Journal: Cancers

Article Title: Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo

doi: 10.3390/cancers13184612

Figure Lengend Snippet: Metformin and everolimus inhibited CaSki and C33A cell growth. Inhibition effects of metformin (10, 20 mM) or everolimus (10, 20 μM) on ( A ) CaSki and ( B ) C33A cell growth measured using a CCK-8 assay. Cells were exposed to metformin with or without everolimus for 48 h, then subjected to CCK-8 assay. CIs of metformin combined with everolimus were calculated using Chou–Talay analyses. Values represent the mean ± SD from three replicates. *, †, γ, ** and δ p < 0.05 compared with CON, Met 10- or Eve 10- or Met 20- or Eve 20-treated groups. CON, 0.1% DMSO; M10, 10 mM metformin; M20, 20 mM metformin; E10, 10 μM everolimus; E20, 20 μM everolimus. CI, combination index; Fa, affected fraction.

Article Snippet: CaSki cells were cultured in an RPMI 1640 medium (1-41P05-K, BioConcept, Amimed, Allschwil, Switzerland), and C33A cells in Eagle’s Minimum Essential Medium (SH30024.02, MEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (SH30396.03, FBS, Cytiva) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Inhibition, CCK-8 Assay

Journal: Cancers

Article Title: Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo

doi: 10.3390/cancers13184612

Figure Lengend Snippet: Metformin and everolimus induced apoptosis in cervical cancer cells. CaSki and C33A cells were treated with metformin (10 mM) with or without everolimus (20 μM) for 48 h. ( A ) Apoptosis profile assessed using an Annexin V/PI assay by flow cytometry. ( B ) Quantitative results of apoptotic cells (Annexin V- and PI-stained cells) presented in the bottom plot. Values represent the mean ± SD from three replicates. *, † and δ p < 0.05 compared with CON, Met 10- or Eve 20-treated groups. CON, 0.1% DMSO; M10, 10 mM metformin; E20, 20 μM everolimus.

Article Snippet: CaSki cells were cultured in an RPMI 1640 medium (1-41P05-K, BioConcept, Amimed, Allschwil, Switzerland), and C33A cells in Eagle’s Minimum Essential Medium (SH30024.02, MEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (SH30396.03, FBS, Cytiva) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Flow Cytometry, Staining

Journal: Cancers

Article Title: Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo

doi: 10.3390/cancers13184612

Figure Lengend Snippet: Metformin and everolimus arrested the cell cycle in the sub-G1 and G0/G1 phases. ( A ) CaSki and ( B ) C33A cells were treated with metformin (10 mM) with or without everolimus (20 μM) for 48 h. Cell-cycle progression was assessed using a PI assay via flow cytometry. Quantitative results for the cell-cycle phase distribution (sub-G1, G0/G1, S, and G2/M phases) are presented in the bottom plot. Values represent the mean ± SD from three replicates. *, † and δ p < 0.05 compared with the CON, Met 10- or Eve 20-treated groups. CON, 0.1% DMSO; M10, 10 mM metformin; E20, 20 μM everolimus.

Article Snippet: CaSki cells were cultured in an RPMI 1640 medium (1-41P05-K, BioConcept, Amimed, Allschwil, Switzerland), and C33A cells in Eagle’s Minimum Essential Medium (SH30024.02, MEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (SH30396.03, FBS, Cytiva) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Flow Cytometry

Journal: Cancers

Article Title: Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo

doi: 10.3390/cancers13184612

Figure Lengend Snippet: Metformin and everolimus enhanced mitoROS-mediated OXPHOS expression in cervical cancer cells. CaSki and C33A cells were treated with metformin (10 mM) with or without everolimus (20 μM) for 48 h. ( A ) Mitochondrial ROS levels measured using MitoSOX Red reagent via flow cytometry. ( B ) Quantitative results of mitochondrial ROS levels presented in the bottom plot. ( C , D ) Protein expression levels of OXPHOS (complex I–V) measured using Western blotting. Quantitative results presented in the bottom plot. Values represent the mean ± SD from three replicates. *, † and δ p < 0.05 compared with CON, Met 10- or Eve 20-treated groups. CON, 0.1% DMSO; M10, 10 mM metformin; E20, 20 μM everolimus.

Article Snippet: CaSki cells were cultured in an RPMI 1640 medium (1-41P05-K, BioConcept, Amimed, Allschwil, Switzerland), and C33A cells in Eagle’s Minimum Essential Medium (SH30024.02, MEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (SH30396.03, FBS, Cytiva) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Cancers

Article Title: Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo

doi: 10.3390/cancers13184612

Figure Lengend Snippet: Metformin and everolimus induced apoptosis through mitochondrial and caspase-mediated pathways. Representative Western blot data showing expressions of apoptosis-related proteins in ( A ) CaSki and ( B ) C33A cells treated with metformin (10 mM) with or without everolimus (20 μM) for 48 h. GAPDH served as the loading control. Quantitative results are presented in the bottom plot. Values represent the mean ± SD from three replicates. *, † and δ p < 0.05 compared with the CON, Met 10- or Eve 20-treated groups. CON, 0.1% DMSO; M10, 10 mM metformin; E20, 20 μM everolimus.

Article Snippet: CaSki cells were cultured in an RPMI 1640 medium (1-41P05-K, BioConcept, Amimed, Allschwil, Switzerland), and C33A cells in Eagle’s Minimum Essential Medium (SH30024.02, MEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (SH30396.03, FBS, Cytiva) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Western Blot, Control

Journal: Cancers

Article Title: Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo

doi: 10.3390/cancers13184612

Figure Lengend Snippet: Metformin and everolimus induced apoptosis through the PI3K/AKT and JNK/p38 MAPK signaling pathways in cervical cancer cells. Western blotting was used to detect PIK3CA, p-AKT, AKT, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK1/2, and JNK1/2 levels in ( A ) CaSki and ( B ) C33A cells treated with metformin (10 mM) with or without everolimus (20 μM) for 48 h. GAPDH served as the loading control. Quantitative results are presented in the bottom plot. Values represent the mean ± SD from three replicates. *, † and δ p < 0.05 compared with CON, Met 10- or Eve 20-treated groups. CON, 0.1% DMSO; M10, 10 mM metformin; E20, 20 μM everolimus.

Article Snippet: CaSki cells were cultured in an RPMI 1640 medium (1-41P05-K, BioConcept, Amimed, Allschwil, Switzerland), and C33A cells in Eagle’s Minimum Essential Medium (SH30024.02, MEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (SH30396.03, FBS, Cytiva) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Protein-Protein interactions, Western Blot, Control

Journal: Cancers

Article Title: Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo

doi: 10.3390/cancers13184612

Figure Lengend Snippet: z-VAD-fmk and BIRB796 altered metformin- and everolimus-induced apoptosis-related protein expressions. CaSki and C33A cells were pretreated with or without z-VAD-fmk (10 μM) and BIRB796 (5 μM) for 2 h, then treated with or without metformin (10 mM) and everolimus (20 μM) for 48 h. ( A ) CaSki and ( B ) C33A cell viability was determined using a CCK-8 assay. Protein expression levels of PARP-1, Bcl-2, cleaved caspase-3, p-AKT, AKT, p-p38, p38, p-JNK1/2, and JNK1/2 were assessed using Western blotting. GAPDH served as the loading control. Quantitative results are presented in the bottom plot. Values represent the mean ± SD from three replicates. * and † p < 0.05 compared with the CON and Met + Eve-treated groups. CON, 0.1% DMSO; Comb, M10 + E20, 10 μM metformin + 20 μM everolimus; Comb + z-VAD, 10 μM metformin + 20 μM everolimus + 2.5 μM z-VAD-fmk; Comb + BIRB798, 10 μM metformin + 20 μM everolimus + 5 μM BIRB798.

Article Snippet: CaSki cells were cultured in an RPMI 1640 medium (1-41P05-K, BioConcept, Amimed, Allschwil, Switzerland), and C33A cells in Eagle’s Minimum Essential Medium (SH30024.02, MEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (SH30396.03, FBS, Cytiva) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: CCK-8 Assay, Expressing, Western Blot, Control

Journal: Cancers

Article Title: Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo

doi: 10.3390/cancers13184612

Figure Lengend Snippet: Metformin and everolimus inhibited tumor growth of C33A xenografts in vivo. BALB/c nude mice were subcutaneously injected with C33A cells (1 × 10 7 cells). When the subcutaneous tumor volume reached ~200 mm 3 , metformin (150 mg/kg), everolimus (5 mg/kg), or vehicle control alone (10% DMSO) was intraperitoneally injected every day. ( A ) Schedule of metformin combined with everolimus treatment, ( B ) representative image of a tumor, ( C ) average tumor volume and ( D ) body weight. *, † and δ p < 0.05 compared with CON, Met- or Eve-treated groups. ( E ) Tumor tissue samples were extracted and subjected to HE and immunohistochemical staining to examine the histopathology and expression levels of ki67, cleaved-caspase-3, and p-p38 (shown as brown staining), and quantitative data are presented (H&E, 200×, bar = 50 μm; IHC, 400×, bar = 20 μm). Values represent the mean ± SD ( n = 4); ** p < 0.01, *** p < 0.001 compared with CON, control. Met, metformin; Eve, everolimus; Comb., metformin combined with everolimus.

Article Snippet: CaSki cells were cultured in an RPMI 1640 medium (1-41P05-K, BioConcept, Amimed, Allschwil, Switzerland), and C33A cells in Eagle’s Minimum Essential Medium (SH30024.02, MEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (SH30396.03, FBS, Cytiva) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: In Vivo, Injection, Control, Immunohistochemical staining, Staining, Histopathology, Expressing