Activation of dynamin-related protein 1 - dependent mitochondria fragmentation and suppression of osteosarcoma by cryptotanshinone
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Discovering how to regulate mitochondrial function to reduce cancer growth holds great potential for future cancer therapy development. Here we explore the effects of cryptotanshinone (CPT), a natural product derived from Salvia miltiorrhiza, on mitochondria of osteosarcoma (OS) both in vitro and in vivo, and further elucidate the underlying molecular mechanisms.
Cytotoxicity in the CPT treated OS cells was analyzed by flow cytometry, CCK8, TUNEL assay and colony formation assays. Flow cytometric analysis was performed to evaluate the effect of CPT on cell cycle of OS cells. Mitochondrial morphology was examined by staining with the mitochondrial membrane potential -sensitive fluorochrome, MitoTracker Red (CMXRos). Immunoblotting, confocal-immunofluorescence staining, co-immunoprecipitation were used to examine the expression and interaction between CPT-mediated Drp1 and Bax. Finally, the synergistic effect of CPT on OS cells was validated using a mouse xenograft tumor model.
In this study, we found CPT treatment induced S-phase arrest, apoptosis, and mitochondrial fragmentation in OS cells. CPT also effectively activated caspase-dependent apoptosis, which could be blocked by pan-caspase inhibitor Z-VAD-FMK. Moreover, we herein provide evidence that treatment with CPT resulted in mitochondrial fragmentation, which is mediated by dynamin-related protein 1 (Drp1), a key mediator of mitochondrial fission. Pursuing this observation, downregulation of Drp1 via silencing RNA could abrogate the induction of apoptosis and mitochondrial fragmentation induced by CPT. Finally, we demonstrate that CPT induced Drp1, which interacted directly with Bcl-2-associated X protein (Bax), which contributed to driving Bax translocation from the cytosol to the mitochondria.
Our findings offer insight into the crosstalk between mitochondrial fragmentation and inhibition of osteosarcoma cell growth in response to CPT.
KeywordsCryptotanshinone (CPT) Drp1 Mitochondria fragmentation Osteosarcoma
Cell counting kit-8
Dynamin-related protein 1
Hematoxylin and eosin stain
5,5′, 6,6′-tetrachloro-l,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide
- Mfn1 and 2
Mitofusin1 and 2
Mitochondrial outer membrane permeabilization
- MT stain
Masson’s trichrome stain
Outer mitochondrial membrane
Small interfering RNA
Terminal deoxynucleotidyl transferase dUTP nick end labeling
Mitochondrial membrane potential
Osteosarcoma is the most commonly occurring form of malignant bone tumor. Specifically, osteosarcoma is the development of cancer in areas of postnatal bone growth and bone remodeling. Although osteosarcoma can affect people of all ages, it most often occurs in children and teens who are still growing, which indicates genetic and molecular alterations that disrupt osteoblast differentiation are important factors in the etiology of the disease. Tumors occur predominantly around the knee, in either the femur (thighbone) or tibia (shinbone) . However, osteosarcoma can also affect other parts of the body and can even develop outside of bones, in soft tissues (extraskeletal osteosarcoma), especially in elderly patients. Treatments of osteosarcoma include surgery, radiation, chemotherapy, or a combination of radiotherapy and chemotherapy; however, such therapies are often negatively characterized by toxicity and side effects. Additionally, considering the long-term and short-term toxicities of chemotherapeutic agents commonly used to treat osteosarcoma, a more promising approach targets the development of effective, and nontoxic therapeutic strategies, using active constitutive agents extracted from natural sources.
Mitochondria play an important role in the production of energy within cells, and are essential in the regulation of cellular life and death. In most healthy mammalian cells, mitochondria exhibit tubular, reticular, or networked morphology which is regulated by dynamic remodeling via the balance between fusion and fission events [2, 3]. Mammalian large GTPases regulate mitochondrial fusion: mitofusins (Mfn) 1 and 2 mediate outer mitochondrial membrane (OMM) fusion, while optic atrophy 1 (Opa1) is responsible for inner mitochondrial membrane (IMM) fusion. Conversely, dynamin-related protein 1 (Drp1) is the master regulator of mitochondrial division in most eukaryotic organisms . Together, Drp1 with its OMM receptors Fission 1 (Fis1), mitochondrial fission factor (MFF), and mitochondrial elongation factor 1 (Mief1) mediates mitochondrial fission [5, 6, 7]. Inhibition of Drp1 by either expression of a Drp1 dominant mutant or RNA interference leads to increased length and interconnectivity of mitochondrial tubules, thereby inhibiting the fission process and preventing cell death . Importantly, excessive mitochondrial fission and mitochondrial structural disarray has been linked to increased mitochondrial production of reactive oxygen species (ROS), impaired function, and activation of cell death [9, 10]. Evidence has emerged indicating that Drp1 and Bax mitochondrial translocation is a crucial step for induction of apoptosis [11, 12, 13].
Danshen root, has been used in traditional Chinese medicine to treat coronary heart disease for thousands of years [14, 15]. Cryptotanshinone (CPT), a natural quinoid diterpene isolated from Danshen root, has been reported to exhibit inhibitory effects on STAT3 activation; furthermore, it has demonstrated other pharmacological effects in the treatment of cardiovascular diseases , anti-inflammation , and neuron protection . Recently, CPT has has been shown to display diverse anticancer properties against many tumors occurring in humans, such as prostate cancer, leukemia, gliomas, lung carcinomas, hepatic carcinomas, pancreatic cancer, breast cancer, colorectal cancer, and melanoma cancer [19, 20, 21, 22, 23]. However, the specific effects of CPT on osteosarcoma have yet to be elucidated. The purpose of the present study is to investigate the mitochondrial morphology-function relationship to gain insight into the anticancer effects of CPT.
Materials and methods
All experiments were done under Institutional Animal Care and Use Committee approval at China Medical University (Taichung, Taiwan) (2017–077). NOD/SCID (NOD CB17-Prkdcscid/NcrCrl, male, 5 weeks of age) mice were obtained from BioLASCO Taiwan Co., Ltd. All mice were housed in specific pathogen−free conditions. During the entire maintenance period, all mice were permitted free cage activity without joint immobilization. The initial body weights of the mice were between 20 and 23 g. After subcutaneous injection of 143B osteosarcoma cells into the back of NOD/SCID mice, the mice were treated with or without CPT (10 or 20 mg/kg). CPT was diluted in DMSO: Ethanol: Normal Saline: Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) = 1:3:3:3 and heated to 60 °C before injection to mice. Seven days after 143B osteosarcoma cell injection, IP injection with CPT was carried out every other day followed by sacrifice at day 45 of tumor cell inoculation. The tumors were removed, weighed, and fixed for use in immunohistochemical experiments. All experiments were carried out using 5 mice in each group, with three independent experiments.
Cryptotanshinone (C5624), JC-1 Dye (T3168), MitoTracker Red CMXRos (M7512) and Z-VAD-FMK (V116) were purchased from Sigma-Aldrich (USA). The primary antibodies against β-actin (ab151318), α-tubulin (ab4074), Caspase-3 (ab44976), Caspase-8 (ab25901), Caspase-9 (ab184786), Bcl-2 (ab182858), Bax (ab32503), Bid (ab10640), Bad (ab62465), Drp1 (ab56788)), Opa1 (ab119685), Mfn-1 (ab57602), Mfn-2 (ab124773), and Hsp60 (ab46798) were purchased from Abcam (UK). Annexin V apoptosis detection kit (55647) was purchased from BD (USA).
The human osteosarcoma (OS) cell line 143B and MG63 cells were grown in DMEM supplemented with 10% FBS and 100 units/ml penicillin and 100 μg/ml streptomycin (HyClone, USA) at 37 °C in a 5% CO2 incubator. To examine whether CPT could induce cell death in osteosarcoma, cells were treated with different doses of CPT for 24 h.
Cell Counting Kit-8 (CCK-8) was obtained from Dojindo (Dojindo Co. Ltd., Japan). Briefly, cells were plated in 96-well plates at a density of 1 × 104 cells per well and cultured in the growth medium. At the indicated time points, the number of cells in triplicate wells was measured using the absorbance at 450 nm of reduced WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt).
The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to evaluate the apoptotic response of tumor cells with a kit from Roche Applied Science (Germany). The cells grew on coverslips fixed by 4% PFA for 30 min at room temperature and washed 3 times by PBS and then incubated with 0.1% Triton X-100 for 2 min and washed by PBS. TUNEL assay was performed according to the manufacturer’s instruction (Roche Applied Science, Germany). After washing in PBS, nick-end labeling was visualized by immersing sections in DAB solution with 3% hydrogen peroxide and counterstained with methyl green. Finally, the sections were counterstained with Mayer’s hematoxylin, washed in water, and mounted. All slides were observed under light microscopy.
The histological observation was performed by staining with hematoxylin/eosin (H&E), Giemsa, and trichrome stains using paraffin sections. For H&E staining, paraffin-embedded sample slides were de-paraffinized, hydrated, and then stained with hematoxylin for 1 min. After rinse, the slides were stained with eosin for 5 min, rinsed, and sealed with cover slips. Tissue sections from tumor mass, kidneys, spleen, liver, heart, and lungs were used. The slides were counterstained with hematoxylin and mounted. To determine the effect of CPT on expression of Ki67, PCNA, Caspase-3, Caspase-8, Caspase-9, Bcl-2, Bax, Bid, Bad, Drp1, Opa1, Mfn1, and Mfn2 by immunohistochemistry, the slides were blocked in 5% bovine serum for 15 min, followed by incubation with the primary antibody at 4 °C overnight in a moist chamber. The sections were then incubated with the corresponding secondary antibodies. The antigen-antibody complex was detected by Dako Liquid DAB + Substrate-Chromogen System (Dako, Carpinteria, CA). All slides were examined under light microscopy. Giemsa stain and trichrome stain were performed according to the manufacturer’s instruction of Giemsa Stain Kit (ab150670) and Trichrome Stain Kit (ab150686) from Abcam (UK). All slides were examined under light microscopy.
Short interfering RNA (siRNA) transfection
Non-targeting siRNA and siRNAs targeting human Drp1 (H00010059-R01) were obtained from Abnova Corporation (Taipei City, Taiwan) (Additional file 1: Table S1). The siRNAs were transfected into the cells using siRNA transfection reagent (Santa Cruz Biotechnology; cat. no. sc-29,528) according to the manufacturer’s protocol. After overnight incubation, cells were treated with or without CPT for 24 h.
Cells were placed in 6-well plates and treated with different combinations for 24 h. The cells were harvested, washed twice with cold PBS, and stained with FITC-conjugated annexin V and propidium iodide for 15 min (BD, USA) in the dark, or stained with 10 μg/mL JC-1 (Thermo Fisher Scientific, MA, USA) in DMEM medium at 37 °C for 30 min. For cell cycle analysis, the cells were then fixed with cold 70% ethanol overnight at − 20 °C, and propidium iodide staining was performed. The stained cells were assessed by flow cytometry (BD FACSLyric, USA), and analyzed by FlowJo V10 software.
Western blot analysis
The tumor masses or CPT-treated cells were harvested and total cell protein was extracted using whole cell lysis buffer. The protein concentrations were determined by the Bradford method (Bio-Rad, CA, USA). Samples with equal amount of protein were subjected to 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidenedifluoride (PVDF) (Millipore, Bedford, MA, USA) membrane. The membrane was incubated at room temperature in blocking solution (5% nonfat milk) for 1 h followed by incubation for 2 h in blocking solution containing an appropriate dilution of anti- Caspase-3, Caspase-8, Caspase-9, Bcl-2, Bax, Bid, Bad, Drp1, Opa1, Mfn1, Mfn2 antibody (Abcam, UK). After washing, blots were then probed with appropriate secondary horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and detected by an ECL detection system (Millipore) and scanned by MultiGel-21 (Top Bio, Taiwan). β-actin served as internal control. Cytosolic and mitochondrial protein extractions were performed according to manufacturer’s protocol from Thermo Scientific (Waltham, MA, USA). HSP60 and α-tubulin were used as mitochondrial and cytosolic markers, respectively.
Confocal laser microscopy
In order to measure transitions in the mitochondrial morphology, the CPT-treated cells were reacted with 10 nM MitoTracker Red CMXRos probe (Invitrogen Corp., Carlsbad, CA, USA) for 20 min at 37 °C, according to the manufacturer’s instructions. After being washed twice in cold PBS, the live cells were visualized under a Leica confocal laser scanning microscope (TCS SP8, Wetzlar, Germany). MitoTracker Red was monitored at an excitation wavelength of 579 nm to locate mitochondria. Fragmented mitochondria were shortened, punctate, and sometimes spherical, whereas filamentous mitochondria showed a long thread-like tubular structure .
All experiments were performed in triplicate and data presented in a representation of three individual experiments. Statistical analyses were performed using GraphPad Prism statistical software (version 6, GraphPad Software, Inc., San Diego, CA). Data was represented as means ± standard deviation (SD) of three independent experiments. One-way ANOVA was carried out when multiple comparisons were evaluated. Values were considered to be significant at p less than 0.05.
CPT induced OS cell death and cell cycle arrest
CPT treatment inhibited osteosarcoma progression in NOD-SCID mice
CPT induced mitochondrial fragmentation in OS cells
CPT induced activation of apoptotic pathway in osteosarcoma cells
Drp1 is required for Bax-mediated intrinsic apoptosis
CPT induced a time-associated increase in the interaction between active Bax and Drp1
CPT is a potent inducer of intrinsic apoptotic pathway and mitochondrial fission in vivo
Investigations into mitochondrial biology and tumorigenic signaling are revealing novel approaches to developing targeted cancer therapy. The present study provides evidence that CPT exposure led to pronounced mitochondrial fragmentation in 143B and MG63 osteosarcoma cells. Additionally, CPT-triggered mitochondrial fragmentation preceded mitochondrial damage in vitro and was associated with CPT-induced cancer cell apoptosis in vivo. We report for the first time that CPT promotes Drp1 interaction with Bax, triggering Bax translocation into mitochondria, which is crucial for CPT-triggered cancer cell death. Autophagy-related protein associated with antitumor properties of CPT, in particular, needs further investigation (Additional file 6: Figure S5).
The core components of the fusion and fission machineries/mechanisms have been identified as the dynamin-related GTPases Drp1, mitofusins (Mfn) 1 and 2, and Opa1 . Multiple studies have demonstrated that enhanced fission or reduced fusion, high expression or enhanced activation of Drp1, and/or downregulation of MFN2 are linked to several cancer-related phenotypes [28, 29], indicating that cancer cells often exhibit fragmented mitochondria . However, Drp1−/− embryos showed considerably weaker signals and decreased numbers of caspase-3–positive cells, demonstrating the physiological role of Drp1 as facilitating developmentally-regulated apoptosis during neural tube formation . The role of Drp1 in tumorigenesis thus may appear to be paradoxical, since mitochondrial fission plays a key mediator of two very distinct processes, cellular apoptosis and cell mitosis . It has been demonstrated that mitochondrial dynamics (both fission and fusion) act as a rheostat that determines apoptotic susceptibility, as loss of Drp1 postpones cytochrome c release and apoptotic induction, while a follow-up study indicated that fission was not required for Bax/Bak-mediated apoptosis . Instead, a GTPase-independent function of Drp1 in membrane remodeling and hemi-fusion results in Bax oligomerization and subsequent MOMP, indicating that the death function of Drp1 can promote apoptosis independent of fission . The role of Drp1 has been detected in complexes with Bax at mitochondria. In response to many apoptotic stimuli, activation of the pro-apoptotic Bax results from a highly regulated multistep process involving its translocation from the cytosol to the OMM, where it integrates and oligomerizes . Although the exact mechanism by which Bax actively moves from the cytosol to the mitochondria is still unclear, recent studies have suggested that irradiation could induce a time-associated increase in the interaction between active Bax and Drp1, then Bax-Drp1complex translocate to discrete foci on the mitochondria, where mitochondrial Bax stabilizes Drp1 [11, 36, 37]. Our results support that CPT acted as an effective Drp1 activator, capable of inducing cancer cell death via direct interaction with Bax to participate in apoptotic fragmentation of mitochondria. Furthermore, 143B cells was more sensitive towards the toxicity of CPT than MG63 cells (Fig. 1). Our results indicated that Bcl-2 was decreased in a dose dependent manner after 24 h exposure of CPT in 143B but not MG63 cells. However, a declining trend of Bcl-2 expression in MG63 cells was observed following long-term exposure (36 h) of CPT (Additional file 4: Figure S3C). This discrepancy may be due to the cell-specific expression of Bcl-2.
Aside from apoptosis, the cell cycle is documented to alter mitochondrial dynamics. Mitochondrial fission increasingly occurs during cellular division, thus assuring equal segregation of mitochondrial contents in daughter cells. Drp1 has been recognized to be functionally or molecularly linked to Cyclin B, E, and D [38, 39, 40]. As a previous study has suggested, during mitosis, CDK1/cyclin B phosphorylates Drp1 at Ser616 to induce mitochondrial fission and proper organelle segregation . On the other hand, mitochondria morphology was found to regulate the cell cycle, as the genetic inhibition of Drp1 and the use of Drp-1 inhibitor (mitochondrial division inhibitor 1, Mdivi-1) have led to a decrease in the number of cells in S phase and an increase in the number of cells in G2 phase . The G2/M cell cycle arrest and aneuploidy were also observed in Drp1-deficient cells . Crosstalk between the mitochondrial fission protein, Drp1, and the cell cycle is identified to play a critical role in the regulation of cell cycle progression. In response to CPT treatment, the present study found that osteosarcoma cells accumulated in S phase and significantly increased apoptosis rates, which could be rescued by knockdown Drp1 expression. Our results provide evidence that CPT-induced Drp1 expression is correlated with CPT-mediated S phase arrest and apoptosis induction.
Translocation, foci formation, and Bax activation, on the OMM leads to permeabilization and causes the release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol . Interestingly, not only does Bax promote the foci formation of Drp1, it also forms apoptotic mitochondrial localized foci that colocalized with Mfn1 and Mfn2 , preventing further fusion . We herein found increased Drp1 in response to CPT treatment in both 143B and MG63 cells, while the expressions of Mfn1 and Mfn2 were decreased in 143B but not MG63 cells. We suppose the discrepancy may be a result of the differing proliferation rates of osteosarcoma. Drp1 expression patterns associated with cancer have been documented in several tumor models [32, 45, 46]. Tanwar et al. claimed that Drp1-based-gene-expression-signature could be used to recognize patients with poor survival possibilities from the primary tumors . Also, Drp1 is essential in Ras-driven tumor growth and poor survival rate is associated with an increased Drp1 level reveal that Drp1 interacts with different biological processes in the tumorigenesis context . Inhibition of cancer cell growth and/or enhanced spontaneous apoptosis induced by Drp1 inhibition have been observed both in vitro and in vivo in several cancer types [28, 42, 49]. The researchers found that treatment with Mdivi-1 resulted in mitochondrial hyper fusion and chronic elevation of cyclin E, which prevented the progression of the cell cycle in human colorectal carcinoma cell line . In agreement with this result, cell apoptosis induced by Mdivi-1 has been reported in human ovarian, breast cancer cell lines and xenograft models of lung cancer [28, 51, 52]. Consistent with this notion, we did find Mdivi-1 treatment caused a marked reduction of xenograft tumors developed from 143B cells (Fig. 8). These results suggest that Drp1 contributes to initial tumor growth rather than later phase of tumor progression. Intriguingly, investigations have revealed that abnormal mitochondrial fission, mediated by Drp1, leads to excessive mitochondrial fragmentation, which appears to be a requisite step in intrinsic apoptosis pathways [26, 53]. A possible explanation might be that upregulated mitochondrial fission may function as an important point of convergence in mediating oncogenic signaling and promoting cancer cell growth. We supposed that CPT induced Drp1 activation caused the imbalance of fission and fusion impacting mitochondrial function which attributes to apoptotic signaling of cancer cell death. As enhanced mitochondrial fission and impaired fusion appear to contribute fundamentally to the inhibition of certain cancers, Drp1-mediated mitochondrial fission thus may represent a promising novel therapeutic target for cancers demonstrating excessive mitochondrial fission.
In summary, our study provides further evidence that CPT triggers Drp1 expression to activate mitochondrial fission, which results in Bax activation and downstream intrinsic apoptosis, effectively inhibiting osteosarcoma growth. Therefore, investigation into CPT-induced inhibition of osteosarcoma cell growth suggests that influencing mitochondrial fission/fusion machinery may offer a novel approach to the development of future therapeutic cancer treatments.
The authors would like to thank James Waddell for the critical reading and revision of our manuscript.
This work was supported and funded by Ministry of Science and Technology with MOST 106–2320-B-039-022, National Research Institute of Chinese Medicine, Ministry of Health and Welfare (MOHW106-NRICM-C-104-000002), Health and welfare surcharge of tobacco products, China Medical University Hospital Cancer Research Center of Excellence (MOHW107-TDU-B-212-114024) China Medical University Hospital (DMR-107-003, DMR-107-006 and DMR-107-164) and Chinese Medicine Research Center, China Medical University under the Higher Education Sprout Project, Ministry of Education (CMRC-CHM-1) in Taiwan.
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Material is available upon request.
JHY wrote the paper, and amended the references. JHY and HSH performed the experiments and analyzed the data. STH designed, conceived the study and amended the manuscript. JHY, HSH and STH approved the final manuscript.
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