer patients are similar to reports in other tumor sites. Considering that patients who used ESA were more likely to experience recurrence and death and to have decreased survival, the use of ESA in ovarian cancer patients should be limited.

Colorectal cancer screening practices in Alabama: a survey of primary care physicians.

In order to inform efforts to increase screening rates for colorectal cancer (CRC), we conducted a survey of Alabama primary care physicians regarding CRC screening practices, educational preferences, and perceptions of obstacles to screening. A mail survey of 2,378 Alabama physicians in Family Medicine, Internal Medicine, and Obstetrics & Gynecology was conducted. Many physicians are not fully up-to-date with current CRC screening practices that could improve patient compliance with screening guidelines. One example is the potential use of high-sensitivity stool tests, such as the fecal immunochemical test, instead of the no longer recommended low-sensitivity guaiac fecal occult blood tests. In addition, enhanced multimedia and web-based approaches to educating physicians and patients could be more fully utilized. Further, greater use of health information technologies could increase screening rates. Enhancing primary care physicians' knowledge of screening modalities and increasing their use of electronic technology could significantly improve colorectal cancer screening outcomes.

The ERCC1 N118N polymorphism does not change cellular ERCC1 protein expression or platinum sensitivity.

Genetic polymorphisms in ERCC1 are thought to contribute to altered sensitivity to platinum-based chemotherapy. Although ERCC1 N118N (500 C>T, rs11615) is the most studied polymorphism, the impact of this polymorphism on platinum-based chemotherapy remains unclear. This is the first study in which the functional impact of ERCC1 N118N on gene expression and platinum sensitivity was explored. The aim of this study is to investigate if the reduced codon usage frequency of AAT, which contains the variant allele of the silent mutation, has functional impact on ERCC1 in a well-controlled biological system. Specifically, the ERCC1 cDNA clone with either the C or T allele was introduced into an ERCC1 deficient cell line, UV20, and assayed for the effect of the two alleles on ERCC1 transcription, translation and platinum sensitivity. Both ERCC1 mRNA and protein expression levels increased upon cisplatin treatment, peaking at 4h post-treatment, however there were no differences between the two alleles (p>0.05). Cells complemented with ERCC1 showed significantly higher survival proportion than the parental cell line following platinum exposure (p<0.0001), although no differences were observed between the cells transfected with the wild type or the polymorphic allele. These data suggest that N118N itself is not related to the phenotypic differences in ERCC1 expression or function, but rather this polymorphism may be linked to other causative variants or haplotypes.

Double prophylaxis for deep venous thrombosis in patients with gynecologic oncology who are undergoing laparotomy: does preoperative anticoagulation matter?

OBJECTIVE: Double prophylaxis for deep venous thrombosis (DVT) with thromboprophylaxis plus sequential compression devices (SCDs) is recommended for high-risk surgical patients with gynecologic oncology. Despite the use of preoperative thromboprophylaxis in clinical trials, the schedule of perioperative low molecular-weight heparin varies widely. We sought to determine the effectiveness and adverse effects of a preoperative dose of anticoagulation in patients with gynecologic oncology. METHODS: A multi-institutional chart review from January 2006 to July 2008 was performed. Patients with gynecologic oncology who received double prophylaxis for laparotomy were eligible. The patients were grouped according to whether they received preoperative anticoagulation (YES PREOP vs NO PREOP). All patients received postoperative low molecular-weight heparin for thromboprophylaxis and SCDs until discharge. Demographic, surgicopathologic, and complication data were collected. RESULTS: A total of 239 patients were identified: YES PREOP (n = 101) and NO PREOP (n = 138). Groups were similar with respect to demographics, diagnosis, and length of hospital stay. There were 2 DVTs in the YES PREOP group compared with 11 in the NO PREOP group (P = 0.04; relative risk, 0.77). There were also fewer DVT-attributable deaths in the YES PREOP group (0 vs 2; P < 0.001). Postoperative hematocrit (30.2% vs 31.4%; P = 0.42) and number of transfusions (26 vs 14; P = 0.31) were similar. CONCLUSION: The use of preoperative anticoagulation seems to significantly decrease the risk of DVT in this patient population, and complication rates are not increased. Patients receiving double prophylaxis should receive a preoperative dose of anticoagulation for maximum benefit.

beta-Elemene, a novel plant-derived antineoplastic agent, increases cisplatin chemosensitivity of lung tumor cells by triggering apoptosis.

beta-Elemene, a new plant-derived anticancer agent with low toxicity, has been reported to be effective in the treatment of leukemia and solid tumors. In the current study, we explored the therapeutic application of beta-elemene in sensitizing lung cancer cells to cisplatin. beta-Elemene considerably enhanced the inhibitory effect of cisplatin on cell proliferation in a time- and dose-dependent manner in the human non-small cell lung cancer (NSCLC) cell lines H460 and A549. Furthermore, this effect of beta-elemene on cisplatin activity occurred through the induction of apoptosis in NSCLC cells, as assessed by an ELISA-based assay, TUNEL assay and annexin V binding assay. Consistent with these results, the protein levels of Bax and phospho-Bcl-2 increased and those of Bcl-2 and XIAP decreased in cells treated with beta-elemene in combination with cisplatin, compared with the levels in cells treated with either agent alone. Finally, beta-elemene augmented the cisplatin-induced increases in caspase-3, -7, -9 and -10 activities and cleaved caspase-3, -9 and poly(ADP-ribose) polymerase levels in NSCLC cells. These observations suggest that beta-elemene sensitizes NSCLC cells to cisplatin via a mitochondria-mediated intrinsic apoptosis pathway involving Bcl-2 family proteins and IAPs (inhibitor of apoptosis proteins). Our data provide a rationale for developing a combination of beta-elemene and cisplatin as a regimen for the treatment of lung carcinoma and other cisplatin-resistant tumors.

A Gynecologic Oncology Group study of platinum-DNA adducts and excision repair cross-complementation group 1 expression in optimal, stage III epithelial ovarian cancer treated with platinum-taxane chemotherapy.

To determine whether platinum-DNA adducts and/or mRNA expression of the excision nuclease excision repair cross-complementation group 1 (ERCC1) from peripheral blood leukocytes (PBL) were associated with clinical outcome in women with epithelial ovarian cancer (EOC), participants that had previously untreated, optimally resected, stage III EOC were randomized to paclitaxel plus cisplatin or carboplatin. DNA and RNA were extracted from PBLs collected 20 to 28 h post-drug infusion. DNA adducts were measured by atomic absorption spectroscopy. ERCC1 expression was evaluated by reverse transcription-PCR. There were 170 cases fully evaluable for DNA adducts and ERCC1 mRNA expression. Adduct levels ranged from 0.43 to 131 fmol platinum/microg DNA in 140 samples; and adducts were not detectable in 30 samples. ERCC1 mRNA was detectable in 132 samples and undetectable in 38. ERCC1 mRNA expression in PBLs was not associated with any clinical end point measured. The presence of detectable versus undetectable adducts was associated with longer median progression-free survival (20.4 versus 15.6 months; P = 0.084) and overall survival (60.3 versus 36.3 months; P = 0.029), respectively. Unadjusted Cox regression modeling indicated a trend toward a reduced risk of disease progression [hazard ratio (HR), 0.686; 95% confidence interval (95% CI), 0.447-1.054; P = 0.086] and a statistically significant reduction in the risk of death (HR, 0.607; 95% CI, 0.385-0.958; P = 0.032) for women with detectable versus undetectable adducts. After adjusting for clinicopathologic variables, detectable adducts were not an independent predictor of progression-free survival or overall survival. The presence of platinum-DNA adducts, but not ERCC1 mRNA expression, in PBLs was associated with better survival, but was not an independent predictor of clinical outcome in optimal advanced EOC.

Ethnic disparities in Americans of European descent versus Americans of African descent related to polymorphic ERCC1, ERCC2, XRCC1, and PARP1.

Nucleotide excision repair (NER) and base excision repair (BER) pathways are DNA repair pathways that are important in carcinogenesis and in response to DNA-damaging chemotherapy. ERCC1 and ERCC2 are important molecular markers for NER; XRCC1 and PARP1 are important molecular markers for BER. Functional polymorphisms have been described that are associated with altered expression levels of these genes and with altered DNA repair capability. We assayed genomic DNA from 156 Americans of European descent and 164 Americans of African descent for the allelic frequencies of specific polymorphisms of ERCC1 N118N (500C>T), ERCC1 C8092A, ERCC2 K751Q (2282A>C), XRCC1 R399Q (1301G>A), XRCC1 R194W (685C>T), and PARP1 V762A (2446T>C). Differences were observed between Americans of European descent and Americans of African descent in the allelic frequencies of the ERCC1 N118N polymorphism (P < 0.000001). Differences were also observed between these two ethnic groups for ERCC2 K751Q (P = < 0.006675), XRCC1 R399Q (P < 0.000001), and PARP1 V762A (P = 0.000001). The ERCC1 N118N polymorphic variant that is seen most commonly in Americans of European descent is associated with a measurable reduction in NER function. ERCC1-mediated reduction in NER functionality affects the repair of cisplatin-DNA lesions.

Development of a federally funded demonstration colorectal cancer screening program.

Colorectal cancer is the second leading cause of cancer-related mortality among U.S. adults. In 2004, treatment costs for colorectal cancer were $8.4 billion. There is substantial evidence that colorectal cancer incidence and mortality are reduced with regular screening. The natural history of this disease is also well described: most colorectal cancers develop slowly from preexisting polyps. This slow development provides an opportunity to intervene with screening tests, which can either prevent colorectal cancer through the removal of polyps or detect it at an early stage. However, much less is known about how best to implement an effective colorectal cancer screening program. Screening rates are low, and uninsured persons, low-income persons, and persons who have not visited a physician within a year are least likely to be screened. Although the Centers for Disease Control and Prevention (CDC) has 15 years of experience supporting the National Breast and Cervical Cancer Early Detection Program for the underserved population, a similar national program for colorectal cancer is not in place. To explore the feasibility of implementing a national program for the underserved U.S. population and to learn which settings and which program models are most viable and cost-effective, CDC began a 3-year colorectal cancer screening demonstration program in 2005. This article describes briefly this demonstration program and the process CDC used to design it and to select program sites. The multiple-methods evaluation now under way to assess the program's feasibility and describe key outcomes is also detailed. Evaluation results will be used to inform future activities related to organized screening for colorectal cancer.

Self-test device for cytology and HPV testing in rural Appalachian women: an evaluation.

OBJECTIVE: To determine the efficacy of a self-test device for obtaining vaginal/cervical samples for HPV DNA and cytologic testing. STUDY DESIGN: We recruited 878 Appalachian women; 775 were able to submit a self-test sample and a provider-collected sample. Each specimen was assessed for cytologic abnormalities and for high-risk HPV infection. RESULTS: Specimen adequacy: 869 of 878 (99%) women had sufficient material for cytologic analysis in provider-collected samples compared to 771 of 775 (99%) self-collected specimens (p = NS). There was sufficient cellular material (DNA) for HPV testing in 724 of 834 (87%) provider-collected samples compared to 690 of 736 (94%) self-collected specimens (p <0.04). In the provider-collected samples, 654 of 869 (75%) women had endocervical cells compared to 103 of 771 (13%) self-collected specimens (p <0.001). HPV assessment: 142 (19%) had high-risk HPV DNA detected. Of those women, 28 (20%) had HPV infection detected on the provider-collected specimen only, 51 (36%) on the self-collected specimen only, and 63 (44%) had HPV DNA on both specimens. CONCLUSION: Our device is sufficient to obtain an adequate number of epithelial cells for cytologic and HPV testing.

Irofulven induces replication-dependent CHK2 activation related to p53 status.

CHK2 and p53 are frequently mutated in human cancers. CHK2 is known to phosphorylate and stabilize p53. CHK2 has also been implicated in DNA repair and apoptosis induction. However, whether p53 affects CHK2 activation and whether CHK2 activation modulates chemosensitivity are unclear. In this study, we found that in response to the DNA damage agent, irofulven, CHK2 activation, rather than its expression, is inversely correlated to p53 status. Irofulven inhibits DNA replication and induces chromosome aberrations (breaks and radials) and p53-dependent cell cycle arrest. Pretreatment of cells with the DNA polymerase inhibitor, aphidicolin, resulted in reduction of irofulven-induced CHK2 activation and foci formation, indicating that CHK2 activation by irofulven is replication-dependent. Furthermore, by using ovarian cancer cell lines expressing dominant-negative CHK2 and CHK2-knockout HCT116 cells, we found that CHK2 activation contributes to the control of S and G2/M cell cycle arrests, but not chemosensitivity to irofulven. Overall, this study demonstrates that in response to irofulven-induced DNA damage, the activation of CHK2 is dependent on DNA replication and related to p53 status. By controlling cell cycle arrest and DNA replication, p53 affects CHK2 activation. CHK2 activation contributes to cell cycle arrest, but not chemosensitivity.

In vitro combination characterization of the new anticancer plant drug beta-elemene with taxanes against human lung carcinoma.

Beta-elemene has recently raised interest in P.R. China as a novel antitumor plant drug isolated from the Chinese medicinal herb Zedoary. To explore potentially useful combinations of beta-elemene with taxanes in the clinic, we characterized the effects of beta-elemene combined with taxanes in human lung cancer cells using a median effect analysis, micronucleus assay, apoptotic detection, and determination of gene expression in the signaling pathways of apoptosis. The synergistic analysis indicated that the interactions of beta-elemene with paclitaxel or docetaxel ranged from slight synergism to synergism. Combinations of beta-elemene with docetaxel induced much stronger synergistic interactions in p53 mutant H23 cells and p53 null H358 cells than in p53 wild-type H460 and A549 cells. Similar synergistic interactions were observed by micronucleus assay, apoptotic detection, and determination of apoptotic gene expression. Our findings indicate that the synergistic effects achieved with combinations of beta-elemene and taxanes are related to the augmented cytotoxic efficacy of taxanes owing to the action of beta-elemene. In H460 and A549 cells, dose-dependent upregulation of p53 protein expression was observed in cultures treated with docetaxel alone and with docetaxel plus beta-elemene, whereas no significant change in p53 expression was observed in any of the treatment groups in H23 cells. Fas revealed no alteration of expression with any of the treatments in this study. However, the combination treatments induced increased cytochrome c release from mitochondria, significant caspase-8 and -3 cleavage, and downregulation of Bcl-2 and Bcl-XL expression. These results suggest that, although p53 plays an important role in taxane-induced cell death, apoptosis induced by beta-elemene or in combination with docetaxel thereof seems to be initiated through a p53- and Fas-independent pathway via mitochondria in our lung cancer cells. The suppression of specific 'survival' gene expression appears to be the key action leading to the synergistic effect of combination treatments with beta-elemene and taxanes. Finally, the beta-elemene-induced alteration of cell membrane permeability, which has potential to result in enhanced cellular uptake of taxanes, may also contribute to the synergistic interactions of the combination treatments.

Vascular endothelium summary statement III: Cancer prevention and control.

The biology of vascular endothelium can be modulated through the use of pharmacologic agents. Whereas this has been explored to reasonable extent in cardiovascular disease, it is clear that the ability to modulate vascular endothelium for the purpose of cancer prevention is in its early stages. Among the unanswered questions, we do not understand under what circumstances such pharmacologic interventions might best be started; nor, how long such interventions might be used for a given individual. We present concepts for further exploration in the use of endothelial-active agents for cancer prevention and control; and, strategies for public health practice and research in this area.

Vascular endothelium summary statement VI: Research directions for the 21st century.

Effective translation of research advances from the bench to clinical and public health practice at the bedside and in the community at large represents an important step in the health research discovery enterprise. Increasingly, the gap in translating these advances into practice is being recognized. Successfully addressing this translational gap for the prevention and control of chronic diseases will require the development of novel, innovative, and, if necessary, nontraditional approaches. Participants in the 8th International Conference on Vascular Endothelium discussed a variety of novel approaches that have significant promise. Three of these approaches-vaccine development, genomics and proteomics, and tissue engineering-are highlighted in this position statement and strategies for public health practice and research are suggested.

Fanconi anemia D2 protein confers chemoresistance in response to the anticancer agent, irofulven.

The Fanconi anemia-BRCA pathway of genes are frequently mutated or epigenetically repressed in human cancer. The proteins of this pathway play pivotal roles in DNA damage signaling and repair. Irofulven is one of a new class of anticancer agents that are analogues of mushroom-derived illudin toxins. Preclinical studies and clinical trials have shown that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. Previously, we have shown that irofulven activates ATM and its targets, NBS1, SMC1, CHK2, and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and FANCD2 may play an important role in modulating cellular responses and chemosensitivity in response to irofulven treatment. By using cells that are proficient or deficient for FANCD2, ATR, or ATM, we showed that irofulven induces FANCD2 monoubiquitination and nuclear foci formation. ATR is important in mediating irofulven-induced FANCD2 monoubiquitination. Furthermore, we showed that FANCD2 plays a critical role in maintaining chromosome integrity and modulating chemosensitivity in response to irofulven-induced DNA damage. Therefore, this study suggests that it might be clinically significant to target irofulven therapy to cancers defective for proteins of the Fanconi anemia-BRCA pathway.

MZF1 possesses a repressively regulatory function in ERCC1 expression.

ERCC1 is a critical gene within the nucleotide excision repair pathway. Overexpression of ERCC1 through promoter-mediating transcriptional regulation is associated with repair of cisplatin-induced DNA damage and clinical resistance to platinum-chemotherapy. Several transcriptional repressors and activators within the 5'-flanking region of the ERCC1 gene may be involved in the up-regulation of this gene. Minimal sequence within the promoter region required for ERCC1 transcription was analyzed by CAT assay and demonstrated that the region of -220 to -110 is essential to constitutive expression of ERCC1 gene in ovarian cancer cell line A2780/CP70. A more forward upstream region seems to be responsible for cisplatin-induced expression. Study of the functional cis-element in this region by electrophoretic mobility shift assay indicates that a MZF1-like site as well as an AP1-like site responded in a time-dependent manner to cisplatin stimulation with altered binding activities. EMSA with MZF1 ZN1-4 consensus oligonucleotides suggests that the MZF1 N-terminal domain of zinc finger cluster may bind to the MZF1-like site of the ERCC1 promoter region. MZF1 mRNA in A2780/CP70 cells decreased upon cisplatin exposure as analyzed by quantitative PCR, suggesting that MZF1 may mediate cisplatin-invoked gene expression in these cells. Overexpression of MZF1 repressed the ERCC1 promoter activity as determined in co-transfection assay, suggesting that MZF1 might be a repressor of ERCC1 transcription upon cisplatin exposure. In summary, our studies revealed a core promoter region and adjacent drug-responsible region within the ERCC1 promoter. The drug-responsible region contains cis-elements of activator, AP1 and repressor, MZF1. In response to cisplatin treatment, decreased MZF1 and increased AP1 binding activities appear to be the leading mechanism of up-regulation of ERCC1 expression. Our findings imply potential therapeutic strategies to antagonize drug resistant mechanisms in treatment of human ovarian cancer.

Antisense telomerase RNA inhibits the growth of human glioma cells in vitro and in vivo.

Telomerase is implicated in the development of cellular immortality and oncogenesis. It has been shown that telomerase activity is considerably higher in the tissue of many different cancers than in normal tissue, and that the inhibition or downregulation of telomerase activity can prevent the malignant proliferation of tumor cells. Antisense oligonucleotides have been widely used in suppressing the expression of genes and, therefore, in the present research, we evaluated the effect of antisense human telomerase RNA (hTR) on glioma cell growth in vitro and in vivo. We showed that antisense hTR cDNA significantly inhibited TJ905 human glioma cell proliferation in vitro and tumor growth in vivo, as determined by MTT assay and by measuring the volume of glioma in nude mice. Consistent with these results, we found that telomerase activity and the mRNA levels of hTR and hTERT (human telomerase reverse transcriptase) expression were markedly decreased in tumor cells treated with antisense hTR cDNA, as assessed by TRAP (telomeric repeat amplification protocol) assay and RT-PCR (reverse transcription-polymerase chain reaction) analysis. Our study conclusively demonstrates that antisense hTR effectively inhibits the growth of human glioma cells in vitro and in vivo and, thus, may be potentially used for gene therapy of malignant gliomas and other cancers.

Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways.

Apigenin is a nontoxic dietary flavonoid that has been shown to possess anti-tumor properties and therefore poses special interest for the development of a novel chemopreventive and/or chemotherapeutic agent for cancer. Ovarian cancer is one of the most common causes of cancer death among women. Here we demonstrate that apigenin inhibits expression of vascular endothelial growth factor (VEGF) in human ovarian cancer cells. VEGF plays an important role in tumor angiogenesis and growth. We found that apigenin inhibited VEGF expression at the transcriptional level through expression of hypoxia-inducible factor 1alpha (HIF-1alpha). Apigenin inhibited expression of HIF-1alpha and VEGF via the PI3K/AKT/p70S6K1 and HDM2/p53 pathways. Apigenin inhibited tube formation in vitro by endothelial cells. These findings reveal a novel role of apigenin in inhibiting HIF-1 and VEGF expression that is important for tumor angiogenesis and growth, identifying new signaling molecules that mediate this regulation.

Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells.

High-fidelity maintenance of genomic integrity in eukaryotes is ensured by cell cycle checkpoints and DNA repair. The checkpoint kinase, Chk2, has been implicated in both of these responses. In response to DNA damage, Chk2 is initially phosphorylated at Thr-68, which leads to its full activation. The fully activated Chk2 then phosphorylates downstream substrates of cell cycle control. However, the mechanism of inactivation of Chk2 is still unknown. Protein phosphatase type 2A (PP2A) plays an essential role in cell cycle regulation and induction of G2 arrest by a mechanism of phosphorylation/dephosphorylation with a variety of protein kinases. Data from our investigation provide evidence that, in response to cisplatin exposure, PP2A associates with Chk2 as a complex in cells and functions as a negative regulator of Chk2 activation by dephosphorylating p-Chk2. Results from immunostaining and coimmunoprecipitation demonstrate that Chk2 and PP2A can colocalize in cells, and the holoenzyme of PP2A (subunits A, B and C) coimmunoprecipitates with p-Chk2. Further, inhibition of PP2A by okadaic acid, an inhibitor of PP2A, and by small interfering RNA (siRNA) to PP2A results in enhanced Chk2 phosphorylation, implicating a direct enzyme-substrate relationship. An in vitro PP2A dephosphorylation assay shows that PP2A dephosphorylates p-Chk2 in a cell-free system. These findings suggest that the protein serine/threonine kinase, Chk2, is activated after cisplatin exposure and negatively regulated by a tightly associated protein serine/threonine phosphatase, PP2A.