Implication from thyroid function decreasing during chemotherapy in breast cancer patients: chemosensitization role of triiodothyronine

A total of 685 female patients with breast diseases admitted in the First Affiliated Hospital of Chongqing Medical University, with median age of 49 years (range 17–81 years) was studied, including 316 cases of breast benign lesions, such as fibroadenoma, mastopathy, intraductal papilloma, lipoma, etc. and 369 cases of breast carcinoma, among which 224 cases were initially diagnosed with breast carcinoma. All breast diseases were confirmed by the pathology. No history of thyroidal diseases or liver, renal dysfunction was found in these patients. All breast cancer patients were treated with standard chemotherapy. A total of 1698 samples were collected from all patients on the first admission and / or 1 to 3 days before and / or 1 to 3 days after each cycle chemotherapy.

Ethical approval for this study and agreement by all patients were obtained from the Biomedicine Ethics Committee of The First Affiliated Hospital of Chongqing Medical University. Each subject signed an agreement of participation in this study that was approved by Biomedicine Ethics Committee of The First Affiliated Hospital of Chongqing Medical University and consented to publish. The protocol of the study adhered to the tenets of the Declaration of Helsinki and was approved by the local ethics committee.

T₃, T₄, free triiodothyronine (FT₃), free thyroxine (FT₄), TSH were detected by UnicelTM DXI 800 with chemiluminescence methods in the Laboratory of Endocrinology of the First Affiliated Hospital of Chongqing Medical University.

The MCF-7, MDA-MB-231 breast cancer cell lines were supported by Molecular Oncology and Epigenetics Laboratory, the First Affiliated Hospital of Chongqing Medical University. The cell lines were maintained in RPMI 1640 (Gibco-BRL, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories, Linz, Austria), 100 U/ml penicillin and 100 mg/ml streptomycin, at 37°C in a humidified atmosphere containing 5% CO₂.

The chemosensitization role of T₃ (Sigma Aldrich, Italy) is reflected by changes of sensitivity of breast cancer cells to 5-fluorouracil (5-Fu) (Xu Dong, China) or Taxol (De Bao, China). The concentration of T₃ used for chemosensitization test ranged from 2 ng/ml to 32 ng/ml, 5-Fu and Taxol were used at the IC₅₀ concentration for each cell line (5-Fu: 13 μmol/L for MCF-7, 8 μmol/L for MDA-MB-231; Taxol: 0.4 μmol/L for MCF-7, 0.5 μmol/L for MDA-MB-231). According to different concentrations of T₃, the chemosensitization test comprised nine independent tests, each contained eight groups with a specific T₃ concentration (Figure 1). Before chemosensitization test began, the MCF-7 cells in logarithmic growth phase were trypsinized and seeded at a density of 3000/well into 96-well plates at 37°C in 5% incubator. After 21 h, the chemosensitization test began.

The 1st to 3rd groups were pretreated with T₃ for 24 h, then treated with 5-Fu for 48 h, the depriving times of T₃ were different, the 1st group was kept with T₃ until test end, T₃ of the 2nd group was deprived at the start of 5-Fu, and T₃ of the 3rd group was deprived after using 5-Fu for 24 h.

After 24 h without T₃ pretreatment, the 4th to 6th groups were also treated with 5-Fu for 48 h. The 4th group was added with T₃ at the start of 5-Fu until test end, T₃ of the 5th group was added at the start of 5-Fu and deprived after using for 24 h, T₃ of the 6th group was added when 5-Fu was added for 24 h.

After 24 h without T₃ pretreatment , the 7th group was treated with 5-Fu only for 48 h, and without treating with T₃. The 8th group was set as control group, without treating with T₃ or 5-Fu.

Before cell proliferation assay, wells of all groups were washed with PBS for two times, replaced with fresh culture medium, and set one new group with culture medium only as blank control. Cell counting kit-8 (CCK8) (Dojindo Laboratories, Japan) was used to evaluate the viability of cells in all groups according to the instructions of the manufacturer. The final concentration used for assay was 10%, after incubation with cells for 1 h at 37°C to allow CCK8 to form formazan crystals by reacting with metabolical active cells, the OD value was measured in a microplate reader at 450 nm. The inhibition ratio was calculated by following formula: 1-OD value of treated group / OD value of control group. All experiments were repeated at least three times by same person.

Flow cytometry analysis of cell cycle was implemented as previously reported [10]. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol and stained with propidium iodide (PI). The cell cycle profiles were assayed using the Elite ESP flow cytometry at 488 nm, and the data were analyzed using the CELL Quest software (BD Biosciences, San Jose, CA). Flow cytometric analysis was immediately performed.

A total of 224 serum samples of breast cancer patients and 316 samples of breast benign lesion patients at initial diagnosis were analyzed for thyroid hormones. Thirty-seven of 224 breast cancer patients were diagnosed as NTIS (either FT₃ or FT₄ is below the normal), of which the morbidity was higher than that in breast benign lesions patients (16.5% vs 7.3%). The mean concentrations of T₃, FT₃ in breast cancer patients were lower than that in breast benign lesions patients, however, only difference of T₃ between them was statistically significant (P<0.05). On contrary, the mean concentrations of T₄ and TSH were higher in breast cancer patients, but only difference of T₄ between them was statistically significant (P≤0.05) (Table 1).

Blood samples from 180 cases of breast cancer patients were analyzed in this part. Eighty-two point two percent (82.2%) patients suffered NTIS during chemotherapy. T₃, T₄, FT₃, TSH decreased and FT₄ increased significantly during chemotherapy compared with prechemotherapy (P<0.05) (Table 2).

A total of 456 samples were analyzed between two consecutive prechemotherapies, no significant differences of thyroid function were found (P>0.05) (Table 3).

The thyroid functions decreasing during chemotherapy indicated adding thyroid hormones may enhance the sensitivity of tumor cells to chemotherapy, as T₃ can induce cells progression from G0-G1 phase to S phase [11] or enhance mitochondrial activity [12] which makes tumor cells more sensitive to chemotherapy. So, as the specific chemotherapeutic reagent for killing tumor cells in S phase, 5-Fu was choosed to study the change of sensitivity of breast cancer cells when T₃ was present. Three groups were set up, cells were seeded in quintuplicate in 96-well plate at a density of 3×10³ cells/well. The 1st group was treated with T₃ and 5-Fu, the 2nd group was treated with 5-Fu only, the 3rd group was set as control group. We firstly used 4 ng/ml T₃ to pretreat MCF-7 or MDA-MB-231 for 24 h, then 5-Fu was added and T₃ were deprived, values of OD after 48 h were measured, the results showed that both MCF-7 and MDA-MB-231 pretreated with T₃ were more sensitive to 5-Fu than those with 5-Fu only, without T₃ pretreatment (P<0.05) (Figure 2A and 2B).

The 5-Fu especially targets tumor cells in S phase, to study whether enhancement of sensitivity of tumor cells to 5-Fu was related with increasing proportion of breast cancer cells in S phase, we further investigated the effect of T₃ on MCF-7 cell cycle. Representative results of cell-cycle distribution in MCF-7 pretreated with T₃ or without T₃ were shown in Figure 2C. Flow cytometry analysis revealed a statistically significant increase in the number of T₃ pretreated MCF-7 with S phase (P<0.05), accompanied with the increase in G2-M phase and decrease in G0-G1 phase (P<0.05) (Figure 2D). So, T₃ enhanced the sensitivity of MCF-7 to 5-Fu, through promoting the cell cycle progression of MCF-7 from G0-G1 phase to S phase.

Besides discussing the effect of thyroid hormone on 5-Fu, we also observed whether T₃ has the chemosensitization role for Taxol. Taxol works differently by arresting the cells in G2-M phase, considering a higher concentration of T₃ may be more efficient in promoting breast cancer cells proliferation, we used 24 ng/ml T₃ to pretreat MCF-7 or MDA-MB-231, the results showed that both MCF-7 and MDA-MB-231 pretreated with T₃ were more sensitive to Taxol than those with Taxol only, without T₃ pretreatment (P<0.05) (Figure 2E and 2F).

Considering the thyroid function decrease during chemotherapy, which may induce breast cancer cells retardant in G0 phase, and T₃ could enhance chemosensitivity of MCF-7 through increasing proportion of cells in S phase, we thought T₃ may be used as a new adjuvant therapy for breast cancer, and started to study the efficacy of different T₃ using modes on chemosensitivity of MCF-7.

Eight groups were set as described in methods. As 4 ng/ml T₃ had been proved to have chemosensitization role by us, we then firstly use 4 ng/ml with different usage modes to study the efficacy of T₃ on chemosensitivity of MCF-7. Surprisingly, only sensitivity of MCF-7 to 5-Fu in the first three groups pretreated with T₃ significantly increased (P<0.05), no chemosensitization was found in the 4th to 6th groups (Figure 3A).

Whether elevating the T₃ dose can enhance the chemosensitivity of MCF-7 in 4th to 6th groups, those without T₃ pretreatment, and decreasing T₃ could reduce the chemosensitivity in the 1st to 3rd groups, those were pretreated with T₃, were still unclear. So, we performed eight other chemosensitization tests with T₃ at 2 ng/ml, 8 ng/ml, 12 ng/ml, 16 ng/ml, 20 ng/ml, 24 ng/ml, 28 ng/ml, 32 ng/ml, using the concentrations of T₃ more than 4 ng/ml (8 ng/ml to 32 ng/ml) is to imitate the hyperthyroidism in choriocarcinoma patients receiving chemotherapy. The results showed 2 ng/ml T₃ was not efficient enough to enhance the chemosensitivity of MCF-7 to 5-Fu in any group (P>0.05) (Figure 3B), which indicated the suitable dose of T₃ is essential point for chemosensitization. On the contrary, the MCF-7 in 4th to 6th groups successively showed significantly increased chemosensitivity, compared with that of 7th group (P<0.05), as the T₃ dose elevating from 8 ng/ml to 32 ng/ml (Figure 3C).

Many non-thyroidal illness may affect the thyoid function and induce NTIS [13], which is also called euthyroid sick syndrome (ESS) [14], low T₃ or T₄ syndrome [15], marked by reductions in both thyroid function and peripheral conversion of T₄ to T₃, presumed to reflect a homeostatic mechanism to conserve energy. TSH levels tend to be normal or slightly decreased in these patients, but the underlying mechanism of reduced thyroid function is unknown. In this study, the NTIS prevalence in breast cancer patients at initial diagnosis was higher than that in breast benign lesions patients (16.5% vs 7.3%). The high NTIS prevalence in breast cancer patients at initial diagnosis suggests that NTIS may be related with malignancy. Furthermore, it was found in this study that the mean concentration of FT₃ in breast cancer patients at initial diagnosis was significantly lower than that in breast benign lesions patients, suggesting that thyroid hypofunction may occur in breast cancer patients. Galton VA [16] studied the effect of malignant tumor on the thyroid function in rats, implanted with Walker 256 carcinosarcoma cell line, results showed a reduction in the concentration of T₄ in tumorous rats serum, compared with corresponding control rats, due to the increased deiodination and urinary excretion.

The causes for hypothyroidism in cancer patient may not only be associated with tumor necrosis factor, interleukin-1, interferon-γ [17, 18], which inhibits the function of hypothalamus-pituitary-thyroid axis and peripheral conversion of T₄ to T₃, but also be associated with the self-protection mechanism, reducing the tissue metabolism to suppress the tumor growth.

Chemotherapy is one of the most effective systemic therapies for cancers. In cancer patients, thyroid function is thought to be vulnerable to chemotherapy, as hypothalamic-pituitary axis is active and chemotherapy is systemic therapy for patients. The influence of chemotherapy on thyroid function was just seen as a late effect, mainly presenting hypothyroidism. Only few studied the alterations of thyroid function during chemotherapy, which induced abnormalities in thyroid hormones metabolism with a significant decrease in serum T₃ concentration (NTIS) instead of overt thyroid disease in malignant hematologic disease [19]. Thyroid hypofunction was thought to be associated with inhibition of the liver thyroglobulin secretion [20] and hypothalamus-pituitary-thyroid axis function [7] by chemotherapy.

In this study, NTIS prevalence in breast cancer patients obviously increased during chemotherapy, compared with that at initial diagnosis (87.1% vs 16.5%) and then decreased to 15.4% before the next chemotherapy (prechemotherapy). The NTIS prevalence reduction from 16.5% to 15.4% indicated that chemotherapy alleviated the NTIS prevalence, as thyroid hypofunction is considered as risk for breast cancer occurrence [21] and poor prognosis [22], this also suggests that NTIS alleviation may be a good efficacy by chemotherapy. With significantly increased NTIS prevalence during chemotherapy, whether controlling it is better for enhancing chemotherapeutic efficacy is unclear.

Furthermore, during chemotherapy for breast cancer patients in this study, significant decreases of T₃, T₄, FT₃ and TSH were found, compared with prechemotherapy (P<0.05). After chemotherapy, thyroid function began to recover, and there was no significant difference between two consecutive prechemotherapies (P>0.05), which indicated obvious thyroid function decreasing caused by chemotherapy mainly occurred during chemotherapy.

While there is still no detailed study on whether decrease of thyroid function goes against breast cancer chemotherapy or not. Decrease of thyroid function does not occur in all patients with malignant tumors during chemotherapy. In a prospective study, effects of chemotherapy on thyroid function in patients with non-seminoma testicular carcinoma were evaluted: Serum HCG was present in sixteen patients and absent in fifteen. HCG levels ranged from 10 to 30,000 μg/l, with a median value of 520 μg/l. And during chemotherapy, T₄, T₃ and rT₃ levels increased significantly, but basal TSH levels and the TSH response to thyrotropin releasing hormone (TRH) decreased. Furthermore, after chemotherapy the increased T₄ and lowered TSH levels returned to normal, while the FT₃ level did not change either during or after chemotherapy [3], which suggested an unaltered hypothalamic / pituitary axis. Non-seminomatous germ-cell tumors (NSGCT) can comprise several different histological components, among which, choriocarcinoma, produces HCG. In NSGCT patients with high-serum HCG levels, hyperthyoidism has been recognized and is considered to be a paraneoplastic phenomenon. Hyperthyroidism frequently accompanies NSGCT with highly elevated HCG [23]. Recent investigations have clarified the structural homology not only in the HCG and TSH molecules but also in their receptors, and this homology suggests the basis for the reactivity of HCG with the TSH receptor [24, 25]. Hyperthyroidism or increased thyroid function has been reported in many patients with trophoblastic tumors [26], either hydatidiform mole [27, 28] or choriocarcinoma [25], for which the principal therapy is chemotherapy. With effective chemotherapy, their long term survival exceeds 95% [24, 29]. Furthermore, hyperthyroidism can be cured after the healing of the trophoblastic tumors [30]. Meister LH et al. reported a 26-year-old pregnant woman suffered from choriocarcinoma with metastases to both lungs. HCG were more than 2.5×10⁶ mU/ml. Consistent with HCG-induced hyperthyroidism, the patient suffered from thyroid crisis with fever of 38.6°C, worsening respiratory distress, tachycardia of 140 bpm and mental confusion during the first cycle of chemotherapy. Then the patient was treated with antithyroid crisis, symptomatic and supportive treatments. After the 10th cycle of chemotherapy the patient was in good condition and free of metastatic lesions in her chest X-ray [5]. It was reported that thyroid hormones could significantly promote the tumor growth and metastases [31]. The effective chemotherapy of curable tumors (such as malignant hydatidiform mole, choriocarcinoma, etc.) may be associated with hyperthyroidism during chemotherapy, which may enhance the chemotherapeutic sensitivity by inducing tumor cells progression from G0-G1 phase into S phase or elevating the mitochondrial activity. While, in this study, thyroid function decreased significantly and majority of the breast cancer patients suffered NTIS during chemotherapy, which may be associated with decreased chemotherapy sensitivity. Therefore, based on the above analysis, it was hypothesized that increasing the thyroid function by giving thyroid hormones before and / or during chemotherapy to imitate the hyperthyroidism or high thyroid function state of some choriocarcinoma patients during chemotherapy may enhance the chemotherapeutic efficacy in breast cancer and other malignant tumor patients who suffer obviously decreased thyroid function or NTIS during chemotherapy.

The effect of thyroid hormones on chemotherapeutic efficacy has been rarely researched before, and whether adding thyroid hormones during chemotherapy is suitable for breast cancer patients is still unknown. Some studies found hypothyroidism may be the protective factor for cancers, as hypothyroidism can suppress tumor growth [32], in addition, Hercbergs et al. found that hypothyroidism could reduce the insulin-like growth factor 1, the antagonist of tamoxifen-induced cytotoxicity, to prolong the survival in recurrent high grade glioma patients [33]. This above mentioned perspective is different from our hypotheses, since we think hyperthyroid function is beneficial for treatment efficacy when patients receive chemotherapy. If patients of cancers reveive no chemotherapy, the status of hypothyroidism will be better for prognosis. Choriocarcinoma is curable with chemotherapy only, long term survival of these patients exceeds 95%, the underlying mechanism may refer with high levels of HCG, which can induce hyperthyroidism [6]. In contrast, we firstly found thyroid hormones decreasing and NTIS prevalence increasing during chemotherapy in breast cancer patients, however, growth of breast cancer cells is regulated by thyroid hormones [34], thyroid hormones are considered to be the growth factor for glioma and thyroid cancer [35], the absence of thyroid hormones in cells could provoke a proliferation arrest in G0-G1 [36] or weak mitochondrial activity [12], which makes tumor cells insensitive to chemotherapy.

Basing on these points, we performed T₃ chemosensitization test in breast cancer cells. The normal FT₃ concentration in human body is 2.2-4.2 ng/ml, we take the physiological concentration range of FT₃ as the reference standard. Therefore, 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. 5-Fu, the chemotherapeutic regent mainly focusing on tumor cells in S phase, is a usual component in chemotherapy for breast cancer. The T₃ pretreatment induced MCF-7 increased progression from G0-G1 phase to S phase, with elevated chemosensitivity to 5-Fu, the similar result was found in MDA-MB-231. Taxol works differently by arresting the breast cancer cells in G2-M phase, it 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. To prove T₃ can sensitize the breast cancer cells to Taxol, we used 24 ng/ml T₃ to pretreat MCF-7 or MDA-MB-231, the results showed that both MCF-7 and MDA-MB-231 pretreated with T₃ were more sensitive to Taxol than those with Taxol only, without T₃ pretreatment.

Both MCF-7 and MDA-MB-231 express the thyroid hormone receptor, the chemosensitization role of T₃ in these two cell lines is thought to be mediated by the proliferation efficacy of T₃. In MCF-7, an estrogen receptor 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 [37]. 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 [11]. Dinda et al. [38] found thyroid hormone not only enhances the phosphorylation of pRb, but also induces the c-Myc expression and mutant P53 expression, this can also explain the cell proliferation efficacy of thyroid hormone. MDA-MB-231 is acknowledged harboring P53 mutations, which further demonstrates that thyroid hormone may promote the cell proliferation. Furthermore, thyroid hormone was found to sensitize the mitochondrial pathway to enhance the chemotherapeutic efficacy in MDA-MB-231 [12].

Thyroid hormone exerts its non-genomic and genomic actions in breast cancer cells [12]. For non-genomic action, which is characterized by combination of thyroid hormone and membrane receptors such as ER to elucidate the downstream signaling cascade, and is independent of transcriptional activity [39]. For genomic action, thyroid hormone primary mode is by binding to the thyroid hormone receptor in the nucleus then influencing the transcription and expression patterns of target genes. In MCF-7, which expresses ER and thyroid receptor, the chemosensitization role of T₃ is mediated through the genomic and non-genomic actions of T₃, while in MDA-MB-231, which expresses thyroid receptor, the enhanced chemotherapeutic efficacy can also be contributed to the genomic and non-genomic actions of T₃, like the influence on genes expression and the elevated mitochondrial activity mentioned above.

In light of the findings mentioned above, T₃ may be used as adjuvant therapy for breast cancer patients in the future, the main points for its application are usage dose and how to combine it with chemotherapy. To imitate different usage modes of T₃ in clinic, six individual groups with treatment by T₃ and 5-Fu were set up to separately compare with control group treated with 5-Fu only. It is indicated in this results that, to get chemosensitization efficacy, pretreatment with lower dose of T₃, using higher dose of T₃ together with 5-Fu or during chemotherapy with 5-Fu were all thought to be available. In clinic, however, using higher dose of T₃ will increase the risk of suffering thyroid crisis, and in most of T₃ chemosensitization tests from 4 ng/ml to 32 ng/ml, the relative inhibition ratio in 1st group was highest, except T₃ at 16 ng/ml and 24 ng/ml. So, pretreatment with lower dose of T₃ until the end of chemotherapy may be a safer and more efficient therapy for breast cancer patients. In addition, only a suitable dose of T₃ could have chemosensitization role in chemotherapy, treatment with extremely low dose of T₃ for long time (72 h) still could not enhance the chemosensitivity of MCF-7 to 5-Fu.

Using T₃ for chemosensitization in breast cancer therapy is to simulate the high thyroid function in choriocarcinoma patients during chemotherapy, based on the new findings that thyroid hormones decreasing during chemotherapy, in which chemotherapeutic regents mainly take effect.

In summary, we studied the thyroid hormones and NTIS in breast patients at initial diagnosis and during chemotherapy, in which with a thyroid function significant decreasing, and found T₃ enhanced the chemosensitivity of MCF-7 to 5-Fu. Moreover, we think the chemotherapeutic efficacy partly depends on the thyroid function, checking the thyroid function in breast cancer patients is important for elevating chemotherapeutic efficacy.