Soft Agar Colony Formation Assay Research Articles

Evaluation for Inhibitory Effects of Hybrid Liposomes on the Growth of Breast Cancer Stem Cells using Soft Agar Colony Formation Assay

Evaluation for Inhibitory Effects of Hybrid Liposomes on the Growth of Breast Cancer Stem Cells using Soft Agar Colony Formation Assay

Reduction of pl-CSA through ChSy-2 knockout inhibits tumorigenesis and metastasis of choriocarcinoma in JEG3 cells.

Background: Placental-like chondroitin sulfate A (pl-CSA) is exclusively expressed in cancerous and placental tissues and is highly correlated with the degree of malignancy. However, the mechanism through which pl-CSA regulates tumorigenesis and metastasis in choriocarcinoma remains unclear.Methods: Stable transfectants of the JEG3 choriocarcinoma cell line, including a negative control (NC) line and a cell line with knockout of the biosynthetic enzyme CS synthase-2 (ChSy-2) (ChSy-2-/-), were obtained using CRISPR/Cas9 systems and identified by immunofluorescence, flow cytometry, western blots and enzyme-linked immunosorbent assays (ELISAs). The proliferation, migration, invasion and colony formation of the cells were determined by a cell counting kit, scratch-wound assays, transwell assays and soft agar colony formation assays in vitro, respectively. The tumorigenesis and metastasis of choriocarcinoma were also investigated through two xenograft models in vivo.Results: The ChSy-2 protein in the ChSy-2-/-group was below the detection threshold, which was accompanied a significant reduction in the pl-CSA level. Reducing pl-CSA through ChSy-2 knockout significantly inhibited cell proliferation, migration, invasion and colony formation in vitro and tumorigenesis and metastasis of choriocarcinoma, with deceases in tumor volume and metastatic foci and a high percent survival compared to the NC in vivo.Conclusion: pl-CSA, as a necessary component of JEG-3 cells, was efficiently reduced through ChSy-2 knockout, which significantly inhibited the tumorigenesis and metastasis of choriocarcinoma. ChSy-2/pl-CSA could be alternative targets for tumor therapy.

Anticancer effects of 6-shogaol via the AKT signaling pathway in oral squamous cell carcinoma.

Objective Oral squamous cell carcinoma (OSCC) is one of the common type of cancer that leads to death; and is becoming a global concern. Due to the lack of efficient chemotherapeutic agents for patients with oral cancer, the prognosis remains poor. 6-shogaol, a bioactive compound of ginger, has a broad spectrum of bioactivities and has been widely used to relieve many diseases. However, its effects on human oral cancer have not yet been fully evaluated. In our study, we investigated the anticancer effects of 6-shogaol on the proliferation, migration, invasion, apoptosis, and underlying mechanisms within human OSCC cell lines.Methodology We investigated the effect of 6-shogaol on the growth of OSCC cells by cell viability and soft agar colony formation assay. Migration and invasion assays were conducted to confirm the effect 6-shogaol on OSCC cell metastasis. Apoptosis was detected by flow cytometry and the underlying mechanism on the antigrowth effect of 6-shogaol in OSCC cells was assessed using western blotting.Results In our results, 6-shogaol not only suppressed proliferation and anchorage-independent cell growth in OSCC cells, but also induced apoptosis by regulating the apoptosis-associated factors such as p53, Bax, Bcl-2, and cleaved caspase-3. Migration and invasion of OSCC cells were inhibited following the regulation of E-cadherin and N-cadherin by 6-shogaol. Additionally, 6-shogaol treatment significantly inhibited the PI3K/AKT signaling pathway.Conclusion Therefore, our results may provide critical evidence that 6-shogaol can be a potential new therapeutic candidate for oral cancer.

C-MYC-directed NRF2 drives malignant progression of head and neck cancer via glucose-6-phosphate dehydrogenase and transketolase activation.

Rationale: NRF2, a redox sensitive transcription factor, is up-regulated in head and neck squamous cell carcinoma (HNSCC), however, the associated impact and regulatory mechanisms remain unclear.Methods: The protein expression of NRF2 in HNSCC specimens was examined by IHC. The regulatory effect of c-MYC on NRF2 was validated by ChIP-qPCR, RT-qPCR and western blot. The impacts of NRF2 on malignant progression of HNSCC were determined through genetic manipulation and pharmacological inhibition in vitro and in vivo. The gene-set enrichment analysis (GSEA) on expression data of cDNA microarray combined with ChIP-qPCR, RT-qPCR, western blot, transwell migration/ invasion, cell proliferation and soft agar colony formation assays were used to investigate the regulatory mechanisms of NRF2.Results: NRF2 expression is positively correlated with malignant features of HNSCC. In addition, carcinogens, such as nicotine and arecoline, trigger c-MYC-directed NRF2 activation in HNSCC cells. NRF2 reprograms a wide range of cancer metabolic pathways and the most notable is the pentose phosphate pathway (PPP). Furthermore, glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) are critical downstream effectors of NRF2 that drive malignant progression of HNSCC; the coherently expressed signature NRF2/G6PD/TKT gene set is a potential prognostic biomarker for prediction of patient overall survival. Notably, G6PD- and TKT-regulated nucleotide biosynthesis is more important than redox regulation in determining malignant progression of HNSCC.Conclusions: Carcinogens trigger c-MYC-directed NRF2 activation. Over-activation of NRF2 promotes malignant progression of HNSCC through reprogramming G6PD- and TKT-mediated nucleotide biosynthesis. Targeting NRF2-directed cellular metabolism is an effective strategy for development of novel treatments for head and neck cancer.

20(S)-Ginsenoside Rh2 Suppresses Oral Cancer Cell Growth by Inhibiting the Src-Raf-ERK Signaling Pathway.

20(S)-Ginsenoside Rh2 (G-Rh2) has demonstrated therapeutic effects in many types of cancers. We aimed to investigate the potential anticancer activity and underlying mechanisms of G-Rh2 in oral cancer cells. The antigrowth effect of G-Rh2 in oral cancer cells was stimulated by cell proliferation, soft agar colony formation, and migration and invasion assay. The cell cycle and apoptosis were detected by flow cytometry. The underlying mechanism of G-Rh2 in oral cancer cells was explored by immunoblotting. G-Rh2 significantly inhibited oral cancer cell growth by inducing apoptosis and cell cycle G0/G1-phase arrest. G-Rh2 inhibited oral cancer cell migration and invasion through regulation of epithelial-mesenchymal transition (EMT)-related proteins. G-Rh2 inhibited the Src/Raf/ERK signaling pathway in YD10B and Ca9-22 cells. G-Rh2 exerted anticancer activity in vitro by inhibiting the Src/Raf/ERK signaling pathway in oral cancer. G-Rh2 is a potential therapeutic drug for oral cancer treatment.

Reactive Oxygen Species-Mediated Autophagy by Ursolic Acid Inhibits Growth and Metastasis of Esophageal Cancer Cells.

Ursolic acid (UA) possesses various pharmacological activities, such as antitumorigenic and anti-inflammatory effects. In the present study, we investigated the mechanisms underlying the effects of UA against esophageal squamous cell carcinoma (ESCC) (TE-8 cells and TE-12 cells). The cell viability assay showed that UA decreased the viability of ESCC in a dose-dependent manner. In the soft agar colony formation assay, the colony numbers and size were reduced in a dose-dependent manner after UA treatment. UA caused the accumulation of vacuoles and LC3 puncta, a marker of autophagosome, in a dose-dependent manner. Autophagy induction was confirmed by measuring the expression levels of LC3 and p62 protein in ESCC cells. UA increased LC3-II protein levels and decreased p62 levels in ESCC cells. When autophagy was hampered using 3-methyladenine (3-MA), the effect of UA on cell viability was reversed. UA also significantly inhibited protein kinase B (Akt) activation and increased p-Akt expression in a dose-dependent manner in ESCC cells. Accumulated LC3 puncta by UA was reversed after wortmannin treatment. LC3-II protein levels were also decreased after treatment with Akt inhibitor and wortmannin. Moreover, UA treatment increased cellular reactive oxygen species (ROS) levels in ESCC in a time- and dose-dependent manner. Diphenyleneiodonium (an ROS production inhibitor) blocked the ROS and UA induced accumulation of LC3-II levels in ESCC cells, suggesting that UA-induced cell death and autophagy are mediated by ROS. Therefore, our data indicate that UA inhibits the growth of ESCC cells by inducing ROS-dependent autophagy.

Bruceine D inhibits Cell Proliferation Through Downregulating LINC01667/MicroRNA-138-5p/Cyclin E1 Axis in Gastric Cancer

Objective: Gastric cancer is one of the most common malignant tumors. Bruceine D (BD) is one of the extracts of Brucea javanica. In recent years, it has been reported that BD has anti-tumor activity in some human cancers through different mechanisms. Here, this study try to explore the effect of BD on gastric cancer and its regulatory mechanism. Methods: Cell proliferation ability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays, 5-bromo-2-deoxyuridine (BrdU) staining and soft agar colony formation assay, respectively. The tumor xenograft model was used to verify the effect of BD on the tumorigenicity of gastric cancer cells in vivo. Flow cytometry analysis and Western blot assay were performed to detect cell cycle and apoptosis. Gastric cancer cells were analyzed by transcriptome sequencing. The interaction between LINC01667, microRNA-138-5p (miR-138-5p) and Cyclin E1 was verified by dual luciferase experiment and RT-PCR assays. Results: We found that BD significantly inhibited cell proliferation and induced cell cycle arrest at S phase in gastric cancer cells. Transcriptome analysis found that the expression of a long non-coding RNA, LINC01667, were significantly down-regulated after BD treatment. Mechanically, it was discovered that LINC01667 upregulated the expression of Cyclin E1 by sponging miR-138-5p. Furthermore, BD enhanced the chemosensitivity of gastric cancer cells to doxorubicin, a clinically used anti-cancer agent. Conclusion: BD inhibit the growth of gastric cancer cells by downregulating the LINC01667/miR-138-5p/Cyclin E1 axis. In addition, BD enhances the chemosensitivity of gastric cancer cells to doxorubicin. This study indicates that BD may be used as a candidate drug for the treatment of patients with gastric cancer.

MiR-326 regulates the proliferation and apoptosis of endometrial cancer by targeting Bcl-2.

MiR-326 has been investigated to be correlated with multiple types of malignancies; however, the role of miR-326 in endometrial cancer (EC) remains rarely reported. The aim of our research is to investigate the functions of miR-326 in EC and the potential molecular mechanism. RT-qPCR was performed to compare the expression of miR-326 and Bcl-2 in normal endometrial epithelial cell line (End1/e6e7) and EC cells lines (HEC-1A, Ishikawa), respectively. Bioinformatic analysis and luciferase assay verified the relationship between miR-326 and the 3'-UTR of Bcl-2. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, soft agar colony formation assay and the flow cytometry were performed to investigate the functions of miR-326 and Bcl-2 on proliferation and apoptosis in EC. Western blotting was employed to explore the expression of Bcl-2, Bcl2-associated X (Bax) and caspase-3. The expression of miR-326 decreased in EC cell lines compared to normal endometrial epithelial cell line, while Bcl-2 expression was increased in EC cells. Results of MTT and soft agar colony formation assays showed that miR-326 suppressed proliferation in EC cells. In addition, flow cytometry revealed that miR-326 promoted apoptosis in EC cells. Western blotting showed that silencing miR-326 promoted the expression of Bcl-2. Bioinformatics analysis and luciferase assay verified the 3'-UTR of Bcl-2 was a target of miR-326. Furthermore, MTT assay, soft agar colony formation assay and the flow cytometry proved that miR-326 acts as tumor suppressor via inhibiting the expression of Bcl-2. MiR-326 acts as a cancer suppressor to inhibit proliferation and promote apoptosis via targeting Bcl-2 axis in EC.

Chondrosarcoma-derived Mutation in IDH1 Promotes Tumorigenesis by Inducing HIF-1α

IDH mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into an oncometabolite, D-2-hydroxyglutarate (D-2HG). IDH mutations occur in more than 50% of patients with conventional or dedifferentiated chondrosarcoma. However, the function of these mutations in the pathogenesis of chondrosarcoma is still unclear. In this study, we aimed to establish a mutant IDH1 knockout model in a chondrosarcoma cell line and determine the role of mutant IDH1 in this malignancy. In our study, we employed a human chondrosarcoma cell line, JJ012 which harbors an endogenous IDH1R132G mutation. As a control, we also used an immortalized chondrocyte cell line, C28 which expresses wildtype IDH1 only. IDH1 was knocked out via CRISPR/Cas9 technology. Quantitative analyses of D-2HG were conducted by tandem mass spectrometry. RNA-Seq analysis was performed to identify differentially expressed genes between the mutant IDH1 knockout (KO) cells and the parental cells. RNA-Seq data were validated by real time PCR. Colony-forming ability of KO cells under hypoxia and normoxia conditions was evaluated using soft agar assay. The effect of mutant IDH1 on chondrosarcoma growth was analyzed in murine xenograft models. The expression of HIF1-1α and its targeted genes were examined in cells and tumor tissues by western blot and Immunohistochemistry (IHC). Mutant IDH1 was successfully knocked out in JJ012 cells. Single cell-derived KO clones were then identified and developed. As expected, D-2HG production is almost completely depleted in these KO clones. Using these genetically modified cells, we found that knockout of mutant IDH1 remarkably inhibited colony formation by 40% under normoxia condition compared to the parental control (p<0.05). Intriguingly, we observed that the HIF1-α levels were significantly reduced in the JJ012 KO cells compared with the parental cells. Moreover, RNA-seq data showed a widespread downregulation of HIF1-1α targeted genes such as VEGFA, VEGFC, SLC2A3, EDN1 in response to the knockout (FDR-adjusted p value < 0.01). These data were confirmed by qRT-PCR. The soft-agar colony formation assay further showed a significantly increased colony number and diameter for JJ012 KO cells under hypoxia condition compared with those under normoxia condition (P<0.05). Furthermore, we observed that loss of mutant IDH1 led to a marked attenuation of chondrosarcoma formation in the murine xenograft model. Immunoblotting and IHC revealed that HIF-1α and its targeted genes were dramatically downregulated in the KO cell-derived tumors. We found that mutant IDH1 contributes to tumorigenicity of chondrosarcoma cells, at least partially through the upregulation of HIF1-α. This suggests that targeting HIF1-α signaling pathway is promising for combinatorial regimens with mutant IDH inhibitors for patients with IDH1-mutant chondrosarcoma.

Tumor suppressive function of Matrin 3 in the basal-like breast cancer

BackgroundBasal-like breast cancer (BLBC) or triple-negative breast cancer (TNBC) is an aggressive and highly metastatic subtype of human breast cancer. The present study aimed to elucidate the potential tumor-suppressive function of MATR3, an abundant nuclear protein, in BLBC/TNBC, whose cancer-relevance has not been characterized.MethodsWe analyzed in vitro tumorigenecity by cell proliferation and soft agar colony formation assays, apoptotic cell death by flow cytometry and Poly (ADP-ribose) polymerase (PARP) cleavage, epithelial-mesenchymal transition (EMT) by checking specific EMT markers with real-time quantitative PCR and in vitro migration and invasion by Boyden Chamber assays. To elucidate the underlying mechanism by which MATR3 functions as a tumor suppressor, we performed Tandem affinity purification followed by mass spectrometry (TAP-MS) and pathway analysis. We also scrutinized MATR3 expression levels in the different subtypes of human breast cancer and the correlation between MATR3 expression and patient survival by bioinformatic analyses of publicly available transcriptome datasets.ResultsMATR3 suppressed in vitro tumorigenecity, promoted apoptotic cell death and inhibited EMT, migration, and invasion in BLBC/TNBC cells. Various proteins regulating apoptosis were identified as MATR3-binding proteins, and YAP/TAZ pathway was suppressed by MATR3. MATR3 expression was inversely correlated with the aggressive and metastatic nature of breast cancer. Moreover, high expression levels of MATR3 were associated with a good prognosis of breast cancer patients.ConclusionsOur data demonstrate that MATR3 functions as a putative tumor suppressor in BLBC/TNBC cells. Also, MATR3 potentially plays a role as a biomarker in predicting chemotherapy-sensitivity and patient survival in breast cancer patients.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

SummaryProlactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Establishment and characterization of a new gastric cancer cell line, XGC-1

BackgroundTo establish a primary human gastric cancer cell line.MethodsFresh gastric cancer tissue samples were separated into a cell suspension, and DMEM/F12 medium containing 10% foetal bovine serum was used for primary culture and subculture. The morphology of the cells was observed under a light microscope, and the cell growth curve was plotted. A soft agar colony formation assay was used to detect the colony formation ability of the cell line. Immunohistochemical methods were used to detect cytokeratin, vimentin and Ki-67, the chromosome G banding method was used to analyse the karyotype of the cells, and the tumourigenic ability of the cells was detected by subcutaneous inoculation of BALB/C nude mice.ResultsWe established a gastric cancer cell line from a 68-year-old male patient. This gastric cancer cell line was named XGC-1 and had a doubling time of approximately 48 h. The cell line displayed strong colony formation ability and tumourigenicity in BALB/C nude mice and had complicated chromosomal abnormalities. When nutrients were insufficient, the cells shed and floated in the medium, but adherent growth was observed in nutrient-rich conditions.ConclusionsThe XGC-1 cell line will be useful for future studies of gastric cancer development, progression, metastasis and therapy.

GOLPH3 inhibition reverses oxaliplatin resistance of colon cancer cells via suppression of PI3K/AKT/mTOR pathway

ObjectiveTo explore whether GOLPH3 regulated oxaliplatin (L-OHP) resistance of colon cancer cells via PI3K/AKT/mTOR pathway. MethodsHCT116/L-OHP cells were divided into Blank, Control/GOLPH3 shRNA, BEZ235 (a PI3K/AKT/mTOR inhibitor), and GOLPH3 + BEZ235 groups followed by the detection with MTT, soft agar colony formation, flow cytometry and TUNEL assays. Mice bearing HCT116/L-OHP xenografts were randomized into Control, L-OHP, NC/GOLPH3 shRNA, L-OHP + NC/GOLPH3 shRNA groups. The expressions of Ki67, Caspase-3, and PI3K/AKT/mTOR pathway proteins were examined by immunohistochemistry. ResultsHCT116/L-OHP cells had increased GOLPH3 expression compared to HCT116 cells, which positively regulated PI3K/AKT/mTOR pathway in HCT116/L-OHP cells. BEZ235 declined IC50 of HCT116/L-OHP cells to L-OHP, decreased the expressions of ABCB1, ABCC1, ABCG2, ATP7A, ATP7B, MATE1, p-gp, MRP1 and BCRP, induced cell apoptosis, reduced cell proliferation, and arrested cells at G0/G1, which was reversed by GOLPH3 overexpression. L-OHP and GOLPH3 shRNA decreased tumor volume and reduced expression of Ki67 in tumor tissues with the increased Caspase-3. Meanwhile, the combined treatment had the better treatment effect. ConclusionGOLPH3 inhibition reduced proliferation and promoted apoptosis of HCT116/L-OHP cells, and also reversed the L-OHP resistance of HCT116/L-OHP, which may be associated with the suppression of P13K/AKT/mTOR pathway.

Evaluation of Impacts of Cellular Metabolism on the Migration of Ovarian Cancer Cells by Two in Vitro Assays: A Method Comparison Study

Background: Alteration of metabolic pathways in cancer cells can intensely modulate their migration as an important step in invasion and metastasis. Ketogenic diet showed some contradictory results in cancer patients. In this study the impact of metabolic reprogramming of A2780CP as a model of ovarian cancer stem-like cells on cell migration by two in vitro methods: wound healing and soft agar colony-forming assays. Materials and Methods: short term and long term metabolic reprogramming were done by restriction of glucose to 250mg/L with or without enrichment with beta-hydroxybutyrate (5 milimolar) for 48 hours and 30 days, respectively. Wound healing assay was done and the wound ratio was calculated for 24 and 48 hours. Soft agar colony formation assay was also done in treated and control cells. For method comparison, ten biological replicates were analyzed in triplicate. Results: Migration of A2780CP ovarian cancer stem-like cells were significantly alleviated by long term glucose restriction but no significant changes were observed in short term study. Beta-hydroxybutyrate enrichment did not produce significant impacts on glucose restriction in short or long term studies. Conclusion: The results of colony formation in soft agar and wound or scratch healing assay were in good correlation and convergence which could be used interchangeably in the investigation of metabolic reprogramming in cancer cells. [GMJ.2020;9:e1831]

Evaluation of Impacts of Cellular Metabolism on the Migration of Ovarian Cancer Cells by Two in Vitro Assays: A Method Comparison Study.

Background: Alteration of metabolic pathways in cancer cells can intensely modulate their migration as an important step in invasion and metastasis. Ketogenic diet showed some contradictory results in cancer patients. In this study the impact of metabolic reprogramming of A2780CP as a model of ovarian cancer stem-like cells on cell migration by two in vitro methods: wound healing and soft agar colony-forming assays. Materials and Methods: short term and long term metabolic reprogramming were done by restriction of glucose to 250mg/L with or without enrichment with beta-hydroxybutyrate (5 milimolar) for 48 hours and 30 days, respectively. Wound healing assay was done and the wound ratio was calculated for 24 and 48 hours. Soft agar colony formation assay was also done in treated and control cells. For method comparison, ten biological replicates were analyzed in triplicate. Results: Migration of A2780CP ovarian cancer stem-like cells were significantly alleviated by long term glucose restriction but no significant changes were observed in short term study. Beta-hydroxybutyrate enrichment did not produce significant impacts on glucose restriction in short or long term studies. Conclusion: The results of colony formation in soft agar and wound or scratch healing assay were in good correlation and convergence which could be used interchangeably in the investigation of metabolic reprogramming in cancer cells.

Abstract 548: Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis

Abstract Background: Apoptosis resistance is a hallmark of cancer, and apoptosis regulators have been targeted for cancer treatment. A class of such regulators is inhibitor of apoptosis proteins (IAPs), which suppress caspase activity by inducing degradation of active caspases or blocking interaction of caspases with their substrates. Dysregulated IAP expression has been reported in many cancers, including breast cancer, and is involved in chemoresistance. IAPs have therefore become attractive targets for cancer treatment. Birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspase (SMAC, an endogenous antagonist of IAPs), has been shown to effectively activate apoptotic signaling by targeting IAPs and has potential application for treatment of multiple cancers. In addition, birinapant showed synergistic anti-tumor effects with several widely used chemotherapeutic agents in various cancers. Here, we assessed whether birinapant synergizes with commonly used anti-cancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), everolimus (mTOR inhibitor), and gemcitabine, in triple-negative breast cancer (TNBC). Among these drugs, gemcitabine strongly synergized with birinapant (combination index [CI] &amp;lt;1). We hypothesize that birinapant enhances anti-tumor efficacy of gemcitabine in TNBC by inducing intrinsic pathway-dependent apoptosis. Methods: The in vitro anti-tumor efficacy of birinapant, gemcitabine, and their combination was determined using CellTiter-Blue viability and soft agar colony formation assays. The CI (CalcuSyn software) was used to quantify the synergy. The effects of drug treatment on cell cycle distribution were analyzed by flow cytometry; the effects on apoptosis were assessed by annexin V-PE and 7-AAD staining, and induction of apoptosis was further confirmed by pan-caspase inhibitor treatment; and the effects on IAP degradation were examined by Western blotting. The in vivo antitumor efficacy was determined using TNBC xenograft mouse models. Results: Birinapant as a single agent had varying anti-proliferation effects in TNBC cells (IC50 = 0.21 to &amp;gt;20 µM). It had no synergistic effects with entinostat, cisplatin, paclitaxel, voxtalisib, dasatinib, or everolimus but strongly synergized with gemcitabine (CI &amp;lt;1 µM) in tested TNBC cells. Birinapant and gemcitabine synergistically inhibited the growth of TNBC cells by activating the intrinsic apoptosis pathway, accomplished by inducing degradation of cIAP2 and XIAP, which led to apoptotic cell death. Furthermore, birinapant significantly enhanced anti-tumor effectiveness of gemcitabine in TNBC xenograft mouse models (n = 10; P &amp;lt; 0.05) by inducing apoptosis. Conclusion: Our findings demonstrate the therapeutic potential of birinapant for enhancing the anti-tumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins. Citation Format: Xuemei Xie, Jangsoon Lee, Troy Pearson, Alexander Y. Lu, Debu Tripathy, Gayathri R. Devi, Chandra Bartholomeusz, Naoto T. Ueno. Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 548.

Demethylation of the NRF2 Promoter Protects Against Carcinogenesis Induced by Nano-SiO2

Nano silicon dioxide (Nano-SiO2) has been widely used in industries such as the field of biomedical engineering. Despite the existing evidence that Nano-SiO2 exposure could induce oxidative stress and inflammatory responses in multiple organ systems, the carcinogenicity of Nano-SiO2 exposure has rarely been investigated. Thus in this study, two types of human bronchial epithelial cell lines (16HBE and BEAS-2B) were selected as in vitro models to investigate the carcinogenicity of Nano-SiO2. Our results revealed that Nano-SiO2 induces a malignant cellular transformation in human bronchial epithelial cells according to the soft agar colony formation assay. The carcinogenesis induced by Nano-SiO2 was also confirmed in nude mice. By using immunofluorescence assay and high-performance capillary electrophoresis (HPCE), we observed a genome-wide DNA hypomethylation induced by Nano-SiO2. Besides the reduced enzyme activity of total DNMTs upon Nano-SiO2 treatment, altered expression of DNMTs and methyl-CpG binding proteins were observed. Besides, we found that the expression of NRF2 was activated by demethylation of CpG islands within the NRF2 promoter region and the overexpression of NRF2 could alleviate the carcinogenesis induced by Nano-SiO2. Taken together, our results suggested that Nano-SiO2 induces malignant cellular transformation with a global DNA hypomethylation, and the demethylation of NRF2 promoter activates the expression of NRF2, which plays an important role in protecting against the carcinogenesis induced by Nano-SiO2.

ARPP-19 Mediates Herceptin Resistance via Regulation of CD44 in Gastric Cancer.

PurposeAs the first-line drug for treatment of HER2-positive metastatic gastric cancer (GC), Herceptin exhibits significant therapeutic efficacy. However, acquired resistance of Herceptin limits the therapeutic benefit of gastric cancer patients, in which the molecular mechanisms remain to be further determined.MethodsQuantitative real-time polymerase chain reaction was performed to detect the mRNA levels of ARPP-19 and CD44 in GC cells. Protein levels were determined using Western blot and IHC staining. MTT and soft agar colony formation assays were used to measure cell proliferation. Xenograft model was established to verify the functional role of ARPP-19 in Herceptin resistance in vivo. Sphere formation assay was conducted to determine cell stemness.ResultsWe observed ARPP-19 was up-regulated in Herceptin resistance gastric cancer cells NCI-N87-HR and MKN45-HR. The forced expression of ARPP-19 promoted, whereas the silencing of ARPP-19 impaired Herceptin resistance of HER2-positive gastric cancer cells both in vitro and in vivo. Moreover, ARPP-19 significantly enhanced the sphere formation capacity and CD44 expression, CD44 was also a positive factor of Herceptin resistance in HER2-positive gastric cancer cells. In addition, high level of ARPP-19 was positively associated with Herceptin resistance and poor survival rate of gastric cancer patients.ConclusionWe have demonstrated that ARPP-19 promoted Herceptin resistance of gastric cancer via up-regulation of CD44, our study suggested that ARPP-19 could be a potential diagnostic and therapeutic candidate for HER2-positive gastric cancer.

CD109 mediates tumorigenicity and cancer aggressiveness via regulation of EGFR and STAT3 signalling in cervical squamous cell carcinoma

BackgroundCD109 was involved in the tumorigenesis and progression of various cancers via TGF-β1 signalling and STAT3 activation. As CD109 is strongly expressed in cervical squamous cell carcinoma, this study was conducted to investigate its functional characteristics in cervical cancer.MethodsCD109 expression was examined by immunohistochemistry (IHC) with cervical tissue microarray. The effects of CD109 expression were examined on migration, cell proliferation, spheroid formation and soft-agar colony-formation assay. Meanwhile, cervical cancer cell lines with high CD109 expression were chosen for the functional study using siRNA knockdown and CRISPR/Cas9 knockout.ResultsIHC demonstrated an upregulation of CD109 in the cell membrane of cervical squamous cell carcinoma. CD109( + ) cells isolated by flow-cytometric sorting displayed enhanced migration, cell proliferation, sphere-forming and anchorage-independent cell growth ability. In contrast, silencing of CD109 expression could reverse the in vitro and in vivo tumorigenic and aggressive properties. Furthermore, CD109 induced EGFR-mediated STAT3 phosphorylation known to be responsible for cell migration, proliferation and maintenance of CSC phenotype.ConclusionAbundant CD109( + ) populations in cervical cancer cells potentially contributed to carcinogenesis and aggressiveness, whereas silencing of CD109 expression could reverse those properties. CD109 mediates cervical tumorigenicity and aggressiveness via CD109/EGFR/STAT3 signalling.

PSAT1 Regulated Oxidation-Reduction Balance Affects the Growth and Prognosis of Epithelial Ovarian Cancer.

IntroductionA growing number of studies have found that the serine-glycine biosynthesis pathway is highly activated for biosynthesis in cancer progression and metastasis. Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second step of the serine-glycine biosynthesis pathway; the effects and mechanism of PSAT1 in epithelial ovarian cancer (EOC) remains unclear.Materials and MethodsThe expression of PSAT1 in clinical EOC samples and normal ovarian tissues was conducted by RT-PCR, Western blot, and immunohistochemical staining. Survival analysis of PSAT1 in ovarian cancer was performed by using the public database. Following the downregulation of PSAT1, the cell growth, cell apoptosis, and cell cycle in ovarian cancer cells were respectively examined by the soft agar colony formation assay and flow cytometry analysis. Then the glutathione (GSH) levels, the GSH/GSSG ratio, the NADPH/NADP ratio, and the cellular reactive oxygen species (ROS) levels were tested to analyze the oxidation-reduction balance in PSAT1-depleted ovarian cancer cells.ResultsPSAT1 is markedly over-expressed in clinical EOC samples (n = 90) compared to that in normal ovarian tissues (n = 10), and the expression of PSAT1 is correlated with histological subtype, FIGO stage, histological grade, lymph node metastasis, distant metastasis and the presence of ascites. Public database analysis shows that higher PSAT1 indicates poor survival in EOC patients. Downregulation of PSAT1 in EOC cells inhibits growth, induces apoptosis and cell cycle arrest in vitro. EOC cells with high PSAT1 levels have increased a higher GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio and lower reactive oxygen species (ROS) content. The cancer-killing effects of PSAT1 knockdown are reversed by exogenous glutathione. PSAT1 participates in cancer growth by regulating oxidation-reduction balance.ConclusionTherefore, these results highlight the potential of PSAT1 inhibitors or metabolic substrate deprivation as therapeutic strategies for treating patients with EOC, especially those with advanced stages of cancer.