Mouse Subcutaneous Tumor Research Articles

Histotripsy ablation stimulates the innate immune system and modulates anti-tumor immunity in pancreatic cancer

Abstract With an overall 5-year survival rate of less than 9%, pancreatic cancer is a substantial cause of cancer related death in the United States. This high mortality has been associated with the immunosuppressive tumor microenvironment (TME) and the shortage of effective late-stage therapeutic options. Histotripsy is a non-thermal, non-ionizing, non-invasive cancer ablation modality that rapidly lyses targeted cells. To investigate how histotripsy can change the pancreatic TME, a Pan02 cell culture model of pancreatic adenocarcinoma was employed to determine the neoantigen release following histotripsy treatment and other standard ablation modalities. Histotripsy was found to release potential neoantigens at a level similar to other non-thermal ablations and superior to thermal ablation, which paralleled the increased innate immune system activation in vivo. For in vivo studies, subcutaneous Pan02 tumors in mice were treated with histotripsy. To monitor changes in the TME after treatment, rtPCR and flow cytometry were used and compared to untreated animals. Progression-free and overall survival were significantly improved with histotripsy treatment. Within 24 hours of treatment there was increased activation of the innate immune system and by 14 days there were decreased tumor-associated immune cell populations. This work illustrates the feasibility of using histotripsy to treat pancreatic cancer and shift the TME as well as providing mechanistic understanding into the activation of the innate immune system post-treatment. Future studies are needed to establish the mechanism of immunomodulation of the TME and the extent of potential systemic effects.

Establishment of Nude Mice Lacking NK Cells and Their Application for Human Tumor Xenografts

Objective:Nude mice are used as a recipient for human tumor cell xenografts. However, the success rate of xenotransplantation is unsatisfactory due to high natural killer (NK) activity. To overcome this limitation, we established nude mice with no NK cells, and compared the transplantation efficiency with that in nude mice.Methods: BALB/c Nude Jak3-/- (Nude-J) mice were established by crossing BALB/c Nude mice and BALB/c Jak-3-/- mice. Hematopoietic malignant cell lines (BCBL-1 and Z138) were implanted subcutaneously to compare the tumorigenicity in Nude-J mice with Nude Rag-2/Jak3 double deficient (Nude RJ) mice and nude mice. Results:Nude-J mice showed complete loss of NK and T lymphocytes, whereas B lymphocytes remained. Both BCBL-1 and Z138 human lymphoid malignant cell lines formed almost the same sizes of subcutaneous tumors in Nude-J and Nude RJ mice, whereas they formed no or only small tumors in nude mice. Splenocytes from Nude-J mice showed no cytotoxic activity in vitro. Conclusion:Nude-J mice can be a valuable tool for human tumor cell transplantation studies.

The Effect of Sirtuin 2 (Sirt2) Overexpressing Bone Marrow Mesenchymal Stem Cells on the Growth of Human Epidermal Growth Factor Receptor 2 (Her-2) Breast Cancer Cells and Its Mechanism

Our study investigates the effect of high expression of Sirt2 in MSCs (MSCs-Sirt2) on Her-2 breast cancer cell proliferation. A mouse subcutaneous xenograft tumor model was established and MSCssirt2 analysis was performed on nude mice. TUNEL staining, flow cytometry, western-blot, real-time PCR and immunohistochemistry were used to detect cancer cell apoptosis. The number of NK cells infiltrated by flow cytometry detected the tumor tissue of tumor-bearing mice, and its killing activity on tumor-bearing mice was detected by isotope labeling and release method. The levels of TNF-α, IFN-γ, IL-8, IL-6 and IL-10 were detected by ELISA. Caspase-3 level was decreased in the MSCs group (P <0.01) while increased in the MSCs-sirt2 group (P <0.001). However, PCNA expression showed an opposite profile in the Her-2 group and MSCs-sirt2 group compared to Caspase-3 level (P <0.01). The tumor volume and weight in the MSCs-sirt2 group was significantly reduced (P < 0.01), while increased in the MSCs group significantly (P < 0.05). The number of Ki-67-positive tumor cells in MSCs-sirt2 group was significantly reduced (P <0.01) and increased in MSCs group (P < 0.001) with oppositive number of TUNEL-positive tumor cells in the MSCs-sirt2 group and MSCs group (P <0.01). IFN-γ level showed an upward trend (P <0.001). The NK cell toxicity of MSCs-Sirt2 group was significantly higher (P <0.001). MSCs-Sirt2 has an inhibitory effect on Her-2 breast cancer cell growth by enhancing the local inflammatory response of NK cells.

In vivo imaging of neuroblastomas using GD2-targeting graphene quantum dots

BackgroundPatients with neuroblastoma, a common childhood malignancy, often have poor prognosis. It is mandatory to develop an accurate and efficient diagnostic tool for neuroblastomas, so that the treatment can be started early. Graphene quantum dot (GQD), a nanomaterial, can be used to carry proteins, genetic materials, or drugs. GD2, a disialoganglioside, is a surface antigen expressed on neuroblastoma. This study investigated the in vivo targeting and imaging of neuroblastomas using GD2-targeting GQDs. MethodsGQDs were synthesized and conjugated with anti-GD2 antibody (anti-GD2/GQDs). In vitro cytotoxicity of GQDs and anti-GD2/GQDs was studied in human neuroblastoma cells by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)-based colorimetric assay. The tumor tracking and imaging of anti-GD2/GQDs in mice were investigated by in vivo imaging system (IVIS). ResultsTreatment with GQDs or anti-GD2/GQDs induced no or mild cytotoxicity in fibroblasts and neuroblastoma cells. After co-incubation, GQDs and anti-GD2/GQDs were located in the cytoplasm and nucleus of neuroblastoma cells, with GQDs showing a blue fluorescence and anti-GD2/GQDs an orange/red emission. The IVIS images demonstrated accumulation of the fluorescence of anti-GD2/GQDs in the subcutaneous tumors in mice 24 h after intravenous injection of anti-GD2/GQDs. ConclusionsAnti-GD2/GQDs may potentially be used for the targeting and imaging of neuroblastomas in vivo.

Baicalin triggers apoptosis, inhibits migration, and enhances anti-tumor immunity in colorectal cancer via TLR4/NF-κB signaling pathway.

Aberrant activation of the nuclear factor-kappa B (NF-κB) signaling pathway is closely implicated in colorectal cancer (CRC) growth, metastasis, and immune escape. In the present study, we reported natural derived compound of baicalin (BA), an efficient inhibitor of NF-κB, with good anti-tumor effect on CRC. CCK8 and colony formation assays showed that Baicalin significantly inhibit viability and proliferation in HCT-116 and CT26 cells. Additionally, Baicalin dramatically triggers mitochondria-mediated apoptosis in both HCT-116 and CT-26 cells, which is evidenced by loss of mitochondrial membrane potential and elevated cellular reactive oxygen species level. Treatment with Baicalin suppresses migration and invasion of CT26 cells by impairing TLR4/NF-κB signaling pathway. What's more, administration of Baicalin significantly retarded tumor growth rate in a subcutaneous xenograft tumor mouse model of CT26 cells. Treatment with Baicalin could ameliorate tumor immunosuppressive environment by downregulation of PD-L1 expression and proportion of myeloid-derived suppressor cells (MDSCs) and upregulation of percent of CD4+ and CD8+ T cells in CT26 tumors, thus improving anti-tumor immunity. In conclusion, our study demonstrated that baicalin triggers apoptosis, inhibits migration, and enhances anti-tumor immunity in colorectal cancer via TLR4/NF-κB signaling pathway, suggesting it might serve as a potential candidate drug for the treatment of CRC. PRACTICAL APPLICATIONS: In the present study, we reported natural derived compound of baicalin (BA), an efficient inhibitor of NF-κB, with good anti-tumor effect on CRC. We demonstrated that baicalin triggers mitochondria-mediated apoptosis, inhibits migration, and improves anti-tumor immunity in colorectal cancer via TLR4/NF-κB signaling pathway.

Gastric cancer-secreted exosomal X26nt increases angiogenesis and vascular permeability by targeting VE-cadherin.

Angiogenesis is closely associated with tumorigenesis, invasion, and metastasis by providing oxygen and nutrients. Recently, increasing evidence indicates that cancer‐derived exosomes which contain proteins, coding, and noncoding RNAs (ncRNAs) were shown to have proangiogenic function in cancer. A 26‐nt‐long ncRNA (X26nt) is generated in the process of inositol‐requiring enzyme 1 alpha (IRE1α)‐induced unspliced XBP1 splicing. However, the role of X26nt in the angiogenesis of gastric cancer (GC) remains largely unknown. In the present study, we found that X26nt was significantly elevated in GC and GC exosomes. Then, we verified that X26nt could be delivered into human umbilical vein endothelial cells (HUVECs) via GC cell exosomes and promote the proliferation, migration, and tube formation of HUVECs. We revealed that exosomal X26nt decreased vascular endothelial cadherin (VE‐cadherin) by directly combining the 3′UTR of VE‐cadherin mRNA in HUVECs, thereby increasing vascular permeability. We further demonstrated that X26nt accelerates the tumor growth and angiogenesis in a mouse subcutaneous tumor model. Our findings investigate a unique intercellular communication mediated by cancer‐derived exosomes and reveal a novel mechanism of exosomal X26nt in the regulation of tumor vasculature.

Predicting Schwannoma Growth in a Tumor Model Using Targeted Imaging.

Vestibular schwannoma (VS) is a common pathology encountered in neurotology clinics. Many patients are observed with a "wait and scan" approach. Previous efforts to determine radiographic indicators of future growth have been unsuccessful. Using a mouse subcutaneous tumor model, we seek to determine if fluorescent imaging with directed immunotargets could be used to predict schwannoma growth rate. Anti-VEGFR2 and anti-Her2/Neu monoclonal antibodies were covalently linked to a near-infrared probe (IRDye800). Immunodeficient mice underwent subcutaneous injections with a rat-derived schwann (R3) cell line. When tumor growth was evident, either Anti-VEGFR2-IRDye800, anti-Her2/Neu-IRDye800, or Immunoglobulin G (IgG) Isotype-IRDye800 (control) were injected via tail vein. The mice were serially imaged in a closed field near-IR device. Fluorescent data were analyzed for tumor signal and correlated with tumor sie and growth rate. Heterogeneity of fluorescent tumor signal was also assessed. In both anti-VEGFR2 and anti-Her2/Neu groups, there were strong correlations between day 1 mean tumor fluorescence and eventual maximum tumor volume (p = 0.002, 0.001; r2 = 0.92, 0.86). There was also strong correlation with maximum tumor signal on day 1 and maximum tumor volume (p = 0.003, 0.008; r2 = 0.90, 0.91). There was no such correlation in the control group (p = 0.99, 0.75; r2 = 0.0002, 0.028). Given the potential morbidity in VS intervention, observation is an appropriate approach for patients with slow-growing or stagnant tumors. We seek to identify immunotargets in a murine model that show promise in predicting schwannoma growth with advanced imaging techniques. Both Her2/Neu and VEGFR2 correlated strongly wth tumor size and growth rates and are promising targets that merit further investigation.

STK17B promotes the progression of ovarian cancer.

BackgroundProtein kinase is increasingly receiving widespread attention because of its role in the tumor progression. Serine/threonine protein kinase (STK) is an important family involved in the development of a variety of cancers. Many studies have shown that serine/threonine kinase 17B (STK17B) is highly expressed in a variety of malignant tumors and participate in proliferation and metastasis. However, the exact function of STK17B remains uncertain in ovarian cancer. Our study aims to investigate whether STK17B plays a role in the occurrence and development of epithelial ovarian cancer.MethodsWe employed quantitative reverse transcription polymerase chain reaction to detect the relative expression of STK17B in ovarian cancer tissues. STK17B was down-regulated and up-regulated in ovarian cancer cell lines by small interfering RNA and overexpressed plasmid, respectively. The effects of STK17B on proliferation, invasion and migration of ovarian cancer cells in vitro were analyzed by CCK-8 test, Transwell test, scratch test and EDU test. The tumorigenicity of subcutaneous xenograft tumor in nude mice to study the role of STK17B in tumorigenesis in vivo. Western Blotting analysis revealed that STK17B and EMT.ResultsSTK17B expression was significantly increased in ovarian cancer tissues. The STK17B silencing suppressed cell progression, while the overexpression of STK17B promoted progression in vivo or in vitro. Western bolt showed that STK17B increased the invasion and migration of ovarian cancer cell by promoting the EMT process.ConclusionsSTK17B was highly expressed in epithelial ovarian cancer tissues and increased the proliferation, invasion and migration of ovarian cancer cells by promoting EMT process.

AMD1 upregulates hepatocellular carcinoma cells stemness by FTO mediated mRNA demethylation.

BackgroundS‐adenosylmethionine decarboxylase proenzyme (AMD1) is a key enzyme involved in the synthesis of spermine (SPM) and spermidine (SPD), which are associated with multifarious cellular processes. It is also found to be an oncogene in multiple cancers and a potential target for tumor therapy. Nevertheless, the role AMD1 plays in hepatocellular carcinoma (HCC) is still unknown.MethodsHCC samples were applied to detect AMD1 expression and evaluate its associations with clinicopathological features and prognosis. Subcutaneous and orthotopic tumor mouse models were constructed to analyze the proliferation and metastasis of HCC cells after AMD1 knockdown or overexpression. Drug sensitive and tumor sphere assay were performed to investigate the effect of AMD1 on HCC cells stemness. Real‐time quantitative PCR (qRT‐PCR), western blot, immunohistochemical (IHC) and m6A‐RNA immunoprecipitation (Me‐RIP) sequencing/qPCR were applied to explore the potential mechanisms of AMD1 in HCC. Furthermore, immunofluorescence, co‐IP (Co‐IP) assays, and mass spectrometric (MS) analyses were performed to verify the proteins interacting with AMD1.ResultsAMD1 was enriched in human HCC tissues and suggested a poor prognosis. High AMD1 level could promote SRY‐box transcription factor 2 (SOX2), Kruppel like factor 4 (KLF4), and NANOG expression of HCC cells through obesity–associated protein (FTO)‐mediated mRNA demethylation. Mechanistically, high AMD1 expression increased the levels of SPD in HCC cells, which could modify the scaffold protein, Ras GTPase‐activating‐like protein 1 (IQGAP1) and enhance the interaction between IQGAP1 and FTO. This interaction could enhance the phosphorylation and decrease the ubiquitination of FTO.ConclusionsAMD1 could stabilize the interaction of IQGAP1 with FTO, which then promotes FTO expression and increases HCC stemness. AMD1 shows prospects as a prognostic predictor and a therapeutic target for HCC.

Apigenin and Temozolomide Synergistically Inhibit Glioma Growth Through the PI3K/AKT Pathway.

Background: Glioma is a devastating disease with the worst prognosis among human malignant tumors. Although temozolomide (TMZ) improves the overall survival of glioma patients, there are still many glioma patients who are resistant to TMZ. In this study, we focused on the effect of apigenin (API) and TMZ on glioma cells in vitro and in vivo, and we studied the underlying molecular mechanisms. Materials and Methods: To investigate the effect of API on glioblastoma cell proliferation, cell viability was assessed after glioma cells were incubated with various concentrations of API with or without TMZ using MTT assays. Then, we explored the synergistic effect of API and TMZ on glioma cell cycle, apoptosis, and migration. To investigate the molecular mechanism behind the synergism of API and TMZ, we examined the related genes of the major signaling pathways involved in glioma pathogenesis by Western blotting. Results: In this study, we found that API significantly suppressed the proliferation of glioma cells in a dose- and time-dependent manner. Combining API and TMZ significantly induced glioma cells arrest at the G2 phase and inhibited glioma cells proliferation compared with API or TMZ alone. In addition, API promoted the ability of TMZ to induce glioma cells apoptosis and inhibit glioma cells invasion. Furthermore, compared with treatment with individual agents, the combination of API and TMZ significantly inhibited the growth of subcutaneous tumors in mice. These results implied that API could synergistically suppress the growth of glioma cells when combined with TMZ. Combining API and TMZ significantly inhibited the protein expression of p-AKT, cyclin D1, Bcl-2, Matrix Metallopeptidase 2, and Matrix Metallopeptidase 9. Conclusion: API and TMZ synergistically inhibited glioma growth through the PI3K/AKT pathway.

Effects of different combined regimens of cisplatin, metformin, and quercetin on nasopharyngeal carcinoma cells and subcutaneous xenografts

Cisplatin, metformin, and quercetin are all reliable anticancer drugs. However, it is unclear how effective their different combination regimens are on the growth of nasopharyngeal carcinoma cell line Sune-1 and subcutaneous xenograft in nude mice. This study evaluated the effects of single-drug, two-drug, and three-drug simultaneous or sequential combined application of these drugs on the growth of Sune-1 cells and subcutaneous xenograft tumors in nude mice. The results showed that the different combination regimens of cisplatin, metformin and quercetin all had significant inhibitory effects on the proliferation of Sune-1 cells and the growth of subcutaneous xenografts in nude mice (P < 0.01), and the inhibition rate of the three drugs simultaneous combined application was significant Higher than the two-drug combination or single-drug application (P < 0.05), the contribution level of each drug in the three-drug combination application from high to low were cisplatin > metformin > quercetin. In summary, our results indicate that the simultaneous combination of cisplatin, metformin, and quercetin may synergistically inhibit the growth of Sune-1 cells and subcutaneous xenografts in nude mice through their different anticancer mechanisms, which may be clinically refractory and provide reference for chemotherapy of patients with recurrent nasopharyngeal carcinoma.

Antitumor effect and molecular mechanism of fucoidan in NSCLC

BackgroundFucoidan, a water-soluble polysaccharide, exerts anticoagulant and antiviral functions. It was recently reported that fucoidan also exerts an antitumor function. Lung cancer is one of the most common cancers in the world. The aim of this study was to investigate anti-tumor,apoptosis and anti-metastasis effects of fucoidan in both cell-based assays and mouse xenograft model, as well as to clarify possible role of m-TOR pathway in the protection.MethodsIn vitro: Different concentrations of fucoidan were given to act on non-small cell lung cancer (NSCLC) cell lines A549 and H1650. The effects of fucoidan on cell proliferation were observed by detecting cyclin expression levels, CCK8 and EDU experiments and cloning experiments. The apoptotic level was detected by flow cytometry and the apoptotic protein level was detected by Westernblot. By detecting the expression of adhesion molecules, the expression of matrix metalloproteinase (MMP) family, and Transwell cell invasion and migration experiment, the effect of fucoidan on cell adhesion, invasion and migration was observed. Meanwhile the effect of fucoidan on angiogenesis was observed by detecting the expression of vascular endothelial growth factor (VEGF).In vivo experiment: An animal model of NSCLC cell mouse subcutaneous xenograft tumor was established to analyze the correlation between the consumption of fucoidan and the size and volume of xenograft tumor through gross observation. Through immunohistochemical staining and immunofluorescence double staining, ki67 and cell adhesion molecules (E-cadherin, N-cadherin and CD31) and VEGF-A in the tumor were detected, and the correlation between the amount of fucoidan and the above indexes was analyzed.ResultsFucoidan inhibited the proliferation and angiogenesis of NSCLC cells via the mTOR pathway and promoted their apoptosis by increasing the Bax/Bcl-2 ratio. Not only that, fucoidan inhibited NSCLC cell invasion via epithelial–mesenchymal transformation (EMT). The mice fed fucoidan exhibited significant reductions in tumor volumes and weights. These indicators (Ki67, VEGF-A,N-cadherin) were decreased and E-cadherin expression was up-regulated in A549 mice that treated with fucoidan. The results showed that fucoidan inhibited tumor proliferation in vivo by affecting the expression of related proteins.ConclusionFucoidan conveys antitumor effects and our results represent an ideal therapeutic agent for NSCLC.

MiR-145 inhibits mitochondrial function of ovarian cancer by targeting ARL5B

Metabolic reprogramming refers to the transformation of the whole metabolic network including glycolysis and mitochondrial metabolism, mainly manifested in Warburg effect and mitochondrial metabolic reprogramming. The roles of miR-145 in glycolysis have been established in ovarian cancer cells. Howerer, its roles in mitochondrial metabolic reprogramming are still unclear. This study aims to identify whether miR-145 regulates mitochondrial metabolic reprogramming in ovarian cancer cells. First, functional experiment showed that overexpression of miR-145 inhibited mitochondrial function in ovarian cancer cells, evident by the decreased mtDNA copy numbers, ATP level, mitochondrial membrane potential, and the expression levels of mitochondrial markers. Mechanistically, miR-145 inhibited mitochondrial function by targeting ARL5B directly. Futhermore, miR-145 overexpression decreased ARL5B expression in ovarian cancer tissue subcutaneous tumors of nude mice. In conclusion, we have highlighted that miR-145 inhibits mitochondrial function and achieves this by targeting ARL5B directly for the first time. The results provides a more adequate theoretical basis for understanding the molecular pathology of ovarian cancer, and provides the necessary basic data for miR-145 as a potential diagnosis and treatment target for ovarian cancer.

FSTL1 increases cisplatin sensitivity in epithelial ovarian cancer cells by inhibition of NF-κB pathway.

To investigate the effects of FSTL1-mediated NF-κB signaling pathway on cisplatin (DDP) sensitivity of EOC cells. FSTL1 expression was determined in epithelial ovarian cancer (EOC) tissues and corresponding adjacent tissues using immunohistochemistry. SKOV3 and SKOV3/DDP cells were transfected and grouped into Blank, Vector, and FSTL1 groups. The sensitivity and 50% inhibitory concentration (IC50) of cells treated with different concentrations of DDP were detected by MTT assay. SKOV3/DDP cells were treated with 20μM DDP, followed by evaluation of cell proliferation, cell apoptosis and determination of NF-κB pathway-related proteins while SKOV3 cells without. FSTL1 expression in EOC tissues and cells was significantly down-regulated, especially decreased in DDP-resistant EOC cells SKOV3/DDP. In SKOV3 cells and SKOV3/DDP cells, the cell viability was reduced and the DDP sensitivity was improved with the decreased IC50 after over-expressing FSTL1. Compared with Blank group, FSTL1 group had declined number of SKOV3 cell colonies and increased cell apoptosis, with obvious up-regulations of FSTL1, Bax/Bcl-2 and cleaved caspase-3 expression and the down-regulations of p-IκBα, p-p65 and survivin expression. Combination of up-regulation of FSTL1 and DDP treatment can also effectively reduce cell colony forming, increase cell apoptosis, and inhibit NF-κB pathway activity of SKOV3/DDP cells. Moreover, this combination can also significantly suppress the growth of subcutaneous xenograft tumors in nude mice. FSTL1 may inhibit NF-κB signaling pathway to suppress the growth and promote the apoptosis of epithelial ovarian cancer cells, and thereby enhancing its DDP sensitivity.

Exosomal Circ_0125473 Drives Macrophages Polarization Toward M2 Through miR-5787/Wnt1/β-Catenin Signaling Pathway in Colorectal Cancer

Background: Accumulating studies have suggested exosomes participate in regulating cancer initiation and progression by delivering bioactive molecules during cell-to-cell communications. Exosomal circRNAs play critical roles in the intercellular communication between colorectal cancer (CRC) cells and macrophages, whereas the molecular mechanism remains largely unclear. Methods: We quantitatively measured the expression of circR_0125473 in serum samples from CRC patients. The effect of exosomal circR_0125473 on CRC cell proliferation and invasion and macrophages polarization was investigated. The molecular mechanisms underlying the oncogenesis and regulating M1/M2 balance in tumor immune microenvironment of exosomal circR_0125473 were explored in cell line models and subcutaneous tumors formation nude mice. Findings: In this study, exosomal circR_0125473 was upregulated in CRC and positively associated with CRC metastasis. Besides, exosomal circR_0125473 promoted CRC cells proliferation and invasion. CRC exosomes-derived circ_0125473 drived macrophages polarization towards M2 through miR-5787/Wnt1/β-catenin signaling pathway. Moreover, exosomal circR_0125473 also significantly promoted the growth of subcutaneous tumors in nude mice, although CRC cells were transplanted with exosomes-pretreated monocyte-derived macrophages (MDMs) subcutaneously. Accordingly, exosomes delivering circR_0125473 participates in regulating M1/M2 balance in tumor immune microenvironment, thereby influencing CRC tumorigenesis and progression. Funding: This study is supported by funds from the National Natural Science Foundation, China (82003042), Shandong Natural Science Foundation (ZR2020KC001 and ZR2019QH012), and Weifang Science and Technology Development Program, China (2019GX031 and 2019YX020). Declaration of Interest: All authors declare no competing interests. Ethical Approval: Animal studies were conducted under the government of the Animal Ethics Committee of Weifang Medical University.

Lobaplatin Enhances Radioactive 125I Seed-Induced Apoptosis and Anti-Proliferative Effect in Non-Small Cell Lung Cancer by Suppressing the AKT/mTOR Pathway.

IntroductionIn recent years, radioactive 125I seed implantation combined with chemotherapy has been regarded as a safe and effective treatment for advanced non-small cell lung cancer (NSCLC). However, the mechanism underlying this success is still unclear.MethodsIn this study, we investigated the apoptosis and anti-proliferative effect induced by 125I in A549, H1975, and H157 cells and determined whether a sensitizing concentration of lobaplatin (LBP) could enhance these effects. We performed in vitro experiments on A549, H1975, and H157 cells; we investigated the effects of 125I or lobaplatin (LBP) alone, or in combination, on cellular apoptosis and proliferation by performing flow cytometry, Bax/Bcl2 ratio, TUNEL, cell viability assay, cell cycle, and EdU. To further verify our findings, a subcutaneous tumor mouse model was established. Moreover, AKT/mTOR pathway was detected to determine whether this pathway was involved in the anti-cancer effect of 125I and LBP by up-regulating or down-regulating the expression of mTOR.ResultsBased on our results, the sensitizing concentration of LBP could enhance the 125I-induced apoptosis and anti-proliferation effect. Furthermore, the subcutaneous tumor mouse model obtained the consistent results. More importantly, the AKT/mTOR pathway was down-regulated after the treatment of 125I and LBP, and the anti-cancer effect of 125I and LBP could be compromised by up-regulating the mTOR expression.ConclusionOur study proved that LBP promotes the apoptotic and anti-proliferative effects of 125I in NSCLC cells by inhibiting the AKT/mTOR pathway and provides a foundation for future studies and enhanced combinatorial approaches for NSCLC in the clinical setting.

Effect of CMNa combined with radiotherapy on the tumor immune microenvironment of mouse cervical cancer cell transplantation tumor model

ABSTRACT In this study, we construct a subcutaneous tumor mice model of U14 cells, observe the tumor growth, and detect the expression of Foxp3 and VISTA in cervical cancer tissues and adjacent tissues during CMNa-enhancing radiotherapy.From the 15th day, compared with the control group, the tumor volume changes in each treatment group were significant (P < 0.01). CMNa combined with radiotherapy had an interactive effect and a positive effect in inhibiting tumor volume growth. There was no significant difference in the expression of Foxp3 and VISTA in mouse cervical cancer tissues and adjacent tissues in each group. The Foxp3 level in the RT group was the highest, and the CMNa group was the lowest. The VISTA level of the CMNa+RT group was the highest, the RT group is followed by, and the Control group is the lowest. The Foxp3 level of the CMNa group did not change much at each different point. The Foxp3 level in RT and CMNa+RT group gradually decreased after a transient increase, and the VISTA level in the CMNa+RT group increased more.Our results show that CMNa can enhance the efficacy of radiotherapy, and at the same time can reduce the compensatory increase in regulatory T cell Foxp3 levels caused by radiotherapy, and reduce the radiotherapy response. However, in the course of the treatment of the two, there may be a substantial increase in the level of VISTA, and the combined application of VISTA inhibitors may increase the anti-tumor response.

Chlorogenic acid induces apoptosis, inhibits metastasis and improves antitumor immunity in breast cancer via the NF‑κB signaling pathway.

Breast cancer which is the most common type of diagnosed cancer among women worldwide possesses metastatic potential, multi-drug resistance, and high mortality. The NF-κB signaling pathway has been revealed to be abnormally activated in breast cancer cells and closely associated with high metastasis and poor prognosis. In the present study, it was reported that chlorogenic acid (CGA), a potent NF-κB inhibitor derived from coffee, exerted antitumor activity in breast cancer. MTT and colony formation assays were conducted and it was revealed that CGA inhibited viability and proliferation in breast cancer cells. Additionally, CGA significantly induced apoptosis and suppressed migration and invasion in breast cancer cells. Notably, immunofluorescence analysis confirmed that CGA could efficiently suppress nuclear transcription of NF-κB p65. In addition, results of western blotting demonstrated that CGA markedly impaired the NF-κB and EMT signaling pathways. The antitumor effect of CGA was evaluated in a subcutaneous tumor mouse model of 4T1 cells, and the results revealed that CGA markedly retarded tumor growth and prolonged the survival rate of tumor-bearing mice. Notably, CGA inhibited pulmonary metastasis of 4T1 cells by enhancing the proportion of CD4+ and CD8+ T cells in spleens of mice, which indicated an improvement of antitumor immunity. In conclusion, the present present study demonstrated that CGA improved antitumor immunity, exerting antitumor and anti-metastatic effects by impairing the NF-κB/EMT signaling pathway, suggesting that CGA may serve as a potential candidate for therapy of breast cancer.

MicroRNA-27 Inhibits Autophagy and Promotes Proliferation of Multiple Myeloma Cells by Targeting the NEDD4/Notch1 Axis.

Multiple myeloma (MM) is a malignant tumor disease that seriously affects the health of patients. Previous studies have shown the crucial role of autophagy in the development of MM. Therefore, the study aimed to study the effect of miR-27 on autophagy in MM via NEDD4/Notch1 axis. RT-qPCR or western blot analysis was used to detect the expression of miR-27, NEDD4, and Notch1 in bone marrow tissues and CD138+ plasma cells of patients and MM cells. After gain- and loss-of-function assays in MM cells, proliferation and invasion were assessed by clone formation and Transwell assays. Meanwhile, expression of autophagy-related proteins was measured by western blot analysis, followed by evaluation of autophagosomes and autophagic flow. The targeting relationship was evaluated by luciferase reporter assay, whereas the binding of NEDD4 to Notch1 protein was analyzed by co-immunoprecipitation. The ubiquitination level of Notch1 protein was detected. A nude mouse tumor model was established to determine the role of miR-27 in MM in vivo. miR-27 and Notch1 upregulation and NEDD4 downregulation were observed in bone marrow tissues and CD138+ plasma cells of patients and MM cells. miR-27 negatively targeted NEDD4, while NEDD4 could specifically bind to Notch1 protein to increase Notch1 ubiquitin degradation in MM cells. miR-27 or Notch1 overexpression or NEDD4 silencing diminished autophagy but enhanced proliferation and invasion of MM cells. miR-27 upregulation promoted the formation of subcutaneous tumor in nude mice. Collectively, miR-27 elevated Notch1 expression by targeting NEDD4 and promoted the development of MM by inhibiting cell autophagy, which provides a new idea and basis for MM treatment.

Electroporation outperforms in vivo-jetPEI for intratumoral DNA-based reporter gene transfer

Intratumoral delivery of drug-encoding plasmid DNA (pDNA) enables localised in vivo expression of biological drugs, offering an attractive alternative to conventional protein treatment. However, this requires physical or chemical methods to enhance the low transfection efficiency of naked pDNA. Electroporation and complexation with the polycation in vivo-jetPEI are both evaluated in the clinic for intratumoral pDNA delivery, but lack head-to-head comparison. This study therefore compared both methods for intratumoral DNA-based reporter gene transfer in a subcutaneous mouse tumour model. Intratumoral electroporation resulted in strong reporter expression that was restricted to the tumour area and persisted for at least ten days. Intratumoral expression after injection of pDNA-jetPEI complexes was two to three logs lower, did not exceed the background in most mice, and lasted less than five days even with repeated dosing. Remarkably, reporter expression was primarily detected in the lungs, presumably due to leakage of pDNA-jetPEI complexes into the systemic circulation. In conclusion, electroporation enabled more efficient, prolonged and tumour-specific reporter expression compared to intratumoral injection of pDNA complexed with in vivo-jetPEI. These results favour the use of electroporation for intratumoral DNA-based gene transfer, and suggest further optimisation of pDNA-jetPEI complexes is needed to improve their efficacy and biosafety.