Subcutaneous Xenografts In Nude Mice Research Articles

Targeting TRIM24 promotes neuroblastoma differentiation and decreases tumorigenicity via LSD1/CoREST complex.

High-risk neuroblastoma (NB) still has an unfavorable prognosis and inducing NB differentiation is a potential strategy in clinical treatment, yet underlying mechanisms are still elusive. Here we identify TRIM24 as an important regulator of NB differentiation. Multiple datasets and clinical specimens were analyzed to define the role of TRIM24 in NB. The effects of TRIM24 on differentiation and growth of NB were determined by cell morphology, spheres formation, soft agar assay, and subcutaneous xenograft in nude mice. RNA-Seq and qRT-PCR were used to identify genes and pathways involved. Mass spectrometry and co-immunoprecipitation were used to explore the interaction of proteins. Trim24 is highly expressed in spontaneous NB in TH-MYCN transgenic mice and clinical NB specimens. It is associated with poor NB differentiation and unfavorable prognostic. Knockout of TRIM24 in neuroblastoma cells promotes cell differentiation, reduces cell stemness, and inhibits colony formation in soft agar and subcutaneous xenograft tumor growth in nude mice. Mechanistically, TRIM24 knockout alters genes and pathways related to neural differentiation and development by suppressing LSD1/CoREST complex formation. Besides, TRIM24 knockout activates the retinoic acid pathway. Targeting TRIM24 in combination with retinoic acid (RA) synergistically promotes NB cell differentiation and inhibits cell viability. Our findings demonstrate that TRIM24 is critical for NB differentiation and suggest that TRIM24 is a promising therapeutic target in combination with RA in NB differentiation therapy.

Long non-coding RNA PANDAR promoted radiation and cisplatin-induced DNA damage repair through ATR/CHK1 in NSCLC.

DNA-damaging agents, including radiation and platinum-based chemotherapy, are indispensable treatments for non-small cell lung cancer (NSCLC) patients. However, cancer cells tend to be resistant to both radiation and chemotherapy, thus resulting in treatment failure or recurrence. The purpose of this study was to explore the effect and mechanism of long non-coding RNA (lncRNA) PANDAR (promoter of CDKN1A antisense DNA damage-activated RNA) on NSCLC sensitivity to radiation and chemotherapy. Cell counting kit (CCK-8), colony formation and flow cytometry were respectively performed to determine the cell cycle and apoptosis of NSCLC cells treated with γ-ray radiation and cisplatin. The extent of DNA damage was evaluated using a comet assay and immunofluorescence staining against γH2AX. In addition, we explored the role of PANDAR in DNA damage response pathways through western blot analysis. Finally, a nude mouse subcutaneous xenograft model was established to assess the sensitivity to radiation and chemotherapy in vivo. In cell experiments, PANDAR knockdown can increase the sensitivity of NSCLC cells to radiation and cisplatin. The CCK-8 results showed that cell viability was significantly increased in the overexpression group after radiation and cisplatin treatments. The overexpression group also showed more colonies, less apoptosis and DNA damage, and G2/M phase arrest was aggravated to provide the time necessary for DNA repair. Contrary to PANDAR overexpression, the trends were reversed in the PANDAR knockdown group. Furthermore, PANDAR knockdown inhibited radiation and cisplatin-activated phosphorylation levels of ATR and CHK1 in NSCLC cells. Finally, our in vivo model showed that targeting PANDAR significantly sensitized NSCLC to radiation and cisplatin. Our study showed that PANDAR knockdown promoted sensitivity to radiation and cisplatin in NSCLC by regulating the ATR/CHK1 pathway, thus providing a novel understanding as well as a therapeutic target for NSCLC treatment. In NSCLC cells, lncRNA PANDAR negatively regulates sensitivity to radiation and cisplatin. PANDAR can promote the repair of radiation and cisplatin-induced DNA damage and activation of the G2/M checkpoint through the ATR/CHK1 pathway. PANDAR knockdown results in defects in DNA damage repair accompanied by more cell apoptosis.

Computable structured aptamer for targeted treatment of ovarian cancer.

Nucleolin protein expression is higher on the ovarian cancer cell surface. AS1411, a DNA aptamer, can bind with nucleolin protein specifically. In this study, we developed HA and ST DNA tiles to assemble six AS1411 aptamers to deliver doxorubicin. In addition, to superior serum stability and drug loading, HA-6AS and ST-6AS outperformed TDN-AS in cellular uptake. HA-6AS and ST-6AS exhibited satisfactory targeted cytotoxicity and achieved resounding lysosomal escape. Moreover, when injected into nude mice subcutaneous xenograft models, HA-6AS reached the peak in tumor more quickly than ST-6AS, and better expressed the active targeting ability of AS1411. Our study suggests that designing appropriate DNA tiles to assemble different aptamers to deliver different chemotherapeutic drugs is a promising treatment for ovarian cancer.

HIF-1α/YAP Signaling Rewrites Glucose/Iodine Metabolism Program to Promote Papillary Thyroid Cancer Progression.

Background: The management of aggressive and progressive metastatic papillary thyroid cancer (PTC) is very difficult. An inverse relationship between radioiodine and F-18 fluorodeoxyglucose (FDG) uptake (''flip-flop'' phenomenon) is described for invasive PTC during dedifferentiation. However, no satisfactory biologic explanation for this phenomenon. Hypoxia is an important microenvironmental factor that promotes cancer progression and glycolysis. The Hippo-YAP is a highly conserved tumor suppressor pathway and contributes to cancer metabolic reprogramming. Thus, we investigated the underlying molecular mechanisms of glucose/iodine metabolic reprogramming in PTC, focusing on the tumor hypoxia microenvironment and Hippo-YAP signaling. Methods: Immunohistochemistry staining was conducted to evaluate the expressions of hypoxia-inducible factor 1α (HIF-1α), yes-associated protein (YAP), glucose transporters 1 (GLUT1) and sodium iodine symporter (NIS) in matched PTC and the adjacent noncancerous tissues. PTC cell lines were cultured under normoxic (20% O2) and hypoxic (1% O2) conditions and the glycolysis level and NIS expression were measured. Further, we characterized the molecular mechanism of glucose/iodine metabolic reprogramming in PTC cell. Finally, we validated the results in vivo by establishing subcutaneous xenografts in nude mice. Results: The expression levels of HIF1-α, YAP and GLUT1 were upregulated in PTC tissues and YAP expression was positively associated with HIF-1α, GLUT1 and TNM stages. Meanwhile, the expression of NIS was negatively correlated with YAP. Further, in vitro studies indicated that hypoxia-induced YAP activation was critical for accelerating glycolysis and reducing NIS expression in PTC cells. Inhibition of YAP had the opposite effects in vitro and tumorigenicity in vivo. Hypoxia inhibited the Hippo signaling pathway resulting in the inactivation of YAP phosphorylation, further promoting the nuclear localization of YAP in PTC cells. The mechanism is that hypoxic stress promoted YAP binding to HIF-1α in the nucleus and maintained HIF-1α protein stability. The YAP/HIF-1α complex bound and directly activated the GLUT1 transcription to accelerate glycolysis. Meanwhile, HIF-1α/YAP signaling might indirectly reduce the expression of NIS by promoting the output of MAPK signaling. In vivo studies confirmed the YAP-mediated reprogramming of glucose/iodine metabolism promoted PTC progression. Conclusions: Collectively, our data revealed a novel regulatory mechanism of the glucose/iodine metabolic program rewritten by HIF-1α/YAP signaling in PTC. Inhibition of HIF-1α/YAP signaling alone or in combination with other potential markers may effectively combat aggressive PTC.

Interaction of MRPL9 and GGCT Promotes Cell Proliferation and Migration by Activating the MAPK/ERK Pathway in Papillary Thyroid Cancer.

Thyroid cancer remains the most common endocrine malignancy worldwide, and its incidence has steadily increased over the past four years. Papillary Thyroid Cancer (PTC) is the most common differentiated thyroid cancer, accounting for 80–85% of all thyroid cancers. Mitochondrial proteins (MRPs) are an important part of the structural and functional integrity of the mitochondrial ribosomal complex. It has been reported that MRPL9 is highly expressed in liver cancer and promotes cell proliferation and migration, but it has not been reported in PTC. In the present study we found that MRPL9 was highly expressed in PTC tissues and cell lines, and lentivirus-mediated overexpression of MRPL9 promoted the proliferation and migration ability of PTC cells, whereas knockdown of MRPL9 had the opposite effect. The interaction between MRPL9 and GGCT (γ-glutamylcyclotransferase) was found by immunofluorescence and co-immunoprecipitation experiments (Co-IP). In addition, GGCT is highly expressed in PTC tissues and cell lines, and knockdown of GGCT/MRPL9 in vivo inhibited the growth of subcutaneous xenografts in nude mice and inhibited the formation of lung metastases. Mechanistically, we found that knockdown of GGCT/MRPL9 inhibited the MAPK/ERK signaling pathway. In conclusion, our study found that the interaction of GGCT and MRPL9 modulates the MAPK/ERK pathway, affecting the proliferation and migration of PTC cells. Therefore, GGCT/MRPL9 may serve as a potential biomarker for PTC monitoring and PTC treatment.

MiR-188-5p and Host MALAT1 Regulate RBE Cell Migration, Invasion, and Apoptosis via Up-regulating PSMD10 in Cholangiocarcinoma.

The study is designed to explore the regulatory network that MALAT1 competitively binds with miR-188-5p to up-regulate PSMD10 to facilitate cholangiocarcinoma cell migration and invasion and suppress apoptosis. qRT-PCR and fluorescence in situ hybridization (FISH) were used to examine the expression and positive signal of MALAT1 and miR-188-5p in cholangiocarcinoma tissues and HIBEC, HCCC-9810, RBE, and QBC939 cells. Western blot, qRT-PCR, and immunohistochemistry were selected to detect PSMD10 expression in cholangiocarcinoma tissues and cell lines. Dual luciferase reporter gene assay was adopted to verify that miR-188-5p targeted MALAT1 and PSMD10. qRT-PCR, pull down, and western blot were used to examine the regulation of MALAT1-miR-188-5p-PSMD10 axis. Transwell, wound healing assay, and Tunel cell apoptosis were adopted to respectively detect the regulatory abilities of MALAT1-miR-188-5p-PSMD10 axis on cell invasion, migration, and apoptosis. Western blot was used to detect the regulation mechanism of MALAT1 on Bax, Bcl-2, and caspase-3 proteins. Nude mice subcutaneous xenograft model of cholangiocarcinoma was established to examine the impacts of MALAT1 on subcutaneous tumor growth. Immunohistochemistry was adopted to examine the positive indicator of Ki67 antibodies and SMD10 antibodies in each group. MALAT1 and PSMD10 were highly expressed in cholangiocarcinoma tissues and cell lines, while miR-188-5p was lowly expressed. MALAT1 could competitively bind to miR-188-5p, and miR-188-5p could negatively regulate PSMD10. MALAT1, In-miR-188-5p, and PSMD10 could facilitate cell invasion and migration and inhibit apoptosis, while siMALAT1, miR-188-5p, and siPSMD10 produced an opposite result. MALAT1-miR-188-5p-PSMD10 axis could promote RBE cell invasion and migration and inhibit apoptosis, whereas siMALAT1-In-miR-188-5p-siPSMD10 axis showed an opposite result. On the other hand, it was verified that up-regulation/down-regulation of MALAT1 can inhibit/promote Bax and caspase-3 proteins and promote/inhibit the expression of Bcl-2 protein. MALAT1 could facilitate subcutaneous tumor growth and enhance cell proliferation and positive signal of PSMD10, while miR-188-5p worked in an opposite direction. MALAT1 competitively binds to miR-188-5p to up-regulate mRNA translation and protein expression of PSMD10, thereby facilitating cholangiocarcinoma cell invasion and migration and inhibiting its apoptosis. However, interfering MALAT1-miR-188-5p-PSMD10 axis could inhibit the occurrence and development of cholangiocarcinoma.

Nanoacetylated N-(4-Hydroxyphenyl) Retinamide Modulates Histone Acetylation-Methylation Epigenetic Disparity to Restrict Epithelial-Mesenchymal Transition in Neuroblastoma.

Neuroblastoma (NB) is an extracranial pediatric tumor with highly invasive growth of cancer biomass and frequent metastases. During the differentiation process in embryonic development, altered epigenetic modifications lead to dysregulated expression of pluripotency markers, resulting in epithelial-mesenchymal transition (EMT) progression. Currently, available chemotherapies have provided a limited solution to this problem due to systemic toxicities and drug resistance. Epigenetic therapeutic molecules like histone deacetylase inhibitors are still in the initial stages of development. We have developed a retinoid (N-(4-hydroxyphenyl) retinamide, 4HPR) loaded acetylated human serum albumin (HSA) nanoformulation to address the epigenetic imbalance and chemoresistance in NB. The idea was conceived to deliver an acetyl pool along with a chemotherapeutic drug, 4HPR, to restrict the invasiveness of NB by maintaining the balance between histone acetylation and trimethylation. The therapeutic efficacy of the formulation was successfully evaluated in the in vitro and in vivo xenograft mouse model system of neuroblastoma. The synthesized nanoparticles show high biocompatibility and therapeutic efficacy in treating neuroblastoma subcutaneous xenografts in nude mice.

Niuhuang (Bovis Calculus)-Shexiang (Moschus) combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway

ObjectiveTo investigate the effects of Niuhuang (Bovis Calculus, BC) and Shexiang (Moschus) (BC-Moschus) on human hepatocellular carcinoma (HCC) cells SMMC-7721 and a nude mouse model of subcutaneous xenografts, and to explore its anti-HCC mechanism. MethodsThe BC-Moschus combination was applied to two liver cancer models in vivo and in vitro. SMMC-7721 was divided into the BC-Moschus group and the control group, and different doses (rude drug dosage 0.625, 1.25, 2.5, and 5 mg/mL) of BC-Moschus extract were used for the intervention. The proliferation ability of HCC cells was detected using the Cell Counting Kit-8 (CCK-8) assay, and the migration ability was detected by a wound healing assay. A subcutaneous xenograft model was prepared using nude mice with human HCC. Specific pathogen-free-grade BALB/c nude mice (5-week-old) were randomly divided into the following groups (n = 6 per group): control (0.9% physiological saline 0.2 mL/d), BC-Moschus [BC 45.5 mg/(kg·d)+ Moschus 13 mg/(kg·d)], and cisplatin (DDP, intraperitoneal injection 5 mg/kg per week) groups. All groups were administered for 14 d. The volume and mass of the subcutaneous xenografts in nude mice were observed. The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, apoptosis-associated factor p70 S6 Kinase (S6K), Bax, Bcl-2, caspase-3, and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR (RT-qPCR) and Western blot. Terminal Deoxynucleotidy Transferase-Mediated dUTP Nick-End Labeling (TUNEL) was used for quantitative analysis of apoptotic cells. ResultsThe CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone, and the inhibition effect was dose- and time-dependent (P < 0.01). The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration (P < 0.01). In the subcutaneous xenograft model of nude mice with human HCC, we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group (P < 0.01). The levels of the PI3K/AKT/mTOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased (P < 0.01). The expression level of the anti-apoptotic gene Bcl-2 was downregulated (P < 0.05), and the expression of the pro-apoptotic gene Bax and apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated (P < 0.01). The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC (P < 0.01). ConclusionThe BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice. The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway, regulation of apoptosis-related protein caspase-3, caspase-9, Bcl-2, and Bax expression, and promotion of apoptosis.

Targeting visualization of malignant tumor based on the alteration of DWI signal generated by hTERT promoter–driven AQP1 overexpression

To specifically diagnose malignant tumors in DWI using the human telomerase reverse transcriptase (hTERT) promoter-driven AQP1 expression. The human telomerase reverse transcriptase (hTERT) promoter-driven AQP1 gene overexpression lentivirus system (hTERT-AQP1) and cytomegalovirus (CMV) promoter-driven AQP1 gene overexpression lentivirus system (CMV-AQP1) were prepared, and transduced into telomerase-positive and -negative cells. The AQP1 expression and DWI signal intensity (SI) change in transduced cells were analyzed. Balb/C nude mice subcutaneous xenograft models derived from lentivirus-transduced telomerase-positive and -negative cells were used to evaluate AQP1 expression and DWI SI change in vivo. We further established another group of subcutaneous xenograft model using pristine telomerase-positive and -negative cells, followed by injecting the lentiviral vectors intratumorally or intravenously, to determine the malignant tumor-targeted imaging of hTERT-AQP1. The hTERT-AQP1 and CMV-AQP1 were successfully prepared. After transduction, hTERT-AQP1 could induce the specific overexpression of AQP1 in telomerase-positive cells. Compared with untransduced cells, all CMV-AQP1-pretransduced cells and hTERT-AQP1-pretransduced telomerase-positive cells showed decreased SI and increased apparent diffusion coefficient (ADC) in DWI, while hTERT-AQP1-pretransduced telomerase-negative cells showed no obvious SI and ADC change. Correspondingly, hTERT-AQP1-transduced telomerase-positive tumors and CMV-AQP1-transduced telomerase-positive and -negative tumors showed decreased DWI SI and increased ADC, while hTERT-AQP1-transduced telomerase-negative tumor had no SI and ADC changes. After intratumoral or intravenous injection, CMV-AQP1 could upregulate AQP1 expression and induce DWI SI and ADC alteration in both telomerase-positive and -negative tumors, while hTERT-AQP1 worked in telomerase-positive tumors specifically. Cancers can be specifically visualized based on the DWI signal alteration which triggered by hTERT-AQP1 lentivirus system that combined AQP1 gene and hTERT promoter.

Tumor-associated macrophages modulate angiogenesis and tumor growth in a xenograft mouse model of multiple myeloma

Tumor-associated macrophages (TAMs) are closely associated with poor multiple myeloma (MM) prognosis. Therefore, in-depth understanding of the mechanism by which TAM supports MM progression may lead to its effective treatment. We used the MM nude mouse subcutaneous xenograft model to evaluate the efficacy of the macrophage-depleting agent clodronate liposome (Clo) against MM and elucidate the mode of action of this therapy. At the same time, observe whether the elimination of TAM in vivo while silencing the expression of VEGFA has the same effect as in vitro experiments. We also used Clo to eliminate macrophages and reinjected M1 or M2 TAM through mouse tail veins to investigate the effects of various macrophage subtypes on MM xenograft tumor growth. We applied qRT-PCR, immunohistochemistry, and enzyme-linked immunosorbent assay to quantify VEGFA, CD31, and CD163 expression in tumor tissues and sera. Removal of TAMs from the tumor microenvironment impeded tumor growth. The combination of Clo plus VEGFA siRNA had a stronger inhibitory effect on tumor growth than Clo alone, and M2 and M1 macrophages promoted and inhibited tumor growth, respectively. Macrophage depletion combined with cytokine blocking is a promising MM treatment. Targeted M2 macrophage elimination together with cytokine block may be more effective at inhibiting MM growth than either treatment alone. The results of the present study lay an empirical foundation for the development of novel therapeutic strategies for MM.

TRIM22 inhibits the proliferation of gastric cancer cells through the Smad2 protein

TRIM22 is involved in tumorigenesis and development, but its mechanism is not clear. In this study, we investigated the expression and biological role of TRIM22 in gastric cancer. We found that TRIM22 mRNA and protein expression was abnormally low in gastric cancer tissues and cells and correlated with tumor size and depth of invasion. Overexpression of TRIM22 significantly inhibited the proliferation, colony formation, and migration of gastric cancer cells and downregulated the expression of HSPA6. However, the HSPA6-siRNA complementation test showed that TRIM22 did not regulate cell proliferation through HSPA6. Furthermore, overexpression of TRIM22 downregulated the phosphorylation of Smad2 and Smad3. In addition, TRIM22 directly binds to Smad2, and overexpression of Smad2 can reverse the inhibition of cell proliferation and migration induced by TRIM22. In vivo, overexpression of TRIM22 significantly inhibited the growth of subcutaneous xenografts in nude mice. Our study indicates that TRIM22 has an important role in the development of gastric cancer and may inhibit the proliferation of gastric cancer cells through Smad2.

Hypoxic exosomal HIF-1α-stabilizing circZNF91 promotes chemoresistance of normoxic pancreatic cancer cells via enhancing glycolysis.

Research has indicated that hypoxia profoundly contributes to chemoresistance of pancreatic cancer (PC), while the precise mechanism has not been fully elucidated. In this study, we report a hypoxic exosomal circular RNA (circRNA)-mediated mechanism of conferred chemoresistance in PC cells. Gemcitabine (GEM) resistance was enhanced in normoxic PC cells incubated with exosomes derived from hypoxic PC cells. CircRNA microarray displayed that circZNF91 was remarkably increased in hypoxic exosomes of PC cells compared with normoxic exosomes. Overexpression of circZNF91 obviously stimulated chemoresistance in PC cells, while knockdown of circZNF91 retarded the hypoxic exosome-transmitted chemoresistance. Mechanistically, the hypoxic-induced exosomal circZNF91 transmitted into normoxic PC cells could competitively bind to miR-23b-3p, which deprives the inhibition of miR-23b-3p on expression of deacetylase Sirtuin1 (SIRT1). Consequently, the upregulated SIRT1 enhanced deacetylation-dependent stability of HIF-1α protein, leading to glycolysis and GEM chemoresistance of recipient PC cells. In addition, we revealed that the increased circZNF91 in hypoxic exosome was attributed to the transcriptional regulation by HIF-1α. Coincidently, transmission of hypoxic exosomes into subcutaneous xenografts in nude mice obviously facilitated the chemoresistance of transplanted PC tumor, which could be reversed by depletion of circZNF91 or upregulation of miR-23b-3p. Furthermore, clinical data showed that circZNF91 was significantly upregulated in PC tissues and correlated with overexpression of glycolytic enzymes and short overall survival time. Collectively, exosomal circZNF91 can function as a cargo mediating the signal transmission between hypoxic and normoxic tumor cells to promote GEM chemoresistance of PC and may potentially serve as a therapeutic target.

Gαi1 Promoted Proliferation, Migration and Invasion via Activating the Akt-mTOR/Erk-MAPK Signaling Pathway in Renal Cell Carcinoma.

BackgroundRenal cell carcinoma (RCC) accounts for about 2–3% of all adult malignancies. G protein alpha inhibitory subunit 1 (Gαi1) plays a key role in mediating PI3K-Akt signaling upon activation of receptor tyrosine kinases (RTKs). However, little is known about its expression, regulation and biological function in RCC.MethodsGαi1 expression in RCC tissues and cells was detected by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry (IHC). The effect of Gαi1 silence on cell proliferation and apoptosis of 786-O and ACHN cells was detected by CCK-8 assay and flow cytometry. Wound-healing assay and Transwell assays were used to detect the cell invasion in RCC cells. The expression of CDK4, cyclin D1, MMP-2, MMP-9, Bax, Bcl-2, p/t-Akt, p/t-S6 and p/t-Erk was detected by Western blot and qRT-PCR. Furthermore, a nude mouse subcutaneous xenograft model was used to further evaluate the potential effects of Gail in vivo.ResultsIn the present study, our data showed that Gαi1 expression was dramatically increased in RCC tissues compared with normal renal tissues. In addition, knocking down the expression of Gαi1 subsequently inhibited proliferation, migration and invasion of RCC cells in vivo and vitro. Furthermore, the expression of CDK4, cyclin D1, MMP-2 and MMP-9 was significantly reduced upon Gαi1 inhibition. Gαi1 positively regulates the activation of the mTOR and Erk pathways.ConclusionIn conclusion, this study reveals Gαi1 promoted proliferation via activating the Akt-mTOR and Erk-MAPK signaling pathways in RCC, and Gαi1 may be a therapeutic and prognostic target for RCC.

Cimicifuga dahurica extract inhibits the proliferation, migration and invasion of breast cancer cells MDA-MB-231 and MCF-7 in vitro and in vivo

Ethnopharmacological relevanceCimicifuga dahurica (Turcz.) Maxim (C. dahurica) has a long history of treating breast cancer. From the Qing Dynasty to the Tang Dynasty and even earlier, C. dahurica has been documented in the treatment of breast carbuncle (Breast cancer is classified as breast carbuncle in Chinese medicine). In traditional prescriptions such as “Sheng Ge Decoction”, “Sheng Ma Powder” and “Breast Carbuncle Pill”, as the main medicine, C. dahurica plays an important role. At present, the systematic studies on the in vitro and in vivo effects of Cimicifuga against breast cancer are rare, especially the C. dahurica. Aim of the studyIn this article, we evaluated the in vitro activity and in vivo effects of CREE (extract of the root of C. dahurica) against breast cancer, and discussed the possible mechanism of CREE in promoting breast cancer cell apoptosis. Materials and methodsThe main component in the CREE was analyzed by HPLC. The effects of CREE on the proliferation, migration and invasion of human breast cancer cells were evaluated through SRB, colony assay, LDH release, wound healing and transwell assay. The pro-apoptotic effect of CREE was investigated in Hochest33342 and Annexin V-FITC/PI assay. To verify the results of CREE in vivo effects, we applied nude mice subcutaneous xenograft experiments. The possible mechanism of CREE treating breast cancer was investigated through mitochondrial membrane potential and western blot experiments. ResultsCREE contains cycloartane triterpene saponins. CREE can significantly inhibit the proliferation, migration and invasion of human breast cancer MCF-7 and MDA-MB-231 cells in vitro and it can effectively inhibit the growth of MDA-MB-231 cell subcutaneous tumors in vivo. Besides, we also found that CREE up-regulated the expression levels of Bax, caspase-9/3 and cytochrome C, and down-regulated the expression of Bcl-2. Therefore, regulation of the mitochondrial pathway may be one of the mechanisms by which CREE promotes breast cancer cell apoptosis. ConclusionsCREE exhibits sufficient anti-breast cancer activity in vivo and in vitro, this study provides persuasive evidence for the further research and development of C. dahurica.

MiRNA-223-3p Affects Mantle Cell Lymphoma Development by Regulating the CHUK/NF-ƘB2 Signaling Pathway.

BackgroundMantle cell lymphoma (MCL) is an aggressive malignancy that accounts for 5–10% of non-Hodgkin’s lymphoma. MiRNA-223-3p has been demonstrated to be down-regulated in MCL and is a useful prognostic factor. However, little is known about underlying molecular mechanism of miRNA-233-3p in MCL.MethodsThe expression levels of miRNA-223-3p and CHUK mRNA in MCL cells were detected by real-time quantitative PCR (RT-qPCR). The effects of miRNA-223-3p/CHUK overexpression/knockdown on MCL cell proliferation and apoptosis were measured by CCK-8 assay and annexin V PE/7-AAD-based flow cytometry/TUNEL assay, respectively. A nude mouse subcutaneous xenograft model was used to further evaluate the potential effects in vivo. Dual-luciferase reporter assay was used to verify the inhibitory effect of miRNA-223-3p on CHUK. Furthermore, the regulatory function of miRNA-223-3p on the CHUK/NF-ƘB2 axis was assessed by RT-qPCR, western blot and immunofluorescence.ResultsIn the present study, miRNA-223-3p overexpression inhibited proliferation and accelerated apoptosis of MCL cells in vitro and in vivo. The results of Luciferase reporter assay showed that CHUK was a direct target of miRNA-223-3p in HEK293T cells. Furthermore, the results of RT-qPCR, western blot confirmed that CHUK was targeted and negatively regulated by miRNA-223-3p for repressing NF-ƘB2 pathway activation in MCL cells. Importantly, CHUK overexpression promoted proliferation and suppressed apoptosis of MCL cells, whereas CHUK knockdown reversed down-regulated miRNA-223-3p -accelerated cell proliferation in vitro.ConclusionIn conclusion, miRNA-223-3p affects MCL development by regulating the CHUK/NF-ƘB2 signaling pathway, which is crucial to provide a novel therapeutic strategy.

CNR1 may reverse progesterone-resistance of endometrial cancer through the ERK pathway

Endocrine therapy is a promising treatment for endometrial cancer (EC) that preserves fertility, however, progesterone-resistance is currently the major challenges. The Cancer Genome Atlas (TCGA) database analysis showed that CNR1 was closely have a negative correlation with overall survival (OS) and relapse-free survival (RFS) in endometrial cancer. To explore the role of CNR1 in progesterone resistance and possible molecular regulation mechanism, we established stable progesterone-resistant cell lines (IshikawaPR) via progesterone tolerance of ordinary cancer cells (Ishikawa). The difference of CNR1 level in two cell lines was assessed by MTT, RT-PCR, Western blot, immunofluorescence. Then, lentiviruses constructed CNR1-knockdown with GV248 as the tool vector were used to transfect IshikwaPR cells, and the changes of biological behavior and progesterone sensitivity was verified respectively through plate cloning experiment, EdU assay, flow cytometry cycle analysis, transwell, Scratch test, etc. We founded after CNR1 was knocked down, the proliferative activity and ability to migrate of IshikawaPR cells decreased, progesterone-response sensitivity could be improved. Moreover, knockdown of CNR1 can also down-regulate ERK and NFκ B expression and activation. Furthermore, subcutaneous xenograft in nude mice was tested similarly in vivo.The above datas suggest that targeting CNR1 may reverse the progesterone resistance in endometrial cancer and may coordinate the role of ERK pathway activation.

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.

Very long-chain acyl-CoA synthetase 3 mediates onco-sphingolipid metabolism in malignant glioma.

Gliomas are the largest category of primary malignant brain tumors in adults, and glioblastomas account for nearly half of malignant gliomas. Glioblastomas are notoriously aggressive and drug-resistant, with a very poor 5 year survival rate of about 5%. New approaches to treatment are thus urgently needed. We previously identified an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), as a potential therapeutic target in glioblastoma. Using the glioblastoma cell line U87MG, we created a cell line with genomic deletion of ACSVL3 (U87-KO) and investigated potential mechanisms to explain how this enzyme supports the malignant properties of glioblastoma cells. Compared to U87MG cells, U87-KO cells grew slower and assumed a more normal morphology. They produced fewer, and far smaller, subcutaneous xenografts in nude mice. Acyl-CoA synthetases, including ACSVL3, convert fatty acids to their acyl-CoA derivatives, allowing participation in diverse downstream lipid pathways. We examined the effect of ACSVL3 depletion on several such pathways. Fatty acid degradation for energy production was not affected in U87-KO cells. Fatty acid synthesis, and incorporation of de novo synthesized fatty acids into membrane phospholipids needed for rapid tumor cell growth, was not significantly affected by lack of ACSVL3. In contrast, U87-KO cells exhibited evidence of altered sphingolipid metabolism. Levels of ceramides containing 18-22 carbon fatty acids were significantly lower in U87-KO cells. This paralleled the fatty acid substrate specificity profile of ACSVL3. The rate of incorporation of stearate, an 18-carbon saturated fatty acid, into ceramides was reduced in U87-KO cells, and proteomics revealed lower abundance of ceramide synthesis pathway enzymes. Sphingolipids, including gangliosides, are functional constituents of lipid rafts, membrane microdomains thought to be organizing centers for receptor-mediated signaling. Both raft morphology and ganglioside composition were altered by deficiency of ACSVL3. Finally, levels of sphingosine-1-phosphate, a sphingolipid signaling molecule, were reduced in U87-KO cells. We conclude that ACSVL3 supports the malignant behavior of U87MG cells, at least in part, by altering cellular sphingolipid metabolism.

Long non-coding RNA SNHG25 promotes epithelial ovarian cancer progression by up-regulating COMP.

Long non-coding RNAs (lncRNAs) play a pivotal role in the genesis and development of cancer. The role and molecular mechanisms of SNHG25 in epithelial ovarian cancer (EOC) have not been investigated. In the present study, we showed that SNHG25 expression was up-regulated in EOC tissues relative to normal ovarian tissues. In vitro, functional experiments demonstrated that high expression of SNHG25 promoted proliferation, migration and invasion, and decreased apoptosis, in ovarian cancer cell lines. In vivo, downregulation of SNHG25 inhibited the growth (tumor volume) of subcutaneous xenografts in nude mice. High-throughput sequencing and western blot analysis showed a significant decrease in the expression of COMP mRNA and protein in SNHG25 knockdown compared to control ovarian cancer cells. These data suggest that SNHG25 promotes EOC progression by regulating COMP, serving as a potential biomarker for EOC.

Exosomal miR-500a-5p derived from cancer-associated fibroblasts promotes breast cancer cell proliferation and metastasis through targeting USP28.

The tumor microenvironment contributes to tumor progression and metastasis. Cancer-associated fibroblasts (CAFs) form a major cellular component of the tumor microenvironment. In this study, we further explored the mechanisms underlying the tumor-promoting roles of CAFs.Methods: Patient-derived CAFs and normal fibroblasts (NFs) were isolated from breast carcinomas and adjacent normal breast tissue. Exosomes were isolated by ultracentrifugation and CAF-derived exosomal microRNAs were screened using next-generation sequencing technology. MiR-500a-5p expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization; Tumor cell proliferation was determined by MTT assays and three-dimensioned (3D) cultures, and tumor metastasis was determined by Transwell assays in vitro. In vivo assays were performed in a nude mouse subcutaneous xenograft model.Results: We confirmed that CAF-derived exosomes significantly promoted the proliferation and metastasis of breast cancer cells. MiR-500a-5p was highly expressed in MDA-MB-231 and MCF7 cells treated with CAF-derived exosomes. The upregulation of miR-500a-5p was also confirmed in CAFs and CAF-derived exosomes. MiR-500a-5p was transferred from CAFs to the cancer cells, and subsequently promoted proliferation and metastasis by binding to ubiquitin-specific peptidase 28 (USP28).Conclusions: The present study demonstrates that CAFs promote breast cancer progression and metastasis via exosomal miR-500a-5p and indicate that inhibiting CAF-derived miR-500a-5p is an alternative modality for the treatment of breast cancer.