The role of mitochondrial dysfunction in the cytotoxic synergistic effect of gemcitabine and arsenic on breast cancer

Abstract

Breast cancer is the most common type of cancer in women worldwide. A common approach to cancer treatment in clinical practice is to use a combination of drugs to enhance the anticancer activity of drugs while reducing their side effects. In this regard, we evaluated the effectiveness of combined treatment with gemcitabine (GCB) and arsenic (ATO) and how they affect the cell death pathway in cancer cells. Cytotoxic activity of drugs individually or combined against MDA-MB-231 and MCF-7 was performed by MTT method and isobolographic analysis was used to determine the interaction between these factors. The combination of ATO and GCB showed synergistic anti-cancer activity (CI < 1) in both cancer cell lines. The combination of ATO and GCB induced sub-G1 phase arrest, apoptosis and death rates in MCF-7 and MDA-MB-231 cells. The apoptotic response induced by the combination of GCB and ATO was dependent on caspase 3/7. Combined treatment with mitochondrial membrane potential (MMP) reduction and increased reactive oxygen species (ROS) production caused mitochondrial dysfunction. Co-treatment significantly reduced catalase (CAT) activity in both cancer cells compared to the control group and cells treated with each monotherapy. A significant decrease in cellular GSH was observed in cancer cells treated with ATO and GCB. In addition, migration and invasion were significantly reduced in breast cancer cells treated with the combination of ATO and GCB compared to cells treated with ATO and GCB. In conclusion, the combined treatment of ATO and GCB synergistically increased the anti-cancer activity, and these findings provide an effective approach for the treatment of breast cancer. To the best of our knowledge, this is the first study showing promising results for combination therapy with ATO and GCB in breast cancer.

Introduction

Breast cancer is one of the most common cancers in women, which is generally very aggressive with high rate of recurrence [1]. Surgery, chemotherapy, radiotherapy, hormone therapy and immunotherapy are common treatments for breast cancer. Despite significant advances in the treatment of breast cancer, it remains a major threat to women’s health [2]. Combined treatment has been considered significantly in clinics due to the reduction of problems related to chemotherapy drugs [3]. Combinations of drugs are usually more effective since each drug compensates for the other. Moreover, the side effects related to high doses of single drug can be eliminated, because a combination of drugs synergistically improves different biological signaling pathways of drugs and allows the use of low doses of each drug [3,4].

Gemcitabine (GCB), a hydrophilic drug is extensively used in the treatment of different solid tumors such as breast cancer. It is a specific analogue of deoxycytidine that inhibits ribonucleotide reductase, dCMP-deaminase and CTP synthetase and eventually DNA damage [5–8].

Arsenic trioxide (ATO) is an approved anti-cancer agent for the treatment of patients with acute promyelocytic leukemia (APL). Furthermore, it has been reported that ATO shows significant anti-cancer activity on some solid tumors including; gastric, breast, colon, liver, esophageal and lung cancer [9,10]. ATO triggers apoptosis in cancer cells through numerous mechanisms, including induction of oxidative stress, activation of the JNK (c-Jun N-terminal kinase) signaling cascade and suppression of AKT activity [5]. However, higher level of ATO is required for apoptosis induction in cancer cells. Treatment with ATO alone is also restricted in the clinical applications due to its toxicity and low efficacy. Therefore, combining ATO with other anti-cancer drugs may provide a new approach for cancer treatment [5,9].

In the current study, we evaluated whether the combination therapy of ATO and GCB are effective against the breast cancer cell lines. To the best of our knowledge, this is the first study indicated promising results for ATO combined with GCB in the treatment of breast cancer.

Material and methods

MDA‐MB‐231 (human breast cancer cell) and MCF-7 (human breast cancer) were obtained from Pasteur Institute, Iran. Dulbecco’s modified eagle medium (DMEM), penicillin-streptomycin (Pen-Strep) and phosphate buffered saline (PBS) were purchased from Bioidea, Iran. Fetal bovine serum (FBS) was obtained from Gibco, USA. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], arsenic trioxide (ATO), gemcitabine (GCB), dimethyl sulfoxide (DMSO), propidium iodide (PI), fluorescein diacetate (FDA), dichloro-dihydro-fluorescein diacetate (DCFH-DA), rhodamine 123, sulfosalicylic acid and O-phthalaldehyde (OPT) were purchased from Sigma-Aldrich Co, USA. RNase A and Matrigel were acquired from Bio basic, Canada and Corning Life Sciences, USA, respectively. There were no human or animal studies.

Cytotoxicity assay

The cytotoxicity of ATO and GCB were determined by MTT assay. MDA‐MB‐231 and MCF-7 cells were grown at 37°C, 5% CO2 and 95% relative humidity in high‐glucose DMEM supplemented with 10% FBS and 1% Pen-Strep. The cells were seeded in a 96-well plate (1×10⁴ cells/well) and allowed to attach and grow for 24 h. Then, cells were treated with different concentrations of ATO (1.25, 2.5, 5, 10 and 20 μM) and GCB (10, 20, 40, 80, 160 and 320 μM) for 48 h. Cells were incubated with MTT (5 mg/ml) at 37˚C for 4 h and 100 μl of DMSO was added to the wells and plates were incubated at 37˚C for 30 min. The cell viability percentages were calculated according the following Eq (1) and IC₅₀ was determined by GraphPad Prism version (8.0.1).

(1)

Combination Index (CI) analysis

The isobolographic analysis was performed to determine the interactions between drugs, which allows their stratification as synergistic, additive and antagonistic. For this purpose, the trypan blue exclusion test was used for evaluation the combination effects of ATO and GCB on breast cancer cells. The cells were cultured under the IC₅₀ concentration ratio at 0.0625x (5.625:0.625), 0.125x (11.25:1.25), 0.25x (22.5:2.5), 0.5x (45:5), 1x (90:10) μM of GCB and ATO, respectively for 48 h. Then, cells were centrifuged at 1500 rpm for 5 min and cell pellets resuspend in 1 ml serum-free complete medium. 1-part cell suspension and 1-part of 0.4% trypan blue were mixed. The unstained (viable) and stained (nonviable) cells separately were counted by the hemocytometer. The viability percentage of cells were calculated as follows (Eq 2):(2)

Using this analysis method, the combination index (CI) value was calculated according to the levels of growth inhibition by each agent individually and combination of GCB with ATO. The CI values of <1, >1 and = 1 indicate synergism, antagonism and additive effect of drugs, respectively.

Apoptosis and cell cycle analysis

MDA‐MB‐231 and MCF-7 cells (5 × 10⁵ cells/well) were seeded in to 6‐well plate and treated with GCB, ATO and GCB+ATO for 48 h. Annexin V-FITC and propidium iodide (PI) were added to each well according to the manufacturer protocol of IQ Products (IQP-116F, Netherlands) kit. Flow cytometry was performed using BD FACSCanto^TM II (BD Biosciences, San Jose, CA, USA). For cell cycle analysis, MDA‐MB‐231 and MCF-7 cells (5 × 10⁵ cells/well) were seeded in to 6‐well plate and treated with GCB, ATO and GCB+ATO for 48 h. Then cells were suspended in 70% cold ethanol and incubated in 4°C for 4 h. After centrifugation, the cell pellets were resuspended with PI (50 μg/ml), RNase A (10 μg/ml) and PBS (940 μl) and incubated in the dark for 30 min in 37°C. Cell cycle distributions were also determined using BD FACSCanto^TM II.

Fluorescein Diacetate (FDA) and Propidium Iodide (PI) double staining

MDA‐MB‐231 and MCF-7 cells (5 × 10⁵ cells/well) were seeded in to 6‐well plate and treated with GCB, ATO and GCB+ATO for 48 h. Then, cells were washed with PBS and stained with FDA (10 μg/ml) and PI (50 μg/ml) on ice for 5 min. After that, FDA-PI working solution was aspirated, then cold PBS was added and images were taken by fluorescent microscopy (Olympus IX71, Japan).

Caspase 3/7 activity assay

Caspase-3/7 activity was determined using the kit of Kiazist (KCAS96, Iran) according to the manufacturer’s protocol. 1 × 10⁶ cells of MDA‐MB‐231 and MCF-7 were treated with GCB, ATO and GCB+ATO for 48 h. Cells were harvested and incubated with 500 μl of caspase lysis buffer for 20 min at 4°C. Then, cells were centrifuged at 12000 rpm for 15 min at 4°C and supernatants were used to measure caspase 3/7 activity. The assay is based on the formation of the chromophore p-nitroaniline (p-NA) formed by cleavage from the labeled substrate DEVD-pNA. The p-NA can be quantified using a spectrophotometer or a microtiter plate reader reading absorbance at 405 nm.

Detection of intracellular ROS

Intracellular ROS was detected using DCFH-DA. After treatment cells with GCB, ATO and GCB+ATO for 48 h, cells were incubated with 20 μmol/L DCFH-DA at 37°C for 30 min in the dark. The fluorescence intensity of 2,7-dichlorofluorescein (DCF) as fluorescent compound (that produced due to deacetylated a DCFH-DA by intracellularly esterase) was detected by spectrofluorometer (CYTATION 3, Imaging reader, USA).

Analysis of mitochondrial membrane potential

Cells were treated with GCB, ATO and GCB+ATO for 48 h. Then, cells were incubated with 20 μM of rhodamine 123 dye for 40 min at 37°C. After incubation, the emission and excitation signals were detected at 535 and 490 nm, respectively using a spectrofluorometer (CYTATION 3, Imaging reader, USA).

Intracellular GSH and CAT activity assay

The GSH content of cells were measured as previous described [11]. Briefly, 1 × 10⁶ cells of MDA‐MB‐231 and MCF-7 were treated with GCB, ATO and GCB+ATO for 48 h. Then, cells were harvested and sonicated to lyse. Samples were centrifuged at 18000 g for 10 min at 4°C and the supernatants collected and mix with 5% of sulfosalicylic acid and centrifuged again at 9000 g for 10 min at 4°C. For GSH assessment, O-phthalaldehyde was added to the supernatant and kept at room temperature for 10 min. Then, GSH activity was assessed at excitation of 355 nm and emission of 420 nm using a spectrofluorometer (CYTATION 3, Imaging reader, USA). Catalase (CAT) activity was also determined using the kit of Kiazist (KCAT96, Iran) according to the manufacturer’s protocol.

Invasion assay

24 transwells cell culture insert with 8 μm pore size were used for invasion assay. At the first, 40 μl Matrigel was added to a 24 transwell insert and incubated in a 37°C for 30 min in order to solidification. MDA‐MB‐231 and MCF-7 cells (3 × 10⁴ cells/well) were seeded into 24‐well plate and treated with GCB, ATO and GCB+ATO for 48 h. Cells were harvested and resuspended in 200 μl serum‐free DMEM placed into the upper chamber of each insert. DMEM with 20% FBS, as chemoattractant was added to the bottom chamber. After 24 h, remaining cells and Matrigel were removed with gently swabbing and cells were fixed with 70% ethanol for 10 min to allow cell fixation. Then, cells were stained with 0.2% crystal violet for 15 min. The crystals violet from the top of the membrane were gently removed and allow the transwell membrane to dry. The numbers of invaded cells were counted under an inverted microscope (Olympus IX71. Japan) and counted the number of cells in different fields of view to got an average sum of cells that have migrated through the membrane.

Migration assay

For migration assay MDA‐MB‐231 and MCF-7 cells were seeded into 6‐well plate (5 × 10⁵ cells/well) with high‐glucose DMEM supplemented with 1% FBS and incubated overnight. When cells reached 90% confluency, using a 10 μl pipette tip, a straight scratch wound was created in each well. Then, wells were gently washed to remove cell debris. This point was considered the “0 h,” and the “zero wound” was captured using a microscope (Olympus IX71) and cells were treated with GCB, ATO and GCB+ATO for 48 h. After that, images of scratch wounds of control and treated groups were captured in phase contrast. Wound areas were measured using the Image J software (National Institutes of Health, Bethesda, MD, USA). The closure of the scratch was quantified according to the following Eq (3):(3)

Statistical analysis

Statistical analysis performed using one-way analysis of variance by GraphPad Prism version (8.0) followed by post hoc Tukey’s test. Results were reported as mean ± SD. Flow jo software version 7.6.1 was also used to analyze flow cytometry data. Statistical significance was indicated as *p < 0.05, **p < 0.01 and ***p < 0.001.

Results and discussion

Cytotoxicity of GCB and ATO on breast cancer cell lines

MTT assay was used for cytotoxic evaluation of ATO and GCB on MCF7 and MDA‐MB‐231 cells. After treatment of MCF7 and MDA-MB-231 cells for 48 h with ATO, IC₅₀s were obtained at 9.75 ± 2.35 μM and 7.64 ± 1.36 μM, respectively (Fig 1Aa and 1Ba). The IC₅₀s of MCF-7 and MDA‐MB‐231 cells treated with GCB were 90.78 ± 14 and 99.64 ± 8.24 μM, respectively (Fig 1Ab and 1Bb). According to the obtained results, cell viability decreased significantly with increasing concentration of drugs. In other words, the cytotoxic effects of ATO and GCB were dose-dependent. Therefore, the combination of 10 μM ATO and 90 μM GCB (diluted concentration till 1:16 ratio) was used for combination treatment. The combination index (CI) was also used for analyzing results. Isobolographic analysis is a statistical method that has been widely used to assess drug interactions. This method is based on the cytotoxicity experiments completed on treated cells with both drugs and the mixture [12]. The ATO and GCB combination showed synergistic anti-cancer activity (CI < 1) in both cancer cell lines (CI: 0.83 for MCF-7 and 0.88 for MDA-MB-231). This finding is supported by data derived via trypan blue staining (Table 1), which indicated a decreased viability of MCF-7 and MDA-MB-231 cells treated with both ATO and GCB. Therefore, 4x dilution of IC₅₀ (2.5 μM ATO and 22.5 μM GCB) was used for further assessment. According to these findings, the combination of ATO and GCB significantly reduced the IC₅₀ of each agent alone. These results showed that by combining ATO and GCB, the side effects of both drugs can be significantly decreased by reducing the dose of both drugs and at the same time maintaining their clinical effectiveness.

[Figure omitted. See PDF.]

Cytotoxicity activity of (Aa) ATO on MCF-7 cells, (Ab) GCB on MCF-7 cells, (Ba) ATO on MDA-MB-231 cells and (Bb) GCB on MDA-MB-231 cells. Data are presented as mean ± SD (n = 3).

[Figure omitted. See PDF.]

Combination effect of ATO and GCB on apoptosis induction

As shown in Fig 2, combination of ATO and GCB induced higher apoptotic rate in MCF-7 cells as compared to each drugs alone. The rate of apoptosis in MCF-7 cells treated with ATO+GCB was 49.02 ± 5.05% vs control 1.88 ± 1.24% (p < 0.0001). The rate of apoptosis in MDA‐MB‐231 cells was 48.57 ± 11.69% vs control 4.27 ± 3.54%. As shown in Fig 3, ATO+GCB significantly increased apoptosis in MDA‐MB‐231 cells than other groups (control group and groups treated with ATO and GCB alone). These findings showed that combined treatment with ATO and GCB led to a synergistic effect in causing apoptosis in cells. However, it should be noted that treatment with ATO and GCB induced both types of cell death, necrosis and apoptosis, but strongly induced cell death toward apoptosis. Apoptosis is one of the most important pathways of programmed cell death, which plays an important role in cancer prevention [13]. One of the main goals of cancer treatment is to initiate the apoptotic pathway because apoptotic cells are eventually removed by phagocytosis, thus preventing damage to the surrounding tissues. However, the necrosis pathway induces an inflammatory response that leads to damage to adjacent healthy cells.

[Figure omitted. See PDF.]

Data are presented as mean ± SD. ****: Significant difference with control, ATO and GCB group (p < 0.0001). &: Significant difference with control group (p < 0.05).

[Figure omitted. See PDF.]

Data are presented as mean ± SD (n = 3). ***: Significant difference with control group (p < 0.001). **: Significant difference with ATO, GCB group (p < 0.01). &&&: Significant difference with control group (p < 0.001).

We further assayed the effects of ATO+GCB on cell cycle arrest using PI staining on both cell lines. As shown in Fig 4A and 4B, in comparison with control cells, the percentage of cells in sub-G1 phase was clearly increased in MCF-7 cells (ATO+GCB 35.82 ± 6.9% vs control 0.43 ± 0.38%), while the percentage of G0/G1 phase cells was noticeably reduced (ATO+GCB 44.9 ± 4.84% vs control 61.45 ± 2%). Moreover, the percentage of cells in sub-G1 phase was remarkably increased in MDA‐MB‐231 (ATO+GCB 30.75 ± 4.03% vs control 0.85 ± 0.21%), whereas the percentage of cells in G0/G1 phase was decreased (ATO+GCB 45.10 ± 10.35% vs control 69.29 ± 2.72%) when compared with control cells or cells treated with either ATO or GCB alone. Cell cycle is crucial for cell growth, which controls cell proliferation [14]. These results demonstrated that combination of ATO and GCB synergistically increased sub-G1 phase arrest in MCF-7 and MDA‐MB‐231 cells, representing the apoptosis induction owing to DNA fragmentation which seems to be the main anti-cancer mechanism of ATO and GCB [15,16].

[Figure omitted. See PDF.]

Cell cycle arrest after treatment of A) MCF-7 and B) MDA-MB-231 cells with ATO (2.5 μM) and GCB (22.5) μM for 48 h at 37°C. Data are presented as mean ± SD (n = 3). ***: Significant difference with control group (p < 0.001). **: Significant difference with control, ATO, GCB group (p < 0.01). &: Significant difference with control group (p < 0.05). $: Significant difference with control group (p < 0.01).

FDA-PI staining and viability of cells

In viable cells, fluorescein diacetate (FDA, a nonpolar ester) can easily cross the cell membranes and after being hydrolyzed by free exoenzymes and membrane-bound enzymes, fluorescein is released and remains in the cells with healthy membranes [17]. Propidium Iodide (PI) interacts with DNA only in damaged cells; therefore, it is used for detection of dead cells [18]. In the current study, FDA-PI double staining was used to detect cell viability by evaluating the dead and viable cells. According to the obtained results, combination of ATO+GCB remarkably increased the rate of death in both cancer cells more than cells treated with ATO and GCB alone (Fig 5A and 5B).

[Figure omitted. See PDF.]

Mean fluorescence intensity of (Aa) FDA, (Ab) PI, cell viability as determined by FDA-PI staining. (Ba) MCF-7 and (Bb) MDA-MB-231 cells stained with FDA/PI (alive cells stained green, whereas dead cells stained red).

Caspase 3/7 activity assay

To determine whether the induction of apoptosis after treatment with GCB and ATO is dependent on caspase 3/7, cells were treated with the IC₅₀ of GCB and ATO. As shown in Fig 6, monotherapy with ATO or GCB significantly increased the activity of caspase 3/7 in MCF-7 and MDA‐MB‐231 cells more than control group. Interestingly, the combined therapy of ATO+GCB remarkably elevated the activity of caspase 3/7 in cancer cells compared to those of cells treated with each monotherapy. Caspases are a unique family of cysteine proteases that trigger apoptosis in cells [19] and caspase 3/7 plays a crucial role in the process of apoptosis [20]. According to obtained results, the apoptotic response induced by GCB and ATO was dependent on caspase 3/7. These findings are consistent with the results of other researchers that reported different combinations of chemotherapeutic agents can significantly induce caspase 3/7 activity in cancer cells [21,22].

[Figure omitted. See PDF.]

Caspase 3/7 activity after treatment of A) MCF-7 and B) MDA-Mb-231 with ATO (2.5 μM) +GCB (22.5 μM) for 48 h at 37°C (n: 3). Data are presented as mean ± SD. ***: Significant difference with control group (p < 0.001). **: Significant difference with control group (p < 0.01). &: Significant difference with control group (p < 0.05). $: Significant difference with control group (p < 0.05).

Determination of intracellular ROS and MMP

ROS generation in cells treated with ATO+GCB combination was measured to evaluate the oxidative stress status. As illustrated in Fig 7A and 7B, ATO+GCB combination significantly increased the ROS level in cancer cells compared to both drugs alone. It has been reported that high level of ROS causes disruption of mitochondrial membrane potential (MMP) and leads to mitochondrial dysfunction. Mitochondrial dysfunction triggers apoptosis through the cytochrome c release, which promotes the caspase cascade [23]. Consequently, we determined the levels of MMP in MCF-7 and MDA-Mb-231 cells treated with ATO+GCB. It was found that the levels of MMP in co-treatment groups was significantly lower than other groups (p < 0.001) (Fig 7C and 7D). These results indicated that combination treatment triggers mitochondrial dysfunction through reduced levels of MMP owing to increased ROS production. Our results are in accordance with Wu et al. findings which observed that co-treatment of ATO and paclitaxel reduced MMP and increased ROS generation [24] and also with the results of Lee et al. which found that combined therapy of GCB and Ivermectin induced apoptotic pathway in cancer cells via the overproduction of ROS and disrupting MMP [25].

[Figure omitted. See PDF.]

Relative ROS production in A) MCF-7 and B) MDA-Mb-231 cells treated with ATO, GCB and ATO + GCB and measurement of MMP in C) MCF-7 and D) MDA-Mb-231 cells treated with ATO, GCB and ATO + GCB (ATO: 2.5 μM+GCB: 22.5 μM, 48 h at 37°C). Data are presented as mean ± SD. ***: Significant difference with control group (p < 0.001). **: Significant difference with control group (p < 0.01). &&: Significant difference with control group (p < 0.01). &: Significant difference with control group (p < 0.05). $: Significant difference with control group (p < 0.05). $ $: Significant difference with control group (p < 0.01).

Intracellular CAT and GSH activity assay

Catalase decomposes hydrogen peroxide (H₂O₂) into water and oxygen, which protects cells from the toxic effects of ROS [26]. ATO+GCB combination caused a statistically significant decrease in the CAT activity in both cancer cells than the control group and cells treated with each monotherapy (Fig 8A and 8B). It has been observed that ATO reduced catalase expression in breast cancer cells [27,28]. Increased levels of catalase have been reported to decrease the effective levels of ROS; consequently, leads to resistance to GCB in cancer cells [29]. According to the results of this study, it seems that ATO decreases catalase activity in breast cancer cells and sensitizes them to GCB.

[Figure omitted. See PDF.]

The CAT activity in A) MCF-7 and B) MDA-Mb-231 cells treated with ATO, GCB and ATO + GCB and GSH content in C) MCF-7 and D) MDA-Mb-231 cells treated with ATO, GCB and ATO + GCB (ATO: 2.5 μM+GCB: 22.5 μM), for 48 h at 37°C. Data are presented as mean ± SD. ***: Significant difference with control and GCB group (p < 0.001). **: Significant difference with control, ATO, GCB group (p < 0.01). &: Significant difference with control group (p < 0.05). #: Significant difference between GCB and ATO group (p < 0.05). ##: Significant difference between GCB and ATO group (p < 0.01). $: Significant difference with control group (p < 0.05). $ $: Significant difference with control group (p < 0.01).

A significant depletion in cellular GSH was observed in cancer cells treated with ATO+GCB (Fig 8C and 8D). GSH is the most abundant cellular antioxidant that plays an important role in promoting cell survival. Moreover, increasing intracellular GSH is related to resistance of cells to anti-cancer drugs [30,31]. Thus, reducing intracellular GSH enhances ROS production and increases chemotherapy sensitivity. As shown in Fig 8C and 8D, GCB treatment induced GSH synthesis in cancer cells, which was declined by co-treatment with ATO. Previous studies demonstrated that GCB induces GSH synthesis via activating of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant pathway to counteract the effects of ROS [32]. On the other hand, ATO can reduce GSH synthesis by decreasing intracellular cysteine and glutamate levels [33]. These findings support our hypothesis that ATO might improve the efficacy of GCB on cancer cells by inhibition of GSH synthesis.

Effect of ATO and GCB combination on the invasion and migration of cancer cells

Migration and invasion ability of cancer cells allow them to alter their position in tissues and leads to metastasis, which is the main cause of death in cancer patients [34]. Accordingly, in this study, we evaluated whether the combination of ATO and GCB can improve the ability of both drugs to inhibit breast cancer cells invasion and migration. It was observed that the combination treatment with ATO and GCB could remarkably reduce the rate of invasion in MCF-7 and MDA‐MB‐231 cells (Fig 9A and 9B). Notch signaling pathway plays an oncogenic role in breast cancer progression. It has been reported that ATO inhibits the Notch pathway by down-regulating the expression of Bcl-2 and NF-κB, which leads to inhibition of invasion [35,36]. It was also observed that migration was significantly reduced in MCF-7 and MDA‐MB‐231 cells treated with ATO+GCB combination as compared to those treated with ATO or GCB (Fig 10A and 10B). These results indicate that the combination of ATO and GCB significantly enhances the inhibitory effect on migration and invasion of breast cancer cells. These findings are in agreement with Lin et al. results that reported co-treatment of ATO and berberine increased the inhibition of migration and invasion on cancer cells [37]. In addition, this is the first report of a strategy involving the combination of ATO and GCB in the treatment of breast cancer cells to reduce migration and invasion, which leads to inhibition of metastasis and increases the efficacy of treatment.

[Figure omitted. See PDF.]

Inhibition of the invasion of A) MCF-7 and B) MDA-Mb-231 cells after treatment with ATO (2.5 μM) and GCB (22.5 μM) for 48 h at 37°C. Data are presented as mean ± SD. ****: Significant difference with control, ATO, GCB group (p < 0.0001). $ $ $: Significant difference with control group (p < 0.001). &: Significant difference with control group (p < 0.05). &&: Significant difference with control group (p < 0.01).

[Figure omitted. See PDF.]

Inhibition of the migration of A) MCF-7 and B) MDA-Mb-231 cells after treatment with ATO (2.5 μM) and GCB (22.5 μM) for 48 h at 37°C. Data are presented as mean ± SD. ****: Significant difference with control, ATO, GCB group (p < 0.0001). $ $ $ $: Significant difference with control group (p < 0.0001). &&&&: Significant difference with control group (p < 0.0001).

Breast cancer is one of the main causes of death in women worldwide, which has become a serious threat to women’s health. GCB is widely used in the treatment of different solid tumors such as breast cancer. ATO also displays significant anti-cancer activity on some solid tumors including breast cancer. However, treatment with GCB or ATO alone is restricted in the clinical applications due to their toxicity and low efficacy. In order to reduce the problems related to chemotherapy drugs, combination therapy has been significantly considered in clinics. It is reported that ATO and GCB reduced cell viability in a manner that was both dose- and time-dependent and also triggering cell cycle arrest and apoptosis in Lymphoma cell lines [38]. The S-phase kinase associated protein 2 (Skp2), which belongs to the F-box protein family, plays a key role in regulating cell cycle progression and is notably overexpressed in pancreatic cancer (PC). In a study, it was demonstrated that ATO inhibited cell growth and invasion by downregulating Skp2 in PC cells. Emerging evidence indicates that Skp2 is critical in mediating drug resistance across various cancer types. Also, it was found that ATO could enhance the sensitivity of PC cell lines to GCB. The results showed that the combination of ATO and GCB produced significant antitumor effects in Patu8988 and Panc-1 PC cells. Furthermore, ATO enhanced the efficacy of GCB through the downregulation of the Skp2 pathway in these cells [39].

In the current study, isobologram analysis was performed to evaluate whether the biological responses resulting from a mixture of agents are superior, less or equal than what would be expected based on the individual activities of each agent alone. According to the obtained results, combination of ATO and GCB showed synergistic anti-cancer activity (CI < 1) in breast cancer cells. Co-administration of ATO and GCB triggered apoptosis via the mitochondrial pathway due to increasing the level of ROS, led to disruption of MMP which promoted the caspase cascade in cancer cells. Combination of ATO and GCB significantly improved the inhibitory effect on migration and invasion of breast cancer cells that can be more effective in inhibiting metastatic progression in patients. In addition, the main finding has been that the combination of ATO and GCB has the ability to reduce the dose of each drug alone, their effectiveness is also maintained, and as a result, side effects will be less. In conclusion, the combined treatment of ATO and GCB may be a promising therapeutic strategy to increase the survival rate of breast cancer patients. The proposed mechanism of anti-tumor activity for synergistic effect of ATO and GCB in breast cancer cells is presented schematically in Fig 11.

[Figure omitted. See PDF.]

Conclusion

The aim of this study was to investigate whether the combination of GCB and ATO shows a stronger therapeutic effect than these agents alone in breast cancer. GCB with ATO showed a synergistic effect on anti-tumor response in cancer cells. According to the findings, it is suggested that the synergistic anti-cancer effect of GCB and ATO on breast cancer cells is done through apoptosis due to increased ROS production, decreased MMP and increased caspase activity. In addition, a significant decrease in cellular GSH, migration and invasion were observed in cancer cells treated with the combination of ATO and GCB. To the best of our knowledge, this is the first report to determine the synergistic effect of ATO and GCB in breast cancer. On the other hand, due to the need for a low dose of GCB and ATO and thus reducing side effects, this combination therapy can be a promising platform for the treatment of breast cancer.

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* PubMed/NCBI

* Google Scholar

9. 9. Nasrollahzadeh A., Bashash D., Kabuli M., Zandi Z., Kashani B., Zaghal A., et al, Arsenic trioxide and BIBR1532 synergistically inhibit breast cancer cell proliferation through attenuation of NF-κB signaling pathway, Life sciences, 257 (2020) 118060.

* View Article

* Google Scholar

10. 10. Yang Y., Qi J., Wang H., Zhao Y., Wu Y., Wu Z., et al, Ameliorative effects of Danshensu from the functional food Salvia miltiorrhiza against arsenic trioxide-induced cardiac toxicity in vivo and in vitro: Involvement of inhibiting the AKT/IKK/NF-κB signaling pathway, Journal of Functional Foods, 99 (2022) 105304.

* View Article

* Google Scholar

11. 11. Singh V., Gera R., Purohit M.P., Patnaik S., Ghosh D., Fluorometric estimation of glutathione in cultured microglial cell lysate, Bio-protocol, 7 (2017) e2304–e2304. pmid:34541073

* View Article

* PubMed/NCBI

* Google Scholar

12. 12. Bobiński M., Okła K., Łuszczki J., Bednarek W., Wawruszak A., Moreno-Bueno G., et al, Gemcitabine and Selected mTOR Inhibitors in Uterine Sarcomas and Carcinosarcoma Cells-an Isobolographic Analysis, International Journal of Medical Sciences, 17 (2020) 2987. pmid:33173419

* View Article

* PubMed/NCBI

* Google Scholar

13. 13. Sarvestani N.N., Sepehri H., Delphi L., Farimani M.M., Eupatorin and salvigenin potentiate doxorubicin-induced apoptosis and cell cycle arrest in HT-29 and SW948 human colon cancer cells, Asian Pacific Journal of Cancer Prevention: APJCP, 19 (2018) 131. pmid:29373904

* View Article

* PubMed/NCBI

* Google Scholar

14. 14. Li P., Zhong D., Gong P.-y., Synergistic effect of paclitaxel and verapamil to overcome multi-drug resistance in breast cancer cells, Biochemical and biophysical research communications, 516 (2019) 183–188. pmid:31204050

* View Article

* PubMed/NCBI

* Google Scholar

15. 15. Yedjou C., Tchounwou P., Jenkins J., McMurray R., Basic mechanisms of arsenic trioxide (ATO)-induced apoptosis in human leukemia (HL-60) cells, Journal of hematology & oncology, 3 (2010) 1–9.

* View Article

* Google Scholar

16. 16. Nakashima M., Adachi S., Yasuda I., Yamauchi T., Kawaguchi J., Itani M., et al, Phosphorylation status of heat shock protein 27 plays a key role in gemcitabine-induced apoptosis of pancreatic cancer cells, Cancer letters, 313 (2011) 218–225. pmid:21999932

* View Article

* PubMed/NCBI

* Google Scholar

17. 17. Chen Y., Sun L., Wang Y., Zhao X., A dual-fluorescent whole-well imaging approach for screening active compounds against doxorubicin-induced cardiotoxicity from natural products, RSC advances, 5 (2015) 106431–106438.

* View Article

* Google Scholar

18. 18. Kocabey S., Ekim Kocabey A., Schneiter R., Rüegg C., Membrane-interacting DNA nanotubes induce cancer cell death, Nanomaterials, 11 (2021) 2003. pmid:34443832

* View Article

* PubMed/NCBI

* Google Scholar

19. 19. Pilco-Ferreto N., Calaf G.M., Influence of doxorubicin on apoptosis and oxidative stress in breast cancer cell lines, International journal of oncology, 49 (2016) 753–762. pmid:27278553

* View Article

* PubMed/NCBI

* Google Scholar

20. 20. Shim M.K., Yoon H.Y., Lee S., Jo M.K., Park J., Kim J.-H., et al, Caspase-3/-7-specific metabolic precursor for bioorthogonal tracking of tumor apoptosis, Scientific reports, 7 (2017) 1–15.

* View Article

* Google Scholar

21. 21. Sagwal S.K., Pasqual-Melo G., Bodnar Y., Gandhirajan R.K., Bekeschus S., Combination of chemotherapy and physical plasma elicits melanoma cell death via upregulation of SLC22A16, Cell Death & Disease, 9 (2018) 1179. pmid:30518936

* View Article

* PubMed/NCBI

* Google Scholar

22. 22. Ma L., Wei J., Su G.H., Lin J., Dasatinib can enhance paclitaxel and gemcitabine inhibitory activity in human pancreatic cancer cells, Cancer biology & therapy, 20 (2019) 855–865. pmid:30866697

* View Article

* PubMed/NCBI

* Google Scholar

23. 23. Handali S., Moghimipour E., Kouchak M., Ramezani Z., Amini M., Angali K.A., et al, New folate receptor targeted nano liposomes for delivery of 5-fluorouracil to cancer cells: strong implication for enhanced potency and safety, Life sciences, 227 (2019) 39–50. pmid:31002921

* View Article

* PubMed/NCBI

* Google Scholar

24. 24. Wu P.-J., Hsin I.-L., Hung W.-L., Lee M.-S., Wang P.-H., Ko J.-L., Combination treatment with cyclosporin A and arsenic trioxide induce synergistic cell death via non-apoptotic pathway in uterine cervical cancer cells, Chemico-Biological Interactions, 368 (2022) 110177. pmid:36100036

* View Article

* PubMed/NCBI

* Google Scholar

25. 25. Lee D.E., Kang H.W., Kim S.Y., Kim M.J., Jeong J.W., Hong W.C., et al, Ivermectin and gemcitabine combination treatment induces apoptosis of pancreatic cancer cells via mitochondrial dysfunction, Frontiers in Pharmacology, (2022) 3403. pmid:36091811

* View Article

* PubMed/NCBI

* Google Scholar

26. 26. Pieniążek A., Czepas J., Piasecka-Zelga J., Gwoździński K., Koceva-Chyła A., Oxidative stress induced in rat liver by anticancer drugs doxorubicin, paclitaxel and docetaxel, Advances in medical sciences, 58 (2013) 104–111. pmid:23612702

* View Article

* PubMed/NCBI

* Google Scholar

27. 27. Glorieux C., Calderon P.B., Catalase down-regulation in cancer cells exposed to arsenic trioxide is involved in their increased sensitivity to a pro-oxidant treatment, Cancer Cell International, 18 (2018) 1–9.

* View Article

* Google Scholar

28. 28. Glorieux C., Calderon P.B., Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach, Biological chemistry, 398 (2017) 1095–1108. pmid:28384098

* View Article

* PubMed/NCBI

* Google Scholar

29. 29. Xiang L., Wang Y., Lan J., Na F., Wu S., Gong Y., et al HIF-1-dependent heme synthesis promotes gemcitabine resistance in human non-small cell lung cancers via enhanced ABCB6 expression, Cellular and Molecular Life Sciences, 79 (2022) 343.

* View Article

* Google Scholar

30. 30. Espinosa-Diez C., Miguel V., Mennerich D., Kietzmann T., Sánchez-Pérez P., Cadenas S., et al, Antioxidant responses and cellular adjustments to oxidative stress, Redox biology, 6 (2015) 183–197. pmid:26233704

* View Article

* PubMed/NCBI

* Google Scholar

31. 31. Traverso N., Ricciarelli R., Nitti M., Marengo B., Furfaro A.L., Pronzato M.A., et al, Role of glutathione in cancer progression and chemoresistance, Oxidative medicine and cellular longevity, 2013 (2013). pmid:23766865

* View Article

* PubMed/NCBI

* Google Scholar

32. 32. Yang J., Xu J., Zhang B., Tan Z., Meng Q., Hua J., et al, Ferroptosis: At the crossroad of gemcitabine resistance and tumorigenesis in pancreatic cancer, International Journal of Molecular Sciences, 22 (2021) 10944. pmid:34681603

* View Article

* PubMed/NCBI

* Google Scholar

33. 33. Ran S., Liu J., Li S., A systematic review of the various effect of arsenic on glutathione synthesis in vitro and in vivo, BioMed Research International, 2020 (2020). pmid:32802886

* View Article

* PubMed/NCBI

* Google Scholar

34. 34. Friedl P., Wolf K., Tumour-cell invasion and migration: diversity and escape mechanisms, Nature reviews cancer, 3 (2003) 362–374. pmid:12724734

* View Article

* PubMed/NCBI

* Google Scholar

35. 35. Shi Y., Cao T., Huang H., Lian C., Yang Y., Wang Z., et al, Arsenic trioxide inhibits cell growth and motility via up-regulation of let-7a in breast cancer cells, Cell Cycle, 16 (2017) 2396–2403. pmid:28980872

* View Article

* PubMed/NCBI

* Google Scholar

36. 36. Xia J., Li Y., Yang Q., Mei C., Chen Z., Bao B., et al, Arsenic trioxide inhibits cell growth and induces apoptosis through inactivation of notch signaling pathway in breast cancer, International journal of molecular sciences, 13 (2012) 9627–9641. pmid:22949821

* View Article

* PubMed/NCBI

* Google Scholar

37. 37. Lin T.-H., Kuo H.-C., Chou F.-P., Lu F.-J., Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide, BMC cancer, 8 (2008) 1–15.

* View Article

* Google Scholar

38. 38. Zhang M., Xu X., Ye Y., Guo Z., Bai X., Wang J., et al, Synergistic Lethal Effects Between Gemcitabine and Arsenic Trioxide on Non-Hodgkin Lymphoma Cell Lines Is Associated with Modulation of PI3K/Akt Signaling Pathway, American Society of Hematology Washington, DC, 2014.

* View Article

* Google Scholar

39. 39. Gao J., Wang G., Wu J., Zuo Y., Zhang J., Chen J., Arsenic trioxide inhibits Skp2 expression to increase chemosensitivity to gemcitabine in pancreatic cancer cells, American Journal of Translational Research, 11 (2019) 991. pmid:30899398

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Citation: Maleki F, Handali S, Rezaei M (2025) The role of mitochondrial dysfunction in the cytotoxic synergistic effect of gemcitabine and arsenic on breast cancer. PLoS ONE 20(1): e0312424. https://doi.org/10.1371/journal.pone.0312424

About the Authors:

Farshid Maleki

Roles: Investigation, Methodology

Affiliation: Faculty of Medical Sciences, Department of Toxicology, Tarbiat Modares University, Tehran, Iran

Somayeh Handali

Roles: Investigation, Writing – original draft, Writing – review & editing

E-mail: [email protected] (MR); [email protected] (SH)

Affiliation: Medical Biomaterials Research Center (MBRC), Tehran University of Medical Sciences, Tehran, Iran

Mohsen Rezaei

Roles: Funding acquisition, Investigation, Supervision, Writing – review & editing

E-mail: [email protected] (MR); [email protected] (SH)

Affiliations: Faculty of Medical Sciences, Department of Toxicology, Tarbiat Modares University, Tehran, Iran, Institute for Natural Products and Medicinal Plants (INPMP), Tarbiat Modares University, Tehran, Iran

ORICD: https://orcid.org/0000-0001-6823-2463

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8. Xie Z., Zhang Y., Jin C., Fu D., Gemcitabine-based chemotherapy as a viable option for treatment of advanced breast cancer patients: a meta-analysis and literature review, Oncotarget, 9 (2018) 7148. pmid:29467957

9. Nasrollahzadeh A., Bashash D., Kabuli M., Zandi Z., Kashani B., Zaghal A., et al, Arsenic trioxide and BIBR1532 synergistically inhibit breast cancer cell proliferation through attenuation of NF-κB signaling pathway, Life sciences, 257 (2020) 118060.

10. Yang Y., Qi J., Wang H., Zhao Y., Wu Y., Wu Z., et al, Ameliorative effects of Danshensu from the functional food Salvia miltiorrhiza against arsenic trioxide-induced cardiac toxicity in vivo and in vitro: Involvement of inhibiting the AKT/IKK/NF-κB signaling pathway, Journal of Functional Foods, 99 (2022) 105304.

11. Singh V., Gera R., Purohit M.P., Patnaik S., Ghosh D., Fluorometric estimation of glutathione in cultured microglial cell lysate, Bio-protocol, 7 (2017) e2304–e2304. pmid:34541073

12. Bobiński M., Okła K., Łuszczki J., Bednarek W., Wawruszak A., Moreno-Bueno G., et al, Gemcitabine and Selected mTOR Inhibitors in Uterine Sarcomas and Carcinosarcoma Cells-an Isobolographic Analysis, International Journal of Medical Sciences, 17 (2020) 2987. pmid:33173419

13. Sarvestani N.N., Sepehri H., Delphi L., Farimani M.M., Eupatorin and salvigenin potentiate doxorubicin-induced apoptosis and cell cycle arrest in HT-29 and SW948 human colon cancer cells, Asian Pacific Journal of Cancer Prevention: APJCP, 19 (2018) 131. pmid:29373904

14. Li P., Zhong D., Gong P.-y., Synergistic effect of paclitaxel and verapamil to overcome multi-drug resistance in breast cancer cells, Biochemical and biophysical research communications, 516 (2019) 183–188. pmid:31204050

15. Yedjou C., Tchounwou P., Jenkins J., McMurray R., Basic mechanisms of arsenic trioxide (ATO)-induced apoptosis in human leukemia (HL-60) cells, Journal of hematology & oncology, 3 (2010) 1–9.

16. Nakashima M., Adachi S., Yasuda I., Yamauchi T., Kawaguchi J., Itani M., et al, Phosphorylation status of heat shock protein 27 plays a key role in gemcitabine-induced apoptosis of pancreatic cancer cells, Cancer letters, 313 (2011) 218–225. pmid:21999932

17. Chen Y., Sun L., Wang Y., Zhao X., A dual-fluorescent whole-well imaging approach for screening active compounds against doxorubicin-induced cardiotoxicity from natural products, RSC advances, 5 (2015) 106431–106438.

18. Kocabey S., Ekim Kocabey A., Schneiter R., Rüegg C., Membrane-interacting DNA nanotubes induce cancer cell death, Nanomaterials, 11 (2021) 2003. pmid:34443832

19. Pilco-Ferreto N., Calaf G.M., Influence of doxorubicin on apoptosis and oxidative stress in breast cancer cell lines, International journal of oncology, 49 (2016) 753–762. pmid:27278553

20. Shim M.K., Yoon H.Y., Lee S., Jo M.K., Park J., Kim J.-H., et al, Caspase-3/-7-specific metabolic precursor for bioorthogonal tracking of tumor apoptosis, Scientific reports, 7 (2017) 1–15.

21. Sagwal S.K., Pasqual-Melo G., Bodnar Y., Gandhirajan R.K., Bekeschus S., Combination of chemotherapy and physical plasma elicits melanoma cell death via upregulation of SLC22A16, Cell Death & Disease, 9 (2018) 1179. pmid:30518936

22. Ma L., Wei J., Su G.H., Lin J., Dasatinib can enhance paclitaxel and gemcitabine inhibitory activity in human pancreatic cancer cells, Cancer biology & therapy, 20 (2019) 855–865. pmid:30866697

23. Handali S., Moghimipour E., Kouchak M., Ramezani Z., Amini M., Angali K.A., et al, New folate receptor targeted nano liposomes for delivery of 5-fluorouracil to cancer cells: strong implication for enhanced potency and safety, Life sciences, 227 (2019) 39–50. pmid:31002921

24. Wu P.-J., Hsin I.-L., Hung W.-L., Lee M.-S., Wang P.-H., Ko J.-L., Combination treatment with cyclosporin A and arsenic trioxide induce synergistic cell death via non-apoptotic pathway in uterine cervical cancer cells, Chemico-Biological Interactions, 368 (2022) 110177. pmid:36100036

25. Lee D.E., Kang H.W., Kim S.Y., Kim M.J., Jeong J.W., Hong W.C., et al, Ivermectin and gemcitabine combination treatment induces apoptosis of pancreatic cancer cells via mitochondrial dysfunction, Frontiers in Pharmacology, (2022) 3403. pmid:36091811

26. Pieniążek A., Czepas J., Piasecka-Zelga J., Gwoździński K., Koceva-Chyła A., Oxidative stress induced in rat liver by anticancer drugs doxorubicin, paclitaxel and docetaxel, Advances in medical sciences, 58 (2013) 104–111. pmid:23612702

27. Glorieux C., Calderon P.B., Catalase down-regulation in cancer cells exposed to arsenic trioxide is involved in their increased sensitivity to a pro-oxidant treatment, Cancer Cell International, 18 (2018) 1–9.

28. Glorieux C., Calderon P.B., Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach, Biological chemistry, 398 (2017) 1095–1108. pmid:28384098

29. Xiang L., Wang Y., Lan J., Na F., Wu S., Gong Y., et al HIF-1-dependent heme synthesis promotes gemcitabine resistance in human non-small cell lung cancers via enhanced ABCB6 expression, Cellular and Molecular Life Sciences, 79 (2022) 343.

30. Espinosa-Diez C., Miguel V., Mennerich D., Kietzmann T., Sánchez-Pérez P., Cadenas S., et al, Antioxidant responses and cellular adjustments to oxidative stress, Redox biology, 6 (2015) 183–197. pmid:26233704

31. Traverso N., Ricciarelli R., Nitti M., Marengo B., Furfaro A.L., Pronzato M.A., et al, Role of glutathione in cancer progression and chemoresistance, Oxidative medicine and cellular longevity, 2013 (2013). pmid:23766865

32. Yang J., Xu J., Zhang B., Tan Z., Meng Q., Hua J., et al, Ferroptosis: At the crossroad of gemcitabine resistance and tumorigenesis in pancreatic cancer, International Journal of Molecular Sciences, 22 (2021) 10944. pmid:34681603

33. Ran S., Liu J., Li S., A systematic review of the various effect of arsenic on glutathione synthesis in vitro and in vivo, BioMed Research International, 2020 (2020). pmid:32802886

34. Friedl P., Wolf K., Tumour-cell invasion and migration: diversity and escape mechanisms, Nature reviews cancer, 3 (2003) 362–374. pmid:12724734

35. Shi Y., Cao T., Huang H., Lian C., Yang Y., Wang Z., et al, Arsenic trioxide inhibits cell growth and motility via up-regulation of let-7a in breast cancer cells, Cell Cycle, 16 (2017) 2396–2403. pmid:28980872

36. Xia J., Li Y., Yang Q., Mei C., Chen Z., Bao B., et al, Arsenic trioxide inhibits cell growth and induces apoptosis through inactivation of notch signaling pathway in breast cancer, International journal of molecular sciences, 13 (2012) 9627–9641. pmid:22949821

37. Lin T.-H., Kuo H.-C., Chou F.-P., Lu F.-J., Berberine enhances inhibition of glioma tumor cell migration and invasiveness mediated by arsenic trioxide, BMC cancer, 8 (2008) 1–15.

38. Zhang M., Xu X., Ye Y., Guo Z., Bai X., Wang J., et al, Synergistic Lethal Effects Between Gemcitabine and Arsenic Trioxide on Non-Hodgkin Lymphoma Cell Lines Is Associated with Modulation of PI3K/Akt Signaling Pathway, American Society of Hematology Washington, DC, 2014.

39. Gao J., Wang G., Wu J., Zuo Y., Zhang J., Chen J., Arsenic trioxide inhibits Skp2 expression to increase chemosensitivity to gemcitabine in pancreatic cancer cells, American Journal of Translational Research, 11 (2019) 991. pmid:30899398