Fig 1: Schematic overview of the potential non-genomic P4 action in colorectal cancer. P4 may initiate rapid non-classical signaling through the complex of PGRMC1 and NENF, leading to increased proliferation and invasion of colorectal cancer cells. However, P4 cannot activate the classical genomic signaling pathway due to weak PGR expression in colorectal cancer cells (arrow: ↓PGR—weak PGR expression). P4 or NENF may significantly increase the release of IL-8 by colorectal cancer cells (arrow: ↑IL-8—increased release of IL-8). P4 significantly up-regulates mPRα and mPRγ expression in colorectal cancer cells (arrow: ↑mPR-α, ↑mPR-γ—increased expression of mPR-α and mPR-γ). NENF, neuron-derived neurotrophic factor; P4, progesterone; PGR, nuclear progesterone receptor; and PGRMC1, progesterone receptor membrane component 1.
Fig 2: Characterization of PGR expression levels in colorectal cancer and DLD-1 and HT-29 cell lines. PGR expression at gene, (a) and protein (b,c) levels in NM (n = 10) and colorectal cancer (CRC) tissues (n = 20). Original magnification, 20×; scale bar, 20 µm. The columns represent the mean ± SEM relative to PPIA. The Mann–Whitney test was used to compare NM vs. CRC results. Statistical significance of NM vs. CRC: **** p ≤ 0.0001, ** p ≤ 0.01. PGR expression after treatment with 1 µM of P4 and 1 µg/mL of NENF in DLD-1 cell line (d) and HT-29 cell line (e) (n = 3 independent experiments). The Mann–Whitney test was used to compare C vs. P4 and C vs. NENF of DLD-1 and HT-29 cells. The differences are statistically non-significant. Abbreviations: C, control/non-treated group; CRC, colorectal cancer; NENF, neudesin; NM, normal mucosa; P4, progesterone; PGR, nuclear progesterone receptor; PPIA, peptidylprolyl isomerase A; SEM, standard error of the mean; and vs., versus.
Fig 3: Characterization of NENF expression and release in colorectal cancer patients and DLD-1 and HT-29 cell lines. NENF expression at gene (a) and protein (b,c) levels in NM (n = 10) and colorectal cancer (CRC) tissues (n = 20). Original magnification, 20×; scale bar, 20 µm. The columns represent the mean ± SEM relative to PPIA. The Mann–Whitney test was used to compare NM vs. CRC results. The differences are statistically non-significant. Serum NENF concentration in colorectal cancer patients (n = 41) compared to the control group (n = 15) (d). The Mann–Whitney test was used to compare serum NENF concentration in C vs. CRC results. Statistical significance: * p ≤ 0.05. NENF expression after treatment with 1 µM of P4 in DLD-1 cell line (e) and HT-29 cell line (f) (n = 3 independent experiments). The Mann–Whitney test was used to compare NENF expression in C vs. CRC results in DLD-1 and HT-29 cells. The differences are statistically non-significant. NENF concentration in the medium after treatment with 1 µM of P4 in DLD-1 cell line (g) (n = 6) and HT-29 cell line (h) (n = 6). The Mann–Whitney test was used to compare NENF concentration in the medium C vs. P4 results of DLD-1 cells, statistical significance: ** p ≤ 0.01. Double staining for PGRMC1 and NENF in NM tissues (n = 10) and colorectal cancer (n = 20) (i). PGRMC1-positive cells are in red, NENF-positive cells are in green, nucleus localization in cells is in blue, and PGRMC1 and NENF merged staining is in orange, scale bar, 20 μm. Abbreviations: C, control/non-treated group; CRC, colorectal cancer; NENF, neudesin; NM, normal mucosa; P4, progesterone; PGRMC1, progesterone receptor membrane component 1; PPIA, peptidylprolyl isomerase A; SD, standard deviation; SEM, standard error of the mean; and vs., versus.
Fig 4: Characterization of IL-8 and CXCR1 expression and release in colorectal cancer patients and DLD-1 and HT-29 cell lines. IL-8 expression in NM (n = 10) and colorectal cancer (n = 20) tissues (a). The columns represent the mean ± SEM relative to PPIA. The Mann–Whitney test was used to compare NM vs. CRC results, statistical significance of NM vs. CRC for IL-8: *** p ≤ 0.001. IL-8 expression after treatment with 1 µM of P4 or with 1 µg/mL of NENF in DLD-1 (b) and HT-29 (c) cell lines (n = 3 independent experiments). The Mann–Whitney test was used to compare C vs. P4 and C vs. NENF results of DLD-1 and HT-29 cells, statistical significance of C vs. NENF: * p ≤ 0.05, ** p ≤ 0.01. IL-8 concentration in the medium of DLD-1 cell line after treatment with 1 µM of P4 or 1 ng/mL of NENF without or with 1 µM of AG-205 (d) (n = 6). The Mann–Whitney test was used to compare C vs. P4 without or with 1 µM of AG-205, C vs. NENF without or with 1 µM of AG-205, and also results of P4 without AG-205 vs. P4 with AG-205 of DLD-1 or NENF without AG-205 vs. NENF with AG-205 of DLD-1 cells, statistical significance: ** p ≤ 0.01. CXCR1 expression in NM (n = 10) and colorectal cancer (n = 20) tissues (e). The Mann–Whitney test was used to compare NM vs. CRC results. The differences are statistically non-significant. CXCR1 expression after treatment with 1 µM of P4 or with 1 µg/mL of NENF in DLD-1 (f) and HT-29 (g) cell lines (n = 3 independent experiments). The Mann–Whitney test was used to compare C vs. P4 and C vs. NENF results of DLD-1 and HT-29 cells. The differences are statistically non-significant. Abbreviations: AG-205, PGRMC1 inhibitor; C, control/non-treated group; CRC, colorectal cancer; CXCR1, C-X-C Motif Chemokine Receptor 1; IL-8, interleukin 8; NENF, neudesin; NM, normal mucosa; P4, progesterone; PGRMC1, progesterone receptor membrane component 1; PPIA, peptidylprolyl isomerase A; SEM, standard error of the mean; and vs., versus.
Fig 5: Characterization of mPRα, mPRβ, and mPRγ expression levels in colorectal cancer and DLD-1 and HT-29 cell lines. mPRα, mPRβ, and mPRγ expression at gene (a) and protein (b,c) levels in NM (n = 10) and colorectal cancer (CRC) tissues (n = 20). Original magnification, 20×; scale bar, 20 µm. The columns represent the mean ± SEM relative to PPIA. The Mann–Whitney test was used to compare NM vs. CRC results. Statistical significance of NM vs. CRC for mPRβ, mPRγ: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. mPRα, mPRβ, and mPRγ expression after treatment with 1 µM of P4 and 1 µg/mL of NENF in DLD-1 cell line (d) and HT-29 cell line (e) (n = 3 independent experiments). The Mann–Whitney test was used to compare C vs. P4 and C vs. NENF. Statistical significance of C vs. P4 for mPRα, mPRγ of HT-29 cells: * p ≤ 0.05. Other differences are statistically non-significant. Abbreviations: C, control/non-treated group; CRC, colorectal cancer; mPRα, membrane progesterone receptor alfa; mPRβ, membrane progesterone receptor beta; mPRγ, membrane progesterone receptor gamma; NENF, neudesin; NM, normal mucosa; P4, progesterone; PPIA, peptidylprolyl isomerase A; SEM, standard error of the mean; and vs., versus.
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