Inhibition of store-operated calcium entry contributes to the anti-proliferative effect of non-steroidal anti-inflammatory drugs in human colon cancer cells

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation and angiogenesis in colorectal cancer. We examined a possible involvement of store-operated calcium (SOC) entry in human colon carcinoma cells (HRT-18), which require calcium for proliferation. Acetyl-salicylic-acid (ASA), mefenamic acid (MEF) and sulindac sulfide (SUS) inhibited cell proliferation with the following order of potency: SUS > MEF > ASA. SUS but not MEF and ASA induced apoptosis following low-dose treatment. Furthermore, SUS and MEF significantly altered the cell cycle distribution. The ability of NSAIDs to inhibit SOC entry was assessed by measuring the intracellular calcium concentration ([Ca2+]i) in response to calcium store depletion using the endoplasmic calcium ATPase inhibitor thapsigargin. SUS and MEF, but not ASA significantly inhibited SOC entry. A causal link between SOC entry inhibition and anti-proliferative activity was tested using the inorganic SOC entry inhibitor La3+ and the specific organic inhibitor N-1-n-octyl-3,5-bis-(4-pyridyl)triazole (DPT). Both La3+ and DPT inhibited cell proliferation and SOC entry. Analogous to MEF, the anti-proliferative effect of DPT was mediated by cell cycle arrest and not by induction of apoptosis. These data indicate a role of SOC entry for cell proliferation in cancer cells and suggest a novel anti-proliferative NSAID mechanism in addition to its known influence on lipid metabolism.     

Musculoskeletal desmoid tumours: Pre‐ and post‐treatment radiological appearances

This study was aimed to illustrate the pre‐ and post‐treatment imaging findings of musculoskeletal desmoid tumours and describe current treatment methods. Imaging of histologically proven cases of desmoid tumours at St. Vincent's Hospital, Melbourne, were obtained via picture archiving communication system (PACS) and then assessed by two musculoskeletal radiologists. Suitable imaging both pre‐ and post‐treatment were then obtained from PACS. All imaging chosen were de‐identified. Ninety‐two patients were found to have histologically proven cases of desmoid tumours between January 2000 and December 2013. Six patients with extra‐abdominal tumours were selected, where pre‐ and post‐treatment imaging was available. Desmoid tumours can occur in many areas of the body. Treatment of desmoids are varied. Although wide‐margin surgery has been the traditional form of treatment, it still cannot guarantee absence of tumour recurrence despite microscopically tumour‐free margins. Other forms of treatment such as non‐steroidal anti‐inflammatory drugs, radiotherapy, chemotherapy, tyrosine kinase inhibitors and also the conservative ‘watch and wait’ approach have been suggested, which show varying results.     

Induction of apoptosis by cyclo-oxygenase-2 inhibitor NS398 through a cytochrome C-dependent pathway in esophageal cancer cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) can induce tumor cells to undergo apoptosis in vitro. They have also shown cancer-preventive activity in vivo. The mechanism of their effects is, however, not well defined. We investigated the mechanism by which a new NSAID, NS398, induces apoptosis in esophageal cancer cell lines. NS398 decreased cell viability in 2 cyclo-oxygenase-2-positive (COX-2(+)) esophageal cancer cell lines but not in a COX-2(-) cell line. DNA fragmentation and TUNEL assays demonstrated that NS398 induced the 2 COX-2(+) cancer cell lines to undergo apoptosis. The percentage of apoptosis induced by NS398 was associated with the level of COX-2 expression. Further investigation showed that the cytochrome c pathway was responsible for NS398-induced apoptosis; i.e., cytochrome c was released from mitochondria, caspase-9 and caspase-3 were activated and finally poly(ADP-ribose)polymerase (PARP) was cleaved. Furthermore, the effect of NS398 was inhibited by the caspase inhibitor Z-DEVD-FMK and prostaglandin E(2). In contrast, bcl-2, bax, c-myc, Fas and Fas-ligand showed minor changes. Altogether, our data suggest that induction of apoptosis by NS398 is associated with COX-2 expression and occurs through the cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3 and cleaves PARP.     

Biomarkers of inflammation are associated with colorectal cancer risk in women but are not suitable as early detection markers

Initial studies have investigated the association between inflammation and colorectal cancer (CRC) using C‐reactive protein (CRP) as a proinflammatory biomarker and have noted inconsistent results among women. We here report the findings from a large prospective study with repeat measurements of CRP, as well as serum amyloid A (SAA), an additional biomarker of inflammation, and risk of CRC. In the Women's Health Initiative Observational Study, we examined associations of CRP and SAA with CRC using repeat assessments (baseline and 3‐year follow‐up) among 953 matched case–control pairs for CRP and 966 pairs for SAA. Multivariate‐adjusted conditional‐logistic regression models were used with two‐sided tests of significance. Receiver operating characteristic (ROC) curve analysis assessed their utility as early detection markers. Colon cancer risk (odds ratio [OR] and 95% confidence intervals) among women in the highest quintiles of CRP or SAA compared to those in the lowest quintiles was OR_colon/CRP = 1.37 (0.95–1.97, p‐trend = 0.04) and OR_colon/SAA = 1.26 (0.88–1.80, p‐trend = 0.10), respectively. Women with elevated concentrations of both CRP and SAA had an increased risk of OR_colon = 1.50 (1.12–2.00, p‐value = 0.006) compared to those with low concentrations. No positive associations were observed with rectal cancer and weaker associations for CRC overall. Temporal changes in biomarkers more than 3 years did not predict risk. The area under the 6‐month ROC curve for CRP+SAA was 0.62 (95% confidence interval = 0.55–0.68). Elevated inflammatory biomarkers are associated with an increased risk of CRC, mainly colon cancer. Nevertheless, changes in the biomarkers over time do not suggest that they merit consideration as early detection markers for CRC.     

Reversal of inflammation-associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin

Dihydrodiol dehydrogenase (DDH) is a member of the aldo-keto reductases superfamily (AKR1C1-AKR1C4), which plays central roles in the metabolism of steroid hormone, prostaglandin and xenobiotics. We have previously detected overexpression of DDH as an indicator of poor prognosis and chemoresistance in human non-small lung cancer (NSCLC). We also found DDH expression to be closely related to chronic inflammatory conditions. The aim of this study was to investigate the links between inflammation, DDH expression and drug resistance in NSCLC cells. We showed that pro-inflammatory mediators including interleukin-6 (IL-6) could induce AKR1C1/1C2 expression in NSCLC cells and increase cellular resistance to cisplatin and adriamycin. This effect was nullified by Safingol, a protein kinase C inhibitor. Moreover, the expression of AKR1C1/1C2 was inversely correlated to NBS1 and apoptosis-inducing factor (AIF). We also showed that IL-6-induced AKR1C1/1C2 expression and drug resistance were inhibited by wogonin and chrysin, which are major flavonoids in Scutellaria baicalensis, a widely used traditional Chinese and Japanese medicine. In conclusion, this study demonstrated novel links of pro-inflammatory signals, AKR1C1/1C2 expression and drug resistance in NSCLC. The protein kinase C pathway may play an important role in this process. Overexpression of AKR1C1/1C2 may serve as a marker of chemoresistance. Further studies are warranted to evaluate wogonin and chrysin as a potential adjuvant therapy for drug-resistant NSCLC, especially for those with AKR1C1/1C2 overexpression.     

Selective inhibition of Rab prenylation by a phosphonocarboxylate analogue of risedronate induces apoptosis, but not S-phase arrest, in human myeloma cells

Bisphosphonates (BPs) are widely used in the treatment of osteolytic bone disease associated with multiple myeloma, and have been demonstrated to exert antitumor effects both in vitro and in vivo. However, the precise molecular mechanisms involved in the direct antitumor effects of BPs in vitro are not known. Nitrogen-containing BPs, such as risedronate (RIS), act by inhibiting protein prenylation. A phosphonocarboxylate analogue of RIS, 3-PEHPC, has previously been shown in osteoclasts and macrophages to specifically inhibit prenylation of Rab GTPases. The aim of this study was to identify the molecular targets of RIS and 3-PEHPC in human myeloma cells and to determine the cellular effects of selective inhibition of Rab prenylation by 3-PEHPC as compared to nonspecific inhibition of protein prenylation by RIS in human myeloma cells. RIS dose-dependently inhibited prenylation of both Rap1A and Rab6, whereas 3-PEHPC only inhibited Rab6 prenylation. Both RIS and 3-PEHPC dose-dependently increased apoptosis in human myeloma cells. RIS induced an accumulation of cells in the S-phase of the cell cycle, associated with inhibition of DNA replication. In contrast, 3-PEHPC did not cause cell-cycle arrest. Furthermore, geranylgeraniol could prevent inhibition of prenylation, induction of apoptosis, and cell-cycle arrest in response to RIS, but not inhibition of Rab prenylation and apoptosis induced by 3-PEHPC, consistent with specific inhibition of Rab geranylgeranyl transferase by 3-PEHPC. In conclusion, our studies demonstrate that selective inhibition of Rab prenylation induces apoptosis, but not S-phase arrest, thus identifying distinct molecular pathways that mediate the antimyeloma effect of nitrogen-containing BPs.     

Serum concentrations of Dickkopf-1 protein are increased in patients with multiple myeloma and reduced after autologous stem cell transplantation

Dickkopf-1 (DKK-1) protein, a soluble inhibitor of Wnt signalling, has been implicated in the pathogenesis of myeloma bone disease through the suppression of osteoblast differentiation. In this study, serum concentrations of DKK-1 were measured in 50 myeloma patients (32 at diagnosis and 18 before and after autologous stem cell transplantation (ASCT), 18 patients with monoclonal gammopathy of undetermined significance (MGUS), and 22 healthy controls. Serum DKK-1 levels were increased in MM at diagnosis compared with MGUS (mean +/- SD: 67 +/- 54 ng/mL vs. 38 +/- 13 ng/mL; p = 0.006) and controls (31 +/- 11 ng/mL; p = 0.02), while there was no difference between MGUS patients and controls. Although patients with stage 2 and 3 myeloma had higher DKK-1 values than stage 1 patients (79 +/- 63 vs. 40 +/- 13; p = 0.005), no significant correlation between serum DKK-1 and myeloma bone disease was observed. Myeloma patients before ASCT also had increased levels of DKK-1 (63 +/- 77 ng/mL; p = 0.03) compared with controls, supporting the notion that DKK-1 may be responsible for the suppressed osteoblast activity even in patients with low tumor burden. After ASCT, there was a sustained decrease in DKK-1 levels over time, while bone formation markers elevated, suggesting that the reduction of DKK-1 levels after ASCT may correlate with the normalization of osteoblast function. These results could provide the basis for developing agents that block DKK-1, thus restoring osteoblast function and counteracting the increased osteoclastogenesis observed in myeloma.     

The next generation of epidermal growth factor receptor tyrosine kinase inhibitors in the treatment of lung cancer

The discovery of “driver” genomic alterations in patients with non‐small cell lung cancer (NSCLC) has dramatically changed the field of thoracic oncology in recent years. The best understood of these molecular drivers are those involving the epidermal growth factor receptor (EGFR), which when aberrantly activated are integral to the development of a subset of NSCLC tumors. First‐generation and second‐generation tyrosine kinase inhibitors (TKIs) specific to the activated EGFR have shown significant efficacy and have brought about the era of targeted therapy for NSCLC. The most common resistance mechanism is a threonine‐to‐methionine substitution (T790M) in exon 20 of the EGFR gene. Although the previous standard of care in patients with EGFR‐mutated NSCLC that progressed on initial TKI therapy was chemotherapy, third‐generation EGFR TKIs have now been developed and have yielded promising results for this population of patients with NSCLC. This article reviews the emerging data regarding third‐generation agents in the treatment of patients with advanced NSCLC. Cancer 2015;121:E1–E6. © 2014 American Cancer Society.     

Circulating human papillomavirus type 16 specific T-cells are associated with HLA Class I expression on tumor cells, but not related to the amount of viral oncogene transcripts

Human papillomavirus (HPV) is a necessary factor in the pathogenesis of cervical cancer. Circulating HPV-specific T-cells responding to the E6 and E7 HPV proteins can be detected only in half of cervical cancer patients. Potential explanations for the absence of this response are lack of sufficient amounts of antigen to activate the immune response or local immune escape mechanisms. We studied the relationship between HPV 16 E6/E7 oncogene mRNA expression, human leukocyte antigen (HLA) expression on tumor cells and the presence of circulating E6- and E7-specific T-cell responses in cervical cancer patients. The amount of antigen was assessed by HPV E6/E7 mRNA expression levels measured by quantitative polymerase chain reaction. HLA Class I and Class II expression on tumor cells was analyzed by immunohistochemistry. A proliferative HPV-specific T-cell response was detected in 15/29 patients. The amount of HPV E6/E7 mRNA was not related to the presence of immune response. HLA Class I expression was downregulated in 19 patients and completely lost in 7 patients. HLA Class II expression was upregulated in 18 patients. HLA Class I expression on tumor cells showed a strong correlation with immunity (p = 0.001). Explicitly, all patients with complete HLA loss lacked HPV specific T-cell responses. The presence of circulating HPV-specific T-cells might reflect ongoing antitumor response that is sustained by CD8+ T-cells killing HLA Class I positive cancer cells. We hypothesize that HLA Class I expression status on tumor cells might as well influence the response to HPV E6/E7 directed immunotherapy.     

Management of acquired resistance to epidermal growth factor receptor kinase inhibitors in patients with advanced non‐small cell lung cancer

The widespread adoption of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors for the first‐line treatment of patients with advanced EGFR‐mutated non‐small cell lung cancer has resulted in acquired tyrosine kinase inhibitor resistance becoming a ubiquitous clinical problem. The identification of specific mechanisms of acquired resistance has allowed a better understanding of the biology and natural history of resistant disease, but is only now starting to impact treatment decisions. Strategies for managing acquired resistance in patients with advanced non‐small cell lung cancer are complex and must be adapted to the individual characteristics of each patient's cancer. Although combination chemotherapy is the presumed standard of care for most patients, prospective trial data are lacking, highlighting the importance of offering patients participation in clinical trials in this setting. Emerging data from trials of third‐generation mutant‐specific EGFR kinase inhibitors suggests particular promise with this class of agents. Cancer 2014;120:2289–2298. © 2014 American Cancer Society.     

Release of nucleophosmin from the nucleus: Involvement in aloe-emodin-induced human lung non small carcinoma cell apoptosis

Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone) is one of the active constituents from the root and rhizome of Rheum palmatum. Our previous study has demonstrated that aloe-emodin induced a significant change in the expression of lung cancer cell apoptosis-related proteins compared to those of control cells. However, the molecular mechanisms underlying the biological effects of aloe-emodin still remain unknown. Based on these reasons, we were interested in the change of aloe-emodin-induced total protein expression by the proteomics technique during aloe-emodin-induced lung cancer cell apoptosis. Our study applied 2D electrophoresis to analyze the proteins involved in aloe-emodin-induced apoptosis in H460 cells. We found that the release of nucleophosmin from the nucleus to the cytosol and the degradation of nucleophosmin were associated with aloe-emodin-induced H460 cell apoptosis. Our study also demonstrated that the gene expression of nucleophosmin remained unchanged after treatment with aloe-emodin. The aloe-emodin-caused increase in the amount of proform and fragment of nucleophosmin in cytoplasm may be one of the important events for aloe-emodin-induced H460 cell apoptosis.     

CHOP or THP‐COP regimens in the treatment of newly diagnosed peripheral T‐cell lymphoma,not otherwise specified: a comparison of doxorubicin and pirarubicin

The CHOP regimen consisting of cyclophosphamide, doxorubicin (DOX), vincristine and prednisolone has been the most used regimen for peripheral T‐cell lymphoma, not otherwise specified (PTCL‐NOS). Pirarubicin [tetrahydropyranyladriamycin (THP)], a derivative of DOX, is an anthracycline with reportedly less cardiotoxicity than DOX. Here, we confirmed the efficacy of THP‐COP using THP instead of DOX in the treatment of PTCL‐NOS. The study protocol employed a retrospective, consecutive entry design. We retrospectively analysed 56 patients with PTCL‐NOS who had received THP‐COP or CHOP. These regimens were performed every 21 days. Twenty‐nine patients received THP‐COP, and 27 received CHOP. There were no significant differences in known prognostic factors, including in the International Prognostic Index (IPI) and the prognostic index for T‐cell lymphoma (PIT), between the two groups. Complete remission rates in patients with THP‐COP and CHOP were 52% in both groups; the 3‐year overall survival (OS) rates were 67% and 52% (p = 0.074), and the 3‐year progression‐free survival (PFS) rates were 51% and 29% (p = 0.070), respectively. In patients with low IPI (low or low‐intermediate), THP‐COP had significantly better 3‐year OS (100% vs. 64%; p < 0.001) and 3‐year PFS (75% vs. 33%; p < 0.05) than CHOP. Similar differences between THP‐COP and CHOP were observed in patients with a low PIT (groups 1 or 2). Our study showed that THP‐COP produced results equivalent to CHOP regarding efficacy and safety in patients with PTCL‐NOS. In patients with low IPI or PIT, THP‐COP resulted in significantly better prognosis. Copyright © 2015 John Wiley & Sons, Ltd.     

Increased HMGB1 and cleaved caspase-3 stimulate the proliferation of tumor cells and are correlated with the poor prognosis in colorectal cancer

Background

Dying tumor cells after irradiation could promote the proliferation of living tumor cells might cause tumor relapse and treatment failure. Our previous study showed that activated caspase-3 after irradiation probably participates in tumor repopulation. In this study, we investigated whether high mobility group box 1(HMGB1) is also involved in tumor repopulation.

Methods

Colorectal tumor cells were irradiated. The cleaved caspase-3 (CC3) in irradiated tumor cells and HMGB1 in the supernatant of irradiated tumor cells were detected by Western blot. A large number of irradiated colorectal tumor cells (feeder cells) were then co-cultured with a small number of luciferase-labeled living colorectal tumor cells (reporter cells) and proliferation of reporter cells was measured by bioluminescence imaging. The CC3 and HMGB1 protein expression in colorectal tumor and peritumoral tissues were detected by immunohistochemistry and their correlation with prognosis were analyzed.

Results

The irradiated colorectal tumor cells underwent apoptosis and necrosis and produced CC3 in tumor cells and HMGB1 in the supernatant of cultured cells. The increased expression of secretory HMGB1 correlated with CC3 level and proliferating cell nuclear antigen (PCNA) after irradiation in vitro. The irradiated dying cells remarkably stimulated living tumor cell proliferation. Interestedly, immunohistochemistry staining showed that positive HMGB1, CC3, and Ki67 expression were significantly higher in colorectal tumor tissues than in peritumoral tissues (p <0.01). The Kaplan-Meier survival analysis revealed that high HMGB1, CC3, and Ki67 levels were significantly associated with poor prognosis (p <0.05, p <0.01). Multivariate analysis using Cox proportional hazards model showed that TNM staging and HMGB1 were independent prognostic factors in patients with colorectal cancer (CRC) (p <0.01, p <0.001).

Conclusion

Both apoptotic and necrotic cells could stimulate proliferation of living tumor cells, and the increased expression of CC3 and HMGB1 in tumor cells could be new markers for poor prognosis in colorectal cancer patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0166-1) contains supplementary material, which is available to authorized users.     

Soluble and membrane levels of molecules involved in the interaction between clonal plasma cells and the immunological microenvironment in multiple myeloma and their association with the characteristics of the disease

Clonal plasma cells (PC) from different types of monoclonal gammopathies (MG) display distinct phenotypes consistent with an increased antigen‐presentation and T‐cell costimulation in MG of undetermined significance that deteriorates in malignant conditions. Expression of other cell surface and soluble molecules (e.g. adhesion/proliferation molecules) involved in the interaction between clonal PC and the bone marrow (BM) microenvironment has also been related to malignant PC, although the exact clinical significance of their expression remains largely unknown. Analysis of cell surface levels of several of these molecules in multiple myeloma (MM) patients shows an association between lower expression on BMPC of the HLA‐I and β2‐microglobulin antigen‐presenting molecules, the CD126 and CD130 IL6 receptor (IL6R) chains, and CD38, and adverse prognostic features of the disease. Likewise, patients showing higher soluble levels of antigen‐presenting molecules (HLA‐I and β2‐microglobulin), IL6R and CD95 tended to be associated with more aggressive disease behavior. In contrast, CD40, CD86, CD56, CD19, and CD45 were not associated with patients' outcome. Interestingly, upon considering the ratio between the soluble and PC membrane expression of each molecule, an increased adverse prognostic impact was observed for both HLA‐I and β2‐microglobulin, but not for the other molecules. Multivariate analysis confirmed the independent prognostic value of cell surface expression of CD126 on BMPC together with serum β2‐microglobulin and LDH. In summary, our results show an abnormal distribution of the cellular and soluble compartments of the HLA‐I, IL6R, and to a lower extent, CD95 molecules, in MM, associated with the clinical characteristics and behavior of the disease. © 2008 Wiley‐Liss, Inc.     

Kinetics of the early recruitment of leukocyte subsets at the sites of tumor cells in the lungs: natural killer (NK) cells rapidly attract monocytes but not lymphocytes in the surveillance of micrometastasis

Early host defense mechanisms play a critical role for the outcome of metastatic disease but most of the initial steps of such responses against tumor cells are still unknown. Here, the specificity and kinetics of leukocyte subsets in response to intravenous inoculation of vital dye labeled Fischer 344 rat syngeneic MADB106 tumor cells were monitored in lungs in situ by immunohistochemistry and image analysis over a time-period of 6 hr. In comparison with sham injections, tumor cell inoculation induces a dynamic sequence of rapidly increasing granulocyte (+40% at 5 min), NK and T cell (+60% at 15 min) as well as monocyte (+100% at 30 min) numbers in lung tissue. Already within the first minutes frequent colocalizations of granulocytes and NK cells with tumor targets were found in situ. Within the first hour NK cells selectively kill tumor targets, because depletion of NK cells in vivo drastically increases both the number of MADB106 cells retained in lungs and the emerging numbers of lung tumor colonies. In addition, the tumor-cell-induced increase of monocytes strictly depends on the presence of NK cells because NK-depletion completely abrogates the time specific response of monocytes. Under NK depleted conditions the tumor-induced recruitment of CD4(+) T cells is more pronounced suggesting a compensatory mechanism. In contrast, B cell numbers progressively decrease within hours after cell inoculation. These findings demonstrate that NK and T cells mediate the initial steps in the surveillance of lung metastasis. NK cells rapidly kill tumor cells and subsequently recruit monocytes in vivo.