Tumor Cells Of Various Origins Research Articles

In vitro and in vivo evaluation of a paclitaxel conjugate with the divalent peptide E-[c(RGDfK)2] that targets integrin αvβ3

The αvβ3 integrin is overexpressed on proliferating endothelial cells such as those present in growing tumors as well as on tumor cells of various origins. Tumor-induced angiogenesis can be inhibited in vivo by antagonizing the αvβ3 integrin with small peptides containing the arginyl-glycyl-aspartic acid (RGD) amino acid sequence. The divalent cyclic peptide E-[c(RGDfK)2] is a novel ligand-based vascular-targeting agent that binds integrin αvβ3 and demonstrated high uptake in OVCAR-3 xenograft tumors. In this work, we coupled the 2′-OH-group of paclitaxel through an aliphatic ester to the amino group of E-[c(RGDfK)2] or the control peptide c(RADfK), thus obtaining the derivatives E-[c(RGDfK)2]-paclitaxel and c(RADfK)-paclitaxel. Subsequently, we investigated the activity of the paclitaxel derivatives using several well-established in vitro angiogenesis assays: using a standard 72h endothelial cell proliferation assay, we showed that both E-[c(RGDfK)2]-paclitaxel and c(RADfK)-paclitaxel inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) in a similar manner as free paclitaxel (IC50 value ∼0.4nM), an observation that can be explained by the half-life of the paclitaxel ester bond in the conjugates of ∼2h at pH 7. In contrast, a 30-min exposure of the cells to the three drugs showed a clear difference between free paclitaxel, E-[c(RGDfK)2]-paclitaxel and c(RADfK)-paclitaxel with IC50 values of 10nM, 25nM, and 60nM, respectively. These differences are very likely due to the different routes of cellular entry of these three molecules. While the hydrophobic paclitaxel diffuses rapidly through the cell membrane, the charged peptide-containing derivative E-[c(RGDfK)2]-paclitaxel binds to the overexpressed αvβ3 integrin in order to enter the cells via receptor-mediated endocytosis. The differences between the derivatives were further demonstrated using an endothelial cell adhesion assay. Inhibition of cell attachment was observed only with the E-[c(RGDfK)2]-paclitaxel derivative indicating its specificity to the growing endothelial cells. Furthermore, E-[c(RGDfK)2]-paclitaxel inhibited both endothelial cells migration and capillary-like tube formation. These results further demonstrate their antiangiogenic properties. In vivo studies in an OVCAR-3 xenograft model demonstrated no antitumor efficacy for either E-[c(RGDfK)2] or E-[c(RGDfK)2]-paclitaxel compared to moderate efficacy for paclitaxel.

The nuclear DNA repair protein Ku70/80 is a tumor‐associated antigen displaying rapid receptor mediated endocytosis

To be of therapeutic relevance, a tumor-associated antigen should be expressed on the surface of neoplastic cells but not, or to a significantly lower extent, on cells of non-transformed nature. The Ku heterodimer (Ku70/80) is involved in DNA double strand break recognition and repair and is ubiquitously expressed in the nucleus of all cells. However, its exclusive nuclear localization has been reassessed by studies that demonstrate Ku to be expressed on the surface of tumor cell lines, displaying functions in cell adhesion, migration and invasion. In this study, we add another feature to the pluripotent role of Ku70/80 by showing that, upon binding the novel human recombinant antibody INCA-X, the Ku70/80 heterodimer is internalized into pancreatic carcinoma cells. The receptor-mediated endocytosis of Ku70/80 is rapid (t(1/) (2) 12 min) and extensive (90% of the receptor pool inside the cell after 100 min) as measured by rotating radioimmunoassay. Ku70/80 was also successfully used as a port of entry for cytotoxic payloads to tumor cells of various origin, as determined by indirect immunotoxin administration of a saporin-conjugated, secondary anti-human antibody. Thus, the internalization properties of the Ku70/80 suggest a potential role of this tumor associated antigen in selective drug-delivery in several human malignancies.

Aberrant hypermethylation of the promoter region of the CHFR gene is rare in primary breast cancer

Taxanes are among the most active agents and they are now known to be an indispensable component in chemotherapy for breast cancer. However, some patients are resistant to taxanes and the identification of the molecular characteristics that can predict the sensitivity to taxanes would be useful in selecting the most appropriate patients to receive taxane therapy. Taxanes are antimicrotubular agents that promote microtubular assembly and stabilize microtubules by preventing depolymerization. They interfere with normal mitotic transition and causes cell cycle arrest in the G2-M metaphase. CHFR (checkpoint with forkhead-associated and ring finger) is a recently identified gene, which functions as an important checkpoint protein early in G2-M transition. Its activation delays the cell cycle in prophase and promotes cell survival in response to the mitotic stress induced by either nocodazole or taxane. CHFR is frequently downregulated in human cancers, mostly owing to the hypermethylation of its promoter region. CHFR downregulation has been found in primary cancers or in the established tumor cells of various origins, such as the lung, colon, esophagus, and stomach. The aberrant hypermethylation of CHFR promoter appears to be a good molecular marker to predict sensitivity to taxanes in gastric, lung, and colon cancer. A downregulation of CHFR was observed in breast cancer cells, however, no apparent promoter hypermethylation has yet been reported. In addition, an alteration of the CHFR expression or aberrant promoter hypermethylation in primary breast cancer has not been fully investigated. In this study, we examined the methylation status of the promoter region of CHFR gene in 110 primary breast cancers. We observed the hypermethylation of the CHFR promoter region in only one case (0.9%). We herein show that the aberrant hypermethylation of this region is quite a rare event in primary breast carcinoma.

The In vitro and In vivo Effects of JNJ-7706621: A Dual Inhibitor of Cyclin-Dependent Kinases and Aurora Kinases

Modulation of aberrant cell cycle regulation is a potential therapeutic strategy applicable to a wide range of tumor types. JNJ-7706621 is a novel cell cycle inhibitor that showed potent inhibition of several cyclin-dependent kinases (CDK) and Aurora kinases and selectively blocked proliferation of tumor cells of various origins but was about 10-fold less effective at inhibiting normal human cell growth in vitro. In human cancer cells, treatment with JNJ-7706621 inhibited cell growth independent of p53, retinoblastoma, or P-glycoprotein status; activated apoptosis; and reduced colony formation. At low concentrations, JNJ-7706621 slowed the growth of cells and at higher concentrations induced cytotoxicity. Inhibition of CDK1 kinase activity, altered CDK1 phosphorylation status, and interference with downstream substrates such as retinoblastoma were also shown in human tumor cells following drug treatment. Flow cytometric analysis of DNA content showed that JNJ-7706621 delayed progression through G1 and arrested the cell cycle at the G2-M phase. Additional cellular effects due to inhibition of Aurora kinases included endoreduplication and inhibition of histone H3 phosphorylation. In a human tumor xenograft model, several intermittent dosing schedules were identified that produced significant antitumor activity. There was a direct correlation between total cumulative dose given and antitumor effect regardless of the dosing schedule. These results show the therapeutic potential of this novel cell cycle inhibitor and support clinical evaluation of JNJ-7706621.

Expression of dengue ApoptoM sequence results in disruption of mitochondrial potential and caspase activation

Apoptotic cell death has been involved as a cytopathologic mechanism in response to dengue (DEN) virus infection. Little information exists about how DEN virus replication triggers apoptosis in infected cells. We reported that a nine-residue sequence of the DEN M protein referred to as ApoptoM has proapoptotic properties in transformed and tumor cells of various origins. The aim of the present study was to investigate whether ApoptoM-induced apoptosis is associated to mitochondrial dysfunction and requires caspase activation. Intracellular expression of ApoptoM provokes the disruption of the mitochondrial transmembrane potential without subsequent generation of reactive oxygen species. We showed that ApoptoM-induced apoptosis involves the activation of a caspase-like protease pathway. Caspase-3 like activity was detected in ApoptoM-expressing cells. However, there was no role for caspase-9 in ApoptoM-mediated cell death. Our data suggest that a particular mitochondrion-dependent apoptotic pathway may be involved in induction of apoptosis by ApoptoM.

Adenovirus expressing p53.

Mutation in the p53 gene is the most frequently found genetic abnormality in human tumors, resulting in increased resistance to chemo- and radiotherapy (1,2). The underlying rationale for p53-mediated cancer gene therapy is to restore the ability of p53 mutant tumors to undergo apoptosis. When the wild-type p53 gene is delivered into tumor cells of various origins by adenoviral vector, the cells become more sensitive to cisplatin and radiation therapy (3). In addition, adenovirus expressing p53 is a great laboratory tool for the determination of function, for conveniently producing large quantities of p53 protein, and for determining the effect of p53 on cellular gene expression using microarray technology (4).

Enhanced ganciclovir killing and bystander effect of human tumor cells transduced with a retroviral vector carrying a herpes simplex virus thymidine kinase gene mutant.

Gene transfer of the herpes simplex virus thymidine kinase (TK) gene associated with ganciclovir (GCV) treatment can lead to death of TK-expressing cells, and of neighboring TK- cells because of the bystander effect. Thus, a small proportion of TK+ cells in a tumor can lead to its complete regression after GCV treatment. However, a lack of efficacy of gene transfer into tumors associated with low GCV sensitivity and poor bystander effect of human cancer cells currently limit the clinical use of this suicide gene therapy approach. To increase the potency of suicide gene therapy, we have tested the GCV sensitivity and the bystander effect of TK mutants that have been previously described. After retroviral transduction of the TK mutants into human tumor cells of various origins, we have found a strong killing effect of GCV with cells expressing the mutants TK30 or TKF161C. The GCV sensitivity of several human tumor cell types expressing TK30 was 9- to 500-fold higher than cells containing wild-type TK. Furthermore, TK30-expressing cells were able to kill bystander cells much more efficiently than TK-expressing cells. Thus, TK30 mutant is a promising candidate for suicide gene therapy clinical trials.

Cytotoxic T cell immunity against telomerase reverse transcriptase in humans.

Telomerase is a ribonucleoprotein enzyme which has been linked to malignant transformation in human cells. Telomerase activity is increased in the vast majority of human tumors, making its gene product the first molecule common to all human tumors. The generation of endogenously processed telomerase peptides bound to Class I MHC molecules could therefore target cytotoxic T lymphocytes (CTL) to tumors of different origins. This could advance vaccine therapy against cancer provided that precursor CTL recognizing telomerase peptides in normal adults and cancer patients can be expanded through immunization. We demonstrate here that the majority of normal individuals and patients with prostate cancer immunized in vitro against two HLA-A2.1 restricted peptides from telomerase reverse transcriptase (hTRT) develop hTRT-specific CTL. This suggests the existence of precursor CTL for hTRT in the repertoire of normal individuals and in cancer patients. Most importantly, the CTL of cancer patients specifically lysed a variety of HLA-A2(+) cancer cell lines, demonstrating immunological recognition of endogenously processed hTRT peptides. Moreover, in vivo immunization of HLA-A2.1 transgenic mice generated a specific CTL response against both hTRT peptides. Based on the induction of CTL responses in vitro and in vivo, and the susceptibility to lysis of tumor cells of various origins by hTRT CTL, we suggest that hTRT could serve as a universal cancer vaccine for humans.

Syndecan-1 (CD 138) in Myeloma and Lymphoid Malignancies: A Multifunctional Regulator of Cell Behavior Within the Tumor Microenvironment

Syndecan-1 is a transmembrane proteoglycan expressed on the surface of tumor cells of various origins including myeloma, Hodgkin's disease, and certain human immunodeficiency virus (HIV) associated lymphomas. Functional studies in myeloma reveal that syndecan-1 may act as a multifunctional regulator of cell behavior in the tumor microenvironment; it mediates cell-cell adhesion, binding of myeloma cells to type I collagen, and inhibits tumor cell invasion into collagen gels. In addition, syndecan-1 is released from the surface of myeloma cells and this shed form of the molecule inhibits growth and induces apoptosis of myeloma cells and may modulate myeloma bone disease by inhibiting osteoclast formation and promoting osteoblast formation. In view of its effects on tumor cell growth, survival, adhesion and invasion and on bone cell differentiation, syndecan-1 may be an important potentially beneficial regulator of myeloma pathobiology. Further studies are needed to define the clinical significance of syndecan-1 in myeloma and to examine its functional significance in other lymphoid malignancies.

Antitumor activity of conjugates of the oncofetal protein alpha-fetoprotein and phthalocyanines in vitro.

The several conjugates of aluminium and cobalt complexes of phthalocyanines with human alpha-fetoprotein have been synthesized. Their cytotoxic activity against tumor cells and human peripheral blood lymphocytes was studied. The experimental data demonstrate that the cytotoxic activity of alpha-fetoprotein-phthalocyanine conjugates against three types of tumor cells of various origin is much higher (for aluminium and cobalt complexes more than 1000 and 50 times, respectively) in comparison with phthalocyanines themselves. The application of phthalocyanines as conjugates with alpha-fetoprotein makes it possible to markedly enhance the selective toxicity of phthalocyanines against human tumor cells.

Antitumor activity of conjugates of the oncofetal protein alpha-fetoprotein and phthalocyanines in vitro.

The several conjugates of aluminium and cobalt complexes of phthalocyanines with human alpha-fetoprotein have been synthesized. Their cytotoxic activity against tumor cells and human peripheral blood lymphocytes was studied. The experimental data demonstrate that the cytotoxic activity of alpha-fetoprotein-phthalocyanine conjugates against three types of tumor cells of various origin is much higher (for aluminium and cobalt complexes more than 1000 and 50 times, respectively) in comparison with phthalocyanines themselves. The application of phthalocyanines as conjugates with alpha-fetoprotein makes it possible to markedly enhance the selective toxicity of phthalocyanines against human tumor cells.

Cellular immune response to human renal‐cell carcinomas: Definition of a common antigen recognized by HLA‐A2‐restricted cytotoxic T‐Lymphocyte (CTL) clones

Cytotoxic T lymphocyte (CTL) clones directed against autologous renal-cell carcinoma (RCC) cell lines were generated by mixed lymphocyte/tumor-cell culture (MLTC) using peripheral blood lymphocytes (PBL). A CD8+, CD4- CTL clone MZ1257-CTL 5/30 with high cytolytic activity for the autologous tumor cell line MZ1257-RCC was established. No lysis of the autologous EBV-transformed B lymphocytes (EBV-B) or K562 cells was observed. A panel of HLA-A2-matched allogeneic RCC lines was recognized by CTL 5/30. Further specificity analysis showed a cross-reactivity with HLA-A2-matched allogeneic tumor cells of various origins, especially melanoma. CTL 5/30 was also cross-reactive with several HLA-A2-positive allogeneic normal kidney cells in culture. The restriction element identified for CTL 5/30 was HLA-A2, as shown by blocking of cytotoxicity using an anti-HLA-A2 monoclonal antibody (MAb) and by resistance of an HLA-A2-negative melanoma variant SK29-MEL. 1.22 against lysis by CTL 5/30. In this report we demonstrate HLA-A2-restricted recognition of a T-cell-defined antigen on autologous renal-cancer cells. This antigen is also expressed and recognized in association with HLA-A2 on normal kidney cells in culture and other HLA-A2-positive tumor cells. It may therefore be a normal differentiation antigen to which tolerance is incomplete in the renal-cell cancer system investigated.