Calcium Supplements and Risk of CVD: A Meta-Analysis of Randomized Trials.

Background: Vitamin D supplements may only be beneficial for the prevention of osteoporotic fractures when administered with calcium and in individuals with low blood levels of 25(OH)D, but possible hazards of calcium supplements on CVD cannot be excluded. Objectives: We conducted a meta-analysis of all placebo-controlled randomized trials assessing the effects of calcium supplements alone or with vitamin D on CHD, stroke, and all-cause mortality. Methods: A meta-analysis of 11 trials included 7 comparisons of calcium alone compared with control (n = 8634) and 6 comparisons of calcium plus vitamin D compared with control (n = 46,804). Aggregated study-level data were obtained from individual trials and combined using a fixed-effects meta-analysis. The main outcomes included MI, CHD death, any CHD, stroke, and all-cause mortality. Results: Among trials of calcium alone (mean daily dose 1 g), calcium was not significantly associated with any excess risk of MI (RR, 1.15; 95% CI: 0.88, 1.51; n = 219 events), CHD death (RR, 1.24; 95% CI: 0.89, 1.73; n = 142), any CHD (RR, 1.01; 95% CI: 0.75, 1.37; n = 177), or stroke (RR, 1.15; 95% CI, 0.90, 1.46, n = 275). Among 6 trials of combined treatment, supplementation with calcium plus vitamin D was not significantly associated with any excess risk of MI (RR, 1.09; 95% CI: 0.95, 1.25; n = 854), CHD death (RR, 1.04; 95% CI: 0.85, 1.27; n = 391), any CHD (RR, 1.05; 95% CI: 0.93, 1.19; n = 1061), or stroke (RR, 1.02; 95% CI: 0.89, 1.17; n = 885). Likewise, calcium alone, or with vitamin D had no significant associations with all-cause mortality. Conclusions: This meta-analysis demonstrated that calcium supplements were not associated with any significant hazard for CHD, stroke, or all-cause mortality and excluded excess risks above 0.3%-0.5% per year for CHD or stroke. Further trials of calcium and vitamin D are required in individuals with low blood levels of 25(OH)D for the prevention of fracture and other disease outcomes.

The 17-gene Genomic Prostate Score assay as a predictor of biochemical recurrence in men with intermediate and high-risk prostate cancer.

The validated 17-gene Oncotype DX Genomic Prostate Score® (GPS™) assay risk-stratifies prostate-cancer patients with localized disease. The assay has primarily been utilized in lower risk patients deciding between active surveillance versus definitive therapy. In this retrospective cohort study, we analyze the association of the GPS result with time to biochemical recurrence post-prostatectomy in patients with National Comprehensive Cancer Network® (NCCN) intermediate and higher risk prostate cancer. The 141 patients included in the study were from the NorthShore University HealthSystem diagnosed 2014-2019 with NCCN intermediate (n = 109) or higher risk (n = 32) prostate cancer, treated with radical prostatectomy 2015-2019. The association of GPS result with time to biochemical recurrence was evaluated using univariable and multivariable Cox proportional hazards models in 120 patients with unfavorable intermediate or higher risk. Median (interquartile range) follow-up time was 28 (20 to 38) months. The GPS result was significantly associated with time to biochemical recurrence as both a continuous and dichotomous variable in univariable (hazard ratio [HR] per 20 GPS units 2.36, 95% CI 1.45-3.80, p < 0.001; HR for GPS result 41-100 vs 0-40 3.28, 95% CI 1.61-7.19, p < 0.001) and in multivariable models accounting for NCCN risk group (HR per 20 GPS units 2.14, 95% CI 1.31-3.46, p = 0.003; HR for GPS result 41-100 vs 0-40 3.00, 95% CI 1.43-6.72, p = 0.003) or biopsy Gleason Score and diagnostic PSA or PSA density. These results indicate that the GPS assay was a strong predictor of biochemical recurrence after radical prostatectomy in this unfavorable intermediate and higher risk prostate cancer patient population.

Anti-tumor NAMPT inhibitor, KPT-9274, mediates gender-dependent murine anemia and nephrotoxicity by regulating SIRT3-mediated SOD deacetylation.

KPT-9274 is a phase 1 first-in-class dual PAK4/NAMPT inhibitor for solid tumor and non-Hodgkin's lymphoma. It demonstrates pre-clinical efficacy toward a broad spectrum of acute myeloid leukemia (AML) subtypes by inhibiting NAMPT-dependent NAD+ production. NAMPT is the rate-limiting enzyme in the salvage metabolic pathway leading to NAD+ generation. Tumor cells which are deficient in de novo pathway enzyme NAPRT1 are addicted to NAMPT. In clinical trials, treatment with NAMPT inhibitors resulted in dose-limiting toxicities. In order to dissect the mechanism of toxicity, mice were treated with KPT-9274 and resulting toxicities were characterized histopathologically and biochemically. KPT-9274 treatment caused gender-dependent stomach and kidney injuries and anemia. Female mice treated with KPT-9274 had EPO deficiency and associated impaired erythropoiesis. KPT-9274 treatment suppressed SIRT3 expression and concomitantly upregulated acetyl-manganese superoxide dismutase (MnSOD) in IMCD3 cells, providing a mechanistic basis for observed kidney toxicity. Importantly, niacin supplementation mitigated KPT-9274-caused kidney injury and EPO deficiency without affecting its efficacy. Altogether, our study delineated the mechanism of KPT-9274-mediated toxicity and sheds light onto developing strategies to improve the tolerability of this important anti-AML inhibitor.

Age of menopause and fracture risk in postmenopausal women randomized to calcium + vitamin D, hormone therapy, or the combination: results from the Women's Health Initiative Clinical Trials.

OBJECTIVE: We previously reported that in the absence of hormone therapy (HT) or calcium/vitamin D (Ca/D) supplementation, earlier menopause age was associated with decreased bone mineral density and increased fracture risk in healthy postmenopausal women. Treatment with HT and Ca/D is protective against fractures after menopause. In this analysis, we asked if the age of menopause onset alters fracture risk in healthy postmenopausal women receiving HT, Ca/D, or a combination. METHODS: Hazard ratios (HRs) for any fracture among 21,711 healthy postmenopausal women enrolled in the Women's Health Initiative Clinical Trial, who were treated with HT, Ca/D, or HT + Ca/D, and who reported age of nonsurgical menopause of <40, 40 to 49, and ≥50 years, were compared. RESULTS: Women with menopause <40 years had significantly higher HR for fracture than women with menopause 40 to 49 or ≥50 years, regardless of treatment intervention (HR [95% CI]: menopause <40 y vs ≥50 y, 1.36 [1.11-1.67]; menopause <40 y vs 40-49 y, 1.30 [1.06-1.60]). CONCLUSIONS: In the overall Women's Health Initiative Clinical Trial cohort and within each treatment group, women with younger menopause age (<40 y) had a higher risk of any fracture than women reporting older menopause ages. The effect of menopause age on fracture risk was not altered by any of the treatment interventions (HT, Ca/D, HT + Ca/D), suggesting that early age of menopause is an independent contributor to postmenopausal fracture risk.

Ibrutinib treatment ameliorates murine chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell-driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell-mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials.

The proteasome inhibitor carfilzomib functions independently of p53 to induce cytotoxicity and an atypical NF-κB response in chronic lymphocytic leukemia cells.

PURPOSE: The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2, and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells. EXPERIMENTAL DESIGN: Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction, and electrophoretic mobility shift assays. In addition, a p53 dominant-negative construct was generated in a human B-cell line. RESULTS: Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells versus CLL. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IκBα, phosphorylation of IκBα, and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription. CONCLUSIONS: Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support phase I investigation of CFZ in this disease.

Potentiating effect of the flavonolignan (-)-hydnocarpin in combination with vincristine in a sensitive and P-gp-expressing acute lymphoblastic leukemia cell line.

The potentiating action of the flavonolignan, (-)-hydnocarpin, in combination with vincristine was evaluated in the 697 acute lymphoblastic leukemia cell line and a P-gp-expressing variant, 697-R. Vincristine at 3 nM caused nearly complete growth inhibition in 697 cells versus a 17% growth inhibition in 697-R cells. When combined with (-)-hydnocarpin at concentrations of 10 and 5 µM, vincristine-mediated growth inhibition in the 697-R cells increased significantly over the sum of the individual agents to 72% (p ≤ 0.0001) and 41% (p = 0.0256), respectively. Vincristine at 1.5 nM (66% growth inhibition) and 0.75 nM (39% growth inhibition) combined with (-)-hydnocarpin at 10 µM (42% growth inhibition) in the 697 cells caused a significant increase in growth inhibition to 83% (p = 0.03) and to 61% (p < 0.0001), respectively, when compared to vincristine treatment as a single agent. To investigate the mechanism for the vincristine re-sensitization caused by (-)-hydnocarpin, the P-gp inhibitory effect of (-)-hydnocarpin was evaluated.

The inhibition of early stages of HER-2/neu-mediated mammary carcinogenesis by dietary n-3 PUFAs.

SCOPE: We previously demonstrated that lifelong feeding of diets enriched in n-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) significantly inhibits HER-2/neu-mediated mammary tumorigenesis in mice. Of interest is whether dietary n-3 fatty acids exert effects at early stages of mammary carcinogenesis. METHODS AND RESULTS: Seven-week-old female MMTV-HER-2/neu transgenic mice were randomized to AIN-based semipurified diets containing either fish or corn oil at 25% energy. Mice were evaluated at 25, 30, and 35 weeks with analysis of mammary glands for atypical ductal hyperplasia (hematoxylin and eosin), cell proliferation (Ki67 immunostaining), and fatty acid synthase and cyclooxygenase-2 gene expression (qRT-PCR). Tissue fatty acid profiles were quantitated by GC. Atypia grade decreased significantly in mice fed fish oil (p = 0.002). Mammary epithelial cells in mammary glands from mice fed fish oil also had an eightfold lower percentage of Ki67 expression. COX-2 expression in mammary fat-pads significantly decreased in mice fed fish versus corn oil enriched diets. CONCLUSION: Dietary fish oil inhibits atypical ductal hyperplasia at early stages of HER-2/neu-mediated mammary carcinogenesis relative to corn oil diets. This histologic change is associated with suppression of mammary epithelial cell proliferation and decreased COX-2 expression in mammary tissue.

The novel deacetylase inhibitor AR-42 demonstrates pre-clinical activity in B-cell malignancies in vitro and in vivo.

BACKGROUND: While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies. PRINCIPAL FINDINGS: In mantle cell lymphoma (JeKo-1), Burkitt's lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC(50) (50% growth inhibitory concentration) of AR-42 is 0.61 microM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC(50) (concentration lethal to 50%) of AR-42 is 0.76 microM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity. CONCLUSIONS/SIGNIFICANCE: Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.

Omega-3 fatty acid supplements in women at high risk of breast cancer have dose-dependent effects on breast adipose tissue fatty acid composition.

BACKGROUND: Preclinical evidence of the preventive benefits of omega-3 (n-3) polyunsaturated fatty acids (PUFAs) in breast cancer continues to fuel interest in the potential role of dietary fat content in reducing breast cancer risk. The dose of fish-oil/omega-3 PUFAs needed to achieve maximal target tissue effects for breast cancer prevention remains undefined. OBJECTIVE: To determine the dose effects of omega-3 fatty acids on breast adipose tissue fatty acid profiles, we conducted a study of 4 doses of omega-3 PUFAs in women at high risk of breast cancer. DESIGN: In this 6-mo randomized open-label study, 48 women with increased breast cancer risk received 1, 3, 6, or 9 capsules/d of an omega-3 PUFA supplement that provided 0.84, 2.52, 5.04, and 7.56 g docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) daily, respectively. Subjects made monthly visits, at which time pill counts were made and fasting blood samples were collected to determine fatty acid profiles; anthropometric measurements were made, breast adipose tissue samples were collected, and laboratory tests of toxicity (alanine aminotransferase, LDL cholesterol, and platelet function) were made at baseline and at 3 and 6 mo. RESULTS: All doses led to increased serum and breast adipose tissue EPA and DHA concentrations, but the response to 0.84 g DHA+EPA/d was less than the maximum possible response with > or = 2.52 g/d. Body mass index attenuated the dose response for serum tissue DHA and EPA (P = 0.015 and 0.027, respectively) and breast adipose tissue DHA (P = 0.0022) in all of the treatment groups. The incremental increase in DHA and EPA correlated inversely with baseline fat and serum values. Compliance over 6 mo was 92.9 +/- 9.2% and was unaffected by treatment arm. No severe or serious toxicities were reported. CONCLUSIONS: Daily doses up to 7.56 g DHA+EPA were well tolerated with excellent compliance in this cohort at high risk of breast cancer. Body mass index and baseline fatty acid concentrations modulated the dose-response effects of omega-3 PUFA supplements on serum EPA and DHA and breast adipose tissue DHA.