Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6 (11), 397

Doxycycline Induces Apoptosis via ER Stress Selectively to Cells With a Cancer Stem Cell-Like Properties: Importance of Stem Cell Plasticity

Affiliations

Doxycycline Induces Apoptosis via ER Stress Selectively to Cells With a Cancer Stem Cell-Like Properties: Importance of Stem Cell Plasticity

Takashi Matsumoto et al. Oncogenesis.

Abstract

Tumor heterogeneity can be traced back to a small subset of cancer stem cells (CSCs), which can be derived from a single stem cell and show chemoresistance. Recent studies showed that CSCs are sensitive to mitochondrial targeting antibiotics such as doxycycline. However, little is known about how cancer cells undergo sphere formation and how antibiotics inhibit CSC proliferation. Here we show that under sphere-forming assay conditions, prostate cancer cells acquired CSC-like properties: promoted mitochondrial respiratory chain activity, expression of characteristic CSC markers and resistance to anticancer agents. Furthermore, those CSC-like properties could reversibly change depending on the culture conditions, suggesting some kinds of CSCs have plasticity in tumor microenvironments. The sphere-forming cells (i.e. cancer stem-like cells) showed increased contact between mitochondria and mitochondrial associated-endoplasmic reticulum (ER) membranes (MAM). Mitochondrial targeting doxycycline induced activating transcription factor 4 (ATF4) mediated expression of ER stress response and led to p53-upregulated modulator of apoptosis (PUMA)-dependent apoptosis only in the cancer stem-like cells. We also found that doxycycline effectively suppressed the sphere formation in vitro and blocked CD44v9-expressing tumor growth in vivo. In summary, these data provide new molecular findings that monolayer cancer cells acquire CSC-like properties in a reversible manner. These findings provide important insights into CSC biology and a potential new treatment of targeting mitochondria dependency.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Sphere-forming PC-3 cells shows the CSC-like properties with plasticity
a Immunofluorescence staining of CD44v9 and E-cadherin in monolayer and sphere-forming PC-3 cells. Scale bar = 10 μm. b CD44v8-10 and CD44s mRNA expression in monolayer and sphere-forming cells were performed by RT-PCR. c Immunoblotting analysis of E-cadherin, c-MYC, Bcl-xL and β-tubulin protein in the change of monolayer cells (3 days) to sphere-forming cells (3 days). M indicates monolayer, S indicates sphere-forming. d Relative mRNA expression of CSC marker in the change of monolayer (3 days) to sphere forming cells (3 days). Data were normalized to the expression level in monolayer for each RNA species. M indicates monolayer cells, S indicates sphere-forming cells. Data shows the mean ± SD of triplicates. *p < 0.05. e Relative cell number using MTS assay for monolayer and sphere-forming cells treated with 100 nM paclitaxel and 10 nM docetaxel for 3 days. Data were normalized to the absorbance level in monolayer for each species. Data shows the mean ± SD of triplicates. **p < 0.01. f OCR was measured using the Seahorse XF24 analyzer for monolayer and sphere-forming cell. Oligomycin, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, and antimycin A (AA) were added at the same time point for each experiment. Data shows the mean ± SD of quadruplicates. *p < 0.05
Fig. 2
Fig. 2. Doxycycline decreases OCR and induces the apoptosis in sphere-forming PC-3 cells
a Relative cell number compared to no drug treatment by MTS assay in monolayer and sphere forming cells treated with various concentrations of doxycycline (Dox) and chloramphenicol (CA) for 3 days. Data shows the mean ± SD of triplicates. *p < 0.05, **p < 0.01. b Immunoblotting analysis of mtDNA-encoded COX1, COX2 and COX3, nuclear-encoded COX4 and β-actin protein in the monolayer and sphere-forming cells treated with 40 μM doxycycline for 48 h. c OCR was measured using the Seahorse XF24 Analyzer in monolayer and spheres with 40 μM doxycycline for 24 h. Data shows the mean ± SD of triplicates. **p < 0.01. d Flow cytometric analysis of apoptosis in monolayer and sphere-forming cells treated with 40 μM doxycycline for 24 h. In the graph on the right, the rates of the population of AnnexinV( + )/PI( + ) are indicated. A representative experiment out of three is shown. All data shows the mean ± SD of triplicates. **p < 0.01
Fig. 3
Fig. 3. MAM formation promote doxycycline-induced apoptosis in sphere-forming cells
a Immunoblotting analysis of FACL4 in monolayer and spheres. β-actin served as an internal loading control. b Representative immunostaining of FACL4 (Green) and TOM20 (Red) in monolayer and sphere forming cells. Colocalized image (white dot) was confirmed in three-dimensional reconstructions from the raw confocal image using IMARIS. Relative colocalized voxel normalized in monolayer was indicated in right panel. Scale bar = 10 μm. c Western blot analysis of MFN2, OPA1, Parkin, and PINK1 protein in monolayer and sphere-forming cells treated with 100 μM doxycycline for 24 h. β-actin served as an internal loading control. d Flow cytometric analysis of apoptosis in monolayer and sphere-forming cells with transfected with MFN2 siRNAs as indicated and treated with 40 μM doxycycline for 24 h. In the graph on the right, the rates of the population of AnnexinV(+)/PI(+) are indicated. Immunoblotting analysis of Mfn2 protein (on the right) indicates the efficacy of the siRNA
Fig. 4
Fig. 4. Doxycycline induces ER stress response and PUMA expression in sphere-forming cells
a Relative ATF4 and PUMA mRNA expression in monolayer and sphere forming cells treated with 40 μM doxycycline for 24 h. Data were normalized to the expression level in monolayer for each RNA species. Data shows the mean ± SD of triplicates. *p < 0.05, **p < 0.01. b Relative mRNA expression of ATF4 and PUMA for spheres transfected with ATF4 (#1 and #2) or control siRNA. Data were normalized to the expression level in monolayer for each RNA species. **p < 0.01. c Flow cytometric analysis of apoptosis in sphere-forming cells transfected with ATF4#1 siRNA and treated with 40 μM doxycycline for 24 h. In the right panel, the rates of subpopulations of Annexin V ( + )/PI ( + ) are shown. *p < 0.05, **p < 0.01. d Flow cytometric analysis of apoptosis in monolayer and sphere-forming cells treated with 1 μM thapsigargin (Tg) for 24 h. In the right panel, the rates of subpopulation of Annexin V (+)/PI (+) are shown. **p < 0.01
Fig. 5
Fig. 5. Doxycycline inhibits CD44v9 positive cell proliferation in the xenograft model
a The monolayer or sphere-forming PC-3 cells with or without doxycycline (40 μM) pretreatment were injected into nude mice (Balb/c-nu) and the tumor volumes were measured at a 19 days. n = 3 for each group. **p < 0.01. b Relative intensity area of CD44v9 expression analyzed by Keyence software in the xenograft model implanted with monolayer or spheres. Data were normalized to the expression level in monolayer. c Changes in tumor volume in Balb/c-nu mice at 15 days after the xenograft. n = 4 for each group. The mice were treated with 60 mg/kg of doxycycline or saline by intraperitoneal administration every day after sphere implantation. In the right panel, the tumor volume indicates chronological change. *p < 0.05. d The % cell number of CD44v9 staining after doxycycline were estimated. e Immunofluorescence staining of CD44v9, Ki67 and DAPI in the xenograft model treated with saline or doxycycline for 15 days. Each arrow indicates the cells with Ki67 and CD44v9 co-staining. Scale bar = 10 μm. f The % cell number of CD44v9 and Ki67 co-staining are estimated. *p < 0.05
Fig. 6
Fig. 6
Scheme of CSC-like properties between monolayer and sphere formation and doxycycline induces apoptosis via ER stress (i) sphere-forming cultures of prostate cancer cells showed increased mitochondrial OCR, CD44v9 expression and resistance to anticancer agents; (ii) during sphere formation, cancer cells display CSC-like properties with plasticity; (iii) sphere-forming cancer cells show increased MAM formation; (iv) doxycycline disrupts MAM formation leading to ER stress response and apoptosis in sphere forming cells (CSC-like properties)

Similar articles

See all similar articles

Cited by 5 articles

References

    1. Bjerkvig R, Tysnes BB, Aboody KS, Najbauer J, Terzis AJ. Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat. Rev. Cancer. 2005;5:899–904. doi: 10.1038/nrc1740. - DOI - PubMed
    1. Pattabiraman DR, Weinberg RA. Tackling the cancer stem cells - what challenges do they pose? Nat. Rev. Drug. Discov. 2014;13:497–512. doi: 10.1038/nrd4253. - DOI - PMC - PubMed
    1. Wulg GG, et al. A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood. 2001;98:1166–1173. doi: 10.1182/blood.V98.4.1166. - DOI - PubMed
    1. Dylla SJ, et al. Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy. PLoS. ONE. 2008;3:e2428. doi: 10.1371/journal.pone.0002428. - DOI - PMC - PubMed
    1. Todaro M, et al. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell. Stem. Cell. 2007;1:389–402. doi: 10.1016/j.stem.2007.08.001. - DOI - PubMed
Feedback