Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript discusses a pertinent study concerning the integration of natural compounds into the gemcitabine/cisplatin-based treatment regimen. However, I have several comments:

1. I suggest including a graphical abstract to improve the article's visibility.
2. Line 81, in vitro in italic form.
3. Line 83, in vitro and in vivo in italic form.
4. The authors used subcultures derived from 7 to 24 passages. Do they consider a reliable passage range for the experiments?
5. It’s important for the scientific community to be aware of the dilutions used for antibodies.
6. For images generated with the fluorescence microscope, I recommend that authors describe the laser exposure time and include scales so readers can size the cells.
7. Lines 103, 109, and 114. I suggest using Figures.
8. Lines 126, 131 and 134. I suggest using Figures.
9. Considering that the authors developed the study with cell lines. What is the reason for the experiments to only have n=3?
10. The supplementary material sent with the Blotting originals does not specify which groups were analyzed.
11. Finally, I recommend that authors send blots stained with Coomassie Blue and Ponceau.

Author Response

Comment 1: I suggest including a graphical abstract to improve the article's visibility.

Our answer: A graphical abstract has been added.

Comment 2: Line 81, in vitro in italic form. Line 83, in vitro and in vivo in italic form.

Our answer: The manuscript has been checked and in vitro and in vivo are now italized.

Comment 3: The authors used subcultures derived from 7 to 24 passages. Do they consider a reliable passage range for the experiments?

Our answer: Culturing and passaging tumor cell lines over an extended period of time can cause selective pressure, leading to altered cell growth behavior and/or the generation of subclones within the overall cell population. Based on former experiments (e.g. Rutz et al. 2022, DOI: 10.3390/ijms231910996) we therefore established a passage number cutoff for the bladder cancer cell lines used here, with a maximum passage number of 24. This concords with Prasad et al. who recommends performing experiments with cancer cell lines under passage 25 (Prasad et al. Passage number of cancer cell lines: Importance, intricacies, and way-forward. Biotechnol Bioeng. 2023. doi: 10.1002/bit.28496).

We have addressed this comment in „Materials and Methods” section “Cell culture” which now reads (lines 442-444): “Subcultures from passages 7–24 were used since culturing and passaging tumor cell lines over an extended period of time can cause undesired alterations of their genetic and molecular profiles [72]”.

Comment 4: It’s important for the scientific community to be aware of the dilutions used for antibodies.

Our answer: dilutions are now included. “Materials and Methods”, section “Western Blot Analysis” which now reads (lines 546-555): “FAK (clone 77, dilution 1∶1000), phospho-specific FAK (pFAK (pY397); clone 18, dilution 1∶1000), ILK (clone 3, dilution 1∶1000; all from BD Biosciences), Ezrin (polyclonal, dilution 1∶1000), E-cadherin (clone 24E10, dilution 1∶1000), N-cadherin (clone 13A9, dilution 1∶1000), vimentin (clone D21H3, dilution 1∶1000), talin (clone C45F1, dilution 1∶1000; all from Cell Signaling, Leiden, The Netherlands). HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (dilution 1:5000, both from Cell Signaling) served as secondary antibodies”. [….] “β-Actin (clone AC-15, dilution 1∶1000, Cell Signaling) served as the internal control”.

Our answer: Amount of monoclonal antibodies is specified in “Materials and Methods”, section “Integrin expression” (lines 505-521): “The cells were then incubated for 60 min at 4 °C with phycoerythrin (PE)-conjugated monoclonal antibodies targeting integrin β1 (anti-integrin β1 IgG1; clone MAR4, 20 µl), integrin β4 (anti-integrin β4 IgG2a; clone 439–9B, 20 µl (all obtained from BD Biosciences, Heidelberg, Germany). Anti-integrin β5 (IgG2a; clone REA718, 2 µl) was from Miltenyi Biotech, Bergisch Gladbach, Germany. Anti-phospho-integrin β1 (Thr788/789, 2.5 µl; Merck KGaA, Darmstadt, Germany) was labeled with allophycocyanin (APC)”.

“Materials and Methods”, chapter “CD44 expression” now reads: “Tumor cells were then stained with anti-CD44s (clone SFF-2, 2.5 µl), anti-CD44v3 (clone VFF-327v3, 2.5 µl), anti-CD44v4 (clone VFF-11, 5 µl ), anti-CD44v5 (clone VFF-8, 25 µl), anti-CD44v6 (clone VFF-7, 2.5 µl), and anti-CD44v7 (clone VFF-9, 2.5 µl); all from eBioscience, ThermoFisher, Darmstadt, Germany)”.

Comment 5: For images generated with the fluorescence microscope, I recommend that authors describe the laser exposure time and include scales so readers can size the cells.

Our answer: The images were generated by a fluorescence microscope, not by a confocal scanning microscope. Therefore, information on the laser exposure time cannot be provided. Scale bars are now included in the images (figure 7).

Comment 6: Lines 103, 109, and 114. I suggest using Figures. Lines 126, 131 and 134. I suggest using Figures.

Our answer: This comment is not clear to us. In fact, the respective data are shown in figures 1, 2 and 3.

Comment 7: Considering that the authors developed the study with cell lines. What is the reason for the experiments to only have n=3?

Our answer: There is no fixed consensus on a standard protocol for a given tumor cell line (Hirsch & Schildknecht 2019. In Vitro Research Reproducibility: Keeping Up High Standards. 2019. doi: 10.3389/fphar.2019.01484.). However, at least three biological replicates are recommended to allow reliable statistical analysis (e.g. Chan et al. Replicates in stem cell models-How complicated! 2020. doi: 10.1002/stem.3237. Niepel et al. A Multi-center Study on the Reproducibility of Drug-Response Assays in Mammalian Cell Lines. 2019. doi: 10.1016/j.cels.2019.06.005.). In fact, based on the analysis of cell culture based articles published between 2015 and 2025, experimental assays were documented to be commonly repeated three times (Hassan et al. Three Fundamental Statistical Elements in Cell Culture-Based Anticancer Studies. 2025. Doi 10.37231/ajmb.2025.9.2.785). Low passage tumor cells were used in our assays to guarantee low-variance populations (please see our answer to comment 3). Additionally, cells were derived from the same cell stock and culture conditions were kept constant. It should also be noted that multiple cell lines were treated with different compounds and then subjected to several assays, resulting in a large number of performed experiments.

Comment 8: The supplementary material sent with the Blotting originals does not specify which groups were analyzed.

Our answer: This has been corrected.

Comment 9: Finally, I recommend that authors send blots stained with Coomassie Blue and Ponceau.

Our answer: β-actin serves as a well-established specific housekeeping protein, often used as a loading control in western blots. Still, we agree, that although β-actin represents a highly conserved protein, its expression can be changed during cell manipulation. In fact, drug treatment may alter protein concentration, and hence cause a different intensity of β-actin bands in treated samples, compared to untreated controls. We do agree that Coomassie staining allows a more exact data interpretation in such cases, since it stains proteins in total. Unfortunately, all experiments have already been finalized, and all blots have been discarded. Therefore, we cannot retrospectively re-evaluate the protein content. However, we would like to point out that figure 8 documents equal β-actin expression in treated versus non-treated cells, making it unlikely that expression of the proteins evaluated was unspecifically altered.

Reviewer 2 Report

Comments and Suggestions for Authors

In section 2.5: Influence of BITC, AITC and PEITC on protein expression.  On line 200, the authors state that "pFAK was reduced in all sublines by AITC".  I recommend changing this to ?pFAK was reduced in all resistant sublimes by AITC" for clarity. Similarly resistant should be added before sublines on line 212.  It may seem redundant, but I think that will add clarity to the wording.

A minor note in the introduction. I think "western" in western blotting should not be capitalized in the middle of the sentence.  The names for western and northern blotting were given for ease, homogeneous and humor after Edwin Southern invented Southern blotting.  No Dr. Northern or Western were involved.

Materials and Methods. Statistics.  Where the overall ANOVA is significant, appropriate multiple comparison methods should be used since multiple t tests can lead to a greater chance of type I errors. 

Author Response

Comment 1: In section 2.5: Influence of BITC, AITC and PEITC on protein expression.  On line 200, the authors state that "pFAK was reduced in all sublines by AITC".  I recommend changing this to “pFAK was reduced in all resistant sublimes by AITC" for clarity. Similarly resistant should be added before sublines on line 212. It may seem redundant, but I think that will add clarity to the wording.

Our answer: The referee is correct, pFAK was potently reduced in the resistant sublimes by AITC. Still, we would like to point to RT112 cells, where AITC also reduced pFAK in the controls. Since this phenomenon was not seen in TCCSup, we have now altered the respective phrase (2.5. Influence of BITC, AITC, and PEITC on protein expression) which now reads (lines 195-196): "pFAK was reduced in all resistant cell sublines by AITC, excluding TCCSUP controls where AITC did not alter pFAK expression". As suggested resistant has been added on line 207.

Comment 2: A minor note in the introduction. I think "western" in western blotting should not be capitalized in the middle of the sentence.  The names for western and northern blotting were given for ease, homogeneous and humor after Edwin Southern invented Southern blotting.  No Dr. Northern or Western were involved.

Our answer: We have corrected it. “Western” is now in lower case except at the beginning of a sentence.

Comment 3: Materials and Methods. Statistics. Where the overall ANOVA is significant, appropriate multiple comparison methods should be used since multiple t tests can lead to a greater chance of type I errors.

Our answer: We apologize that “Statistics” does not explain which test was used for which assay. We have further refined the statistics section, which now reads (lines 567-568): “All experiments were performed three times. Statistical significance was calculated using ANOVA for adhesion and migration, and t-test for integrin expression and functional blockade. Differences were considered statistically significant at p < 0.05”. The figure legends have been improved as well:

Figure 1: “Adhesion is given as mean relative to the respective untreated control (set to 100%)”.

Fig 9: “Error bars show standard deviation. Significant difference to related unblocked controls: * = p < 0.05; ** = p < 0.01; n = 3”.

Reviewer 3 Report

Comments and Suggestions for Authors

 The manuscript investigates the effects of three plant-derived isothiocyanates on the adhesion, migration, and invasion properties of cisplatin- and gemcitabine-resistant bladder cancer cell lines. The authors demonstrate that these compounds significantly inhibit adhesion to extracellular matrix proteins, suppress chemotaxis and migration, and alter integrin expression and FAK signaling. However, the manuscript needs minor revision in some sections and different aspects to publishing in Molecules. For the manuscript “Influence of plant derived natural isothiocyanates on adhesion and invasion of gemcitabine and cisplatin resistant bladder cancer cell lines”, I suggest the following things:

 

Major things:

1. Refine the title for conciseness and clarity.
2. The abstract includes excessive result details and lacks a coherent flow to emphasize the key mechanisms.
3. More recent studies on the three plant-derived isothiocyanates should be introduced in the manuscript.
4. Figure 7, lack scale bars.
5. The concentrations used are relatively high, especially 40 µM AITC, why?
6. Figure 9, Why do β1 and β4 antibodies fail to block resistant RT112 chemotaxis when these integrins are expressed?
7. Can CD44 translocation be blocked, and does this rescue the invasive phenotype?

 

Minor things:

1. Line 416, “TCCSupp”, typo.

Author Response

Comment 1: Refine the title for conciseness and clarity.

Our answer: The title now reads: “Natural isothiocyanates block adhesion and invasion of gemcitabine and cisplatin resistant bladder cancer cell lines”.

Comment 2: The abstract includes excessive result details and lacks a coherent flow to emphasize the key mechanisms.

Our answer: The abstract has been shortened to the 200 word limit and and incorporates a coherent flow emphasizing key mechanisms. It now reads:

Aggressive metastatic progression often develops in bladder cancer patients with acquired cisplatin or gemcitabine resistance.  The potential of the natural isothiocyanates allyl-isothiocyanate (AITC), butyl-isothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC) to inhibit adhesion and migration of cisplatin- or gemcitabine-resistant and sensitive RT112, T24, and TCCSUP bladder cancer cell lines was investigated. Parameters determined were: cell interaction with collagen or fibronectin, chemotaxis, membrane receptors involved in adhesion (total and activated integrins β1, β4, β5, CD44s, and CD44v3-v7). CD44s location and adhesion and migration related signaling proteins were determined. AITC blocked adhesion of almost all sensitive and resistant cancer cells. PEITC and BITC suppressed fibronectin interaction of sensitive and resistant RT112. All three isothiocyanates diminished chemotaxis in all cell lines. Integrin expression was differentially altered but CD44s and CD44v were not altered. BITC and PEITC translocated CD44s from the cell membrane to cytoplasm. The tumor suppressor E-cadherin increased, whereas focal adhesion kinase (FAK), linked to integrin signaling, was deactivated after isothiocyanate treatment. Blocking FAK, β1, β4, or β5 was associated with reduced chemotaxis. Thus, AITC, BITC, and PEITC blocked adhesion and migration in cisplatin and gemcitabine resistant bladder cancer cells. This was associated with altered integrin expression and signaling, CD44s translocation, and enhanced E-cadherin.

Comment 3: More recent studies on the three plant-derived isothiocyanates should be introduced in the manuscript.

Our answer: We have added some recent studies concerning the potential of ITCs to resensitize tumor cells to cisplatin treatment. To our knowledge, all relevant articles dealing with ITCs and bladder cancer have been considered in this manuscript. In fact, we are presently preparing a review article, concentrating on the relevance of ITCs in treating different drug-resistant tumors.  “Discussion”, last paragraph, now reads:

“Based on the in vitro data presented here, we conclude that the ITCs may be potent natural compounds worth integrating into a GC-based treatment regimen for bladder cancer. ITCs have been shown to resensitize other drug resistant tumor cells. Long-term treatment with a panel of structurally diverse ITCs reversed the epithelial-mesenchymal phenotype and reduced the migratory potential of cisplatin resistant lung cancer cells compared to parental cells. Reduced migration was accompanied with an increased expression of E-cadherin. Most importantly, the ITCs mediated a decrease in ATP Binding Cassette Subfamily C Member 1 (ABCC1) and aldehyde dehydrogenase (ALDH) 3 protein levels whose overexpression has been associated with drug resistance and cancer progression [67]. Similar effects have been noted with PEITC, which reduced the population of ALDH1 expressing cancer stem cells and suppressed the downstream multidrug resistance protein, P-glycoprotein (ABCB1) [68,69]. PEITC has also been shown to reverse cisplatin resistance in non-small cell lung cancer cells by depleting cellular glutathione (GSH). GSH mediated export of cisplatin is a further key resistance mechanism with high expression correlated to a strong decrease in intracellular cisplatin, due to drug efflux [70]. PEITC has also been shown to restore chemosensitivity in cisplatin-resistant non-small cell lung cancer by targeting c-Myc and decreasing the Akt/mTOR signaling pathway [71]. This is important, since not only PEITC but BITC and AITC also decreased phosphorylated Akt in cisplatin and gemcitabine resistant RT112 and T24 bladder cancer cells [14]. Since AITC, BITC, and PEITC reverse chemoresistance in the bladder cancer cell model, bladder cancer patients may also profit from integrating ITCs into their treatment, once cisplatin and/or gemcitabine treatment is no longer effective”.

Comment 4: Figure 7, lack scale bars.

Our answer: Scale bars have been added.

Comment 5: The concentrations used are relatively high, especially 40 µM AITC, why?

Our answer: We did address the high concentration of AITC in the “Discussion”, second para, which reads: “The reason for the different efficacy of AITC compared to BITC/PEITC is not clear. BITC and PEITC, but not AITC, clearly inhibited tubulin polymerization in cancer cells. Thus, BITC and PEITC functioned as anti-mitotic agents and the investigators assumed that the inferior effect of AITC on tubulin might be the reason why higher AITC concentrations were necessary to induce similar anti-proliferative activity [29]. Possibly, these differences in effective inhibitive concentrations might be due to the lipophilic and hydrophilic properties of the ITCs. BITC and PEITC are more lipophilic, while AITC is more hydrophilic [30]. The higher the lipid solubility, the higher the intracellular uptake. This would make BITC and PEITC more effective at lower concentrations than AITC”. Overall, the concentrations of the isothiocyanates we used were within the ranges applied by others.

Comment 6: Figure 9, Why do β1 and β4 antibodies fail to block resistant RT112 chemotaxis when these integrins are expressed?

Our answer: Why this occured can only be speculated upon. Integrins determine cell adhesion and migration but also initiate signaling cascades responsible for proliferation, apoptosis, and epithelial-mesenchymal transition. We and others have shown that integrins are linked to the Akt-mTOR pathway in cancer cells that force progressive tumor growth (e.g. Juratli et al. Integrin α2 and β1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells. 2022. doi: 10.3390/cancers14102430. Siech et al. Insulin-like Growth Factor-1 Influences Prostate Cancer Cell Growth and Invasion through an Integrin α3, α5, αV, and β1 Dependent Mechanism. 2022 doi: 10.3390/cancers14020363). Therefore, failure of β1 and β4 antibodies to block resistant RT112 chemotaxis does not mean that β1 and β4 are not important for these cells. Rather, an influence of β1 and β4 on RT112 growth and proliferation cannot be excluded. However, integrin β1 knockdown has recently been shown by others to reduce proliferation of RT112 cells (Wang et al. Knockdown of integrin β1 inhibits proliferation and promotes apoptosis in bladder cancer cells. 2025. doi: 10.1002/biof.2150). There is also evidence that resistance may cause a functional switch of integrins so that integrin blocking may induce a different or even opposing response in sensitive versus resistant cells (e.g. Juengel et al. Resistance to the mTOR inhibitor temsirolimus alters adhesion and migration behavior of renal cell carcinoma cells through an integrin α5- and integrin β3-dependent mechanism. 2014. doi: 10.1016/j.neo.2014.03.011. Tsaur et al. Resistance to the mTOR-inhibitor RAD001 elevates integrin α2- and β1-triggered motility, migration and invasion of prostate cancer cells. 2012. doi: 10.1038/bjc.2012.313). The integrins β1 and β4 expression levels distinctly differ among the cell lines and between sensitive versus resistant cells (please see figure 4 in the manuscript). The same is true with respect to E–and N-cadherin expression (please see figure 8 in the manuscript), proteins which have been shown to communicate with integrins (Srinivasan et al. Epithelial-Mesenchymal Transition in Cancer: Insights Into Therapeutic Targets and Clinical Implications. 2020. doi: 10.1002/mco2.70333. de Abreu Pereira et al. Proteomic Analysis of HCC-1954 and MCF-7 Cell Lines Highlights Crosstalk between αv and β1 Integrins, E-Cadherin and HER-2. 2022. doi: 10.3390/ijms231710194.). Considering all of these disparate findings it does not surprise us that integrin β1 (as well as integrin β4) blockade caused different responses in different cell lines.

We have considered this issue in more detail. “Discussion”, page 14, now reads (lines 338-356):

“Blocking integrin β1 and β4 suppressed chemotaxis of the parental RT112 and TCCSup cells, indicating a principal role of these receptors in tumor cell migration. Unexpectedly, β1 and β4 blockade down-regulated chemotaxis of the resistant TCCSup but not of the resistant RT112 cells. The reason for this difference is not clear. The integrin β1 expression level distinctly differed among the cell lines and between sensitive and resistant cells. Integrin β1 was lower in resistant RT112 cells but elevated in resistant TCCSUP cells, each compared to their respective sensitive sublines. We assume that different basal integrin expression levels in both cell lines, with disparate alterations after resistance develops, activate adhesion and invasion related down-stream signaling differently. Gou et al. has shown that integrin β1 alterations correlate with chemotherapy resistance in a subset of urothelial carcinoma cells with enhanced EMT features [41]. This is important since integrins communicate with E–and N-cadherin [42,43]. We did not investigate EMT proteins in detail. However, drug resistant TCCSUP cells were characterized by a loss of E-cadherin and high expression of N-cadherin. This contrasts to RT112 cells with high E-cadherin and low N-cadherin expression. Therefore, the different E- and N-cadherin expression levels during cadherin-integrin interaction may also explain why β1 blockade down-regulated chemotaxis of the resistant TCCSUP but not of the resistant RT112 cells. Resistance development has been associated with a functional switch of integrins so that integrin blocking may induce a different or even opposing response in sensitive versus resistant cells [44,45]. A functional switch of integrin β1 might contribute to the different role of this receptor in sensitive versus resistant RT112 cells as well. Although speculative, transcriptomic data of 262 gastric carcinoma samples has revealed an inverse correlation between E-cadherin and integrin β1 function [46], supporting our hypothesis“.

Comment 7: Can CD44 translocation be blocked, and does this rescue the invasive phenotype?

Our answer: In our opinion, there is no method to block receptor translocation. Life cell imaging e.g. by a confocal scanning microscope might be a way to depict protein movement (given stability of the fluorescence marker used). Blocking CD44-translocation would require a highly specific stimulus to activate translocation that could then be stopped by blocking the stimulus. Whether this strategy is associated with cell movement must be explored. Indeed it is intriguing to deal with the question presented by the referee. For certain though, establishing a method to block CD44 translocation will be very challenging.

Comment 8: Line 416, “TCCSupp”, typo.

Our answer: This has been corrected (line 447).