Effects of Curcumin on Sodium Arsenite Induced Neoplastic Cell Transformation in Balb/c 3T3 Cells

HIGHLIGHTS Sodium arsenite can cause neoplastic transformation in cells. Curcumin reduced cell viability and increased LDH activity in transformed Balb/c 3T3 cells. Curcumin caused DNA damage in transformed Balb/c 3T3 cells. Curcumin may play a protective role in sodium arsenite-induced toxicity.

Abstract Arsenic is a toxic substance that spreads widely around the environment and accumulates as metalloid in the earth's crust. Arsenic and its derivatives are found in drinking water, nutrients, soil, and air. Exposure to arsenic is associated with lung, blood, skin cancer and various lesions. Curcumin is a polyphenolic compound derived from Curcuma longa (turmeric) rhizome and is one of the main curcuminoids. Curcumin is known to be antioxidant, antibacterial, anti-inflammatory, analgesic effects. This study aimed to investigate the potential of sodium arsenite to transform embryonic fibroblast cells and to evaluate the cytotoxic and genotoxic effects of curcumin in neoplastic transformed cells. Neoplastic cells transformation was induced by sodium arsenite in Balb/c 3T3 cells at the end of 32 days. After transformation assay, the transformed cells were treated with various concentration of curcumin to evaluate cell viability, lactate dehydrogenase activity and DNA damage for 24h. The results revealed that curcumin decreased cell viability and increased the activity of lactate dehydrogenase enzyme in neoplastic transformed Balb/c 3T3 cells. In conclusion, the results demonstrated that curcumin has an anticancer effect on neoplastic transformed Balb/c 3T3 cells by causing DNA damage.

Efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L / In vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the rhizome of Curcuma longa L

RESUMEN Objetivos: Determinar el efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L. Materiales y métodos: El efecto citotóxico fue evaluado utilizando líneas celulares DU-145, HT-29, 3T3 BALB/c. Se hallaron los porcentajes de crecimiento en 48 horas y se determinó la concentración inhibitoria 50 (CI50). El efecto genotóxico en el ADN genómico humano se determinó mediante el método Tomasevich. Resultados: El extracto crudo produjo una CI50 de 12,98 ± 0,21 μg/mL para la línea celular tumoral HT-29, que es inferior a DU-145 con una CI50 de 36,77 ± 9,12 μg/mL; el extracto etanólico presentó una CI50 de 13,24 ± 0,77 y 20,54 ± 2,58 µg/mL para ambas líneas celulares, respectivamente; el compuesto estándar curcumina presentó una CI50 de 3,96 ± 0,60 y 13,94 ± 2,79 μg/mL, respectivamente. El extracto crudo a concentraciones de 50 y 100 mg/mL fragmentó entre el 40% a 95% de ADN genómico humano; mientras que, a 200 mg/mL, la fragmentación fue mayor al 95%. El extracto etanólico a todas las concentraciones no fragmentó el ADN. La curcumina a 200 mg/mL fragmentó menos del 5% de ADN genómico humano. Conclusiones: Los extractos crudo y etanólico de Curcuma longa L. demuestran efecto citotóxico in vitro diferencial para la línea celular tumoral humana DU-145 y HT29 semejante al compuesto estándar curcumina. El extracto crudo de Curcuma longa L. presenta una potente actividad genotóxica in vitro frente al ADN genómico humano, esta actividad está ausente en el extracto etanólico.

ABSTRACT Objectives: To determine the in vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the Curcuma longa L. rhizome. Materials and methods: The cytotoxic effect was evaluated using DU-145, HT-29, 3T3 BALB/c cell lines. The growth percentages in 48 hours; and the half maximal inhibitory concentration (IC50) were determined. The genotoxic effect on human genomic DNA was determined using the Tomasevich method. Results: Crude extract produced an IC50 of 12.98 ± 0.21 μg/mL for the HT-29 tumor cell line, which is lower than the value obtained for DU-145, with an IC50 of 36.77 ± 9.12 μg/mL. The ethanolic extract presented an IC50 of 13.24 ± 0.77 and 20.54 ± 2.58 μg/mL for both cell lines, respectively; the curcumin standard compound presented an IC50 of 3.96 ± 0.60 and 13.94 ± 2.79 μg/mL, respectively. Crude extract concentrations of 50 and 100 mg/mL fragmented between 40% to 95% of human genomic DNA; while at 200 mg/mL, fragmentation was greater than 95%. The ethanolic extract at all concentrations did not fragment the DNA. Curcumin at 200 mg/mL fragmented less than 5% of human genomic DNA. Conclusions: The crude and ethanolic extracts of Curcuma longa L. demonstrate different in vitro cytotoxic effects for the human tumor cell lines DU-145 and HT-29; similar to the standard curcumin compound. The crude extract of Curcuma longa L. shows a potent genotoxic in vitro activity against human genomic DNA; this type of effect is not produced by the ethanolic extract.

Comparison of prediction performance of PAHs carcinogenicity between a BALB/c-E6E7 cell transformation assay and a BALB/c 3T3 cell transformation assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To predict the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) by cell transformation assay using BALB/c 3T3 cells and HPV16-E6E7-transfected BALB/c 3T3 cells (BALB/c-E6E7 cells).</p><p><b>METHODS</b>The cell transformation assays induced by PAHs using BALB-E6E7 cells and BALB/c 3T3 cells.</p><p><b>RESULTS</b>The initiating and promoting activities of PAHs examined in a BALB-E6E7 cell transformation assay were similar to in a BALB/c 3T3 cell transformation assay, which was up to the standard of agents classified by the IARC. There were much more transformed foci appeared and much shorter time consumed to accomplish phenotypic alterations in the BALB/c-E6E7 cell transformation assay than in the BALB/c 3T3 cell transformation assay. The BALB/c-E6E7 cell transformation assay was superior to the BALB/c 3T3 cell transformation assay in cost and labor performance, the sensitivity of transformation response.</p><p><b>CONCLUSION</b>The BALB/c-E6E7 cell transformation assay, with a satisfied prediction performance of initiating activity and promoting activity, would improve the overall process of safety and risk assessment of carcinogenicity.</p>

Mechanism of inhibitory effect of P7 on 3T3 cell proliferation induced by basic fibroblast growth factor / 药学学报

To investigate the mechanism of inhibitory effect of a novel bFGF antagonist peptide isolated from the phage display random heptapeptide library on cell proliferation induced by basic fibroblast growth factor. The effect of P7 on cell morphology was observed under an inverted microscope. Flow cytometry was applied to analyze the effect of P7 on cell cycle progress of bFGF-stimulated cells. The effect of P7 on bFGF-induced activation of MEK and Erk1/2 in MAPK pathway was detected by Western blotting. The results showed that no significant cell morphology change was observed in the range of detected concentrations of P7. Cell cycle analysis showed that P7 decreased S-phase cell population and arrested cell cycle at the G0/G1 phase of bFGF-stimulated cells. The results of MAP kinase activation assay indicated that P7 decreased bFGF-induced MEK and Erk1/2 phosphorylation in a dose-dependent manner. P7 inhibited proliferation of bFGF-stimulated Balb/c 3T3 cells possibly via cell cycle arrest at the G0/G1 phase and down-regulation of signal molecular activation in MAPK pathway.

An improved BALB/c 3T3 cell transformation assay and its application in the cocarcinogenesis study / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To improve the protocol of BALB/c 3T3 cell transformation assay, and apply it to the cocarcinogenesis study.</p><p><b>METHOD</b>Appropriate serum concentration, culture media and method of administration were selected by testing their effects on the growth and transformation of BALB/c 3T3 cells. The co-carcinogenic activity between diethylnitrosamine (DEN) and microcystin-LR (MC-LR) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined using the improved cell transformation assay. The malignant characteristics of transformed cells were verified by neoplasia in SCID mice.</p><p><b>RESULTS</b>There were strong co-carcinogenic activity between DEN and TCDD. On the contrary, although MC-LR has strong ability to induce cell transformation, the effect was markely inhibited by DEN. The transformed cells show some malignant characteristics.</p><p><b>CONCLUSION</b>The improved BALB/c 3T3 cell transformation assay is reliable and time-saving, and can be efficiently used in the study of cocarcinogenesis.</p>

Prokaryotic expression, purification of prM of JEV and preparation of monoclonal antibody / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare monoclonal antibody (mAb) against prM epitope.</p><p><b>METHODS</b>The gene encoding prM was isolated using RT-PCR from brain of JEV infected mouse and cloned into prokaryotic expression vector pET-32a. Recombinant plasmid was transformed into E.coli BL21/DE3/LysS, then the transformed cells were expressed with the induction of IPTG. The expression and purification of the prM protein was analyzed by SDS-PAGE. The BALB/c mice were immunized with purified prM protein. Hybridoma cell lines secreting monoclonal antibodies against prM were established after cell fusion of mouse splenic cell and P3-X63-Ag8.653 cells. The specificity of mAb was identified by ELISA, Western Blot and Immunohistochemistry assay.</p><p><b>RESULTS</b>mAb against prM epitope of JEV was prepared successfully.</p><p><b>CONCLUSION</b>The obtained prM specific mAb was valuable for the prevention and dignosis of Japanese encephalitis.</p>

A discussion on the biological evaluation method for nanosilver antibiotic devices for gynecological external use / 中国医疗器械杂志

Nanosilver antibiotic devices for gynecological external use are the third-class products of medical devices, whose biological safety and efficiency should be strictly controlled. But there is not yet the national standard or industry standard for the products to control the production process, so their testing method of biological evaluation mainly refers to GB/T16886 "The Guide to Implementation of Biological Evaluation of Medical Devices". To control the biological safety effectively, it's necessary to work out the testing items and methods of the biological evaluation for such products.

The protein X4 of severe acute respiratory syndrome-associated coronavirus is expressed on both virus-infected cells and lung tissue of severe acute respiratory syndrome patients and inhibits growth of Balb/c 3T3 cell line / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection.</p><p><b>METHODS</b>The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles.</p><p><b>RESULTS</b>We expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays.</p><p><b>CONCLUSION</b>The results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.</p>

Gene expression profiles in early stage of BALB/c 3T3 cells' transformation promoted with 12-O-tetradecanoylphorbol-13-acetate / 中华预防医学杂志

<p><b>OBJECTIVE</b>To elucidate the potential molecular mechanism responsible for the early time of tumor promotion, gene expression profile was studied in the transformed BALB/c 3T3 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).</p><p><b>METHODS</b>The two-stage cell transformation model was established by using the initiator of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promoter of TPA. Cell proliferation was measured by trypan blue staining and cell cycle analysis was carried out by flow cytometry assay. A cDNA microarray representing 1 152 genes was used to investigate the gene expression profiles of BALB/c 3T3 cells exposed to TPA at 4 h and 24 h respectively.</p><p><b>RESULTS</b>TPA could effectively inhibit cell proliferation and induce the G1 and S cell cycle arrested in the early time. Moreover 19 genes were found differentially expressed at least twofold in the TPA treated cells as compared with the control cells, 9 of them were upregulated and 10 downregulated. Most of the differentially expressed genes were involved in cell proliferation, differentiation or apoptosis, and related to ras or p53 signal transduction pathway.</p><p><b>CONCLUSION</b>TPA could influence the transcriptional expression of some genes related to cell cycle modulation and ultimately result in the cell growth arrest.</p>

Effect of aberrant DNA methylation on the expression of cancer-related genes in Cd-transformed cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study aberrant DNA methylation potentially resulting in changes in the expression of cancer-related genes as a possible epigenetic mechanism for cadmium carcinogenesis.</p><p><b>METHODS</b>Genomic DNA isolated from CdCl(2)-transformed BALB/c-3T3 cells was digested with Mse1 (methylation non-sensitive) alone or with Mse1 and BstU1 (methylation sensitive). The resulting DNA was analyzed for aberrant methylation using PCR-based technique-Methylation-Sensitive Restriction Fingerprinting (MSRF). Several DNA fragments differentially methylated in the transformed cells identified by MSRF were confirmed by Southern hybridization analysis using the aberrantly methylated DNA fragments as the probes.</p><p><b>RESULTS</b>Aberrant DNA methylation was identified in the transformed cells. DNA sequencing and sequence similarity analysis identified one of the aberrantly methylated DNA fragments as the p16 tumor suppressor gene.</p><p><b>CONCLUSION</b>DNA hypermethylation is known to result in gene silencing, it appears that hypermethylation of p16 gene may represent a possible epigenetic mechanism for Cd-induced cell transformation and carcinogenesis.</p>

Synergistic regulation of the acute phase protein SIP24/24p3 by glucocorticoid and pro-inflammatory cytokines / 生理学报

SIP24/24p3 is a secreted murine acute phase protein which has been speculated to play an anti-inflammatory role in vivo. Recently SIP24/24p3 has been found to be able to specifically induce apoptosis in leukocytes. By using (35)S metabolic labeling method, we studied the regulation of SIP24/24p3 by glucocorticoid and pro-inflammatory cytokines IL-6 and TNF-alpha in cultured Balb/c 3T3 and BNL cells. The following results were observed: (1) dexamethasone induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells, the induction was more significant in BNL cells; (2) dexamethasone and IL-6 synergistically induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells; (3) in Balb/c 3T3 cells dexamethasone and TNF-alpha acted synergistically to induce the expression of SIP24/24p3, whereas in BNL cells dexamethasone and TNF-alpha induced the expression of SIP24/24p3 in an additive manner; (4) dexamethasone and IL-6/TNF-alpha acted synergistically in Balb/c 3T3 cells and additively in BNL cells to induce the expression of SIP24/24p3. The inducibility of SIP24/24p3 by multiple factors will help to explain its highly specific expression in vivo. The difference in the expression patterns of SIP24/24p3 in different cell types is also suggestive to its expression and regulation in hepatic and extrahepatic tissues. Finally, the fact that SIP24/24p3 protein can be induced by both pro-inflammatory as well as anti-inflammatory factors is indicative of the important role of SIP24/24p3 in the entire acute phase response process.