Fig. 7: Berberine inhibits the MT-CO2-GLS1 axis to suppress oncogenic Ras-induced glutaminolysis and tumor growth.
a, b Indicated cells were treated with an indicated concentration of berberine for 24 h prior to western blot analyses (a) or were treated with berberine (5 μM, and thereafter) in the presence of 500 μM dimethyl α-Ketoglutaric acid (DM-αKG) upon Glc−/Gln+ condition for 24 h, followed by examining cell viability (b, n = 3 independent experiments). c, d H292-KRasG12V cells were treated with berberine for 24 h prior to western blot analyses (c) or were treated with berberine in the presence of 500 μM DM-αKG upon Glc-/Gln+ condition for 24 h, followed by examining cell viability (d, n = 3 independent experiments). The samples derive from the same experiment but different gels for MT-CO2, GLS1, GAPDH, another for pERK, Ras, and another for ERK were processed in parallel (c). e–l H292-KRasG12V (8 ×105, e–h) or A549 (1 ×106, i–l) cells were subcutaneously inoculated into the right scruff of the female BALB/c nude mouse (n = 6/group, H292; n = 8/group, A549). On day 14 (H292) or 22 (A549) after inoculation, mice were given berberine (40 mg/kg) by oral gavage daily. The tumor volumes on indicated days after gavage were examined (e, i). On day 16 (H292) or 12 (A549) after gavage, tumors were weighed (f, j). Immunohistochemistry staining was performed to examine MT-CO2 and GLS1 expression (g, h, k, l). m–o Lewis lung carcinoma (LLC, 5 ×105) cells were subcutaneously inoculated into the right scruff of each C57BL/6 mice (n = 7/group). On day 7 after inoculation, mice were given berberine (40 mg/kg) by oral gavage daily. The tumors volume on indicated days after gavage were examined (m). On day 16 after gavage, tumors were weighed (n). The mice’s overall survival (Tumor volume >1500 mm3 is defined as ethical death) was examined (n = 7/group) (o). p A model depicting the role of MT-CO2 in tumor cell survival upon glucose deprivation. These experiments have been repeated three times with similar results (a, c). Data were presented as mean ± SD (b, d) or SEM (e–n). Comparisons were performed with one-way ANOVA with Tukey’s test (b, d, f–h), two-way ANOVA with Tukey’s (e) or Bonferroni’s (i, m) test, unpaired two-tailed Student’s t test (j–l, n), and Log-rank (Mantel–Cox) test (o).
