Cervical cancer, resulting from infection with human papillomavirus (HPV)16, remains the fourth most common cancer in women worldwide. Recently, three prophylactic HPV vaccines targeting high-risk HPVs (particularly HPV16 and HPV18) have been implemented to protect younger women. However, individuals with pre-existing infections have no benefit from prophylactic vaccines. Thus, there is an urgent need to develop therapeutic vaccines. HPV16 E7 has been widely utilized as a target for immune therapy of HPV16-associated lesions or cancers, reflecting the sustained existence of this virus in cancerous cells. We developed mannosylated HPV16 E7 (mE7) expressed from Pichia pastoris as a therapeutic vaccine against HPV16-associated cancer. Unmannosylated E7 (E7) was also generated from Pichia pastoris as a control. Mannosylation enhanced the uptake of mE7 by mannose receptors of bone marrow-derived dendritic cells (BMDCs), while the uptake of E7 was unaffected. mE7-uptake BMDCs in vitro induced more IFN-gamma secretion by splenocytes of immunized mice than E7. Vaccination of C57BL/6 mice with mE7 combined with adjuvant monophosphoryl lipid A (MPL) elicited stronger Th1 (type 1 T helper cell) responses and E7-specific T cell responses than E7. The mE7 vaccine induced the increased production of IFN-gamma, IL-2 and TNF-alpha, elicited more E7-specific IFN-gamma-secreting CD8(+) T cells in spleen and peripheral blood mononuclear cells (PMBCs) and promoted stronger E7-specific cytotoxic CD8(+) T cell responses compared with E7. Furthermore, TC-1 tumor challenged mice were used to confirm the antitumor activity of the vaccines. As a result, mE7 generated complete antitumor activity against TC-1 tumors, while E7 only provided partial antitumor activity. Taken together, mE7 can be a promising immunotherapy for treating cervical cancer.
Melanoma is the seventh most common malignancy in females and the fifth most common cancer in males worldwide. An increasing number of studies have reported that microRNA (miRNA) dysregulation is frequently observed in various types of human cancers, including melanoma. Abnormally expressed miRNAs play an important role in melanoma formation and progression by serving as potential biomarkers and therapeutic targets. Recently, miRNA-326 (miR-326) has been reported to be differentially expressed in various types of tissues and play important roles in tumouri-genesis and tumour development. However, the expression levels, biological roles and underlying mechanisms of miR-326 in melanoma remain unknown. In the present study, we demonstrated that miR-326 was significantly downregulated in melanoma tissues and cell lines. Functional assays revealed that the enforced expression of miR-326 suppressed melanoma cell proliferation and invasion and increased cell apoptosis in vitro. Using bioinformatic analysis, Kirsten rat sarcoma viral oncogene homolog (KRAS) was predicted as a potential target of miR-326. Luciferase reporter assay confirmed that miR-326 could directly target the 3'-untranslated region of KRAS. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis revealed that miR-326 upregulation decreased the KRAS expression in melanoma cells at both the mRNA and protein level. Furthermore, KRAS was upregulated in melanoma tissues and inversely correlated with miR-326 expression. In addition, the KRAS knockdown phenocopied the tumour-suppressing effects of miR-326 overexpression on melanoma cells. The restoration of the expression of KRAS markedly reversed the antitumour effects induced by miR-326 overexpression in melanoma cells. Further experiments indicated that miR-326 inactivated the AKT and ERK signalling pathways in melanoma. Collectively, these results revealed that miR-326 serves as a tumour suppressor in melanoma by targeting KRAS and regulating the AKT and ERK signalling pathways, indicating that miR-326 may be a promising therapeutic target for melanoma patients.
ITR-284 is a carboxamide analog that can inhibit proliferation in human promyelocytic leukemia HL-60 cells. To understand the effects and molecular mechanisms of ITR-284 in human erythromyeloblastoid leukemia, we treated K562 cells with different concentrations of ITR-284 (0, 2, 4, 6, 8 and 10 nM) and all-trans retinoic acid (ATRA) (0, 0.1, 0.5, 1, 5 and 10 mu M) for 24 h. The IC50 of ITR-284 was similar to 10 nM in K562 cells treated for 24 h as determined by MTT assay. May-Grunwald-Giemsa staining and nitro blue tetrazolium (NBT) assays were used to determine cell morphology changes and differentiation after ITR-284 and ATRA treatment. In addition, mRNA expression levels of hematopoietic factors, including GATA-1, NF-E2 and GATA-2, were elevated, while expression levels of BCR-ABL were downregulated in K562 cells after 24 h of treatment with ITR-284 as determined by quantitative reverse transcription polymerase chain reaction. In addition, western blot analyses showed that FOXM1, GLI 1 and c-MYC protein levels were decreased by ITR-284. Taken together, our data show that ITR-284 induced K562 cell differentiation, which led to decreased tumorigenesis. Our findings suggest that ITR-284 could be a potential candidate for treating chronic myelogenous leukemia.
Liver kinase B1 (LKB1) regulates a variety of cellular functions, including cell polarity, energy metabolism and cell growth, by targeting multiple signaling pathways such as AMPK/mTOR and p53. LKB1 functions as a tumor suppressor in sporadic cancers including lung cancer. Extracellular vesicles such as exosomes secreted by cancer cells modulate the tumor microenvironment and progression by targeting both tumor cells (autocrine actions) and other types of cells associated with tumors (paracrine actions). While the roles of LKB1 in cellular signaling in general is well-studied, its specific role in exosome-mediated signaling remains to be explored. To this purpose, we reintroduced LKB1 into H460 and A549 lung cancer cells that are endogenously deficient in LKB1 expression. Notably, we found that while restoration of LKB1 significantly reduced lung cancer cell growth as expected, it greatly promoted cell motility and enhanced the release of exosomes. In addition, exosomes isolated from H460 cells with stable restoration of LKB1 had much higher ability in stimulating lung cancer cell migration than did those from H460 cells lacking LKB1. Mechanistically, restoration of LKB1 in H460 cells inhibited cellular expression and exosomal secretion of migration-suppressing microRNAs (miRNAs), including miR-125a, miR-126 and let7b. Taken together, the present study revealed a new role for LKB1 in promoting cell motility by downregulating migration-suppressing miRNA expression and exosome secretion.
Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is a tissue-specific gene of nasopharyngeal tissue, and has been recognized as a potential tumor-suppressor gene in nasopharyngeal carcinoma. As a secreted protein, SPLUNC1 plays an important role in innate immunity including antimicrobial and host defense. However, the related immune cells which are regulated by SPLUNC1 remain elusive. In the present study, an acute lung injury (ALI) mouse model was established by administration of lipopolysaccharide (LPS) intraperitoneal injections to wild-type and SPLUNC1(-/-) mice (5 mg/kg). Pathologic results showed that the SPLUNC1(-/-) group appeared to have more severe pulmonary damage and infiltrated inflammatory cells compared with the WT group after LPS treatment for 24, 48, 72 and 96 h. The mRNA expression levels of interleukin-6 (IL-6), chemokine (C-C motif) ligand-2 (CCL-2), chemokine (C-C motif) ligand-3 (CCL-3) and chemokine (C-X-C motif) ligand-1 (CXCL-1) in lungs of the SPLUNC1(-/-) group were higher than these levels in lungs of the WT group at different time points after LPS injection. The percentage of splenic CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) in the SPLUNC1(-/-) mice was higher than this percentage in the WT mice at the time points of 72 and 96 h post LPS injection (P<0.05). These findings demonstrated that SPLUNC1 had a certain protective effect on the LPS-induced ALI mouse model as well as it was found to inhibit the recruitment of MDSCs to the spleen in this model.
Epigenetic abnormalities as well as genetic abnormalities may play a vital role in the tumorigenesis of papillary thyroid cancer (PTC). The present study aimed to analyze the function and methylation status of the HOXD10 gene in PTC and aimed to identify relationships between HOXD10 methylation, HOXD10 expression, BRAF mutation and clinicopathological characteristics of PTC. A total of 152 PTC patients were enrolled in the present study. The methylation status of the HOXD10 promoter was analyzed by quantitative methylation-specific polymerase chain reaction (Q-MSP). BRAF(V600E) mutation status was analyzed by polymerase chain reaction (PCR) followed by DNA sequencing. HOXD10 mRNA expression level was analyzed by real-time polymerase chain reaction (RT-PCR). 5-Aza-2-deoxycytidine (5-Aza) treatment was performed in 4 PTC cell lines to observe the change in HOXD10 expression. Transwell, cell cycle and apoptosis assays were then performed in an HOXD10overexpressing PTC cell line. Furthermore, we analyzed the associations between HOXD10 methylation, HOXD10 expression, BRAF mutation and clinicopathological characteristics in PTC. Overexpression of HOXD10 suppressed the migration of PTC cells, and promoted cell apoptosis. Q-MSP showed that methylation levels of the HOXD10 promoter were significantly higher in PTC tissues than levels in the adjacent normal thyroid tissues (P=0.02). In addition, expression of HOXD10 was decreased in the PTC cell lines and PTC tissues compared with that noted in the adjacent normal thyroid tissues (P=0.008). However, BRAF(V600E) mutation was detected in 42.1% of PTC patients enrolled. In addition, the BRAF mutation status was associated with the methylation and expression level of HOXD10 in PTC. We then observed that 5-Aza treatment could revert the expression of HOXD10 in PTC cell lines. Moreover, the hypermethylation of HOXD10 was associated with invasion of the primary tumor and age >45. In conclusion, the HOXD10 gene may act as a tumor suppressor in PTC. The aberrant hypermethylation and decreased expression of the HOXD10 gene were shown in PTC patients, particularly in those with BRAF(V600E) mutation. The epigenetic suppression of the HOXD10 gene may play a role in the tumorigenesis of PTC, and it is a prospective biomarker for the diagnosis and prognosis of PTC.
This study investigated how miR-106b-5p/PTEN signaling affects the cell cycle of malignant melanoma (MM) cells. miR-106b-5p mRNA was identified with qRT-PCR. Through transient transfection, miR-106b-5p or PTEN was upregulated and downregulated in MM cells. With such transfected cells, MTT assay, colony formation assay and flow cytometry were carried out to investigate the role of miR-106b-5p in cell cycle progression after the transfected cells were treated with reverse-regulation of miR-106b-5p or PTEN. Western blot analysis was used to quantify all proteins, and a luciferase reporter assay was carried out to validate miR-106b-5p targeting PTEN. miR-106b-5p mRNA was overexpressed in MM tissues and cell lines. MM cells with upregulated miR-106b-5p presented faster growth and shorter cell cycles, while those with knockdown of miR-106b-5p presented the opposite trend. PTEN was subject to post-transcriptional regulation of miR-106b-5p. Based on such a finding, further exploration was carried out to investigate the interaction between cyclin D1 and P27(Kip1), with the finding that miR-106b-5p can stimulate cyclin D1 and suppress P27(Kip1) via the Akt/ERK pathway. The results of this study suggest that miR-106b-5p may be a promoter in MM progression, possibly by targeting PTEN and thus regulating the downstream cell-cycle-related proteins and Akt/ERK pathway.
The present study was aimed to identify proteins associated with signaling pathways involved in chemoresistance, and establish a predictive model for chemoresistance in gastric cancer patients after radical surgery. A total of 140 clinically-staged III gastric cancer samples from patients after D2 radical gastrectomy were enrolled in the present study. Protein Pathway Array (PPA) and 286 antibodies were used to assess the protein expression in tumor tissues of patients. The Significance Analysis of Microarray (SAM) software and clustering and discriminant analysis were used to identify differentially expressed proteins between chemosensitive and chemoresistant subsets, and a predictive model for chemoresistance was established using the independent predictive factors. The Ingenuity Pathway Analysis (IPA) software was also used to investigate the relationship between proteins and the signaling transduction network. A total of 23 proteins were differentially expressed between 67 chemosensitive and 73 chemoresitant tumor tissues. Six proteins including PLK1 and DACH1 were independent risk factors for chemoresistance. A predictive model for chemoresistance by these proteins was established, and the accuracy, the sensitivity, and the specificity of this modal was 89.3, 90.3 and 88.2%, respectively. In addition, the present study revealed that differentially expressed proteins were closely related to cellular activity, DNA methylation and DNA damage and repair, and also involved in the ERK/MAPK, Wnt/beta-catenin, PI3K/AKT, apoptosis and p53 signaling pathways. In conclusion, the predictive model established by PPA may be an effective detection system for predicting the chemosensitivity of gastric cancer patients after D2 gastrectomy.
Vacuolar ATPase (V-ATPase), widespread in eukaryotic cells, is extensively expressed in many highly metastatic tumors, of which the V-ATPase c subunit ATP6V0C is particularly associated with the invasion and metastasis of cancer. ATP6V0C was directly found to interact with LASS2/TMSG1 which is a new tumor metastasis inhibitory gene identified by our laboratory in 1999. In order to study the role of ATP6V0C, we generated small interference RNA (siRNA) targeting ATP6V0C and investigated its function on the invasion of human prostate cancer cell line PC-3M-1E8 with high metastatic potential and its interplay with LASS2/TMSG1. We found that the expression of ATP6V0C was higher in prostate cancer cell lines PC-3M-1E8 and PC-3M with high metastatic potential than that from cell lines PC-3M-2B4 and PC-3 with low metastatic potential, indicating that ATP6V0C enhanced metastatic capacity in prostate cancer cells. Furthermore, silencing of ATP6V0C in PC-3M-1E8 cells inhibited V-ATPase activity (by similar to 5-fold), decreased extracellular hydrogen ion concentration and successively decreased activation of secreted MMP-9 (by similar to 3.6-fold), which coincided with the inhibition of cell migration and invasion in vitro, as well as a marked decrease in the expression of LASS2/TMSG1 probably through positive feedback. Thus we concluded that silencing of the ATP6V0C gene effectively suppressed the migration and invasion of prostate carcinoma cells through the inhibition of the function of V-ATPase, not through a LASS2/TMSG1-dependent manner. Therefore ATP6V0C inhibitors are promising therapeutic targets for advanced prostate cancer.
The purpose of the present study was to examine the function of microRNA-95-3p on cell growth of osteosarcoma and to investigate its mechanism. Compared to healthy controls, the serum expression of microRNA-95-3p was effectively upregulated in patients with osteosarcoma. MicroRNA-95-3p expression in patients with osteosarcoma had significant association with clinical stage. Downregulation of microRNA-95-3p expression suppressed cell growth, induced apoptosis, increased caspase-3 and caspase-9 activities and Bax/Bcl-2 protein expression in osteosarcoma cells. The anticancer effects of microRNA-95-3p on cell growth of osteosarcoma cell suppressed TGF-beta and p-Smad2 protein expression, induced p21 protein expression and suppressed cyclin D1 protein expression in osteosarcoma cells. Whereas, overexpression of microRNA-95-3p increased cell growth, and inhibited apoptosis of osteosarcoma cells through TGF-beta/CDKN1A/p21/cyclin D1 expression. After promotion of TGF-beta, the anticancer effects of microRNA-95-3p were effectively reversed in osteosarcoma cells. Taken together, our results demonstrate that downregulation of microRNA-95-3p suppresses cell growth of osteosarcoma via CDKN1A/p21 expression.
Xanthohumol (XN), a prenylflavonoid found in the hop plant, Humulus lupulus, exhibits a variety of biological activities. Numerous studies have reported that XN inhibits the growth of many types of cancer cells, but the effects of XN on tumor immunity have not yet been studied. We explored the effect of XN on Th1/Th2 balance and the underlying mechanism based on a BALB/c-4T1 breast cancer mouse model. The results showed that XN significantly slowed down tumor growth and inhibited expression of antitumor proliferation protein Ki-67 as well as breast cancer-specific marker cancer antigen 15-3 (CA15-3). Flow cytometric analysis revealed that XN enhanced the secretion of perforin, granzyme B and increased the ratio of CD8(+)/CD25(+). ELISA analysis of cytokine results demonstrated that XN obviously upregulated Th1 cytokines, while downregulated Th2 cytokines. Th1/Th2 ratio analysis by flow cytometry illustrated that XN regulated the balance drift to Th1 polarization. Western blotting and immunohistochemistry (IHC) results manifested that XN induced expression of T-bet, a Th1-specific transcription factor. Furthermore, we found that XN significantly promoted the phosphorylation of signal transducer and activator of transcription (STAT)4. Our results demonstrated that XN promoted Th1/Th2 balance towards Th1 polarization, and STAT4 may play a positive role in the regulation of Th1/Th2 cytokines by XN.
As discovered by Warburg 80 years ago most malignant cells rely more on glycolysis than normal cells. The high rate of glycolysis provides faster ATP production and greater lactic acid for tumor proliferation and invasion, thus indicating a potential target in anticancer therapy. Our previous studies demonstrated that 3-bromopyruvate (3-BrPA) and sodium citrate (SCT) inhibited tumor cell proliferation in vitro. However, the underlying mechanisms still warrant further investigation. In the present study, we employed the human SGC-7901 gastric cancer cell line, built an orthotopic xenograft model in nude mice, examined the treatment response by F-18-FDG PET/CT and investigated the mechanisms of 3-BrPA and SCT in vivo. Our results demonstrated that glycolysis and tumor growth were inhibited by intraperitoneal injection of 3-BrPA and SCT, which were imaged using an F-18-FDG PET/CT scanner. In addition, apoptosis induced by 3-BrPA and SCT was initiated by the upregulation of Bax and downregulation of Bcl-2, which promote cytochrome c release and subsequently activate caspase-9 and -3, and ultimately execute mitochondria-mediated apoptosis. Furthermore, apoptosis was also modulated by the generation of ROS and inhibition of survivin. Accordingly, 3-BrPA and SCT can inhibit glycolysis and induce gastric cancer apoptosis through the mitochondrial caspase-dependent pathway.
Despite increasing advances in the diagnosis and treatment for pancreatic cancer, the mortality rate remains high world-wide. There is an urgent need for new therapies to improve survival and quality of life for pancreatic cancer patient. Epigenetic therapeutic agents such as 5-Aza-CdR and suberoylanilide hydroxamic acid (SAHA) have shown therapeutic effects for human cancers. We evaluated the efficacy of 5-Aza-CdR or SAHA and their combination as potential therapies for pancreatic cancer in vitro. Treatment with 5-Aza-CdR or SAHA inhibited pancreatic cancer cell proliferation, migration and induced cell arrest. However, 5-Aza-CdR alone can not induce cell apoptosis. Combination of the two agents enhanced the proliferation and migration inhibition, and induced more cells to G2 arrest and increased the cell apoptosis proportion. Furthermore, combination treatment with SAHA and 5-Aza-CdR significantly increased expression of TP53 and P16. The possible mechanism might be that the two agents inhibited the PI3K/AKT/PTEN signaling pathway. In conclusion, these data demonstrate a potential role for epigenetic modifier drugs for the management of pancreatic cancer.
To investigate the expression pattern, clinical significance and functional roles of microRNA (miR)-615-5p in human esophageal squamous cell carcinoma (ESCC),, quantitative real-time PCR was performed to detect expression levels of miR-615-5p in ESCC tissues and cell lines. Associations between miR-615-5p expression and various clinicopathological features of ESCC patients were also statistically evaluated. The candidate targets of miR-615-5p were identified by integrating bioinformatics miRNA target prediction, western blot analysis and luciferase reporter assay. Moreover, the functions of miR-615-5p in ESCC cell migration and invasion were determined using the transfection of miRNA mimics, or co-transfected with miRNA mimics and the expression vector of its target gene. As a result, miR-615-5p expression in ESCC tissues and cells were markedly lower than those in non-cancerous esophageal mucosa and human normal esophageal cells, respectively (both P<0.001). miR-615-5p downregulation was significantly associated with advanced tumor-node-metastasis stage, positive lymph node metastasis and moderate-poor differentiation. Functionally, the re-expression of miR-615-5p suppressed the invasion and migration of ESCC cells in vitro. Interestingly, insulin-like growth factor 2 (IGF2) was identified as a direct target gene of miR-615-5p, and the inhibitory effects of miR-615-5p in ESCC cell motility were reversed by the restoration of IGF2 expression. In conclusion, miR-615-5p downregulation may be an underlying molecular mechanism of development and progression of ESCC, and may function as a potential therapeutic target of this malignancy. Also, we illustrate that the miR-615-5p/IGF2 axis may bring important contributions to cell motility of human ESCC.
Autophagy is critical for the metastasis of cancer cells through induction of epithelial-to-mesenchymal transition (EMT). Activation of TGF-beta signaling plays a key role in regulating autophagy. miR-16 may be associated with non-small cell lung carcinoma (NSCLC) progression. However, the role of miR-16 in NSCLC cell autophagy in the presence of TGF-beta and the underlying mechanism are still unclear. To test whether miR-16 targets ATG3 which is involved in autophagy of NSCLC cells, we studied the expression levels of miR-16 and ATG3 in NSCLC patients, verified the targeting of ATG3 by miR-16 by luciferase reporter gene system, and investigated the role of miR-16 in the autophagy of NSCLC cells. Results revealed that miR-16 was significantly downregulated, and ATG3 was significantly upregulated in NSCLC patient tissue samples. ATG3 was found to be a direct target of miR-16. TGF-beta 1 significantly downregulated the expression of miR-16 and ATG3 mRNA. Using transmission electron microscopy, we observed that TGF-beta 1 treatment reduced autophagosomes in the A549 cells, and miR-16 mimics increased the autophagosomes in the presence of TGF-beta 1. Acridine orange (AO) staining and expression of LC3B II/I and p62 confirmed the inhibition of autophagy by TGF-beta 1, and the recovery of TGF-beta 1-mediated inhibition of autophagy by miR-16 mimics. Finally, miR-16 mimics inhibited TGF-beta 1-induced EMT, and this effect was attenuated by autophagy inhibitor 3-MA. Taken together, miR-16 mimics inhibited TGF-beta 1-induced EMT via activation of autophagy.