Tsung-Ying Yang

Surgical results of metastatic spinal cord compression secondary to non-small cell lung cancer

Study Design. The results for 37 surgical interventions in 31 consecutive patients with non-small cell lung cancer (NSCLC) with symptomatic spinal cord compression were reviewed retrospectively. Objectives. To evaluate postoperative outcomes and survival rates of NSCLC patients surgically treated for symptomatic spinal metastasis. Summary of Background Data. For patients with spinal cord compression secondary to lung cancer, the prognosis is usually poor. However, with the development of new chemotherapeutic drugs and targeted therapeutic agents, the survival rate may be better. Methods. From November 2000 to March 2005, 31 patients with symptomatic metastatic spinal cord compression secondary to NSCLC underwent palliative surgery using a posterolateral transpedicular approach (PTA) or combined posterior and anterior procedures. The indication for surgery was neurologic progression due to spinal cord compression. Results. The patients ranged in age from 20 to 81 years (mean, 61.4 years). Twenty-eight patients (90%) underwent PTA, and 3 patients had combined posterior and anterior procedures. Neurologic improvement by at least one Frankel grade was noted in 25 of 31 cases (80%). Overall, 74% of patients (23 of 31) were able to walk after surgery. There was no case of intraoperative mortality, but two deaths occurred in the postoperative period. Median survival time was 8.8 months. Conclusions. Even though lung cancer is considered an aggressive tumor, it is justifiable to aggressively treat patients with symptomatic spinal cord compression. Surgery by PTA can lead to good results in these patients.

Sustained activation of ERK and Cdk2/cyclin-A signaling pathway by pemetrexed leading to S-phase arrest and apoptosis in human non-small cell lung cancer A549 cells

Pemetrexed, a multitargeted antifolate with the ability to inhibit several enzymes involved in purine and pyrimidine syntheses, has demonstrated clinical activity in non-small cell lung cancer cells, as well as in a broad array of other solid tumors. In this study, we show that inducing cell cycle S-phase arrest and apoptosis in human lung adenocarcinoma A549 cells with pemetrexed is associated with increased cyclin-A and cyclin-dependent kinase 2 (Cdk2) protein and Cdk2/cyclin-A kinase activity. Knockdown of cyclin-A using small interfering RNA (siRNA), and inhibiting Cdk2 activity with flavopiridol, strikingly reduced S-phase arrest and apoptosis. Moreover, pemetrexed induced sustained activation of extracellular signal-regulated kinase1/2 (ERK1/2). Knockdown of ERK1/2 using specific siRNA, as well as known inhibitors (PD98059 and U0126), effectively suppressed the expression of cyclin-A and Cdk2, and reduced S-phase arrest and apoptosis induced by pemetrexed. These data provide the first evidence that pemetrexed-induced S-phase arrest and apoptosis is associated with an increase in Cdk2 and cyclin-A expression and activation, which is ERK-dependent and upstream of caspase-3. Our findings suggest that the ERK-mediated Cdk2/cyclin-A signaling pathway is an important regulator of pemetrexed-induced S-phase arrest and apoptotic cell death.

Activity of gefitinib in advanced non-small-cell lung cancer with very poor performance status

Advanced non-small-cell lung cancer (NSCLC) patients with poor performance status (PS) are less likely to respond to chemotherapy, or to have an improvement in survival, but more likely to experience toxicity. We retrospectively evaluated the efficacy and tolerability of gefitinib in patients with advanced NSCLC and very poor PS in Taiwan. Patients with stage IIIB, IV NSCLC with an Eastern Cooperative Oncology Group (ECOG) PS of 3–4 received oral gefitinib 250 mg once daily. Totally, 52 patients were included (25 men, 27 women). Forty-three patients (82.7%) were in a PS of 3. Tumor response rate was 25.0% (13/52). Tumor response rate to gefitinib was highest in chemonaive patients 38.1% (8/21) vs. failed 1 chemotherapy regimen 13.3% (2/15) vs. failed 2 or more chemotherapy regimens 18.8% (3/16), p = 0.015. The median overall survival was 2.5 months (response group 9.1 months, stable disease 3.1 months, and progressive group 0.8 month, p < 0.001). Adverse events, mainly skin reactions and diarrhea, were generally mild (grade 1 or 2) except paronychia and acne. Thus, gefitinib has clinically antitumor activity and good tolerability in {Taiwan} patients with advanced NSCLC and very poor performance status, with a higher response rate than that seen Europe or in European heritage Americans. Chemonaive patients responded better than patients with prior chemotherapy. Formal clinical trials are warranted to evaluate the role of gefitinib in this situation.

L-type calcium channel blockers reverse docetaxel and vincristine-induced multidrug resistance independent of ABCB1 expression in human lung cancer cell lines

Multidrug resistance (MDR) of cancer cells to cytotoxic drugs significantly impedes chemotherapeutic treatment. The purpose of this study is to characterize docetaxel (DOC) or vincristine (VCR) selected A549 and H1299 non-small cell lung cancer (NSCLC) sublines that exhibit MDR phenotypes and followed by re-sensitization study. Although all drug resistant sublines showed cross-resistance to DOC, VCR, and doxorubicin (DXR), the expression of ATP-binding cassette (ABC) transporter B1 (ABCB1) gene was found to be strongly induced in DOC but not in VCR resistant A549 sublines by quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR). In DOC and VCR resistant H1299 sublines, moderate expression of ABCB1 was detected. The levels of ABCB1 protein and efflux activities were further examined by immunoblotting and rhodamin-123 staining assay. The results showed that both ABC and non-ABC mediated MDR are existed. Furthermore, verapamil (VER), an inhibitor of ABCB1 and an L-type calcium channel blocker, is capable of reversing the resistance in all drug-resistant sublines independent of ABCB1 expression. Importantly, VER only sensitizes resistant sublines but has no effect on parental cancer cells. Other L-type calcium channel blockers, such as diltiazem (DIL) and nifedipine (NIF), also sensitize MDR sublines without interfering with ABCB1 activity but with lower efficacy than VER. Our data showed that in addition to ABCB1, calcium channel activity may play a crucial role in DOC- and VCR-acquired MDR. Therefore, inhibition of calcium influx may provide a new target to modulate MDR in chemotherapy.

Retrospective study of erlotinib in patients with advanced squamous lung cancer.

BACKGROUNDnThe effective targeted therapy for lung squamous cell carcinoma (SCC) is needed. The epidermal growth factor receptor (EGFR) mutation rate is low in lung SCC. The aim of this study was to evaluate the status of erlotinib treatment and EGFR mutation in lung SCC patients.nnnMETHODSnWe retrospectively enrolled lung cancer patients with SCC histology and history of erlotinib treatment. The primary objective was to assess overall response rate (ORR) and disease control rate (DCR) and the secondary objective was to assess progression-free survival (PFS) and overall survival (OS). EGFR mutations were assessed in parts of patients using both direct sequencing and protein nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp methods.nnnRESULTSnIn total, 92 patients were analyzed (75 men and 17 women, median age 69 years, and 74 current or former smokers). Sixteen patients achieved partial response and 9 had stable disease. The ORR was 17.4% and the DCR was 27.2%. The PFS and OS were longer in patients with disease control than with progressive disease (PFS 7.8 versus 1.3 months and OS 20.7 versus 2.7 months, both p<0.0001). The 1-year survival rate was 21.7%. In 27 patients with adequate specimens for molecular analysis (including 4 PR and 4 SD), two (7.4%) had EGFR complex mutations. One patient experienced response to erlotinib and the other did not.nnnCONCLUSIONSnA significant proportion of lung SCC patients would derive a clinical benefit from erlotinib treatment. The relatively higher response rate than the EGFR mutation rate in present study needs further evaluation.

Regulation of chemosensitivity and migration by clusterin in non-small cell lung cancer cells

PurposeIn terms of drug resistance, cancer cells usually benefit from high clusterin (CLU) expression on chemotherapy. In contrast, CLU expression has been found to be a favorable prognostic factor in lung cancer patients. The aims of this study are to determine the association between CLU expression and chemotherapeutic sensitivity and the potential role of CLU in migration in human non-small-cell lung cancer (NSCLC) cell lines.MethodsThe levels of clusterin in NSCLC cell lines were altered by short hairpin RNA interference (shRNAi) and overexpression on chemosensitivity assay. Migratory ability of these cell lines was also investigated.ResultsH1355 cells with the highest level of CLU demonstrated the lowest sensitivities to Adriamycin (ADR), docetaxel (DOC), and gemcitabine (GEM) treatment. Inhibition of CLU expression in H1355 cells resulted in higher chemosensitivities. When CLU was stably overexpressed in A549 and H1299 cells, only the chemosensitivity to ADR was reduced. The migratory ability of CLU-overexpressing cells significantly decreased. Moreover, MMP2 transcription was inhibited in CLU-overexpressing H1299 cells. These results indicated lower metastatic potential for cancer cells with high CLU level.ConclusionLung cancer cells with high level of CLU have reduced chemosensitivity. High level of CLU may result in migratory inhibition and thus favorable prognosis in lung cancer.

Immunomodulatory Protein from Ganoderma microsporum Induces Pro-Death Autophagy through Akt-mTOR-p70S6K Pathway Inhibition in Multidrug Resistant Lung Cancer Cells.

Chemoresistance in cancer therapy is an unfavorable prognostic factor in non-small cell lung cancer (NSCLC). Elevation of intracellular calcium level in multidrug resistant (MDR) sublines leads to sensitization of MDR sublines to cell death. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on Annexin-V assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers.

Good response to gefitinib in a lung adenocarcinoma harboring a heterozygous complex mutation of L833V and H835L in epidermal growth factor receptor gene.

An 89-year-old man presented with chest tightness and progressive body weight loss of 7 kilograms during a 6-month period. He had been a light smoker, at three packs per year, and he quit smoking more than 50 years ago. The chest x-ray revealed a ground-glass lesion in the left lower lobe (LLL) and a well-defined nodule in the right lower lobe (RLL) of the lung. Chest computed tomography (CT) scan showed a bulla and adjacent consolidative mass in the LLL approximately 4.5 2.5 cm in size and several small metastatic nodules in both lower lobes, with the largest one, 1.5 1.3 cm in size, in the RLL. The pathologic examination of the CT-guided lung biopsy from the nodule in the RLL proved well-differentiated adenocarcinoma. The diagnosis of adenocarcinoma of the LLL with multiple lung metastases was made. Because of the patient’s age, he received five cycles of singleagent chemotherapy with gemcitabine, with stable disease as the best response. The disease progressed 6 months after chemotherapy completion. He then received gefitinib 250 mg per day as the second-line therapy. The follow-up chest CT scan revealed the nodule in the RLL (Fig 1A) had almost disappeared (Fig 1B) after gefitinib treatment for 8 weeks. Because of severe xeroderma and malaise, the dose of gefitinib was reduced to 250 mg every other day. The disease progressed after 34 weeks of gefitinib treatment. A complex mutation of L833V and H835L in exon 21 of the epidermal growth factor receptor (EGFR) gene was detected from the tumor specimen by direct sequencing of EGFR from exon 18 to exon 21, as described previously. The common drug-sensitive mutations, including in-frame deletions in exon 19, G719X in exon 18, and L858R and L861Q in exon 21, were not found with the sensitive DxS amplification refractory mutation system (ARMS) EGFR 29 mutationdetection kit (DxS, Manchester, United Kingdom). The complex mutation of L833V and H835L in the EGFR gene is rare. From our database, which contains 420 mutations in EGFR exons 18 to 21 found by direct sequencing from 1,090 patients with non–small-cell lung cancer (NSCLC), four H835L mutations and five L833V mutations were found. All 4 H835L mutations were combined with L833V mutations. The other L833V mutation was combined with the G719S mutation. All of these five patients had adenocarcinoma. This might suggest that L833V-H835L complex mutations are unique mutational types in NSCLC. To verify the allele status of this complex mutation, the EGFR exon 21 of each of these four patients was amplified by polymerase chain reaction (PCR) from the tumor DNA with proofreading DNA polymerase (TaKaRa LA) and was cloned into TOPO TA cloning vectors (Invitrogen) and then transfected into Escherichia coli. The clones with a correct insert were sequenced with an ABI 3730 DNA analyzer. For each patient, there were clones revealing L833V and H835L in the same sequence tract (Fig 2A); others showed only the normal sequence of exon 21 (Fig 2B). This indicated the co-occurrence of L833V and H835L mutations on the same allele in the tumors of all four patients. This study was approved by the institutional review board, and written informed consent was obtained from all patients. EGFR tyrosine kinase inhibitors (TKIs) have become one of the major agents in the treatment of advanced NSCLC. Somatic mutations in the EGFR kinase domains are found in a subset of NSCLC and are associated with sensitivity to the EGFR TKIs. The common drugsensitive mutations include point mutations in exon 18 (G719A/C) and exon 21 (L858R and L861Q), and in-frame deletions in exon 19. Three known kinase domain mutations are associated with drug resistance: an exon 19 point mutation (D761Y), an exon 20 point mutation (T790M), and an exon 20 insertion (D770_N771insNPG). Although these mutations account for approximately 90% of EGFR mutations in NSCLC, the sensitivity of many uncommon mutations to EGFR TKIs treatment is not clear, only with sensitivity of some rare mutations tested in vitro. To our knowledge, this is the first report of a patient with lung adenocarcinoma harboring complex mutations of L833V and H835L in EGFR exon 21 that responded to EGFR TKI. Complex mutations are not uncommon in the EGFR tyrosine kinase domain in NSCLC. The frequency of doublet mutations is as

Effect of folic acid and vitamin B12 on pemetrexed antifolate chemotherapy in nutrient lung cancer cells.

Pemetrexed (MTA) is a multitargeted antifolate drug approved for lung cancer therapy. Clinically, supplementation with high doses of folic acid (FA) and vitamin B12 (VB12) lowers MTA cytotoxicities. An antagonistic effect of FA/VB12 on MTA efficacy has been proposed. However, patients who receive FA/VB12 show better tolerance to MTA with improved survival. The aims of this study are to investigate the modulation of FA and VB12 on MTA drug efficacy in human nonsmall cell lung cancer (NSCLC) cell lines. The sensitivities of cells, apoptosis, and MTA-regulated proteins were characterized to determine the possible effects of high doses of FA and VB12 on MTA efficacy. MTA has the lowest efficacy under 10% serum conditions. However, supplementation with FA and VB12 individually and additively reversed the insensitivity of NSCLC cells to MTA treatment with 10% serum. The enhanced sensitivities of cells following FA/VB12 treatment were correlated with increasing apoptosis and were specific to MTA but not to 5-fluorouracil (5-FU). Enhanced sensitivity was also associated with p21WAF1/Cip1 expression level. Our results revealed no antagonistic effect of high doses of FA/VB12 on MTA efficacy in cancer cells grown in nutrient medium. Furthermore, these data may partially explain why supplementation of FA and VB12 resulted in better survival in MTA-treated patients.

Tissue transglutaminase 2 expression is epigenetically regulated in human lung cancer cells and prevents reactive oxygen species-induced apoptosis

Purpose Tissue transglutaminase 2 (TG2) is a stress-regulated protein and associated with cancer cell survival. However, the effects of TG2 expression in human non-small-cell lung cancer (NSCLC) cells on reactive oxygen species (ROS) production and redox homeostasis have not been fully elucidated. Materials and methods We investigated the TG2 expression and activity in A549, H1299, H1355, and H460 lung cancer cells by Western blots and quantitative polymerase chain reaction assay. The enzyme-linked immunosorbent assay was used for transglutaminase activity. The epigenetic expression was characterized with histone deacetylase inhibitor trichostatin A and DNA methyltransferase inhibitor 5-Aza treatment. TG2 expression was inhibited by siRNA transfection and the intracellular calcium was measured by Flow-3AM assay, apoptosis was analyzed by Annexin V/propidium iodide assay, and intracellular ROS was detected by fluorescence-activated cell sorting analysis. The ROS scavenger N-acetyl-L-cysteine (NAC) was applied to reduce TG2-knockdown-induced oxidative stress. Results Only A549 cells expressing high levels of TG2 correlated with high TG2 activity. The expression of TG2 can be regulated by epigenetic regulation in A549, H1299, and H1355 cells. The data also show that TG2 reduction induces apoptosis in A549 and H1299 cells. Furthermore, increased intracellular ROS and calcium levels were both detected in TG2-reduced cells. Moreover, endoplasmic reticulum stress inhibitor (salubrinal) and antioxidant NAC were able to reduce ROS and calcium levels to recover cell viability. Interestingly, the extrinsic and intrinsic apoptosis pathways were activated with a p53 independence upon TG2 reduction. TG2 reduction not only attenuated AKT activation but also reduced superoxide dismutase 2 (SOD2) expression. Exogenous NAC partially recovered SOD2 expression, indicating that mitochondrial-mediated apoptosis accounts for a part of but not all of the TG2-reduction-related death. Conclusion TG2 plays a protection role in NSCLC cell lines. Regardless of the endogenous level of TG2 and p53 status, reduction of TG2 may result in oxidative stress that induces apop-tosis. Therefore, target TG2 expression represents a logical strategy for NSCLC management.