Mistletoe Extracts from Different Host Trees Disparately Inhibit Bladder Cancer Cell Growth and Proliferation

Round 1

Reviewer 1 Report

Eva Juengel et al. investigated the effect of different mistletoe extracts in bladder cancer cell lines and confirmed their inhibitory activity in tumor growth and proliferation. They observed the changes in the BrdU incorporation by ELISA and the cell cycling/apoptosis by Flow cytometry, induced by mistletoe extracts. They also explored the Integrins and CD44 receptor expression changes after extract treatment. This study is well-written and designed scientifically. This study is a relatively complete investigation of mistletoe extract’s potential in bladder cancer treatment in vitro. However, there still are some concerns before publication.

Please address the below concerns:

 

1. Italicize all the species' taxonomic names like Line 15,19.  check the whole manuscript.
2. Line 20 “as was”?
3. Line 48 typing error for Nonetheless.
4. Line 85 Please give a few sentences more in the background introduction of Integrins and CD44 receptors related to cancer and move into the middle of the Background.
5. Better separate the blocking study and knockdown study methods.
6. Line 209 control is included in figure 1, actually. Please in figure legend, indicate control in figure 1 is which type of control, I assume is medium control.
7. Figures 3 and 4 can be merged into one, even moved into supplementary data.
8. Figure 6A, better to show a graph data with triplicate samples, not just a representative raw data.  6B, better to have exact average percentage values labeled in column or an addition graph.
9. Figure 7, are these siRNA done separately? It’s inappropriate to share one actin band in different siRNA-transfected samples in WB data.
10. Sometimes, describing the figure in text, better to have some exact values when you highlight the significant comparison, like decrease 60%, or enhance 1.5-fold then plus the P value with it.
11. After reading the discussion, the novelty of this manuscript is decreasing and not highlighted. Please rephrase, and add some sentences highlighting the novel work in this study, not reported in other papers.
12. Line 461, content in conclusion, more likely the discussion. Please either add more conclusion sentences in the Results part to summarize each experiment or replace the Conclusions content with your specific findings.
13. Figure S1, please label the standard protein size near your target protein.
14. Have you considered or tried to further study in vivo on mice? 

Author Response

Answers to the comments of referee 1

Comment 1: Italicize all the species' taxonomic names like Line 15,19.  check the whole manuscript.

Our answer: This has been done.

Comment 2: Line 20 “as was”?

Our answer: The sentence now reads (Line 19): “Tumor cell growth and proliferation, apoptosis induction, and cell cycle progression were then evaluated.”

Comment 3: Line 48 typing error for Nonetheless.

Our answer (Line 47): The mistake has been corrected.

Comment 4: Line 85 Please give a few sentences more in the background introduction of Integrins and CD44 receptors related to cancer and move into the middle of the Background.

Our answer: We have added more information about integrins and CD44 to the “Introduction”.  Lines 83-91 now read: Integrins are heterodimeric transmembrane glycoprotein signaling receptors composed of an α and β subunit. In mammals, 18 α and 8 β subunits have been identified that impact a wide range of cellular functions including cell migration, growth, proliferation, and apoptosis. CD44 is a non-kinase cell surface transmembrane glycoprotein, largely ex-pressed in the CD44s form, encoded by constant exons. However, post-translational alternative splicing may occur in variant exons to form different CD44v types (CD44v1-10). Along with integrin subtypes, CD44 members are involved in the fate of tumor cells and are associated with bladder cancer size, tumor grade, and recurrence [18].”

Comment 5: Better separate the blocking study and knockdown study methods.

Our answer: We have separated, “2.9. Blocking studies” and “2.10. Knockdown studies” (lines 186-203).

Comment 6: Line 209 control is included in figure 1, actually. Please in figure legend, indicate control in figure 1 is which type of control, I assume is medium control.

Our answer: That is correct. Indeed, “Results”, section 3.1, reads: “No differences were seen between the cell growth activity in the presence of isotonic solvent or cell culture medium alone (solvent control values are not included in the figure)”. We have now added to the legend of figure 1: “Control indicates medium control” (lines 223-4).

Comment 7: Figures 3 and 4 can be merged into one, even moved into supplementary data.

Our answer: As suggested, figure 3 and 4 have been merged. We would, however, like to keep this figure in the manuscript to provide the reader with an easy overview of the receptor expression.

Comment 8: Figure 6A, better to show a graph data with triplicate samples, not just a representative raw data. 6B, better to have exact average percentage values labeled in column or an addition graph.

Our answer: Mean values have now been added to the figure (now figure 5A). Exact average percentage values from figure 5B are now included in the manuscript text (lines 291-294), which reads: “Cell cycle analysis in the presence of dilutions of 1:160,000 showed a significant increase in G0/G1 phase cells (Medium control: 51.7+/- 4.4%, Populi: 58.1+/-4.8%, Salicis: 59.7+/-6.6%) , accompanied by a loss of S-phase cells (Medium control: 16.7+/- 2.9%, Populi: 13.1+/-4.0%, Salicis: 12.2.+/-1.6%) (figure 5B)”.

Comment 9: Figure 7, are these siRNA done separately? It’s inappropriate to share one actin band in different siRNA-transfected samples in WB data.

Our answer: Yes, the siRNA experiments were done separately, all combined with their respective β-actin controls. The figure (now figure 6) initially showed one representative β-actin band. This has been corrected.

Comment 10: Sometimes, describing the figure in text, better to have some exact values when you highlight the significant comparison, like decrease 60%, or enhance 1.5-fold then plus the P value with it.

Our answer: We have now modified the text in section 3.1 (Lines 230-236) which now reads:  “Employing the 1:8,000 dilution, Populi and Crategi reduced BrdU incorporation by about 90% in RT112 and UMUC3 but by only 50% in TCCSup. BrdU incorporation was completely blocked in RT112 and UMUC3 cells in the presence of Salicis, whereas the same extract diminished BrdU in TCCSup by only 65%. Distinct differences were also seen when the tumor cells were exposed to Populi, Salicis, and Crataegi at a 1:160,000 dilution with a suppression of 75% in RT112 and 65% in UMUC3, in contrast to only 12% suppression in TCCSup.” Percentage values indicating change have now been included in sections 3.1 and 3.2.

Comment 11: After reading the discussion, the novelty of this manuscript is decreasing and not highlighted. Please rephrase, and add some sentences highlighting the novel work in this study, not reported in other papers. (Also Comment 12: Line 461, content in conclusion, more likely the discussion. Please either add more conclusion sentences in the Results part to summarize each experiment or replace the Conclusions content with your specific findings.

Our answer: We would like to confirm that our paper presents novel insights into the mode of action of several mistletoe extracts on bladder cancer cells. Only references 22 (from 2007) and 24 (from 2006) are related to mistletoe and bladder cancer. Otherwise, few articles have been published dealing with the influence of mistletoe on growth or apoptosis of other tumor entities. To our knowledge, no data have been published so far, pointing to integrins, CD44 receptors and the CDK-Cyclin axis as relevant targets of mistletoe. We have now highlighted this aspect in “Conclusions” (Lines 486-497) which now reads: “Mistletoe extracts derived from different host trees significantly suppressed the growth of different bladder cancer cells and induced apoptosis in vitro. This is the first report identifying integrin adhesion receptors and CD44 as targets of mistletoe extracts relevant for modulating growth and proliferation of bladder cancer cells. Integrin α5 was strongly diminished by Salicis and Populi on both RT112 and UMUC3, and was moderately down-regulated on TCCSup. Receptor alterations due to the influence of mistletoe were not all the same but rather depended on the origin of the extract and the tumor cell type. The same was true with respect to the Cyclin-CDK-axis which was also disparately targeted by mistletoe extracts. Mistletoe from Salicis specifically targeted CDK1 and 2, and Cyclin A, whereas mistletoe from Populi acted via CDK2, Cyclin A and B. Low grade bladder cancer appears to be more sensitive to mistletoe extracts than does high grade bladder cancer. ”

Comment 13: Figure S1, please label the standard protein size near your target protein.

Our answer: This has been done.

Comment 14: Have you considered or tried to further study in vivo on mice?

Our answer: We intend to start in vivo experiments using both drug-sensitive as well as drug-resistant cell lines for injection. Currently, we are in a pilot phase establishing the optimum number of cells to be injected.

 

 

Reviewer 2 Report

the article investigate the Mistletoe extracts from different host trees disparately inhibit bladder cancer cell growth and proliferation. 

there still some concerns below:

1. have we first test the toxicity to the cells using IC  50 testing?

2. for the cell type selection, as we know, bladder cancer cell has other types like T24.

3. add some explanation about why tested the CD44 background information for advice.

 

Author Response

Answers to the comments of referee 2

Comment 1: have we first test the toxicity to the cells using IC 50 testing?

Our answer: Our experiments were based on extract mother solutions which were further diluted 10 or 20fold to reach the final dilutions indicated in figure 1. Although the data may allow calculation of IC50-values for each extract and each tumor cell line, we used 1:8,000 and 1:160,000 dilutions continuously throughout the study, since these concentrations exerted significant effects on tumor growth (figure 1). 

 

Comment 2: for the cell type selection, as we know, bladder cancer cell has other types like T24.

Our answer: RT112, UMUC3, and TCCSup cell lines were chosen for the present study due to their different grading and staging. This strategy allowed reflection of mistletoes’ relevance in the treatment of bladder cancer during epithelial-mesenchymal transition, e.g., at different stages of tumor progression. It is correct that T24 cells, derived from a poorly differentiated (grade 3) bladder carcinoma, may also be good candidates (Please see our publications “Allyl-, Butyl- and Phenylethyl-Isothiocyanate Modulate Akt-mTOR and Cyclin-CDK Signaling in Gemcitabine- and Cisplatin-Resistant Bladder Cancer Cell Lines”. Doi: 10.3390/ijms231910996. “Plant-Derived Sulforaphane Suppresses Growth and Proliferation of Drug-Sensitive and Drug-Resistant Bladder Cancer Cell Lines In Vitro”. Doi: 10.3390/cancers14194682.). To address the aspect of cell type selection we have added a sentence to the last paragraph of the discussion (lines 480-484): “More studies should be directed towards examining cell signaling processes in other bladder cancer cell lines, not only to verify our observations but also to exclude undesired events or undesired protein-protein cross-communication that could reduce the tumor cell response to mistletoe or even re-activate oncoproteins.”

 

Comment 3: add some explanation about why tested the CD44 background information for advice.

Our answer: We have now added to the “Introduction”, last paragraph (lines 83-91): “Integrins are heterodimeric transmembrane glycoprotein signaling receptors composed of an α and β subunit. In mammals, 18 α and 8 β subunits have been identified that impact a wide range of cellular functions including cell migration, growth, proliferation, and apoptosis. CD44 is a non-kinase cell surface transmembrane glycoprotein, largely ex-pressed in the CD44s form, encoded by constant exons. However, post-translational alternative splicing may occur in variant exons to form different CD44v types (CD44v1-10). Along with integrin subtypes, CD44 members are involved in the fate of tumor cells and are associated with bladder cancer size, tumor grade, and recurrence [18].”

Reviewer 3 Report

The article by Eva et al. explores the potential effects of mistletoe extracts, derived from various host trees, on the growth, proliferation, apoptosis, cell cycle, and receptor expression of bladder cancer cells. The study adeptly illustrates the anticancer properties of these extracts, albeit with varying degrees of efficacy depending on the specific extract and cell line in question. Furthermore, the manuscript posits that mistletoe could potentially enhance current bladder cancer therapies. Overall, I found the manuscript to be well-articulated and systematically organized, with the data effectively substantiating the conclusions drawn.

 

However, I would like to suggest a few areas where the manuscript could benefit from further refinement:

 

Figure 1 - Concentration Representation: It would be more conventional and clearer to represent the data in terms of concentration instead of dilution ratio. 

 

Figure 1 - Baseline Comparison: The choice to set the 24-hour values as 100% is not clearly justified in the manuscript. It might be more insightful to compare the data with the 0-hour mark or a control group (medium devoid of any drug) to provide a more comprehensive view of the effects over time.

 

Consistency in Error Bars: I noticed that the standard deviation (SD) bars in the figures are inconsistently placed - appearing above, below, or on both sides of the data points. Standardizing the presentation of these bars will enhance the visual clarity and coherence of the data representation.

 

Evaluation of Potential Toxicity: The manuscript currently does not address the potential toxicity or adverse effects of mistletoe extracts on normal cells or tissues. 

Minor editing of English language required

Author Response

Referee 3

Comment 1: Figure 1 - Concentration Representation: It would be more conventional and clearer to represent the data in terms of concentration instead of dilution ratio.

Our answer: We agree that it would be clearer to show data in terms of concentration and would certainly make dose comparison with previously published investigations easier. However, the mistletoe mother tincture we used was prepared and delivered as a solution (please see reference 19), and not as a dried extract. Unfortunately, it is therefore not possible to exactly calculate the extract concentration in weight units/volume.

 

Comment 2: Figure 1 - Baseline Comparison: The choice to set the 24-hour values as 100% is not clearly justified in the manuscript. It might be more insightful to compare the data with the 0-hour mark or a control group (medium devoid of any drug) to provide a more comprehensive view of the effects over time.

Our answer: Initially, at 0 h, each well of a 96-well plate was filled with 5,000 cells. Not all of these cells settle down and attach to the bottom of the plates, and even those cells that establish an initial loose contact do not always proceed to establish firm contact (indicated by altered cell shape, from round to flattened). This must be taken into account since firm adhesion is necessary to activate growth-related intracellular signaling. It is also important to note that the tumor cells need time to “recover” following enzymatic detachment from the culture flask. Aside from this the referee suggests including a control group (devoid of any drug). This has already been done by evaluating the number of tumor cells incubated with cell culture medium alone (please see figure 1 and legend of figure 1). Materials and Methods, section 2.2. (lines 109-10) read: “Isotonic solvent or cell culture medium alone were used as controls”.

To make our approach clearer, we have now added in the methods section 2.3 “Tumor cell growth” (lines 112-118): “Cell growth was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Tumor cells were detached from the culture flask by enzymatic treatment using accutase (PAA Laboratories, Cölbe, Germany). Each well of a 96-well plate was then filled with 5,000 cells and exposed to a mistletoe extract or control medium without extract (each in triplicate). Cells were allowed to settle down to establish firm adhesion contact and to recover from enzymatic treatment overnight. Tumor cell number was then counted after 72 h”.

Comment 3: Consistency in Error Bars: I noticed that the standard deviation (SD) bars in the figures are inconsistently placed - appearing above, below, or on both sides of the data points. Standardizing the presentation of these bars will enhance the visual clarity and coherence of the data representation.

Our answer: The referee is correct (figure 1, figure 6). We did this to prevent overlapping of the SD bars. Upper lines mostly show positive error bars, lower lines negative error bars. We would prefer to maintain this approach which prevents bar overlap (not in all but most cases). However, we are prepared to alter the figure if the referee finds it necessary.

 

Comment 4: Evaluation of Potential Toxicity: The manuscript currently does not address the potential toxicity or adverse effects of mistletoe extracts on normal cells or tissues.

Our answer: Indeed, this is an important issue. We have addressed potential toxicity and added pertinent information to the discussion (lines 475-484): “Side-effects from mistletoe including local skin reactions at the injection site and systemic reactions such as fever, fatigue, rash, and flu-like symptoms [53,54] have been documented. When mistletoe was applied intravenously to cancer patients, treatment had to be discontinued due to toxicity in 15% of cases (grade 3 fatigue, grade 3 alanine aminotransferase elevation or grade 3 dyspnea/flank pain) [52]. Hence, establishing appropriate dosage is important and requires further study. More studies should be directed towards examining cell signaling processes in other bladder cancer cell lines, not only to verify our observations but also to exclude undesired events or undesired protein-protein cross-communication that could reduce the tumor cell response to mistletoe or even re-activate oncoproteins.”