ONCOLOGY REPORTS 42: 1125-1132, 2019

Hypoxia‑induced mitochondrial translocation of DNM1L increases and triggers mPTP opening in HCC cells via activation of HK2

Min Cai1, Ping He2 and Dian‑Liang Fang2

Departments of 1Nephrology, 2Gastroenterology, Yongchuan Hospital of Chongqing Medical University, Chongqing 402160, P.R. China

Received February 14, 2019; Accepted June 18, 2019

DOI: 10.3892/or.2019.7213

Abstract. Disturbed mitochondrial dynamics are closely of the mitochondrial membrane, inhibition of the respiratory associated with the progression of different types of chain, reduction in enzyme activity, and mitochondrial DNA including hepatocellular carcinoma (HCC). However, the (mtDNA) damage, which can lead to a number of diseases manner in which mitochondrial dynamics are regulated in including cancer (1). Mitochondrial functions are also regulated HCC remains largely unclear. In the present study, via immu- by mitochondrial dynamics (2,3). Under normal physiological nofluorescence, real‑time PCR and western blot analysis, conditions, mitochondria are highly dynamic and constantly the effects of dynamin‑1‑like (DNM1L) on mitochondrial undergo fusion and fission, but the equilibrium is disrupted translocation and the mitochondrial permeability transition under pathological conditions, when mitochondria fuse with pore (mPTP) were investigated in HCC cells under hypoxic each other into a long chain, ring, or mesh structure, or are split conditions, and the underlying molecular mechanisms were into smaller rod‑ or point‑like structures. Increasing evidence explored. Our data revealed that hypoxic treatment decreased has shown a strong association between cancer progression and the mitochondrial membrane potential, elevated generation of unbalanced mitochondrial dynamics (2,3). Some studies have , and promoted mitochondrial fission shown that disruption of the mitochondrial network strongly in a DNM1L‑dependent manner. Instead of changing the levels affects the of cancer cells, including lung, bladder of DNMlL, hypoxia increased the translocation of DNM1L to and colon cancer cells (2). Additionally, in lung cancer, clinical mitochondria, leading to excessive mitochondrial fission and prognosis is significantly associated with abnormalities in decreased the viability of HCC cells. In addition to the effects governing mitochondrial dynamics (4). In hepatocel- on mitochondrial dynamics, DNM1L also regulated mPTP lular carcinoma (HCC), mitochondrial fission is frequently opening in HCC. IP analysis revealed that DNM1L interacted upregulated, significantly contributing to poor patient prog- with the enzyme hexokinase 2 (HK2), and was involved in its nosis (5). Increased mitochondrial fission increases HCC cell , resulting in HK2 detachment from the mito- survival, primarily by facilitating autophagy and inhibiting chondria and consequently mPTP opening. mitochondrial‑dependent apoptosis, which are mediated via elevated production of reactive oxygen species (ROS) and Introduction subsequent degradation of and activation of nuclear factor RETRACTED(NF)‑κB (5). Moreover, mitochondrial fission activates calcium Mitochondria, the main subcellular organelles for energy signaling in HCC cells, which in turn promotes mitochondrial metabolism in eukaryotes, play extremely important roles fission in a positive feedback loop (6). However, the manner in in several physiological and pathological processes (1). which mitochondrial dynamics are regulated in HCC remains Mitochondrial dysfunction mainly refers to an energy metabo- largely unclear. lism disorder caused by various factors including dissipation Mitochondrial dynamics can be regulated by mitochon- drial fusion proteins, such as mitofusin 1 (MFN1), mitofusin 2 (MFN2) and OPA1 mitochondrial dynamin like GTPase (OPA1), as well as mitochondrial fission proteins, such as dynamin‑1‑like (DNM1L), mitochondrial fission factor (MFF), fission 1 (FIS1) Correspondence to: Dr Dian‑Liang‑Fang, Department of and mitochondrial dynamics 49/51 (MID49/51) (3). Gastroenterology, Yongchuan Hospital of Chongqing Medical University, Chongqing 402160, P.R. China MFN1 and MFN2 together promote fusion of the outer mito- E‑mail: [email protected] chondrial membrane (OMM), while OPA1 regulates fusion of the inner mitochondrial membrane (IMM) (3). In mitochondrial Key words: DNM1L, HK2, mPTP opening, mitochondrial dynamics, fission, FIS1 protein is integrated into the OMM and acts as a HCC receptor for DNM1L, which translocates from the to the OMM, and forms a ring that drives fission of the organ- elle (7). DNM1L is essential for the process of mitochondrial 1126 CAI et al: ROLE OF DNM1L IN HCC fission, whereas other fission proteins are replaceable as corre- quantified using ImageJ software (US National Institutes of sponding receptors required for DNM1L binding to the OMM Health, Bethesda, MD, USA; imagej.nih.gov/ij) following the (8). Therefore, aberrant expression of DNM1L causes severe WB quantification guidelines. disruptions in mitochondrial dynamics leading to pathologies, such as neurodegenerative, neoplastic, endocrine and cardio- Real‑time PCR. Total RNA was extracted from cells using vascular diseases (8,9). Decreasing DNM1L in cardiomyocytes TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according induces mitochondrial elongation, accumulation of damaged to the manufacturer's instructions. RNA was then reverse mitochondria and increased apoptosis (8). Furthermore, DNM1L transcribed and the resulting cDNA was used as a template knockdown has been shown to significantly enhance spontaneous for quantitative PCR using a Takara RNA PCR kit and SYBR apoptosis in vitro and in vivo in several cancer types (10,11). In Premix Ex Taq (Takara Bio Inc., Kusatsu, Japan) in accordance HCC, DNM1L is aberrantly upregulated, and forced expression with the manufacturer's instructions. GAPDH was used as an of DNM1L increases mitochondrial fission and promotes the internal control. The following primers were used: human survival of HCC cells (6,11). The expression of DNM1L may be HIF1A, sense, 5' to 3'‑GAACGT​ CGA​ AAA​ GAA​ AAG​ TCT​ CG​ regulated by both cytosolic Ca2+ signaling and p53/AR signaling and antisense, 5' to 3'‑CCT​TAT​CAA​GAT​GCG​AAC​TCA​CA; (6). Cadmium levels have also been reported to regulate mito- human DNM1L, sense, 5' to 3'‑CTG​CCT​CAA​ATC​GTC​GTA​ chondrial translocation of DNM1L (9). However, the manner in GTG and antisense, 5' to 3'‑GAG​GTC​TCC​GGG​TGA​CAA​ which the effect of DNM1L is regulated in HCC cells remains TTC; human MID51, sense, 5' to 3'‑CAC​GGC​CAT​TGA​CTT​ to be elucidated. TGT​GC and antisense, 5' to 3'‑TCG​TAC​ATC​CGC​TTA​ACT​ In this study, we investigated the effects of DNM1L on GCC; human GAPDH, sense, 5' to 3'‑GAG​TCA​ACG​GAT​TTG​ mitochondrial translocation and mitochondrial permeability GTC​GT and antisense, 5' to 3'‑TTG​ATT​TTG​GAG​GGA​TCT​ transition pore (mPTP) opening in HCC cells under hypoxia, CG; human FIS1, sense, 5' to 3'‑GTC​CAA​GAG​CAC​GCA​GTT​ and explored the underlying molecular mechanisms and TG and antisense, 5' to 3'‑ATG​CCT​TTA​CGG​ATG​TCA​TCA​ potential therapeutic applications. Our findings contribute TT; human MFF, sense, 5' to 3'‑ACT​GAA​GGC​ATT​AGT​CAG​ to the understanding of the pathological roles of mitochon- CGA and antisense, 5' to 3'‑TCC​TGC​TAC​AAC​AAT​CCT​CTC​ drial dynamics and provide strong evidence to support a C. The RT‑qPCR protocol was: i) 95˚C for 2 min; ii) 95˚C for 5 novel strategy for HCC treatment targeting proteins of the sec, 60˚C for 30 sec, 72˚C for 30 sec, 40 cycles; iii) dissociation mitochondrial fission machinery. curve. All results were calculated using the 2‑ΔΔCq method (12).

Materials and methods siRNA. The siRNA sequence (5'‑aatatccatctctggcca‑3') for DNM1L was purchased from Sigma‑Aldrich; Merck KGaA. A Cell lines. The Huh7 cell line was obtained from the Shanghai scrambled siRNA precursor was used as the negative control. Cell Bank of the Chinese Academy of Sciences (Shanghai, siRNAs were transfected into cells using Lipofectamine® 2000 China). Cells were cultured in Dulbecco's modified Eagle's (Thermo Fisher Scientific, Inc.) following the manufacturer's medium (DMEM) (Sigma‑Aldrich; Merck KGaA, Darmstadt, instructions. After 48 h, the transfection efficiency was Germany) supplemented with 10% fetal bovine serum (FBS) assessed using qPCR and WB, and the cells were used for (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in a humidified further experiments.

5% CO2 incubator. Overexpression of DNM1L. The coding sequences of DNM1L Immunoprecipitation and western blotting. Immunoprecipitation were constructed into p3xFlag‑CMV7 (Sigma‑Aldrich; Merck (IP) was carried out using the Protein G IP Kit (Roche, Basel, KGaA). Transfection of siRNA and overexpression plasmids Switzerland) according to the manufacturer's instructions. was carried out using Lipofectamine® 2000 (Invitrogen; SDS‑PAGE was used for western blotting (WB) in order to Thermo Fisher Scientific, Inc.) according to the manufacturer's separate proteins extractedRETRACTED from tissues or cells, and proteins protocol. In the overexpression assays, p3xFlag‑CMV7 vectors were transferred onto polyvinylidene fluoride membranes. were used as a control. Membranes were incubated first with primary antibodies against DNM1L (dilution 1:1,000; cat. no. ab184247; Abcam), MID51 Immunofluorescence staining (IMF). Cells were seeded onto (dilution 1:100; cat. no. ab220672; Abcam), FIS1 (dilution coverslips coated with poly‑L‑lysine, and fixed with 4% para- 1:1,000; cat. no. ab71498; Abcam), mitochondrial fission factor formaldehyde before staining. Cells were permeabilized with (MFF) (dilution 1:1,000; cat. no. ab81127; Abcam), hypoxia 0.5% Triton X‑100 in phosphate‑buffered saline (PBS) at room inducible factor 1 subunit α (HIF1A) (dilution 1:1,000; cat. no. temperature for 5 min, and blocked with 1% bovine serum ab187524; Abcam, Cambridge, MA, USA), voltage‑dependent albumin. Then, cells were incubated with primary antibodies anion channel (VDAC) (dilution 1:1,000; cat. no. 4661; Cell (anti‑DNML1 and/or anti‑HK2), secondary antibodies, and Signaling Technology, Danvers, MA, USA), adenine nucleo- then stained with DAPI and photographed with a TCS SP5 tide translocator (ANT) (dilution 1:1,000; cat. no. 14671; Cell imaging system (Leica Microsystems, Wetzlar, Germany). For Signaling Technology), cyclophilin D (CypD) (dilution 1:1,000; mitochondrial morphology, cells were incubated with 100 nM cat. no. ab110324; Abcam), hexokinase 2 (HK2) (dilution 1:1,000; MitoTracker Deep Red (Invitrogen; Thermo Fisher Scientific, cat. no. 2867; Cell Signaling Technology), and GAPDH (dilution Inc.) at 37˚C for 10 min. 1:10,000; cat. no. 60004‑1‑Ig; Proteintech, Chicago, IL, USA), followed by HRP‑conjugated secondary antibody. Enhanced Mitochondrial transmembrane potential assays. Mitochondrial chemiluminescence was used for detection. WB results were transmembrane potential was determined using JC‑1 staining ONCOLOGY REPORTS 42: 1125-1132, 2019 1127

(Sigma‑Aldrich; Merck KGaA) according to the manufacturer's mitochondrial function, mitochondrial membrane potential instructions. Briefly, cells were incubated with JC‑1 for 20 min and ROS generation, in hypoxia‑exposed Huh7 cells. JC‑1 at 37˚C. JC‑1 monomers were stained green and JC‑1 aggregate fluorescence, which was used to determine the mitochondrial was stained red. Cell images were obtained and analyzed membrane potential, was significantly decreased by hypoxia, by IMF. whereas ROS generation was significantly elevated (Fig. 1A and B). Meanwhile, a significant increase in mitochondrial ROS detection. 2,7‑Dichlorofluorescein diacetate (DCFH‑DA; fragments was observed in hypoxia‑exposed Huh7 cells (Fig. Sigma‑Aldrich; Merck KGaA) was used to determine intracel- 1C), suggesting that mitochondrial dynamic equilibrium of lular ROS levels according to the manufacturer's instructions. fusion and fission in Huh7 cells is disrupted by hypoxia. Next Briefly, cells were incubated with 20 µM DCFH‑DA for 30 we determined whether regulators of mitochondrial fission, min at 37˚C. After washing the cells with PBS three times, such as DNM1L, MID51, FIS1 and MFF, are affected in Huh7 images were obtained and analyzed by IMF. cells under hypoxia. HIF1A was used as positive control under hypoxia. No significant changes were observed for any of mPTP opening test. Briefly, treated cells were incubated with these regulators at the mRNA or protein levels (Fig. 1D and 2 µM calcein (Sigma‑Aldrich; Merck KGaA) and 100 nM E). Since the effect of DNMlL depends on their translocation MitoTracker Deep Red for 30 min in the absence of light. into mitochondria, we also determined their levels in mito-

Cells were subsequently exposed to hypoxia (1% O2, 5% CO2, chondrial and cytoplasmic fractions. Among these proteins, 94% N2) for 24 h. After washing the cells with PBS three only DNM1L was decreased in the cytoplasm and increased times, images were obtained and analyzed using IMF. in mitochondria under hypoxic conditions (Fig. 1F). In addi- tion, confocal images verified that hypoxia increased the Mitochondrial isolation. Mitochondrial proteins were isolated recruitment of DNM1L into the mitochondria (Fig. 1G). Taken from tumor cells using Mitochondria Isolation Kit (MP‑007; together, these data indicate that hypoxia induces mitochon- Invent Biotechnologies, Plymouth, MN, USA) according to drial translocation of DNM1L in HCC cells. the manufacturer's instructions. Briefly, the cell lysate was centrifuged at 20,000 x g for 1 min and the precipitate was DNM1L translocation leads to excessive mitochondrial suspended briefly. The floccule was centrifuged for 1 min fission and decreased viability of HCC cells. Our confocal at 1,500 x g after overhanging and then the supernatant was images revealed that hypoxia triggered mitochondrial mixed by vortexing with 400 µl buffer. After centrifugation at fission and aggravated mitochondrial fragmentation (Fig. 1). 20,000 x g for 10 min, the supernatant was removed completely To determine whether DNM1L is critical for this process, and the pellet was suspended in 200 µl buffer. After a further DNM1L was knocked down in Huh7 cells. Knockdown effi- 30 min of centrifugation at 20,000 x g, the supernatant was ciency was determined by qPCR and WB analysis (Fig. 2A). discarded and the pellet comprised isolated mitochondria. DNM1L knockdown significantly elevated the mitochondrial membrane potential and significantly reduced ROS levels in Cell viability assay. Cell viability was quantified with a Cell both hypoxia‑exposed and unexposed Huh7 cells, and signifi- Counting Kit‑8 (CCK‑8) (Dojindo, Tokyo, Japan), according cantly alleviated hypoxia‑induced elevation of ROS generation to the manufacturer's instructions. The cells were plated at a and reduction of mitochondrial membrane potential (Fig. 2B density of 3,000 cells per well in 96‑well plates after transfec- and C). The alleviation was also observed with treatment of tion with siRNA in a 10 cm dish for 48 h. CCK‑8 assays were Huh7 cells with the DNM1L translocation inhibitor Mdivi assessed at 12, 24 and 48 h after transfection by measuring the (Fig. 2B and C). These data demonstrated the important role of absorbance at 450 nm. DNM1L in hypoxia‑induced excessive mitochondrial fission and the subsequent mitochondrial dysfunction. Since mito- Statistical analysis. All data are presented with error bars chondrial dynamics have been reported to regulate apoptosis denoting standard deviation.RETRACTED All statistical data are based and proliferation of HCC cells, we also determined the effects on experiments conducted in triplicate. Statistical analysis of DNM1L knockdown on the viability of Huh7 cells under was performed with GraphPad Prism 5 software (GraphPad hypoxia. DNM1L knockdown decreased the viability of Huh7 Software, Inc., La Jolla, CA, USA). Differences between two cells without hypoxic treatment, whereas hypoxia reversed groups were examined by Student's two‑tailed t‑test; two‑way the effects of DNM1L knockdown on viability, indicating that analysis of variance was used when comparing more than two DNM1L alleviated the suppressive effects of hypoxia on tumor groups. For comparisons among multiple groups, one‑way cell viability, and the oncogenic effects of DNM1L depends on analysis of variance (ANOVA) followed by Scheffe post hoc hypoxic condition (Fig. 2D). test was used. Correlations between two groups were analyzed using nonparametric Spearman's r‑test. Two‑sided P‑values DNM1L also regulates mPTP opening. To note, compared <0.05 were considered statistically significant. to Mdivi treatment, DNM1L knockdown conferred stronger effects on mitochondrial function (Fig. 2B and C), suggesting Results that DNM1L regulates mitochondrial function in ways other than mitochondrial fission. Since DNM1L has been reported to Hypoxia induces mitochondrial translocation of DNM1L in regulate calcium signaling in HCC (6), and calcium signaling is HCC cells. Mitochondrial function is affected by hypoxic closely associated with mPTP opening (13), we speculated that exposure in different types of cells. To determine the effect DNM1L may also be able to regulate mPTP opening in HCC of hypoxia on HCC cells, we measured two indicators of cells. We analyzed the mPTP opening levels in calcein‑CoCl2 1128 CAI et al: ROLE OF DNM1L IN HCC

Figure 1. Hypoxia induces mitochondrial translocation of DNM1L in HCC cells. (A) Mitochondrial membrane potential in hypoxia‑exposed Huh7 cells. Both representative images of JC‑1 fluorescence (aggregates, red; monomers, green) and statistical results of the aggregate/monomer ratio are shown. (B) ROS generation of hypoxia‑exposed Huh7 cells. Both representative images and statistical data are shown. (C) Mitochondrial fragmentation in hypoxia‑exposed Huh7 cells. Both representative images of Mitotracker staining and statistical results of the fragmented percentage are shown. (D and E) Changes in the levels of HIF1A, DNM1L, MID51, FIS1 and MFF in hypoxia‑exposed Huh7 cells. Both mRNA and protein levels were determined. (F) Subcellular translocation of DNM1L, MID51, FIS1 and MFF in hypoxia‑exposed Huh7 cells as determined by WB. (G) Subcellular translocation of DNM1L in hypoxia‑exposed Huh7 cells as determined by IMF. ***P<0.001 vs. normoxia condition; n.s., not significant. HCC, hepatocellular carcinoma; ROS, reactive oxygen species; IMF, immunofluorescence; WB, westernRETRACTED blotting; DNM1L, dynamin‑1‑like; MID51, mitochondrial dynamic protein of 51 kDa; HIF1A, hypoxia inducible factor 1 subunit α; FIS1, fission 1; MFF, mitochondrial fission factor. tests, and found that DNM1L knockdown significantly inhib- these proteins were not altered after DNM1L knockdown ited mPTP opening in hypoxia‑exposed Huh7 cells (Fig. 3A). in hypoxic Huh7 cells (Fig. 4A). Meanwhile, as control, no Further, we overexpressed DNM1L in Huh7 cells (Fig. 3B) and changes in mitochondrial translocation were observed for treated these cells with CsA (an mPTP opening antagonist). mitochondrial proteins CypD, VDAC, and ANT, whereas CsA treatment alleviated the effects of DNM1L on mitochon- mitochondrial HK2 was increased after DNM1L knockdown drial membrane potential, ROS generation and cell viability (Fig. 4A), indicating that instead of regulating the expression (Fig. 3C‑E). of mPTP‑related proteins, DNM1L regulates mitochondrial detachment of HK2 in hypoxia‑exposed Huh7 cells. This DNM1L increases mitochondrial detachment of hexokinase effect of DNM1L on HK2 was demonstrated in our confocal 2 (HK2) to regulate mPTP opening. To further investigate images (Fig. 4B). To determine how DNM1L regulates HK2 the regulatory mechanism of DNM1L on mPTP opening, the translocation, we performed IP analysis and found a slight expression and mitochondrial translocation of several mPTP interaction between DNM1L and HK2 in Huh7 cells (Fig. 4C). structural proteins, including VDAC, ANT, CypD and HK2 Of note, hypoxia enhanced their binding affinity (Fig. 4C). were assessed. WB analysis showed that the total levels of Moreover, hypoxia increased the phosphorylation of HK2, ONCOLOGY REPORTS 42: 1125-1132, 2019 1129

Figure 2. DNM1L leads to excessive mitochondrial fission and decreased viability of HCC cells. (A) DNM1L knockdown (siDNM1L) efficiency in Huh7 cells as determined by qPCR and WB analysis. (B) Mitochondrial membrane potential in hypoxia‑exposed Huh7 cells with DNM1L knockdown or Mdivi (3 µM) treatment. Both representative images of JC‑1 fluorescence (aggregates, red; monomers, green) and statistical results of aggregate/monomer ratio are shown. (C) ROS generation of hypoxia‑exposed Huh7 cells with DNM1L knockdown or Mdivi (3 µM) treatment. Both representative images and statistical data are shown. (D) Cell viability of hypoxia‑exposed Huh7 cells with DNM1L knockdown as determined using the CCK‑8 assay at 12, 24 and 48 h after transfection by measuring the absorbance at 450 nm. *P<0.05, **P<0.01, ***P<0.001. HCC, hepatocellular carcinoma; ROS, reactive oxygen species; WB, western blotting; DNM1L, dynamin‑1‑like.

which was attenuated by DNM1L knockdown (Fig. 4C). These response to hypoxic stress, DNM1L is recruited by the outer data indicate that DNM1L interacts with HK2 and mediates mitochondrial membrane (OMM) protein FUNDC1 to drive the activation of HK2, which induces mitochondrial detach- mitochondrial fission (15). Hypoxia also induces HIF‑1α acti- ment, thus regulating the mPTP (Fig. 4D). vation which in turn activates DNM1L S616 phosphorylation RETRACTED(16). Hypoxia‑induced DNM1L recruitment is often observed Discussion in disorders of the neuronal and vascular systems (17,18). Recently, the effects of hypoxia on mitochondrial fission were and fission dynamically remodel the also found to promote migration, invasion, and tube forma- morphology of mitochondria within cells, and dysregulation of tion in breast cancer, glioblastoma, and endothelial progenitor the fusion/fission process has been linked to the tumorigenesis cells (19‑21). Meanwhile, DNM1L‑dependent mitochondrial of different types of including hepatocellular carcinoma fission was shown to regulate hypoxia‑induced migration of (HCC) (14). Recently, it was demonstrated that mitochondrial breast cancer cells, and to increase their sensitivity to chemo- fission is frequently upregulated in HCC tissues mainly therapeutic agents (20). However, although DNM1L‑mediated because of elevated expression of dynamin‑1‑like (DNM1L), mitochondrial fission was found to promote proliferation and which also was found to contribute to the poor prognosis of inhibit apoptosis in most previous studies (10,11), some reports HCC patients (5,6,11). However, the manner in which DNM1L also suggested that increased mitochondrial fission could be and mitochondrial dynamics regulate HCC cells remains positively related with cell death in HCC, and the effect of largely unclear. In the present study, we found that hypoxia hypoxia‑induced DNM1L translocation may reduce tumor could induce the dissociation of DNM1L from mitochondria, cell viability (21). Here, our results revealed that, in contrast and the resulting excessive mitochondrial fission promoted the to the effect in non‑hypoxic conditions, DNM1L knockdown viability of HCC cells. It is well established that hypoxia can increased the viability of HCC cells in hypoxic conditions induce mitochondrial fragmentation and (15). In and alleviated the suppressive effects of hypoxia on tumor 1130 CAI et al: ROLE OF DNM1L IN HCC

Figure 3. DNM1L regulates mPTPRETRACTED opening. (A) Levels of mPTP opening in hypoxia‑exposed Huh7 cells with DNM1L knockdown (siDNM1L). Both repre- sentative images and statistical data are shown. (B) DNM1L overexpressing (DNM1L) efficiency in Huh7 cells as determined by qPCR and WB analysis. (C) Mitochondrial membrane potential in hypoxia‑exposed Huh7 cells with DNM1L overexpression and/or CsA (1 µM) treatment. Both representative images of JC‑1 fluorescence (aggregates, red; monomers, green) and statistical results of the aggregate/monomer ratio are shown. (D) ROS generation of hypoxia‑exposed Huh7 cells with DNM1L overexpression and/or CsA (1 µM) treatment. Both representative images and statistical data are shown. (E) Cell viability of hypoxia‑exposed Huh7 cells with DNM1L overexpression and/or CsA (1 µM) treatment as determined using the CCK‑8 test. *P<0.05, ***P<0.001; n.s., not significant. DNM1L, dynamin‑1‑like; mPTP, mitochondrial permeability transition pore; WB, western blotting; ROS, reactive oxygen species. cell viability. These data indicated that DNM1L promotes that regulate mPTP opening, including elevated mitochondrial cell viability in normal conditions, but hypoxia reverses this ROS, excessive mitochondrial calcium levels, mitochondrial effects. DNA damage, and cytoplasmic signal transduction (13). The In addition to the effects on mitochondrial dynamics, our liberation of HK2 from mitochondria has also been demon- data showed that DNM1L also regulated the mitochondrial strated to regulate mPTP opening (23). HK2 binds to the permeability transition pore (mPTP) opening in HCC cells. OMM via association with VDAC, which interacts with ANT, Excessive opening of mPTP produces osmotic dysregulation forming the mPTP (24). HK2 detachment from mitochondria of the inner mitochondrial membrane (IMM), loss of mito- induces a conformational change of the molecular complex, chondrial and NADPH, uncoupling of oxidative leading to mitochondrial depolarization and cell death (25). phosphorylation, and cell death (22). There are many factors A recent study in neonatal cardiac myocytes revealed a ONCOLOGY REPORTS 42: 1125-1132, 2019 1131

Figure 4. DNM1L increases mitochondrial detachment of HK2. (A) Mitochondrial levels of HK2, CypD, VDAC and ANT in hypoxia‑exposed Huh7 cells as determined by WB. (B) HK2 detachment from mitochondria in hypoxia‑exposed Huh7 cells with or without DNM1L knockdown. ***P<0.001 vs. the scramble control. (C) Interaction between DNM1L and HK2 in Huh7 cells as determined by IP analysis. (D) Schematic illustration of the functions of DNM1L in hypoxia‑exposed HCC cells. DNM1L, dynamin‑1‑like; WB, western blotting; IP, immunoprecipitation; HCC, hepatocellular carcinoma; HK2, hexokinase 2; CypD, cyclophilin D; VDAC, voltage‑dependent anion channel; ANT, adenine nucleotide translocator. reciprocal interaction between DNM1L and HK2 binding to mitochondrial fission and decreased viability of HCC cells. mitochondria, and demonstrated that DNM1L knockdown In hypoxia‑exposed HCC cells, DNM1L also regulated mPTP prevented the loss of HK2 as well as mPTP opening (26). It opening via increasing mitochondrial detachment of HK2. has been suggested that the opposite translocation of DNM1L and HK2 may reflect a shared but competing binding region Acknowledgements on the OMM, thus regulating mPTP opening (27). These effects of DNM1L on mPTPRETRACTED opening have been demonstrated Not applicable. in ischemia and hypoxia‑exposed myocytes (27). In HCC cells, DNM1L‑mediated mitochondrial fission has also been Funding reported to increase the cytosolic calcium concentration and promote calcium oscillation (6), which might induce excessive This study was supported by the Science and Technology mPTP opening (13). Here, we provided direct evidence for Research Project of Chongqing Education (grant no: a connection between DNM1L and mPTP opening in HCC. KJ1600215) and the Preliminary National Natural Science Both DNM1L depletion and Mdivi treatment were able to Foundation of China at Chongqing Medical University (grant improve mPTP opening. However, the opening of mPTP chan- no: NSFYY201519). nels following hypoxia was not fully restored after DNM1L deletion or inhibition. A similar phenomenon was reported in Availability of data and materials dilated cardiomyopathy where mPTP channels remained open in DNM1L‑deleted myocardial mitochondria (28), indicating Not applicable. that critical factors other than DNM1L are involved in regu- lating mPTP opening in hypoxia‑exposed HCC cells. This Authors' contributions possibility requires further investigation. In conclusion, hypoxia induced mitochondrial trans- MC, PH and DLF conceived and designed the study. location of DNM1L in HCC cells, leading to excessive DLF prepared the manuscript. MC and PH performed the 1132 CAI et al: ROLE OF DNM1L IN HCC experiments. All authors participated in the discussion and 12. Livak KJ and Schmittgen TD: Analysis of relative expres- sion data using real‑time quantitative PCR and the 2(‑Delta Delta data interpretation. 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