| Abstract: | 口腔黏膜下纖維化（OSF）是一種口腔內之疾病，其特徵為組織間隙膠原纖維的發炎及過度堆積。據估計吃檳榔的人口中有10%至25%會有OSF之產生，而此可能為癌症前期的表現，OSF形成病理機制尚未完成了解。突變因子、致癌因子、營養因素以及免疫因素都可能造成OSF，吃檳榔已被認為是個很重要的危險因素，某些腫瘤抑制基因和其蛋白質產物造成OSF之癌前情況是值得我們去探討的。OSF細胞和正常人類纖維母細胞(HGF)培養於加入10%小牛胚胎血清之DMEM培養液中，置於37℃培養箱內培養。淬取培養後細胞之genomic DNA，經由聚合鏈反應擴大選取APC腫瘤抑制基因。又將培養細胞溶解後取出之野生型p53蛋白質以西方墨漬方法來分析。所得資料非參數樣本符號檢定及變異分析來分析。結果顯示OSF細胞APC基因第498密碼組CGA轉為GGA造成Arg變成Gly之誤異突變(8/8) (p<0.01)，而HGF則無突變發生。又OSF之8個樣本中有7個在密碼組504、DNA序列1494被刪除而轉成停止密碼組（p<0.01）於是造成提早產生結束訊息，而形成一個含503個氨基酸之殘餘物。又HGF細胞之野生型p53蛋白質比OSF細胞野生型p53蛋白質高（p<0.01）。結論：由於APC基因以及野生型p53腫瘤抑制基因的改變使OSF病患可能惡化變成口腔癌症的危機。
利用熱處理治療腫瘤的方法已存在數個世紀。熱緊迫蛋白質(heat shock proteins，HSPs)為近年來的研究主題。HSPs包括許多的蛋白質，當細胞遭受未達致死程度之熱緊迫時可誘發HSPs表現以防止蛋白質凝集(aggregation)及將組態改變之可溶性蛋白質分子重新摺疊成具功能性組態。其依據分子大小可分為四個主要的族群，HSP27、HSP60、HSP70及HSP90，每一族群包含數個成員且各自展現特異的表現型態（恆定的或誘發性）。HSP70的族群中HSP73恆定的表現於所有細胞中，然而HSP72則由緊迫誘發表現。HSP於腫瘤細胞之功能仍莫衷一是。利用特定的寡核酸(oligonuclotide)序列阻斷HSP70及HSP72之表現可誘發腫瘤細胞凋亡(apoptosis)。其他學者研究顯示HSPs的表現可抑制或誘發許多不同型態的細胞凋亡。前人研究指出熱處理誘發腫瘤抑制基因p53蛋白質的積存及促使細胞週期(cell cycle)停滯或細胞凋亡。已知p53具有許多的生理功能包括DNA複製及修補、DNA受損後的細胞週期停滯與細胞凋亡。此等功能乃來自於誘發其下游基因之表現。當細胞遭受相當程度的緊迫時p53蛋白質會磷酸化以增加安定性及積存量。其後，致活其標的基因特別是p21及Bax導致細胞週期停滯於G1期及細胞凋亡。此研究之目的乃在探討熱處理誘發之HSP70於p53訊息傳導中所扮演之角色。我們的結果發現熱處理可誘發p53表現與磷酸化，轉而誘發p21與Bax/Bcl-2表現增加。熱處理可誘發HSP70結合於p53蛋白質，此一作用可能有助於p53蛋白質的安定與生理功能之維持。
清胃散是傳統中藥處方之一其中含升麻、牡丹皮、黃連、當歸、生地黃等成分，主要於牙齦潰爛口內炎、牙齒疼痛、牙齦腫痛、牙齦流血、咽喉腫痛的症狀時使用。但是清胃散是否可應用於口腔癌的治療，卻很少有這方面的研究報告。我們對清胃散在治療主要症狀之餘是否具備引起細胞凋亡或使用清胃散處方時機制有所改變具高度的興趣。我們使用清胃散水溶物置於含OC2及TSCCa細胞株內，其濃度分別為0、10、25、50、75及100μl/ml，用MTT測試法來計量細胞活性和生存率，在洋菜膠電泳凝膠做DNA梯狀脆片測試，用免疫墨漬技術來表達bax蛋白質。再把資料以NIH影像1.56軟體掃瞄分析。形態上大的改變、細胞生存率下降和細胞核內DNA梯狀脆片形成都証明清胃散對OC2及TSSCa細胞都具引起細胞凋亡的能力。同時bax蛋白質隨清胃散量增加而有顯著增加，所以清胃散在此兩種口腔細胞株是經由依bax傳導路徑來引起細胞凋亡。
Oral submucous fibrosis (OSF) is a chronic disease of the oral cavity characterized by inflammation and excessive accumulation of interstitial collagenous fibers. OSF affects an estimated 10% to 25% of individuals who are habitual areca nut—chewers worldwide and may be precancerous. The mechanism for the pathogenesis of OSF is at present not fully understood. Mutagenesis, carcinogenesis, and nutritional and immunologic factors may contribute to the genesis of OSF, and chewing of betel quid has been demonstrated to be one of the important risk factors. It would be worthwhile to explore the
possible contributions of certain tumor suppressor genes and their protein products to the precancerous aspect of OSF. Cells from OSF and control subjects were cultured in Dulbecco modified Eagle medium with 10% fetal bovine serum at 37°C. Genomic DNA was extracted from cultured cells and used as a template for polymerase chain reaction amplification of the APC tumor suppressor gene. The presence of wild-type p53 protein in cell lysates of cultured cells was analyzed by Western blot. Data were analyzed by the sign test for nonparametric samples and by analysis of variance. The results showed that the APC gene of explant cultured cells from OSF patients (8/8) had a CGA-to-GGA transition mutation at codon 498 that resulted in an Arg-to-Gly missense mutation (P < .01). All (8/8) normal HGF cultures revealed expression of the wild-type APC protein. Cells cultured from 7 of 8 OSF patients were also found to have a single nucleotide deletion at nucleotide 1494 that resulted in creating a stop codon (TGA) at codon 504 (P < .01). This created a premature signal for the endpoint of translation and thus resulted in the generation of a truncated protein product that encodes a polypeptide of 503 amino acid residue. It was found that wild- type p53 protein in human gingival fibroblast cell cultures was significantly higher than in OSF cells (P < .01). Our conclusion is alterations of the APC and wild-type p53 tumor suppressor genes in OSF may imply a risk for progression to oral cancer.
Heat treatment used in tumor therapy existed for centuries. The heat shock proteins (HSPs) are the most subjects in these years. HSPs are a group of proteins, function as molecular chaperone to preventing protein aggregation and refolding soluble misfolded protein moleculars, induced in cells exposed to sublethal heat shock or stress. They can be classified according to their molecular mass into four major families, the HSP27 family, HSP60 family, HSP70 family and HSP90 family. Each family is comprised of several members and exhibits a distinct constitutive and inducible expression pattern. The HSP70 family is constitutive expressed (HSP73) in all cells and can be induced (HSP72) by stress. Using specific oligonuclotides to block the expression of HSP70 and 72 triggered cancer cell apoptosis. It seems not surprising that heat shock proteins make cells more resistant to apoptosis. However, the function of HSPs in tumor cells remains controversial. Previous studies showed that HSPs expression had been correlated with a number of cell types to resist or induce apoptosis. Heat shock induced the tumor suppressor gene p53 accumulation and contributed to cell cycle arrest or apoptosis. It well known that p53 has several biological effects involving DNA replication and repair, cell cycle arrest and apoptosis after DNA damage. When cells exposed to an appropriate stress, p53 will be phosphorylated and results in increasing p53 stability and accumulation. Subsequent activation of p53-target genes, particularly the cyclin-dependent-kinase inhibitor p21 and bax, results in the arrest of cell cycle in the G1 phase and activation of apoptosis. The aim of this study is to determine the role of heat-induced chaperone hsp70 on p53 signaling pathway. We found that heat shock triggered HSP70 conformation changed and induced HSP70-p53 complex formation, it assist to stable p53 protein and maintain p53 physiological function.
Chingwaysan, a Chinese herbal formula, contains Cimicfugae Rhizoma, Rehmanniae Radixet Rhizoma, Moutan Radicis Cortex, Coptidis Rhizoma and Angelicae Sinensis Radix. This medicine is well-known for its curing power for ulcerated gums, toothaches, cheek boils and bleeding gingiva. However, no reports can be found on its application in the treatment of oral cancers. We are therefore interested in whether Chingwaysan is capable of causing abnormal apoptosis processes, and whether this condition can be rectified through Chingwaysan herb treatment. We used aqueous extract to treat OC2 and TSCCa cells (both are human oral cancer cell lines) with different Chingwaysan concentrations (0, 10, 25, 50, 75 and 100 µl/ml). The MTT (3, (4, 5-dimethyl-thiazol) 2, 5-diphenyl-tetraxolium bromide) reduction assay was employed to quantify the differences in cell activity and viability. DNA ladder formation on agarose electrophoresis was also performed. The bax expression level was monitored using immunoblotting techniques. The patterns of the changes in expression were scanned and analyzed by NIH image 1.56 software. Taken together, drastic morphological changes, reduced cell viability and the presence of inter-nucleosomal DNA fragmentation all indicated that Chingwaysan is capable of inducing apoptosis in OC2 and TSCCa cell lines. Furthermore, the accumulation of wild type bax protein significantly increased in a dose-dependent manner upon treatment with Chingwaysan. In conclusion, Chingwaysan can induce apoptosis via a bax-dependent pathway in cells from these two particular oral cancer cell lines. |
