Potent Antiproliferative Action Research Articles

Targeting the IGF-IR system in pituitary tumors in vitro: antiproliferative action & pitfalls

IGF-I receptors (IGF-IR) and their aberrant signaling cascade contribute to the pathogenesis of several solid cancers. Pituitary adenomas express IGF-IR and present with overactivation of the IGF-IR pathway. The aim of the study was to identify the potential antiproliferative action of small molecule inhibitors targeting IGF-IR signaling cascade in pituitary adenomas. The IGF-IR tyrosine kinase inhibitor NVP-TAE226, the PI3K/mTOR inhibitor NVP_BEZ235 and the allosteric mTOR inhibitor everolimus were used on acromegalic (n=11) and nonfunctioning pituitary adenomas (NFPA; n=15) in primary cell culture. Changes in cell viability were determined by thymidine incorporation and a nonradioactive colorimetric assay. TAE226at the 100nM concentration suppressed cell viability by more than 20% in 9 out of 15 NFPA and in 10 acromegalic tumors, while at 10nM it was efficacious in 8 NFPA and 4 acromegalic tumors. Inhibiting the PI3K pathway in the same tumors, using BEZ235 suppressed cell viability at 10nM concentration in all NFPA and in two acromegalic tumors. In contrast, mTOR inhibition with everolimus was efficacious in all but one acromegalic cases, but in only 4 out of 15 NFPA. IGF-IR inhibition in acromegalic tumors in vitro was accompanied by increased GH secretion, which was evidenced when GH got normalized to cell viability assay counts. Similar GH increase was observed after mTOR but not in the same extend after PI3K inhibition (P=0.043), indicating a previously unsuspected role for the mTOR pathway on GH synthesis. Altogether, these data indicate a tumor specific dependency to the different IGF-IR signaling branches, with NFPA being sensitive to PI3K inhibition and acromegalic tumors to the mTOR inhibition. Targeting IGF-IR pathways have a potential as antiproliferative agents for the treatment of NFPA. In contrast their potent antiproliferative action in acromegalic tumors is compromised by the concomitant increase in GH secretion.

Ethyl acetate fraction of Radix rubiae inhibits cell growth and promotes terminal differentiation in cultured human keratinocytes

Ethnopharmacological relevanceIn Chinese medicine practice, Radix rubiae, the dry root of Rubia cordifolia L. is commonly used for the treatment of psoriasis. Aim of the studyPsoriasis is a chronic inflammatory skin disorder characterized by hyperproliferation and aberrant differentiation of epidermal keratinocytes. Our previous studies identified Radix rubiae to have potent antiproliferative action on cultured HaCaT keratinocytes and to induce keratinocyte differentiation in mouse tail model. The present study aimed to investigate whether Radix rubiae could also induce terminal differentiation in cultured human keratinocytes. Methods and resultsThe cornified envelope (CE) formation assay showed that ethyl acetate (EA) fraction of Radix rubiae significantly accentuated the CE formation, a well-recognized marker of terminal differentiation, in cultured HEK and HaCaT cells in a dose and time dependent manner. Western blot analyses demonstrated that EA fraction of Radix rubiae at a concentration of 3.2μg/ml significantly increased transglutaminase type I and involucrin expression in both HEK and HaCaT keratinocytes after 96h treatment, a response similar to that of Ca2+ positive control. Moreover, the expression level of cytokeratin 5/14, which is specifically related to cell proliferation, was significantly downregulated while terminal differentiation markers cytokeratin 1/10 were markedly increased by Radix rubiae treatment in both HEK and HaCaT cells. ConclusionThe present experimental findings unequivocally confirmed the keratinocyte terminal differentiation promoting capacity of Radix rubiae, and strongly suggest that Radix rubiae is a promising antipsoriatic agent warranting further clinical development for psoriasis treatment.

Ethyl acetate fraction of the root of rubia cordifolia L. inhibits keratinocyte proliferation in vitro and promotes keratinocyte differentiation in vivo: potential application for psoriasis treatment

Psoriasis is a skin disease associated with hyperproliferation and aberrant differentiation of keratinocytes. Our previous studies have identified the root of Rubia cordifolia L. as a potent antiproliferative and apoptogenic agent in cultured HaCaT cells (IC(50) 1.4 microg/ml). In the present study, ethanolic extract of Radix Rubiae was fractioned sequentially with hexane, ethyl acetate (EA), n-butanol and water. EA fraction was found to possess most potent antiproliferative action on HaCaT cells (IC(50) 0.9 microg/ml). Mechanistic study revealed that EA fraction induced apoptosis on HaCaT cells, as it was capable of inducing apoptotic morphological changes. Annexin V-PI staining assay also demonstrated that EA fraction significantly augmented HaCaT apoptosis. In addition, EA fraction decreased mitochondrial membrane potential in a concentration- and time-dependent manner. The standardized EA fraction was formulated into topical gel and its keratinocyte-modulating action was tested on mouse tail model. EA fraction dose-dependently increased the number and thickness of granular layer and epidermal thickness on mouse tail skin, indicative of the keratinocyte differentiation-inducing activity. Taking the in vitro and in vivo findings together, the present preclinical study confirms that EA fraction is a promising antipsoriatic agent warranting further development for psoriasis treatment.

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Δ 12,14-prostaglandin J2

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 μM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPγS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.

Chronic allograft nephropathy – a clinical syndrome: early detection and the potential role of proliferation signal inhibitors

Chronic allograft nephropathy (CAN) leads to the majority of late graft loss following renal transplantation. Detection of CAN is often too late to permit early intervention and successful management. Most current strategies for managing CAN rely on minimizing or eliminating calcineurin inhibitors (CNIs) once CAN has become established. The proliferation signal inhibitors everolimus and sirolimus have potent immunosuppressive and antiproliferative actions, with the potential to alter the natural history of CAN by reducing CNI exposure whilst avoiding acute rejection. Whilst data will be forthcoming from a number of clinical trials investigating this potential, we discuss early detection of CAN and the rationale for a role for this class of agent.

Realgar-mediated growth inhibition on HaCaT human keratinocytes is associated with induction of apoptosis

Traditional Chinese medicine has long been used to treat a variety of ailments including skin diseases. Our previous study has revealed the ethanolic extract of realgar, a common ingredient used in psoriasis treatment in Chinese medicine, to possess potent anti-proliferative action on cultured HaCaT cells of human keratinocyte origin. In the present study, the mechanisms of action of the observed growth inhibitory action of realgar were investigated. Several bioassay methods were employed to elucidate whether cellular apoptosis is involved in the realgar-induced growth inhibition of the skin cells. Morphologically, nuclear condensation and DNA fragmentation were observed when HaCaT cells were exposed to the realgar extract. DNA fragmentation induced by the treatment of realgar was also evident as detected by gel electrophoresis and the TUNEL method. Cell cycle analysis by propidium iodide (PI) staining demonstrated the appearance of sub-G1 peak and cell cycle arrest at the G1 phase upon realgar treatment. Quantitative analysis by annexin V-PI staining revealed that the realgar-induced apoptotic event was dose-dependent. Furthermore, realgar was able to activate caspase-3 expression when examined by Western blot analysis. Our experimental data unambiguously confirm that induction of cellular apoptosis is mainly responsible for the observed growth inhibition brought about by realgar on the HaCaT keratinocytes, and this finding helps place the traditional use of this mineral for psoriasis treatment on a scientific footing.

Arsenic Trioxide, Arsenic Pentoxide, and Arsenic Iodide Inhibit Human Keratinocyte Proliferation through the Induction of Apoptosis

Arsenic compounds have been traditionally used to treat a variety of ailments, including skin diseases. Our previous study identified the extract of realgar to possess potent antiproliferative action on HaCaT cells. The present study aimed at evaluating whether several inorganic arsenics found in realgar also possess similar antiproliferative properties. The results showed that arsenic trioxide, arsenic pentoxide, and arsenic iodide had significant antiproliferative action on HaCaT cells, with IC(50) values at 2.4, 16, and 6.8 microM, respectively. However, these compounds only modestly inhibited the growth of Hs-68 cells, a normal human skin fibroblast cell line, with IC(50) values at 43.4, 223, and 89 microM, respectively, conferring a favorable toxicity profile. In mechanistic studies, all three compounds caused DNA fragmentation as demonstrated by gel electrophoresis and the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling method. Morphologically, nuclear condensation and DNA fragmentation were observed when the cells were exposed to arsenic compounds. Cell cycle analysis with propidium iodide (PI) staining demonstrated the appearance of sub-G(1) peak and cell arrest at the G(1) phase in the presence of these compounds. Quantitative analysis by annexin V-PI staining revealed that the arsenic-induced apoptotic event was dose-dependent. Moreover, the arsenic compounds were able to activate caspase-3 expression when examined by Western blot analysis. Our experimental data unambiguously demonstrated that induction of cellular apoptosis was mainly responsible for the observed antiproliferation brought about by the arsenic compounds on HaCaT keratinocytes, suggesting that these arsenic compounds are putative agents from which psoriasis-treating topical formulae could be developed.

Study of protein targets for covalent modification by the antitumoral and anti‐inflammatory prostaglandin PGA1: focus on vimentin

Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A(1) (PGA(1)) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA(1) has been used as an inhibitor of transcription factor NF-kappaB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA(1) could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA(1) have been identified. In previous work, we have observed that a biotinylated analog of PGA(1) that retains the cyclopentenone moiety (PGA(1)-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA(1)-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA(1) addition. These results may contribute to a better understanding of the mechanism of action of PGA(1) and the potential of cyPG-based therapeutic strategies.

Mechanism and Potential of the Growth-Inhibitory Actions of Vitamin D and Analogs

1alpha,25-Dihydroxyvitamin D(3) [1,25-(OH) (2)D(3)] can exert its biological actions through binding with the nuclear vitamin D receptor (VDR), a ligand-activated transcription factor. Next to control of bone and mineral homeostasis, these actions include an immunomodulatory effect and a potent growth-inhibitory, antiproliferative or prodifferentiating action on a wide variety of cell types. The molecular mechanisms underlying this antiproliferative action form an intriguing research topic and they remain, although thoroughly studied, not completely understood. Important cell cycle regulators are involved such as cyclins, cyclin dependent kinases and their corresponding inhibitors as well as E2F transcription factors and accompanying pocket proteins. Whether 1,25-(OH)(2)D(3) influences the expression of all these proteins directly through the nuclear VDR or rather in an indirect manner is not always clear. The antiproliferative action makes 1,25-(OH) (2)D(3) a possible therapeutic tool to treat hyperproliferative disorders, among which different types of cancer. Clinical application, however, is severely hampered by calcemic effects such as hypercalcemia, hypercalciuria and increased bone resorption. Rational design of chemically modified 1,25-(OH) (2)D(3)-analogs tries to overcome this problem. As such, several thousands of analogs have been synthesized and evaluated, some of which display the desired dissociation between beneficial antiproliferative and unwanted calcemic effects. A number of those analogs are 'superagonistic' and have a several-fold stronger antiproliferative action than the parent compound. This review focuses on recent findings about the complex mechanisms behind the antiproliferative and prodifferentiating effect of 1,25-(OH) (2)D(3). Furthermore, the mode of action and possible clinical application of chemically modified 1,25-(OH) (2)D(3)-analogs will be discussed.

Sirolimus-Associated Proteinuria and Renal Dysfunction

Sirolimus is a novel immunosuppressant with potent antiproliferative actions through its ability to inhibit the raptor-containing mammalian target of rapamycin protein kinase. Sirolimus represents a major therapeutic advance in the prevention of acute renal allograft rejection and chronic allograft nephropathy. Its role in the therapy of glomerulonephritis, autoimmunity, cystic renal diseases and renal cancer is under investigation. Because sirolimus does not share the vasomotor renal adverse effects exhibited by calcineurin inhibitors, it has been designated a 'non-nephrotoxic drug'. However, clinical reports suggest that, under some circumstances, sirolimus is associated with proteinuria and acute renal dysfunction. A common risk factor appears to be presence of pre-existing chronic renal damage. The mechanisms of sirolimus-associated proteinuria are multifactorial and may be due to an increase in glomerular capillary pressure following calcineurin inhibitor withdrawal. It has also been suggested that sirolimus directly causes increased glomerular permeability/injury, but evidence for this mechanism is currently inconclusive. The acute renal dysfunction associated with sirolimus (such as in delayed graft function) may be due to suppression of compensatory renal cell proliferation and survival/repair processes. Although these adverse effects occur in some patients, their occurrence could be minimised by knowledge of the molecular effects of sirolimus on the kidney, the use of sirolimus in appropriate patient populations, close monitoring of proteinuria and renal function, use of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers if proteinuria occurs and withdrawal if needed. Further long-term analysis of renal allograft studies using sirolimus as de novo immunosuppression along with clinical and laboratory studies will refine these issues in the future.

Altered Vitamin D receptor–coactivator interactions reflect superagonism of Vitamin D analogs

The active form of Vitamin D, 1α,25-dihydroxyvitamin D 3 [1,25-(OH) 2D 3], has potent antiproliferative actions on various normal and malignant cells. Calcemic effects, however, hamper therapeutic application of 1,25-(OH) 2D 3 in hyperproliferative diseases. Two 14-epi-analogs of 1,25-(OH) 2D 3 namely 19-nor-14-epi-23-yne-1,25-(OH) 2D 3 (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH) 2D 3 (TX527), display reduced calcemic effects coupled to an (at least 10-fold) increased antiproliferative potency when compared with 1,25-(OH) 2D 3. Altered cofactor recruitment by the Vitamin D receptor (VDR) might underlie the superagonism of these 14-epi-analogs. Therefore, this study aims to evaluate their effects at the level of VDR–coactivator interactions. Mammalian two-hybrid assays with VDR and the coactivators TIF2 and DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR–coactivator interactions than 1,25-(OH) 2D 3. TX522 and TX527 require 30- and 40-fold lower doses to obtain the VDR–DRIP205 interaction induced by 1,25-(OH) 2D 3 at 10 −8 M. Evaluation of additional 1,25-(OH) 2D 3-analogs and their impact on VDR–coactivator interactions revealed a strong correlation between the antiproliferative potency of an analog and its ability to induce VDR–coactivator interactions. In conclusion, these data show that altered coactivator binding by the VDR is one possible explanation for the superagonistic action of the two 14-epi-analogs TX522 and TX527.

Antiproliferative Action of an Angiotensin I-Converting Enzyme Inhibitory Peptide, Val-Tyr, via an L-Type Ca2+ Channel Inhibition in Cultured Vascular Smooth Muscle Cells

Recent antihypertensive studies have demonstrated that small peptides with angiotensin I-converting enzyme (ACE) inhibitory activity had an ability to lower or to modulate a pressor blood pressure response in mild hypertensive subjects. However, the underlying mechanisms still remain unclear. Based on our previous finding that a small peptide, Val-Tyr (VY), was accumulated in the rat aorta and kidney as well as in the circulating blood system, we here investigated whether antihypertensive small peptides exert an antiproliferative effect on serum- or mitogen-induced human vascular smooth muscle cells (VSMCs). Treatment with some ACE inhibitory small peptides (VY, Ile-Trp [IW], and Ile-Val-Tyr [IVY]) had diverse effects on serum-stimulated VSMC proliferation that were independent of their ACE inhibitory activity, though only VY exerted a potent antiproliferative action. VY also showed a greater inhibition of WST-8 incorporation in response to angiotensin (Ang) II-stimulation than the other two small peptides. The attenuation of Ang II-stimulated WST-8 incorporation by VY was not affected by Ang II receptor antagonists (losartan and saralasin ([Sar1, Ile8]-Ang II)), indicating that the antiproliferative action of VY may not be due to the peptide's antagonistic effect against Ang II receptors. Treatment with VY had a significant inhibitory effect on the WST-8 incorporation induced by the stimulation of a voltage-gated L-type Ca2+ channel agonist, Bay K 8644. Even in the presence of a K+ channel blocker (paxillin) the inhibition was apparent, suggesting that VY inhibited the proliferation of VSMCs by serving as a natural L-type Ca2+ channel blocker, but not as a K+ channel agonist.

Expression of somatostatin in the adult and developing mouse kidney

Somatostatin (somatotropin release inhibiting factor) (SRIF) has potent antiproliferative and antisecretory actions. In the adult kidney, somatostatin alters renal blood flow, ion transport, and water permeability. While some evidence suggests that SRIF may be produced by adult kidney tubular cells, the specific tubules generating SRIF are unknown. Somatostatin has also been detected in a variety of embryonic tissues, although it has not been described in the developing kidney. Our objective was to determine the expression pattern of SRIF in both the adult and embryonic mouse kidney. We performed reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence for SRIF in developing and adult mouse kidney tissues. We localized SRIF by dual or serial labeling immunofluorescence with specific markers. Somatostatin mRNA was present in kidneys throughout embryogenesis and into adulthood. Starting at embryonic day (E) 12.5, SRIF was strongly expressed at the interface of the metanephric mesenchymal cells and the basolateral surfaces of ureteric bud trunks. Starting at E16.5, the staining at the interface was confined to the peripheral ureteric bud trunks and the clefts of newly dividing ureteric bud ampullae. In older embryos, SRIF also appeared in medullary tubules that appeared to be maturing thin descending limbs of Henle. In the adult kidney, SRIF proteins localized exclusively to medullary thin descending limbs of the Henle loop. In embryonic kidneys, SRIF is expressed first at the interface of the metanephric mesenchyme and basolateral ureteric bud and later in maturing thin descending limbs of Henle. Expression in the thin descending limb persists in the adult kidney.

Vitamin D and placental-decidual function.

The active form of vitamin D (1,25-dihydroxyvitamin D(3), 1,25[OH](2)D(3)) has well-established effects on bone metabolism and mineral homeostasis. However, recently it has become clear that 1,25(OH)(2)D(3) has potent antiproliferative and immunomodulatory actions that are not immediately linked to its role as a skeletal regulator. Both the nuclear receptor for 1,25(OH)(2)D(3) (vitamin D receptor, VDR) and the vitamin D-activating enzyme 1alpha-hydroxylase are expressed in a wide variety of nonclassic tissues, highlighting the potential for local autocrine-paracrine responses rather than traditional endocrine effects. Prominent among the tissues that express 1alpha-hydroxylase is the placenta-decidua, and this has raised important questions concerning the potential role of locally generated 1,25(OH)(2)D(3) as a modulator of fetal-placental development and function. When bound to the VDR, 1,25(OH)(2)D(3) regulates key target genes associated with implantation, such as HOXA10, whereas the potent immunosuppressive effects of 1,25(OH)(2)D(3) suggest a role in implantation tolerance. These observations are further supported by data from our group showing increased expression of 1alpha-hydroxylase and VDR in first-trimester trophoblast and decidua from human pregnancies. Studies by other groups have reported abnormal expression of 1alpha-hydroxylase in preeclamptic pregnancies, revealing a potential role for 1,25(OH)(2)D(3) as a regulator of placentation. The effect of vitamin D on reproduction has been further endorsed by murine gene knockout models for 1alpha-hydroxylase and VDR, both of which are infertile. These observations and others are discussed in this article in which we postulate an active role for 1,25(OH)(2)D(3) in placenta-decidua. In particular, we describe how induction of the vitamin D-activating enzyme 1alpha-hydroxylase in early gestation might provide a mechanism by which environmental or dietary vitamin D can influence fetal-placental development.

Microarray analysis of 1α,25-dihydroxyvitamin D 3-treated MC3T3-E1 cells

The active form of Vitamin D, 1α,25-dihydroxyvitamin D 3 [1,25-(OH) 2D 3], demonstrates potent antiproliferative actions on normal as well as on malignant cell types by blocking the transition from the G1- to the S-phase of the cell cycle. Key target genes for 1,25-(OH) 2D 3 in this non-classic effect remain largely unknown. Therefore, this study aims to identify genes that, through changes in expression after 1,25-(OH) 2D 3 treatment, contribute to the observed antiproliferative effect. cDNA microarrays containing 4600 genes were used to investigate changes in gene expression in MC3T3-E1 mouse osteoblasts at 6 and at 12 h after treatment with 1,25-(OH) 2D 3 (10 −8 M), preceding (6 h) or coinciding with (12 h) the G1/S block in these cells. Approximately one fifth of the genes that were significantly down-regulated after a 12 h incubation period with 1,25-(OH) 2D 3 were genes involved in the DNA replication process, a basic process for cell growth that starts at the end of G1-phase and continues in S-phase. Down-regulation of these genes by 1,25-(OH) 2D 3 was confirmed by quantitative RT-PCR in MC3T3-E1. In conclusion, cDNA microarrays revealed that treatment of MC3T3-E1 cells with 1,25-(OH) 2D 3 resulted in the down-regulation of DNA replication genes in parallel with the observed G1/S-arrest.

The effects of 1alpha,25-dihydroxyvitamin D3 on the expression of DNA replication genes.

To identify key genes in the antiproliferative action of 1,25(OH)2D3, MC3T3-E1 mouse osteoblasts were subjected to cDNA microarray analyses. Eleven E2F-driven DNA replication genes were downregulated by 1,25(OH)2D3. These results were confirmed by quantitative RT-PCR in different cell types, showing the general nature of this action of 1,25(OH)2D3. 1Alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] has a potent antiproliferative action characterized by a blocked transition from the G1- to the S-phase of the cell cycle. This study aims to identify genes whose expression is markedly altered after 1,25(OH)2D3 treatment in parallel with or preceding the observed G1-arrest. The cDNA microarray technique was used, and the expression of approximately 4600 genes in MC3T3-E1 mouse osteoblasts was studied 6 and 12 h after treatment with 10(-8) M 1,25(OH)2D3. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed on MC3T3-E1 cells and on wildtype and vitamin D receptor (VDR) knockout primary murine epidermal keratinocytes (VDRwt MEKs, VDR-/- MEKs) and murine mammary tumor cells (GR) to confirm the microarray data. After 12 h of treatment, in parallel with the 1,25(OH)2D3-induced G1 arrest, a particular set of DNA replication genes including a cell division cycle 6 homolog, a DNA polymerase alpha subunit, proliferating cell nuclear antigen, two DNA polymerase delta subunits, and flap-structure specific endonuclease 1, was downregulated at least 2-fold. These genes are known targets of the E2F family of transcription factors, which are probably the central mediators of this action of 1,25(OH)2D3. Indeed, as shown by transfection assays with an E2F reporter construct, 12- and 24-h treatment of MC3T3-E1 cells with 1,25(OH)2D3 reduced E2F activity by 49% and 73%, respectively. Quantitative RT-PCR analyses confirmed the downregulation of these DNA replication genes by 1,25(OH)2D3 in MC3T3-E1, GR, and VDRwt MEKs cells, but not in VDR-/- MEKs cells, showing that this 1,25(OH)2D3-driven antiproliferative action is of a general nature and depends on a functional VDR.

A low-calcemic vitamin D analog (Ro 25-4020) inhibits the growth of LNCaP human prostate cancer cells with increased potency by producing an active 24-oxo metabolite (Ro 29-9970).

In this study, we have characterized a novel less-calcemic vitamin D analog Ro 25-4020 (1alpha, 25 dihydroxy-16-ene-5,6-trans-vitamin D3) and investigated the mechanisms underlying its enhanced growth inhibitory properties. We found that Ro 25-4020 (IC50 = 0.3 nM) exhibited greater inhibitory activity than 1,25(OH)2D3 (IC50 = 1 nM) on LNCaP human prostate cancer cell growth. However, Ro 25-4020 was tenfold less active than 1,25(OH)2D3 in receptor-binding assays, ligand-induced heterodimerization and transactivation assays using VDR. HPLC and GC-MS analyses revealed that 1,25(OH)2D3 is converted to a 24-hydroxy metabolite, which has been shown to be less potent than 1,25(OH)2D3. In contrast, Ro 25-4020 was converted to a major 24-oxo metabolite that was more stable. Ligand-binding assays reveal that both Ro 25-4020 and its 24-oxo metabolite have similar affinity for VDR. Synthetic 24-oxo-Ro 25-4020, however, inhibited LNCaP cell proliferation as potently as 1,25(OH)2D3 and was more potent in transactivation of two out of three vitamin D target genes tested. These results suggest that conversion of Ro 25-4020 into an active and more stable 24-oxo metabolite with longer half-life contributes significantly to its potent antiproliferative actions on the LNCaP cells.

Relationship of bursal anti-steroidogenic peptide (BASP) and histone H1

Previous in vitro research from our laboratory has demonstrated the existence of a protein purified from the chicken bursa of Fabricius, with potent antisteroidogenic and antiproliferative action on granulose cells and lymphocytes, respectively called Bursal anti-steroidogenic peptide (BASP). This protein is heat-labile, basic, and amino- and carboxy-terminus blocked. In highly purified form, the protein presents as a doublet on SDS–PAGE electrophoresis with an apparent MW of ∼29 and ∼32 kDa. Recently, Nanoflow Q–TOF Mass Spectrometry amino acid sequencing allowed determination of a convincing partial amino acid sequence, strongly suggesting a probable relationship of BASP with histone H1. Bursal cDNA expression library screening, using an antibody produced against BASP, also identified a clone with a sequence matching histone H1. Presently, we have demonstrated that SDS–PAGE electrophoresis of highly purified and bioactive BASP, and commercially-available calf thymus derived histone H1, produced similar doublets at approximately the same apparent MW, and that the electrophoretic profile of these 2 preparations were strikingly similar following 2 dimensional gel electrophoresis. The BASP doublet produced on SDS–PAGE was recognized by a commercially available monoclonal antibody recognizing a highly conserved region of histone H1. Furthermore, calf thymus histone H1 was found to suppress mitogen-stimulated chicken B-cell proliferation in a concentration-related manner, similar to the action of BASP. These data indicate that BASP shares substantial structural homology with, and may be identical to, histone H1.

Receptorphin: A conserved peptide derived from the sequence of the opioid receptor, with opioid displacement activity and potent antiproliferative actions in tumor cells

BackgroundIn addition to endogenous opioids, a number of peptide sequences, derived from endogenous (hemorphins, alphaS1-casomorphin), and exogenous proteins (casomorphins, exorphins) have been reported, possessing opioid activity. In the present work, we report the identification of a new peptide, receptorphin (Tyr-Ile-Phe-Asn-Leu), derived from the sequence of the second transmembrane loop of the opioid receptor. This sequence is unique for the opioid receptor, and conserved in all species and receptor-types.Results and DiscussionReceptorphin competes for opioid binding, presenting a kappa-receptor interaction, while it binds equally to delta- and mu- opioid and somatostatin-binding sites, and inhibits the cell proliferation of a number of human cancer cell lines, in a dose-dependent and reversible manner, at the picomolar or the nanomolar range. Receptorphin shows a preferential action on prostate cancer cells.ConclusionOur work identifies, for the first time a peptide, in a receptor sequence, possessing ligand-agonistic activities. A hypothesis, based on receptorphin liberation after cell death, is presented, which could tentatively explain the time-lag observed during opioid antiproliferative action.

Somatostatin inhibits follicle-stimulating hormone-induced adenylyl cyclase activity and proliferation in immature porcine Sertoli cell via sst2 receptor.

The potential involvement of somatostatin (SRIF) in testicular function was studied by using as a model primary cultures of purified immature porcine Sertoli cells. In the present report we show that Sertoli cells express mRNA for sst2 SRIF receptor and display SRIF-sensitive adenylyl cyclase. Sensitivity of adenylyl cyclase to SRIF and its analogues is compatible with the pharmacological profile of this receptor type. Relevant cAMP production is similarly inhibited by SRIF in both basal and stimulated (by gonadotropin FSH or by forskolin) conditions. Moreover, the observed SRIF actions on Sertoli cells require functional coupling of specific membrane receptors to adenylyl cyclase via Gi proteins because pertussis toxin prevents SRIF-dependent inhibition of adenylyl cyclase in either basal or FSH-stimulated conditions. Given the potent antiproliferative actions of SRIF in other cell types, we further assessed the possible SRIF-dependent modulation of [(3)H]thymidine incorporation by Sertoli cells. Our data point to SRIF-mediated inhibition of both basal and FSH-stimulated [(3)H]thymidine uptake. This inhibition of Sertoli cell proliferation is, at least in basal conditions, also blocked by pertussis toxin pretreatment. Altogether, these data suggest that SRIF may play a role as an (local) inhibitor of FSH actions in testicular development.