Mukonal Inhibits Cell Proliferation, Alters Mitochondrial Membrane Potential and Induces Apoptosis and Autophagy in Human CNE1 Nasopharyngeal Carcinoma Cells
Authors: Shuaishuai Ma
Fang Guo
Jie Bai
Zhiling Zhu
Yanru Hao
Guofang Guan
Yingyuan Guo
Journal: Medical Science Monitor (2019)
Discipline: CLINICAL MEDICINE
Keywords: Nasopharyngeal carcinoma; Mukonal; Cell proliferation; Apoptosis; Autophagy
Affiliations: Department of Otolaryngology, The Second Hospital of Jilin University, Changchun, Jilin, China (mainland).

Abstract:  BACKGROUND Nasopharyngeal carcinoma results in high patient morbidity and mortality, due to early metastasis, and toxicity due to chemotherapy. Mukonal is plant-derived carbazole alkaloid that has been used in traditional Chinese medicine to treat several types of cancer. This study aimed to investigate the effects of mukonal on cell proliferation, apoptosis, autophagy, and the mitochondrial membrane potential of nasopharyngeal carcinoma cells in vitro. MATERIAL AND METHODS CNE1 human nasopharyngeal carcinoma cells and NP69 normal nasopharyngeal epithelial cells were cultured with and without treatment with increasing doses of mukonal. Cell viability was determined by the MTT assay. Fluorescence microscopy was used to detect reactive oxygen species (ROS), mitochondrial membrane potential, and the release of cytochrome C. Flow cytometry was used to examine changes in the cell cycle, electron microscopy examined cell autophagy, and Western blot was performed to measure levels of proteins associated with autophagy and apoptosis. RESULTS Mukonal had an antiproliferative effect on CNE1 cells, with an IC₅₀ of 9 µM and there were effects of toxicity on normal NP69 cells. Mukonal triggered ROS-mediated changes in mitochondrial membrane potential which was also accompanied by the discharge of cytochrome C in the CNE1 cells. Mukonal activated autophagy and apoptosis in CNE1 cells, which was also associated with upregulation of the autophagy-related proteins, LC3 II and beclin-1, as well as apoptosis-associated proteins, Bax, cleaved caspase-3 and -9. Mukonal treatment also resulted in CNE1 cells cycle arrest at G₂/M. CONCLUSIONS Mukonal inhibited the growth of human CNE1 nasopharyngeal carcinoma cells in vitro. 

Retracted: Yes

PubPeer comments:
 Figure 6. The four TEM images in this paper look remarkably similar to the panels in Figure 2 from a paper by different authors at a different institution, where also represent completely different cells and experiments. That other paper is: Xiangxiang Zhu et al., JBUON 2020, PMID: 32277665. ![file] , 
 Fig 2.  Fluorescence microscopy of mukonal-treated CNE1 nasopharyngeal carcinoma cells shows an increase in reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential. Overlap marked. ![file] , 
 Figure 8.  Flow cytometry shows the effects of mukonal on the distribution of the CNE1 nasopharyngeal carcinoma cells in different phases of the cell cycle. ![file] Histograms contain areas of unexpected similarity. , 
 Figure 9.  Western blot shows the effect of mukonal on the expression cell cycle-associated proteins. ![file] Familiar from Fig 8A,B of [Luo et al (2019)] : ![] There. colored rectangles mark bands that also appear in *[left]* Figure 8 of [Pan Zhang (2019)] .  Effect of indicated concentrations of Linalool on the PI3K AKT signalling pathway as indicated by western blotting. The Figure depicts that Linalool blocks the PI3K AKT pathway in OECM-1 cells concentration-dependently. *[right]* Figure 5 from [Zhang et al (2018)] .  Western blots showing the effect of ludartin on cell cycle proteins in Saso-2 osteosarcoma cells. (A) Cdc25c, pCdc25c, Cdc2, pCdc2, p21WAFI, and cyclin B1, and (B) p21, p53, and p27. Ludartin treatment increased the expression of cyclin-dependent kinase inhibitor p21 (WAF1) or p21WAF1 in Saso-2 osteosarcoma cells but did not alter the expression of p27 or p53 tumor suppressor proteins . ![] UPDATE: Another sighting! Figure 7B from [Xiong et al (2019)] .  The effects of pancratistatin on the cell cycle and expression of cell cycle-associated proteins in colorectal cancer cells in vitro. .. (B) Western blot shows that pancratistatin is associated with a dose-dependent decrease in the expression of the cell cycle-associated proteins . ![file] , 
  Plant-derived molecules have been reported to affect ROS levels in the cancer cells [15]. In the present study, mukonal was shown to trigger the formation ROS in CNE1 cells, ... The reference is to  15. Hua F, Li CH, Chen XG et al: Daidzein exerts anticancer activity towards SKOV3 human ovarian cancer cells by inducing apoptosis and cell cycle arrest, and inhibiting the Raf MEK ERK cascade. Int J Mol Med, 2018; 41(6): 3485u201392 . Its relevance is not obvious, as [Hua et al (2018)] **do not mention** ROS levels. ------------------------------------------------ The influence of [Hua et al (2018)] is surprisingly far-reaching. Google Scholar informs us that their in-vitro study of Daidzein has been cited **71** times. Many of these are review papers, but we also find a surprising number of papers using it in  Apoptosis Assay paragraphs to illustrate the use of flow-cytometry and Annexin V PI staining to quantify cell apoptosis. Indeed, Hua et al did apply the method, but they were not pioneers, nor is their explanation superior to many others. Alternatively, the paper is cited as a precedent for a phytochemical (daidzein) inhibiting cancer proliferation by interrupting the cell cycle. Given all the earlier precedents, the choice again invites some explanation. It is almost as if all these papers were prepared by a third-party service (a papermill ), following a template from one paper to the next, without checking its contents. Consistent with this notion, the papers came to my attention by recycling the same figures (despite a lack of authors in common). - [Jia et al (2019)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR3 cells was performed as described previously [17]. - [Luo et al (2019)] .  Many plant-derived molecules have been reported to interfere with the **ROS** level in the cancer cells [13]. ... The slides were then covered with a covers lip and examined with a fluorescent microscope. Then 4u2019,6-diamidino-2-phenylindole (DAPI) and annexin V propidium iodide (PI) staining was performed as described previously [13]. - [Zeng et al (2019)] .  The slides were then covered with a covers lip and examined with a fluorescent microscope (BDBiosciences, San Jose, CA, USA). The annexin V PI staining was performed as described previously [8]. - [Wu et al (2019)] *[retracted]*,  The slides were covered with a coverslip and examined with a fluorescent microscope (BD Biosciences, San Jose, CA, USA). Annexin V PI staining was performed as described previously [13]. - [Yang, Chen Wang (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as described previously 16 . - [Yang et al (2019)] .  The slides were covered with cover slips and examined with a fuorescent microscope. DAPI and annexin V propidium iodide (PI) staining were performed as described previously [14] *[14 is a misprint for 13]*. - [Huang et al (2019)] .  The slides were covered with cover slip and examined under fluorescence microscope. Annexin V propidium iodide (PI) staining of the Icariin-treated HeLa cells was performed as described previously [18]. - [Liu et al (2019)] .  The distribution of the breast cancer SK-BR-3 cells in different cycle phases was performed by flow cytometry after PI staining, following the method reported by Hua et al [11]. ...After overnight incubation at 4u00b0C, the cells were subjected to PI staining and flow cytometry. - [Liu et al (2019)] .  After covering the glass slides with cover slips, the cells were analyzed under a fluorescent microscope (Nikon Instruments, Inc., NY, USA). For determining percentage of apoptotic cells, Annexin V PI staining assay using a FACSCalibur flow cytometry (BD Biosciences, NY, USA) was carried out using a previously reported method [12] - [Yin et al (2019)] .  The slides were covered with covers slips and examined with a fluorescent microscope. Annexin V PI staining of the SW-948 cells was performed as previously described [13] . - [Zhang et al (2019)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining of the SW48 cells was performed as described previously [11] . - [Ma et al (2019)] .  The slides were cover-slipped and examined under a fluorescent microscope. Annexin V PI staining was performed as described previously [15]. - [Li et al (2019)] .  The slides were cover-slipped and assessed by fluorescent microscopy. ROS estimation was performed as described previously [13]. - [Zhang et al (2019)] .  The slides were covered with a cover slip and examined with a fluorescent microscope. Annexin V PI staining was performed as previously described [12]. - [Wu et al (2020)] .  The slides were covered with a covers lip and examined with a fluorescent microscope. Annexin V PI staining of the AGS cells was performed as described previously [15]. - [Yu et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [9]. - [Yin et al (2020)] .  The slides were cover-slipped and examined with a fluorescent microscope. Annexin V PI staining was performed as described previously [10]. - [Wei et al (2019)] .  The slides were covered with cover slips and examined with a fluorescent microscope. Annexin V PI staining of the OVACAR-3 cells was performed as described previously [11]. - [Wen et al (2020)] .  The glass slides laden with cell cultures were then covered with cover slips and then using a fluorescence microscope (BioTek Instruments, Inc., Winooski, VT, USA,) the cells were analysed. Quantification of apoptosis was done with annexin V PI staining assay which was performed as reported already (Hua et al., 2018). -------------------------------------------------------------- Cell-cycle arrest: - [Li Han (2019)] .  Cell cycle arrest is another important mechanism by which anticancer molecules exert their effects [16] . - [Mao et al (2019)] .  The arrest of the cancer cells at G2 M phase prevents the cancer cells from entering mitosis and as such the molecules that trigger the cell cycle arrest prevent the proliferation and tumourigenesis of cancer cells (Hua et al., 2018). - [Si et al (2019)] .  Previous studies have shown that depletion of cyclin A and B1 and upregulation of p27 and p21 results in the G2 M arrest of the cells during cell division [21]. - [Zhou et al (2019)] .  The arrest of cancer cells at G2 M phase prevents the cancer cells from entering mitosis, so the molecules that trigger cell cycle arrest prevent the proliferation and tumorigenesis of cancer cells [20]. - [Tan et al (2019)] .  Cell cycle arrest caused by several anticancer agents is an essential mechanism in the growth inhibition of cancer cells [19] . - [Wei et al (2019)] .  Furthermore, arrest of the cells at different phases of the cell cycle is an important route by which anticancer agents halt the growth of cancer cells [18]. - [Zeng et al (2019)] .  Besides autophagy, cell cycle arrest is another mechanism by which anticancer agents exert their anticancer effects [19] . -------------------------------------------------- Another list of papers cite Hua et al (2018) as precedents for methods which those authors did not in fact apply. - [Zhang et al (2019)] .  For determination of the ROS and mitochondrial membrane potential levels, the A549 cells were treated with 0, 9, 18, and 36 u00b5M concentrations of murrayanine for 24 hours and then the ROS and mitochondrial membrane potential levels in the A549 cells were determined, as described previously [11] *(in fact Hua et al did not measure or even mention ROS or mitochondrial membrane potentials)*. - [Guo et al (2019)] .  Plant-derived molecules have been reported to affect ROS levels in the cancer cells [15] . - [Xu et al (2019)] . Hua et al appear in the References but not in the text at all. - [Du et al (2019)] .  **Osteosarcoma** causes significant mortality world over and the treatments available are not efficient and are associated with a number of side-effects [11]. - [Yu et al (2019)] .  Several of the natural products have been reported to induce apoptosis and autophagy through production of ROS [18]. - [Liu et al (2019)] .  The colony formation assay was carried out as described previously [15] *[Hua et al did not perform colony-formation assays]* - [Zhu et al (2020)] .  Estimation of ROS was measured according to a method previously described [8]. - [Du et al (2020)] . Hua et al appear in the References but not in the text at all. , 
  Mukonal, a carbazole alkaloid derived from Clausena guillauminii has recently been shown to have anticancer effects in different types of cancer [9]. Mukonal has been reported to induce apoptosis and cell cycle arrest of laryngeal cancer cells [9]. ... . Also, mukonal has previously been shown to inhibit the growth of laryngeal cancer cells, which is a finding that supports the results of this study [9]. Ref [9] is  Li L, Huizhi L, Binu W et al: Anticancer activity of mukonal against human laryngeal cancer cells involves apoptosis, cell cycle arrest, and inhibition of PI3K AKT and MEK ERK signalling pathways. Med Sci Monit, 2018; 24: 7295u2013302 . [PubPeer thread here] . , 
 Retracted [25 March 2021] .  Retracted, due to breach of publishing guidelines, following the identification of non-original content. ,