Cell proliferation and apoptosis in rat mammary cancer after electrochemical treatment (EChT)

Abstract

Background: Several authors have recently reported encouraging results from Electrochemical treatment (EChT) in malignant tumours. However, EChT is not established and mechanisms are not completely understood. In vivo studies were conducted to evaluate the toxic changes and effectiveness of EChT on an animal tumour model.

Methods: Tumours were induced by injecting cells from the R3230AC rat mammary tumour cell line clone D subcutaneously, in 28 female Fischer 344 rats. EChT was conducted by inserting a platinum electrode into the tumours. The positive and negative control groups were subjected to the same conditions but without current. The rats were kept for 0, 7 or 14 days post-treatment. Three hours prior to euthanasia an i.p. injection of Bromodioxyuridine (BrdU) was given. The rats were euthanized, the lesions extirpated and samples were collected for histopathological, and immunohistochemical examination.

Results: Significant changes in cell proliferation rate were seen both in the cathode and anode regions. Apoptosis were induced in the anodic treated area outside the primary necrosis, detected with the TUNEL method.

Discussion: The results suggest that secondary cell destruction was caused by necrosis with cathodic EChT and apoptosis or necrosis with anodic EChT.

Fig. 1

A schematic of how the cell proliferation labelling index (LI) was determined. In the seven directions indicated by the arrows each of the zones numbered 1 through 10 was evaluated, starting at the border of destruction (zone 1).

Fig. 2

Average tumour size after EChT compared to untreated control. In the beginning the anodic treatment gives rise to an immediate decrease in tumour volume due to dehydration. At day 0 the initial tumour volume and the one after treatment is the same for the control. Hence, the difference in volume becomes 0. There is an illusionary prominent increase in “tumour volume” immediately after EChT at the cathode due to the formation of oedema (electro-osmosis). Error bars are showing the standard deviation (SD), which is the percentage increase/decrease from start of the experiment (e.g. day 21) until termination (0, 7 or 14 days after EChT).

Fig. 3

Histopathological examination of the R3230AC Rat mammary tumour clone D after EChT. At the anodic treatment (20 C 7d) the border of destruction is clearly visible (A, magnification 212×) and a marked central necrosis has developed with a secondary inflammation (B, magnification 212×). The untreated control has a prominent capsule (C, magnification 100×) and contains a few minor local necroses (D, magnification 212×). Arrows marking the area of necrosis.

Fig. 4

Chart showing the cumulative incidence of tumour cells in S-phase from the border of destruction and out towards “normal” tumour tissue. Note that the cell proliferation is significantly decreased even far from the actual EChT destruction, suggesting that additional impact on tumour proliferation is achieved, apart from the primary necrosis. Initially (0d) the decrease in cell proliferation rate is more prominent close to the border of destruction, due to toxic impact from the produced hydrochloric acid, oxygen, and chlorine gas (anode) or sodium hydroxide (cathode; not shown). Further away from the necrotic area it becomes higher than for the 7d group. Error bars are showing the standard deviation (SD).

Fig. 5

Cell proliferation after EChT. Ten high power fields were counted in the light microscope after staining for BrdU incorporation (brown nuclei). Note the decrease in proliferation far from the border of destruction in the cathodic treatment (A, magnification 212×) compared to the untreated control (B, 212×).

Fig. 6

Immunohistochemistry using the TUNEL method shows the induction of apoptosis after EChT (10 C 7d). In fluorescence microscope the distinct apoptotic nuclei at the border of destruction at the anode (A, objective 64×) is compared to the untreated control (C, objective 64×) where few apoptotic nuclei are found and the cathodic treatment (E, objective 64×) where a general necrosis is present. After staining with peroxidase technique the apoptosis can be seen in the light microscope (B, D and F; magnificaion 212×). The light microscope view is placed beside the corresponding immunofluorescence picture. Arrows mark the border of destruction or necrotic margin.