Proteomic and Functional Analyses Reveal the Potential Involvement of Endoplasmic Reticulum Stress and α-CP1 in the Anticancer Activities of Oridonin in HepG2 Cells

Abstract

Oridonin has been shown to exhibit therapeutic effects against hepatocellular carcinoma (HCC) in vitro and in vivo. This study aimed to identify the anti-HCC mechanisms of oridonin in HepG2 cells using proteomic and functional analyses. MTT assay showed that oridonin treatment for 24 hours dose-dependently inhibited cell growth with an IC 50 value of 40.4 mM. Treatment with 40 mM oridonin for 24 hours induced apoptosis determined by nuclear morphologic changes of DAPI-stained cells and flow cytometric analysis of annexin V-FITC/PI-stained cells, which was accompanied by Grp78 upregulation and a-CP1 downregulation identified by proteomic analysis. Immunoblot analysis for the endoplasmic reticulum (ER) stressrelated proteins demonstrated that the expression levels of phosphorylated PERK (p-PERK) and CHOP were increased, whereas PERK, ATF-6, and IRE-1 expression levels were decreased. Knockdown of a-CP1 expression with siRNA significantly increased cell death and apoptosis in control and oridonin-treated HepG2 cells. Together, these data provide proteomic and functional evidence for the potential involvement of ER stress and a-CP1 in the antiproliferative and apoptotic activities of oridonin in HepG2 cells, which shed new light on the action mechanisms of oridonin in HCC management.

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“…Rabdosia rubescens is an effective traditional Chinese medicine for the treatment of hepatoma. 9 As its main component, oridonin has been proven to inhibit the progression of HCC. The potential mechanism may be associated with the regulation of mitochondrial ROS and ES stress to induce cell apoptosis.…”

“…The potential mechanism may be associated with the regulation of mitochondrial ROS and ES stress to induce cell apoptosis. For example, it has been reported that oridonin activates oxidative stress pathways leading to HepG2 cell death, Hsp70-1 is a key protective protein in oridonin-mediated oxidative stress, 14 oridonin modulates ER stress and α-CP1, leading to proliferation inhibition and apoptosis induction in HCC cells, 9 and oridonin-induced HCC cell apoptosis is mediated by mitochondrial ROS through PARP activation, p53, and the MAPK pathway. 15 , 16 In addition, considering that oridonin could effectively inhibit HCC, many researchers have extracted oridonin derivatives, which have also been proven to have significant inhibitory effects on the progression of HCC.…”

“… 8 Oridonin, the major bioactive component of Rabdosia rubescens , shows favorable therapeutic effects against many cancer types, such as breast cancer, ovarian cancer, colon cancer, osteosarcoma, gastric cancer, lung cancer, esophageal squamous cell carcinoma and hepatocellular carcinoma. 9 In addition, oridonin shows synergistic anticancer effects when combined with other treatments. For example, cotreatment with oridonin and cisplatin inhibited the transcriptional activity of NF-κB and induced apoptosis in HCC, 10 oridonin enhanced radiation-induced cell death in lung cancer, 11 and oridonin intensified the toxicity of 5-FU in renal carcinoma.…”

Mitochondrial ROS contribute to oridonin-induced HepG2 apoptosis through PARP activation

“…In HepG2 cells, multiple mechanisms, including the inhibition of telomerase, induction of endoplasmic reticulum stress and regulation of p53, MAPK and mitochondrial signaling pathways, have been implicated in the apoptotic activity of oridonin ( 25 , 49 , 50 ). Whether other mechanisms, such as PARP activation, are associated with the anti-HepG2 activity of oridonin remains unknown.…”

Direct cellular targets and anticancer mechanisms of the natural product oridonin

“…Among the pharmacological activities mentioned above, the anticancer effect of oridonin has drawn increasing attention in recent years. Over the past decades, oridonin and its analogs have been demonstrated to exhibit potent anticancer activities by promoting apoptosis, 49 inducing cell cycle arrest, 50 inhibiting angiogenesis, 51 and suppressing metastasis 52 in a variety of cancer types, such as lung carcinoma, 53 hepatocellular carcinoma, 54 gastric cancer, 55 colorectal cancer, 56 breast cancer, 57 pancreatic cancer, 58 prostatic cancer, 59 and leukemia 60 . Furthermore, oridonin can induce autophagy, which contributes to apoptosis induction and inhibition of cancer progression in several cancer cell types, such as breast cancer cells 57 and colorectal cancer cells 56 .…”