A novel IRAK4/PIM1 inhibitor ameliorates rheumatoid arthritis and lymphoid malignancy by blocking the TLR/MYD88-mediated NF-κB pathway.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal enzyme in the Toll-like receptor (TLR)/MYD88 dependent signaling pathway, which is highly activated in rheumatoid arthritis tissues and activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Inflammatory responses followed by IRAK4 activation promote B-cell proliferation and aggressiveness of lymphoma. Moreover, proviral integration site for Moloney murine leukemia virus 1 (PIM1) functions as an anti-apoptotic kinase in propagation of ABC-DLBCL with ibrutinib resistance. We developed a dual IRAK4/PIM1 inhibitor KIC-0101 that potently suppresses the NF-κB pathway and proinflammatory cytokine induction in vitro and in vivo. In rheumatoid arthritis mouse models, treatment with KIC-0101 significantly ameliorated cartilage damage and inflammation. KIC-0101 inhibited the nuclear translocation of NF-κB and activation of JAK/STAT pathway in ABC-DLBCLs. In addition, KIC-0101 exhibited an anti-tumor effect on ibrutinib-resistant cells by synergistic dual suppression of TLR/MYD88-mediated NF-κB pathway and PIM1 kinase. Our results suggest that KIC-0101 is a promising drug candidate for autoimmune diseases and ibrutinib-resistant B-cell lymphomas.

Anticancer activity of IRAK-4 inhibitors against canine lymphoid malignancies.

The interleukin-1 receptor-related kinase 4 (IRAK4), downstream of myd88, plays an essential role in hyperactive TLR signalling seen in some B-cell lymphomas. In particular, efficient IRAK4 inhibitors of activated B-cell subtype of human diffuse large B-Cell lymphoma (DLBCL) are being developed. However, the anticancer effect of IRAK-4 inhibitors in veterinary medicine has not been elucidated. It is therefore explored in this study involving the GL-1 and CL-1 canine lymphoma cell lines in vitro. MyD88 expression was analysed using polymerase chain reaction. GL-1 and CL-1 cells were subjected to concentration- and time-dependent treatment with an IRAK-4 inhibitor and assessed for viability, TLR signalling association and apoptosis using a cell counting Kit-8 assay, Western blotting and flow cytometry. The GL-1 and CL-1 cells exhibited enhanced MyD88 expression, however, canine peripheral blood mononuclear cells (cPBMCs) did not. The IRAK-4 inhibitor reduced cell viability in a dose- and time-dependent manner, significantly reduced the phosphorylation of molecules associated with TLR signalling at IC50 such as IRAK1, IRAK4, NF-κB and STAT3, and induced apoptosis in GL-1 and CL-1 cells. The anticancer effect of the IRAK-4 inhibitor on canine lymphoma cells is mediated by apoptosis via downregulation of TLR signalling.

A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations.

Perturbations in cellular chloride concentrations can affect cellular pH and autophagy and lead to the onset of apoptosis. With this in mind, synthetic ion transporters have been used to disturb cellular ion homeostasis and thereby induce cell death; however, it is not clear whether synthetic ion transporters can also be used to disrupt autophagy. Here, we show that squaramide-based ion transporters enhance the transport of chloride anions in liposomal models and promote sodium chloride influx into the cytosol. Liposomal and cellular transport activity of the squaramides is shown to correlate with cell death activity, which is attributed to caspase-dependent apoptosis. One ion transporter was also shown to cause additional changes in lysosomal pH, which leads to impairment of lysosomal enzyme activity and disruption of autophagic processes. This disruption is independent of the initiation of apoptosis by the ion transporter. This study provides the first experimental evidence that synthetic ion transporters can disrupt both autophagy and induce apoptosis.

Mechanical compression induces VEGFA overexpression in breast cancer via DNMT3A-dependent miR-9 downregulation.

Tumor growth generates mechanical compression, which may trigger mechanotransduction in cancer and stromal cells and promote tumor progression. However, very little is known about how compression stimulates signal transduction and contributes to tumor progression. In the present study, we demonstrated that compression enhances a tumor progression phenotype using an in vitro compression model, and validated the results from the in vitro model with high- and low-compressed breast cancer tissues. Mechanical compression induced miR-9 downregulation by DNMT3A-dependent promoter methylation in the MDA-MB-231 and BT-474 breast cancer cell lines and in cancer-associated fibroblasts. The overexpression of miR-9 target genes (LAMC2, ITGA6, and EIF4E) was induced by miR-9 downregulation, which eventually enhanced vascular endothelial growth factors production. Demethylation and decompression could reverse compression-induced miR-9 downregulation and following overexpression of miR-9 target genes and VEGFA.

Synthetic aminopyrrolic receptors have apoptosis inducing activity.

We report two synthetic aminopyrrolic compounds that induce apoptotic cell death. These compounds have been previously shown to act as receptors for mannosides. The extent of receptor-induced cell death is greater in cells expressing a high level of high-mannose oligosaccharides than in cells producing lower levels of high-mannose glycans. The ability of synthetic receptors to induce cell death is attenuated in the presence of external mannosides. The present results provide support for the suggestion that the observed cell death reflects an ability of the receptors to bind mannose displayed on the cell surface. Signaling pathway studies indicate that the synthetic receptors of the present study promote JNK activation, induce Bax translocation to the mitochondria, and cause cytochrome c release from the mitochondria into the cytosol, thus promoting caspase-dependent apoptosis. Such effects are also observed in cells treated with mannose-binding ConA. The present results thus serve to highlight what may be an attractive new approach to triggering apoptosis via modes of action that differ from those normally used to promote apoptosis.

Selective homocysteine turn-on fluorescent probes and their bioimaging applications.

The design and development of new pyrene-based fluorescent probes, P-Hcy-1 and P-Hcy-2, which display selective fluorescence enhancements in response to homocysteine (Hcy), are described. The distinctly different fluorescence responses of P-Hcy-1 and P-Hcy-2 to Hcy vs. Cys are explained by theoretical calculations. Finally, the results of cell experiments show that these probes can be used to selectively detect Hcy in mammalian cells.

Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models.

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5ß1 and αvß3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis.

Human breast cancer-associated fibroblasts enhance cancer cell proliferation through increased TGF-α cleavage by ADAM17.

We demonstrate here increased expression of ADAM17 protein in cancer-associated fibroblasts (CAFs) extracted from human breast carcinomas compared with donor-matched normal fibroblasts, and TGF-α secretion positively correlates with ADAM17 expression in these cells. In SK-BR-3 cells co-cultured with CAFs, CAF-secreted TGF-α promotes cell proliferation by activation of EGFR, Akt, and ERK, but it does not promote cell migration. Furthermore, anti-TGF-α neutralizing antibodies antagonize the CAF-dependent increase in proliferation and activation of EGFR, Akt and ERK. Thus, pharmacologic inhibition of ADAM17 and TGF-α may have therapeutic potential for the treatment of breast cancer when fibroblast-directed therapy is considered.

CD24⁺ ovary cancer cells exhibit an invasive mesenchymal phenotype.

We recently reported that the subset of CD24(+) cells in ovarian cancer possesses various cancer stem cell properties. In this study, we further show that this subpopulation of ovarian cancer cells exhibits an epithelial-mesenchymal transition (EMT) phenotype, high invasive capacity, and CXCR4/SDF-1-mediated chemotactic migration. We evaluated CD24 expression in various ovarian cancer cell lines by flow cytometric analysis. CAOV3 and a primary ovarian cancer cell line Clone 4 were sorted into CD24(+) and CD24(-) subpopulations by FACS and Western blot, cell invasion, adhesion, and in vitro chemotaxis assays were performed with these two subpopulations. We also assessed the effects of shRNA depletion of CD24 in CAOV3 and Clone 4 cells by Western blot and cell invasion assays. CD24 expression in ovarian cancer cell lines correlated with aggressive histologic subtypes of epithelial ovarian cancer. The CD24(+) subpopulation was also more invasive than the CD24(-) subpopulation and showed higher CXCR4/SDF-1-mediated chemotactic migration. CD24(+) cells exhibited an EMT phenotype as characterized by loss of E-cadherin expression and gain of vimentin, Twist, and Snail1 expression. In addition, CD24(+) cells stimulated cell attachment to fibronectin through the activation of ß1 integrin. Depletion of CD24 expression by CD24 shRNA efficiently suppressed cell invasion and induced downregulation of CXCR4 as well as loss of the EMT phenotype. In conclusion, CD24 expression in ovarian cancer may be related to tumor aggressiveness, in particular cell invasion and chemotactic migration. Therefore, CD24 may be a good candidate for a therapeutic target for ovarian cancer.

Targeting ILK and ß4 integrin abrogates the invasive potential of ovarian cancer.

Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of ß1 and ß4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of ß1 and ß4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of ß4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of ß4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting ß4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

In situ identification and localization of IGHA2 in the breast tumor microenvironment by mass spectrometry.

Modifications in the tumor microenvironment (TME) play a major role in the establishment, progression, and metastasis of cancer. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique that enables the simultaneous identification and localization of biological compounds within tissues. To detect markers of early TME remodeling in invasive breast cancer, we used MALDI-MSI to compare the molecular profiles of tissues from the breast cancer interface zone, tumor zone, and normal-tissue zone. Using direct-tissue MALDI tandem mass spectrometry (MS/MS), we identified immunoglobulin heavy constant alpha 2 (IGHA2) as a new, zone-specific protein in the breast TME. The zone-specific expression of IGHA2 was verified by immunoblotting and immunohistochemical analysis. IGHA2 expression was consistently positive in tumor cells that were metastatic to regional nodes, with intense expression along the cytoplasmic borders. As a factor related to an increased percentage of nodes with tumor metastasis, IGHA2 expression was upregulated 3.745-fold in cases with an increased number of cancerous nodes (p = 0.0468). Our results provide the first evidence of IGHA2 as a marker of the early process of TME remodeling in invasive breast cancer. Furthermore, IGHA2 may be a novel marker for regional metastases in the lymph nodes of patients with breast cancer.

Invasive breast cancer induces laminin-332 upregulation and integrin ß4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype during tissue remodeling.

INTRODUCTION: Although development of anoikis-resistant myofibroblasts during tissue remodeling is known to be associated with tumor invasion, the mechanism by which myofibroblasts become resistant to anoikis is unknown. We previously demonstrated laminin-332 upregulation in the fibrosis around invasive ductal carcinoma (IDC). Because laminin-332 promotes cell survival through binding to integrins, we hypothesized that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts by upregulating laminin-332 expression during tissue remodeling. Here, we demonstrate that invasive breast cancer cells induce laminin-332 upregulation and integrin ß4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype. METHODS: Three types of fibroblasts were isolated from the tumor burden, the fibrosis, and normal tissue of patients with early stage IDC (less than 10 mm diameter), designated cancer-associated fibroblasts (CAFs), interface fibroblasts (InFs), and normal breast fibroblasts (NBFs), respectively. To investigate direct and indirect crosstalk with tumor cells, fibroblasts were co-cultured with invasive MDA-MB-231 or noninvasive MCF7 cells or in conditioned medium. Anoikis resistance of fibroblasts was measured by cell viability and caspase-3 activity after incubation on poly-HEMA coated plates for 72 hours. Involvement of laminin-332/integrin α3ß1 or α6ß4 signaling in anoikis resistance was confirmed by treatment with purified laminin-332 or blocking antibodies against laminin-332, integrin ß1, or integrin ß4. RESULTS: MDA-MB-231 cells induced laminin-332 upregulation and integrin ß4 neoexpression in fibroblasts, leading to anoikis resistance. InFs showed a higher endogenous level of laminin-332 than did CAFs and NBFs. After stimulation with MDA-MB-231-conditioned medium, laminin-332 expression of InFs was dramatically increased and maintained under anoikis conditions. Laminin-332 upregulation was also observed in CAFs and NBFs, but at a lower level than in InFs. Laminin-332 induced Akt (Ser473) phosphorylation by binding to integrin α3ß1. Integrin ß4 neoexpression induced laminin-332-independent Rac1 activation and promoted anoikis resistance in fibroblasts approximately twofold more effectively than did laminin-332, regardless of the type of fibroblast. In addition, integrin ß4 expression suppressed fibroblast aggregation in conditions of anoikis. CONCLUSION: Invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin ß4 neoexpression. Interface fibroblasts appear to be the primary myofibroblasts that interact with invasive tumor cells during tissue remodeling.

Nuclear localization of Nm23-H1 in head and neck squamous cell carcinoma is associated with radiation resistance.

BACKGROUND: Although radiation resistance is a primary issue in radiation therapy, attempts to find predictors of radiation resistance have met with little success. The authors therefore aimed to determine predictors for radiation resistance to improve the prognosis of head and neck squamous cell carcinoma (HNSCC). METHODS: HNSCC cell lines, SCC15, SCC25, and QLL1, irradiated with an acute dose of 4 grays (Gy) (RR-4), a cumulative dose of 60 Gy (RR-60), and a booster dose of 4 Gy over 60 Gy (RR-60 + 4), were used with nonirradiated cell lines. Those were used in cDNA microarray, proteomics, Western blotting, and immunofluorescence, respectively. One hundred five HNSCC tissue samples with radiation resistance were analyzed by immunohistochemistry. RESULTS: Western blot analysis of RR-60 cell lines was identical to the data of Nm23-H1 overexpression by cDNA array and proteomic screening. Immunofluorescence demonstrated significant nuclear translocation of Nm23-H1 in RR-4 and RR-60 cell lines, and less but still intense nuclear shuttling in RR-60 + 4. Similarly, Nm23-H1 nuclear localization was observed in 20% (21 of 105) of tissue samples. Univariate analysis demonstrated that Nm23-H1 nuclear localization was strongly associated with overall and recurrence-free survival. Multivariate stepwise Cox regression analysis showed that Nm23-H1 nuclear localization (odds ratio [OR], 7.48) and N stage (OR, 2.13) were associated with overall survival, and Nm23-H1 nuclear localization (OR, 3.02), T stage (OR, 1.43), and insufficient tumor margin (OR, 3.27) were associated with recurrence-free survival. CONCLUSIONS: Overexpression of Nm23-H1, specifically its nuclear translocation, may be a powerful predictor of radiation resistance in HNSCC.

Molecular portraits of intratumoral heterogeneity in human ovarian cancer.

One of the most common characteristic profiles of cancer is intratumoral heterogeneity (ITH). We aimed to clarify the molecular profiles and biological significance of ITH with relation to cancer stem cell (CSC). We analyzed five primary cultured clones generated from different spatial zones, front and rear zone, of a fresh-frozen ovarian tumor tissue, performing ATP-CRA, conventional RT-PCR, side population (SP) analysis, flow cytometry immunophenotyping, and cell proliferation assays. We also carried out array CGH and Ingenuity Pathways Analysis (IPA) between SP and non-SP (NSP) cells. Clones from tumor front zone showed phenotypically and genetically distinct subpopulations with relatively higher SP proportions, CD24(+) and CD117(+) expression, and chemotherapeutic resistance. We demonstrate that phenotype of SP cells in heterogeneous clones of human ovarian cancer was closely related to CD24(+), CD117(+), and combined CD117(+)/CD24(+) fractions. Chromosomal alterations in SP cells relative to NSP cells were closely related to the novel core networks of cancer stem cell-like cells (CSCs), such as cycle checkpoint regulation, notch, PTEN, wnt/ß-catenin, PI3K/AKT, integrin, and cytokine and chemokine signaling. ITH could arise from clonal diversity closely related to CSC-like molecules, as evidenced by accumulated genetic, transcriptional and gene products alterations in SP.

Laminin-332-rich tumor microenvironment for tumor invasion in the interface zone of breast cancer.

Dense fibrosis, which is caused by desmoplastic reaction, is usually found in invasive ductal carcinoma and may represent the alteration of the tumor microenvironment preceding tumor invasion. Thus, the dense fibrotic zone around invasive ductal carcinoma can be considered to be the actual tissue site of tumor microenvironment, where the precedent alterations for tumor invasion occur. To characterize the dense fibrotic zone, we classified invasive ductal carcinoma tissue into a tumor zone, a normal zone, and the novel interface zone (IZ), which shows dense fibrosis. The postulated IZ is a 5-mm-wide belt that circles the tumor margin and overlaps with normal tissue. Of the extracellular matrix components, laminin-332 was specifically overexpressed in the IZ. Events that appear to be similar to the epithelial-mesenchymal transition, a novel source of myofibroblast formation from epithelial cells, were observed in the IZ, according to the following characteristics: overexpression of matrix metalloproteinase 3, membrane type 1-matrix metalloproteinase, snail, and zinc finger E-box-binding homeobox 1, and the gain of N-cadherin expression, as well as the down-regulation of miR200c. The myofibroblasts isolated from the IZ, which were designated interface zone-fibroblast, displayed laminin-332 and membrane type 1-matrix metalloproteinase overexpression, in contrast with both cancer-associated fibroblasts and normal breast fibroblasts. Taken together, our results suggest that the IZ, which shows dense fibrosis, may provide a specialized microenvironment for guiding tumor invasion: the fibrosis caused by laminin-332 overexpressing myofibroblast formation (interface zone-fibroblast) via epithelial-mesenchymal transition.

Stromal fibroblasts from the interface zone of human breast carcinomas induce an epithelial-mesenchymal transition-like state in breast cancer cells in vitro.

Fibroblasts were extracted from tissue in tumor burden zones, distal normal zones and interface zones between tumor and normal tissue of human breast carcinomas, and the corresponding fibroblasts were designated as cancer-associated fibroblasts (CAFs), normal zone fibroblasts (NFs) and interface zone fibroblasts (INFs). The crosstalk between three types of fibroblasts and breast cancer cells was evaluated using an in vitro direct co-culture model. We found that INFs grew faster and expressed higher levels of fibroblast activation protein than did NFs and CAFs. Compared with CAFs and NFs, INFs grown with breast cancer cells were significantly more effective in inducing an epithelial-mesenchymal transition (EMT) in cancer cells, as indicated by induction of vimentin and N-cadherin and downregulation of E-cadherin. This EMT process was also accompanied by activation of extracellular signal-regulated kinase (ERK) and modulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Additionally, INFs promoted breast cell migration to a larger extent compared with NFs and CAFs. Taken together, these findings indicate that INFs isolated from the tumor interface zone exhibited more robust biological modulatory activity than did NFs and CAFs isolated from normal and tumor zones of the same tumor tissue, suggesting that the interface zone of the tumor represents a dynamic region vital to tumor progression.

Proteomic molecular portrait of interface zone in breast cancer.

Surgical tumor margins are intended to encompass residual tumor cells but may not always accurately delineate the boundary between tumor and normal tissue. Efforts to define tumor margins based on molecular analysis have achieved limited success. Furthermore, no clinical trials have addressed the scope of the tumor microenvironment. Here, we considered the tumor cell population and surrounding microenvironment in delineating tumor margins, classifying breast cancer into tumor and normal zones, and introducing the concept of an interface zone, the region between the invading tumor front and normal tissue, which develops during tumor invasion and metastasis through remodeling of the tumor microenvironment. Pathological signatures of invasion markers in tumor tissues are most dynamic within the invading tumor front. We compared protein profiles of tumor, normal, and interface zones using MALDI-MS. Proteins upregulated in the interface zone were identified by peptide mass fingerprinting and confirmed by database searching with chemically assisted MALDI-PSD spectra. Upregulation was confirmed for RhoGDIα, CAPG, WDR1, and CK8 by Western and immunohistochemical analyses. Our results demonstrate that the molecular profile of the interface zone is unique and suggest that upregulation of proteins here may be related to progression and metastasis of breast carcinomas.

Panel of candidate biomarkers for renal cell carcinoma.

The timely diagnosis and therapeutic monitoring of human renal cell carcinoma (RCC) is limited by the lack of specific biomarkers. To identify candidate RCC biomarkers, we used 2-DE gel electrophoresis with mass spectrometry and 2-DE spot intensity-based ROC analysis to analyze 18 sets of paired normal and RCC tumor tissue including conventional, papillary, and chromophobe subtypes. Validation was performed with RCC patient plasma samples and confirmed by clustergram, shRNA, and immunohistochemistry assays. Cardinal candidates were evaluated by ELISA. The leading candidate biomarker that was upregulated in RCC samples according to the clustergram and validation analysis was nicotinamide N-methyltransferase (NNMT) (13/15, P < 0.0001). Other upregulated candidate biomarkers that were identified by this method include ferritin, hNSE, NM23, secretagogin, and L-plastin. The upregulation of NNMT in RCC was confirmed by immunoblotting and immunohistochemistry. Analysis of fractionated membrane-associated proteins identified CAP-G, mitofillin, tubulin alpha, RBBP7, and HSP27. Of these, RBBP7 and HSP27 were highly expressed in the chromophobe subtype of RCC (3/3) but were absent from conventional RCC (0/3). The triple combination of the NNMT, FTL, and hNSE biomarkers had the highest predictive capacity of 0.993, while NNMT was the single, most powerful candidate diagnostic biomarker for all types of RCC.

Induction of cell apoptosis in non-small cell lung cancer cells by cyclin A1 small interfering RNA.

Cyclin A1 and cyclin B1 are overexpressed in various tumors but are present at low levels in normal tissues. Cyclin A1 is restricted to germ cells undergoing meiosis. In order to explore the possibility of using cyclin A1 and cyclin B1 as anticancer targets, we knocked them down in two lung cancer cell lines, H157 and H596, using siRNA. As with cyclin A1 siRNA in lung cancer cell lines, cyclin B1, Cdc2 and CDK2 were all significantly downregulated. The S phase fraction increased significantly, and they eventually underwent apoptosis by way of downregulated intrinsic apoptotic pathways and modulators with upregulated extrinsic apoptotic pathways. Our study suggests that cyclin A1 might be a promising anticancer target specific to lung cancer.

Methylation of p16INK4a is a non-rare event in cervical intraepithelial neoplasia.

The cell cycle inhibitor, p16INK4a may be a useful surrogate biomarker of cervical intraepithelial neoplasia (CIN); however, there is currently no consensus of p16INK4a genetic alterations throughout the multiple step process of CIN. Our goal was to identify the methylation frequency of p16INK4a in each step of CIN that is associated with human papillomavirus (HPV) infection, using several different detection methods of p16INK4a methylation to correlate the data. The present study included a total of 43 patients, including 38 with CIN, and 5 normal patients. Three different methods were used to detect hypermethylation of CpG islands, methylation-specific PCR (MSP) amplification of different primer sets of M1, M2, and M3, pyrosequencing of each forward primer region, and immunohistochemistry of p16INK4a. Analysis of MSP showed that 20 of the 38 CIN patients (52.6%) revealed hypermethylation in at least 1 primer set of the p16INK4a promoter. A complete loss of p16INK4a protein expression was observed in 11 cases (28.9%). There was no observed association of methylation of the p16INK4a gene with either CIN grading (P=0.0698) or HPV status (P=0.2811): specifically 42.9% (3/7) was found in CIN 1, 57.1% (8/14) in CIN 2, and 52.9% (9/17) in CIN 3. In concordance with immunohistochemistry results, hypermethylation of the p16INK4a promoter was significantly correlated with a lack of p16 protein expression (P=0.0145). All positive peaks from pyrosequencing matched the MSP results, which ranged from 6.3% to 24.5%. In conclusion, p16INK4a gene silencing during CIN was not determined to be a particularly rare event; however, it does not correlate with either HPV status or CIN grading.