Background
Breast cancer has attracted great scientific attention because it is the most common cancer type and the second-largest cause of death in the United States [
1]. Breast cancer incidence has grown by 0.5% annually [
1]. This alarming increase emphasizes the need to identify the underlying processes of cancer formation and develop efficient treatments [
2]. Breast cell malignancy evolved from disturbance of cell signaling and uncontrolled growth factors through cascades of abnormal genetic events [
3]. Molecular subtypes of breast cancer can be classified into luminal A, luminal B, Human epidermal growth receptor-2 (HER2)-enriched, and triple-negative subtypes based on the levels of mRNA gene expression [
4]. Treatment options vary for breast cancer patients ranging from surgical removal of breast mass, pharmacotherapy, or using radiotherapy to kill the cancer cells. Patients with HER2-enriched and triple-negative tumors have the worst prognosis, and they should precede surgery with neoadjuvant chemotherapy regimens [
4,
5].
Commonly, a regimen of double or triple chemotherapeutic drugs is initiated as soon as the diagnosis is verified [
4]. For most advised regimens, doxorubicin and cyclophosphamide are used to treat breast cancer [
6‐
10]. Despite the adequate response, these chemotherapeutic agents result in different side effects that impair the patient’s tolerability to the treatment. Identifying novel therapeutic additives is necessary to reduce the chemotherapy dose and the potential side effects.
Drug repositioning aims to discover new efficacies and direct well-known drugs for other diseases based on the pharmacological aspect of the drug to escape the conundrum of drug discovery economics and safety issues [
10‐
12]. An example of drug repurposing is using statins in breast cancer. Cholesterol is usually essential for cell regulatory functions, conserving membrane integrity, and interacting with the extracellular matrix [
13,
14]. Feedback mechanisms could closely control cholesterol levels depending on the amount of cholesterol in the cells. Unfortunately, these feedback mechanisms are impaired in cancer cells with high proliferation rates leading to the accumulation of intracellular cholesterol and activation of the HMG-CoA-enzyme [
15]. Statins significantly impact cancer cells in addition to their hypocholesterolemic action, primary and secondary prevention of cardiovascular illnesses [
16,
17]. Statins can inhibit tumor necrosis factor-alpha (TNF a), inhibiting angiogenesis [
18]. Additionally, statins can prevent the expression of matrix metalloproteinases 2 and 9 (MMP2 & MMP9) and lower the likelihood of metastasis [
19]. Moreover, statins have a suppressive effect on the cell cycle and cause G1 phase arrest, interfering with cell proliferation and migration activity [
20]. It was also discovered that statins can oblige cancerous cells to evade the Warburg effect and continue through oxidative phosphorylation and activation of the tricarboxylic acid cycle (TCA) [
21].
Statin members have varying degrees of physicochemical properties, including their solubility. They are divided into hydrophilic statins with hepatic availability and lipophilic statins that exert higher extrahepatic concentrations, making lipophilic members better candidates for anti-cancer agents [
22]. Pitavastatin and simvastatin are members of statins that have displayed the highest anticancer activity [
23]. A recent study indicated that the concomitant use of Pitavastatin with standard neoadjuvant chemotherapy protocols may improve neoadjuvant chemotherapy responses in patients with breast cancer [
24]. However, further studies are required to compare the effect of different statins and to explore the molecular mechanism of action.
The current study aimed to examine whether Pitavastatin or simvastatin can enhance the anti-cancer activity of the doxorubicin/cyclophosphamide combination in breast cancer cell lines and to explore their prospective molecular mechanism of action.
Methods
Drugs and chemicals
In this study, doxorubicin 50 mg/25 ml vial (Dox, Adriamycin, Hikma specialized, USA) and cyclophosphamide (Cyclo, Endoxan 1gm IV vial, Baxter Oncology, USA) were used. Simvastatin and Pitavastatin were generous grants from EVA Pharma, Egypt. Other used materials include Dulbecco’s modified eagle’s medium high glucose enriched medium (DMEM, Lonza, Verviers, Belgium), fetal bovine serum (FBS, Sera laboratories international, Ltd., Brazil EU grade), phosphate buffer saline (PBS, Lonza, Verviers, Belgium) streptomycin and penicillin (Lonza, Verviers, Belgium), favor-PrepTM blood/cultured cell total RNA purification mini kit (Favorgen Biotech Corp., Ping-Tung, Taiwan), Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA), HERAPLUS SYBR® Green qPCR Kit (Willowfort, Nottingham, UK) and propidium iodide (PI, ab14083, Abcam).
Experimental cell lines
Breast cancer cell lines (M.D. Anderson - Metastatic Breast 231 (MDA-MB-231) & Michigan Cancer Foundation-7 (MCF7)) were purchased from Nawah Scientific (Almokattam, Cairo, Egypt) and grown in DMEM medium enforced with 10% FBS and 1% streptomycin/penicillin incubated under standard conditions (37oC humidified air and 5% CO2 pressure).
Ethical approval
The ethical committee of the Faculty of Pharmacy, Mansoura University (Ref. No. 2020 − 176) approved this study.
Cell viability analysis
MDA-MB-231 and MCF7 cells were seeded in 96-well plates with 20,000 cells/well under standard conditions. The plates were incubated to allow cell growth for 24 h before stimulation. The next day, cells were stimulated with doxorubicin (50, 25, 12.5, 6.25, 3.125 µM), cyclophosphamide (100, 50, 25, 12.5, 6.25 µM), Pitavastatin (200, 100, 50, 25, 12.5 µM), simvastatin (200, 100, 50, 25, 12.5 µM) in triplicates. Drugs were used with doxorubicin combination at concentrations around or below the resultant IC50; cyclophosphamide 100 µM, Pitavastatin 50 µM, or simvastatin 25 µM. The percentage of viable cells was detected using the crystal-violet assay technique 24 h after stimulation using a microplate reader (Bio Tek ELx800, USA) at a wavelength of 570 nm. The results are expressed as the percent of viable cells compared with the living control group (100% viability); cells grown with standard media without added drugs, negative control group (0%viablility); cells treated with a mixture of toxic compounds containing doxorubicin, dimethyl sulfoxide, sodium azide.
Quantitative real-time PCR (qPCR)
Cells were cultivated in 6 well plates with 1 × 10
6 cells/well in triplicates, then incubating the cells in standard conditions for 24 h before stimulation. Cells were treated with doxorubicin (10 µg/ml), cyclophosphamide (100 µM), Pitavastatin (50 µM), simvastatin (25 µM), or their combinations for gene expression. After stimulation for 24 h, cells were washed twice using cold PBS, scraped from the flask, transferred into Eppendorf tubes, centrifuged to get the precipitated cells, and discarded the supernatant. Total ribonucleic acid (RNA) was extracted using the Favor-PrepTM Blood/Cultured cell total RNA purification mini kit (Favorgen Biotech Corp., Ping-Tung, Taiwan). The first-strand cDNA was formed using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). HERAPLUS SYBR® Green qPCR Kit (Willowfort, Nottingham, UK) was used in (qPCR) following the manufacturer protocol. Using the 2
−∆∆ct method, gene expression fold changes were determined and presented as an average of three independent experiments (Livak and Schmittgen, 2001). Table
1. shows the primer sequences used in qPCR for caspase-3, Bax, BCL-2, and cyclin D1.
Table 1
Primer sequences used in qPCR
Caspase-3 | 5’-ACATGGAAGCGAATCAATGGACTC-3’ | 5’-AAGGACTCAAATTCTGTTGCCACC-3’ |
Cyclin D1 | 5’-AGACCTGCGCGCCCTCGGTG-3’ | 5’-GTAGTAGGACAGGAAGTTGTTC-3’ |
Bax | 5’-CCCGAGAGGTCTTTTTCCGAG-3’ | 5’-CCAGCCCATGATGGTTCTGAT-3’ |
BCL-2 | 5’-TGTGGCCTTCTTTGAGTTCGGTG-3’ | 5’-GGTGCCGGTTCAGGTACTCAGTCA-3’ |
Cell cycle analysis
Cells were seeded into 6 well plates at a density of 1 × 10
6 well, maintained in standard conditions for 24 h to allow their adhesion. Following centrifugation at 1800 rpm and removal of the supernatant, cells were permeabilized and fixed in 1 ml of cold 96% or absolute ethanol in ice, which was added dropwise, while vortexing to ensure the fixation of all cells with minimum clumping. The tubes stood for 15 min before centrifugation for 10 min at 1800, followed by aspiration of alcohol without disturbing the pellets [
25]. Then, pellets were resuspended in 1 ml of propidium iodide (PI) buffer (25 µg/mL PI, 500 mg sodium citrate, and 0.5 ml Triton X-100 to 500 ml distilled water). Cells were suspended in 1 ml of the staining solution at 4 °C for 30 min and maintained in ice. Afterward, they were filtered through 30 μm nylon mesh to remove nuclear aggregates in another 5 ml tube. DNA content was measured using Accuri™ C6, and G0/G1, S, and G2/M cells were appropriately gated using Accuri™ C6 Software.
Statistical analysis
Data were expressed as mean ± standard error of the mean (SEM). One-way ANOVA followed by Tukey-Kramer, multiple comparison test, was performed using GraphPad Prism version 9.0.0 for Windows; “GraphPad Software, San Diego, California USA,
www.graphpad.com”. The significance level was at a
P-value of ˂0.05.
Discussion
Doxorubicin is frequently used as a chemotherapeutic agent in different regimens for treating breast cancer in combination with cyclophosphamide in each cycle [
6,
10,
26]. Comprehensive strategies with minimal adverse effects and maximum therapeutic response can be evolved from combination strategies of different therapeutic agents at lower doses to result in better response and decreased drug resistance [
27]. In addition, drug repurposing sheds light on statins as anti-cancer agents, and using them in combination with conventional chemotherapy would offer many benefits for the patients [
28‐
30].
This study evaluated the effect of two lipophilic statin members (simvastatin and Pitavastatin) with the most popular chemotherapy regimen of doxorubicin/ cyclophosphamide in ER-positive breast cancer cells (MCF7) and triple-negative breast cancer cells (MDA-MB-231).
According to our results, the
IC50 of analyzed drugs was higher in the MCF7 cell line than in MDA-MB-231, indicating that MCF7 may be more resistant than MDA-MB-231 cells against the analyzed drugs in this study. A previous study mentioned that the positivity of hormonal receptor expression lowers the chemotherapy treatment response [
31]. This was in concordance with another study on different cell lines and had revealed that
IC50 was lower for triple-negative cell lines, represented here by MDA-MB-231 cells than non-triple negative cells (MCF7) [
32,
33]. Consequently, the MDA-MB-231 cell line showed lower viability than MCF7 after treatment with combination therapies. Treatment of cells with doxorubicin/ cyclophosphamide with Pitavastatin or simvastatin decreased the cellular resistance to chemotherapy resulting in the lowest cell viability percentage on treatment, especially for MDA-MB-231 cells. However, this contradicts Rezano et al. findings, which stated that simvastatin’s synergistic activity with doxorubicin was produced in MCF7 but not MDA-MB-231 cells [
33]. The difference may be attributed to the presence of cyclophosphamide in the treatment combination in our study. The findings of this study mean that combination treatment with statin members would offer more cell death in MDA-MB-231 cells than MCF7. The observed difference in the response of MCF7 and MDA-MB-231 cells to statins may be due to the variation in receptor expression status between the two cell types. Specifically, MCF7 cells express estrogen receptor (ER), whereas MDA-MB-231 cells do not. This confirmed that statins can decrease cell proliferation and progression mainly on ER-negative breast cancer subtypes [
23,
34]. Furthermore, MDA-MB-231 cells harbor a mutation in the p53 gene and exhibit overexpression of the mevalonate pathway, rendering them more susceptible to the effects of statins [
32]. Moreover, MDA-MB-231 cells express pituitary tumor transforming gene 1 (PTTG1), which is markedly suppressed using statins, leading to diminished cell invasion due to decreased matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) activity [
35,
36].
Both Pitavastatin and simvastatin produced antiproliferative activity, evidenced by the decreased expression level of cyclin D1 than the untreated cells. Cyclin D1 is implicated in regulating cell division and G1/S transition [
37], and its overexpression is related to malignant transition [
38]. The current study found that simvastatin and Pitavastatin in combination with doxorubicin/ cyclophosphamide significantly reduced cyclin D1 expression levels more than combined doxorubicin/ cyclophosphamide in the MDA-MB-231 cell line. In this context, the flow cytometric analysis of the cell cycle demonstrated that the cell-cycle progression of MDA-MB-231 cells was arrested in the G1 phase and accumulated in the G0/1 phase, and the arrest was significant in the triple combination-treated group rather than in other treated groups. These findings demonstrate that simvastatin and Pitavastatin inhibit MDA-MB-231 cell proliferation by inducing cell-cycle arrest. This was compatible with a study that stated that statin could upregulate cyclin-dependent kinase inhibitors causing G1/S arrest [
39].
Apoptosis, as a naturally orchestrated mechanism, occurs physiologically with a pivotal role in tumor preventive effect and also can participate in the chemotherapeutic response by playing an important target for treatment strategies resulting in the activation of different pathways inhibiting the malignant transformation of different cells and hence preventing resistance [
40]. The present study showed that Pitavastatin or simvastatin alone increases the expression of the apoptotic markers to a degree that is comparable to that induced by chemotherapeutic agents such as doxorubicin and cyclophosphamide. Furthermore, combining Pitavastatin or simvastatin with doxorubicin/ cyclophosphamide enhanced apoptotic activity in MDA-MB-231 and MCF7 cells. These findings are in agreement with the results reported by Buranrat et al. [
41]. Apoptosis is evidenced by a significantly decreased expression of the antiapoptotic Bcl2 gene with increased expression of proapoptotic Bax and subsequently increase in the ratio of Bax/Bcl2. Moreover, there was a maximal increase in the activity of caspase-3 when statins were combined with doxorubicin/cyclophosphamide, as demonstrated by our prior clinical study where the addition of Pitavastatin to a doxorubicin/cyclophosphamide regimen resulted in a rise in the serum level of caspase-3 when compared to patients who received chemotherapy regimen alone [
24]. The elevated apoptotic activity was higher in the simvastatin combination with doxorubicin/ cyclophosphamide than in the Pitavastatin combination. This may indicate that simvastatin would minimize the dosage of chemotherapy and hence decrease the toxicity. These findings were confirmed by cell cycle analysis, which revealed a rise in the proportion of apoptotic cell fragments in the sub-G0 phase. While the percentage of apoptotic cells did not differ significantly between doxorubicin/cyclophosphamide co-treatment and doxorubicin treatment alone, the administration of either Pitavastatin or simvastatin in combination with doxorubicin/cyclophosphamide resulted in a notable increase in apoptotic fragments. Furthermore, triple treatment with doxorubicin/ cyclophosphamide with Pitavastatin or simvastatin resulted in arresting cell progression at the G0/1 phase in both cell lines, similarly in other cancer cells such as hepatocellular carcinoma and prostate cancer [
42,
43].
In agreement with our study, statins can induce apoptosis by activating the intrinsic mitochondrial pathway which involves reducing mitochondrial membrane potential and releasing the mitochondrial activator of caspases, Smac/DIABLO [
44].
Additionally, they upregulate the expression of proapoptotic proteins Bax and activate procaspases 3, 7, 8, and 9, and Bim while downregulating the antiapoptotic protein Bcl2 [
44‐
47].
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