Choline suppresses hepatocellular carcinoma progression by attenuating AMPK/mTOR-mediated autophagy via choline transporter SLC5A7 activation

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. Emerging evidence suggests that autophagy plays a critical role in HCC tumorigenesis, metastasis, and prognosis. Choline is an essential nutrient related to prolonged survival and reduced risk of HCC. However, it remains unclear whether this phenomenon is mediated by autophagy.

Methods: Two HCC cell lines (HUH-7 and Hep3B) were used in the present study. Cell growth was evaluated by cell counting kit 8 (CCK-8), colony formation, and in vivo mouse xenografts assays. Cell motility was calculated by wound healing and transwell assays. Autophagosomes were measured by transmission electron microscope (TEM), and autophagy flux was detected by mRFP-GFP-labeled LC3 protein. The mRNA level of genes was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels were detected by Western blotting (WB).

Results: We found that choline inhibited the proliferation, migration, and invasion of HCC cells by downregulating autophagy in vitro and in vivo. Upregulated expression of the solute carrier family 5 member 7 (SLC5A7), a specific choline transporter, correlated with better HCC prognosis. We further discovered that choline could promote SLC5A7 expression, upregulate cytoplasm p53 expression to impair the AMPK/mTOR pathway, and attenuate autophagy. Finally, we found that choline acted synergistically with sorafenib to attenuate HCC development in vitro and in vivo.

Conclusions: Our findings provide novel insights into choline-mediated autophagy in HCC, providing the foothold for its future application in HCC treatment.

Keywords: Hepatocellular carcinoma (HCC); autophagy; choline; solute carrier family 5 member 7 (SLC5A7).

Figure 1

Choline inhibits HCC development in vitro and in vivo. (A) HUH-7 and Hep3B cells were treated with different choline concentrations for 24 h, and cell viability was detected by CCK-8 assay. (B) Dose-dependent inhibitory curve of choline against HUH-7 and Hep3B cells. (C) HUH-7 and Hep3B cells were subjected to 20 µM of choline, and cell viability was detected every 24 h by CCK-8 assay. (D-F) The effects of choline on HCC cell colony formation, migration, and invasion were measured by colony formation, wound healing, and transwell assays, respectively. Cells in colony formation and transwell assays were stained by 0.1% crystal violet. Scale bar =100 µm. (G-J) Choline inhibited the growth of HUH-7 xenograft tumors (n=5). Tumor volume and mice body weight were measured every two days, and tumor weight was recorded after sacrificing. (K) The level of proteins (Ki-67 and MMP2) in the control and choline groups was analyzed by WB assay, quantified by optical density and normalized to ACTB levels of the same lane. Choline group vs. control group: *, P<0.05; **, P<0.01; ***, P<0.001. OD, optical density; CCK-8, cell counting kit 8; HCC, hepatocellular carcinoma; WB, Western blotting; ACTB, beta-actin.

Figure 2

Choline attenuates autophagy activation. (A) WB assay was used to measure the effect of choline on the expression of autophagy-related markers LC3 II, p62, and BECN1, quantitated by optical density and normalized to ACTB levels of the same lane. (B) WB assay was used to detect the effect of choline on starvation-induced autophagy in HCC cells. The expression of LC3 II was quantitated and normalized to ACTB levels of the same lane. (C) Representative images of autophagosomes (red arrows) in HCC cells in response to choline treatment captured by TEM (magnification: left picture =8,000×, right picture =20,000×). (D) mCherry-GFP-LC3 was transfected to HUH-7 and Hep3B cells to detect the influence of choline on autophagy flux. The red puncta represented the autophagolysosomes, and the green puncta represented the autophagosomes. Scale bar =10 µm. Choline group vs. control group: *, P<0.05; **, P<0.01; ***, P<0.001. EBSS + choline group vs. EBSS group: ^##, P<0.01. EBSS, Earle’s balanced salt solution; GFP, green fluorescent protein; WB, Western blotting; ACTB, beta-actin; HCC, hepatocellular carcinoma; TEM, transmission electron microscope.

Figure 3

Autophagy induction impairs the tumor suppressive roles of choline on HCC cells. (A) WB analysis of LC3 in HUH-7 and Hep3B cells with choline alone and choline combined with rapamycin. Expression of LC3 II was quantitated based on optical density and normalized to ACTB levels of the same lane. (B) Representative images of autophagosomes (red arrows) in HUH-7 and Hep3B cells treated with choline alone and choline combined with rapamycin were captured with TEM (magnification: left picture =8,000×, right picture =20,000×). (C) mCherry-GFP-LC3 was transfected to HUH-7 and Hep3B cells to detect the influence of choline and choline combined with rapamycin on autophagy flux. The red puncta represented the autophagolysosomes, and the green puncta represented the autophagosomes. Scale bar =10 µm. (D,E) HUH-7 and Hep3B cells were subjected to different treatments in these three groups: control group, choline group, and Choline + Rapa (rapamycin) group. Cell viability was detected by CCK-8 and colony formation assays. Cells in colony formation assay were stained by 0.1% crystal violet. (F,G) Transwell and wound healing assays were used to detect cell migration and invasion under different treatments. Cells in transwell assay were stained by 0.1% crystal violet. Scale bar =100 µm. (H-K) Rapamycin reversed the suppressive role of choline on the growth of HUH-7 xenograft tumors (n=5). Tumor volume and mice body weight were measured every two days, and tumor weight was recorded after sacrificing. (L,M) WB analysis of Ki-67 and MMP2 in HCC cells and HUH-7 xenograft tissues with choline alone and combined with rapamycin treatment. Expression of Ki-67 and MMP2 was quantitated based on optical density and normalized to ACTB levels of the same lane. Choline group vs. control group: *, P<0.05; **, P<0.01; ***, P<0.001.Choline + Rapa group vs. choline group: ^#, P<0.05; ^##, P<0.01; ^###, P<0.001. ACTB, beta-actin; OD, optical density; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; WB, Western blotting; TEM, transmission electron microscope; CCK-8, cell counting kit 8.

Figure 4

Choline triggers anti-tumor effect by stimulating SLC5A7. (A,B) The expression of SLC5A7 in tumor and paired non-tumor tissues of HCC patients was analyzed by ShinyGEO (GSE14520) and TNMplot. (C) The proteins from 14 paired HCC tumor, and non-tumor tissues were extracted for WB detection. The level of SLC5A7 was measured by optical density and normalized to ACTB levels of the same lane. (D) Overall survival curves for HCC patients stratified by high and low expression of SLC5A7 were analyzed by the online KMplotter tool. (E) Overall survival curves for 74 HCC patients in GLCC stratified by high and low expression of SLC5A7. (F) The correlation of SLC5A7 level with LC3B and p62 expression in HCC using microarray data of GSE14520. (G,H) WB analysis of SLC5A7 in HCC cells and HUH-7 xenograft tissues with choline treatment. Expression of SLC5A7 was normalized to ACTB levels of the same lane. (I) WB analysis of LC3 in HCC cells treated with choline alone and choline combined with si-SLC5A7. Expression of LC3 was quantitated based on optical density. The level of LC3 was normalized to ACTB levels of the same lane. (J) Representative images of autophagosomes (red arrows) in HUH-7 and Hep3B cells treated with choline alone and combined with si-SLC5A7 were captured with TEM (magnification: left picture =8,000×, right picture =20,000×). (K) mCherry-GFP-LC3 was transfected to HUH-7 and Hep3B cells to detect the influence of choline and choline combined with si-SLC5A7 on autophagy flux. The red puncta represented the autophagolysosomes, and the green puncta represented the autophagosomes. Scale bar =10 µm. Choline group vs. control group: *, P<0.05; **, P<0.01; ***, P<0.001. Choline + si-SLC5A7 group vs. choline group: ^#, P<0.05; ^##, P<0.01. TNM, tumor node metastasis; GLCC, Guangdong Liver Cancer Cohort; KM, Kaplan-Meier; HR, hazard ratio; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; WB, Western blotting; ACTB, beta-actin; TEM, transmission electron microscope.

Figure 5

Choline inhibits autophagy through SLC5A7/AMPK/mTOR pathway. (A) WB analysis of total p53 and cytoplasm p53 in HCC cells treated with choline. Expression of total p53 and cytoplasm p53 was quantitated based on optical density. The level of total p53 and cytoplasm p53 was normalized to ACTB levels of the same lane. (B) WB analysis of total p53 and cytoplasm p53 in HCC cells treated with choline alone and combined with si-SLC5A7. Expression of total p53 and cytoplasm p53 was quantitated based on optical density. The level of total p53 and cytoplasm p53 was normalized to ACTB levels of the same lane. (C,D) WB analysis of p-AMPK, total AMPK, p-mTOR, total mTOR, p-P70S6K, total P70S6K, p-4EBP1, and total 4EBP1 in HCC cells and HUH-7 xenograft tissues with choline treatment. Expression of p-AMPK, total AMPK, p-mTOR, total mTOR, p-P70S6K, total P70S6K, p-4EBP1, and total 4EBP1 was quantitated based on optical density and normalized to ACTB levels of the same lane. (E) WB analysis of p-AMPK, total AMPK, p-mTOR, total mTOR, p-P70S6K, total P70S6K, p-4EBP1, and total 4EBP1 in HCC cells with choline alone and choline combined with si-SLC5A7. Expression of p-AMPK, total AMPK, p-mTOR, total mTOR, p-P70S6K, total P70S6K, p-4EBP1, and total 4EBP1, was quantified based on optical density and normalized to ACTB levels of the same lane. (F) HUH-7 and Hep3B cells were subjected to four groups: Control group, Choline group, Choline + si-SLC5A7 group, and Choline + si-SLC5A7 + si-AMPK group. WB analysis of p-AMPK, total AMPK, p-mTOR, total mTOR, p-P70S6K, total P70S6K, p-4EBP1, total 4EBP1, and LC3 in HCC cells. Expression of p-AMPK, total AMPK, p-mTOR, total mTOR, p-P70S6K, total P70S6K, p-4EBP1, total 4EBP1, and LC3 was quantitated based on optical density and normalized to ACTB levels of the same lane. Choline group vs. control group: *, P<0.05; **, P<0.01; ***, P<0.001. Choline + si-SLC5A7 group vs. choline group: ^#, P<0.05; ^##, P<0.01; ^###, P<0.001. Choline + si-SLC5A7 + si-AMPK group vs. choline + si-SLC5A7 group: ^&, P<0.05; ^&&, P<0.01; ^&&&, P<0.001. ACTB, beta-actin; WB, Western blotting; HCC, hepatocellular carcinoma.

Figure 6

Choline enhances the anti-tumor effects of sorafenib. (A,B) HUH-7 and Hep3B cells were subjected to four groups: control group, Sora (sorafenib) group, choline group and Choline + Sora group. Cell growth was detected by CCK-8 and colony formation assays in vitro. Cells in colony formation assay were stained by 0.1% crystal violet. (C,D) Wound healing and transwell assays were used to detect cell migration and invasion under different treatments, respectively. Cells in transwell assay were stained by 0.1% crystal violet. Scale bar =100 µm. (E-H) Choline synergized the antitumor effect of sorafenib in HUH-7 xenograft tumors (n=5). Tumor volume and mice body weight were measured every two days, and tumor weight was recorded after sacrificing. (I,J) WB analysis of Ki-67 and MMP2 in HCC cells and HUH-7 xenograft tissues treated with sorafenib alone or in combination with choline. Expression of Ki-67 and MMP2 was quantitated based on optical density and normalized to ACTB levels of the same lane. Sora group vs. control group: *, P<0.05; **, P<0.01; ***, P<0.001. Choline group vs. control group: ^#, P<0.05; ^##, P<0.01; ^###, P<0.001. Choline + Sora group vs. Sora group: ^&&, P<0.01; ^&&&, P<0.001. OD, optical density; ACTB, beta-actin; CCK-8, cell counting kit 8; WB, Western blotting; HCC, hepatocellular carcinoma.

Figure 7

Schematic diagram of the inhibitory effect of choline on HCC progression. HCC, hepatocellular carcinoma.