Abstract
Background/Aim: Metformin is a widely used drug for type 2 diabetes mellitus and has recently attracted broad attention for its therapeutic effects on many cancers. This study aimed to investigate the molecular mechanism of metformin’s anticancer activity. Materials and Methods: Cell viability was measured by MTT assay. Gene and protein expression levels were determined by reverse transcription-polymerase chain reaction and western blot analyses, respectively. Results: Metformin and phenformin markedly induced NUPR1 expression in a dose- and time-dependent manner in H1299 non-small-cell lung cancer (NSCLC) cells. The silencing of NUPR1 in H1299 NSCLC cells enhanced cell sensitivity to metformin or ionizing radiation. Our previous report showed that metformin induces AKT serine/threonine kinase (AKT) activation in an activating transcription factor 4 (ATF4)-dependent manner and that the inhibition of AKT promotes cell sensitivity to metformin in H1299 NSCLC cells. Interestingly, ATF4-induced AKT activation in H1299 NSCLC cells treated with metformin was suppressed by the knockdown of NUPR1. Conclusion: Targeting NUPR1 could enhance the sensitivity of H1299 NSCLC cells to metformin by AKT inhibition.
Nuclear protein 1 (NUPR1, also known as com1 and p8) was originally identified in rat pancreatitis acinar cells, and has been shown to regulate pancreatitis progression (1). It is a stress-induced transcription factor that is implicated in diverse functions, including matrix remodeling, cell cycle regulation, DNA repair response, autophagy, senescence, and apoptosis (2-7). Furthermore, it is over-expressed in various types of cancers and is involved in the resistance to some chemotherapeutic drugs (8, 9). Moreover, NUPR1 has been reported to be a potential therapeutic target, since its genetic inactivation suppresses the growth of cancer cells, including pancreatic ductal adenocarcinoma, non-small-cell lung cancer (NSCLC), hepatocellular carcinoma, cholangiocarcinoma, glioblastoma, ovarian cancer, and gastric cancer (6, 10-17).
Metformin is a safe, inexpensive, and off patent biguanide compound that is the most widely prescribed drug for type 2 diabetes mellitus. Metformin inhibits mitochondrial electron transport chain complex I, resulting in an increased intracellular AMP/ATP ratio and AMP-activated protein kinase (AMPK) activation (18). Multiple clinical and epidemiological studies have suggested that metformin inhibits cancer cell growth and reduces the risk of development and progression of various tumors (19, 20). Metformin exerts its antitumor effects mainly by activating AMPK and inhibiting the mTOR signaling pathway in vitro and in vivo, causing apoptosis of cancer cells (21). More interestingly, metformin can not only induce cell death synergistically with traditional therapies, but it can also overcome therapeutic resistance (22-24). However, the underlying mechanism by which metformin exerts its anticancer effects on NSCLC cells remains unclear.
The present study aimed to clarify the function of NUPR1 in H1299 NSCLC cells treated with metformin. We showed that knockdown of NUPR1 enhanced the sensitivity of H1299 NSCLC cells to metformin. Furthermore, knockdown of NUPR1 down-regulated AKT activation in H1299 NSCLC cells treated with metformin. These results suggest that NUPR1 might serve as a potential anticancer target for NSCLC treatment with metformin.
Materials and Methods
Cell culture and reagents. H1299 non-small-cell lung cancer (NSCLC) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI 1640 medium (Welgene, Gyeongsangbuk-do, Republic of Korea) with 10% fetal bovine serum (FBS; Corning, NY, USA) in an incubator at 37°C with 5% CO2. Metformin, phenformin, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Gamma radiation was delivered using a dual-source 137Cesium unit at a dose rate of 3.2 Gy/min with a GC-3000 Elan irradiator (MDS Nordion, Ottawa, Canada).
MTT assay of cell viability. Cell viability was measured by using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. Cells were seeded in a 60 mm dish and grown overnight until they reached approximately 50% cell confluence. At the end of treatment, the cells were treated with MTT solution (final concentration 0.5 mg/ml) for 1 h at 37°C. The formazan crystals were dissolved in isopropanol, and the absorbance was measured at 570 nm. The results are expressed as the percent reduction in MTT, and the absorbance of the control cells was assumed to be 100%. The MTT experiments were repeated three times.
Transient transfection. NUPR1 (#1: 5’- AGGUCGCACCAAGAGAG AAdTdT-3’, #2: 5’-CUGGUGACCAAGCUGCAGAdT dT-3’, #3: 5’- GGAGGACCCAGGACAGGAUdTdT-3’) (25) and control (5’- CCUACGCCACCAAUUUCGUdTdT-3’) small interfering RNAs (siRNAs) were synthesized by Bioneer Corporation (Daejeon, Republic of Korea). Activating transcription factor 4 (ATF4) and control siRNAs were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The expression plasmid encoding mouse wild-type ATF4 (pEF/mATF4-myc) was kindly provided by Dr. Jawed Alam (26). Transfection with siRNAs and plasmids in H1299 cells was performed using Lipofectamine RNAiMAX and Lipofectamine Plus, respectively, according to the manufacturer’s instructions (Invitrogen; Thermo Fisher Scientific, MA, USA). Twenty-four hours after transfection, the cells were treated with 10 mM metformin or exposed to 10 Gy ionizing radiation. After 24 or 30 h of incubation, the cells were harvested for reverse transcription-polymerase chain reaction (RT-PCR) or western blot analysis, and cell viability was measured by MTT assay.
RNA extraction and RT–PCR analysis. RNA was extracted from H1299 cells using TRIzol reagent according to the manufacturer ‘s instructions (Invitrogen; Thermo Fisher Scientific). cDNA primed with oligo dT was prepared from 2 μg total RNA using M-MLV reverse transcriptase (Invitrogen; Thermo Fisher Scientific).
The following primers were used for PCR: NUPR1: 5’- GAGACGGGACTGCGGAGGAAG-3’ and 5’-GTTGCTGCCA CCCTGGAGGA-3’ (27), 242 bp product; β-Actin (ACTB) (5’- GGATTCCTATGTGGGCGACAG-3’ and 5’-CGCTCGGTGAGGA TCTTCATG-3’; 438 bp) (28). Amplification of NUPR1 was performed for 32 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 30 s. Amplification of ACTB was performed for 25 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 30 s. The PCR products were separated by 2% agarose gel electrophoresis. The PCR products on gels were stained with ethidium bromide, and the band intensities were quantified using ImageJ software (version 1.52a; NIH; National Institutes of Health, Bethesda, MD, USA).
Western blot analysis. Proteins from cell lysates were separated using 11% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes followed by immunoblotting with the specified primary and horseradish peroxidase-conjugated secondary antibodies. Protein bands were developed using SuperSignal West Pico Chemiluminescent Substrates (Thermo Scientific Pierce, Rockford, IL, USA) and the chemiluminescence signal was captured on X-ray film. The following antibodies were used: anti-ATF4 antibody obtained from Santa Cruz Biotechnology; anti-AKT and anti-p-AKT (Ser473) antibodies obtained from Cell Signaling Technology (Beverly, MA, USA); and anti-β-Actin obtained from Sigma-Aldrich (Merck KGaA).
Statistical analysis. The data are expressed as the mean±standard deviation. Statistical differences were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism software (version 9.0, San Diego, CA, USA); differences at p<0.05 were considered statistically significant.
Results
Metformin treatment increased NUPR1 expression levels in H1299 NSCLC cells. NUPR1 is a stress-inducible protein and, as mentioned before, one possible mechanism of action of sorafenib is induction of ER stress response. We first examined NUPR1 mRNA expression in metformin-treated H1299 NSCLC cells. H1299 cells were treated with increasing metformin doses (0, 2.5, 5, and 10 mM) for 8 h, and NUPR1 mRNA levels were analyzed by reverse transcription-polymerase chain reaction (RT–PCR). As shown in Figure 1A, metformin dose-dependently upregulated the mRNA levels of NUPR1. Metformin also increased NUPR1 mRNA expression in a time-dependent manner, as H1299 cells were treated with 10 mM metformin for 0, 6, 12, and 24 h (Figure 1B). Phenformin, a biguanide derivative similar to metformin, also increased NUPR1 mRNA expression in a dose- and time-dependent manner (Figure 1C and D). These data suggest that metformin induces NUPR1 expression in H1299 NSCLC cells.
Metformin and phenformin induce NUPR1 mRNA expression in H1299 non-small cell lung cancer cells. A, C) H1299 cells were treated with the indicated concentrations of metformin or phenformin for 8 h. B, D) H1299 cells were treated with 10 mM metformin or 200 μM phenformin for the indicated time. NUPR1 mRNA levels were estimated by reverse transcription-polymerase chain reaction. β-Actin was used as a loading control. The blot is representative of three independent experiments. NUPR1 expression was quantified using ImageJ software, the values were normalized against those of β-actin expression, and the fold change data were plotted.
Knockdown of NUPR1 enhanced the sensitivity of H1299 NSCLC cells to metformin or ionizing radiation. NUPR1 plays an essential role in the regulation of tumor cell growth and chemoresistance (8, 9). To investigate the functional role of NUPR1 in metformin-treated H1299 cells, H1299 cells were transfected with NUPR1 siRNAs, and the effects on cell viability were evaluated after metformin treatment. The three NUPR1 siRNAs led to considerable knockdown of NUPR1 mRNA expression in metformin-treated H1299 cells (Figure 2A). NUPR1 siRNA reduced cell viability after 48 h of transfection, and this effect was enhanced after 72 h of transfection (Figure 2B). NUPR1 siRNA enhanced metformin-mediated inhibition of cell viability (Figure 2C). Furthermore, the knockdown of NUPR1 resulted in enhanced cell viability inhibition in the cells treated with ionizing radiation (IR) (Figure 2D). These data suggest that the knockdown of NUPR1 enhances the sensitivity of H1299 NSCLC cells to metformin or IR.
Knocking down NUPR1 enhances the cell sensitivity to metformin or ionizing radiation in H1299 non-small cell lung cancer cells. A) H1299 cells were transfected with control or three different NUPR1 siRNAs for 24 h and then treated with 10 mM metformin for 24 h. NUPR1 mRNA levels were estimated by reverse transcription-polymerase chain reaction. β-Actin was used as a loading control. B) H1299 cells were transfected with control or NUPR1 siRNA and then incubated for the indicated times. C) H1299 cells were transfected with control or NUPR1 siRNA for 24 h and then treated with 10 mM metformin for 24 h. D: H1299 cells were transfected with control or NUPR1 siRNA for 24 h and then exposed to 10 Gy of ionizing radiation for 48 h. C, D) Cell viability was measured using MTT assay. Data are presented as the mean±SD (n=3; ***p<0.001). CTL: Control; IR: ionizing radiation.
Knockdown of NUPR1 suppressed the AKT activation induced by metformin. In our previous study, we observed that metformin induces AKT serine/threonine kinase (AKT) activation and that AKT inhibition enhances the sensitivity of H1299 NSCLC cells to metformin (29). AKT has also been reported to contribute to chemoresistance and radioresistance in NSCLC cells (30). Thus, we investigated whether the knockdown of NUPR1 would abolish metformin-induced AKT activation. As we previously reported (29), metformin induced AKT phosphorylation at Ser473 (Figure 3A). The knockdown of NUPR1 by siRNA resulted in a decrease in AKT phosphorylation at Ser473 induced by metformin (Figure 3B). These data suggest that NUPR1 knockdown reduces metformin-induced AKT activation.
Knocking down NUPR1 suppresses metformin-induced AKT phosphorylation in H1299 NSCLC cells. A) H1299 cells were treated with the indicated concentrations of metformin for 8 h. B) H1299 cells were transfected with control or NUPR1 siRNA for 24 h and then treated with 10 mM metformin for 24 h. The indicated protein levels were estimated by western blot analysis. β-Actin was used as a loading control. The blot is representative of three independent experiments. CTL: Control.
Metformin-induced AKT activation was regulated by ATF4-mediated NUPR1 expression. We have previously reported that metformin induces AKT activation by ATF4 in H1299 NSCLC cells (29). Thus, we investigated whether ATF4 knockdown abolished metformin-induced NUPR1 expression and AKT activation in these cells. Metformin induced ATF4 mRNA and protein expression in a time-dependent manner (Figure 4A). The over-expression of Myc-mATF4 resulted in increased levels of NUPR1 expression and AKT Ser473 phosphorylation compared with cells transfected with an empty vector (Figure 4B). The knockdown of ATF4 blocked metformin-induced NUPR1 expression and AKT phosphorylation at Ser473 (Figure 4C). ATF4 siRNA significantly reduced the viability of H1299 NSCLC cells exposed to metformin (Figure 4D). These data suggest that ATF4 knockdown enhances H1299 NSCLC cell sensitivity to metformin by down-regulating NUPR1 expression and AKT activation.
Knocking down ATF4 enhances cell sensitivity to metformin by reducing NUPR1 and AKT activation. A) H1299 cells were treated with 10 mM metformin for the indicated time. B) H1299 cells were transfected with an empty vector or a myc-tagged mATF4 (mATF4-myc) plasmid for 24 h and then treated with 10 mM metformin for 12 h. C) H1299 cells were transfected with control or ATF4 siRNA for 24 h and then treated with 10 mM metformin for 12 h. A-C) The indicated mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. β-Actin was used as a loading control. A representative of three independent experiments is shown. D) H1299 cells were transfected with control or ATF4 siRNA for 24 h and then treated with 10 mM metformin for 24 h. Cell viability was measured using MTT assay. Data are presented as the mean±SD (n=3; **p<0.01, ***p<0.001). CTL: Control.
Discussion
Metformin is a safe and inexpensive biguanide compound that has been widely used for the treatment of type 2 diabetes mellitus. Clinical and epidemiological reports have revealedthat the oral administration of metformin reduces the risk of the development and progression of various tumors (19). However, the underlying mechanism by which metformin exerts anticancer effects on cancer cells has not yet been fully elucidated. In this study, we found that metformin induced NUPR1 mRNA expression in a dose- and timedependent manner in H1299 non-small-cell lung cancer (NSCLC) cells (Figure 1). Phenformin, a biguanide derivative similar to metformin, also increased NUPR1 mRNA expression (Figure 1).
NUPR1 has recently received considerable attention due to its function in promoting cancer development and progression (8). NUPR1 is over-expressed in various types of cancers and confers resistance to chemotherapeutic drugs, including gemcitabine, paclitaxel, and doxorubicin (8, 9). Several studies have demonstrated that NUPR1 silencing suppresses the growth of cancer cells (6, 10-17). In this study, transfection of NUPR1 siRNA in H1299 NSCLC cells reduced cell viability and promoted cell sensitivity to metformin or ionizing radiation (Figure 2). These results indicate that NUPR1 expression is associated with a resistance to chemotherapy or radiotherapy in NSCLC cells.
Our previous studies showed that metformin induces AKT activation by ATF4 and that AKT inhibition enhances cell sensitivity to metformin in H1299 NSCLC cells (29, 31). Thus, we investigated whether the knockdown of NUPR1 abolishes metformin-induced AKT activation. As shown in Figure 3B, metformin-induced AKT activation was markedly suppressed by siRNA targeting NUPR1. Targeting NUPR1 could enhance the sensitivity of H1299 NSCLC cells to metformin by down-regulating AKT activation.
Acknowledgements
This research was supported by grants from the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by the Ministry of Science and ICT (MSIT) (50544-2022; 50531-2022) and from the National Research Foundation of Korea (NRF), funded by the MIST (NRF-2020M2D9A3094178), Republic of Korea.
Footnotes
Authors’ Contributions
Hyeon-Ok Jin and In-Chul Park developed the concept and designed the study. Yu Jin Kim, Se-Kyeong Jang, Sung-Eun Hong and Hyeon-Ok Jin carried out the experiments. Ki Soo Park and Chun-Ho Kim provided technical support and conceptual advice. Yu Jin Kim, Hyeon-Ok Jin, and In-Chul Park wrote the manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received May 12, 2022.
- Revision received June 7, 2022.
- Accepted June 8, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
