Podophyllotoxin Isolated from Podophyllum peltatum Induces G2/M Phase Arrest and Mitochondrial-Mediated Apoptosis in Esophageal Squamous Cell Carcinoma Cells
, Mee-Hyun Lee 2,3,†, Ah-Won Kwak 1, Ha-Na Oh 1, Seung-Sik Cho 1, Joon-Seok Choi 4, Kangdong Liu 2,3, Jung-Il Chae 5,* and Jung-Hyun Shim 1,2,*Round 1
Reviewer 1 Report
At line 55 the authors should introduce the concepts of natural products in management of health diseases. Some examples and related references should be added. The aim should be rewritten. Results in Figure 1 should be reported as more Figures and should be better described in the text. Figure 2 should be better decscribed in the text. The subparagraph "PT induces intracellular ROS generation and activates ROS-dependent MAPK pathway" should be implemented.
Discussion and Conclusion should be greatly implemented.
Author Response
Reviewer #1 Question:
At line 55 the authors should introduce the concepts of natural products in management of health diseases. Some examples and related references should be added. The aim should be rewritten. Results in Figure 1 should be reported as more Figures and should be better described in the text. Figure 2 should be better decscribed in the text. The subparagraph "PT induces intracellular ROS generation and activates ROS-dependent MAPK pathway" should be implemented.
▶ Response to Question:
We have added more information line 56-58.
Line 56-58 Natural products such as plant extracts or isolated show benefits less toxicity and low cost, and moreover have indicate anticancer, antioxidant, anti-inflammatory, anti-inflammatory and antibacterial [4-6]. We added results and figure legend text of Figure 1 and Figure 2. Line 188-190 PT (0.1, 0.2, 0.3 and 0.4 μM) treatment for 24 h and 48 h significantly decreased cell proliferation of ESCC cells KYSE 30, KYSE 70, KYSE 410, KYSE 450 and KYSE 510 in a concentration-dependent manner (Figure. 1B-F). Line 202, 203 Thus, our data indicate that treatment with PT reduced cell viability and promoted apoptosis in ESCC cells. Line 207-209 (B-E) MTS cell proliferation assays for P KYSE 30, KYSE 70, KYSE 410, KYSE 450 and KYSE 510 cells treated with increasing concentrations of the indicated PT (0.1 μM, 0.2 μM, 0.3 μM and 0.4 μM) for 24 h and 48 h. Experiments were replicated in triplicate as the means ± SD. *P < 0.05. Line 224-226 Compared with the untreated control, PT treatment accumulated the number of cells in the G0/G1 phase as well as resulting in an appreciable arrest of ESCC cells at G2/M phase of the cell cycle (Figure. 2A). “PT induces intracellular ROS generation and activates ROS-dependent MAPK pathway” replaced to “PT induces intracellular ROS generation and activates JNK and p38 MAPK pathway”
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The authors here present for the first time the effects of the nutraceutical Podophyllotoxin in inducing cell apoptosis, ROS production in human esophageal squamous cell carcinoma. Such effects are mediated via MAPK signaling pathway.
Some aspects of the study should be however clarified:
The role of MAPK and ROS production, both driven by Podophyllotoxin was described in human leukemia cells, despite the neoplastic setting is here different, the reference should be included in the text together with the other reported (PMID: 27484511) Authors decide to complete all the experiments using only 2 of the 5 cell lines used in the proliferation assay. Despite the choice could comprehensibly due to easily compare few cell lines instead of all 5, authors should clarify the reason why cell lines were diminished and why KYSE30 and KYSE450 were chosen. Which cells were used as control? Has PT been used on normal cells to test its cytotoxic effects? The fig. 2C is erroneously indicated as 2B (line 228) In fig. “2C” authors show the decreased expression in WB of cyclin B1 inversely correlated with increasing concentration of Podophyllotoxin. However, in cell line KYSE 30 the expression of Cyclin B1 at 0.3 uM seems even higher than 0.2 uM. How do you explain that? Authors investigated the multi-caspases activity by means of cytofluorimetry to explain the increased levels of apoptosis after PT treatment. The results shown in fig 1H demonstrate an overall degree of apoptosis (early and late apoptosis) of 10.11% +/- 0,86% at the concentration of 0.3 uM that is rather closely related to the apoptotic levels seen at 0.2 uM. Multi-caspases activation levels raised to 24,90% +/- 0,64% at the concentration of 0.3 uM, how authors explain the such marked difference between the levels of apoptosis and multi-caspases activity that should be somehow similar? Moreover, the levels of apoptosis seems to be quite low at the same concentration of IC50 (0.3 uM), especially for KYSE450, how do you explain that?
Author Response
Reviewer #2 Question:
The authors here present for the first time the effects of the nutraceutical Podophyllotoxin in inducing cell apoptosis, ROS production in human esophageal squamous cell carcinoma. Such effects are mediated via MAPK signaling pathway.
Some aspects of the study should be however clarified:
The role of MAPK and ROS production, both driven by Podophyllotoxin was described in human leukemia cells, despite the neoplastic setting is here different, the reference should be included in the text together with the other reported (PMID: 27484511) Authors decide to complete all the experiments using only 2 of the 5 cell lines used in the proliferation assay. Despite the choice could comprehensibly due to easily compare few cell lines instead of all 5, authors should clarify the reason why cell lines were diminished and why KYSE 30 and KYSE 450 were chosen. Which cells were used as control? Has PT been used on normal cells to test its cytotoxic effects? The fig. 2C is erroneously indicated as 2B (line 228) In fig. “2C” authors show the decreased expression in WB of cyclin B1 inversely correlated with increasing concentration of Podophyllotoxin. However, in cell line KYSE 30 the expression of Cyclin B1 at 0.3 uM seems even higher than 0.2 uM. How do you explain that? Authors investigated the multi-caspases activity by means of cytofluorimetry to explain the increased levels of apoptosis after PT treatment. The results shown in fig 1H demonstrate an overall degree of apoptosis (early and late apoptosis) of 10.11% +/- 0,86% at the concentration of 0.3 uM that is rather closely related to the apoptotic levels seen at 0.2 uM. Multi-caspases activation levels raised to 24,90% +/- 0,64% at the concentration of 0.3 uM, how authors explain the such marked difference between the levels of apoptosis and multi-caspases activity that should be somehow similar? Moreover, the levels of apoptosis seems to be quite low at the same concentration of IC50 (0.3 uM), especially for KYSE450, how do you explain that?
▶ Response to Question:
We added a reference line 67 (PMID: 27484511). We added to the discussion section (line 330-332) why we selected KYSE 30 and KYSE 450 cell lines from five esophageal cancer cell lines. Among the five ESCC cell lines, KYSE 30 and KYSE 450 have analogical genetic background and characteristics [26], and have been selected for in-depth analysis because they show similar responses to PT treatment. Normal esophageal cell lines could not be obtained from ATCC. Thus, we selected and experimented commercially available esophageal cancer cell lines through screening. We added to the discussion section (line 320-321). Line 330-332 Among the five ESCC cell lines, KYSE 30 and KYSE 450 have analogical genetic background and characteristics [26], and have been selected for in-depth analysis because they show similar responses to PT treatment. Line 320-321 According to a 2018 study, weight loss and side effects were not observed when 15 mg/Kg Free PT was treated for 30 days in mice xenografts to assess antitumor activity [21]. We changed Figure 2. Please see the attachment. We conclude that there is no significant difference in cyclinB1 expression at 0.2 μM and 0.3 μM PT concentrations by western blot experiments. We have performed three time indepedent experiments. All multi-caspase experiments obtained similar results. Annexin V, ROS, cell cycle, MMP, and multi-caspase experiments did not proceed simultaneously, resulting in slightly different results. The results of the multi-caspase experiments were used as data because it showed a tendency of concentration-dependent increase. In the future, we will proceed an in-depth study.
Author Response File: Author Response.pdf
