| Abstract: | Voltage-dependent anion channel 1 (VDAC1)主要位於細胞粒線體外膜，組合成受電壓影響而改變離子通透之孔道。VDAC1可能因與pro-apoptotic或anti-apoptotic protein結合，而影響子宮頸癌細胞凋亡、生長或存活。我們由基因晶片實驗發現，human nonmetastatic clone 23 type 1 (nm23-H1)會影響VDAC1表現，且nm23-H1在子宮頸癌細胞癌化、移動、侵犯能力、臨床病理特徵及復發存活扮演重要角色。VDAC1的失衡除表現量有差異外，尚有可能因為不同alternative splicing或缺氧產生truncated VDAC1，所以探討子宮頸癌細胞是否因VDAC1過量表現或產生truncated VDAC1，而影響其癌化和進展。而nm23-H1和nucleoside diphosphate磷酸化成nucleoside triphosphate及DNA形成和細胞增生有關，可能影響VDAC1失衡；VDAC1失衡和ADP/ATP能量代謝異常有關，影響粒線體通透性開啟，造成粒線體腫脹破裂，釋放出reactive oxygen species (ROS)；而hypoxia inducible facter-1A (HIF-1A)和ROS有關。ROS的增加或NM23-H1的變化可能和signal transducer and activator of transcription 3的磷酸化(p-STAT3)有關，並加重HIF-1A表現和影響VDAC1失衡或truncated VDAC1之形成及vascular endothelial growth factor的產生，促使癌細胞增生和進展。此外探討阻斷或過度表現VDAC1，是否影響癌細胞對化療藥物如cisplatin之反應，並分析VDAC1、nm23-H1和子宮頸癌病人臨床病理特徵及復發存活之關係。在人類許多癌症，hexokinase和anti-apoptotic Bcl-2會過度表現，而這些蛋白會與VDAC1結合，抑制細胞凋亡。因此將VDAC1當作子宮頸癌治療之標靶，利用VDAC1-based peptides來阻斷這些蛋白和VDAC1之結合，抑制子宮頸癌細胞生長或轉移等表徵，更是本研究之重要目標。
Voltage-dependent anion channel 1 (VDAC1) is mainly located in outer mitochondrial membrane and forms as a voltage-dependent ion channel. Because VDAC1 can interact with pro-apoptotic or anti-apoptotic proteins, it may affect the apoptosis, growth and survival of cancer cells of uterine cervix. Our previous experiment, using genechips, revealed that human nonmetastatic clone 23 type 1 (nm23-H1) has an impact on the expression of VDAC1. We previously demonstrated that nm23-H1 has a critical role in cervical carcinogenesis and is associated with cancer cell migration and invasion as well as clinicopathological variables, recurrence and survival of cancer patients. In addition to its abnormal expression, VDAC1 imbalance means the formation of truncated VDAC1, which may be produced by alternative splicing or induced by hypoxia. Therefore, we investigate whether VDAC1 is overexpressed or the truncated VDAC1 is formed in cervical cancer cells, which affects tumorigenesis or cancer progression. Nm23-H1 phosphorylates nucleoside diphosphate into nucleoside triphosphate and is correlated with DNA synthesis and cell proliferation. It may influence VDAC1 imbalance. VDAC1 imbalance is associated with abnormal ADP/ATP energy mataboism and it affects mitochondrial permeability and leads to mitochondrial swelling and rupture, which enhances the release of reactive oxygen species (ROS). However, hypoxia indubible-1A (HIF-1A) is associated with ROS. Concerned with the phosphorylation of signal transducer and activator of transcription 3, enhanced ROS production or nm23-H1 change may aggravate HIF-1A expression and VDAC1 imbalance or formation of truncated VDAC1 and increase the production of vascular endothelial growth factor and thereafter promote cell proliferation and progression of cervical cancer. Furthermore, we investigate whether the reduced expression of VDAC1 (by VDAC1 knock-down) or overexpresed VDAC1 (by transfection) affect the sensitivity of cervical cancer cells to chemotherapeutic drug such as cisplatin and relate the expression of VDAC1 or nm23-H1 with clinicopathological variables, recurrence and survival of cancer patients. In a variety of human cancers, the overexpression of hexokinase and anti-apoptotic Bcl-2 is found. They may interact with VDAC1 and control cell apoptois. Therefore, we regard VDAC1 as a therapeutic target of cervical cancer and develop VDAC1-based peptides to block their interaction and inhibit growth or metastasis of cervical cancer cells. We knock down the VDAC1 gene by Lentivirus system and overexpress it by transfection in SiHa and Caski cervical cancer cells and analyze cell proliferation using cell counts and MTT assay, and apply flow cytometry to detect cell cycle and DNA fragmentation sub-G1 phase distribution and the change of ROS (oxidation of dihydroethidine to ethidium) as well as mitochondrial membrane potential [by 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) or Millipore Millicell® ERS-2], and check the expression of p-STAT3, HIF-1A and VEGF and autophagy-related proteins using Western blot and further detect cancer cells sensitivity to cisplatin as compared to SiHa and Caski cancer cells in the control group. We also detect the impact of VDAC1-based peptides on these cancer cells for the VDAC1 target therapy. We establish tissue microarrays and utilize semi-quantitative method by immunohistochemistry to associate these biomarkers, especially VDAC1, with clinicopathological variables, recurrence and survival of patients with cervical cancer. |
