Estrogen receptor‐α36 is involved in diallyl sulfide‐induced inhibition of malignant growth of HepG2 and Huh7 hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma (HCC) is a highly malignant disease that currently lacks effective treatment. Epidemiological studies have suggested the preventive role of raw garlic intake in different tumors, such as HCC. Although diallyl sulfide (DAS), the main component of garlic extracts, has been reported to inhibit the growth of HCC cells, the underlying mechanism remains elusive. This study aimed to investigate the inhibitory effect of DAS on the growth of HepG2 and Huh7 hepatocellular carcinoma cells and its underlying mechanism. HepG2 and Huh7 cells were treated with DAS and nude mice were intrahepatically injected with human HCC HepG2 cells and maintained with or without DAS administration for 28 days. MTS and clonogenic assays revealed that DAS inhibited the growth and clonogenicity of HepG2 and Huh7 hepatocellular carcinoma cells. Furthermore, DAS inhibited the growth of xenograft tumors accompanied by a decreased rate of pathological karyomitosis as observed by H&E staining. The expression levels of estrogen receptor-α36 (ER-α36) and epidermal growth factor receptor (EGFR) in HepG2 and Huh7 cells and in xenograft tumors derived from HepG2 cells after DAS treatment were detected by immunohistochemistry and western blotting. We found that DAS disrupted the positive regulatory loop between ER-α36 and EGFR, and decreased the phosphorylation of AKT at Ser 473 both in vivo and in vitro. DAS also induced cell apoptosis, as evidenced by Hoechst and TUNEL staining. Western blotting revealed activation of caspase3, increased BAX and decreased Bcl-2 expression. However, the ER-α36 expression knockdown attenuated DAS-induced ERK and AKT phosphorylation in HCC cells. DAS was also able to inhibit ER-α36-mediated activation of the MAPK/ ERK signaling induced by estrogen. Thus, our results indicate that ER-α36 signaling is involved in DAS-induced inhibition of HCC cell growth both in vitro and in vivo.

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“…Western blot analysis was performed as described previously 31–33 . Total protein from treated HEC1B and KLE cells was extracted using ice‐cold lysis buffer (Beyotime, Shanghai, China).…”

“…Western blot analysis was performed as described previously. [31][32][33] Total protein from treated HEC1B and KLE cells was extracted using ice-cold lysis buffer (Beyotime, Shanghai, China). The concentration of the extracted protein was examined using a BCA protein assay kit (Keygentec, Nanjing, China).…”

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“…37 Therefore, apoptosis induced by 2 may be a concentrationdependent mechanism; however, more studies are needed to clarify this aspect of its biochemical pharmacology. DAS (4), a fat-soluble compound present in garlic, has demonstrated anticancer activity against hepatocellular, 38 breast, 39−41 skin, 42 and prostate 43 carcinomas. In vivo studies carried out in nude mice injected with human hepatocellular carcinoma (HCC) HepG2 cells and treated with 4 revealed that it inhibited the growth and clonogenicity of HepG2 and Huh7 HCC cells.…”

“…It works by activating procaspase-3 to caspase-3 by chelating zinc, as procaspase-3 is known to be inhibited by low levels of this metal. PAC-1 (38) exhibits IC 50 values between 0.003 μM and 1.41 μM in various cancer cells, while the IC 50 values in non-cancerous cells were 7 times higher.…”

“…DAS ( 4 ), a fat-soluble compound present in garlic, has demonstrated anticancer activity against hepatocellular, breast, − skin, and prostate carcinomas. In vivo studies carried out in nude mice injected with human hepatocellular carcinoma (HCC) HepG2 cells and treated with 4 revealed that it inhibited the growth and clonogenicity of HepG2 and Huh7 HCC cells.…”