DNA vaccine elicits an efficient antitumor response by targeting the mutant Kras in a transgenic mouse lung cancer model.

Mutant Kras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is observed in more than 20% of non-small-cell lung cancers; however, no effective Kras target therapy is available at present. The Kras DNA vaccine may represent as a novel immunotherapeutic agent in lung cancer. In this study, we investigated the antitumor efficacy of the Kras DNA vaccine in a genetically engineered inducible mouse lung tumor model driven by Kras(G12D). Lung tumors were induced by doxycycline, and the therapeutic effects of Kras DNA vaccine were evaluated with delivery of Kras(G12D) plasmids. Mutant Kras(G12D) DNA vaccine significantly decreased the tumor nodules. A dominant-negative mutant Kras(G12D)N17, devoid of oncogenic activity, achieved similar therapeutic effects. The T-helper 1 immune response was enhanced in mice treated with Kras DNA vaccine. Splenocytes from mice receiving Kras DNA vaccine presented an antigen-specific response by treatment with peptides of Kras but not Hras or OVA. The number of tumor-infiltrating CD8(+) T cells increased after Kras vaccination. In contrast, Kras DNA vaccine was not effective in the lung tumor in transgenic mice, which was induced by mutant L858R epidermal growth factor receptor. Overall, these results indicate that Kras DNA vaccine produces an effective antitumor response in transgenic mice, and may be useful in treating lung cancer-carrying Ras mutation.

Prognostic factors in pathological stage IB nonsmall cell lung cancer greater than 3 cm.

Significant heterogenity of stage IB (sixth edition of the TNM staging system) nonsmall cell lung cancer (NSCLC) has been identified, and further subclassification according to tumour size has been proposed. The aim of this study is to evaluate the prognostic factors in patients with resected stage IB NSCLC > 3 cm. From January 1980 to December 2000, 525 patients underwent surgical resection for stage IB NSCLC > 3 cm at Taipei Veterans General Hospital, Taipei, Taiwan. The clinicopathological characteristics of these patients were retrospectively reviewed. The 5- and 10-yr overall survival rates were 44.9% and 27.3%, respectively. Age (p < 0.001), tumour size (p = 0.002), extent of pulmonary resection (p = 0.002), histological type (p = 0.005) and number of mediastinal lymph nodes dissected/sampled (p = 0.004) were significant predictors for overall survival in multivariate analysis. Patients with tumour size >7 cm, or > 5 to ≤ 7 cm, had a worse survival than those with tumour size > 3 to ≤ 5 cm. However, visceral pleural invasion did not influence overall survival. Stage IB NSCLC with a diameter > 3 cm may be subclassified according to tumour size regardless of visceral pleural invasion.

Mercury distribution and speciation in two lagoons with different pollution and eutrophication conditions in Taiwan.

This study explored biogeochemical processes controlling the distribution of mercury (Hg) species in two lagoons with different pollution and eutrophication conditions in southwestern Taiwan. The eutrophication and pollution levels were higher in the Dapeng Bay than in the Chiku Lagoon, engendering a higher particulate Hg concentration and enrichment factor in the Dapeng Bay. The concentration range of total dissolved Hg (HgTD) and reactive Hg (HgR) was comparable between the lagoons, but the concentration of particulate Hg (HgP) was higher in the Dapeng Bay. HgR and HgTD abundance was primarily controlled by the availability of dissolved oxygen (DO) and biological absorption. In addition to pollution (which elevated HgP concentration), biological absorption and/or adsorption rather than lithogenic processes more likely regulated the HgP concentration. The effect of Hg pollution may superimpose on that of DO on the distributions of HgR and HgTD and may enhance HgP formation in the Dapeng Bay.

Prognostic significance of hypoxia-inducible factor-1alpha, TWIST1 and Snail expression in resectable non-small cell lung cancer.

BACKGROUND: Metastasis is the most common cause of disease failure and mortality for non-small cell lung cancer (NSCLC) after surgical resection. Snail and TWIST1 are epithelial-mesenchymal transition (EMT) regulators which induce metastasis. Intratumoral hypoxia followed by stabilisation of hypoxia-inducible factor 1alpha (HIF-1alpha) promotes metastasis through regulation of certain EMT regulators. The aim of this study was to evaluate the prognostic value of HIF-1alpha, TWIST1 and Snail expression in patients with resectable NSCLC. METHODS: A retrospective analysis of 87 patients with resectable NSCLC from Taipei Veterans General Hospital between 2003 and 2004 was performed using immunohistochemistry to analyse HIF-1alpha, TWIST1 and Snail expression. The association between HIF-1alpha, TWIST1 and Snail expression and patients' overall and recurrence-free survivals was investigated. RESULTS: Overexpression of HIF-1alpha, TWIST1 or Snail was shown in 32.2%, 36.8% and 55.2% of primary tumours, respectively. Overexpression of HIF-1alpha, TWIST1 or Snail in primary NSCLCs was associated with a shorter overall survival (p = 0.005, p = 0.026, p = 0.009, respectively), and overexpression of HIF-1alpha was associated with a shorter recurrence-free survival (p = 0.016). We categorised the patients into four groups according to the positivity of HIF-1alpha/TWIST1/Snail to investigate the accumulated effects of these markers on survival. Co-expression of more than two markers was an independent prognostic indicator for both recurrence-free survival and overall survival (p = 0.004 and p<0.001, respectively, by multivariate Cox proportional hazards model). CONCLUSIONS: Co-expression of more than two markers from HIF-1alpha, TWIST1 and Snail is a significant prognostic predictor in patients with NSCLC.

Post-recurrence survival in completely resected stage I non-small cell lung cancer with local recurrence.

OBJECTIVE: Resection is the best treatment for patients with stage I non-small cell lung cancer (NSCLC). Patterns of disease recurrence after complete resection in stage I NSCLC have not been well demonstrated. The aim of this study was to evaluate the prognostic predictors of post-recurrence survival in patients with resected stage I NSCLC with local recurrence. METHODS: The clinicopathological characteristics of 123 patients with local recurrence after complete resection of stage I NSCLC in Taipei Veterans General Hospital between 1980 and 2000 were retrospectively reviewed. Post-recurrence survival and their predictors were analysed. RESULTS: The patterns of local recurrence included local only in 74 (60.2%) and both local and distant in 49 (39.8%) patients. The 1 and 2 year post-recurrence survival rates for the 74 patients with local only recurrence were 48.7% and 17.6%, respectively. Tumour size (p = 0.033) and treatment for initial recurrence (p<0.001) were significant predictors for post-recurrence survival in 74 patients with local only recurrence in univariate analyses. The hazard of death was greater in patients with larger tumour size. Treatment for initial recurrence (p = 0.001) was still a significant prognostic indicator in multivariate analyses. Patients who underwent reoperation after local recurrence survived longer than those who received chemotherapy and/or radiotherapy and those that received no treatment. CONCLUSIONS: Treatment for initial recurrence is a prognostic predictor for post-recurrence survival in resected stage I NSCLC with local recurrence. Complete surgical resection should be considered in selected candidates with resectable local recurrent disease.

EGF-mediated inhibition of ubiquitin-specific peptidase 24 expression has a crucial role in tumorigenesis.

In this study, several cancer-related proteins (Bax, p300, E2F4 and securin) have been proven to be substrates of ubiquitin-specific peptidase 24 (USP24), and relevance has been shown between USP24 and its substrates in samples from clinical lung cancer patients. Silencing USP24 increases the cancer formation by inhibiting cellular apoptosis and increasing cellular proliferation. Epidermal growth factor (EGF) treatment, and the KrasG12D and EGFRL858R mutations decrease USP24 protein stability via EGF- or CDK1-mediated phosphorylation at Ser1616, Ser2047 and Ser2604. Knockdown of USP24 decreases Bax and p300 levels, and reduces Ku70 acetylation, thereby preventing cancer cell apoptosis. In addition, knockdown of USP24 increases cell cycle progression by enhancing the G1-S transition and metaphase-anaphase transition. The molecular mechanism involves a decrease in the USP24 level, which reduces the expression of E2F4 and its partner TFDP1, and thus increases the G1/S transition. In conclusion, the USP24 level was decreased during the early stage of cancer and the mitotic stage of the cell cycle to regulate its substrates p300, Bax, E2F4 and securin, resulting in decreased cell apoptosis and increased cell cycle progression and, thus, cancer formation.

Upregulation of CYP17A1 by Sp1-mediated DNA demethylation confers temozolomide resistance through DHEA-mediated protection in glioma.

Steroidogenesis-mediated production of neurosteroids is important for brain homeostasis. Cytochrome P450 17A1 (CYP17A1), which converts pregnenolone to dehydroepiandrosterone (DHEA) in endocrine organs and the brain, is required for prostate cancer progression and acquired chemotherapeutic resistance. However, whether CYP17A1-mediated DHEA synthesis is involved in brain tumor malignancy, especially in glioma, the most prevalent brain tumor, is unknown. To investigate the role of CYP17A1 in glioma, we determined that CYP17A1 expression is significantly increased in gliomas, which secrete more DHEA than normal astrocytes. We found that as gliomas became more malignant, both CYP17A1 and DHEA were significantly upregulated in temozolomide (TMZ)-resistant cells and highly invasive cells. In particular, the increase of CYP17A1 was caused by Sp1-mediated DNA demethylation, whereby Sp1 competed with DNMT3a for binding to the CYP17A1 promoter in TMZ-resistant glioma cells. CYP17A1 was required for the development of glioma cell invasiveness and resistance to TMZ-induced cytotoxicity. In addition, DHEA markedly attenuated TMZ-induced DNA damage and apoptosis. Together, our results suggest that components of the Sp1-CYP17A1-DHEA axis, which promotes the development of TMZ resistance, may serve as potential biomarkers and therapeutic targets in recurrent glioma.

Variants of ubiquitin-specific peptidase 24 play a crucial role in lung cancer malignancy.

Ubiquitin is a critical modifier regulating the degradation and function of its target proteins during posttranslational modification. Here we found that ubiquitin-specific peptidase 24 (USP24) is highly expressed in cell lines with enhanced malignancy and in late-stage lung cancer clinical samples. Studying single-nucleotide polymorphisms (SNPs) of USP24 using genomic DNA of lung cancer patients revealed an increase in SNP 7656C/T. When using RNA specimens instead of the genomic DNA of lung cancer patients, we found significant increases in the ratios of variants 930C/T and 7656T/C, suggesting that variants at these two sites are not only caused by the SNP of DNA but also by the RNA editing. USP24-930T and USP24-7656C increase USP24 expression levels by increasing RNA stability. Knocking down USP24 increased Suv39h1 level through a decrease in mouse double-minute 2 homolog levels, thus enhancing lysine-9 methylation of histone H3, and resulting in the prevention of lung cancer malignancy. In conclusion, as USP24 variant analysis revealed a higher ratio of variants in blood specimens of lung cancer patients than that in normal individuals, USP24-930T and USP24-7656C might be useful as diagnostic markers for cancer detection.

MMP7-mediated cleavage of nucleolin at Asp255 induces MMP9 expression to promote tumor malignancy.

Nucleolin (NCL) participates in DNA transcription, ribosomal biogenesis and the regulation of RNA stability. However, the contribution of NCL to tumor development is still not clear. Herein, we found that NCL expression correlated with poor prognosis in lung cancer patients. Overexpressed NCL was predominantly cleaved to C-terminal truncated NCL (TNCL). In lung cancer formation, activation of the epidermal growth factor receptor pathway induced NCL expression, and also the expression of matrix metalloproteinase (MMP) 7, which then cleaved NCL at Asp255 to generate TNCL of 55 kDa. TNCL increased the expression of several oncogenes, including MMP9, anaplastic lymphoma kinase (ALK), HIF1a and CBLB, and decreased the expression of tumor suppressors including BRD4, PCM1, TFG and KLF6 by modulating mRNA stability through binding to the 3'-untranslated regions of their transcripts, thus ultimately enhancing metastasis activity. In conclusion, this study identified a novel role of the cleavage form of NCL generated by MMP7 in stabilizing MMP9 mRNA. We also provide a new insight that MMP7 not only cleaves the extracellular matrix to promote tumor invasion but also cleaves NCL, which augment oncogenesis. Blocking NCL cleavage may provide a useful new strategy for lung cancer therapy.

Organotin concentrations in three intertidal neogastropods from the coastal waters of Taiwan.

This study was aimed to determine organotin concentrations in the muricids Thais clavigera, Thais tuberosa and Morula granulata on an island wide scale and to find an adequate bioindicator species for long-term monitoring purpose. Samples were collected from the coast of Taiwan and vicinity islands with organotin analysis by graphite furnace atomic absorption spectrophotometry. Because triphenyltin (TPT) could not be discriminated from tributyltin (TBT) by the extraction method, organotins were measured as TBT + TPT and total hexane extractable organotins. Severity of imposex in the muricid population samples expressed as relative penis size (RPS) indices were calculated and compared to determine a suitable bioindicator species. In T. clavigera, T. tuberosa and M. granulata, TBT + TPT were 17-157, 1-44 and 117-1343 ng Sn g(-1) dry wt, respectively. And total organotins varied in the range of 181 to 1125, 23 to 44 and 229 to 1402 ng Sn g(-1) dry wt, respectively. A positive correlation was found between TBT + TPT and total organotins in M. granulata (TBT + TPT = Total organotins x 1.01-110.79; R2=0.97; p<0.001). At the site of Dapaisha, total organotins in M. granulata was 10 times higher than in T. tuberosa (i.e. 242 versus 23 ng Sn g(-1) dry wt). No signs of imposex were found in T. tuberosa (a single site in two subsequent years) and in M. granulata (seven collection sites). The degree of imposex (RPS) of T. clavigera varied from 0.2 to 38.1%. The RPS indices from fishing harbors were not higher than from rocky shores and oyster culture sites. No correlation was found between RPS indices and concentrations of TBT + TPT or total organotins. M. granulata seemed to be insensitive to organotin pollution although organotins were also detected. Our results indicate that organotin pollution is wide spread in coastal waters of Taiwan and pollutants at least include butyltins and phenyltins. As a bioindicator species, T. clavigera is more appropriate than T. tuberosa and M. granulata to monitor organotin contamination.

Post-traumatic, high-flow priapism treated with selective cavernous artery embolization and intracavernous streptokinase irrigation: a case report.

A 54-year-old man developed priapism shortly after a blunt perineal trauma. An arteriocavernous fistula caused the high-flow priapism, and was detected on both color Doppler sonography and selective phaloarteriography. Selective embolization of the left cavernous artery with Gelfoam was performed to seal the fistula, resulting in immediate detumescence. However, the penis remained firm despite returning to almost normal size. No fistula was detected by subsequent color Doppler sonography and phaloarteriography examination. Intracavernous irrigation with 200,000 U streptokinase was applied to treat residual firmness 2 weeks after embolization. Successful sexual intercourse was reported 3 months later. The combination of selective cavernous artery embolization and intracavernous streptokinase irrigation was effective for the treatment of the high-flow priapism in this case.

Present state and historical changes of trace metal pollution in Kaoping coastal sediments, southwestern Taiwan.

Surface and gravity-cored sediments were collected from the Kaoping coastal area off southwestern Taiwan to determine particle size, organic carbon, trace metal concentration and enrichment factor (EF), 210Pb dating, and 206Pb/207Pb ratio for determining present and historical metal pollution. Surface distributions of trace metals ranged from 0.02 to 0.13 mg/kg for Cd (EF: 0.16-1.65), from 35 to 189 mg/kg for Cr (EF: 0.85-2.92), from 25 to 64 mg/kg for Ni (EF: 0.53-2.38), from 10 to 32 mg/kg for Pb (EF: 1.20-4.94), and from 29 to 129 mg/kg for Zn (EF: 1.18-3.50). Trace metal concentrations correlate closely with distributions of mud (<63 microm) and organic carbon which accumulate largely around river mouths and within the Kaoping Canyon. With the exception of Cd, Cr and Ni in certain areas with rather coarse sediments, metals were generally elevated above the baseline levels over the studied area. Metals were also relatively enriched in areas with high contents of mud and organic carbon. Sedimentation rates derived from the excess 210Pb data in core sediments sampled from the canyon illustrate metal pollution beginning around 1970 that is corresponding to the booming time of economic growth in Taiwan. Meanwhile, the status of Pb pollution in core sediments is verified by an inversed correlation between 206Pb/207Pb ratios and Pb concentrations. Anthropogenic Pb and other metals in the studied area were apparently derived primarily from the Kaoping River and accumulated around the river mouth and within the Kaoping Canyon. The Kaoping Canyon appears to act as a major sink for river borne trace metals.

Treatment results of the TPOG-NHL92 protocols for childhood non-Hodgkin's lymphomas in Taiwan: a report from the Taiwan Pediatric Oncology Group (TPOG)

A nation-wide chemotherapeutic trial for childhood non-Hodgkin's lymphoma (NHL) was conducted by the Taiwan Pediatric Oncology Group (TPOG). Four TPOG-NHL92 protocols based on stage and histology were activated in 1992: TPOG-92LD (treatment duration: 8 months) was used for localized (stages I/II) NHL with any histology, 92LB (2 years), 92SNC (5 months), and 92LC (1 year) for advanced (stages III/IV) lymphoblastic (LB), small non-cleaved cell (SNC), and large cell (LC) lymphoma, respectively. From January 1992 through June 1998, 200 children with newly diagnosed NHL from 13 member hospitals of TPOG were enrolled. There were 140 boys and 60 girls. Their ages at diagnosis ranged from 2.4 months to 18.3 years with a median of 8.2 years. There were 54 (27.3%) patients with LB, 94 (47.5%) with SNC including B-cell acute lymphoblastic leukemia (B-ALL), and 50 (25.2%) with LC. Stages I, II, III, and IV (including B-ALL) of the disease comprised 5%, 10%, 43%, and 42% of cases, respectively. There were 176 patients eligible for evaluation of treatment results. The remission rate of induction was 82.4%, induction failed in 22 (12.5%) patients, and nine patients died during induction. As of August 31, 1999, 26 patients relapsed, six died during remission, one patient developed secondary acute myelomonocytic leukemia, and 105 patients remained in continuous remission with a median remission duration of 49 months. The event-free survival (EFS) at 7 years was 63.5%, 61.5% and 65% for LB, SNC, and LC groups (P = 0.8298). The 7-year EFS for stages I/II, III, and IV of the disease was 73%, 68.9%, and 50.3% (P = 0.0212), respectively. We concluded that following the strategy of stratification of therapy, only disease stages had prognostic significance in this study. More efforts are needed to improve our treatment results.

Sp1 expression regulates lung tumor progression.

The role of specificity protein 1 (Sp1) in controlling gene expression in lung tumor development and metastasis is not well understood. In this study, we showed that the Sp1 level was highly increased and required for lung tumor growth in transgenic mice bearing Kras-induced lung tumors under the control of doxycycline. Furthermore, the Sp1 level was highly upregulated in lung adenocarcinoma cells with low invasiveness and in patients with stage I lung cancer. We also demonstrated that Sp1 was downregulated in lung adenocarcinoma cells with high invasiveness and in patients with stage IV lung adenocarcinoma. Moreover, Sp1 inversely regulated migration, invasion and metastasis of lung adenocarcinoma cells in vivo. In addition, a decrease in the Sp1 level in highly invasive lung adenocarcinoma cells resulted from instability of the Sp1 protein. Furthermore, overexpression of Sp1 in highly invasive lung adenocarcinoma cells increased expression of E-cadherin, a suppressor of metastasis, and attenuated the translocation of ß-catenin into the cellular nucleus that leads to tumor malignancy. Taken together, Sp1 level accumulated strongly in early stage and then declined in late stage, which is important for lung cancer cell proliferation and metastasis during tumorigenesis.

Sp1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells.

Sp1 is important for the transcription of many genes. Our previous studies have shown that Sp1 is degraded in normal cell, but it is preserved in cancer cells during mitosis and exists a priori in the daughter cells, ready to engage in gene transcription and thereby contributes to the proliferation and survival of cancer cells. The mechanism by which Sp1 is preserved in cancer cells during mitosis remains unknown. In this study, we observed that Sp1 strongly colocalized with cyclin-dependent kinase 1 (CDK1)/cyclin B1 during mitosis. Moreover, we showed that Sp1 is a novel mitotic substrate of CDK1/cyclin B1 and is phosphorylated by it at Thr 739 before the onset of mitosis. Phospho-Sp1 reduced its DNA-binding ability and facilitated the chromatin condensation process during mitosis. Mutation of Thr739 to alanine resulted in Sp1 remaining in the chromosomes, delayed cell-cycle progression, and eventually led to apoptosis. Screening of Sp1-associated proteins during mitosis by using liquid chromatography/mass spectrometry indicated the tethering of Sp1 to myosin/F-actin. Furthermore, phospho-Sp1 and myosin/F-actin appeared to exist as a congregated ring at the periphery of the chromosome. However, at the end of mitosis and the beginning of interphase, Sp1 was dephosphorylated by PP2A and returned to the chromatin. These results indicate that cancer cells use CDK1 and PP2A to regulate the movement of Sp1 in and out of the chromosomes during cell-cycle progression, which may benefit cancer-cell proliferation.

Pancoast syndrome caused by metastasis to the superior mediastinum of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is common in Taiwan. Recently, extrahepatic metastases have developed more frequently in patients due to prolonged survival rates to which improved diagnostic and treatment methods have contributed. We present here a case of a 46-year-old man with an unusual metastasis to the superior mediastinum causing Pancoast syndrome eight months after a right hepatic lobectomy for HBV-related HCC. Early detection and prompt treatment of paraspinal metastasis are important to relieve symptoms and prevent permanent loss of neurological functions.

Role of transcriptional factors Sp1, c-Rel, and c-Jun in LPS-induced C/EBPdelta gene expression of mouse macrophages.

Transcription factor C/EBPs are involved in the regulation of various cellular responses. Here, it was suggested that C/EBPdelta gene was activated by lipopolysaccharide (LPS) through transcription factors Sp1, c-Rel, and c-Jun. Assay of the luciferase reporter vectors containing a 5'-deletion of the C/EBPdelta gene promoter indicated that a LPS-responsive element was positioned between -345 and -35 bp of mouse C/EBPdelta gene promoter. Transcription factors Sp1, c-Rel, and c-Jun bound to this region were identified using both in vivo chromatin immunoprecipitation and in vitro DNA-protein binding assays. LPS enhanced the proteins and DNA binding capacities of c-Rel and c-Jun, and the downstream Sp1 site was essential for LPS-induced C/EBPdelta gene. Treatment of cells with ERK/JNK/p38 inhibitors or NF-kappaB inhibitor inhibited the LPS-induced C/EBPdelta gene expression by inhibiting c-Jun, c-Rel, and p300 binding to DNA. Our findings provide a better understanding of LPS-induced C/EBPdelta gene expression.