Tumor Model In Nude Mice Research Articles

Novel Frog Skin-Derived Peptide Dermaseptin-PP for Lung Cancer Treatment: In vitro/vivo Evaluation and Anti-tumor Mechanisms Study.

Lung cancer is the major cause of cancer deaths worldwide, and it has the highest incidence and mortality rate of any cancer among men and women in China. The first-line therapy for lung cancer treatment is platinum-based chemotherapy drugs such as cisplatin. However, the application of present chemotherapies is limited by severe side effects, which stimulates the discovery of new drugs with new anti-tumor mechanisms and fewer side effects. Beneficially, many antimicrobial peptides (AMPs) from frog skin have been reported to exhibit potent anti-cancer activities with low toxicity, high selectivity and a low propensity to induce resistance. In this study, we first reported an AMP named Dermaseptin-PP, from a rarely studied frog species, Phyllomedusa palliata. Dermaseptin-PP exhibited selective cytotoxicity on H157, MCF-7, PC-3, and U251 MG cancer cells instead of normal HMEC-1 cells with low hemolytic effect. Furthermore, on subcutaneous H157 tumor model of nude mice, Dermaseptin-PP was found to display potent in vivo anti-tumor activity in a dose-related manner without obvious hepatopulmonary side effects. It is widely accepted that AMPs usually work through a membrane disruptive mode, and the confocal laser microscope observation confirmed that Dermaseptin-PP could destroy H157 cell membranes. Further investigation of mechanisms by flow cytometry assay and immunohistochemical analysis unraveled that Dermaseptin-PP also exerted its anti-tumor activity by inducing H157 cell apoptosis via both endogenous mitochondrial apoptosis pathway and exogenous death receptor apoptosis pathway. Herein, we emphasize that the membrane disrupting and the apoptosis activation effects of Dermaseptin-PP both depend on its concentration. Overall, a novel frog skin-derived AMP, named Dermaseptin-PP, was identified for the first time. It possesses strong antimicrobial activity and effective anti-tumor activity by distinct mechanisms. This study revealed the possibility of Dermaseptin-PP for lung cancer treatment and provided a new perspective for designing novel AMP-based anti-tumor candidates with low risk of cytotoxicity.

STAT3 Inhibition by Centipede Scolopendra Extract in Liver Cancer HepG2 Cells and Orthotopic Mouse Models of Hepatocellular Carcinoma

ObjectiveTo observe the effects of Centipede Scolopendra extraction (CSE) on human liver cancer HepG2 cells and the nude mouse tumor model of liver orthotopic transplantation, and to explore the anti-liver cancer mechanism of the extract. MethodsHepG2 cells were respectively treated with CSE250 (250 μg/mL), CSE500 (500 μg/mL) and 5-FU, and control group was established. An enzymatic hydrolysis and acetone precipitation method was used to separate and purify CSE, which was then used to treat HepG2 cells. The CCK8 assay was used to detect the inhibition of cell proliferation and the half maximal inhibitory concentration (IC50) was calculated. Flow cytometry was used to analyze the cell cycle, and western blot was used to detect the expression of signal transduction and activator of transcription 3 (STAT3) pathway-related proteins in HepG2 cells treated with CSE. A nude mouse model with an orthotopic liver tumor was prepared. The mice were randomly divided into four groups, each containing 12 animals: the model group, the 5-FU group, the CSE10 group [10 mg/(kg·d)] and the CSE50 group [50 mg/(kg·d)]. The volume and mass changes in the nude mice with orthotopic transplanted tumors were observed. Western blot method was used to test the protein expression levels of p-STAT3 and p38 mitogen-activated protein kinase (p38MAPK). Tissues from the liver of mice in the model group and the CSE50 group were analyzed by using a protein tyrosine kinase (PTK) chip, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) function enrichment analysis of the differentially expressed proteins was performed. ResultsThis study showed that CSE significantly inhibited the proliferation of HepG2 cells (P < 0.05). After 48 h of CSE treatment, the cell cycle of HepG2 cells manifested as S phase and G2/M phase; p-STAT3 protein levels in the CSE groups were significantly lower than that in the control group (P < 0.05). Analysis of the tumor inhibition in the mice showed that the tumor masses and volume in CSE groups were lower (P < 0.05). The protein levels of p-STAT3 and p38MAPK in CSE50 group and 5-FU group decreased significantly (P < 0.05). PTK antibody chip screening results showed that CSE groups had a bidirectional regulation trend, and there were 23 up-regulated PTKs and six down-regulated PTKs. The GO and KEGG analyses showed that CSE exerted its anticancer effects through regulation of biological processes, including mitogen-activated protein kinase (MAPK) cascade, chemotaxis, cell invasion, cell adhesion, angiogenesis and other biological processes, and through signaling pathways, including the MAPK, phosphatidylinositol-3-kinase/serine threonine protein kinase (PI3K/AKT), and RAS signaling pathways. ConclusionsCSE can effectively inhibite the proliferation of HepG2 cells and effectively inhibite the growth of liver cancer orthotopic transplantation tumor. Its mechanism may be closely related to the regulation of STAT3, MAPK and PI3K/AKT signaling pathways.

RETRACTED ARTICLE: Circular RNA circ-CSPP1 regulates CCNE2 to facilitate hepatocellular carcinoma cell growth via sponging miR-577

ObjectiveCirc-centro-some/spindle pole-associated protein (CSPP1) has been confirmed to be characterized in diverse human malignancies and its ectopic expression may regulate tumor progression and development. However, in hepatocellular carcinoma (HCC), its biological role, clinical significance and molecular mechanism are still unclear.MethodsCirc-CSPP1 expression and its prognostic values in HCC tissues were detected by qRT-PCR or in situ hybridization (ISH), and enriched by using Rnase R. The functional experiments (Circ-CSPP1 was overexpressed or knocked down) were performed in HCC cells. The HCC cell growth was analyzed by CCK-8 assay, transwell, wound healing and colony formation assays. The interation between circ-CSPP1 and miR-577/miR-577 and cyclin E2 (CCNE2) were determined by dual luciferase assay or RNA binding protein immunoprecipitation (RIP) assay. The RNA fluorescence in situ hybridization (FISH) assay was used to detect the subcellular distribution. Finally, an in vivo nude mouse tumor model was constructed.ResultsIn HCC patients and cells, circ-CSPP1 was aberrantly expressed, and its upregulation predicted poor prognosis, and closely correlated with tumor size and TNM stage. Circ-CSPP1 resisted RnaseR digestion, indicating it is a circular RNA structure. Moreover, overexpression of circ-CSPP1 promoted HCC cell viability, colony formation, migration, and invasion in vitro. Knockdown of circ-CSPP1 showed contrary results. Circ-CSPP1 acts as a miR-577 sponge and positively regulated the target of miR-577, CCNE2. Besides, miR-577 inhibitor rescued the suppressive effects of circ-CSPP1 knockdown on HCC cell growth, whereas was completely reversed by silencing of CCNE2. Finally, the in vivo experiments confirmed that circ-CSPP1 knockdown regulated xenograft tumor volume and downregulated CCNE2, p-Rb, E2F1 and c-myc expression.ConclusionThese findings revealed that circ-CSPP1 contributed to HCC progression by positively regulating CCNE2 via miR-577, thus established its potential as new a prognostic and therapeutic marker for HCC patients.

Pharmacological effects of vinorelbine in combination with lenvatinib in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is a rare neoplasia with a poor prognosis. Proliferation and apoptosis assays were performed on ATC cell lines (8305C, 8505C) exposed to vinorelbine, lenvatinib, as well as to concomitant combinations. ABCB1, ABCG2 and CSF-1 mRNA expression was evaluated by real time PCR. The relative levels of pospho Akt were investigated as part of a human phospho-kinase array analysis, and CSF-1 and VEGFR-2 protein levels were measured by ELISA. The intracellular concentration of lenvatinib in ATC cells was measured by combined reversed-phase liquid chromatography-tandem mass spectrometry. An ATC subcutaneous xenograft tumor model in nude mice was treated with vinorelbine, lenvatinib, or vinorelbine plus lenvatinib.After treatment with vinorelbine, lenvatinib, a significant antiproliferative effect in ATC cell lines was observed. The concomitant treatment of vinorelbine and lenvatinib revealed synergism for all the fractions of affected cells. A decrease in ABCB1 expression was reported in both ATC cell lines treated with the lenvatinib plus vinorelbine combination, as was an increase in the intracellular concentration of lenvatinib. The combination caused a decrease in Akt, GSK3α/β, PRAS40 and Src phosphorylation, and in both CSF-1 mRNA and protein levels. In the subcutaneous tumor model, the combination reduced the tumor volume during the treatment period.Our results establish the synergistic ATC antitumor activity of a vinorelbine and lenvatinib combination.

Knock-down of the TIM/TIPIN complex promotes apoptosis in melanoma cells.

The Timeless (TIM) and it's interacting partner TIPIN protein complex is well known for its role in replication checkpoints and normal DNA replication processes. Recent studies revealed the involvement of TIM and TIPIN in human malignancies; however, no evidence is available regarding the expression of the TIM/TIPIN protein complex or its potential role in melanoma. Therefore, we investigated the role of this complex in melanoma.To assess the role of the TIM/TIPIN complex in melanoma, we analyzed TIM/TIPIN expression data from the publicly accessible TCGA online database, Western blot analysis, and RT-qPCR in a panel of melanoma cell lines. Lentivirus-mediated TIM/TIPIN knockdown in A375 melanoma cells was used to examine proliferation, colony formation, and apoptosis. A xenograft tumor formation assay was also performed.The TIM/TIPIN complex is frequently overexpressed in melanoma cells compared to normal melanocytes. We also discovered that the overexpression of TIM and TIPIN was significantly associated with poorer prognosis of melanoma patients. Furthermore, we observed that shRNA-mediated knockdown of TIM and TIPIN reduced cell viability and proliferation due to the induction of apoptosis and increased levels of γH2AX, a marker of DNA damage. In a xenograft tumor nude mouse model, shRNA-knockdown of TIM/TIPIN significantly reduced tumor growth.Our results suggest that the TIM/TIPIN complex plays an important role in tumorigenesis of melanoma, which might reveal novel approaches for the development of new melanoma therapies. Our studies also provide a beginning structural basis for understanding the assembly of the TIM/TIPIN complex. Further mechanistic investigations are needed to determine the complex’s potential as a biomarker of melanoma susceptibility. Targeting TIM/TIPIN might be a potential therapeutic strategy against melanoma.

Circ_0001023 Promotes Proliferation and Metastasis of Gastric Cancer Cells Through miR-409-3p/PHF10 Axis.

BackgroundCircular RNAs (circRNAs) have been well documented to regulate the gene expression via sponging microRNA (miRNA) in diverse neoplasms including gastric cancer (GC).MethodsIn the present study, the expressions of circ_0001023, miR-409-3p, and plant homeodomain finger 10 (PHF10) in GC tissues were detected by qRT-PCR. Chi-square test was performed to analyze the associations between circ_0001023 and pathological parameters. Cell Counting Kit-8 assay, colony formation assay, flow cytometry, and transwell assay were adopted to detect the role of circ_0001023/miR-409-3p axis in the proliferation, apoptosis, and migration of GC cells, respectively. The targeting relationship between circ_0001023 and miR-409-3p was investigated by dual-luciferase gene reporter gene assay. Additionally, subcutaneous xenotransplanted tumor model in nude mice was established to detect the function of circ_0001023 on GC growth in vivo.ResultsCompared with adjacent tissues, the expression of circ_0001023 was significantly upregulated and correlated with lymph node invasion and higher T stage of GC patients. It has also been proved that circ_0001023 could target miR-409-3p. Silencing circ_0001023 can impede the proliferation of GC cells and promote apoptosis, while miR-409-3p inhibitors can partially reverse the biological behavior of GC cells mentioned above. Moreover, the expression of circ_0001023 was reversely associated with miR-409-3p expression but positively correlated with PHF10, a downstream oncogene of miR-409-3p.ConclusionCollectively, it is concluded that circ_0001023 promotes the progression of GC via regulating miR-409-3p/PHF10 axis.

Long intergenic noncoding RNA 00844 promotes apoptosis and represses proliferation of prostate cancer cells through upregulating GSTP1 by recruiting EBF1.

Accumulating evidence have suggested the function of long noncoding RNAs as crucial players in the pathogenesis of prostate cancer (PC), a urologic tumor in male with poor prognosis. This study was designed to explore the functions of long intergenic noncoding RNA 00844 (LINC00844) in PC progression. The expression of LINC00844 and glutathione S-transferase P1-1 (GSTP1) was detected by reverse transcription quantitative polymerase chain reaction, followed by the identification of the relationship among LINC00844, GSTP1, and early B cell factor 1 (EBF1) by dual luciferase reporter gene assay, RNA immunoprecipitation assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. Using loss- and gain-of-function assays, the effects of LINC00844, GSTP1, and EBF1 on the biological characteristics of PC cells were assessed by cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry. Lastly, the results from in vitro experiments were verified in vivo by establishing a xenograft tumor model in nude mice. LINC00844 and GSTP1 both displayed low expression in PC tissues and cells. LINC00844 positively regulated the expression of GSTP1 via recruiting EBF1. Overexpression of LINC00844 reduced proliferation and elevated apoptosis of PC cells through recruiting EBF1, which subsequently upregulated GSTP1. In vivo experiments confirmed that LINC00844 or GSTP1 upregulation attenuated tumor growth. LINC00844 elevated GSTP1 expression by recruiting EBF1 to the promoter region of GSTP1, thereby suppressing PC progression. Hence, LINC00844 is a novel therapeutic target for the development of new treatment protocols for PC.

Dendrobium officinalis inhibited tumor growth in non-small cell lung cancer.

BackgroundLung cancer is the most common and lethal tumor in the world, and the number of patients who die from lung cancer is growing steadily. Because of conventional chemotherapy drugs’ poor tumor selectivity, side effects are significant. Conducting relevant studies and developing highly efficient and low toxicity anti-cancer drugs are urgently needed. Dendrobium officinale, which belongs to Orchidaceae aerophyte, has the characteristic of slow growth and lower natural propagation rate. In China, Dendrobium officinale has a very high value and is often referred to as the “gold of herbs”. According to reports in the literature, the active ingredients of Dendrobium officinale have anticancer activity and inhibit neovascularization’s potential. This study aimed to investigate the inhibitory effect of Dendrobium officinale in A549 lung cancer cells and its potential involvement in slowing tumor growth.MethodsWe cultured A549 cells and established a cancer xenograft model in nude mice. Infused stomach with Dendrobium officinale was applied to the nude mouse model. Tumor volume and body weight were recorded.ResultsThe results show that, compared with the negative control group, the gross tumor volume (GTV) of treatment groups decreased (all P<0.05), while the effect of the high concentration of the Dendrobium officinale was more significant than that found in the medium and low group. We believe that Dendrobium officinale exhibits a promising antitumor effect in the nude mouse tumor model. The best treatment concentrations for the nude mouse tumor model were achieved when treatment with the drug began about 7–15 days, and was more significant in high concentrations.ConclusionsDendrobium officinale has potent effects of inhibiting tumor on the nude mouse tumor model.

Cisplatin Nano-Liposomes Promoting Apoptosis of Retinoblastoma Cells Both In Vivo and In Vitro

This study was established to investigate the effects of cisplatin nano-liposomes on the apoptosis of the human retinoblastoma (RB) cell line Y79 in vitro and in vivo. Y79 cells were cultured and then exposed to Annexin V/PI to test their apoptosis, tested with the Caspase-3 activity detection kit to examine the change in activity of Caspase-3, and subjected to western blotting to test Bcl-2 and Bax protein expression. Y79-cell-transplanted tumor model in nude mice was also established and divided into three groups, with five nude mice in each. Cisplatin nano-liposomes were applied to the experimental group, cisplatin was injected into the control group, while saline was administered to the blank group, after which the nude mice were killed and the tumor was removed. Tumor volumes and weights in the three groups were compared. Nucleic acid extraction from magnetic beads was adopted to extract DNA, RT-PCR was employed to test Bcl-2 and Bax mRNA levels in tumor tissues, and in situ cell death assay kit was applied to test apoptotic cells. In comparison to the cisplatin solution and DMSO groups, the cisplatin liposome group showed higher Y79 apoptotic rate, Caspase-3 activity, and Bax protein expression, and lower Bcl-2 protein expression (all P &lt; 0 05). In comparison with the control and blank groups, the experimental group showed lower tumor volume, weight, and Bcl-2 mRNA level of nude mice. In addition, in comparison with the control group, the experimental group showed higher cellular apoptotic rate and Bax mRNA level. In terms of the clinical effects of cisplatin nano-liposomes on a tumor transplant in nude mice with cervical cancer, they were shown to promote tumor apoptosis.

Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer.

The human positive coactivator 4 (PC4) was originally identified as a multi-functional cofactor capable of mediating transcription activation by diverse gene- and tissue-specific activators. Recent studies suggest that PC4 might also function as a novel cancer biomarker and therapeutic target for different types of cancers. siRNA knockdown studies indicated that down-regulation of PC4 expression could inhibit tumorigeneicity of A549 non-small cell lung cancer tumor model in nude mice. Here we show that AG-1031, a small molecule identified by high throughput screening, can inhibit the double-stranded DNA binding activity of PC4, more effectively than its single-stranded DNA binding activity. AG-1031 also specifically inhibited PC4-dependent transcriptional activation in vitro using purified transcription factors. AG-1031 inhibited proliferation of several cultured cell lines derived from non-small cell lung cancers (NSCLC) and growth of tumors that formed from A549 cell xenografts in immuno-compromised mice. Moreover, pre-injection of AG-1031 in these mice not only reduced tumor size, but also prevented tumor formation in 20% of the animals. AG-1031 treated A549 cells and tumors from AG-1031 treated animals showed a significant decrease in the levels of both PC4 and VEGFC, a key mediator of angiogenesis in cancer. On the other hand, all tested mice remained constant weight during animal trials. These results demonstrated that AG-1031 could be a potential therapy for PC4-positive NSCLC.

Phospholipid-Coated Guanosine Diphosphate Auxiliary CaP Active Nanoparticles Can Systematically Improve the Efficiency of Gene Therapy for Cancer Disease.

Endogenous active substance guanosine diphosphate (GDP) is involved in the physiological process of DNA transfection and expression in the cytoplasm by binding to Ran proteins. To substantially improve the gene delivery efficiency of nanoparticles, phospholipid-coated Ca(P-GDP)/pDNA/NLS hybrid nanoparticles were prepared using GDP as a common biophosphorus source based on the biological process of exogenous gene expression in the cells. This nanoparticle has a relative uniform particle size distribution and in vitro stability. The addition of GDP in nanoparticles significantly enhanced the gene expression efficiency with good biocompatibility. Moreover, an in vivo study further verified that hybrid nanoparticles were more effective in increasing the p53 gene expression, thus significantly inhibiting the tumor growth in the heterotopic tumor model of nude mice. These results demonstrated that phospholipid-coated Ca(P-GDP) nanoparticles were a potential nonviral gene vector to promote gene expression. The experimental results confirmed the feasibility of designing a delivery system based on active substances and provided a new solution to improve the transfection efficiency of gene drugs.

Patient-derived xenograft model engraftment predicts poor prognosis after surgery in patients with pancreatic cancer

ObjectivesTo establish and evaluate a first generation patient-derived xenograft (PDX) model in nude mice using tumors resected from pancreatic cancer (PC) patients for the identification of key factors that influence xenograft success and prediction of patient prognosis. MethodsPrimary tumor samples harvested from PC patients who underwent curative resection between May 2016 and April 2018 at our hospital were xenografted into nude mice. Tumor size was evaluated for 2 months. Patients’ baseline characteristics and follow-up data were analyzed. ResultsTumor xenograft models were generated from 67 patients; 30 (44.8%) were successful and 37 (55.2%) failed. Xenograft models could recapitulate the pathology and genetic information of the primary tumors. Univariate analysis identified tumor engraftment, post-operation CA19-9, tumor size, lymph node status, and lymphovascular invasion as significant predictors (P=0.000, 0.023, 0.004, 0.035 and 0.005, respectively) of disease-free survival (DFS). Multivariate Cox regression analysis confirmed tumor engraftment, tumor size and lymphovascular invasion function as independent risk factors for DFS (P=0.000, 0.039 and 0.025, respectively). The hazard ratio of tumor engraftment for DFS was 0.239 (95% confidence interval, 0.109 to 0.524). Kaplan–Meier analysis of DFS indicated an unfavorable outcome in the engraftment group compared to that in the failed engraftment group (6.2 vs. 12.2 months, log rank P=0.000). ConclusionThe pathology and genetic information of primary PC tumors are recapitulated in the PDX tumor model in nude mice. Furthermore, engraftment success is an effective predictor of disease recurrence in patients after surgery.

MicroRNA‑145‑5p inhibits osteosarcoma cell�proliferation by targeting E2F transcription factor 3

Osteosarcoma is a common type of bone tumor that primarily occurs in children and young adults. MicroRNA (miRNA/miR) dysregulation is associated with the progression of osteosarcoma; therefore, the aim of the present study was to investigate the biological functions and molecular mechanisms of miR-145-5p in osteosarcoma. The expression of miR-145-5p in osteosarcoma tissues and cell lines was quantified using reverse transcription-quantitative PCR (RT-qPCR). The effect of miR-145-5p on the proliferation of osteosarcoma cells was detected using Cell Counting Kit-8 and colony formation assays, as well as cell cycle distribution analysis. The effect of miR-145-5p on tumor growth was further investigated in vivo using a subcutaneous tumor model in nude mice. The interaction between miR-145-5p and E2F transcription factor 3 (E2F3) was determined using bioinformatics analysis, a luciferase assay, RT-qPCR and western blotting. The results revealed that miR-145-5p expression was decreased in osteosarcoma cell lines and tissues compared with the corresponding normal controls. Increased miR-145-5p expression inhibited the proliferation and colony formation ability of osteosarcoma cells, and induced G1 phase arrest. Furthermore, mice injected with tumor cells overexpressing miR-145-5p exhibited smaller tumors than those in the control group. Further investigation revealed that miR-145-5p binds to and decreases the expression of E2F3. In addition, the mRNA levels of E2F3 were negatively associated with miR-145-5p in osteosarcoma tissues, and increasing E2F3 expression abrogated the inhibitory effects of miR-145-5p on osteosarcoma cells. Collectively, the results obtained in the present study suggest that miR-145-5p may suppress the progression of osteosarcoma, and may serve as a useful biomarker for the diagnosis of osteosarcoma, as well as a therapeutic target.

Abstract P4-14-13: Concurrent exercise and chemotherapy in preclinical breast cancer models

Abstract The benefits of exercise following a cancer diagnosis is increasingly recognized. Increased physical activity is associated with reduced breast cancer recurrence and breast cancer specific mortality. However, the mechanisms underpinning this effect are still under investigation. The role of exercise as an adjunct to systemic therapy for breast cancer remains unclear. We hypothesize that exercise may exert an anti-tumor effect and a change in tumor immune cell infiltration. Methods We evaluated the effect of exercise alone and in combination with chemotherapy in preclinical patient-derived TNBC xenografts (PDX) established in Nude mice and PyMT mouse tumor models. Mice were individually housed in boxes equipped with running wheels and randomized to 1) clamped wheels (sedentary controls), 2) doxorubicin (Dox, 2mg/kg/week), 3) exercise (Ex) and 4) Ex + Dox. Daily distance run was measured. One week after randomization (acclimatization period), the intervention was commenced. Body composition was measured at randomization and at end point. Tumors were harvested after 5 weeks of intervention or at ethical endpoint. Tumor immune infiltrates were analyzed, and transcriptomic analysis performed. Results In the TNBC PDX model, there was no difference in tumor volume at randomization (p=0.96), or cumulative distance run after 1 week of acclimatization to the running wheel (p=0.47). At 5 weeks, Ex alone significantly reduced tumor growth rate compared with controls (relative reduction 10%, p=0.025). There was no difference between the other interventions. Mice randomized to Ex + Dox ran a shorter cumulative distance over 5 weeks compared with Ex alone (103.6 ± 16.2km vs 168.8 ±23km, p=0.028). There was no correlation between distance run and tumor volume in either of the treatment cohorts involving exercise (p=0.39). PyMT, transcriptomic and immune cell infiltration analysis will be reported. At baseline, there was no significant difference in mean total body mass (TBM), lean mass (LM) or fat mass (FM) between the intervention groups (p&amp;gt;0.05). At 5 weeks, the mean TBM and LM in both groups treated with Dox was significantly lower than Ex only and controls. The Ex and the control mice gained weight (11%), compared with Dox only and Ex + Dox mice which did not gain weight (0% and -6% respectively). Therefore exercise had no significant impact on TBM or LM at 5 weeks, but Dox resulted in loss of TBM and LM compared with mice not treated with Dox. Conclusion Exercise significantly reduced tumor growth compared with sedentary controls in our preclinical TNBC PDX model, however there was not a synergistic effect seen with Dox. Cumulative doses of Dox resulted in weight loss and loss of lean mass, and reduced the cumulative running distance, compared with mice not treated with Dox. Citation Format: Sara Wahlroos, Ashleigh Wilkinson, Heloisa Milioli, Neil Portman, David Gallego-Ortega, Elgene Lim. Concurrent exercise and chemotherapy in preclinical breast cancer models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-14-13.

Asiatic Acid Interferes with Invasion and Proliferation of Breast Cancer Cells by Inhibiting WAVE3 Activation through PI3K/AKT Signaling Pathway.

Objective To explore the ability of asiatic acid to interfere with the invasion and proliferation of breast cancer cells by inhibiting WAVE3 expression and activation through the PI3K/AKT signaling pathway. Methods The MDA-MB-231 cells with strong invasiveness were screened by transwell assay, and plasmids with high expression of WAVE3 were constructed for transfection. The transfection effect and protein expression level of plasmids were verified by PCR and WB. The effects of asiatic acid on cell proliferation and invasion were investigated by flow cytometry. The xenografted tumor models in nude mice were established to study the antitumor activity of asiatic acid. Results Asiatic acid significantly inhibited the activity of MDA-MB-231 cells, and the expression level of WAVE3 increased significantly in the tissue of ductal carcinoma in situ and was lower than that in the metastasis group. After plasmid transfection, the mRNA and protein expression of WAVE3 increased significantly in the cells. Asiatic acid at different concentrations had an impact on cell apoptosis and invasion and could significantly inhibit the expression of WAVE3, P53, p-PI3K, p-AKT, and other proteins. The T/C(%) of asiatic acid (50 mg/kg) for MDA-MB-231(F10) xenografted tumor in nude mice was 46.33%, with a tumor inhibition rate of 59.55%. Asiatic acid could significantly inhibit the growth of MDA-MB-231 (F10) xenografted tumors in nude mice (p < 0.05). Conclusions Asiatic acid interferes with the ability of breast cancer cells to invade and proliferate by inhibiting WAVE3 expression and activation and the mechanism of action may be related to the PI3K/AKT signaling pathway.

Knock-Down of the TIM/TIPIN Complex Promotes Apoptosis in Melanoma Cells

Background: The Timeless (TIM) and its interacting partner TIPIN protein complex is well known for its role in replication checkpoint and normal DNA replication processes. TIM appears to be an essential component of the DNA damage response. Recent studies revealed the involvement of TIM and TIPIN in human malignancies; however, no evidence is available regarding TIM/TIPIN protein complex expression and its potential role in melanoma. Therefore, we investigated the role of TIM as well as TIPIN in melanoma. Methods: To assess the role of TIM/TIPIN in melanoma, we analyzed TIM/TIPIN expression data from the publicly accessible TCGA online database, Western blot analysis, and RT-qPCR in a panel of melanoma cell lines. Lentivirus-mediated TIM/TIPIN knockdown in A375 melanoma cells was used to examine cell proliferation, colony formation, and apoptosis. A xenograft tumor formation assay was performed using shTIM, shTIPIN, and shMock cells. Findings: The TIM/TIPIN complex was frequently overexpressed in melanoma cells compared to normal melanocytes. We also discovered that the overexpression of TIM and TIPIN was significantly associated with poorer prognosis of melanoma patients. Furthermore, we observed that shRNA-mediated knockdown of TIM and TIPIN reduced cell viability and proliferation due to the induction of apoptosis and increased levels of γH2AX, a marker of DNA damage. In a xenograft tumor nude mouse model, shRNA-knockdown of TIM/TIPIN significantly reduced tumor growth. Interpretation: Our results suggest that TIM and TIPIN play an important role in tumorigenesis of melanoma, which might reveal novel approaches for the development of new melanoma therapies. Further mechanistic investigations are needed to determine this complex’s potential as a biomarker of melanoma susceptibility. Targeting TIM/TIPIN might be a potential therapeutic strategy against melanoma. Funding Statement: This work was supported by the Hormel Foundation (Z. Dong). Declaration of Interests: All authors have agreed to publish this manuscript and declare no competing interests. Ethics Approval Statement: All animal studies were performed following the guidelines approved by the University of Minnesota Institutional Animal Care and Use Committee (Protocol ID: 1803-35739A).

Effects of evodiamine on PI3K/Akt and MAPK/ERK signaling pathways in pancreatic cancer cells.

The effective antitumor drug evodiamine (EVO) is attracting increased attention. Therefore, the present study aimed to investigate the effects of EVO on the proliferation, apoptosis and autophagy of human pancreatic cancer (PC) cell lines in vitro and in vivo. Human PANC-1 and SW1990 PC cell lines were treated with different concentrations of EVO and proliferation was detected using a Cell Counting Kit (CCK)-8 assay. Colony formation and wound-healing assays showed that EVO inhibited PC cell viability and migration, and apoptosis was detected using flow cytometry. Western blotting and immunofluorescence detected the expression of proteins in PANC-1 and SW1990 cells. The PANC-1 cells were used to establish an orthotopic pancreatic tumor model in nude mice. Tumor-bearing nude mice were administered with different concentrations of EVO, and growth was monitored. High-resolution positron emission tomography and fluorine-18-labeled fluorodeoxyglucose were used to monitor the tumor/non-tumor (T/NT) ratio and standard uptake value (SUV) of the mice, which were subsequently sacrificed to measure the transplanted tumor weight. Apoptosis increased with increasing EVO concentration. The EVO-treated PC cells exhibited significantly higher expression of LC3II than the controls cells. EVO decreased LC3II, enhanced P62 and inhibited the expression of Akt, extracellular-signal-regulated protein kinase (ERK)1/2 and p38. Compared with the control group, the T/NT ratio, SUV and tumor weight decreased more markedly in the EVO-treated group. The tumor expression of phosphorylated AKT, detected using immunohistochemistry, decreased with increasing EVO doses in vivo. EVO induced PC cell apoptosis by inhibiting phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase/ERK and inhibiting the phosphorylation of signal transducer and activator of transcription activator 3 in PC cells to inhibit autophagy, suggesting that EVO may be considered as a novel PC treatment.

GR silencing impedes the progression of castration-resistant prostate cancer through the JAG1/NOTCH2 pathway via up-regulation of microRNA-143-3p.

BACKGROUND: Despite notable progression from a therapeutic point of view, castration resistant prostate cancer (CRPC) remains a clinical significant stumbling block. The current study aimed to elucidate the functional role of the gene glucocorticoid receptor (GR) in CRPC, and identify the contributions of the GR gene in CRPC in connection with microRNA-143-3p (miR-143-3p)/Jagged1 (JAG1)/NOTCH2.METHODS: The expression of GR and miR-143-3p in CRPC tissues and cells as well as JAG1/NOTCH2 expression in CRPC tissues was initially determined by quantitative polymerase chain reaction and Western blot analyses. The relationship among GR, JAG1, NOTCH2 and miR-143-3p was subsequently verified using the dual-luciferase reporter gene assay. ChIP assay confirmed the binding of GR to miR-143-3p promoter. Gain- and loss-function approaches were applied to ascertain the role of GR and miR-143-3p in progression of CRPC. Additionally, xenograft tumor models in nude mice were established to further confirm our results.RESULTS: GR was found to be highly expressed while miR-143-3p was lowly expressed in the CRPC tissues and cells. Silencing GR reduced migration, invasion, proliferation and increased apoptosis of CRPC cells. GR was enriched in the miR-143-3p promoter region and could down-regulate miR-143-3p expression. The overexpression of miR-143-3p led to a reduction in the migration, invasion, proliferation and increased apoptosis of CRPC cells. JAG1 and NOTCH2 were the target genes of miR-143-3p, and GR up-regulated the JAG1/NOTCH2 expression by down-regulating miR-143-3p. Silencing JAG1/NOTCH2 inhibited epithelial-mesenchymal transition and CRPC progression in vitro. Furthermore, the in vitro findings were reproduced in the in vivo experiments.CONCLUSION: The key findings of the current study demonstrated that silencing GR suppressed the progression of CRPC through the JAG1/NOTCH2 pathway via up-regulation of miR-143-3p.

Anti-osteosarcoma effect of hydroxyapatite nanoparticles both in vitro and in vivo by downregulating the FAK/PI3K/Akt signaling pathway.

Numerous studies have reported that hydroxyapatite nanoparticles (nano-HAPs) can inhibit the proliferation of a variety of tumor cells and this effect is different in different carcinoma cells. However, the effect of nano-HAPs on osteosarcoma cell proliferation has not been well understood thus far. In this study, we first showed that our synthesized nano-HAPs reduced cell viability and inhibited migration and invasion of OS-732 cells in a concentration-dependent manner. Using a BALB/c nude mouse tumor model, we demonstrated that nano-HAPs could effectively suppress tumor growth in vivo. We also performed RNA-seq analysis to investigate the underlying mechanism of these effects and discovered that treatment of OS-732 cells with nano-HAPs significantly downregulated the FAK/PI3K/Akt signaling pathway. Collectively, our study suggests that treatment with nano-HAPs can inhibit osteosarcoma cell growth, migration and invasion in vitro and suppress osteosarcoma in vivo.

Optimized Anti-Prostate-Specific Membrane Antigen Single-Chain Variable Fragment-Loaded Nanobubbles as a Novel Targeted Ultrasound Contrast Agent for the Diagnosis of Prostate Cancer.

ObjectivesTo prepare optimized prostate‐specific membrane antigen (PSMA) single‐chain variable fragment (scFv)–loaded nanobubbles (NBs) as a novel targeted ultrasound (US) contrast agent for diagnosis and treatment of prostate cancer (PCa).MethodsProstate‐specific membrane antigen scFv‐loaded NBs were prepared by membrane hydration and biotin‐streptavidin conjugation. Flow cytometry was used to observe the binding rate of the targeted NBs to PSMA‐expressing cells. Contrast‐enhanced US was used to monitor targeted and nontargeted NBs administered to nude mice with 22RV1, LNCaP, and PC‐3 xenograft tumors. The specific binding ability of the targeted NBs was further examined by fluorescence imaging of tumor cryosections.ResultsUniformly sized targeted NBs were successfully prepared (mean ± SD, 485.3 ± 28.4 nm). The NBs showed good stability and bound specifically to LNCaP and 22RV1 cells with high PSMA expression in vitro but did not bind to PC‐3 cells without PSMA expression. The targeted NBs presented good US enhancement, and the results of the in vivo xenograft tumor nude mouse model showed that the peak contrast intensity in LNCaP and 22RV1 cells was significantly higher for the targeted NBs than the nontargeted NBs (P < .05), whereas there was no significant difference in PC‐3 cells. Immunofluorescence results obtained from tumor sections confirmed that the targeted NBs were capable of targeting PSMA‐expressing tumor cells.ConclusionsThese novel PSMA scFv‐loaded NBs have proven to be an excellent US contrast agent for imaging PSMA‐expressing PCa and have the potential to not only enable efficient and safe molecular imaging but also to serve as a delivery system for targeted PCa therapies.