Analysis of DOX uptake by fluorescence microscopy and flow cytometry.

<p>A) Observation of cellular uptake of apatite-DOX by fluorescence microscopy in SW480 cells. Cells were grown (5×10⁵ cells/dish) on 60 mm culture dishes. After 1 day, the medium was replaced by 1 ml of fresh medium and free DOX or DOX-loaded apatite particles (200 nM DOX equivalents) were added. After 4 h, the culture dishes were washed 3 times with PBS and the extracellularly bound particles were removed by 5 mM EDTA in PBS. Scale bar, 200 μm. B) Analysis of cellular uptake of apatite-DOX by flow cytometry in NIH:OVCAR-3 cell line. The cells were seeded (0.5×10⁶cells/dish) in a 60 mm tissue culture dish, incubated overnight. 5 ml of DOX-loaded particles prepared with 200 nM DOX concentration in medium was introduced to each well and incubated for 4 h. The cells were then trypsinized, washed three times with PBS solution and then fixed with 10% formaldehyde. After filtering through a nylon mesh, cell fluorescence was measured by flow cytometry.</p

Morphology and size distribution of apatite-DOX complexes.

<p>Different techniques like A–C) AFM, D) DLS, and E) TEM were applied to characterize the nanoparticles. AFM study revealed that the maximum size was 69.8 nm. The cross-sectional curve profile was drawn between A and B as shown in figure A) where as B) shows the enlarged image focusing on the central area showed single spherical particles as well as the combined bodies consisting of several single particles and C) reveals the height distribution of nanoparticles obtained from the cross-sectional curve between A and B.</p

<i>In vitro</i> cytotoxic effect of DOX and apatite-DOX.

<p>The cytotoxicity in response to different concentrations of free and apatite loaded drugs towards A) SW480 colon carcinoma cells, B) ovarian carcinoma cells NIH:OVCAR-3 estimated by MTT cell proliferation assay. Data represent mean value ± SE (n = 3). C) Microscopic observation on HCT116 colon cancer cells after 72 h (DOX dose: 150 nM). Magnifications: x50.</p

The FTIR spectrum of pure DOX, apatite and apatite-DOX complexes.

<p>After generation of apatite-DOX nanopaticles, the centrifuged pellet of the apatite particles was repeatedly washed by ddH₂O and then lyophilized by freeze drying and checked the spectrum under FT/IR-230, JASCO.</p

Esophageal squamous cell carcinoma with low mitochondrial copy number has mesenchymal and stem-like characteristics, and contributes to poor prognosis

<div><p>Alterations in mitochondrial DNA (mtDNA) copy numbers in various human cancers have been studied, but any such changes in esophageal squamous cell carcinoma (ESCC) are not established. In the present study, we investigated the correlation of mtDNA copy number with clinicopathologic features, prognosis, and malignant potential of ESCC. MtDNA copy numbers of resected specimens from 80 patients treated with radical esophagectomy were measured by quantitative real-time PCR analyses. Human ESCC cells, TE8 and TE11, were cultured, and depletion of mtDNA content was induced by knockdown of mitochondrial transcription factor A expression or treatment with ethidium bromide. The mRNA and protein expression, proliferation, invasion, and cell cycle were investigated. The results showed that the mtDNA copy number of cancerous portions was 56.0 (37.4–234.5) percent that of non-cancerous parts and significantly lower (p<0.01). Low mtDNA copy number in resected cancerous tissues was significantly correlated with pathological depth of tumor invasion (p = 0.045) and pathological stage (p = 0.025). Patients with lower mtDNA copy number had significantly poorer 5-year overall survival compared to patients with higher levels (p<0.01). The mtDNA-depleted TE8 and TE11 cells had morphological changes and proliferated more slowly than control cells under normoxia but proliferated at almost the same rate under hypoxic conditions. In mtDNA-depleted cells, E-cadherin mRNA expression was decreased, and N-cadherin, vimentin, zeb-1, and cd44 mRNA expression was increased. Immunoblotting and flow cytometry analysis also showed downregulated E-cadherin and upregulated N-cadherin and CD44 protein in mtDNA-depleted cells. Moreover, mtDNA-depleted cells had enhanced invasion, migration, and sphere formation abilities, and the cell cycle arrest at G0/G1 phase was induced in these cells. These results suggested that mtDNA-depleted ESCC cells had mesenchymal characteristics, cancer stemness, and tolerance to hypoxia, which played important role in cancer progression. In conclusion, a low copy number of mtDNA is associated with tumor progression in ESCC.</p></div

<i>In vitro</i> pH-responsive drug release behaviors of apatite-DOX nanoparticles.

<p>The release media of different pH values which were used to simulate the alkalescent conditions in normal tissues and blood (pH∼7.4) and the acidic conditions in tumor (pH = 4∼6.8). The nano apatite-DOX hardly released DOX in the pH∼7.4 release medium (PBS), but responsively released DOX in pH 5.5 acidic media (sodium acetate). Data represent mean value ± SE (n = 3).</p

Observation of cellular uptake behavior of carbonate apatite by SEM.

<p>Scanning electron microscopy revealed a process of uptake behavior into HCT116 colon cancer cells. A) The control HCT116 devoid of apatite particles. B) The particles attached on the cell. C) The particles were getting internalized at as early as 45 min, and D) further going into the cells at 90 min.</p

Cell proliferation and mRNA expression of mtDNA-depleted ESCC.

<p><b>(A)</b> Cell proliferation of mtDNA-depleted cells and control cells were investigated under normoxia (20% O₂) and hypoxia (1% O₂). Under normoxia, the proliferation rates of mtDNA-depleted cells were significantly lower than control cells at 24 (TE8 tfam-sh1 or tfam-sh2 vs control-sh, p<0.01; TE11 tfam-sh1 vs control-sh, p = 0.011; TE11 tfam-sh2 vs control-sh, p<0.01), 48 (p<0.01), and 72 h (p<0.01). <b>(B)</b> Under hypoxia, the proliferation rate of control cells was decreased, but mtDNA-depleted cells proliferated at almost the same rate as under normoxia. <b>(C)</b> Hypoxia-resistant rates in mtDNA-depleted cells, as assessed by the ratio of the proliferation rate under hypoxia to that under normoxia, were significantly higher at 24, 48, and 72 h (p<0.01).</p

mtDNA-depleted TE8 and TE11 cells by knockdown of TFAM gene expression.

<p><b>(A)</b> By short hairpin RNA knockdown of TFAM gene expression, the mRNA expression of TFAM was silenced to about 50% compared with non-target control cells in both TE8 and TE11 cells. <b>(B)</b> Immunoblotting showed downregulation of TFAM expression in tfam-sh cells. <b>(C)</b> Silencing TFAM reduced mtDNA copy number to about 40% in TE8 and about 60–70% in TE11 compared with non-target control cells. <b>(D)</b> The extracellular lactate concentrations of mtDNA-depleted cells were higher than control cells under normoxia (TE8: 1.34±0.03 or 1.44±0.01 vs 1.13±0.01 μg/μL, p<0.01; TE11: 1.34±0.03 (p<0.01) or 1.26±0.01 (p = 0.011) vs 0.83±0.16). <b>(E)</b> The TFAM knockdown cells showed spindle cell transformation. <b>(F)</b> The TFAM knockdown cells showed mitochondrial swelling and dissolved cristae compared with the control cells.</p

Inhibition of colorectal tumor growth by the carbonate apatite-mediated doxorubicin delivery.

<p>HCT116 cells were inoculated s.c. in the both left and right flanks of the mice (n = 6). When the diameter of tumors reached φ5 mm, Free-DOX at dose of 0.33 mg/kg/day or Apatite-DOX containing the same amount of DOX were administered by i.v. injection into the tail vein on days 0,1,2,7,8,9,14,15, and 16. Saline was administrated in case of control animals. Significant differences in the tumor volume were found between Control or Free-DOX and Apatite-DOX on day 12 and 19 (p<0.05). Data represent mean value ± SE (n = 6).</p