膳食類黃酮木犀草素與槲皮素調控氧化還原恆定性與抑制A431癌細胞侵襲能力之探討

Abstract

癌細胞從人體原發處擴散到他處組織或器官的惡性轉移一直是促成癌症病人死亡率居高不下的常見主因。上皮間質轉化(Epithelial-mesenchymal transition, EMT)是癌細胞後續得發展惡性轉移的重要且必須的環節之一。最近的研究發現氧化壓力的刺激不僅調節癌細胞的生長與增殖，在癌細胞的上皮間質轉化過程中亦扮演著重要的調節角色，而癌細胞亦可在不同階段與時期選擇性調控不同抗氧化物酶，以因應其所遭受到的氧化壓力，促進上皮間質轉化過程並使其獲得具有或更強的侵襲能力，這也使得這些抗氧化物酶非可單純歸類視為腫瘤抑制或致癌蛋白質，從而取決癌細胞正處於何種發展階段。在不同組織的癌細胞與臨床腫瘤樣本中可發現具有高度表現量的牛皮癬素(Psoriasin)，並促使不同組織的癌細胞獲得較強的遷移及侵襲能力，有助於參與癌症的惡性轉移。相較於牛皮癬素所調控的下游其他蛋白質訊息傳遞路徑而強化癌細胞的侵襲能力，其所受到的上游調控分子機制較不明朗。類黃酮(flavonoids)廣泛並豐富存在於各種植物體及其相關食品中，透過參與細胞中各種不同的分子訊息傳遞路徑，類黃酮可對癌細胞於癌症進展中起多重抑制作用，顯著的展現其有效的抗癌活性，包括對上皮間質轉化及細胞侵襲能力的抑制影響，然而類黃酮所參與的相關分子訊息傳遞路徑相當廣泛且複雜，仍需待更多的研究揭露之，以有助於發展相關癌症的預防或治療方法。本研究分為二部分探討木犀草素(luteolin) 和槲皮素 (quercetin)等兩種類黃酮類，透過不同方式對抑制上皮間質轉化相關的A431子宮頸癌細胞之癌遷移和侵襲的影響。 在本研究的第一部分中，發現具有不同侵襲能力的A431子宮頸癌細胞可選擇性調控錳超氧化物歧化酶(Manganese superoxide dismutase, MnSOD)及過氧化氫酶(Catalase)的表現及活性，而使其具有不同的過氧化氫的含量並與癌細胞侵襲能力具有正向關聯。抑制錳超氧化物歧化酶所增加癌細胞內過氧化氫(H2O2)的氧化壓力及相關基質金屬蛋白酶(Matrix metalloproteinas)的表現及活性可強化癌細胞的細胞遷移及侵襲能力，然而活性氧物質抑制物N-acetyl-L-cysteine (NAC) 及 diphenyleneiodonium (DPI) 可降低抑制錳超氧化物歧化酶所增加癌細胞內過氧化氫的氧化壓力並進而顯著反轉其所造成癌細胞的細胞遷移及侵襲能力。木犀草素和槲皮素可正向調控具有高侵襲能力癌細胞內的過氧化氫酶表現量而顯著降低原有的高含量過氧化氫。本研究的第一部份結果顯示癌細胞的侵襲能力不僅受不同表現量的抗氧化酶所導致的氧化壓力正向調控外，也可受到抑制錳超氧化物歧化酶而造成細胞內氧化還原失衡所影響。另外，類黃酮類可透過正向調控過氧化氫酶的表現量而降低癌細胞內的過氧化氫含量進而達成其抗癌的活性。在本研究的第二部分中，發現在具高侵襲能力的A431子宮頸癌細胞株有較高表現量的牛皮癬素，並同時伴有較高Src及Signal transducer and activator of transcription 3 (Stat 3)蛋白質磷酸化現象，而亦發現Stat3蛋白質可正向調控牛皮癬素的核糖核酸轉錄活性。然而Src及Stat3蛋白質的化學抑制物SU6656 及 S3I-201可顯著降低牛皮癬素表現量，且同時使具高侵襲能力的A431子宮頸癌細胞株降低原有的高侵襲能力。在木犀草素與槲皮素等類黃酮處理下，發現癌細胞內的Src及Stat3蛋白質磷酸化現象及牛皮癬素表現量有所降低，且牛皮癬素的核糖核酸轉錄活性亦受到抑制。另亦發現木犀草素與槲皮素與SU6656 及 S3I-201相同，皆可降低A431子宮頸癌細胞株原有的高侵襲能力並調控有關上皮間質轉化相關的蛋白質，以抑制上皮間質轉化過程。透過人為調控牛皮癬素的表現量，發現牛皮癬素可調控有關上皮間質轉化相關的蛋白質，以促使癌細胞的上皮間質轉化過程。另亦透過癌細胞異種移植斑馬魚模式動物實驗，更進一步證明木犀草素與槲皮素等類黃酮可經由Src/Stat3訊息傳遞路徑抑制癌細胞的惡性轉移能力。 透過本研究可瞭解子宮頸癌細胞藉由選擇性調控抗氧化物酶及活化Src/Stat3訊息傳遞路徑分別正向影響癌細胞內過氧化氫的氧化壓力及牛皮癬素的表現量，促使子宮頸癌細胞進行上皮間質轉化及提升侵襲能力與後續惡性轉移，然而木犀草素與槲皮素可有效阻斷其機制並發揮類黃酮的抑癌功效，將有助於為癌症研究及發展與類黃酮相關藥物上提供癌症治療的新策略。

Malignant metastasis from the occurred place originally to disperse to another tissues or organs is always the major reason commonly to result in high mortality of patients suffering cancers globally. Epithelial-mesenchymal transition (EMT) is the one of crucial links for metastasis to make the cancer cells gain the migratory ability and invasive potential. Oxidative stress not only affects the growth and proliferation of cancer cells, but also plays important role in regulating EMT. Various antioxidant enzymes in different stages of cancer development could be controlled selectively by cancer cells to response the suffered oxidative stress and promote EMT. Depending on the role in the developmental stage of cancer, those enzymes could not simply be considered as tumor suppressors or oncogenes. The upregulated expression of psoriasin (S100A7) has been found in cancer cells and tumor specimens clinically from different tissues, and correlated with resulting in cancer cells with higher capability of migration and invasion for metastatic promotion. However, there is little known about the upstream regulation of S100A7 compared with the downstream regulations by S100A7. Enriched flavonoids in various plants and related foods and involving in different and complicated signaling pathway broadly in cancers show their multiple antitumor activities significantly including EMT and invasive potential of cancer cells, but many mechanisms correlated with the effect of flavonoids are still to elucidate further to develop the effective ways for cancer-related prevention and therapies. This research was divided into two parts to study the inhibitory effect of the two flavonoids, luteolin and quercetin, on the EMT-correlated migration and invasion through different ways in A431 cervical cancer cell line. In the first part, A431 cells with different potential of invasion have been found the different content of hydrogen peroxide to correlate with the invasive potential positively through the selective regulation in the expression and activities of manganese superoxide dismutase (MnSOD) and catalase. Increased content of hydrogen peroxide and upregulation of matrix metalloproteinases (MMPs) resulted from MnSOD knockdown facilitated the migratory ability and invasive potential of A431 cells. But ROS inhibitors, N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI) relieved the MnSOD knockdown-induced augmentation of hydrogen peroxide to reverse significantly these aggressive phenotypes of A431 cells. Moreover, luteolin and quercetin were found to upregulate catalase expression to further decrease significantly the high content of hydrogen peroxide in A431 cells with higher invasive potential. The results in the first part showed the invasive potential of cervical cancer cells was affected positively not only by oxidative stress resulted from differential expression of antioxidant enzymes, but also by redox imbalance resulted from MnSOD knockdown; moreover, flavonoids relieved the oxidative stress through regulation of catalase expression to exhibit their anticancer activities. In the second part, A431 cells with higher invasive potential exhibited S100A7 upregulation accompanied with higher phosphorylation of Src and Signal transducer and activator of transcription 3 (Stat3) proteins. Moreover, the transactivation activity of S100A7 was also found to be regulated positively by Stat3 transcription factor, but S100A7 was significantly decreased by the SU6656 and S3I-201, the chemical inhibitors of Src and Stat3 protein, respectively. S100A7 downregulation correlated with decrease of aggressive potential and reduction of EMT-related proteins were found simultaneously in the A431 cell treated with SU6656 or S3I-201. The lower phosphorylation of Src and Stat3 accompanied with decreased expression and transactivation activity of S100A7 were also observed in the A431 cells treated with luteolin and quercetin. In the same as the chemical inhibitors of Src and Stat3 protein, both luteolin and quercetin decreased the invasive potential and EMT process through regulation of the proteins involving in EMT. Knockdown and overexpression of S100A7 was also suggested to regulate EMT-related proteins to promote EMT. Furthermore, both luteolin and quercetin were demonstrated to suppress the malignant metastasis in vivo through Src/Stat3/S100A7 signaling using the zebrafish xenograft. The results in the second part showed S100A7 could be regulated positively by Src/Stat3 signaling to further promote the EMT process and invasive potential of A431 cancer cells. In addition, flavonoids inhibited the aggression and metastasis of cervical cancer cells in vivo to display their antitumor functions via regulation of Src/Stat3/S100A7 signaling negatively. This study revealed oxidative stress and S100A7 expression could be regulated by selectively control of antioxidant enzymes and activation of Src/Stat3 signaling, respectively to facilitate EMT and further metastasis of cervical cancer cells. Conversely, both luteolin and quercetin suppressed these molecular mechanisms effectively to highlight their multiple and pleiotropic anticancer activities and helpfully provide new strategies to advance cancer researches and develop new drugs correlated with flavonoids.