Fig 2: Curcumae radix extract treatment exhibited anti-metastatic effects on primary tumor of MMTV-PyMT transgenic mouse. (A) Quantitative analysis of real-time PCR was done using genes associated with breast cancer metastasis. The expression levels of Ccr7, c-Fos, Mmp9, Ccl21, and Ccl19 were determined by qRT-PCR. The Hprt gene was used as input control, respectively, * p < 0.05 and ** p < 0.005 vs. control. (B) Western blot of proteins extracted from tumor tissues in the PyMT transgenic model against Ccr7 and Gapdh. Histogram data was presented as mean ± SEM. * p < 0.05 vs. control. (C) Western blot of proteins extracted from tumor tissues against Phosphorylated Akt, Phosphorylated Ampk, Her2, ERalpha, Gapdh and β-actin. (D) Quantitative analysis of real time PCR was done by using genes associated with apoptosis. The expression levels of Jun, Akt, and Tnf were determined by qRT-PCR. Data are representative of three independent experiments and are expressed as the mean ± SD.

Fig 3: Downregulation of CCR7 gene by siRNA inhibits gene expression of MMP9, c-FOS, c-JUN and CCL21 in vitro. (A) Western blot of proteins extracted from MCF7 cell against CCR7 and GAPDH. (B)To investigate level of transcripts associated with CCR7, MMP9, CCR7, c-FOS, c-JUN and CCL21 were monitored in MCF7 cell transfected with CCR7 siRNA by using qPCR. Β-ACTIN and 18S mRNA was used as an internal control in real time PCR. Data are representative of four independent experiments and are expressed as the mean ± SD; * p < 0.05 vs. negative control.

Fig 4: Anti-migration effects of Curcumae radix extract on human breast cancer MCF7 cells. (A) Cells were incubated with broad and narrow range of CRE concentrations and cell viabilities were measured by XTT assay at 72 h. Concentrations up to 25 µg/mL Curcumae radix extract did not significantly change cell viability in vitro. Absorbance was expressed as A450 nm. Data are representative of three independent experiments and are expressed as the mean ± SD; * p < 0.05 vs. vehicle. (B) Scratch wound healing assay was performed with MCF7 cells in the two doses of Curcumae radix extract. Representative images were taken at 0, 24, and 48 h after wound scratch. Red horizontal lines indicate wounded area borders at 0 hour. White lines indicate wounded area borders at 24 and 48 h. Scale bars = 100 px. The graphic represents quantitative analysis of cell migration of MCF7 cells by calculating percentage of unrecovered area after 48 hours by using Image J analysis. We can observe that CRE treated cells slowed down the migration and closed the wound slower than control group. Data are representative of three independent experiments and are expressed as the mean ± SD; * p < 0.05 vs. vehicle. (C) To investigate level of transcripts associated with breast cancer metastasis, MMP9, CCR7 and COX2 expression levels were monitored in MCF7 cell incubated with CRE. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control in real time PCR. Data are representative of four independent experiments and are expressed as the mean ± SD; * p < 0.05 and ** p < 0.005 vs. vehicle. (D) MCF7 cells were maintained in medium containing 40 µg/mL CRE. Levels of CCR7 protein, a metastasis marker, was analyzed by Western blot. GAPDH was used as an internal control. Values represent means ± SD of at least 3 experiments; * p < 0.05 vs. vehicle. (E) To investigate the level of transcripts associated with apoptosis and inflammation, c-JUN and TNF-α were monitored in MCF7 cell incubated with CRE. GAPDH mRNA was used as an internal control in real time PCR. Data are representative of three independent experiments and are expressed as the mean ± SD; * p < 0.05 vs vehicle. (F) To investigate level of transcripts associated with metastasis markers, CCL21 and NFAT1 were monitored in MCF7 cells incubated with CRE. GAPDH mRNA was used as an internal control in real time PCR. Values represent means ± S.E.M. n = 3, * p < 0.05 vs. vehicle.

Fig 5: Gross and histological appearance of lung excised from 13 weeks of age PyMT transgenic females. CRE treated PyMT transgenic females showed decreased frequency of mammary tumor-derived lung metastases. (A) Representative photos of whole lung excised from 13 weeks age of control group (upper side) and CRE group (bottom side). Larger and more visible metastatic foci were observed in control group. (B) Hematoxylin and eosin (H&E) staining was done with lung samples excised from 13 weeks of age PyMT transgenic females with/without CRE treatment. Slides were microscopically examined and images taken. The number and size of metastases significant decreased in PyMT transgenic females with CRE treatment Scale bars = 400 µm. Curcumae radix extract suppresses frequency of PyMT mammary tumor-derived lung metastases and tumorigenesis by inhibiting CCR7 and its downstream genes in lung of PyMT transgenic mouse. (C) The distribution of sizes of metastatic foci in control group (n = 7) and CRE group (n = 6) lungs as mean ± SEM. * p < 0.05 vs. control. (D) Immunohistochemistry staining against Ccr7 in lung tissues was done and representative images of tissue sections from each group were taken at 200X and 40X magnification. Scale bars = 50µm. Histogram data was presented as mean ± SEM. * p < 0.05 vs. control. (E) Western blot of proteins extracted from lung tissues against ERalpha and β-actin. * p < 0.05 vs. control. (F) Level of Ccr7 protein in lung tissues was analyzed by Western blot. Gapdh was used as an internal control. * p < 0.05 vs. control. (G) To investigate level of transcripts associated with breast cancer metastasis the expression levels of Ccr7, c-Fos, Mmp9, Ccl21, Ccl19, Ps2, Fsp1 and Jun were monitored by qRT-PCR. β-actin gene was used as an internal control in real time PCR. Values represent means ± S.E.M. n = (3); * p < 0.05 and ** p < 0.005 vs. control.