Title: Combination of a third generation bisphosphonate and replication-competent adenoviruses augments the cytotoxicity on mesothelioma

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1 Author s response to reviews Title: Combination of a third generation bisphosphonate and replication-competent adenoviruses augments the cytotoxicity on mesothelioma Authors: Masatoshi Tagawa (mtagawa@chiba-cc.jp) Yuanyuan Jiang (himiya666@hotmail.com) Boya Zhong (bellazhongboya@hotmail.com) Kiyoko Kawamura (kkawamur@chiba-cc.jp) Takao Morinaga (tmorinaga@chiba-cc.jp) Masato Shingyoji (mshingyoji@chiba-cc.jp) Ikuo Sekine (isekine@md.tsukuba.ac.jp) Yuji Tada (ytada25@yahoo.co.jp) Koichiro Tatsumi (tatsumi@faculty.chiba-u.jp) Hideaki Shimada (hideaki.shimada@med.toho-u.ac.jp) Kenzo Hiroshima (kenzo@tymc.twmu.ac.jp) Version: 1 Date: 15 Mar 2016 Author s response to reviews: Replies to reviewers Referee 2 Minor Revisions

2 1) p12 line 8: "211H cell" should be MSTO-211H. 2) p12 line 8: Describe about NCI-H28 data in Figure 6 in this paragraph Many thanks for pointing out our mistakes. We corrected cell name as the reviewer indicated and describe the data of NCI-H28 in this paragraph. We also revised the part to make our point clear. (Before Revision) in page 12 line 8 There was no change in 211H cell, but ZOL treatment significantly suppressed viral propagation of Ad-delE1B55. The enhanced cell death by the combination was not thereby due to increased production of infectious Ad progenies on MSTO-211H cells, but could be linked with a new mechanism by ZOL that could influence Ad-delE1B55 replication on mesothelioma. (After Revision) in page 18 line 9-13 The viral production in MSTO-211H cells remained unchanged except ZOL-treated cells at 72 hrs which decreased the production. In contrast, ZOL treatments in NCI-H28 cells augmented the viral propagations. Enhanced cytotoxicity by the combination in MSTO-211H cells were thereby irrelevant to production of infectious Ad progenies, whereas that in NCI-H28 cells could be attributable to the enhanced production of viral progenies. Referee 3 Major Points: 1. The synergistic effects of the combination treatment (ZOL + Ad-delE1B55) is questionable. There is a dramatic discrepancy between the effect of the adenovirus treatment alone shown in figure 1B and the effect shown in figure 2A. In the first figure a dose of 5x104 causes almost nearly 100% cell death, while in Fig2B that dose causes only 40% cell death. It seems that different culture conditions may introduce variables to the results and alter the sensitivity to these

3 reagents, obscuring the true efficacy of the treatments. Therefore experiments should be repeated using uniform conditions to be able to draw more solid conclusions. Moreover while a combinatorial effect of the two treatments is observed in Fig 2A, the effect is not evident when looking at the survival data shown in Fig 2B where, in both NCI-H226 and NCI-H28, the addition of ZOL does not seem to improve the already profound effect of the Adenovirus. The reviewer referred some discrepancy of Ad-induced cytotoxicity between Figure 1B and Figure 2A. We found that Ad-delE1B55 doses in MSTO-211H and NCI-H226 cells (as well as in NCI-H28 cells with a topological error), and ZOL concentrations in NCI-H226 and NCI-H28 cells were erroneously described in Figure 2A. We are terribly sorry for our mistakes. We therefore corrected Figure 2A. We also tested the cytotoxicity of Ad-delE1B55 to MSTO-211H and NCI-H226 cells at lower vp doses (shown in revised Figure 1B) to match Ad-delE1B55 ranges used in Figure 2A. We presume that these corrections in Figure 2A and additional experiments in Figure 1B solved the inconsistent susceptibility of these cells to Ad-delE1B55. In addition, we conducted the Figure 2A experiments 3 times and obtained similar data. We used CalcuSyn software since it is widely used to analyze interactions between 2 agents. As for Figure 2B is concerned, we repeated twice for revision and replaced with new data. Consequently, we showed in the revised Figure 2B that the combination produced greater cytotoxicity than a single agent with statistical significance. We also conducted cell cycle analysis to demonstrate that both agents in the combination produced greater effects than a single agent. We cannot use the CalcuSyn software for cell proliferation test (Figure 2B) or cell cycle analysis (Figure 3, Table 1 and 2), and a possible interactions between the two agents can be tested only with the WST assay. We presume under the experimental conditions that the two agents produced combinatory effects, additive and at least partly synergistic effects. We therefore described combinatory effects or additive or synergistic effects rather than simply synergistic effects in the revised manuscript wherever appropriate. Ad doses and drug concentrations were not completely identical in respective experiments since we needed to modify the optimal conditions in these experiments as follows. In NCI-H226 and NCI-H28 cells, ZOL concentrations used in cell proliferation and cell cycle were higher than those in the WST assay since induction of cell death in a short period needs relatively high concentrations. In contrast, a high ZOL concentrations were too toxic to MSTO-211H cells for proliferation and cell cycle, and we used a relative low concentrations. We explained the differential use of ZOL in Results in page 12 line 16 page 13 line 5. In the combination study

4 with the WST assay, we used low doses of Ad-delE1B55 since high doses killed all the cells and left few cells killed by ZOL. In the revised manuscript, we replaced Figure 1B, 2A (for correction) and 2B and revised corresponding description in Results and Figure legends as shown in the highlighted portions. (Revised portions) Results section page 12 line 6-7, and page 12 line 11- page 13 line 11. Discussion page 20 line The synergistic effect observed in vitro is of questionable significance; therefore a true antitumor effect produced by the combination of the two agents should be validated with an in vivo model. We believe that the revised manuscript convinced the reviewer of the combinatory effects, additive or synergistic effects. The reviewer also mentioned an in vivo model, which makes the combinatory effects more complex. Many factors are involved in the anti-tumor effects including anti-angiogenesis, immune responses such as sensitivity to natural killer cells in nude mice (the cell lines used in the present study are human tumors), and interactions of tumors with stroma in the vicinity of the tumors. In that sense, anti-tumor effects produced in animal experiments does not directly indicate a possible combination of two agents in the strict sense. We presume that cell cycle analysis and cell proliferation can be a more direct way to show the combinatory antitumor effects. We also used combinatory effects or additive or synergistic effects rather than synergistic effects in the revised manuscript since we did not show an evidence of synergism except the WST assay with a limited Fa points (see reply to No1 comment above). 3. Considering the proposed application of a replication-competent virus, the reviewer is concerned about the complete lack of explanation for the clinical application of this therapeutic strategy.

5 The reviewer seems to be negative for a clinical application with replication-competent viruses. However, US FDA already approved replication-competent herpesviruses (talimogene laherparepvec) for melanoma patients. In addition, Ad-delE1B55 has already approved in China for head and neck cancer. We added some clinical aspects about a possible therapy with replication-competent Ad with a molecular target agent in Discussion section (page 24 line 17- page 25 line 4). (After Revision) We added the followings. Ad-delE1B55 has been approved in China as an anti-cancer agent for head and neck cancer [27] and ZOL is used in a clinical setting in many countries. We has also started a phase I clinical trial with an intrapleural injection of ZOL for mesothelioma patients, which can maintains a local concentration of ZOL to achieve cytotoxicity [28]. The present results under these circumstances will be one of the preclinical or relevant studies for a future clinical trial with replicationcompetent Ad and an inhibitor for small G proteins. 4. ZOL and Ad-delE1B55 effects are initially described in a few different cell lines, while later on, the authors focus mainly one (MSTO-211H) or sometimes two cell lines (MSTO-211H and NCI-H28) to describe the mechanism. Unfortunately the behavior of the chosen lines upon single and combination treatments is very different, making difficult to draw any conclusion about the possible mechanism for the observed cytotoxic effect of the combination treatments. A more extensive validation should be performed in other MM lines to understand what is the predominant mechanism of action, or to understand the molecular determinants that drive the same response through different mechanism in different cell lines. Many thanks for pointing out an important issue. We revised the manuscript to make our points clear. We used 5 cell lines in Figure 1A and 1B, all of which are wild-type p53 in the genotype. However, NCI-H2452 cells have truncated p53 and EHMES-10 cells were resistant to AddelE1B55. Moreover, nutrin-3a, which augments endogenous p53 expression through inhibiting MDM2 functions, induced cell death in MSTO-211H, NCI-H28, NCI-H226 and NCI-H28 cells, but not in EHMES-10 and NCI-H2452 cells (the data not included in the manuscript). We thereby did not investigate both EHMES-10 and NCI-H2452 cells from the standpoint of a

6 possible p53 involvement in the combination. We mentioned the above in Results section (page 12 line 6-10). We also did not analyze cell cycle with NCI-H226 cells since we always detected a high rate of sub-g1 populations in the cells even without any treatments, which makes us difficult precisely estimate increased sub-g1 populations in the combination. We mention this in Results section (page 13 line 15). We also believe that it is not unusual to focus on small numbers of cell lines in order to further investigate the mechanism underlying the phenomena even though one started to examine a wide variety of cells at the initial stage. Narrowing down cell numbers for further studies is rather a common way in a number of publications. The point in the manuscript is to demonstrate a possible combinatory use of Ad-delE1B55 and ZOL in human mesothelioma and the possible mechanisms of actions. The numbers of cell lines tested cannot determine what is predominant or a minor mechanism. At this point, we can concluded that at least 2 mechanisms are involved in the combination, augmentation of apoptosis and increased infectivity followed by up-regulated production of viral progenies. We therefore revised manuscript clearly mention the above mechanism (Results section page [Molecular changes induced by combination of ZOL and Ad-delE1B55] and page [Effects of ZOL on viral proliferations of Ad-delE1B55] as well as increased sub-g1 fraction in page [Cell cycle changes induced by ZOL and Ad-delE1B55]) with additional figures (Figure 4, 7B, and Supplementary Figure 4) and tables (Table 3 and Supplementary Table 1). 5. The study describes a few different mechanisms to explain the cytotoxic effect of the combination treatment, including apoptosis, S-phase arrest, phosphorylation of p53, and induction of viral replication. The apoptotic phenotype shown with the PI staining is confirmed by detection of cleaved caspase 3 and PARP. However, the results shown for Caspase 8 and 9 are not convincing. Moreover decrease of the pro-apoptotic Bid protein is shown. The authors claim that this is not followed by an increase in tbid, but this data is not shown. In the overall context of induction of apoptosis, the latter results do not fit. In Fig4, ZOL is shown to slightly enhance the adenovirus-induced phosphorylation of p53, at one of the time points. In discussion the authors conclude that the data presented "suggest a crucial role of p53 induction in the combination mediated cytotoxicity". However in another previous publication the same authors indicated while ZOL induces p53 and p53 phosphorylation in MM cell, ZOL pro-apoptotic effects were independent on p53. Genetic knock-down of p53 should be performed in combination with the treatments to validate the conclusion drawn here.

7 The reviewer raised several points and these were not convincing. (A) Caspase-8 and -9 data in Figure 5B (in Figure 4B in the original manuscript): Cleavage of both caspase-8 and -9 was not significantly enhanced in the combination but the cleavage of caspase-3 at 24 and 48 hrs and that of PARP at hrs was augmented in the combination. Both caspase-3 and PARP were mapped in downstream pathways of caspase-8 and -9. We therefore can conclude that caspase-mediated apoptosis contributed to the combinatory cytotoxicity. We also presume that cleavage of caspase-8 in the combination and that of caspase- 9 in Ad and ZOL-treatments were clearly detected. (B) tbid expression: Thank you for the reviewer s comment. We used anti-bit Ab which could detect t-bid as well (mentioned in Materials section). We therefore indicated the t-bid site in Figure 5B. (C) Genetic knock-down of p53: We previously shown that ZOL augmented up-regulated p53 and consequently activates the downstream pathways. However, knock-down of p53 levels with p53-sirna but did not influence ZOL-mediated cytotoxicity. These data indicated that ZOL surely augments p53 levels, perhaps due to DNA damages (as evidenced by augmented phosphorylated H2AX level in Figure 5B), but the cytotoxicity itself was not p53-dependent. On the other hand, we previously demonstrated that Ad-delE1B55 as well as Ad-p53 (replicationcompetent Ad expressing wild-type p53) (Cancer Gene Ther 19: , 2012) augmented p53 levels in p14/p16-defective and p53-wild-type mesothelioma and induced apoptosis in these cells. We therefore presume that ZOL-mediated augmentation of p53 contributed to the p53 activation-induced cell death. In fact, our previous study showed that combination of ZOL and cisplatin produced synergistic cytotoxicity in the mesothelioma (Reference 11). In that study, we showed that both ZOL and cisplatin augmented p53 levels and that knock-down of p53 levels with the sirna depleted the combinatory cytotoxic effects. These data indicate that ZOLinduced p53 is functionally active and contributes to cell death through up-regulated p53. Therefore, we believe it acceptable to mention, "suggest (but not conclude) a crucial role of p53 induction in the combination mediated cytotoxicity", in Discussion section even without including p53-sirna experiments in the manuscript. We revised the corresponding part and described more in detail to avoid unnecessary misunderstanding, referring a role of p53 in the combinatory cytotoxicity of ZOL and Ad-delE1B55 (see Discussion page 21 line 8-16 and page 24 line 2-16).

8 6. Other signaling pathways are not thoroughly addressed and/or the results are not carefully pondered. E.g. Beclin seem to be decreased by all treatment at 24 and 48 ours, but this data is not discussed. The addition of Ad-delE1B55 seems to inhibit the LC3A/B induced by ZOL, but this result is not addressed. Thank you for the comments about these points. We revised our description on western blot analysis and mentioned reduced Beclin-1 by Ad-delE1B55, ZOL and the combination at 24 and 48hrs, and Ad-del E1B55 at 72 and 96 hrs, Ad-delE1B55-mediated suppression of ZOLmediated LC3A/B induction in Results section (page 15 line 8-10, line 16-17). We mentioned a possible involvement of autophagy in page 15 line 17-page 16 line Material and Methods should be more carefully described as many major details are missing, e.g. the dose of ZOL and Adenovirus used to produce the data shown in Fig 2B is not mentioned. The gating strategy to produce the cell cycle data is not shown, nor explained. This is important, as the presence of doublets may compromise cell population accounted as hyperploid. Thank you for the comments. We added dose information of ZOL and Ad-delE1B55 in the figure legends and revised Methods sections. We also confirmed these doses in all the experiments were described. We showed gating data for cell cycle analysis in Supplementary Figure 2. The data clearly showed that we ruled out the presence of doublets (page 9 line 7-8). Consequently, the hyperploid populations were not due to the doublets. 8. Authors described induction of hyperploidy and S-phase fraction. To better characterize this phenomenon, the authors should use PI staining in combination with BrdU. The reviewer suggested to characterize hyperploidy and S-phase with other methods. We conducted western blot analysis to detect cyclin A and E expression instead of BrdU (antibody against BrdU was unfortunately not good for S-phase defection). Cell cycle analyses showed that NCI-H28 cells increased S-phase fractions in ZOL- and Ad-delE1B55-treated cells in particular

9 at 48 hrs. We detected decrease of cyclin E and increase of cyclin A in the treated cells (shown in Supplementary Figure 3). These molecular analysis supported the increased S-phase fractions detected with cell cycle analysis. We also conducted Giemsa staining to demonstrate the hyperploidy in cells infected with Ad-delE1B55 but not treated with ZOL. The staining showed increased DNA contents followed by pyknotic changes in Ad-delE1B55 but not ZOL-treated cells (in new Figure 4). The new data indicated that induction of hyperploid fraction and subsequent cell death. We mentioned the western blot analysis (page 14 line 5-12) and Giemsa staining data (page 14 line 15-page 15 line 2) in Results section. 9. Authors describe effect of ZOL on integrin expression. The rationale is not addressed, the flow cytometry data are not quantified and the results are questionable: the induction of integrin αvβ5 by ZOL treatment in MSTO does not seem any greater than that of integrin αvβ3, however the authors conclude that αvβ 5 only is induced in MSTO and inhibited in NCI-H28. The results are inconclusive. We prepared Table 3 and Supplementary Table 1 to quantitate the staining profiles with different doses and compared the mean fluorescence intensity between untreated and ZOL-treated cells. The Table 3 showed that mean fluorescence intensity was different among the molecules and between the cells. We also showed relative changes of the expression after the ZOL treatment. The Supplementary Table 1 indicated dose-dependent decrease of integrin expressions. These data indicated that ZOL decreased both αvβ3 and αvβ5 expression levels in NCI-H28 cells with a dose dependent manner, while CAR expression levels remained almost unchanged. MSTO-211H cells were however sensitive to ZOL at a high dose, which prevent us to examine ZOL-mediated effects further. We revised the corresponding parts in Results (page 17 line 6-13) and Discussion (page 22 line 10-page 23 line12) sections to make more these point clear. 10. Authors show an interesting enhancement in the viral replication upon ZOL treatment in NCI-H28. This is the most significant result shown in this study. The potential mechanism for this is briefly hypothesized in the discussion. Investigation of the possible mechanisms leading to this result should be pursued.

10 Viral production is influenced by many factors. In the present study we showed that ZOL treatments increased production of viral particles in NCI-H28 but not in MSTO-211H cells. We also added additional data in new Figure 7B, which demonstrated that ZOL enhanced expression of E1A, one of the early gene product, and hexon, a structure protein expressed as a late gene product, in NCI-H28 cells but rather decreased in METO-211H cells. The additional data supported that viral replication was enhanced in NCI-H28 cells but not in MSTO-211H cells. We thereby discussed possible reasons in Discussion as follows. (1) Increased infectivity: Figure 6B showed that ZOL increased infectivity of Ad in NCI-H28 but not in MSTO-211H cells, and a new Supplementary Figure 4 showed that increased infectivity in NCI-H28 cells was dose-dependent of ZOL. Figure 7A showed that ZOL increased production of Ad progenies in NCI-H28 cells but not in MSTO-211H cells. We consequently presume that increased viral infection augmented viral production accordingly. (2) Differential cell cycle changes: Cell cycle distribution data showed that the combination of Ad-delE1B55 and ZOL induced sub-g1 populations in MSTO-211H cells and S-phase in NCI- 211H. Induction of apoptosis during the viral proliferation is one of the negative factors for virus production since early cell death terminates the production. In contrast, viral replication is active during S-phase since the viral replication correlates with DNA synthesis in host cells. A new Figure 7B demonstrated that both E1A and hexon expression levels were enhanced after ZOL treatments in NCI-H28 but decreased in MSTO-211H cells, which was correlated with increased viral production in NCI-H28 but not in MSTO-211H cells and with enhanced apoptosis in MSTO-211H cells. In fact, MSTO-211H cells up-regulated apoptosis-linked molecules in western blot analysis such as cleavage of caspases. These data collectively indicated that combinatory effects in MSTO-211H cells were attributable to apoptotic cell death and NCI-211H cells to enhanced virus replication. We mentioned these data in Results sections and in Discuss sections (page 20 line 4-6, page 24 line 2-16) 11. The manuscript contains many language and grammar mistakes, which impair the comprehension of the study rationale, and of the result explanation and discussion, and should be revised.

11 Numerous grammatical, stylistic and lexical changes were made with the assistance of a native editor of scientific publications. We also revised the manuscript in Results and Discussion sections to improve the quality. We do hope that the revised manuscript is satisfactory. Minor Points 1. Authors seem to indicate that the error bars represent the SD of technical triplicates performed in a single experiment. This is acceptable only if the experiments have been repeated multiple times and the authors clearly indicate that the results shown are a representation of n. experiment preformed. We conducted experiment in general 3 times in most of the data and showed representative data. We indicated the numbers of experiments in Figure legends whenever possible. 2. The discrepancy in the observed behavior of the two lines may be due to their intrinsic differences in the genetic makeup: e.g. the author mentions that most MM are p53 wt but luck of CDKN2A. While it is mentioned that the cell lines used in this study are p53 wt, no direct evidence of the p53 and CDKN2A status of these lines is reported here, nor a reference to a publication, which addressed this question, is listed. We added the data to show lack of p14 and p16 gene or the expression in Supplementary Figure 1. We sequenced the p53 gene of the cell lines and mentioned the data in Methods sections (page 8 line 8-11).

Supplementary Figure 1: Overexpression of EBV-encoded proteins Western blot analysis of the expression levels of EBV-encoded latency III proteins in

Supplementary Figure 1: Overexpression of EBV-encoded proteins Western blot analysis of the expression levels of EBV-encoded latency III proteins in BL2 cells. The Ponceau S staining of the membranes or