Co-Treatments of Edible Curcumin from Turmeric Rhizomes and Chemotherapeutic Drugs on Cytotoxicity and FLT3 Protein Expression in Leukemic Stem Cells

Round 1

Reviewer 1 Report

Some comments are given below: 1. The author should describe the dose of doxorubicin and idarubicin for clinical use in the Introduction. 2. The paragraph of Line 208-213 was suggested to be included in Introduction section. 3. The abstract should be rewritten and reorganized, because the novelty and value of present work were rare mentioned, and the expression of some sentences is inadequate. 4. The results of western blot in Figure 3 should be rechecked, because the band is blurred in the graph. 5. Please indicate the band of FLT3 exactly in all blots. 6. Conclusion should address the benefit for co-treatments of edible curcumin and chemotherapeutic drugs in cancer therapy.

Author Response

Reviewer #1

Some comments are given below:

1. The author should describe the dose of doxorubicin and idarubicin for clinical use in the Introduction.

Answer: The dose of doxorubicin and idarubicin for clinical use have been added in the introduction at line numbers 72-76 (track changes) with references 13-15.

2. The paragraph of Line 208-213 was suggested to be included in Introduction section.

Answer: The paragraph of line numbers 208-213 was moved to the introduction already at line numbers 110-115 (track changes), page number 3.

3. The abstract should be rewritten and reorganized, because the novelty and value of present work were rare mentioned, and the expression of some sentences is inadequate.

Answer: Abstract was rewritten with the novelty and value at line numbers 28-34 (track changes), page 1, “In summary, this study exhibits a novel report of Dox-Cur co-treatment in both enhancing cytotoxicity of Dox and inhibiting cell proliferation via FLT3 protein expression in leukemia stem cells and leukemic cells. This is the option of leukemia treatment with reducing side effects of chemotherapeutic drugs to leukemia patients.”.

4. The results of Western blot in Figure 3 should be rechecked, because the band is blurred in the graph.

Answer: Figure 3 has been changed.

5. Please indicate the band of FLT3 exactly in all blots.

Answer: Bands of FLT3 were indicated exactly already in all blots. Anti-mouse FLT3 (MERCK) stained both mature (155 kDa) and premature (130 kDa) forms of FLT3 protein.

6. Conclusion should address the benefit for co-treatments of edible curcumin and chemotherapeutic drugs in cancer therapy.

Answer: Conclusion was addressed at line numbers 595-599 (track changes), page 13-14, “This study is benefit for co-treatment of edible curcumin and chemotherapeutic drugs (Dox and Ida) in leukemia therapy due to decreasing doses and side effects of chemotherapeutic drugs. Adding of nontoxic doses of edible Cur in chemotherapeutic drugs can enhance cytotoxicity of Dox and suppress leukemic stem cell proliferation.”.

Reviewer 2 Report

                The manuscript entitled “Co-treatments of Edible Curcumin from Turmeric Rhizomes and Chemotherapeutic Drugs on Cytotoxicity and FLT3 Protein Expression in Leukemic Stem Cells” reports several studies regarding the possibility to enhance efficacy and reduce toxicity of the combination treatment of a drug and curcumin on leukemic stem cell and leukemic cell lines. In particular, doxorubicin-curcumin co-treatment showed additive effects in several conditions. The effect of this co-treatment in different case decreased FLT3 protein levels and total cell numbers in all the cell lines examined.

            Considering my opinion, several points must be addressed before a consideration of the article for its publication on “Molecules”.

 

Hereafter a list of points is indicated:

 

• The sentence “Previous studies reported that Cur exhibited a potent cytotoxic effect, induced…..” (lines 84-87) is supported only by references from the Authors themselves. Are there no other references in the literature?
• The authors should provide some information on the amount of chemotherapeutic agents used in vivo and the doses tested in the in vitro experiments performed.
• The authors used the same percentage of polyacrylamide to detect via Wester FLT3 protein and GAPDH ?
• What is the total protein content loaded in SDS-PAGE for Western? The same for each experiment shown? or different? The content should be appropriately mentioned in the legends
• The files molecules-1370288-non-published and molecules-1370288-supplementary are identical. The authors make it clear.
• At the line 541 in paragraph title “Synergistic effects of combination treatment”, what is the meaning of [41]?.

Author Response

Reviewer#2

1. The sentence “Previous studies reported that Cur exhibited a potent cytotoxic effect, induced…..” (lines 84-87) is supported only by references from the Authors themselves. Are there no other references in the literature?

Answer: Other references were added to support the previous studies already as follows.

27. Kong, Y.; Ma, W.; Liu, X.; Zu, Y.; Fu, Y.; Wu, N.; Liang, L.; Yao, L.; Efferth, T. Cytotoxic Activity of Curcumin towards CCRF-CEM Leukemia Cells and Its Effect on DNA Damage. Molecules 2009, 14, 5328-5338.
28. Seghetti, F.; Di Martino, R.M.C.; Catanzaro, E.; Bisi, A.; Gobbi, S.; Rampa, A.; Canonico, B.; Montanari, M.; Krysko, D.V.; Papa, S., et al. Curcumin-1,2,3-Triazole Conjugation for Targeting the Cancer Apoptosis Machinery. Molecules 2020, 25, 3066.
29. Gao, S.-m.; Yang, J.-j.; Chen, C.-q.; Chen, J.-j.; Ye, L.-p.; Wang, L.-y.; Wu, J.-b.; Xing, C.-y.; Yu, K. Pure curcumin decreases the expression of WT1 by upregulation of miR-15a and miR-16-1 in leukemic cells. J. Exp. Clin. Cancer Res. 2012, 31, 1-9.

2. The authors should provide some information on the amount of chemotherapeutic agents used in vivo and the doses tested in the in vitro experiments performed.

Answer: Information on the amount of chemotherapeutic agents used in vivo and the doses tested in the in vitro experiments were added at line numbers 410-416 (tracking changes) of discussion section, page 10, “Ida and Dox doses in a previous report in vitro were 1-100 ng/mL and 0.1-1.5 mg/mL, respectively in normal and leukemic human bone marrow progenitors [42]. Drug doses were within the range of our studies. However, doses used in this study showed at low levels when compared to the previous report. Intravenous injections of Ida and Dox in rat model, the maximum tolerated doses of both drugs were 3 mg/kg and 0.75 mg/kg per injection. Moreover, the cardiac toxicity of Ida remained significantly lower than that of Dox [43].”.

3. The authors used the same percentage of polyacrylamide to detect via Wester FLT3 protein and GAPDH ?

Answer: Percentage of polyacrylamide for FLT3 protein and GAPDH detections was 7.5% SDS PAGE as shown at line number 573, page 13. After transferring proteins from gel to PVDF membrane of blotting process, PVDF membrane was cut to separate FLT3 (target protein) and GAPDH and stained with rabbit polyclonal anti-FLT3 at a dilution 1:1,000, whereas the part containing GAPDH was probed with rabbit polyclonal anti-GAPDH antibody at a dilution of 1:1,000.

4. What is the total protein content loaded in SDS-PAGE for Western? The same for each experiment shown? or different? The content should be appropriately mentioned in the legends

Answer: Total proteins were the same for each experiment (80 mg/lane). The content was added in all legends of protein Western blotting as shown at line number 262, page 6; line number 287, page 7; line number 306, page 8/ line number 398, page 10.

5. The files molecules-1370288-non-published and molecules-1370288-supplementary are identical. The authors make it clear.

Answer: Thank you very much

6. At the line 541 in paragraph title “Synergistic effects of combination treatment”, what is the meaning of [41]?.

Answer: Reference number 41 (or 49 after revision) was moved to line number 554, page 13.