PMC

Sorbitol and lactate levels in tumors of mouse mammary tumor virus-neu mice from the withaferin A (WA) treatment group and the control group. Results shown are means and their 95% confidence intervals (error bars). Statistical significance of differences was determined by two-sided Student t test.

Withaferin A (WA) administration inhibits incidence of lung metastasis in mouse mammary tumor virus–neu mice. A) Representative hematoxylin and eosin–stained lung sections exhibiting metastasis in representative mice of the WA treatment group and the control group (×100 magnification, scale bars = 200 µm). B) Incidence of lung metastasis. Results shown are the percentages of mice with lung metastasis. The P value was calculated by two-sided Fisher exact test. C) Multiplicity of lung metastasis. Results show mean number of lung metastasis per mouse with 95% confidence intervals (error bars). Statistical significance of differences was determined by two-sided Student t test. D) Area of lung metastatic foci. Results represent the mean areas of lung metastatic tumor with their corresponding 95% confidence intervals (error bars). Statistical significance of differences was determined by two-sided Student t test.

Withaferin A (WA) administration inhibits activity of complex III in the tumor of mouse mammary tumor virus–neu mice. A) Activity of complex III in tumor lysates from mice of the WA treatment group and the control group. Complex III activity was measured using oxidized cytochrome c and ubiquinol as substrates. Results represent mean complex III activity (n = 10) with their 95% confidence intervals (error bars). B) Activity of complex IV in tumor lysates from mice of the WA treatment group and the control group. Complex IV activity was measured using reduced cytochrome c as the substrate. Results represent mean complex IV activity (n = 10) with their 95% confidence intervals (error bars). C) 8-Hydroxy-2′-deoxyguanosine (8-OHdG) level by immunohistochemistry in representative tumors of a mouse from the WA treatment group and the control group. Immunohistochemical staining was performed with anti-8-OHdG antibody and analyzed by nuclear algorithm using Aperio ImageScope software (×200 magnification, scale bars = 100 μm). Bar graph shows mean H-score for 8-OHdG (n = 7) with their 95% confidence intervals (error bars). D) 8-OHdG level by immunohistochemistry in representative normal mammary ducts of a mouse from the WA treatment group and the control group. Immunohistochemical staining was performed with anti-8-OHdG antibody and analyzed by nuclear algorithm using Aperio ImageScope software (×200 magnification, scale bars = 100 μm). Bar graph shows mean H-score for 8-OHdG (n = 7) with their 95% confidence intervals (error bars). Statistical significance of differences was determined by two-sided Student t test (A–D).

Withaferin A (WA) administration results in apoptosis induction in the tumor of mouse mammary tumor virus–neu mice. A) Proliferating cell nuclear antigen (PCNA) expression revealing cell proliferation in representative tumor of a mouse from the WA treatment group and the control group. Immunohistochemical staining was performed with anti-PCNA antibody and analyzed by nuclear algorithm using Aperio ImageScope software (×200 magnification, scale bars = 100 μm). Results shown are mean H-score for PCNA expression with their 95% confidence intervals (error bars, n = 7). B) Terminal deoxynucleotidyl transferase-mediated deoxyuridine-5’-triphosphate nick-end labeling (TUNEL)–positive apoptotic cells in representative tumor of a mouse from the WA treatment group and the control group. Apoptotic cells were visualized by TUNEL staining and analyzed by nuclear algorithm using Aperio ImageScope software (×200 magnification, scale bars = 100 μm). Results represent mean TUNEL-positive cells per high-power field (n = 7) with their 95% confidence intervals (error bars). C) CD31-positive blood vessels in representative tumor of a mouse from the WA treatment group and the control group. Immunohistochemical staining was performed with anti-CD31 antibody and quantified using Image ProPlus 5.0 software (×200 magnification, scale bars = 100 μm). Results represent mean CD31-positive cells per high-power field (n = 7) with their 95% confidence intervals (error bars). Statistical significance of differences was determined by two-sided Student t test (A–C).

Withaferin A (WA)–mediated alterations in the levels of tricarboxylic acid (TCA) cycle intermediates in the plasma and/or tumor of mouse mammary tumor virus-neu mice. A) TCA cycle. B) Levels of TCA cycle intermediates in plasma and/or tumors of mice from the WA treatment group and the control group. Plasma and tumor samples from 8 different mice of each group were outsourced to Metabolon (Durham, NC) for metabolic profiling. The letters P (purple) and T (purple) represent plasma and tumor, respectively. A red arrow denotes P less than .05, and a green arrow denotes P greater than or equal to .05 and P less than .10. TCA cycle intermediates with differences between the WA treatment group and the control group are indicated with red font. TCA cycle–related proteins with differences in expression between the WA treatment group and the control group are indicated with green font. Bar graphs represent the mean scaled intensity of the intermediates (n = 8) with their 95% confidence intervals (error bars). acetyl CoA = acetyl coenzyme A; succinyl CoA = succinyl conenzyme A; succinyl CoA synthase = succinyl coenzyme A synthase.

Withaferin A (WA)–mediated alterations in levels of glycolysis intermediates in the plasma and tumor of mouse mammary tumor virus-neu mice. A) Glycolysis pathway. B) Levels of glycolysis intermediates in plasma and/or tumors of mice from the WA treatment group and the control group. Plasma and tumor samples from eight different mice of each group were outsourced to Metabolon, Inc (Durham, NC) for metabolic profiling. The letters P (purple) and T (purple) represent plasma and tumor, respectively. A red arrow denotes P less than .05, and a green arrow denotes P greater than or equal to .05 and P less than .10. Glycolysis intermediates with significant differences between the WA treatment group and the control group are indicated with red font. Glycolysis-related proteins with significant differences in expression between the WA treatment group and the control group are indicated with green font. Bar graphs represent the mean scaled intensity of the intermediates (n = 8) with their 95% confidence intervals (error bars) acetyl CoA = acetyl coenzyme A; DHAP = dihydroxyacetone phosphate; LDH = lactate dehydrogenase; TPI = triose phosphate isomerase.

Withaferin A (WA) administration decreases burden of macroscopic (tumor size) and microscopic tumors (tumor area) in mouse mammary tumor virus–neu (MMTV-neu) transgenic mice. A) Chemical structure of WA. B) Representative hematoxylin and eosin–stained sections of mammary glands showing ductal carcinoma in situ (DCIS), papillary tumor, and focus of invasive carcinoma around papillary tumor (×50 magnification, scale bars = 400 µm). C) Overall tumor incidence in MMTV-neu mice of the WA treatment group and the control group. The P value was calculated by two-sided Fisher exact test. D) Macroscopic tumor burden. Results represent the mean weight of palpable tumors (macroscopic tumors) with a cutoff of ≥.05g with corresponding 95% confidence intervals (error bars). Statistical significance of differences was determined by two-sided Student t test. E) Microscopic tumor burden (area) of DCIS plus papillary tumors and invasive carcinoma lesions. Results represent the mean areas of DCIS plus papillary tumors and invasive carcinoma with their corresponding 95% confidence intervals (error bars). Statistical significance of differences was determined by two-sided Student t test.