Metformin increases sensitivity of osteosarcoma stem cells to cisplatin by inhibiting expression of PKM2

  • Authors:
    • Depeng Shang
    • Ju Wu
    • Lianyi Guo
    • Yanju Xu
    • Lezi Liu
    • Jianmin Lu
  • View Affiliations

  • Published online on: April 5, 2017     https://doi.org/10.3892/ijo.2017.3950
  • Pages: 1848-1856
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Abstract

Multiple drug resistance is reported to be a major obstacle in treatment of osteosarcoma (OS). Research has demonstrated that small subsets of cells called cancer stem cells (CSCs) are responsible for multiple drug resistance. CSCs are potential targets for reversing chemoresistance. In the present study, we compared cisplatin sensitivity between OS stem cells and OS non-stem cells. We confirmed that OS stem cells showed significant cisplatin-resistance compared with the OS non-CSCs. Mechanically, we proved that overexpression of the pyruvate kinase isoenzyme M2 (PKM2) was responsible for the resistance to cisplatin in OS stem cells. As a potential strategy, we found that co-treatment with metformin significantly decreased the half maximal inhibitory concentration (IC50) of cisplatin to HOS OS stem cells by downregulating the expression of PKM2. PKM2 downregulation resulted in, metformin inhibited glucose uptake, lactate production and ATP production in HOS CSCs. Therefore, metformin impaired the resistance of HOS CSCs to cisplatin and promoted cisplatin-induced apoptosis. In addition, antitumor effects of other chemotherapeutic drugs such as doxorubicin and 5-fluorouracil were proved to be enhanced by metformin on OS stem cells.

Introduction

Osteosarcoma (OS) is a malignant bone cancer commonly diagnosed in children and adolescents (1). At present, chemotherapy is still an effective treatment to kill OS cells and prolong the life of patients. However, repeated use of chemotherapeutic drugs induces inevitable multiple drug resistance in cancer cells (2,3). Reliable strategies are urgently required to reverse the multiple drug resistance of OS.

Cancer stem cells (CSCs) are small cell subsets existing in cancer. Characteristics of CSCs include high self-renewal capacity and multilineage differentiation potential. They are important for tumor formation and development (4,5). CSCs are considered to be responsible for induction of acquired drug resistance. Continuous chemotherapy induces stemness in OS cells (68). CD133, a glycoprotein on cell surface, is identified as the molecular marker of cancer stem cells in many tumors including OS (9,10). CD133-positive OS cells are considered as effective targets for reducing chemoresistance (11).

Metformin is an anti-diabetic drug, which is the first-choice for patients with type-II diabetes (12). Of note, metformin therapy is shown to reduce cancer incidence in patient with diabetes. Metformin treatment reduces the risk of many tumors including breast cancer, lung cancer and colorectal cancers (1316). However, the mechanism by which metformin inhibits malignancies still remains unclear. Some studies demonstrate that metformin can reduce the production of ATP in cancer cells, therefore inhibiting cell cycle and proliferation of cancer (17,18). In this study, we investigated the potential role of metformin in OS stem cells. We report that metformin treatment can inhibit glucose metabolism and increase sensitivity of OS stem cells to chemotherapy.

Materials and methods

Cell culture

Human osteosarcoma cell lines HOS, Saos-2 and MG-63 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). For separation of cancer stem cells from OS cell lines, HOS, Saos-2 and MG-63 cells were incubated with fluorescein isothiocyanate (FITC) conjugated CD133 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 20 min at room temperature. Then, cells were washed with cold PBS twice followed by sorting the CD133-positive OS cells and CD133-negative OS cells on a FACS vantage (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). CD133-positive OS cells were considered as the OS stem cells, whereas the CD133-negative OS cells were identified as the OS non-stem cells (9). All of these cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco-Invitrogen, Carlsbad, CA, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNAs were extracted from HOS, Saos-2 and MG-63 OS cells using Trizol reagent (Invitrogen). cDNA of these cells was synthesized using M-MLV Reverse Transcriptase (Invitrogen) by taking total RNAs as templates. For detecting the expression of PKM2, Real-time PCR was performed using SYBR Premix Ex Taq (Takara, Shiga, Japan) on ABI PRISM 7900 sequence detection system (Applied Biosystems, Carlsbad, CA, USA). GAPDH gene was used as the internal reference to determine the relative expression of PKM2.

PKM2 plasmid and siRNA

To investigate the role of PKM2 in OS stem cells, we overexpressed or knocked down the PKM2 by transfecting with PKM2 plasmid or small interfering RNA (siRNA), respectively. For conducting PKM2 eukaryotic expression plasmid, open reading frame of PKM2 gene was amplified by PCR followed by linking the PCR products to pcDNA3.1 plasmid (Invitrogen). For knockdown of PKM2, siRNA targets to PKM2 was purchased from Genechem Co., Ltd. (Shanghai, China). For transfection, 2 μg/ml PKM2 plasmid or 50 pmol/ml PKM2 siRNA were transfected into the OS stem cells by using Lipofectamine 2000 (Invitrogen).

Cell viability and IC50

OS cells (1×104) were seeded in 96-well plates and cultured at 37°C. Cells were transfected with PKM2 vector or PKM2 siRNA. At 24 h after transfection, the cells were treated with 2 mM metformin and 8 μM cisplatin for 48 h, cell viability was measured by Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) assays. The absorbance was measured at 450 nm by using an ELISA microplate reader (Sunrise microplate reader; Tecan, Mannedorf, Switzerland). Half maximal inhibitory concentration (IC50) was calculated according to the results of CCK-8 assays.

Western blot analysis

Total proteins were extracted from OS stem cells and OS non-CSCs using radio immunoprecipitation assay (RIPA) lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). The proteins in cell lysates were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on the gels were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% non-fat dry milk for 2 h followed by incubating with primary antibodies against PKM2, pro-caspase-9, pro-caspase-3, cleaved caspase-9, cleaved caspase-3 and β-actin (Cell Signaling Technology, Inc.) overnight. Then, the proteins on PVDF were incubated with appropriate HRP-conjugated secondary antibodies followed by detecting the protein signals by using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Analysis of apoptosis

HOS CSCs were transfected with PKM2 vector followed by treating with 2 mM metformin and 8 μM cisplatin. The cells were stained using the Annexin V-FITC apoptosis detection kit (Sigma-Aldrich, St. Louis, MO, USA) and subsequently analyzed using flow cytometry (Becton-Dickinson, San Jose, CA, USA).

Proliferation assay

HOS CSCs were transfected with PKM2 vector followed by treating with 2 mM metformin and 8 μM cisplatin. Proliferation of HOS CSCs was measured by using 3H-thymidine incorporation assay.

Glucose uptake measurement

HOS CSCs were transfected with PKM2 vector followed by treating with 2 mM metformin and 8 μM cisplatin. Relative glucose uptake was measured using an Amplex Red Glucose/Glucose Oxidase assay kit (Molecular Probes, Camarillo, CA, USA) according to the manufacturer's instructions.

Detection of lactate production

HOS CSCs were transfected with PKM2 vector followed by treating with 2 mM metformin and 8 μM cisplatin. Production of lactate in HOS CSCs was detected by using a Lactate assay kit (BioVision, Milpitas, CA, USA) according to the manufacturer's protocol.

ATP measurement

HOS CSCs were transfected with PKM2 vector followed by treating with 2 mM metformin and 8 μM cisplatin. After treatment, cells were harvested and washed with PBS twice. NP40 solution (1%) was used to split the cells. ATP in the cell lysates were measured by using ATP Colorimetric/Fluorometric Assay kit (BioVision).

Tumor growth in nude mice

Four-week-old female immunodefcient nude BALB/c mice were purchased from Shanghai Super-B&K Laboratory Animal Corp., Ltd. (Shanghai, China). For xenografts, mice were injected subcutaneously into the right armpit with 5×106 HOS CSCs. Cisplatin (5 mg/kg) and metformin (200 mg/kg) were administered by intraperitoneal injection twice a week. Tumor size was measured every five days. Tumor volume was calculated according to the following formula: volume (V) = 1/2 × length × width2. For detection of PKM2 expression in tumor tissues, collagenase type III (Worthington Biochemical Corp., Lakewood, NJ, USA) was used to digest the tissues. The animal care and experimental protocols were approved by the Animal Care Committee of The Affiliated Zhongshan Hospital of Dalian University and complied with the recommendations of the Chinese guidelines for the care and use of laboratory animals. The study was approved by ethics committee of The Affiliated Zhongshan Hospital of Dalian University.

Statistical analysis

Data are presented as mean ± SD and analyzed by using SPSS 14.0. Two-tail Student's t-test and ANOVA were performed to determine the differences. A value of P<0.05 was considered to indicate a statistically significant difference. All the experiments were independently repeated at least 3 times.

Results

Osteosarcoma stem cells are resistant to cisplatin treatment

To investigate the cisplatin sensitivity of OS stem cells and OS non-stem cells, we first separated them from the HOS, Saos-2 and MG-63 OS cell lines. As shown in Fig. 1A, the separation efficiency was evaluated by flow cytometry. CD133-positive OS cells were considered as the OS stem cells. Whereas, the CD133-negative OS cells were identified as the OS non-stem cells. Results of CCK-8 assays showed that the sensitivity of OS stem cells (HOS CSCs, Saos-2 CSCs and MG-63 CSCs) to cisplatin was significantly lower than the OS non-stem cells (HOS non-CSCs, Saos-2 non-CSCs and MG-63 non-CSCs) (Fig. 1B). IC50 of cisplatin to OS CSCs (28.4 μM to HOS CSCs, 32.9 μM to Saos-2 CSCs and 26.3 μM to MG-63 CSCs) was significantly higher than their corresponding OS non-CSCs (6.1 μM to HOS non-CSCs, 8.8 μM to Saos-2 non-CSCs and 7.9 μM to MG-63 non-CSCs) (Fig. 1C). Taken together, we demonstrated that osteosarcoma stem cells showed resistance to cisplatin treatment.

Cisplatin-resistance in OS stem cells is dependent on the overexpression of PKM2

PKM2 plays an important role in tumor growth, development and chemoresistance (19,20). In our study, we found that the expression of PKM2 was significantly overexpressed in OS stem cells compared with that in their corresponding OS non-stem cells at mRNA (Fig. 2A) and protein levels (Fig. 2B). To investigate the role of the overexpressed PKM2, we transfected the HOS, Saos-2 and MG-63 CSCs with PKM2 siRNA. Effects of PKM2 siRNA on OS stem cells are shown in Fig. 2C. Noteworthy, we observed that knockdown of PKM2 by the specific siRNA restored the sensitivity of OS stem cells to cisplatin treatment (Fig. 2D). These results emphasized the key role of PKM2 in cisplatin-resistance in OS stem cells.

Metformin resensitizes HOS CSCs to cisplatin by down-regulating the expression of PKM2 in vitro

To investigate the potential role of metformin in the sensitivity of OS stem cells to cisplatin, we treated the HOS CSCs and non-CSCs with 8 μM cisplatin and 2 mM metformin for evaluating their synergistic effects. We found that metformin markedly enhanced the cytotoxicity of cisplatin to HOS CSCs, whereas the effect of metformin was slight on enhancing the cytotoxicity of cisplatin on HOS non-CSCs (Fig. 3A). Combination with metformin decreased the IC50 of cisplatin to HOS CSCs by 74.9%. In contrast, the reduction of cisplatin IC50 to HOS non-CSCs was 31.2% (Fig. 3B). These data demonstrated that metformin was able to enhance the antitumor effect of cisplatin, and HOS CSCs were more sensitive to metformin-promoted cell death than their corresponding HOS non-CSCs. Mechanically, results of western blot analysis showed that metformin treatment significantly downregulated the expression of PKM2 in HOS CSCs (Fig. 3C). We therefore inferred that synergistic effect of metformin on cisplatin-induced cell death was dependent on the inhibition of PKM2. To prove this inference, we transfected the HOS CSCs with PKM2 expression vector. We found that enforced expression of PKM2 'rescued' the HOS CSCs from the combination treatment with cisplatin and metformin (Fig. 3D). These results indicated that metformin had the ability to resensitize OS stem cells to cisplatin by targeting PKM2.

Metformin resensitizes OS CSCs to cisplatin in vivo

To investigate whether metformin enhanced the antitumor effect of cisplatin on OS CSCs in vivo, we established the in vivo model of OS by using HOS CSCs. We observed that single administration with cisplatin slightly reduced the tumor sizes in mice. Of note, although metformin single treatment did not reduce the tumor volume obviously, it strongly enhanced the antitumor effect of cisplatin on HOS CSC model (Fig. 4A and B). After analyzing the expression of PKM2 in tumor tissues formatted by HOS CSCs, we found that metformin significant decreased the expression of PKM2 in HOS CSC in vivo model (Fig. 4C). These results suggested that metformin have to the ability to sensitize the OS CSCs to cisplatin by inhibiting the expression of PKM2 in vivo.

Metformin weakens the glucose metabolism

As PKM2 is a key regulator of glucose metabolism in tumor cells (21), we next studied the effect of metformin on glucose metabolism in HOS CSCs. As shown in Fig. 5A and B, cisplatin treatment did not obviously influence the glucose metabolism in HOS CSCs. However, metformin treatment significantly decreased the consumption of glucose as well as increased the production of lactate in HOS CSCs. In addition, transfection with PKM2 vector restored the glucose uptake and lactate production in HOS CSCs treated with metformin. These results demonstrated that metformin had the ability to weaken the glucose metabolism by inhibiting the expression of PKM2. Furthermore, as the results of glucose metabolism reduction, metformin decreased the production of ATP in HOS CSCs (Fig. 5C).

Combination with metformin and cisplatin induce proliferation inhibition and apoptosis in HOS CSCs

In tumor cells, glucose metabolism and intracellular ATP regulate cell proliferation and chemoresistance (22,23). In our study, we found that combination with metformin and cisplatin induced drastic proliferation inhibition in HOS CSCs. However, enforced expression of PKM2 restored the proliferation of HOS CSCs which were co-treated with metformin and cisplatin (Fig. 6A). Furthermore, we observed that metformin treatment reduced the resistance of HOS CSCs to cisplatin-induced apoptosis. On the other hand, transfection with PKM2 vector impaired the effect of cisplatin plus metformin (Fig. 6B and C). Taken together, we demonstrated that metformin promoted cisplatin-induced proliferation inhibition and apoptosis in HOS CSCs by downregulating the expression of PKM2.

Effect of metformin on multidrug resistance in OS stem cells

Multidrug resistance is a major obstacle for chemotherapy of OS. We therefore treated OS stem cells (HOS CSCs, Saos-2 CSCs and MG-63 CSCs) with three kinds of chemotherapeutic drugs (cisplatin, doxorubicin and 5-fluorouracil). As shown in Fig. 7A, co-treatment with metformin significantly increased the sensitivity of HOS CSCs, Saos-2 CSCs and MG-63 CSCs to cisplatin, doxorubicin and 5-fluorouracil. Metformin treatment obviously decreased the IC50 of cisplatin, doxorubicin and 5-fluorouracil to HOS, Saos-2 and MG-63 OS stem cells (Fig. 7B). We demonstrated that metformin can increase the sensitivity of OS stem cells to chemotherapy.

Discussion

Cisplatin is a cytotoxic drug that is used for treatment of OS (24,25). Mechanism by which cisplatin kills tumor cells is dependent on the formation of cross-linking DNA by interacting with it. When the DNA repair mechanism in tumor fails to neutralize the DNA damage induced by cisplatin, apoptosis occurs (26,27). However, chemotherapy resistance often occurs in OS due to the repeated use of cisplatin (28). Recent studies demonstrate that one important factor in tumor recurrence is the existence of cancer stem cells (CSCs) which are resistant to chemotherapy (29,30). Similarly in the present study, HOS, Saos-2 and MG-63 CSCs exhibited significant resistance to cisplatin compared with their corresponding non-CSCs. We proved that OS stem cells showed high IC50 of cisplatin. In view of the high proliferation and oncogenicity of stem cells, increasing sensitivity of OS stem cells to chemotherapy may represent an effective strategy against treatment failure.

Pyruvate kinase isoenzyme M2 (PKM2) in cancer cells catalyzes the final step reaction of glycolysis by converting phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP. Therefore PKM2 is a key regulator of glucose metabolism in tumor cells (31). Recent studies indicate that overexpression of PKM2 is associated with chemoresistance. PKM2 inhibition can restore sensitivity of colon cancer cells, liver cancer cells and multiple myeloma cells to chemotherapy (20,32,33). In our study, we observed significant overexpression of PKM2 in OS stem cells rather than the non-CSCs. As PKM2 siRNA enhanced the cisplatin-induced cell death of HOS, Saos-2 and MG-63 CSCs, we proved that knockdown of PKM2 can restore the sensitivity of OS stem cells to cisplatin treatment. Metformin has been reported to show biological effects of reducing the occurrence of some cancers, and the mechanism is associated with the inhibition of PKM2 induced by metformin (34). Consistent with this, our results proved that metformin can decrease the expression of PKM2 in OS stem cells. Sensitization of metformin to cisplatin-induced cell death is dependent on the inhibition of PKM2.

PKM2 regulates glucose metabolism and ATP production in tumor cells (31). Previous research has demonstrated that intracellular ATP levels are the switch to chemoresistance. Bioprocesses of drug efflux, drug inactivation and DNA damage repair require the consumption of ATP. Therefore low-levels of ATP reduce the resistance of drug-induced apoptosis (23,35).

Herein, we demonstrated that metformin treatment reduced the production of ATP in OS stem cells by inhibiting the PKM2 expression. Subsequently, metformin significantly sensitized the OS stem cells to cisplatin-induced apoptosis and activation of caspase-9 and caspase-3. Furthermore, resistance of OS stem cells to some other chemotherapeutic agents such as doxorubicin and 5-fluorouracil was also inhibited by metformin co-treatment. It is proved that metformin/PKM2 axis is associated with chemosensitivity in OS stem cells.

In conclusion, we provide strong evidence that metformin mediates chemosensitivity in OS stem cells by targeting PKM2. These findings suggest that combination with metformin and chemotherapeutic agents is a promising strategy for treatment of OS.

Acknowledgments

Thanks are due to all the contributors who assisted with this study.

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May-2017
Volume 50 Issue 5

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Spandidos Publications style
Shang D, Wu J, Guo L, Xu Y, Liu L and Lu J: Metformin increases sensitivity of osteosarcoma stem cells to cisplatin by inhibiting expression of PKM2. Int J Oncol 50: 1848-1856, 2017
APA
Shang, D., Wu, J., Guo, L., Xu, Y., Liu, L., & Lu, J. (2017). Metformin increases sensitivity of osteosarcoma stem cells to cisplatin by inhibiting expression of PKM2. International Journal of Oncology, 50, 1848-1856. https://doi.org/10.3892/ijo.2017.3950
MLA
Shang, D., Wu, J., Guo, L., Xu, Y., Liu, L., Lu, J."Metformin increases sensitivity of osteosarcoma stem cells to cisplatin by inhibiting expression of PKM2". International Journal of Oncology 50.5 (2017): 1848-1856.
Chicago
Shang, D., Wu, J., Guo, L., Xu, Y., Liu, L., Lu, J."Metformin increases sensitivity of osteosarcoma stem cells to cisplatin by inhibiting expression of PKM2". International Journal of Oncology 50, no. 5 (2017): 1848-1856. https://doi.org/10.3892/ijo.2017.3950