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Oxalate Induces Breast Cancer

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Oxalate Induces Breast Cancer

Andrés M Castellaro et al. BMC Cancer.

Abstract

Background: Microcalcifications can be the early and only presenting sign of breast cancer. One shared characteristic of breast cancer is the appearance of mammographic mammary microcalcifications that can routinely be used to detect breast cancer in its initial stages, which is of key importance due to the possibility that early detection allows the application of more conservative therapies for a better patient outcome. The mechanism by which mammary microcalcifications are formed is still largely unknown but breast cancers presenting microcalcifications are more often associated with a poorer prognosis.

Methods: We combined Capillary Electrochromatography, histology, and gene expression (qRT-PCR) to analyze patient-matched normal breast tissue vs. breast tumor. Potential carcinogenicity of oxalate was tested by its inoculation into mice. All data were subjected to statistical analysis.

Results: To study the biological significance of oxalates within the breast tumor microenvironment, we measured oxalate concentration in both human breast tumor tissues and adjoining non-pathological breast tissues. We found that all tested breast tumor tissues contain a higher concentration of oxalates than their counterpart non-pathological breast tissue. Moreover, it was established that oxalate induces proliferation of breast cells and stimulates the expression of a pro-tumorigenic gene c-fos. Furthermore, oxalate generates highly malignant and undifferentiated tumors when it was injected into the mammary fatpad in female mice, but not when injected into their back, indicating that oxalate does not induce cancer formation in all types of tissues. Moreover, neither human kidney-epithelial cells nor mouse fibroblast cells proliferate when are treated with oxalate.

Conclusions: We found that the chronic exposure of breast epithelial cells to oxalate promotes the transformation of breast cells from normal to tumor cells, inducing the expression of a proto-oncogen as c-fos and proliferation in breast cancer cells. Furthermore, oxalate has a carcinogenic effect when injected into the mammary fatpad in mice, generating highly malignant and undifferentiated tumors with the characteristics of fibrosarcomas of the breast. As oxalates seem to promote these differences, it is expected that a significant reduction in the incidence of breast cancer tumors could be reached if it were possible to control oxalate production or its carcinogenic activity.

Figures

Fig. 1
Fig. 1
Human breast tumor tissues have higher concentration of total Oxalate than non-cancerous breast tissues. 13 samples of human breast tumor tissues (Tumor samples) and 12 samples of tumor-adjacent non-cancerous breast tissues (Control samples) were homogenized in 2.75 M hydrochloride acid. The supernatant fractions were analyzed by Capillary Electrochromatography to establish the total concentration of oxalate present in each sample. Results of oxalate concentrations are expressed as μg oxalate/mg of tissue (n = 13 and n = 12 were analyzed for tumor and non-tumor samples, respectively); statistical significance was calculated using Student’s two tailed t -test. ****P value < 0.0001
Fig. 2
Fig. 2
Oxalate induces breast cancer cell proliferation. Proliferation was performed using a colorimetric assay and following the manufacturing instructions (CyQUANT, Life Technologies) in cells (a) after 3 days of treatment of MCF-7, MDA-MB231, HEK-293 or 7 days of treatment of MCF-10A cells (a) as indicated with oxalate or with acetic acid, or (b) after 3 weeks of treatment in of MCF-7 cells. Cells were cultured in DMEM medium plus an additional specific reagent or not, according to each condition, as indicated. Con: Control, no additional reagent was added; FBS: fetal bovine serum, Ox: oxalic acid or A.A.: acetic acid were added to the culture medium. Results are expressed as DNA content (arbitrary units) found after seeding 4 x103 cells/well.. Bars represent the standard error of the mean of four independent experiments performed in triplicate. A.U.: arbitrary units. Statistical significance determined by Two-way ANOVA with Holm-Sidak’s multiple comparison test (α = 0.05) were performed in experiments graphed in Fig. 2a. One- way ANOVA with Holm-Sidak’s test was performed in Fig. 2b. ****:P value <0.001; **P value <0.01; *P value < 0.05
Fig. 3
Fig. 3
c-Fos is over-expressed in human breast tumor tissues. Western Blot of Breast tumor tissue (T) and paired tumor-adjacent non-cancerous tissue (NA) from a patient, together with a non-paired non-cancerous breast tissue (NC) (as an additional control) were fractionated in 12 % SDS-PAGE gel and immunoblotted using anti-c-Fos and anti α-Tubulin antibodies. α-Tubulin was used as loading control. The blot shown is representative of five independent experiments performed that gave similar results
Fig. 4
Fig. 4
Oxalate induces expression of c-Fos in MCF-7 cells but not in HEK-293 cells. Cells were plated in six wells and grown to 80 % of confluence. Then cells were starved to achieve quiescence (see M & M). After that, each experimental condition was achieved by the stimulation with the specified reagent during 1.5 h. MCF-7 (a) or HEK-293 (b) cells were lysed, then the supernatant fractions were separated in 12 % SDS-PAGE gel and immunoblotted using anti-c-Fos antibody. α-Tubulin was used as loading control. c-fos expression was measured in MCF-7 (c) or HEK-293 (d) cells by qRT-PCR and normalized against housekeeping genes (GAPDH for MCF-7 cells and RPLPO for HEK-293 cells) using the Sequence Detection Software v1.4. Shown are the mean values of 3 independent determinations performed in quadruplicate. Con: Control, No reagent addition FBS: fetal bovine serum. Ox: oxalic acid. A.A.: acetic acid. Statistical significance determined by One- way ANOVA with Holm-Sidak’s test was performed in experiments shown in Fig. 4c and d, **** P value < 0.0001
Fig. 5
Fig. 5
Oxalate induces tumor formation and short-term survival in mice. a Mice were injected every 3 or 4 days into mammary fat pad to reach 9 doses (one dose every 3.2 days on average). The experimental group (Ox Breast) received 50 μL of a solution with microcrystals of calcium oxalate (oxalic acid 810 μM in a carrier solution containing CaCl2 1.8 mM). The two control groups received 50 μL of either carrier solution containing CaCl2 1.8 mM (Con Breast) or a solution of carrier solution plus acetic acid 810 μM (A.A. Breast). Each group consisted of 8 mice. b Mice were injected every 2 or 3 days to reach 7 doses (one dose every 2.6 days on average). Experimental groups received injections with 50 μL of saline solution containing potassium oxalate 810 μM either at the mammary fat pad (Ox Breast) or in the Back (Ox Back). Control groups were injected at the same places as the experimental ones with saline solution only into mammary fat pad (Con Breast) or into the back (Con Back). Each group consisted of 5 mice. Red arrows: times of injection. Pink arrows: time of the first tumor appearance in Ox Breast group. Blue arrows: time at which all mice of Ox breast group have at least one tumor. Statistical significance between survival curves was analyzed by LogRank (Mantel-Cox) test. ****P value < 0.0001
Fig. 6
Fig. 6
Oxalate-treated mice generated tumors. Mice were injected every 2 or 3 days with 50 μL of saline solution containing oxalate 810 μM at into mammary fat pad. Control groups were injected at the same places than the experimental ones with saline solution. Tumor volume (v) of the mice treated were measured and calculated in accordance with the method of Attia and Weiss [35]. To the right it has been included a photograph of a typical tumor induced by oxalate in mice. Each experimental group consisted of 9 animals per group. Results were analyzed for statistical significance using Student’s two tailed t -test. ****P value < 0.0001
Fig. 7
Fig. 7
Mice breast tumor tissues contain higher concentrations of total Oxalate than non-cancerous breast tissues. Seven samples of mice breast tumor tissues (Tumor samples) and seven samples of non-cancerous breast tissues (Control samples) were homogenized in 2.75 M HCl. The supernatant fractions were analyzed by Capillary Electrochromatography to establish the total concentration of Oxalate present in each sample. Results expressed as μg/mg tissue were analyzed for statistical significance by Student’s two tailed t -test. All graph were performed using GraphPad Prism version 6.0e for Mac OS X (GraphPad Software, La Jolla California USA) ****P value <0.001
Fig. 8
Fig. 8
Oxalate-treated mice generated undifferentiated and highly aggressive tumors. H&E staining of two representative sections of both non-cancerous breast tissue (a and b) and breast mice tumor tissues (d and e), generated after treatment with oxalate, are shown. Panels c and f are enlargements of the areas delimited in panels b and e respectively. Panel f mitotic figures (arrowheads) and abnormal epithelial breast duct cells (arrow) are marked. A total of 13 tumors and of 13 non-tumor tissues were examined. In all cases, animals treated with oxalate generated malignant, undifferentiated tumors with the characteristics of fibrosarcoma of the breast. Scale bar 50 μm
Fig. 9
Fig. 9
Mice breast tumor tissues express higher amounts of c-Fos than non-cancerous breast tissues. a Sections of both breast tumor tissues (n = 6) and non-cancerous breast tissues (n = 6) were adhered to uncharged slides. c-Fos expression was seen by immunohistochemical staining (green) and nuclei were visualized with DAPI (blue). Shown is a representative figure of all slides analyzed. A minimum if 3 sections were examined per tissue sample. b c-fos expression was measured in breast tumor tissues (n = 5) and non-cancerous breast tissues (n = 5) by qRT-PCR and normalized against the housekeeping gene Tbp using the Sequence Detection Software v1.4. A difference in c-fos expression was statistically analyzed by Student’s two tailed t -test. ****P value <0.001

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