Lipid Droplets in Lung Cancers Are Crucial for the Cell Growth and Starvation Survival.

For rapid and unlimited cell growth and proliferation, cancer cells require large quantities of nutrients. Many metabolic pathways and nutrient uptake systems are frequently reprogrammed and upregulated to meet the demand from cancer cells, including the demand for lipids. The lipids for most adult normal cells are mainly acquired from the circulatory system. Whether different cancer cells adopt identical mechanisms to ensure sufficient lipid supply, and whether the lipid demand and supply meet each other, remains unclear, and was investigated in lung cancer cells. Results showed that, despite frequent upregulation in de novo lipogenesis and the lipid transporter system, different lung cancer cells adopt different proteins to acquire sufficient lipids, and the lipid supply frequently exceeds the demand, as significant amounts of lipids stored in the lipid droplets could be found within lung cancer cells. Lipid droplet surface protein, PLIN3, was found frequently overexpressed since the early stage in lung cancer tissues. Although the expression is not significantly associated with a specific gender, age, histology type, disease stage, and smoking habit, the frequently elevated expression of PLIN3 protein indicates the importance of lipid droplets for lung cancer. These lipid droplets are not only for nutrient storage, but are also crucial for tumor growth and proliferation, as well as survival in starvation. These results suggest that manipulation of lipid droplet formation or TG storage in lung cancer cells could potentially decrease the progression of lung cancer. Further exploration of lipid biology in lung cancer could help design novel treatment strategies.

A highly sensitive and specific real-time quantitative PCR for BRAF V600E/K mutation screening.

Mutations that lead to constitutive activation of key regulators in cellular processes are one of the most important drivers behind vigorous growth of cancer cells, and are thus prime targets in cancer treatment. BRAF V600E mutation transduces strong growth and survival signals for cancer cells, and is widely present in various types of cancers including lung cancer. A combination of BRAF inhibitor (dabrafenib) and MEK inhibitor (trametinib) has recently been approved and significantly improved the survival of patients with advanced NSCLC harboring BRAF V600E/K mutation. To improve the detection of BRAF V600E/K mutation and investigate the incidence and clinicopathological features of the mutation in lung cancer patients of southern Taiwan, a highly sensitive and specific real-time quantitative PCR (RT-qPCR) method, able to detect single-digit copies of mutant DNA, was established and compared with BRAF V600E-specific immunohistochemistry. Results showed that the BRAF V600E mutation was present at low frequency (0.65%, 2/306) in the studied patient group, and the detection sensitivity and specificity of the new RT-qPCR and V600E-specific immunohistochemistry both reached 100% and 97.6%, respectively. Screening the BRAF V600E/K mutation with the RT-qPCR and V600E-specific immunohistochemistry simultaneously could help improve detection accuracy.

MET exon 14 skipping mutations and gene amplification in a Taiwanese lung cancer population.

Somatic mutations of MET gene are emerging as important driver mutations for lung cancers. To identify the common clinicopathological features of MET exon 14 skipping mutations and amplification and clarify whether the two MET gene alterations cause protein overexpression were investigated using 196 lung cancer samples of Taiwan through real time-qPCR/sequencing, fluorescence in situ hybridization, and immunohistochemistry. The two MET gene alterations are both present in low frequency, ~1%, in the studied lung cancer population of Taiwan. MET exon 14 skipping mutations were identified from two early-stage patients, who were both relatively advanced in age, and did not carry other driver mutations. One was an adenocarcinoma and the other was a rare carcinosarcoma. Three gene amplifications cases were identified. Neither of the two MET gene alterations would lead to protein overexpression; hence, direct detection in nucleic acid level would be a preferred and straightforward solution for the identification of skipping mutations. The presence of MET exon 14 mutations in minor histological types of lung cancers urge to extend screening scope of this mutation in lung cancer and treatment response evaluation in clinical trials. These would be important next steps for the success of MET target therapy in clinical practice.

Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells.

Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future.

A sensitive and high throughput TaqMan-based reverse transcription quantitative polymerase chain reaction assay efficiently discriminates ALK rearrangement from overexpression for lung cancer FFPE specimens.

OBJECTIVES: ALK fusion gene is an oncogenic driver in lung cancer with low prevalence, which can be ameliorated by crizotinib. Currently, ALK fusion gene can be diagnosed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), but inconstistnt results between the two methods are encountered regularly. To make the ALK fusion gene screening more efficient and to provide a simple solution to clarify the discrepancy between FISH and IHC results, a sensitive TaqMan-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was established. MATERIALS AND METHODS: The 3-plex TaqMan-based RT-qPCR assay was established and performed on 102 archived formalin-fixed, paraffin-embedded (FFPE) NSCLC samples to detect ALK rearrangement and overexpression. Break-apart FISH and automatic immunohistochemistry based ALK assays were performed side by side using tissue microarray. RESULTS: The RT-qPCR was performed successfully for 80 samples and 10 of them showed positive signals. Three out of the 10 qPCR positive cases were further confirmed by FISH and IHC test. Two others were IHC positive and FISH negative, and expressed full-length ALK transcript. The rest were neither FISH nor IHC positive and their ALK expression level was significantly lower than those FISH or IHC positive cases. CONCLUSION: Our RT-qPCR assay demonstrates that the capability and reliability of ALK detection is comparable to FISH and IHC, but it is more effective at discriminating ALK rearrangement from overexpression. The RT-qPCR assay easily clarifies the discrepancy between FISH and IHC, and can be incorporated into routine ALK screening for lung cancer.

Epidermal growth factor receptor mutation enhances expression of vascular endothelial growth factor in lung cancer.

Epidermal growth factor receptor (EGFR) activation has been demonstrated to have a critical role in tumor angiogenesis. In the present study, the correlation between EGFR mutations and vascular endothelial growth factor (VEGF) was investigated in lung cancer cell lines and non-small-cell lung cancer (NSCLC) tumor tissues. VEGF levels were significantly increased in culture medium of lung cancer cells and NSCLC tissues with EGFR mutations (H1650 vs. A549, P=0.0399; H1975 vs. A549, P<0.0001). Stable lung cancer cell lines expressing mutant (exon 19 deletion, E746-A750; exon 21 missense mutation, L858R) and wild-type EGFR genes were established. Significantly increased expression of VEGF and stronger inhibitory effects of gefitinib to VEGF expression were observed in exon 19 deletion stable lung cancer cells (exon 19 deletion vs. wild-type EGFR, P=0.0005). The results of the present study may provide an insight into the association of mutant EGFR and VEGF expression in lung cancer, and may assist with further development of targeted therapy for NSCLC in the future.

Mechanism of maprotiline-induced apoptosis: role of [Ca2+](i), ERK, JNK and caspase-3 signaling pathways.

Antidepressants are generally used for treatment of various mood and anxiety disorders. Several studies have shown the anti-tumor and cytotoxic activities of some antidepressants, but the underlying mechanisms were unclear. Maprotiline is a tetracyclic antidepressant and possesses a highly selective norepinephrine reuptake ability. We found that maprotiline decreased cell viability in a concentration- and time-dependent manner in Neuro-2a cells. Maprotiline induced apoptosis and increased caspase-3 activation. The activation of caspase-3 by maprotiline appears to depend on the activation of JNK and the inactivation of ERK. Maprotiline also induced [Ca(2+)](i) increases which involved the mobilization of intracellular Ca(2+) stored in the endoplasmic reticulum. Pretreatment with BAPTA/AM, a Ca(2+) chelator, suppressed maprotiline-induced ERK phosphorylation, enhanced caspase-3 activation and increased maprotiline-induced apoptosis. In conclusion, maprotiline induced apoptosis in Neuro-2a cells through activation of JNK-associated caspase-3 pathways. Maprotiline also evoked an anti-apoptotic response that was both Ca(2+)- and ERK-dependent.

Analysis of the size of DNA packaged by the human JC virus-like particle.

Previously, it has been demonstrated that the JC virus-like particle (VLP) is able to package DNA in E. coli and deliver the DNA into human colon cancer cells for gene expression. In this study, the maximum size of DNA packaged by the VLP was determined further. Plasmid DNAs with various sizes were packaged by the VLP in E. coli. Human neuroblastoma cells were then infected with the VLPs containing the various sizes of DNA to allow gene expression. In addition, plasmid DNAs packaged in the VLPs were extracted and retransformed back into E. coli under selection to determine the size of the DNA packaged. The results showed that the JC VLP was able to package plasmid DNA in E. coli up to at least 9.4 kbp in size and this size of DNA could be delivered successfully into human neuroblastoma cells for gene expression. The JC VLP is able to package exogenous DNA up to at least 9.4 kbp in size for gene transduction. These findings will help with the development of gene delivery systems using the JC VLP as the gene delivery vector.

Inorganic arsenic in drinking water accelerates N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder tissue damage in mice.

Epidemiological studies have revealed that exposure to an arsenic-contaminated environment correlates with the incidence of bladder cancer. Bladder cancer is highly recurrent after intravesical therapy, and most of the deaths from this disease are due to invasive metastasis. In our present study, the role of inorganic arsenic in bladder carcinogenesis is characterized in a mouse model. This work provides the first evidence that inorganic arsenic in drinking water promotes N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder tissue damage, including the urothelium and submucosal layer. This damage to the bladder epithelium induced by BBN includes thickening of the submucosal layer, the loss of the glycosaminoglycan layer and an increase in both the deoxyguanosine oxidation and cytosine methylation levels in the DNA. Further, when 10ppm inorganic arsenic is combined with BBN, the number of bladder submucosal capillaries is increased. In addition, inorganic arsenic also increases the deoxyguanosine oxidation level, alters the cytosine methylation state, decreases the activities of glutathione reductase and glucose-6-phosphate dehydrogenase, decreases the protein expression of NAD(P)H quinone oxidoreductase-1 (NQO-1) and increases the protein expression of specific protein 1 (Sp1) in bladder tissues. In summary, our data reveal that inorganic arsenic in drinking water promotes the BBN-induced pre-neoplastic damage of bladder tissue in mice, and that the 8-hydroxy-2'-deoxyguanosine, 5-methylcytosine, NQO-1 protein and Sp1 protein levels may be pre-neoplastic markers of bladder tumors.

Human JC virus-like particles as a gene delivery vector.

INTRODUCTION: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy. AREAS COVERED: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed. EXPERT OPINION: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.

Prevalence and genotype identification of human JC virus in colon cancer in Taiwan.

Although JC virus (JCV), a human polyomavirus, has been detected in colon cancers, the association between JCV and colon cancer remains controversial. In Taiwan, the prevalence of JCV infection in colon cancer patients has not been reported. Thus, the purpose of this study was to investigate JCV infection in colon cancers in Taiwan. Formalin-fixed, paraffin-embedded tissues from 22 colon cancer patients were examined in this study. Nested PCR was performed to detect viral genomic DNA. The product of the nested PCR flanking the JCV regulatory region was sequenced further. Viral large tumor protein, LT, and late capsid protein, VP1, were examined by immunohistochemistry (IHC). Nested PCR revealed JCV genomic DNA in 86.4% (19/22) of the colon cancer tissue samples. Both rearranged and archetypal genotypes of JCV were identified. Expression of JCV LT was positive in 63.6% (14/22) of the examined colon cancer tissue samples but not in any adjacent normal region. Expression of viral capsid protein VP1 was not detected in any of the tissues examined. The current study demonstrates that JCV genomic DNA was present in the examined colon cancer tissues. The genotypes of JCV in colon cancer tissues were also identified. Expression of viral early protein but not structural capsid protein was detected in the examined colon cancer tissues. Furthermore, a high prevalence of JCV infection in colon cancer tissues in Taiwan was also demonstrated.

The impact factors on prognosis of patients with pT3 upper urinary tract transitional cell carcinoma.

PURPOSE: Stage 3 upper urinary tract transitional cell carcinoma is a heterogeneous disease including different tumor locations (pelvis vs ureter) and invasion patterns (renal parenchyma, peripelvic fat and periureteral fat). Unfortunately the outcomes of patients with pT3 disease with different invasion pattern are largely unknown. This study presents the clinical outcome of patients with pT3 disease with upper urinary tract transitional cell carcinoma. MATERIALS AND METHODS: We retrospectively reviewed the medical records of all patients with pT3 disease with upper urinary tract transitional cell carcinoma. Four patient groups were classified according to tumor location and tumor invasion pattern. Prognostic factors including age, gender, tumor grade, tumor size, tumor number, tumor location and microscopic finding of vascular invasion were analyzed with respect to disease recurrence and survival. RESULTS: A total of 72 patients were included in this study. The most common complaint and tumor relapse pattern were painless gross hematuria and distant metastasis, respectively. Patients with pT3 disease with superficial parenchymal invasion had better disease-free and recurrence-free survival than the other 3 groups. Initial tumor location (p = 0.02) and vascular invasion (p = 0.02) were independent factors for disease-free survival, and vascular invasion (p = 0.001) was the only predictive factor for recurrence-free survival. CONCLUSIONS: The present study demonstrated that patients with pT3 disease with superficial parenchymal invasion should be considered to have lower stage disease, and that vascular involvement is the only independent prognostic factor for patients with pT3 disease for disease-free and recurrence-free survival. Systemic adjuvant therapy should be recommended for patients with pT3 disease with vascular involvement.

Observation of biochemical imaging changes in human pancreatic cancer tissue using Fourier-transform infrared microspectroscopy.

Fourier-transform infrared (FT-IR) microspectroscopic mapping can be used to distinguish between different tissue structures, and to increase the image contrast between normal and cancerous regions of a given tissue sample. This study demonstrates the biochemical changes associated with a consistent link between cancerous tissue and various molecular changes in the IR spectra of human pancreatic cancer tissue using FT-IR mapping. Tissue samples were obtained immediately after resection in a patient who underwent a distal pancreatectomy including the pancreatic body and tail. The biochemical imaging changes of lipids, proteins, and nucleic acids in human pancreatic cancer tissue were analyzed via FT-IR microspectroscopy, using imaging, mapping, and line scan techniques. The intensities and frequencies of the absorption bands in the IR spectra of human pancreatic cancerous tissue were markedly reduced and shifted, particularly in the amide bands of protein and CH2 and CH3 stretching vibrations of lipids. The cancerous tissue contained significant protein content, and the distributions of DNA and lipid were very low, indicating low amounts of nucleic acids and lipids in human pancreatic cancer tissue. The analytical results indicate that these FT-IR microspectroscopic biochemical images reflect the distribution of cell components, which could be correlated with stained tissue in adenocarcinoma in pancreatic tissues. This study with samples of noncancerous and cancerous pancreatic tissues has clearly demonstrated that FT-IR microspectroscopy using the mapping method can be used for diagnosis.

Rhabdomyolysis, acute renal failure, and multiple focal neuropathies after drinking alcohol soaked with centipede.

Many Chinese like to drink alcohol soaked with creatures for promoting health. This study reports a 49-year-old male who presented with multiple focal neuropathies of the upper limbs, coagulopathy, erythematous swelling of the bilateral upper extremities and trunk with bullous skin lesions, and rhabdomyolysis associated with acute renal failure after drinking alcohol soaked with centipede. Soaking a centipede, Scolopendra subspinipes mutilans, in 53% alcohol, produced the wine. Supportive treatment was administered, and the skin lesions and renal failure improved with subsequent neurologic deficit during the week following initial presentation. Alcohol binge or immobilization was the likely cause of neuropathy, bullous skin lesions and rhabdomyolysis in the patient. However, there is a possibility that centipede venom also contributed to the illness in this patient.

Clinical significance of signet ring cell rectal carcinoma.

BACKGROUND AND AIMS: Signet ring cell carcinoma of the rectum (SCCR) is a rare type of rectal carcinoma. This study examined the clinical significance of SCCR. PATIENTS AND METHODS: From our medical records we retrospectively identified 61 SCCR patients and compared their clinical data and outcomes to those of 144 consecutive patients with non-SCCR mucinous rectal adenocarcinomas (NSMR) and 2,414 consecutive patients with nonmucinous rectal adenocarcinomas (NMR). RESULTS: . The incidence of SCCR was 1.39% of rectal cancers. Mean patient age at onset of SCCR (48.1years, range 15-80) was significantly lower than that for NSMR (57.4 years, 9-88) and NMR (62.6 years, 12-94). The proportion of late stage (TNM III+IV) tumors was significantly higher in SCCR (90%) than in NSMR (69%) and NMR (48%). There were more tumors located in the lower rectum in SCCR (46%) than in NSMR (34%) and NMR (29%). SCCR tumors were significantly larger (5.68+/-3.84 cm) than NSMR (4.27+/-1.78 cm) and NMR tumors (3.76+/-1.71 cm). A higher percentage of patients with SCCR (42.6%) received abdominoperineal resection for treatment. In tumors with TNM stage IV the rate of tumor spread via the hematogenous route was significantly lower in SCCR (18.5%) than in NSMR (43.5%) and in NMR (69%). The rate of tumor spread via seeding to the peritoneum was lower in SCCR (22.2%) than in NSMR (43.5%) but higher than in NMR (2.7%). The rate of tumor spread via the lymphatic route was higher in SCCR (44.4%) than in NSMR (26.1%) and significantly higher than in NMR (12.3%). The 1-, 2-, and 5-year overall SCCR survival rates were 73.9%, 36.3%, and 23.3% respectively, which were significantly poorer than those of NSMR and NMR. For the 28 stage III and R0 SCCR tumors the 1-, 2-, and 5-year disease-free survival rates of SCCR were 84.0%, 44.2%, and 30.3%, respectively, which are comparable with general data of stage III rectal cancer in the world. CONCLUSION: Diffuse infiltration of signet ring cells enhances the tendency of mucinous carcinomas of the rectum in more local extension and easier lymphatic spreading but not at peritoneal seeding. Although SCCR had the poorest prognosis, this outcome may be due to the advanced tumor stage rather than histology itself.

Concurrent cisplatin, 5-fluorouracil, leucovorin, and radiotherapy for invasive bladder cancer.

PURPOSE: To investigate the tolerance and efficacy of a modified concurrent chemoradiation (CCRT) protocol for patients with invasive bladder cancer "unfit" for radical cystectomy. METHODS AND MATERIALS: Twenty-three muscle-invasive bladder cancer patients who were unfit for or unwilling to receive radical cystectomy were enrolled in this study. All patients had transitional cell carcinoma of bladder, and distribution of stage was 14 (61%), 1 (4%), and 8 (35%) for T3a, T3b, and T4, respectively. This study included a relatively old-age population, with the median age being 75 and 70% of patients over 70 years old. Patients were treated with maximal transurethral resection of the bladder tumor followed by curative CCRT. The chemotherapy (C/T) regimen was comprised of cisplatin, 50 mg/m(2) intravenously (i.v.) on Day 1; 5-fluorouracil (5-FU), 500 mg/m(2)/day by continuous i.v. infusion on Days 1-3; and leucovorin, 50 mg/day by continuous i.v. infusion on Days 1-3. Chemotherapy course was repeated at 21-day intervals. The radiation dose was 44-45 Gy to whole pelvis and 60-61.2 Gy to bladder, with a daily fraction of 1.8-2 Gy. The completeness of the CCRT protocol was defined as patients receiving at least 55 Gy of radiotherapy to the whole bladder and at least three courses C/T. RESULTS: Seventy-four percent of patients (17/23) completed the CCRT protocol. Radiation Therapy Oncology Group (RTOG) Grade 3 acute toxicities were observed in 4 patients, which included leucopenia, vomiting, genitourinary (GU) tract infection, and diarrhea. No treatment-related deaths occurred during the CCRT period. RTOG Grade 3 or more late complications were observed in 3 patients; one of them died of radiation cystitis superimposed with GU infection. Of the 18 patients whose response to CCRT was evaluated, a complete tumor response was documented in 16 patients (89%). With a median follow-up of 3 years, the 3-year overall survival (OS) and disease-free survival (DFS) for all patients was 69% and 65% respectively. Meanwhile, the 3-year overall and DFS rates for patients who completed CCRT vs. those who did not complete CCRT were 82% vs. 33% and 75% vs. 33%, respectively (p = 0.18 for OS and p = 0.04 for DFS). CONCLUSIONS: Concurrent cisplatin, 5-FU, leucovorin, and radiotherapy for treatment of invasive bladder cancer is a feasible and promising treatment even for relatively old patients. Our results are comparable to those in recent studies by using combined modality treatment or neoadjuvant chemotherapy plus radical cystectomy. Consequently, this novel protocol warrants a prospective clinical trial and may be a safe, effective alternative to radical cystectomy.

Angiomyolipoma of the colon: report of a case and review of the literature.

Angiomyolipoma of the colon is very rare. Only three cases have been reported in the literature. Here we report the case of a 54-year-old male, who presented with a progressive abdominal cramping pain. We performed left hemicolectomy under the impression of a cancerous mass over the splenic flexure of the colon. Histology revealed an angiomyolipoma of the colon. In addition, we review the literature.

Leiomyomatosis of mesenteric lymph nodes associated with duodenal adenocarcinoma.

Leiomyomatosis of lymph nodes is an extremely rare disease. Only a few cases have previously been reported in pelvic lymph nodes. They were related to a benign uterine leiomyoma, a metastasizing uterine leiomyoma, an endometrial adenocarcinoma, and an ovarian endometrioid carcinoma. We report on a case of leiomyomatosis of the mesenteric lymph nodes associated with a duodenal adenocarcinoma with no history of uterine leiomyoma or any gynecological malignancy. The patient, a 56-year-old woman, was found to have an adenocarcinoma of the duodenum. All mesenteric lymph nodes removed showed leiomyomatosis, which was verified by immunohistochemical study showing positive immunostaining for smooth muscle actin, desmin, and vimentin, but negative staining for HMB-45. It is necessary to make a differential diagnosis from other examples of spindle cell proliferation involving lymph nodes such as a hemorrhagic spindle cell tumor with amianthoid fibers (palisade myofibroblastoma), angiomyolipoma, lymphangiomyomatosis, inflammatory pseudotumor, and Kaposi's sarcoma.