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ABSTRACT Purpose: This study aimed to optimize the effective doses of mitomycin C, 5-fluorouracil, and their combination on cultivated basal cell carcinoma. Methods: Cultivated basal cell carcinoma and fibroblastic cells were treated with different concentrations of mitomycin C, 5-fluorouracil, and their combination. Cell viability, cell cycle, apoptosis, and expression levels of TP53, CDKN1A, and CDK6 were investigated. The most effective drug with its optimum dosage was administered via multiple intralesional injections to a 65-year-old woman with advanced periorbital nodulo-ulcerative BCC. Results: The concentrations of 0.00312 and 0.312 mg/mL were considered optimum for mitomycin C and 5-fluorouracil, respectively. The mean viabilities of basal cell carcinoma treated with mitomycin C alone and its combination with 5-fluorouracil were significantly less than those of the controls (p=0.002 and p=0.04, respectively). The cell cycle of all the treated basal cell carcinoma groups was arrested in the S phase. The apoptotic rates (p=0.002) of mitomycin C treated basal cell carcinoma were higher than those of the other treated cells, and their TP53 was significantly upregulated (p=0.0001). Moreover, CDKN1A was upregulated, whereas CDK6 was downregulated in basal cell carcinoma treated with either 5-fluorouracil (p=0.0001 and p=0.01, respectively) or the combination of 5-fluorouracil and mitomycin C (p=0.007 and p=0.001, respectively). Basal cell carcinoma lesions were significantly alleviated following mitomycin C injections in the reported patient. Conclusion: Our in vitro results revealed that the effective doses of mitomycin C and 5-fluorouracil on cultivated basal cell carcinoma were optimized. Mitomycin C was more effective in inducing the apoptosis of basal cell carcinoma than 5-fluorouracil and their combination. The intralesional injections of the optimum dose of mitomycin C could be proposed for the nonsurgical treatment of advanced eyelid basal cell carcinoma.
RESUMO Objetivo: Otimizar a dose efetiva de mitomicina C, 5fluorouracil e da combinação de ambos em culturas de células de carcinoma basocelular (CBC). Métodos: Culturas de células de células de carcinoma basocelular e de fibroblastos foram tratadas com diferentes concentrações de mitomicina C, 5fluorouracil e combinação de ambos. Além disto, foram investigados a viabilidade celular, o ciclo celular, a apoptose e a expressão dos genes TP53, CDKN1A e CDK6. O medicamento mais eficaz, em sua dosagem otimizada, foi administrado em últiplas injeções intralesionais em uma mulher de 65 anos com carcinoma basocelular nódulo-ulcerativo periorbital avançado. Resultados: A concentração de 0,00312 mg/mL de mitomicina C e a de 0,312 mg/mL de 5fluorouracil foram consideradas as ideias. A viabilidade média das células de carcinoma basocelular tratadas com mitomicina C isoladamente e em combinação foi significativamente menor que nas células de controle (respectivamente, p=0,002 e p=0,04). Todos os grupos de carcinoma basocelular tratados demonstraram interrupção do ciclo celular na fase S. As células de carcinoma basocelular tratadas com mitomicina C mostraram maiores taxas de apoptose (p=0,002) e significativa regulação positiva do gene TP53 (p=0,0001). Além disso, o gene CDKN1A foi positivamente regulado e o gene CDK6 foi negativamente regulado em células de carcinoma basocelular tratadas com 5fluorouracil (respectivamente, p=0,0001 e p=0,01) ou com a combinação de medicamentos (respectivamente, p=0,007 e p=0,001). Injeções posteriores de mitomicina C na paciente em questão levaram à melhora significativa da lesão do carcinoma basocelular. Conclusão: Nossos resultados in vitro otimizaram as doses efetivas de mitomicina C e 5fluorouracil na cultura de células de carcinoma basocelular e mostraram que a mitomicina C tem mais eficácia na apoptose de células de carcinoma basocelular do que o 5fluorouracil e a combinação de ambos. Injeções intralesionais de doses otimizadas de mitomicina C podem ser propostas para o tratamento não cirúrgico do células de carcinoma basocelular avançado de pálpebra.
Subject(s)
Aged , Female , Humans , Skin Neoplasms , Carcinoma, Basal Cell , Carcinoma, Basal Cell/drug therapy , Antineoplastic Combined Chemotherapy Protocols , Survival Analysis , Mitomycin , FluorouracilABSTRACT
The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells. Methods: BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array. Results: BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05). Conclusion: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.(AU)
Subject(s)
Snake Venoms , Biomarkers , Bothrops , Carcinoma, Hepatocellular , Hep G2 Cells , EpigenomicsABSTRACT
@#[Abstract] Objective: To investigate the expression of long non-coding RNA LINC01001 in breast cancer tissues and its effect on the proliferation of breast cancer MCF-7 cells. Methods: The expression levels of lncRNA LINC01001 were analyzed in 12 cases of cancer and para-cancer tissues from breast cancer patients, who underwent surgical resection in Affiliated People’s Hospital of Hubei University of Medicine from March 2016 to June 2017. The plasmid over-expressing LINC01001 was transfected into human breast cancer MCF-7 cells. The cell cycle distribution and proliferation ability of MCF-7 cells were detected by flow cytometry and MTT assay, respectively. The mRNA expressions of miR-485-5p and CDKN1A mRNA were detected by qRT-PCR, and the protein expressions of CDKN1A, CDK4, CDK6 and Cyclin D1 were detected by Western blotting. Results: The expression level of lncRNA LINC01001 in breast cancer tissues was lower than that in para-cancer tissues (P<0.01). LINC01001 recombinant plasmid transfection significantly inhibited cell cycle progression (P<0.05) and cell proliferation (P<0.05) of MCF-7 cells. qRT-PCR showed that the expression level of miR-485-5p was decreased (P<0.01) and the expression level of CDKN1A mRNA was increased (P<0.01) after over-expressing LINC01001. Western blot results confirmed that over-expression of LINC01001 could promote the expression of CDKN1A protein, but decrease the expressions of CDK4, CDK6 and Cyclin D1 proteins. Conclusion: The expression of LINC01001 in breast cancer tissues was decreased. LINC01001 may down-regulate the expression of miR-485-5p to up-regulate the expression of CDKN1A, and further to inhibit the proliferation of breast cancer MCF-7 cells, providing experimental basis for the clinical application of lncRNA.
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@# Objective: To study the effects of microRNA-1180-5p (miR-1180-5p) on malignant biological behaviors of prostate cancer VCAP and LNCaP cells and the possible mechanisms. Methods: dsControl (dsControl group) and miR-1180-5p (miR-1180-5p group) were constructed and then transfected into two prostate cancer cell lines VCAPand LNCaP. qPCR and Western blotting were used to analyze the changes in mRNA and protein expressions of CDKN1A, Cyclin D1 and CDK6 after transfection. Cell cycle distribution, proliferation activity, clone formation capacity, cell migration and invasion ability were detected by flow cytometry, MTT assay, colony culture assay and Transwell assay, respectively. Results: qPCR results showed that compared with dsControl, CDKN1A mRNA levels in VCAP and LNCaP cells transfected with miR-1180-5p were up-regulated significantly, while the mRNA expressions of Cyclin D1 and CDK6 were significantly down-regulated (all P<0.01). Western blotting result was consistent with that of qPCR. The percentage of cells in G0/G1 phase was increased after transfection of miR-1180-5p (P<0.05), but the proportion of cells in S phase and G2/M phase was decreased and the cell cycle was arrested at G0/G1 phase (P<0.05). The proliferation activity of the two prostate cancer cells was significantly lower than that of the dsControl group after miR-1180-5p transfection (P<0.05), and the number of colonies in the miR-1180-5p group was significantly lower than that in the dsControl group (P<0.01). In the meanwhile, the cell migration and invasion ability in miR-1180-5p group was decreased (P<0.01). Conclusion: miR-1180-5p can significantly activate CDKN1A gene expression in prostate cancer cells and further inhibit the proliferation, migration and invasion of prostate cancer cells.
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Background and purpose: Accumulating evidence has revealed that long non-coding RNA (lncRNA) is correlated with carcinogenesis and tumor development. Recent literature suggested that lncRNA promoter of CDKN1A antisense DNA damage activated RNA (PANDAR) was involved in the development of various cancers. However, the functional role of PANDAR in colorectal cancer (CRC) has not been elucidated yet. The present study aimed to explore the functional role of lncRNA PANDAR in promoting CRC metastasis and its mechanism.Methods: The expression of lncRNA PANDAR in CRC cell lines and tissues was detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR), and the correlation between lncRNA PANDAR expression and CRC clinicopathological characteristics was statistically analyzed. Then, lncRNA PANDAR stably silencing CRC cells (HCT116-shPANDAR), overexpression cells (DLD1-PANDAR) and control vector cells (HCT116-shNC and DLD1-vector) were established using lentiviral vectors. Moreover, Transwell assay and Matrigel assay were performed to investigate the function of lncRNA PANDAR in CRC migration and invasion. Furthermore, the expression of transcriptional factors mediating epithelial-mesenchymal transition of lncRNA PANDAR overexpression cells were monitored by RTFQ-PCR assay, and the function of the target gene in modulating lncRNA PANDAR mediated CRC metastasis was also explored. Results: The expression levels of lncRNA PANDAR in normal colorectal epithelial cells were much lower than in CRC cell. The levels of lncRNA PANDAR in tumor-adjacent tissues were verified to be much lower than in CRC tissues [(171.52±97.80)% vs (100.00±63.18)%, P<0.05]. Moreover, the expression of lncRNA PANDAR was detected to be significantly correlated with CRC TNM stage, lymph node metastasis and distant metastasis (P<0.05). Besides, lncRNA PANDAR deficiency significantly reduced the migration [100.00% vs (42.08±4.77)%, P<0.05] and invasion [100.00% vs (39.14±3.81)%, P<0.05] capabilities in CRC cells, in contrast, the migration [100.00% vs (194.12±9.33)%, P<0.05] and invasion [100.00% vs (204.08±12.27)%, P<0.05] capa-bilities of CRC cells were obviously increased with lncRNA PANDAR overexpression. Furthermore, zinc-finger E-box binding homeobox 1 (ZEB1) expression was detected to be positively correlated with lncRNA PANDAR expression, and ZEB1 silencing could significantly reverse the increased migration and invasion capabilities induced by lncRNA PANDAR in CRC cells. Conclusion: LncRNA PANDAR could promote CRC metastasis by potentially targeting ZEB1. LncRNA PANDAR might be a promising diagnostic marker and therapeutic target for CRC patients.
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Aims: Progressive loss of cell cycle control is an important feature on the colorectal cancer. CDKN1A gene encoded p21 protein that’s one of the cyclin-dependent kinase inhibitors, plays a key role in regulating the cell cycle. The aim of this study was toinvestigate associations of the CDKN1A gene polymorphism rs762624 and rs3176336 with risk of colorectal cancer in an Iranian population. Methods: A case-controls study was conducted to investigate the association of polymorphism rs3176336 and rs762624, with colorectal cancer risk in Iranian population. In this study 150 cases of sporadic CRC and 150 healthy controls were recruited, genomic DNA were extracted from peripheral blood, the genotypes were determined using the polymerase chain reaction and restriction fragment length polymorphism (PCRRFLP) method and the result was validated by direct sequencing. Results: The rs762624 frequencies of the AA, AC, and CC genotypes among cases were 9.3%, 74.7%, and 16%, respectively, while in controls genotype frequencies were 10%, 74%, and 16%, respectively. The rs3176336 frequencies of the AA, AC, and CC genotypes among cases were 29.3%, 18% and 52.7%, respectively, while in controls genotype frequencies were18%, 20%, and 62%, respectively. No association was found for the CDKN1A rs3176336 AT/AA genotype (Adjusted odds ratio (OR), 0.726, 95% confidence interval (CI), 0.365–1.443 for AT genotype; OR, 1.67, 95% CI, 0.754–3.702 for AA genotype) with risk of colorectal cancer, compared with the TT genotype. In our research, we could not found significant relation between stage of colorectal cancer and genotypes of rs762624 and rs3176336 polymorphisms (p=0.081, p=0.988). Conclusion: Present data do not confirm association of rs3176336 and rs762624 polymorphisms with susceptibility of Iranian to colorectal cancer.
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Objective:To explore the ameliorative effect and machanism of MSCs conditioned medium on the ovarian granulosa cells damage induced by triptolide.Methods: Cell Counting Kit 8 assay was used to examine the cell vitality of KGNs with the treatment of triptolide.The mixed enzyme digestion method were used for the isolation of human umbilical cord mesenchymal stem cells (MSCs),and flow cytometry was used for the subsequent immunotype identification.MSCs conditioned medium was collected ,and Cell Counting Kit 8 assay and PI staining was used to analyse the effect of MSCs conditioned medium on the cell vitality and cell cycle distri -bution of triptolide-damaged KGN.Real-time PCR method was used to examine the expression of cell cycle related gene CDKN1A.Results:Triptolide can inhibit KGN cell growth with the inhibition of cell vitality and cell cycle of KGN.MSCs conditioned medium did not influence the proliferation and cell cycle of normal KGN , but improved the triptolide-induced vitality inhibition and extent of S-phase arrest, and inhibit the abnormal up-regulation of CDKN1A in KGN.Conclusion: MSCs conditioned medium ameliorated the KGN cell damage induced by triptolide.
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Histone deacetylase inhibitors (HDIs), a new class of anti-cancer agents, have been reported to suppress formation of osteoclast precursors and their fusion into multinucleated cells. However, little is known about the effect of HDIs on mature osteoclasts, which may have significance for their therapeutic use. Here, we demonstrate a novel action of HDIs on osteoclast apoptosis. Primary multinucleated mature osteoclasts were prepared from mouse bone marrow cells. Treatment of osteoclasts with the HDI trichostatin A (TSA) caused apoptosis, as confirmed by annexin V staining and caspase activation. TSA caused the upregulation of p21WAF1 in osteoclasts. To understand the role of p21(WAF1) upregulation in TSA-treated osteoclasts, shRNA against p21(WAF1)-containing lentivirus was introduced into osteoclasts. The suppression of p21(WAF1) decreased TSA-directed osteoclast apoptosis. Collectively, our results provide evidence that TSA causes osteoclast apoptosis, which involves, in part, TSA-induced upregulation of p21(WAF1), and strongly supports HDIs as potential therapeutic agents for excessive bone resorption.