Cancer Drug Resistance: Targeting Proliferation or Programmed Cell Death

Abstract

The development of resistance to chemotherapy is one of the main problems for effective cancer treatment. Drug resistance may result from disturbances in two important physiological processes-cell proliferation and cell death. Importantly, both processes characterize alterations in cell metabolism, the level of which is often measured using MTT/MTS assays. To examine resistance to chemotherapy, different cancer cell lines are usually used for the in vitro modulation of developing resistance. However, after the creation of resistant cell lines, researchers often have difficulty in starting investigations of the mechanisms of insensitivity. In the first stage, researchers should address the question of whether the drug resistance results from a depression of cell proliferation or an inhibition of cell death. To simplify the choice of research strategy, we have suggested a combination of different approaches which reveal the actual mechanism. This combination includes rapid and high-throughput methods such as the MTS test, the LIVE/DEAD assay, real-time cell metabolic analysis, and Western blotting. To create chemoresistant tumor cells, we used four different cancer cell lines of various origins and utilized the most clinically relevant pulse-selection approach. Applying a set of methodological approaches, we demonstrated that three of them were more capable of modulating proliferation to avoid the cytostatic effects of anti-cancer drugs. At the same time, one of the studied cell lines developed resistance to cell death, overcoming the cytotoxic action.

Keywords: cell death; chemotherapy; proliferation; resistance.

Figure 1

Development of a cisplatin-resistant cell line to study the biological changes leading to resistance. Design for the creation of resistant cell lines (for details, see Section 2).

Figure 2

Response of wild-type and cisplatin-resistant cells to treatment with various concentrations of cisplatin. The MTS assay values obtained from wild-type and cisplatin-resistant A549 (A) and U1810 (B), SKOV3 (C), and SW620 (D) cells were normalized to the untreated sample. The data are presented as the mean ± standard deviation (N = 3). “WILD”—wild-type cells; “RES”—cells resistant to cisplatin; “cis”—cisplatin; * p < 0.05; ** p < 0.01.

Figure 3

Cytotoxic and cytostatic effects of cisplatin treatment in SW620, SKOV3, A549, and U1810 cell lines according to the LIVE/DEAD assay. Wild-type and cisplatin-resistant A549 (A), U1810 (B), SKOV3 (C), and SW620 (D) cells were treated with cisplatin (5 µM for SW620 and SKOV3, 10 µM—for U1810 and A549 cell line) in the presence or absence of 25 μM of Q-VD for 72 h. Calcein-AM (viable cell) and EthD-1 (dead cell) signals were obtained via the LIVE/DEAD assay. The data are normalized to the respective “Contr” samples; the lines and whiskers indicate the mean ± standard deviation (N = 4). “WILD”—wild-type cells; “RES”—cells resistant to cisplatin; “cis”—cisplatin; * p < 0.05; ** p < 0.01.

Figure 4

Clonogenic assay of parental and resistant cell lines A549 (A), U1810 (B), SKOV3 (C), and SW620 (D). The data are presented as a rate (WILD/RES cells) of the number of colonies, normalized to wild-type cells (N = 4); n.s.—non-significant difference. “WILD”—wild-type cells; “RES”—cells resistant to cisplatin; ns, non-significant difference, * p < 0.05.

Figure 5

Western blot analysis of protein levels in lysates of wild-type and cisplatin-resistant cells. Wild-type and cisplatin-resistant A549 and U1810 (A), SKOV3 and SW620 (B) cells were treated with cisplatin (10 and 20 µM for U1810 and A5495; 10 µM for SW620 and SKOV3 cell line) for 72 h. The samples are indicated above; proteins of interest are indicated on the left; molecular weight markers are indicated on the right. “Contr”—control sample; “Cis”—cisplatin. “WILD”—wild-type cells; “RES”—cells resistant to cisplatin.

Figure 6

Assessments of the basal respiration rate (A) and basal glycolysis rate (B) of wild-type A549, U1810, SKOV3, and SW620 cell lines using the Seahorse XF Extracellular Flux Analyzer, measured from 4 independent experiments. One-way ANOVA with Sidak’s multiple comparison test was used for statistical analysis; *** p < 0.001.

Figure 7

Assessment of the basal respiration rates of A549 (A), U1810 (B) SKOV3 (C), and SW620 (D) using the Seahorse XF Extracellular Flux Analyzer. The data were normalized to wild-type cells and presented as the WILD/RES rates (from 3 independent experiments). Two-way ANOVA with Sidak’s multiple comparison test was used for statistical analysis. “WILD”—wild-type cells; “RES”—cells resistant to cisplatin. “Contr”—control sample; “Cis”—cisplatin; ** p < 0.01; *** p < 0.001; **** p < 0.0001.