This study investigated the effects and action mechanism of tannins from Galla chinensis cream(TGCC) on the skin wound of rat tail. Male Sprague Dawley(SD) rats were randomly divided into a control group, model group, model+low-dose TGCC(50 mg per rat) group, model+high-dose TGCC group(100 mg per rat), and model+TGC+FAK inhibitor(Y15) cream(100 mg+10 mg per rat) group, with 10 rats in each group. After the rat tail skin injury model was successfully constructed, in the treatment group, corresponding drugs were applied to the wound surface, while in the control and model groups, the same amount of cream base as the TGCC group was applied by the same method. Then, sterile gauze was wrapped around the wound edge, and these operations were performed three times a day for 28 consecutive days. The wound healing status at the third, seventh, eleventh, fourteenth, twenty-first, and twenty-eighth days was recorded, and the wound healing rate and healing time were calculated. On the day after the last dose of medication, rat serum and tail skin wound tissue were collected for analyzing the activities of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), creatinine(CREA), urea, reactive oxygen species(ROS), interferon gamma(IFN-γ), interleukin(IL)-1β, IL-6, IL-4, IL-10, tumor necrosis factor(TNF)-α, as well as catalase(CAT), glutathione(GSH), lactate dehydrogenase(LDH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC), platelet endothelial cell adhesion molecule-1(CD31), and leukocyte differentiation antigen 34(CD34) in the wound tissue of rat tail skin. Hematoxylin-eosin, Masson, and sirius red staining were used to observe the morphological changes in the wound tissue of rat tail skin. The thickness of the epidermis, the number of fibroblasts and blood vessels, and the contents of collagen fibers, typeⅠ collagen(COLⅠ), and COLⅢ were calculated. The mRNA expressions of keratin 10(KRT10), KRT14, vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF), epidermal growth factor(EGF), CD31, CD34, matrix metallopeptidase-2(MMP-2), MMP-9, COLⅠ, COLⅢ, desmin, fibroblast specific protein 1(FSP1), IFN-γ, IL-1β, TNF-α, IL-4, IL-6, and IL-10 in skin wound tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expressions of KRT10, KRT14, VEGF, FGF, EGF, MMP-2, MMP-9, COLⅠ, COLⅢ, desmin, FSP1, focal adhesion kinase(FAK), phosphorylated focal adhesion kinase(p-FAK), phosphatidylin-ositol-3-kinase(PI3K), phosphorylated phosphatidylin-ositol-3-kinase(p-PI3K), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated mammalian target of rapamycin(p-mTOR). The results manifest that TGCC can dramatically elevate the healing rate of rat tail wounds and shorten wound healing time. Besides, it can reduce serum ROS levels, the contents of MDA, MPO, and LDH in the rat skin wound tissue, as well as the serum IFN-γ, IL-1β, IL-6, and TNF-α levels and the mRNA expression levels of IFN-γ, IL-1β, IL-6, and TNF-α in the skin wound tissue. It can elevate the activities of CAT, GSH, SOD, and T-AOC in wound tissue, the IL-4 and IL-10 contents in serum, and the mRNA expressions of IL-4 and IL-10 in the wound tissue. In addition, TGGC can inhibit inflammatory cell infiltration and increase the epidermal thickness, counts of fibroblasts and blood vessels, and contents of collagen fibers, COLⅠ, and COLⅢ. Besides, TGCC can elevate the mRNA and protein expressions of epidermal differentiation markers(KRT10 and KRT14), endothelial cell markers(CD31 and CD34), angiogenesis and fibroblast proliferation, differentiation markers(VEGF, FGF, EGF, COLⅠ, COLⅢ, desmin, and FSP1), reduce the mRNA and protein expressions of gelatinases(MMP-2 and MMP-9), and increase protein expressions of p-FAK, p-PI3K, p-Akt, p-mTOR, as well as ratios of p-FAK/FAK, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR. These results suggest that TGCC can significantly facilitate skin wound healing, and its mechanism may be related to the activation of the FAK/PI3K/Akt/mTOR signaling pathway, inhibition of inflammatory cell infiltration in skin wound tissue, elevation of epidermal thickness, counts of fibroblasts and vessels, and contents of collagen fiber, COLⅠ, and COLⅢ, and reduction of MMP-2 and MMP-9 expressions, thus accelerating wound healing.
Animals ; Male ; Wound Healing/drug effects* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; TOR Serine-Threonine Kinases/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Skin/metabolism* ; Proto-Oncogene Proteins c-akt/genetics* ; Tannins/pharmacology* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Focal Adhesion Kinase 1/genetics*

Insufficient alveolar bone thickness increases the risk of periodontal dehiscence and fenestration, especially in orthodontic tooth movement. Abaloparatide (ABL), a synthetic analog of human PTHrP (1-34) and a clinical medication for treating osteoporosis, has recently demonstrated its potential in enhancing craniofacial bone formation. Herein, we show that intraoral submucosal injection of ABL, when combined with mechanical force, promotes in situ alveolar bone thickening. The newly formed bone is primarily located outside the original compact bone, implying its origin from the periosteum. RNA sequencing of the alveolar bone tissue revealed that the focal adhesion (FA) pathway potentially mediates this bioprocess. Local injection of ABL alone enhances cell proliferation, collagen synthesis, and phosphorylation of focal adhesion kinase (FAK) in the alveolar periosteum; when ABL is combined with mechanical force, the FAK expression is upregulated, in line with the accomplishment of the ossification. In vitro, ABL enhances proliferation, migration, and FAK phosphorylation in periosteal stem cells. Furthermore, the pro-osteogenic effects of ABL on alveolar bone are entirely blocked when FAK activity is inhibited by a specific inhibitor. In summary, abaloparatide combined with mechanical force promotes alveolar bone formation via FAK-mediated periosteal osteogenesis. Thus, we have introduced a promising therapeutic approach for drug-induced in situ alveolar bone augmentation, which may prevent or repair the detrimental periodontal dehiscence, holding significant potential in dentistry.
Osteogenesis/drug effects* ; Periosteum/cytology* ; Parathyroid Hormone-Related Protein/administration & dosage* ; Animals ; Focal Adhesion Protein-Tyrosine Kinases/metabolism* ; Alveolar Process/drug effects* ; Cell Proliferation/drug effects* ; Phosphorylation ; Rats ; Male ; Humans ; Focal Adhesion Kinase 1/metabolism* ; Cell Movement/drug effects*

Based on the focal adhesion kinase(FAK)/steroid receptor coactivator(Src)/extracellular regulated protein kinase(ERK) pathway, this study explored the effects of Xihuang Pills on angiogenesis, invasion, and metastasis in prostate cancer. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) was used to analyze and identify the active ingredients of Xihuang Pills. Bioinformatics techniques, including R language and Perl programs, were employed to analyze the interactions between prostate cancer-related targets and the potential targets of Xihuang Pills. A subcutaneous transplantation tumor model of prostate cancer was established in nude mice using PC3 cells to verify the efficacy and molecular mechanisms of Xihuang Pills. In vitro cellular experiments, including cell proliferation assays(CCK-8), Transwell assays, scratch assays, real-time quantitative reverse transcription PCR, and Western blot, were used to detect the effects of Xihuang Pills on the proliferation, invasion, and migration of prostate cancer cells, as well as on FAK/Src/ERK pathway-related targets. LC-MS/MS identified 99 active ingredients in Xihuang Pills, including gallic acid, gentisic acid, artemisinin, corilagin, phenylbutazone-glucoside, thujic acid, and arecoic acid B. Network pharmacological analysis of the active ingredients in Xihuang Pills revealed that the FAK/Src/ERK signaling pathway was a key pathway in its anti-prostate cancer effects. In vivo and in vitro experiments confirmed that Xihuang Pills significantly inhibited the proliferation, invasion, and migration of PC3 and LNCaP cells, suppressed the growth of PC3 subcutaneous tumors, and reduced the protein expression levels related to the FAK/Src/ERK signaling pathway. In conclusion, the inhibition of angiogenesis, invasion, and metastasis by regulating the FAK/Src/ERK pathway is one of the mechanisms by which Xihuang Pills exert anti-prostate cancer effects.
Humans ; Male ; Prostatic Neoplasms/enzymology* ; Drugs, Chinese Herbal/chemistry* ; Animals ; Mice ; Cell Proliferation/drug effects* ; Mice, Nude ; Cell Movement/drug effects* ; Cell Line, Tumor ; src-Family Kinases/genetics* ; Neovascularization, Pathologic/metabolism* ; Neoplasm Metastasis ; Neoplasm Invasiveness ; Focal Adhesion Kinase 1/genetics* ; Extracellular Signal-Regulated MAP Kinases/genetics* ; MAP Kinase Signaling System/drug effects* ; Focal Adhesion Protein-Tyrosine Kinases/genetics* ; Signal Transduction/drug effects* ; Angiogenesis

Objective: To uncover the time-dependent expression pattern of ptk2b gene and ptk2b-encoded protein, protein tyrosine kinase 2 beta(PTK2B), in the brain tissues of transgenic animal models of Alzheimer's disease (AD) and its relationship with the levels of Aβ_1-42, phosphorylation of Tau (p-Tau) and low density lipoprotein receptor-related protein-1(LRP-1) in blood and brain tissues. Methods: In this study, 5-, 10- and 15-month-old APPswe/PS1dE9 double-transgenic mice harboring the genotype of AD confirmed by the gene test were divided into the 5-, 10- and 15-month-old experiment groups, and simultaneously, age-matched C57BL/6J mice were placed into the corresponding control groups, with 8 mice in each group. All mice were subjected to the Morris Water Maze for test of cognitive and behavioral ability. Expression profiles of PTK2B, Aβ_1-42, p-Tau/Tau and LRP-1 in the hippocampus or blood of mice were quantified by using the immunohistochemistry staining, Western blot or enzyme-linked immunosorbent assay (ELISA), while the mRNA expression of ptk2b in the hippocampus was quantified by using the real-time quantitative polymerase chain reaction (qRT-PCR). Results: Results of experiment groups demonstrated that as mice aged, the expression levels of PTK2B, ptk2b mRNA, Aβ_1-42 and p-Tau/Tau in the hippocampus were increased, and the expression of LRP-1 was decreased gradually. While in the blood, the level of Aβ_1-42 was decreased, and the cognitive and behavioral ability was decreased in an age-dependent manner (all P＜ 0.05). However, comparisons among the control groups, only the age-dependent downregulation of LRP-1 were observed in hippocampus(P＜0.05), but other indicators had no significant differences (P＞0.05). Conclusion: In the hippocampus of APP/PS1 double-transgenic mice, the expressions of PTK2B, Aβ_1-42 and p-Tau/Tau are upregulated, LRP-1 is downregulated, while cognitive and behavioral ability is decreased, and such changes are presented in a time-dependent manner.
Alzheimer Disease/metabolism* ; Amyloid beta-Peptides ; Amyloid beta-Protein Precursor/genetics* ; Animals ; Focal Adhesion Kinase 2/metabolism* ; Hippocampus/metabolism* ; Low Density Lipoprotein Receptor-Related Protein-1 ; Maze Learning ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; RNA, Messenger

This study was designed to evaluate ERK5 expression in lung cancer and malignant melanoma progression and to ascertain the involvement of ERK5 signaling in lung cancer and melanoma. We show that ERK5 expression is abundant in human lung cancer samples, and elevated ERK5 expression in lung cancer was linked to the acquisition of increased metastatic and invasive potential. Importantly, we observed a significant correlation between ERK5 activity and FAK expression and its phosphorylation at the Ser
A549 Cells ; Animals ; Cell Movement ; Epithelial-Mesenchymal Transition/genetics* ; Focal Adhesion Kinase 1/metabolism* ; Humans ; Lung Neoplasms/pathology* ; MAP Kinase Signaling System ; Mice ; Mitogen-Activated Protein Kinase 7/metabolism* ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Proteins/metabolism*

Laryngeal squamous cell carcinoma (LSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC) worldwide. Protein phosphatase 2A (PP2A) dysfunction has been widely reported in a broad range of malignancies due to its distinctive role in miscellaneous cellular processes. However, it is poorly understood whether aberrant alterations of PP2A are involved in the network of oncogenic events in LSCC. Here, we detected a panel of PP2A-associated proteins using western blot in both laryngeal squamous cell carcinoma tissues and paired adjacent normal tissues from patients (Data S1). We found that phospho-PP2A/C (Y307), α4, cancerous inhibitor of protein phosphatase 2A (CIP2A), Akt, ezrin, phospho-ezrin (T567), 14-3-3, and focal adhesion kinase (FAK) showed increased expression levels in carcinoma tissues relative to normal tissues, while phospho-Akt (T308) showed decreased levels. Our study, thus, provides a rationale for targeting PP2A to develop novel therapies and proposes a combination of interrelated biomarkers for the diagnostic evaluation and prognosis prediction in LSCC.
Autoantigens/metabolism* ; Biomarkers, Tumor/metabolism* ; Carcinoma, Squamous Cell/metabolism* ; Case-Control Studies ; Cytoskeletal Proteins/metabolism* ; Focal Adhesion Kinase 1/metabolism* ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; Intracellular Signaling Peptides and Proteins/metabolism* ; Laryngeal Neoplasms/metabolism* ; Larynx/metabolism* ; Membrane Proteins/metabolism* ; Phosphorylation ; Protein Phosphatase 2/metabolism*

Metastasis is responsible for the majority of cancer-related deaths and prevention of metastasis remains a big challenge for cancer therapy. Cucurbitacin B (Cuc B) is a natural triterpenoid with potent anticancer activities while its effect on metastasis remains unclear. In the present study, the inhibitory effect and mechanisms of Cuc B on metastasis were investigated in MDA-MB-231 breast cancer cells. The cells were treated with or without Cuc B, and the cytotoxicity was determined by MTT assay. The effect of Cuc B on metastasis was evaluated with wound healing, transwell, and adhesion assays. Furthermore, the adhesion of cancer cells to endothelial cells was determined. The protein expression was determined by Western blotting. Cuc B (< 100 nmol·L) showed no obvious cytotoxicity to MDA-MB-231 cells, but significantly inhibited migration, invasion, and adhesion to Matrigel, fibronectin, type I collagen, and endothelial cells. Cuc B dramatically inhibited the phosphorylation of focal adhesion kinase (FAK) and paxillin in dose- and time-dependent manners. Furthermore, Cuc B induced intracellular reactive oxygen species (ROS) generation, which could be reduced by N-acetyl-l-cysteine (NAC). In addition, NAC pretreatment could reverse Cuc B-induced suppression of migration and adhesion, expression of FAK, but showed no effect on paxillin expression. In summary, Cuc B suppressed ROS-dependent metastasis through FAK pathway in breast cancer MDA-MB-231 cells, demonstrating novel mechanisms for the anticancer effects of Cuc B.
Acetylcysteine ; pharmacology ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; enzymology ; metabolism ; pathology ; physiopathology ; Cell Adhesion ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Collagen Type I ; metabolism ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Female ; Fibronectins ; metabolism ; Focal Adhesion Kinase 1 ; metabolism ; Humans ; Neoplasm Invasiveness ; pathology ; Neoplasm Metastasis ; pathology ; Paxillin ; metabolism ; Phosphorylation ; drug effects ; Reactive Oxygen Species ; metabolism ; Triterpenes ; antagonists & inhibitors ; chemistry ; pharmacology

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.
AMP-Activated Protein Kinases/antagonists & inhibitors/chemistry/*metabolism ; Aminoimidazole Carboxamide/analogs & derivatives/pharmacology ; Angiotensin II/*pharmacology ; Angiotensin II Type 1 Receptor Blockers/pharmacology ; Animals ; Blotting, Western ; Cell Line ; Cell Nucleus/metabolism ; Crk-Associated Substrate Protein/*metabolism ; Cytoplasm/metabolism ; Focal Adhesion Kinase 1/metabolism ; Losartan/pharmacology ; Metformin/pharmacology ; Mice ; Microscopy, Confocal ; Podocytes/cytology/drug effects/metabolism ; Protein Kinase Inhibitors/*pharmacology ; Ribonucleotides/pharmacology ; Signal Transduction/*drug effects

Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP (an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.
A549 Cells ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Chemokine CCL5 ; metabolism ; Focal Adhesion Kinase 1 ; metabolism ; Humans ; Lung Neoplasms ; Marsdenia ; chemistry ; Phosphorylation ; Plant Extracts ; pharmacology ; Receptors, CCR5 ; metabolism ; rho GTP-Binding Proteins ; metabolism ; rhoC GTP-Binding Protein

OBJECTIVETo investigate the effects of sphingosine kinase 1 (SphK1) on the proliferation, migration and invasion of human colon cancer LoVo cells, and to explore the related mechanisms.

METHODSHuman colon cancer LoVo cells were divided into three groups: phorbol 12-myristate 13-acetate (PMA) was used to induce the activation of SphK1 in the PMA group, N,N-dimethylsphingosine (DMS) used to suppress the activity of SphK1 in DMS group, and the cells treated with equal amount of 0.9 % NaCl instead of drugs served as the control group. The activity of SphK1 was assayed by autoradiography, the cell proliferation was assessed by MTT assay, cell migration and invasion were examined by Boyden chamber assay, concentrations of sICAM-1 and sVCAM-1 were assayed by ELISA, and RT-PCR and Western blot were used to evaluate the mRNA and protein expression in the cells.

RESULTSThe activity of SphK1 was efficiently induced by PMA and significantly suppressed by DMS. PMA induced cell proliferation in a time- and dose-dependent manner. On the contrast, DMS suppressed cell proliferation in a time- and dose-dependent manner. After treating with PMA, the number of migrating and invasing cells were increased to 143.36 ± 8.73 and 118.46 ± 6.25, significantly higher than those of the control group (75.48 ± 6.12 and 64.19 ± 5.36). After treating with DMS, the number of migrating and invasing cells were decreased to 38.57 ± 3.24 and 32.48 ± 4.27, significantly lower than those of the control group (P < 0.01). The relative expression levels of FAK, ICAM-1 and VCAM-1 mRNA in the PMA group were 0.82 ± 0.06, 0.74 ± 0.05 and 0.89 ± 0.09, and those in the DMS group were 0.23 ± 0.02, 0.26 ± 0.03 and 0.37 ± 0.04, with significant differences between the PMA, DMS and control groups (P < 0.01). Compared with the control group, the relative expression levels of FAK and p-FAK proteins in the PMA group (0.52 ± 0.06 and 0.51 ± 0.06) were significantly elevated, and those of the DMS group (0.20 ± 0.03 and 0.09 ± 0.02) were significantly decreased. In addition, the concentrations of sICAM-1 and sVCAM-1 were significantly elevated with the activation of SphK1. On the contrary, those of the DMS group were significantly reduced with the suppression of SphK1 (Both P < 0.01).

CONCLUSIONSSphK1 may enhance the proliferation, migration and invasion of colon cancer LoVo cells through activating FAK pathway and up-regulating the expression of ICAM-1 and VCAM-1.

Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; enzymology ; metabolism ; pathology ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Neoplasm Invasiveness ; Phosphorylation ; drug effects ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; Sphingosine ; analogs & derivatives ; pharmacology ; Tetradecanoylphorbol Acetate ; pharmacology ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism