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Background: Germline mutations in CDH1 are associated with hereditary and early onset- diffuse gastric cancer. However, the frequency of CDH1 germline mutation in unselected gastric cancer cases is not well established. Aim: The aim of this study was to investigate the frequency and clinical characteristics of germline CDH1 V832M mutation carriers in unselected Korean gastric cancer cases. Methods: Direct sequencing was performed to determine the presence of CDH1 V832M in 305 unselected Korean gastric cancer patients. Lauren's histologic type, family history of gastric cancer, and age of cancer diagnosis were compared between V832M carriers and non-carriers. Results: In the study population, seven gastric cancer patients (7/305, 2.29%) were found to have the CDH1 V832M mutation. The CDH1 V832M mutation carrier state was not significantly associated with phenotypes including Lauren's histologic type, family history of gastric cancer, age of cancer diagnosis, and other cancer history in a patient. Conclusion: This study demonstrates that the germline CDH1 V832M mutation is common in sporadic, late onset, and intestinal type gastric cancer as well as familial, early onset, and diffuse type gastric cancer. Our finding suggests that guidelines for managing CDH1 mutation carriers should be refined through additional data on penetration according to CDH1 mutation type in sporadic cases.

Precision medicine has received increased attention as an effective approach for the treatment of cancer patients. Because of challenges associated with the availability of archived tissue, liquid biopsies are often performed to detect cancer-specific mutations. One of the major advantages of the liquid biopsy is that the treatment can be monitored longitudinally, even after the tumor tissue is no longer available. In a clinical setting, one component of precision medicine is the detection of cancer-specific mutations using archived samples. In this study, we evaluated the epidermal growth factor receptor (EGFR) mutation status of samples of lung cancer patients stored before introduction of the plasma EGFR test at our institution. The aim of this study was to validate the utility of archived plasma samples for detection of the EGFR mutation in nonsmall cell lung cancer (NSCLC) patients. The Cobas® EGFR Mutation Test v2 was the first liquid biopsy test approved as a companion diagnostic test for patients with NSCLC treated with tyrosine kinase inhibitors. We tested for the EGFR mutation in 116 plasma samples archived in the biobank, and the results were compared with those obtained in the tissue or cytology EGFR mutation test. The EGFR mutation-positive rate from archived plasma was lower than that determined from tissue or cytology at 19.0% and 53.4%, respectively, and the concordance rate between the two tests was 58.6%. Of interest, five (4.3%) samples showed the T790M mutation in the plasma test, whereas this mutation was only detected in two (1.7%) tissue/cytology samples. Five (4.3%) samples were additionally positive in the plasma test. Overall, these results indicate that archived plasma samples can serve as an alternative source for the plasma EGFR mutation test when tissue samples are not available, and can improve precision medicine and long-term follow-up in a noninvasive manner.

In the era of precision medicine, the prediction of ovarian function recovery from chemotherapy-induced amenorrhoea using feasible biological markers may be helpful to optimise the treatment strategy for young patients with hormone receptor-positive breast cancer. The purpose of this study was to investigate the accuracy of post-chemotherapy biological markers for predicting the recovery of ovarian function in breast cancer patients of the ASTRRA trial, with chemotherapy-induced amenorrhoea. Using data of 82 participants from a single institution in the ASTRRA trial, the post-chemotherapy serum levels of the anti-Müllerian hormone (AMH), oestradiol, inhibin B and other clinical factors associated with chemotherapy-induced amenorrhoea were evaluated. Recovery of ovarian function was defined by the resumption of menstruation manifested by vaginal bleeding. Fifty-two patients regained menstruation within 55 months after enrolment. In univariate analysis, P = 0.009), oestradiol ≥37 pg/mL (P = 0.003) or AMH ≥800 pg/mL (P = 0.026) were associated with recovery of menstruation. On multivariate analysis, oestradiol (hazard ratio: 3.171, 95% CI: 1.306–7.699, P = 0.011) and AMH (hazard ratio: 2.853, 95% CI: 1.011–8.046, P = 0.048) remained as significant independent predictors for resumption of menstruation. The diagnostic accuracy of age, oestradiol and AMH in predicting the resumption of menstruation was 38.3, 23.3 and 86.7%, respectively. In conclusion, post-chemotherapy AMH level might be a relatively accurate predictor of the recovery of ovarian function, presented by resumption of menstruation in breast cancer patients with chemotherapy-induced amenorrhoea.

Introduction: Desmoid tumor is a rare aggressive tumor where there is no satisfying systemic treatment. Previous studies showed increased expression of transforming growth factor-β (TGF-β) in the desmoid tumor. Here, we analyzed genomic and transcriptomic data of tumor samples from patients with desmoid tumor to evaluate therapeutic applicability of inhibition of TGF-β response. Methods: We collected pre-treatment desmoid tumor samples from 31 patients in Yonsei cancer center, (YCC)-desmoid cohort and performed targeted next-generation sequencing and whole RNA sequencing. To compare the data with other cancer types, we used whole exome sequencing and whole RNA sequencing data from sarcoma patients from Yonsei Cancer Center (YCC-sarcoma cohort, n = 17 and n =53, respectively) and The Cancer Genome Atlas (TCGA, n = 9,235). Transcriptomic data was normalized throughout the three cohorts to reduce batch effect. To evaluate the enrichment of TGF-β responsive signature (TBRS) in tumor and microenvironment, we calculated the mean expression values of fibroblast-TBRS (F-TBRS) from previous study. Results: In the two sarcoma cohorts (YCC-desmoid and YCC-sarcoma cohorts), desmoid tumor samples had the highest F-TBRS score among the various types of sarcoma. When compared with other cancer types in TCGA, desmoid tumor samples showed the higher F-TBRS than all other cancer types except for pancreatic adenocarcinoma. In the desmoid tumor samples, CTNNB1, GNAQ, and APC mutations were the top 3 frequent mutations (23, 19, and 2 samples, respectively). In the merged two sarcoma cohorts, CTNNB1/APC mutation and GNAQ mutation were both associated with higher enrichment in F-TBRS (p < 0.001, respectively). Conclusion: Desmoid tumors are enriched with expression of genes associated with TGF-β response of fibroblast compared with other cancer types including other sarcomas. In addition, CTNNB1, APC and GNAQ mutations were associated with higher enrichment in TBRS. Therapeutic intervention to decrease the TGF-β response of fibroblast by using TGF-β receptor inhibitor may show clinical benefit in patients with desmoid tumors. Citation Format: Changhee Park, Kum-Hee Yun, Sanghoon Song, Jin Kyung Lee, Jiyeon Ryu, Bitna Oh, Sunjin Hwang, Ki Baik Hahm, Seung-Jin Kim, Seung Hyun Kim, Chan-Young Ock, Hyo-Song Kim. Genomic and transcriptomic analyses of desmoid tumor reveals enrichment of transforming growth factor beta responsive signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2250.

Although it has been proposed that the beneficial effect of HER2-targeted therapy in HER2-negative breast cancer is associated with the molecular subtype conversion, the underlying mechanism and the clinical biomarkers are unclear. Our study showed that breast cancer stem cells (BCSCs) mediated HER2 subtype conversion and radioresistance in HER2-negative breast cancer cells and evaluated serum HER2 as a clinical biomarker for HER2 subtype conversion. We found that the CD44+/CD24-/low BCSCs from HER2-negative breast cancer MCF7 cells overexpressed HER2 and EGFR and showed the radioresistant phenotype. In addition, we showed that trastuzumab treatment sensitized the radioresistant phenotype of the CD44+/CD24-/low cells with decreased levels of HER2 and EGFR, which suggested that HER2-targeted therapy in HER2-negative breast cancer could be useful for targeting BCSCs that overexpress HER2/EGFR. Importantly, our clinical data showed that serial serum HER2 measurement synchronously reflected the disease relapse and the change in tumor burden in some patients who were initially diagnosed as HER2-negative breast cancer, which indicated that serum HER2 could be a clinical biomarker for the evaluation of HER2 subtype conversion in patients with recurrent HER2-negative breast cancer. Therefore, our data have provided in vitro and in vivo evidence for the molecular subtype conversion of HER2-negative breast cancer.

// Sang Hyeok Woo 1, * , Sung-Keum Seo 1, * , Yoonhwa Park 1, 2 , Eun-Kyu Kim 3 , Min-Ki Seong 4 , Hyun-Ah Kim 4 , Jie-Young Song 1 , Sang-Gu Hwang 1 , Jin Kyung Lee 5 , Woo Chul Noh 4 , In-Chul Park 1 1 Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Nowon-gu, Seoul, 01812, Republic of Korea 2 School of Life Science and Biotechnology, Korea University, Seongbuk-gu, Seoul, 02841, Republic of Korea 3 Department of Surgery, Breast Cancer Center, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Bundang-gu, Seongnam, 13620, Republic of Korea 4 Department of Surgery, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, Nowon-gu, Seoul, 01812, Republic of Korea 5 KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, Nowon-gu, Seoul, 01812, Republic of Korea * These authors contributed equally to this work Correspondence to: In-Chul Park, email: parkic@kcch.re.kr Keywords: tamoxifen, breast cancer, dichloroacetate, epidermal growth factor receptor, pyruvate dehydrogenase kinase Received: May 18, 2016 Accepted: July 22, 2016 Published: August 01, 2016 ABSTRACT Metabolic reprogramming in cancer cells has recently been recognized as an essential hallmark of neoplasia. In this context, metabolic alterations represent an attractive therapeutic target, and encouraging results with drugs targeting various metabolic processes have been obtained in preclinical studies. Recently, several studies have suggested that dichloroacetate (DCA), a specific pyruvate dehydrogenase kinase inhibitor, may be a potential anticancer drug in a large number of diverse tumors. However, the precise mechanism is not fully understood, which is important for the use of DCA in cancer treatment. In the present study, we found that DCA sensitized MCF7 breast cancer cells to tamoxifen-induced cell death by decreasing epidermal growth factor receptor (EGFR) expression. The downregulation of EGFR was caused by degradation of the protein. Furthermore, p38 mitogen-activated protein kinase played an important role in DCA/tamoxifen-induced EGFR degradation. Finally, DCA also promoted comparable tamoxifen-induced cell death in tamoxifen-resistant MCF7 cells, which were established by long-term treatment with tamoxifen. In summary, our results suggest that DCA is an attractive potential drug that sensitizes cells to tamoxifen-induced cell death and overcome tamoxifen resistance via downregulation of EGFR expression in breast cancer cells.

Melatonin is implicated in various physiological functions, including anticancer activity. However, the mechanism(s) of its anticancer activity is not well understood. In the present study, we investigated the combined effects of melatonin and arsenic trioxide (ATO) on cell death in human breast cancer cells. Melatonin enhanced the ATO-induced apoptotic cell death via changes in the protein levels of Survivin, Bcl-2, and Bax, thus affecting cytochrome c release from the mitochondria to the cytosol. Interestingly, we found that the cell death induced by co-treatment with melatonin and ATO was mediated by sustained upregulation of Redd1, which was associated with increased production of reactive oxygen species (ROS). Combined treatment with melatonin and ATO induced the phosphorylation of JNK and p38 MAP kinase downstream from Redd1 expression. Rapamycin and S6K1 siRNA enhanced, while activation of mTORC1 by transfection with TSC2 siRNA suppressed the cell death induced by melatonin and ATO treatment. Taken together, our findings suggest that melatonin enhances ATO-induced apoptotic cell death via sustained upregulation of Redd1 expression and inhibition of mTORC1 upstream of the activation of the p38/JNK pathways in human breast cancer cells.

Recently, targeting deregulated energy metabolism is an emerging strategy for cancer therapy. In the present study, combination of DCA and metformin markedly induced cell death, compared with each drug alone. Furthermore, the expression levels of glycolytic enzymes including HK2, LDHA and ENO1 were downregulated by two drugs. Interestingly, HIF-1α activation markedly suppressed DCA/metformin-induced cell death and recovered the expressions of glycolytic enzymes that were decreased by two drugs. Based on these findings, we propose that targeting HIF-1α is necessary for cancer metabolism targeted therapy.

Redd1 is a stress response protein that functions as a repressor of mTORC1, a central regulator of protein translation, resulting in the inhibition of cell growth and metabolism. However, paradoxically, high Redd1 expression favors cancer progression and generates resistance to cancer therapy. Herein, we revealed that constitutive overexpression of Redd1 induced HSP27 and HSP70 expression in lung cancer cells. The expression of Redd1, HSP27 and HSP70 was highly increased in lung cancer tissues compared with that in normal lung tissues. Inhibition of HSP27 or HSP70 suppressed AKT phosphorylation, which was induced by constitutive overexpression of Redd1 and enhanced the inhibitory effects on viability of Redd1‑overexpressing cells. Inhibition of AKT phosphorylation resulted in a decrease of HSP27 and HSP70 expression in Redd1‑overexpressing cells. These data indicated that HSPs and AKT in Redd1‑overexpressing cells positively regulated the function and expression of each other and were involved in lung cancer cell survival. Knockdown of HSP27, HSP70 or AKT enhanced ionizing radiation (IR) sensitivity, particularly in lung cancer cells in which Redd1 was stably overexpressed. Collectively, constitutive overexpression of Redd1 led to HSP27 and HSP70 induction and AKT activation, which were involved in lung cancer cell survival and resistance to IR, suggesting that Redd1 may be used as a therapeutic target for lung cancer.

Heat shock protein 90 (HSP90) regulates the stability of various proteins and plays an essential role in cellular homeostasis. Many client proteins of HSP90 are involved in cell growth, survival, and migration; processes that are generally accepted as participants in tumorigenesis. HSP90 is also up-regulated in certain tumors. Indeed, the inhibition of HSP90 is known to be effective in cancer treatment. Recently, studies showed that HSP90 regulates transforming growth factor β1 (TGF-β1)-induced transcription by increasing the stability of the TGF-β receptor. TGF-β signaling also has been implicated in cancer, suggesting the possibility that TGF-β1 and HSP90 function cooperatively during the cancer cell progression. Here in this paper, we investigated the role of HSP90 in TGF-β1-stimulated Mv1Lu cells. Treatment of Mv1Lu cells with the HSP90 inhibitor, 17-allylamino-demethoxy-geldanamycin (17AAG), or transfection with truncated HSP90 (ΔHSP90) significantly reduced TGF-β1-induced cell migration. Pretreatment with 17AAG or transfection with ΔHSP90 also reduced the levels of phosphorylated Smad2 and Smad3. In addition, the HSP90 inhibition interfered the nuclear localization of Smads induced by constitutively active Smad2 (S2EE) or Smad3 (S3EE). We also found that the HSP90 inhibition decreased the protein level of importin-β1 which is known to regulate R-Smad nuclear translocation. These data clearly demonstrate a novel function of HSP90; HSP90 modulates TGF-β signaling by regulating Smads localization. Overall, our data could provide a detailed mechanism linking HSP90 and TGF-β signaling. The extension of our understanding of HSP90 would offer a better strategy for treating cancer.

Two cases of hematological malignancies were reported in an industrial radiography company over a year, which were reasonably suspected of being consequences of prolonged exposure to ionizing radiation because of the higher incidence than expected in the general population. We analyzed chromosomal aberrations in the peripheral blood lymphocytes from the other workers who had been working under similar circumstances as the patients in the company. Among the subjects tested, 10 workers who belonged to the highest band were followed up periodically for 1.5 years since the first analysis. The aim of this study was to clarify pertinence of translocation analysis to an industrial set-up where chronic exposure was commonly expected. To be a useful tool for a retrospective biodosimetry, the aberrations need to be persistent for a decade or longer. Therefore we calculated the decline rates and half-lives of frequency for both a reciprocal translocation and a dicentric chromosome and compared them. In this study, while the frequency of reciprocal translocations was maintained at the initial level, dicentric chromosomes were decreased to 46.9% (31.0-76.5) of the initial frequency over the follow-up period. Our results support the long-term stability of reciprocal translocation through the cell cycle and validate the usefulness of translocation analysis as a retrospective biodosimetry for cases of occupational exposure.

The unique metabolic profile of cancer (aerobic glycolysis) is an attractive therapeutic target for cancer. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, has been shown to reverse glycolytic phenotype and induce mitochondrion-dependent apoptosis. In the present study, we investigated the effects of S6 kinase 1 (S6K1) inhibition on DCA-induced cell death and the underlying mechanisms in breast cancer cells. Cell death was evaluated by annexin V and PI staining. The synergistic effects of DCA and PF4708671 were assessed by isobologram analysis. Small interfering RNA (siRNA) was used for suppressing gene expression. The mRNA and protein levels were measured by RT-PCR and Western blot analysis, respectively. PF4708671, a selective inhibitor of S6K1, and knockdown of S6K1 with specific siRNA enhanced DCA-induced cell death. Interestingly, a combination of DCA/PF4708671 markedly reduced protein expression of a glycolytic enzyme, hexokinase 2 (HK2). Suppression of HK2 activity using specific siRNA and 2-deoxyglucose (2-DG) further enhanced cell sensitivity to DCA/PF4708671. Overexpression of Myc-tagged HK2 rescued cell death induced by DCA/PF4708671. Based on these findings, we propose that inhibition of S6K1, in combination with the glycolytic inhibitor, DCA, provides effective cancer therapy.

Dear Editor, T lymphoblastic leukemia (T-ALL) comprises 10-15% of pediatric ALL cases and is known to be a clinically and genetically heterogeneous disease [1]. Recent studies have presented various cytogenetic abnormalities in T-ALL. The alterations often present as reciprocal translocations implicated in several transcriptional factor genes and lead to arrest during T-cell differentiation [1,2]. Although cytogenetic aberrations are known to be critical in leukemogenesis of many hematologic malignancies, the relationships between these aberrations and the prognosis are not well understood in T-ALL except for t(11;19), a mixed-lineage leukemia gene (MLL)-ENL fusion related to favorable prognosis [3]. Here, we report a very rare case of pediatric T-ALL with t(11;17)(q23;q21) involving MLL rearrangement, which has not been previously reported in Korea. A 16-month-old boy was admitted to our hospital because of fever, cough, rhinorrhea, and poor oral intake. Physical examination revealed anemic conjunctiva, cervical lymph node enlargement, and splenomegaly. Chest radiography showed no abnormal findings. The complete blood count (CBC) results were as follows: hemoglobin, 8.2 g/dL; leukocytes, 41.3×109/L; platelets, 177×109/L; and reticulocyte count, 3.4%. The blood smear showed increased blasts (66%). The bone marrow (BM) specimen contained 65% blasts and showed a hypercellular pattern. Small-to-medium sized blasts were observed with condensed nuclear chromatin and an irregular nuclei margin. Immunophenotyping using the BM aspirate was brightly positive for CD2 (86%), cCD3 (91%), CD5 (91%), CD7 (92%), and CD34 (76%). Serum lactate dehydrogenase was increased to 638 IU/L. Cytogenetic studies on the BM revealed the karyotype of 46,XY,t(11;17)(q23;q21)[17]/46,XY[3] (Fig. 1A). The patient was diagnosed with T-ALL according to the 2008 WHO classification [4]. Fig. 1 Bone marrow karyogram at diagnosis and interphase FISH analysis after induction chemotherapy. (A) G-banded karyogram showing t(11;17)(q23;q21). The arrows denote the rearranged chromosomes. (B) FISH analysis using 11q23 break-apart probe revealed MLL ... The patient began receiving induction chemotherapy comprised of prednisolone, vincristine, L-asparaginase, intrathecal cytosine arabinoside, and methotrexate. He seemed to respond to chemotherapy, with decreasing leukocyte counts and spleen size. However, the BM aspirate performed on the 7th day showed increased blasts to 69%. Moreover, interphase FISH analysis using an 11q23 break-apart probe revealed MLL rearrangement (158 of 200, 79%) (Fig. 1B). He was switched to more intensive induction chemotherapy including doxorubicin and cyclophosphamide. The patient achieved complete remission after 28 days of re-induction chemotherapy. Because of his high-risk features, the patient received cord blood stem cell transplantation. At the time of writing, he is alive and has been in a continuous complete remission state for 26 months. MLL/11q23 rearrangement is intricate in many hematologic malignancies such as ALL, AML, and MDS. The 11q23 translocation, which is rarely identified in T-ALL, is a distinct subtype characterized by differentiation arrest at an early stage of thymocyte differentiation [5]. Since MLL fusion partners are also known to have an important role in leukemogenesis, recent efforts to unravel MLL fusion partners using long-distance inverse-PCR (LDI-PCR) as compensatory diagnostic method have been implemented by using current molecular diagnostic tools [6,7]. Not only scarce MLL rearrangement but also few fusion genes are involved in T-ALL, despite that 79 different MLL fusion partner genes have been identified in other hematologic malignancies [6,8]. When balanced t(11;17)(q23;q21) involving MLL rearrangement was searched on the molecular level, three different fusions with MLL at 11q23 were generated by proximally clustered genes at the same chromosomal loci: MLLT6/AF17, ACACA, and LASP1 at 17q21 [8]. Thus far, MLL with each of the other three fusion partner genes at 17q21 has been identified mainly in AML and rarely in ALL [8]. Unfortunately, the fusion partner gene in this case could not be confirmed by using LDI-PCR because of specimen problems. A literature search revealed only two cases regarding t(11;17)(q23;q21) in adults with T-ALL [9,10] (Table 1). Table 1 Reported cases of T-ALL with t(11;17)(q23;q21) Prior studies have suggested that immunophenotypic markers are correlated with genetic alterations in pediatric acute leukemia [2]. The immunophenotype of leukemic cells in our patient could be classified into the pre-T stage and immature stage according to the European Group for the Immunological Characterization of Leukaemias and T-cell receptor classification system [2]. The immature stage of T-ALL was reported with respect to poor outcome and the lack of a common genetic lesion [1,2]. In several studies, MLL-rearranged T-ALL overexpressed multiple HOX, which are transcription factors that initiate T-cell differentiation [5]. Additional cases involving genetic profiling of t(11;17)(q23; q21) involving MLL rearrangement will help assess whether this rare fusion impacts the disease progression and reveals prognostic information.

Nutrient-limiting conditions are frequently encountered by tumor cells in poorly vascularized microenvironments. These stress conditions may facilitate the selection of tumor cells with an inherent ability to decrease apoptotic potential. Therefore, selective targeting of tumor cells under glucose deprivation conditions may provide an effective alternative strategy for cancer therapy. In the present study, we investigated the effects of S6 kinase 1 (S6K1) inhibition on glucose deprivation-induced cell death and the underlying mechanisms in MCF-7 breast cancer cells. PF4708671, a selective inhibitor of S6K1, and knockdown of S6K1 with specific siRNA enhanced cell death induced under glucose deprivation conditions. Moreover, inhibition of S6K1 led to apoptosis in glucose-starved MCF-7 cells via downregulation of the anti-apoptotic proteins, Mcl-1 and survivin. Further experiments revealed that sorafenib, shown to be involved in Mcl-1 and survivin downregulation via mTOR/S6K1 inhibition significantly promotes cell death under glucose deprivation conditions. These findings collectively suggest that S6K1 plays an important role in tumor cell survival under stress conditions, and thus inhibition of S6K1 may be an effective strategy for sensitizing cells to glucose deprivation.

Previous studies have shown that hypoxia can reverse DCA/metformin-induced cell death in breast cancer cells. Therefore, targeting hypoxia is necessary for therapies targeting cancer metabolism. In the present study, we found that TRAIL can overcome the effect of hypoxia on the cell death induced by treatment of DCA and metformin in breast cancer cells. Unexpectedly, DR5 is upregulated in the cells treated with DCA/metformin, and sustained under hypoxia. Blocking DR5 by siRNA inhibited DCA/metformin/TRAIL-induced cell death, indicating that DR5 upregulation plays an important role in sensitizing cancer cells to TRAIL-induced cell death. Furthermore, we found that activation of JNK and c-Jun is responsible for upregulation of DR5 induced by DCA/metformin. These findings support the potential application of combining TRAIL and metabolism-targeting drugs in the treatment of cancers under hypoxia.

As the activation of autophagy contributes to the efficacy of many anticancer therapies, deciphering the precise role of autophagy in cancer therapy is critical. Here, we report that the dual mTORC1/2 inhibitors PP242 and OSI-027 decreased cell viability but did not induce apoptosis in the non-small cell lung cancer (NSCLC) cell lines H460 and A549. PP242 induced autophagy in NSCLC cells as demonstrated by the formation of massive vacuoles and acidic vesicular organelles and the accumulation of LC3-II. JNK was activated by PP242, and PP242-induced autophagy was blocked by inhibiting JNK pathway with SP600125 or JNK siRNA, suggesting that JNK activation is required for the mTORC1/2 inhibitor-mediated induction of autophagy in NSCLC cells. Inhibiting JNK or autophagy increased the sensitivity of H460 cells to mTORC1/2 inhibitors, indicating that JNK or autophagy promoted survival in NSCLC cells treated with mTORC1/2 inhibitors. Together, these data suggest that combining mTORC1/2 inhibitors with inhibitors of JNK or autophagy might be an effective approach for improving therapeutic outcomes in NSCLC.

Purpose: We did this retrospective study to explore the association between epidermal growth factor receptor (EGFR) mutation and clinical features in postoperative recurrent female non-small-cell lung cancer (NSCLC). Materials and Methods: We reviewed clinical data on 86 female patients who had postoperative recurrent disease between December 1992 and July 2007. The start of tyrosine kinase inhibitor therapy was treated as a censoring event. Corresponding surgical specimens of primary tumors were used to test for EGFR mutations. Results: Thirty patients presented with local recurrence and distant recurrence was identified in 56. Thirty-four of the 86 patients (40%) harbored EGFR mutations. Patients with distant recurrence were more likely to have EGFR mutations than patients with local recurrence (48% versus 23%; P = 0.024). On multivariate analysis, distant recurrence was associated with a high frequency of EGFR mutations (OR, 3.3; P = 0.028). Survival analysis showed poor survival of patients with mutated EGFR (HR, 2.3; P = 0.017) or with non-adenocarcinoma histology (HR, 3.3; P = 0.001). Conclusion: The association between recurrence pattern and EGFR mutation status was suggested in recurrent female NSCLC patients. In addition, our data indicate unfavorable disease process of EGFR mutated tumors. Further studies need to be conducted to validate these findings.

Redd1 acts as a negative regulator of mTOR in response to various stress conditions, but its specific physiological role is currently unclear. In the present study, we showed that Redd1 inhibits the invasive activity of non-small cell lung cancer (NSCLC) cells. Interestingly, expression of Redd1 was extremely low in H1299 cells displaying high invasiveness, compared with that in H460 cells with lower invasive activity. Overexpression of Redd1 inhibited the invasive activity of H1299 cells, while suppression with specific siRNAs enhanced the invasiveness of H460 cells. Knockdown of the mTOR downstream substrate, S6K, resulted in a decrease in the invasive property of H1299 cells. Our results provide preliminary evidence that Redd1 inhibits the invasive activity of NSCLC cells via suppression of the mTOR downstream pathway.

Multidrug resistance-associated protein (Mrp) 2-deficient (TR(-)) Wistar rats have been used to elucidate the role of Mrp2 in drug disposition. Decreased breast cancer resistance protein (Bcrp) levels were reported in sandwich-cultured hepatocytes (SCH) from TR(-) rats compared with those from wild-type (WT) rats. This study was designed to characterize hepatic Bcrp expression and function in TR(-) rats, using nitrofurantoin and pitavastatin as substrates. Bcrp was knocked down by RNA interference in rat SCH. Antibody BXP53, but not BXP21, specifically detected Bcrp knockdown in SCH. Bcrp protein levels were decreased markedly in TR(-) but not Mrp2-deficient Sprague-Dawley [Eisai hyperbilirubinemic rats (EHBR)] rats. Bcrp mRNA levels were decreased significantly in TR(-) livers as determined by TaqMan real-time reverse transcriptase-polymerase chain reaction. Biliary excretion of nitrofurantoin, a specific Bcrp substrate, was decreased significantly in SCH and isolated perfused livers from TR(-) rats compared with those from WT controls, indicating that hepatic Bcrp function is decreased in TR(-) rats. In Bcrp knockdown SCH, the biliary excretion index and in vitro biliary clearance of pitavastatin were decreased significantly to ∼ 58 and ∼ 52% of control, respectively, indicating that Bcrp plays a role in pitavastatin biliary excretion. Pitavastatin biliary excretion was decreased significantly in perfused livers from TR(-) compared with those from WT rats. In conclusion, expression and function of hepatic Bcrp are decreased significantly in TR(-) rats. The potential role of both Bcrp and Mrp2 should be considered when data generated in TR(-) rats are interpreted. TR(-) and EHBR rats in combination may be useful in differentiating the role of Mrp2 and Bcrp in drug/metabolite disposition.

Inversion of chromosome 16 [inv(16)(p13.1q22)] and t(16;16)(p13.1;q22) are associated with acute myelomonocytic leukemia (AMML) with eosinophilia and a favorable prognosis. On the other hand, patients with del(16)(q22) usually present with MDS or chronic myelomonocytic leukemia (CMML), which can evolve to AMML without eosinophilia, and this chromosomal aberration is associated with older age, a complex karyotype, and a poor prognosis. We report a case of AML with del(16)(q22) which showed a complex karyotype, absence of eosinophilia in bone marrow study and a poor response to chemotherapy.

Sulindac is a commonly used nonsteroidal anti-inflammatory drug. This study tested the hypothesis that sulindac-mediated drug-drug interactions and/or hepatotoxicity may be caused, in part, by inhibition of proteins responsible for the hepatic transport of drugs and/or bile acids by sulindac and/or sulindac metabolites [sulindac sulfone (S-sulfone) and sulindac sulfide (S-sulfide)]. The uptake and excretion of model substrates, [(3)H]taurocholate (TC), [(3)H]estradiol 17-beta-glucuronide (E217G), and nitrofurantoin (NF), were investigated in rat and human suspended and sandwich-cultured hepatocytes (SCH). In suspended rat hepatocytes, S-sulfone and S-sulfide inhibited Na(+)-dependent TC initial uptake (IC(50) of 24.9 +/- 6.4 and 12.5 +/- 1.8 microM, respectively) and Na(+)-independent E217G initial uptake (IC(50) of 12.1 +/- 1.6 and 6.3 +/- 0.3 microM, respectively). In rat SCH, sulindac metabolites (100 microM) decreased the in vitro biliary clearance (Cl(biliary)) of TC, E217G, and NF by 38 to 83%, 81 to 97%, and 33 to 57%, respectively; S-sulfone and S-sulfide also decreased the TC and NF biliary excretion index by 39 to 55%. In suspended human hepatocytes, S-sulfone and S-sulfide inhibited Na(+)-dependent TC initial uptake (IC(50) of 42.2 and 3.1 microM, respectively); S-sulfide also inhibited the TC Cl(biliary) in human SCH. Sulindac/metabolites markedly inhibited hepatic uptake and biliary excretion of E217G by 51 to 100% in human SCH. In conclusion, sulindac and metabolites are potent inhibitors of the uptake and biliary clearance of bile acids in rat and human hepatocytes and also inhibit substrates of rat breast cancer resistance protein, rat and human organic anion-transporting polypeptides, and human multidrug resistance-associated protein 2. Inhibition of multiple hepatic transport proteins by sulindac/metabolites may play an important role in clinically significant sulindac-mediated drug-drug interactions and/or liver injury.

Tetraploidy or near-tetraploidy is a rare cytogenetic abnormality found in AML, and is divided into primary and secondary forms. The secondary tetraploidy or near-tetraploidy found in AML is known to be specifically associated with t(8;21). In this case report, FISH analysis detected RUNX1-RUNX1T1 gene rearrangement in the absence of cytogenetic abnormality of t(8;21), which suggests the presence of unvailed t(8;21). This is the first case report of tetraploidy or near-tetraploidy AML with cryptic RUNX1/RUNX1T1 in Korea. Although the prognosis of tetraploidy or near- tetraploidy with t(8;21) is known to be poor, this patient shows a relatively good clinical course compared to other reported cases.

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) seems almost inevitable, even in patients with lung cancer that initially respond well to EGFR-TKIs. MET amplification was recently found to be a mechanism of escape from the anticancer effect of EGFR inhibitors. In the present study, we investigated the means whereby MET affects sensitivity to EGFR-TKIs in PC-9 cells. Gefitinib- or erlotinib-resistant sublines were established by exposing the parental PC-9 cell line to chronic, repeated treatments with these drugs. These resistant sublines showed more than 100-fold more resistance to gefitinib and erlotinib and acquired cross-resistance to other EGFR-TKIs. The T790M EGFR mutation was found by pyrosequencing, and this seemed to be the cause of drug resistance. Resistant cells also showed MET activation, although gene amplification was not detected. Furthermore, the induction of MET activity was not found to be associated with sensitivity to EGFR-TKIs. Interestingly, increased passage number without exposure to gefitinib or erlotinib caused MET activation, but this did not affect sensitivity to EGFR-TKIs. In addition, hepatocyte growth factor was found to block the ability of EGFR-TKIs to inhibit MET activation. However, sustained MET activation by hepatocyte growth factor did not modulate the cellular effects of gefitinib or erlotinib. Rather, activated MET enhanced migration and invasion abilities. Summarizing, MET activation may be acquired during cancer cell proliferation and enhances migratory and invasive abilities without affecting cellular sensitivity to EGFR-TKIs. Accordingly, the present study suggests that MET activation caused by factors other than MET gene amplification is not a suitable surrogate marker of resistance to EGFR-TKIs. (Mol Cancer Res 2009;7(10):1736–43)

Breast cancer resistance protein (BCRP, ABCG2) is expressed in the hepatic canalicular membrane and mediates biliary excretion of xenobiotics including sulfate and glucuronide metabolites of some compounds. Hepatic Bcrp expression is sex-dependent, with higher expression in male mice. The hypothesis that sex-dependent Bcrp expression influences the hepatobiliary disposition of phase II metabolites was tested in the present study using acetaminophen (APAP) and the generated APAP glucuronide (AG) and sulfate (AS) metabolites in single-pass in situ perfused livers from male and female wild-type and Abcg–/– (Bcrp-deficient) mice. Pharmacokinetic modeling was used to estimate parameters governing the hepatobiliary disposition of APAP, AG, and AS. In wild-type mice, the biliary excretion rate constant was 2.5- and 7-fold higher in males than in females for AS and AG, respectively, reflecting male-predominant Bcrp expression. Sex-dependent differences in AG biliary excretion were not observed in Bcrp-deficient mice, and AS biliary excretion was negligible. Interestingly, sex-dependent basolateral excretion of AG (higher in males) and AS (higher in females) was noted in wild-type mice with a similar trend in Bcrp-deficient mouse livers, reflecting an increased rate constant for AG formation in male and AS formation in female mouse livers. In addition, the rate constant for AS basolateral excretion was increased significantly in female mouse livers compared with that in male mouse livers. It is interesting to note that multidrug resistance-associated protein 4 was higher in female than in male mouse livers. In conclusion, sex-dependent differences in conjugation and transporter expression result in profound differences in the hepatobiliary disposition of AG and AS in male and female mouse livers.

Epithelial to mesenchymal transition (EMT) has been reported to be related with reduced sensitivity to EGFR tyrosine kinase (EGFR-TK) inhibitors. We performed this study to investigate whether this phenomenon would play a role in acquired resistance to gefitinib. In this study, we established a gefitinib-resistant subline (A549/GR), which was derived from the parental A549 cell line by chronic, repeated exposure to gefitinib. Compared with the A549 cells, the A549/GR cells were approximately 7.7-fold more resistant to gefitinib and they showed the cross-resistance against other EGFR-TK inhibitors, including CL-387,758, erlotinib and ZD6478. Phenotypic changes such as a spindle-cell shape and increased pseudopodia formation suggesting EMT was present in the A549/GR cells. These changes were accompanied by a decrease of E-cadherin and an increase of vimentin, which is a mesenchymal marker. In addition, the ability of invasion and migration was increased in the A549/GR cells. TGF-beta1 treatment for 72 h also induced EMT in the A549 cells and this transition led to resistance to gefitinib. Conversely, this was reversed through the removal of TGF-beta1. In conclusion, induction of EMT may contribute to the decreased efficacy of therapy in primary and acquired resistance to gefitinib.

이지원∙이진경∙홍영준∙홍석일∙장윤환 원자력병원 진단검사의학과 Background : Immunoglobulin heavy chain (IGH ) gene rearrangement, 13q14 deletion and trisomy 1q are frequently observed in Korean patients with multiple myeloma. The purpose of our study was to analyze the statistical correlation between chromosomal aberrations and routine laboratory test results as prognostic markers and to evaluate the potential of chromosomal aberrations for the indirect assessment of prognosis in multiple myeloma patients. Methods : We investigated the prevalence of cytogenetic aberrations in 41 patients with newly diagnosed multiple myeloma. Cytogenetic analysis was conducted by conventional karyotyping and FISH for the presence of IGH/CCND1 translocation, 13q14 deletion, and trisomy 1q using bone marrow aspirates. The records of routine laboratory tests were reviewed and their correlation with cytogenetic abnormalities was investigated. Results : Sixteen (39.0%) of 41 patients had at least one cytogenetic abnormalities in conventional karyotyping or FISH. In FISH analysis of 37 patients, 8 (21.6%) showed positive result for IGH/CCND1 translocation, 8 (21.6%) for trisomy 1q, and 5 (13.5%) for 13q14 deletion. Cytogenetic abnormalities, especially trisomy 1q, were associated with significantly lower hemoglobin level and significantly higher bone marrow plasma cell percentage and β 2-microglobulin level. Conclusions : Statistical correlation between the presence of trisomy 1q and prognostic markers suggests that the evaluation of trisomy 1q in multiple myeloma patients may be used for the indirect assessment of prognosis in these patients. (Korean J Lab Med 2008;28:413-8)

Silymarin, an extract from seeds of Silybum marianum, is used by 8 to 33% of patients to self-treat chronic viral hepatitis C in the United States. Studies in humans and rodents suggest that biliary excretion of glucuronide and sulfate conjugates is the major route for silymarin's elimination. To determine the role of multidrug resistance-associated protein 2 (Mrp2) (Abcc2) in the biliary excretion of silymarin, the hepatobiliary disposition of the six major silymarin flavonolignans was studied using isolated perfused livers (IPRLs) from wild-type (WT) and Mrp2-deficient (TR(-)) Wistar rats. For all the flavonolignans, approximately 96% of the dose was cleared from perfusate within 30 min in both WT and TR(-) livers, and

This study aimed to assess the antimicrobial efficacy of a novel polymeric chlorhexidine-controlled release device as an intracanal medicament. One hundred cylindrical dentin blocks prepared from human single-rooted teeth were inoculated with Enterococcus faecalis for 3 weeks. The intracanal medicaments tested were calcium hydroxide, a polymeric chlorhexidine-controlled release device (PCRD), a polymeric controlled release device without chlorhexidine (CHx), 0.2% CHx solution, and sterile saline. Dentin samples (at 200-mum and 400-mum depths) were collected from the medicated canal lumens after 1 week of medication with sterile LightSpeed files and placed in growth medium. Bacterial growth was assessed spectrophotometrically by analysis of optical density (OD) after 24 hours of incubation. The OD values at both depths were significantly lower in the PCRD group than in the other experimental groups (P < .001). These results indicate that a PCRD can be an effective intracanal medicament against E. faecalis.

Summary Diffuse bronchiolo-alveolar cell carcinoma (BAC) with endolymphatic tumor emboli involving whole lung fields was found in a 25-year-old male. He had a history of 6 pack years of smoking. After a thoracoscopic lung biopsy, his condition was aggravated leading to imminent respiratory failure. Although gefitinib is not usually recommended as a first-line chemotherapeutic drug, we decided to use it considering that the added toxicity of conventional drugs would be fatal to him. Surprisingly, the patient showed the dramatic improvement and was able to exercise 1 week after the treatment. The immunostainings for p-Akt and E-cadherin were strongly positive. A deletion mutation (delE746–750) was detected in exon 19 by sequencing. The fluorescent in situ hybridization (FISH) was negative as low trisomy suggesting that the epidermal growth factor receptor (EGFR) mutation is a more reliable finding for prediction of the response to this novel drug, even in a patient with unfavorable clinical characteristics such as male gender and smoker. An EGFR–tyrosine kinase inhibitor (EGFR–TKI), either alone or combined with other chemotherapeutic drugs, should be considered an initial therapeutic option in patients with disseminated lung cancer requiring urgent treatment.

Spinal metastatic disease is characterised by the preservation of the intervertebral disc structure, even after severe destruction of the vertebral body by neoplastic tissues. Anatomical features of the discs are thought to be the reason for the disc’s resistance to metastatic cancer. However, little is known about the biochemical mechanism to prevent or attenuate the local invasion of cancer cells into the discs. The purpose of this study was to investigate the hypothesis that Fas ligand (FasL) produced by nucleus pulposus cells can kill Fas-expressing cancer cells infiltrating into the discs by the activation of caspases. Fas-expressing MCF-7 breast cancer cells were cultured with (experimental group) and without (control group) supernatant of nucleus pulposus cells containing FasL (50 pg/ml) for 48 h. The apoptosis of MCF-7 breast cancer cells was determined by the TUNEL technique. In addition, the activation of caspase-8, -9 and -3 was investigated by Western blot analysis. After treatment with supernatant of the nucleus pulposus cells containing FasL, the apoptosis of MCF-7 breast cancer cells was significantly increased, along with the activation of caspase-8, -9 and -3 compared with those of the control group. Our results suggest that the Fas/FasL interaction of nucleus pulposus and cancer cells might be a potential mechanism of the disc’s resistance to metastatic cancer.

Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H+ ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC.

Background: Apoptosis is thought to be a critical component of disc degeneration. Two main pathways of Fas-mediated apoptosis have been identified: Type I, which is the death-inducing signaling complex pathway, and Type II, which is the mitochondrial pathway. The apoptotic pathway for anulus fibrosus cells, which is phenotypically different from that of nucleus pulposus cells, has not been elucidated to our knowledge. The ultimate initiators or executioners of apoptosis are caspases. There are also inhibitors of caspases, which have the potential of being used as anti-apoptotic therapeutic agents. We therefore undertook this study to determine (1) the apoptotic pathway of anulus fibrosus cells and (2) the anti-apoptotic potential of caspase inhibitors. Methods: Rat anulus fibrosus cells were isolated, cultured, and placed in either 0% (apoptosis-promoting condition) or 10% (normal control) fetal bovine serum. We identified and quantified the presence of apoptotic cell death, caspase activities, and loss of mitochondrial membrane potential. In addition, we examined the cells for the expression of Fas, procaspases, and cytochrome-c. Finally, we analyzed the degree of anti-apoptotic effects of caspase inhibitors on the cells in 1% fetal bovine serum. Results: The percentage of apoptosis and Fas expression in the cells incubated in 0% fetal bovine serum were increased compared with those in the cells incubated in 10% fetal bovine serum (both p < 0.001). Caspase-8, 9, and 3 activities were increased and expression of procaspases was decreased in the 0% fetal bovine serum compared with those in the 10% fetal bovine serum (all p < 0.001). In contrast, the loss of mitochondrial membrane potential and cytochrome-c release into the cytosol were unchanged in the 0% fetal bovine serum. Pancaspase and caspase-8 inhibitors reduced apoptotic cell death (p < 0.001 and p < 0.05, respectively), but caspase-9 inhibitor did not reduce apoptotic cell death. Conclusions: Our results suggest that, unlike nucleus pulposus cells, anulus fibrosus cells are Fas Type-I cells, which undergo apoptosis through the death-inducing signaling complex. We also found that apoptosis of intervertebral disc cells can be attenuated by caspase inhibitors. Clinical Relevance: Caspase inhibitors may play a therapeutic role in slowing disc degeneration that is due to inappropriate or excessive apoptosis of intervertebral disc cells.

A new virus with a dsRNA genome was isolated and characterized from the Suhan-:neutari strain (ASI 2504) of Pleurotus ostreatus, which was characterized as long and slightly bent with small caps on the stipe of fruit body. Thirty nm isometric viruses with three dsRNA segments (approximately 2.0, 1.84 and 1.82 kb in sizes) were isolated by ultracentrifugation in sucrose gradients. Western analysis of protein extracted purified viruses with anti-virus polyclonal antibody confirmed that viruses have two specific proteins (36 and 68 kDa). Computer analysis of 2.0 kb segment shows that high. sequence identity with RNA-dependent RNA polymerase (RdRp) of partitiviruses, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis, OMDV was most related to Pleurotus ostreatus virus 1.

Yuzu (Cittus junos) trees were examined from six geographically separate provinces in the Republic of Korea, including four islands (Geoje, Namhae, Wan, and Jeju), 1 peninsula (Goheung), and 1 inland area (Boseong). The population of sequence variants of citrus tristeza virus (CTV) was isolated and analyzed by single-strand conformation polymorphism (SSCP) analysis of cDNA from thep20 gene. SSCP profiles of 65 PCR products showed different band patterns but with similar intensities. Sixteen haplotypes were subgrouped according to their SSCP profiles and severity of symptoms. Their genomes were sequenced and compared. DNA analysis of thep20 genes revealed nucleotide identities ranging from 88-99.8%. Based on SSCP analysis, the pathologically mild isolates of CTV yielded two to three DNA bands, whereas the most virulent isolates contained more than two bands. Comparisons of these physically separate haplotypes suggest that CTV isolates with multiple SSCP profiles could have arisen as a result of a mixed infection and genetic recombination of two divergent isolates. Plants with severe disease symptoms, such as stem pitting, closely corresponded to a CTV strain showing typical SSCP profiles in Florida (USA) and Japan.

Piperlongumine, a natural alkaloid isolated from the long pepper, selectively increases reactive oxygen species production and apoptotic cell death in cancer cells but not in normal cells. However, the molecular mechanism underlying piperlongumine-induced selective killing of cancer cells remains unclear. In the present study, we observed that human breast cancer MCF-7 cells are sensitive to piperlongumine-induced apoptosis relative to human MCF-10A breast epithelial cells. Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1). Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells. However, knockdown of HO-1 expression and pharmacological inhibition of its activity abolished the ability of piperlongumine to induce apoptosis in MCF-7 cells, whereas those promoted apoptosis in MCF-10A cells, indicating that HO-1 has anti-tumor functions in cancer cells but cytoprotective functions in normal cells. Moreover, it was found that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell apoptosis are not dependent on the generation of reactive oxygen species. Instead, piperlongumine, which bears electrophilic α,β-unsaturated carbonyl groups, appears to inactivate Kelch-like ECH-associated protein-1 (Keap1) through thiol modification, thereby activating the Nrf2/HO-1 pathway and subsequently upregulating HO-1 expression, which accounts for piperlongumine-induced apoptosis in cancer cells. Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.

The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes.

The PI3K/Akt/mTOR axis in lung cancer is frequently activated and implicated in tumorigenesis. Specific targeting of this pathway is therefore an attractive therapeutic approach for lung cancer. However, non-small cell lung cancer cells are resistant to BEZ235, a dual inhibitor of PI3K and mTOR. Interestingly, blockage of Stat3 with a selective inhibitor, S3I-201, or siRNA dramatically sensitized the BEZ235-induced cell death, as evident from increased PARP cleavage. Furthermore, inhibition of Stat3 led to enhancement of cell death induced by LY294002, a PI3K inhibitor. Treatment of cells with a combination of BEZ235 and S3I-201 significantly induced the proapoptotic transcription factor, CHOP, and its targets, Bim and DR4. Knockdown of CHOP or Bim suppressed cell death stimulated by the combination treatment, implicating the involvement of these BEZ235/S3I-201-induced factors in pronounced cell death. Moreover, the BEZ235/S3I-201 combination enhanced TRAIL-induced cell death. Our results collectively suggest that blockage of Stat3 presents an effective strategy to overcome resistance to PI3K/Akt/mTOR inhibition.

Study design The concentration of transforming growth factor-beta 1 (TGF-beta1) was examined in the ligamentum flavum of lumbar spinal stenosis and disc herniation. Objective To investigate the role of TGF-beta1 on hypertrophy of the ligamentum flavum in lumbar spinal stenosis compared with that of lumbar disc herniation. Summary of background data The hypertrophy of the ligamentum flavum is known to be related to degenerative changes that are secondary to the aging process or mechanical instability. However, there has been no study to investigate the effect of biochemical factors, such as growth factors, associated with hypertrophy of the ligamentum flavum. Methods The concentrations of TGF-beta1 were analyzed in the surgically obtained ligamentum flavum specimens from lumbar spinal stenosis (n = 10; mean age 62.8 years) and disc herniation (n = 10; mean age 35.6 years) by enzyme-linked immunosorbent assay. The localization of TGF-beta1 within the ligamentum flavum was determined using immunohistochemical study. The thickness of the ligamentum flavum was measured with axial T1-weighted magnetic resonance imaging. The biochemical and radiologic results were compared for these two conditions. Results The mean concentration of TGF-beta1 was 123.78 pg/100 microg protein (range 11-374 pg/100 microg protein) in lumbar spinal stenosis and 38.56 pg/100 microg protein (range 0-155 pg/100 microg protein) in lumbar disc herniation; the difference between lumbar spinal stenosis and disc herniation was statistically significant (P = 0.029). The mean thickness of the ligamentum flavum was 4.44 mm (range 3.4-5.4 mm) in lumbar spinal stenosis and 2.44 mm (range 1.8-4.0 mm) in lumbar disc herniation; the difference between lumbar spinal stenosis and disc herniation was statistically significant (P = 0.001). On immunohistochemical study TGF-beta1 was positively stained on the fibroblasts within the ligamentum flavum specimens. Conclusion The current results suggest that higher expression of TGF-beta1 by fibroblasts might be related to the development of hypertrophy of the ligamentum flavum in lumbar spinal stenosis.

Aminoalcoholphosphotransferase catalyzes the synthesis of phosphatidyicholine and phosphatidylethanolamine from diacylglycerol plus CDP-choline or CDP-ethanolamine as the phosphobase donor. We screened a cDNA library to isolate a clone for use in studying the structure and expression pattern of this enzyme fromPimpinella brachycarpa. TheP. brachycarpa aminoalcoholphosphotransferase cDNA contains an open reading frame of 1,170 bp coding for a protein of 389 amino acids. The deduced amino acid sequence shares over 90% similarity with other aminoalcoholphosphotransferase sequences. Hydropathy profile analysis suggests that the secondary structure ofP. brachycarpa aminoalcoholphosphotransferase is very similar to that of soybean and Chinese cabbage enzymes, having an overall hydrophobicity and the same number of predicted transmembrane helices. The catalytic domain contains the CDP-alcohol phosphotransferase motif with two aspartate residues. Reverse transcriptase-PCR analysis indicates that the expression ofP. brachycarpa AAPT is regulated by temperature.

Introduction The aggregation of mixed bacterial flora into sessile biofilms on root canal surfaces can be one of the causes of persistent apical periodontitis. The aim of this study was to evaluate the antibacterial efficacy of human β-defensin-3 (HBD3) peptide on multispecies biofilms by using confocal laser scanning microscopy. Methods Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were cultured in a peptone-yeast-glucose broth, and their culture suspensions were combined in equal proportions. The mixed bacteria were inoculated on sterile coverslips placed into the wells of tissue culture plates to permit the formation of mixed species biofilm. After incubation for 3 weeks, the samples were treated for 24 hours with saline (control), saturated calcium hydroxide solution (CH), 2% chlorhexidine solution (CHX), and 50 μg/mL HBD3 solution. A commercial biofilm/viability assay kit was used to assess cell viability and analyze the 3-dimensional architecture of biofilms. The percentage of dead cells was determined from the ratio of biovolumes for the red subpopulation and the total biofilm. Results Three medication groups showed a significant reduction of biovolume within the biofilms compared with the control group (P .05). Conclusions HBD3 peptide exhibited more antibacterial activity against mature multispecies biofilms in vitro than either CH or CHX. © 2013 American Association of Endodontists.

Purpose: The measurement of serum human epidermal growth factor receptor 2 (HER2) extracellular domain levels is a wellestablished method for evaluating whether a metastatic HER2positive breast cancer patient will respond to HER2-targeted treatment. However, little is known about the value of serum HER2 for detecting disease relapse following curative surgical treatment in breast cancer patients. The purpose of this study was to evaluate the sensitivity of serum HER2, carcinoembryonic antigen (CEA), and carcinoma antigen 15-3 (CA 15-3) for the detection of disease recurrence in postoperative breast cancer patients with a primary HER2-positive tumor. Methods: Serial measurements were taken of serum HER2, CEA, and CA 15-3 levels in patients with primary invasive HER2-positive breast cancer who underwent curative surgical treatment between January 2008 and December 2010. Following treatment, serum HER2 levels were monitored every 6 months using a chemiluminescence immunoassay. Results: Overall, 264 patients were analyzed in this retrospective study. The median follow-up period was 27.7 months, and 24 patients relapsed during follow-up. The sensitivity of serum HER2, CEA, and CA 15-3 for the detection of disease recurrence was 37.5%, 25.1%, and 12.5%, respectively. Sensitivity increased to 45.8% when all three tumor markers were combined in the analysis. In a subgroup of patients without liver disease, the sensitivity of serum HER2, CEA, and CA 15-3 was 57.1%, 21.4%, and 14.3%, respectively. Of the 264 patients in this study, 80 patients had chronic hepatitis, liver cirrhosis, or abnormal aspartate aminotransferase or alanine aminotransferase levels during the follow-up period. Following the exclusion of these patients, the sensitivity of serum HER2 for the detection of disease recurrence increased to 57.1%. Conclusion: Serial serum HER2 measurement may be useful for the detection of disease relapse in patients with HER2-positive breast cancer. Abnormal liver function can result in elevated serum HER2 in the absence of disease recurrence.

Sorafenib is an orally active tyrosine kinase inhibitor used in the treatment of renal and hepatocellular carcinoma. This study was designed to establish whether transport proteins are involved in the hepatic uptake of sorafenib and to determine the extent of biliary excretion of sorafenib and its metabolites in human hepatocytes. Initial uptake was assessed in freshly isolated, suspended human hepatocytes in the presence of inhibitors and modulators. [(14)C]Sorafenib (1 µM) uptake at 4°C was reduced by about 61-63% of the uptake at 37°C, suggesting a high degree of passive diffusion. Hepatocyte uptake of [(14)C]sorafenib was not Na(+) dependent or influenced by the organic anion transporter 2 inhibitor ketoprofen. However, initial [(14)C]sorafenib hepatocyte uptake was reduced by 46 and 30% compared with control values in the presence of the organic anion transporting polypeptide inhibitor rifamycin SV and the organic cation transporter (OCT) inhibitor decynium 22, respectively. [(14)C]Sorafenib (0.5-5 µM) uptake was significantly higher in hOCT1-transfected Chinese hamster ovary cells compared with mock cells, and inhibited by the general OCT inhibitor, 1-methyl-4-phenylpryidinium. OCT1-mediated uptake was saturable with a Michaelis-Menten constant of 3.80 ± 2.53 µM and a V(max) of 116 ± 42 pmol/mg/min. The biliary excretion index and in vitro biliary clearance of sorafenib (1 µM) in sandwich-cultured human hepatocytes were low (∼11% and 11 ml/min/kg, respectively). Results suggest that sorafenib uptake in human hepatocytes occurs via passive diffusion, by OCT1, and by organic anion transporting polypeptide(s). Sorafenib undergoes modest biliary excretion, predominantly as a glucuronide conjugate(s).

S6 kinase 1 (S6K1) was suggested to be a marker for endocrine therapy resistance in breast cancer. We examined whether tamoxifen’s effect can be modulated by S6K1 inhibition. S6K1 inhibition by PF4708671, a selective inhibitor of S6K1, acts synergistically with tamoxifen in S6K1-high MCF-7 cells. Similarly, the knockdown of S6K1 with small interfering RNA (siRNA) significantly sensitized MCF-7 cells to tamoxifen. Inhibition of S6K1 by PF4708671 led to a marked decrease in the expression levels of the anti-apoptotic proteins Mcl-1 and survivin, which was not related to mRNA levels. In addition, suppression of Mcl-1 or survivin, using specific siRNA, further enhanced cell sensitivity to tamoxifen. These results showed that inhibition of S6K1 acts synergistically with tamoxifen, via translational modulation of Mcl-1 and survivin. Based on these findings, we propose that targeting S6K1 may be an effective strategy to overcome tamoxifen resistance in breast cancer.

Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.

Individuals who traveled to contaminated areas after the Fukushima nuclear accident have concerns about the health effects. However, medical follow-up for any adverse health effects will be difficult without personal dose measurements. Cytogenetic biodosimetry is a reasonable method of assessing absorbed doses retrospectively. We analyzed dicentric chromosomes for 265 Fukushima travelers, mostly journalists and rescue workers, who had been dispatched to northeastern Japan during the nuclear emergency. As a control group, 37 healthy volunteers who had not visited Japan since the accident were enrolled. Yields of dicentrics and absorbed doses calculated from a dose-response calibration curve for travelers and the control group were compared. The cut-off level for dicentric chromosomes in the controls was 3.5 per 1000 cells. Of the 265 travelers, 31 had elevated numbers of dicentrics (High-Dics group) while 234 were below the cut-off (Normal-Dics group). All but one of the individuals in the High-Dics group also reported a significantly higher number of medical exposures to radiation within the past three years compared with the Normal-Dics or control groups. The 225 travelers with no history of medical exposure showed no difference of dicentrics yield compared to the control group. Our data indicate that Fukushima travel alone did not enhance the yield of dicentrics.

We performed this retrospective study to assess the association of epidermal growth factor receptor (EGFR) with metastatic presentations in advanced non-small cell lung cancer (NSCLC). The data from 125 patients with stage III or IV NSCLC were analyzed. We detected EGFR mutations in 36 NSCLC patients. EGFR mutations were predominant in never-smokers ( 𝑃 < . 0 0 1 ) , patients with adenocarcinomas ( 𝑃 < . 0 0 1 ) , and female patients ( 𝑃 < . 0 0 1 ) . When the metastatic sites were analyzed, pleural metastases were associated with a high incidence of EGFR mutations ( 𝑃 = . 0 2 8 ) . Particularly, pleural metastases with minimal effusion (PMME) were associated with EGFR mutational status ( 𝑃 = . 0 0 1 ) . Patients with N3 lesions were less likely to harbor EGFR mutations ( 𝑃 = . 0 3 3 ) . On multivariate analysis, N3 lesions ( 𝑃 = . 0 1 7 ) and PMME ( 𝑃 < . 0 0 1 ) remained significant factors for EGFR mutations. EGFR mutations may be associated with different presentations of pleural and N3 nodal metastases.

Aims and backgroundColorectal cancer (CRC) is one of the commonest malignant disorders and frequently associated with high expression of epidermal growth factor receptor (EGFR), resulting in advanced disease and a poor prognosis. In this study, we investigated the radiosensitizing effects of the selective EGFR inhibitor cetuximab in human CRC cell lines.MethodsFour human CRC cell lines, CaCo-2, HCT-8, LoVo, and WiDr, were treated with cetuximab and/or radiation. The effects on cell proliferation and viability were measured by MTT and annexin-V staining, and clonogenic survival assay. The in vivo effect on the growth of CRC xenografts was assessed in athymic nude mice.ResultsCetuximab in combination with radiation significantly inhibited the in vitro proliferation of CRC cells, with a concomitant increase in cell death, except in WiDr cells. Clonogenic survival assay confirmed that cetuximab worked as a radiosensitizer in three cetuximab-sensitivie CRC cells. However, no correlations were found between the radiosensitivity and EGFR expression level or mutation status of EGFR signaling molecules. In nude mice bearing CRC cell xenografts, cetuximab plus radiation significantly inhibited the tumor growth over either agent alone. Interestingly, the WiDr xenograft was also sensitive to cetuximab and/or radiation in vivo, suggesting host-mediated effects of cetuximab.ConclusionsCetuximab enhanced the radiosensitivity of CRC cells in vitro and efficiently inhibited xenograft tumor growth. This study provided a rationale for the clinical application of the selective EGFR inhibitor cetuximab in combination with radiation in CRC. Free full text available at www.tumorionline.it

A polymerase chain reaction-restriction fragment mass polymorphism (PCR-RFMP) assay protocol using PGMY09/11 primers for the detection and identification of human papillomavirus (HPV) has recently been developed. The present study evaluated the analytical sensitivity and clinical utility of HPV genotyping employing PCR-RFMP as compared to direct sequencing. Serial dilutions of cloned HPV DNA were analyzed in order to assess the limit of detection (LOD) and three sets of HPV clone mixtures (types 16+18, 16+11 and 18+11) were used to assess the accuracy of the genotyping assays. For 423 cervical specimens that were cytologically categorized as normal or cancer, the concordance between the two assays was evaluated. Clinical sensitivity was calculated by evaluating 101 histologically confirmed cases. The PCR-RFMP HPV assay had a lower LOD and 100% accuracy when detecting double HPV infection. Agreement between the two assays upon 423 clinical specimens was 91.0% with a κ-value of 0.86. The incidence of multiple HPV infections among HPV-positive patients was 19.0% by PCR-RFMP and 5.4% by sequencing. The clinical sensitivity of PCR-RFMP and sequencing was 92% and 84%, respectively. In conclusion, the PCR-RFMP assay for HPV genotyping correlated well with direct sequencing, provides high analytical and clinical sensitivity, and is advantageous in the detection of multiple HPV infections.