| 摘要: | 神經膠質母細胞瘤是腦部腫瘤中最致命的癌症。神經膠質母細胞瘤患者的標準治療是以手術切除腫瘤，接著施以放射線治療與帝盟多進行化療。然而，由於神經膠質母細胞瘤容易產生抗藥性，致使神經膠質母細胞瘤總是會復發。我們的研究顯示，在具帝盟多抗性的神經膠質母細胞瘤中，腫瘤細胞合成大量的神經類固醇，包括脫氫表雄酮與17β-雌二醇。因此，我們試圖釐清神經類固醇在神經膠質母細胞瘤產生抗藥性的過程中扮演的角色。
為了評估神經類固醇生成是否參與神經膠質母細胞瘤的生長與抗藥性的發展，我們整理了Oncomine數據資料庫分析中神經類固醇的生合成酵素的表現量。結果顯示，轉化17β-雌二醇的酵素-芳香酶，相較於正常的腦組織，其在神經膠質母細胞瘤中其有較高的表現。進一步，我們利用超高壓液相層析儀串聯式質譜儀，以質譜法建立更靈敏與更高通量的神經類固醇檢驗方法，該方法可以在同一個樣品中檢測15種神經類固醇的標準品，且證實包含17β-雌二醇、脫氫表雄酮、睪酮、別孕烷醇酮、二氫睪酮、孕烯醇酮與17α-羥孕烯醇酮在具帝盟多抗藥性的神經膠質母細胞瘤細胞或復發的神經膠質母細胞瘤病人檢體樣品中濃度異常增加。
根據過往的研究，脫氫表雄酮的合成酵素17α-羥化酶於神經膠質母細胞瘤中調控脫氫表雄酮的生成並保護神經膠質母細胞瘤使其降低帝盟多誘導的細胞凋亡。為了進一步釐清其機制，我們使用17α-羥化酶的抑製劑阿比特龍，其已被應用於臨床治療前列腺癌之化療藥物。以阿比特龍抑制17α-羥化酶於神經膠質母細胞瘤中的表現，結果顯示，阿比特龍誘導內質網逆境，並導致活性氧分子的積累，進而造成Secretion associated Ras related GTPase 1（SAR1）a/b的泛素化，最終導致細胞凋亡。相反的，若過量表現17α-羥化酶與SAR1a/b則會阻止阿比特龍造成的內質網逆境，降低活性氧分子累積於神經膠質母細胞瘤，顯示17α-羥化酶調控SAR1以維持神經膠質母細胞瘤的內質網功能與氧化還原代謝的健全，進而影響神經膠質母細胞瘤的細胞活性。
另一方面，我們也發現從正常星形細胞瘤發展至神經膠質母細胞瘤的過程中，17β-雌二醇的合成酵素芳香酶的表現量逐漸增加，進而增加17β-雌二醇在神經膠質母細胞瘤細胞的濃度，並降低帝盟多誘導的細胞死亡。進一步分析，17β-雌二醇減少粒線體產生的活性氧分子，並透過17β-雌二醇的調控以增加超氧化物歧化酶、過氧化氫酶與Nuclear factor erythroid 2-related factor 2（NRF2）的表現，進而減輕氧化逆境。我們也發現神經膠質母細胞瘤中，NRF2的表現對於17β-雌二醇減少帝盟多誘導的氧化逆境，進而產生抗藥性的機制是重要的。
總體而言，我們之前和目前的研究結果顯示，神經膠質母細胞瘤產生抗藥性需要多種神經類固醇的產生，這顯示抑制神經類固醇合成與功能是治療神經膠質母細胞瘤的潛在方法。我們的研究為開發有效的臨床干預措施提供了新的線索，希望可以改善神經膠質母細胞瘤患者的病情並延長其存活期。
Glioblastoma multiforme (GBM) is the most fatal cancer among brain tumors, and the standard treatment of GBM patients is surgical tumor resection followed by radiotherapy and temozolomide (TMZ) chemotherapy. However, tumors always recur due to the developing drug resistance. It has been shown that neurosteroids, including dehydroepiandrosterone (DEHA) and 17β-estradiol (E2), are synthesized in TMZ-resistant GBM tumors. Therefore, we sought to explore the possible role of DHEA and E2 in the development of drug resistance in GBM.
In order to evaluate whether neurosteroids production is involved in the growth of GBM and the development of drug resistance, we have compiled the mRNA expression of neurosteroid biosynthetic enzymes in the analysis of the Oncomine database. In order to evaluate whether neurosteroid production is involved in the growth and resistance development of GBM, we compiled the mRNA expression of neurosteroid biosynthetic enzymes in the Oncomine database analysis. The results show that Aromatase that synthesizes E2 has a higher Aromatase mRNA expression in GBM. Furthermore, we use UPLC/MS to establish a more sensitive and higher-throughput detection method for analyzing neurosteroid, which can detect 15 kinds of neurosteroids standards in the same sample, and it is confirmed that including E2, DHEA, Testosterone, Allopregnanolone, Dihydrotestosterone, Pregnenolone and 17α-Hydorxypregnenolone , the concentration of these neurosteroids abnormally increased in TMZ-resistant GBM cells or GBM-relapsed samples.
According to previous studies, CYP17A1, the synthase of DHEA, regulates the production of DHEA in GBM and protects GBM to reduce TMZ-induced apoptosis. To further clarify the mechanism in CYP17A1-mediated MZ resistance, we used CYP17A1 inhibitor, Abiraterone (Abi), to inhibit CYP17A1 expression in GBM. The results show that Abi induces endoplasmic stress and leads to the accumulation of reactive oxygen species (ROS), which in turn causes the ubiquitination of SAR1a/b, and ultimately leads to apoptosis in GBM. On the contrary, overexpression of CYP17A1 and SAR1a/b prevents the endoplasmic reticulum adversity caused by Abi and reduces the accumulation of ROS in GBM cells. This shows that CYP17A1 regulates SAR1 to maintain endoplasmic reticulum health and redox metabolism in GBM, thereby affecting The activity of GBM cells.
Furthermore, we also found Aromatase, the synthase of E2, which express gradually increased during the development of normal astrocytoma to GBM. Lead to increase the concentration of E2 and attenuated TMZ-induced cell death and ROS species production by mitochondria. In addition, E2 attenuated oxidative stress by increasing the expression of superoxide dismutase (SOD) 1/2, catalase, and nuclear factor erythroid 2-related factor (NRF) 2. We found that NRF2 expression was essential for the induction of drug resistance by 17β-estradiol through the reduction of oxidative stress in GBM.
In general, our previous and current research results show that GBM resistance requires the production of multiple neurosteroid, which shows that inhibition of neurosteroid synthesis and function is a potential method for the treatment of GBM. Our research provides new clues for the development of effective clinical interventions, hoping to improve the condition of GBM patients and prolong their survival. |
