Dynasore suppresses proliferation and induces apoptosis of the non- small-cell lung cancer cell line A549
Feifei Shen, Junda Gai, Jilin Xing, Jingqian Guan, Lin Fu, Qingchang Li*
Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, China

a r t i c l e i n f o

Article history:
Received 6 November 2017 Received in revised form 15 November 2017
Accepted 17 November 2017 Available online xxx

Keywords:
Lung cancer Dynasore Cisplatin Dynamin
Dynamin-related protein 1 Autophagy
Apoptosis Proliferation
a b s t r a c t

Lung cancer is the leading cause of cancer death worldwide, and most of all cases are non-small-cell lung cancer. Lung cancer is associated with dysregulation of mitochondrial fusion and fission, and inhibition of the fission regulator Dynamin-related protein 1 (Drp1) reduces proliferation and increases apoptosis of lung cancer cells. Dynasore is a small molecule non-selective inhibitor of the GTPase activity of dynamin 1, dynamin 2, and Drp1 in vivo and in vitro. Here, we investigated the effects of dynasore on the pro- liferation and apoptosis of A549 lung cancer cells, alone and in combination with the chemotherapeutic drug cisplatin. We found that cisplatin increased mitochondrial fission and dynamin 2 expression, whereas dynasore had the opposite effects. However, both cisplatin and dynasore independently induced mitochondrial oxidative stress, leading to mitochondrial dysfunction, reduced cell proliferation, and enhanced apoptosis. Importantly, dynasore significantly augmented the anti-cancer effects of cisplatin. To the best of our knowledge, this is the first report that dynasore inhibits proliferation and induces apoptosis of lung cancer cells, and enhances the inhibitory effects of cisplatin.
© 2017 Elsevier Inc. All rights reserved.

⦁ Introduction

Non-small-cell lung cancer accounts for 85% of all cases of lung cancer, the principal cause of cancer-related deaths worldwide [1]. Cisplatin is widely used as a first-line chemotherapy in the treat- ment of lung cancer [2], and acts by inhibiting cell proliferation, invasion, and metastasis [3,4]. However, cisplatin resistance is common, reducing its efficacy and limiting its clinical applications [5]. Therefore, identifying new therapies for lung cancer is likely to have a major impact on clinical practice.
Mitochondria are dynamic organelles that undergo frequent fission and fusion and play crucial roles in the regulation of cell metabolism, growth, differentiation, proliferation, and apoptosis [6,7]. Recent studies have revealed that mitochondrial dynamics also regulate the cell cycle [8], and play a role in the genomic instability, migration, and apoptosis of cancer cells [9]. Dynamin- related protein 1 (Drp1), a member of the dynamin family of GTPases, is required for mitochondrial fission and is upregulated in multiple cancers, including lung [6], breast [10], and ovarian

* Corresponding author. China Medical University, No. 77 Pu River Road, Shenbei New Area, Shenyang, 110000, China.
E-mail address: [email protected] (Q. Li).
cancers [11]. In addition, the proliferation, invasion, and metastasis of some cancers can be reduced by inhibition of Drp1-mediated mitochondrial fission, demonstrating that Drp1 could be an important target for treating cancer [6,9,10].
Autophagy maintains cell homeostasis by degrading intracel- lular macromolecules and damaged organelles [12], and may be involved in cisplatin-induced apoptosis and its resistance in lung cancer [4,5,13]. Dynamins are large multi-domain GTPases involved in the regulation of clathrin-mediated endocytosis [14], autophagy (both directly and indirectly) [15,16], and cell migration [17]. Deletion of dynamin has been shown to influence autophagy dysfunction [15]. Dynasore is a small molecule non-selective in- hibitor of the GTPase activity of dynamin 1, dynamin 2, and Drp1 in vivo and in vitro [18,19]. In addition, More recent studies have shown that dynasore may have protective effects in heart ischemia/ reperfusion injury [20] and Alzheimer’s disease [21]. Interestingly, Yamada et al. reported that dynasore inhibits lung cancer cell in- vasion by destabilizing actin filaments [18]. However, there have been few studies on the effects of dynasore on lung cancer apoptosis and proliferation. The goal of this study was to determine whether dynasore, alone or in combination with cisplatin, affected the proliferation and apoptosis of the human lung cancer cell line A549.

https://doi.org/10.1016/j.bbrc.2017.11.109

0006-291X/© 2017 Elsevier Inc. All rights reserved.

¼
Fig. 1. Dynasore inhibits Drp1 and dynamin 2 expression in A549 cells. (A) Western blot analysis of Drp1 and dynamin 2 expression in A549 cells. (B and C) Quantitative analysis of Drp1 (B) and dynamin 2 (C) levels. (D and F) Immunofluorescence staining of Drp1 (D, green) and dynamin 2 (F, red) in A549 cells. Scale bars, 200 mm (Drp1) and 50 mm (dynamin 2). (E and G) Quantitative analysis of the mean fluorescence intensity of Drp1 (E) and dynamin 2 (G). Data are presented as the means ± SD, n 6. aP < 0.05 vs. control, bP < 0.05 vs. cisplatin, cP < 0.05 vs. dynasore. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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⦁ Methods and materials

⦁ Cells and reagents

Human non-small-cell lung cancer A549 cells were purchased from Shanghai Cell Bank (Shanghai, China). The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin (Sigma, St. Louis, MO, USA), and 100 mg/mL
streptomycin (Sigma) in a humidified 5% CO2 atmosphere at 37 ◦C.
Cisplatin was purchased from Qilu Pharmaceutical Co., Ltd. (Shan- dong, China) and dynasore was purchased from Selleck Chemicals (Houston, TX, USA). Experiments were performed on four groups of cells treated with 3% dimethylsulfoxide (DMSO, control group), cisplatin 30 mM, dynasore 80 mM [18], or dynasore 80 mM þ cisplatin 30 mM.

⦁ Detection of mitochondrial function

The Mitochondrial membrane potential (MMP) was measured using the membrane-permeant JC-1 dye according to the manu- facturer's instructions (Beyotime, Shanghai, China). Levels of adenosine triphosphate (ATP), malondialdehyde (MDA), and reduced glutathione (GSH) were measured using specific assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Levels of reactive oxygen species (ROS) were measured with a kit from Baomanbio (Shanghai, China) according to the manufacturer's recommendations. Levels of ATP, MDA, GSH, and ROS were normalized to protein concentrations. Each experiment was per- formed six times.

⦁ MTT assay for cell proliferation

A549 cells were seeded in 96-well plates at approximately
×
3 103 cells/well and incubated at 37 ◦C in a 5% CO2 atmosphere for 24 h. The cells were then treated with DMSO, dynasore, and/or cisplatin and incubated for an additional 48 h. An aliquot of 10 mL of
×
¼
5 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) was added to each well and the cells were incubated for 4 h. DMSO (100%, 100 mL) was then added to each well and the plates were agitated for 10 min. Absorbance at 490 nm was read and cell proliferation was calculated as: (%) (A490 [drug intervention group]/A490 [control group]) 100. Each experiment was performed six times [3].

⦁ Colony-forming assay

A549 cells were pretreated with DMSO, dynasore, and/or cisplatin for 48 h, transferred to 6-cm cell culture plates (500 cells per dish) and incubated for 2 weeks. The cells were then stained with Giemsa (0.1%, 1 mL/well) and viewed by microscopy. The plates were photographed and the colonies were enumerated using the counting function of Adobe Photoshop CS3. The experiments were performed six times.

⦁ Western blot analysis

Western blotting was performed as previously described [22]. In brief, mitochondrial, cytoplasmic, and nuclear fractions of A549 cells were isolated using a mitochondria isolation kit and nuclear- cytosol extraction kit according to the manufacturer's instructions (Applygen Technologies, Beijing, China). Proteins were separated by
SDS-PAGE and transferred to PVDF membranes. After blocking, the membranes were incubated overnight at 4 ◦C with primary anti- bodies against proliferating cell nuclear antigen (PCNA, 2586),

cytochrome c (cytC; 11940T), apoptosis inducing factor (AIF, 5318T), voltage-dependent anion channel (VDAC, 4661T), and b-actin (3700T) (all 1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), DRP1 (ab56788), dynamin 2 (ab3457), active caspase-3 (ab32042), and lamin A (ab26300) (all 1:1000 dilution; Abcam, Cambridge, UK). The membrane was then washed and incubated at room temperature for 2 h with the appropriate secondary antibody. The protein bands were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Protein expression levels were calculated by comparison to the corresponding internal con- trols: b-actin for total or cytoplasmic proteins, VDAC for mito- chondrial proteins, and lamin A for nuclear proteins. The experiments were performed six times.

⦁ Immunofluorescence labeling

Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, incubated with 0.1% Triton X-100 for 10 min, and then blocked for 2 h at room temperature with 5% goat serum (005-000- 121, Jackson ImmunoResearch Laboratories, Inc West Grove, PA, USA). The cells were then incubated overnight at 4 ◦C with primary
antibodies against the following proteins: DRP1 (ab56788), active caspase-3 antibody E83-77 (ab32042), and dynamin 2 (ab3457) (all 1:200 dilution; Abcam), and PCNA (PC10, 2586) (1:500 dilution; Cell Signaling Technology). The cells were then incubated at room temperature for 1 h in the dark with Alexa Fluor-conjugated goat anti-mouse IgG (A32723) and goat anti-rabbit IgG (R37117) sec- ondary antibodies (all 1:250; Thermo Fisher Scientific, Waltham, MA, USA). Finally, nuclei were counterstained with 40,6-diamidino-
¼ ×
2-phenylindole (DAPI). The cells were visualized under a fluores- cent microscope (Leica DMI4000B, Germany). Drp1, dynamin 2, and PCNA expression was quantified as the mean fluorescence intensity (integrated density/area) using Image J. Cells positive for cleaved caspase-3 were enumerated using the counting function in Adobe Photoshop CS3. The percentage of positive cells was calculated as (%) (100 [cleaved caspase-3-positive cells/total cells]). The experiments were performed six times [23].

⦁ Statistical analysis

All data are expressed as mean ± standard deviation (SD). Dif- ferences were analyzed using one-way analysis of variance (ANOVA) and the least significant difference t-test. SPSS version
19.0 (IBM, Armonk, NY, USA) was used for the analyses. P < 0.05 was considered statistically significant.

⦁ Results

⦁ Dynasore inhibits Drp1 and dynamin 2 expression in A549 cells

Human lung cancer A549 cells were treated with vehicle, dynasore, and/or cisplatin for 48 h and the expression of Drp1 and dynamin 2 was assessed by western blotting and immunofluores- cence microscopy. Western blotting showed that cisplatin increased, whereas dynasore decreased, the expression of both proteins compared with control cells (P < 0.05, Fig. 1AeC). Notably, incubation of cells with a combination of dynasore and cisplatin reduced, but did not completely reverse, the effects of cisplatin on Drp1 and dynamin 2 expression (P < 0.05, Fig. 1AeC).
Immunofluorescence staining of Drp1 and dynamin 2 supported the western blotting results (P < 0.05, Fig. 1DeG). These data sug- gest that mitochondrial fission and autophagy are enhanced by cisplatin but inhibited by dynasore, and dynasore partially reverses cisplatin-induced mitochondrial fission and autophagy.

4 F. Shen et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e9

Fig. 2. Dynasore inhibits A549 cell proliferation in vitro. (A) Western blot analysis of PCNA expression in A549 cells. (B) Quantitative analysis of PCNA levels normalized to b-actin.
(C) Immunofluorescence staining of PCNA (green) in A549 cells. Scale bar, 50 mm. (D) Quantitative analysis of mean fluorescence intensity of PCNA. (EeG) A549 cell viability assay
(G) and colony-forming assays (F and G). Data are presented as means ± SD, n ¼ 6. aP < 0.05 vs. control, bP < 0.05 vs. cisplatin, cP < 0.05 vs. dynasore. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

⦁ Dynasore inhibits A549 cell proliferation in vitro

Western blot analysis of the proliferation marker PCNA [6]
revealed that dynasore or cisplatin alone significantly reduced PCNA expression. Consistent with our previous results, PCNA expression was further suppressed by dynasore and cisplatin in

F. Shen et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e9 5

Fig. 3. Dynasore induces mitochondrial dysfunction in A549 cells in vitro. (A) MMP, (B) ATP, (C) ROS, (D) MDA, and (E) GSH were measured in A549 cells. Data are presented as means ± SD, n ¼ 6. aP < 0.05 vs. control, bP < 0.05 vs. cisplatin, cP < 0.05 vs. dynasore.

6 F. Shen et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e9

Fig. 4. Dynasore induces A549 cell apoptosis via the mitochondrial-associated pathway in vitro. (A, D, and G) Western blotting of cytC, AIF, and cleaved caspase-3 in A549 cells. (B, C, E, F, and H) Quantitative analysis of cytC, AIF, and cleaved caspase-3 levels normalized to VDAC, lamin A, or b-actin. Mito: mitochondria; Cyto: cytoplasm; Nu: nucleus. (I) Immunofluorescence staining of cleaved caspase-3 (red) in A549 cells. Scale bars, 50 mm. (J) Quantitative analysis of mean fluorescence intensity of cleaved caspase-3. Data are presented as means ± SD, n ¼ 6. aP < 0.05 vs. control, bP < 0.05 vs. cisplatin, cP < 0.05 vs. dynasore. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

F. Shen et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e9 7

combination (P < 0.05, Fig. 2A and B). Similar results were observed when PCNA expression was examined by immunofluorescence staining (P < 0.05, Fig. 2C and D).
Finally, we examined the viability and colony-forming ability of A549 cells and found the same pattern of effects of dynasore and cisplatin. Thus, while either agent alone significantly reduce both the viability and colony formation, the inhibitory effects were even greater when both agents were present simultaneously (P < 0.05, Fig. 2EeG).

⦁ Dynasore induces mitochondrial dysfunction in A549 cells

A normal MMP must be maintained for optimal mitochondrial oxidative phosphorylation and ATP production [24]. To determine whether cisplatin and/or dynasore induce oxidative stress in A549 cells, we measured MMP, ATP, and ROS levels as well as MDA and GSH, which are widely used markers of oxidative stress [25]. We found that, compared with the control conditions, treatment with dynasore or cisplatin alone significantly increased ROS and MDA levels (Fig. 3C and D) and decreased GSH, MMP, and ATP levels (P < 0.05; Fig. 3A, B, and E). Interestingly, these changes were significantly more marked when cells were incubated with both dynasore and cisplatin (P < 0.05, Fig. 3). Thus, while dynasore and cisplatin both increase oxidative stress leading to mitochondrial dysfunction, the effects are more prominent when both com- pounds are present.

⦁ Dynasore induces A549 cell apoptosis via the mitochondrial- associated pathway in vitro

We next examined the expression of key apoptotic proteins to determine whether dynasore-induced disruption of mitochondrial function in A549 cells led to apoptosis. The results of western blot analysis revealed that treatment with dynasore or cisplatin signif- icantly increased the expression of cytoplasmic cytC, nuclear AIF, and total cleaved caspase-3, but significantly decreased the expression of mitochondrial cytC and AIF (P < 0.05, Fig. 4AeF). Notably, these effects were further enhanced when dynasore and cisplatin were added in combination (P < 0.05, Fig. 4AeH). Mean- while, immunofluorescence staining of cleaved caspase-3 sup- ported the western blotting results (P < 0.05, Fig. 4I and J). Collectively, these results indicate that dynasore and cisplatin alone induce A549 cell apoptosis by activating the intrinsic mitochondrial apoptotic pathway, and that dynasore enhances the pro-apoptotic effect of cisplatin.

⦁ Discussion

Lung cancer is now the leading cause of cancer death worldwide [1]. Cancer is often the result of abnormalities in cell signaling pathways that lead to aberrant proliferation, invasion, and metas- tasis [26]. Chemotherapy is a fundamental treatment for lung cancer, with cisplatin being one of the most commonly used and effective drugs [2]. Cisplatin suppresses lung cancer proliferation, invasion, and metastasis [3] through multiple mechanisms, the most important of which is induction of DNA damage leading to activation of the mitochondrial apoptotic pathway [3,25]. However, the emergence of cisplatin-resistant cells reduces the efficacy and limits the utility of cisplatin as a lung cancer treatment [27]. There is thus an urgent need to discover new treatments for lung cancer. Current work suggests that mitochondria are highly dynamic organelles that undergo frequent fission, fusion, and movement [7]. Such dynamics play roles in mitochondrial function and morphology [22,28], cell division [29], and apoptosis [7]. Mitofusin proteins initiate mitochondrial fusion by tethering adjacent

mitochondria [30], while Drp1 induces mitochondrial fission by forming circumferential structures around mitochondria and then segmenting the organelle into discrete fragments [31]. Some studies have suggested that inhibition of Drp1-mediated mito- chondrial fission may be an effective therapy for cancers [6,9,32]. Lung adenocarcinoma cells have been shown to undergo decreased mitochondrial fusion and increased fission [6], and human lung cancer tissues express significantly higher levels of Drp1 than do normal lung tissues [33]. Notably, enhancement of fusion and in- hibition of fission in A549 cancer cells can reduce proliferation and increase apoptosis in vitro and suppress tumor growth in vivo [6]. Since dynasore inhibits the GTPase activity of Drp1 and dynamin 1/ 2 [34], it was not clear whether dynasore would have anti-cancer effects. Our results demonstrate that mitochondrial fragmenta- tion in A549 cells was enhanced by cisplatin and reduced by dynasore; furthermore, dynasore suppressed cisplatin-induced mitochondrial fission in these cells. These findings are consistent with the studies described above, and will form the foundation for further research on dynasore [18,35,36].
Abnormalities in mitochondrial fission lead to mitochondrial dysfunction and apoptosis [6,25]. An intact MMP is necessary for mitochondrial oxidative phosphorylation and ATP production [24,37]. Our results showed that cisplatin and, to a lesser extent, dynasore increased oxidative stress and mitochondrial dysfunction in A549 cells. Moreover, despite the smaller impact of dynasore, it significantly enhanced the anti-cancer effect of cisplatin. Further- more, the same enhancing effects were observed for the release of mitochondrial cytC and AIF and the expression of active caspase-3, suggesting that dynasore increased A549 cell apoptosis. It is possible that these effects result from dynasore-mediated mito- chondrial fission abnormalities that activate the mitochondrial apoptotic pathway [6]. However, Li et al. reported that Mdivi-1- mediated inhibition of mitochondrial fission can protect mito- chondrial function, suppress mitochondrial oxidative stress, and inhibit apoptosis after spinal cord injury [22]. These results suggest that mitochondrial fission may have different effects in different tissues. Further studies will be necessary to address this question. Nevertheless, our results showing that the combination of dyna- sore and cisplatin had better anti-cancer activity than either drug alone suggests that this combination may be a useful therapy for lung cancer.
Mitochondria grow continuously throughout the cell cycle, and tight control of the mitochondrial network is maintained at different stages of the cell cycle [9]. Recent findings have revealed that mitochondrial fusion and fission cycles participate in the regulation of cell cycle progression [8,38]. Drp1-mediated mito- chondrial fission is necessary for proper progression through the cell cycle following G1/S transition. Sustained mitochondrial fusion beyond the G1/S border induces replication stress, leading to an ATM-dependent G2/M delay and chromosomal instability during mitosis [8]. Replication stress results from continuous signaling of replication initiation by the hyperfused mitochondria [8,38]. Pro- longed mitochondrial fusion also causes mitochondrial bridges between daughter cells, resulting in defective cytokinesis, unequal distribution of mitochondria, and even missegregation of chro- mosomes causing aneuploidy, which inhibits cell division [39]. Our results show that dynasore inhibits PCNA expression, cell viability, and colony formation of A549 cells, and enhances cisplatin- mediated inhibition of the same processes. Thus, dynasore alone decreases A549 proliferation via inhibition of mitochondrial fission. It is possible that inhibition of Drp1-mediated fission leads to excessive mitochondrial fusion beyond the G1/S border, dysregu- lating proliferation [8,38,39].
Since dynasore inhibits the GTPase activity of not only Drp1
but also dynamin 1 and dynamin 2 [34]. In our present study, we

8 F. Shen et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e9

also found that autophagy was enhanced by cisplatin and reduced by dynasore in A549 cells; furthermore, dynasore suppressed cisplatin-induced autophagy in these cells. Another study also demonstrated that dynasore itself can reduce autophagy by inhibiting the GTPase activity of dynamin 2 in vitro [19]. On the contrary, one latest research reported that dynasore can inhibit the mechanistic target of rapamycin complex 1 (mTORC1) acti- vation by amino acids and not via inhibition of dynamin [16]. However, mTORC1 inhibition by dynasore does not promote autophagy as expected [16,40,41], but rather inhibits autophagy [16]. So Persaud Avinash et al. [16] thought it might be likely due (at least in part) to its inhibitory effect on dynamin. Altogether, dynasore may regulate autophagy via different pathways, even two completely opposite pathways, but its ultimate effect is in- hibition of autophagy.
Cisplatin is often effective in controlling cancer at early stages of treatment but the frequent emergence of drug resistance often leads to therapeutic failure. Some studies have suggested that enhanced autophagy may be involved in cisplatin resistance in lung cancer [5,13]. Other studies have shown that cisplatin in- duces autophagy and apoptosis in A549 lung cancer cells and that inhibition of autophagy promotes cisplatin-induced apoptosis [3,4]. Our results showed that cisplatin inhibits pro- liferation and induces apoptosis of A549 cells, which supports these studies [3,4]. Interestingly, we found that dynasore inhibits autophagy induced by cisplatin and enhances the anti-cancer effects of cisplatin in A549 cells, which support the study [16]. We speculate that the enhancing effect of dynasore results from the suppression of autophagy induced by cisplatin. In addition, when the two drugs were combined for the treatment of lung cancer, dynasore may have the potential to reduce cisplatin resistance. However, autophagy can also lead to cell death under other conditions [42] that may differ in distinct cancer cell types. Further studies will be necessary to address the role of auto- phagy in lung cancer cells.
In conclusion, to our knowledge, this is the first demonstration that dynasore increases lung cancer cell apoptosis, inhibits pro- liferation, and enhances the anti-cancer effect of cisplatin. How- ever, the specific mechanism of action of dynasore in A549 cells is far from clear, and analyses of mitochondrial morphology and autophagosomes by electron microscopy will be useful future endeavors. Since the treatment of non-small-cell and small-cell lung cancer differs, it will be of great interest to determine whether dynasore also has anti-cancer effects on small-cell lung cancer.

Conflicts of interest

The authors declare that they have no competing interests.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant No. 81672964). The authors declare that they have no competing interests.

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