Effects of pressure on the survival and viability of cancer cells in vitro: An analytical study

Abstract

Intense cancer research is underway to discover possible therapies but no major breakthrough appears to be in sight in terms of its cure when diagnosed late. The cytostatic and growth inhibitory effect of high pressure on cells is well documented. In the present study, two cancer cell lines viz. MDA-MB-231 (breast carcinoma) and A549 (lung carcinoma) and one normal cell line (Vero) were subjected to increased pressure of 18 psi in a specially constructed pressure chamber. It was found that a pressure of 18 psi induced a significant change in the growth parameters of both cancer cell lines versus normal cells. Exposure to increased pressure greatly increased the proportion of MDA-MB-231 cells in the S phase while concurrently reducing the number of cells in the G0/G1 phase as compared to their untreated counterparts. SEM and AFM analysis revealed presence of characteristic 'pores' and 'pits' on the cell surface of pressure-treated versus untreated cancer cells. TEM analysis also revealed significant intracellular differences between pressure-treated and untreated cancer cells. Hyperbaric nitrogen therapy is proposed as a novel cancer-treatment modality involving administration of N2 at the tumor site in murine models of breast and lung cancer. This would eventually pave the way for development of a device effective treatment strategy for human tumors in future.

Fig 1

(a). Schematic drawing of the experimental setup. (b). Designed Pressure Chamber.

Fig 2. Mica chips for sample immobilization in AFM.

Fig 3

Morphological analysis of control versus experimental MDA-MB-231 cells; (A-C) Controls showing MDA-MB-231 human breast cancer cells at 14.7 psi after 24, 48 and 72h respectively (D-F) Cells maintained at 18 psi as per setup 1 described above (Magnification 10x).

Fig 4

Morphological analysis of control versus experimental A549 cells; (A-C) Controls showing A549 human lung cancer cells at 14.7 psi after 24, 48 and 72 h respectively (D-F) Cells maintained at 18 psi as per setup 1 described above (Magnification 10x).

Fig 5

Morphological analysis of control versus experimental Vero cells; (A-C) Controls showing Vero normal kidney epithelial cells at 14.7 psi after 24, 48 and 72 h (D-F) Cells maintained at 18 psi as per setup 2 described above (Magnification 10x).

Fig 6

Effect of increased pressure (18 psi) on the growth of (a) MDA-MB-231 (b) A549 and (c) Vero cells (c) versus their respective controls at normal pressure (14.7 psi). Results are expressed as mean ± SD of three independent observations.

Fig 7. Effect of hyperbaric treatment on cancer cell cycle progression and DNA content by flow-cytometry.

Pictorial graph showing mean proportion of cells in different phases of cell cycle (A) Control MDA-MB-231 cells at normal pressure (14.7 psi) and (B) those subjected to 18 psi pressure for 48h; stained with PI and analyzed using flow cytometry. (C-E) The percentage of cell frequency is graphed along the y-axis. The data are representative of three independent experiments. Significance of the differences between control cancer cells as compared to pressure treated cancer cells was of P value = 0.0002 (***) and P value = 0.0010 (**), for G0/G1 and S phase respectively. There was no significance of difference (P value = 0.2148) in G2/M phase.

Fig 8. Apoptosis assay using Annexin V/PI to determine cell viability and health.

(a) MDA-MB-231 cells at 14.7 psi (controls) and (b) MDA-MB-231 cells maintained at 18 psi (experimentals) for 48 h (c) Vero cells at 14.7 psi (controls) and (d) Vero cells maintained at 18 psi (experimentals) for 48 h. Green fluorescent cells were labeled with Annexin and designated apoptotic; PI-labeled cells fluoresced red and were dead; yellow fluorescent cells were labeled with both Annexin and PI and were also counted as dead; whereas unstained cells were counted as live.

Fig 9. Effects of hyperbaric treatment on MDA-MB-231 cells.

Scanning electron micrographs showing MDA-MB-231 cells at 14.7 psi (a1-a4) and 18 psi pressure (b1-b4) at various magnifications. (a1) large number of cells seen (a2) cell membranes appeared well-defined and mostly regular; no lamellipodia or invadopodia could be made out at this magnification; no spikes or tentacles seen (a3, a4) normal looking multiple cells seen (b1, b2) cells appeared almost similar as in controls (a1, a2) because of unappreciated differences (b3) surface of cells appeared granular at this magnification; cell membranes showed pore-like structures, no other details could be made out at this magnification (b4) cell surface from a different area; pore-like structures were more prominent; granular congregations were more apparently defined (a1, a2: 30 and 2 μm bars; a3 and a4: 5 μm bars b1, b2: 30 and 4 μm bars; b3 and b4: 4 μm bars).

Fig 10. Effects of hyperbaric treatment (18psi) on A549 cells.

Scanning electron micrographs showing A549 cells at 14.7 psi (a1-a4) and 18 psi pressure (b1-b4) at various magnifications. (a1-a4) Membrane surface showed irregularities; no lamellipodia or invadopodia could be made out at this magnification (b1-b3) Cells in cluster; although outer membrane appeared irregular in a majority of cells, the surface showed several pits or depression like areas which were irregular and not communicating with each other; no other structural details of surface membrane like ruffling, etc., could be made out; no lamellipodia or invadopodia could be seen (b4) very sharp pits and well-defined membranes could be seen at this magnification; smaller pits well-defined as compared to larges ones (a1, a2: 40 and 30 μm bars; a3 and a4: 20 and 10μm bars; b1, b2: 40 and 30 μm bars; b3 and b4: 20 and 10 μm bars).

Fig 11. Effects of hyperbaric treatment on MDA cells.

(a1, a2) TEM images of untreated MDA controls showed almost normal morphology; the nuclear membrane showed mild chromatin thickening and at one place there appeared a membrane discontinuity (a1); liposomes and other vesicles appeared normal (a2) (b1-b4) TEM images of pressure-treated MDA cells displayed thickened and disrupted nuclear membrane with condensed chromatin (b1, b2), no well-defined cellular vesicles were seen (b3, b4) (ND: Nuclear details, VD: Vesicular details).

Fig 12. Effects of hyperbaric treatment on A549 cells.

(a1-a4) TEM images of untreated A549 control cells displayed very well-defined cell nuclei with intact and continuous membranes; vesicles were numerous and well-defined (b1, b2) TEM images of pressure-treated A549 cells showed nuclear pores and nuclei with abnormal morphology with prominent leakage of nuclear proteins which needs further biochemical assessment. Chromatin appeared disrupted and condensed and nuclear membrane showed irregular thickening. No mitochondria, microvilli, vesicles or Golgi apparatus could be seen in the pictures (ND: Nuclear details, VD: Vesicular details).

Fig 13

High-resolution AFM of membrane of (a) untreated (b) pressure-treated MDA cells; and (c) microsomal fraction of pressure-treated MDA cells. (a) cell membranes appeared well-defined and mostly regular except for a few irregularities (b) cell surface appeared to be granular and cell membranes showed characteristic pore-like structures (c) cell surface appeared more irregular and granular and more were more pronounced and conspicuous. Contact mode AFM topographs were recorded at scan rate 0.59 Hz, scan size: 10μm, cantilever spring constant: 1.0 N/m, as mentioned din materials and methods (Whole cell membrane details: CM, Microsomal fraction details: MF).