RESUMEN
Cyclopropane fatty acid synthase (CFAS) catalyzes the transfer of a methylene group from S-adenosyl methionine to an unsaturated fatty acid, generating a cyclopropane fatty acid (CFA). The gene encoding CFAS is present in many bacteria and several Leishmania spp. including Leishmania mexicana, Leishmania infantum and Leishmania braziliensis. In this study, we characterised the CFAS-null and -overexpression mutants in L. mexicana, the causative agent for cutaneous leishmaniasis in Mexico and central America. Our data indicate that L. mexicana CFAS modifies the fatty acid chain of plasmenylethanolamine (PME), the dominant class of ethanolamine glycerophospholipids in Leishmania, generating CFA-PME. While the endogenous level of CFA-PME is extremely low in wild type L. mexicana, overexpression of CFAS results in a significant increase. CFAS-null mutants (cfas-) exhibit altered cell shape, increased sensitivity to acidic pH, and aberrant growth in serum-free media. In addition, the CFAS protein is preferentially expressed during the proliferative stage of L. mexicana and is required for the cell membrane targeting of lipophosphoglycan. Finally, the maturation and localization of CFAS protein are dependent upon the downstream sequence of the CFAS coding region. Without the downstream sequence, the mis-localised CFAS protein cannot fully rescue the defects of cfas-. Our data suggest that CFA modification of phospholipids can significantly affect the parasite's response to certain adverse conditions. These findings are distinct from the roles of CFAS in L. infantum, highlighting the functional divergence in lipid modification among Leishmania spp.
Asunto(s)
Ácidos Grasos/biosíntesis , Leishmania mexicana/metabolismo , Metiltransferasas/metabolismo , Animales , Southern Blotting , Western Blotting , Ciclopropanos , Concentración de Iones de Hidrógeno , Leishmania mexicana/citología , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/genética , Leishmaniasis Cutánea/parasitología , Lípidos/análisis , Macrófagos/parasitología , Metiltransferasas/química , Metiltransferasas/genética , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Plasmalógenos/química , Plasmalógenos/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
BACKGROUND AND OBJECTIVE: Long non-coding RNAs can regulate tumorigenesis of various cancers. Dys-regulation of lncRNA-AFAP1-AS1 has not been studied in colorectal carcinoma (CRC). This study was to examine the function involvement of AFAP1-AS1 in tumor growth and metastasis of CRC. METHODS: Relative expression of AFAP1-AS1 in CRC tissues and CRC cells lines was determined using quantitative real-time PCR (qRT-PCR). Functional involvement of AFAP1-AS1 in tumor proliferation and metastasis was evaluated in AFAP1-AS1-specific siRNA-treated CRC cells and in CRC cell xenograft. Expression of epithelial-mesenchymal transition (EMT)-related gene expression was determined using western blot. RESULTS: Relative expression of AFAP1-AS1 was significantly elevated in CRC tissues and CRC HCT116 and SW480 cell lines. AFAP1-AS1 knock-down suppressed SW480 cell proliferation, colony formation, migration and invasion. Also AFAP1-AS1 knock-down inhibited tumor metastasis-associated genes expression in terms of EMT. This carcinostatic action by AFAP1-AS1 knock-down was further confirmed by suppression of tumor formation and hepatic metastasis of CRC cells in nude mice. CONCLUSION: lncRNA-AFAP1-AS1 knock-down exhibits antitumor effect on colorectal carcinoma in respects of suppression of cell proliferation and metastasis of cancer cells.
Asunto(s)
Carcinoma/secundario , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/secundario , ARN Largo no Codificante/metabolismo , Animales , Western Blotting , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patología , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Neoplasias Hepáticas/genética , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , ARN Largo no Codificante/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Tumorales CultivadasRESUMEN
BACKGROUND AND OBJECTIVE: Long non-coding RNAs can regulate tumorigenesis of various cancers. Dys-regulation of lncRNA-AFAP1-AS1 has not been studied in colorectal carcinoma (CRC). This study was to examine the function involvement of AFAP1-AS1 in tumor growth and metastasis of CRC. METHODS: Relative expression of AFAP1-AS1 in CRC tissues and CRC cells lines was determined using quantitative real-time PCR (qRT-PCR). Functional involvement of AFAP1-AS1 in tumor proliferation and metastasis was evaluated in AFAP1-AS1-specific siRNA-treated CRC cells and in CRC cell xenograft. Expression of epithelial-mesenchymal transition (EMT)-related gene expression was determined using western blot. RESULTS: Relative expression of AFAP1-AS1 was significantly elevated in CRC tissues and CRC HCT116 and SW480 cell lines. AFAP1-AS1 knock-down suppressed SW480 cell proliferation, colony formation, migration and invasion. Also AFAP1-AS1 knock-down inhibited tumor metastasis-associated genes expression in terms of EMT. This carcinostatic action by AFAP1-AS1 knock-down was further confirmed by suppression of tumor formation and hepatic metastasis of CRC cells in nude mice. CONCLUSION: lncRNA-AFAP1-AS1 knock-down exhibits antitumor effect on colorectal carcinoma in respects of suppression of cell proliferation and metastasis of cancer cells.
Asunto(s)
Humanos , Animales , Masculino , Carcinoma/secundario , Neoplasias Colorrectales/patología , ARN Largo no Codificante/metabolismo , Neoplasias Hepáticas/secundario , Células Tumorales Cultivadas , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Movimiento Celular , Western Blotting , Células HCT116 , Proliferación Celular , Técnicas de Silenciamiento del Gen , Transición Epitelial-Mesenquimal , Reacción en Cadena en Tiempo Real de la Polimerasa , ARN Largo no Codificante/análisis , Neoplasias Hepáticas/genética , Ratones Endogámicos C57BL , Ratones DesnudosRESUMEN
OBJECTIVES: This study was designed to investigate the activation of the unfolded protein response (UPR) in tumor associated endothelial cells (TECs) and its association with chemoresistance during acidic pH stress. MATERIALS AND METHODS: Endothelial cells from human oral squamous cell carcinomas (OSCC) were excised by laser capture microdissection (LCM) followed by analysis of UPR markers (Grp78, ATF4 and CHOP) using quantitative PCR. Grp78 expression was also determined by immunostaining. Acidic stress was induced in primary human dermal microvascular endothelial cells (HDMECs) by treatment with conditioned medium (CM) from tumor cells grown under hypoxic conditions or by adjusting medium pH to 6.4 or 7.0 using lactic acid or hydrochloric acid (HCl). HDMEC resistance to the anti-angiogenic drug Sunitinib was assessed with SRB assay. RESULTS: UPR markers, Grp78, ATF4 and CHOP were significantly upregulated in TECs from OSCC compared to HDMECs. HDMECs cultured in acidic CM (pH 6.0-6.4) showed increased expression of the UPR markers. However, severe acidosis led to marked cell death in HDMECs. Alternatively, HDMECs were able to adapt when exposed to chronic acidosis at pH 7.0 for 7 days, with concomittant increase in Grp78 expression. Chronic acidosis also confers drug resistance to HDMECs against Sunitinib. Knockdown of Grp78 using shRNA resensitizes HDMECs to drug treatment. CONCLUSIONS: UPR induction in ECs under acidic pH conditions is related to chemoresistance and may contribute to therapeutic failures in response to chemotherapy. Targeting Grp78, the key component of the UPR pathway, may provide a promising approach to overcome ECs resistance in cancer therapy.