Effects And Mechanisms Of Translationally Controlled Tumor Protein In Cancer Cells Of Chemoresistance

Chemotherapy is one of the necessary methods of clinical cancer treatment. Althoughmost malignancies initially respond to chemotherapeutic treatments, after an unpredictableperiod, developed chemoresistance and recurrence of tumor cells could always lead to thefailure of treatment. Multiple mechanisms which cancer cells may use to developresistance to the cancer treatment have been proposed. Notably, Cancer Stem Cells (CSCs),also named tumor-initiating cells and stem-like cancer cells (SLCCs) have been postulated recently as responsible for recurrent tumours after chemotherapy. CSCs not only have theability of self-renewal and proliferation, but also have developed highly resistant toionizing radiation and chemical drugs. Current studies showed that, during the process ofchemo-or radio-therapy, while DNA damage induced by ionizing radiation or chemicalsmay eliminate tumor cells, it may aggravate the instability of genome and promote thetumor cells to stem-like cancer cells（SLCCs）. The molecular mechanism is still unclear.Translationally controlled tumor protein（TCTP）is a small molecular, which is highlyconservative in evolution and widely expressed in many tissues. It has been shown thatTCTP is involved in regulating a variety of important physiological processes, includingthe cell cycle, proliferation, anti-apoptosis and development of cancer. Our previous studyconfirmed that TCTP is one of the key molecules in the maintenance of genome stabilityand plays a critical role in DNA damage repair. Other studies have also shown that TCTPis involved in the regulation of tumor cell malignant transformation; knocking-down ofTCTP can effectively promote the malignant reversion of breast cancer cells and thenreduce the amount of the SLCCs. Interestingly, the change of TCTP expression in tumorcells has been suggested to associate with the chemoresistance, but its mechanisms hasnot yet been reported. The above studies implicates that TCTP may be involved in theprocess of SLCCs generation during chemotherapy.To learn the distribution characteristics and significance of TCTP in different clinicalclassification of tumor, we initiated our study by detecting the tissue and tissue chips ofglioma and breast cancer. Using5tumor cell lines, with different potential metastasis fromglioma and breast cancer, we set up9kinds of chemoresistant models through continualselection in6generation under different drug pressure. Then, we observed thecharacteristics of the generation of SCLLs which may be induced durng chemotherapy andfurther studied the correlation of the TCTP expression change and SCLLs biologicalcharacteristics.Results1. Clinical tissue chips detection shows that, the expression of TCTP increased withtumor malignant degree in different clinical classification of breast cancer and glioma tissues; Also, Western blot confirms that the amount of protein expression of TCTPincreased with tumor malignant degree.2. Through MTT analysis, we optimized the selection concentration of drugs,5-FU：1μg/ml，ADM：0.08-0.1μg/ml and TMZ:500μM-1000μM, respectively. As ABCG2expression changes the main resistance markers, compared with the control group,After continual selection through6generation, the expression of ABCG2, one of themarkers of chemoresistance, increased significantly in all9selected cell lines,which manifests that the chemoresistant cell models of breast cancer and glioma areestablished successfully.3. The study of biological characteristics of chemoresistant tumor cell lines shows that,①Compared with the control group, while interstitial cells emerge in two breastcancer resistant cells in morphology appearance, no obvious changes in threeTMZ-resistant glioma cells;②Compared with the control group, scratch woundhealing ability of five kinds of drug-resistant cells were significantly increased,meanwhile, Transwell Chambers in membrane the populations of cells through theTranswell Chambers membrane increased, which implicated that the migration andinvasion abilities of drug-resistant cell lines increased significantly;③Cloneformation assay shows that the proliferation ability of drug-resistant cells enhanced inthe culture medium containing drugs and3%FBS.4.Western blot analysis of SLCCs showed that, the expression of the stem cell markersOCT4in five chemoresistant cell lines were significantly increased, along with theup-regulation of TCTP; flow cytometry results show that,compared with the controlgroup, the percentage of CD44+/CD24-cells significantly increased inchemoresistant breast cancer cells. In vivo experiment in nude mice demonstrated that,compared with the control5. group, the growth rate of chemoresistant tumor cells which were subcutaneouslyinjected is much faster; immunohistochemical staining of tumor tissues show that thepositive staining particles of TCTP in groups of chemoresistant tumor cells weresignificantly higher than respective control groups. The above results suggested that TCTP expression is closely related to the proliferation ability of chemoresistant tumorcells.6. In chemoresistant breast cancer cells, down-regulation of TCTP by siRNA plasmidtransfection technology could also induce the reduction of Oct4expression. Thechange of TCTP and Oct4shows a positive correlation. Collectively, the results ofexperiments of four to six implicated that TCTP may play an important role inmaintaining the “stem” biological characteristics of SLCCs.7. We detected the radio-resistance of chemoresistant tumor cells by exposing the cellsto the different dose of X-rays. The clone formation analysis showed that thechemoresistant cells also have the stronger radio-resistant potentials. Interfering theexpression of TCTP by siRNA transfection would significantly reduced theproliferation and survival abilities of chemoresistant tumor cells when exposed to4GyX-rays.8. To understand the mechanisms of TCTP involved in maintaining radio-resistance ofchemoresistant tumor cells, we detect the occurrence of apoptosis and LC3Bexpression, marker of autophagy, in cells exposed to X-rays with down-regulation ofTCTP. The expression of caspase-3and caspase-8are increase in somechemoresistant. Our results showed that TCTP participates in radio-resistance ofchemoresistant tumor cells through both anti-apoptosis and anti-autophagy functions.Conclusion1. The expression of TCTP in glioma and breast cancer tissues is mainly located incytoplasm and nucleus. The expression of TCTP increase significantly with the tumormalignant degree in both glioma and breast cancer tissue, which suggested that TCTPcould probably become a new promising marker in clinical prognosis or classificationof tumor.2. The ratio of SLCCs increased significantly when tumor cells experienced thecontinual treatment of chemotherapeutic drugs. The chemoresistant tumor cellsshowed SLCCs biological characteristics, including much higher abilities of cellproliferation, migration and colony-forming, and up-regulation of OCT4. Strikingly, TCTP plays an important role in chemoresistant tumor cells by maintaining theSLCCs biological characteristics, which may relate with the regulation of OCT4.3. Chemoresistant tumor cells also showed significant radio-resistance, TCTP may beinvolved in which through both anti-apoptosis and anti-autophagy functions.