Antioxidant vitamins supplementation and mortality: a randomized trial in head and neck cancer patients

There has been concern that long-term supplementation with high-dose antioxidant vitamins, especially vitamin E (alpha-tocopherol), may increase all-cause mortality. We conducted a randomized controlled trial with alpha-tocopherol (400 IU/day) and beta-carotene (30 mg/day) supplements among 540 head and neck cancer patients treated by radiation therapy. Supplementation with beta-carotene was discontinued during the trial. The supplements were given during radiation therapy and for 3 additional years. During the follow-up (median 6.5 years), 179 deaths were recorded. All death certificates were obtained. All-cause and cause-specific mortality rates were compared between the 2 arms of the trial by Cox regression. All-cause mortality was significantly increased in the supplement arm: hazard ratio: 1.38, 95% confidence interval 1.03-1.85. Cause-specific mortality rates tended to be higher in the supplement arm than in the placebo arm. Our results concur with previous reports to suggest that high-dose vitamin E could be harmful.     

Modulation of lung molecular biomarkers by β‐carotene in the Physicians' Health Study

BACKGROUND:

β‐Carotene supplementation showed neither benefit nor harm among apparently healthy physicians (all men) in the Physicians' Health Study (PHS) trial. The objective of the current investigation was to evaluate how long‐term β‐carotene supplementation affects molecular markers of lung carcinogenesis in the PHS.

METHODS:

The protein levels of total p53, cyclin D1, proliferating cellular nuclear antigen (PCNA), retinoic acid receptor β (RARβ), and cytochrome p450 enzyme 1A1 (CYP1A1) were measured using the immunohistochemical method in 40 available archival lung tissue samples from patients who were diagnosed with lung cancer in the PHS. The protein levels of these markers were compared by category of β‐carotene treatment assignment and other characteristics using unconditional logistic regression models.

RESULTS:

The positivity for total p53, RARβ, cyclin D1, and PCNA was nonsignificantly lower among lung cancer patients who were assigned to receive β‐carotene than those who were assigned to receive β‐carotene placebo. There was a borderline significant difference in CYP1A1 positivity with an OR of 0.2 (95% confidence interval, 0.2‐1.1; P = .06) in a comparison of men who received β‐carotene and men who received β‐carotene placebo.

CONCLUSIONS:

The 50‐mg β‐carotene supplementation on alternate days had no significant influence on molecular markers of lung carcinogenesis that were evaluated in the PHS. This finding provides mechanistic support for the main PHS trial results of β‐carotene, which demonstrated no benefit or harm to the risk of developing lung cancer. Cancer 2009. © 2009 American Cancer Society.     

Effect of α‐tocopherol,N‐acetylcysteine and omeprazole on esophageal adenocarcinoma formation in a rat surgical model

We previously demonstrated that oxidative stress subsequent to gastroesophageal reflux is an important driving force of esophageal adenocarcinoma (EAC) formation in the esophagogastroduodenal anastomosis (EGDA) rat model. This study investigated the possible tumor inhibitory effects of 2 antioxidants, α‐tocopherol (389 and 778 ppm), N‐acetylcysteine (NAC, 500 and 1,000 ppm), and their combination (389 and 500 ppm, respectively), as well as an antacid therapeutic agent, omeprazole (1,400 ppm). The rats were fed experimental diets 2 weeks after EGDA. All the animals were sacrificed 40 weeks after EGDA and the esophagi were harvested for histopathological examination. α‐Tocopherol dose‐dependently decreased the incidence of EAC (p = 0.03), with 778 ppm α‐tocopherol reducing the incidence of EAC to 59% (16/27) in comparison with 84% (26/31) in the control group (p = 0.04). Supplementation of α‐tocopherol also increased the serum concentration of α‐tocopherol. NAC at 500 and 1,000 ppm did not significantly decrease EAC incidence; however, the combination of α‐tocopherol 389 ppm and NAC 500 ppm significantly reduced the incidence of EAC to 55% (15/27) (p = 0.02). α‐Tocopherol alone or in combination with NAC significantly reduced the number of infiltrating cells positively stained for 4‐hydroxynonenal. Omeprazole showed only a slight nonsignificant inhibitory effect at the dose given. Our results suggest that supplementation with α‐tocopherol inhibits the development of EAC in the rat EGDA model and similar inhibitory effect can be achieved when a lower dose of α‐tocopherol is used in combination with NAC. © 2008 Wiley‐Liss, Inc.     

Modulation of TGF‐β activity by latent TGF‐β‐binding protein 1 in human malignant glioma cells

High biological activity of the transforming growth factor (TGF)‐β‐Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF‐β has become a novel target for the experimental treatment of these tumors. TGF‐β is processed by furin‐like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency‐associated peptide (LAP). Latent TGF‐β‐binding protein 1 (LTBP‐1) covalently binds to this small latent TGF‐β complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP‐1 protein in vivo increase with the grade of malignancy in gliomas. LTBP‐1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP‐1 into the medium is decreased by the inhibition of FLP activity. Gene‐transfer mediated overexpression of LTBP‐1 in glioma cell lines results in an increase inTGF‐β activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF‐β activity is enhanced. Conversely, LTBP‐1 gene silencing reduces TGF‐β activity and Smad2 phosphorylation without affecting TGF‐β protein levels. Collectively, we identify LTBP‐1 as an important modulator of TGF‐β activation in glioma cells, which may contribute to the malignant phenotype of these tumors. © 2009 UICC     

γ‐Glutamyltransferase and cancer risk: The Korean cancer prevention study

Elevated serum γ‐glutamyltransferase (GGT) is a marker of hepatic injury and is associated with risk of chronic disease. However, the value of GGT as a biomarker for cancer risk remains unclear. Therefore, we evaluated the association of serum GGT with cancer incidence among more than 1.6 million Koreans. We included 1,662,087 Koreans (1,108,121 men and 553,966 women aged 20–95 years) who received health insurance from the National Health Insurance Service and had a biennial medical evaluation between 1995 and 1998. Follow‐up was through December 2012. Using Cox proportional hazards models, we adjusted for age, smoking status, alcohol consumption, exercise and body mass index after exclusion of early cases (cancer diagnosis or death within 5 years of starting follow‐up) and estimated hazard ratios (HRs) of overall and organ‐specific cancer incidence by GGT quintiles. During the 17‐year follow‐up, 129,087 new cancer cases occurred among the participants. Across levels of GGT, there was a positive gradient of HR and the highest quintile of GGT (≥60 IU/L) had the highest HR for all cancers in both men and women. By cancer site, the association was strongest for liver cancer, comparing the highest and lowest strata in men [HR, 6.67; 95% confidence interval (95%CI), 5.88–7.57] and in women (HR, 7.57; 95%CI, 6.41–8.94). Significant associations were also observed for cancers of the esophagus, larynx, stomach, colorectal, bile duct and lung in men and of the bile duct in women. Increased serum GGT level is independently associated with risk of cancer.     

Cancer incidence and mortality among members of the Danish resistance movement deported to German concentration camps: 65‐Year follow‐up

The widespread belief that a stressful life event increases cancer incidence and mortality was investigated in a unique cohort of all Danish male political prisoners, who survived the extremely stressful experience of life in German concentration camps between 1943 and 1945. A virtually complete cohort of all 1,322 Danish male political prisoners who survived deportation to German concentration camps were followed up for cancer incidence and all‐cause and cancer‐specific mortality from 1946 through 2010. Standardized ratios and 95% confidence intervals were calculated from the observed and expected numbers of cancers or deaths, the latter based on national rates. We observed slightly increased standardized cancer incidence ratio (SIR 1.16; 95% CI, 1.06–1.27), particularly of smoking‐ or alcohol‐related cancers (SIR 1.31; 95% CI, 1.15–1.49) and nonsignificantly increased SIR of immune system‐ and hormone‐related cancers (SIR 1.17; 95% CI, 0.80–1.65 and 1.05; 95% CI, 0.81–1.34 respectively). Both the standardized all‐cause mortality ratio (SMR 1.11; 95% CI, 1.05–1.18) and cancer specific mortality ratio (SCMR 1.17; 95% CI, 1.01–1.26) were slightly increased, particularly from smoking‐ or alcohol‐related cancers (SCMR 1.25; 95% CI, 1.06–1.45). The minor increased cancer incidence and cancer mortality among the survivors is probably not directly associated with exposure to this extreme stressful event, but may be indirectly mediated through behavioral responses to psychological stress, as reflected in the increased incidence of and mortality from tobacco‐ and alcohol‐related cancers.     

Retinoid X receptor α enhances human cholangiocarcinoma growth through simultaneous activation of Wnt/β‐catenin and nuclear factor‐κB pathways

Retinoid X receptor α (RXRα) plays important roles in the malignancy of several cancers such as human prostate tumor, breast cancer, and thyroid tumor. However, its exact functions and molecular mechanisms in cholangiocarcinoma (CCA), a chemoresistant carcinoma with poor prognosis, remain unclear. In this study we found that RXRα was frequently overexpressed in human CCA tissues and CCA cell lines. Downregulation of RXRα led to decreased expression of mitosis‐promoting factors including cyclin D1and cyclin E, and the proliferating cell nuclear antigen, as well as increased expression of cell cycle inhibitor p21, resulting in inhibition of CCA cell proliferation. Furthermore, RXRα knockdown attenuated the expression of cyclin D1 through suppression of Wnt/β‐catenin signaling. Retinoid X receptor α upregulated proliferating cell nuclear antigen expression through nuclear factor‐κB (NF‐κB) pathways, paralleled with downregulation of p21. Thus, the Wnt/β‐catenin and NF‐κB pathways account for the inhibition of CCA cell growth induced by RXRα downregulation. Retinoid X receptor α plays an important role in proliferation of CCA through simultaneous activation of Wnt/β‐catenin and NF‐κB pathways, indicating that RXRα might serve as a potential molecular target for CCA treatment.     

Endometrial cancer invasion depends on cancer‐derived tumor necrosis factor‐α and stromal derived hepatocyte growth factor

Cancer invasion is an outcome of interactions of the cancer and the host cell. It is now becoming increasingly clear that ovarian hormones have a huge influence on such intercommunications in various types of cancers. Estrogen is known to aggravate the aggressiveness of the endometrial cancer whereas progesterone seems to act as a negative factor. Insight into the mode of ovarian hormonal actions could come from the studies of its regulation of the paracrine interactions between the endometrial cancer and the normal stromal cells during the cancer invasion. In this context, we report here that estrogen promotes the endometrial cancer invasion by inducing humoral interactions between the cancer and the stromal cells, i.e., estrogen stimulates tumor necrosis factor‐α expression from the endometrial cancer cells, which, in turn, induces the stromal expression of hepatocyte growth factor (HGF), conferring the enhanced NK4 (HGF‐antagonist/angiogenesis inhibitor)‐sensitive invasion characteristic of the endometrial cancer cells. Additionally, we demonstrate a close correlation of the invasion of endometrial cancer cells with the expression and dimerization of integrin α_vβ₅ as well as the activation of focal adhesion kinase as the consequences of paracrine interactions. Thus, understanding of paracrine interactions of cancer cells with host stromal cells can yield new insight into the architecture and function of cancer invasion and metastasis, leading to a development of a new cancer therapeutic intervention. © 2008 Wiley‐Liss, Inc.     

Evaluation of the safety,pharmacokinetics and treatment effects of an ανβ3 integrin inhibitor on bone turnover and disease activity in men with hormone‐refractory prostate cancer and bone metastases

Aim: This study aimed to evaluate the safety, pharmacokinetics and treatment effects of an α_νβ₃ integrin inhibitor on bone turnover and disease activity in men with hormone‐refractory prostate cancer (HRPC) and bone metastases. Methods: A total of 21 patients with bone metastases and HRPC were randomized to receive MK‐0429 200 mg b.i.d. or 1600 mg b.i.d. for 4 weeks. Toxicity, pharmacokinetics and markers of bone turnover and tumor activity were examined. Results: Nausea was the most common adverse event: one (200‐mg group) and 11 (1600‐mg group) patients. At 4 weeks, mean AUC_{0–12 h} was 210 mmol*h (200‐mg group) and 673 mmol*h (1600‐mg group); mean C_max values were 42 mmol/L (200‐mg group) and 154 mmol/L (1600‐mg group). Urinary cross‐linked N‐telopeptides of type I collagen to creatinine ratio (uNTx), a bone turnover biomarker, showed a change from baseline of ?43.4 percent (200‐mg group) and ?34.1 percent (1600‐mg group). There was an increase in serum prostate specific antigen (PSA), a marker for disease activity, of 54.1 percent (200‐mg group) and 44.5 percent (1600‐mg group). Conclusion: MK‐0429 was generally well tolerated, with the most common side‐effect being nausea. There was some evidence of an early reduction of bone turnover, indicating a potential for clinical use in the treatment of MBD although serum PSA was unexpectedly increased during the study.     

Beta‐carotene supplementation and cancer risk: a systematic review and metaanalysis of randomized controlled trials

The effect of beta‐carotene supplementation on cancer incidence has been investigated in several randomized controlled trials. The objective was to review the effect of beta‐carotene supplementation on cancer incidence in randomized trials by cancer site, beta‐carotene supplementation characteristics and study population. Relevant trials were retrieved by searching PubMed (up to April 2009). Authors involved in selected studies were contacted for additional information. Thirteen publications reporting results from 9 randomized controlled trials were included. Overall, no effect of beta‐carotene supplementation was observed on the incidence of all cancers combined (RR, 1.01; 95% CI, 0.98–1.04), pancreatic cancer (RR, 0.99; 95% CI, 0.73–1.36), colorectal cancer (RR, 0.96; 95% CI, 0.85–1.09), prostate cancer (RR, 0.99; 95% CI, 0.91–1.07), breast cancer (RR, 0.96; 95% CI, 0.85–1.10), melanoma (RR, 0.98; 95% CI, 0.65–1.46) and non melanoma skin cancer (RR, 0.99; 95% CI, 0.93–1.05). The incidence of lung and stomach cancers were significantly increased in individuals supplemented with beta‐carotene at 20–30 mg day⁻¹ (RR, 1.16; 95% CI, 1.06–1.27 and RR, 1.34; 95% CI, 1.06–1.70), in smokers and asbestos workers (RR, 1.20; 95% CI, 1.07–1.34 and RR, 1.54; 95% CI, 1.08–2.19) compared to the placebo group. Beta‐carotene supplementation has not been shown to have any beneficial effect on cancer prevention. Conversely, it was associated with increased risk not only of lung cancer but also of gastric cancer at doses of 20–30 mg day⁻¹, in smokers and asbestos workers. This study adds to the evidence that nutritional prevention of cancer through beta‐carotene supplementation should not be recommended.     

Dkk3, downregulated in cervical cancer,functions as a negative regulator of β‐catenin

The Wnt/β‐catenin signaling pathway is activated during the malignant transformation of keratinocytes that originate from the human uterine cervix. Dkk1, 2 and 4 have been shown to modulate the Wnt‐induced stabilization of the β‐catenin signaling pathway. However, the function of Dkk3 in this pathway is unknown. Comparison of the Dkk3 gene expression profiles in cervical cancer and normal cervical tissue by cDNA microarray and subsequent real‐time PCR revealed that the Dkk3 gene is frequently downregulated in the cancer. Methylation studies showed that the promoter of Dkk3 was methylated in cervical cancer cell lines and 22 (31.4%) of 70 cervical cancer tissue specimens. This promoter methylation was associated with reduced expression of Dkk3 mRNA in the paired normal and tumor tissue samples. Further, the reintroduction of Dkk3 into HeLa cervical cancer cells resulted in reduced colony formation and retarded cell growth. The forced expression of Dkk3 markedly attenuated β‐catenin‐responsive luciferase activity in a dose‐dependent manner and decreased the β‐catenin levels. By utilizing a yeast two‐hybrid screen, βTrCP, a negative regulator of β‐catenin was identified as a novel Dkk3‐interacting partner. Coexpression with βTrCP synergistically enhanced the inhibitory function of Dkk3 on β‐catenin. The stable expression of Dkk3 blocks the nuclear translocation of β‐catenin, resulting in downregulation of its downstream targets (VEGF and cylcin D), whereas knockdown of Dkk3 abrogates this blocking. We conclude from our finding that Dkk3 is a negative regulator of β‐catenin and its downregulation contribute to an activation of the β‐catenin signaling pathway. © 2008 Wiley‐Liss, Inc.     

Identification of integrins α6 and β7 as c‐Jun‐ and transformation‐relevant genes in highly invasive fibrosarcoma cells

Understanding the mechanisms of tumor cell invasion is essential for our attempts to prevent cancer deaths. We screened by DNAmicroarrays the c‐Jun‐ and transformation‐related gene expression changes in S‐adenosylmethionine decarboxylase (AdoMetDC)‐overexpressing mouse fibroblasts that are highly invasive in vivo, and their derivatives expressing a tetracycline‐inducible dominant‐negative mutant of c‐Jun (TAM67) or c‐Jun shRNA. Among the small set of target genes detected were integrins α6 and β7, cathepsin L and thymosin β4, all upregulated in the AdoMetDC‐transformed cells and downregulated upon reversal of transformation by TAM67 or c‐Jun shRNA. The upregulation of integrin α6 subunit, pairing with integrin β1, endowed the transformed cells with the capability to attach to basement membrane laminin and to spread. Further, inhibition of integrin α6 or β1 function with neutralizing antibodies blocked the invasiveness of AdoMetDC‐transformants and human HT‐1080 fibrosarcoma cells in three‐dimensional Matrigel. Moreover, immunohistochemical analyses showed strong integrin α6 staining in high‐grade human fibrosarcomas. Our data show that c‐Jun can regulate all three key steps of invasion: cell adhesion (integrin α6), basement membrane/extracellular matrix degradation (cathepsin L) and cell migration (thymosin β4). In addition, this is the first study to associate integrin β7, known as a leukocyte‐specific integrin binding to endothelial/epithelial cell adhesion molecules, with the transformed phenotype in cells of nonleukocyte origin. As tumor cell invasion is a prerequisite for metastasis, the observed critical role of integrin α6β1 in fibrosarcoma cell invasion/spreading allures testing antagonists to integrin α6β1, alone or combined with inhibitors of cathepsin L and thymosin β4, as chemotherapeutic agents. © 2009 UICC     

Association between activation of atypical NF‐κB1 p105 signaling pathway and nuclear β‐catenin accumulation in colorectal carcinoma

Recent studies have demonstrated that increased expression of coding region determinant‐binding protein (CRD‐BP) in response to β‐catenin signaling leads to the stabilization of β‐TrCP1, a substrate‐specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IκBα and activation of canonical nuclear factor‐κB (NF‐κB) pathway. Here, we show that the noncanonical NF‐κB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with β‐catenin‐mediated increased expression of CRD‐BP and β‐TrCP1. In the carcinoma tissues exhibiting high levels of nuclear β‐catenin the phospho‐p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF‐κB pathway. Knockdown of CRD‐BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF‐κB inhibitory proteins, p105 and IκBα. Furthermore decreased NF‐κB binding activity was observed in CRD‐BP siRNA‐transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF‐κB1 p105 signaling in colorectal carcinoma might be attributed to β‐catenin‐mediated induction of CRD‐BP and β‐TrCP1. © 2009 Wiley‐Liss, Inc.     

Expression of galectin‐1 in carcinoma‐associated fibroblasts promotes gastric cancer cell invasion through upregulation of integrin β1

Increased expression of galectin‐1 (Gal‐1) in carcinoma‐associated fibroblasts (CAFs) has been reported to correlate with progression and prognosis in many cancers. However, rarely have reports sought to determine whether high Gal‐1 expression in CAFs in gastric cancer is involved in the tumor process, and the specific mechanism by which it promotes the evolution of gastric cancer is still unknown. In this study, we cultured gastric cancer CAFs, which showed strong expression of Gal‐1, and established a co‐culture system of CAFs with gastric cancer cells. Specific siRNA and in vitro migration and invasion assays were used to explore the effects of the interaction between Gal‐1 expression of CAFs and gastric cancer cells on cell migration and invasion. We found that the overexpression of Gal‐1 in CAFs enhanced gastric cancer cell migration and invasion, and these stimulatory effects could be blocked by specific siRNA which reduced the Gal‐1 expression level. A set of cancer invasion‐associated genes were then chosen to identify the possible mechanism of Gal‐1‐induced cell invasion. Among these genes, integrin β1 expression in cancer cells was considered to be associated with Gal‐1 expression. Pre‐blocking of the integrin β1 expression in gastric cancer cells with siRNA could interrupt the invasion‐promoting effect of CAFs with high Gal‐1 expression. Furthermore, immunohistochemical assay confirmed a positive correlation between Gal‐1 and integrin β1 expression. Our results showed that high expression of Gal‐1 in CAFs might facilitate gastric cancer cell migration and invasion by upregulating integrin β1 expression in gastric cancer.     

Phenotypic characterization and prognostic impact of circulating γδ and αβ T‐cells in metastatic malignant melanoma

Human T cells carrying γδ T‐cell receptors (TCRs) represent a minor population relative to those with αβ TCRs. There has been much interest recently in the possibility of using these γδ T‐cells in cancer therapy because they can kill tumor cells in vitro in an MHC‐unrestricted manner, and possess potential regulatory capability and antigen‐presenting capacity. The presence of γδ T‐cells in late‐stage melanoma patients and their relationship with survival has not been extensively explored, although relatively lower percentages of total γδ T‐cells and Vδ2+ cells have been reported. Here, we present a detailed analysis of associations of γδ T‐cell subsets and differentiation stages with survival in Stage IV patients, compared with CD4+ and CD8+ αβ T‐cells. We found an increased Vδ1:Vδ2‐ratio and a decreased CD4:CD8‐ratio in patients compared to healthy controls, on the basis both of relative frequencies and absolute cell counts per μL blood. Nonetheless, Kaplan–Meier analyses showed that a higher than median frequency of Vδ1+ cells was negatively associated with survival, whereas there were no positive or negative associations with frequencies of Vδ2+ cells. Correlations of cell differentiation status with survival revealed a negative association of early‐differentiated Vδ1+ T cells with survival, both on the basis of relative frequencies and absolute counts. There was also a positive correlation between the frequencies of early‐differentiated CD8+ αβ T‐cells and survival. Our findings suggest peripheral blood frequencies of Vδ1+ T‐cells as a potential prognostic marker in melanoma. The mechanisms by which higher abundance of Vδ1+ cells are associated with poorer survival require determination.     

Evaluation of trace elements,oxidant/antioxidant status,vitamin C and β‐carotene in dogs with dermatophytosis

The aim of the study was to determine zinc, copper and iron levels, erythrocyte oxidant/antioxidant status, vitamin C and β‐carotene in dogs with dermatophytosis. A total of 23 dogs with clinically established diagnosis of dermatophytosis by trichogram and positive fungal culture and six dogs as control were included in this study. On cultural examination 52.17% fungal isolates were found to be Microsporum canis, 30.43% were Trichophyton mentagrophytes and 17.39% were M. gypseum. In comparison to healthy control, the dogs with dermatophytosis had significantly lower levels of zinc (P < 0.01), copper (P < 0.05), β‐carotene and vitamin C levels (P < 0.05) and activities of superoxide dismutase (SOD) (P < 0.05) and catalase (P < 0.01), whereas the iron (P < 0.05) and malondialdehyde (MDA) (P < 0.01) levels were significantly increased. On correlation analysis, SOD activity was observed to be positively correlated (P < 0.05) with zinc and copper in both healthy and dermatophytosis affected dogs. In dermatophytosis affected dogs the MDA levels were negatively correlated (P < 0.05) with iron, β‐carotene levels and the activities of antioxidant enzymes; SOD and catalase. Our results demonstrated that dermatophytosis in dogs is associated with significant alteration in oxidant/antioxidant balance and trace elements. It might be secondary consequence of dermatophytosis infection or contributing factor in its pathogenesis.