Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region.

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.

Disulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platform.

Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 1011. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and in vitro folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease. Through affinity maturation strategies, we have transformed initially weak binders against Notch2 with micromolar Kd values to high-affinity ligands in the nanomolar range. This process highlights a viable hit-to-lead progression. Overall, our platform holds significant potential to greatly enhance the discovery of peptide therapeutics.

The Long Non-coding RNA NEAT1/miR-224-5p/IL-33 Axis Modulates Macrophage M2a Polarization and A1 Astrocyte Activation.

To identify potential regulators and investigate the molecular mechanism of macrophage polarization affecting astrocyte activation from the perspective of non-coding RNA regulation, we isolated mouse bone marrow mononuclear cells (BMMNCs)-induced macrophages toward M1 or M2a polarization. Long non-coding RNA NEAT1 and IL-33 expression levels were significantly upregulated in M2a macrophages; NEAT1 knockdown in M2a macrophages markedly reduced the protein levels of IL-33 and M2a markers, IL-4 and IL-13 concentrations, and the bacterial killing capacity of M2a macrophages. NEAT1 acted as a competing endogenous RNA (ceRNA) to regulate IL-33 expression by sponging miR-224-5p in M2a macrophages; NEAT1 knockdown upregulated miR-224-5p expression, while miR-224-5p inhibition increased the protein content and concentration of IL-33. miR-224-5p inhibition exerted the opposite effects on the protein levels of IL-33 and M2a markers, IL-4 and IL-13 concentrations, and the bacterial killing capacity of M2a macrophages compared to NEAT1 knockdown; the effects of NEAT1 knockdown were significantly reversed by miR-224-5p inhibition. M2a macrophage conditioned medium (CM) significantly suppressed the activation of A1 astrocytes. NEAT1 knockdown M2a macrophage CM led to enhanced A1 astrocyte activation while miR-224-5p-silenced M2a macrophage CM led to a blockade of A1 astrocyte activation; the effects of NEAT1 knockdown M2a macrophage CM on A1 astrocyte activation were significantly reversed by miR-224-5p inhibition in M2a macrophages. The NEAT1/miR-224-5p/IL-33 axis modulates macrophage M2a polarization, therefore affecting A1 astrocyte activation.

PLAP -CAR T cells mediate high specific cytotoxicity against colon cancer cells.

Placental alkaline phosphatase, PLAP encoded by ALPP gene in humans is mainly expressed in placenta and testis, and not expressed in any other normal tissues. PLAP is overexpressed in colorectal cancers which makes it an attractive target for CAR (chimeric antigen receptor)-T cell therapy. PLAP mRNA expression was detected in 21.5% (25 out of 116) of colorectal cancer cell lines and this expression was confirmed by FACS at the protein level. In addition, IHC staining on primary colorectal cancer tumors demonstrated PLAP expression in >20% of colorectal cancer tumors. We generated mouse and humanized PLAP ScFv-CAR-T cells and demonstrated high specificity against PLAP-positive colon cancer cells using RTCA (real-time cytotoxicity assay) and IFN-gamma secretion. In addition, humanized-CAR-T cells significantly decreased Lovo xenograft tumor growth in vivo. The combination of hPLAP-CAR-T cells with PD-1, PD-L1 or LAG-3 checkpoint inhibitors significantly increased the activity of hPLAP-CAR-T cells. This study demonstrates ability of novel PLAP-CAR-T cells to kill colorectal cancers and that the extent of killing can be increased by combination with checkpoint inhibitors.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth.

The cell-surface protein B cell maturation antigen (BCMA, CD269) has emerged as a promising target for CAR-T cell therapy for multiple myeloma. In order to create a novel BCMA CAR, we generated a new BCMA monoclonal antibody, clone 4C8A. This antibody exhibited strong and selective binding to human BCMA. BCMA CAR-T cells containing the 4C8A scFv were readily detected with recombinant BCMA protein by flow cytometry. The cells were cytolytic for RPMI8226, H929, and MM1S multiple myeloma cells and secreted high levels of IFN-γ in vitro. BCMA-dependent cytotoxicity and IFN-γ secretion were also observed in response to CHO (Chinese Hamster Ovary)-BCMA cells but not to parental CHO cells. In a mouse subcutaneous tumor model, BCMA CAR-T cells significantly blocked RPMI8226 tumor formation. When BCMA CAR-T cells were given to mice with established RPMI8226 tumors, the tumors experienced significant shrinkage due to CAR-T cell activity and tumor cell apoptosis. The same effect was observed with 3 humanized BCMA-CAR-T cells in vivo. These data indicate that novel CAR-T cells utilizing the BCMA 4C8A scFv are effective against multiple myeloma and warrant future clinical development.

Mitochondrial Dysfunctions Regulated Radioresistance through Mitochondria-to-Nucleus Retrograde Signaling Pathway of NF-κB/PI3K/AKT2/mTOR.

We investigated the relationship between significantly different genes of the mitochondria-to-nucleus retrograde signaling pathway (RTG) in H1299 ρ0 cells (mtDNA depleted cell) and compared their radiosensitivity to that of parental ρ+ cells, to determine the possible intervention targets of radiosensitization. ρ0 cells were depleted of mitochondrial DNA by chronic culturing in ethidium bromide at low concentration. Radiosensitivity was analyzed using clonogenic assay. Western blot was used to analyze the cell cycle-related proteins, serine/threonine kinase ataxia telangiectasia mutant (ATM), ataxia telangiectasia and Rad3-related protein (ATR) and cyclin B1 (CCNB1). The γ-H2AX foci were detected using confocal fluorescence microscopy. RNA samples were hybridized using the Agilent human genome expression microarray. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for Gene Ontology (GO) Consortium and pathway annotations of differentially expressed genes, respectively. The H1299 ρ0 cells were found to be more radioresistant than ρ+ cells. The ATP production of H1299 ρ0 cells was lower than that of the ρ+ cells before or after irradiation. Both H1299 ρ0 and ρ+ cells had higher ROS levels after irradiation, however, the radiation-induced ROS production in ρ0 cells was significantly lower than in ρ+ cells. In addition, the percentage of apoptosis in H1299 ρ0 cells was lower than in ρ+ cells after 6 Gy irradiation. As for the cell cycle and DNA damage response-related proteins ATM, ATR and CCNB1, the expression levels in ρ0 cells were significantly higher than in ρ+ cells, and there were less γ-H2AX foci in the ρ0 than ρ+ cells after irradiation. Furthermore, the results of the human genome expression microarray demonstrated that the phosphorylated protein levels of the NF-κB/PI3K/AKT2/mTOR signaling pathway were increased after 6 Gy irradiation and were decreased after treatment with the AKT2-specific inhibitor MK-2206 combined with radiation in H1299 ρ0 cells. MK-2206 treatment also led to an increase in pro-apoptotic proteins. In conclusion, these results demonstrate that mtDNA depletion might activate the mitochondria-to-nucleus retrograde signaling pathway of NF-κB/PI3K/AKT2/mTOR and induce radioresistance in H1299 ρ0 cells by evoking mitochondrial dysfunctions.

Serine-arginine protein kinase 1 (SRPK1) is elevated in gastric cancer and plays oncogenic functions.

Serine-arginine protein kinase 1 (SRPK1) phosphorylates proteins involved in the regulation of several mRNA processing pathways including alternative splicing. SRPK1 has been reported to be over-expressed in multiple cancers including prostate, breast, lung and glioma. Several studies further identified that inhibition of SRPK1 showed tumor-suppressive effects, thus raising SRPK1 as a novel candidate chemotherapy target. Interestingly, SRPK1 plays tumor suppressing role in mouse embryonic fibroblasts, on that SRPK1-silencing induces cell transformation. Therefore, the effect of SRPK1 seems heterogeneously in different cell types and tissues. The existence and role of SRPK1 in gastric cancer (GC) hasn't been reported. Here we investigated the expression pattern of SRPK1 in GC by immunohistochemistry and found that it was up-regulated in tumor tissues, where its expression was correlated with tumor grade and prognosis. Further, we explored the signaling mechanism of SRPK1 in promoting GC progression, which revealed that both PP2A and DUSP6 phosphatases impaired the oncogenic effects of SRPK1. However, we didn't find any direct interaction between SRPK1 with PP2A or DUSP6, indicating PP2A and DUSP6 function by regulating the downstream effectors of SRPK1. Our study not only revealed the clinical significance of SRPK1 in GC, but also provided new evidence for its signaling modulation which is invaluable for novel chemotherapy development.

Downregulation of Mitochondrial Single Stranded DNA Binding Protein (SSBP1) Induces Mitochondrial Dysfunction and Increases the Radiosensitivity in Non-Small Cell Lung Cancer Cells.

Radiotherapy is one of the major therapeutic strategies for human non-small cell lung cancer (NSCLC), but intrinsic radioresistance of cancer cells makes a further improvement of radiotherapy for NSCLC challenging. Mitochondrial function is frequently dysregulated in cancer cells for adaptation to the changes of tumor microenvironment after exposure to radiation. Therefore, targeting mitochondrial biogenesis and bioenergetics is an attractive strategy to sensitize cancer cells to radiation therapy. In this study, we found that downregulation of single-strand DNA-binding protein 1 (SSBP1) in H1299 cells was associated with inducing mitochondrial dysfunction and increasing radiosensitivity to ionizing radiation. Mechanistically, SSBP1 loss induced mitochondrial dysfunction via decreasing mitochondrial DNA copy number and ATP generation, enhancing the mitochondrial-derived ROS accumulation and downregulating key glycolytic enzymes expression. SSBP1 knockdown increased the radiosensitivity of H1299 cells by inducing increased apoptosis, prolonged G2/M phase arrest and defective homologous recombination repair of DNA double-strand breaks. Our findings identified SSBP1 as a radioresistance-related protein, providing potential novel mitochondrial target for sensitizing NSCLC to radiotherapy.

Heteroplasmy of mutant mitochondrial DNA A10398G and analysis of its prognostic value in non-small cell lung cancer.

Mitochondrial dysfunction is associated with pathogenic mitochondrial (mt)DNA mutations. The majority of mtDNA point mutations have a heteroplasmic status, which is defined as the coexistence of wild-type and mutated DNA within a cell or tissue. Previous findings demonstrated that certain mtDNA heteroplasmic mutations contribute to widely spread chronic diseases, including cancer, and alterations in the heteroplasmy level are associated with the clinical phenotype and severity of cancer. In the present study, the proportions of mutant mtDNA 10398G were assessed using amplification-refractory mutation system-quantitative polymerase chain reaction (PCR) assay in 129 non-small cell lung cancer (NSCLC) tissue samples. Wild-type and mutant sequences were separately amplified using allele-specific primers and, subsequently, the PCR products containing the mtDNA 10398 site were ligated into vectors to construct a standard plasmid DNA construct. The association between mtDNA A10398G and the prognosis of patients was analyzed by survival analysis and Cox proportional hazards model. For the patient cohort, the median follow-up time and overall survival time were 20.6 and 26.3 months, respectively. The ratios of mutant heteroplasmy ranged between 0.31 and 97.04%. Patients with a high degree of mutant mtDNA 10398G had a significantly longer overall survival time compared with those with a low degree of mutant mtDNA 10398G (28.7 vs. 22.5 months, respectively; P<0.05). In addition, multivariate analysis demonstrated that epidermal growth factor receptor mutation status, tumor stage and the possession of a low degree of mutant 10398G were the three most independent prognostic factors. In conclusion, the present study suggests that, among NSCLC patients, there are large shifts in mutant mtDNA 10398G heteroplasmy and a low degree of mutant mtDNA 10398G heteroplasmy may be a marker of poor prognosis in patients with NSCLC.

D2-resected stage IIIc gastric cancer patients benefit from adjuvant chemoradiotherapy.

Although adjuvant chemoradiotherapy has been an important part in the treatment of gastric cancer, whether or not adjuvant radiation can benefit patients undergoing resection with D2 lymph node dissection remains controversial. This retrospective study aimed to evaluate the role of adjuvant chemoradiotherapy on patients with D2-resected gastric cancer. A total of 337 patients with resected gastric cancer treated at Zhongnan Hospital of Wuhan University from 2004 to 2012 were retrospectively analyzed. Eligible patients were divided into the adjuvant chemoradiotherapy group (CRT; n = 124) and the adjuvant chemotherapy group (CT; n = 213). The primary endpoints were disease-free survival (DFS) and overall survival (OS), with toxicity as the secondary endpoint. A subgroup analysis was performed based on clinical staging. The two groups were comparable in baseline characteristic, except for the number of lymph nodes dissected. The median OSs in the CRT and CT groups were 51.0 months and 48.6 months, respectively (P = 0.251), and the median DFSs were 40.7 months and 31.2 months, respectively (P = 0.112). Subgroup analysis revealed that the median OSs in patients at stage IIIc in the CRT group and CT group were 29.0 and 23.0 months, respectively (P = 0.049), and those of the median DFSs were 21.2 and 15.1 months, respectively (P = 0.015). There was no significant difference in main adverse events between two groups. Collectively, adjuvant chemoradiotherapy in gastric cancer patients with D2 resection was well tolerated. For Stage IIIc patients, the addition of adjuvant chemoradiotherapy was associated with a significant benefit in both OS and DFS.

An association analysis between mitochondrial DNA content, G10398A polymorphism, HPV infection, and the prognosis of cervical cancer in the Chinese Han population.

The aim was to analyze quantitative (mitochondrial DNA (mtDNA) content) and qualitative (G10398A polymorphism) mtDNA alterations as well as human papillomavirus (HPV) infection in cervical cancer prognosis. One hundred and twenty-two cases of formalin-fixed paraffin-embedded cervical carcinoma specimens were collected from the Yichang Tumor Hospital and Zhongnan Hospital of Wuhan University in the recent 10 years together with medical records. A quantitative real-time PCR (RT-PCR) was used to determine the copy number of the mitochondrial DNA and HPV expression levels. G10398A polymorphism was determined by PCR-RFLP assay. The overall survival of patients with higher mtDNA content was significantly reduced compared with lower mtDNA content patients (P = 0.029). But there was no difference of prognosis between the mtDNA 10398 A allele and G allele. However, the Kaplan-Meier survival curve illustrated a significantly reduced overall survival in the patients with 10398A plus high mtDNA copy number compared with the other groups (P < 0.05). Although no association between HPV expression level and cervical cancer prognosis was observed, 10398A got increased mtDNA content compared with 10398G (P < 0.05) and 10398G displayed an increased HPV-positive rate compared with 10398A. Furthermore, HPV-18 and mtDNA content were positively related in the younger subgroup (≤45 years) (correlation coefficient = 0.456, P = 0.022). This study indicated that mtDNA content and HPV infection status are associated with cervical cancer prognosis. High mitochondrial DNA content plus 10398 A may be a marker of poor prognosis in cervical cancer. And mtDNA variation may potentially influence the predisposition to HPV infection and cervical carcinogenesis.

Extrapleural locating method in computed tomography-guided needle biopsies of 1,106 lung lesions.

Transthoracic needle biopsy is considered to be safe and effective for the diagnosis of focal lung lesions. The aim of the present study was to evaluate factors affecting the accuracy and safety of automated cutting needle lung biopsy (ACNB) using a new extrapleural locating (EPL) method. Computed tomography (CT)-guided needle biopsies were performed on 1,065 patients between March 2005 and May 2012 using the EPL method. The locating needle remained in the chest following extrapleural positioning, while the radiologist confirmed the puncture angle and distance between the locating needle and lesion. The biopsy instrument was advanced into the lung, and the core needle was subsequently fired into the lesion based on the direction indicated by the locating needle. Univariate and multivariate regression analyses were used to evaluate the diagnostic accuracy and safety of the procedure. The sensitivity, specificity, positive predictive value and negative predictive value of the extrapleural method were 91.9, 100, 100 and 82.9%, respectively, and the overall diagnostic accuracy was 94.2%. Significant risk factors affecting accuracy were younger age, atelectasis, hemoptysis and lesion depth (P<0.03). Multivariate logistic regression analysis revealed that the risk of malignant lesions receiving a false-negative diagnosis decreased for each additional year of subject age [odds ratio (OR), 0.97; P=0.027] and increased with each millimeter increase in lesion depth (OR, 1.03; P=0.008). Among the 1,106 lesions biopsied, 207 were associated with pneumothorax, 251 with hemorrhage and 58 with hemoptysis. Multivariate analysis revealed that lesion size and emphysema affected pneumothorax incidence, while age, lesion location and depth and emphysema significantly affected hemorrhage incidence (P<0.05). In conclusion, low-dose, CT-guided ACNB with the EPL method provides a safe and accurate diagnosis.

Distinct regulation of Maf1 for lifespan extension by Protein kinase A and Sch9.

The Protein kinase A (PKA) and Sch9 regulates cell growth as well as lifespan in Saccharomyces cerevisiae. Maf1 is a RNA polymerase III (PolIII) inhibitor that tailors 5S rRNA and tRNA production in response to various environmental cues. Both PKA and Sch9 have been shown to phosphorylate Maf1 in vitro at similar amino acids, suggesting a redundancy in Maf1 regulation. However, here we find that activating PKA by bcy1 deletion cannot replace Sch9 for Maf1 phosphorylation and cytoplasmic retention; instead, such modulation lowers Maf1 protein levels. Consistently, loss of MAF1 or constitutive PKA activity reverses the stress resistance and the extended lifespan of sch9Δ cells. Overexpression of MAF1 partially rescues the extended lifespan of sch9Δ in bcy1Δsch9Δ mutant, suggesting that PKA suppresses sch9Δ longevity at least partly through Maf1 abundance. Constitutive PKA activity also reverses the reduced tRNA synthesis and slow growth of sch9Δ, which, however, is not attributed to Maf1 protein abundance. Therefore, regulation of lifespan and growth can be decoupled. Together, we reveal that lifespan regulation by PKA and Sch9 are mediated by Maf1 through distinct mechanisms.

Downregulation of high mobility group box 1 modulates telomere homeostasis and increases the radiosensitivity of human breast cancer cells.

The functions of the high mobility group box 1 (HMGB1) in tumor cells include replenishing telomeric DNA and maintaining cell immortality. There is a negative correlation between human telomerase reverse transcriptase (hTERT) and radiosensitivity in tumor cells. Our aim was to elucidate the relationship among HMGB1, telomere homeostasis and radiosensitivity in MCF-7 cells. In this study, we established stably transfected control (MCF-7-NC) and HMGB1 knockdown (MCF-7-shHMGB1) cell lines. The expression of HMGB1 mRNA and the relative telomere length were examined by real-time PCR. Radiosensitivity was detected by clonogenic assay. The protein expressions were determined by western blot analysis. The telomerase activity was detected by PCR-ELISA. Proliferation ability was examined by CCK-8 assay. Cell cycle and apoptosis were examined by flow cytometry. DNA damage foci were detected by immunofluorescence. ShRNA-mediated downregulation of HMGB1 expression increased the radiosensitivity of MCF-7 cells, and reduced the accumulation of hTERT and cyclin D1. Moreover, knockdown of HMGB1 in MCF-7 cells inhibited telomerase activity and cell proliferation, while increasing the extent of apoptosis. Downregulation of HMGB1 modulated telomere homeostasis by changing the level of telomere-binding proteins, such as TPP1 (PTOP), TRF1 and TRF2. This downregulation also inhibited the ATM and ATR signaling pathways. The current data demonstrate that knockdown of HMGB1 breaks telomere homeostasis, enhances radiosensitivity, and suppresses the repair of DNA damage in human breast cancer cells. These results suggested that HMGB1 might be a potential radiotherapy target in human breast cancer.

Rab1A is an mTORC1 activator and a colorectal oncogene.

Amino acid (AA) is a potent mitogen that controls growth and metabolism. Here we describe the identification of Rab1 as a conserved regulator of AA signaling to mTORC1. AA stimulates Rab1A GTP binding and interaction with mTORC1 and Rheb-mTORC1 interaction in the Golgi. Rab1A overexpression promotes mTORC1 signaling and oncogenic growth in an AA- and mTORC1-dependent manner. Conversely, Rab1A knockdown selectively attenuates oncogenic growth of Rab1-overexpressing cancer cells. Moreover, Rab1A is overexpressed in colorectal cancer (CRC), which is correlated with elevated mTORC1 signaling, tumor invasion, progression, and poor prognosis. Our results demonstrate that Rab1 is an mTORC1 activator and an oncogene and that hyperactive AA signaling through Rab1A overexpression drives oncogenesis and renders cancer cells prone to mTORC1-targeted therapy.

Peripheral blood mitochondrial DNA content, A10398G polymorphism, and risk of breast cancer in a Han Chinese population.

It has been reported that quantitative alterations and sequence variations of mtDNA are associated with the onset and progression of particular types of tumor. However, the relationship between mtDNA content, certain mtDNA polymorphisms in peripheral blood leukocytes and breast cancer risk remain obscure. This study was undertaken to investigate whether mtDNA content and the A10398G polymorphism in peripheral blood leukocytes could be used as risk predictors for breast cancer in Han Chinese women. Blood samples were obtained from a total of 506 breast cancer patients and 520 matched healthy controls. The mtDNA content was measured by using quantitative real-time PCR assay; A10398G polymorphism was determined by PCR-RFLP assay. There was no statistically significant difference between cases and controls in terms of peripheral blood mtDNA content or A10398G polymorphism. However, further analysis suggested that the risk of breast cancer was associated with decreased mtDNA content in premenopausal women (P = 0.001; odds ratio = 0.54; 95% confidence interval, 0.38-0.77), with increased mtDNA content in postmenopausal women (P = 0.027; odds ratio = 1.49; 95% confidence interval, 1.05-2.11). In addition, the associations between mtDNA content and several clinicopathological parameters of cases such as age, menopausal status, and number of pregnancies and live births were observed. This case-control study indicated that the peripheral blood mtDNA content might be a potential biomarker to evaluate the risk of breast cancer for selected Chinese women.

Prognostic value of mitochondrial DNA content and G10398A polymorphism in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality worldwide. Mitochondrial dysfunction has been postulated to render cancer cells resistant to apoptosis based on the Warburg hypothesis. However, few studies have investigated the prognostic value of mitochondrial DNA (mtDNA) content and G10398A polymorphism in NSCLC patients. mtDNA copy number and G10398A polymorphism in 128 NSCLC tissue samples were assessed by real-time PCR (RT-PCR) and PCR-RFLP respectively, and their relationship to prognosis were analyzed by survival analysis and Cox proportional hazards model. In vitro, an mtDNA deletion A549 ρ(0) cell model was utilized to assess the function of mtDNA on radiosensitivity. Cell cycle distribution and reactive oxygen species (ROS) were analyzed to elucidate the potential mechanisms. For the whole group, the median follow-up time and overall survival time were 22.5 and 23.4 months, respectively. Patients with high mtDNA content had a marginally longer survival time than patients with low mtDNA content (P=0.053). Moreover, patients with high mtDNA content plus 10398G had a significantly longer overall survival time compared with those having low mtDNA content plus 10398A (47 vs. 27 months, P<0.05). In addition, multivariate analysis showed that stage and low mtDNA content plus 10398A were the two most independent prognostic factors. In vitro, the A549 ρ(0) cells showed more resistance to radiation than ρ(+) cells. Following radiation, ρ(0) cells showed delayed G2 arrest and lower ROS level as compared to ρ(+) cells. In conclusion, the present study suggests that in patients with NSCLC, low mtDNA content plus 10398A could be a marker of poor prognosis which is associated with resistance to anticancer treatment caused by low mtDNA content plus 10398A polymorphism resulting in mitochondrial dysfunction.

Identification of cisplatin-resistance associated genes through proteomic analysis of human ovarian cancer cells and a cisplatin-resistant subline.

Chemoresistance to cancer therapy is a major obstacle to the effective treatment of human cancers with cisplatin (DDP), but the mechanisms of cisplatin-resistance are not clear. In this study, we established a cisplatin- resistant human ovarian cancer cell line (COC1/DDP) and identified differentially expressed proteins related to cisplatin resistance. The proteomic expression profiles in COC1 before and after DDP treatment were examined using 2-dimensional electrophoresis technology. Differentially expressed proteins were identified using matrix- assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and high performance liquid chromatography-electrospray tandem MS (NanoUPLC-ESI-MS/MS). 5 protein spots, for cytokeratin 9, keratin 1, deoxyuridine triphosphatase (dUTPase), aarF domain containing kinase 4 (ADCK 4) and cofilin1, were identified to be significantly changed in COC1/DDP compared with its parental cells. The expression of these five proteins was further validated by quantitative PCR and Western blotting, confirming the results of proteomic analysis. Further research on these proteins may help to identify novel resistant biomarkers or reveal the mechanism of cisplatin-resistance in human ovarian cancers.