For Reviewer 1
1. We have further demonstrated our findings in MDA-MB-231 cell, whose ER status differs
from MCF-7. The related results were revised in our manuscript.
2. Considering only FT3 can enter into the cells to exert the effect of metabolism adjustment,
while T3 is combined with globulin, therefore we take the physiological concentration range of
FT3 as the reference standard, the normal FT3 concentration in human body is 2.2-4.2 ng/ml.
Hence, 4 ng/ml T3 was used firstly in cell culture medium which contains no thyroid hormones,
to evaluate whether the physiological concentration of T3 can elevate the chemotherapeutic
efficacy. The tumor cells in culture medium without T3 is similar to the breast cancer patients
undergoing hypothyroidism during chemotherapy, for those tumor cells added with 4 ng/ml T3,
this can be seen as we restore the thyroid function of breast cancer patients who underwent
hypothyroidism during chemotherapy.
In our hypotheses, we resume the better chemotherapeutic efficacy of choriocarninoma
patients is due to the hyperthyroid function during chemotherapy. To approve this hypotheses,
we use different concentrations of T3 combined with chemotherapy to treat breast cancer cells,
in order to simulate the hyperthyroid function. This can explain why we use 8, 12, 16, 20, 24,
28, 32 ng/ml T3, which are all higher than the physiological concentration (4 ng/ml). Gradually
elevating the concentration of T3 is to evaluate the chemotherapeutic efficacy of different
usage modes of T3, to clarify whether treating breast cancer cells with higher concentration of
T3 but with shorter time can also achieve chemosensitization efficacy.
3. NTIS means lower levels of T3 and/or lower T4, we reported that 82% of the breast cancer
patients suffered NTIS during chemotherapy, the remaining 18% of patients didn’t suffer
hypothyroid function during chemotherapy, so they are not the relevant study population. We
just consider those patients who have hypothyroid function during chemotherapy as the
relevant study population, aiming to discuss whether elevating the thyroid hormones can make
these patients have a better chemotherapeutic efficacy. Therefore, according to the high
prevalence of NTIS during chemotherapy, we should realize that not only the thyroid diseases
can influence the thyroid function in breast cancer patients, but also the chemotherapy will
effect on the thyroid function. Basing on the basic results in this paper, we think the
chemotherapeutic efficacy partly depends on the thyroid function, so checking the thyroid
function in breast cancer patients is important for elevating chemotherapeutic efficacy.
Taxol works differently by arresting the breast cancer cells in G2/M phase, in addition, we
also know that taxol enhances the polymerization of tubulin to stable microtubules and also
interacts directly with microtubules, stabilizing them against depolymerization by cold and
calcium, which readily depolymerize normal microtubules, so cells treated with taxol are
unable to form a normal mitotic apparatus. In short, taxol significantly inhibits the cell mitosis,
this explains why taxol arrest cancer cells in G2/M phase. For thyroid hormones, there are
large amounts of literatures proving that T3 can promote cell proliferation, in gastric cancer
cells, flow cytometric analysis indicated that the growth stimulatory effect was mainly due to
shortening of G1 phase accompanied by increases in S and G2/M phases of the cell cycle.
Therefore, we thought breast cancer cells stimulated by thyroid hormones are also sensitive to
taxol, because thyroid hormones can promote breast cancer cells proliferation, cells entering
into G2/M phase is included in proliferation.
4. Originally, we thought thyroid hormones promoting breast cancer cells proliferation is
acknowledged, so we didn’t provide the possible mechanisms of T3 action in breast cancer
cells. In MCF-7, an estrogen receptor (ER)-positive breast cancer cell line, the proliferative
effect of thyroid hormone is not only initiated through the combination with the thyroid
hormone receptor, but also through the combination with the estrogen receptor. In
MDA-MB-231, which also expresses thyroid hormone receptor (TRα, TRβ), we think thyroid
hormone can combine with thyroid hormone receptor, to sensitize the mitochondrial pathway
to enhance chemotherapeutic efficacy[1]. As mentioned in the following figure, which is cited
from Dinda’s paper, thyroid hormones not only enhance the phosphorylation of pRb, but also
induce the c-Myc expression and mutant P53 expression[2], this can partly explains the cell
proliferation efficacy of thyroid hormone. MDA-MB-231 is acknowledged harboring P53
mutations[3], which further demonstrates that thyroid hormone may promote cell proliferation
in MDA-MB-231. There are specific mechanisms that explain why thyroid hormone stimulate
cell proliferation in gastric cancer cells, the cyclin/cyclin-dependent kinase levels and activity
are involved[4]. At present, we are still performing the experiments to illustrate the
mechanisms of T3 action in breast cancer cells, the results need to be further demonstrated, we
will provide them as soon as possible.
According to the details mentioned above, we think T3 action observed in this study is
directly correlated with the chemotherapeutic efficacy and not just a coincidental result.
Moreover, we discussed the influence of different concentrations of T3 on chemotherapeutic
efficacy, which further demonstrates T3 action is directly correlated with the chemotherapeutic
efficacy in this study.
5. The comparison of thyroid function between patients of breast cancer and breast benign lesion
at initial diagnosis (Table 1) was performed with Student’s t test. The comparison of thyroid
function between postchemotherapy and prechemotherapy (Table 2), thyroid function between
two consecutive prechemotherapies (Table 3) were performed with paired statistical
comparison, the nonparametric test was used when equal variances is not assumed. The Anova
or the nonparametric tests were applied to compare the proportion of tumor cells in S phase
between treated and control group, inhibition ratios among seven different treated groups.
6. The direct research on effect of thyroid hormones on chemotherapeutic efficacy is rare, the
supplementary article is just the review article presenting the indirect evidences that supports
the chemosensitization role of thyroid hormone, only Suhane et al. studied the
chemosensitization role of thyroid hormone in MDA-MB-231. In addition ,we have cited the
references provided by reviewer in our revision and discussed our results in the light of these
previous findings.
7. We have discussed the results in the light of variations of genomic and non-genomic actions of
T3 in page 17/18.
Reference
[1]
Suhane S,Ramanujan V K. Thyroid hormone differentially modulates Warburg phenotype in
breast cancer cells [J]. Biochem Biophys Res Commun. 2011, 414(1): 73-78.
[2]
Dinda S, Sanchez A,Moudgil V. Estrogen-like effects of thyroid hormone on the regulation of
tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells [J]. Oncogene. 2002,
21(5): 761-768.
[3]
Ostrakhovitch E A,Cherian M G. Inhibition of extracellular signal regulated kinase (ERK)
leads to apoptosis inducing factor (AIF) mediated apoptosis in epithelial breast cancer cells:
the lack of effect of ERK in p53 mediated copper induced apoptosis [J]. J Cell Biochem. 2005,
95(6): 1120-1134.
[4]
Barrera-Hernandez G, Park K S, Dace A, et al. Thyroid hormone-induced cell proliferation in
GC cells is mediated by changes in G1 cyclin/cyclin-dependent kinase levels and activity [J].
Endocrinology. 1999, 140(11): 5267-5274.
For Reviewer 2
We have showed the background of the breast cancer patient and regimens of chemotherapy for
the patients in Tables.
In addition ,to make our theory more intelligible, we added Figure 4 in our revised manuscript,
which is cited from the paper written by us in another journal.