Didymin induces apoptosis through mitochondrial dysfunction and up-regulation of RKIP in human hepatoma cells
Authors: Jun Lin
Xing Lin
Jinlan Nie
Renbin Huang
Facheng Bai
Shengjuan Lu
Chunyuang Lu
Quanfang Huang
Jinbin Wei
Zhongpeng Lu
Journal: Chemico-Biological Interactions (2017)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Didymin; ERK/MAPK pathway; Mitochondrial dysfunction; PI3K/Akt pathway; RKIP.
Affiliations: Guangxi Medical University, Nanning 530021, China., The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China., The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; Department of Biochemistry, University of Arkansas Medical School, 4301 W. Markham, Little Rock, AR 72205-7199, USA., Guangxi Medical University, Nanning 530021, China. Electronic address: .

Abstract:  In the present study, a flavonoid was isolated from Origanum vulgare and identified as didymin. The effect and mechanism of O. vulgare didymin (OVD) on human HepG2 liver carcinoma cell was then assessed. Our results showed that OVD strongly inhibited the viability, clonogenicity and migration of HepG2 cells. OVD significantly induced apoptosis and induced cell cycle arrest at G2/M phase by regulating cyclin B1, cyclin D1 and CDK4. The anti-proliferative and pro-apoptotic effects were associated with changes in the Bcl-2/Bax ratio and induction of caspase-mediated apoptosis. Moreover, OVD attenuated the mitochondrial membrane potential, accompanied by the release of cytochrome c. In addition, OVD inhibited the ERK/MAPK and PI3K/Akt pathways by increasing the level of Raf kinase inhibitor protein (RKIP). Our study indicates that OVD induces apoptosis against of HepG2 cells through mitochondrial dysfunction and inactivation of the ERK/MAPK and PI3K/Akt pathways by up-regulating RKIP. 

Retracted: No

PubPeer comments:
 *[Right]* Fig 8C.  OVD enhanced RKIP expression, but inhibited the ERK MAPK pathway in HepG2 cells. ...The expression of ... **ERK**, ... were detected by western blot, respectively. The bands 1 to 4 represented the control cells group, OVD group, locostatin group, and OVD + locostatin group, respectively. *[Left]* Fig 8 from [Huang et al (2016)] .  The expressions of RKIP, p-RKIP, ERK, **p-ERK** ... in liver fibrotic rats. A: normal control group; B: locostatin control group; C: model group; D: locostatin-treated group . ![file] Lanes overlap, despite completely different conditions and description. , 
 Fig 3B.  OVD inhibited HepG2 cell clonogenicity and migratory potential....(B) OVD treatment resulted in a significant decrease in migration ability.... The numbers 1 to 4 represented the control cells group, OVD group, locostatin group, and OVD + locostatin group, respectively. ![file] Panels 1 and 3 share large areas of cellular texture, but rearranged and duplicated. ![file] The same is true for panels 2 and 4, but I have left them as an exercise for the reader, and only marked some peculiarities in each central scratch. ![file] , 
 *[Right]* Fig 8F. For comparison with *[left]* Fig 8D of [Huang et al (2017)] . ![file] Some adjustment of contrast and scale gives us this: ![file] , 
 More fun with WBs. Upper right, Fig. 8A,D.  OVD enhanced RKIP expression, but inhibited the ERK MAPK pathway in HepG2 cells. (A-E) The expression of RKIP... p-p38... detected by western blot... The bands 1 to 4 represented the control cells group, OVD group, locostatin group, and OVD + locostatin group, respectively . Upper left, Fig 8 from [Huang et al (2016)] .  The expressions of RKIP, p-RKIP, ERK, p-ERK,... in liver fibrotic rats. A: normal control group; B: locostatin control group; C: model group; D: locostatin-treated group. ![file] Below: the two RKIP + p-p38 bands; then enhanced-contrast versions; then enhanced versions, with boxes emphasising similar lanes. , 
 Fig 3A.  OVD inhibited HepG2 cell clonogenicity and migratory potential. (A) Clonogenicity assay showed that OVD suppressed HepG2 cell colony formation.... The numbers 1 to 4 represented the control cells group, OVD group, locostatin group, and OVD + locostatin group, respectively . ![file] Fig 4B from [Huang et al (2016)] .  Down-regulating RKIP expression promoted HSCs proliferation **and colony formation**, and increased the expression of u03b1-SMA in hepatic tissues and primary HSCs. .... The bands 1 to 4 represented the normal control cells, the locostatin control cells, the PDGF-BB-treated cells and the PDGF-BB + locostatin-treated cells, respectively. ![file] Coloured boxes are mine. , 
 *[Right]* Fig 8C. *[left]* Fig 7A from [Zhang et al (2018)] . ![file] p-JNK or p-ERK? , 
 *[right]* Fig 9.  OVD inhibited PI3K Akt pathway in HepG2 cells. PI3K, Akt and p-Akt expression was detected by western blot. The bands 1 to 4 represented the control cells group, OVD group, locostatin group, and OVD + locostatin group, respectively . *[left]* 1B from [Lin et al (2018)] .  Locostatin enhances the reduction of RKIP in liver tissues of mice with acute liver injury. ... (B) Hepatic RKIP expression was assessed by western blot analysis. Lanes: 1, Normal control; 2, locostatin control; 3, TAA model; 4, TL group . ![file] , 
 #4 Fig 8A again. *[right]* Fig 4B from [Lin et al (2018)] .  Inhibition of Raf kinase inhibitor protein by locostatin inhibits Nrf2 and HO-1 expression in the livers of mice with acute liver injury. The expression of (A) Nrf2 protein and (B) HO-1 protein was detected by western blot analysis. Lanes: 1, Normal control; 2, locostatin control; 3, TAA model; 4, TL group. ![file] GAPDH or HO-1? , 
 Corrigendum [25 January 2021] . ------------------------------------------------ The authors regret that the wrong Figs. 3, 6B, 8 and 9 were published. The correct figures are provided in this corrigendum. The authors would like to apologise for any inconvenience caused. ![] Fig. 3. OVD inhibited HepG2 cell clonogenicity and migratory potential. (A) Clonogenicity assay showed that OVD suppressed HepG2 cell colony formation. (B) OVD treatment resulted in a significant decrease in migration ability. (C) OVD treatment decreased the ratio of migration. ![] Fig. 6. OVD induced loss of MMP and mitochondrial cytochrome c release in HepG2 cells. (B) Cytochrome c expression was determined by Western blot analysis. The numbers 1 to 4 represented the control cells group, OVD group, locostatin. Group, and OVD + locostatin group, respectively. ![] Fig. 8. OVD enhanced RKIP expression, but inhibited the ERK MAPK pathway in HepG2 cells. (A to E) The expression of RKIP, MEK, p-MEK, ERK, p-ERK, p38, p-p38, JNK and p-JNK were detected by western blot, respectively. The bands 1 to 4 represented the control cells group, OVD group, locostatin group, and OVD + locostatin group, respectively. *P 0.05 vs. control cells group and u25b2P 0.05 vs. OVD + locostatin group. (F) siRNA assay was used to silence RKIP expression. The bands I to V represented the control cells group, OVD group, Control-siRNA group, RKIP-siRNA group, and OVD + RKIP-siRNA group, respectively. *P 0.05 vs. control cells group, u25b2P 0.05 vs. OVD + RKIP-siRNA group, and u25b3P 0.05 vs. OVD + Control-siRNA group. ![] Fig. 9. OVD inhibited PI3K Akt pathway in HepG2 cells. PI3K, Akt and p-Akt expression was detected by western blot. The bands 1 to 4 represented the control cells group, OVD group, locostatin group, and OVD + locostatin group, respectively. Data were expressed as the means u00b1 SD. *P 0.05 vs. control cells group and u25b2P 0.05 vs. OVD + locostatin group. , 
 #9 I agree with the authors that it was unfortunate that the original Figures 3, 6B, 8 and 9 were published. I feel, though, that the Corrigendum note could be longer. It appears that the authors had prepared one Figs 3 6B 8 9 set which is genuine and features in the correction; and a second set with the appearance of collages or stamp collections, overlapping with images used in other papers; and they used the second set by mistake. I would welcome a fuller explanation of how that second set came to exist. It would also be useful to learn more of the journal editors reasoning, when they decided to accept the replacement. They are not alone in this approach to regrettable illustrations. I compiled the following list of regret, from a range of journals but involving an overlapping roster of authors: -------------------------------------------------------  The authors regret that the wrong Figures 7 and 8 were published . [Hepatoprotective effects of total saponins isolated from Taraphochlamys affinis against carbon tetrachloride induced liver injury in rats] . QuanFang Huang, ShiJun Zhang, Li Zheng, Min He, RenBin Huang, Xing Lin, *Food and chemical toxicology* (2012). -------------------------------------------  The authors regret the incorrect publication of Figs. 3B, 3D, 4, 5B and 5C in the original article . [Antiviral activity of methyl helicterate isolated from Helicteres angustifolia (Sterculiaceae) against hepatitis B virus] . Quanfang Huang , Renbin Huang , Ling Wei , Yongxing Chen , Shujuan Lv , Chunhong Liang , Xuerong Zhang , Fujiang Yin , Hongtao Li , Lang Zhuo , Xing Lin, *Antiviral Research* (2013). ----------------------------------------------  The authors regret that the wrong Fig. 4, Fig. 6, Fig. 7 were published. [Protective effect of madecassoside against cognitive impairment induced by D-galactose in mice] . Xing Lin , Shijun Zhang , Renbin Huang , Ling Wei , Shimei Tan , Chunhong Liang , Shujuan Lv , Yongxin Chen , Shuang Liang , Yuanchun Tian , Zhongpeng Lu , Quanfang Huang, *Pharmacology Biochemistry and Behavior* (2014). --------------------------------------------------  The authors regret for the incorrect publication of Fig. 2, Fig. 5A in the original article. [Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide D-galactosamine induced fulminant hepatic failure in mice] . Xing Lin , Shijun Zhang , Renbin Huang , Shimei Tan , Shuang Liang , Xiaoyan Wu , Lang Zhuo , Quanfang Huang, *International Immunopharmacology* (2014). ------------------------------------------------------  The authors regret that the wrong Figs. 3, 6B, 8 and 9 were published. [Didymin induces apoptosis through mitochondrial dysfunction and up-regulation of RKIP in human hepatoma cells] . Jinbin Wei , Quanfang Huang , Facheng Bai , Jun Lin , Jinlan Nie , Shengjuan Lu , Chunyuang Lu , Renbin Huang , Zhongpeng Lu , Xing Lin, *Chemico-Biological Interactions* (2017). -----------------------------------------------  The authors regret the incorrect publication of Figs. 3A, 4, 7, 8B and 11 in the original article . [Methyl helicterilate ameliorates alcohol-induced hepatic fibrosis by modulating TGF-u03b21 Smads pathway and mitochondria-dependent pathway] . Shujuan Wen , Yuanyuan Wei , Xiaolin Zhang , Facheng Bai , Shimei Tan , Jinlan Nie , Jinbin Wei, Xing Lin, *International Immunopharmacology* (2019). ,