碩士 / 國防醫學院 / 藥理學研究所 / 105 / Background
According to the report of Taiwan Ministry of Health and Welfare, the mortality rate of lung cancer has ranked as top one of cancerous death in Taiwan. During the development of tumor, cancer cells has acquired the ten traits as following: sustaining proliferative signaling, evading growth suppressors, resisting cells death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, avoiding immune destruction, promoting inflammation, genome instability and mutation and deregulating cellular energetics.
Homeostasis of intracellular pH (pHi) regulates many cellular functions, such as cell growth, migration and apoptosis in mammalian cells. Cancer cells have been characterized with alkaline pHi values (≥ 7.2) and acidic extracellular pH (pHe) values (≦7.0). This ‘reversed’ gradient enables cancer progression by promoting proliferation, evasion of apoptosis, metabolic adaptation, migration and invasion. The pHi regulators include, at least, Na+/H+ exchanger (NHE), Na+/HCO3- co-transporter (NBC), Cl-/HCO3- exchanger (AE), Cl-/OH- exchanger (CHE) and monocarboxylate/H+ (MCT) in many mammalian cells.
Isoorientin (C21H2OO11) is the principle flavonoid-like compound found in Lophatherum gracile. It shows hepatoprotective, anti-viral, anti-bacterial, anti-cancer, anti-oxidant, diuretic and hyperglycemic properties. Moreover, daily administration of the extract of crude Lophatherum Herba was shown to inhibit tumor growth. Previous studies show at that inhibition of MCT1and MCT4 activity reduced the pHi and inhibited cancer cell migration. Moreover, studies have demonstrated that isoorientin up-regulated tumor suppressor elements such as MMP2 and MMP9, and further reduced cancer cells migration. However, the relationship between active pHi regulators and tumor suppressors is not clear in human lung cancer tissues and A549 cells (human lung cancer cell line). The aims of the present study were to i) analyze the expression of MCT1/4 and CD147; ii) characterize the functional acid extruding mechanism; iii) examine the concentration-dependent effect of isoorientin (3~300 μM) on activity/expression of MCTs and cell migration, and iv) explore the isoforms of active transmembrane acid extruders in A549 cell line.
Materials and Methods
Human lung cancer tissues were obtained from cancer patients undergoing surgery, with the agreement of patients and the approval of the institutional review committee (TSGHIRG No. 2-106-05-134). The change of pHi in cultured A549 cells was detected by microspectrofluorometry method with a pH-sensitive fluorescent dye, BCECF. In standard HEPES-buffered Tyrode solution, we used lactate perfusion technique to induce intracellular acidosis. The subsequently lactate-induced influx and efflux rate were used to represent the activity of bi-directional lactate-related regulating mechanism. AR-C155858 (a MCT1/2 inhibitor) and SR13800 (a specific MCT1 inhibitor) were used to examine the existence of MCTs. Wound-healing assays and electric cell-substrate impedance sensing (ECIS) were used to evaluate the effects of isoorentin on the migration. The protein expression of possible acid-extruders and related apoptotic proteins were examined by Western blot. Lactate assay was used to detect the intracellular lactate of A549. MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to detect the cell viability of A549 after incubated with isoorientin for designated times. A549 cells used were commercial bought.
Results
1. The significant increase of protein expression of MCT1, MCT4 and CD147 were observed in human lung adenocarcinoma tissues, dectected by using IHC method, while the protein expression of MCT2 and MCT3 remained unchanged significantly.
2. In the HEPES-buffered system, rapidly intracellular acidification and alkalization following superfusion and removal of 10 mM lactate demonstrated that MCT functionally exist in A549. Either adding 0.3 μM AR-C155858 (MCT1/2 inhibitor) or 3 μM SR13800 (MCT1 specific inhibitor) inhibited the lactate transport that pharmacology demonstrated the existance of MCT1 in A549 cells (n=6; p < 0.01).
3. AR-C155858 significantly inhibited cell migration in A549 cells at 12 and 24 hr (-40 and -45%, respectively), respectively, and the saturated inhibition concentration of AR-C155858 is 0.3 μM (n=3; p < 0.001).
4. AR-C155858 (0.1 to 10μM) didn’t change the cell viability in A549 for 12 and 24 hr (n=6; p > 0.05), and demonstrated that inhibition of migration by AR-C155858 was not due to it’s effect on cell viability or death.
5. In HEPES buffered solutions, isoorientin (3 to 300 μM) concentration-dependently decreased pHi lactate influx (-12~-36%, respectively) and efflux rate (-10~-42%, respectively) in A549 cells (n=6; p < 0.01).
6. In the HEPES-buffered system, pre-treatment with isoorientin (3 to 100 μM) for 24 hr significantly inhibited the lactate influx (-20%) and efflux (-25%), and 10 μM is the concentration of saturated effect (n=6; p < 0.01). Moreover, pre-treatment with isoorientin (3~100 μM) for 48 hr also significantly inhibited the lactate influx and efflux, and the concentration of saturated effect is 3 and 100 μM (-10% and -7%, respectively, n=5; p < 0.05).
7. Isoorientin (3 to 300 μM) significantly inhibited A549 cell migration at 12 and 24 hr (-15% and -40%, respectively), and the concentration of saturated effect is 10 μM (n=6; p < 0.001). In addition, administration of isoorientin (3 to 10 μM) also significantly inhibited cell migration. (-15% and -11%, respectively, n=6; p < 0.05).
8. Isoorientin (1 to 300 μM) didn’t change the A549 cell viability for 12, 24, and 48 hr (n=6; p > 0.05), the results showed that inhibition of migration by isoorientin was not due to the effect of cell viability or death.
9. The Western blot analysis showed that the expression of MCT1/4, CD147, MMP2 and MMP9 was significantly inhibited by the treating with isoorientin for 24 hr in A549 cells (n=4, p<0.05). However, treating with isoorientin for 48 hr did not inhibit the MCT1/4, CD147, and MMP2/9 protein expression (n=4, p>0.05).
Conclusions
In conclusion, this study demonstrates for the first time that the protein expression of MCTs1/4 and CD147 specifically upregulated in human lung tumor tissues of Taiwanese patients, while that of MCT2 and MCT3 remained unchanged. In addition, this study revealed the functional existence of MCT responsible for lactate influx and efflux in cultured A549 cells. Moreover, the data showed that isoorientin inhibits cells migration by decreasing metastasis proteins, reducing activity and expression of MCT1/4, MMP2 and MMP9 in A549 cells. Therefore, my present study implicates that isoorientin or potential MCTs inhibitor may be a promising novel targeting agent in the treatment of lung cancer.
| Identifer | oai:union.ndltd.org:TW/106NDMC0550002 |
| Date | January 2018 |
| Creators | LEE, SHIN-YI, 李欣怡 |
| Contributors | Loh, Shih-Hurng, 羅時鴻 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 127 |
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