RESUMO
In the recent decades, ion channels became the focus of cancer biologists, as many channels are overexpressed in tumour tissue and functionally they are linked to abnormal cell behaviour with processes including apoptosis, chemo- and radioresistance, proliferation and migration. KCa3.1 is a Ca2+-activated K+ channel that plays a central role in tumour progression in many cancer types. Therefore, the aim of the present study was to investigate KCa3.1 expression in pancreatic cancer cells and assess possible implications to disease progression. Using qPCR technique, we found abundant expression of KCa3.1 in pancreatic cancer cell lines. Patch clamp measurements on MiaPaCa-2 cells revealed a Ca2+-activated K+ current that matched biophysical characteristics as described for KCa3.1. Moreover, the current was sensitive to the commonly used channel modulators TRAM-34, clotrimazole and DC-EBIO, and it was abolished following transient gene knockdown of KCa3.1. We utilized both pharmacology and RNAi to assess a possible role of the channel in tumour cell behaviour. We found that the channel supported MiaPaCa-2 cell proliferation. Using RNAi protocols, we also identified KCa3.1 as important entity in cell invasion. However, TRAM-34 had unexpected stimulatory effects on cell migration and invasion estimated in various assays. Moreover, TRAM-34 increased intracellular Ca2+. In conclusion, we found prominent functional expression of KCa3.1 in pancreatic cancer cells. We provide evidence that the channel has a key role in cell proliferation and for the first time identify KCa3.1 as important entity in PDAC cell migration. We further reveal anomalous effects of TRAM-34.
Assuntos
Adenocarcinoma/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Neoplasias Pancreáticas/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Pirazóis/farmacologia , Potenciais de Ação , Benzimidazóis/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Clotrimazol/farmacologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca(2+)-activated Cl(-) channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca(2+) and voltage-dependent Cl(-) currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16Ainh-A01, CaCCinh-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.
Assuntos
Adenocarcinoma/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Anoctamina-1 , Cálcio/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Cloretos/metabolismo , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
Members of the TMEM16 family have recently been described as Ca(2+)-activated Cl(-) channels. They have been implicated in cancer and appear to be associated with poor patient prognosis. Here, we investigate the role of TMEM16 channels in cell migration, which is largely unknown. We focused on TMEM16A and TMEM16F channels that have the highest expression of TMEM16 channels in Ehrlich Lettre ascites (ELA) cells. Due to the lack of specific pharmacological modulators, we employed a miRNA approach and stably knocked down the expression of TMEM16A and TMEM16F channels, respectively. Migration analysis shows that TMEM16A KD clones are affected in their directional migration, whereas TMEM16F KD clones show a 40 % reduced rate of cell migration. Moreover, TMEM16A KD clones have a smaller projected cell area, and they are rounder than TMEM16F KD clones. The morphological changes are linearly correlated with the directionality of cells. TMEM16A and TMEM16F, thus, have an important function in cell migration-TMEM16A in directional migration, TMEM16F in determination of the speed of migration. We conclude that TMEM16A and TMEM16F channels have a distinct impact on the steering and motor mechanisms of migrating ELA cells.