All-trans retinoic acid targets gastric cancer stem cells and inhibits patient-derived gastric carcinoma tumor growth.

Gastric carcinoma is the third leading cause of cancer-related death worldwide. This cancer, most of the time metastatic, is essentially treated by surgery associated with conventional chemotherapy, and has a poor prognosis. The existence of cancer stem cells (CSC) expressing CD44 and a high aldehyde dehydrogenase (ALDH) activity has recently been demonstrated in gastric carcinoma and has opened new perspectives to develop targeted therapy. In this study, we evaluated the effects of all-trans-retinoic acid (ATRA) on CSCs in human gastric carcinoma. ATRA effects were evaluated on the proliferation and tumorigenic properties of gastric carcinoma cells from patient-derived tumors and cell lines in conventional 2D cultures, in 3D culture systems (tumorsphere assay) and in mouse xenograft models. ATRA inhibited both tumorspheres initiation and growth in vitro, which was associated with a cell-cycle arrest through the upregulation of cyclin-dependent kinase (CDK) inhibitors and the downregulation of cell-cycle progression activators. More importantly, ATRA downregulated the expression of the CSC markers CD44 and ALDH as well as stemness genes such as Klf4 and Sox2 and induced differentiation of tumorspheres. Finally, 2 weeks of daily ATRA treatment were sufficient to inhibit gastric tumor progression in vivo, which was associated with a decrease in CD44, ALDH1, Ki67 and PCNA expression in the remaining tumor cells. Administration of ATRA appears to be a potent strategy to efficiently inhibit tumor growth and more importantly to target gastric CSCs in both intestinal and diffuse types of gastric carcinoma.

Helicobacter pylori generates cells with cancer stem cell properties via epithelial-mesenchymal transition-like changes.

Helicobacter pylori infection is the major risk factor for gastric adenocarcinoma. The link with gastric adenocarcinoma is partly due to the H. pylori CagA oncoprotein. CagA is responsible for a particular cell phenotype in vitro, the 'hummingbird' phenotype, that corresponds to an elongation of the cells, mimicking an epithelial-mesenchymal transition (EMT). EMT participates in the carcinogenesis process, and is involved in the generation of cancer stem cells (CSCs). However, its involvement in gastric carcinogenesis has yet not been studied. Therefore, the aim of this study was to determine the role of H. pylori in EMT and in the emergence of gastric CSCs. For this purpose, gastric epithelial cells were cocultured with a cagA-positive H. pylori strain or its isogenic-deleted mutants or were transfected with CagA expression vectors. Study of the expression of epithelial and mesenchymal markers showed that H. pylori, via CagA, is responsible for an EMT phenotype associated with an increase in mesenchymal markers as well as CD44 expression, a known gastric CSC marker. Moreover, infection led to an increased ability to migrate, to invade and to form tumorspheres. Cell sorting experiments showed that only the CD44(high) cells induced by H. pylori infection displayed the mesenchymal phenotype and CSC properties in vitro, and had higher tumorigenic properties than CD44(low) cells in xenografted mice. Immunohistochemistry analyses on human and mouse gastric mucosa tissue samples confirmed a high expression of CD44 and mesenchymal markers in H. pylori-infected cases, and in gastric dysplasia and carcinoma. All of these data suggest that H. pylori, via CagA, unveils CSC-like properties by induction of EMT-like changes in gastric epithelial cells.

Helicobacter pylori and gastric cancer: possible role of microRNAs in this intimate relationship.

Chronic infection by Helicobacter pylori is a major risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. H. pylori possesses a set of virulence factors, including the CagA effector, which interferes with intracellular signalling pathways and mediates phenotypic alterations, strongly evoking neoplasic transformation. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression involved in development, cell proliferation and immune responses. miRNAs are frequently altered in cancers, revealing their functions as oncogenes or tumour suppressors. However, the role, if any, that miRNAs play in the host cell responses to H. pylori remains unknown. This review considers the possible involvement of some miRNAs, including miR-146, miR-155, miR-21, miR-27a, miR-106-93-25 and miR-221-222 clusters and the miR-200 family in H. pylori-induced infection and gastric cancers. Further exploration of miRNA-mediated gene silencing, taking into account the relationship between host targets and bacterial effectors, will most certainly bring new insights into the control of gene expression in human gastric cells chronically infected by H. pylori.

Absence of Bcl-xL down-regulation in response to cisplatin is associated with chemoresistance in ovarian carcinoma cells.

OBJECTIVE: Recurrence and subsequent acquired chemoresistance to platinum-based treatments constitute major hurdles to ovarian carcinoma therapy. Our objective was to examine the involvement of Bcl-xL anti-apoptotic protein in resistance to cisplatin. METHODS: We described the effect of cisplatin on cell cycle and apoptosis induction in sensitive (IGROV1 and OAW42) and resistant (IGROV1-R10 and SKOV3) ovarian carcinoma cell lines. We correlated it with Bcl-xL mRNA and protein expression after exposure to cisplatin. We then used bcl-xS gene transfer to impede Bcl-xL activity. RESULTS: Our study showed that Bcl-xL basal expression was high in both sensitive and resistant cell lines, as well as in all the studied ovarian tumor samples. Thus, Bcl-xL basal expression could not allow to predict sensitivity. Wondering whether variation of Bcl-xL level in response to cisplatin could be a better determinant of sensitivity, we investigated the expression of this protein in the cell lines after treatment. Cisplatin-induced down-regulation of Bcl-xL was strictly associated with apoptosis and absence of recurrence in vitro. Conversely, the maintenance of Bcl-xL expression in response to cisplatin appeared as a sine qua non condition to escape to treatment. To try to sensitize SKOV3 cells by impeding anti-apoptotic activity of Bcl-xL, we transfected bcl-xS gene in these cells. Bcl-xS exogenous expression was only slightly cytotoxic on its own, but highly sensitized SKOV3 resistant cells to cisplatin-induced apoptosis, and delayed recurrence. CONCLUSION: This work thus provides one more argument to put Bcl-xL forward as a pertinent target of inhibition to overcome chemoresistance of epithelial ovarian carcinoma.

The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells.

The seriousness of ovarian cancer, which is related to the observed link between recurrency and cell cycle control defect, prompted us to explore the effect of ectopic expression of the cdk inhibitor p21(cip1/waf1) on ovarian carcinoma chemosensitivity. The transfection of p21(cip1/waf1) cDNA into SKOV3 and OVCAR3 cells led to reduction of tumor cell growth, enhanced susceptibility to cisplatin-induced apoptosis, and abolition of recurrency after cisplatin exposure. p21(cip1/waf1) gene transfer allowed a marked reduction of the cisplatin concentration needed to erradicate the tumor cell population. These results suggest exploring the possible use of p21(cip1/waf1) as an adjunctive to conventional chemotherapy.

Human ovarian adenocarcinoma cells synthesize vitronectin and use It to organize their adhesion.

Extracellular matrix components and integrin receptors are frequently altered in cancer, including ovarian adenocarcinoma. Vitronectin (Vn) is a matrix protein mainly synthesized by liver cells; it is present in normal ovarian surface epithelium and differentiated ovarian adenocarcinoma, but is frequently undetectable in undifferentiated carcinoma (F. Carreiras et al., 1996, Gynecol Oncol 62:260-267). Wondering about the cellular origin of Vn in ovarian carcinoma, we searched for evidence of Vn synthesis by these tumors. We demonstrated that three human ovarian adenocarcinoma cell lines were able to synthesize Vn, as revealed by the presence of Vn mRNA and the protein. The Vn matrix promotes adhesion of ovarian tumor cells through alphav integrins. Moreover, during in vitro growth, Vn is progressively organized into a particular pattern in combination with the recruitment of alphav into focal contacts. Our results suggest that Vn synthesis may participate in ovarian adenocarcinoma cell biology and raise the possibility that altered expression of Vn in some ovarian carcinomas could result from a defect in Vn synthesis.

Acquisition of chemoresistance in a human ovarian carcinoma cell is linked to a defect in cell cycle control.

Chemoresistance is a major concern in cancer erradication; it involves various mechanisms, including defects in the apoptosis program induced by anticancer drugs. In order to further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin treatment, we established an in vitro model, mimicking a clinical protocol of administration of cisplatin. Therefore, IGROV1 ovarian carcinoma cells were exposed for 2 hr to the drug and allowed to recover for several weeks; this way of exposure was reiterated with escalating doses. We followed changes in cytotoxicity of the drug, cell cycle kinetics and long-term survival of cells after cisplatin treatment, and found that resistance to cisplatin was not associated with altered apoptosis pathway, since both cisplatin sensitive and resistant cells underwent apoptosis in a similar way. Acquisition of resistance to cisplatin was associated with the ability of the treated cells to progress through the cell cycle beyond the G1/S checkpoint; although most cells died by apoptosis, a few surviving cells proliferated and recolonized the cultures. Compared to sensitive cells, the chemoresistant variants were able to override the G1/S checkpoint whatever the dose, and the recurrent cells recolonized the cultures much faster. Analysis of alterations in gene expression suggests that the defect in cell cycle regulation could take place at the level of the cdk inhibitor p21(CIP1/WAF1).

Lipospermine-mediated gene transfer technique into murine cultured cortical cells.

In order to transfer exogenous DNA into embryonic cortical cells, we have chosen a transfection technique using a synthetic lipospermine (dipalmitoylphosphatidylethanolamylspermine, DPPES) which complexes DNA molecules and allows their penetration into the intracellular compartment. The procedure was optimized after testing several parameters: DPPES/DNA ratio, incubation time, kinetics of transgene expression, and growth medium. The protocol was achieved by following the expression of the E. coli LacZ reporter gene under the control of the cytomegalovirus promoter. The lipopolyamine-mediated transfection is efficient for terminally differentiated cells, since we routinely obtained transfection efficiencies of 30% for neurons.

Expression and localization of alpha v integrins and their ligand vitronectin in normal ovarian epithelium and in ovarian carcinoma.

In an extension of a previous in vitro study [Carreiras et al., Int. J. Cancer 63, 530-536 (1995)] and in an effort to understand the adhesive interactions mediated by integrins within epithelial ovarian tumors, the presence of the alpha v and beta 3 subunits and that of vitronectin (Vn) in ovarian carcinomas at various stages of differentiation and in normal ovarian epithelium were comparatively investigated. The study was performed on material from 34 patients. By immunofluorescence, cryostat sections were analyzed for their expression of alpha v (34 cases), beta 3 (19 cases), and Vn (29 cases). alpha v was expressed in normal epithelium and in highly differentiated tumors as well as in a majority of moderately and poorly differentiated carcinomas with identical staining pattern. beta 3 subunit and Vn were also expressed in normal cases and highly differentiated carcinomas. However, they were lacking in most of the less differentiated tumors. The analysis of cases which were simultaneously tested for the presence of alpha v, beta 3, and Vn revealed that a large proportion of normal ovarian epithelium and highly differentiated tumors simultaneously expressed alpha v, beta 3, and Vn; in contrast, in all moderately and poorly differentiated carcinomas either beta 3 or Vn was absent. The potential role of the alpha v beta 3/Vn system in ovarian epithelium functions is discussed. It is also speculated that modifications of this system in ovarian carcinomas might contribute to tumor progression.

Dietary fish oil and olive oil improve the liver insulin receptor tyrosine kinase activity in high sucrose fed rats.

In order to shed light on the possible beneficial effect of dietary unsaturated fatty acids on insulin binding, the effect of fish oil and olive oil administration on insulin binding, autophosphorylation and tyrosine kinase activity of partially purified liver insulin receptors were investigated. These data were confronted with the parameters of sugar and lipid metabolism (blood glucose, insulin and triglycerides), with liver plasma membrane fluidity and fatty acid composition. High sucrose feeding resulted in the elevation of blood glucose and triglyceride level, while the supplementation of animals with fish oil reduced that of triglycerides and olive oil that of insulin. Any significant changes between experimental groups were not detected either in insulin binding to partially purified liver insulin receptor nor in receptor autophosphorylation. However, the insulin stimulated tyrosine kinase activity towards an exogenous substrate (poly(Glu,Tyr)) was decreased by about 50% in the receptors solubilized from liver membranes of sucrose fed rats. Increased dietary intake of fish oil or olive oil restored the activity of insulin tyrosine kinase towards control values, half maximal effect being obtained at similar insulin concentration in all groups. Such improvement might be due to the induced increase of membrane fluidity by unsaturated fatty acids, and/or to the decrease of insulinemia.

High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine:DNA complexes.

The ability to introduce DNA into mammalian cells has provided a powerful means to examine the regulation of gene expression and the function of gene products. However, the most commonly used techniques for DNA transfection are not always suitable for primary cells. Primary human keratinocytes are particularly stringent in their growth requirements and are also very refractory to transfection, rendering transient gene expression studies difficult. We have investigated the ability of several polycationic lipids to promote DNA uptake into human epidermal keratinocytes, as monitored with the bacterial beta-galactosidase reporter gene. We report that the cationic lipopolyamine dipalmitoyl phosphatidylethanolamine spermine as well as another procedure using Polybrene can achieve a 20% to 30% transfection efficiency, superior to any other agent tested on these cells. Gene transfer was accomplished by a 3-h exposure of monolayer cells to DNA complexes formed with either reagent by simple mixing in a serum-free medium, followed by a brief osmotic shock with glycerol. Neither DNA carrier showed any toxicity at the effective concentrations nor interfered with cell attachment, growth or differentiation. The use of a fully biodegradable lipopolyamine as DNA carrier should make it possible to extend this transfection method to gene transfer for in vivo therapeutic applications.

Reconstitution studies of lipid effects on insulin-receptor kinase activation.

Insulin receptors extracted from human placenta were reconstituted by dialysis into well-characterized lipid vesicles. For all types of lipids studied, vesicles were shown to be unilamellar, about 120 nm in diameter. The incorporation of lectin-purified insulin receptors was assessed by cosedimentation of 125I-insulin binding and [32P]phospholipids in a sucrose gradient. The insulin-binding activity was not modified by the composition of the lipid vesicles. However, tyrosine kinase activation appeared to be more sensitive to its lipid environment. Mixtures of phosphatidylcholine/phosphatidylserine or phospholipids/phosphatidylserine, in ratios of 1-4, increased the insulin-induced tyrosine kinase activation in a dose-dependent manner. In contrast, experiments performed in the presence of phosphatidylinositol showed a decrease in the enzyme stimulation. These results indicate an opposing involvement of these two anionic phospholipids in the kinase activation. Inclusion of cholesterol (10-30%) into phosphatidylcholine vesicles reduced kinase activation, which was drastically inhibited by 30% cholesterol. The effect of a total extract of brain gangliosides was biphasic, stimulatory at low concentration (5-10%), but with a reverse effect at higher concentrations. These results stress the importance of the lipid environment for insulin-receptor signaling, particularly for the insulin-induced activation of its beta-subunit kinase.

Influence of cholesterol derivatives on cytoskeletal organization of human carcinoma cells.

Recent developments of immunotherapeutic approaches have shown that artificial ordering of tumor cell membranes with cholesterol hemisuccinate (CHS) or 25-hydroxycholesterol (25-OH) may significantly enhance the immunogenicity of human renal adenocarcinoma cells. To gain further insight into the molecular mechanism of these sterols, we investigated cytoskeletal modification, which is related to the cell membrane. After treatment of human renal carcinoma cells with these cholesterol (at 10(-6) and 10(-7) M) for 5 days, we observed a disorganization of the submembrane end of the cytoplasmic actin stress fibers by cytofluorescence. The microtubule network was not affected. Thus, in the present study, we found that changes in membrane physicochemical properties impaired the anchorage of actin microfilaments in the plasma membrane of human renal cancer cells. Under the same experimental conditions, such modifications were not observed in normal cells (human fibroblasts) or in human hepatoma cells. We suggest that incubation of cancer cells with these sterols induced a redistribution of the cholesterol-rich membrane microdomains which are linked to the cytoskeleton through submembrane proteins.

Modifications of ependymal cells membranes by galactocerebrosides in cell culture.

In this paper we have demonstrated that treatment of ependymal cells in culture by galactocerebrosides induced a decrease in plasma membrane fluidity and an increase of EGF binding sites. We have shown in a previous work that galactocerebroside in vitro and in vivo caused an important morphological change in ependymal cells that grew into an astrocytic shape after a five day treatment. We discuss the hypothesis that the first event in morphological effect could be a modification of plasma membrane followed by important changes in molecules distribution.

Detergents affect insulin binding, tyrosine kinase activity and oligomeric structure of partially purified insulin receptors.

Insulin receptor activities, i.e., insulin binding and tyrosine kinase activation depend on the lipid environment of the receptor. As detergent may disrupt or interfere with this environment, we investigated the effect of various common detergents on insulin receptor properties. Experiments were carried out (i) on solubilized and partially purified insulin receptor and (ii) on the receptor reconstituted into phosphatidylcholine vesicles. The detergents tested, Triton X-100, octyl-beta-D-glucopyranoside, octyl-beta-D-thioglucopyranoside, 3[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Chaps), and Na deoxycholate affected the insulin receptor properties differently when compared with the control receptor in the absence of detergent. On the partially purified insulin receptor, Na deoxycholate inhibited both insulin receptor activities; octyl-beta-D-glucopyranoside and octyl-beta-D-thioglucopyranoside decreased insulin binding and kinase activation as their concentration increased, particularly above their respective critical micellar concentration (CMC). Triton X-100 was the only detergent which allowed an increase of insulin binding and kinase activation throughout the whole range of concentrations assayed. Reconstitution of the receptor into phosphatidylcholine vesicles protected the receptor from the direct effects of the detergents, for both the stimulation observed with Triton X-100 and the inhibition produced by the other detergents. In order to determine the effect of detergents on the oligomeric forms of the soluble insulin receptor, we investigated a new rapid sucrose gradient centrifugation technique. Insulin receptors were detected on the gradient by 125I insulin binding. For low concentrations of detergent, i.e., near the CMC, octylglucoside, Chaps, and Triton X-100 favored the (alpha 2 beta 2)2 oligomeric form of the receptor. Higher concentrations of Triton X-100 did not modify the polymeric state of the receptor. In contrast, octylglucoside and Chaps induced an increase in the sedimentation coefficient of the receptor which appeared as (alpha 2 beta 2)3 and (alpha 2 beta 2)4 forms. These alterations in the oligomerization status of the insulin receptor may explain the deleterious effects observed with both Chaps and octylglucoside at higher concentrations.

Encapsulation of insulin for oral administration preserves interaction of the hormone with its receptor in vitro.

It has been shown that insulin associated with nanocapsules of isobutylcyanoacrylate retains biological activity after oral administration to diabetic rats from 6 to 21 days. Because part of this action is unexplained, we focused on the interaction of encapsulated insulin with the insulin receptor in vitro. We have shown that encapsulated insulin is able 1) to bind to insulin receptors both in rat liver plasma membranes and after solubilization from Chinese hamster ovary (CHO) cells transfected with the gene of human insulin receptor, 2) to accelerate 125I-labeled insulin dissociation from its receptor, and 3) to ensure transduction of a signal leading to stimulation of the beta-subunit phosphorylation, with parameters similar to those of native insulin. In addition, encapsulated 125I-insulin was rapidly internalized in transfected CHO cells. Analysis of cell-associated radioactivity showed that encapsulated insulin remained largely intact (greater than 80%) after 3 h, whereas native insulin was mostly degraded. These data indicate that encapsulated insulin fulfills all the earliest events at the receptor level leading to biological actions and suggests that encapsulation protects insulin against insulin degradation inside the cells.

Malignant transformation in hepatocytes is associated with the general increase of glycoprotein ligands specifically binding to the endogenous lectin CSL.

Several hepatoma cell lines and hepatic ascite tumour cells were studied for the presence of glycoprotein ligands of an endogenous lectin, the "Cerebellar Soluble Lectin" (CSL). This lectin is also present in hepatocytes in vivo and in vitro and can be detected biochemically and immunologically. In transformed cells, the level of CSL glycoprotein ligands is increased 50-fold as compared to the control cells. Such an increase is not observed for the ligands of the plant lectin, concanavalin A, which is, as CSL, a D-mannose-binding lectin. These results indicated that the changes in glycans during malignant transformation, in these cells, is specifically important for minor glycans binding to CSL.