1-alpha,25-Dihydroxyvitamin D3 regulates inducible nitric oxide synthase messenger RNA expression and nitric oxide release in macrophage-like RAW 264.7 cells

The expression of inducible nitric oxide synthase (iNOS) expression and release of nitric oxide (NO) from macrophages are markedly increased in granulomatous infections. Activation of macrophages 1alpha-hydroxylase results in an increase of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, the significance of this increased production is not completely understood. In this study, we analyzed 1,25(OH)(2)D(3) and NO production in patients with tuberculosis infection and hypercalcemia and used lipopolysaccharide (LPS) to stimulate RAW 264.7 cells in an attempt to assess iNOS expression and gaseous NO production regulated by 1,25(OH)(2)D(3). Peroxynitrite (OONO(-)) production and lactate dehydrogenase activity were also examined. Without additional stimulation, peripheral-blood mononuclear cells (PBMCs) from patients with tuberculosis converted more 25-hydroxyvitamin D(3) to 1,25(OH)(2)D(3) than did those from normal controls. These PBMCs released less NO than did those from control subjects, at baseline and in the stimulated state. We found that 1,25(OH)(2)D(3) dose-dependently inhibited iNOS messenger RNA expression of the LPS-stimulated RAW 264.7 cells and also significantly reduced the gaseous NO release and OONO(-) production. Paralleling the 1,25(OH)(2)D(3)-induced inhibition of NO release were reductions in OONO(-) and LDH production. In conclusion, 1,25(OH)(2)D(3) inhibited iNOS expression and reduced NO production by LPS-stimulated macrophages in the range of physiological doses. Inhibition of the NO surge was coupled with a reduction in OONO(-) and LDH production. Increased 1,25(OH)(2)D(3) production and decreased release of NO from the PBMCs of patients with tuberculosis and hypercalcemia were also noted. We propose that 1,25(OH)(2)D(3) production by macrophages may protect themselves against oxidative injuries caused by the NO burst. In the case of tuberculosis infection, increased 1,25(OH)(2)D(3) synthesis may further contribute to the development of an unwanted phenomenon-hypercalcemia.     

Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia- and serum-induced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells

Green tea extract and its major component (-)-epigallocatechin-3-gallate (EGCG) exhibit antiangiogenic activities in various experimental tumor models. A growing body of evidence has established that hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumor angiogenesis. In this study, we investigated the effect of green tea extract and EGCG on HIF-1alpha and VEGF expression in human cervical carcinoma (HeLa) and hepatoma (HepG2) cells. Our results showed that green tea extract and EGCG significantly inhibited hypoxia- and serum-induced HIF-1alpha protein accumulation in these cancer cells but had no effects on HIF-1alpha mRNA expression. Suppression of HIF-1alpha protein by green tea extract and EGCG also resulted in a drastic decrease in VEGF expression at both mRNA and protein levels. The mechanisms of green tea extract and EGCG inhibition of hypoxia-induced HIF-1alpha protein accumulation seem to involve the blocking of both phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling pathways and the enhancing of HIF-1alpha protein degradation through the proteasome system. In addition, green tea extract and EGCG inhibited serum-induced HIF-1alpha protein and VEGF expression by interfering with the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathways, which play a crucial role in the protein translational machinery cascade. Functionally, green tea extract and EGCG abolished both chemoattractant- and hypoxia-stimulated HeLa cell migration. Our data suggested that HIF-1alpha/VEGF function as therapeutic target for green tea extract and EGCG in the context of cancer chemoprevention and anticancer therapy.     

Inhibition of hypoxia-inducible factor-1alpha in RPMI8226 myeloma cells results in reduced tumor growth in nude mice

OBJECTIVE: To explore the influence of inhibition of hypoxia-inducible factor-1alpha (HIF-1alpha) by RNA interference (RNAi) on tumorigenesis of human myeloma cell line (HMCL) RPMI8226 cells in nude mice. METHOD: RNAi vector of HIF-1alpha was constructed with commercial shRNA expression vector pSilencer 2. 1-U6 hygro. RT-PCR and western blot were used to detect HIF-1alpha mRNA and protein expression respectively. Vascular endothelial growth factor (VEGF) secretion and cell cycle changes were detected by ELISA and flow cytometry respectively. Expression of target gene of HIF-1alpha, VEGF and Glut-1 were tested under hypoxia condition. Tumorigenesis was observed after transfected cells were injected subcutaneously in nude mice. RESULTS: After interference, expression of HIF-1alpha decreased significantly at both mRNA and protein level. Under normoxia condition, VEGF concentrations in HIF-la inhibited cells (RPMI8226-il and RPMI8226-i2) and non-inhibited cells (RPMI8226-c and RPMI8226) showed no differences. While under hypoxia condition, VEGF concentration in the above four cells was (506.0 +/- 53.2), (494.7 +/- 63.1), (984.4 +/- 61.9) and (938.2 +/- 62.2) pg/ml, respectively, being significantly lower in RPMI8226-il and RPMI8226-i2 cells than in RPMI8226-c and RPMI8226 cells (P <0.05). HIF-1alpha interference was found to suppress the cells shift from S-phase to G1 induced by hypoxia. VEGF and Glut-1 expressions were markedly attenuated (P <0.05). The growth rate of HIF-1alpha inhibition tumors in subcutaneous xenograft model decreased drastically. CONCLUSIONS: RNAi inhibits HIF-1alpha expression. Reduced tumor growth by HIF-1alpha inhibition may partly through inhibiton of angiogenesis and glycolysis metabolism.     

Suppression of RelB-mediated manganese superoxide dismutase expression reveals a primary mechanism for radiosensitization effect of 1alpha,25-dihydroxyvitamin D(3) in prostate cancer cells

Nuclear factor-kappaB provides an adaptive response to protect cancer cells against cytotoxicity induced by redox active therapeutics. RelB is uniquely expressed at a high level in prostate cancer with high Gleason scores. Recently, we showed that the level of RelB rapidly increases in androgen-independent prostate cancer cells after exposure to ionizing radiation (IR), leading to a reduction in intrinsic radiosensitivity. Here, we show that interaction of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] with the vitamin D receptor significantly enhances radiosensitivity of prostate cancer cells at clinically relevant radiation doses. The radiosensitization effect of 1alpha,25-(OH)(2)D(3) is mediated, at least in part, by selectively suppressing IR-mediated RelB activation, leading to a reduced expression of its target gene MnSOD, a primary antioxidant enzyme in mitochondria. These results suggest that suppression of manganese superoxide dismutase is a mechanism by which 1alpha,25-(OH)(2)D(3) exerts its radiosensitization effect and that 1alpha,25-(OH)(2)D(3) may serve as an effective pharmacologic agent for selectively sensitizing prostate cancer cells to IR via suppression of antioxidant responses in mitochondria.     

Size at birth, adult intestinal calcium absorption and 1,25(OH)(2) vitamin D

BACKGROUND: Adult bone mineral status is modified by early environmental influences, but the mechanism of this phenomenon is unknown. Intestinal calcium absorption and vitamin D metabolism are integrally involved in bone metabolism and may be programmed during early life. AIM: To examine the early-life influences on calcium absorption and its control in 322 post-menopausal female twins. METHODS: Intestinal calcium absorption was assessed by the stable strontium (Sr) method. Serum PTH, 25(OH) and 1,25(OH)(2) vitamin D were measured and recalled birth weight recorded. RESULTS: Fractional intestinal Sr absorption (alpha Sr) was correlated with serum 1,25(OH)(2) vitamin D (p<0.001), but not with 25(OH) vitamin D. Birth weight was inversely associated with serum 1,25(OH)(2) vitamin D (p=0.04), the association being independent of serum calcium, phosphate, creatinine and PTH. Birth weight was inversely correlated with alpha Sr (p=0.03), this association being independent of age, season, customary calcium intake and serum 25(OH) vitamin D; however, when serum 1,25(OH)(2) vitamin D was added into the model, the association became non-significant, suggesting that the association was partially mediated via serum 1,25(OH)(2) vitamin D. DISCUSSION: We found a significant inverse association between birth weight and intestinal calcium absorption that is partially explained by an association between serum 1,25(OH)(2) vitamin D and birth weight. This suggests a mechanism whereby the intra-uterine environment might affect adult skeletal status.     

Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion

Hypoxia-induced stress plays a central role in retinal vascular disease and cancer. Increased hypoxia-inducible factor-1 alpha (Hif-1 alpha) expression leads to HIF-1 formation and the production of vascular endothelial growth factor (VEGF). Cytokines, including insulin-like growth factor-1 (IGF-1), also stimulate VEGF secretion. In this study, we examined the relationship between IGF-1 signaling, HIF-1 alpha protein turnover and VEGF secretion in the ARPE-19 retinal pigment epithelial cell line. Northern analysis revealed that IGF-1 stimulated Hif-1 alpha message expression, whereas the hypoxia-mimetic CoCl2 did not. CoCl2 treatment increased Hif-1 alpha protein accumulation to a greater extent than IGF-1 treatment. However, IGF-1 stimulated a more significant increase in VEGF secretion. IGF-1-stimulated VEGF promoter activity was phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin)-dependent, whereas VEGF secretion was only partially reduced by inhibition of PI3K/Akt/mTOR and HIF-1 activities. Analysis of VEGF promoter truncation mutants indicated that sensitivity to CoCl2 was hypoxia response element (HRE)-dependent with the region upstream of the HRE conferring IGF-1 sensitivity. In conclusion, IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and -independent pathways.     

Fasudil-induced hypoxia-inducible factor-1alpha degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)-dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1alpha induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1alpha expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1alpha protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1alpha mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1alpha expression without altering HIF-1alpha mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1alpha expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1alpha expression is through preventing HIF-1alpha degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1alpha signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1alpha signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis.     

Comparison of effects of 1 alpha-hydroxy-vitamin D3 and 1,25-dihydroxy-vitamin D3 in man.

The effects of short-term treatment with 1,25-dihydroxy-vitamin D3 [1,25(0H)2D3] or 1 alpha-hydroxy-vitamin D3 [1 alpha(OH)D3] on intestinal absorption of 47Ca were compared in 41 experiments in normals and 72 experiments in patients with chronic renal failure. 11 patients were studied a second time after treatment for 2-5 mo. Doses varied from 0.14 to 5.4 mug/day to establish dose-response relationships. Urinary calcium was monitored in normal subjects, nine of whom received a constant calcium intake on a metabolic unit. There was an increase in intestinal absorption of 47Ca and urinary calcium in normals receiving 1,25 (OH)2D3, 0.14 mug/day or greater, and 0.28 mug/day or greater augmented intestinal absorption of 47Ca in chronic renal failure. In contrast, 2.6 mug/day of 1 alpha (OH) D3 was required to increase intestinal absorption of 47Ca in both groups. The increase in urinary calcium to maximal levels was delayed during treatment with 1 alpha (OH) D3, 5-10 days vs. 2-5 days with 1,25 (OH)2D3. Moreover, half times for urinary calcium to decrease to pretreatment levels after stopping treatment were greater after 1 alpha-(OH) D3 (1.5-2.7 days) than 1,25(OH)2D3 (1.1-2.0 days). With long-term administration there was a progressive increase in intestinal absorption of 47Ca in the patients receiving 1 alpha (OH)D3; this was not observed with 1,25(OH)2D3. The pharmacologic differences between 1 alpha(OH) D3 and 1,25(OH)2D3 may be explained by the requirement for 25-hydroxylation of 1alpha(OH) D3 before biologic effects occur; at low doses (less than 1 mug/day), 1 alpha(OH) D3 competes with vitamin D3 for 25-hydroxylation. With prolonged treatment or larger doses (greater than 2 mug/day),, 1alpha(OH) D3 could accumulate and then be hydroxylated resulting in production of higher levels of 1,25(OH)2D3.     

Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1alpha

We have reported previously that PX-478 (S-2-amino-3-[4'-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1alpha (HIF-1alpha) within the tumor. We now report that PX-478 inhibits HIF-1alpha protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1alpha inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1alpha mRNA and inhibits translation as determined by 35S labeling experiments and reporter assays using the 5' untranslated region of HIF-1alpha. Moreover, to a lesser extent, PX-478 also inhibits HIF-1alpha deubiquitination resulting in increased levels of polyubiquitinated HIF-1alpha. The inhibitory effect of PX-478 on HIF-1alpha levels is primarily due to its inhibition of translation because HIF-1alpha translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1alpha at multiple levels that together or individually may contribute to its antitumor activity against HIF-1alpha-expressing tumors.     

Up-regulation of stanniocalcin 1 expression by 1,25-dihydroxy vitamin D(3) and parathyroid hormone in renal proximal tubular cells

Stanniocalcin 1 and stanniocalcin 2 are two glycoprotein hormones, which act as calcium phosphate-regulating factor on intestine and kidney. We have previously reported that stanniocalcin 2 expression is positively and negatively controlled by 1,25(OH)(2)D(3) and parathyroid hormone in renal proximal tubular cells. However, it has been unclear whether they regulate the stanniocalcin 1 gene expression. In this study, we identified the opossum stanniocalcin 1 cDNA sequence. The opossum stanniocalcin 1 amino acid sequence had 83% homology with human stanniocalcin 1, and has a conserved putative N-linked glycosylation site. Real-time PCR analysis using opossum kidney proximal tubular (OK-P) cells revealed that the mRNA levels of stanniocalcin 1 gene is up-regulated by both 1,25(OH)(2)D(3) and parathyroid hormone in dose-dependent and time-dependent manners. We also demonstrated that the stanniocalcin 1 expression was increased in parathyroid hormone injected rat kidney. Furthermore, the mRNA expression of stanniocalcin 1 and stanniocalcin 2 were oppositely regulated by phorbol 12,13-myristic acetate, a specific PKC activator. Interestingly, the up-regulation of stanniocalcin 1 gene by 1,25(OH)(2)D(3) and phorbol 12,13-myristic acetate were not prevented in the presence of actinomycin D, an RNA synthesis inhibitor. These results suggest that the stanniocalcin 1 gene expression is up-regulated by 1,25(OH)(2)D(3) and parathyroid hormone through mRNA stabilization in renal proximal tubular cells.     

Human alpha1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by alpha1beta1 integrin

Human noncollagenous domain 1 of the alpha1 chain of type IV collagen [alpha1(IV)NC1], or arresten, is derived from the carboxy terminal of type IV collagen. It was shown to inhibit angiogenesis and tumor growth in vivo; however, the mechanisms involved are not known. In the present study we demonstrate that human alpha1(IV)NC1 binds to alpha1beta1 integrin, competes with type IV collagen binding to alpha1beta1 integrin, and inhibits migration, proliferation, and tube formation by ECs. Also, alpha1(IV)NC1 pretreatment inhibited FAK/c-Raf/MEK/ERK1/2/p38 MAPK activation in ECs but had no effect on the PI3K/Akt pathway. In contrast, alpha1(IV)NC1 did not affect proliferation, migration, or the activation of FAK/c-Raf/MEK1/2/p38/ERK1 MAPK pathway in alpha1 integrin receptor knockout ECs. Consistent with this, alpha1(IV)NC1 elicited significant antiangiogenic effects and tumor growth inhibition in vivo but failed to do the same in alpha1 integrin receptor knockout mice. This suggests a highly specific, alpha1beta1 integrin-dependent antiangiogenic activity of alpha1(IV)NC1. In addition, alpha1(IV)NC1 inhibited hypoxia-induced expression of hypoxia-inducible factor 1alpha and VEGF in ECs cultured on type IV collagen by inhibiting ERK1/2 and p38 activation. This unravels a hitherto unknown function of human alpha1(IV)NC1 and suggests a critical role for integrins in hypoxia and hypoxia-induced angiogenesis. Collectively, the above data indicate that alpha1(IV)NC1 is a potential therapeutic candidate for targeting tumor angiogenesis.