| Background:Oral cancer ranked sixth as the most common malignant tumor, afflicting more than300,000people each year with a five-year survival rate of-50%. In United States, more than35,000new cases and nearly8,000deaths were reported in2008alone. The lack of methods for timely detection and recognition of suspicious lesions timely contributes to low survival rates. These lesions appear as atypical, innocuous-appearing mucosal alteration, such as ulceration, leukoplakia and erythema. Conditions conducive for the malignant transformation of premalignant lesion remain unknown. Compared with other deep tumors as hepatocellular carcinoma, oral cancer can be monitored and treated early in its premalignant status because its positions are visible and superficial. Therefore, the biological mechanism of malignant transformation should be clearly understood to block the malignant process before carcinoma development.In this regard, our group is committed to the discovery of new cancer screening methods to improve the quality of life and survivals. Du has studied Rose bengal (RB) staining for oral cancer screening with the diagnostic sensitivity and specificity of93.9%and73.7%. However, RB cannot differentiate some inflammation and cancer. Wang et al, synthesized new materials as RB-Gold nanorods, propose new ideas for oral cancer diagnostic using sensitive optical detection, through redshift or dark-field imaging to distinguish oral cancer cultured cells with normal epithelial cells, but only for laboratory research. But clinical application requires a variety of precision optical instruments and it is expensive. On this basis, this paper combined quick and simple screening method with optical sensitivity requirements, and established a more efficient cancer screening for early oral cancer screening. Besides, we studied the mechanism of malignant changes in the oral mucosa to slow or curb its malignant transformation and search the targeted molecules for the specific diagnosis of oral cancer.Biopsy is the gold standard for clinical diagnosis of cancer for the suspicious lesion excised for a professional pathologist to observe the microscopic cell morphology change. Therefore, it is subjective and invasive. However, only about2%of mucosal changes could be transformed into cancer, repeated operation may cause greater damage for the patient. To reduce the unnecessary suffering of patients, we hope to use surface-enhanced Raman scattering (SERS) to obtain Raman signals by sampling a small amount of tissue or cells before biopsy. The efficacy of spectroscopic techniques as Raman scattering in the diagnostics of cancer such as renal tumors, hepatocellular carcinoma, lung cancer, cervical cancer, and oral cancer has been investigated. And Raman scattering conducted in biopsy in vitro has demonstrated the feasibility of differentiating normal and malignant tissues. However, invasive and massive biopsy is generally needed to obtain the adequate amount of sample on account of small scattering cross section and direct illumination of the suspicious lesion with an intense laser beam may not be preferred. With the development of scientific research, only a few cells need to be collected by SERS using SPR effect of nanoparticles to amplify Raman signals to complete the highly sensitive screening, once become a hot topic again.Surface enhanced Raman scattering (SERS) provides the capability of single molecule detection and owing to the high sensitivity, inherent molecular specificity, and narrow bandwidth, SERS is an excellent diagnostic tool for cancer. For example, specific DNA sequences and mutations have been checked for genetic errors by SERS and ultrasensitive detection of proteins and RNA has been demonstrated. Moreover, immunoassays employing SERS labels have been established to selectively target and quantify biomarkers to facilitate early diagnosis in vitro and cancerous cells have been detected from periphery blood and other media. The multiplexing ability of molecular imaging with encoded SERS nanoprobes have been utilized in living cells and animal tumor models and hence, a technique using SERS to amplify signals may offer cost-effective, sensitive, and specific sensing capability and is of both scientific and clinical interest.The first part of the paper is to invent a new cancer screening method using the plasmonic gold nanorods to amplify the raman signal of a few cell samples. As the clinical manifestation is asystemic, oral cancer can be difficult to notice. Besides, the position of oral cancer is superficial and accessible, and the sampling of pathogenic cells is feasible. Herein, the merits of exfoliative cytology and SERS are combined to establish a sensitive, rapid, and noninvasive platform for cancer screening.The second part of dissertation is to invest the dynamic observation and its overall functions of autophagy in oral tumorigenesis. Many studies have demonstrated that autophagy is involved in the tumorigenesis of various cancers, such as hepatocellular carcinoma, cutaneous malignant melanoma, gastrointestinal cancer, and colorectal cancer. However, divergent results have been reported. Autophagy may be influenced by different stimuli, including reactive oxygen species (ROS) production during tumor formation. Autophagy can spatially and temporarily suppress tumor growth by regulating cell proliferation. Beclin1, provides key autophagic proteins to a pre-autophagosomal structure, to form the core complex consisting of Beclin1, Vps34, and Vps15. Suppression of autophagy-related proteins, such as Beclin1can accelerate tumorigenesis. Anti-apoptotic Bcl-2protein regulates autophagy by binding to Beclin1through a putative a-helical BH3domain, thereby inhibiting the association between Beclin1and hVps34. This interaction inhibits autophagy by preventing Beclin1from assembling the pre-autophagosomal structure. Oncogenic H-Ras activation triggers the release of Beclin1from the Beclin1-Bcl-2complex, thereby limiting clonogenic survival. Moreover, autophagy can promote tumor development in other conditions. Once the tumor is established, autophagy provides energy, and resists various therapeutic measures, such as radiation and chemotherapy. The different functions of autophagy may be due to the context of experimental conditions and different tumors and stages. And the excessive low or high expression of the autophagy related protein is associated with the condition of cancer, exhibiting the restraining the malignant transformation of premalignant lesion or promoting the cancer survivals. And only2%premalignant lesion will transform into malignant condition, and the autophagy may be associated with the possibility of transformation. Thus, autophagy and its overall functions in different tumorigenic stages of oral cancer should be explored dynamically.And the first part has achieved rapid and sensitive cancer screening without molecular target by plasmonic gold nanorods. The second part has invested autophagy in tumorigenesis for appropriate molecular target for cancer screening. And at last, the bonding of SERS and molecular target has been urgent for clinical cancer screening.Currently autophagy was known meaningful in stratifying cancer patients, but the study of autophagy for oral cancer is relatively scarce, the relationship between the autophagy expression and prognosis of oral cancer has not been determined. Therefore, we investigated autophagy through oral tumorigenesis models and to understand the mechanism of oral cancer at an early stage in the process of malignant lesions as a preliminary study. In the following, a lot more experiments for specific molecular screening of autophagy is needed to achieve targeted SERS cancer screening.The first part of the experiment using a plasmonic nanoparticles to achieve SERS label-free oral cancer screening with a small sample of exfoliated cells in differentiating normal and cancer cells with good sensitivity and specificity. However, it is more significant for differentiating cancer and inflammation, precancerous lesions, or for less or more chance of malignant transformation. It still needs further more studies. The great significance of autophagy detection is suitable for stratification of patients with relationship of cancer recurrence and prognosis to achieve the goals. The autophagy-related oral cancer research is less. Therefore, we hope that through first exploration autophagy of oral tumorigenesis for the post-screening appropriate marker. Finally, the implementation still requires a lot of work by binding a marker SERS, using SERS quantify measurements autophagy to achieve better screening results.Part I:Paper-based plasmonic platform for sensitive, noninvasive, and rapid cancer screeningBackground:SERS (surface-enhanced Raman scattering) fingerprints of individual molecules offer the possibility of multiplexing as well as cancer screening. Objective: To establish a new cancer screening by SERS. Methods:A highly sensitive, noninvasive, and rapid cancer screening platform encompassing exfoliative cytology and paper-based SERS technology is described. The SERS substrate which consists of plasmonic gold nanorods (GNRs) adsorbed on a piece of filter paper forms the flexible and three-dimensional heterogeneous scaffold for cancer screening. Results: Different and reproducible SERS spectra are obtained from normal and cancerous cells. Proper monitoring of the Raman scattering features reveals specific biomolecular changes, including the increase in the relative amounts of nucleic acid and proteins as well as decrease in the percentage of lipid in cancerous cells. A diagnostic algorithm based on the ratio of spectra value is adopted to distinguish between the exfoliated cells from normal and cancerous tissues of20patients, and a high sensitivity of100%and specificity of100%are achieved by I1600/1440(the peak ratio of signals at1600cm-1to1440cm-1),/1440/1340(1440cm-1to1340cm-1) better than I14o/1340(1440cm-1to1340cm-1) with a sensitivity of70%and specificity of60%. Conclusion:The combination of exfoliative cytology and paper-based plasmonic technology enables highly sensitive, rapid, and non-invasive cancer screening and has large clinical potential.Part â…¡:Autophagy in oral tumorigenesisBackground and Objective:Autophagy has essential functions in tumorigenesis. However, its temporal expression and function under different status have yet to be determined. This study aims to investigate the temporal expression of autophagy and its possible function in7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch squamous cell tumorigenesis. Methods:The left pouch of hamster was embrocated with0.5%DMBA solution, and then sacrificed after4,8,10, and13weeks. Immunohistochemistry, immunofluorescence and western blot were performed to reveal the temporal expression and distribution of LC3, Beclin1, and Bcl-2. D-NA damage was evaluated by8-oHdG staining. Results:The expression levels of LC3and Beclin1significantly upregulated at week4(p<0.05), but gradually downregulated at week13. By contrast, the expression level of Bcl-2significantly upregulated at week13. The colocalized regions of Bcl-2and Beclin1peaked at week4and then decreased gradually. The DNA damage in epithelial cells slightly at week4and then increased rapidly in the next2months. Conclusion:Autophagy was motivated as a tumor suppressor that diminishes carcinogen-induced DNA damage. However, autophagy was gradually suppressed, which may be attributed to the interaction between Bcl-2and Beclin1. |
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