|Ahead of print publication
Sildenafil enhances cisplatin-induced apoptosis in human breast adenocarcinoma cells
Fariba Hassanvand1, Tannaz Mohammadi2, Negar Ayoubzadeh3, Atieh Tavakoli4, Naiemeh Hassanzadeh5, Nafiseh Sadat Sanikhani6, Ali Ipakchi Azimi7, Hamid Reza Mirzaei8, Mehdi Khodamoradi9, Kazem Abbaszadeh Goudarzi10, Hossein Pourghadamyari11, Mohamad Ali Zaimy12
1 Department of Biological Sciences and Biotechnology, University of Kurdistan, Iran
2 Molecular and Cellular Biology Department, Islamic Azad University Tehran Medical Branch, Tehran, Iran
3 Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
4 Department of Molecular Genetics, Islamic Azad University, Damghan, Iran
5 Department of Biology, Tehran Medical Branch, Islamic Azad University, Tehran, Iran
6 Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
7 Department of Medical Laboratory Science, Medical Science Faculty Babol Islamic Azad University, Babol, Iran
8 Department of Medical Immunology, Tehran University of Medical Sciences, School of Medicine, Tehran, Iran
9 Substance Abuse Prevention Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
10 Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran
11 Department of Clinical Biochemistry, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
12 Department of Medical Genetic, Ilam University of Medical Sciences, Ilam, Iran
|Date of Submission||30-Aug-2019|
|Date of Decision||01-Nov-2019|
|Date of Acceptance||30-Dec-2019|
|Date of Web Publication||03-Nov-2020|
Department of Clinical Biochemistry, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman
Source of Support: None, Conflict of Interest: None
Introduction: Cyclic nucleotide phosphodiesterase (PDE) enzymes are a large superfamily of enzymes that catalyze the conversion reaction of cyclic adenosine monophosphate (AMP) and cyclic guanosine monophosphate (GMP) to AMP and GMP, respectively. In some cancer cells, PDE-5 has been shown to be overexpressed in multiple human carcinomas. It seems that the inhibition of PDE-5 may has anticancer effects. Cisplatin is one of the prevalent chemo-agents to treat solid tumors. However, its clinical usefulness is hindered by dose-limiting toxicities, especially on the kidneys (nephrotoxicity) and ears (ototoxicity). In this study, the antitumor activity of the sildenafil as a PDE-5 inhibitor alone and in combination with cisplatin on human mammary adenocarcinomas and MCF-7 and MDA-MB-468 was assessed.
Materials and Methods: Sildenafil as PDE type 5 (PDE5) inhibitor is the drugs that we combined with the cisplatin (chemotherapeutic agent), in vitro. Human mammary adenocarcinomas and MCF-7 and MDA-MB-468 cell lines were cultured in standard conditions. At time point, following 24 h and 48 h incubation, the cell lines were treated by cisplatin in the presence/absence of sildenafil. Cell viability, apoptosis, and reactive oxygen species (ROS) were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, real-time polymerase chain reaction, and Western blot; and fluorimetric methods, respectively. Statistical analysis was performed using SPSS software SPSS (SPSS Inc., Chicago, IL, USA).
Results: In MCF-7 cell line, following 24 h incubation, combinations of sildenafil with cisplatin (P < 0.001) showed decreased cell viability when compared to sildenafil and cisplatin alone. Moreover in MDA-MB-468 cell line, following 24 h incubation, data did not show any significant changes on cell viability when treated with cisplatin, in the presence or absence of sildenafil. However, following 48 h incubation, combinations of cisplatin with sildenafil (P < 0.001) were showed decreased cell viability when compared to cisplatin and sildenafil alone in both MCF-7 and MDA-MB-468 cell lines. Concerning the ROS production and apoptosis, data showed that both processes increase significantly in the presence of the sildenafil in comparison absent it.
Conclusion: Our data showed that the combination of sildenafil with cisplatin can improve cell toxicity and anticancer effect of cisplatin. And also sildenafil as a PDE-5 inhibitor could be used as additive treatment in combination with cisplatin to make a reduction in cisplatin dosage and its side effects.
Keywords: Antitumor activity, apoptosis, breast cancer, cisplatin, sildenafil
|How to cite this URL:|
Hassanvand F, Mohammadi T, Ayoubzadeh N, Tavakoli A, Hassanzadeh N, Sanikhani NS, Azimi AI, Mirzaei HR, Khodamoradi M, Goudarzi KA, Pourghadamyari H, Zaimy MA. Sildenafil enhances cisplatin-induced apoptosis in human breast adenocarcinoma cells. J Can Res Ther [Epub ahead of print] [cited 2020 Dec 3]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=299888
| > Introduction|| |
In women, the most prevalent invasive cancer is breast cancer, with about 2.4 million annual incidences. It has the second incidence rank in common cancer overall. Breast cancer accounts for about more than 500,000 deaths in a year., Standard treatment of breast cancer is surgery removal followed by radiation and chemotherapy. However, it has been high recurrence rate, and hence, management of tumor is one of the most important challenges in patients with breast cancer.,
Cisplatin is a widely used and successful chemotherapeutic agent, which is intravenously prescribed to treat various cancers, such as breast, bladder, cervical, stomach, and prostate  cancers. Cisplatin can exert its cytotoxic effects by inducing DNA damage through crosslinking of the DNA and generation of free radicals in cancer cells. Although cisplatin as a chemotherapeutic agents can be effective to treat of various cancers, long-term clinical application is hindered by its side effects and complications, such as nephrotoxicity, ototoxicity, and neurotoxicity., Moreover, it is determined that these adverse side effects occurs in a dose-dependent manner. Hence, researchers are looking for compounds that have synergistic effects with cisplatin to reduce its clinical dose, adverse side effects, and improve its safety.,
To catalyze cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and convert them into 5'AMP and 5'GMP, respectively, is conducted by a large and complex superfamily of enzymes named as phosphodiesterases (PDEs). It means that this capability helps to fine-tune cyclic nucleotides in the intracellular levels. According to sequence homology and enzymatic properties, PDEs divided into 11 subfamilies (PDE1 to PDE11). PDEs have a critical role in physiological cell signaling pathways such as adhesion, cell growth, energy homeostasis and neuronal signaling. And also several lines of evidence have recently suggested that PDEs signaling have been altered in some pathophysiological conditions, such as cancer.
There is increasing scientific and clinical interest in targeting PDEs by pharmacological agents, in order to make the desired changes to modulate their effects in pathologic conditions. As important intracellular second messengers, cAMP and cGMP concentration can be increased or decreased by the activation and inhibition of PDEs, respectively. Some studies showed that PDE-5 has high expression in bladder and breast cancers. These observations prompted the PDE inhibitors (PDEIs) used as antitumor compounds alone or in combination., Sildenafil is one of the main representatives as a selective inhibitor of PDE-5 by the hydrolysis of cGMP. To mention the importance to treat diseases such as pulmonary artery hypertension and erectile dysfunction, it has Food and Drug Administration approval., More recently, studies have focused on potential antitumor activity of PDE-5 inhibitors.,, This study was conducted to evaluate sildenafil antitumor activity as a PDE-5 inhibitor, alone and in combination with cisplatin on human breast cancer cell lines.
| > Materials and Methods|| |
Chemical reagents, including Sildenafil, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, and DMSO were provided (Sigma-Aldrich, St. Louis, MO, USA). Cell lines “MCF-7 and MDA-MB-468” were obtained from Iranian Biological Resource Center (Tehran, Iran). The caspase3 antibody was provided from Cell Signaling (cat# 9662S). High-glucose Dulbecco's Modified Eagle's Medium (EMEM), penicillin-streptomycin, and fetal bovine serum were purchased (Gibco, Germany).
Cell lines, including MCF-7 and MDA-MB-468 were provided from Iranian Biological Resource Center (Tehran, Iran). Cells culture were performed in DMEM medium containing fetal bovine serum (10%), 2 mM L-glutamine, 0.01 mg/mL bovine insulin (90%) and also Earle's BSS containing 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, and 0.1 mM nonessential amino acids. Cells were treated by different concentration of drugs used 24 and 72 h as incubation times. For MTT and apoptosis assay, cells were seeded at 7000 and 100,000 per well, respectively. To validate the results precisely, there was a sample as a control. Different treatment was done in triplicate. Of note, cells were treated with sildenafil as a PDE5 inhibitor in combination with cisplatin to investigate the synergistic effect.
To use a modified MTT assay,, the viability of cell was evaluated. In flat-bottom 96-well plate, cells were cultured at 7000 per well to assess the impact of drugs after 24 and 48 h treatment. To determine the cell viability, 10 μL of MTT stock solution at 12 mM concentration, was added to wells and then incubation was done for 4 h at 37°C. In a humidified chamber, after adding 100 μL of sodium dodecyl sulfate (SDS)–HCl solution to each well, plate was incubated for 4–18 h at 37°C. Before final evaluation at 570 nm by ELISA reader (StatFAX303), each well should be precisely mixed using a pipette.
Reverse transcription quantitative polymerase chain reaction
To conduct reverse transcription polymerase chain reaction (RT-PCR), RNA extraction was performed using TRIzol (Sigma, USA). In order to purify total RNA, sample was treated by DNase (Fermentas, Germany) based on the manufacturer's instructions. RNA quality and concentration were evaluated by spectrophotometry at 260 nm, respectively. By random hexamer primers, total RNA (1 μg) was reverse transcribed using reverse transcriptase enzyme (Takara). Following cDNA synthesis, real-time PCR was done. BAX and BCL2 expression levels were measured by specific primers. According to GAPDH transcript as an internal control, the data were normalized [Table 1].
Western blot analysis
After cell centrifugation, protein concentration was evaluated. The separation of proteins was performed by SDS-polyacrylamide gel electrophoresis gel electrophoresis in 12% gel. To do western blot analysis, proteins were transferred to polyvinylidene fluoride membranes. By 5% skim milk, the membrane was blocked in refrigerator for an overnight. Following blocking, to estimate caspase 3, membrane was incubated with antibodies against caspase 3 at room temperature for 2 h. To remove the extra antibodies, membrane washing was done by 0.1% Tween 20 in phosphate buffered saline. With an enhanced chemiluminescence system and goat anti-rabbit secondary antibody, bound antibodies were measured.
Measurement of radical oxygen specie
To determine the levels of intracellular reactive oxygen species (ROS) following treatment with cisplatin in sildenafil presence/absence, both cell lines were seeded at 105 cells per well in a 24-well plates. After treatment with above drug insult with time exposure of 24 and 48 h located in dark place with the presence of 10 μM H2DCF-DA for 30 min at 4°C, the incubation of cells was performed. Based on excitation/emission at 485/530 nm, the intensity of fluorescence was measured.
| > Results|| |
MTT assay was used to detect IC50 concentration of cisplatin. To this end, 5, 10, 15, 20, 25, 30, 35, and 40 μM were used and data showed that IC50s of cisplatin were 15 μM and 22 μM in MCF-7, and MDA-MB-468, respectively [Figure 1].
|Figure 1: Cell survival curve of MCF-7 and MDA-MB-468 cells were measured by the MTT assay. Both cell lines were treated with cisplatin in different doses from 0 to 40 μM for 48 h. IC50 were determined as the drug concentrations that caused inhibition of 50% cell viability (a) 17 μM was determined as IC50 for the MCF7 cell line. (b) 22 μM was determined as IC50 for MDA-MB-468 cell line. Bars represent the means ± standard error of the mean of duplicate determinations of triplicate measurements|
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Synergistic effect of sildenafil with cisplatin
MCF-7 and MDA-MB-468 cells were incubated in 96 well plates with 15 μM and 22 μM cisplatin, respectively, and after 1 h sildenafil at 6.25, 12.5, 25, 50, 75, and 100 μM were added. As shown in [Table 2], combination indexes were calculated by CompuSyn software in both cell lines and shown synergistic effect between sildenafil with cisplatin (If CI <1 shown synergistic effect, CI = 1 drugs have additive effect and when CI > 1, the drugs have antagonism effect). Hence, the selected concentrations for Sildenafil in MCF-7 and MDA-MB-468 cell lines were 50 and 100 μM, respectively.
Phosphodiesterase type 5 inhibitors sensitizes human breast adenocarcinoma cells to cisplatin
Sildenafil as a selective PDE5 inhibitor could enhance cisplatin-induced cytotoxicity in breast adenocarcinoma cell lines. Treatment of MCF-7 and MDA-MB-468 cells with cisplatin was performed based on the sildenafil presence/absence for 24 and 48 h. Cell viability was measured by MTT assay. Our data indicated that cell viability was decreased when incubated with the sildenafil presence/absence [Figure 2]. Based on [Figure 2], cell viability was more decreased in the presence of sildenafil in both cell lines after 48 h ((MCF-7, P < 0.001), (MDA-MB-468, P < 0.01)), but results also did not show any significant differences just in MDA-MB-468 cells (P > 0.05) when treated by cisplatin in the sildenafil presence/absence after 24 h [Figure 2]b.
|Figure 2: Effects of sildenafil on improving cisplatin anticancer effects. (a) MCF-7 cells were treated with cisplatin in the presence and absence of Sildenafil for 24 and 48 h. Subsequently, the cell viability was recorded. (b) MDA-MB-468 cells were treated with cisplatin in the presence and absence of Sildenafil for 24 and 48 h. Subsequently, the cell viability r was recorded. Data represent the average of four independent experiments. The means and standard error mean are shown. Statistically significant differences were considered when P < 0.0. *P < 0.05, **P < 0.01. ***P < 0.001|
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Effect of phosphodiesterase type 5 inhibitors on reactive oxygen species production
ROS production was measured when the cells incubated with cisplatin in the presence or absence of sildenafil after 24 and 48 h. In the both breast adenocarcinomas cell lines, data showed that treatment with cisplatin could increase the ROS levels released in the culture medium after 24 and 48 h compared with control. And also the results indicated more increase of the ROS levels released in the culture medium when the cell lines treated with cisplatin in the presence of sildenafil after 48 h. Whereas, after 24 h, a significant rise in the amount of ROS just seen in MCF-7 cell line when it was treated by cisplatin in the presence of sildenafil [Figure 3].
|Figure 3: Effects of sildenafil on reactive oxygen species production MCF-7 and MDA-MB-468 cell lines at 24 and 48 h. (a) Bar graph shows the difference ROS production in the presence and absence of sildenafil in MCF-7 cell line. (b) Bar graph shows the difference ROS production of cisplatin in the presence and absence of Sildenafil in MDA-MB-468 cell line. Bars represent the means ± standard error of the mean, **P < 0.01. ***P < 0.001|
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Our results indicated that sildenafil as a selective PDE5 inhibitor could enhance release ROS into the extracellular environment in both breast adenocarcinomas cell lines.
Combined effect of cisplatin with sildenafil on apoptotic induction
To evaluate the impact of sildenafil on cisplatin-induced cytotoxicity in breast adenocarcinoma cell lines, the mRNA expression of BAX and BCL2 as key regulators of apoptosis agents was analyzed using RT-quantitative PCR (qPCR) method. To evaluate, MCF-7 and MDA-MB-468 cells were treated with cisplatin, in the presence or absence of sildenafil for 48 h and mRNA expression analysis was performed. Data showed that relative BAX expression levels were significantly increased in presence of sildenafil in both breast adenocarcinoma cell lines in comparison to the absence of sildenafil [Figure 4]. Findings showed a significant reduction in the expression levels of BCL2 mRNA in presence of sildenafil in MCF-7 cell line compared to absence of it. However, data did not indicate any significant changes in BCL2 mRNA expression levels in MDA-MB-468 cell line in the presence or absence of Sildenafil for 48 h [Figure 4].
|Figure 4: BAX and BCL2 mRNA expression in MCF-7 and MDA-MB-468 cell lines. (a) BAX relative mRNA expression in the presence and absence of Sildenafil in MCF-7 cells. (b) BAX relative mRNA expression in the presence and absent of Sildenafil in MDA-MB-468 cells. (c) BCL2 relative mRNA expression in the presence and absence of sildenafil in MCF-7 cells. (d) BCL2 relative mRNA expression in the presence and absence of sildenafil in MDA-MB-468 cells. mRNA levels were quantified normalized relative to GAPDH mRNA expression. Bars represent the means ± standard error of the mean, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001|
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Furthermore, to investigate sildenafil effect on cisplatin-induced cytotoxicity in breast adenocarcinoma cell lines, the expression levels of caspase-3 were evaluated using by western blot. The results showed that caspase 3 levels significantly increased when combination treatment of sildenafil and cisplatin performed on MCF-7 and MDA-MB-468 cells in comparison to cisplatin by itself [Figure 5].
|Figure 5: Effect of sildenafil on Caspase 3 protein expression in MCF-7 and MDA-MB-468 cell lines. Protein expression was determined by Western blot analysis and Band intensity was quantified by image j software. (a) Caspase 3 protein expression in the presence and absence of Sildenafil in MCF-7 cells. (b) Caspase 3 protein expression in the presence and absence of Sildenafil in MDA-MB-468 cells. Band intensity was quantified by image j software. Bars represent the mean ± standard error of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001|
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| > Discussion|| |
Despite the advances in cancer treatment and diagnosis, we are not able to fight against various types of cancer; however, chemotherapy is still one of the most promising therapeutic approaches that available to treat most cancers., To treat various solid tumors, cisplatin as an effective chemotherapeutic agent has been widely used. However, a major problem with the application of cisplatin is unwanted side effects, including nephrotoxicity, ototoxicity, and neurotoxicity. Over the recent years, more attention has been paid to combination chemotherapy to reduce the dosage of cisplatin. Several studies reported that PDE 5 has high expression in some cancer tissues. In this contest, we evaluate the sildenafil as a PDE5 inhibitor in combination with cisplatin on human adenocarcinoma breast cancer cell lines.,,
Findings of this experiment showed the sensitivity of different breast cancer cell lines to cisplatin is different from each other. Of note, in this study, the IC50s concentration of MCF-7 and MDA-MB-468 were determined 15 μM and 22 μM, respectively [Figure 1]. There are different reports in respect to cisplatin IC50 concentrations in different cancer cell lines. The results obtained from different studies, suggested that cisplatin dose should be determined based on the type and even subtype of cancers.
The results revealed that sildenafil significantly enhanced the anticancer effect of cisplatin after 24 h and 48 h in MCF-7 cell line [Figure 2]a. And also data showed sildenafil could significantly increase anticancer effect of cisplatin just after 48 h in MDA-MB-468 cell line [Figure 2]b. However, results indicated that 24 h was not a sufficient time for sildenafil to enhance anticancer effect of cisplatin in MDA-MB-468 cell line [Figure 2]b. Thus, it seems that sildenafil improve anticancer effect of cisplatin in time dependent manner in the breast adenocarcinoma cells.
In the current study, the findings have consistency with those of Mei, Xiao-Long, et al. who reported that sildenafil enhances anticancer effect of cisplatin in the dose and time-dependent manners in human colorectal cancer cells.
It is worth noted that ROS and the other oxidants have a critical role in pathology of several diseases and even minimal change of them can impact on the severity of these diseases. Moreover, in both breast cancer cell lines, it has been shown that sildenafil in combination with cisplatin also can significantly increase ROS production compared to cisplatin by itself in time dependent manner [Figure 3]. In different cancer cell lines, studies showed that sildenafil is capable to increase the ROS level., And also, it has been reported that sildenafil as a PDEI can enhance anticancer effects of other chemotherapeutic agents such as, doxorubicin, celecoxib, vinblastine, docetaxel, and paclitaxel.
Moreover, gene expression analysis by RT-qPCR have been indicated that BCL2 mRNA expression as an anti-apoptotic agent was significantly decreased in the presence of sildenafil compared to cisplatin alone, and on the other hand, utilizing sildenafil in combination with cisplatin could considerably increase BAX mRNA expression as a proapoptotic gene in the cells in comparison with cisplatin treatment alone. In relation to protein expression level, data showed that caspase 3 as a key mediator of apoptosis process, significantly increased in the presence of sildenafil in both human mammary adenocarcinoma cell lines. These results have consistency with several experiments showed sildenafil can improve anticancer effects of various chemotherapeutic agents through activation of apoptosis pathways in some cancer cell lines.
Collectively, with respect to high expression of PDE-5 in cancerous cell and tissues, it seems that PDE-5 inhibitors can be used to reduce dosage and side effects of chemotherapeutic agents and enhance their anticancer effects. Our data demonstrated that sildenafil through increasing the production of ROS, is able to improve cisplatin anticancer effect in a time and dose-dependent manner, and subsequently induces apoptosis in the adenocarcinoma breast cancer cell lines. However, further data collection is required to exactly determine how sildenafil effects on cisplatin anticancer properties.
This work was supported by Student Research Committee, Kerman University of Medical Sciences (grant number: 97000025).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]