In recent years, there have been significant developments using combined therapies in cancer treatment. The present study aimed to determine the effects of using crizotinib alone and in combination with butyric acid on different types of breast cancer cells. A total of three different breast cancer models were used: MDA-MB-231, a triple negative model; MCF-7, a Luminal A model; and SKBR-3 cell line, a human epidermal growth factor receptor 2 positive model. In the experiments, proliferation rates and cell index values were obtained using the xCELLigence RTCA DP System, and mitotic index, bromodeoxyuridine labeling index and caspase activity were evaluated as cell kinetics parameters. The results showed that while proliferation rates, cell index values, mitotic index and bromodeoxyuridine labeling index decreased, caspase activity values increased. These results demonstrated that the combined application was more effective than the monotherapy application and could be used at lower concentrations than those drugs applied as monotherapy.
breast cancercrizotinibsodium butyratein vitroScientific Research Projects Coordination Unit of Istanbul UniversityFBA-2022-38767The present study was supported by the Scientific Research Projects Coordination Unit of Istanbul University (grant no. FBA-2022-38767).Introduction
Breast cancer, a heterogeneous disease associated with impaired cell proliferation, is the second most prevalent cause of cancer-related deaths globally and the most common cancer in women (1,2). It is a heterogeneous disease with several morphological and molecular features, as well as clinical outcomes. The majority of breast cancer cases are hormone-dependent, and a positive relationship has been reported between long-term exposure to high concentrations of estrogen and breast cancer incidence (3). However, there are also types of breast cancer that occur and develop independently of hormones. Treatment approaches also differ in breast cancers that differ in terms of formation and development mechanisms (4).
Currently, the parameters used to determine the classification of breast carcinomas are as follows: estrogen receptor (ER) and progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/cErbB2) overexpression and/or HER2 gene amplification and Ki-67 proliferation index (5). Accordingly, invasive breast carcinoma can be classified as hormone receptor-positive, (HER2)-positive and triple-negative, and these subtypes have their own specific treatment approaches (6–11).
Luminal A is the most common subgroup and constitutes the majority of all breast cancers. Whilst these tumors are ER and/or PR positive, they are HER2 negative. Ki-67 level is also generally <20%. The Luminal A subgroup has the best prognosis of all breast cancers and is generally characterized by low-grade, slow-growing tumors with a high survival rate. Furthermore, relapse rates are lower compared with that of other subgroups (12). In the treatment of hormone receptor-positive breast cancer, selective estrogen receptor modulators (such as tamoxifen and its derivatives) that affect the effect of endogenous estrogens via the receptor, and selective estrogen enzyme modulators (such as formestane and letrozole) that affect the activity of enzymes involved in the synthesis of estrogens, are used (13,14).
HER2-positive tumors constitute 13–20% of invasive breast cancers, and ~50% of them are hormone receptor-negative (11). Whilst the presence of HER2 was used as an indicator of poor prognosis in the past, this view has changed with the use of recombinant humanized anti-HER2 antibody (transtuzumab) (7).
In the triple negative subgroup, all three hormone receptors (ER, PR and HER2) are detected as negative. A distinguishing feature from other subgroups is that it is more common in women aged <40 years and constitutes ~20% of all breast cancers (15).
The majority of cancer drugs used today are tyrosine kinase (TK) inhibitors. This is a family of enzymes involved in signal transduction in human cells. TKs are necessary for normal physiology and regulate signal transduction mechanisms that serve a role in cell homeostasis, cell proliferation, growth arrest and apoptosis. The disruption of these functions causes abnormal cell activity and immunological, neurological, metabolic and infectious diseases, especially cancer. TKIs stop the cell cycle by preventing protein phosphorylation catalyzed by TKs and cause tumor cell apoptosis; it is used in cancer treatment with these effects (16,17). The MET gene (c-MET) encodes a receptor tyrosine kinase (RTK) known as MET (18). A significant factor in the onset and spread of many cancers, including breast cancer, is abnormal MET activation (19,20). Crizotinib acts as a MET inhibitor and crizotinib activity has been reported in patients with MET amplification (21).
Sodium butyrate (SB), as the sodium salt of butyrate, a short-chain fatty acid, causes the activation of genes related to apoptosis due to its histone deacetylase (HDAC) inhibitor (HDACi) activity (22). The HDACi feature, one of the functions of SB, is a subject that has been studied extensively in terms of its effects on tumor formation and development. Acetylated histones form a loose chromatin structure suitable for the transcription initiation complex. In this respect, deacetylation inhibited by SB causes a global increase in transcription (23). It has been reported that SB triggers apoptosis when applied in combination with different molecules such as quercetin (24,25).
The present study aimed to compare the use of Crizotinib as monotherapy and in combination with butyric acid under in vitro conditions using cell lines from different breast cancer types and different cell kinetic parameters, assessing whether the combined use is more effective than its use alone.
Materials and methodsCell culture
MCF-7, MDA-MB-231 and SKBR-3 were used as different breast cancer models. All cells were adherent cell lines growing in a single layer. The MCF-7 and SKBR3 cell lines were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) medium containing 2 mM L-glutamine and the MDA-MB-231 cell line was cultured in high glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.). In order for the cells to proliferate, 10% (v/v) fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) was added to all media. A total of 100 µg/ml streptomycin (streptomycin sulphate; IE Ulugay Ilac Sanayi TAS), 100 IU/ml penicillin (Pronapen; Pfizer, Inc.), amphotericin B (Merck KGaA) were added to the media. All cells were grown at 37°C in a humidified 5% CO2 atmosphere.
Cell index
For all three cell lines, 100 µl medium was added to each well of the 16-well e-plates (special cell culture containers containing microelectrodes). All cells were grown at 37°C in a humidified 5% CO2 atmosphere. Subsequently, cell counting was performed. After the plate was placed in the station, measurements were taken by the device every 15 min and continued until the end of the experiment. A total of 100 µl cells per well were seeded on e-plates, with 10×103 cells for MCF-7, 8×103 cells for SKBR3 and 5×103 cells for MDA MB-231 in each well. After 24 h, the cells were treated with appropriate concentrations of substances (crizotinib: 5, 10 and 15 µM; sodium butyrate: 300, 400 and 500 µM; combined concentrations: 10 µM crizotinib + 50 µM SB, 10 µM crizotinib + 100 µM SB, 10 µM crizotinib + 200 µM SB, 400 µM SB + 2.5 µM crizotinib, 400 µM SB + 5 µM crizotinib and 400 µM SB + 7.5 µM crizotinib) and changes in cell proliferation were observed using the xCELLigence RTCA DP System (Agilent Technologies, Inc.) in the incubator in special cell culture dishes containing microelectrodes.
Bromodeoxyuridine (BrdU) incorporation assay
The Colorimetric BrdU Cell Proliferation Assay Kit (cat. no. 2750; Millipore Sigma) was used to determine the proliferation levels in the MCF-7, MDA-MB-231 and SKBR-3 cells. This method was based on the principle of marking BrdU, which is incorporated into genomic DNA as a thymidine analogue in proliferating cells, using antibody probes and detecting cells in S phase. The BrdU test was performed following the instructions for use provided by the manufacturer. BrdU-labeled cells were evaluated at 370 nm in a microplate reader spectrophotometer.
Mitotic index
Mitotic index values, which demonstrate mitotic activity (the division rate of cells), were evaluated using the Mitotic Assay Kit (cat. no. 18021; Active Motif, Inc.). The mitotic activity assay was performed following the instructions for use provided by the manufacturer. Mitotic cells were evaluated at 450 nm in a microplate reader spectrophotometer within 5 min.
Caspase activity
The CaspaTag Caspase 3,7 In Situ Assay Kit (cat. no. APT403; MilliporeSigma) was used to determine active caspase-3 or −7 in cells undergoing apoptosis. The methodology was based on Fluorochrome Inhibitors of Caspases (26). Caspase activity was performed following the instructions for use provided by the manufacturer, and the activity was assessed at 490 nm excitation wavelength and 520 nm emission wavelength (λex 490, λem 520) using a BioTek Agilent FLx-800 Fluorescent Plate Reader (Agilent Technologies, Inc.).
Statistical analysis
The experiments were performed in three replicates. The data in the present study is presented as the mean ± standard deviation. Comparisons between groups were performed using one-way ANOVA and Dunnett's tests. Statistical evaluations of the cell index were made by the xCelligence device. Dunnett's post hoc test was used for all other parameters. Statistical analyses were reviewed twice in line with referee opinions. Experiments were performed in triplicate. The statistical analyses were performed using SPSS statistics software (v22.0; IBM). P<0.05 was considered to indicate a statistically significant difference.
ResultsCell index
Crizotinib and sodium butyrate were applied alone and in combination at different concentrations on three different cell lines: Crizotinib alone at concentrations of 5, 10 and 15 µM, SB alone at concentrations of 300, 400 and 500 µM, and combined concentrations of 10 µM crizotinib + 50 µM SB, 10 µM crizotinib +100 µM SB, 10 µM crizotinib +200 µM SB, 400 µM SB + 2.5 µM crizotinib, 400 µM SB + 5 µM crizotinib and 400 µM SB + 7.5 µM crizotinib. The cell index was measured for each cell line. Both single and combined applications of crizotinib and sodium butyrate showed cytostatic effects in MCF-7 cell lines (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9).
Mitotic index
The mitotic index values obtained following the application of crizotinib and SB alone and in combination to MCF-7, MDA-MB-231 and SKBR-3 cells are presented in Table I. As a result of the mitotic index application of critotinib and SB alone and in combination, mitotic index values in all cell lines decreased significantly over time, in comparison with the control (P<0.05). Moreover, the drug combinations were more successful in reducing the mitotic index values than single applications and were associated with a greater decline in cell proliferation.
BrdU cell proliferation test (labelling index)
The labeling index values obtained following the application of crizotinib and SB alone and in combination to MCF-7, MDA-MB-231 and SKBR-3 cells are presented in Table II. As a result of the BrdU cell proliferation application of critotinib and SB alone and in combination, labeling index values in all cell lines decreased significantly over time, in comparison with the control (P<0.05). Moreover, the drug combinations were more successful in reducing the labeling index values compared with that of the single applications and were associated with blockages in the synthesis phase of the cells.
Caspase activity
The caspase activity values obtained following the application of crizotinib and SB alone and in combination to MCF-7, MDA-MB-231 and SKBR-3 cells are presented in Table III. As a result of the caspase activity application of critotinib and SB alone and in combination, caspase activity values in all cell lines increased significantly over time, compared with the control (P<0.05). Furthermore, the drug combinations were associated with a greater increase in caspase activity compared with that of the single applications, and this caused the cells to undergo apoptosis.
Discussion
In the present study, the effectiveness of crizotinib as a monotherapy and when used with SB, which acts as a HDACi, was evaluated in different types of breast cancer cell lines.
Although there are different treatment approaches in cancer treatment, a single definitive treatment method cannot be focused on as each method has its own advantages and disadvantages, cancer is a disease specific to each individual, and treatments may differ from person to person (27). Furthermore, combination treatments, which have many advantages over monotherapy applications, are important in increasing treatment response and reducing drug resistance (28). The combination therapy approach allows the use of lower doses than monotherapy applications. Therefore, it increases the effectiveness of treatment and reduces the toxicity caused by high doses (29).
RTKs are associated with the development and progression of most types of cancer in humans (30). Therefore, targeting RTKs has become a therapeutic target for the treatment of cancer (30–32). Among RTKs, MET serves an important role during tumor development and progression (33,34). High levels of MET in breast cancer have been associated with a high tumor grade and poor prognosis (35–37). Results from clinical studies have associated MET overexpression with increased recurrence rates and decreased survival in patients with breast cancer (38,39). Furthermore, tissue microarray analyses and expression data have reported associations of MET overexpression with basal and ER/HER2-negative forms of breast cancer in humans (32,35,38–40). Animal experiments have also reported that MET activation induces mammary tumors with different characteristics (38,39).
The therapeutic use of anti-MET drugs has attracted interest due to the possible role of the hepatocyte growth factor/MET axis in the development of breast carcinomas and other malignancies (31,41). Studies have reported the development and assessment of small molecule MET tyrosine kinase inhibitors as a monotherapy or in combination with other targeted medicines (42). Anaplastic lymphoma kinase, receptor tyrosine kinase C-Ros oncogene 1 and MET are all targets of the multitarget RTK inhibitor crizotinib (43,44).
MET activation promotes cancer cell spread, migration and invasion (45). The findings from the present study with MDA-MB-231 cells demonstrated crizotinib administration dose-dependently inhibited the proliferation of these cells. Moreover, decreased MET activation may explain the anti-proliferative, anti-migratory and anti-invasive effects of crizotinib in triple-negative breast cancer (46).
Furthermore, it has been reported that MET overexpression is associated with decreased treatment sensitivity in different cancer types (47–50). Therefore, it may be beneficial to use crizotinib together with different therapeutic targets to increase treatment sensitivity (46).
SB is a short-chain fatty acid and a byproduct of carbohydrate metabolism in the intestine and. It is considered a potent HDACi that has emerged as an anticancer agent for certain cancers (51). Studies with SB have reported that it is associated with a reduction in the rate of breast cancer cells depending on dose and time (52,53). The inhibitory effect of SB on hormone-dependent and independent cell lines, and its ability to induce apoptosis through cell cycle disruption in hormone-dependent cell lines, suggests that it may have important implications for the treatment of human tumors, including breast cancer (53). Stockhammera et al (54) reported that HDAC inhibition sensitized highly resistant PF240-PE tumor cells to crizotinib and may overcome treatment resistance. Moreover, Fukuda et al (55) reported that the HDACi quinostat induced mesenchymal-epithelial transition by reducing zinc finger E-Box binding homeobox 1 expression in clones in vitro and therefore, restoring sensitivity to crizotinib.
The results of the present study, in which crizotinib was used together with a HDACi, are important in terms of creating more effective treatment strategies in different molecular subtypes of breast cancer when crizotinib is used with different therapeutic targets. The results demonstrated that combined applications, in which the concentrations of both crizotinib and SB were reduced, resulted in more effective results in different cell kinetic parameters than monotherapy applications. This is important in terms of reducing the side effects of the drugs used and reducing treatment resistance. Furthermore, when evaluated clinically, the results of the present study suggest that the MET inhibitor crizotinib may be more effective in breast cancers with different molecular subtypes when used together with SB. However, the absence of healthy breast cells in the present study makes it difficult to estimate clinical efficacy, as it could not be observed at which dose ranges the combined applications would cause toxic effects.
Acknowledgements
Not applicable.
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
MT, İÇ, EP and MÇ performed the experiments. MT, İÇ, EP and MÇ wrote and edited the manuscript. All authors confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Cell index of MCF-7 cells treated with 5, 10 and 15 µM crizotinib (1, Control; 2, 5 µM; 3, 10 µM; and 4, 15 µM). All concentrations were demonstrated to have cytostatic effects.
Cell index of MCF-7 cells treated with 300, 400 and 500 µM sodium butyrate (1, Control; 2, 300 µM; 3, 400 µM; and 4, 500 µM). All concentrations were demonstrated to have cytostatic effects.
Cell index of MCF-7 cells treated with combined concentrations of crizotinib and SB (1, Control; 2, 10 µM crizotinib + 50 µM SB; 3, 10 µM crizotinib + 100 µM SB; 4, 10 µM crizotinib + 200 µM SB; 5, 400 µM SB + 2.5 µM crizotinib; 6, 400 µM SB + 5 µM crizotinib; and 7, 400 µM SB + 7.5 µM crizotinib). All concentrations were demonstrated to have cytostatic effects. SB, sodium butyrate.
Cell index of MDA-MB-231 cells treated with 5, 10 and 15 µM crizotinib (1, Control; 2, 5 µM; 3, 10 µM; and 4, 15 µM). All concentrations were demonstrated to have cytostatic effects, the 5-µM concentration.
Cell index of MDA-MB-231 cells treated with 300, 400 and 500 µM sodium butyrate (1, Control; 2, 300 µM; 3, 400 µM; and 4, 500 µM). All concentrations were demonstrated to have cytostatic effects.
Cell index of MDA-MB-231 cells treated with combined concentrations of crizotinib and SB (1, Control; 2, 10 µM crizotinib + 50 µM SB, 3, 10 µM crizotinib +100 µM SB; 4, 10 µM crizotinib + 200 µM SB; 5, 400 µM SB + 2.5 µM crizotinib; 6, 400 µM SB + 5 µM crizotinib; and 7, 400 µM SB + 7.5 µM crizotinib). All concentrations were demonstrated to have cytostatic effects. SB, sodium butyrate.
Cell index of SKBR3 cells treated with 5, 10 and 15 µM crizotinib (1, Control; 2, 5 µM; 3, 10 µM; and 4, 15 µM). All concentrations were demonstrated to have cytostatic effects.
Cell index of SKBR3 cells treated with 300, 400 and 500 µM sodium butyrate (1, Control; 2, 300 µM; 3, 400 µM; and 4, 500 µM). All concentrations were demonstrated to have cytostatic effects.
Cell index of SKBR3 cells treated with combined concentrations of crizotinib and SB (1, Control; 2, 10 µM crizotinib + 50 µM SB; 3, 10 µM crizotinib + 100 µM SB; 4, 10 µM crizotinib + 200 µM SB; 5, 400 µM SB + 2.5 µM crizotinib; 6, 400 µM SB + 5 µM crizotinib; and 7, 400 µM SB + 7.5 µM crizotinib). All concentrations were demonstrated to have cytostatic effects. SB, sodium butyrate.
Mitotic index values of different breast cancer cells.
A, MCF-7 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
158±3
102±4a
112±6a
54±2a
59±2a
48
213±6
94±5a
105±2a
48±1a
46±3a
72
257±7
86±1a
96±2a
39±2a
37±4a
B, MDA-MB-231 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
121±3
73±2a
68±1a
45±2a
41±2a
48
134±5
64±4a
54±4a
38±3a
33±3a
72
149±4
59±6a
47±2a
35±4a
28±3a
C, SKBR-3 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
133±4
69±2a
65±3a
47±2a
45±1a
48
142±7
57±1a
59±2a
40±3a
41±1a
72
153±6
55±2a
54±3a
34±2a
33±2a
P<0.05 vs. control. Cri, crizotinib; SB, sodium butyrate.
Bromodeoxyuridine labelling index values of different breast cancer cells.
A, MCF-7 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
621±10
453±6a
401±4a
234±3a
251±5a
48
652±6
441±5a
396±2a
225±4a
234±3a
72
658±9
387±7a
383±8a
198±2a
201±4a
B, MDA-MB-231 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
404±7
296±4a
288±3a
199±2a
195±4a
48
416±8
287±5a
224±4a
145±3a
151±3a
72
438±6
265±7a
178±5a
121±5a
112±6a
C, SKBR-3 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
514±12
316±7a
321±6a
251±11a
235±7a
48
523±15
304±9a
301±8a
226±7a
197±6a
72
543±13
287±5a
244±10a
184±8a
152±5a
P<0.05 vs. control. Cri, crizotinib; SB, sodium butyrate.
Caspase activity values of MCF-7 cells.
A, MCF-7 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
164±11
278±23a
254±16a
356±19a
364±22a
48
165±9
302±21a
289±22a
368±24a
379±25a
72
167±12
325±18a
314±14a
398±22a
387±29a
B, MDA-MB-231 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
216±14
321±13a
343±23a
514±38a
523±33a
48
214±11
335±16a
544±26a
534±41a
544±29a
72
215±13
358±12
365±22a
575±37a
565±31a
C, SKBR-3 cells
Time, h
Control
10 µm Cri
400 µm SB
10 µm Cri + 50 µm SB
400 µm SB + 2.5 µm Cri
24
198±11
325±22a
351±20a
543±29a
554±39a
48
198±14
367±19a
403±19a
547±36a
568±42a
72
200±17
374±24a
433±18a
575±44a
579±26a
P<0.05 vs. control. Cri, crizotinib; SB, sodium butyrate.