Study The Role Of TROP2 In Biological Behavior Of Cervical Cancer And Anti-TROP2 Conjugated Hollow Gold Nanospheres As A Novel Nanostructure For Phototherinal Destruction Of Cervical Cancer Cells

Cervical cancer is a seriously threat to women’ healthy, it is also the second prevalent malignancy only to breast cancer among women worldwide, with an estimated approximately 500000 new cases one year, and more than one-third occurred in China. Present study showed that high-risk human papillomavirus infection is the most important factor for cervical cancer, but HPV infection to the development of cervical cancer is considered to be a common result of multiple genes involved, multi-stage, and the exact molecular mechanism is not yet fully clear. At present, the main treatment for cervical cancer include surgery, chemotherapy and radiotherapy, with the development of improved surgical skills and emerging chemotherapy, the cure rate of patients with early cervical cancer has been improved evidently, but cure rate of patients with late phase and recurrent carcinoma remained at a low level.Therefore, there is an urgent need to look for novel biomarkers as a complementary predictive indicator for early diagnosis and accurate prognosis assessment, which would be helpful in targeting therapies of cervical cancer.Trophoblast cell surface antigen 2 (TROP2) is a 323 amino acids transmembrane glycoprotein belonging to tumor-associated calcium signal transducer (TACSTD) gene family, it has been proved that TROP2 plays a role in the regulation of Ca2+. Structurally, it comprises a cytoplasmic tail PIP2 (phosphatidylinositol diphosphate) binding domain and tyrosine and serine phosphorylation sites, offer a possible target for TROP2 participation in malignant cell signal transduction pathway. Present study confirmed that TROP2 showed high expression of in a variety of malignant tumors, low expression in normal tissues, and is closely associated with poor prognosis of tumors. TROP2 is thought to play an important role in tumor cell proliferation, apoptosis and invasion, and it may has some interaction with ERK/MAPK signaling pathways. Based on the characteristics of TROP2 in tumor cells, it is considered to be a potential predictor of early tumors and a new therapeutic target.Hyperthermia therapy refers to a method of eliminating tumor cells by heating, once the temperature is maintained at 42.5℃-43.5 ℃, sustained 60min-120min, can achieve the purpose of killing tumor cells without damaging to normal tissue (the safety temperature boundary for normal cells is 45℃±1℃), hyperthermia is the fifth therapy for cancer, followed by surgery, chemotherapy, radiotherapy and immunotherapy. With the development of nanotechnology, nano-materials attract more and more interest by researchers based on the laser-assisted thermotherapy. When the laser irradiation on the surface of the metal nanoparticles, it can lead to resonance, resulting in the free electron absorbs light and convert it to heat. Compared to the conventional light-sensitive materials, gold nanoparticles showed the capacity to absorb 4-5 times in atomic and free electron interaction, the strong absorption from the energy of the laser makes a strong invasion force accurately locate therapeutic targets by extremely short time. Our former study showed that TROP2 was highly expressed in cancer cells, we developed a new synthesis of nanomaterials, hollow gold nanospheres (HGNs), on this basis, coupled with anti-TROP2 targeting monoclonal antibody, to form a composite functional nanoparticles. In our vitro study, we aimed to find the effect that the targeted HGNs combined with photothermal therapy killing cancer cells, and further explore the mechanism of photothermal ablation, which could provide a new theoretical basis.Part I The expression of TROP2 in cervical cancers and the relationship of TROP2 with the expression of Ki-67Objective:Detect and analyze the the expression of TROP2 in cervical cancers, and explore the role of TROP2 expression in cancer cell proliferation. Evaluate the relationship of TROP2 expression and clinicopathologic features and prognosis of patients.Methods:Immunohistochemistry was used to detect the expression of TROP2 and Ki-67 in 20 cases of normal cervical tissue,34 cases of cervical intraepithelial neoplasia (CIN) and 106 cases of cervical cancer.The chi-square test analysis was used to analyze the correlation between the expression level of TROP2 and clinicopathological characteristics. Spearman correlation test analyze the relationship of TROP2 and Ki-67 expression; Kaplan-Meier method was used to draw survival curves, univariate and multivariate Cox regression analysis were used to evaluate the prognostic significance of TROP2 expression and other factors. Log-rank test analysis was used to compare the difference between the overall survival rate and progression-free survival.Results:1.TROP2 was mainly expressed in the cell membrane of cervical tissues. In normal cervical tissue,8 cases (40%) showed weak staining, one case (5%) moderate staining, no strong expression. The rate of TROP2 positive expression in CIN group showed an increasing trend, with CIN I was 50%, CINⅡ was 33 66.7%, CINⅢ positive rate was 76.9%, and the difference was statistically significant (p=0.037). The highest expression of TROP2 was in cervical cancer,the positive rate was 88.7%, significantly higher than CIN and normal cervical tissue(p<0.001).2.The expression of TROP2 was significantly associated with histological grade, lymphatic metastasis, invasive interstitial depth, FIGO stage and histological-subtype. However, there was no significant associations between TROP2 expression and age (p= 0.100), tumor size (p=0.255).3.The positive staining rate of Ki-67 was gradually increased from normal cervical tissue, CIN and cervical cancer,the rate was 25%(5/20),55.9%(19/34),79.2%(84/106) respectively, three group difference was statistically significant (p<0.001).Statistical analysis revealed that there was a significant correlation between increased TROP2 staining and Ki67-positive proliferating cells in cervical cancer specimens (r=0.440, p <0.001)4.TR0P2 high expression compared to patients with low expression in the patient, exhibit lower OS and PFS. Multivariate Cox regression analysis showed that, TROP2 protein expression, histological grade and lymph node metastasis were independent prognostic factors of cervical cancer.Kaplan-Meier survival curves showed that patients with positive TROP2 expression had poorer OS compared to those without TROP2 expression (p,0.01), PFS also gradually declined with increasing TROP2 expression scores (p,0.01). Cox regression model analysis. TROP2 protein overexpression, along with histological grade and lymphatic metastasis were identified to be independent predictive factors for poor OS (p=0.022, p=0,023 and p=0.002, respectively) and PFS (p=0.016, p=0.04 and p=0.005, respectively).Conclusions:1.The positive staining rate of TROP2 was gradually increased from normal cervical tissue, CIN and cervical cancer, and the overexpression of TROP2 was significantly associated with the poor clinicopathological characteristics, which indicated that TROP2 plays a crucial role as an oncogene in the development of cervical cancer.2.The expression of TROP2 and Ki-67 was positively correlated, indicating TROP2 may be involved in the growth and proliferation of cervical cancer cells.3.The overexpression of TROP2 was related with the poor prognosis of cervical cancer patients, and is identified to be an independent factor to prognosis evaluation. analysis revealed that there was a significant correlation between increased TROP2 staining and Ki67-positive proliferating cells in cervical cancer specimens (r=0.440, p <0.001)4.TR0P2 high expression compared to patients with low expression in the patient, exhibit lower OS and PFS. Multivariate Cox regression analysis showed that, TROP2 protein expression, histological grade and lymph node metastasis were independent prognostic factors of cervical cancer.Kaplan-Meier survival curves showed that patients with positive TROP2 expression had poorer OS compared to those without TROP2 expression (p,0.01), PFS also gradually declined with increasing TROP2 expression scores (p,0.01). Cox regression model analysis. TROP2 protein overexpression, along with histological grade and lymphatic metastasis were identified to be independent predictive factors for poor OS (p=0.022, p=0,023 and p=0.002, respectively) and PFS (p=0.016, p=0.04 and p=0.005, respectively).Conclusions:1.The positive staining rate of TROP2 was gradually increased from normal cervical tissue, CIN and cervical cancer, and the overexpression of TROP2 was significantly associated with the poor clinicopathological characteristics, which indicated that TROP2 plays a crucial role as an oncogene in the development of cervical cancer.2.The expression of TROP2 and Ki-67 was positively correlated, indicating TROP2 may be involved in the growth and proliferation of cervical cancer cells.3.The overexpression of TROP2 was related with the poor prognosis of cervical cancer patients, and is identified to be an independent factor to prognosis evaluation.Part Ⅱ The effect of overexpression of TROP2 on biological behavior of cervical cancer and the study of related mechanismsObjective:To investigate the roles of TROP2 expression on proliferation,apoptosis,cell cycle, invasion and Chemoresistance,explore the molecular mechanisms in the pathogenesis of cervical cancer.Methods:Silencing the expression of TROP2 in Siha and CaSki cells by siRNA interference, pcDNA3.1 TROP2 plasmid transfection was used to elevate the expression of TROP2 in HeLa and C33A cells. Western blot was used to confirm the interference efficiency. CCK8 assay was used to detect the cell proliferation after the changes of TROP2 expression; The cell apoptosis was detected by AnnexinV-FITC/PI double staining; flow cytometry was used to determine cell cycle; Wound healing and transwell assay was used to determine the cell migration and invasion; Western blot analyzed some protein related with apoptosis, cycle and invasion, and association with ERK/MAPK signaling pathway correlation. Different concentrations of cisplatin was used to treat with Siha and CaSki cells after the TROP2 gene silencing, CCK-8 assay was used to draw cell survival curves, calculated the IC50, and analyze the correlation between drug resistance and ERK signaling pathway.Results:1.Western blot analysis confirmed that TROP2 was expressed in Siha, HeLa, CaSki and C33A cells. The sequence of siRNA-1100 and siRNA-550 can effectively knockdown the expression TROP2, interference efficiency was about 70%; The plasmid pcDNA3.1 TROP2 transfection evidently elevate the TROP2 protein expression.2.CCK-8 assay found that knockdown of TROP2 expression decreased the cell proliferation ability(p<0.05), whereas overexpression of TROP2 of HeLa and C33A significantly increased proliferative capacity (p<0.05)3.The cell apoptosis was detected by AnnexinV-FITC/PI double staining by flow cytometry, after knockdown of TROP2 expression, the early and late apoptosis rates was significantly higher compared with negative control group in both Siha and CaSki cells. On the contrary, enforced expression of TROP2 significantly inhibited cell apoptosis. Western blot results showed that the expression of bcl-2 protein was obviously decreased while bax was markedly enhanced in both Siha and CaSki transfected cells. In contrast, HeLa and C33A cells transfected with pcDNA3.1-TROP2 exhibited the opposite effect.4.The cell cycle was detected by flow cytometry, after knockdown of TROP2 expression, the cells in G1 phase was increased, while cells was significantly reduced in the S phase (p<0.05). After elevate the TROP2 expression, HeLa and C33A cells in G1 phase was significantly reduced, while the S-phase cells increased, the difference was statistically significant. Knockdown of TROP2 result in decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and elevated expression of cyclin-dependent kinase inhibitor p27; In contrast, elevated expression of TROP2 showed the opposite results, cyclinD, cyclinE, CDK2 and CDK4 expression was increased, while p27 expression was reduced.5.The wound healing test test confirmed after TROP2 gene silencing, cell migration was significantly decreased. And elevated expression of TROP2 significantly increased cell migration. By Transwell assay we found that knockdown of TROP2 reduces cell invasion capacity, while elevating of TROP2 expression enhance cell invasion. Knockdown of TROP2 increased the expression of E-cadherin; While elevated expression of TROP2 decreased the expression of E-cadherin.6.Knockdown of TROP2 inhibits p-ERK1/2 expression, but does not affect the expression of total-ERK1/2 in. And elevated expresson of TROP2 in C33A and HeLa cells increased the expression of p-ERK1/2 increases, which indicated there are some relationship between TROP2 and ERK signaling pathway.7.CCK-8 results showed that the cell survival rate appeared to show a dose -dependent manner in response to cisplatin treatment, and down-regulation of TROP2 protein resulted in enhanced sensitivity to CDDP treatment in both Siha and CaSki cells. The mean IC50 of transfected cells was significantly lower than untransfected and negative control cells. We found that the number of apoptotic cells was evidently increased in TROP2 siRNA group as compared with that of control groups by Hoechst staining. By western blot we found that cisplatin inhibited phosphorylation of ERK in Siha cells, however, suppression of TROP2 expression induced even lower pERK1/2 expression, while the total ERK1/2was unaffected. Then we used specific ERK inhibitor U0126 to determine the influence of ERK 1/2 activity on cell viability, the results showed that U0126 effectively strengthened TROP2siRNA mediated cisplatin sensitivity of Siha cells.Conclusion:1.TROP2 expression plays a role in cancer cell proliferation, apoptosis,cell cycle,invasion and other malignant behaviors,knockdown its expression increased the sensitivity to cisplatin. Indicated that TROP2 plays an important role in the development of cervical cancer, which prvide a theoretical basis for targeted therapy.2.There are some association between TROP2 and ERK/MAPK signaling pathway in cervical cancer cells, which may plays a role in tumor cell proliferation, apoptosis and chemoresistance and some other malignant behaviors. however, suppression of TROP2 expression induced even lower pERK1/2 expression, while the total ERK1/2was unaffected. Then we used specific ERK inhibitor U0126 to determine the influence of ERK 1/2 activity on cell viability, the results showed that U0126 effectively strengthened TROP2siRNA mediated cisplatin sensitivity of Siha cells.Conclusion:1.TROP2 expression plays a role in cancer cell proliferation, apoptosis,cell cycle,invasion and other malignant behaviors,knockdown its expression increased the sensitivity to cisplatin. Indicated that TROP2 plays an important role in the development of cervical cancer, which prvide a theoretical basis for targeted therapy.2.There are some association between TROP2 and ERK/MAPK signaling pathway in cervical cancer cells, which may plays a role in tumor cell proliferation, apoptosis and chemoresistance and some other malignant behaviors.Part Ⅲ Anti-TROP2 conjugated hollow gold nanospheres as a novel nanostructure for targeted photothermal destruction of cervical cancer cellsObjective:The aim of the study is to develop a new kind of hollow gold nanospheres with specific cancer cell targeting and to study its hyperthermia effect to cervical cancer cells,further reveal the mechanism of hyperthermia.Methods:The silver colloids were first prepared, hollow gold nanospheres were prepared using the galvanic replacement reaction between HAuC14 and silver colloid in an aqueous solution.Bi-functional SH-PEG-COOH used as a linker to conjugate antibodies to HGNs. The HGNs was characterized by UV-vis spectrum and TEM. CCK-8 assay was used to detect the cytotoxicity of HGNs. The cellular uptake and intracellular location of these functionalized HGNs were assayed by both phase contrast microscopy and TEM. The cell viability was assessed using CCK-8 assay after HeLa cells were incubated with either PEGylated HGNs or anti-TROP2 conjugated HGNs for three hours, and then exposed to 808 nm NIR laser, within 3 h after irradiation. More accurate viability assays were conducted by observing the morphological changes and double Hoechst-PI dyes staining. Flow cytometry was used to determine the amount of cell apoptosis occurrence. Western blot was used to determine the expression of bcl-2 and bax after hyperthermia.Confocal microscopy was used to detect the phosphorylation of histone-H2AX at serine-139 through measuring the discrete nuclear foci.Results:1.The HGNs synthesized in our experiment have a mean diameter of 51.6±7 nm and wall thickness of 4.67±1.52 nm calculated based on measurements made by TEM. We can clearly see the hollow interior and thin but robust shell. UV-vis extinction spectrum revealed that the plasma resonance peak for HGNs was approximately 780 nm.Bi-functional SH-PEG-COOH used as a linker to conjugate antibodies to HGNs. Meanwhile the shape of HGNs remains unchanged after conjugation with antibody based on the transmission electron microscopy, but an increase in the hydrodynamic diameter of the HGNs was observed.ions. A red-shift (from 780 nm to 818nm) in the localized surface plasmon resonance was detected after conjugation with antibody.2.CCK-8 assay found that HGNs did not show obvious toxicity with the concentration of 0.3nM,1 nM,2 nM and 3 nM HGNs and incubation for 3-48h.3.We could see that both PEGylated HGNs and anti-TROP2 conjugated HGNs were rapidly internalized into the cells after 1h incubation, from the TEM image, we found that revealed that anti-TROP2 conjugated HGNs were absorbed in HeLa cells and were mainly located at the cytoplasm, in either endosomes or lysosomes, functional HGNs were present in the cell cytosols and were embedded inside the cells in aggregated states.4.The numbers of HGNs internalized by the HeLa cells was quantified using ICP-MS. We found that the uptake of both PEGylated HGNs and anti-TROP2 conjugated HGNs increased with increased incubation time during the first 48 h. The peak uptake incubation time for both PEGylated HGNs and immuno HGNs was observed at 24 h. HeLa cells absorbed much more conjugates than PEGylated HGNs at each time interval.5.HeLa cells were incubated with either 2nM PEGylated HGNs or anti-TROP2 conjugated HGNs for three hours, and then exposed to 808 nm NIR laser. The cell viability was assessed using CCK-8 assay within 3 h after irradiation. The combination treatment showed that the anti-TROP2 conjugated HGNs plus NIR laser radiation have a much stronger effect on reducing HeLa cells viability compared to PEGylated HGNs, while the cells treated with PEGylated HGNs or NIR laser radiation alone did not induced significantly growth inhibition. Hoechst staining showed that the untreated cells were found to be intact and exhibited normal proliferation behavior, but the cells treated with anti-TROP2 conjugated HGNs plus laser radiation turned rounded, exhibited unclear boundary, membranous vesicles and fragmentation demonstrating drastic apoptotic features.6.Flow cytometry was used to determine the amount of cell apoptosis occurrence after hyperthermia.Anti-TROP2 conjugated HGNs subject to laser irradiation induced a significant increase in apoptosis compared to irradiation alone. Western blot analysis showed that there was a significant up-regulation of bax and down-regulation of bcl-2 after anti-TROP2 conjugated HGNs’ treatment together with laser radiation.7. Using confocal microscopy, we found that cells treated with NIR laser radiation alone did not show significant expression of γ-H2AX, which indicated that the laser irradiation did not induce apparent DNA damage. However, immuno HGNs’ administration together with NIR laser evidently increased the density of the y-H2AX foci compared to cells receiving irradiation alone, suggesting that anti-TROP2 conjugated HGNs can significantly induced DSBs in HeLa cells.Conclusion:1.The experiment successfully prepared hollow gold nanosphere, covalently conjugated with anti-TROP2 antibodies, HeLa cells absorbed much more conjugates than PEGylated HGNs.2.There is no obviously toxicity with the low concentration of HGNs.3.The combination treatment showed that the anti-TROP2 conjugated HGNs plus NIR laser radiation have a much stronger effect on reducing HeLa cells viability compared to PEGylated HGNs,4.Anti-TROP2 conjugated HGNs plus NIR laser radiation play a role in apoptosis by regulating the expression of the Bcl-2 family of proteins and inducing the DNA double-strand breaks.