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. 2024 Feb 16:15:1345516.
doi: 10.3389/fphar.2024.1345516. eCollection 2024.

New insights into the anticancer effects of Polycladia crinita aqueous extract and its selenium nanoformulation against the solid Ehrlich carcinoma model in mice via VEGF, notch 1, NF-кB, cyclin D1, and caspase 3 signaling pathway

Badriyah S Alotaibi  1 , Thanaa A El-Masry  2 , Hend Selim  3 , Maisra M El-Bouseary  4 , Mostafa M El-Sheekh  5 , Mofida E M Makhlof  6 , Maysa M F El-Nagar  2
Affiliations

New insights into the anticancer effects of Polycladia crinita aqueous extract and its selenium nanoformulation against the solid Ehrlich carcinoma model in mice via VEGF, notch 1, NF-кB, cyclin D1, and caspase 3 signaling pathway

Badriyah S Alotaibi et al. Front Pharmacol. .

Abstract

Background: Phaeophyceae species are enticing interest among researchers working in the nanotechnology discipline, because of their diverse biological activities such as anti-inflammatory, antioxidant, anti-microbial, and anti-tumor. In the present study, the anti-cancer properties of Polycladia crinita extract and green synthesized Polycladia crinita selenium nanoparticles (PCSeNPs) against breast cancer cell line (MDA-MB-231) and solid Ehrlich carcinoma (SEC) were investigated. Methods: Gas chromatography-mass spectroscopy examinations of Polycladia crinita were determined and various analytical procedures, such as SEM, TEM, EDX, and XRD, were employed to characterize the biosynthesized PCSeNPs. In vitro, the anticancer activity of free Polycladia crinita and PCSeNPs was evaluated using the viability assay against MDA-MB-231, and also cell cycle analysis by flow cytometry was determined. Furthermore, to study the possible mechanisms behind the in vivo anti-tumor action, mice bearing SEC were randomly allocated into six equal groups (n = 6). Group 1: Tumor control group, group 2: free SeNPs, group 3: 25 mg/kg Polycladia crinita, group 4: 50 mg/kg Polycladia crinita, group 5: 25 mg/kg PCSeNPs, group 6: 50 mg/kg PCSeNPs. Results: Gas chromatography-mass spectroscopy examinations of Polycladia crinita extract exposed the presence of many bioactive compounds, such as 4-Octadecenoic acid-methyl ester, Tetradecanoic acid, and n-Hexadecenoic acid. These compounds together with other compounds found, might work in concert to encourage the development of anti-tumor activities. Polycladia crinita extract and PCSeNPs were shown to inhibit cancer cell viability and early cell cycle arrest. Concentrations of 50 mg/kg of PCSeNPs showed suppression of COX-2, NF-кB, VEGF, ki-67, Notch 1, and Bcl-2 protein levels. Otherwise, showed amplification of the caspase 3, BAX, and P53 protein levels. Moreover, gene expression of caspase 3, caspase 9, Notch 1, cyclin D1, NF-кB, IL-6, and VEGF was significantly more effective with PCSeNPs than similar doses of free extract. Conclusion: The PCSeNPs mediated their promising anti-cancerous action by enhancing apoptosis and mitigating inflammation, which manifested in promoting the total survival rate and the tumor volume decrease.

Keywords: anticancer; brown algae; macroalgae; selenium nanoparticles; solid Ehrlich carcinoma.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

None
SeNPs, selenium nanoparticles; P. crinita, Polycladia crinita; and PCSeNPs, Polycladia crinita selenium nanoparticles.
FIGURE 1
FIGURE 1
(A) GC-MS chromatogram of Polycladia crinita aqueous extract. (B) TEM imaging of Polycladia crinita mediated selenium nanoparticles (PCSeNPs), showing different particle sizes and morphology at scale bar 100 nm. (C) SEM imaging of Polycladia crinita mediated selenium nanoparticles (PCSeNPs), showing different particle sizes and morphology at scale bar 500 nm. (D) The EDX analysis of Polycladia crinita mediated selenium nanoparticles (PCSeNPs). (E) Polycladia crinita-mediated selenium nanoparticles (PCSeNPs) produced some background noise in the XRD pattern.
FIGURE 2
FIGURE 2
The breast cancer cell line (MDA-MB-231) cell-cycle distribution. Control (A), after treatment with Polycladia crinita extract (B), and PCSeNPs (C), analyzed using flow cytometric analysis on the IC50 concentrations detected by MTT assay.
FIGURE 3
FIGURE 3
(A) The survival rate of mice in each group described by Kaplan-Meier survival plots. The survival rate of each group was calculated according to the formula: survival rate = number of surviving mice/total number of mice during the experiment (28 days) after 1, 2, 3, and 4 weeks. (B) Effect of treatments tumor weight. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus the free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, and bmeans significant versus 50 mg/kg PCSeNPs group. SeNPs: selenium nanoparticles, Polycladia crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05. (C) Effect of various treatments on tumor volume. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, and bmeans significant versus 50 mg/kg PCSeNPs group, SeNPs: selenium nanoparticles, P. crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05.
FIGURE 4
FIGURE 4
Effect of treatments on VEGF (A), NF-кB (B), Notch 1 (C), and Cyclin D1 (D) protein levels using ELISA technique at the end of the experiment (28 days). Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, bmeans significant versus 50 mg/kg PCSeNPs group, and ns: means non-significant versus control group. SeNPs: selenium nanoparticles, Polycladia crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05.
FIGURE 5
FIGURE 5
Effect of treatments on Caspase 3 (A), Caspase 9 (B), Notch1 (C), Cyclin D1 (D), NF-кB (E), IL-6 (F) and VEGF (G) gene expression using the qRT-PCR technique at the end of the experiment (28 days). Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, bmeans significant versus 50 mg/kg PCSeNPs group, and ns: means non-significant versus a control group. SeNPs: selenium nanoparticles, Polycladia crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05.
FIGURE 6
FIGURE 6
Histopathological findings (100x, scale bar = 200 μm). (i): Tumor control group showed solid sheets of malignant cells (black arrows), many tumor giant cells (blue arrow) with a mild area of necrosis grade 1 (red arrows), and mild lymphocytic infiltrate (green arrow). (ii): Free SeNPs group showed malignant cells (black arrows), many tumor giant cells (blue arrows) surrounded by mild tumor necrosis (red arrows) [necrosis grade 1], and mild lymphocytic infiltrate (green arrows). (iii): 25 mg/kg Polycladia crinita group showed malignant cells (black arrows), some tumor giant cells (blue arrows) surrounded by moderate tumor necrosis (red arrows) [necrosis grade 2], and mild lymphocytic infiltrate (green arrows). (iv): 50 mg/kg Polycladia crinita group showed malignant cells (black arrows), some tumor giant cells (blue arrows) surrounded by marked tumor necrosis (red arrows) [necrosis grade 3], and moderate lymphocytic infiltrate (green arrows). (v): 25 mg/kg PCSeNPs group showed malignant cells (black arrows), a few tumor giant cells (blue arrows) surrounded by marked tumor necrosis (red arrows) [necrosis grade 3], and moderate lymphocytic infiltrate (green arrows). (vi): 50 mg/kg PCSeNPs group showed shadows of necrotizing malignant and giant cells (black arrows) surrounded by diffuse tumor necrosis (red arrows) [necrosis grade 4] and moderate lymphocytic infiltrate (green arrow). (vii): Necrosis area (%) in tumor sections stained with H&E staining. Quantification of area% was done by ImageJ software; version 1.54 D, Java 1.8.0_354. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, bmeans significant versus 50 mg/kg PCSeNPs group, and ns: means non-significant versus control group. SeNPs: selenium nanoparticles, Polycladia crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05.
FIGURE 7
FIGURE 7
(A). COX-2 immunohistochemical expression (100x, scale bar = 200 μm). (i): Tumor control group showed moderate immune reaction (arrows) all over the tumor and in the vicinity of fat cells (arrowhead) invading the tumor. (ii): Free SeNPs group showed moderate immune reaction (arrows) all over the tumor and in the vicinity of fat cells (arrowhead) invading the tumor. (iii): 25 mg/kg Polycladia crinita group showed slightly moderate immune reaction (arrows) all over the tumor. (iv): 50 mg/kg Polycladia crinita group showed mild immune reaction (arrows). (v): 25 mg/kg PCSeNPs group showed mild immune reaction (arrows). (vi): 50 mg/kg PCSeNPs group showed very weak immune reaction (arrows) all over the tumor. (vii): COX-2 immunohistochemical positive area (%). Quantification of area% was done by ImageJ software; version 1.54 D, Java 1.8.0_354. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, and bmeans significant versus 50 mg/kg PCSeNPs group. SeNPs: selenium nanoparticles, Polycladia crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05. (B). Ki-67 immunohistochemical expression (100x, scale bar = 200 μm). (i): Tumor control group showed severely positive immune reaction (arrows), in the vicinity of fat cells (arrowheads) and necrotic muscle fibers (curved arrows). (ii): Free SeNPs group showed moderately positive immune reaction (arrows), in the vicinity of fat cells (arrowheads) and muscle fibers (curved arrows). (iii): 25 mg/kg P. crinita group showed moderately positive immune reaction (arrows), in the vicinity of fat cells (arrowheads) and muscle fibers (curved arrows). (iv): 50 mg/kg P. crinita group showed mild immune reaction (arrows), in the vicinity of fat cells (arrowheads) and muscle fibers (curved arrows). (v): 25 mg/kg PCSeNPs group showed mild positive immune reaction (arrows), in the vicinity of fat cells (arrowheads). (vi): 50 mg/kg PCSeNPs group showed a negative immune reaction of the tumor muscle fibers (curved arrows) which are invaded by fat cells (arrowheads). (vii): Ki-67 immunohistochemical positive area (%). Quantification of area% was done by ImageJ software; version 1.54 D, Java 1.8.0_354. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, and ameans significant versus 25 mg/kg PCSeNPs group. SeNPs: selenium nanoparticles, P. crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05. (C). Bcl-2 immunohistochemical expression (100x, scale bar = 200 μm). (i): Tumor control group had severe positive reactions (arrows) and in the vicinity of fat cells (arrowheads). (ii): Free SeNPs group showed strongly positive reaction (arrows) around and in-between necrotic muscle fibers (curved arrows). (iii): 25 mg/kg P. crinita group showed a moderately positive reaction (arrows) in the vicinity of necrotic muscle fibers (curved arrows). (vi): 50 mg/kg P. crinita group showed moderately positive reaction (arrows) around and in-between fat cells (arrowheads) invading the tumor. (v): 25 mg/kg PCSeNPs group showed mildly positive immune reaction (arrows) around and in-between fat cells (arrowheads) invading the tumor. (vi): 50 mg/kg PCSeNPs group showed weak immune reaction (arrows) around and in-between fat cells (arrowheads) invading the tumor. (vii): Bcl-2 immunohistochemical positive area (%). Quantification of area% was done by ImageJ software; version 1.54 D, Java 1.8.0_354. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, and bmeans significant versus 50 mg/kg PCSeNPs group. SeNPs: selenium nanoparticles, P. crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05. (D). Caspase 3 immunohistochemical expression (100x, scale bar = 200 μm). (i): Tumor control group showed weak positive reaction (arrows) in the vicinity of fat cells (arrowheads) all over the tumor and in-between and fat cells (arrowheads) invading the tumor. (ii): Free SeNPs group showed mild positive reaction (arrows) all over the tumor. (iii): 25 mg/kg P. crinita group showed mild positive reaction (arrows) all over the tumor, in-between muscle fibers (curved arrows) and fat cells (arrowheads) invading the tumor. (vi): 50 mg/kg P. crinita group showed strong moderate positive reaction (arrows) all over the tumor. (v): 25 mg/kg PCSeNPs group strong positive reaction (arrows) all over the tumor, in-between muscle fibers (curved arrows) and fat cells (arrowheads) invading the tumor. (vi): 50 mg/kg PCSeNPs group showed strong positive reaction (arrows) all over the tumor, in-between muscle fibers (curved arrows) and fat cells (arrowheads) invading the tumor. (vii): Caspase 3 immunohistochemical positive area. Quantification of area% was done by ImageJ software; version 1.54 D, Java 1.8.0_354. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, and bmeans significant versus 50 mg/kg PCSeNPs group. SeNPs: selenium nanoparticles, P. crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05. (E). BAX immunohistochemical expression (100x, scale bar = 200 μm). (i): Tumor control group showed weak mild immune reaction (arrows) all over the tumor. (ii): Free SeNPs group showed weak mild immune reaction (arrows) all over the tumor and in the vicinity of fat cells (arrowheads). (iii): 25 mg/kg P. crinita group showed mild positive immune reaction (arrows) all over the tumor and in-between muscle fibers (arrowheads). (vi): 50 mg/kg P. crinita group showed strong moderate immune reaction (arrows) all over the tumor, in the vicinity of fat cells (arrowheads) and in-between muscle fibers (curved arrows). (v): 25 mg/kg PCSeNPs group showed moderate immune reaction (arrows) all over the tumor and in the vicinity of muscle fibers (arrowheads). (vi): 50 mg/kg PCSeNPs group showed severe immune reaction (arrows) all over the tumor. (vii): BAX immunohistochemical positive area. Quantification of area% was done by ImageJ software; version 1.54 D, Java 1.8.0_354. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, and bmeans significant versus 50 mg/kg PCSeNPs group. SeNPs: selenium nanoparticles, P. crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05. (F). P53 immunohistochemical expression (100x, scale bar = 200 μm). (i): Tumor control group showed mild positive reactions (arrows) all over the tumor. (ii): Free SeNPs group showed mild positive reaction (arrows) all over the tumor. (iii): 25 mg/kg P. crinita group showed moderate positive reaction (arrows) all over the field and in the vicinity of fat cells (arrowhead). (iv): 50 mg/kg P. crinita group showed moderate positive reaction (arrows) all over the tumor. (v): 25 mg/kg PCSeNPs group showed strong moderate positive reaction (arrows) all over the tumor. (vi): 50 mg/kg PCSeNPs group showed strong positive reactions (arrows) all over the tumor and in the vicinity of fat cells (arrowheads). (vii): P53 immunohistochemical positive area (%). Quantification of area% was done by ImageJ software; version 1.54 D, Java 1.8.0_354. Data are expressed as mean ± SD, n = 6. *means significant versus the tumor control group, #means significant versus free SeNPs group, ameans significant versus 25 mg/kg PCSeNPs group, and bmeans significant versus 50 mg/kg PCSeNPs group. SeNPs: selenium nanoparticles, P. crinita: Polycladia crinita, and PCSeNPs: Polycladia crinita selenium nanoparticles. Each group differed significantly from the others at p ≤ 0.05.
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