Induction of Mitochondrial Dysfunction and Oxidative Damage by Antibiotic Drug Doxycycline Enhances the Responsiveness of Glioblastoma to Chemotherapy

Qian Tan, Xiaoqiong Yan, Lin Song, Hongxiang Yi, Ping Li, Guobin Sun, Danfang Yu, Le Li, Zheng Zeng, Zhenlin Guo, Qian Tan, Xiaoqiong Yan, Lin Song, Hongxiang Yi, Ping Li, Guobin Sun, Danfang Yu, Le Li, Zheng Zeng, Zhenlin Guo

Abstract

BACKGROUND Inducing mitochondrial dysfunction has been recently demonstrated to be an alternative therapeutic strategy for cancer treatment. Doxycycline is an antibiotic that has been shown to have anti-cancer activities in various cancers by way of targeting mitochondria. In this work, we examined whether doxycycline can be repurposed for glioblastoma treatment. MATERIAL AND METHODS The effects of doxycycline on the growth, survival, and mitochondrial metabolisms of glioblastoma were investigated. The efficacy of a combination of doxycycline with temozolomide was examined using xenograft mouse model in total number of 40 mice. RESULTS Doxycycline targeted glioblastoma cell lines, regardless of their origin, through inhibiting growth and inducing cell death, accompanied by a significant decrease in proliferating cell nuclear antigen (PCNA) and increase in cleaved caspase-3. In addition, doxycycline significantly sensitized glioblastoma cell response to temozolomide in vitro and in vivo. Mechanistically, doxycycline disrupted mitochondrial functions through decreasing mitochondrial membrane potential and mitochondrial respiration. Inducing mitochondrial dysfunctions by using doxycycline led to energy crisis, oxidative stress, and damage as shown by the decreased levels of ATP and the elevated levels of mitochondrial superoxide, intracellular ROS, 8-OHdG, protein carbonylation, and lipid peroxidation. An antioxidant N-acetyl-L-cysteine (NAC) significantly abolished the anti-proliferative and pro-apoptotic effects of doxycycline, demonstrating that doxycycline acts on glioblastoma via inducing oxidative stress. CONCLUSIONS In our study, we show that the antibiotic doxycycline is effective in targeting glioblastoma through inducing mitochondrial dysfunctions and oxidative stress. Our work also demonstrated the importance of mitochondrial metabolism in glioblastoma.

Conflict of interest statement

Conflict of interest

All authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Doxycycline significantly inhibits proliferation and induces apoptosis in glioblastoma cancer cells. Doxycycline inhibits proliferation (A) and induces apoptosis (B) of two glioblastoma cell lines A172 and U87 in a dose-dependent manner. (C) Representative Western blot image showing the levels of PCNA and cleaved caspase 3 in A172 and U87 exposed to doxycycline. These data were derived from three independent experiments. * p<0.05 compared to control.
Figure 2
Figure 2
Doxycycline significantly induces mitochondrial dysfunction and oxidative stress in glioblastoma cells. Doxycycline significantly decreases mitochondrial membrane potential (A), basal (B) and maximal OCR (C), and ATP levels (D) in A172 and U87 cells. Doxycycline significantly increases mitochondrial superoxide (E) and intracellular ROS (F) levels in A172 and U87. These data were derived from three independent experiments. The concentrations of pyrvinium and paclitaxel used in the combination studies were 0.1 μM and 0.5 μM, respectively. * p<0.05 compared to control.
Figure 3
Figure 3
Doxycycline significantly induces oxidative damage in glioblastoma cells. Doxycycline significantly increases levels of 8-OHdG (A), carbonyls (B) and malondialdehyde (MDA, C) in A172 and U87 cells. * p<0.05 compared to control.
Figure 4
Figure 4
NAC significantly reveres the effects of doxycycline in glioblastoma cells. The effects of doxycycline in increasing ROS levels (A), inhibiting proliferation (B) and inducing apoptosis (C) in A172 and U87 cells were abolished by NAC. 10 mM NAC and 40 μM doxycycline were added to the medium. * p<0.05 compared to control.
Figure 5
Figure 5
Doxycycline significantly enhances the inhibitory effects of temozolomide in glioblastoma in vitro and in vivo. Combination of doxycycline and temozolomide is more superior in inhibiting proliferation (A) and inducing apoptosis (B) than single drug alone in glioblastoma cells. The concentrations of doxycycline and temozolomide used in combination studies were 10 μM and 5 μM, respectively. (C) Combination of doxycycline and temozolomide further inhibits A172 tumor growth in SCID mice compared to single drug alone. 100 mg/kg intraperitoneal doxycycline and 20 mg/kg oral temozolomide or combination of both was given once per day for 30 days. * p<0.05 compared to single arm treatment.

References

    1. Dolecek TA, Propp JM, Stroup NE, Kruchko C. CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2005–2009. Neurooncology. 2012;14(Suppl 5):v1–49.
    1. Gerstner ER, Batchelor TT. Antiangiogenic therapy for glioblastoma. Cancer J. 2012;18(1):45–50.
    1. Patel AP, Tirosh I, Trombetta JJ, et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science. 2014;344(6190):1396–401.
    1. Brennan CW, Verhaak RG, McKenna A, et al. The somatic genomic landscape of glioblastoma. Cell. 2013;155(2):462–77.
    1. Phillips HS, Kharbanda S, Chen R, et al. Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell. 2006;9(3):157–73.
    1. Chopra I, Hawkey PM, Hinton M. Tetracyclines, molecular and clinical aspects. J Antimicrob Chemother. 1992;29(3):245–77.
    1. Duivenvoorden WC, Popovic SV, Lhotak S, et al. Doxycycline decreases tumor burden in a bone metastasis model of human breast cancer. Cancer Res. 2002;62(6):1588–91.
    1. Lamb R, Ozsvari B, Lisanti CL, et al. Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: Treating cancer like an infectious disease. Oncotarget. 2015;6(7):4569–84.
    1. Zhao Y, Wang X, Li L, Li C. Doxycycline inhibits proliferation and induces apoptosis of both human papillomavirus positive and negative cervical cancer cell lines. Can J Physiol Pharmacol. 2016;94(5):526–33.
    1. Yang B, Lu Y, Zhang A, et al. Doxycycline induces apoptosis and inhibits proliferation and invasion of human cervical carcinoma stem cells. PLoS One. 2015;10(6):e0129138.
    1. Ahler E, Sullivan WJ, Cass A, et al. Doxycycline alters metabolism and proliferation of human cell lines. PLoS One. 2013;8(5):e64561.
    1. Varum S, Rodrigues AS, Moura MB, et al. Energy metabolism in human pluripotent stem cells and their differentiated counterparts. PLoS One. 2011;6(6):e20914.
    1. Xie Y, Bergstrom T, Jiang Y, et al. The human glioblastoma cell culture resource: Validated cell models representing all molecular subtypes. EBioMedicine. 2015;2(10):1351–63.
    1. Kudryavtseva AV, Krasnov GS, Dmitriev AA, et al. Mitochondrial dysfunction and oxidative stress in aging and cancer. Oncotarget. 2016;7(29):44879–905.
    1. Kerksick C, Willoughby D. The antioxidant role of glutathione and N-acetyl-cysteine supplements and exercise-induced oxidative stress. J Int Soc Sports Nutr. 2005;2:38–44.
    1. Moreno-Sanchez R, Rodriguez-Enriquez S, Marin-Hernandez A, Saavedra E. Energy metabolism in tumor cells. FEBS J. 2007;274(6):1393–418.
    1. Pedersen PL. Tumor mitochondria and the bioenergetics of cancer cells. Prog Exp Tumor Res. 1978;22:190–274.
    1. Zu XL, Guppy M. Cancer metabolism: Facts, fantasy, and fiction. Biochem Biophys Res Commun. 2004;313(3):459–65.
    1. Zhang X, de Milito A, Olofsson MH, et al. Targeting mitochondrial function to treat quiescent tumor cells in solid tumors. Int J Mol Sci. 2015;16(11):27313–26.
    1. Skrtic M, Sriskanthadevan S, Jhas B, et al. Inhibition of mitochondrial translation as a therapeutic strategy for human acute myeloid leukemia. Cancer Cell. 2011;20(5):674–88.
    1. Wen S, Zhu D, Huang P. Targeting cancer cell mitochondria as a therapeutic approach. Future Med Chem. 2013;5(1):53–67.
    1. Song M, Wu H, Wu S, et al. Antibiotic drug levofloxacin inhibits proliferation and induces apoptosis of lung cancer cells through inducing mitochondrial dysfunction and oxidative damage. Biomed Pharmacother. 2016;84:1137–43.
    1. Miyazaki T, Yamasaki N, Tsuchiya T, et al. Infectious episodes lead to the oxidative stress response after lung transplantation. Am J Case Rep. 2015;16:255–58.
    1. Stefano GB, Samuel J, Kream RM. Antibiotics may trigger mitochondrial dysfunction inducing psychiatric disorders. Med Sci Monit. 2017;23:101–6.

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe