Effect of anticancer drugs on breast cancer cells sensitive and resistant to doxorubicin : expression of mRNAs of TGF-b and its receptors

Aim. Comparative study of the effect of chemotherapeutic drugs (doxorubicin, methotrexate and cisplatin) and TGF-b on the human breast carcinoma MCF-7 cells, sensitive (wt) and resistant (DOX/R) to the doxorubicin action. Methods. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the estimation of expression of mRNAs coding for the TGF-b isoforms (TGF-b1 and TGF-b2) and the TGF-b type I and II receptors (TbRI and TbRII). Trypan blue exclusion method was used for measuring cell number and cell viability. Results. The MCF-7(DOX/R) cells were more refractory to the TGFb1-dependent growth inhibition than the MCF-7(wt) cells. The level of mRNAs coding for TGF-b and its receptors was higher in the untreated MCF-7 (DOX/R) cells comparing to the MCF-7(wt) cells. The expression of mRNA coding for TbRII was decreased in both cell lines treated with doxorubicin, methotrexate and cisplatin, while the down-regulation of mRNA coding for TbRI was revealed only in the MCF-7(DOX/R) cells upon the treatment with doxorubicin and methotrexate. Conclusions. The differential effects of studied anticancer drugs and TGF-b on the doxorubicin-sensitive and -resistant cells have been demonstrated. The elucidation of the molecular mechanisms of escape of the MCF-7 (DOX/R) cells from the growth inhibition by TGF-b requires further investigation.

Introduction.Chemotherapy is a major tool in the treatment of breast cancer.However, the main problem hindering the success of chemotherapy is the development of acquired tumor drug resistance during the treatment.Although the most common chemotherapeutic drugs used for the breast cancer treatment cause DNA damage and induce apoptosis, they exert the cell killing effects through different molecular mechanisms.The biological effects displayed by doxorubicin that may be responsible for the inhibition of tumor cell growth involve the covalent DNA binding and DNA cross-linking, topoisomerase II inhibition, arrest of tumor cell cycle progression in G2 phase, induction of apoptosis and free radicals formation [1,2].The alkylating agent cisplatin binds to DNA bases causing intra-and interstrand cross-links and breaks in DNA strands, and thus, interfering with the DNA replication [3].The antimetabolite methotrexate inhibits the enzyme dihydrofolate reductase preventing normal synthesis of the nucleoside triphosphates [4].
The tumor cells, resistant to a single chemotherapeutic agent, can also become cross-resistant to several other anti-cancer drugs due to the development of their multi-drug resistance.Such resistance was shown to be associated with numerous mechanisms: 1) decreased drug accumulation and/or altered intracellular distribution of drugs; 2) increased activity of the cell detoxification systems; 3) alterations in the cell cycle and signal transduction pathways; 4) increased repair of DNA damage and high anti-apoptotic potential of the tumor cells [5][6][7].
The multi-drug resistance in the MCF-7(DOX/R) cells was shown to be associated with various mechanisms including the over-expression of P-glycoprotein, glutathione S-transferase and glutathione peroxidase, the loss of estrogen responsiveness, the DNA hypomethylation (loss of cytosine methylation of the MDR1, GSTp and MGMT chemoresistance related genes) [6,8], and the changes in the expression of several cell cycle regulators (decreased Ki-67, cyclin D1 and pRb and increased p21 expression) [9].The elucidation of novel mechanisms of drug resistance and further development of the ways of overcoming such resistance or preventing its occurrence might increase the efficiency of treatment of cancer patients.
TGF-b belongs to the most perspective molecular markers which have both prognostic and predictive significance in the breast cancer [10,11].This cytokine is involved either in the regulation of cell proliferation, differentiation, apoptosis or in the intracellular and extracellular processes which contribute to the tumor progression (e. g. pro-tumorigenic effects on the vascular, immune and fibroblastic cells) [10].However, TGF-b may induce the entirely different cellular responses, depending on the cell type and the stimulation context, under both physiological and pathological conditions [12].An alteration in the expression of the TGF-b receptors and the post-receptor signal transduction proteins may play a critical role in the rendering malignant cells resistant to TGF-b [13][14][15].
Thus, there are multiple etiologies of the development of drug resistance, and the role of TGF-b regulatory system in the refractoriness of cancer cells to chemotherapeutic agents is not fully understood.In the present study, we have compared the effect of some of the most common drugs used for breast cancer treatment (doxorubicin, cisplatin and methotrexate) on the expression of mRNAs coding for TGF-b and its receptors in the doxorubicin-sensitive and the doxorubicin-resistant human breast cancer cells.Besides, we have compared the inhibiting effect of TGF-b on cell growth in the tested cell sub-lines in order to check if the tumor cross-refractoriness to both doxorubicin and TGF-b may exist.
Drug treatment.Doxorubicin and cisplatin were purchased from «Ebewe» (Austria), and methotrexatefrom «Leberle» (USA).Each drug was added 24 h after cell plating and each sample was incubated for 24 h with the chemotherapeutic drug of appropriate concentration.Transforming growth factor b 1 was purchased from «R & D Systems, Inc.» (USA).
Measuring cell number and viability.The effect of TGF-b 1 and doxorubicin on cell growth and viability was determined using trypan blue (0.1 % (w/v)) exclusion method.The numbers of dead (stained) cells and alive (unstained) cells were counted in the hemocytometer chamber under the light microscope.
b-Actin was used as an internal control for normalizing the amount of sample loading.
The reaction products were analyzed by electrophoresis in 2 % agarose gels and visualized by ethidium bromide staining of DNA.Gels were examined under UVlight using the transilluminator Macro Vue UV-20 («Hoeffer Pharmacia Biotech Inc.», USA) and photographed using the Olympus C-4000 digital camera.Semiquantitation of the ethidium bromide stained DNA bands was performed using the Gel-Pro Analyzer v3.1.00.00 program («Media Cybernetics, L. P.», USA).

Statistical analysis.
The experiments were repeated 3 times and significance of difference was assessed by Student's t-test.The level of significance was set at 0.05.
Results and discussion.Our previous study has shown that the MCF-7(DOX/R) cells, comparing to the MCF-7(wt) cells, are more refractory to the cytotoxic and cytostatic effects of the studied anticancer drugs [17] and ionizing radiation [18].We also have revealed a higher DNA repair potential in the MCF-7 (DOX/R) cells than in the MCF-7(wt) cells [19].The treatment of the MCF-7 and T47D cell lines of human breast carcinoma with doxorubicin (1-100 µg/ml) induced a secretion of TGF-b 1 by both cell lines in a dose-dependent manner [20].An elevated level of TGF-b 1 was detected in the blood plasma of breast [21], lung [22], colorectal [23,24] cancer patients and metastatic patients with prostate cancer [25].
In order to compare the sensitivity of studied cells to TGF-b 1 , the cells were incubated in the absence (control) or presence of TGF-b 1 (10 ng/ml) for 24 and 48 h.We found that the MCF-7(wt) cells were more sensitive to the growth inhibition by the exogenous TGF-b 1 action than the MCF-7(DOX/R) cells (Table ).The TGF-b 1 inhibited growth and caused cell death (16.5 ± 2.3 %) of the MCF-7(wt) cells (p < 0.001) after a 48 h incubation, whereas in the similarly treated MCF-7 (DOX/R) cells no statistically significant changes were observed in the growth inhibition comparing to the control (untreated) cells of that sub-line.Doxorubicin (5 µg/ml) was used as a positive control of the drug effect on the cell growth and viability of the sensitive and resistant sub-lines of 56 CHORNA I. V. ET AL.We also studied whether the above mentioned anticancer drugs can alter the expression of mRNAs for TGF-b and its receptors in the MCF-7(wt) and the MCF-7(DOX/R) cells.The cells of both sub-lines were exposed to different doses of the anticancer drugs, doxorubicin (5 µg/ml), methotrexate (25 µg/ml) and cisplatin (10 µg/ml) at 24 h exposure.When using b-actin as an internal control for normalizing the amount of sample loading, we found that the levels of mRNAs coding for TGF-b 1 , TGF-b 2 , TbRI and TbRII, were higher in the untreated MCF-7(DOX/R) cells comparing to the untreated MCF-7(wt) cells (Fig. 1 and Fig. 2).It was reported that the expression of mRNAs coding for TGFb 2 and TGF-b receptors (TbRI and TbRII) was higher in most malignant tumor cells, for example, in the breast cancer cells of Hs578T line [28].Thus, we hypothesize that the genes of the TGF-b regulatory system, the ex-pression of which is higher in the doxorubicin-resistant cancer cell line (MCF-7(DOX/R)), may be responsible for the observed chemoresistance via their protein products.
The results presented in Fig. 1 and Fig. 2, A, show that under the doxorubicin action there was no statistically significant increase in the expression of the TGFb 1 mRNA in both cell lines tested, whereas a decrease in the expression of the TGF-b 2 mRNA was revealed in the MCF-7(wt) and MCF-7(DOX/R) cells (1.4 and 2.3 fold, correspondingly) (Fig. 2, B).Methotrexate and cisplatin did not affect the expression of mRNAs coding for TGF-b 1 ligand in both studied cancer cell lines (Fig. 2, A).We found an increase in the TGF-b 2 mRNA expression in the MCF-7(wt) cells treated with methotrexate and in the MCF-7(DOX/R) cells treated with methotrexate and cisplatin (Fig. 2, B).
TGF-bs elicit their effects via binding with receptors on the cellular surface [10].TbRI and TbRII form a heteromeric receptor complex.The direct involvement of both TbRI and TbRII in conferring TGF-bs effects indicates that the loss of either of the functional receptors would contribute to the failure to respond to autocrine and exogenous TGF-b [13,14,29].It has been shown that the mutational inactivation of TbRII occurs frequently in a subset of the microsatellite unstable colon cancers [30].The inactivation of TbRII has also been described in the non-small cell lung adenocarcinomas and the small cell lung carcinomas [31].Several studies describe the mutations in TbRI and TbRII in the adenomas and gliomas [32][33][34] as well as a correlation between higher expression of TbRI and TbRII and more aggressive glioma cell lines and tumors [35,36].
The treatment of cells with doxorubicin and methotrexate decreased the production of mRNA coding for TbRI (2.0 and 1.4 fold, correspondingly) in the doxorubicin-resistant sub-line, while no statistically significant changes in the level of TbRI mRNA were found in the doxorubicin-sensitive sub-line under the same conditions (Fig. 2, C).A two-fold increase of mRNA coding for TbRI was detected in the MCF-7(wt) cells treated with cisplatin.At the moment, the reasons for such dynamics in the TbRI expression in MCF-7(wt) cells under cisplatin treatment are unknown, and additional studies are needed to get a support for such explanation of the detected differences.We also revealed a decreased expression of the mRNA coding for TbRII in both studied cell sub-lines under the action of anticancer drugs used.The most prominent effect on the MCF-7(wt) cells was found for the cisplatin action, while in MCF-7(DOX/R) cells -for the doxorubicin action (Fig. 2, D).Earlier, it has been established that anticancer drugs doxorubicin and cisplatin decrease the expression of the mRNA coding for TbRII in the human lung adenocarcinoma A549 cells, and cause the changes in the post-receptor Smad-dependent TGF-b 1 signaling pathway [37].
In summary, we have got the evidence that the resistance of the human breast cancinoma cells to doxorubicin is a multi-factorial phenomenon.The multi-factorial resistance to doxorubicin might explain the crossresistance of MCF-7(DOX/R) cells to other compounds (methotrexate and cisplatin) which are even not structurally related.
The following conclusions can be suggested taking into account the results obtained: 1) tumor cell refracto-riness of the MCF-7(DOX/R) cells to both doxorubicin and TGF-b 1 was found; 2) the studied anticancer drugs (doxorubicin, methotrexate and cisplatin) can inhibit the transduction of TGF-b regulatory signals in both cell sub-lines tested owing to the violation of the corresponding signal pathway at different levels, particularly by decreasing the expression of the mRNA coding for TbRII; 3) TGF-b might affect the response of the breast cancer cells to the anticancer drugs, thereby, allowing for a possibility of chemotherapeutic modulation.Êëþ ÷å âûå ñëî âà: MCF-7, ÒÔÐ-b, äîê ñî ðó áè öèí, ìå òîò ðåê ñàò, öèñ ïëà òèí, ðå çèñ òåí òíîñòü.

Factor
0.05; **p < 0.001 (in treated versus the respective control (untreated) cells).The effect of TGF-b 1 (10 ng/ml) and doxorubicin (5 µg/ml) treatment on growth and viability of MCF-7(wt) and MCF-7 (DOX/R) cells the MCF-7 cells (Table).It is known that during the early phase of epithelial tumorigenesis, TGF-b inhibits the primary tumor development and growth by inducing the cell cycle arrest and apoptosis.At the late stages of tumor progression when the tumor cells become resistant to the growth inhibition by TGF-b due to inactivation of the TGF-b signaling pathway or aberrant regulation of the cell cycle, TGF-b exhibits a pro-metastatic role[26].Earlier it has been found that the cisplatin-resistant sub-line of murine leukemia L1210 cells possesses a cross-resistance to TGF-b 1 and the mechanism of drug resistance, based on the involvement of TGF-b 1 , has been proposed[27].