Review Reports - General Direct Anticancer Effects of Deer Growing Antler Extract in Several Tumour Cell Lines, and Immune System-Mediated Effects in Xenograft Glioblastoma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is really interesting that extraction deer velvet antler extraction（DVA）can inhibit the proliferation of tumor cells without affecting normal cells. In particular, in vivo experiments demonstrated amazing anti-tumor effects.The authors carried out a large number of experiments with plausible results.But there are still some details to be improved.

1) a detailed description of the Tip and Mid of the antler, or a diagrammed should be given.

2) Figure1, Please standardize GB or GBM for glioblastoma.

3) In Figure 2, Only graphs of statistical results are shown, and it is recommended that the results of the cell migration experiments were shown in complementary figures

4) Line316, need for clearer statements;

5) Figure 4, has a long figure legends, but I still missed the point;

6) Figure5,In addition to the statistical graphs, the actual graphs of the tumors should also be shown;

Author Response

Reviewer 1.

1) a detailed description of the Tip and Mid of the antler, or a diagrammed should be given.

Authors: We have added a detailed explanation in lines 153-163:

“Then, they were divided into portions according to the distance from the extreme of each antler (main beam, bez tine and brow tine were developed with 60 days of age). Tip is considered in every antler as 2.5 cm section at the top. Then, middle portions are considered 5 cm below the tip section, obtaining 2 or 3 Mid sections depending on the deer age. Only the first Mid-section (2.5-7.5 cm from the top) has been used in this study. The Mid portion used in this study has a functional difference with the rest of the antler: according to Muir et al. [35], the transition from mineralized cartilage to trabecular bone occurs in a discrete band located from 5.0 to 7.5 cm below the antler tip (i.e., our mid section). Each portion was separated, and powder was obtained through a grinding process in ball mills until particles were less than 0.18 mm.”

Reviewer 1. 2) Figure1, Please standardize GB or GBM for glioblastoma.

Authors: Page 7, line 321. Figure 1 has been standardised as GBM.

Reviewer 1. 3) In Figure 2, Only graphs of statistical results are shown, and it is recommended that the results of the cell migration experiments were shown in complementary figures.

Authors: The images of the cell migration experiments are shown in supplementary Fig. 2: “Figure S2. Images of Scratch assay”

Reviewer 1. 4) Line 316, need for clearer statements. The reviewer refers likely to this sentence:

“In all cases, the addition of the supernatant of differentiated M1 or M2 macrophages to the U87 reduced the viability further than the use of DVA alone. There was no further reduction of viability when M1 or M2 was added with DVA (only a non-significant trend from 40.6% to 45.1% in M1 at 48 h, whereas a significant slight increase in viability was found for M2 both in 24 and 48 h).”

Authors: We have now increased the clarity of the sentence and this is now:

“In all cases, the addition of the supernatant of differentiated M1 or M2 macrophages to the U87 reduced the viability further than the use of DVA alone. As the media is more than enough to nurture cells for 72h, the reduction in viability of U87 must be produced by some factor released by M1 or M2 during their growth, which affect the viability more than DVA can. The addition of DVA to the supernantant of M1 or M2 did not reduce the viability of U87 further (only a non-significant trend from 40.6% to 45.1% in M1 at 48 h, whereas a significant slight increase in viability was found for M2 both in 24 and 48 h).”

Reviewer 1. 5) Figure 4, has a long figure legends, but I still missed the point.

Authors: Science is an art, and as such, each one of us has its own style. The main writer of this paper likes to write very clearly and long legends, but the referee is right that it was not clear. Now the legend is longer, but much clearer than before. We could cut it if the referee or the editor wants, but it gives now all the information an intelligent but naïve reader needs to understand the experiment (which, by the way, did not go the way we expected). The legend is now:

“Figure 4. Modulation of macrophages M1 (pro-inflammatory, anticancer, top) and M2 (tol-erant and protective of tumour, bottom) effect by DVA in assays with glioblastoma. The bars show the viability of GBM line U87 alone (control), treated with DVA-TIP (1 mg/mL), treated with su-pernatant of macrophage M1 or M2, and the macrophage supernatant with DVA. The reduction in viability of adding the culture media (supernatant) of macrophages M1 or M2 could be produced by some factor released by M1 or M2, or by a reduction in nutrients caused by macrophage growth. However, as the media is more than enough to nurture cells for 72h, the reduction in viability of U87 must be produced by some factor released by M1 or M2. It was expected than M2, inducing tol-erance to tumour growth, may counteract the viability reduction of DVA. (a, b) M1 and (c, d) M2 are differentiated from THP-1 after treatment with PMA towards M0, then polarized towards M1 (LPS) or M2 (IL4 and IL13). The graphic on the left shows the effects at 24 h, whereas the one on the right is at 48 h. Data are shown as mean value ± SD. The brackets show the probability indicated of a t-test comparing U87 (left end of bracket) with treatment at the other end of the bracket (p < 0.001 in all cases). Despite the further reduction in viability of adding DVA-TIP to M1 supernatant at 48 h (compared to M1 supernatant alone: bar next to the left), the test between them did not achieve significance. Error lines show SD. The probability indicated by *, **, and *** corresponds to a t-test at levels p < 0.05 , p < 0.01 and p < 0.001. Asterisks on top of bars show differences with control (C), whereas those above brackets indicate the significance of differences between the bars at both ends of the bracket.”

Reviewer 1. 6) Figure 5. In addition to the statistical graphs, the actual graphs of the tumours should also be shown.

Authors: The referee probably misunderstood that we measured tumour weight, not tumour volume. Tumour volume can be estimated by measuring it in the live animal, but tumour weight can only be weighed when the animal is killed (i.e. only once). Some papers, such as Tang et al. (2018: https://doi.org/10.1080/09168451.2018.1537775), examined tumour volume as this: “Tumor sizes were monitored every two days using digital calipers, and the tumor volumes were calculated according to the formula L × S2 × 0.5, where L and S represent the tumor’s longest and shortest diameter, respectively”.

However, we thought that showing the time change of tumour volume does not bring any additional substantial information over that of the final weight: at the end of the 28 day treatment, there is a 61-66% reduction in weight.

In any case, as we did not measure the volume, we cannot add now this information. We hope both the referee and the editor understand that the figure with the tumour weight provides sufficient and substantial information on the effectivity of the treatments as compared to the control.

What we have added in response to referee comment is the photographs of the tumours for readers to assess the aspect and size rather than simply a graphic. This has been added as Figure S3 (Images of xenograft tumours) in the supplementary figures.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, here are my comments to your very extensive and interesting work:

Please, work on the abstract - this is the weakest part of the manuscript which does not show the multitude of studies that are performed in antlers.

-in the abstract, please, show the full names of the cell lines that were used for the study

- in the abstract add the percentage of spleen weight reduction

- in the abstract mention that it is deer antler that is being tested

For the remaining parts of the mansucript

-please, mention what were the quantities of the dried residue obtained after the evaporation of water from the water extract (the mass of the liophyllized powder) for different parts of the tested antlers.

- please, enrich the discussion and conclusion sections in some thoughts about the sourcing possibilities. is it possible to obtain sufficient quantity of antlers for introducing them to therapeutical strategies?

- what is the efficiency of extraction process...how much extract/lipophyllized powder can we obtain from one animal and which possibilities it gives us for the treatment?

other minor comments:

- the title should not be written in capital letters

- section 2.9. - remove the word ' figure' from the title of the section

Comments on the Quality of English Language

the language is fine, however the abstract should be carefully checked again

Author Response

Reviewer 2.

Comments and Suggestions for Authors

Dear Authors, here are my comments to your very extensive and interesting work:

Reviewer 2. Please, work on the abstract. This is the weakest part of the manuscript which does not show the multitude of studies that are performed in antlers.

-in the abstract, please, show the full names of the cell lines that were used for the study

- in the abstract add the percentage of spleen weight reduction

- in the abstract mention that it is deer antler that is being tested.

Authors: Actually, the abstract is the part of the MS which has been most intensely worked. A previous version of 250 words had to be polished again and again to reduce it below the 200 words allowed in Pharmaceutics. It is extremely difficult to reduce an abstract so much without a significant loss of important information. Because we left it in 167 words, we have now been able to increase the word count to 195 adding the details that the referee asks us. As we still had a few words left, we indicated at the end that “DVA may contain a future wide anticancer medicine without secondary effects” (this is what we foresee as the end objective of this whole research line).

The abstract now is:

Deer antler is the fastest growing tissue. Because is based on proto-oncogenes, to avoid the risk of cancer, antlers evolved strong anticancer mechanisms, and thus its extract (DVA) is effective also against the few human tumours studied so far. We assessed whether DVA is a general anticancer compound testing direct effects in cells of different tumours: glioblastoma (GBM; lines U87MG and U251), colorectal (CRC; lines DLD-1, HT-29, SW480 and SW620), breast cancer (BRCA; lines MCF7, SKBR3 and PA00), and leukaemia (THP-1). DVA reduced viability of tumour but not healthy cells (NHC; lines 293T and HaCaT). Mobility decreased at least at the longest test (72 h). Intraperitoneal/oral 200 mg DVA/kg administration in GBM xenograft mice for 28 d reduced tumour weight 66.3% and 61.4% respectively, and also spleen weight (43.8%). In addition, tumours treated with DVA showed symptoms of liquefactive necrosis. Serum cytokines showed DVA up-regulated factors related with tumour fighting and down-regulated those related with inducing immune tolerance to the tumour. DVA shows general anticancer effects in the lines tested and, in GBM mice, also strong indirect effects apparently mediated by the immune system. DVA may contain a future wide anticancer medicine without secondary effects.

For the remaining parts of the manuscript

Reviewer 2. Please, mention what were the quantities of the dried residue obtained after the evaporation of water from the water extract (the mass of the liophyllized powder) for different parts of the tested antlers.

Authors: We have now added the percentage of weight loss due to water evaporation during freeze-drying in line 163. The new sentence is this:

“The weight loss after freeze-drying (i.e. corresponding to the percentage of water in the fresh sample) was 78.4 ± 2.5 (%) and 74.4 ± 2.7 (%), respectively, for the tip and first 5- cm MID section of the main beam, and 77.9 ± 1.2 (%) and 71.6 ± 1.5 (%) for the tip and first 5- cm MID sections of first and second tine of the antler.”

Reviewer 2. Please, enrich the discussion and conclusion sections in some thoughts about the sourcing possibilities. is it possible to obtain sufficient quantity of antlers for introducing them to therapeutical strategies?

Authors: following the suggestion of the referee, we have now ended the discussion with a paragraph on the potential of DVA or some of its molecules as a future medicine against cancer. We thank him for this suggestion as this enriches the discussion showing its potential applicability and clarifying the aims of this research line: this is not to assess if DVA could become a medicine (it does not comply with the requirements of a modern medicine consisting of one or few principles fully characterised and standardised in composition), but to point that one or more biomolecules having anticancer properties within DVA could become a medicine in the future (not extracted from DVA, but synthesized by a pharmaceutical company).

The paragraph added (at the end of discussion) is this:

“The potential of DVA or its molecules as a future medicine against cancer.

DVA has not the properties required to become a modern medicine, as it is a complex mixture of molecules, some of which are active against cancer, whereas minerals and other molecules may not have effects on health. Furthermore, as a biological extract, it is variable in composition depending on some factors we know affect it (stage of growth, sections of the antler used to create the extract, age of the deer, and probably even size of the antler), whereas others we do not know (there appears to be inter-individual differences in the efficiency of the anticancer effect). A medicine should have one or few active principles of fully known effects (both positive on health as well as its secondary effects), and its content per pill or dose should be clearly defined and without variability. Thus, the final aim of this research line should be to find such molecule or small set of molecules within DVA that have anticancer properties to develop a future medicine that may have wide anticancer activity without secondary effects. What are the candidates for such molecule? According to Sunwoo et al. [52], the tip of the growing antler is composed mostly of protein (69%), followed by lipids (19%) and most of the rest is ash (minerals). The most likely candidate for active molecule based on the fact that is the most abundant, as well as soluble in water (the solvent most often used) is a protein. However, other type of molecules cannot be discarded, as a minor component may be very powerful against cancer despite being in small quantities. Studies assessing anticancer properties have focused on proteins. Thus, Cao et al. [17] isolated from the velvet antler by-product of antler wine a 23.088 kDa protein that had the anticancer and immune system promoting effects reported in this study. In a rather coarser purification method, Yang et al. [11] extracted with 50% water/ethanol solvent from DVA powder a set of proteins ranging from 250 kDa to 35 kDa that they called Sika Deer Antler Protein. The 23 kDa protein of Cao et al.’s [17] appears to be outside this range, but it should be noted that in the first case the species is the red deer, and in the second, it is the sika deer. Equally, the study by Li et al. [15] was a set of soluble proteins of unknown size, although in this case they were dissolved in water”.

Reviewer 2. What is the efficiency of extraction process...how much extract/lipophyllized powder can we obtain from one animal and which possibilities it gives us for the treatment?

Authors: This seems to be very similar to the query above that we have responded with a new sentence in line 163. It is easy for the reader to figure out what is the powder remaining substracting 100% fresh weight minus the water loss. This is about 22-28%. And weight average for 60 days antlers are 35 and 120 g for the main beam (tip and mid1, respectively), 15 and 55 g for bez tine and 13 and 41 g for brow tine.

Reviewer 2. other minor comments:

- the title should not be written in capital letters

Authors: changed as suggested.

Reviewer 2. Section 2.9. - remove the word ' figure' from the title of the section.

Authors: “Figure 7” from the title of section 2.9 has been removed.

Comments on the Quality of English Language

the language is fine, however the abstract should be carefully checked again

Reviewer 3 Report

Comments and Suggestions for Authors

The work reports DVA extracts as anticancer. The investigated topic is interesting, and I suggest publication after revisions detailed below.

- Although the biological effects of DVA extracts on cells is deeply investigated, the DVA extract itself is not characterized. I suggest including some chemical information on the extract.

- Equations should be labeled in a sequential way. See line 188 where a label is missed.

- Fig 7 needs revision. It is impossible for me to read the labels. Please make it with larger size.

- Authors reported that DVA can lead to different activity depending on the antler section included. Apparently also the price and availability of the powder strongly depends on this. The chemical composition should be highlighted, and the different activities discussed. What is the actual active molecule? That is a key question that should be addressed.

Author Response

Reviewer 3.

Comments and Suggestions for Authors

The work reports DVA extracts as anticancer. The investigated topic is interesting, and I suggest publication after revisions detailed below.

Reviewer 3. Although the biological effects of DVA extracts on cells is deeply investigated, the DVA extract itself is not characterized. I suggest including some chemical information on the extract.

Authors: As mentioned in the comment to referee 2, we have added information regarding the composition of the growing antler (content in protein, lipids and ashes, and the evidence that it is a protein or set of water-soluble proteins that may be responsible for the anticancer effect. This is added to the end of the discussion.

Reviewer 3. Equations should be labeled in a sequential way. See line 188 where a label is missed.

Authors: Page 4, line now 190. We have corrected it and it is now: “The equation “Cell viability (%) = (Mean absorbance of the sample)/(Mean absorbance of the control)x100” has been included.”

Reviewer 3. Fig 7 needs revision. It is impossible for me to read the labels. Please make it with larger size.

Authors: The referee is completely right. Rather than having 2 small figures one at the side of the other as an image (therefore, being very small and impossible to read the labels), we have placed two separate images one on top of the other in a larger size. Not it is possible to read the labels clearly.

Reviewer 3. Authors reported that DVA can lead to different activity depending on the antler section included. Apparently also the price and availability of the powder strongly depends on this. The chemical composition should be highlighted, and the different activities discussed. What is the actual active molecule? That is a key question that should be addressed.

Authors: The referee is right in that the key question is to find the biomolecule (or set of molecules, as there might be more than one) that is responsible for the anticancer activity (and related effects such as promoting the response of the immune system towards the tumour). Actually, the importance of the studies assessing the anticancer activities of DVA is to develop a research line that will end up in finding this/these biomolecules which will be able to be produced by a pharmaceutical company to become a medicine for many if not all types of cancers.

As indicated in the comment above of referee 3, and another one on the same line by referee 2, we have added a paragraph in the discussion pointing to the evidence that the anticancer effects of the DVA may be produced by one or few proteins that are water soluble (but there may be other fractions soluble in non-polar solvent which also have anticancer properties).

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

All comments addressed