Targeting Endometrial Cancer Cells With Radiotherapy And Disulfiram

Background:Endometrial cancer(EC) is the most frequent gynecological malignancy with increasing rate of mobility reported worldwide during the recent years. Endometrial cancer is an important public health problem. Via clinicopathological studies, EC is broadly classified into two major types, type I and type II, which correlate with its biological behavior. Type I EC, is usually well differentiated and responds favorably to appropriate therapy. However a small proportion of patients who have risk factors such as low differentiation or non differentiated endometrial adenocarcinoma were classified as high-risk endometrial cancer. Owing to the pathology subtypes, all the type II EC is belonged to be high-risk ednometiral cancer, is more likely to metastasize, and yields a poor prognosis, namely due to the lack of targeted therapies.Radiation therapy(RT), is the main adjuvant therapy for both type II and type I patients who are classified as high-risk endometrial cancer. RT for EC is aimed at eliminating residual cancer cells, in order to achieve better local tumor control. Unfortunately, critical review of the recent clinical trials does not yield convincing evidence of benefit in terms of overall survival. RT can shrink tumor cells, but can not effectively kill those radioresistant pre-existing cancer stem cells(CSCs). The presence of radiation-induced breast and liver cancer stem cells are challenging for a beneficial outcome of RT.The cancer stem cells(CSCs) concept posits the existence of a subpopulation of tumor cells that display “stem cell-like” properties, such as, self-renewal, angiogenesis as well as a high tumorigenic potential, which offers resistance to most currently employed radiotherapies. The experiments confirmed CSCs can be found in EC in vitro and in vivo. However, little is known about the regulation mechanism of cancer stem cells at present. The data showed, stemness gene-encoded transcription factors(TFs) supposed to play a vital role in the determination of stem cell states, with specific transcriptional networks in sustaining the growth and self-renewal of cancer stem cells. SOX9, Nanog, HER2, OCT4 and Slug known as the transcription factors expressed high level in EC, correlated with the worse outcome, may involve in the functional regulation of cancer stem cells. So based on the traditional RT therapy, the combination with the CSCs targeting therapy, offer a major clinical breakthrough towards the treatment of EC.CSCs typically over-express aldehyde dehydrogenase(ALDH). There is compelling evidence showing that the cells expressing elevated levels of intracellular aldehyde dehydrogenase 1(ALDH1) in human and mouse endometrial cancer are endometrial cancer stem cells(ECSCs). ALHD+ cells and these represent promising targets for the development of novel interventions for cancer prevention/treatment. Disulfiram(DSF), is currently FDA approved drug. The clinical data confirmed DSF as one of the safty and less expensive drugs, which can be used for months and years for treating chronic alcoholism. DSF is also as an irreversible pan-ALDH inhibitor, that binds to one molecule of copper(DSF/Cu), can inhibite tumor cells proliferation and induce apoptosis. The noval research revealed that the DSF/Cu complex can specifically target cancer stem cells in breast cancer. Cu2+ is endogenously present in tumors and is accumulated in rich regions of CD133+ cells which have cancer stem cell-like properties. The specific distribution expression of Cu2+ endowed the specific binding capacity of DSF to target cancer stem cells. However, the mechanizing of targeting CSCs of DSF/Cu is still unknown.Our previous research studies have shown that DSF/Cu acts as a potent apoptosis inducer and an effective proteasome inhibitor, thus inhibiting NF-kB activation. NF-kB is a key transcription factor(TF) which regulates the activation of many genes involved HER2, MYC when cells are exposed to RT in breast cancer. NF-kB regulated stemness genes included SOX9, OCT4 and HER2 reported high level expresstion in EC. Whether targeting CSCs in EC with DSF/Cu and radiotherapy via NF-kB regulated stemness genes pathway remains to investigate. So far there has very little studied about the effect of the DSF/Cu and stemness genes on the radiosensitivity of endometrial cancer stem cells.Part ⅠThe effect of radiotherapy for endometrial cancer initiating cell Objectives:Use type I and type II endometrial cancer cell lines to investigate the effect of RT for ECSCs. Methods:1. ALDH, CD133 were used as stem cell markers to investigate the number of CSCs in the type I and type II EC cell lines after and before single radiation by flow cytometry.2. In vitro sphere formation assay and in vivo xenograft mouse tumor modal experiments were used to verify tumorigenicity of the type I and type II EC cell lines after and before single radiation.3. A colony formation assay was used to investigate the inhibition rate of bulk EC cell population after radiotherapy.4. All the analyses were treated by SPSS19.0 statistic software. Comparison of continuous variables was carried out with the use of two-tailed t test. The inhibition rates were analyzed using the Pearson chi square test. ‘Alpha equals 0.05’ was considered to be statistically significant.1. Radiotherapy can increase the percentage number of ALHD+, CD133+ cells in type II endometrial cancer compared to the untreated group, as well as AN 3CA cells in vitro(P < 0.05). Radiotherapy can neither increase the percentage number of ALHD+ nor CD133+ cells in type I endometrial cancer compared to the untreated group(P > 0.05).2. Radiotherapy cannot decrease tumorigenicity in vivo and in vitro compared to the untreated group in AN 3CA cell line(P > 0.05). In type I endometrial cancer Ishikawa cells radiotherapy can reduce the tumorigenicity in vitro compared to the untreated group in Ishikawa cell line(P < 0.05).3. Radiation cannot significantly decrease the inhibition rate of sphere formation compared to clony formation in Ishikawa cell lines. Conclusions:Radiation had no effect on the depleting of Type II endometrial cancer stem cells; Radiation can non-specificlly reduce the number of type I ECSCs. Results: Part ⅡEfficacy of the combination with radiotherapy and DSF in the treatment of endometrial bulk cells and cancer stem cells Objectives:To investigate whether administering RT in combination with DSF can decrease the bulk of tumor as well as targeting CSCs. Methods:1. MTT test was used to identify the cell growth inhibition rate in DSF and DSF/Cu groups.2. ALDH, CD133 stem cell markers were used to investigate the CSC number in serious endometrial cell line and endometrial cell line after treatment with RT+DMSO(DMSO used as a buffer control), RT + DSF/Cu, RT + DSF and untreated group.3. Verification was done via in vitro sphere formation assays, and in vivo xenograft mouse tumor modal experiments.4. A colony formation assay was used to investigate the inhibition rate of bulk EC cell population after combination treatment with RT and DSF/Cu.5. WB was used to test the apoptosis factor Cleaved-PARP and PARP expression in EC cell lines after combination therapy.6. The human EC xenograft mouse model(AN 3CA) was used to test the efficacy of the combination with RT + DSF/Cu or RT + DSF with targeting bulk tumor cells and CSCs7. All the analyses were treated by SPSS19.0 statistic software. Comparison of continuous variables was carried out with the use of two-tailed t test. The inhibition rates were analyzed using the Pearson chi square test. ‘Alpha equals 0.05’ was considered to be statistically significant. Results:1. The cell inhibition rate in AN 3CA and Ishiakwa cell lines were higher in DSF/Cu group than in DSF group(P < 0.05).2. The combination of radiation and DSF/Cu cannot only significantly target the ALHD+ or CD133+ cells, but it can also reduce tumorigenicity in AN 3CA or Ishikawa cell line in vitro and in vivo(P < 0.05). The positive result can not observed in the combination of radiation and DSF group.3. The combination of radiation and DSF/Cu can significantly decrease the inhibition rate of sphere formation compared to clony formation(P < 0.05) in both AN 3CA and Ishikawa cell lines.4. The expression of apoptosis factor Cleaved-PARP increased in AN 3CA and Ishikawa cells by treatment with combination of radiation and DSF/Cu in protein level(P < 0.05).5. The combination of radiation and DSF, but not the combination of radiation and DSF/Cu, can significantly decrease the tumor volume, the tumor weight and increase the mice weight in AN 3CA xenograft mouse model(P < 0.05).The xenograft tumor recurrent rate is lower in the combination of RT+DSF group than the others group, and the mice survival rate is the highest one among the testing groups. Both the combination of radiation and DSF and combination of radiation and DSF/Cu can significantly decrease the sphere formation using mouse tumor cells(P < 0.05). Conclusions:1. The combination of RT and DSF/Cu can not only reduce the bulk of tumor cells, but it can also effectively specifically target the ECSCs in both type I and type II endometrial cancer in vitro.2. The combination of RT and DSF considered to be one of the save and efficient theraputic method to treat endometrial cancer in vivo. Part ⅢThe mechanism of the combination with radiotherapy andDSF in the treatment of endometrial cancer stem cells Objectives:Investigate whether RT can upregulate the transcription factors SOX9, Nanog, HER2, OCT4 and Slug in both type Ⅰ and type II endometrial cancer. Further experiments investigate whether the mechanism of targeting endometrial cancer stem cell with RT and DSF/Cu combination therapy is via downregulation of the NF-kB-stemness genes pathway. Methods:1. Real-time q PCR was used to test the stem cell transcription factors SOX9, Nanog, HER2, OCT4 and Slug m RNA expression level, before and after radiation in both AN 3CA and Ishikawa cell lines.2. Real-time q PCR and WB were tested the stem cell transcription factors which upregulated by radiation after the combination treatment with IR and DSF/Cu in both AN 3CA and Ishikawa cell lines.3. A dual luciferase reporter assay was used to test NF-kB activity change after the combination treatment.4. NF-kB inhibitor IMD-0354 was used to test the stemness genes expression change after exposure to RT.5. All the analyses were treated by SPSS19.0 statistic software. Comparison of continuous variables was carried out with the use of two-tailed t test, ‘Alpha equals 0.05’ was considered to be statistically significant. Results:1. Radiotherapy can increase the expression of the stem cell transcription factor SOX9, OCT4, Slug m RNA and protein levels compared to untreated group in AN 3CA cells(P < 0.05).2. The combination of RT and DSF/Cu can significantly reduce the activity of NF-kB(P < 0.01), and the stemness genes SOX9, OCT4, Slug m RNA and protein levels in both AN 3CA and Ishikawa cell lines(P < 0.05). Also via inhibition of the NF-kBi pathway, DSF/Cu can effectively reduce the m RNA and protein levels of SOX9 and OCT4 in AN 3CA and Ishikawa cells. However, DSF/Cu cannot reduce the m RNA and protein levels of Slug after radiation(P < 0.05). Conclusions:1. Radiotherapy can increase the expression of SOX9, OCT4 and Slug in AN 3CA cells, but not in Ishikawa cells.2. The combination of RT and DSF/Cu target the ECSCs via both the NF-kB /(SOX9, OCT4) pathway and non-NF-kB / Slug pathway.