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. 2022 Mar 7;82(8):1503–1517. doi: 10.1158/0008-5472.CAN-21-1820

Figure 5.

Figure 5. Administering the glycolysis inhibitor metformin based on HK2 circadian rhythm improves trastuzumab efficacy. A and B, Immunoblotting analysis of PER1, AMPK, and p-AMPK proteins in TR cells treated with MET with or without AMPK knockdown (A) or MG132 treatment (B). C, NCI-N87TR cells were subcutaneously injected in nude mice under a 12:12 h light/dark cycle with the lights on from ZT0 to ZT12. Mice were treated with PBS, trastuzumab (TRA, 10 mg/kg), metformin (MET, 250 mg/kg), or TRA combined with MET at ZT6 or ZT18. Immunoblotting analysis was performed to detect PER1, HK2, AMPK, and p-AMPK in PBS and MET groups at ZT6 or ZT18. D, Nude mice were killed at 4 hours intervals across the 24 hours light/dark cycle for two consecutive days. qPCR analysis of relative mRNA levels of clock genes BMAL1, CLOCK, PER1, and PER2 in PBS and MET (250 mg/kg) groups were shown (n = 3). E and F, Tumor volumes (E) and weight of nude mice (F) were calculated (n = 5). One-way ANOVA was used to calculate P values. G, Representative IHC images of Ki67, Cleaved-caspase3, PER1, and HK2 performed on serial sections of subcutaneous tumors of xenograft nude mice; scale bar, 140 µm. Data are graphed as the mean ± SD; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05. AMPK, adenosine monophosphate-activated protein kinase; ANOVA, analysis of variance; HK2, hexokinase 2; IHC, immunohistochemistry; MET, metformin; PBS, phosphate-buffer saline; qPCR, quantitative polymerase chain reaction; SD, standard deviation; TR, trastuzumab-resistant; ZT, Zeitgeber time.

Administering the glycolysis inhibitor metformin based on HK2 circadian rhythm improves trastuzumab efficacy. A and B, Western blot analysis of PER1, AMPK, and p-AMPK proteins in TR cells treated with MET with or without AMPK knockdown (A) or MG132 treatment (B). C, NCI-N87TR cells were subcutaneously injected in nude mice under a 12:12 h light/dark cycle with the lights on from ZT0 to ZT12. Mice were treated with PBS, trastuzumab (TRA; 10 mg/kg), metformin (MET; 250 mg/kg), or TRA combined with MET at ZT6 or ZT18. Western blot analysis was performed to detect PER1, HK2, AMPK, and p-AMPK in PBS and MET groups at ZT6 or ZT18. D, Nude mice were killed at 4 hours intervals across the 24 hours light/dark cycle for two consecutive days. qPCR analysis of relative mRNA levels of clock genes BMAL1, CLOCK, PER1, and PER2 in PBS and MET (250 mg/kg) groups are shown (n = 3). E and F, Tumor volumes (E) and weight (F) of nude mice were calculated (n = 5). One-way ANOVA was used to calculate P values. G, Representative IHC images of Ki67, cleaved-caspase-3, PER1, and HK2 performed on serial sections of subcutaneous tumors of xenograft nude mice. Scale bar, 140 µm. Data are graphed as the mean ± SD; *, P < 0.05; ***, P < 0.001; ns, nonsignificant, P > 0.05.