Pharmacogenomics and chemical library screens reveal a novel SCFSKP2 inhibitor that overcomes Bortezomib resistance in multiple myeloma.

While clinical benefit of the proteasome inhibitor (PI) bortezomib (BTZ) for multiple myeloma (MM) patients remains unchallenged, dose-limiting toxicities and drug resistance limit the long-term utility. The E3 ubiquitin ligase Skp1-Cullin-1-Skp2 (SCFSkp2) promotes proteasomal degradation of the cell cycle inhibitor p27 to enhance tumor growth. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance. SCFSkp2 activity is increased by the Cullin-1-binding protein Commd1 and the Skp2-binding protein Cks1B. Here we observed higher CUL1, COMMD1 and SKP2 mRNA levels in CD138+ cells isolated from BTZ-resistant MM patients. Higher CUL1, COMMD1, SKP2 and CKS1B mRNA levels in patient CD138+ cells correlated with decreased progression-free and overall survival. Genetic knockdown of CUL1, COMMD1 or SKP2 disrupted the SCFSkp2 complex, stabilized p27 and increased the number of annexin-V-positive cells after BTZ treatment. Chemical library screens identified a novel compound, designated DT204, that reduced Skp2 binding to Cullin-1 and Commd1, and synergistically enhanced BTZ-induced apoptosis. DT204 co-treatment with BTZ overcame drug resistance and reduced the in vivo growth of myeloma tumors in murine models with survival benefit. Taken together, the results provide proof of concept for rationally designed drug combinations that incorporate SCFSkp2 inhibitors to treat BTZ resistant disease.

Rational combination treatment with histone deacetylase inhibitors and immunomodulatory drugs in multiple myeloma.

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.

Class IIa HDAC inhibition enhances ER stress-mediated cell death in multiple myeloma.

Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biological role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized that HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming that ATF4 has a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressors, including proteasome inhibitors, to improve patient outcome in MM.

Histone deacetylase 3 as a novel therapeutic target in multiple myeloma.

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.

Small-molecule multi-targeted kinase inhibitor RGB-286638 triggers P53-dependent and -independent anti-multiple myeloma activity through inhibition of transcriptional CDKs.

Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

Neonatal programming of body weight regulation and energetic metabolism.

Programming is an epigenetic phenomena by which nutritional, hormonal, physical psychological and other stressful events acting in a critical period of life, such as gestation and lactation, modifies in a prolonged way certain physiological functions. This process was preserved by natural selection as an important adaptive tool for survival of organisms living in nutritional impaired areas. So, malnutrition during gestation and lactation turns on different genes that provide the organism with a thrifty phenotype. In the case of an abundant supply of nutrients after this period, those organisms that were adapted to a low metabolic waste and higher energy utilization will be in a higher risk of developing metabolic diseases, such as obesity, hyperlipidemia, diabetes mellitus and hypertension. The kind of malnutrition, duration and intensity are important for the type of programming obtained. We discuss some of the hormonal and metabolic changes that occur in gestation or lactation, when malnutrition is applied to the mothers and their offspring. Some of these changes, such as an increase of maternal triiodothyronine (T(3)), leptin and glucocorticoids (GC) and decrease in prolactin are by itself potential programming factors. Most of these hormones can be transfer through the milk that has other important macronutrients composition changes in malnourished dams. We discuss the programming effects of some of these hormones upon body weight and composition, leptin, thyroid and adrenal functions, and their effects on liver, muscle and adipose tissue metabolism and the consequences on thermogenesis.

Malnutrition during lactation in rats is associated with higher expression of leptin receptor in the pituitary of adult offspring.

OBJECTIVE: Recent studies have shown that leptin receptor is expressed in human and rat pituitary glands. The expression of leptin receptor in rats whose dams were malnourished during lactation has not been previously reported. METHODS: We examined leptin receptor expression in the pituitary gland of adult rats whose dams were assigned to one of the following groups during lactation: control diet, protein-restricted diet (8% protein), or energy-restricted diet (the control diet fed in restricted quantities that were calculated according to the mean ingestion of the protein-restricted group). After weaning, all pups had free access to the control diet until they reached adult age, at which time leptin receptor expression in the pituitary was analyzed by immunohistochemistry. RESULTS: Adult animals from protein- and energy-restricted dams had a higher expression of leptin receptor in pituitary tissue, normal serum leptin concentrations, higher serum tri-iodothyronine concentrations, and lower thyroid-stimulating hormone concentrations than did the control rats. CONCLUSIONS: In the fed state, leptin has a stimulatory effect on release of thyroid-stimulating hormone. The higher expression of leptin receptor in the pituitary of animals from protein- and energy-restricted dams may suggest a postreceptor failure in leptin action. This higher receptor expression may have allowed a greater inhibition of release of thyroid-stimulating hormone.

[Hemorrhoidectomy with ropivacaine (Naropin) local anesthesia. Preliminary experience]. / L'emorroidectomia in anestesia locale con ropivacaina (Naropina). Esperienza preliminare.

BACKGROUND: The use of local anesthesia in non-septic anal surgery is now well established. Tolerance to local injection, duration of local effect and the risk of local or systemic complications still represent unsolved issues. Ropivacaine, a new local anesthetic, seems particularly indicated for this kind of surgery because of its pharmacologic properties which reduce patient's discomfort during infiltration and provide good antalgic coverage in the first hours following the operation. METHODS: The first 20 consecutive cases operated with local anesthesia by ropivacaine have been prospectively studied. All patients have been given an 11-point box VAS scale which is used for subjective evaluation of pain. RESULTS: Mean pain score resulted 1.1, 1.6 and 1.4 at 1, 2 and 3 postoperative hours, respectively. Thirty percent of patients subsequently required pain medication up to the first bowel movement. No complications related to the use of ropivacaine has been observed. CONCLUSIONS: This new drug can be safely used in the outpatient or Day-Surgery treatment of hemorrhoids.

Risk factors for acute renal failure in trauma patients.

OBJECTIVE: To elucidate the risk factors for the development of acute renal failure (ARF) in severe trauma. DESIGN: Prospective observational study. SETTING: A general intensive care unit (ICU) of a university hospital. PATIENTS: A cohort of 153 consecutive trauma patients admitted to the ICU over a period of 30 months. RESULTS: Forty-eight (31%) patients developed ARF. They were older than the 105 patients without ARF (p = 0.002), had a higher Injury Severity Score (ISS) (p < 0.001), higher mortality (p < 0.001), a more compromised neurological condition (p = 0.007), and their arterial pressure at study entry was lower (p = 0.0015). In the univariate analysis, the risk of ARF increased by age, ISS > 17, the presence of hemoperitoneum, shock, hypotension, or bone fractures, rhabdomyolysis with creatine phosphokinase (CPK) > 10000 IU/l, presence of acute lung injury requiring mechanical ventilation, and Glasgow Coma Score < 10. Sepsis and use of nephrotoxic agents were not associated with an increased risk of ARF. In the logistic model, the need for mechanical ventilation with a positive end-expiratory pressure > 6 cm H2O, rhabdomyolysis with CPK > 10000 IU/l, and hemoperitoneum were the three conditions most strongly associated with ARF. CONCLUSIONS: The identified risk factors for post-traumatic acute renal failure may help the provision of future strategies.

Effects of hyperbarism on central respiratory drive and respiratory pattern in humans.

The aim of our study was to evaluate the effects of increasing pressure (from 1 to 3 and 6 atm abs) on respiratory drive, respiratory pattern, and inspiratory impedance of the respiratory system. Seven healthy volunteers were studied during a dry compression to 6 atm abs in a hyperbaric multiplace chamber. We observed a significant increase in tidal volume, P0.1 (the pressure generated in the airway after 100 ms of inspiration against a closed inspiratory line), Ttot, and Ti, and a significant respiratory rate reduction with increasing pressure from 1 to 3 atm.abs; P0.1 also increased significantly when comparing 3 and 6 atm abs measurement with 1 atm abs. The P > 0.01 and P0.1/(VT/Ti) showed a significant progressive increase compared with 1 atm abs. In conclusion, the passage from 1 to 3 and 6 atm abs causes, in healthy subjects at rest, an increase in the central respiratory drive activity, evaluated with P0.1 measurement. The response to the respiration system is an increase in tidal volume and Ti with a decrease in respiratory rate.

Urea, creatinine, and glucose determined in plasma and whole blood by a differential pH technique.

We report the conditions (buffer composition and enzyme activity) required for estimating three frequently determined analytes--urea, glucose, and creatinine--by use of an improved version of the differential pH apparatus previously described (Clin Chem 29: 80-85, 1983). For each analyte, we used only one specific enzyme, thus avoiding a chain of auxiliary and indicator reactions. The method requires about a minute for each determination in undiluted plasma or whole blood.

An integrated system for the automation of the clinical chemistry laboratory.

A user programmable microcomputer-based integrated system for automatic analysis in the clinical chemistry laboratory is presented. Friendly user-instrument interaction helps the operator when defining tests and analytical conditions or entering sample data and test requests. Quality control data are retained for the automatic production of various statistical lists and graphs. Routine operator intervention is limited to reagent preparation, sample loading onto the autosampler plate, definition of the tests to be performed on each of the samples and data validation before the generation of reports. Finally, connection with an existing laboratory management system is provided by means of a standard serial interface.