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Akt signaling pathway
Gene 745 (2020) 144623
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Research paper
Metformin enhances the sensitivity of colorectal cancer cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway
T
Pei Zhanga,1, Surong Zhaoa,1, Xingyue Lua, Zongfen Shia, Hao Liua, , Bing Zhub, ⁎
a b
⁎
School of Pharmacy, Bengbu Medical College, Anhui Engineering Technology Research Center of Biochemical Pharmaceuticals, Bengbu 233030, Anhui, China Department of Gastrointestinal Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233000, Anhui, China
ARTICLE INFO
ABSTRACT
Keywords: Colorectal cancer Metformin Cisplatin Apoptosis Reactive oxygen species PI3K/Akt signaling pathway
Metformin and cisplatin have been widely studied as antitumor agents. However, the effect of metformin combined with cisplatin has not been investigated in colorectal cancer (CRC) cells. This study was aimed to explore the effect of metformin or/and cisplatin on cell viability, apoptosis, and the related signaling pathways in CRC SW480 and SW620 cells. We found that metformin or cisplatin inhibited cell viability of SW480 and SW620 cells in a concentration- and time-dependent manner. Furthermore, metformin combined with cisplatin obviously inhibited cell viability, decreased colony formation, induced apoptosis, mediated cleavage of caspase9, caspase-3 and PARP, activated mitochondrial membrane potential, downregulated Mcl-1 and Bcl-2 expression, upregulated Bak and Bax expression, and increased reactive oxygen species (ROS) production, compared to the individual agent in SW480 and SW620 cells, which were attenuated by N-acetyl-L-cysteine (NAC), a ROS scavenger. Moreover, NAC could recover the downregulation of p-PI3K and p-Akt treated with combination of metformin and cisplatin, which subsequently activated the PI3K/Akt signaling pathway. Taken together, our results demonstrated that metformin enhanced the sensitivity of CRC cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway.
1. Introduction Colorectal cancer (CRC) is one of the most common tumor and the rate of death continues to rise all over the world, which is a greatly malignant cancer with aggressive clinical behavior (Wilkins et al., 2018). Up to present, surgery is recommended for the treatment of CRC. However, the prognosis for CRC remains poor despite advances in the methodologies of diagnosis, and approximately 50% of patients diagnosed with CRC will survive 5 years, even with the help of individualized therapy, such as surgery combined with chemotherapy, in particular, platinum-based chemotherapy (Murcia et al., 2016). In clinic, high-dose cisplatin might be beneficial to CRC patients. But the adverse events usually result in intolerable side effects (Sui et al., 2019). Furthermore, patients usually develop resistance after cisplatin therapy. Therefore, to obtain better treatment efficacy in CRC patients, it is very vital to determine the effective therapeutic agents to overcome this cancer and identify new drugs targeting different signaling
pathways. In this study, we used a paired CRC cell line SW480 and SW620 derived from the same patient and shared a common genetic background (Leibovitz et al., 1976), and thus provide an in vitro model of cancer progression to study the cellular changes. Metformin (1,1-dimethylbiguanide hydrochloride) is a safe and inexpensive agent that is broadly applied to treat type II diabetes mellitus in the clinic (Peters et al., 2019). Recent researches have established that metformin effectively represses the growth of many tumors, such as nasopharyngeal carcinoma, pancreatic, breast, lung and melanoma cancer. Moreover, other studies have indicated that metformin can suppress cell proliferation, migration, invasion in vitro, and enhance the apoptotic sensitivity to other chemotherapeutic agents (Zhang and Guo, 2016; Lurati, 2017; Sun et al., 2018; Zheng et al., 2018). Overall, metformin has recently emerged as a potential therapeutic agent for tumor. Apoptosis, a well-known type of programmed cell death, is closely modulated by the B-cell lymphoma/leukaemia-2 (Bcl-2) family
Abbreviations: CRC, colorectal cancer; ROS, reactive oxygen species; MMP, mitochondrial membrane potential; PARP, poly ADP-ribose polymerase; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PVDF, polyvinylidene difluoride; Bcl-2, B-cell lymphoma/leukaemia-2; PI3K, phosphatidylinositol 3 kinase ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Liu), [email protected] (B. Zhu). 1 Pei Zhang and Surong Zhao contributed equally to this study. https://doi.org/10.1016/j.gene.2020.144623 Received 3 November 2019; Received in revised form 8 February 2020; Accepted 24 March 2020 Available online 25 March 2020 0378-1119/ © 2020 Elsevier B.V. All rights reserved.
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molecules, which contains anti-apoptotic and pro-apoptotic members, and the dysregulation of Bcl-2 family members is key to the development of certain human diseases, including cancer (Cheng et al., 2016; Maji et al., 2018; Lee et al., 2019a). However, cancer cells develop various strategies to escape apoptosis by promoting the expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1, or/and reducing the expression of pro-apoptotic proteins, such as Bax and Bak (Jeong et al., 2015). The apoptosis of most cells is mainly induced through the mitochondrial pathway triggered by stress signals (Sharikova et al., 2018). The reduction of mitochondrial membrane potential (MMP) and consequent generation of reactive oxygen species (ROS) is considered as a significant sign of early apoptosis (Chen et al., 2018). ROS regulates the mitochondrial pathway of apoptosis by releasing cytochrome C from mitochondria (Kim et al., 2017b). Recent evidence indicates that the PI3K/Akt signaling pathway has been elucidated to modulate vital aspects of tumor progression, comprising cell growth, survival, and apoptosis (Liang et al., 2016; Liu et al., 2017; Hou et al., 2018). Besides, the PI3K/Akt signaling pathway displays high activation in most human cancers, including CRC. Moreover, suppression of PI3K/Akt pathway induces mitochondrial dysfunction such as decreased MMP and ROS production, leading to the induction of cancer cell apoptosis (Kim et al., 2017a). Although studies on the role of ROS production in the inactivation of PI3K/Akt signaling pathway have not been fully demonstrated, ROS-mediated PI3K/Akt signaling pathway may serve as an attractive therapeutic target for inducing apoptosis in cancer cells (Song et al., 2017; Guo et al., 2018; Zhou et al., 2019). In the present study, we explored the effects of metformin or/and cisplatin on cell viability and apoptosis in human CRC SW480 and SW620 cells, and its underlying mechanisms.
2.4. Colony formation assay The cells were plated in 6-well plates and treated for 24 h, then dealt with metformin or/and cisplatin for 24 h. The cells were cultured for an additional 7 days after the medium was substituted with fresh medium, and then fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and visualized by a camera. 2.5. Apoptosis assay Apoptosis was analyzed with the Annexin V-FITC/PI apoptosis detection kit described in the manufacturer’s instruction. Briefly, the SW480 and SW620 cells (4 × 105 cells/well) were plated and dealt with 10 mM metformin or/and 8 μM cisplatin for 24 h. The cells were harvested, and then resuspended in 500 μL binding buffer composing 5 μL Annexin V-FITC for 20 min, and stained with 5 μL PI for 5 min. Cells were then detected with a BD Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and subsequently analyzed by FlowJo software (Tree Star Inc., Ashland, OR, USA). 2.6. MMP assay The MMP detection was performed using the JC-1 kit as described in the manufacturer’s instruction. Briefly, the SW480 and SW620 cells were plated in 12-well plates and incubated with 10 mM metformin or/ and 8 μM cisplatin for 24 h, and then cells were incubated with JC-1 (10 μM) for 30 min. The JC-1 signal was visualized on a 561 nm (red) and 488 nm (green) wavelength laser by confocal laser scanning microscopy (Nikon, Tokyo, Japan), respectively. 2.7. Intracellular ROS detection
2. Materials and methods
The intracellular ROS level was probed with a fluorescent DCFH-DA kit as recommended by the manufacturer’s instruction. Briefly, the SW480 and SW620 cells were seeded and incubated with 10 mM metformin or/and 8 μM cisplatin for 24 h. The cells were harvested, rinsed with PBS, treated with DCFH-DA (10 μM) for 20 min, and analyzed by flow cytometry. To avoid the generation of ROS, NAC (5 mM), as a ROS scavenger, was added to the cells for 1 h before incubation with metformin or/and cisplatin. The results were evaluated by Cell Quest analysis software.
2.1. Reagents and antibodies Metformin, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), N-acetyl-L-cysteine (NAC) were from Sigma (St. Louis, MO, USA). Cisplatin was from Qilu Pharmaceutical Co., Ltd. (Jinan, China). Annexin V-FITC/PI apoptosis detection kit was acquired from Nanjing KeyGen Biotech (Nanjing, China). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) kit and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) kit were acquired from Beyotime (Wuhan, China). Antibodies against Mcl-1, Bcl-2, Bax, PARP, caspase-3, and β-actin were from Abcam (Cambridge, MA, USA). Antibodies against caspase-9, PI3K, p-PI3K, Akt, p-Akt, and Bak were from Cell Signaling Technology (Danvers, MA, USA), respectively.
2.8. Western blot analysis Cells (5 × 105 cells/well) were plated in 6-well culture plates. After incubation with different treatments, the cells were harvested and lysed in RIPA buffer. Protein concentration was examined with BCA kit (Beyotime, Wuhan, China). Subsequently, total protein (about 40 µg) was subjected to SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% nonfat milk, PVDF membranes were incubated with the primary antibodies overnight at 4˚C, then further incubated with the corresponding secondary antibodies for 1 h at room temperature, finally visualized with ECL chemiluminescent reagent (Millipore, MA, USA), and imaged with a gel imaging equipment (BioRad, Berkeley, CA, USA). β-actin was used as the loading control. The relative protein level was used to show the expression of the studied protein, which was the ratio of the gray value of the studied protein to the gray value of β-actin.
2.2. Cell lines and cell culture Human CRC SW480 and SW620 cells were acquired from the American Type Culture Collection (Manassas, VA, USA), and maintained in Dulbecco’s modified Eagle medium (DMEM) (Grand Island, NY, USA) supplemented with 10% fetal bovine serum and antibiotics (penicillin/streptomycin) at 37 °C, 5% CO2. 2.3. Cell viability assay
2.9. Statistical analysis
Cell viability was measured by MTT assay. The SW480 and SW620 cells (7 × 103 cells/well) were plated in triplicate and dealt with the indicated concentration of metformin or/and cisplatin. After treatment, 15 µL MTT (5 g/L in PBS) was added to each well for 4 h. Subsequently, another 150 µL dimethyl sulfoxide (DMSO) was added, and the optical density (OD) was examined at 490 nm. Cell viability was displayed as a percentage of control.
Numerical data are represented as the mean ± SD. All statistical analyses were performed by the two-tailed Student’s t-test and one-way analysis of variance (ANOVA) followed by LSD test with SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Statistical difference was defined as P < 0.05. 2
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Fig. 1. Metformin elevated the inhibitory effects of CRC cells to cisplatin on cell viability. (A and B) SW480 and SW620 cells were incubated with various concentrations of metformin (1.25, 2.5, 5, 10, and 20 mM), different concentrations of cisplatin (1, 2, 4, 8, and 16 μM) for 24, 48, 72 h, respectively. Cell viability was analyzed using the MTT assay. (C) The cells were incubated with metformin (1.25, 2.5, 5, 10, and 20 mM) with or without cisplatin (4 μM) for 24 h by performing the MTT assay. **P < 0.01 vs metformin alone. (D and E) SW480 and SW620 cells were incubated with metformin (10 mM), cisplatin (8 μM), and metformin (10 mM) plus cisplatin (8 μM), and cell colony formation was analyzed by crystal violet staining. a, control; b, metformin; c, cisplatin; d, combination of metformin and cisplatin. **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. MET, metformin; DDP, cisplatin.
3. Results
3.2. Metformin enhanced cisplatin-mediated apoptosis in CRC cells
3.1. Metformin elevated the inhibitory effect of CRC cells to cisplatin on cell viability
To define the effects of metformin or/and cisplatin on apoptosis, SW480 and SW620 cells were dealt with metformin (10 mM), cisplatin (8 μM), and combination of metformin and cisplatin for 24 h. The apoptosis assay displayed that metformin and cisplatin increased apoptosis of SW480 and SW620 cells, and combination of metformin and cisplatin induced more apoptosis than treatment with metformin or cisplatin alone (Fig. 2A and B). Moreover, the activation of caspase-9, caspase-3 and PARP was assessed to further characterize the apoptotic process. As shown in Fig. 2C–E, metformin combined with cisplatin markedly mediated the cleavage of caspase-9, caspase-3 and PARP, while metformin or cisplatin alone mediated a slight cleavage of caspase-9, caspase-3 and PARP.
To investigate the effect of metformin or/and cisplatin on cell viability, SW480 and SW620 cells were incubated with various concentrations of metformin (1.25, 2.5, 5, 10, and 20 mM), different concentrations of cisplatin (1, 2, 4, 8, and 16 μM), and cisplatin (4 mM) combined with metformin (1.25, 2.5, 5, 10, and 20 mM) by performing the MTT assay. The results demonstrated that metformin and cisplatin attenuated cell viability of SW480 and SW620 cells in a concentrationand time-dependent manner within a certain range of concentration, respectively (Fig. 1A and B). Metformin combined with cisplatin could markedly increase inhibition of cell viability in SW480 and SW620 cells, compared to treatment with metformin alone (Fig. 1C). To further explore the cytotoxicity of metformin and cisplatin in CRC cells, colony formation assay was performed. As shown in Fig. 1D and E, combination of metformin and cisplatin could obviously inhibit the colony formation in SW480 and SW620 cells, compared to treatment with metformin or cisplatin alone. Therefore, metformin combined with cisplatin efficiently suppressed cell viability of CRC cells.
3.3. Metformin and cisplatin decreased the MMP of CRC cells by modulating Bcl-2 family members’ expression Mitochondrial dysfunction has been demonstrated to participate in the induction of apoptosis and even been supposed to be central to the apoptotic pathway (Chen et al., 2019). To confirm whether metformin and cisplatin affect the function of mitochondria in SW480 and SW620 3
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Fig. 2. Metformin enhanced cisplatin-mediated apoptosis in CRC cells. SW480 and SW620 cells were incubated with metformin (10 mM), cisplatin (8 μM), and metformin (10 mM) plus cisplatin (8 μM) for 24 h. (A and B) Apoptosis was detected by flow cytometry, and quantification of the percentage of apoptotic cells. ** P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. (C-E) The protein levels of caspase-9, caspase-3 and PARP were measured by western blot analysis, and quantification. **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. Ctrl, control; MET, metformin; DDP, cisplatin.
cells, JC-1 staining assay was performed. This assay is based on the principle that red fluorescence will be present in areas with high MMP, while green fluorescence will be prevalent in areas with low MMP. We found that combination of metformin and cisplatin triggered a greater decrease of MMP than the treatment with metformin or cisplatin alone (Fig. 3A and B). Moreover, the mitochondrial pathway is a major mediator of apoptosis, which is modulated by the Bcl-2 family proteins. To deeply discuss the mechanism of the apoptosis mediated by metformin and cisplatin, the level of anti-apoptotic proteins Mcl-1, Bcl-2, and pro-apoptotic proteins Bak, Bax were measured. It was shown that the expression of Bcl-2 and Mcl-1 was significantly downregulated in CRC cells treated with combination of metformin and cisplatin compared to metformin or cisplatin alone, whereas the expression of Bak and Bax was upregulated (Fig. 3C–E). We found that metformin combined with cisplatin markedly mediated the cleavage of caspase-9, caspase-3 and PARP, therefore, we hypothesized that the mitochondrial pathway might be involved in metformin-enhanced cisplatin-induced apoptosis in CRC cells.
proliferation and apoptosis) by changing the balance between the cell survival and death progression (Tang et al., 2018a). We evaluated intracellular ROS levels in SW480 and SW620 cells treated with metformin or/and cisplatin by DCFH-DA staining coupled with flow cytometry. As a result, SW480 and SW620 cells showed considerably greater increases of ROS levels after treatment with combination of metformin and cisplatin, while metformin or cisplatin resulted in only a slight promotion of ROS levels (Fig. 4A and B). Meanwhile, pre-treatment of SW480 and SW620 cells with NAC effectively decreased the levels of ROS induced by combination of metformin and cisplatin (Fig. 4C and D). Next, to further explore the effects of ROS on proliferation in SW480 and SW620 cells, the results indicated that NAC could attenuate the inhibitory effect of metformin combined with cisplatin on proliferation (Fig. 4E), suggested that the generation of ROS might play a vital role in anti-proliferative effect of metformin and cisplatin. 3.5. ROS was involved in metformin combined with cisplatin-induced apoptosis in CRC cells
3.4. Metformin and cisplatin induced the production of intracellular ROS in CRC cells
Next, we tested whether ROS inhibition affected apoptosis induced by metformin and cisplatin in CRC cells. We found that the increased apoptosis of SW480 and SW620 cells induced by combination of
The intracellular ROS affect diverse cellular functions (such as 4
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Fig. 3. Metformin and cisplatin decreased the MMP of CRC cells by modulating Bcl-2 family members’ expression. SW480 and SW620 cells were treated with metformin (10 mM), cisplatin (8 μM), and metformin (10 mM) plus cisplatin (8 μM) for 24 h. (A and B) The MMP was determined by JC-1 staining, and quantification via the ratio of red-to-green fluorescence intensity. **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. Scale bar is 50 μm. (C-E) The protein levels of Mcl-1, Bak, Bcl-2, and Bax were detected by western blot analysis, and quantification. *P < 0.05, **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. Ctrl, control; MET, metformin; DDP, cisplatin.
metformin and cisplatin could be blocked by NAC (Fig. 5A and B). Moreover, NAC could abrogate the cleavage of caspase-9, caspase-3 and PARP in SW480 and SW620 cells mediated by metformin combined with cisplatin (Fig. 5C-E). Meanwhile, the attenuation of Mcl-1 and Bcl2 in SW480 and SW620 cells incubated with metformin combined with cisplatin, and the upregulation of Bak and Bax, were also abolished by NAC (Fig. 5F-I), suggested that metformin enhanced cisplatin-induced apoptosis dependent on the generation of ROS.
expression (Fig. 6A-C). To further unveil whether the contribution of ROS is mediated through inactivation of the PI3K/Akt pathway, the effects of metformin combined with cisplatin on p-PI3K and p-Akt were detected when NAC blocked the production of ROS. As shown in Fig. 6D-F, NAC could recover the downregulation of p-PI3K and p-Akt in CRC cells treated with combination of metformin and cisplatin. These results demonstrated that the inactivation of PI3K/Akt pathway relied on the generation of ROS incubated with metformin and cisplatin in CRC cells.
3.6. ROS was critical for the inactivation of PI3K/Akt pathway in CRC cells incubated with metformin and cisplatin
4. Discussion
To discuss the role of PI3K/Akt pathway in the apoptosis of SW480 and SW620 cells incubated with metformin and cisplatin, we measured the level of PI3K, p-PI3K, Akt, and p-Akt. The results displayed that pPI3K and p-Akt expression was evidently decreased in CRC cells dealt with combination of metformin and cisplatin compared to metformin or cisplatin alone, whereas did not significantly change PI3K and Akt
The treatment of cancer (including CRC) is a major concern of the medical profession, the current treatment options mainly include surgery, radiotherapy, and chemotherapy. However, many chemotherapeutic drugs are low-sensitive, highly toxic, and susceptible to develop drug resistance, which leads to the recurrence and metastasis of the tumor (Kartal-Yandim et al., 2016). Thus, combination therapy is an 5
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Fig. 4. Metformin and cisplatin induced the production of intracellular ROS in CRC cells. SW480 and SW620 cells were incubated with metformin (10 mM), cisplatin (8 μM), and metformin (10 mM) plus cisplatin (8 μM) for 24 h. (A and B) The intracellular ROS levels were measured by DCFH-DA staining with flow cytometry, and Quantification via Cell Quest analysis software. *P < 0.05, **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs metformin or cisplatin alone. (C and D) SW480 and SW620 cells were pre-treated with NAC (5 mM) for 1 h, and the intracellular ROS levels were measured by flow cytometry using DCFH-DA staining. **P < 0.01 vs combination of metformin and cisplatin. (E) SW480 and SW620 cells were pre-treated with NAC (5 mM) for 1 h, cell viability was measured using the MTT assay. ** P < 0.01 vs combination of metformin and cisplatin. Ctrl, control; MET, metformin; DDP, cisplatin; NAC, N-acetyl-L-cysteine.
important strategy to improve therapeutic outcomes and reduce the toxic side effects of anticancer drugs. Recent studies have established that metformin effectively inhibits cell proliferation as a promising therapeutic agent for cancer, and increases the apoptotic sensitivity to other chemotherapeutic drugs (Tang et al., 2018b; Zhao et al., 2018; Lee et al., 2019b; Zhao et al., 2019). In this study, we found that metformin combined with cisplatin could evidently increase inhibition of cell viability and apoptosis of SW480 and SW620 cells, compared to treatment with metformin or cisplatin alone. To the best of our knowledge, apoptosis is an activated cellular process, which mainly uses pathological or physiological factors to remove redundant and damaged cells (Xu et al., 2015). The disorder of apoptosis is involved in the occurrence and development of many diseases, including cancer (Pistritto et al., 2016). Apoptosis can be initiated through two classic and correlated pathways in mammalian cells, including the mitochondria- and the death receptor-mediated apoptotic pathway (Sun et al., 2014). Furthermore, induction of the
mitochondrial-mediated apoptotic pathway can trigger the permeability of the mitochondrial membrane to raise. According to the present study, combination of metformin and cisplatin decreased signals in red fluorescence while increased signals in green fluorescence, thus led to loss of MMP. The Bcl-2 family, a well-known family of apoptosis-regulating proteins, exhibits a major role in the mitochondria pathway, and tightly regulates the released cytochrome C from mitochondria, which results in caspase activation and eventually apoptosis. Besides, the release of cytochrome C mediates caspase-9 activation, which participates in the activation of caspase-3, thus, subsequently activates downstream processes, such as cleavage of PARP (Yang et al., 2017; Zhao et al., 2017; Liu et al., 2018). As a result, metformin combined with cisplatin obviously mediated the cleavage of caspase-9, caspase-3 and PARP, while metformin or cisplatin alone mediated a slight proteolytic cleavage of caspase-9, caspase-3 and PARP. Moreover, the balance between proapoptotic and anti-apoptotic molecules influences the occurrence of 6
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Fig. 5. ROS was involved in metformin combined with cisplatin-induced apoptosis in CRC cells. SW480 and SW620 cells were pre-treated with NAC (5 mM) for 1 h, then treated with metformin (10 mM) plus cisplatin (8 μM) for 24 h. (A and B) Apoptosis was analyzed by flow cytometry, and quantification of the percentage of apoptotic cells. **P < 0.01 vs combination of metformin and cisplatin. (C-E) The protein levels of caspase-9, caspase-3 and PARP were measured by western blot analysis, and quantification. **P < 0.01 vs combination of metformin and cisplatin. (F-H) The protein levels of Mcl-1, Bak, Bcl-2, and Bax were detected by western blot analysis, and quantification. **P < 0.01 vs combination of metformin and cisplatin. Ctrl, control; MET, metformin; DDP, cisplatin; NAC, N-acetyl-L-cysteine.
apoptosis, and is related to the success rate of chemotherapy in cancer patients (Arababadi and Asadikaram, 2016). The results showed that Bcl-2 and Mcl-1 were significantly downregulated, while Bak and Bax were evidently upregulated in the cells dealt with metformin combined with cisplatin compared with metformin or cisplatin alone. These data
confirmed that the mitochondrial pathway might be involved in metformin-enhanced cisplatin-induced apoptosis in CRC cells. Accumulating evidences indicate that ROS, typical products of oxidative stress, are mainly generated by mitochondria, function as mediators of various intracellular signaling cascades, and recent 7
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Fig. 6. ROS was critical for the inactivation of PI3K/Akt signaling pathway in CRC cells incubated with metformin and cisplatin. (A-C) SW480 and SW620 cells were incubated with metformin (10 mM), cisplatin (8 μM, and metformin (10 mM) plus cisplatin (8 μM) for 24 h. The protein levels of PI3K, p-PI3K, Akt and p-Akt were measured by western blot assay, and quantification. *P < 0.05, **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. (D-F) SW480 and SW620 cells were pre-treated with NAC (5 mM) for 1 h, then incubated with metformin (10 mM) plus cisplatin (8 μM) for 24 h. The protein levels of PI3K, p-PI3K, Akt and p-Akt were detected by western blot analysis, and quantification. **P < 0.01 vs combination of metformin and cisplatin. Ctrl, control; MET, metformin; DDP, cisplatin; NAC, N-acetyl-L-cysteine.
evidence has revealed their participation in the regulation of apoptosis by causing mitochondrial dysfunction and DNA damage (Chiu et al., 2012; Jiang et al., 2019; Rashmi et al., 2019). Indeed, the mitochondrial dysfunction induced by ROS production leads to loss of MMP, thus induced caspase-9 activation, followed by activation of effector caspases, ultimately resulted in apoptosis (Zhong et al., 2017). In this study, we found that SW480 and SW620 cells showed considerably greater increases of ROS levels after treatment with combination of metformin and cisplatin compared to metformin or cisplatin alone, which was abolished by the pretreatment of cells with NAC. In addition, NAC could attenuate anti-proliferative effect and apoptosis induced by metformin combined with cisplatin. Meanwhile, the activation of caspase-9, caspase-3 and PARP, down-regulation of Mcl-1 and Bcl-2, and up-regulation of Bak and Bax induced by combination of metformin and cisplatin were also blocked by NAC. These data implied that ROS were of fundamental importance to apoptosis of CRC cells induced by metformin and cisplatin. Studies have shown that metformin enhances the effect of cisplatin in various cancers. For example, the development of cisplatin and metformin nano-cubosomes induces CRC cell apoptosis through inhibition of several metabolic pathways, namely, AMPK/mTOR and Akt/ Mtor (Saber et al., 2018). Meanwhile, metformin increases the chemotherapeutic sensitivity of liver cancer cells to cisplatin through the AMPK pathway (Dong et al., 2017), metformin enhances the antitumor activity of cisplatin in gallbladder cancer through PI3K/Akt pathway (Bi et al., 2018), metformin combined with cisplatin induces apoptosis
of human ovarian cancer cells by inactivating ERK1/2 (Dang et al., 2017). But the mechanism by which metformin enhances the anticancer effect of cisplatin in CRC cells is not clear. It is well known that the PI3K/Akt pathway is closely related to apoptosis (Lv et al., 2018). Several explores have elucidated that inactivation of the PI3K/Akt pathway can reverse the sensitivity of diverse cancer cells to chemotherapeutic drugs (You et al., 2018), Akt can phosphorylate members of the Bcl-2 family, thereby inhibiting apoptosis. In addition, Akt can also inhibit the activity of the proteolytic enzyme caspase-9 to prevent the initiation of the apoptotic cascade and thus inhibit apoptosis, indicating that targeting PI3K/Akt signaling can serve as a potential therapeutic strategy for cancer treatment. We observed that the level of p-PI3K and p-Akt was evidently decreased in CRC cells dealt with combination of metformin and cisplatin compared to metformin or cisplatin alone, whereas did not significantly change PI3K and Akt expression. Furthermore, ROS generation functions serves as a second messenger that mediates activation of the PI3K/Akt pathway in various types of cells (Ahn et al., 2017; Han et al., 2017; Deng et al., 2019). Thus, we discussed the connection between ROS and the PI3K/Akt pathway. In our study, NAC could recover the downregulation of pPI3K and p-Akt in CRC cells dealt with combination of metformin and cisplatin, which subsequently activated the PI3K/Akt signaling pathway. In summary, this study demonstrated that metformin enhanced the sensitivity of CRC cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway. This information may be useful for developing a 8
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new therapy for the treatment of CRC in future, and the specific underlying molecular mechanisms remains to be subsequent research.
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CRediT authorship contribution statement Pei Zhang: Formal analysis, Funding acquisition, Writing - original draft, Data curation. Surong Zhao: Methodology, Supervision, Writing - review & editing. Xingyue Lu: Formal analysis, Validation. Zongfen Shi: Methodology, Data curation, Validation. Hao Liu: Project administration, Resources, Supervision. Bing Zhu: Data curation, Resources, Validation, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This study was supported by the National Natural Science Foundation of China (81603155), the Natural Science Foundation of Anhui Province (1708085QH212), and the Key Project of Natural Science Research for College and University of Anhui Province (KJ2018A1028, KJ2016A486). References Ahn, K.I., Choi, E.O., Kwon, D.H., HwangBo, H., Kim, M.Y., Kim, H.J., Ji, S.Y., Hong, S.H., Jeong, J.W., Park, C., Kim, N.D., Kim, W.J., Choi, Y.H., 2017. Induction of apoptosis by ethanol extract of Citrus unshiu Markovich peel in human bladder cancer T24 cells through ROS-mediated inactivation of the PI3K/Akt pathway. Biosci. Trends 11, 565–573. Arababadi, M.K., Asadikaram, G., 2016. Opium induces apoptosis in Jurkat cells via promotion of pro-apoptotic and inhibition of anti-apoptotic molecules. Iran J. Basic Med. Sci. 19, 215–220. Bi, T., Zhu, A., Yang, X., Qiao, H., Tang, J., Liu, Y., Lv, R., 2018. Metformin synergistically enhances antitumor activity of cisplatin in gallbladder cancer via the PI3K/AKT/ERK pathway. Cytotechnology 70, 439–448. Chen, B., Cao, X., Lu, H., Wen, P., Qi, X., Chen, S., Wu, L., Li, C., Xu, A., Zhao, G., 2018. N(3-oxo-acyl) homoserine lactone induced germ cell apoptosis and suppressed the over-activated RAS/MAPK tumorigenesis via mitochondrial-dependent ROS in C. elegans. Apoptosis 23, 626–640. Chen, W., Liu, H., Wang, T., Bao, G., Wang, N., Li, R.C., 2019. Downregulation of AIF-2 inhibits proliferation, migration, and invasion of human glioma cells via mitochondrial dysfunction. J. Mol. Neurosci. 68, 304–310. Cheng, C.H., Cheng, Y.P., Chang, I.L., Chen, H.Y., Wu, C.C., Hsieh, C.P., 2016. Dodecyl gallate induces apoptosis by upregulating the caspase-dependent apoptotic pathway and inhibiting the expression of anti-apoptotic Bcl-2 family proteins in human osteosarcoma cells. Mol. Med. Rep. 13, 1495–1500. Chiu, W.H., Luo, S.J., Chen, C.L., Cheng, J.H., Hsieh, C.Y., Wang, C.Y., Huang, W.C., Su, W.C., Lin, C.F., 2012. Vinca alkaloids cause aberrant ROS-mediated JNK activation, Mcl-1 downregulation, DNA damage, mitochondrial dysfunction, and apoptosis in lung adenocarcinoma cells. Biochem. Pharmacol. 83, 1159–1171. Dang, J.H., Jin, Z.J., Liu, X.J., Hu, D., Wang, J., Luo, Y., Li, L.L., 2017. Metformin in combination with cisplatin inhibits cell viability and induces apoptosis of human ovarian cancer cells by inactivating ERK 1/2. Oncol. Lett. 14, 7557–7564. Deng, S., Dai, G., Chen, S., Nie, Z., Zhou, J., Fang, H., Peng, H., 2019. Dexamethasone induces osteoblast apoptosis through ROS-PI3K/AKT/GSK3beta signaling pathway. Biomed. Pharmacother. 110, 602–608. Dong, H., Huang, J., Zheng, K., Tan, D., Chang, Q., Gong, G., Zhang, Q., Tang, H., Sun, J., Zhang, S., 2017. Metformin enhances the chemosensitivity of hepatocarcinoma cells to cisplatin through AMPK pathway. Oncol. Lett. 14, 7807–7812. Guo, C.L., Wang, L.J., Zhao, Y., Liu, H., Li, X.Q., Jiang, B., Luo, J., Guo, S.J., Wu, N., Shi, D.Y., 2018. A novel bromophenol derivative BOS-102 induces cell cycle arrest and apoptosis in human A549 lung cancer cells via ROS-mediated PI3K/Akt and the MAPK signaling pathway. Mar. Drugs 16. Han, M.H., Lee, D.S., Jeong, J.W., Hong, S.H., Choi, I.W., Cha, H.J., Kim, S., Kim, H.S., Park, C., Kim, G.Y., Moon, S.K., Kim, W.J., Hyun Choi, Y., 2017. Fucoidan induces ROS-dependent apoptosis in 5637 human bladder cancer cells by downregulating telomerase activity via inactivation of the PI3K/Akt signaling pathway. Drug Dev. Res. 78, 37–48. Hou, T., Zhou, L., Wang, L., Kazobinka, G., Chen, Y., Zhang, X., Chen, Z., 2018. Leupaxin promotes bladder cancer proliferation, metastasis, and angiogenesis through the PI3K/AKT pathway. Cell. Physiol. Biochem. 47, 2250–2260. Jeong, J.W., Lee, W.S., Go, S.I., Nagappan, A., Baek, J.Y., Lee, J.D., Lee, S.J., Park, C., Kim, G.Y., Kim, H.J., Kim, G.S., Kwon, T.K., Ryu, C.H., Shin, S.C., Choi, Y.H., 2015.
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P. Zhang, et al. Prevention. Am. Fam. Phys. 97, 658–665. Xu, Y., Wang, C., Su, J., Xie, Q., Ma, L., Zeng, L., Yu, Y., Liu, S., Li, S., Li, Z., Sun, L., 2015. Tolerance to endoplasmic reticulum stress mediates cisplatin resistance in human ovarian cancer cells by maintaining endoplasmic reticulum and mitochondrial homeostasis. Oncol. Rep. 34, 3051–3060. Yang, M., Wang, B., Gao, J., Zhang, Y., Xu, W., Tao, L., 2017. Spinosad induces programmed cell death involves mitochondrial dysfunction and cytochrome C release in Spodoptera frugiperda Sf9 cells. Chemosphere 169, 155–161. You, P., Wu, H., Deng, M., Peng, J., Li, F., Yang, Y., 2018. Brevilin A induces apoptosis and autophagy of colon adenocarcinoma cell CT26 via mitochondrial pathway and PI3K/AKT/mTOR inactivation. Biomed. Pharmacother. 98, 619–625. Zhang, H.H., Guo, X.L., 2016. Combinational strategies of metformin and chemotherapy in cancers. Cancer Chemother. Pharmacol. 78, 13–26. Zhao, Y., Jing, Z., Lv, J., Zhang, Z., Lin, J., Cao, X., Zhao, Z., Liu, P., Mao, W., 2017. Berberine activates caspase-9/cytochrome c-mediated apoptosis to suppress triplenegative breast cancer cells in vitro and in vivo. Biomed. Pharmacother. 95, 18–24. Zhao, Y., Zeng, X., Tang, H., Ye, D., Liu, J., 2019. Combination of metformin and
paclitaxel suppresses proliferation and induces apoptosis of human prostate cancer cells via oxidative stress and targeting the mitochondria-dependent pathway. Oncol. Lett. 17, 4277–4284. Zhao, Y., Zhang, E., Lv, N., Ma, L., Yao, S., Yan, M., Zi, F., Deng, G., Liu, X., He, J., Wu, W., Cai, Z., Yu, R., 2018. Metformin and FTY720 synergistically induce apoptosis in multiple myeloma cells. Cell. Physiol. Biochem. 48, 785–800. Zheng, Y., Zhu, J., Zhang, H., Liu, Y., Sun, H., 2018. Metformin inhibits ovarian cancer growth and migration in vitro and in vivo by enhancing cisplatin cytotoxicity. Am. J. Transl. Res. 10, 3086–3098. Zhong, Y., Jin, C., Gan, J., Wang, X., Shi, Z., Xia, X., Peng, X., 2017. Apigenin attenuates patulin-induced apoptosis in HEK293 cells by modulating ROS-mediated mitochondrial dysfunction and caspase signal pathway. Toxicon 137, 106–113. Zhou, Y.D., Hou, J.G., Yang, G., Jiang, S., Chen, C., Wang, Z., Liu, Y.Y., Ren, S., Li, W., 2019. Icariin ameliorates cisplatin-induced cytotoxicity in human embryonic kidney 293 cells by suppressing ROS-mediated PI3K/Akt pathway. Biomed. Pharmacother. 109, 2309–2317.
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Metformin enhances the sensitivity of colorectal cancer cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway
T
Pei Zhanga,1, Surong Zhaoa,1, Xingyue Lua, Zongfen Shia, Hao Liua, , Bing Zhub, ⁎
a b
⁎
School of Pharmacy, Bengbu Medical College, Anhui Engineering Technology Research Center of Biochemical Pharmaceuticals, Bengbu 233030, Anhui, China Department of Gastrointestinal Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233000, Anhui, China
ARTICLE INFO
ABSTRACT
Keywords: Colorectal cancer Metformin Cisplatin Apoptosis Reactive oxygen species PI3K/Akt signaling pathway
Metformin and cisplatin have been widely studied as antitumor agents. However, the effect of metformin combined with cisplatin has not been investigated in colorectal cancer (CRC) cells. This study was aimed to explore the effect of metformin or/and cisplatin on cell viability, apoptosis, and the related signaling pathways in CRC SW480 and SW620 cells. We found that metformin or cisplatin inhibited cell viability of SW480 and SW620 cells in a concentration- and time-dependent manner. Furthermore, metformin combined with cisplatin obviously inhibited cell viability, decreased colony formation, induced apoptosis, mediated cleavage of caspase9, caspase-3 and PARP, activated mitochondrial membrane potential, downregulated Mcl-1 and Bcl-2 expression, upregulated Bak and Bax expression, and increased reactive oxygen species (ROS) production, compared to the individual agent in SW480 and SW620 cells, which were attenuated by N-acetyl-L-cysteine (NAC), a ROS scavenger. Moreover, NAC could recover the downregulation of p-PI3K and p-Akt treated with combination of metformin and cisplatin, which subsequently activated the PI3K/Akt signaling pathway. Taken together, our results demonstrated that metformin enhanced the sensitivity of CRC cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway.
1. Introduction Colorectal cancer (CRC) is one of the most common tumor and the rate of death continues to rise all over the world, which is a greatly malignant cancer with aggressive clinical behavior (Wilkins et al., 2018). Up to present, surgery is recommended for the treatment of CRC. However, the prognosis for CRC remains poor despite advances in the methodologies of diagnosis, and approximately 50% of patients diagnosed with CRC will survive 5 years, even with the help of individualized therapy, such as surgery combined with chemotherapy, in particular, platinum-based chemotherapy (Murcia et al., 2016). In clinic, high-dose cisplatin might be beneficial to CRC patients. But the adverse events usually result in intolerable side effects (Sui et al., 2019). Furthermore, patients usually develop resistance after cisplatin therapy. Therefore, to obtain better treatment efficacy in CRC patients, it is very vital to determine the effective therapeutic agents to overcome this cancer and identify new drugs targeting different signaling
pathways. In this study, we used a paired CRC cell line SW480 and SW620 derived from the same patient and shared a common genetic background (Leibovitz et al., 1976), and thus provide an in vitro model of cancer progression to study the cellular changes. Metformin (1,1-dimethylbiguanide hydrochloride) is a safe and inexpensive agent that is broadly applied to treat type II diabetes mellitus in the clinic (Peters et al., 2019). Recent researches have established that metformin effectively represses the growth of many tumors, such as nasopharyngeal carcinoma, pancreatic, breast, lung and melanoma cancer. Moreover, other studies have indicated that metformin can suppress cell proliferation, migration, invasion in vitro, and enhance the apoptotic sensitivity to other chemotherapeutic agents (Zhang and Guo, 2016; Lurati, 2017; Sun et al., 2018; Zheng et al., 2018). Overall, metformin has recently emerged as a potential therapeutic agent for tumor. Apoptosis, a well-known type of programmed cell death, is closely modulated by the B-cell lymphoma/leukaemia-2 (Bcl-2) family
Abbreviations: CRC, colorectal cancer; ROS, reactive oxygen species; MMP, mitochondrial membrane potential; PARP, poly ADP-ribose polymerase; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PVDF, polyvinylidene difluoride; Bcl-2, B-cell lymphoma/leukaemia-2; PI3K, phosphatidylinositol 3 kinase ⁎ Corresponding authors. E-mail addresses: [email protected] (H. Liu), [email protected] (B. Zhu). 1 Pei Zhang and Surong Zhao contributed equally to this study. https://doi.org/10.1016/j.gene.2020.144623 Received 3 November 2019; Received in revised form 8 February 2020; Accepted 24 March 2020 Available online 25 March 2020 0378-1119/ © 2020 Elsevier B.V. All rights reserved.
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molecules, which contains anti-apoptotic and pro-apoptotic members, and the dysregulation of Bcl-2 family members is key to the development of certain human diseases, including cancer (Cheng et al., 2016; Maji et al., 2018; Lee et al., 2019a). However, cancer cells develop various strategies to escape apoptosis by promoting the expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1, or/and reducing the expression of pro-apoptotic proteins, such as Bax and Bak (Jeong et al., 2015). The apoptosis of most cells is mainly induced through the mitochondrial pathway triggered by stress signals (Sharikova et al., 2018). The reduction of mitochondrial membrane potential (MMP) and consequent generation of reactive oxygen species (ROS) is considered as a significant sign of early apoptosis (Chen et al., 2018). ROS regulates the mitochondrial pathway of apoptosis by releasing cytochrome C from mitochondria (Kim et al., 2017b). Recent evidence indicates that the PI3K/Akt signaling pathway has been elucidated to modulate vital aspects of tumor progression, comprising cell growth, survival, and apoptosis (Liang et al., 2016; Liu et al., 2017; Hou et al., 2018). Besides, the PI3K/Akt signaling pathway displays high activation in most human cancers, including CRC. Moreover, suppression of PI3K/Akt pathway induces mitochondrial dysfunction such as decreased MMP and ROS production, leading to the induction of cancer cell apoptosis (Kim et al., 2017a). Although studies on the role of ROS production in the inactivation of PI3K/Akt signaling pathway have not been fully demonstrated, ROS-mediated PI3K/Akt signaling pathway may serve as an attractive therapeutic target for inducing apoptosis in cancer cells (Song et al., 2017; Guo et al., 2018; Zhou et al., 2019). In the present study, we explored the effects of metformin or/and cisplatin on cell viability and apoptosis in human CRC SW480 and SW620 cells, and its underlying mechanisms.
2.4. Colony formation assay The cells were plated in 6-well plates and treated for 24 h, then dealt with metformin or/and cisplatin for 24 h. The cells were cultured for an additional 7 days after the medium was substituted with fresh medium, and then fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and visualized by a camera. 2.5. Apoptosis assay Apoptosis was analyzed with the Annexin V-FITC/PI apoptosis detection kit described in the manufacturer’s instruction. Briefly, the SW480 and SW620 cells (4 × 105 cells/well) were plated and dealt with 10 mM metformin or/and 8 μM cisplatin for 24 h. The cells were harvested, and then resuspended in 500 μL binding buffer composing 5 μL Annexin V-FITC for 20 min, and stained with 5 μL PI for 5 min. Cells were then detected with a BD Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and subsequently analyzed by FlowJo software (Tree Star Inc., Ashland, OR, USA). 2.6. MMP assay The MMP detection was performed using the JC-1 kit as described in the manufacturer’s instruction. Briefly, the SW480 and SW620 cells were plated in 12-well plates and incubated with 10 mM metformin or/ and 8 μM cisplatin for 24 h, and then cells were incubated with JC-1 (10 μM) for 30 min. The JC-1 signal was visualized on a 561 nm (red) and 488 nm (green) wavelength laser by confocal laser scanning microscopy (Nikon, Tokyo, Japan), respectively. 2.7. Intracellular ROS detection
2. Materials and methods
The intracellular ROS level was probed with a fluorescent DCFH-DA kit as recommended by the manufacturer’s instruction. Briefly, the SW480 and SW620 cells were seeded and incubated with 10 mM metformin or/and 8 μM cisplatin for 24 h. The cells were harvested, rinsed with PBS, treated with DCFH-DA (10 μM) for 20 min, and analyzed by flow cytometry. To avoid the generation of ROS, NAC (5 mM), as a ROS scavenger, was added to the cells for 1 h before incubation with metformin or/and cisplatin. The results were evaluated by Cell Quest analysis software.
2.1. Reagents and antibodies Metformin, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), N-acetyl-L-cysteine (NAC) were from Sigma (St. Louis, MO, USA). Cisplatin was from Qilu Pharmaceutical Co., Ltd. (Jinan, China). Annexin V-FITC/PI apoptosis detection kit was acquired from Nanjing KeyGen Biotech (Nanjing, China). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) kit and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) kit were acquired from Beyotime (Wuhan, China). Antibodies against Mcl-1, Bcl-2, Bax, PARP, caspase-3, and β-actin were from Abcam (Cambridge, MA, USA). Antibodies against caspase-9, PI3K, p-PI3K, Akt, p-Akt, and Bak were from Cell Signaling Technology (Danvers, MA, USA), respectively.
2.8. Western blot analysis Cells (5 × 105 cells/well) were plated in 6-well culture plates. After incubation with different treatments, the cells were harvested and lysed in RIPA buffer. Protein concentration was examined with BCA kit (Beyotime, Wuhan, China). Subsequently, total protein (about 40 µg) was subjected to SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% nonfat milk, PVDF membranes were incubated with the primary antibodies overnight at 4˚C, then further incubated with the corresponding secondary antibodies for 1 h at room temperature, finally visualized with ECL chemiluminescent reagent (Millipore, MA, USA), and imaged with a gel imaging equipment (BioRad, Berkeley, CA, USA). β-actin was used as the loading control. The relative protein level was used to show the expression of the studied protein, which was the ratio of the gray value of the studied protein to the gray value of β-actin.
2.2. Cell lines and cell culture Human CRC SW480 and SW620 cells were acquired from the American Type Culture Collection (Manassas, VA, USA), and maintained in Dulbecco’s modified Eagle medium (DMEM) (Grand Island, NY, USA) supplemented with 10% fetal bovine serum and antibiotics (penicillin/streptomycin) at 37 °C, 5% CO2. 2.3. Cell viability assay
2.9. Statistical analysis
Cell viability was measured by MTT assay. The SW480 and SW620 cells (7 × 103 cells/well) were plated in triplicate and dealt with the indicated concentration of metformin or/and cisplatin. After treatment, 15 µL MTT (5 g/L in PBS) was added to each well for 4 h. Subsequently, another 150 µL dimethyl sulfoxide (DMSO) was added, and the optical density (OD) was examined at 490 nm. Cell viability was displayed as a percentage of control.
Numerical data are represented as the mean ± SD. All statistical analyses were performed by the two-tailed Student’s t-test and one-way analysis of variance (ANOVA) followed by LSD test with SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Statistical difference was defined as P < 0.05. 2
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Fig. 1. Metformin elevated the inhibitory effects of CRC cells to cisplatin on cell viability. (A and B) SW480 and SW620 cells were incubated with various concentrations of metformin (1.25, 2.5, 5, 10, and 20 mM), different concentrations of cisplatin (1, 2, 4, 8, and 16 μM) for 24, 48, 72 h, respectively. Cell viability was analyzed using the MTT assay. (C) The cells were incubated with metformin (1.25, 2.5, 5, 10, and 20 mM) with or without cisplatin (4 μM) for 24 h by performing the MTT assay. **P < 0.01 vs metformin alone. (D and E) SW480 and SW620 cells were incubated with metformin (10 mM), cisplatin (8 μM), and metformin (10 mM) plus cisplatin (8 μM), and cell colony formation was analyzed by crystal violet staining. a, control; b, metformin; c, cisplatin; d, combination of metformin and cisplatin. **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. MET, metformin; DDP, cisplatin.
3. Results
3.2. Metformin enhanced cisplatin-mediated apoptosis in CRC cells
3.1. Metformin elevated the inhibitory effect of CRC cells to cisplatin on cell viability
To define the effects of metformin or/and cisplatin on apoptosis, SW480 and SW620 cells were dealt with metformin (10 mM), cisplatin (8 μM), and combination of metformin and cisplatin for 24 h. The apoptosis assay displayed that metformin and cisplatin increased apoptosis of SW480 and SW620 cells, and combination of metformin and cisplatin induced more apoptosis than treatment with metformin or cisplatin alone (Fig. 2A and B). Moreover, the activation of caspase-9, caspase-3 and PARP was assessed to further characterize the apoptotic process. As shown in Fig. 2C–E, metformin combined with cisplatin markedly mediated the cleavage of caspase-9, caspase-3 and PARP, while metformin or cisplatin alone mediated a slight cleavage of caspase-9, caspase-3 and PARP.
To investigate the effect of metformin or/and cisplatin on cell viability, SW480 and SW620 cells were incubated with various concentrations of metformin (1.25, 2.5, 5, 10, and 20 mM), different concentrations of cisplatin (1, 2, 4, 8, and 16 μM), and cisplatin (4 mM) combined with metformin (1.25, 2.5, 5, 10, and 20 mM) by performing the MTT assay. The results demonstrated that metformin and cisplatin attenuated cell viability of SW480 and SW620 cells in a concentrationand time-dependent manner within a certain range of concentration, respectively (Fig. 1A and B). Metformin combined with cisplatin could markedly increase inhibition of cell viability in SW480 and SW620 cells, compared to treatment with metformin alone (Fig. 1C). To further explore the cytotoxicity of metformin and cisplatin in CRC cells, colony formation assay was performed. As shown in Fig. 1D and E, combination of metformin and cisplatin could obviously inhibit the colony formation in SW480 and SW620 cells, compared to treatment with metformin or cisplatin alone. Therefore, metformin combined with cisplatin efficiently suppressed cell viability of CRC cells.
3.3. Metformin and cisplatin decreased the MMP of CRC cells by modulating Bcl-2 family members’ expression Mitochondrial dysfunction has been demonstrated to participate in the induction of apoptosis and even been supposed to be central to the apoptotic pathway (Chen et al., 2019). To confirm whether metformin and cisplatin affect the function of mitochondria in SW480 and SW620 3
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Fig. 2. Metformin enhanced cisplatin-mediated apoptosis in CRC cells. SW480 and SW620 cells were incubated with metformin (10 mM), cisplatin (8 μM), and metformin (10 mM) plus cisplatin (8 μM) for 24 h. (A and B) Apoptosis was detected by flow cytometry, and quantification of the percentage of apoptotic cells. ** P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. (C-E) The protein levels of caspase-9, caspase-3 and PARP were measured by western blot analysis, and quantification. **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. Ctrl, control; MET, metformin; DDP, cisplatin.
cells, JC-1 staining assay was performed. This assay is based on the principle that red fluorescence will be present in areas with high MMP, while green fluorescence will be prevalent in areas with low MMP. We found that combination of metformin and cisplatin triggered a greater decrease of MMP than the treatment with metformin or cisplatin alone (Fig. 3A and B). Moreover, the mitochondrial pathway is a major mediator of apoptosis, which is modulated by the Bcl-2 family proteins. To deeply discuss the mechanism of the apoptosis mediated by metformin and cisplatin, the level of anti-apoptotic proteins Mcl-1, Bcl-2, and pro-apoptotic proteins Bak, Bax were measured. It was shown that the expression of Bcl-2 and Mcl-1 was significantly downregulated in CRC cells treated with combination of metformin and cisplatin compared to metformin or cisplatin alone, whereas the expression of Bak and Bax was upregulated (Fig. 3C–E). We found that metformin combined with cisplatin markedly mediated the cleavage of caspase-9, caspase-3 and PARP, therefore, we hypothesized that the mitochondrial pathway might be involved in metformin-enhanced cisplatin-induced apoptosis in CRC cells.
proliferation and apoptosis) by changing the balance between the cell survival and death progression (Tang et al., 2018a). We evaluated intracellular ROS levels in SW480 and SW620 cells treated with metformin or/and cisplatin by DCFH-DA staining coupled with flow cytometry. As a result, SW480 and SW620 cells showed considerably greater increases of ROS levels after treatment with combination of metformin and cisplatin, while metformin or cisplatin resulted in only a slight promotion of ROS levels (Fig. 4A and B). Meanwhile, pre-treatment of SW480 and SW620 cells with NAC effectively decreased the levels of ROS induced by combination of metformin and cisplatin (Fig. 4C and D). Next, to further explore the effects of ROS on proliferation in SW480 and SW620 cells, the results indicated that NAC could attenuate the inhibitory effect of metformin combined with cisplatin on proliferation (Fig. 4E), suggested that the generation of ROS might play a vital role in anti-proliferative effect of metformin and cisplatin. 3.5. ROS was involved in metformin combined with cisplatin-induced apoptosis in CRC cells
3.4. Metformin and cisplatin induced the production of intracellular ROS in CRC cells
Next, we tested whether ROS inhibition affected apoptosis induced by metformin and cisplatin in CRC cells. We found that the increased apoptosis of SW480 and SW620 cells induced by combination of
The intracellular ROS affect diverse cellular functions (such as 4
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Fig. 3. Metformin and cisplatin decreased the MMP of CRC cells by modulating Bcl-2 family members’ expression. SW480 and SW620 cells were treated with metformin (10 mM), cisplatin (8 μM), and metformin (10 mM) plus cisplatin (8 μM) for 24 h. (A and B) The MMP was determined by JC-1 staining, and quantification via the ratio of red-to-green fluorescence intensity. **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. Scale bar is 50 μm. (C-E) The protein levels of Mcl-1, Bak, Bcl-2, and Bax were detected by western blot analysis, and quantification. *P < 0.05, **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. Ctrl, control; MET, metformin; DDP, cisplatin.
metformin and cisplatin could be blocked by NAC (Fig. 5A and B). Moreover, NAC could abrogate the cleavage of caspase-9, caspase-3 and PARP in SW480 and SW620 cells mediated by metformin combined with cisplatin (Fig. 5C-E). Meanwhile, the attenuation of Mcl-1 and Bcl2 in SW480 and SW620 cells incubated with metformin combined with cisplatin, and the upregulation of Bak and Bax, were also abolished by NAC (Fig. 5F-I), suggested that metformin enhanced cisplatin-induced apoptosis dependent on the generation of ROS.
expression (Fig. 6A-C). To further unveil whether the contribution of ROS is mediated through inactivation of the PI3K/Akt pathway, the effects of metformin combined with cisplatin on p-PI3K and p-Akt were detected when NAC blocked the production of ROS. As shown in Fig. 6D-F, NAC could recover the downregulation of p-PI3K and p-Akt in CRC cells treated with combination of metformin and cisplatin. These results demonstrated that the inactivation of PI3K/Akt pathway relied on the generation of ROS incubated with metformin and cisplatin in CRC cells.
3.6. ROS was critical for the inactivation of PI3K/Akt pathway in CRC cells incubated with metformin and cisplatin
4. Discussion
To discuss the role of PI3K/Akt pathway in the apoptosis of SW480 and SW620 cells incubated with metformin and cisplatin, we measured the level of PI3K, p-PI3K, Akt, and p-Akt. The results displayed that pPI3K and p-Akt expression was evidently decreased in CRC cells dealt with combination of metformin and cisplatin compared to metformin or cisplatin alone, whereas did not significantly change PI3K and Akt
The treatment of cancer (including CRC) is a major concern of the medical profession, the current treatment options mainly include surgery, radiotherapy, and chemotherapy. However, many chemotherapeutic drugs are low-sensitive, highly toxic, and susceptible to develop drug resistance, which leads to the recurrence and metastasis of the tumor (Kartal-Yandim et al., 2016). Thus, combination therapy is an 5
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Fig. 4. Metformin and cisplatin induced the production of intracellular ROS in CRC cells. SW480 and SW620 cells were incubated with metformin (10 mM), cisplatin (8 μM), and metformin (10 mM) plus cisplatin (8 μM) for 24 h. (A and B) The intracellular ROS levels were measured by DCFH-DA staining with flow cytometry, and Quantification via Cell Quest analysis software. *P < 0.05, **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs metformin or cisplatin alone. (C and D) SW480 and SW620 cells were pre-treated with NAC (5 mM) for 1 h, and the intracellular ROS levels were measured by flow cytometry using DCFH-DA staining. **P < 0.01 vs combination of metformin and cisplatin. (E) SW480 and SW620 cells were pre-treated with NAC (5 mM) for 1 h, cell viability was measured using the MTT assay. ** P < 0.01 vs combination of metformin and cisplatin. Ctrl, control; MET, metformin; DDP, cisplatin; NAC, N-acetyl-L-cysteine.
important strategy to improve therapeutic outcomes and reduce the toxic side effects of anticancer drugs. Recent studies have established that metformin effectively inhibits cell proliferation as a promising therapeutic agent for cancer, and increases the apoptotic sensitivity to other chemotherapeutic drugs (Tang et al., 2018b; Zhao et al., 2018; Lee et al., 2019b; Zhao et al., 2019). In this study, we found that metformin combined with cisplatin could evidently increase inhibition of cell viability and apoptosis of SW480 and SW620 cells, compared to treatment with metformin or cisplatin alone. To the best of our knowledge, apoptosis is an activated cellular process, which mainly uses pathological or physiological factors to remove redundant and damaged cells (Xu et al., 2015). The disorder of apoptosis is involved in the occurrence and development of many diseases, including cancer (Pistritto et al., 2016). Apoptosis can be initiated through two classic and correlated pathways in mammalian cells, including the mitochondria- and the death receptor-mediated apoptotic pathway (Sun et al., 2014). Furthermore, induction of the
mitochondrial-mediated apoptotic pathway can trigger the permeability of the mitochondrial membrane to raise. According to the present study, combination of metformin and cisplatin decreased signals in red fluorescence while increased signals in green fluorescence, thus led to loss of MMP. The Bcl-2 family, a well-known family of apoptosis-regulating proteins, exhibits a major role in the mitochondria pathway, and tightly regulates the released cytochrome C from mitochondria, which results in caspase activation and eventually apoptosis. Besides, the release of cytochrome C mediates caspase-9 activation, which participates in the activation of caspase-3, thus, subsequently activates downstream processes, such as cleavage of PARP (Yang et al., 2017; Zhao et al., 2017; Liu et al., 2018). As a result, metformin combined with cisplatin obviously mediated the cleavage of caspase-9, caspase-3 and PARP, while metformin or cisplatin alone mediated a slight proteolytic cleavage of caspase-9, caspase-3 and PARP. Moreover, the balance between proapoptotic and anti-apoptotic molecules influences the occurrence of 6
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Fig. 5. ROS was involved in metformin combined with cisplatin-induced apoptosis in CRC cells. SW480 and SW620 cells were pre-treated with NAC (5 mM) for 1 h, then treated with metformin (10 mM) plus cisplatin (8 μM) for 24 h. (A and B) Apoptosis was analyzed by flow cytometry, and quantification of the percentage of apoptotic cells. **P < 0.01 vs combination of metformin and cisplatin. (C-E) The protein levels of caspase-9, caspase-3 and PARP were measured by western blot analysis, and quantification. **P < 0.01 vs combination of metformin and cisplatin. (F-H) The protein levels of Mcl-1, Bak, Bcl-2, and Bax were detected by western blot analysis, and quantification. **P < 0.01 vs combination of metformin and cisplatin. Ctrl, control; MET, metformin; DDP, cisplatin; NAC, N-acetyl-L-cysteine.
apoptosis, and is related to the success rate of chemotherapy in cancer patients (Arababadi and Asadikaram, 2016). The results showed that Bcl-2 and Mcl-1 were significantly downregulated, while Bak and Bax were evidently upregulated in the cells dealt with metformin combined with cisplatin compared with metformin or cisplatin alone. These data
confirmed that the mitochondrial pathway might be involved in metformin-enhanced cisplatin-induced apoptosis in CRC cells. Accumulating evidences indicate that ROS, typical products of oxidative stress, are mainly generated by mitochondria, function as mediators of various intracellular signaling cascades, and recent 7
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Fig. 6. ROS was critical for the inactivation of PI3K/Akt signaling pathway in CRC cells incubated with metformin and cisplatin. (A-C) SW480 and SW620 cells were incubated with metformin (10 mM), cisplatin (8 μM, and metformin (10 mM) plus cisplatin (8 μM) for 24 h. The protein levels of PI3K, p-PI3K, Akt and p-Akt were measured by western blot assay, and quantification. *P < 0.05, **P < 0.01 vs control; ##P < 0.01 vs metformin or cisplatin alone. (D-F) SW480 and SW620 cells were pre-treated with NAC (5 mM) for 1 h, then incubated with metformin (10 mM) plus cisplatin (8 μM) for 24 h. The protein levels of PI3K, p-PI3K, Akt and p-Akt were detected by western blot analysis, and quantification. **P < 0.01 vs combination of metformin and cisplatin. Ctrl, control; MET, metformin; DDP, cisplatin; NAC, N-acetyl-L-cysteine.
evidence has revealed their participation in the regulation of apoptosis by causing mitochondrial dysfunction and DNA damage (Chiu et al., 2012; Jiang et al., 2019; Rashmi et al., 2019). Indeed, the mitochondrial dysfunction induced by ROS production leads to loss of MMP, thus induced caspase-9 activation, followed by activation of effector caspases, ultimately resulted in apoptosis (Zhong et al., 2017). In this study, we found that SW480 and SW620 cells showed considerably greater increases of ROS levels after treatment with combination of metformin and cisplatin compared to metformin or cisplatin alone, which was abolished by the pretreatment of cells with NAC. In addition, NAC could attenuate anti-proliferative effect and apoptosis induced by metformin combined with cisplatin. Meanwhile, the activation of caspase-9, caspase-3 and PARP, down-regulation of Mcl-1 and Bcl-2, and up-regulation of Bak and Bax induced by combination of metformin and cisplatin were also blocked by NAC. These data implied that ROS were of fundamental importance to apoptosis of CRC cells induced by metformin and cisplatin. Studies have shown that metformin enhances the effect of cisplatin in various cancers. For example, the development of cisplatin and metformin nano-cubosomes induces CRC cell apoptosis through inhibition of several metabolic pathways, namely, AMPK/mTOR and Akt/ Mtor (Saber et al., 2018). Meanwhile, metformin increases the chemotherapeutic sensitivity of liver cancer cells to cisplatin through the AMPK pathway (Dong et al., 2017), metformin enhances the antitumor activity of cisplatin in gallbladder cancer through PI3K/Akt pathway (Bi et al., 2018), metformin combined with cisplatin induces apoptosis
of human ovarian cancer cells by inactivating ERK1/2 (Dang et al., 2017). But the mechanism by which metformin enhances the anticancer effect of cisplatin in CRC cells is not clear. It is well known that the PI3K/Akt pathway is closely related to apoptosis (Lv et al., 2018). Several explores have elucidated that inactivation of the PI3K/Akt pathway can reverse the sensitivity of diverse cancer cells to chemotherapeutic drugs (You et al., 2018), Akt can phosphorylate members of the Bcl-2 family, thereby inhibiting apoptosis. In addition, Akt can also inhibit the activity of the proteolytic enzyme caspase-9 to prevent the initiation of the apoptotic cascade and thus inhibit apoptosis, indicating that targeting PI3K/Akt signaling can serve as a potential therapeutic strategy for cancer treatment. We observed that the level of p-PI3K and p-Akt was evidently decreased in CRC cells dealt with combination of metformin and cisplatin compared to metformin or cisplatin alone, whereas did not significantly change PI3K and Akt expression. Furthermore, ROS generation functions serves as a second messenger that mediates activation of the PI3K/Akt pathway in various types of cells (Ahn et al., 2017; Han et al., 2017; Deng et al., 2019). Thus, we discussed the connection between ROS and the PI3K/Akt pathway. In our study, NAC could recover the downregulation of pPI3K and p-Akt in CRC cells dealt with combination of metformin and cisplatin, which subsequently activated the PI3K/Akt signaling pathway. In summary, this study demonstrated that metformin enhanced the sensitivity of CRC cells to cisplatin through ROS-mediated PI3K/Akt signaling pathway. This information may be useful for developing a 8
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new therapy for the treatment of CRC in future, and the specific underlying molecular mechanisms remains to be subsequent research.
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CRediT authorship contribution statement Pei Zhang: Formal analysis, Funding acquisition, Writing - original draft, Data curation. Surong Zhao: Methodology, Supervision, Writing - review & editing. Xingyue Lu: Formal analysis, Validation. Zongfen Shi: Methodology, Data curation, Validation. Hao Liu: Project administration, Resources, Supervision. Bing Zhu: Data curation, Resources, Validation, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This study was supported by the National Natural Science Foundation of China (81603155), the Natural Science Foundation of Anhui Province (1708085QH212), and the Key Project of Natural Science Research for College and University of Anhui Province (KJ2018A1028, KJ2016A486). References Ahn, K.I., Choi, E.O., Kwon, D.H., HwangBo, H., Kim, M.Y., Kim, H.J., Ji, S.Y., Hong, S.H., Jeong, J.W., Park, C., Kim, N.D., Kim, W.J., Choi, Y.H., 2017. Induction of apoptosis by ethanol extract of Citrus unshiu Markovich peel in human bladder cancer T24 cells through ROS-mediated inactivation of the PI3K/Akt pathway. Biosci. Trends 11, 565–573. Arababadi, M.K., Asadikaram, G., 2016. Opium induces apoptosis in Jurkat cells via promotion of pro-apoptotic and inhibition of anti-apoptotic molecules. Iran J. Basic Med. Sci. 19, 215–220. Bi, T., Zhu, A., Yang, X., Qiao, H., Tang, J., Liu, Y., Lv, R., 2018. Metformin synergistically enhances antitumor activity of cisplatin in gallbladder cancer via the PI3K/AKT/ERK pathway. Cytotechnology 70, 439–448. Chen, B., Cao, X., Lu, H., Wen, P., Qi, X., Chen, S., Wu, L., Li, C., Xu, A., Zhao, G., 2018. N(3-oxo-acyl) homoserine lactone induced germ cell apoptosis and suppressed the over-activated RAS/MAPK tumorigenesis via mitochondrial-dependent ROS in C. elegans. Apoptosis 23, 626–640. Chen, W., Liu, H., Wang, T., Bao, G., Wang, N., Li, R.C., 2019. Downregulation of AIF-2 inhibits proliferation, migration, and invasion of human glioma cells via mitochondrial dysfunction. J. Mol. Neurosci. 68, 304–310. Cheng, C.H., Cheng, Y.P., Chang, I.L., Chen, H.Y., Wu, C.C., Hsieh, C.P., 2016. Dodecyl gallate induces apoptosis by upregulating the caspase-dependent apoptotic pathway and inhibiting the expression of anti-apoptotic Bcl-2 family proteins in human osteosarcoma cells. Mol. Med. Rep. 13, 1495–1500. Chiu, W.H., Luo, S.J., Chen, C.L., Cheng, J.H., Hsieh, C.Y., Wang, C.Y., Huang, W.C., Su, W.C., Lin, C.F., 2012. Vinca alkaloids cause aberrant ROS-mediated JNK activation, Mcl-1 downregulation, DNA damage, mitochondrial dysfunction, and apoptosis in lung adenocarcinoma cells. Biochem. Pharmacol. 83, 1159–1171. Dang, J.H., Jin, Z.J., Liu, X.J., Hu, D., Wang, J., Luo, Y., Li, L.L., 2017. Metformin in combination with cisplatin inhibits cell viability and induces apoptosis of human ovarian cancer cells by inactivating ERK 1/2. Oncol. Lett. 14, 7557–7564. Deng, S., Dai, G., Chen, S., Nie, Z., Zhou, J., Fang, H., Peng, H., 2019. Dexamethasone induces osteoblast apoptosis through ROS-PI3K/AKT/GSK3beta signaling pathway. Biomed. Pharmacother. 110, 602–608. Dong, H., Huang, J., Zheng, K., Tan, D., Chang, Q., Gong, G., Zhang, Q., Tang, H., Sun, J., Zhang, S., 2017. Metformin enhances the chemosensitivity of hepatocarcinoma cells to cisplatin through AMPK pathway. Oncol. Lett. 14, 7807–7812. Guo, C.L., Wang, L.J., Zhao, Y., Liu, H., Li, X.Q., Jiang, B., Luo, J., Guo, S.J., Wu, N., Shi, D.Y., 2018. A novel bromophenol derivative BOS-102 induces cell cycle arrest and apoptosis in human A549 lung cancer cells via ROS-mediated PI3K/Akt and the MAPK signaling pathway. Mar. Drugs 16. Han, M.H., Lee, D.S., Jeong, J.W., Hong, S.H., Choi, I.W., Cha, H.J., Kim, S., Kim, H.S., Park, C., Kim, G.Y., Moon, S.K., Kim, W.J., Hyun Choi, Y., 2017. Fucoidan induces ROS-dependent apoptosis in 5637 human bladder cancer cells by downregulating telomerase activity via inactivation of the PI3K/Akt signaling pathway. Drug Dev. Res. 78, 37–48. Hou, T., Zhou, L., Wang, L., Kazobinka, G., Chen, Y., Zhang, X., Chen, Z., 2018. Leupaxin promotes bladder cancer proliferation, metastasis, and angiogenesis through the PI3K/AKT pathway. Cell. Physiol. Biochem. 47, 2250–2260. Jeong, J.W., Lee, W.S., Go, S.I., Nagappan, A., Baek, J.Y., Lee, J.D., Lee, S.J., Park, C., Kim, G.Y., Kim, H.J., Kim, G.S., Kwon, T.K., Ryu, C.H., Shin, S.C., Choi, Y.H., 2015.
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