[Mechanism of PLOD2 induced osimertinib resistance in non-small cell lung cancer HCC827 cells].

Objective: To investigate the effects of osimertinib on proliferation, migration and invasion of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) overexpressing HCC827 cells and explore the potential mechanism of PLOD2 induced osimertinib resistance. Methods: We transfected HCC827 cells with LV-vector and LV-over/PLOD2. The expression of PLOD2 was detected by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. The effects of osimertinib on the proliferation of HCC827-vector and HCC827-PLOD2 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The effects of osimertinib on the migration and invasion of HCC827-vector and HCC827-PLOD2 cells were determined by Transwell assays. The expressions of E-cadherin and vimentin in cells were detected by immunofluorescence (IF). The expressions of epithelial-mesenchymal transition (EMT), FAK-PI3K/AKT and MAPK signal pathway related proteins were detected by western blotting. Results: The MTT assay showed that HCC827-PLOD2 cells were hyposensitive to osimertinib. The 50% inhibitory concentration (IC(50)) and resistance index of osimertinib for HCC827-PLOD2 cells was over 1 000 nmol/L and over 100, respectively. The result of wound healing assay showed that the migration distance of HCC827-PLOD2 was about (2.13±0.21) fold changes as that of HCC827-vector cells. The result of Transwell assay showed that the numbers of HCC827-PLOD2 passing through the matrix membrane were (212.78±10.43), significantly higher than (101.32±12.52) of HCC827-vector cells (P<0.01). The result of IF showed that compared with HCC827-vector cells, the expression of E-cadherin was down-regulated while vimentin was up-regulated in HCC827-PLOD2 cells. Osimertinb downregulated E-cadherin and upregulated vimentin expression in HCC827-vector cells but had limited effect in HCC827-PLOD2 cells. The result of western blotting showed that PLOD2 significantly increased vimentin expression level while decreased E-cadherin expression level. Osimertinib inhibited the expression of p-EGFR, but did not affect the expressions of PLOD2, p-FAK, p-AKT, p-ERK, vimentin and E-cadherin in HCC827-PLOD2 cells. Conclusion: PLOD2 confers resistance to osimertinib in HCC827 cells by regulating EMT, FAK-PI3K/AKT and MAPK signal pathways.

An arabinose-rich heteropolysaccharide isolated from Belamcanda chinensis (L.) DC treats liver cancer by targeting FAK and activating CD40.

An undisclosed polysaccharide, BCP80-2, was isolated from Belamcanda chinensis (L.) DC. Structural investigation revealed that BCP80-2 consists of ten monosaccharide residues including t-α-Araf-(1→, →3,5)-α-Araf-(1→, →5)-α-Araf-(1→, →4)-ß-Xylp-(1→, →3)-α-Rhap-(1→, →4)-ß-Manp-(1→, t-ß-Glcp-(1→, →6)-α-Glcp-(1→, t-ß-Galp-(1→, and→3)-α-Galp-(1→. In vivo activity assays showed that BCP80-2 significantly suppressed neoplasmic growth, metastasis, and angiogenesis in zebrafish. Mechanistic studies have shown that BCP80-2 inhibited cell migration of HepG2 cells by suppressing the FAK signaling pathway. Moreover, BCP80-2 also activated immunomodulation and upregulated the secretion of co-stimulatory molecules CD40, CD86, CD80, and MHC-II. In conclusion, BCP80-2 inhibited tumor progression by targeting the FAK signaling pathway and activating CD40-induced adaptive immunity.

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin ß1/FAK signaling pathway.

BACKGROUND: Temozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PURPOSE: This study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. METHODS: The differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin ß1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. RESULTS: DEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin ß1, FAK, ß-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin ß1, ß-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin ß1/FAK pathway. CONCLUSION: We first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin ß1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

Clinical significance of integrin αV and ß superfamily members and focal adhesion kinase activity in oral squamous cell carcinoma: a retrospective observational study.

Objectives: Integrins are heterodimeric transmembrane plasma membrane proteins composed of α- and ß-chains. They bind to extracellular matrix (ECM) and cytoskeletal proteins as ECM protein receptors. Upon ECM protein binding, integrins activate focal adhesion kinase (FAK) and transduce various signals. Despite their importance, integrin and FAK expression in oral squamous cell carcinoma (OSCC) tissue and the prognosis of patients with OSCC remains elusive. Methods: In a retrospective observational study, we immunohistochemically evaluated integrin αV, ß1, ß3, ß5, ß6, FAK, and phosphorylated-FAK (pFAK) expressions as prognostic predictors in 96 patients with OSCC. Patients were classified as positive or negative based on staining intensity, and clinicopathologic characteristics and survival rates of the two groups were compared. The association between above integrin-related proteins and PD-1 or PD-L1 in OSCC tissues was investigated. Results: We observed immunohistochemical integrin αV, ß1, ß6, ß8, and FAK expressions in the cell membrane and cytoplasm but not integrin ß3 and ß5 in the OSCC tissues. pFAK was expressed in the cytoplasm of OSCC cells. The overall survival rate significantly decreased in pFAK-positive OSCC patients compared to the negative group, and cervical lymph node metastasis significantly increased in integrin ß8-positive patients with OSCC (p < 0.05). No association between integrin-related proteins and PD-1 or PD-L1 in OSCC tissues was observed. Conclusion: Our results indicate that pFAK and integrin ß8 are prognostic factors for OSCC. Therefore, pFAK- and integrin ß8-targeting new oral cancer diagnostic and therapeutic methods hold a promising potential.

Upregulation of CPNE3 suppresses invasion, migration and proliferation of glioblastoma cells through FAK pathway inactivation.

Glioblastoma (GBM) is a deadly brain tumor with a bleak prognosis. In recent years, the copine III (CPNE3) protein was discovered to be associated to metastasis across various types of malignancies. Nevertheless, its function has not been well documented in glioma. This study characterizes CPNE3 expression in GBM along with its impact and underlying molecular mechanism with regards to cellular migration, invasion and proliferation. Immunohistochemistry was used to characterizes CPNE3 expression in the glioma tissues. Then, knockdown of CPNE3 expression was used to analyze the role of CPNE3 in GBM cell viability, migration, invasion. Western blot analysis was performed to measure the protein levels of FAK signaling pathway. We found that GBM tissues had higher CPNE3 expressions as compared to those in normal brain tissues. CPNE3 silencing in GBM cells impaired the migratory, invasive and proliferative abilities of GBM cells that can be attributed to inactivation of the FAK signaling pathway. Collectively, these findings highlight the role of CPNE3 as a new biomarker, offering deeper insights into its carcinogenic role in GBM.

Knockdown of lncRNA MIR4435­2HG and ST8SIA1 expression inhibits the proliferation, invasion and migration of prostate cancer cells in vitro and in vivo by blocking the activation of the FAK/AKT/ß­catenin signaling pathway.

Prostate cancer is a main health risk for males with a high incidence and mortality. The present study aimed to examine the effects of long non­coding RNA (lncRNA) MIR4435­2HG binding with ST8SIA1 on the proliferation, invasion and migration of prostate cancer cells via the activation of the FAK/AKT/ß­catenin signaling pathway. The expression of MIR4435­2HG and ST8SIA1 in prostate cancer cell lines, and the transfection efficacy were analyzed by RT­qPCR. The proliferation, clone formation ability, and the invasion and migration of transfected cells were detected by CCK­8 assay, clone formation assay, Transwell assay and wound healing assay, respectively. Plasmids were injected subcutaneously into mice to construct a xenograft tumor model. The expression levels of proteins related to proliferation, apoptosis, invasion and migration, and the FAK/AKT/ß­catenin pathway were detected by western blot analysis. The results revealed that MIR4435­2HG expression was increased in the prostate cancer cell lines and MIR4435­2HG expression was the highest in the PC­3 cells. Interference with MIR4435­2HG inhibited the proliferation, clone formation ability, and the invasion and migration of PC­3 cells, as well as tumor growth by suppressing the activation of the FAK/AKT/ß­catenin signaling pathway. MIR4435­2HG was demonstrated to target ST8SIA1. ST8SIA1 expression was also increased in the prostate cancer cell lines and MIR4435­2HG expression was the highest in the PC­3 cells. Interference with ST8SIA1 inhibited the promoting effects of MIR4435­2HG on the proliferation, invasion and migration of PC­3 cells, as well as tumor growth by suppressing the activation of the FAK/AKT/ß­catenin signaling pathway. On the whole, the present study demonstrates that interference with MIR4435­2HG, combined with ST8SIA1, inhibits the proliferation, invasion and migration of prostate cancer cells in vitro and in vivo by blocking the activation of the FAK/AKT/ß­catenin signaling pathway.

Dietary flavonoid myricetin inhibits invasion and migration of radioresistant lung cancer cells (A549-IR) by suppressing MMP-2 and MMP-9 expressions through inhibition of the FAK-ERK signaling pathway.

Myricetin is a commonly found dietary flavonoid. In the present study, we investigated the effects of myricetin on migration and invasion of radioresistant lung cancer cells (A549-IR). Transcriptome analysis of A549-IR cells identified several differentially expressed genes (DEGs) in A549-IR cells compared to parental A549 cells. Functional enrichment analysis revealed that most of the DEGs were linked with PI3K-AKT signaling, proteoglycans, focal adhesion, and ECM-receptor interactions. A549-IR cells demonstrated enhanced migratory potential with increased expression of vimentin, snail and slug, and reduced expression of E-cadherin. A549-IR cells exposed to myricetin displayed reduced migration and suppressed MMP-2 and MMP-9 expression. Notably, myricetin inhibited the phosphorylation of focal adhesion kinase (FAK) and altered the F-actin/G-actin ratio in A549-IR cells, without modulation of EMT markers. These findings suggest that myricetin can inhibit migration of A549-IR cells by suppressing MMP-2 and MMP-9 expressions through inhibition of the FAK-ERK signaling pathway.

Inhibition of cancer cell migration with CuS@ mSiO2-PEG nanoparticles by repressing MMP-2/MMP-9 expression.

The metastasis of cancer cells is a vital aspect of disease progression and therapy. Although a few nanoparticles (NPs) aimed at controlling metastasis in cancer therapy have been reported, the NPs are normally combined with drugs, yet the direct therapeutic effects of the NPs are not reported. To study the direct influence of NPs on cancer metastasis, the potential suppression capacity of CuS@mSiO2-PEG NPs to tumor cell migration, a kind of typical photothermal NPs, was systemically evaluated in this study. Using CuS@mSiO2-PEG NP stimulation and a transwell migration assay, we found that the migration of HeLa cells was significantly decreased. This phenomenon may be associated with two classical proteins in metastasis: matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9). In addition, the mechanism may closely associate with non-receptor tyrosine kinase protein (SRC)/focal adhesion kinase (FAK) signaling pathway which varies in vivo and in vitro. To confirm the differences in the expression of SRC and FAK, related inhibitors were studied for additional comparison. Also, the results indicated that even though the migration inhibition was closely related to SRC and FAK signaling pathway, there may be another unknown regulation mechanism existing and its metastasis inhibition was significant. Confirmed by long-term survival curve study, CuS@mSiO2-PEG NPs significantly reduced the metastasis of cancer cells and improved the survival rates of metastasis in a mouse model. Thus, we believe that the direct influence of NPs on cancer cell metastasis is a promising study topic.

Effect of Banxia Xiexintang-containing Intestinal Absorption Solution on Migration and Invasion of PMN-MDSCs in Gastric Cancer Microenvironment / 中国实验方剂学杂志

ObjectiveTo observe the effect of Banxia Xiexintang containing intestinal absorption solution （BXCIAS） on migration and invasion of polymorphonuclear myeloid-derived suppressor cells （PMN-MDSCs） in gastric cancer microenvironment. MethodThe complex solution （containing 0.63 g·mL-1 crude drug） was prepared. Gastric cancer cells were subjected to non-contact co-culture with PMN-MDSCs in Transwell chamber to create gastric cancer microenvironment. Cell counting kit-8 （CCK-8） assay was used to screen the optimal intervention concentration and time of BXCIAS on PMN-MDSCs for subsequent experiment. The blank group， model group， FAK inhibitor group， and BXCIAS groups （26%， 18%， and 10%） were designed. Scratch assay and Transwell assay were employed to detect the migration and invasion ability of PMN-MDSCs， and enzyme-linked immunosorbent assay （ELISA） to measure the expression of vascular endothelial cell growth factor （VEGF） and matrix metalloproteinase-9 （MMP-9） in tumor microenvironment. The expression levels of PMN-MDSCs pathway-related proteins FAK， phosphorylated （p）-FAK， protein tyrosine kinase （Src）， and p-Src were detected by Western blot. ResultThe inhibition rates of PMN-MDSCs by 5%， 50%， 75%， and 100% BXCIAS at 48 h were higher than those at 24 h （P<0.05， P<0.01）. The inhibition rate of PMN-MDSCs by 50% BXCIAS at 72 h was lower than that at 48 h （P<0.01）， and the inhibition rates by 5% and 100% BXCIAS at 72 h were higher than those at 48 h （P<0.05， P<0.01）. There was no significant difference in the inhibition rate by other concentration levels at 48 h. The half-maximal inhibitory concentration （IC50） at 48 h was 18.09%， indicating that 18% BXCIAS and 48 h were the optimal concentration and time， respectively. The migration distance of PMN-MDSCs was large （P<0.01）， and the number of migrating and invading cells increased （P<0.01） in the mode group compared with those in the blank group. Compared with model group， FAK inhibitor and BXCIAS at different concentration decreased the migration distance of PMN-MDSCs （P<0.01）， and the number of migrating and invading cells （P<0.01）， especially the 26% BXCIAS （P<0.01）. The expression of PMN-MDSCs pathway-related proteins FAK， p-FAK， Src and p-Src （P<0.01） and the expression of VEGF and MMP-9 （P<0.01） were higher in the model group than in the blank group. Compared with model group， FAK inhibitor and BXCIAS （26%， 18%， 10%） decreased the expression of FAK， p-FAK， and Src （P<0.01）， and FAK inhibitor and 18% BXCIAS reduced the expression of p-Src （P<0.01）， and the expression of VEGF and MMP-9 （P<0.01）. ConclusionBXCIAS can inhibit the migration and invasion of PMN-MDSCs by down-regulating the expression of FAK， p-FAK， Src， and p-Src proteins in the FAK signaling pathway of PMN-MDSCs in gastric cancer microenvironment.

Connexin 32 affects doxorubicin resistance in hepatocellular carcinoma cells mediated by Src/FAK signaling pathway.

Doxorubicin (DOX) is first-line chemotherapy for hepatocellular carcinoma (HCC), but the effect is not satisfactory. The resistance of HCC cells to DOX is the main reason leading to treatment failure. Therefore, it is necessary to study the mechanism of DOX resistance in HCC. In this study, expression of connexin (Cx)32 was significantly decreased in HCC tissues compared with corresponding paracancerous tissues, and activity of the Src/focal adhesion kinase (FAK) signaling pathway was significantly enhanced. Expression of Cx32 was closely associated with activity of the Src/FAK signaling pathway, Cx32, and the Src/FAK signaling pathway was also correlated with degree of HCC differentiation. In DOX-resistant HepG2 cells, compared with DOX-sensitive HepG2 cells, expression of Cx32 was significantly reduced and activity of the Src/FAK pathway increased. After silencing Cx32 in HepG2 cells, activity of the Src/FAK pathway increased and sensitivity to DOX decreased. In contrast, overexpression of Cx32 in HepG2/DOX cells decreased activity of the Src/FAK pathway and increased sensitivity to DOX. Dasatinib and KX2-391, inhibitors of the Src/FAK pathway, significantly increased the sensitivity of HepG2/DOX cells to DOX. The results suggest that Src/FAK is a downstream regulator of Cx32 and Cx32 regulates the sensitivity of HCC cells to DOX via the Src/FAK signaling pathway. Our study demonstrates a potential mechanism of DOX resistance in HCC cells and supports that Cx32-Src/FAK is an important target for reversing drug resistance of HCC.

Lewis y enhances CAM-DR in ovarian cancer cells by activating the FAK signaling pathway and upregulating Bcl-2/Bcl-XL expression.

Oligosaccharides on the surface of adhesion molecules may contribute to the process of CAM-DR. To investigate the role of the Lewis y antigen in this process, we established a cell adhesion model mediated by the integrin α5ß1-FN interaction in the ovarian cancer cell line, RMG-1-hFUT, which highly expresses Lewis y by transfection with α1,2-fucosyltransferase into RMG-1 cells. Our results indicate that the rates of carboplatin-induced apoptosis and necrosis are reduced in FN-adhered tumor cells, and carboplatin resistance is significantly decreased in the presence of anti-Lewis y antibody. CAM-DR in tumor cells has been correlated with elevated expression of the nuclear anti-apoptotic proteins Bcl-2 and Bcl-XL. Lewis y promotes the expression of the Bcl-2 and Bcl-XL genes by activating the focal adhesion kinase signaling pathway and accelerating their transcription. Thus, Lewis y leads to inhibition of apoptosis and enhancement of CAM-DR by activation of the FAK signaling pathway and upregulation of Bcl-2/Bcl-XL expression in ovarian cancer cell lines.