Rouslan Kotchetkov

Development of resistance to vincristine and doxorubicin in neuroblastoma alters malignant properties and induces additional karyotype changes: a preclinical model.

Cytotoxic drug treatment of neuroblastoma often leads to the development of drug resistance and may be associated with increased malignancy. To study the effects of long‐term cytotoxic treatment on malignant properties of tumor cells, we established 2 neuroblastoma cell sublines resistant to vincristine (VCR) and doxorubicin (DOX). Both established cell lines (UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100) were highly resistant to VCR, DOX and vice‐versa but retained their sensitivity to cisplatin. UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100 expressed significant amounts of P‐glycoprotein, while parental cells were P‐glycoprotein negative. GD2 expression was upregulated, whereas NCAM expression was decreased in both resistant cells. Spectral karyotype (SKY) analysis revealed complex aberrant karyotypes in all cell lines and additional acquired karyotype changes in both resistant cells. All cell lines harbored high levels of N‐myc amplification. Compared to parental cells, UKF‐NB‐2rVCR20 and UKF‐NB‐2rDOX100 exhibited more than 2‐fold increase in clonal growth in vitro, accelerated adhesion and transendothelial penetration and higher tumorigenicity in vivo. We conclude that development of drug resistance and acquisition of certain karyotypic alterations is associated with an increase of additional malignant properties that may contribute to the poor prognosis in advanced forms of NB. The 2 novel neuroblastoma cell sublines also provide useful models for the study of drug resistance in aggressive forms of neuroblastoma.

Chemoresistance induces enhanced adhesion and transendothelial penetration of neuroblastoma cells by down-regulating NCAM surface expression.

BackgroundDrug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). One explanation for the link between resistance and malignancy might be that resistance facilitates cancer progression and invasion. To investigate this hypothesis, adhesion, transendothelial penetration and NCAM (CD56) adhesion receptor expression of drug-resistant versus drug-sensitive NB tumor cells were evaluated.MethodsAcquired drug resistance was mimicked by exposing parental UKF-NB-2, UKF-NB-3 or IMR-32 tumor cells to increasing concentrations of vincristine- (VCR) or doxorubicin (DOX) to establish the resistant tumor cell sublines UKF-NB-2VCR, UKF-NB-2DOX, UKF-NB-3VCR, UKF-NB-3DOX, IMR-32VCR and IMR-32DOX. Additionally, the malignant behaviour of UKF-NB-4, which already possessed the intrinsic multidrug resistance (MDR) phenotype, was analyzed. UKF-NB-4 exposed to VCR or DOX were designated UKF-NB-4VCR or UKF-NB-4DOX. Combined phase contrast – reflection interference contrast microscopy was used to separately evaluate NB cell adhesion and penetration. NCAM was analyzed by flow cytometry, western blot and RT-PCR.ResultsVCR and DOX resistant tumor sublines showed enhanced adhesion and penetration capacity, compared to their drug naïve controls. Strongest effects were seen with UKF-NB-2VCR, UKF-NB-3VCR and IMR-32DOX. DOX or VCR treatment also evoked increased invasive behaviour of UKF-NB-4. The process of accelerated tumor invasion was accompanied by decreased NCAM surface and protein expression, and down-regulation of NCAM coding mRNA. Transfection of UKF-NB-4VCR cells with NCAM cDNA led to a significant receptor up-regulation, paralleled by diminished adhesion to an endothelial cell monolayer.ConclusionIt is concluded that NB cells resistant to anticancer drugs acquire increased invasive capacity relative to non-resistant parental cells, and that enhanced invasion is caused by strong down-regulation of NCAM adhesion receptors.

Cytomegalovirus infection blocks apoptosis in cancer cells.

Recent pathological findings reveal a higher frequency of human cytomegalovirus (HCMV) in tumor cells from different tumors compared with surrounding tissues. Experimental investigations suggest possible supportive effects of HCMV for tumor development and progression. One HCMV effect on tumor cells is the inhibition of apoptosis, leading to the promotion of tumor cell survival. Decreased sensitivity to treatment-induced tumor cell death is a major reason for failure of anticancer chemotherapy. HCMV infection interferes with both the intrinsic and extrinsic cellular apoptosis pathways. HCMV promotes cell survival signaling influencing the tumor suppressor p53 and its relative p73, and stimulates the antiapoptotic Ras/Raf/MEK/Erk- and PI-3K-signaling pathways. Antiapoptotic effects mediated by HCMV are inhibited by antiviral treatment in cell culture. Therefore, a better understanding of the influence of HCMV infection on tumor cell apoptosis might translate into improved anti-cancer therapy.

Zidovudine Induces Resistance to Antineoplastic Agents and Alterations in Apoptosis in T-Cell Lymphoma Cells

Prolonged treatment of leukemic cell lines with antiretroviral agents such as nucleoside analogues may result in the development of cell resistance against antiviral agents. We tested whether prolonged treatment of H9 cells (T-cell lymphoma cell line) with 3′-azido-3′-deoxythymidine (AZT) interferes with sensitivity to antitumor agents commonly used for treatment of AIDS-associated malignancies. H9 cells grown for more than 2 years in medium containing 250 µM of AZT were at least 100-fold less sensitive to cytotoxic effects of AZT than parental H9 cell line. These cells designated H9rAZT250 were 5- to 20-fold less sensitive to toxic effects of antitumor agents including cisplatin (CP), vincristine (VIN), doxorubicin (DOX) and etoposide (VP-16). The resistance to these drugs was associated with inhibition of apoptosis as demonstrated by the terminal deoxynucleotidyl transferase-mediated nicked-end labelling (TUNEL) assay and DNA fragmentation assay. The expression of genes involved in regulation of apoptosis such bd-2 was enhanced in H9rAZT 250 cells. The results demonstrate that prolonged treatment of tumor cells with AZT may result in the development of resistance to antineoplastic agents due to the inhibition of apoptosis.