Low glucose and metformin-induced apoptosis of human ovarian cancer cells is connected to ASK1 via mitochondrial and endoplasmic reticulum stress-associated pathways
Metformin, a first-line drug for type 2 diabetes, could induce apoptosis in cancer cells. However, the concentration of glucose affects the effect of metformin, especially low glucose in the culture medium can enhance the cytotoxicity of metformin on cancer cells. Since mitochondria and endoplasmic reticulum is vital for maintaining cell homeostasis, we speculate that low glucose and metformin-induced cell apoptosis may be associated with mitochondria and endoplasmic reticulum. ASK1, as apoptosis signaling regulating kinase 1, is associated with cell apoptosis and mitochondrial damage. This study was designed to investigate the functional significance of ASK1, mitochondria and endoplasmic reticulum and underlying mechanism in low glucose and metformin-induced cell apoptosis.
An MTT assay was used to evaluate cell viability in SKOV3, OVCAR3 and HO8910 human ovarian cancer cells. Cell apoptosis was analyzed by flow cytometry. The expression of ASK1 was inhibited using a specific pharmacological inhibitor or ASK1-siRNA. Immunofluorescence was used to detect mitochondrial damage and ER stress. Nude mouse xenograft models were given metformin or/and NQDI-1, and ASK1 expression was detected using immunoblotting. In addition, subcellular fractionation of mitochondria was performed to assay the internal connection between ASK1 and mitochondria.
The present study found that low glucose in culture medium enhanced the anticancer effect of metformin in human ovarian cancer cells. Utilization of a specific pharmacological inhibitor or ASK1-siRNA identified a potential role for ASK1 as an apoptotic protein in the regulation of low glucose and metformin-induced cell apoptosis via ASK1-mediated mitochondrial damage through the ASK1/Noxa pathway and via ER stress through the ROS/ASK1/JNK pathway. Moreover, ASK1 inhibition weakened the antitumor activity of metformin in vivo. Thus, mitochondrial damage and ER stress play a crucial role in low glucose–enhanced metformin cytotoxicity in human ovarian cancer cells.
These data suggested that low glucose and metformin induce cell apoptosis via ASK1-mediated mitochondrial damage and ER stress. These findings indicated that the effect of metformin in anticancer treatment may be related to cell culture conditions.
KeywordsMitochondrial damage ER stress ASK1 Metformin Ovarian cancer
Apoptosis signaling regulating kinase 1
- ER stress
Endoplasmic reticulum stress
- MMP, ΔΨm
Mitochondrial membrane potential
Reactive oxygen species
Ovarian cancer remains one of the most common gynecological tumors . Most patients with ovarian cancer are diagnosed at an advanced stage of III or IV, which hinders effective treatment in the clinic . The first-line chemotherapy for advanced ovarian cancer is cisplatin, but subsequent drug resistance minimizes the effectiveness of cisplatin and many other chemotherapy drugs . Therefore, there is a critical need for novel approaches for the effective treatment of ovarian cancer.
Recent epidemiological evidence has shown that ovarian carcinogenesis is negatively correlated with obesity [4, 5]. Some groups have focused on “reprogramming of energy metabolism” as a hallmark of cancer and found that targeting cancer metabolism inhibits cancer cell growth . Dr. Otto Warburg has previously reported that the underlying metabolism of malignant cancer is different from that of adjacent normal tissue  and that cancer cells are mainly dependent on glycolysis for glucose metabolism even in the presence of oxygen. Glycolysis provides ATP with low efficiency, but it supplies sufficient intermediates for the biosynthesis of nucleotides, NADPH, and amino acids . Thus, a high rate of glucose uptake is required for the survival of cancer cells. As a result, the glucose level influences the effect of cancer treatment. High glucose promotes the proliferation of cancer cells, whereas reduced glucose enhances the cytotoxicity of therapeutic drugs, such as metformin, in several cancers, including ovarian cancer . Moreover, Zhuang Y et al. found low glucose and metformin treatment in cancer cells leads to cell death by decreasing ATP production and inhibiting survival signaling pathways . In general, the culture medium of cancer cells contains high glucose (25 mM), which is the optimal environment facilitating cancer cell growth. The normal level of serum glucose is approximately 4–6 mM, but the glucose level of cancer cell culture medium is decreased to 2.5 mM [9, 10]. Thus, caloric restriction and even starvation can effectively reduce the growth of cancer cells [11, 12]. As a biguanide drug, metformin is commonly considered as an effective treatment for type 2 diabetes, mainly due to its glucose-lowering effect . Studies have confirmed that metformin increases the ratios of both ADP/ATP and AMP/ATP, resulting in a decreased cellular energy level through specific inhibition of mitochondrial respiratory-chain complex 1 [14, 15, 16, 17]. In the response to metformin-induced energetic stress, the byproducts of mitochondrial respiration, reactive oxygen species (ROS), damage cellular components, such as mitochondria, leading to cell death in high concentrations . ROS accumulation-induced cell death is associated with ASK1 . ASK1 is a ubiquitously expressed MAP3K and can be activated by various stressors, such as oxidative stress, lipopolysaccharide and ATP . ASK1 activation selectively results in sustained Jun N-terminal kinase (JNK) activation, which is associated with ER stress.
ER stress can be induced by glucose deprivation, and hypoglycemia induces the ER stress–unfolded protein response (UPR) system in retinal pericytes [20, 21, 22, 23]. ER stress is usually activated in response to various stressors, including low glucose level, and either promotes cell survival or induces cell death in cancer cells [24, 25, 26]. When cells show altered glucose metabolism from glycolysis, ER stress is further exacerbated by glucose insufficiency . Initiation of adaptive ER stress protects stressed cells from apoptosis through maintaining cell homeostasis. Grp78, which is also called immunoglobulin heavy chain binding protein (BiP), is a key factor in the ER stress process and is detected under conditions of ER stress-inducing agents [28, 29]. An elevated level of GRP78 not only indicates elimination of the harmful components of various stresses in cancer cells, but it may also reflect a change in cancer cell metabolism, such as the Warburg effect . However, severe ER stress leads to cell death despite a high Grp78 expression levels. CCAAT/enhancer binding protein homologous transcription factor (CHOP), which is also called growth arrest and GADD153, plays a decisive role in this process . The expression of CHOP in normal cells is difficult to detect, but severe ER stress induces CHOP transcription. The acute increase of CHOP expression leads to activation of the mitochondria-mediated apoptosis pathway . However, the mechanism of ER stress induction under conditions of low glucose and metformin remains poorly understood.
The present study showed that human ovarian cancer cells were insensitive to metformin alone but that low glucose enhanced the cytotoxicity of metformin in these cells. Moreover, the mechanism of low glucose and metformin-induced cell apoptosis was investigated. Low glucose and metformin caused the accumulation of ROS, which led to mitochondrial dysfunction and activation of ASK1 and JNK. Activated ASK1 was involved in low glucose and metformin-induced mitochondrial dysfunction and ER stress. Sustaining ER stress induced ovarian cancer cells to undergo apoptosis. Thus, mitochondrial damage and ER stress together played a crucial role in low glucose–enhanced metformin cytotoxicity in human ovarian cancer cells. Moreover, ASK1 was involved in the anti-tumor effect of metformin in vivo. These data suggested that low glucose and metformin induce cell apoptosis via ASK1-mediated mitochondrial damage and ER stress. These findings suggested that changes in cellular conditions combined with metformin treatment may represent a novel treatment strategy for the clinical treatment of cancer. The present study aided the understanding of the mechanism of metformin resistance.
Materials and methods
SKOV3, OVCAR3 and HO8910 human ovarian cancer cells were purchased from American Tissue Culture Collection (ATCC; Rockville, MD). Cells were cultured in RPMI-1640 medium (GIBCO, Carlsbad, CA), supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37 °C with 5% CO2.
Cell viability assays
SKOV3, OVCAR3 and HO8910 human ovarian cancer cells were seeded into 96-well microplates at a density of 1 × 104 cells/well in 100 μl of complete medium and cultured overnight. Cells were then treated with varying concentrations of glucose and metformin for 24 h or 48 h. An MTT assay was used to detect cell viability by adding 20 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 5 mg/ml in PBS) to each well for 4–6 h. To each well, 150 μl of dimethyl sulfoxide was added (Beijing Chemical Industry Limited Company, China). The absorbance was detected at a wavelength of 570 nm using a Vmax Microplate Reader (Molecular Devices, Sunnyvale, CA).
Immunofluorescent confocal laser microscopy
Cells were seeded into a 24-well microplate and incubated overnight, and cells were then treated with different concentrations of glucose and metformin for the indicated periods. Cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 20 min and blocked with 10% goat serum for 30 min. Cells were then incubated overnight at 4 °C with the following primary antibodies: PDI, GADD153, and cleaved caspase 3 (1:100). Cells were then incubated with FITC/Texas Red-conjugated secondary antibodies (1:200) (Santa Cruz Biotechnology, CA) for 30 min, and cell nuclei were stained with Hoechst 33342 (Sigma–Aldrich, St. Louis, MO) for 2 min. Cells were then washed 3 times with PBS and then visualized using confocal fluorescence microscopy.
Reactive oxygen species (ROS) assays
The intracellular levels of reactive oxygen species (ROS) in cells treated with low glucose and metformin were measured using dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma) and flow cytometric analysis. As DCFH-DA is cell permeable, it interacts with intracellular ROS when it enters cells to generate fluorescent dichlorofluorescein. Cells (1 × 104 cells/well) were seeded into 24-well microplates and incubated overnight. After treating cells with low glucose and metformin, cells were washed 3 times with cold phosphate-buffered saline (PBS) and then incubated with 5 μM DCFH-DA solution for 15 min at 37 °C. Cells were then washed 3 times with PBS and subsequently analyzed by flow cytometry.
Western blot analysis
After harvesting cells treated with low glucose and metformin, cells were washed with cold PBS. Ice-cold RIPA buffer was added to cells, and cells were sonicated for 30 s on ice and lysed at 4 °C for 1 h. After centrifugation at 12,000 g for 45 min, the supernatant was collected. Protein concentration was detected by the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). Proteins (25–45 μg) were separated by SDS electrophoresis and transferred onto PVDF membranes. PVDF membranes were blocked with nonfat dry milk in buffer for 60 min at room temperature and incubated overnight at 4 °C with primary antibodies. After incubating with horseradish peroxidase-conjugated secondary antibody (1:2000; Thermo, Waltham, MA), membranes were washed 3 times with PBST. Immunoreactive proteins were detected using ECL reagents and visualized by Syngene Bio Imaging (Synoptics, Cambridge, UK). Protein levels were quantified using Quantity One software (Bio-Rad).
Apoptosis analysis by flow cytometry
Cell apoptosis was assessed using the FITC-Annexin V Apoptosis Detection Kit (Beyotime Institute of Biotechnology) and flow cytometry according to the manufacturer’s instructions. Cells were seeded into 6-well microplates and incubated overnight, and cells were treated with glucose and metformin for the indicated periods. Cells were collected and then incubated Annexin V-FITC and propidium iodide (PI) (Vybrant; Invitrogen, Karlsruhe, Germany) for 15 min at 37 °C in the dark. Apoptotic cells were quantitated by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA).
Caspase 3 activity assay
Human ovarian cancer cells were treated with low glucose and metformin in different conditions. After harvesting cells, caspase 3 activity was measured using a colorimetric assay kit (Beyotime Biotechnology Limited Company, Shanghai, China) according to the manufacturer’s instructions.
The knockdown of the ASK1 gene in SKOV3 cells was performed using siRNA. The ASK1 siRNA and negative control siRNA were obtained from GenePharma (Shanghai, China). The siRNA sequences were as follows: human ASK1 #1, 5’-GCCAACACUACAGUCAGGAAUUAAU-3′; human ASK1 #2, 5’-UGAAGCUAAGUAGUCUUCUUGGUAA-3′, and control siRNA (Scramble), 5’-ACGUGACACGUUCGGAGAAdTdT-3′ [32, 33]. Cell lines were transfected using Lipofectamine 2000 (Invitrogen).
Mitochondrial membrane potential (MMP, ΔΨm) assay
Human ovarian cancer cells were treated with low glucose and metformin in different conditions. After harvesting, cells were exposed to JC-1 solution (Biotrend, Cologne, Germany) for 30 min at room temperature in the dark. Green fluorescence (520–530 nm) and red fluorescence (> 550 nm) were measured by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA).
After treating with low glucose and metformin, human ovarian cancer cells were harvested, and the pure mitochondrial fraction was extracted using a Mitochondria Isolation Kit (Beyotime Biotechnology, Shanghai, China). The experiment was performed three times.
Ovarian cancer tumor xenografts in female nude mice
Athymic BALB/c female nude mice (4 weeks old and weight of 20 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). A human ovarian cancer xenograft model in female nude mice was established through subcutaneously injecting SKOV3 cells into the right flank of each mouse. Mice were randomly divided into control (n = 5) and treatment groups (n = 5) as follows: control group; NQDI-1 alone (1 mg/kg/day by intraperitoneal injection); metformin alone (200–600 mg/kg/day by gavage); and combination of NQDI-1 and metformin. Treatment was started when the tumor reached approximately 150–200 mm3. Tumor diameter was measured every 3 days using a caliper, and the tumor volume was calculated according to the following formula: length × width × height × 0.5. After treatment with NQDI-1 or metformin, tumors were excised, and tumor weights were measured .
Immunohistochemical staining was performed on ovarian cancer tumor xenografts models . The tissue sections of of xenograft tumour were deparaffinized with xylene and dehydration in a graded alcohol series. After, antigen retrieval by microwaving in pH 6.0 citrate buffer for 10 min, inhibiting endogenous peroxidase activity was performed. After blocking nonspecific binding, the slides were incubated with primary antibody overnight at 4 °C in a humidified container. According to the manufacturer’s recommendations, immunoreactivity was detected through using diaminobenzidine (DAB) staining. Brown signals mainly in the cytoplasm were defined positive reactions.
All data represent at least 3 independent experiments and are presented as the means ± SD. Data were analyzed using one-way ANOVA. P-values less than 0.05 were considered statistically significant.
Low glucose in culture medium enhances sensitivity of ovarian cancer cells to metformin
Low glucose enhances metformin-induced apoptosis through a mitochondria-associated pathway
Low glucose and metformin-induced mitochondrial damage is associated with the ASK1/Noxa pathway
Low glucose and metformin-induced activation of ASK1 is associated with ROS
Low glucose and metformin activate ER stress through ROS and ultimately trigger ER stress-associated apoptosis
Low glucose and metformin activate ER stress through the ROS/ASK1/JNK pathway
ASK1 is involved in the anti-tumor effects of metformin: In vivo xenograft models
Metformin is commonly used in the treatment of cancer and is thought to work through targeting mitochondria. However, recent studies have revealed that its cytotoxicity is enhanced by low glucose in various cancers, including ovarian cancer [9, 14, 47]. Glucose at a concentration of 25 mM, which is the normal level in cancer cell culture, can maintain typical plasma levels of 5–7 mM glucose and provide the optimal environment to promote cancer cell growth. As an anticancer agent, metformin weakly induces cancer cell apoptosis. However, under cell culture conditions with low glucose, the cytotoxicity of metformin increases cell apoptosis. The present study found that ovarian cancer cells were more sensitive to metformin at a glucose concentration of 2.5 mM compared to 25 mM. Glucose deprivation has been previously shown to increase metformin-induced cell death in breast cancer cells [27, 48, 49]. Low glucose and metformin-induced apoptosis of ovarian cancer cells was a consequence of ROS accumulation, which lead to mitochondria damage. ROS accumulation induced activation of ASK1, which, in turn, triggered the localization change in the pro-apoptotic protein, Noxa. Mitochondrial localization of Noxa protein lead to mitochondrial dysregulation, which was a key determinant for the initiation of apoptosis. Moreover, the present study demonstrated that ROS-ASK1 and JNK was associated with the modulation Noxa expression induced by low glucose and metformin. ASK1 activation was involved in the loss of ΔΨm, caspase 3 cleavage, and the subsequent release of cytochrome c. Therefore, the low glucose and metformin-induced mitochondrial associated apoptosis was a result of ROS-mediated ASK1 activation, which induced the phosphorylation of JNK, subsequently triggering the initiation of ER stress.
ER stress is identified as a critical factor in glucose deprivation–induced apoptosis through the ER stress pathway, and sustained and severe ER stress can trigger caspase-mediated apoptosis . Oxidative injury, hypoglycemia, hypoxia, ER-Ca2+ depletion, high-fat diet, and viral infections may lead to an imbalance of ER homeostasis, thereby triggering ER stress. Moreover, the presence of ER stress induces mitochondrial dysfunction, which may activate cascades of the apoptotic signaling pathway [50, 51, 52]. However, the exact mechanisms of cell death induced by low glucose and metformin are unclear. According to the present data, metformin induced the elevation of ROS accumulation under the condition of 2.5 mM glucose, and NAC, a ROS scavenger, significantly reduced the degree of ER stress. Several studies have shown that a metformin-mediated ER stress response does not lead to cell apoptosis . In contrast, other studies have reported that metformin triggers ER stress-dependent apoptosis . The presence of prolonged or sustained ER stress activates the program of cell apoptosis . Metformin reduces cellular respiration through inhibiting the respiratory-chain complex 1 (NADH: ubiquinone oxidoreductase) and, thus, affects mitochondrial function . Moreover, recent studies have found that high glucose in the culture medium may partially cause metformin resistance and decrease the level of glucose-enhanced metformin cytotoxicity in cancer cells [48, 53]. In the present study, under the condition of 25 mM glucose, metformin-induced ER stress did not lead to cell apoptosis. However, low glucose enhanced metformin-mediated ER stress and cell apoptosis. TUDC, an inhibitor of ER stress, significantly decreased apoptosis induced by 2.5 mM glucose and metformin. Moreover, NQDI-1, an inhibitor of ASK1, reduced the ER stress induced by the combination of 2.5 mM glucose and metformin. Moreover, the activation of ASK1 lead to JNK phosphorylation. Consistently, metformin induced ER stress and cell apoptosis, and ASK1 played an important role in the anti-tumor effect of metformin on ovarian cancer tumor xenografts models, but we did not detect this effect in ovarian cancer cells elsewhere. Therefore, low glucose and metformin activated ER stress through the ROS/ASK1/JNK pathway in ovarian cancer cells.
In summary, ovarian cancer cells are insensitive to metformin under high glucose conditions, but low glucose enhances the cytotoxicity of metformin in these cells (Fig. 7g). Low glucose and metformin cause ROS accumulation, which, in turn, activates ASK1 and JNK. The activation of ASK1 triggers Noxa protein localization to mitochondria, further leading to mitochondrial dysregulation and ultimately cell apoptosis. Thus, low glucose and metformin induce mitochondrial dysregulation via ROS/ASK1/Noxa. ASK1-mediated activation of JNK induces sustained ER stress, and severe ER stress leads to apoptosis of ovarian cancer cells. Therefore, low glucose and metformin activate ER stress through the ROS/ASK1/JNK pathway. In conclusion, mitochondrial dysregulation and ER stress play an important role in low glucose–enhanced metformin cytotoxicity in human ovarian cancer cells. These results suggested that cell culture conditions may be an important determinant of metformin resistance in the treatment of some cancers.
The present findings indicated that mitochondrial damage and ER stress play a crucial role in low glucose–enhanced metformin cytotoxicity in human ovarian cancer cells. Moreover, ASK1 is involved in low glucose and metformin-induced mitochondrial damage and ER stress. These results suggested that changes in cellular conditions combined with metformin treatment may represent a novel treatment strategy for the clinical treatment of cancer.
We thank American Journal Experts(https://secure.aje.com/certificate) for editing the English of this manuscript.
The present study was supported by a grant from the National Natural Science Foundation of China (Grant No. 81702780 and U1804172).
Availability of data and materials
All data generated or analyzed during the present study were included. Further materials are available from the corresponding author upon request.
LM and JW designed the experiments and wrote the manuscript. LM conceived the concept. JW and WW performed the immunoblotting and immunofluorescence assays. LW performed the RNA interference experiments. YY and ZY established the animal models. XL analyzed the data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.
Consent for publication
The authors declare that they have no competing interests.
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