Transwell Chamber Research Articles

Human monocytes differentiate into tumor-associated macrophages upon SKOV3 cells coculture and/or lysophosphatidic acid stimulation

BackgroundSerous ovarian carcinoma is the most common type of ovarian carcinoma. Tumor-associated macrophages (TAMs) promote ovarian cancer progression. Most macrophages are generated by monocyte differentiation. Lysophosphatidic acid (LPA) levels are high in blood, tissues and ascites of patients with ovarian cancer. This study investigated whether human monocytes can directly differentiate into TAMs in the serous ovarian carcinoma microenvironment.MethodsHuman monocytes were isolated and purified from umbilical cord blood. A serous ovarian carcinoma-like microenvironment was generated by coculturing monocytes and SKOV3 cells in 0.4-μm-pore-size Transwell chambers. Additionally, the effect of LPA was assessed. The two cultured cell types and supernatants were evaluated.ResultsThe morphology and function of monocytes cocultured with SKOV3 cells and/or stimulated with LPA were significantly changed compared with those of non-stimulated monocytes. The CD14 + CD163 + and CD206 + phenotype indicated that stimulated cells were TAMs. The induced cells promoted SKOV3 cell proliferation and invasion, further proving that they were TAMs. The level of the cytokine interleukin-6R in the supernatant was significantly elevated in the treatment groups compared to the control monocyte group. Pathway enrichment analysis of ELISA results showed a strong influence of interleukin-6 family signaling, especially the JAK-STAT signaling pathway, further confirming the importance of IL-6R.ConclusionMonocytes can differentiate into TAMs under coculture with SKOV3 cells and/or LPA stimulation. The induced TAMs promote SKOV3 cell proliferation and invasion. The cytokine receptor IL-6sR and the JAK-STAT signaling pathway play an important role in the differentiation of monocytes into TAMs.

The Regulatory Role of SNORD35A in Pancreatic Cancer Involves the HGF/C-Met Pathway.

Background: Pancreatic adenocarcinoma (PAAD) is a type of malignant tumors in the digestive tract. It is extremely aggressive. However, the molecular mechanism of the occurrence and development of pancreatic cancer has not yet been elucidated. New evidence shows that the dysregulation of small nucleolar RNAs (SnoRNAs) plays an important role in tumorigenesis and has a certain connection with tumor stem cells. In this study, the authors screened differentially expressed SnoRNAs in pancreatic cancer, further explored whether the HGF/C-Met pathway is involved in the regulation of SNORD35A in pancreatic cancer stem cells. Materials and Methods: AffymetrixmiRNA 4.0 and QRT-PCR was used for differential screening of SnoRNA. CCK8, wound healing assay and TransWell chamber were used to detect cell proliferation, migration and invasion. QRT-PCR was used to detect the changes of epithelial - mesenchymal Transition (EMT) related genes of tumors. The authors detected the expression levels of HGF/C-Met pathway and its related proteins by Western blotting. Result: The authors found that SNORD35A is significantly overexpressed in pancreatic cancer. After disturbing the expression of SNORD35A, the epithelial markers increased and the mesenchymal markers decreased during the EMT process. At the same time, down-regulation of SNORD35A inhibited the proliferation, migration and invasion of pancreatic cancer stem cells in cellular level. In nude mouse transplanted tumor models, low expression of SNORD35A reduced tumor growth volume and attenuated its pathological features. Finally, the authors found that silencing SNORD35A reduced the expression levels of C-Met and its phosphorylated proteins. Conclusion: These results suggest that the regulation of SNORD35A on proliferation, migration, invasion and EMT of pancreatic cancer stem cells involves HGF/C-Met signaling pathway. SNORD35A has carcinogenic effects in pancreatic cancer and may become a prognostic biomarker and therapeutic target for pancreatic cancer patients.

LncRNA ZFAS1 Promotes HMGCR mRNA Stabilization via Binding U2AF2 to Modulate Pancreatic Carcinoma Lipometabolism

Being one of the most lethal malignant tumors worldwide, pancreatic carcinoma (PC) shows strong invasiveness and high mortality. In tumorigenesis and progression, the role played by long-chain noncoding RNAs (lncRNAs) cannot be ignored. This article mainly probes into the function of lncRNA ZFAS1 in PC. ZFAS1 expression in PC and normal counterparts retrieved from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Atlas (TCGA) database was analysed by GEPIA2. Its expression profile in clinical specimens and human PC cell strains was quantified using qRT-PCR. Measurements of BxPC-3 cell multiplication and invasiveness employed CCK-8, plate clone formation test, and Transwell chamber assay. ZFAS1's impact on lipid content in BxPC-3 cells was detected. RNA pulldown and RIP assays analyzed the interaction of ZFAS1 with U2AF2 and HMGCR in BxPC-3 cells. Finally, the impacts of U2AF2 and HMGCR on the biological behavior of BxPC-3 were observed. ZFAS1 was kept at a high level in PC tissues versus the normal counterparts. ZFAS1 gene knockout remarkably suppressed PC cell multiplication and invasiveness and decreased the contents of free fatty acids, total cholesterol, triglycerides, and phospholipids. Mechanistically, ZFAS1 stabilized HMGCR mRNA through U2AF2, thus increasing HMGCR expression and promoting PC lipid accumulation. Meanwhile, reduced PC cell viability and invasiveness were observed after downregulating U2AF2 and HMGCR. As an oncogene of PC, ZFAS1 can modulate lipometabolism and stabilize HMGCR mRNA expression by binding with U2AF2 in PC, which is a candidate target for PC diagnosis and treatment.

HECTD3 enhances cell radiation resistance and migration by regulating LKB1 mediated ZEB1 in glioma.

Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3) has been reported to play a role in carcinogenesis. Here, we explored the role of HECTD3 in regulating the radiation resistance of glioma, and the underlying mechanism. HECTD3 expressions in glioma tissues were assessed using Western blotting, quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and immunohistochemistry. Glioma cells were exposed to 2-, 4-, 6- or 8-Gy X-ray to mimic the radiation treatment. Cell count kit-8 (CCK-8), clone formation assay, flow cytometry assay, transwell chambers and animal assay were used to test cell viability, apoptosis, migration, invasiveness and tumourigenesis, respectively. HECTD3 expression was increased in glioma tissues, especially from patients with radiation resistance. Knockdown of HECTD3 promoted cell apoptosis and inhibited cell viability under the condition of 8-Gy X-ray, as well as suppressed cell migration and invasiveness. In mechanism, HECTD3 positively regulated ZEB1 (zinc finger E-box binding hemeobox 1) expression through regulating the ubiquitination of liver kinase B1 (LKB1) protein. Overexpression of ZEB1 significantly abolished the effects of HECTD3 downregulation in inhibiting the radiation resistance and migration of glioma cells. Moreover, downregulation of HECTD3 further enhanced the anti-tumour effect of X-ray on glioma growth in vivo. In conclusion, HECTD3 was overexpressed in glioma patients with radiation resistance. Knockdown of HECTD3 sensitized glioma cells to radiation and inhibited cell migration by downregulating ZEB1 expression via regulating the ubiquitination of LKB1 protein. This study reveals that HECTD3 might be a potent target to enhance the radiation sensitivity of glioma.

Evaluation of potential anti-metastatic and antioxidative abilities of natural peptides derived from Tecoma stans (L.) Juss. ex Kunth in A549 cells.

BackgroundTecoma stans (L.) Juss. ex Kunth is a well-known medicinal plant found in tropical and subtropical regions. It contains a broad range of bioactive compounds that exhibit many biological effects, including antidiabetic, antibacterial, and antioxidative activities. However, the effect of natural peptides from T. stans against cancer progression and free radical production is unknown. This study aims to evaluate the cytotoxic, anti-metastatic, and antioxidative activities of natural peptides from T. stans on A549 cells.MethodsThe natural peptides were extracted from the flower of T. stans using the pressurized hot water extraction (PHWE) method, followed by size exclusion chromatography and solid-phase extraction-C18. The cytotoxic and anti-metastatic effects of natural peptides were evaluated using MTT and transwell chamber assays, respectively. The free radical scavenging activity of natural peptides was determined using ABTS, DPPH, and FRAP assays. The cells were pretreated with the IC50 dosage of natural peptides and stimulated with LPS before analyzing intracellular reactive oxygen species (ROS) and proteomics.ResultsNatural peptides induced cell toxicity at a concentration of less than 1 ng/ml and markedly reduced cell motility of A549 cells. The cells had a migration rate of less than 10% and lost their invasion ability in the treatment condition. In addition, natural peptides showed free radical scavenging activity similar to standard antioxidants and significantly decreased intracellular ROS in the LPS-induced cells. Proteomic analysis revealed 1,604 differentially expressed proteins. The self-organizing tree algorithm (SOTA) clustered the protein abundances into eleven groups. The volcano plot revealed that the cancer-promoting proteins (NCBP2, AMD, MER34, ENC1, and COA4) were down-regulated, while the secretory glycoprotein (A1BG) and ROS-reducing protein (ASB6) were up-regulated in the treatment group.ConclusionThe anti-proliferative and anti-metastatic activities of natural peptides may be attributed to the suppression of several cancer-promoting proteins. In contrast, their antioxidative activity may result from the up-regulation of ROS-reducing protein. This finding suggests that natural peptides from T. stans are viable for being the new potential anti-cancer and antioxidative agents.

Biosynthesized tin oxide-sodium alginate-polyethylene glycol-carvacrol nanocomposite shows anticancer activity on esophagus squamous carcinoma cells

Esophagus cancer is the sixth leading cause of cancer-related mortalities worldwide. In this study, we synthesized a biocompatible nanocomposite with green synthesized tin oxide using carvacrol, sodium allyl, and polyethylene glycol. Carvacrol is a phenolic monoterpenoid extracted from the essential oils of the plants belonging to the Lamiaceae family. Carvacrol possesses immense pharmacological properties such as antioxidant, antiviral, anticancer, etc. In this study, we assessed the efficacy of Tin oxide-Sodium alginate-Polyethylene glycol-Carvacrol (SnO2-SAPG-Carvacrol) nanocomposite. The synthesized nanocomposite was subjected to spectroscopic analysis, FTIR spectroscopic analysis, FESEM & EDAX analysis, TEM, and XRD & DLS analysis. The antimicrobial efficacy of SnO2-SAPG-Carvacrol nanocomposite against various microorganisms was analyzed using the disc diffusion method. MTT assay was performed to detect the cytotoxic effect of SnO2-SAPG-Carvacrol nanocomposite on KYSE-150–7 cells esophagus squamous carcinoma cell line. The apoptotic efficacy of SnO2-SAPG-Carvacrol nanocomposite was assessed with staining techniques such as DCFH-DA, Rhodamine 123 staining, and AO/EtBr staining. Oxidative stress markers were quantified to detect the anticancer property of SnO2-SAPG-Carvacrol nanocomposite. To analyze the anti-migratory effect we have done a transwell chamber assay. To confirm the anticancer potency of SnO2-SAPG-Carvacrol nanocomposite the cell cycle regulatory and apoptotic regulatory proteins were quantified. Characterization analysis of SnO2-SAPG-Carvacrol nanocomposite confirms it has a potent nanocomposite that can be utilized as a drug. MTT assay and staining results of SnO2-SAPG-Carvacrol nanocomposite treated KYSE-150–7 cells prove the apoptotic property of nanocomposite synthesized. SnO2-SAPG-Carvacrol nanocomposite treatment on KYSE-150–7 cells significantly increased oxidative stress thereby inducing apoptosis in KYSE-150–7 cells. The anti-migratory property of synthesized nanocomposite was confirmed with the transwell chamber assay. The ELISA results of cell regulatory and apoptotic regulatory protein confirm the anticancer activity of SnO2-SAPG-Carvacrol nanocomposite. Overall our results confirm that SnO2-SAPG-Carvacrol nanocomposite is a potent anticancer drug.

Relationship of the Circulating Endothelial Progenitor Cells to the Severity of a Coronary Artery Lesion in Unstable Angina.

The number and function of circulating endothelial progenitor cells (EPCs) decreased in stable coronary artery disease. Nevertheless, there were few studies that explored the variation of EPC and the relationship with the severity of coronary artery lesions in unstable angina (UA). Therefore, this leaves an area for the investigation of the difference in the number and activity of circulating EPCs and the relationship with the Gensini score in unstable angina. Fluorescence-activated cell sorter analysis, as well as DiI-acLDL and lectin fluorescent staining measure the number of circulating EPCs. The transwell chamber assay and MTT were evaluated by the migration and proliferation of circulating EPCs. In addition, the flow-mediated dilation (FMD), Gensini score, and IL-6 levels in plasma were determined. We found that UA patients had the higher number and lower function of circulating EPCs. With the increase in severity of coronary artery lesions, the migration and proliferation of EPCs were decreased. Moreover, the function of the circulating EPCs was negatively associated with severity of coronary artery lesions in unstable angina. In addition, UA patients presented elevated IL-6, which was negatively correlated with the function of circulating EPCs and FMD and positively correlated with the severity of coronary artery lesions evaluated by the Gensini score. These findings revealed the decline in the function of circulating EPCs was associated with the severity of coronary artery disease, which may be related to systemic inflammation.

FOCAD/miR-491-5p, downregulated by EGR1, function as tumor suppressor by inhibiting the proliferation and migration of gastric cancer cells

Gastric cancer is a common malignant tumor in China; however, its carcinogenesis remains unknown. Focadhesin (FOCAD) is a tumor suppressor gene in gliomas, its expression, role, and mechanism in gastric cancer have not been defined. The aim of the present study was to explore the expression pattern of FOCAD in human normal tissues and cancer tissues and elucidate the role and regulatory mechanism of Early Growth Response 1 (EGR1) in FOCAD and its intron, miR-491-5p, in gastric cancer. Immuno histochemical staining revealed that FOCAD is widely and highly expressed in normal gastric mucosa, but is absent in gastric cancer tissue. Based on an association analysis FOCAD expression was found to be negatively associated with lymph node metastasis (P = 0.004); higher FOCAD levels were associated with longer survival in patients with gastric cancer (P = 0.001). MTT, colony, Transwell chamber, and flow cytometry assays revealed that siFOCAD promoted cell proliferation, growth, and migration, and inhibited apoptosis. Furthermore, bioinformatic analysis, Fluorescence reporter gene and chromatin immunoprecipitation analyses confirmed that EGR1 binds to the promoter and negatively regulates FOCAD and miR-491-5p at the transcriptional level. The overexpression of EGR1 was also found to promote cell proliferation, growth, and migration, and inhibit apoptosis. Overall, FOCAD is specifically overexpressed in the gastric mucosa and is significantly downregulated in gastric cancer. To our knowledge, this is the first study to demonstrate that FOCAD is a tumor suppressor, higher FOCAD levels might be a better prognostic marker of gastric cancer, and FOCAD/miR-491-5p may be negatively regulated by EGR1.

Elevated LINC00894 relieves the oncogenic properties of thyroid cancer cell by sponging let-7e-5p to promote TIA-1 expression

LINC00894 plays an important role in cancer cell proliferation and invasion in breast and kidney cancer. However, its role in thyroid cancer proliferation and metastasis remains unclear. In this study, data on LINC00894 expression in thyroid cancer tissues were obtained from GEPIA2. miRNA expression in thyroid cancer tissues was obtained from starBase 3.0 and OncomiR. Cell proliferation was evaluated using CCK-8, and Transwell chambers were used for the migration and invasion assays. LINC00894 and let-7e-5p expressions in thyroid cancer cells were measured using qRT–PCR. Meanwhile, TIA-1 expression in thyroid cancer cells was analyzed via western blotting. We found that LINC00894 expression was markedly reduced in thyroid cancer tissues and cells, and low expression of LINC00894 was associated with poor prognosis in thyroid cancer. LINC00894 overexpression inhibited the proliferation, migration, and invasion of CAL-62 and TPC-1 cells. Additionally, let-7e-5p expression was substantially enhanced in CAL-62 and TPC-1 cells. LINC00894 overexpression promoted TIA-1 expression by acting as a sponge of let-7e-5p. Finally, let-7e-5p weakened the function of LINC00894 in thyroid cancer cells via reduction in TIA-1 levels. In conclusion, our data suggest that increased LINC00894 expression reduces the oncogenic properties of thyroid cancer cells by sponging let-7e-5p to promote TIA-1 expression.

CircKIF5B Promotes Hepatocellular Carcinoma Progression by Regulating the miR-192 Family/XIAP Axis.

BackgroundThe long-term prognosis of HCC (hepatocellular carcinoma) with metastasis remains extremely poor. CircRNAs are promising as critical biological markers in identifying disease mechanisms and developing new effective treatments. However, the role of the aberrant expression of circRNAs in HCC progression remains largely unknown.MethodsCircKIF5B location was investigated by RNA fluorescence in situ hybridization (RNA-FISH). For circRNA determination, RNase R treatment and Real-Time Quantitative RT-PCR (qRT-PCR) were performed. Transwell chamber assays examined the chemotactic migration and invasion of liver cancer cells.ResultsThis study identified the circRNA circKIF5B originating from exons 1, 2, and 3 of the KIF5B gene. Importantly, we found that circKIF5B circRNA, rather than KIF5B linear mRNA, was notably upregulated in liver cancer cell lines and tissues. Moreover, we found that silencing circKIF5B markedly reduced the proliferation, invasion, and metastasis of liver cancer cells by sponging the miR-192 family, thus decreasing the expression of X-linked inhibitor of apoptosis (XIAP).ConclusionOur data demonstrate that circKIF5B can regulate XIAP expression by sponging miR-192 and miR-215 competing for the ceRNA mechanism, indicating that circKIF5B may act as an essential upstream regulator and providing mechanistic evidence to support the view that circKIF5B/miR-192s/XIAP is a promising therapeutic target for treating liver cancer.

Combination therapy of doxorubicin and Sildenafil inhibits the growth of pediatric rhabdomyosarcoma

BackgroundOverexpression of phosphodiesterase type 5 (PDE5) has been detected in many types of malignant tumors. Sildenafil, a potent and selective inhibitor of a cGMP-specific PDE5, has been found to enhance the cytotoxic activity of different chemotherapeutic agents including doxorubicin. The combined therapy of doxorubicin with Sildenafil might reduce the possible side effects of chemotherapy while maintaining synergistic anticancer activity. The present study explored for the first time the effects of Sildenafil, alone and in combination with doxorubicin, on pediatric RMS cells.MethodsHuman RMS cells lines RH30 and RD were used. Western blotting and RT-PCR were performed to analyze PDE5 expression in RMS cells. Cell viability was determined using MTT assay. Cell migration was analyzed via transwell chambers, clonal growth and mitotic cell death were analyzed using the clonogenic assay. FACS analysis was performed to evaluate reactive oxygen species (ROS) and apoptosis.ResultsSildenafil significantly decreased cell viability and migration of RD and RH30 cells. The exposure of RMS cells to doxorubicin resulted in a dose-dependent decrease in their viability. Simultaneous administration of Sildenafil enhanced this effect. The incubation of the RMS cells with Sildenafil in the presence of doxorubicin significantly increased the proportion of apoptotic cells and ROS production compared to the treatment with Sildenafil alone.ConclusionsThe results of our study indicate a link between PDE5 inhibition by Sildenafil and decreased calcium signalling leading to significantly impaired viability, migration, and colony forming of RMS cells. Increased ROS production and apoptosis are mechanisms relevantly contributing to this observation.

Bisdemethoxycurcumin suppresses human brain glioblastoma multiforme GBM 8401 cell migration and invasion via affecting NF‐κB and MMP‐2 and MMP‐9 signaling pathway in vitro

Human glioblastoma (GBM) is one of the common cancer death in adults worldwide, and its metastasis will lead to difficult treatment. Finding compounds for future to develop treatment is urgent. Bisdemethoxycurcumin (BDMC), a natural product, was isolated from the rhizome of turmeric (Curcuma longa), which has been shown to against many human cancer cells. In the present study, we evaluated the antimetastasis activity of BDMC in human GBM cells. Cell proliferation, cell viability, cellular uptake, wound healing, migration and invasion, and western blotting were analyzed. Results indicated that BDMC at 1.5-3μM significantly decreased the cell proliferation by MTT assay. BDMC showed the highest uptake by cells at 3h. After treatment of BDMC at 12-48 h significantly inhibited cell motility in GBM 8401 cells by wound healing assay. BDMC suppressed cell migration and invasion at 24 and 48 h treatment by transwell chamber assay. BDMC significantly decreased the levels of proteins associated with PI3K/Akt, Ras/MEK/ERK pathways and resulted in the decrease in the expressions of NF-κB, MMP-2, MMP-9, and N-cadherin, leading to the inhibition of cell migration and invasion. These findings suggest that BDMC may be a potential candidate for the antimetastasis of human GBM cells in the future.

Myrcene exerts anti-tumor effects on oral cancer cells in vitro via induction of apoptosis

Purpose: To investigate the anticancer potential of myrcene against oral cancer cells.Methods: The effect of myrcene on oral cancer cell growth were studied using CCK-8 proliferation andclonogenic assays, while Annexin V-FITC/PI and DAPI staining methods were used for the determination of its effect on cell apoptosis. Protein expression levels were assayed by western blotting technique. Wound healing and Transwell chamber assays were used to determine the effect of myrcene on oral cancer cell migration and invasion, respectively.Results: Myrcene significantly and dose-dependently inhibited oral cancer cell growth (p < 0.05). The anti-proliferative effect of myrcene was comparatively lower on Hs27 human fibroblast cells than on SCC9 oral cancer cells. Myrcene stimulated apoptosis in human oral cancer cells via enhancement of Bax expression and repression of Bcl-2 expression. The percentage of early and late apoptotic cells increased from 4.03 in untreated control to 27.77 when treated with 20 μM myrcene. Moreover, myrcene in vitro significantly suppressed the migration and invasion of oral cancer cells (p < 0.05).Conclusion: These results indicate that myrcene exerts potent anticancer effects on oral cancer cells via induction of apoptosis, and should be further investigated in vivo for this purpose.

Abstract 2192: HSF1 downregulation of CCL5 reduces CD8 + T cell trafficking in breast cancer

Abstract Breast cancer is the second leading cause of cancer-related death in women. The presence of cytotoxic immune cells, specifically CD8+ T cells, in breast tumors is associated with better patient outcomes. Understanding the mechanisms that regulate the infiltration of CD8+ T cells into breast tumors could improve the treatment and enhance patient overall survival. Utilizing a novel HSF1 activity gene signature, we observed that HSF1 activity was negatively associated with the presence of CD8+ T cells in breast cancer patients. Both CIBERSORT analysis of TCGA data as well as direct assessment of patient primary specimens demonstrated that patient tumors with high HSF1 activity had lower numbers of CD8+ T cells. To functionally test this relationship, HSF1 was knocked down in 4T1 breast cancer cells and grown orthotopically in BALB/c mice. Tumors with HSF1 knockdown showed lower tumor volumes and increased CD8+ T cell infiltration. We further tested the effect of CD8+ T cells on the growth of HSF1 knockdown tumors by depleting CD8+ T cells in BALB/c mice. HSF1 knockdown tumors were significantly larger with CD8+ T cell depletion suggesting a functional role for HSF1 to inhibit CD8+ T cell infiltration and protect the tumor from immune-mediated killing. To investigate the mechanism by which HSF1 affects CD8+ T cells, we investigated whether HSF1 affects chemotactic cytokines. We found that loss of HSF1 significantly increased secretion of CCL5, a known chemoattractant for CD8+ T cells. Loss of HSF1 also increased mRNA levels of CCL5, suggesting a transcriptional effect on CCL5. To test the importance of CCL5 in the phenotype of HSF1 knockdown, a transwell migration assay was performed wherein T cells from BALB/c mice were placed in the upper chamber of a transwell chamber and the lower chamber contained conditioned medium from 4T1 cells with knockdown of HSF1 with or without knockdown of CCL5. The lower chamber was subjected to flow cytometry for CD3/CD8 to identify CD8+ T cells and we observed that loss of HSF1 increased migration of CD8+ T cells and this was ablated with additional loss of CCL5. These results suggest CCL5 is a significant mediator for the recruitment of CD8+ T cells when HSF1 is lost in cancer cells. We propose the model whereby high HSF1 activity in breast cancer cells suppresses expression and secretion of the chemo-attractant cytokine CCL5 that ultimately leads to a decrease in CD8+ T cells in breast tumors microenvironment. Citation Format: Curteisha LeeAnn Jacobs, Sakhi Shan, Wen-Cheng Lu, Richard Carpenter. HSF1 downregulation of CCL5 reduces CD8 + T cell trafficking in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2192.

Abstract 2443: Knocking out SNCA in SKMEL28 melanoma cells suppresses EMT and prevents invasion

Abstract Malignant melanoma is one of the most aggressive and fifth most prevalent cancer among men and women. Clinical and epidemiological studies shows that Parkinson’s disease (PD) patients have higher risk of developing invasive melanoma, and, reciprocally, patients with invasive melanoma have a higher risk of developing PD. Alpha-synuclein (α-syn; SNCA), an intrinsically disordered protein expressed in neurons, is also highly expressed in malignant melanoma and is a common molecular signature between Parkinson’s disease and melanoma. We have previously reported that SK-Mel-28 SNCA-knockout (KO) cells exhibit suppressed tumor growth and proliferation in vivo and in vitro, and that putting back SNCA via lenti virus restored the phenotype to that of the control cells. The present study is designed to investigate the role of α-syn in melanoma progression in SK-MEL-28 cells in the context of metastasis and the epithelial-to-mesenchymal transition (EMT). The SK-Mel-28 SNCA-KO clones showed a significant decrease in the cell-surface protein L1CAM, a major cell adhesion molecule highly expressed in various melanoma and correlates with melanoma progression, whereas the knock in clones resembled the control. Migration and invasion assay were performed using transwell chambers showed a profound migratory and invasive defect in SNCA-KO clones. To explore further whether this is a proliferative or migratory defect, a single cell phagokinetic motility assay was performed with colloidal gold which showed a marked decrease in cell motility in knockout clones compared to control and knock in clones, which was consistent with our migration and invasion experiments. To determine the proteins involved in the inhibition of invasion mediated by α-syn, we immunoblotted for the mesenchymal markers N-cadherin and vimentin, and these were significantly downregulated in the SNCA-KO clones. These data reinforce that knocking out SNCA impedes melanoma cell migration and invasion in SK-Mel- 28 cell line, implying that α-syn might be a novel therapeutic target for malignant melanoma. Citation Format: Nithya Gajendran, Santhanasabapathy Rajasekaran, Nirjhar M. Aloy, Sahar Shekoohi, Stephan N. Witt. Knocking out SNCA in SKMEL28 melanoma cells suppresses EMT and prevents invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2443.

CFTR Suppresses Neointimal Formation Through Attenuating Proliferation and Migration of Aortic Smooth Muscle Cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) plays important roles in arterial functions and the fate of cells. To further understand its function in vascular remodeling, we examined whether CFTR directly regulates platelet-derived growth factor-BB (PDGF-BB)-stimulated vascular smooth muscle cells (VSMCs) proliferation and migration, as well as the balloon injury-induced neointimal formation. The CFTR adenoviral gene delivery was used to evaluate the effects of CFTR on neointimal formation in a rat model of carotid artery balloon injury. The roles of CFTR in PDGF-BB-stimulated VSMC proliferation and migration were detected by mitochondrial tetrazolium assay, wound healing assay, transwell chamber method, western blot, and qPCR. We found that CFTR expression was declined in injured rat carotid arteries, while adenoviral overexpression of CFTR in vivo attenuated neointimal formation in carotid arteries. CFTR overexpression inhibited PDGF-BB-induced VSMC proliferation and migration, whereas CFTR silencing caused the opposite results. Mechanistically, CFTR suppressed the phosphorylation of PDGF receptor β, serum and glucocorticoid-inducible kinase 1, JNK, p38 and ERK induced by PDGF-BB, and the increased mRNA expression of matrix metalloproteinase-9 and MMP2 induced by PDGF-BB. In conclusion, our results indicated that CFTR may attenuate neointimal formation by suppressing PDGF-BB-induced activation of serum and glucocorticoid-inducible kinase 1 and the JNK/p38/ERK signaling pathway.

Expression of LATS1 in head and neck squamous cell carcinoma and its effect on tumor cell proliferation and invasion.

Nowadays, the incidence and mortality of head and neck tumors are gradually increasing. Head and neck malignant tumors (such as laryngeal cancer, hypopharyngeal cancer, oral cancer, nasopharyngeal cancer, oropharyngeal cancer, and other head and neck malignancies) are more common among systemic tumors. The most common pathological head and neck tumor type is squamous cell carcinoma, accounting for about 90%. In this study, immunohistochemical methods were used to collect the normal squamous epithelial tissues of the head and neck, atypical hyperplasia tissues, and head and neck squamous cell carcinomas on a tissue chip for detection. The recombinant LATS1 overexpression plasmid was prepared and transferred into B88 cells. Western blotting, MTT, and Transwell chamber methods were used to detect the effects of LATS1 proliferation, migration, and B88 cell overexpression. The experimental results showed that in head and neck squamous cell carcinoma, the expression of LATS1 protein decreased from 59.3% to 11.3%. At the same time, this protein inhibited the proliferation, migration, and invasion of head and neck squamous epithelial cells and also inhibited epithelium- Interstitial transformation exerts its tumor suppressor effect, indicating that LATS1 may play a tumor suppressor effect as a tumor suppressor gene. An in-depth study of the role and mechanism of LATS1 protein in the occurrence of head and neck squamous cell carcinoma may provide new opportunities for the treatment of head and neck squamous cell carcinoma in the future.

FTX Regulated miR-153-3p/FOXR2 to Promote Cisplatin Resistance in Ovarian Cancer.

Purpose The present study was aimed at exploring the role of FTX in cisplatin (DDP) resistance in ovarian cancer (OC). Methods QPCR was applied to evaluate mRNA expression in OC tissue and cells. CCK-8 assay was conducted to evaluate cell proliferation. Transwell chamber assay was performed to evaluate invasion of SKOV3/DDP cells. The protein expression was evaluated via western blot assay. Flow cytometry was performed to evaluate the apoptosis of SKOV3/DDP cells. Results The expression of FTX in DDP-resistant cells was observably higher in contrast to DDP-sensitive cells and normal ovarian cells. FTX was higher expressed in DDP-resistant tissues by comparison with DDP-sensitive tissues. Knockdown of FTX obviously suppressed the proliferation ability invasion ability of SKOV3/DDP cells. Knockdown of FTX obviously enhanced apoptosis of SKOV3/DDP cells. miR-153-3p was proved to be directly regulated by FTX via the luciferase reporter assays. By comparison with normal cells, miR-153-3p was lower expressed in OC cells. miR-153-3p was lower expressed in SKOV3/DDP cells in contrast to SKOV3 cells. More interestingly, FTX reversed the inhibiting influence of miR-153-3p on cisplatin resistance of OC cells. Moreover, miR-153-3p was proved to directly regulate FOXR2. Knockdown of miR-153-3p attenuated the inhibitory influence of knockdown FOXR2 on cisplatin resistance of OC cells. Conclusion FTX regulated miR-153-3p/FOXR2 to promote cisplatin resistance via inhibiting the apoptosis and promoting the viability and invasion in OC.

SCARA3 inhibits cell proliferation and EMT through AKT signaling pathway in lung cancer

BackgroundScavenger receptor class A member 3 (SCARA3) is decreased in prostate cancer and myeloma. However, functions of SCARA3 in various cancers remain unclear. In this study, we tried to evaluate the functional study of SCARA3 in lung cancer.MethodsThe expression level of SCARA3 in the TCGA-database, lung cancer tissue microarray and lung cancer cells and the prognosis of lung cancer patients were measured. Lung cancer tissue microarray was analyzed pathologically using immunohistochemistry, and quantitative analysis of SCARA3 in normal lung cells and lung cancer cells was analyzed using western blot analysis. Survival curves for lung cancer patients were prepared with the Kaplan-Meier method. Migration and invasion of SCARA3 overexpressed lung cancer cells were determined using a Transwell chamber system. Proliferation of lung cancer cells was determined based on cell viability assay using cell culture in vitro and a tumorigenicity model of BALB/C nude mouse in vivo.ResultsThe expression of SCARA3 was abnormally reduced in TCGA-database, lung tissue microarray, and various lung cancer cells. However, overexpression of SCARA3 reduced the proliferation of lung cancer. The ability of SCARA3 to inhibit cancer cell proliferation was maintained even in vivo using a mouse xenograft model. In addition, overexpression of SCARA3 reduced migration and invasion ability of lung cancer cells and induced decreases of EMT markers such as β-catenin, vimentin, and MMP9. We aimed to prove the role of SCARA3 in the treatment of Lung cancer, and shown that the expression level of SCARA3 is important in cancer treatment using cisplatin. The enhancement of the effect of cisplatin according to SCARA3 overexpression is via the AKT and JNK pathways.ConclusionsThis study confirmed an abnormal decrease in SCARA3 in lung cancer. Overexpression of SCARA3 potently inhibited tumors in lung cancer and induced apoptosis by increasing sensitivity of lung cancer to cisplatin. These results suggest that SCARA3 is a major biomarker of lung cancer and that the induction of SCARA3 overexpression can indicate an effective treatment.

Treatment of Triple Negative Cell Lines with Olaparib to Block DNA Repair.

The most aggressive breast cancer is the triple negative histological type, and the gold standard for its treatment is platinum salts, such as carboplatin. Due to high recurrence, there is a need to test new drugs, such as PARP inhibitors (PARPi), that induce lethality in cells with DNA damage. Olaparib is a PARPi, already used in some tumors but not tested in canine species. Thus, the aim of this study was to demonstrate the efficacy of olaparib in inhibiting DNA repair and control disease progression by decreasing the migration capacity of mammary tumor cells. The cell lines CF41.Mg and MDA-MB-468 were cultured and MTT was performed to define the best dose of carboplatin. Next, the cells were treated with 10 μM carboplatin, olaparib, and with a combination of both for 24 hours. PARP-1 protein and gene expression were evaluated by immunofluorescence, western blotting, and qRT-PCR, respectively. The analysis of cell migration was performed in transwell chambers. For CF41.Mg and MDA-MB-468 cell lines, there was a decrease in PARP-1 protein and gene expression after treatment with carboplatin, olaparib, and both in combination compared to the group without treatment (control) (p<0.05). Moreover, in both lines, a reduction in invasion rate was observed after treatment with carboplatin, olaparib and when combined, compared to the control group (p<0.05). Our data suggest that carboplatin and olaparib were able to block DNA repair and control the cancer invasion, especially when used in combination. The results with olaparib in the canine line are unpublished. The olaparib should be a possible agent against human breast cancer and canine mammary tumors.