Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 3. Combination of Aloin CPT-11 downregulates IGF1R and its downstream MEK/ERK and PI3K/AKT/mTOR pathways. (A) The effect of the Aloin-CPT- 11 combination on IGF1R expression in CRC cells (Lovo, Caco2 and SW620). (B-C) The effect of Aloin and CPT-11 combination on the two main IGF1R downstream pathways determined by western blot analysis. (D) IGF1R was knocked down to determine its role in CRC in the context of cell viability and apoptosis. (E-F) Knockdown of IGF1R reduced cell viability. Co-treatment of Aloin-CPT combination with siRNA-IGF1R reduced cell viability even further. (G-H) Aloin-CPT-11 enhanced apoptosis. Treatment of Aloin-CPT-11 combination with IGF1R knockdown intensified apoptosis even further. Results are presented as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Expressing, Western Blot, Knockdown, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 4. MicroRNA-133b is upregulated by Aloin-CPT-11 combination, targets IGF1R and reduces cell viability in LoVo cells. (A) MiRNAs that are predicted to target IGF1R were identified using miRTARGETLink2.0 2. Forty-seven miRNAs were initially identified. (B) The 47 miRNAs were further filtered and trimmed to 17 miRNAs. These miRNA were screened by RT-qPCR in LoVo cells after treatment with Aloin-CPT-11 combination. (C) The three miRNAs that showed the best sig nificant upregulation with Aloin-CPT-11 combination were identified. (D).The statistically significant upregulated miRNA-133b was adopted and Target Scan indicated that miR-133b binds the 3′ UTR region between 4007 and 4013 of the IGF1R. (E) Cells were transfected with miR-133b mimic and inhibitor and the transfection efficiency was determined by RT-qPCR. (F) Transfection of miR-133b mimic downregulated IGF1R while inhibitor upregulated it. (G-H) miRNA-133b targets IGF1R. The increase in cell viability induced by miRNA-133b inhibitor was reversed by Aloin-CPT-11 combination treatment. (I) IGF1R downregulation by miRNA-133b mimic was further enhanced with an addition of Aloin-CPT-11 combination treatment. Results are presented as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Quantitative RT-PCR, Transfection, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 5. Over expression of miRNA-133b reversed the effect of IGF1R over expression. (A) Co-transfection of cells with pcDNA-IGF1R and miR-133b mimic resulted in the downregulation of pcDNA-induced IGF1R overexpression. (B) pcDNA- induced IGF1R overexpression was reversed by treatment with Aloin-CPT-11 combination. (C) IGF1R-overexpressed cells showed an increase in cell viability but this phenomenon was reversed when treated with Aloin-CPT-11 combination. (D) Flow cytometry: combination of Aloin and CPT-11 enhanced LoVo cell apoptosis in presence of IGF1R upregulation. Results are presented as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Over Expression, Cotransfection, Flow Cytometry, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 6. Aloin and CPT-11 attenuated and reversed CPT-11 resistance in LoVo CPT-11 resistant cells. (A). CPT-11-resistant cells were established. Their response to CPT-11 were compared with that of parental LoVo cells. MTT cell viability had both cells respond to CPT-11 dose dependently. The response by the resistant cell was poor with IC50 three times more than parental cell (35.6 μМ against 12.0 μM). (B) Cell resistant characteristics were confirmed by IGF1R expression and stem cell markers. (C) The response of resistant cells to CPT-11 was again confirmed by western blot with apoptotic markers. (D-E) Expression of miR-133b and IGF1R mRNA in resistant cells was determined by RT-qPCR. (F-G) The effect of Aloin-CPT-11 combination in resistant cells was assessed through western blot focusing on IGF1R expression and apoptosis. Results are presented as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 7. Aloin and CPT-11 combination exhibited improved anti-tumor efficacy against murine colorectal cancer in BALB/c nude mice. (A) Schematic illustration of the animal model timeline and experimental plan. (B) Tumor volumes were recorded twice a week until the end of the study and were statistically compared between groups. (C) The physical sizes of the tumors were compared against each group after mice were sacrificed. (D-E) Tumor sizes and weights were measured and statistically compared. (F) Survival times and percentages were predicted and calculated using Kaplan-Meier estimator. (G-J). The in vivo effect of the Aloin-CPT-11 combination was determined by estimating the expression levels of IGF1R and its downstream pathways, as well as apoptotic markers in tumor xe nografts for each treatment group. (K) Eosin & Hematoxylin stain was used to asses mophological and poliferative changes in tumos. Immunohistochemestly was used to determine the expression levels of IGF1R and Ki67 in tumor xenograft. Results are shown as means ± standard deviation from three independent experi ments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Animal Model, In Vivo, Expressing, Staining, Standard Deviation