Journal: International Journal of Molecular Sciences

Article Title: Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids

doi: 10.3390/ijms27010123

Figure Lengend Snippet: mCHT treatment suppresses expression of stemness-related genes in TNBC cell line-derived spheroids. RNA was extracted from BT-549 ( A ) and MDA-MB-231 ( B ) surviving cells recovered from the culture of 1st- and 2nd-generation spheroids treated with vehicle alone (NT) or treated with either STD or mCHT 5-FU+VNR schedule, as described in . 1st-generation spheroids are shown in ( A , C ), and 2nd-generation spheroids are shown in ( B , D ). Expression levels of stemness-related genes (CD44, CD133, NOTCH-1, and MYC) were normalized to GAPDH and reported as relative fold changes. Statistical analysis was performed using two-tailed T test. Values represent mean ± SEM of three separate experiments. Asterisks indicate p < 0.05 between NT and mCHT- or STD-treated cells.

Article Snippet: All TaqMan probes, CD133 (Hs00608272_m1), CD44 (Hs01075861_m1), NOTCH-1 (Hs01062014_m1), MYC (Hs00153408_m1) and GAPDH (Hs99999905_m1) were purchased from Life Technologies (Thermo Fisher Scientific, Monza, Italy).

Techniques: Expressing, Derivative Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids

doi: 10.3390/ijms27010123

Figure Lengend Snippet: mCHT is more effective than STD schedule in decreasing stemness-related gene expression in both in 1st- and 2nd-generation spheroids derived from TNBC commercial cell lines. (( A , B ) Top ) Representative Western blots (from a single experiment) using proteins extracted from BT-549 ( A ) and MDA-MB-231 ( B ) surviving cells recovered from the culture of 1st- and 2nd-generation spheroids at the end of STD and mCHT treatments. NT indicates lysates from cells treated with vehicle alone. cl-NOTCH-1 indicates the cleaved form of NOTCH-1 that corresponds to the transcriptionally active intracellular domain of NOTCH-1. GAPDH was used as a loading control and to perform densitometric evaluation of relative protein levels. (( A , B ) Bottom ). Graphs plotting the densitometric quantitation of protein levels reported relative to NT (set to 1). Statistical analysis was performed using two-tailed T test. Values represent mean ± SEM of three experiments. Asterisks indicate p < 0.05.

Article Snippet: All TaqMan probes, CD133 (Hs00608272_m1), CD44 (Hs01075861_m1), NOTCH-1 (Hs01062014_m1), MYC (Hs00153408_m1) and GAPDH (Hs99999905_m1) were purchased from Life Technologies (Thermo Fisher Scientific, Monza, Italy).

Techniques: Gene Expression, Derivative Assay, Western Blot, Control, Quantitation Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids

doi: 10.3390/ijms27010123

Figure Lengend Snippet: mCHT reduces the ability to form 1st-generation spheroids and the expression of stemness markers in MS-186 cells (( A ) Top ) 1st-generation spheroids derived as described in were treated with vehicle alone (NT) or under the STD or mCHT schedule. (( A ) Bottom ) quantification of spheroid area (mm 2 ) in MS-186 1st-generation spheroids under NT, STD, and mCHT treatment (10× magnification) For each treatment, but for mCHT (where very few spheroids were formed), 10 spheroids were evaluated. Statistical analysis was performed using two-tailed T test. Values represent mean ± SEM of three experiments. Asterisks indicate p < 0.05. ( B ) Relative gene expression analysis (RT-qPCR) of the stemness-associated markers CD44, CD133, NOTCH-1, and MYC. Statistical analysis was performed using two-tailed T test. Values represent mean ± SEM of three experiments. Asterisks indicate p < 0.05 between NT and mCHT- or STD-treated cells.

Article Snippet: All TaqMan probes, CD133 (Hs00608272_m1), CD44 (Hs01075861_m1), NOTCH-1 (Hs01062014_m1), MYC (Hs00153408_m1) and GAPDH (Hs99999905_m1) were purchased from Life Technologies (Thermo Fisher Scientific, Monza, Italy).

Techniques: Expressing, Derivative Assay, Two Tailed Test, Gene Expression, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids

doi: 10.3390/ijms27010123

Figure Lengend Snippet: mCHT downregulates stemness-associated protein and epigenetic regulators in 1st- and 2nd-generation spheroids derived from MS-186 cells. (( A , B ), Top ) Representative Western blots using proteins extracted from MS-186-derived 1st- and 2nd-generation surviving spheroids recovered at the end of STD and mCHT treatments. Cl-NOTCH-1 indicates the cleaved form of NOTCH-1 that corresponds to the intracellular domain of NOTCH-1. NT indicates lysates from cells treated with vehicle alone. GAPDH was used as a loading control and to perform densitometric evaluation of relative protein levels. (( A , B ), Bottom ). Graphs plotting the densitometric quantitation of protein levels reported relative to NT (set to 1). Statistical analysis was performed using two-tailed T test. Values represent mean ± SEM of three experiments. Asterisks indicate p < 0.05.

Article Snippet: All TaqMan probes, CD133 (Hs00608272_m1), CD44 (Hs01075861_m1), NOTCH-1 (Hs01062014_m1), MYC (Hs00153408_m1) and GAPDH (Hs99999905_m1) were purchased from Life Technologies (Thermo Fisher Scientific, Monza, Italy).

Techniques: Derivative Assay, Western Blot, Control, Quantitation Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids

doi: 10.3390/ijms27010123

Figure Lengend Snippet: Summary of the results obtained on 2nd-generation spheroids derived from primary TNBC cell line MS-186, disaggregated from 1st-generation spheroids after STD or mCHT treatments. In the absence of treatment (NT), CSCs continue to proliferate and maintain stemness markers (CD44, CD133, MYC, cleaved NOTCH-1), epigenetic regulators (H3K27ac, EZH2), and signaling molecules (p-STAT3/STAT3). The STD treatment partly reduces the mass and the number of spheroids but induces high levels of stemness markers, which continue to activate stemness pathways and sustain drug resistance. In contrast, mCHT dramatically impairs spheroid formation, epigenetic signaling, and associated markers, thereby improving treatment responses.

Article Snippet: All TaqMan probes, CD133 (Hs00608272_m1), CD44 (Hs01075861_m1), NOTCH-1 (Hs01062014_m1), MYC (Hs00153408_m1) and GAPDH (Hs99999905_m1) were purchased from Life Technologies (Thermo Fisher Scientific, Monza, Italy).

Techniques: Derivative Assay