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Retinoblastoma (RB) is the most common malignant intraocular tumor in early childhood. Imminent chemotherapy resistance diminishes the clinical-therapeutic options and emphasizes the necessity for new therapeutic approaches. The present study aimed at characterizing and comparing etoposide and cisplatin-resistant human RB cell lines with regard to changes in proliferation and apoptosis levels, anchorage independent growth behavior
Retinoblastoma (RB) is the most common intraocular malignant neoplasm of infancy and childhood. Although enucleation is an effective therapy for children with RB, one has to consider the resulting visual impairment and cosmetic deformity. External beam radiation therapy (EBRT) was proven to be the first effective eye salvage therapy for advanced RB, but it increased the risk of secondary tumors in children with germline
DNA topoisomerase (topo) enzymes regulate DNA metabolism and affect replication, transcription, recombination, chromatin assembly, DNA repair and ultimately cell division. Important chemotherapeutic agents target these enzymes. Inhibitors of topo II enzymes, such as etoposide, stabilize DNA-topo II complexes by blocking DNA relegation. Trapping the enzyme in a complex with cleaved DNA causes direct double-strand DNA damage that then leads to p53 stabilization, finally causing apoptosis (
The DNA-damaging agent cisplatin is likewise used extensively as a chemotherapeutic drug. Since 1994 chemotherapy with cisplatin and vincristine combined with focal therapy has been successfully used for RB treatment. Cisplatin acts as an alkylating or chelating agent, capable of forming adducts with macromolecules such as cellular DNA. This results in DNA cross-links and induces cell cycle arrest (
Another commonly used drug regiment includes a combination of vincristine, etoposide and carboplatin (VEC) for intravenous administration (
In the present study, we set out to characterize three etoposide- and three cisplatin-resistant RB cell lines with regard to morphological and functional changes compared to their respective parental, chemosensitive counterparts.
The human RB cell line RB-355, established and first described by Griegel
The cell lines were cultivated as suspension cultures in Dulbecco's modified Eagle's medium (DMEM) with 15% fetal bovine serum (FBS) (both from PAN-Biotech GmbH, Aidenbach, Germany), 100 U penicillin/ml and 100 µg streptomycin/ml, 4 mM L-glutamine (both from Gibco, Karlsruhe, Germany), 50 µM β-mercaptoethanol (Carl Roth, Karlsruhe, Germany) and 10 µg insulin/ml (PAN-Biotech) at 37°C, 10% CO2 and 95% humidity. No approval from an Ethics Committee was required for work with the human cell lines.
All chemoresistant RB cell lines characterized were generously provided by Dr H. Stephan. To generate these cell lines, established Y-79, WERI-Rb1 and RB-355 cells (see above) were continuously treated with consecutively increasing concentrations of etoposide or cisplatin (both from Teva, Berlin, Germany) until the chemoresistant sublines exhibited a at least 10-fold higher IC50 value in WST-1 viability assays than the respective parental controls (
Cell proliferation was determined by 5-bromo-2′-deoxyuridine (BrdU; Sigma, Steinheim, Germany) incorporation. For BrdU immunocytochemistry 10 µM BrdU was added to the cells 4 h prior to paraformaldehyde (PFA; Sigma) fixation. Cells were incubated with a rat anti-BrdU antibody (1:1,000; ab6326; Abcam, Cambridge, UK) and proliferating cells were visualized using a goat anti-rat antibody labelled with Alexa Flour® 488 (1:1,000; A11006; Life Technologies, Darmstadt, Germany). In order to determine changes in apoptosis levels, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma) or Click-iT® Plus TUNEL assay for
To determine growth kinetics, 3×105 RB cells were seeded in 500 µl DMEM with supplements in a 24-well plate and vital cells were counted manually using the trypan blue exclusion method. Cells were seeded in triplicates and counted at several time points (24, 48, 72 and 96 h). In the case of the extremely slow-growing Y-79 cisplatin-resistant RB cell line, we recorded long-term growth curves over a period of 336 h with longer counting intervals and plotted the values logarithmically to visualize differences between resistant and control cells.
Soft agarose assays were performed as previously described in detail (
In order to test for changes in the migration and tumor formation capacity following etoposide and cisplatin resistance, RB cells were grafted onto the chick chorioallantoic membrane (CAM) as described in a recent publication by our group (
The duration of the chick CAM assay is limited to a 7–9 day window prior to hatching. Seven days after grafting (E10-17) chick embryos were anesthetized by cooling on ice and sacrificed by decapitation. CAM tumors were excised, measured, photographed and fixed for 1 h at 4°C in 4% PFA in 0.1 M phosphate buffer (pH 7.4). For cryo-embedding, the tumor tissue was incubated for 30 min in PBS (pH 7.3) containing 15% sucrose, followed by a 30-min incubation in PBS containing 30% sucrose and finally embedded in OCT compound (Tissue-Tek; Germany), and sectioned at 10 µm using a cryostat. Images and measurements of tumors forming on the upper CAM were captured with a Nikon stereo dissecting microscope SMZ 1000 equipped with a Nikon digital camera and Nikon EclipseNet software. Exemplarily, images were captured with a 3D-Profilomter VR-3200 microscope (Keyence, Neu-Isenburg, Germany) to visualize changes in the 3D volume of the tumors.
The localization of the tumors forming in the upper CAM after grafting was visualized on hematoxylin and eosin (H&E)-stained cryosections. The human origin of the tumors forming in the chicken CAM after grafting human RB cells was verified using a mouse anti-human nuclear antibody (MAB 128; Merck Millipore, Darmstadt, Germany) at a dilution of 1:100 in PBS containing 0.1% Triton, 4% bovine serum albumin (BSA) and 1% normal goat serum (NGS) overnight at 4°C. The reaction was visualized using a goat anti-mouse antibody labelled with Alexa Flour® 488 (A11001; Molecular Probes, Camarillo, CA, USA), diluted 1:1,000 in PBS with 1% BSA for 2 h at room temperature.
For β-tubulin stainings, 1×105 cells were stained on coverslips as previously described (
For measurements of cell and nuclear sizes, images from β-tubulin-stained cells on coverslips were acquired using a Nikon Eclipse E600 microscope equipped with a digital camera. For each cell line, 5 equally distributed, x-shape rendered visual fields/coverslip were captured, and the cytoplasmic and nuclear outline of 7 cells/visual field were measured using the Nikon Eclipse net measurement software.
All assays were performed at least in triplicate. Statistical analyses were conducted using GraphPad Prism 6. Data represent means ± SEM of 2 to 5 independent experiments from independent RB cell cultures. Results were analyzed by a Students t-test or one-way ANOVA and Newman-Keuls post hoc test and considered significantly different at *P<0.05, **P<0.01 or ***P<0.001. Statistics on the growth curves was performed using a free web interface
In order to check for morphological changes following etoposide and cisplatin resistance, we compared the appearance of parental and chemoresistant Y-79, WERI-Rb1 and RB-355 cells a) in cell culture (
Measurements of cell and nuclear size revealed that compared to their parental counterparts, cisplatin-resistant cells were significantly bigger and had larger nuclei (
The etoposide-resistant cell lines Y-79 and WERI-Rb1, established from two well-established RB cell lines, originally derived from unilateral RB tumors (
As revealed by WST-1 assays, etoposide resistance resulted in a significant increase in Y-79 cell viability. WERI-Rb1 etoposide-resistant cells displayed a slightly, but not significantly increased viability, whereas etoposide resistance did not significantly alter the viability of RB-355 cells compared to the parental controls (
Cell proliferation was significantly increased in all three etoposide-resistant RB cell lines analyzed as reflected by significantly higher numbers of BrdU-positive cells (
Etoposide treatment still induces apoptosis in etoposide-resistant RB cell lines. As revealed by DAPI cell counts (
Photo-documentation (
The localization of the tumors developing in the upper chicken CAM at the border between CAM ectoderm and mesoderm 7 days after grafting human RB cells was visualized by H&E staining of tumor cryosections (
Etoposide resistance changed the anchorage independent growth of Y-79 and WERI-Rb1 cells as reflected by a significant decrease in their colony formation capacity in soft agarose (
All cisplatin-resistant cell lines analyzed displayed significantly lower growth kinetics compared to the control cells (
As revealed by DAPI cell counts (
Photo-documentation (
Soft agarose assays revealed that cisplatin resistance significant decreased the colony formation capacity of all cisplatin-resistant RB cell lines analyzed (
Drug resistance and relapse are the major issues associated with chemotherapy, which is regarded as the mainstay of globe preserving treatment in retinoblastoma (RB). In the present study presented, we provide a morphological and functional characterization of three etoposide- and three cisplatin-resistant RB cell lines.
Regarding morphological changes, we observed a significant increase in cell and nuclear size in the cisplatin-resistant RB cells, but no significant changes in the etoposide-resistant cells. In this context, Żuryń
We showed that compared to the cells of origin, etoposide-resistant RB cell lines were highly proliferative, displayed a significantly increased tumor formation capacity and formed larger tumors. Thus, these cells obviously become more aggressive than their parental counterparts. Surprisingly, etoposide treatment still induced apoptosis in the etoposide-resistant RB cell lines. In line with our finding, it has been shown that etoposide-resistant melanoma cells likewise display reduced but still detectable apoptotic activities, but activation of the mitochondrial pro-apoptotic pathway was no longer detectable after exposure to etoposide (
In contrast to etoposide resistance, we found cisplatin resistance to significantly lower growth kinetics and to induce tumors with equal or diminished weights. Thus, compared to the cells of origin, cisplatin-resistant RB cells do not display increased tumorigenicity and aggressiveness.
Soft agarose assays of the present study presented revealed that etoposide- and cisplatin-resistant RB cell lines exhibited significantly reduced colony formation capacities and formed considerably smaller colonies compared to their parental counterparts. As RB cells normally grow as aggregates (
Among other mechanisms, ATP-binding cassette (ABC) transporters were found to contribute to the process of drug resistance in cancer (
Hypoxic tumor microenvironment is another factor that determines the therapeutic response in many tumors. It has been shown that hypoxia attenuates etoposide mediated G2/M arrest and apoptosis induction and thus promotes etoposide resistance in neuroblastoma cells (
It has been reported that clusterin, a cytoprotective chaperone protein known to protect various retinal cells, is overexpressed in several types of malignant tumors. Song
Finally, microRNAs (miRNAs) have been identified to directly or indirectly influence the development of cancer drug resistance [for review see (
After a first, more descriptive functional characterization in the present study, ongoing experiments may address the question of which mechanisms underlie the development of etoposide and cisplatin resistance in RB cell lines and how these can be circumvented. Currently, one can state that etoposide-resistant RB cells display therapeutically undesirable features such as fast growth, high proliferation rate and a significantly increased tumor formation capacity compared to the tumor cells of origin. Thereby, etoposide resistance seems to aggravate the course of the disease and potentially worsens patient prognosis. By contrast, compared to their parental counterparts, cisplatin-resistant RB cells continue to exhibit higher apoptosis rates, decreased growth kinetics and equal or diminished tumor weights. It takes an extended treatment period to induce cisplatin resistance and cisplatin-resistant RB cells do not display more aggressive features than the cells of origin. Thus, finally cisplatin resistance has less severe consequences for RB patients.
Effects of etoposide and cisplatin resistance on RB cell line morphology. (A) Images of parental (Ctr), etoposide (Etop) and cisplatin-resistant (CisPt) Y-79, WERI-Rb1 and RB-355 RB cells in culture. (B) Images of parental, etoposide- and cisplatin-resistant RB cell lines after seeding on coverslips and immunocytochemical staining with β-tubulin (green fluorescence) and DAPI counterstaining (blue fluorescence). Morphological comparison revealed that parental controls frequently form chain-like structures, whereas chemoresistant RB cell lines tend to form clusters.
Changes in (A) RB cell and (B) nuclei size following cisplatin resistance. Compared to their parental counterparts (Ctr), cisplatin-resistant cells (CisPt) were significantly bigger (A) and had larger nuclei (B). *P<0.05, **P<0.01, ***P<0.001 statistical differences compared to the control group calculated by Student's t-test.
Effects of etoposide resistance on RB cell growth, viability, proliferation and apoptosis. (A-C) Growth curves, (D) WST-1 assays, (E) BrdU counts and (F) DAPI cell counts of etoposide-resistant (Etop) Y-79 (A), WERI-Rb1 (B) and RBL-355 cells (C) vs. control cells (Ctr) revealed that etoposide resistance led to a significant increase in growth kinetics of Y-79 and WERI-Rb1 cells (A and B). Accordingly, cell proliferation was significantly increased in all three etoposide-resistant cell lines (E). Values are means of at least 3 independent experiments ± SEM; *P<0.05, **P<0.01, ***P<0.001; ns, no statistical differences compared to the control group calculated by Student's t-test.
Effect of etoposide and cisplatin resistance on apoptosis levels in retinoblastoma cell lines. (A) Comparative quantification of DAPI and TUNEL cell counts of etoposide-resistant RB cell lines (Etop) compared to their parental counterparts (Ctr). (B) Comparative quantification of DAPI and TUNEL cell counts of cisplatin-resistant RB cell lines (CisPt) compared to their parental counterparts (Ctr). Values are means from 3 independent experiments ± SEM; **P<0.01, ***P<0.001 compared to the control group calculated by Student's t-test.
Effects of etoposide resistance on tumor formation capacity of different RB cell lines. (A) Images of tumors
Tumor formation in the chicken CAM. (A) H&E-stained cryosection of a tumor developing at the border between CAM ectoderm (e) and mesoderm (m) 7 days after grafting human Y-79 control RB cells on the upper chicken CAM. (B) Staining with a human-specific anti-nuclear antibody clearly proved the human RB cell origin of the tumor. (C) DAPI counterstaining. (D) Merged images (green fluorescence, anti-nuclear antibody; blue fluorescence, DAPI) arrowheads, outline of tumor front. Scale bar, 100 µm (B, applies to all figures).
Effect of etoposide resistance on RB cell colony formation capacity. (A) Quantification of soft agarose assays showing a significant lower capacity of etoposide-resistant RB cell lines to form colonies. (B) Images captured from soft agarose colonies at the day of seeding (day 0) and after 3 weeks in culture revealing that etoposide-resistant (Etop) RB cells formed considerably smaller colonies than control cells (Ctr). Values are means from 3 independent experiments ± SEM; **P<0.01 compared to the control group calculated by Student's t-test, ***P<0.001.
Effects of cisplatin resistance on RB cell growth, viability, proliferation and apoptosis. (A-C) Growth curves, (D) WST-1 assays, (E) BrdU counts and (F) DAPI cell counts of cisplatin-resistant (CisPt) Y-79 (A), WERI-Rb1 (B) and RBL-355 cells (C) vs. control cells (Ctr) revealed that cisplatin resistance led to a significant decrease in growth kinetics (A-C), cell viability (D) and proliferation (E). Cisplatin treatment, however, still significantly increased apoptosis levels in all cisplatin-resistant cell lines (F). Values are means of at least 3 independent experiments ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared to the control group calculated by Student's t-test.
Effects of cisplatin resistance on tumor formation capacity of different RB cell lines. (A) Images of tumors
Effect of cisplatin resistance on RB cell colony formation capacity. (A) Quantification of soft agarose assays showing a significant lower capacity of cisplatin-resistant RB cell lines to form colonies. (B) Images taken from soft agarose colonies at the day of seeding (day 0) and after 3 weeks in culture revealed that cisplatin-resistant (CisPt) RB cells formed considerably smaller colonies than control cells (Ctr). Values are means from 3 independent experiments ± SEM; ***P<0.001 compared to the control group calculated by Student's t-test.
Concentrations of the chemotherapeutic agents used to treat the RB cell lines.
Cytostatic drug | Cell line | Final concentration (µmol/ml) |
---|---|---|
Cisplatin | WERI-Rb1 | 8 |
(1 mg/ml) | Y-79 | 5 |
RB-355 | 6 | |
Etoposide | WERI-Rb1 | 5 |
(20 mg/ml) | Y-79 | 3 |
RB-355 | 1 |