Flow cytometric evaluation of measurable residual disease in chronic lymphocytic leukemia: Where do we stand?

Measurable residual disease (MRD) has emerged as a relevant parameter of response to therapy in chronic lymphocytic leukemia (CLL). Although several methods have been developed, flow cytometry has emerged as the most useful and standardized approach to measure and quantify MRD. The improved sensitivity of MRD measurements has been paralleled by the development of more effective therapeutic strategies for CLL, increasing the applicability of MRD detection in this setting. Chemotherapy and chemoimmunotherapy have firstly demonstrated their ability to obtain a deep MRD. Combined targeted therapies are also demonstrating a high molecular response rate and prospective trials are exploring the role of MRD to guide the duration of treatment in this setting. In this review we briefly summarize what we have learned about MRD with emphasis on its flow cytometric detection.

Cytotoxic Potential of the Marine Diatom Thalassiosira rotula: Insights into Bioactivity of 24-Methylene Cholesterol.

Marine microalgae are receiving great interest as sustainable sources of bioactive metabolites for health, nutrition and personal care. In the present study, a bioassay-guided screening allowed identifying an enriched fraction from SPE separation of the methanolic extract of the marine diatom Thalassiosira rotula with a chemically heterogeneous composition of cytotoxic molecules, including PUFAs, the terpene phytol, the carotenoid fucoxanthin and the phytosterol 24-methylene cholesterol (24-MChol). In particular, this latter was the object of deep investigation aimed to gain insight into the mechanisms of action activated in two tumour cell models recognised as resistant to chemical treatments, the breast MCF7 and the lung A549 cell lines. The results of our studies revealed that 24-MChol, in line with the most studied ß-sitosterol (ß-SIT), showed cytotoxic activity in a 3-30 µM range of concentration involving the induction of apoptosis and cell cycle arrest, although differences emerged between the two sterols and the two cancer systems when specific targets were investigated (caspase-3, caspase-9, FAS and TRAIL).

MicroRNA-33 and SIRT1 influence the coronary thrombus burden in hyperglycemic STEMI patients.

Primary percutaneous coronary intervention (PPCI) is a pivotal treatment in ST-segment elevation myocardial infarction (STEMI) patients. However, in hyperglycemic-STEMI patients, the incidence of death is still significant. Here, the involvement of sirtuin 1 (SIRT1) and miR33 on the pro-inflammatory/pro-coagulable state of the coronary thrombus was investigated. Moreover, 1-year outcomes in hyperglycemic STEMI in patients subjected to thrombus aspiration before PPCI were evaluated. Results showed that hyperglycemic thrombi displayed higher size and increased miR33, reactive oxygen species, and pro-inflammatory/pro-coagulable markers. Conversely, the hyperglycemic thrombi showed a lower endothelial SIRT1 expression. Moreover, in vitro experiments on endothelial cells showed a causal effect of SIRT1 modulation on the pro-inflammatory/pro-coagulative state via hyperglycemia-induced miR33 expression. Finally, SIRT1 expression negatively correlated with STEMI outcomes. These observations demonstrate the involvement of the miR33/SIRT1 pathway in the increased pro-inflammatory and pro-coagulable state of coronary thrombi in hyperglycemic STEMI patients.

Hybrid Complexes of High and Low Molecular Weight Hyaluronans Highly Enhance HASCs Differentiation: Implication for Facial Bioremodelling.

BACKGROUND/AIMS: Adipose-derived Stem Cells (ASCs) are used in Regenerative Medicine, including fat grafting, recovery from local tissue ischemia and scar remodeling. The aim of this study was to evaluate hyaluronan based gel effects on ASCs differentiation and proliferation. METHODS: Comparative analyses using high (H) and low (L) molecular weight hyaluronans (HA), hyaluronan hybrid cooperative complexes (HCCs), and high and medium cross-linked hyaluronan based dermal fillers were performed. Human ASCs were characterized by flow cytometry using CD90, CD34, CD105, CD29, CD31, CD45 and CD14 markers. Then, cells were treated for 7, 14 and 21 days with hyaluronans. Adipogenic differentiation was evaluated using Oil red-O staining and expression of leptin, PPAR-γ, LPL and adiponectin using qRT-PCR. Adiponectin was analyzed by immunofluorescence, PPAR-γ and adiponectin were analyzed using western blotting. ELISA assays for adiponectin and leptin were performed. RESULTS: HCCs highly affected ASCs differentiation by up-regulating adipogenic genes and related proteins, that were also secreted in the culture medium. H-HA and L-HA induced a lower level of ASCs differentiation. CONCLUSION: HCCs-based formulations clearly enhance adipogenic differentiation and proliferation, when compared with linear HA and cross-linked hyaluronans. Injection of HCCs in subdermal fat compartment may recruit and differentiate stem cells in adipocytes, and considerably improving fat tissue renewal.

Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering.

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration.

Biotechnological Chondroitin a Novel Glycosamminoglycan With Remarkable Biological Function on Human Primary Chondrocytes.

Cartilage tissue engineering, with in vitro expansion of autologus chondrocytes, is a promising technique for tissue regeneration and is a new potential strategy to prevent and/or treat cartilage damage (e.g., osteoarthritis). The aim of this study was (i) to investigate and compare the effects of new biotechnological chondroitin (BC) and a commercial extractive chondroitin sulfate (CS) on human chondrocytes in vitro culture; (ii) to evaluate the anti-inflammatory effects of the innovative BC compared to extractive CS. A chondrogenic cell population was isolated from human nasoseptal cartilage and in vitro cultures were studied through time-lapse video microscopy (TLVM), immunohistochemical staining and cytometry. In order to investigate the effect of BC and CS on phenotype maintainance, chondrogenic gene expression of aggrecan (AGN), of the transcriptor factor SOX9, of the types I and II collagen (COL1A1 and COL1A2), were quantified through transcriptional and protein evaluation at increasing cultivation time and passages. In addition to resemble the osteoarthritis-like in vitro model, chondrocytes were treated with IL-1ß and the anti-inflammatory activity of BC and CS was assessed using cytokines quantification by multiplex array. BC significantly enhances cell proliferation also preserving chondrocyte phenotype increasing type II collagen expression up to 10 days of treatment and reduces inflammatory response in IL-1ß treated chondrocytes respect to CS treated cells. Our results, taken together, suggest that this new BC is of foremost importance in translational medicine because it can be applied in novel scaffolds and pharmaceutical preparations aiming at cartilage pathology treatments such as the osteoarthritis. J. Cell. Biochem. 117: 2158-2169, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

In vitro analysis of the effects on wound healing of high- and low-molecular weight chains of hyaluronan and their hybrid H-HA/L-HA complexes.

BACKGROUND: Recent studies have reported the roles of Hyaluronic acid (HA) chains of diverse length in wound repair, especially considering the simultaneous occurrence in vivo of both high- (H-HA) and low-molecular weight (L-HA) hyaluronan at an injury site. It has been shown that HA fragments (5 ≤ MW ≤ 20 kDa) usually trigger an inflammatory response that, on one hand, is the first signal in the activation of a repair mechanism but on the other, when it's overexpressed, it may promote unwanted side effects. The present experimental research has aimed to investigate H-HA, L-HA and of a newly developed complex of the two (H-HA/L-HA) for stability (e.g. hyaluronidases digestion), for their ability to promote wound healing of human keratinocytes in vitro and for their effect on cellular biomarker expression trends. RESULTS: Time-lapse video microscopy studies proved that the diverse HA was capable of restoring the monolayer integrity of HaCat. The H-HA/L-HA complex (0.1 and 1%w/v) proved faster in regeneration also in co-culture scratch test where wound closure was achieved in half the time of H-HA stimulated cells and 2.5-fold faster than the control. Gene expression was evaluated for transformation growth factor beta 1 (TGF-ß1) proving that L-HA alone increased its expression at 4 h followed by restoration of similar trends for all the stimuli. Depending on the diverse stimulation (H-HA, L-HA or the complex), metalloproteinases (MMP-2, -9, -13) were also modulated differently. Furthermore, type I collagen expression and production were evaluated. Compared to the others, persistence of a significant higher expression level at 24 h for the H-HA/L-HA complex was found. CONCLUSIONS: The outcomes of this research showed that, both at high and low concentrations, hybrid complexes proved to perform better than HA alone thus suggesting their potential as medical devices in aesthetic and regenerative medicine.

Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: evidence for HDAC2 involvement.

Adult mesenchymal stem cells, such as dental pulp stem cells, are of great interest for cell-based tissue engineering strategies because they can differentiate into a variety of tissue-specific cells, above all, into osteoblasts. In recent years, epigenetic studies on stem cells have indicated that specific histone alterations and modifying enzymes play essential roles in cell differentiation. However, although several studies have reported that valproic acid (VPA)-a selective inhibitor of histone deacetylases (HDAC)-enhances osteoblast differentiation, data on osteocalcin expression-a late-stage marker of differentiation-are limited. We therefore decided to study the effect of VPA on dental pulp stem cell differentiation. A low concentration of VPA did not reduce cell viability, proliferation, or cell cycle profile. However, it was sufficient to significantly enhance matrix mineralization by increasing osteopontin and bone sialoprotein expression. In contrast, osteocalcin levels were decreased, an effect induced at the transcriptional level, and were strongly correlated with inhibition of HDAC2. In fact, HDAC2 silencing with shRNA produced a similar effect to that of VPA treatment on the expression of osteoblast-related markers. We conclude that VPA does not induce terminal differentiation of osteoblasts, but stimulates the generation of less mature cells. Moreover, specific suppression of an individual HDAC by RNA interference could enhance only a single aspect of osteoblast differentiation, and thus produce selective effects.

Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization.

Primary tumors are responsible for 10% of cancer deaths. In most cases, the main cause of mortality is the formation of metastases. Accumulating evidence suggests that a subpopulation of tumor cells with distinct stem-like properties is responsible for tumor initiation, invasive growth, and metastasis formation. This population is defined as cancer stem cells (CSCs). Existing therapies have enhanced the length of survival after diagnosis of cancer but have completely failed in terms of recovery. CSCs appear to be resistant to chemotherapy, may remain quiescent for extended periods, and have affinity for hypoxic environments. The CSCs can be identified and isolated by different methodologies, including isolation by CSC-specific cell surface marker expression, detection of side population phenotype by Hoechst 33342 exclusion, assessment of their ability to grow as floating spheres, and aldehyde dehydrogenase (ALDH) activity assay. None of the methods mentioned are exclusively used to isolate the solid tumor CSCs, highlighting the imperative to delineate more specific markers or to use combinatorial markers and methodologies. This review provides an overview of the main characteristics and approaches used to identify, isolate, and characterize CSCs from solid tumors.

Isolating Cancer Stem Cells from Solid Tumors.

Over the past 20 years, there has been a lot of interest in the study and investigation of cancer stem cells (CSCs) or tumor-initiating cells (TICs). CSCs are rare, dormant cells and able to self-renew and maintain tumor development and heterogeneity. A new age of basic and clinical cancer research, reclassification of human tumors, and the development of novel therapeutic approaches will undoubtedly result from a better knowledge of CSCs. In order to develop effective and therapeutic strategies to treat cancer, it is crucial to understand the basic characteristics of CSCs, their importance to cancer therapy, and methodologies to isolate, detect, and characterize them. Here, we outline the main methods and protocols to identify, isolate, and culture CSCs from primary tumors.

ALDH Activity Assay: A Method for Cancer Stem Cell (CSC) Identification and Isolation.

Cancer stem cells (CSCs) are a small tumor cell subpopulation, driving cancer initiation, progression, multidrug resistance, and metastasis. Several methods are used to detect and isolate CSCs by flow cytometry. Among these, measurement of aldehyde dehydrogenase (ALDH) activity within the cell is an assay widely used to identify and isolate CSCs from different types of solid tumors. The aldehyde dehydrogenase (ALDH) is a polymorphic enzyme responsible for the oxidation of aldehydes to carboxylic acids, overexpressed both in normal and cancer stem cells. In this chapter, it is described how CSCs are detected and isolated by using ALDH activity assay.

ITGB1 and DDR activation as novel mediators in acquired resistance to osimertinib and MEK inhibitors in EGFR-mutant NSCLC.

Osimertinib is a third-generation tyrosine kinase inhibitor clinically approved for first-line treatment of EGFR-mutant non-small cell lung cancer (NSCLC) patients. Although an impressive drug response is initially observed, in most of tumors, resistance occurs after different time and an alternative therapeutic strategy to induce regression disease is currently lacking. The hyperactivation of MEK/MAPKs, is one the most common event identified in osimertinib-resistant (OR) NSCLC cells. However, in response to selective drug pressure, the occurrence of multiple mechanisms of resistance may contribute to treatment failure. In particular, the epithelial-to-mesenchymal transition (EMT) and the impaired DNA damage repair (DDR) pathways are recognized as additional cause of resistance in NSCLC thus promoting tumor progression. Here we showed that concurrent upregulation of ITGB1 and DDR family proteins may be associated with an increase of EMT pathways and linked to both osimertinib and MEK inhibitor resistance to cell death. Furthermore, this study demonstrated the existence of an interplay between ITGB1 and DDR and highlighted, for the first time, that combined treatment of MEK inhibitor with DDRi may be relevant to downregulate ITGB1 levels and increase cell death in OR NSCLC cells.

PBMCs as Tool for Identification of Novel Immunotherapy Biomarkers in Lung Cancer.

BACKGROUND: Lung cancer (LC), including both non-small (NSCLC) and small (SCLC) subtypes, is currently treated with a combination of chemo- and immunotherapy. However, predictive biomarkers to identify high-risk patients are needed. Here, we explore the role of peripheral blood mononuclear cells (PBMCs) as a tool for novel biomarkers searching. METHODS: We analyzed the expression of the cGAS-STING pathway, a key DNA sensor that activates during chemotherapy, in PBMCs from LC patients divided into best responders (BR), responders (R) and non-responders (NR). The PBMCs were whole exome sequenced (WES). RESULTS: PBMCs from BR and R patients of LC cohorts showed the highest levels of STING (p < 0.0001) and CXCL10 (p < 0.0001). From WES, each subject had at least 1 germline/somatic alteration in a DDR gene and the presence of more DDR gene mutations correlated with clinical responses, suggesting novel biomarker implications. Thus, we tested the effect of the pharmacological DDR inhibitor (DDRi) in PBMCs and in three-dimensional spheroid co-culture of PBMCs and LC cell lines; we found that DDRi strongly increased cGAS-STING expression and tumor infiltration ability of immune cells in NR and R patients. Furthermore, we performed FACS analysis of PBMCs derived from LC patients from the BR, R and NR cohorts and we found that cytotoxic T cell subpopulations displayed the highest STING expression. CONCLUSIONS: cGAS-STING signaling activation in PBMCs may be a novel potential predictive biomarker for the response to immunotherapy and high levels are correlated with a better response to treatment along with an overall increased antitumor immune injury.

Mitochondrial transfer from Adipose stem cells to breast cancer cells drives multi-drug resistance.

BACKGROUND: Breast cancer (BC) is a complex disease, showing heterogeneity in the genetic background, molecular subtype, and treatment algorithm. Historically, treatment strategies have been directed towards cancer cells, but these are not the unique components of the tumor bulk, where a key role is played by the tumor microenvironment (TME), whose better understanding could be crucial to obtain better outcomes. METHODS: We evaluated mitochondrial transfer (MT) by co-culturing Adipose stem cells with different Breast cancer cells (BCCs), through MitoTracker assay, Mitoception, confocal and immunofluorescence analyses. MT inhibitors were used to confirm the MT by Tunneling Nano Tubes (TNTs). MT effect on multi-drug resistance (MDR) was assessed using Doxorubicin assay and ABC transporter evaluation. In addition, ATP production was measured by Oxygen Consumption rates (OCR) and Immunoblot analysis. RESULTS: We found that MT occurs via Tunneling Nano Tubes (TNTs) and can be blocked by actin polymerization inhibitors. Furthermore, in hybrid co-cultures between ASCs and patient-derived organoids we found a massive MT. Breast Cancer cells (BCCs) with ASCs derived mitochondria (ADM) showed a reduced HIF-1α expression in hypoxic conditions, with an increased ATP production driving ABC transporters-mediated multi-drug resistance (MDR), linked to oxidative phosphorylation metabolism rewiring. CONCLUSIONS: We provide a proof-of-concept of the occurrence of Mitochondrial Transfer (MT) from Adipose Stem Cells (ASCs) to BC models. Blocking MT from ASCs to BCCs could be a new effective therapeutic strategy for BC treatment.

Human Ng2+ adipose stem cells loaded in vivo on a new crosslinked hyaluronic acid-Lys scaffold fabricate a skeletal muscle tissue.

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross-linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2(+) ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2(-) ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT-PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut-4, and PPAR-γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2(+) ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre-differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration.

Human adipose CD34+ CD90+ stem cells and collagen scaffold constructs grafted in vivo fabricate loose connective and adipose tissues.

Stem cell based therapies for the repair and regeneration of various tissues are of great interest for a high number of diseases. Adult stem cells, instead, are more available, abundant and harvested with minimally invasive procedures. In particular, mesenchymal stem cells (MSCs) are multi-potent progenitors, able to differentiate into bone, cartilage, and adipose tissues. Human adult adipose tissue seems to be the most abundant source of MSCs and, due to its easy accessibility; it is able to give a considerable amount of stem cells. In this study, we selected MSCs co-expressing CD34 and CD90 from adipose tissue. This stem cell population displayed higher proliferative capacity than CD34(-) CD90(-) cells and was able to differentiate in vitro into adipocytes (PPARγ(+) and adiponectin(+)) and endothelial cells (CD31(+) VEGF(+) Flk1(+)). In addition, in methylcellulose without VEGF, it formed a vascular network. The aim of this study was to investigate differentiation potential of human adipose CD34(+) /CD90(+) stem cells loaded onto commercial collagen sponges already used in clinical practice (Gingistat) both in vitro and in vivo. The results of this study clearly demonstrate that human adult adipose and loose connective tissues can be obtained in vivo, highlighting that CD34(+) /CD90 ASCs are extremely useful for regenerative medicine.

ß2-AR inhibition enhances EGFR antibody efficacy hampering the oxidative stress response machinery.

The ß2-Adrenergic receptor (ß2-ARs) is a cell membrane-spanning G protein-coupled receptors (GPCRs) physiologically involved in stress-related response. In many cancers, the ß2-ARs signaling drives the tumor development and transformation, also promoting the resistance to the treatments. In HNSCC cell lines, the ß2-AR selective inhibition synergistically amplifies the cytotoxic effect of the MEK 1/2 by affecting the p38/NF-kB oncogenic pathway and contemporary reducing the NRF-2 mediated antioxidant cell response. In this study, we aimed to validate the anti-tumor effect of ß2-AR blockade and the synergism with MEK/ERK and EGFR pathway inhibition in a pre-clinical orthotopic mouse model of HNSCC. Interestingly, we found a strong ß2-ARs expression in the tumors that were significantly reduced after prolonged treatment with ß2-Ars inhibitor (ICI) and EGFR mAb Cetuximab (CTX) in combination. The ß2-ARs down-regulation correlated in mice with a significant tumor growth delay, together with the MAPK signaling switch-off caused by the blockade of the MEK/ERK phosphorylation. We also demonstrated that the administration of ICI and CTX in combination unbalanced the cell ROS homeostasis by blocking the NRF-2 nuclear translocation with the relative down-regulation of the antioxidant enzyme expression. Our findings highlighted for the first time, in a pre-clinical in vivo model, the efficacy of the ß2-ARs inhibition in the treatment of the HNSCC, remarkably in combination with CTX, which is the standard of care for unresectable HNSCC.

CD133 and CD44 cell surface markers do not identify cancer stem cells in primary human gastric tumors.

Emerging evidence suggests that tumors contain and are driven by a cellular component that displays stem cell properties, the so-called cancer stem cells (CSCs). CSCs have been identified in several solid human cancers; however, there are no data about CSCs in primary human gastric cancer (GC). By using CD133 and CD44 cell surface markers we investigated whether primary human GCs contain a cell subset expressing stem-like properties and whether this subpopulation has tumor-initiating properties in xenograft transplantation experiments. We examined tissues from 44 patients who underwent gastrectomy for primary GC. The tumorigenicity of the cells separated by flow cytometry using CD133 and CD44 surface markers was tested by subcutaneous or intraperitoneum injection in NOD/SCID and nude mice. GCs included in the study were intestinal in 34 cases and diffuse in 10 cases. All samples contained surface marker-positive cells: CD133(+) mean percentage 10.6% and CD133(+)/CD44(+) mean percentage 27.7%, irrespective of cancer phenotype or grade of differentiation. Purified CD133(+) and CD133(+)/CD44(+) cells, obtained in sufficient number only in 12 intestinal type GC cases, failed to reproduce cancer in two mice models. However, the unseparated cells produced glandular-like structures in 70% of the mice inoculated. In conclusion, although CD133(+) and CD133(+)/CD44(+) were detectable in human primary GCs, they neither expressed stem-like properties nor exhibited tumor-initiating properties in xenograft transplantation experiments.

MDR1-P-glycoprotein behaves as an oncofetal protein that promotes cell survival in gastric cancer cells.

P-glycoprotein (P-gp), traditionally linked to cancer poor prognosis and multidrug resistance, is undetectable in normal gastric mucosa and overexpressed in gastric cancer (GC). We propose that P-gp may be involved in Helicobacter pylori (Hp)-related gastric carcinogenesis by inhibiting apoptosis. Aim of the study was to evaluate the expression of P-gp in fetal stomach and in Hp-related gastric carcinogenesis, the epigenetic control of the multi-drug resistance-1 (MDR1) gene, the localization and interaction between P-gp and Bcl-x(L) and the effect of the selective silencing of P-gp on cell survival. P-gp and Bcl-xl expression was evaluated by immunohistochemistry on 28 spontaneously abortive human fetuses, 66 Hp-negative subjects, 138 Hp-positive chronic gastritis (CG) of whom 28 with intestinal metaplasia (IM) and 45 intestinal type GCs. P-gp/Bcl-x(L) colocalization was investigated by confocal immunofluorescence microscopy and protein-protein interaction by co-immunoprecipitation, in basal conditions and after stress-induced apoptosis, in GC cell lines AGS and MKN-28 and hepatocellular carcinoma cell line Hep-G2. The role of P-gp in controlling apoptosis was evaluated by knocking down its expression with a specific small interfering RNAs in stressed AGS and MKN-28 cell lines. P-gp is expressed in the gastric mucosa of all human fetuses while, it is undetectable in adult normal mucosa and re-expressed in 30/110 Hp-positive non-IM-CG, 28/28 IM-CG and 40/45 GCs. P-gp expression directly correlates with that of Bcl-x(L) and with the promoter hypomethylation of the MDR1 gene. In GC cell lines, P-gp is localized on the plasma membrane and mitochondria where it colocalizes with Bcl-x(L). Co-immunoprecipitation confirms the physical interaction between P-gp and Bcl-x(L) in AGS, MKN-28 and Hep-G2, at both basal level and after stress-induced apoptosis. The selective silencing of P-gp sensitizes GC cells to stress-induced apoptosis. P-gp behaves as an oncofetal protein that, by cross-talking with Bcl-x(L), acts as an anti-apoptotic agent in Hp-related gastric carcinogenesis.