Zoledronic acid targets chemo-resistant polyploid giant cancer cells

Abstract

Although polyploid giant cancer cells (PGCCs) are known as a key source of failure of current therapies, sufficient drugs to target these cells are not yet introduced. Considering the similarities of polyploid cells in regeneration and cancer, we hypothesized that zoledronic acid (ZA), an osteoclast-targeting agent, might be used to eliminate PGCCs. The 5637-bladder cancer cell line was treated with various doses of cisplatin to enrich polyploid cells and the efficacy of different concentrations of ZA in reducing this population was assessed. The metabolic profile of PGCCs was investigated with gas chromatography-mass spectrometry. Lipid profiles, mitochondrial density, and ROS content were also measured to assess the response of the cells to ZA. Cancer cells surviving after three days of exposure with 6 μM cisplatin were mainly polyploid. These cells demonstrated special morphological features such as fusion with diploid or other polyploid cells and originated in daughter cells through budding. ZA could substantially eradicate PGCCs with the maximal effect observed with 50 μM which resulted in the drop of PGCC fraction from 60 ± 7.5 to 19 ± 1.7%. Enriched PGCCs after cisplatin-treatment demonstrated a drastic metabolic shift compared to untreated cancer cells with an augmentation of lipids. Further assays confirmed the high content of lipid droplets and cholesterol in these cells which were reduced after ZA administration. Additionally, the mitochondrial density and ROS increased in PGCCs both of which declined in response to ZA. Taken together, we propose that ZA is a potent inhibitor of PGCCs which alters the metabolism of PGCCs. Although this drug has been successfully exploited as adjuvant therapy for some malignancies, the current evidence on its effects on PGCCs justifies further trials to assess its potency for improving the success of current therapies for tackling tumor resistance and relapse.

Figure 1

Cisplatin-induced polyploid cells demonstrate a dynamic polyploidization and depolyploidization characteristic. Cisplatin administration resulted in significant cell death and the enrichment of large polyploid cells. (A) Representative images of cells treated with 3, 6, and 13 μM cisplatin for 72 h are shown. In this panel, all pictures are captured with 20X objective lens except the third one (Cis 6 μM) which is taken with 10X. (B) DNA content analysis of treated cells with different doses of cisplatin indicated that the 6 μM cisplatin results in the highest rate of polyploidy. (C) Assessment of different recovery periods following the three days of 6 μM cisplatin administration revealed that polyploidy is maximal with a 7 days recovery time. (D) Time-lapse imaging of cisplatin-treated PGCCs demonstrated considerable changes in the genome content and cytoplasm morphology. White arrowheads: fusion with a diploid cell; orange arrowheads: fusion with another PGCC; yellow arrowheads: budding of daughter cells. Nuclei and cell membrane are stained with DAPI and CM-Dil, respectively; Cis: Cisplatin; * p-value < 0.05; ** p-value < 0.01; error bars: mean ± SEM.

Figure 2

Zoledronic acid declines the population of PGCCs pre- and post-cisplatin administration. (A) DNA content analysis of 5637 bladder cancer cells treated with different doses of ZA indicates the reduction of PGCCs post-treatment. (B) Cisplatin administration enriches PGCCs and ZA at different doses from 10–600 μM declines the PGCC population with the most profound effect being observed at 50 μM. *p-value < 0.05; ** p- value < 0.01; error bars: mean ± SEM.

Figure 3

Cisplatin-treated PGCC-enriched cells have a distinct metabolic profile. The metabolome of cisplatin-treated (orange peaks) and untreated bladder cancer cells (gray peaks) were assessed by gas chromatography-mass spectrometry (GC–MS).

Figure 4

PGCCs have augmented lipid metabolism. (A) Oil Red O staining demonstrated the accumulation of lipid droplets in PGCCs surviving after cisplatin treatment which partly declined after ZA treatment. Nuclei are stained with haematoxylin. (B) Quantification of the red signals in the images of Oil Red O-stained cells in three independent experiments confirmed the microscopic observations. (C) Total cholesterol content of cancer cells increased after cisplatin treatment. * p-value < 0.05; error bars: mean ± SEM.

Figure 5

Cisplatin-surviving cells have high mitochondrial density and ROS content, both of which are reduced by ZA. (A) Representative images of mitochondrial staining of cancer cells treated with cisplatin or cisplatin + ZA are demonstrated. (B) Representative flow-cytometry graphs and (C) quantification of four independent experiments demonstrated the augmentation of mitochondrial density after cisplatin and the partial declining effect of ZA. (D, E) ROS content of cisplatin-survived cells is increased and is significantly diminished after ZA administration. (F) The effect of ZA in declining ROS in cisplatin-treated cells is observed both in diploid and polyploid sub-populations. *p- value < 0.05; ** p- value < 0.01; error bars: mean ± SEM.

Figure 6

Cytoplasmic vacuolization is a common morphology in cisplatin-induced PGCCs. (A) Representative images of vacuolated cells (arrowheads) in cisplatin and cisplatin + ZA treated groups are shown. Nuclei are stained with DAPI. (B) Time-series quantification of microscopic observations in three independent experiments demonstrated the accumulation of these vacuoles in cisplatin-treated cells. The asterisk indicates the significant decline of these vacuoles in Cis + ZA group compared to Cis (p-value < 0.05); error bars: mean ± SEM.