| ObjectivePrevious proteomic study found that YiQiChuTan Fang(YQCTF) could significantly reduce expression of GRP78 and vimentin in mice Lewis lung tissue. Vimentin is a key marker in epithelial-mesenchymal transition (EMT), suggesting that YQCTF may be reversed EMT by decreasing the expression of GRP78. This research would explore the mechanism of GRP78 high expression induced EMT by constructing the A549 cell hypoxia model inducible expression of GRP78 and constructing GRP78 reduced interference targeting siRNA as control. Adopting specific protein inhibitor of SRC, MAPK, PI3K, AKT, smad2/3 to explore downstream pathways of GRP78 expression causing EMT. Using in vivo and in vitro experiments to explore the mechanism of YQCTF through side down GRP78 to inhibit EMT. This research may reveal the molecular mechanisms of tumor cells happen EMT in hypoxic microenvironment and provide theoretical and experimental evidence of TCM suppress EMT.Methods1. Cell experiment:Using YQCTF tool drugs, A549 cell as target cells, using hypoxia hypoxia incubator constructing A549 cell hypoxia model; annexin V/PI test apoptosis; immunofluorescence observe the distribution and expression of GRP78 in A549 cells; RT-PCR test EMT-related transcription factor (Snail1, Snail2, Twist, ZEB1, ZEB2); western blot test protein expression of EMT-related markers ((E-cadherin, vimentin and fibronectin)), and the expression of GRP78, SRC, MAPK, PI3K, AKT, smad2/3 and the phosphorylation proteins. Constructing GRP78 plasmid to silence GRP78 expression in A549 cells, combined with SRC, MAPK, PI3K, AKT, smad2/3 protein-specific inhibitors to explore molecular mechanisms of EMT in hypoxic microenvironment and the mechanism of YQCTF reversing EMT.2. Animal experiment:Building A549 lung cancer xenograft model in nude mice, and randomly divided models into control group and YQCTF group, and then intragastric administration. Measure mice weight and tumor volume every 3 days during the time of intragastric administration. In the end of intragastric administration, measuring tumor weight to calculate inhibition rate; observing lung surface metastatic nodules to calculate the rate of metastasis suppressor. Test expression of EMT-related markers ((E-cadherin, vimentin and fibronectin)), GRP78, SRC, MAPK, PI3K, AKT, smad2/3 and the phosphorylation protein in tumor tissue by immunohistochemistry.ResultsWhen Culturing A549 cells in hypoxic environment, epithelial cell morphology changed as noticeably elongated spindle-shaped stromal cell morphology. At the same time, representatives epithelial phenotype of E-cadherin protein decreased, while representatives stromal phenotypes of vimentin and fibronectin protein expression was significantly increased, EMT-associated transcription factor (Snaill, Snail2, ZEB1, ZEB2, Twist) expression was significantly increased (P<0.05), suggesting A549 cells happaned epithelial-mesenchymal transformation in hypoxic microenvironment. When adding YQCTF, interstitial cells decreased, and the degree of spindle cell morphology change was significantly reduced. At the same time, the degree of E-cadherin decrease, degree of vimentin and fibronectin increased was significantly reduced compared with hypoxia model group, EMT-associated transcription factor (Snaill, Snail2, ZEB1, ZEB2 and Twist) expression was significantly decreased (P<0.05), suggesting YQCTF could inhibit A549 cells occuring EMT.In hypoxia environment GRP78, SRC, MAPK, smad2/3 and the phosphorylated proteins increased (P<0.05), suggesting GRP78, smad2/3, SRC, P38, ERK and JNK signaling proteins may play important role in EMT. When adding YQCTF, GRP78, SRC, PI3K, AKT, MAPK, smad2/3 and the phosphorylated proteins decreased (P<0.05), suggesting YQCTF may through inhibit these protein signaling pathways to inhibit EMT.Results of immunofluorescence shows GRP78 evenly distributed in the cytoplasm and significantly high expressed in hypoxic conditions. When adding YQCTF, GRP78 significantly decreased, suggesting GRP78 play an important role in the pathogenesis of EMT.This study successfully constructed GRP78 plasmid and transfected to A549 cells, and got stably transfected cell lines by resistance screening. Applicating stably transfected A549 cell line to silent GRP78 express, combining with smad2/3, P13K, AKT, SRC, P38, ERK and JNK inhibitors to observe the effects of different signaling pathways in EMT. The results showed that when GRP78, smad2/3, SRC, P38, ERK and JNK protein was inhibited, the extent of EMT was weakened in A549 cells (P<0.05), and the impact on EMT was most obvious when silent GRP78, effect of smad2/3 and SRC in EMT took second place. While PI3K and AKT inhibitors had no effect in EMT.Further analysis showed that when silenting GRP78, compared with hypoxia model group, smad2/3, SRC, P38, ERK, JNK phosphorylation and protein expression decreased (P<0.05), while PI3K, AKT did not change significantly (P>0.05), suggesting that the mechanism of GRP78 affect EMT might connect with SRC, smad2/3, JNK, P38, ERK signaling pathway. In smad2/3 inhibitor group, GRP78 protein expression increased (P<0.05), suggesting smad2/3 activate EMT may contact with GRP78, consider smad2/3 down regulated when GRP78 silence, suggesting smad2/3 may be downstream signaling protein of GRP78, when smad2/3 was inhibition, resulting in GRP78 elevated by negative feedback.In SRC inhibitor group, P38, ERK, JNK phosphorylation and expression decreased (P<0.05), while GRP78 increased (P＜0.05), suggesting SRC activated EMT activate EMT may contact with GRP78, consider SRC down-regulated when GRP78 silence, suggesting that SRC may be another downstream signaling protein of GRP78 in EMT. In P38, ERK, JNK inhibitor groups, SRC and its phosphorylated protein were up-regulated (P<0.05), consider P38, ERK, JNK down-regulation when SRC was inhibited, suggesting that P38, ERK, JNK may be downstream signaling proteins of SRC in EMT.PCR results showed that EMT key transcription factor (Snaill, Snail2, Twist, ZEB1, ZEB2) decreased in GRP78 plasmid group, smad2/3, SRC, P38, ERK and JNK inhibitor groups(P＜0.01), suggesting GRP78, smad2/3, SRC, P38, ERK and JNK play important role in EMT when A549 cells live in hypoxic microenvironment. In the PI3K and AKT inhibitor groups, expression of Snaill, Snail2, Twist, ZEB1, ZEB2 had no significant difference compare with hypoxia model group (P>0.05).Further analysis showed that GRP78 plasmid transfection group, Snaill, Snail2, Twist, ZEB1, ZEB2 decline significantly more than smad2/3, SRC, P38, ERK and JNK inhibitor groups (P<0.05). In smad2/3, SRC inhibitor groups, Snaill, Snail2, Twist, ZEB1, ZEB2 decline more than P38, ERK, JNK inhibitor groups(P<0.05), suggesting that the impact on EMT was most obvious when silent GRP78, effect of smad2/3 and SRC in EMT took second placeAnimal experiment show that YQCTF could inhibit tumor volume and tumor weight (P<0.05), tumor inhibited rate was 27.6%. the lung metastatic nodules in YQCTF group was significantly lower than control group (P<0.05), suggesting YQCTF could inhibit tumor cell growth and inhibit metastasis of lung cancer cells in vivo.Immunohistochemistry showed that E-cadherin protein increased (P<0.01), whereas vimentin and fibronectin decreased significantly in YQCTF group (P<0.01), suggesting YQCTF could inhibit EMT occur in vivo. In YQCTF group, smad2/3, SRC, P38, ERK, JNK phosphorylation and expression decreased (P<0.05), suggesting YQCTF in vivo inhibit EMT may relate to suppress GRP78, smad2/3, SRC, JNK, P38 and ERK, consistent with the results of cell experiment. Immunohistochemistry results also show that, in YQCTF group, expression and phosphorylation of PI3K and AKT decreased compared with control group (P<0.05), suggesting YQCTF in vivo may inhibit tumor grow by inhibiting PI3K/AKT proteins signaling pathway.Conclusion1. lung cancer cell may occur epithelial-mesenchymal transition when living in hypoxic microenvironment, GRP78 play important role in the process of EMT.2. GRP78/smad2/3 and GRP78/SRC/MAPK may be important protein signaling pathways in the process of tumor cells occur EMT in hypoxic microenvironment.3. YQCTF inhibit tumor cells occur EMT in vitro and in vivo, reducing invasion and metastasis of cancer cells.4. YQCTF may through multiple inhibit GRP78/smad2/3 and GRP78/SRC/MAPK signaling pathway to achieve the reverse effect of EMT. |
PDF Full Text Request