Anticancer activity of isolated compounds from twigs of Koelreuteria elegans cultivated in Egypt

Altered gene products are associated with activation of cellular ras K genes in human lung and colon carcinomas

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell- cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, we engineered E-cadherin-positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.

Modulation by d,l-buthionine- S,R-sulphoximine of etoposide cytotoxicity on human non-small cell lung, ovarian and breast carcinoma cell lines

Splice variants of CD44 are overexpressed in human lung, breast, and colon carcinoma cell lines. This study was conducted to clarify the association between the expression of CD44 variant exons 8–10 and metastatic potential in human colorectal cancer. We found that the expression of a CD44 splice variant containing exons v8–10 was increased in all of 60 colorectal cancer specimens examined compared with matched normal colorectal mucosa, as determined by Northern blotting. Expression of CD44 variant exons 8–10 did not significantly correlate with histological type, depth of tumor invasion, lymphatic invasion, venous invasion, or lymph node metastasis. However, the level of CD44 variant exon 8–10 expression was significantly higher in carcinomas associated with liver metastasis than in those without liver metastasis. In addition, expression of CD44 variant exons 8–10 in the liver metastases was more intense than that in the primary colorectal cancers. These findings indicated that this domain of the CD44 glycoprotein encoded by exons v8–10 may play an important role in tumor hematogenous metastasis of human colorectal cancer.

Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can inhibit tumor cell growth in vitro, and preliminary in vivo studies of the flavone L86-8275 have shown growth inhibition of LX529 and A549 lung carcinomas. L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] is a flavone of novel structure. The purpose of this study was to determine in vitro whether L86-8275 is a more potent inhibitor of growth in breast carcinoma and lung carcinoma cells than quercetin or genistein. We studied the effects of L86-8275 on cell growth in seven breast carcinoma cell lines and five lung carcinoma cell lines. MDA468 breast carcinoma was then selected for further study. Cell proliferation was measured by a colorimetric dye reduction assay; synthesis of DNA, RNA, and protein by incorporation of the radioactive metabolic precursors thymidine, uridine, or leucine, respectively; adenosine triphosphate (ATP) content by a luciferase-mediated bioluminescence reaction; and cell cycle progression by the use of cell-synchronizing drugs (aphidicolin and nocodazole) and flow cytometry. L86-8275 was not cytotoxic to stationary-phase cells but reversibly inhibited the growth of cells in exponential growth phase. At concentrations of 25-160 nM, L86-8275 inhibited growth of human breast and lung carcinoma cell lines by 50%. MDA468 breast carcinoma cells were 60-fold and 400-fold more sensitive to L86-8275 than to quercetin and genistein, respectively. By 24 hours after addition of L86-8275, DNA synthesis in MDA468 cells was inhibited by greater than 95%, protein synthesis by 80%, and RNA synthesis by 40%-60%, under conditions that preserved cellular ATP levels at approximately 80%-90% of control values. When MDA468 cells released from aphidicolin-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed the S phase but arrested in G2. When cells released from nocodazole-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed mitosis but arrested in G1. L86-8275 is a potent, yet reversible, growth-inhibitory flavone that can selectively block cell cycle progression in vitro at more than one point in the cell cycle. These findings suggest that L86-8275 is a candidate for further preclinical development, as well as a model for the synthesis of other flavonoids that might potently delay cell cycle progression to achieve inhibition of tumor growth. Future studies need to address optimal schedules for antiproliferative activity in vivo and inhibition of clonogenic activity.

1012 EGF-related peptides in cancer

The hydromethanolic extract of Sapindus saponaria L. aerial parts was investigated for antimicrobial activity (against several Gram-positive and Gram-negative bacteria and fungi) and capacity to inhibit the growth of different human tumor cell lines as also nontumor liver cells. The evaluated extract was further characterized in terms of phytochemicals using UV, 1H-NMR, 13C-NMR, and MS spectroscopic tools. The extract has shown a significant antimicrobial activity on all tested bacterial and fungal species. The best activity was achieved against Bacillus cereus and Staphylococcus aureus among bacteria and against all three Penicillium species tested. It also revealed cytotoxicity against human colon (HCT-15), cervical (HeLa), breast (MCF-7), and lung (NCI-H460) carcinoma cell lines, with HeLa being the most susceptible tumor cell line. The extract was not toxic for nontumor liver cells. Chromatographic separation of the extract resulted in the isolation and identification of stigmasterol, oleanolic acid, luteolin, luteolin 8-C-β-glucoside (orientin), luteolin 6-C-β-glucoside (isoorientin), luteolin 7-O-β-glucuronide, and rutin. The results of the present findings may be useful for the discovery of novel antitumor and antimicrobial agents from plant origin.

Voacanga globosa (Blanco), a plant endemic to the Philippines, is traditionally used especially by indigenous people of Bataan in the treatment of ulcers, wounds and tumorous growths. This study aimed to provide scientific evidence to therapeutic properties by determining cytotoxic and pro-apoptotic activity of HPLC fractions from leaves on HCT116 human colon carcinoma and A549 human lung carcinoma cell lines. Ethanolic extraction was performed on V globosa leaves followed by hexane and ethyl acetate partitioning. Silica gel column chromatography and high performance liquid chromatography (HPLC) produced MP1, MP2 and MP3 fractions. Cytotoxic activity of the fractions was determined through MTT assay against the cancer cell lines HCT116 and A549 and the non-cancer AA8 Chinese hamster ovarian cell line. Pro-apoptotic activities of the most active fractions were further assessed through DAPI staining, TUNEL assay and JC-1 mitochondrial membrane potential assay with HCT116 cells. While the MP1 fraction exerted no significant activity against all cell lines tested, MP2 and MP3 fractions demonstrated high toxicity against HCT116 and A549 cells. The MP3 fraction induced formation of apoptotic bodies, condensed DNA and other morphological changes consistent with apoptosis of HCT116 cells and TUNEL assay showed significant increase in DNA fragmentation over time. In these cells, the MP3 fraction also induced mitochondrial membrane destabilization, which is generally associated with the beginning of apoptosis. Phytochemical analysis demonstrated the presence only of saponins and terpenoids in the MP3 fraction. The results indicate that the MP3 fraction exerts cytotoxic activity on HCT116 cells via induction of apoptosis triggered by loss of mitochondrial membrane potential crucial for cell survival.

The extensive C-terminal molecular heterogeneity of alpha- and beta-tubulin is a consequence of multiple isotypes, the products of distinct genes, that undergo several posttranslational modifications. These include polyglutamylation and polyglycylation of both subunits, reversible tyrosination and removal of the penultimate glutamate from alpha-tubulin, and phosphorylation of the beta III isotype. A mass spectrometry-based method has been developed for the analysis of the C-terminal diversity of tubulin from human cell lines. Total cell extracts are resolved by SDS--PAGE and transferred to nitrocellulose, and the region of the blot corresponding to tubulin (approximately 50 kDa) was excised and digested with CNBr to release the highly divergent C-terminal tubulin fragments. The masses of the human alpha- and beta-tubulin CNBr-derived C-terminal peptides are all in the 1500--4000 Da mass range and can be analyzed directly by MALDI-TOF mass spectrometry in the negative ion mode without significant interference from other released peptides. In this study, the tubulin isotype diversity in MDA-MB-231, a human breast carcinoma cell line, and A549, a human non-small lung cancer cell line, is reported. The major tubulin isotypes present in both cell lines are k-alpha 1 and beta 1. Importantly, we report a previously unknown alpha isotype present at significant levels in both cell lines. Moreover, the degree of posttranslational modifications to all isotypes was limited. Glu-tubulin, in which the C-terminal tyrosine of alpha-tubulin is removed, was not detected. In contrast to mammalian neuronal tubulin which exhibits extensive polyglutamylation, only low-level monoglutamylation of the k-alpha 1 and beta 1 isotypes was observed in these two human cell lines.

Thrombospondin (TSP) is a member of a family of extracellular matrix glycoproteins that may participate in multiple aspects of the metastatic cascade. We report an inverse correlation of steady-state Thbs-1 mRNA and protein expression with malignant progression among murine melanoma and human lung and breast carcinoma cell lines. Murine K-1735 melanoma cell lines of low metastatic potential, including K-1735 lines transfected with the murine nm23-1 cDNA, expressed higher TSP levels than related highly metastatic lines. In a model system of lung carcinoma malignant progression, immortalized human bronchial epithelial cells expressed higher TSP levels than v-Ki-ras, v-Ha-ras or n-ras transfectants, which in turn expressed higher TSP levels than tumor-derived, more aggressive variants. Among 3 unrelated breast carcinoma cell lines, Thbs-1 steady-state mRNA levels were greater in the 2 non-metastatic lines than the metastatic line. Our data show that malignant progression in some cell lines is associated with reduced TSP expression. The suppressive effects of nm23-1 transfection on metastatic potential are also associated with increased TSP expression; ras transfection, which results in increased tumorigenesis, is associated with decreased TSP expression.

Amooranin (AMR), a plant terpenoid, isolated from Amoora rohituka, was investigated for its ability to overcome multidrug resistance in human leukemia and colon carcinoma cell lines. AMR IC(50) values of multidrug-resistant leukemia (CEM/VLB) and colon carcinoma (SW620/Ad-300) cell lines were higher (1.9- and 6-fold) than parental sensitive cell lines (CEM and SW620). AMR induced G(2)+M phase-arrest during cell cycle traverse in leukemia and colon carcinoma cell lines and the percentage of cells in G(2)+M phase increased in a dose-dependent manner. Coincubation of tumor cells with both DOX and AMR reversed DOX resistance in 104-fold DOX-resistant CEM/VLB and 111-fold DOX-resistant SW620/Ad-300 cell lines with a dose modification factor of 50.9 and 99.6, respectively. Flow cytometric assay showed that AMR causes enhanced cellular DOX accumulation in a dose-dependent manner. AMR inhibits photolabeling of P-glycoprotein (P-gp) with [(3)H]-azidopine and the blocking effect enhanced with increasing concentrations of AMR. Our results show that AMR competitively inhibits P-gp-mediated DOX efflux, suggestive of a mechanism underlying the enhanced DOX accumulation and reversal of multidrug resistance by AMR.

Identification of Genes (SPON2 and C20orf2) Differentially Expressed between Cancerous and Noncancerous Lung Cells by mRNA Differential Display

The antineoplastic activity of 5-aza-2'-deoxycytidine (5-AZA-CdR) and docetaxel (Taxotere, Taxo) alone or in combination against human MDA-MB-231 breast, Calu-6 lung and DU-145 prostate carcinoma cell lines was evaluated by clonogenic assay. We also investigated by RT-PCR the capacity of these agents to re-activate the expression of E-cadherin and maspin, two tumor suppressor genes that were silenced by DNA methylation. 5-AZA-CdR and Taxo in combination produced a greater loss of clonogenicity than either agent alone. In MDA-MB-231 breast carcinoma cells, Taxo did not interfere with the re-activation of E-cadherin and maspin genes by 5-AZA-CdR. These results provide a rationale for clinical trials on the combination of 5-AZA-CdR and Taxo in patients with advanced cancer.

Apoptosis and cell cycle disturbances induced by coumarin and 7-hydroxycoumarin on human lung carcinoma cell lines

BackgroundNumerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R) agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues.MethodsBreast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and for TPO-R protein expression by immunohistochemistry (IHC). Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot.ResultsMPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43%) and malignant (3-17%) breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines.ConclusionsMultiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and megakaryocyte precursors.