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Shikonin Inhibits the Warburg Effect, Cell Proliferation, Invasion and Migration by Downregulating PFKFB2 Expression in Lung Cancer

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Shikonin Inhibits the Warburg Effect, Cell Proliferation, Invasion and Migration by Downregulating PFKFB2 Expression in Lung Cancer MOLECULAR MEDICINE REPORTS 24: 560, 2021 Shikonin inhibits the Warburg effect, cell proliferation, invasion and migration by downregulating PFKFB2 expression in lung cancer LIYING SHA1, ZHIQIANG LV1, YUJUN LIU1, YUN ZHANG2, XIN SUI1, TENG WANG1 and HUI ZHANG1 Departments of 1Pharmacy and 2Business, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China Received June 24, 2020; Accepted February 4, 2021 DOI: 10.3892/mmr.2021.12199 Abstract. Lung cancer is one of the most lethal diseases differences in lung cancer cell migration, apoptosis, glucose and therefore poses a significant threat to human health. uptake, lactate levels and ATP levels between cells with The Warburg effect, which is the observation that cancer knocked down PFKFB2 expression or treated with shikonin cells predominately produce energy through glycolysis, even and the knockdown of PFKFB2 in cells treated with shikonin. under aerobic conditions, is a hallmark of cancer. 6‑phospho‑ In conclusion, the results of the present study revealed that fructo‑2‑kinase/fructose‑2,6‑biphosphatase 2 (PFKFB) is an shikonin inhibited the Warburg effect and exerted antitumor important regulator of glycolysis. Shikonin is a Traditional activity in lung cancer cells, which was associated with the Chinese herbal medicine, which has been reported to exert downregulation of PFKFB2 expression. antitumor effects. The present study aimed to investigate the anticancer activity of shikonin in lung cancer. Cell Counting Introduction Kit‑8 (CCK‑8) and colony formation assays were used to analyze proliferation in A549 and H446 cells. Wound healing Lung cancer is one of the most common causes of cancer‑related and Transwell assays were used to measure migration and mortality, accounting for 18.4% of cancer‑related deaths invasion in A549 and H446 cells. Cell apoptosis was analyzed worldwide (1). In total, ~85% of lung cancer cases are diag‑ using flow cytometry. Lactate levels, glucose uptake and nosed as non‑small cell lung cancer (NSCLC) and ~15% of cellular ATP levels were measured using their corresponding lung cancer cases are diagnosed as small cell lung cancer (2). commercial kits. Western blotting was performed to analyze At present, the most common treatment methods for lung the protein expression levels of key enzymes involved in cancer include surgical resection, chemotherapy, radiotherapy, aerobic glucose metabolism. Reverse transcription‑quantita‑ immunotherapy and targeted drugs (3). Although targeted tive PCR was used to analyze the mRNA expression levels therapies, such as EGFR inhibitors, and immunotherapy drugs, of PFKFB2. The results of the present study revealed that such as anti‑programmed cell death protein 1/programmed PFKFB2 expression levels were significantly upregulated in death‑ligand 1, have significantly improved the treatment of NSCLC tissues. Shikonin treatment decreased the prolifera‑ lung cancer, only a small number of patients are eligible to be tion, migration, invasion, glucose uptake, lactate levels, ATP treated with targeted therapies, thus the global 5‑year survival levels and PFKFB2 expression levels and increased apoptosis rate of lung cancer remains at ~20% (4‑8). Therefore, iden‑ in lung cancer cells in a dose‑dependent manner. The over‑ tifying specific and sensitive prognostic markers, therapeutic expression of PFKFB2 increased the proliferation, migration, targets and devising novel therapeutic drugs to improve the glucose uptake, lactate levels and ATP levels in lung cancer survival time and quality of life of patients with lung cancer cells, while the knockdown of PFKFB2 expression exerted remains a priority. the opposite effects. Moreover, there were no significant The Warburg effect is defined by the altered metabolism of glucose, whereby tumor cells produce large amounts of lactate, even in the presence of oxygen and fully functioning mito‑ chondria (9). The intrinsic mechanism of the Warburg effect is complex, and is associated with the activation of numerous Correspondence to: Dr Hui Zhang, Department of Pharmacy, The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Shinan, oncogenes, inactivation of numerous tumor suppressor genes, Qingdao, Shandong 266003, P.R. China the abnormal expression of glycosylase and changes in the E‑mail: [email protected] tumor microenvironment, amongst other factors (10‑12). However, the specific mechanism requires further investiga‑ Key words: shikonin, Warburg effect, 6‑phosphofructo‑2‑ tion. It is well established that the cell death program (apoptosis) kinase/fructose‑2,6‑biphosphatase 2, migration, lung cancer exerts an anticancer effect (13). The Warburg effect is closely associated with the occurrence and development of cancer, as it provides a growth advantage to tumor cells, helping them to 2 SHA et al: ROLE OF SHIKONIN IN LUNG CANCER escape apoptosis and create a suitable environment for tumor ATT‑3'), siRNA‑PFKFB2 (sense, 5'‑AUU GUC AUG CCG AAA metastasis to occur (14‑16). The inhibition of the Warburg GAA GUC‑3' and antisense, 5'‑CUU CUU UCG GCA UGA effect promotes cell apoptosis and inhibits tumor proliferation CAA UGA‑3'), pcDNA3.1‑PFKFB2 and pcDNA3.1‑NC (all and metastasis (17). Several key metabolic enzymes [pyruvate Shanghai GenePharma Co., Ltd.) were used to knockdown kinase M2 (PKM2), pyruvate dehydrogenase kinase 1 (PDK1) or overexpress PFKFB2, respectively. Cells were transfected and hexokinase 2] have been found to play an important role with siRNA (30 nM) or overexpression vectors (1 µg) using in the Warburg effect (18‑20). The changes in key enzymes Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher can lead to an enhanced glycolytic ability, promote glucose Scientific, Inc.) for 48 h at 37˚C. Cells were harvested 48 h uptake into tumor cells and increase the accumulation of post‑transfection for use in subsequent experiments. lactic acid, thus further supporting tumor growth and develop‑ ment (21). 6‑Phosphofructo‑2‑kinase/fructose‑2,6‑biphosphatase Reverse transcription‑quantitative PCR (RT‑qPCR). (PFKFB2) is an enzyme that regulates the synthesis and Transfected or untransfected A549 and H446 cells were degradation of fructose‑2,6‑bisphosphate (Fru‑2, 6‑P2), treated with or without 50 µM shikonin (Shanghai Yuanye which is widely expressed in a variety of cancer cells, such as Biotechnology Co., Ltd.) for 24 h at 37˚C. Total RNA was ovarian, breast and pancreatic cancer cells (22,23). By regu‑ extracted from clinical specimens or cells using TRIzol® lating the expression of Fru‑2, 6‑P2 (a signaling metabolite reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according that participates in glycolysis), the PFKFB family has been to the manufacturer's protocol. Total RNA was reverse found to regulate intracellular glycolysis in cancer by acting as transcribed into cDNA using a PrimeScript RT kit (Takara an oncogene (24,25). Biotechnology Co., Ltd.), according to the manufacturer's Shikonin is a naphthalene quinone compound extracted protocol. qPCR was subsequently performed on an ABI 7500 from Lithospermum erythrorhizon (an herbaceous plant), Real‑Time PCR system (Applied Biosystems; Thermo Fisher which has demonstrated anti‑inflammatory, antiviral, anti‑ Scientific, Inc.) using a SYBR® Premix Ex Taq™ kit (Takara tumor and wound healing potential, amongst other biological Biotechnology Co., Ltd.). The following thermocycling condi‑ activities (26). The present study aimed to investigate the tions were used for the qPCR: Initial denaturation at 95˚C for effects of shikonin on cell proliferation, migration, inva‑ 3 min; followed by 40 cycles at 95˚C for 5 sec and 60˚C for sion, apoptosis and aerobic glycolysis in lung cancer cells, 30 sec. The following primers pairs (Sangon Biotech Co., Ltd.) and further determined the potential underlying molecular were used for the qPCR: PFKFB2 forward, 5'‑GCT GCT TGG mechanisms of shikonin. The current findings may provide a TGG GAG TGA TAA‑3' and reverse, 5'‑TGA GAA GCC AAG foundation for the clinical use of shikonin in the treatment of TGT CAG GG‑3'; and β‑actin forward, 5'‑GAG GAC CCT GGA lung cancer. TGT GAC AG‑3' and reverse, 5'‑AAG ACC TGT ACG CCA ACA CA‑3'. Relative mRNA expression levels were quantified using Materials and methods the 2‑ΔΔCq method (27) and normalized to β‑actin. Patient studies. A total of 20 NSCLC and adjacent normal Western blotting. A549 and H446 cells were treated with tissues were obtained from patients (10 women and 10 men; shikonin [0 (control), 10, 20 or 50 µM] for 24 h at 37˚C, then age range, 50‑79 years) who underwent surgical resection at total protein was extracted from cells using RIPA lysis buffer The Affiliated Hospital of Qingdao University (Qingdao, (Santa Cruz Biotechnology, Inc.) supplemented with protease China) between January 2019 and December 2019. Patients and phosphatase inhibitor mixture. Total protein was quanti‑ who had received chemotherapy or radiotherapy prior to fied using a BCA assay and 50 µg protein/lane was separated surgery were excluded from the study. Patients who were via 10% SDS‑PAGE. The separated proteins were subse‑ diagnosed by positron emission tomography/CT scan and quently transferred onto PVDF membranes (EMD Millipore) were eligible for radical surgery were included in the study. and blocked with 5% skim milk for 2 h at room temperature. All samples were stored at ‑80˚C until required for RNA The membranes were then incubated with the following extraction. The study protocols were approved by the Ethics primary antibodies at 4˚C overnight: Anti‑PFKFB2 Committee of The Affiliated Hospital of Qingdao University, (1:1,000; cat. no. ab234865; Abcam), anti‑PDK1 (1:2,000; and written informed consent was obtained from all patients cat. no. ab202468; Abcam), anti‑glucose transporter 1 (GLUT1; prior to participation. 1:200; cat. no. ab150299; Abcam), anti‑phosphoglycerate kinase 2 (PGK2; 1:1,000; cat. no. ab183031; Abcam), anti‑lactate Cell lines and culture. The Beas‑2B lung epithelial cell line dehydrogenase A (LDHA; 1:1,000; cat. no. ab101562; Abcam), and lung cancer cell lines, A549 and H446, were obtained anti‑PKM2 (1:1,000; cat.
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