Sandra Rizk

The Antiproliferative Effect of Kefir Cell-Free Fraction on HuT-102 Malignant T Lymphocytes

Kefir is produced by adding kefir grains (a mass of proteins, polysaccharides, bacteria, and yeast) to pasteurized milk; it has been shown to control several cellular types of cancer, such as Sarcoma 180 in mice, Lewis lung carcinoma, and human mammary cancer. Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia, which is a fatal disease with no effective treatment. The current study aims at investigating the effect of a cell-free fraction of kefir on HuT-102 cells, which are HTLV-1-positive malignant T-lymphocytes. Cells were incubated with different kefir concentrations: the cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir cell-free fraction on the proliferation of HuT-102 cells was then assessed. The levels of transforming growth factor (TGF)-alpha mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction. Finally, the growth inhibitory effects of kefir on cell cycle progression and/or apoptosis were assessed by flow cytometry. The maximum cytotoxicity recorded at 80 microg/microL for 48 hours was only 43%. The percent reduction in proliferation was very significant, dose and time dependent, and reached 98% upon 60-microg/microL treatment for 24 hours. Kefir cell-free fraction caused the downregulation of TGF-alpha, which is a cytokine that induces the proliferation and replication of cells. Finally, a marked increase in cell cycle distribution was noted in the pre-G1 phase. In conclusion, kefir is effective in inhibiting proliferation and inducing apoptosis of HTLV-1-positive malignant T-lymphocytes. Therefore, further in vivo investigation is highly recommended.

Kefir induces cell-cycle arrest and apoptosis in HTLV-1-negative malignant T-lymphocytes

Background: Adult lymphoblastic leukemia (ALL) is a malignancy that occurs in white blood cells. The overall cure rate in children is 85%, whereas it is only 40% in adults. Kefir is an important probiotic that contains many bioactive ingredients, which give it unique health benefits. It has been shown to control several cellular types of cancer. Purpose: The present study investigates the effect of a cell-free fraction of kefir on CEM and Jurkat cells, which are human T-lymphotropic virus type I (HTLV-1)-negative malignant T-lymphocytes. Methods: Cells were incubated with different kefir concentrations. The cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir on the proliferation of CEM and Jurkat cells was then assessed. The levels of transforming growth factor-alpha (TGF-α), transforming growth factor- beta1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), and MMP-9 mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, the growth inhibitory effects of kefir on cell-cycle progression/apoptosis were assessed by Cell Death Detection (ELISA) and flow cytometry. Results: The maximum cytotoxicity recorded after 48-hours treatment with 80 μg/μL kefir was only 42% and 39% in CEM and Jurkat cells, respectively. The percent reduction in proliferation was very significant, and was dose-, and time-dependent. In both cell lines, kefir exhibited its antiproliferative effect by downregulating TGF-α and upregulating TGF-β1 mRNA expression. Upon kefir treatment, a marked increase in cell-cycle distribution was noted in the preG1 phase of CEM and Jurkat cells, indicating the proapoptotic effect of kefir, which was further confirmed by Cell Death Detection ELISA. However, kefir did not affect the mRNA expression of metalloproteinases needed for the invasion of leukemic cell lines. Conclusion: In conclusion, kefir is effective in inhibiting proliferation and inducing apoptosis of HTLV-1-negative malignant T-lymphocytes. Therefore, further in vivo investigation is highly recommended.

Kefir exhibits anti‑proliferative and pro‑apoptotic effects on colon adenocarcinoma cells with no significant effects on cell migration and invasion

Kefir, a fermented milk product, exhibits anti‑tumoral activity in vivo; yet its mechanism of action remains elusive. Recent studies have focused on the mechanism of action of kefir on cancer cells in vitro. The current study aims at examining the effect of kefir on cell survival, proliferation, and motility of colorectal cancer (CRC) cells. Kefirs anti‑cancer potential was tested on CRC cell lines, Caco‑2 and HT‑29, through cytotoxicity, proliferation, and apoptotic assays. The expression of certain genes involved in proliferation and apoptosis was measured using reverse transcriptase‑polymerase chain reaction (RT‑PCR) and western blotting. To assess the effect of kefir on cancer metastasis, wound‑healing and time‑lapse movies, in addition to collagen‑based invasion assay, were used. The results show that cell‑free fractions of kefir exhibit an anti‑proliferative effect on Caco‑2 and HT‑29 cells. Analysis of DNA content by flow cytometry revealed the ability of kefir to induce cell cycle arrest at the G1 phase. Kefir was also found to induce apoptosis, as seen by cell death ELISA. Results from RT‑PCR showed that kefir decreases the expression of transforming growth factor α (TGF‑α); and transforming growth factor‑β1 (TGF‑β1) in HT‑29 cells. Western blotting results revealed an upregulation in Bax:Bcl‑2 ratio, confirming the pro‑apoptotic effect of kefir, and an increase in p53 independent‑p21 expression upon kefir treatment. MMP expression was not altered by kefir treatment. Furthermore, results from time‑lapse motility movies, wound‑healing, and invasion assays showed no effect on the motility of colorectal as well as breast (MCF‑7 and MB‑MDA‑231) cancer cells upon kefir treatment. Our data suggest that kefir is able to inhibit the proliferation and induce apoptosis in HT‑29 and Caco‑2 CRC cells, yet it does not exhibit a significant effect on the motility and invasion of these cells in vitro.

A modified lipid composition in Fabry disease leads to an intracellular block of the detergent-resistant membrane-associated dipeptidyl peptidase IV

Fabry disease is an X-linked lysosomal storage disorder that leads to abnormal accumulation of glycosphingolipids due to a deficiency of alpha-galactosidase A (AGAL). The consequences of these alterations on the targeting of membrane proteins are poorly understood. Glycosphingolipids are enriched in Triton-X-100- resistant lipid rafts [detergent-resistant membranes (DRMs)] and play an important role in the transport of several membrane-associated proteins. Here, we show that In fibroblasts of patients suffering from Fabry disease, the colocalization of AGAL with the lysosomal marker LAMP2 is decreased compared with wild-type fibroblasts concomitant with a reduced transport of AGAL to lysosomes. Furthermore, overall composition of membrane lipids in the patients’ fibroblasts as well as in DRMs reveals a substantial increase in the concentration of glycolipids and a slight reduction of phosphatidylethanolamine (PE). The altered glycolipid composition in Fabry fibroblasts is associated with an intracellular accumulation and impaired trafficking of the Triton-X-100 DRM-associated membrane glycoprotein dipeptidyl peptidase IV (DPPIV) in transfected Fabry cells, whereas no effect could be observed on the targeting of aminopeptidase N (ApN) that is not associated with this type of DRM. We propose that changes in the lipid composition of cell membranes in Fabry disease disturb the ordered Triton X-100 DRMs and have implications on the trafficking and sorting of DRM-associated proteins and the overall protein–lipid interaction at the cell membrane. Possible consequences could be altered signalling at the cell surface triggered by DRM-associated proteins, with implications on gene regulation and subsequent protein expression.

Thymoquinone from Nigella sativa Seeds Promotes the Antitumor Activity of Noncytotoxic Doses of Topotecan in Human Colorectal Cancer Cells in Vitro

Topotecan, a topoisomerase I inhibitor, is an anticancer drug widely used in the therapy of lung, ovarian, colorectal, and breast adenocarcinoma. Due to the primary dose-limiting toxicity of topotecan, which is myelosuppressive, it is necessary to identify other chemotherapeutic agents that can work synergistically with topotecan to increase its efficacy and limit its toxicity. Many studies have shown synergism upon the combination of topotecan with other chemotherapeutic agents such as gemcitabine. Other studies have demonstrated that pre-exposing cells to naturally occurring compounds such as thymoquinone, followed by gemcitabine or oxaliplatin, resulted in higher growth inhibition compared to treatment with gemcitabine or oxaliplatin alone. Our aim was to elucidate the underlying mechanism of action of topotecan in the survival and apoptotic pathways in human colon cancer cell lines in comparison to thymoquinone, to study the proapoptotic and antiproliferative effects of thymoquinone on the effectiveness of the chemotherapeutic agent topotecan, and to investigate the potential synergistic effect of thymoquinone with topotecan. Cells were incubated with different topotecan and thymoquinone concentrations for 24 and 48 hours in order to determine the IC50 for each drug. Combined therapy was then tested with ± 2 values for the IC50 of each drug. The reduction in proliferation was significantly dose- and time-dependent. After determining the best combination (40 µM thymoquinone and 0.6 µM topotecan), cell proteins were extracted after treatment, and the expression levels of B-cell lymphoma 2 and of its associated X protein, proteins p53 and p21, and caspase-9, caspase-3, and caspase-8 were studied by Western blot. In addition, cell cycle analysis and annexin/propidium iodide staining were performed. Both drugs induced apoptosis through a p53-independent mechanism, whereas the expression of p21 was only seen in thymoquinone treatment. Cell cycle arrest in the S phase was detected with each compound separately, while combined treatment only increased the production of fragmented DNA. Both compounds induced apoptosis through the extrinsic pathway after 24 hours; however, after 48 hours, the intrinsic pathway was activated by topotecan treatment only. In conclusion, thymoquinone increased the effectiveness of the chemotherapeutic reagent topotecan by inhibiting proliferation and lowering toxicity through p53- and Bax/Bcl2-independent mechanisms.

The Anti-Proliferative and Pro-apoptotic Effect of Topotecan in Combination With Thymoquinone in Acute Myelogenous Leukemia

BACKGROUND Topotecan has shown promising antineoplastic activity in solid tumors and acute leukemia. Because of the primary dose-limiting toxicity of topotecan, it is necessary to identify other agents that can work synergistically with topotecan, potentially increasing its efficacy while limiting its toxicity. Many studies showed synergism in combination of topotecan with gemcitabine and bortezomib. Other studies report the increase in growth inhibition of gemcitabine or oxaliplatin when cells were preexposed to naturally occurring drugs such as thymoquinone. The aim of this project was to study the mode of action of topotecan along with thymoquinone, on survival and apoptosis pathways in acute myelogenous leukemia (AML) cell lines, and to investigate the potential synergistic effect of thymoquinone on topotecan. MATERIALS AND METHODS U937 cells were incubated with different topotecan and thymoquinone concentrations for 24 and 48 hours, separately and in combination. Cell proliferation was determined using WST-1 (Roche) reagent. The effect of the compounds on protein expression of Bax, Bcl2, p53, caspase-9, -8, and -3 was determined using Western blot analysis. Cell cycle analysis was performed in addition to annexin/propidium iodide staining. RESULTS Thymoquinone and topotecan exhibited antiproliferative effects on U937 cells when applied separately. In combination, the reduction in proliferation was extremely significant with a major increase in the expression levels of Bax/Bcl2, p53, and caspase-3 and -9. Preexposure with thymoquinone resulted in an increase in cell growth inhibition compared with topotecan treatment. CONCLUSION Thymoquinone, when combined with topotecan in noncytotoxic doses, produced synergistic antiproliferative and proapoptotic effects in AML cells. Preexposure to thymoquinone seems to be more effective than simultaneous application with topotecan.

Protocadherin of the Liver, Kidney, and Colon Associates with Detergent-resistant Membranes during Cellular Differentiation

Protocadherin LKC (PLKC) is a member of the heterogeneous subgroup of protocadherins that was identified and described as a potential tumor-suppressor gene involved in contact inhibition (Okazaki, N., Takahashi, N., Kojima, S., Masuho, Y., and Koga, H. (2002) Carcinogenesis 23, 1139–1148 and Ose, R., Yanagawa, T., Ikeda, S., Ohara, O., and Koga, H. (2009) Mol. Oncol. 3, 54–66). Several aspects of the structure, posttranslational processing, targeting, and function of this new protocadherin are still not known. Here, we demonstrate that the expression of PLKC at the apical membrane domain and its concentration at regions of cell-cell contacts occur concomitantly with significant elevation of PLKC-mRNA levels. Furthermore, it can be found within the adherens junctions, but it does not colocalize with tight junctions proteins ZO-1 and occludin, respectively. Additionally, unlike E-cadherin, PLKC is not redistributed upon Ca2+ removal. Biosynthetic labeling revealed N- and O-glycosylation as posttranslational modifications as well as a fast transport to the cell surface and a low turnover rate. During differentiation, PLKC associates with detergent-resistant membranes that trigger its redistribution from intracellular membranes to the cell surface. This association occurs concomitant with alterations in the glycosylation pattern. We propose a role for PLKC in the establishment of a proper epithelial cell polarity that requires O-linked glycosylation and association of the protein with detergent-resistant membranes.

Cadherin-related protein 24 induces morphological changes and partial cell polarization by facilitating direct cell-cell interactions.

Abstract Cadherin-related protein 24 (CDHR24) is a potential tumor suppressor located apically as well as laterally in polarized cells. Here, the role of CDHR24 in contributing to cell morphology and polarity is examined. CDHR24 was predominantly localized at the nonattached part of nonpolarizing cells while another apically sorted protein, aminopeptidase N, was equally distributed over the plasma membrane. Furthermore, CDHR24 expression induced cell aggregation capacity, indicating direct cell-cell interaction. The transepithelial resistance, however, was elevated in polarized MDCK cells, but not in nonpolarizing CHO cells. Our data propose a model in which CDHR24 is directly involved in cell and tissue morphogenesis.

Identification of EDTA-Soluble Polysaccharides from Pea Epicotyl Cell Walls and Their Interaction with Xyloglucan

Nascent pectin and glucuronoxylan were prepared from membrane-bound enzymes obtained from pea epicotyls. They had previously been shown to exhibit a protein- and pH-dependent pattern of binding to cell wall ghosts and to xy-loglucan extracted from cell walls prepared from pea epicotyls; maximum binding required a pH of 3-4, and the pres-ence of cell wall proteins, namely assemblins. To determine whether wall polysaccharides deposited in cell walls be-have in the same manner as nascent polymers, radioactively labeled EDTA-soluble polymers were prepared from newly-deposited pea epicotyl cell walls. Different enzyme treatments followed by column chromatography, in addition to complete acid hydrolysis followed by paper and thin layer chromatography, indicated the presence of pectin, to-gether with smaller amounts of glucuronoxylan, in this EDTA-soluble extract. These radioactively labeled polysaccha-rides were found to bind to cell wall ghosts and to xyloglucan extracted from the second and third internodes of pea epicotyls cell walls in a pH-dependent manner, similar to the binding pattern obtained with nascent polymers. Maxi-mum binding occurred at pH 3-4, and also required the presence of protein.