Morin loaded mesoporous molecular sieves as novel devices to the potential treatment of tumor pathologies.

Morin is an antioxidant and anticancer flavonoid, extracted from natural sources, that may exert beneficial effects for several pathologies. Despite this, the administration of morin represents a challenge due to its low aqueous solubility. Mesoporous silica materials have emerged as biocompatible tools for drug delivery, as their pore size can be modulated for maximum surface area to volume ratio. In this contribution, we evaluate the ability of iron-modified mesoporous materials, for morin loading and controlled delivery. The SBA-15 and MCM-41 sieves were synthesized and modified with iron (metal content 4.02 and 6.27 % wt, respectivily). Characterization by transmission electron microscopy, XRD and UV-Vis revealed adequate pore size and agglomerates of very small metallic nanospecies (nanoclusters), without larger iron oxide nanoparticles. FT-IR spectra confirmed the presence of silanol groups in the solid hosts, which can interact with different groups present in morin molecules. SBA-15 materials were more efficient in terms of morin loading capacity (LC) due to their larger pore diameter. LC was more than 35% for SBA-15 materials when adsorptions studies were carried out with 9 mg of drug. Antioxidant activity were assayed by using DPPH test. Free iron materials presented a significate improvement as antioxidants after morin incorporation, reaching a scavenging activity of almost a 90%. On the other hand, in iron modified mesoporous materials, the presence of morin did not affect the scavenging activity. The results could be related with the formation of a complex between the flavonoid and the iron. Finally, biosafety studies using normal epithelial cells revealed that neither the loaded nor the unloaded materials exerted toxicity, even at doses of 1 mg/ml. These findings expand knowledge about mesoporous materials as suitable carriers of flavonoids with the aim of improving therapies for a wide range of pathologies.

How the interplay among the tumor microenvironment and the gut microbiota influences the stemness of colorectal cancer cells.

Colorectal cancer (CRC) remains the third most prevalent cancer disease and involves a multi-step process in which intestinal cells acquire malignant characteristics. It is well established that the appearance of distal metastasis in CRC patients is the cause of a poor prognosis and treatment failure. Nevertheless, in the last decades, CRC aggressiveness and progression have been attributed to a specific cell population called CRC stem cells (CCSC) with features like tumor initiation capacity, self-renewal capacity, and acquired multidrug resistance. Emerging data highlight the concept of this cell subtype as a plastic entity that has a dynamic status and can be originated from different types of cells through genetic and epigenetic changes. These alterations are modulated by complex and dynamic crosstalk with environmental factors by paracrine signaling. It is known that in the tumor niche, different cell types, structures, and biomolecules coexist and interact with cancer cells favoring cancer growth and development. Together, these components constitute the tumor microenvironment (TME). Most recently, researchers have also deepened the influence of the complex variety of microorganisms that inhabit the intestinal mucosa, collectively known as gut microbiota, on CRC. Both TME and microorganisms participate in inflammatory processes that can drive the initiation and evolution of CRC. Since in the last decade, crucial advances have been made concerning to the synergistic interaction among the TME and gut microorganisms that condition the identity of CCSC, the data exposed in this review could provide valuable insights into the biology of CRC and the development of new targeted therapies.

Tumor microenvironment involvement in colorectal cancer progression via Wnt/ß-catenin pathway: Providing understanding of the complex mechanisms of chemoresistance.

Colorectal cancer (CRC) continues to be one of the main causes of death from cancer because patients progress unfavorably due to resistance to current therapies. Dysregulation of the Wnt/ß-catenin pathway plays a fundamental role in the genesis and progression of several types of cancer, including CRC. In many subtypes of CRC, hyperactivation of the ß-catenin pathway is associated with mutations of the adenomatous polyposis coli gene. However, it can also be associated with other causes. In recent years, studies of the tumor microenvironment (TME) have demonstrated its importance in the development and progression of CRC. In this tumor nest, several cell types, structures, and biomolecules interact with neoplastic cells to pave the way for the spread of the disease. Cross-communications between tumor cells and the TME are then established primarily through paracrine factors, which trigger the activation of numerous signaling pathways. Crucial advances in the field of oncology have been made in the last decade. This Minireview aims to actualize what is known about the central role of the Wnt/ß-catenin pathway in CRC chemoresistance and aggressiveness, focusing on cross-communication between CRC cells and the TME. Through this analysis, our main objective was to increase the understanding of this complex disease considering a more global context. Since many treatments for advanced CRC fail due to mechanisms involving chemoresistance, the data here exposed and analyzed are of great interest for the development of novel and effective therapies.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

BACKGROUND: Parathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found that in colorectal cancer (CRC) HCT116 cells, the binding of PTHrP to its receptor PTHR type 1 (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cell xenografts, PTHrP modulates the expression of molecular markers linked to tumor progression. Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells. AIM: To elucidate the relationship among PTHR1, PTHrP, and Met in CRC models. METHODS: For in vitro assays, HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP (1-34) (10-8 M). Where indicated, cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide, the vehicle of the inhibitors. The protein levels were evaluated by Western blot technique. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the changes in gene expression. Wound healing assay and morphological monitoring were performed to evaluate cell migration and changes related to the epithelial-mesenchymal transition (EMT), respectively. The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan (CPT-11), oxaliplatin (OXA), or doxorubicin (DOXO) with or without PTHrP. For in vivo tests, HCT116 cell xenografts on 6-wk-old male N:NIH (S)_nu mice received daily intratumoral injections of PTHrP (40 µg/kg) in 100 µL phosphate-buffered saline (PBS) or the vehicle (PBS) as a control during 20 d. Humanitarian slaughter was carried out and the tumors were removed, weighed, and fixed in a 4% formaldehyde solution for subsequent treatment by immunoassays. To evaluate the expression of molecular markers in human tumor samples, we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr. José Penna (Bahía Blanca, Buenos Aires, Argentina) and the Hospital Provincial de Neuquén (Neuquén, Neuquén, Argentina) from January 1990 to December 2007. Seven cases with normal colorectal tissues were assigned to the control group. Tumor tissue samples and clinical histories of patients were analyzed. Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique; subsequently, representative histological samples were selected from each patient. From each paraffin block, tumor sections were stained for immunohistochemical detection. The statistical significance of differences was analyzed using proper statistical analysis. The results were considered statistically significant at P < 0.05. RESULTS: By Western blot analysis and using total Met antibody, we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 min (P < 0.01) and at 20 h (P < 0.01) whereas the levels diminished at 3 min (P < 0.05), 10 min (P < 0.01), and 1 h to 5 h (P < 0.01) of PTHrP treatment. Using an active Met antibody, we found that where the protein levels of total Met decreased (3 min, 10 min, and 60 min of PTHrP exposure), the status of phosphorylated/activated Met increased (P < 0.01) at the same time, suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP. The increment of its protein level after these decreases (at 30 min and 20 h) suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis (P < 0.05). We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/ activation of Met induced by PTHrP in HCT116 cells. By Western blot technique, we observed that PP1, a specific inhibitor of the activation of the proto-oncogene protein tyrosine kinase Src, blocked the effect of PTHrP on Met phosphorylation (P < 0.05). Furthermore, the selective inhibition of the ERK 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation (P < 0.05). Using SU11274, the specific inhibitor of Met activation, and trypan blue dye exclusion test, Western blot, wound healing assay, and morphological analysis with a microscope, we observed the reversal of cell events induced by PTHrP such as cell proliferation (P < 0.05), migration (P < 0.05), and the EMT program (P < 0.01) in HCT116 cells. Also, PTHrP favored the chemoresistance to CPT-11 (P < 0.001), OXA (P < 0.01), and DOXO (P < 0.01) through the Met pathway. Taken together, these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells. By immunohistochemical analysis, we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met (0.190 ± 0.014) compared to tumors from control mice (0.110 ± 0.012; P < 0.05) and of its own receptor (2.27 ± 0.20) compared to tumors from control mice (1.98 ± 0.14; P < 0.01). Finally, assuming that the changes in the expression of PTHrP and its receptor are directly correlated, we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis. Comparing histologically differentiated tumors with respect to those less differentiated, we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner, respectively (P < 0.05). CONCLUSION: PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model. More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.

Chitosan-Based Formulations Intended as Protective Spray for Mask Surfaces in Prevention of Coronavirus Dissemination.

The extraordinary occurrence of COVID-19 by the fast expansion of viral infections has propelled particular interest in developing novel antiviral and virucidal agents to guarantee personal security. The main objective of this work is to propose novel formulations able to optimize the use of personal protection elements. In recent years, chitosan (CH) has attracted attention for being an interesting multifunctional, biodegradable, non-antigenic, non-toxic, and biocompatible natural polymer with antimicrobial properties. In this work, formulations based on a CH matrix containing silver, and Copper based nanoparticles have been developed. The novelty of this proposal is that almost liquid formulations have been reached, possessing verified properties to inhibit evolved virus such as herpes simplex type 1 (HSV-1) and bovine betacoronavirus (BCoV), the latter belonging to the same family of the well-known the well-known SARS-CoV-2. Besides antibacterial bioactivity; as well as the ability of these formulations to be easily sprayed on various surfaces, including conventional face masks, have been verified and discussed. The results presented in this contribution provide strong evidence on CH films as an ideal biosafe surface-protective for several daily used materials including the conventional face masks.

Involvement of parathyroid hormone-related peptide in the aggressive phenotype of colorectal cancer cells.

Colorectal cancer (CRC) remains one of the leading causes of mortality from malignant diseases worldwide. In general terms, CRC presents high heterogeneity due to the influence of different genetic and environmental factors; also, the neoplastic cells are strongly influenced by the extracellular matrix and several surrounding cells, known together as the tumor microenvironment (TME). Bidirectional communication takes place between the tumor and the TME through the release of autocrine and paracrine factors. Parathyroid hormone-related peptide (PTHrP) is a cytokine secreted by a wide variety of tissues and is able to regulate several cellular functions both in physiological as well as in pathological processes. It exerts its effects as a paracrine/autocrine factor, although its mode of action is mainly paracrine. It has been shown that this peptide is expressed by several tumors and that the tumor secretion of PTHrP is responsible for the malignant humoral hypercalcemia. Eight years ago, when our research group started studying PTHrP effects in the experimental models derived from intestinal tumors, the literature available at the time addressing the effects of PTHrP on colorectal tumors was limited, and no articles had been published regarding to the paracrine action of PTHrP in CRC cells. Based on this and on our previous findings regarding the role of PTH in CRC cells, our purpose in recent years has been to explore the role of PTHrP in CRC. We analyzed the behavior of CRC cells treated with exogenous PTHrP, focalizing in the study of the following events: Survival, cell cycle progression and proliferation, migration, chemoresistance, tumor-associated angiogenesis, epithelial to mesenchymal transition program and other events also associated with invasion, such us the induction of cancer stem cells features. This work summarizes the major findings obtained by our investigation group using in vitro and in vivo CRC models that evidence the participation of PTHrP in the acquisition of an aggressive phenotype of CRC cells and the molecular mechanisms involved in these processes. Recently, we found that this cytokine induces this malignant behavior not only by its direct action on these intestinal cells but also through its influence on cells derived from TME, promoting a communication between CRC cells and surrounding cells that contributes to the molecular and morphological changes observed in CRC cells. These investigations establish the basis for our next studies in order to address the clinical applicability of our findings. Recognizing the factors and mechanisms that promote invasion in CRC cells, evasion to the cytotoxic effects of current CRC therapies and thus metastasis is decisive for the identification of new markers with the potential to improve early diagnosis and/or to predict prognosis, to predetermine drug resistance and to provide treatment guidelines that include targeted therapies for this disease.

Survival effect of probiotics in a rat model of colorectal cancer treated with capecitabine.

BACKGROUND: Probiotics are used to manage a number of gastrointestinal disorders due to their beneficial properties. Clinical reports showed that probiotics also improve the life quality of patients with colorectal cancer (CRC) subjected to oncologic treatment. In a CRC animal model, probiotics supplementation has the potential to decrease the formation of aberrant crypts and ameliorate tumor malignancy, enhancing the antitumor effect of 5-fluorouracil (5-FU) chemotherapy. Based on these data, we hypothesize that the administration of probiotics impact positively in the overall survival and life quality of rats with CRC under the treatment of capecitabine, which is the pro drug of 5-FU. AIM: To evaluate the probiotics effects in a rat CRC model treated with capecitabine and followed until the end of life. METHODS: 1,2-Dimethylhidrazine dihydrochloride (1,2-DMH) was employed as carcinogen inductor of CRC. Fifty male Wistar-Lewis rats were randomly assigned to one of five following groups: Control (n = 5), Control + probiotics (Control-P group, n = 5), 1,2-DMH alone (DMH group, n = 10), 1,2-DMH + capecitabine (DMH-C group, n = 10), 1,2-DMH + probiotics (DMH-P group, n = 10) and 1,2-DMH + capecitabine + probiotics (DMH-C-P group, n = 10). All parametric data were expressed as the mean ± SD. The statistical significance of differences was analyzed using one-way ANOVA. Data were analyzed with InfoStat software. The results were considered statistically significant at P < 0.05. Overall survival was evaluated with the Kaplan-Meier estimator with the log-rank test. RESULTS: The data of mean overall survival for DMH, DMH-P, DMH-C, DMH-C-P, Control and Control-P groups were 250 d [95% confidence interval (CI): 242.5-253.1], 268 d (95%CI: 246.3-271.4), 380 d (95%CI: 337.8-421.9), 480 d (95%CI: 436.9-530.7), 588 d (95%CI: 565.8-609.3) and 590 d (95%CI: 564.3-612.9), respectively, with a significant difference between DMH-C and DMH-C-P groups (P = 0.001). Comparing all groups by Kaplan-Meier estimator, we found a significantly different in the overall survival of DMH and DMH-P groups respect to DMH-C (P = 0.001) and DMH-C-P (P = 0.001) groups; interestingly, there were no meaningful differences between Control, Control-P and DMH-C-P groups (P = 0.012). The tendency of change in body weight gain of the rats at 90 d of finishing DMH administration was similar in Control group compared with DMH-C and DMH-C-P groups; however, and of relevance, DMH-C-P group has experienced a higher body weight gain at the end of animal's life than DMH-C group (P = 0.001). In DMH-C-P group we found a positive effect of probiotics in clinical manifestations since diarrhea, constipation and blood stool were absenting. Also, the tumor burden was lower in DMH-C-P than DMH-C, DMH-P or DMH groups (1.25 vs 1.81 vs 3.9 vs 4.8 cm2, respectively). DMH-C and DMH-C-P groups showed only mucinous carcinoma type while in other DMH groups the tumor types were variable. However, mucinous carcinoma from DMH-C-P group showed invasion until muscularis propria layer. Interestingly, metastatic lymph node was observed in DMH, DMH-C and DMH-P groups but not in DMH-C-P. All animals in Control group died from natural causes without objective injuries. All animals of DMH and DMH-P groups died from tumor complications (i.e., obstruction or intestinal perforation); however, this cause was seen only in 44.5% of DMH-C and DMH-C-P groups. CONCLUSION: Probiotics administration improves life quality of rats with CRC under capecitabine treatment and also has a positive effect in the overall survival of these animals treated with this drug.

Role of SPARC in the epithelial-mesenchymal transition induced by PTHrP in human colon cancer cells.

Parathyroid hormone-related peptide (PTHrP) exerts its effects on cells derived from colorectal cancer (CRC) and tumor microenvironment and is involved in processes requiring the epithelial-mesenchymal transition (EMT). Here, we report that PTHrP modulates factors expression and morphological changes associated with EMT in HCT116 cells from CRC. PTHrP increased the protein expression of SPARC, a factor involved in EMT, in HCT116 cells but not in Caco-2 cells also from CRC but with less aggressiveness. PTHrP also increased SPARC expression and its subsequent release from endothelial HMEC-1 cells. The conditioned media of PTHrP-treated HMEC-1 cells induced early changes related to EMT in HCT116 cells. Moreover, SPARC treatment on HCT116 cells potentiated PTHrP modulation in E-cadherin expression and cell migration. In vivo PTHrP also increased SPARC expression and decreased E-cadherin expression. These results suggest a novel PTHrP action on CRC progression involving the microenvironment in the modulation of events associated with EMT.

Doxorubicin delivery by magnetic nanotheranostics enhances the cell death in chemoresistant colorectal cancer-derived cells.

Colorectal cancer (CRC) is a major cause of cancer death with a high probability of treatment failure. Doxorubicin (DOXO) is an efficient antitumor drug; however, most CRC cells show resistance to its effects. Magnetic nanoparticles (MNPs) are potential cancer management tools that can serve as diagnostic agents and also can optimize and personalize treatments. This work aims to evaluate the aptitude of magnetic nanotheranostics composed of magnetite (Fe3O4) nanoparticles coated with folic acid intended to the sustained release of DOXO. The administration of DOXO by means of these MNPs resulted in the enhancement of cell death respect to the free drug administration. Chromatin compaction and cytoplasmic protrusions were observed. Mitochondrial transmembrane potential disruption and increased PARP protein cleavage confirmed apoptosis. The nanosystem was also tested as a vectoring tool by exposing it to the stimuli of a static magnetic field in vitro. CRC-related magnetic nanotechnology still remains in pre-clinical trials. In this context, this contribution expands the knowledge of the behavior of MNPs in contact with in vitro models and proposes the nanodevices studied here as potential theranostic agents for the monitoring of the progress of CRC and the evolution of its treatment.

In vitro Biological Tests as the First Tools To Validate Magnetic Nanotheranostics for Colorectal Cancer Models.

Colorectal cancer (CRC) remains a leading cause of cancer death. Nanotechnology has focused on reaching more effective treatments. In this concern, magnetic nanoparticles (MNPs) have been studied for a wide range of biomedical applications related to CRC, such as diagnostic imaging, drug delivery and thermal therapy. However, limited research is currently found in the open literature that refers to nanosystems combining all these mentioned areas (theranostics). When developing nanosystems intended as theranostics applied to CRC, possible variations between patients must be considered. Therefore, multiple in vitro assays are required as guidance for future preclinical and clinical trials. The objective of this contribution is to evaluate the available and recent literature regarding the interactions of MNP and CRC models, aiming to critically analyze the information given by the commonly used assays and evaluate the data provided by each one with a view to implementing this novel technology in CRC diagnostics and therapy.

PTHrP treatment of colon cancer cells promotes tumor associated-angiogenesis by the effect of VEGF.

We showed that Parathyroid Hormone-related Peptide (PTHrP) induces proliferation, migration, survival and chemoresistance via MAPKs and PI3K/AKT pathways in colorectal cancer (CRC) cells. The objective of this study was to investigate if PTHrP is also involved in tumor angiogenesis. PTHrP increased VEGF expression and the number of structures with characteristics of neoformed vessels in xenografts tumor. Also, PTHrP increased mRNA levels of VEGF, HIF-1α and MMP-9 via ERK1/2 and PI3K/Akt pathways in Caco-2 and HCT116 cells. Tumor conditioned media (TCMs) from both cell lines treated with PTHrP increases the number of cells, the migration and the tube formation in the endothelial HMEC-1 cells, whereas the neutralizing antibody against VEGF diminished this response. In contrast, PTHrP by direct treatment only increased ERK1/2 phosphorylation and the HMEC-1 cells number. These results provide the first evidence related to the mode of action of PTHrP that leads to its proangiogenic effects in the CRC.

Selective contrast agents with potential to the earlier detection of tumors: Insights on synthetic pathways, physicochemical properties and performance in MRI assays.

Magnetic iron oxide nanoparticles (MNPs) have been prepared and stabilized with three organic acids (tartaric, malic and ascorbic) in order to obtain biocompatible and water dispersible MNPs with potential to bind specifically to tumoral cancer cells. An in deep characterization was performed aiming to verify the presence and effect of the coating and stabilizer on MNPs surface. Besides the mechanisms followed by the different acids to bind MNPs were elucidated and used to justify the differences in the physicochemical properties of each formulation. Data related to characterization revealed that MNPs coated with ascorbic acid (MNPs-AA) resulted the most suitable in terms of their size, surface charge and stability along the time. Besides, ascorbic acid may be recognized by GLUTs receptors that are overexpressed in several kinds of tumoral cells. Therefore, MNPs-AA was selected to explore its performance in both MRI and in vitro assays using human colon cancer cells HCT 116. MRI experiments were performed in clinical equipment using a series of aqueous dispersions of MNPs-AA that were evaluated as T2 contrast agent. The T2- weighted images obtained as well as the calculated r2, indicated that MNPs-AA could act as efficient T2 contrast agent for MRI. Regarding in vitro assays, MNPs-AA did not alter the cellular function neither exert cytotoxicity using the three explored doses. The internalization of the nanoparticles on the cellular structure was confirmed quanti and qualitatively using atomic absorption spectroscopy and Prussian blue techniques respectively. From these results, it emerges that ascorbic acid coated-magnetite nanoparticles may be used as alternative contrast agent to avoid or minimize some toxicological issues related to the widely used gadolinium.

Potential therapeutic targets for growth arrest of colorectal cancer cells exposed to PTHrP.

Although PTHrP is implicated in several cancers, its role in chemoresistance is not fully elucidated. We found that in CRC cells, PTHrP exerts proliferative and protective effects and induces cell migration. The aim of this work was to further study the effects of PTHrP in CRC cells. Herein we evidenced, for the first time, that PTHrP induces resistance to CPT-11 in Caco-2 and HCT116 cells; although both cell lines responded to the drug through different molecular mechanisms, the chemoresistance by PTHrP in these models is mediated through ERK, which in turn is activated by PCK, Src and Akt. Moreover, continue administration of PTHrP in nude mice xenografts increased the protein levels of this MAPK and of other markers related to tumorigenic events. The understanding of the molecular mechanisms leading to ERK 1/2 activation and the study of ERK targets may facilitate the development of new therapeutic strategies for CRC treatment.

Case Report: Penile Squamous Cell Carcinoma Associated With Severe Hypercalcemia and High Levels of Parathyroid Hormone-related Peptide Expressed in Metastatic Skin Tissue.

It is known that high levels of parathyroid hormone-related protein (PTHrP) correlate with a bad prognostic in malignancies. Here we present a patient with advanced penile cancer (PC) without antecedents of human papillomavirus infections and bone metastases but with severe hypercalcemia. By quantitative polymerase chain reaction, we observed high levels of PTHrP messenger RNA in metastatic cutaneous tissue. This is the first reported case in Argentina of hypercalcemia induced by PTHrP in human PC. Furthermore, the association of PTHrP and this disease through quantitative polymerase chain reaction allows us to consider this molecular technique as a novel tool for diagnosis in patients with PC.

RSK activation via ERK modulates human colon cancer cells response to PTHrP.

Parathyroid hormone-related peptide (PTHrP) is associated with several human cancers such as colon carcinoma. This disease is a complex multistep process that involves enhanced cell cycle progression and migration. Recently we obtained evidence that in the human colorectal adenocarcinoma Caco2 cells, exogenous PTHrP increases the proliferation and positively modulates cell cycle progression via ERK1/2, p38 MAPK and PI3K. The purpose of this study was to explore if the serine/threonine kinase RSK, which is involved in the progress of many cancers and it is emerging as a potential therapeutic target, mediates PTHrP effects on cancer colon cells. Western blot analysis revealed that PTHrP increases RSK phosphorylation via ERK1/2 signaling pathway but not through p38 MAPK. By performing subcellular fractionation, we found that the peptide also induces the nuclear localization of activated RSK, where many of its substrates are located. RSK participates in cell proliferation, in the upregulation of cyclin D1 and CDK6 and in the downregulation of p53 induced by PTHrP. Wound healing and transwell filter assays revealed that cell migration increased after PTHrP treatment. In addition, the hormone increases the protein expression of the focal adhesion kinase FAK, a regulator of cell motility. We observed that PTHrP induces cell migration and modulates FAK protein expression through ERK/RSK signaling pathway but not via p38 MAPK pathway. Finally, in vivo studies revealed that the hormone activates RSK in xenografts tumor. Taken together, our findings provide new insights into the deregulated cell cycle and migration that is characteristic of tumor intestinal cells.

Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.

Molecular mechanisms associated with PTHrP-induced proliferation of colon cancer cells.

Parathyroid Hormone-related Protein (PTHrP) is normally produced in many tissues and is recognized for its endocrine, paracrine, autocrine and intracrine modes of action. PTHrP is also implicated in different types of cancer and its expression correlates with the severity of colon carcinoma. Using the human colon cell line Caco-2 we recently obtained evidence that PTHrP, through a paracrine pathway, exerts a protective effect under apoptotic conditions. However, if exogenous PTHrP is able or not to induce the proliferation of these intestinal tumor cells is not known. We found that PTHrP treatment increases the number of live Caco-2 cells. The hormone induces the phosphorylation and nuclear translocation of ERK 1/2, α p38 MAPK, and Akt, without affecting JNK phosphorylation. In addition, PTHrP-dependent ERK phosphorylation is reverted when PI3K activity was inhibited. Following MAPKs nuclear translocation, the transcription factors ATF-1 and CREB were activated in a biphasic manner. In addition PTHrP induces the translocation into the nucleus of ß-catenin, protein that plays key role in maintaining the growth and proliferation of colorectal cancer, and increases the amount of both positive cell cycle regulators c-Myc and Cyclin D. Studies with ERK1/2, α p38 MAPK, and PI3K specific inhibitors showed that PTHrP regulates Caco-2 cell proliferation via these signaling pathways. In conclusion, the results obtained in this work expand our knowledge on the role of exogenous PTHrP in intestinal tumor cells and identify the signaling pathways that are involved in the mitogenic effect of the hormone on Caco-2 cells.

Role of PTHrP in human intestinal Caco-2 cell response to oxidative stress.

We have previously demonstrated that parathyroid hormone (PTH) induces apoptosis in human colon adenocarcinoma Caco-2 cells but the effects of its tumoral analog PTH-related peptide (PTHrP) in this cell line are still unknown. In the present work we investigated whether PTHrP, as PTH, is able to induce Caco-2 cell apoptosis or if it exerts protective effects under apoptotic conditions. Using Caco-2 cells cultured under serum deprivation in the presence or absence of PTHrP we demonstrated that, differently to PTH, its analog employed at the same concentration (10(-8)M) is not a pro-apoptotic hormone. Cells were exposed to an oxidative insult in the form of hydrogen peroxide to induce apoptosis, which leads to a 50% loss of cell viability determined by MTS assay, morphological changes observed under fluorescence microscopy and Western blot analysis. Herein we demonstrate, for the first time, that pre-treatment with PTHrP prior to H2O2 incubation, prevents cell death induced by the apoptotic inductor; and using specific inhibitors we evidenced that protein kinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and p38 mitogen-activated protein kinase (MAPK) mediate this anti-apoptotic effect. Also, we found that PTHrP decreases the pro-apoptotic protein BAX levels and increases the protein expression of the anti-apoptotic HSP27. Immunoblot analysis revealed that H2O2 increases the phosphorylation levels of AKT and MAPKs, exhibiting a cellular defense response; and consequently increases phospho-BAD levels. The H2O2-induced activation of protein kinases is reverted when cells are pre-treated with PTHrP. Altogether these results evidence a protective effect of PTHrP under apoptotic conditions in intestinal cells, which may be mediated by AKT and MAPKs.

Parathyroid hormone and the regulation of cell cycle in colon adenocarcinoma cells.

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In this study, we investigated the role of PTH in the regulation of the cell cycle in human colon adenocarcinoma Caco-2 cells. Flow cytometry analysis revealed that PTH (10(-8)M, 12-24h) treatment increases the number of cells in the G0/G1 phase and diminishes the number in both phases S and G2/M. In addition, analysis by Western blot showed that the hormone increases the expression of the inhibitory protein p27Kip1 and diminishes the expression of cyclin D1, cyclin D3 and CDK6. However, the amounts of CDK4, p21Cip1, p15INK4B and p16INK4A were not different in the absence or presence of PTH. Inhibitors of PKC (Ro-318220, bisindolylmaleimide and chelerythine), but not JNK (SP600125) and PP2A (okadaic acid and calyculin A), reversed PTH response in Caco-2 cells. Taken together, our results suggest that PTH induces G0/G1 phase arrest of Caco-2 intestinal cells and changes the expression of proteins involved in cell cycle regulation via the PKC signaling pathway.