2.2. The Cellular Expression of PrPC in Different Stages of Human Bladder Carcinoma (Figure 1)
We hypothesized that there could be a strong correlation between an increased intensity of cellular expression of PrP
C and a progressive increase in BC stages in urinary bladder cancer patients. Therefore, we acquired a tissue array from US Biomax Inc., and performed an IHC staining. The analytical results illustrated that (1) the cellular expression of PrP
C in BC tissue was significantly increased when compared with the normal bladder specimen; (2) when we looked at the correlation between BC stage (i.e., TNM staging system) and the expression of PrP
C, we found that the cellular expression of PrP
C was significantly upregulated in patients with stage II BC when compared to those with Stage I BC, suggesting that our hypothesis was reasonable. However, for the tissue array specimens, we could only obtain two patients with stage III BC and no stage IV patient who received an operation to provide a BC specimen (i.e., due to advanced BC that was not suitable for surgical intervention). Accordingly, we had no adequate sample sizes of stages III and IV of BC for statistical analysis. (3) When we examined the tumor grading and the
TNM staging system, we also found that there was a strong correlation among these two BC classifications and the expression of PrP
C; (4) two interesting findings were observed during analysis. First, we identified that the PrP
C expression in nuclei was remarkably increased compared to its expression in the cytoplasm of BC cells. Second, we also found that the expression of PrP
C intensity was significantly enhanced in the elder patients than in the younger patients. To the best of our knowledge, the significance of these two findings has yet to be explored. Our findings from the tissue array may suggest that the PrP
C might correlate with BC growth and proliferation (
Figure 1).
Figure 1.
The cellular expression of PrPC in human bladder cancer patients. (A) Immunohistochemical results of cellular prion protein (PrPC) on bladder cancer (BC) tissue arrays from 38 BC patients. Columns a to d represented harvested BC tissue; e to f represented normal specimens (i.e., normal part of BC), i.e., indicating that (a) represented a BC specimen and (d) corresponded to a normal specimen from the same BC patient. The green, blue, and red circles represent Stage I, II, and III classifications (i.e., TNM staging system), respectively. (B) The expression of PrPC in the tumor part was remarkably increased when compared to the normal part from the same patient. *** represents the p value < 0.001 by paired t-tests. N = normal; T = tumor. (C) The expression of PrPC in Stage II was significantly more enhanced than the Stage I in the tumor part. *** represents the p value < 0.001 by unpaired t-tests. (D) The expression of PrPC was notably and gradually increased from grade 1 to grade 3. * and *** represent the p value < 0.05 and <0.001 by one-way ANOVA analysis. (E) The expression of PrPC in Stage 3 (T stage in TNM classifications) was significantly stronger than in Stages I and II. * represents the p value < 0.05 by one-way ANOVA analysis. (F) Immunohistochemical result of PrPC identified that the nuclear translocation in PrPC is strongly expressed in the tumor part rather than in the normal part of the same patient. (G) The PrPC expression was remarkably increased in nuclear (Nu) translocation rather than in cytoplasm (Cyto). ** represents the p value < 0.01 in unpaired t-tests. (H) The expression of PrPC was significantly increased in older patients when compared to younger ones. The classifications of high vs. low PrPC expression were separated by mean ± 1 S.E.M. * represents the p value < 0.05 by unpaired t-tests.
Figure 1.
The cellular expression of PrPC in human bladder cancer patients. (A) Immunohistochemical results of cellular prion protein (PrPC) on bladder cancer (BC) tissue arrays from 38 BC patients. Columns a to d represented harvested BC tissue; e to f represented normal specimens (i.e., normal part of BC), i.e., indicating that (a) represented a BC specimen and (d) corresponded to a normal specimen from the same BC patient. The green, blue, and red circles represent Stage I, II, and III classifications (i.e., TNM staging system), respectively. (B) The expression of PrPC in the tumor part was remarkably increased when compared to the normal part from the same patient. *** represents the p value < 0.001 by paired t-tests. N = normal; T = tumor. (C) The expression of PrPC in Stage II was significantly more enhanced than the Stage I in the tumor part. *** represents the p value < 0.001 by unpaired t-tests. (D) The expression of PrPC was notably and gradually increased from grade 1 to grade 3. * and *** represent the p value < 0.05 and <0.001 by one-way ANOVA analysis. (E) The expression of PrPC in Stage 3 (T stage in TNM classifications) was significantly stronger than in Stages I and II. * represents the p value < 0.05 by one-way ANOVA analysis. (F) Immunohistochemical result of PrPC identified that the nuclear translocation in PrPC is strongly expressed in the tumor part rather than in the normal part of the same patient. (G) The PrPC expression was remarkably increased in nuclear (Nu) translocation rather than in cytoplasm (Cyto). ** represents the p value < 0.01 in unpaired t-tests. (H) The expression of PrPC was significantly increased in older patients when compared to younger ones. The classifications of high vs. low PrPC expression were separated by mean ± 1 S.E.M. * represents the p value < 0.05 by unpaired t-tests.
2.3. The Cell Viability and Wound Healing Rate (Figure 2)
To test the impact of PrPC overexpression (i.e., PRNP gene overexpression in T24 cells, denoted as PrPC-OE-T24), Mel, and cisplatin on T24 cell viability, the MTT assay was adopted in the present study. The result showed that, when compared with the control (i.e., SV-HUC-1), the cell viability was notably increased in T24 (G1) cells at the time intervals of 24/48/72 h for the cell culture. However, this parameter was significantly inverted by Mel (G2) and more remarkably inverted by cisplatin (G3) at these time points. When looked at the situation of PrPC overexpression in T24 cells (i.e., PrPC-OE-T24), we found that the cell viability was significantly increased in PrPC-OE-T24 cells (G4) when compared to SV-HUC-1 and G1 to G3 at all time intervals of 24, 48, or 72 h, indicating that the condition of PrPC-OE would accelerate the speed of cell proliferation. The important finding was that this cell viability was significantly suppressed by Mel (G5) and more significantly suppressed by cisplatin (G6).
To test the wound-healing process, i.e., an indicator of cell proliferation, growth, and expansion rate in the above-mentioned conditions, the cells were incubated for 24 h. As we expected, the result of the wound-healing process exactly followed a similar pattern of cell viability among the SV-HUC-1 and G1 to G6 groups (
Figure 2).
Figure 2.
The cell viability and wound healing rate in different situations. (A) Analysis of the cell survival rate by 24 h (i.e., cell viability), * vs. different symbols (†, ‡, §, ¶, α, and β), p < 0.0001. (B) Analysis of the cell survival rate by 48 h (i.e., cell viability), * vs. different symbols (†, ‡, §, ¶, α, and β), p < 0.0001. (C) Analytical result of the cell survival rate by 72 h (i.e., cell viability), * vs. different symbols (†, ‡, §, ¶, α, and β), p < 0.0001. (D1–D4,E1–E7) The microscopic findings (40×) at 0 h (i.e., (D1–D7)) and 24 h (i.e., (E1–E7)) for illustrating the “wound healing” process (cell amount: 5.0 × 104/well) between the seven groups. (F) Analysis of percentage of wound healing at 24 h, * vs. different symbols (†, ‡, §, ¶, α, and β), p < 0.0001. Symbols: (*, †, ‡, §, ¶, α, and β) or letters (E1–E7) indicate significance for each other (at 0.05 level). All statistical analyses were conducted by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). The formula for the calculation of the wound healing process (%) = cell migrated area at 24 h/the original migrated area at 0 h. PrPC = cellular prion protein. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 μM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 μM treated for 24 h).
Figure 2.
The cell viability and wound healing rate in different situations. (A) Analysis of the cell survival rate by 24 h (i.e., cell viability), * vs. different symbols (†, ‡, §, ¶, α, and β), p < 0.0001. (B) Analysis of the cell survival rate by 48 h (i.e., cell viability), * vs. different symbols (†, ‡, §, ¶, α, and β), p < 0.0001. (C) Analytical result of the cell survival rate by 72 h (i.e., cell viability), * vs. different symbols (†, ‡, §, ¶, α, and β), p < 0.0001. (D1–D4,E1–E7) The microscopic findings (40×) at 0 h (i.e., (D1–D7)) and 24 h (i.e., (E1–E7)) for illustrating the “wound healing” process (cell amount: 5.0 × 104/well) between the seven groups. (F) Analysis of percentage of wound healing at 24 h, * vs. different symbols (†, ‡, §, ¶, α, and β), p < 0.0001. Symbols: (*, †, ‡, §, ¶, α, and β) or letters (E1–E7) indicate significance for each other (at 0.05 level). All statistical analyses were conducted by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). The formula for the calculation of the wound healing process (%) = cell migrated area at 24 h/the original migrated area at 0 h. PrPC = cellular prion protein. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 μM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 μM treated for 24 h).
2.5. Cell Proliferation, Cell Stress, and Cell Cycle Signaling, Oxidative Stress, and Cell Apoptosis (Figure 4, Figure 5, Figure 6 and Figure 7)
In addition to an assessment of the impacts of PrPC, Mel, and cisplatin on the T24 cellular-level expressions, we also investigated the impact of these three factors on the protein expressions of the signaling of T24 cells in the same situations, i.e., the cells were categorized from G1 to G6.
The result of the Western blot analysis revealed that the protein levels of PrP
C, PI3K, p-Akt, and p-m-TOR, four cell-proliferation biomarkers, and MMP-9, an indicator of a cell migratory helper, were significantly increased in G1 when compared to the control (i.e., SV-HUC-1) (
Figure 4). However, these biomarkers were remarkably inverted in G2 and more remarkably inverted in G3 (
Figure 4). Additionally, we further recognized that these biomarkers were significantly upregulated in G4 (i.e., PrP
C-OE-T24 cells) than in those of the control and G1 to G3, implying that the condition of PrP
C-OE would upregulate the cell-survival/proliferative signaling (
Figure 4). Of notable significance was that these parameters were notably suppressed in G5 (i.e., by Mel treatment) and were more significantly suppressed in G6 (i.e., by cisplatin treatment) (
Figure 4).
Figure 4.
The protein levels of cell proliferation markers in different situations. (A) Protein level of PrPC: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (B) Protein level of phosphorylated (p)-PI3K: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (C) Protein level of p-Akt: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (D) Protein level of p-m-TOR: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (E) Protein level of MMP-9: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. n = 6 for each group. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 uM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 uM treated for 24 h).
Figure 4.
The protein levels of cell proliferation markers in different situations. (A) Protein level of PrPC: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (B) Protein level of phosphorylated (p)-PI3K: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (C) Protein level of p-Akt: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (D) Protein level of p-m-TOR: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (E) Protein level of MMP-9: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. n = 6 for each group. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 uM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 uM treated for 24 h).
Next, when we looked at the protein expressions of cyclin-D1, cyclin-E1, ckd2, ckd4, and the mitochondrial cytochrome, C, five biomarkers of cell cycle/mitochondrial functional integrity and the protein expression of PINK1, a mediator of mitophagy/fission (
Figure 5), as well as the protein levels of RAS, c-RAF, p-MEK1/2, and p-ERK1/2 (four indices of cell stress biomarkers) (
Figure 6), all exhibited a similar manner of cell proliferation signaling among the groups of SV-HUC-1 and G1 to G6.
Figure 5.
The protein levels of cell cycle signaling in different situations. (A) Protein level of cyclin D1: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (B) Protein expression of cyclin E1: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (C) Protein level of ckd2: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (D) Protein level of phosphatase and tensin homolog (p-TEN): 1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (E) Protein level of ckd4: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (F) Protein level of mitochondrial cytochrome C (mit-CytoC): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (G) Protein level of PTEN-induced kinase 1 (PINK1): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. n = 6 for each group. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 uM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 uM treated for 24 h).
Figure 5.
The protein levels of cell cycle signaling in different situations. (A) Protein level of cyclin D1: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (B) Protein expression of cyclin E1: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (C) Protein level of ckd2: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (D) Protein level of phosphatase and tensin homolog (p-TEN): 1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (E) Protein level of ckd4: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (F) Protein level of mitochondrial cytochrome C (mit-CytoC): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (G) Protein level of PTEN-induced kinase 1 (PINK1): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. n = 6 for each group. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 uM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 uM treated for 24 h).
Figure 6.
Protein expressions of cell stress signaling in different situations. (A) Protein level of RAS: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (B) Protein level of p-MEK1/2): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (C) Protein level of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (D) Protein level of proto-oncogene c-Raf (c-Raf): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. n = 6 for each group. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 μM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 uM treated for 24 h).
Figure 6.
Protein expressions of cell stress signaling in different situations. (A) Protein level of RAS: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (B) Protein level of p-MEK1/2): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (C) Protein level of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (D) Protein level of proto-oncogene c-Raf (c-Raf): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. n = 6 for each group. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 μM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 uM treated for 24 h).
Further, when we inspected the protein expressions of NOX-1 and NOX-2 (
Figure 7), two indices of oxidative stress, and the protein levels of mitochondrial Bax, cleaved (c)-PARP, and c-caspase3 (
Figure 7), three indices of apoptosis, all revealed an identical manner of cell stress biomarkers among the groups.
Figure 7.
Protein levels of oxidative stress and apoptosis in different situations. (A) Protein level of NOX-1: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (B) Protein level of NOX-2: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (C) Protein level of mitochondrial Bax (mit-Bax): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (D) Protein level of cleaved (c) caspase 3 (c-Casp3): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (E) Protein expression of c-PARP: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. n = 6 for each group. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 uM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 uM treated for 24 h).
Figure 7.
Protein levels of oxidative stress and apoptosis in different situations. (A) Protein level of NOX-1: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (B) Protein level of NOX-2: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (C) Protein level of mitochondrial Bax (mit-Bax): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. (D) Protein level of cleaved (c) caspase 3 (c-Casp3): (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values < 0.0001. (E) Protein expression of c-PARP: (1) * vs. different symbols (†, ‡, and §) and (2) ¶ vs. different symbols (α and β), all p values <0.0001. n = 6 for each group. Control (Clt) = V-HUC-1; G1 = T24 only; G2 = melatonin (Mel); G3 = cisplatin; G4 = PrPC overexpression in T24 cells (i.e., PrPC-OE-T24); G5 = PrPC-OE-T24 cells + Mel (100 uM treated for 24 h); G6 = PrPC-OE-T24 cells + cisplatin (6.0 uM treated for 24 h).
2.6. Serial Changes of Tumor Volume and Tumor Weight at Day 28 after UMUC3 Bladder Cancer Cell Line Implantation into Nude Mouse Backs (Figure 8)
To evaluate the implanted tumor development in the living nude mice, the assessment of tumor dimensions occurred on days 7, 14, 21, and 28 after UMUC3 cancer cell implantation into the left and right backs of the nude mice. The results revealed that the total tumor volume (i.e., the summation of the left and right side) was notably gradually increased in group 1 (UMUC3 cells) from day 7 to day 28 after UMUC3 cell implantation. Additionally, by days 7 and 14 after UMUC3 cell implantation, the tumor volume showed no difference among the four groups. However, by days 21 and 28 after UMUC3 cell implantation, the tumor volume was remarkably diminished in group 2 (UMUC3 cell + Mel), further remarkably diminished in group 3 (UMUC3 cells + cisplatin) and further remarkably diminished in group 4 (UMUC3 cells + Mel + cisplatin) in these two time intervals when compared to group 1. Additionally, by day 28 after the tumor cell implantation, the harvested tumor weight displayed an identical pattern of day 28 tumor volume among the four groups. Our findings implied that the combined therapy of Mel and cisplatin offers the greatest benefit for suppressing the tumor growth (
Figure 8).
Figure 8.
Serial changes of tumor volume and tumor weight on day 28 after UMUC3 bladder cancer cell engraftment into nude mouse backs. (A–D) Showing the grossly anatomical appearance of tumor masses in UMUC3 (A), UMUC3 + Mel (20 mg/kg/day) (B), UMUC3 + cisplatin (1 mg/kg/day) (C) UMUC3 + combined Mel-cisplatin (D) groups. The tumor size was remarkably diminished in UMUC3 +Mel, further remarkably diminished in UMUC3 + cisplatin and furthermore remarkably diminished in UMUC3 + combined Mel–cisplatin when compared to UMUC3 only by days 14, 21, and 28, respectively. (E) The analyses of the time points of tumor volume on days 7, 14, 18, 21, and 28. By days 7 and 14, the tumor volume did not differ among the four groups. On day 21, * vs. different symbols (†, ‡, and §), p < 0.0001. On day 28, α vs. different symbols (ß, γ, and κ), p < 0.0001. (F) Revealing the time courses of the growth of tumor volume size. The analytical results on the time intervals of days 7, 14, 18, 21, and 28 were exactly identical to (E). (G) On day 28, the tumor weight, * vs. different symbols (†, ‡, and §), p < 0.0001. n = 10 for each group. Mel = melatonin.
Figure 8.
Serial changes of tumor volume and tumor weight on day 28 after UMUC3 bladder cancer cell engraftment into nude mouse backs. (A–D) Showing the grossly anatomical appearance of tumor masses in UMUC3 (A), UMUC3 + Mel (20 mg/kg/day) (B), UMUC3 + cisplatin (1 mg/kg/day) (C) UMUC3 + combined Mel-cisplatin (D) groups. The tumor size was remarkably diminished in UMUC3 +Mel, further remarkably diminished in UMUC3 + cisplatin and furthermore remarkably diminished in UMUC3 + combined Mel–cisplatin when compared to UMUC3 only by days 14, 21, and 28, respectively. (E) The analyses of the time points of tumor volume on days 7, 14, 18, 21, and 28. By days 7 and 14, the tumor volume did not differ among the four groups. On day 21, * vs. different symbols (†, ‡, and §), p < 0.0001. On day 28, α vs. different symbols (ß, γ, and κ), p < 0.0001. (F) Revealing the time courses of the growth of tumor volume size. The analytical results on the time intervals of days 7, 14, 18, 21, and 28 were exactly identical to (E). (G) On day 28, the tumor weight, * vs. different symbols (†, ‡, and §), p < 0.0001. n = 10 for each group. Mel = melatonin.
2.7. Cellular Expressions of MMPs and PrPC in Harvested Tumors on Day 28 after UMUC3 Cell Engraftment into the Mouse Backs (Figure 9)
It is well-known that activation of proteolytic enzymes such as matrix metalloproteinases (MMPs) for cleavage of extra-cellular matrix (ECM) serves as the cardinal role for the invasion and migration of malignancy [
30]. In the present study, we observed that as to compare with the group 1, the expressions of MMP-2 and MMP-9, two indices of MMPs for ECM proteolysis/degradation, were remarkably reduced in group 2, further remarkably reduced in group 3 and furthermore remarkably reduced in group 4. Additionally, the cellular expression of PrP
C illustrated a similar manner of MMPs between the groups (
Figure 9).
Figure 9.
Cellular expressions of MMPs and PrPC in harvested tumors on day 28 after UMUC3 cell engraftment into nude mouse backs. (A–D) Demonstrating the microscopic finding (400×) of IHC staining for identifying MMP-2 (gray color) expression. (E) Analysis of expression of intensity (i.e., scores) of positively stained MMP-2, * vs. different symbols (†, ‡, and §), p < 0.0001. (F–I) Showing the microscopic finding (400×) of IHC staining for identifying MMP-2 (gray color) expression. (J) Analysis of expression of intensity (i.e., scores) of positively stained MMP-9, * vs. different symbols (†, ‡, and §), p < 0.0001. (K–N) Demonstrating the microscopic finding (400×) for identifying cellular prion protein (PrPC) expression (gray color). (O) Analysis of expression of intensity (i.e., scores) of positively stained PrPC, * vs. different symbols (†, ‡, and §), p < 0.0001. Scale bars in right lower corner represent 20 µm. n = 8 for each group. Mel = melatonin.
Figure 9.
Cellular expressions of MMPs and PrPC in harvested tumors on day 28 after UMUC3 cell engraftment into nude mouse backs. (A–D) Demonstrating the microscopic finding (400×) of IHC staining for identifying MMP-2 (gray color) expression. (E) Analysis of expression of intensity (i.e., scores) of positively stained MMP-2, * vs. different symbols (†, ‡, and §), p < 0.0001. (F–I) Showing the microscopic finding (400×) of IHC staining for identifying MMP-2 (gray color) expression. (J) Analysis of expression of intensity (i.e., scores) of positively stained MMP-9, * vs. different symbols (†, ‡, and §), p < 0.0001. (K–N) Demonstrating the microscopic finding (400×) for identifying cellular prion protein (PrPC) expression (gray color). (O) Analysis of expression of intensity (i.e., scores) of positively stained PrPC, * vs. different symbols (†, ‡, and §), p < 0.0001. Scale bars in right lower corner represent 20 µm. n = 8 for each group. Mel = melatonin.
2.8. The Protein Levels of Cell Survival/Cell Proliferation, Cell Stress and Oxidative-Stress/Mitochondrial Autophagic Biomarkers on Day 28 after UMUC3 Cell Engraftment into Nude Mouse Backs (Figure 10, Figure 11, Figure 12 and Figure 13)
After harvesting the tumor specimens, we employed a Western blot analysis to clarify the amounts of the three signaling biomarkers. The results of the Western blot analysis showed that the protein levels of p-PI3K, p-Akt, p-m-TOR, MMP-9, and PrP
C, five indicators of cell proliferation, were remarkably and gradually reduced from groups 1 to 4 (
Figure 10).
Figure 10.
Protein expressions of cell survival/cell proliferation signaling on day 28 after UMUC3 engraftment into nude mouse backs. (A) Protein level of phosphorylated (p)-PI3K, * vs. different symbols (†, ‡, and §), p < 0.0001. (B) Protein level of p-Akt, * vs. different symbols (†, ‡, and §), p < 0.0001. (C) Protein level of p-m-TOR, * vs. different symbols (†, ‡, and §), p < 0.0001. (D) Protein level of matrix metalloproteinase (MMP)-9, * vs. different symbols (†, ‡, and §), p < 0.0001. (E) Protein level of PrPC, * vs. different symbols (†, ‡, and §), p < 0.0001. n = 6 for each group. Mel = melatonin.
Figure 10.
Protein expressions of cell survival/cell proliferation signaling on day 28 after UMUC3 engraftment into nude mouse backs. (A) Protein level of phosphorylated (p)-PI3K, * vs. different symbols (†, ‡, and §), p < 0.0001. (B) Protein level of p-Akt, * vs. different symbols (†, ‡, and §), p < 0.0001. (C) Protein level of p-m-TOR, * vs. different symbols (†, ‡, and §), p < 0.0001. (D) Protein level of matrix metalloproteinase (MMP)-9, * vs. different symbols (†, ‡, and §), p < 0.0001. (E) Protein level of PrPC, * vs. different symbols (†, ‡, and §), p < 0.0001. n = 6 for each group. Mel = melatonin.
Additionally, the Western blot analysis demonstrated that the protein levels of cyclin-D1, clyclin-E1, ckd2, and ckd4, four indices of cell cycle, and PINK1, an indicator of mitophagy, were also remarkably and gradually reduced from groups 1 to 4 (
Figure 11). On the other hand, the protein level of P-TEN, an index of tumor suppression, revealed an inverse manner of cell cycling between the groups (
Figure 11).
Figure 11.
Protein expressions of cell cycle biomarkers on day 28 after UMUC3 engraftment into nude mouse backs. (A) Protein level of cyclin-D1, * vs. different symbols (†, ‡, and §), p < 0.0001. (B) Protein level of clyclin-E1, * vs. different symbols (†, ‡, and §), p < 0.0001. (C) Protein level of ckd2, * vs. different symbols (†, ‡, and §), p < 0.0001. (D) Protein level of ckd4, * vs. different symbols (†, ‡, and §), p < 0.0001. (E) Protein level of PINK1, * vs. different symbols (†, ‡, and §), p < 0.0001. (F) Protein level of mitochondrial cytochrome C (mit-CytoC), * vs. different symbols (†, ‡, and §), p < 0.0001. (G) Protein level for P-TEN, * vs. different symbols (†, ‡, §), p < 0.0001. n = 6 for each group. Mel = melatonin.
Figure 11.
Protein expressions of cell cycle biomarkers on day 28 after UMUC3 engraftment into nude mouse backs. (A) Protein level of cyclin-D1, * vs. different symbols (†, ‡, and §), p < 0.0001. (B) Protein level of clyclin-E1, * vs. different symbols (†, ‡, and §), p < 0.0001. (C) Protein level of ckd2, * vs. different symbols (†, ‡, and §), p < 0.0001. (D) Protein level of ckd4, * vs. different symbols (†, ‡, and §), p < 0.0001. (E) Protein level of PINK1, * vs. different symbols (†, ‡, and §), p < 0.0001. (F) Protein level of mitochondrial cytochrome C (mit-CytoC), * vs. different symbols (†, ‡, and §), p < 0.0001. (G) Protein level for P-TEN, * vs. different symbols (†, ‡, §), p < 0.0001. n = 6 for each group. Mel = melatonin.
Furthermore, the Western blot analysis showed that that protein expressions of RAS, c-RAF, p-MEK1/2 and p-ERK1/2, four biomarkers of cell-stress signaling, displayed a similar pattern of cell cycle among the groups (
Figure 12).
Figure 12.
Protein levels of cell stress signaling on day 28 after UMUC3 embedded into nude mouse back. (A) Protein level of RAS, * vs. different symbols (†, ‡, and §), p < 0.0001. (B) Protein level of c-RAF, * vs. different symbols (†, ‡, and §), p < 0.0001. (C) Protein level of p-MEK1/2, * vs. different symbols (†, ‡, and §), p < 0.0001. (D) Protein level of p-ERK1/2, * vs. different symbols (†, ‡, and §), p < 0.0001. n = 6 for each group. Mel = melatonin.
Figure 12.
Protein levels of cell stress signaling on day 28 after UMUC3 embedded into nude mouse back. (A) Protein level of RAS, * vs. different symbols (†, ‡, and §), p < 0.0001. (B) Protein level of c-RAF, * vs. different symbols (†, ‡, and §), p < 0.0001. (C) Protein level of p-MEK1/2, * vs. different symbols (†, ‡, and §), p < 0.0001. (D) Protein level of p-ERK1/2, * vs. different symbols (†, ‡, and §), p < 0.0001. n = 6 for each group. Mel = melatonin.
On the other hand, the protein levels of mitochondrial Bax, c-caspase 3, and c-ARP, three indices of apoptosis, the protein levels of NOX-1 and NOX-2, two indicators of oxidative stress, and the protein level of p-DRP1, an index of fission (i.e., implicated mitochondrial damage biomarker) expressed an inverse manner of cell stress signaling (
Figure 13).
Figure 13.
Protein levels of oxidative stress/mitochondrial damage apoptotic markers on day 28 after UMUC3 embedded into nude mouse back. (A) Protein level of mit-Bax, * vs. different symbols (†, ‡, and §), p < 0.0001. (B) Protein level of c-caspase 3 (c-Casp3), * vs. different symbols (†, ‡, and §), p < 0.0001. (C) Protein level of c-PARP, * vs. different symbols (†, ‡, and §), p < 0.0001. (D) Protein level of NOX-1, * vs. different symbols (†, ‡, and §), p < 0.0001. (E) Protein level of NOX-2, * vs. different symbols (†, ‡, and §), p < 0.0001. (F) Protein level of p-DRP1, * vs. different symbols (†, ‡, and §), p < 0.0001. n = 6 for each group. Mel = melatonin.
Figure 13.
Protein levels of oxidative stress/mitochondrial damage apoptotic markers on day 28 after UMUC3 embedded into nude mouse back. (A) Protein level of mit-Bax, * vs. different symbols (†, ‡, and §), p < 0.0001. (B) Protein level of c-caspase 3 (c-Casp3), * vs. different symbols (†, ‡, and §), p < 0.0001. (C) Protein level of c-PARP, * vs. different symbols (†, ‡, and §), p < 0.0001. (D) Protein level of NOX-1, * vs. different symbols (†, ‡, and §), p < 0.0001. (E) Protein level of NOX-2, * vs. different symbols (†, ‡, and §), p < 0.0001. (F) Protein level of p-DRP1, * vs. different symbols (†, ‡, and §), p < 0.0001. n = 6 for each group. Mel = melatonin.