Impact of nitric oxide in liver cancer microenvironment.

The pro- or antitumoral properties of nitric oxide (NO) are dependent on local concentration, redox state, cellular status, duration of exposure and compartmentalization of NO generation. The intricate network of the tumor microenvironment (TME) is constituted by tumor cells, stromal and immune cells surrounded by active components of extracellular matrix that influence the biological behavior and, consequently, the treatment and prognosis of cancer. The review describes critical events in the crosstalk of cellular and stromal components in the TME, with special emphasis in the impact of NO generation in the regulation of hepatocellular carcinoma (HCC). The increased expression of nitric oxide synthase (NOS) in tumors and NO-end products in plasma have been associated with poor prognosis of cancer. We have assessed the level of the different isoforms of NOS in tumors and its relation to cell proliferation and death markers, and cell death receptor expression in tumors, and apoptotic markers and ligands of TNF-α receptor family in blood from a cohort of patients with HCC from different etiologies submitted to orthotopic liver transplantation (OLT). The high levels of NOS2 in tumors were associated with low plasma concentration of apoptotic markers (M30 and M65), FasL and TNF-α in HCV patients. By contrast, the low levels of NOS2 in tumors from alcohol-derived patients was associated with increased Trail-R1 expression in tumors, and circulating Trail levels compared to observed in plasma from HCV- and alcohol + HCV-derived patients. This study reinforces the association between increased NOS2 expression and potential risk of low patients' survival in HCC. However, a differential functional relevance of NOS expression in HCC seems to be influenced by etiologies.

Long non-coding RNA H19 as a biomarker for hepatocellular carcinoma.

BACKGROUND AND AIMS: Liver cancer stem cells (CSCs) could be involved in the carcinogenesis, recurrence, metastasis and chemoresistance of hepatocellular carcinoma (HCC). The aim of this study was to explore the role of lncRNA-H19 as a biomarker for liver cancer. METHODS: LncRNA-H19 expression levels and the functional assays were conducted in EpCAM+ CD133+ CSCs and C57BL/6J mice fed with a high-fat high-cholesterol carbohydrate (HFHCC) or standard diet for 52 weeks. Liver tissue and plasma samples from patients with cirrhosis, with or without HCC, were used for the analyses of gene expression and circulating lncRNA-H19 levels in an estimation and validation cohort. RESULTS: EpCAM+ CD133+ cells showed a stem cell-like phenotype, self-renewal capacity, upregulation of pluripotent gene expression and overexpressed lncRNA-H19 (p < .001). Suppression of lncRNA-H19 by antisense oligonucleotide treatment significantly reduced the self-renewal capacity (p < .001). EpCAM, CD133 and lncRNA-h19 expression increased accordingly with disease progression in HFHCC-fed mice (p < .05) and also in liver tissue from HCC patients (p = .0082). Circulating lncRNA-H19 levels were significantly increased in HCC patients in both cohorts (p = .013; p < .0001). In addition, lncRNA-H19 levels increased accordingly with BCLC staging (p < .0001) and decreased after a partial and complete therapeutic response (p < .05). In addition, patients with cirrhosis who developed HCC during follow-up showed higher lncRNA-H19 levels (p = .0025). CONCLUSION: LncRNA-H19 expression was increased in CSCs, in liver tissue and plasma of patients with HCC and decreased after partial/complete therapeutic response. Those patients who developed HCC during the follow-up showed higher levels of lncRNA-H19. LncRNA-H19 could constitute a new biomarker of HCC.

Validation of a Novel Fecal Incontinence Scale: The Rapid Assessment Fecal Incontinence Score (RAFIS).

OBJECTIVE: The development and validation of a new version of the fecal incontinence (FI) scale "Rapid Assessment Fecal Incontinence Score" (RAFIS) incorporating domains for severity, type of stool loss, and global perception of the effect of incontinence on quality of life (QoL). BACKGROUND: FI negatively impacts on QoL. Currently used incontinence questionnaires have outstanding limitations on the global assessment of the impact of the disease on QoL that patients perceive. We developed a new version of RAFIS with a more complete questionnaire. MATERIALS AND METHODS: A 3-phase study was performed to evaluate the applicability and reliability of our questionnaire as a tool for assessing FI. Our score was completed by 98 patients (78 women; mean age: 57±13 y) who presented with FI and who were referred from 4 colorectal surgery centers. The RAFIS was assessed for internal consistency, test-retest reliability, and sensitivity to change. A multivariate analysis was performed. Comparisons were made with the Wexner Cleveland Clinic Incontinence Score and the Fecal Incontinence Quality of Life Scale. RESULTS: The RAFIS showed good internal consistency and test-retest reliability, differentiating the severity of incontinence but not the etiology. There was a moderate-high correlation between the new scale and the reference scales. Sensitivity to change, compared with the Wexner Score, was moderate. Comparison with established QoL instruments showed a moderate negative correlation. Logistic regression of the RAFIS discriminated between mild and moderate-severe impact on QoL. No correlation was detected with the new score to the presence of an anal sphincter defect or sphincter hypotonia. CONCLUSION: The RAFIS scale is easy to administer and compares well with other validated incontinence instruments.

Molecular characterization of autophagic and apoptotic signaling induced by sorafenib in liver cancer cells.

Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c-Jun-N-terminal kinase (JNK), Akt, and 5'AMP-activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3-12 hr) ER stress characterized by an increase of Ser51 P-eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of Thr183,Tyr185 P-JNK1/2/JNK1/2, Thr172 P-AMPKα, Ser413 P-Foxo3a, Thr308 P-AKt/AKt and Thr32 P-Foxo3a/Foxo3a ratios, and reduction of Ser2481 P-mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim EL , Beclin-1, Bcl-xL, Bcl-2, autophagy markers, and reduction of myeloid cell leukemia-1 (Mcl-1) expression. The progressive increase of CHOP expression, and reduction of Thr308 P-AKt/AKt and Ser473 P-AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim EL expression and caspase-3 activity (24 hr). Small interfering-RNA (si-RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase-3 in sorafenib-treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor-derived xenograft model. In conclusion, the early sorafenib-induced ER stress and regulation of JNK and AMPK-dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK-CHOP-dependent rise of Bim EL , which was associated with the shift from autophagy to apoptosis. The kinetic of Bim EL expression profile might also be related to the tight balance between AKt- and AMPK-related signaling leading to Foxo3a-dependent BIM EL upregulation.

The search for novel diagnostic and prognostic biomarkers in cholangiocarcinoma.

The poor prognosis of cholangiocarcinoma (CCA) is in part due to late diagnosis, which is currently achieved by a combination of clinical, radiological and histological approaches. Available biomarkers determined in serum and biopsy samples to assist in CCA diagnosis are not sufficiently sensitive and specific. Therefore, the identification of new biomarkers, preferably those obtained by minimally invasive methods, such as liquid biopsy, is important. The development of innovative technologies has permitted to identify a significant number of genetic, epigenetic, proteomic and metabolomic CCA features with potential clinical usefulness in early diagnosis, prognosis or prediction of treatment response. Potential new candidates must be rigorously evaluated prior to entering routine clinical application. Unfortunately, to date, no such biomarker has achieved validation for these purposes. This review is an up-to-date of currently used biomarkers and the candidates with promising characteristics that could be included in the clinical practice in the next future. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Clinical decisions in pancreatic cancer surgery: a national survey and case-vignette study.

Very few surveys have been carried out of oncosurgical decisions made in patients with pancreatic cancer (PC), or of the possible differences in therapeutic approaches between low/medium and high-volume centers. A survey was sent out to centers affiliated to the Spanish Group of Pancreatic Surgery (GECP) asking about their usual pre-, intra- and post-operative management of PC patients and describing five imaginary cases of PC corresponding to common scenarios that surgeons regularly assess in oncosurgical meetings. A consensus was considered to have been reached when 80% of the answers coincided. We received 69 responses from the 72 GECP centers (response rate 96%). Pre-operative management: consensus was obtained on 7/16 questions (43.75%) with no significant differences between low- vs high-volume centers. Intra-operative: consensus was obtained on 11/28 questions (39.3%). D2 lymphadenectomy, biliary culture, intra-operative biliary margin study, pancreatojejunostomy, and two loops were significantly more frequent in high-volume hospitals (p < 0.05). Post-operative: consensus was obtained on 2/8 questions (25%). No significant differences were found between low-/medium- vs high-volume hospitals. Of the 41 questions asked regarding the cases, consensus was reached on 22 (53.7%). No differences in the responses were found according to the type of hospital. Management and cases: consensus was reached in 42/93 questions (45.2%). At GECP centers, consensus was obtained on 45% of the questions. Only 5% of the answers differed between low/medium and high-volume centers (all intra-operative). A more specific assessment of why high-volume centers obtain the best results would require the design of complex prospective studies able to measure the therapeutic decisions made and the effectiveness of their execution. Clinicaltrials.gov identifier: NCT04755036.

Emergency adrenalectomy due to acute heart failure secondary to complicated pheochromocytoma: a case report.

Pheochromocytomas are catecholamine producing tumors arising mostly from chromaffin cells of the adrenal medulla. The most common clinical presentation is hypertension, mainly in the form of paroxymal episodes. Cardiovascular manifestations include malignant arrhythmia and catecholamine cardiomyopathy, mimicking acute coronary syndromes and acute heart failure.There are reports of pheochromocytomas presenting as acute coronary syndrome and rapidly leading to cardiogenic shock; the failure of intensive medical treatment in these cases has prompted the need for emergency adrenalectomy as the only remaining option. We report on a case of complicated pheochromocytoma presenting as cardiogenic shock, in which emergency adrenalectomy was performed following a total lack of response to intensive medical treatment.

The stromal-vascular fraction of adipose tissue contributes to major differences between subcutaneous and visceral fat depots.

Adipose tissue represents a complex tissue both in terms of its cellular composition, as it includes mature adipocytes and the various cell types comprising the stromal-vascular fraction (SVF), and in relation to the distinct biochemical, morphological and functional characteristics according to its anatomical location. Herein, we have characterized the proteomic profile of both mature adipocyte and SVF from human visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) fat depots in order to unveil differences in the expression of proteins which may underlie the distinct association of VAT and SAT to several pathologies. Specifically, 24 proteins were observed to be differentially expressed between SAT SVF versus VAT SVF from lean individuals. Immunoblotting and RT-PCR analysis confirmed the differential regulation of the nuclear envelope proteins lamin A/C, the membrane-cytoskeletal linker ezrin and the enzyme involved in retinoic acid production, aldehyde dehydrogenase 1A2, in the two fat depots. In sum, the observation that proteins with important cell functions are differentially distributed between VAT and SAT and their characterization as components of SVF or mature adipocytes pave the way for future research on the molecular basis underlying diverse adipose tissue-related pathologies such as metabolic syndrome or lipodystrophy.

Effects of capecitabine and celecoxib in experimental pancreatic cancer.

INTRODUCTION: Pancreatic cancer is a major health problem because of its aggressiveness and the lack of effective systemic therapies. The aim of the study was the identification of beneficial properties of combined celecoxib and capecitabine treatment during an experimental pancreatic cancer model. METHODS: N-nitrosobis (2-oxopropyl)amine (BOP) was used as a tumoral agent for 12 weeks. Celecoxib and capecitabine were administered either as monotherapy or combined 12 weeks after cancer induction for a period of 24 weeks. The presence of well-developed or moderate adenocarcinoma was evaluated in the pancreas. Several markers of stress, such as lipoperoxides, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GHS-Px) were determined. RESULTS: BOP induced the presence of pancreatic tumors associated with a rise in lipoperoxides and the reduction of the antioxidant status in the pancreas. The administration of celecoxib and capecitabine reduced the number of animals with tumors (33 and 66%, respectively). This antitumoral effect was associated with a recovery of GSH, SOD and CAT activity in the pancreas of BOP-treated animals. The combined treatment exerted a synergic antitumoral effect and reduced pancreatic oxidative stress. CONCLUSION: The combined administration of celecoxib and capecitabine exerted a synergistic antitumoral effect and increased the antioxidant status restoration in pancreatic cancer.

Melatonin and celecoxib improve the outcomes in hamsters with experimental pancreatic cancer.

Pancreatic cancer is a major health problem because of the aggressiveness of the disease and the lack of effective systemic therapies. Melatonin (MEL) has antioxidant activity and prevents experimental genotoxicity. The specific inhibitor of cyclooxygenase-2 (COX-2), celecoxib (CEL), increases the efficacy of chemoradiotherapy in advanced pancreatic cancer. The objective of the study was the comparison and synergic effect of MEL and CEL during either the induction or progression phases of the tumor process, measuring parameters of oxidative stress, number of tumor nodules and survival of animals with pancreatic cancer. Pancreatic cancer was induced by N-nitrosobis (2-oxopropyl)amine) (BOP) in Syrian hamsters. Melatonin and/or CEL were administered during the induction, postinduction as well as during both phases. The presence of tumor nodules were observed macroscopically in pancreatic and splenic areas, and the levels of lipoperoxides (LPO), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in pancreatic tissue were measured. The increases in tumor nodules and LPO as well as the reductions in GSH and enzymatic antioxidants in the pancreas induced by BOP were related to a lower survival rate of animals. The administration of MEL exerted a more potent beneficial effect than CEL treatment on the reduction in tumor nodules, oxidative stress and death of experimental BOP-treated animals. The combined treatment only exerted a synergistic beneficial effect when administered during the induction phase. Melatonin by itself had significant beneficial actions in improving the survival of hamsters.

Differential effectiveness of tyrosine kinase inhibitors in 2D/3D culture according to cell differentiation, p53 status and mitochondrial respiration in liver cancer cells.

Sorafenib and Regorafenib are the recommended first- and second-line therapies in patients with advanced hepatocellular carcinoma (HCC). Lenvatinib and Cabozantinib have shown non-inferior antitumoral activities compared with the corresponding recommended therapies. The clinical trials have established recommended doses for each treatment that lead different blood concentrations in patients for Sorafenib (10 µM), Regorafenib (1 µM), Lenvatinib (0.1 µM), and Cabozantinib (1 µM). However, very low response rates are observed in patients attributed to intrinsic resistances or upregulation of survival signaling. The aim of the study was the comparative dose-response analysis of the drugs (0-100 µM) in well-differentiated (HepG2, Hep3B, and Huh7), moderately (SNU423), and poorly (SNU449) differentiated liver cancer cells in 2D/3D cultures. Cells harbors wild-type p53 (HepG2), non-sense p53 mutation (Hep3B), inframe p53 gene deletion (SNU423), and p53 point mutation (Huh7 and SNU449). The administration of regular used in vitro dose (10 µM) in 3D and 2D cultures, as well as the dose-response analysis in 2D cultures showed Sorafenib and Regorafenib were increasingly effective in reducing cell proliferation, and inducing apoptosis in well-differentiated and expressing wild-type p53 in HCC cells. Lenvatinib and Cabozantinib were particularly effective in moderately to poorly differentiated cells with mutated or lacking p53 that have lower basal oxygen consumption rate (OCR), ATP, and maximal respiration capacity than observed in differentiated HCC cells. Sorafenib and Regorafenib downregulated, and Lenvatinib and Cabozantinib upregulated epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor receptor (c-Met) in HepG2 cells. Conclusions: Sorafenib and Regorafenib were especially active in well-differentiated cells, with wild-type p53 and increased mitochondrial respiration. By contrast, Lenvatinib and Cabozantinib appeared more effective in moderately to poorly differentiated cells with mutated p53 and low mitochondrial respiration. The development of strategies that allow us to deliver increased doses in tumors might potentially enhance the effectiveness of the treatments.

Melatonin exerts a more potent effect than S-adenosyl-l-methionine against iron metabolism disturbances, oxidative stress and tissue injury induced by obstructive jaundice in rats.

Melatonin and S-adenosyl-l-methionine (SAMe) prevent oxidative stress and tissue dysfunction in obstructive jaundice (OJ). Lipid peroxidation is exacerbated in the presence of trace amounts of iron (Fe). The study investigated the regulation by melatonin and SAMe the induction of oxidative stress, iron metabolism disturbances and tissue injury in an experimental model of OJ. Different parameters of lipid peroxidation, antioxidant status, tissue injury and Fe metabolism were determined in liver and blood. OJ induced Fe accumulation in liver, and increased transferrin (Tf) saturation and loosely bound Fe content in blood. Melatonin, and SAMe at lesser extent, enhanced protein Tf content in liver and blood, that reduced loosely bound Fe content in blood. Melatonin and SAMe did not affect ferritin (FT) and Tf mRNA expression, but reduced Tf receptor (TfR) mRNA expression in liver. In conclusion, the effect of melatonin and SAMe on Fe metabolism may be included in the beneficial properties of these agents on lipid peroxidation and tissue injury induced by OJ.

Simvastatin and metformin inhibit cell growth in hepatitis C virus infected cells via mTOR increasing PTEN and autophagy.

Hepatitis C virus (HCV) infection has been related to increased risk of development of hepatocellular carcinoma (HCC) while metformin (M) and statins treatment seemed to protect against HCC development. In this work, we aim to identify the mechanisms by which metformin and simvastatin (S) could protect from liver cancer. Huh7.5 cells were infected with HCV particles and treated with M+S. Human primary hepatocytes were treated with M+S. Treatment with both drugs inhibited Huh7.5 cell growth and HCV infection. In non-infected cells S increased translational controlled tumor protein (TCTP) and phosphatase and tensin homolog (PTEN) proteins while M inhibited mammalian target of rapamycin (mTOR) and TCTP. Simvastatin and metformin co-administered down-regulated mTOR and TCTP, while PTEN was increased. In cells infected by HCV, mTOR, TCTP, p62 and light chain 3B II (LC3BII) were increased and PTEN was decreased. S+M treatment increased PTEN, p62 and LC3BII in Huh7.5 cells. In human primary hepatocytes, metformin treatment inhibited mTOR and PTEN, but up-regulated p62, LC3BII and Caspase 3. In conclusion, simvastatin and metformin inhibited cell growth and HCV infection in vitro. In human hepatocytes, metformin increased cell-death markers. These findings suggest that M+S treatment could be useful in therapeutic prevention of HCV-related hepatocellular carcinoma.

Role of p63 and p73 isoforms on the cell death in patients with hepatocellular carcinoma submitted to orthotopic liver transplantation.

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) submitted to orthotopic liver transplantation (OLT) have a variable 5-year survival rate limited mostly by tumor recurrence. The etiology, age, sex, alcohol, Child-Pugh, and the immunesuppressor have been associated with tumour recurrence. The expression of ΔNp73 is related to the reduced survival of patients with HCC. The study evaluated the expression of p63 and p73 isoforms and cell death receptors, and their relation to tumour recurrence and survival. The results were in vitro validated in HCC cell lines. METHODS: HCC sections from patients submitted to OLT were used. The in vitro study was done in differentiated hepatitis B virus (HBV)-expressing Hep3B and control HepG2 cells. The expression of cell death receptors and cFLIPS/L, caspase-8 and -3 activities, and cell proliferation were determined in control and p63 and p73 overexpressing HCC cells. RESULTS: The reduced tumor expression of cell death receptors and TAp63 and TAp73, and increased ΔNp63 and ΔNp73 expression were associated with tumor recurrence and reduced survival. The in vitro study demonstrated that HBV-expressing Hep3B vs HepG2 cells showed reduced expression of p63 and p73, cell death receptors and caspase activation, and increased cFLIPL/cFLIPS ratio. The overexpression of TAp63 and TAp73 exerted a more potent pro-apoptotic and anti-proliferative effects in Hep3B than HepG2-transfected cells which was related to cFLIPL upregulation. CONCLUSIONS: The reduction of TAp63 and TAp73 isoforms, rather than alteration of ΔN isoform expression, exerted a significant functional repercussion on cell death and proliferation in HBV-expressing HepB cells.

Role of serotonin in cerebral oxidative stress in rats.

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter synthesized by the aromatic amino acid decarboxylase using 5-hydroxytryptophan (5-HTP) as a substrate. It was recently shown that serotonin and its precursor have powerful antioxidant properties. The aim of this study was to evaluate the effect of reduction in 5-HT levels by parachlorophenylalanine (pCPA) and their restoration by 5-HTP administration on lipid peroxidation and antioxidant status in rat brain. Serotonin levels were decreased by p-chlorophenylalanine administration. The effect of p-chlorophenylalanine was counteracted by the intraperitoneal administration of 5-hydroxytryptophan. We evaluated the concentration of serotonin, malonyl dialdehyde and the status of antioxidants (GSH, catalase and superoxide dismutase) in brain. The results showed that p-chlorophenylalanine (300 mg/kg) induced a depletion of serotonin concentration and antioxidant status, as well as enhancing malonyl dialdehyde concentration in brain. The exogenous administration of 5-hydroxytryptophan prevented all effects induced by p-chlorophenylalanine in brain tissue. The recovery of the neurotransmitter concentration in brain was related to the reduction of lipid peroxide generation and improved antioxidant status. In conclusion, our study supports the view that the antioxidant properties of serotonin protect against basal oxidative stress in brain.

Antitumoral Activity of Sorafenib in Hepatocellular Carcinoma: Effects on Cell Survival and Death Pathways, Cell Metabolism Reprogramming, and Nitrosative and Oxidative Stress.

Sorafenib is an oral multikinase inhibitor approved for the treatment of hepatocellular carcinoma (HCC). Its antitumor activity is attributed to inhibition of tyrosine kinase receptors (VEGFR, PDGFR, c-kit) and intracellular serine/threonine kinases (Raf), which alter gene expression to promote apoptosis and downregulate survival and angiogenesis pathways. The beneficial properties of sorafenib have also been related to a reduction in liver fibrosis trough regulation of TGF-ßR-related STAT3 signaling. Sorafenib plays a role in the regulation of mitochondrial function, ATP, and autophagy, a process leading to either survival or apoptotic cell death depending on its intensity and duration, by altering several cellular pathways such as mTOR, AMPK, activating endoplasmic reticulum stress responses, and deregulating miRNAs that modulate autophagy. Sorafenib reduces S-nitrosation of cell death receptors and caspase-3, triggering a switch to caspase-3 from caspase-8. In this paper, we review the antitumor effects of sorafenib by interaction with cell survival and apoptosis pathways, metabolic reprogramming, and effect on oxidative and nitrosative stress, along with different mechanisms that might be involved in resistance to the drug.

Differential Antitumoral Properties and Renal-Associated Tissue Damage Induced by Tacrolimus and Mammalian Target of Rapamycin Inhibitors in Hepatocarcinoma: In Vitro and In Vivo Studies.

Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with potential portal hypertension and/or bilirubinemia, but without vascular-associated diseases. The patients are receiving immunosuppressive therapy to reduce graft rejection, but differential side effects have been related to calcineurin and mTOR inhibitor administration regarding tumor recurrence and nephrotoxicity. The in vitro studies showed that Tacrolimus exerted a more potent pro-apoptotic effect than Everolimus (Huh 7>Hep 3B>HepG2), being sirolimus only active in Hep3B cell line. Tacrolimus and Everolimus exerted potent antiproliferative properties in Huh 7 and Hep3B in which cells Sirolimus was inactive. Interestingly, Tacrolimus- and Everolimus-dependent G0/G1 cell accumulation occurred as a consequence of drastic reduction in S, as well as in S and G2+M phases, respectively. The in vivo studies support data on the more effective antitumoral properties of Everolimus, eventual risk of pro-angiogenic tumoral properties and nephrotoxicity of Tacrolimus, and pro-proliferative properties of Sirolimus in tumors developed in nude mice.