Anticancer activity of 23,24-dihydrocucurbitacin B against the HeLa human cervical cell line is due to apoptosis and G/M cell cycle arrest
Authors: Chun-Yan Hu
Guang-Hua Chu
Jun-Xiao Zhang
Hong Wei-Tan
Li-Hui Wei
Liu Chen
Wei-Qiang Wang
Journal: Experimental and Therapeutic Medicine (2018)
Discipline: CLINICAL MEDICINE
Keywords: 23,24-dihydrocucurbitacin B; cervical carcinoma; mechanistic target of rampamycin; reactive oxygen species.
Affiliations: Department of Gynecology, Northwest Women and Children's Hospital, Xi'an, Shaanxi 710061, P.R. China., Department of Gynecology, Northwest Women and Children's Hospital, Xi'an, Shaanxi 710061, P.R. China, Department of Gynecology, Peking University People's Hospital, Beijing 100044, P.R. China.

Abstract:  Cervical cancer is one of the primary causes of cancer-associated mortality worldwide. Due to the increasing incidence of cervical cancer, multiple treatment options are required. Initial responses to chemotherapy and surgical interventions are generally positive, however patients often experience relapse and tumor recurrence. Currently, the effects of cucurbitacins on different types of cancer are being investigated, as they exhibit a wide variety of bioactivities. The anticancer activity of the cucurbitacin 23,24-dihydrocucurbitacin B against a panel of human cervical cancer cell lines was investigated in the current study. Cell viability was determined using an MTT assay and apoptosis was detected using DAPI staining. The proportion of apoptotic cells, cell cycle distribution, mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were estimated using flow cytometry. Protein expression was determined using western blot analysis. The results of the current study indicated that 23,24-dihydrocucurbitacin B inhibited the viability of human cervical cancer cell lines and had an IC50 of 40-60 µM. However, its cytotoxic effects were much less pronounced in normal epithelial fr2 and HerEpiC cells, where it had an IC50 of 125 µM. The underlying mechanisms of this were further studied and the results demonstrated that 23,24-dihydrocucurbitacin B induced apoptosis in HeLa cells and caused ROS-mediated shifts in the ΔΨm. Additionally, it caused the cell cycle arrest of HeLa cells at the G2/M checkpoint. The phosphoinositide 3 kinase/protein kinase B/mechanistic target of rampamycin (PI3K/AKT/mTOR) cascade may serve an important role in cancer tumorigenesis, progression and resistance to chemotherapy. The results indicated that 23,24-dihydrocucurbitacin B significantly decreased the expression of important proteins in the PI3K/Akt/mTOR cascade. Taken together, these results suggest that 23,24-dihydrocucurbitacin B may be novel method of treating cervical cancer. 

Retracted: Yes

PubPeer comments:
 Fig 4A.  Estimation of apoptotic population of HeLa cells treated with 0, 20, 40 and 80 u00b5M 23,24-dihydrocucurbitacin B using Annexin V-FITC PI staining and flow cytometry. (A) Flow cytometric analysis. ![file] For comparison: *[left]* Figure 4 from [Wu et al (2018)] .  Estimation of apoptotic cell populations at indicated concentrations of u03b1-Bisabolol by annexin V PI staining followed flow cytometry. ... The Figure shows that the apoptotic cell populations increase with increase in the concentrations of u03b1-Bisabolol . *[right]* Figure 4 from [Liu et al (2019)] .  Annexin V PI assay showing percentage of apoptotic HL-60 cells at indicated concentrations of helenalin. The percentage of apoptotic leukemia cells increased in a concentration-dependent manner. ![file] , 
 *[left]* Figure 7 from [Zhang et al (2018)] .  Effect of 0, 20, 40 and 80 u00b5M 23,24-dihydrocucurbitacin B on the expression of mTOR PI3K Akt signaling pathway proteins in HeLa cells . *[right]* Figure 7 frpm [Zhang, Zhang Ni (2020)] .  Effect of indicated doses of eriodictyol on key proteins of PI3K AKT mTOR signalling pathway . ![file] Unrelated papers used the same b-actin loading-control band. How did this happen? , 
 #2 Repeated: *[left]* Figure 7 from [Zhang et al (2018)] .  Effect of 0, 20, 40 and 80 u00b5M 23,24-dihydrocucurbitacin B on the expression of mTOR PI3K Akt signaling pathway proteins in HeLa cells . *[right]* Figure 7 from [Zhang, Zhang Ni (2020)] .  Effect of indicated doses of eriodictyol on key proteins of PI3K AKT mTOR signalling pathway . ![file] For comparison with Figs 4 and 6B from [Xing et al (2020)] .  Western blot analysis showing the effect of Fisetin at indicated concentrations on the expression of Bcl-2, Bax and Caspase-3. The Figure depicts that the expression of the Bax and Caspase-3 increases and Bcl-2 decreases with increase concentration of Fisetin.  Western blotting showing the effect of Fisetin on the expression of cyclin B1. The Figure depicts that the expression of cyclin B1 decreases dose dependently. ![file] , 
 *[left]* Figure 7 from [Zhang et al (2018)] .  Effect of 0, 20, 40 and 80 u00b5M 23,24-dihydrocucurbitacin B on the expression of mTOR PI3K Akt signaling pathway proteins in HeLa cells . *[right]* Fig 3B,C from [Chen et al (2019)] . ![file] I have concerns. , 
 #2 Fig 7 again. ![] More sightings of the bands in Figure 7 of [Wang et al (2019)] .  Effect of Acetylshikonin on the expression of Bax and Bcl-2 proteins as depicted by western blot analysis. The Figure shows that the expression of Bax increased while of Bcl-2 decreased with increased doses of Acetylshikonin . ![] Figs 4 and 6B from [Xing et al (2020)] .  Western blot analysis showing the effect of Fisetin at indicated concentrations on the expression of Bcl-2, Bax and Caspase-3. The Figure depicts that the expression of the Bax and Caspase-3 increases and Bcl-2 decreases with increase concentration of Fisetin.  Western blotting showing the effect of Fisetin on the expression of cyclin B1. The Figure depicts that the expression of cyclin B1 decreases dose dependently. ![file] , 
 *[left]* Figure 2A.  Effect of 23,24-dihydrocucurbitacin B on colony formation in HeLa cells. (A) Images of the colony formation potential of HeLa cells treated with 0, 20, 40 and 80 u00b5M 23,24u2011dihydrocucurbitacin B. *[right]* Figure 1C from [Yeng Zhang (2018)](http: www.ijcem.com files ijcem0051920.pdf).  Colony forming potential of lung cancer A549 cells at varied doses of 6.2, 12.5 and 25 u00b5M of globularifolin. The journal here is Int J Clin Exp Med, from  e-Century . No DOI or PMID, so no separate PubPeer entry for the paper. ![file] The use of the same colony-formation images is calling out for an explanation. , 
 Retracted [12 April 2021] . Following the publication of the above paper, a concerned reader drew to the Editoru2019s attention that several figures contained data that bore striking similarities to data published in other papers; notably, the flow cytometric data shown in Fig. 4A appeared to be identical with data published in the original (and now archived) version of another paper published around the same time, although by different authors from different research institutions [Lin J, Zhao Y, Shi Z and Bai Y: Antitumor effects of helenalin in doxorubicin-resistant leukemia. *JBUON* 24: 2068-2074, 2019]. After having conducted an independent investigation in the Editorial Office, the Editor of *Experimental and Therapeutic Medicine* has determined that this article should be retracted from the Journal on account of a lack of confidence concerning the originality and the authenticity of the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office never received any reply. The Editor regrets any inconvenience that has been caused to the readership of the Journal. ,